MBL

Volume 178

THE

Number 1

BIOLOGICAL
BULLETIN

MAR

FEBRUARY, 1990

Published by the Marine Biological Laboratory

THE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

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Reference: liiol Hull 178: 1-9. (February, 1990)

The Role of Arachidonic Acid and Eicosatrienoic Acids

in the Activation of Spermatozoa in Arenicola

marina L. (Annelida: Polychaeta)

M. G. BENTLEY 1 *, S. CLARK 2 , AND A. A. PACEY 1

^Gatty Marine Laboratory, University of St. Andrews, St Andrews, Fife, KY16 8LB. Scotland, U.K.,
and 2 Dove Marine Laboratory and Department of Biology, University of Newcastle upon Tyne,

Newcastle upon Tyne, NE1 7RU, U.K.

Abstract. Partial purification of a sperm maturation
factor (SMF) in the intertidal polychaete Arenicola ma-
rina has implicated arachidonic acid, an arachidonic me-
tabolite, or a similar substance as the active factor from
the prostomium. The effects of a number of 20-carbon
fatty acids on inactive spermatozoa are investigated, and
this reveals that only arachidonic acid and 8,1 1,14-eico-
satrienoic acid cause sperm activation. The use of argen-
tation thin-layer chromatography to separate fatty acids
with varying degrees of unsaturation reveals a compo-
nent in prostomial lipid extract, which co-migrates with
eicosatrienoic acids. Investigations using cyclooxygenase
and lipoxygenase result in a loss of sperm-activating
properties of both prostomial extract and fatty acids. The
use of cyclooxygenase and lipoxygenase inhibitors has no
effect. Bovine serum albumin (BSA) reduces the sperm
activating properties of both fatty acids and prostomial
extract in a dose-dependant way. Additional purification
procedures using: (a) organic solvents and aqueous buff-
ers and (b) ODS silica cartridges, demonstrate that the
active fraction of prostomial extract co-elutes at every
step with the 8,1 1,14-eicosatrienoic acid standard. Gas
chromatography of methyl esters of prostomial lipid ex-
tract reveals the presence of a peak with an identical re-
tention time to the methyl ester of authentic 8,1 1,14-ei-
cosatrienoic acid standard. The results described here
provide strong evidence that the active SMF in prosto-
mial homogenate is not a fatty acid metabolite but the
parent acid 8,1 1,14-eicosatrienoic acid. These results
could only be made unequivocal by full structural analy-

Received 28 August 1989; accepted 30 November 1989.
* To whom all correspondence should be addressed.

sis using mass spectrometry and NMR following capil-
lary gas-liquid chromatography.

Introduction

Arenicola marina is a common intertidal polychaete.
Its reproductive cycle is annual, with most populations
found around the coasts of the British Isles spawning in
the autumn or early winter (Howie, 1959). The repro-
ductive biology of this species has been reviewed by
Howie (1984). In both sexes, gamete proliferation occurs
in the gonads, and early germ cells are released into the
coelomic fluid where gametogenesis proceeds (Ash-
worth, 1904; Newell, 1948). Females approaching matu-
rity are characterized by many oocytes that have com-
pleted vitellogenesis but are arrested in prophase of mei-
osis I; maturing males have many sperm morulae
(Howie, 1959). Sperm morulae are plates of several hun-
dred fully differentiated immotile spermatozoa, which
are bound together at both the head and distal ends of
the flagella (Newell, 1948; Bentley 1985, 1986a, b; Bent-
ley and Pacey, 1989).

Spawning in both male and female Arenicola marina
is a direct consequence of the maturation of the gametes.
The maturation of the oocytes (entry into metaphase of
meiosis I), and the breakdown of the sperm morulae to
free-swimming spermatozoa, results in the immediate
shedding of these from the ciliated funnels of the ne-
phromixia (Howie, 196 Ib, c). Oocytes mature by the ac-
tion of a maturation hormone (Howie, 1963, 1966) from
the prostomium, which induces germinal vesicle break-
down in v;/ro(Meijerand Durchon, 1977). The dissocia-
tion of the sperm morulae in males is also brought about

M. G. BENTLEY ET AL

by a prostomial maturation hormone (sperm maturation
factor) (Howie, 1963, 1966), which is a lipid (Howie,
1961a;Bentley, 1985).

Bentley (1985) began purifying the sperm matura-
tion factor (SMF) using thin-layer chromatography, in-
dicating that it was a relatively polar lipid. Lipids recov-
ered from the TLC plates were tested for SMF activity in
an in vitro assay. Biological activity was recovered from
areas of the TLC plates where a number of pharmacolog-
ically active, non-steroid lipids are found. Further
TLC studies led Bentley (1986a) to suggest that SMF
may be a metabolite of the 20-carbon polyunsaturated
fatty acid arachidonic acid. Arachidonic acid (5,8,1 1,14-
eicosatetraenoic acid) is one of a number, and probably
the most important, of 20-carbon polyunsaturated fatty
acids that are naturally occurring precursors of a wide
range of extremely biologically active compounds. These
include the prostaglandins, HETE's (hydroxy-eicosatet-
raenoic acids), and leukotrienes. Roles for these com-
pounds have been identified in a wide range of verte-
brates and invertebrates. Their roles in invertebrates
have been recently reviewed (Stanley-Samuelson, 1987)
and we will not discuss them further here. However, it
should be noted that arachidonic acid is metabolized by
starfish oocytes ( Meijer and Guerrier, 1984; Meijer ct at. ,
1984; Meijer el a/., 1986), and that this results in the
breakdown of the germinal vesicle prior to fertilization.

In light of the information available on the chemical
nature of SMF, the present investigation examines in de-
tail the possible role of arachidonic acid and the related
8,11,1 4-eicosatrienoic acid in the activation of spermato-
zoa in Arenicola marina

Materials and Methods

Gravid individuals of Arenicola marina were collected
by digging in sand during low water of spring tides at St.
Andrews Bay, Fife, Scotland, and Fairlie Sands, Ayr-
shire, Scotland. Specimens were maintained individually
in seawater at 5C in the laboratory until use. Sperm
samples for bioassay use were removed from the coe-
lomic cavity as described previously (Bentley and Pacey,
1989). In vitro assays of sperm morula suspensions were
performed as described by Bentley (1985).

Preparation of prostomial lipid extracts

Prostomial lipid extracts were prepared from mature
specimens of Arenicola marina. The prostomia were re-
moved using iridectomy scissors, and were homogenized
using an MSE Soniprep 150 ultrasonic disintegrator at
0C. The lipid fraction was partitioned from the sample
using an equal volume of chlorofornrmethanol (2:1 v/
v). The organic layer from several extractions was re-
moved, pooled, and dried over anhydrous sodium sul-

phite. The samples were then concentrated by removing
the solvent mixture in a rotary evaporator. The dried
lipid residues were redissolved in methanol before being
assayed for biological activity or used in subsequent ana-
lytical procedures.

Thin-layer chromatography (TLC)

A sample of total lipid was applied to 20 X 20 X 0.25
cm pre-coated silica gel F 254 TLC plates (Merck) using
a 100 M' disposable micropipette. Prior to applying the
sample, the plates were cleaned of any lipid contami-
nants by running the blank plate, in the solvent system
to be used, for its full length. After allowing the solvent
to evaporate, the plate was activated in an oven at 1 20C
for 30 min. The solvent system used was the upper phase
of: ethyl acetate:2,2,4-trimethylpentane:acetic acid:wa-
ter (45:25: 10:50 v/v) (Salmon and Flower, 1982) and the
plates were run in a vertical chamber until the solvent
front had moved 12 cm up the plate. The solvent was
then allowed to evaporate from the plate in a fume cup-
board, and the spots were visualized by spraying the plate
with 10% phosphomolybdic acid in ethanol. A second
plate was run simultaneously with the above, but was not
sprayed with phosphomolybdic acid. Areas correspond-
ing to the visualized spots on the first TLC plate were
scraped off the plate and the lipids eluted in methanol
and tested for biological activity as described above.

Argentation TLC

A sample of total prostomial lipid was spotted on to
two further activated TLC plates impregnated with 5%
AgNO, in acetone (see Christie, 1982). The plates were
developed as for the TLC described above. Free fatty acid
standards were also applied to the plates. When the sol-
vent front had reached the 12-cm mark, the plates were
removed, the solvent evaporated, and the plates washed
with distilled water to remove the AgNO,. One of the
plates was sprayed with phosphomolybdic acid and the
second plate was used for recovering the lipids for bio-
assay.

In vitro assay of 20-carbon fatty acids

Free fatty acids: eicosanoic, 1 1-eicosenoic, 1 1,14-eico-
sadienoic, 8,1 1,1 4-eicosatrienoic acid, 1 1,14, 17-eicosa-
trienoic, 5,8,1 1,1 4-eicosatetraenoic (arachidonic), and
5,8,1 1,14, 1 7-eicosapentaenoic acids, were obtained
from Sigma Chemical Co., and 1 X 10~ 2 M stock solu-
tions prepared in HPLC grade methanol (BDH). For use
in bioassay, aliquots of these stock solutions were diluted
100 fold to give a final free acid concentration of 1
X 10~ 4 A/ and a negligible residual solvent concentration.
Double dilutions of the free acids were then used to de-

FATTY ACID ACTIVATION OF SPERMATOZOA

termine the biological activity of each acid in the activa-
tion of spermatozoa in vitro.

Effect ofcydooxygenase and lipoxygenase

pathway inhibitors

Stock solutions ( 1 mM) of the cyclooxygenase inhibi-
tors aspirin, indomethacin, and tolazoline, were pre-
pared in TFSW (triple filtered seawater). A 1-mM
solution of butylated-hydroxytoluene, a lipoxygenase in-
hibitor, was also prepared. Prostomia were then homoge-
nized in solutions of each inhibitor before bioassay for
SMF activity. Control experiments were carried out in
which the inhibitors of cyclooxygenase or lipoxygenase
were added to prostomial extract after homogenization.
or TFSW prior to bioassay. This permits the distinction
to be made between metabolism of fatty acid substrate
by the prostomial homogenate and metabolism by the
spermatozoa themselves.

Incubation of biologically active fatty acid with
cyclooxygenase and lipoxygenase

(A) Arachidonic acid was incubated with fresh bovine
lung homogenate to obtain products from the cyclooxy-
genase pathway as described by Powell (1982). One gram
of bovine lung tissue was homogenized on ice in 5 ml
0.05 A/ Tris-HCl buffer, pH 7.4. One ml of the homoge-
nate was incubated with arachidonic acid at a final con-
centration of 1 X 10~ 2 Mat 37C for 5 min. The reaction
was terminated by adding 5 ml ethanol, then adding 16
ml H 2 O, and centrifuging at 400 X g for 10 min. The
supernatant was removed and assayed for biological ac-
tivity.

(B) One-mi aliquots each containing 1.8 mg (c.
250,000 units) of soybean lipoxygenase was incubated
with arachidonic acid at a final concentration of 5 X 10 3
M at 25C for 1 5 min. After incubation, the reaction was
terminated by heating, the extract was centrifuged, and
the supernatant was assayed for biological activity. Incu-
bations containing denatured lipoxygenase and lacking
lipoxygenase were also carried out.

Incubation of prostomial extract with lipoxygenase

One-mi aliquots each containing the equivalent of
0.36 prostomium were incubated with 1.8 mg (c.
250,000 units) of lipoxygenase, for 60 min at 20C. After
incubation, the reaction was stopped and the sample
treated as described above. Incubations containing dena-
tured lipoxygenase and lacking lipoxygenase were car-
ried out in parallel.

Incubations of biologically active fatty acid with BSA

Prostomial homogenate and 8,1 1 , 1 4-eicosatrienoic
acid were bioassayed in the presence of dissolved BSA

(bovine serum albumin). BSA solutions were freshly pre-
pared in TFSW to give a final concentration in the assay
ofO, 100, 1000 Mg- ml" 1 , and 10 mg- ml ', respectively.

Extraction of SMF by organic solvents and
aqueous buffers

Extraction of SMF from biologically inactive lipid
constituents of prostomial extracts were carried out as
described by Jouvenaz el al. (1970) and Van Dorp
( 197 1 ). This allows larger quantities of starting material
to be purified, and permits the separation of free fatty
acids from their often biologically active, but extremely
labile, metabolites. This procedure involves the initial
preparation of ethanolic lipid extract, washing the resi-
due with ethanol:diethyl ether (1:1 v/v), followed by add-
ing saline. The extract is then reduced in volume to 2.5
ml, acidified to pH 4 with citric acid, and contaminating
lipids removed with petroleum ether. The remaining lip-
ids are then taken up into ethyl acetate concentrated to
about 5 ml total volume. Tris buffer (1.5 ml, pH 7.8) is
then added to take up any prostaglandins present. All the
organic and aqueous fractions obtained throughout this
procedure were bioassayed for SMF activity.

Extraction of SMF on ODS silica cartridges

Freshly prepared prostomial homogenate and free
fatty acid standards were separately applied to pre-wet
Sep-Pak Ci 8 Cartridges (Waters Associates) in 10%
aqueous ethanol with the pH adjusted to 4.0 using a 1-
M stock solution of citric acid. The Sep-Pak was pre-
wetted using 2 ml of methanol followed by 5 ml of H 2 O
before applying the sample or standard. Fractions were
partitioned using the following solvent mixtures (Powell,
1982): aqueous ethanol (20 ml, 10%), 20 ml H 2 O, 10 ml
petroleum ether, 10 ml petroleum etherxhloroform (65:
35 v/v), and 10 ml methyl formate. The Sep Pak was
regenerated using 10 ml of 80% aqueous ethanol. The
fraction obtained using each solvent was collected and
prepared for bioassay as described above.

Gas-liquid chromatography of prostomial lipids

Prostomial lipid extracts were prepared as described
above, and methylated using a method modified from
Christie (1982). The sample was dissolved in 1 ml of di-
chloromethane, and refluxed for 2 h with 2% methanolic
H 2 SO 4 . After cooling, 4 ml of saturated NaCl was added,
and the fatty acid methyl esters extracted with 2 ml pe-
troleum ether (40-60C). GC analysis was performed
using a Hewlett Packard 5 890 A gas chromatograph fitted
with a flame ionisation detector. Samples were separated
on a capillary non-polar column (fused silica, 25 m
X 0.25 mm i.d., 0.12 df, CP-Sil 5CB) following on-col-

M. G. BENTLEY ET AL.

1.0

g w 49 49 ^^ SMF activity

Rf value 0.6

0.4

0.2

0.0

1 : 11, 14 - eicosadienoic acid

2 8, 11, 14 - eicosatrienoic acid

3 11, 14, 17 - eicosatrienoic acid

4 8, 11, 14, 17, - eicosatetraenoic acid

5 5, 8, 11, 14. 17, - eicosapentaenoic acid

6 : Prostomial lipids

Figure 1. Separation of 20-carbon fatty acids (spots I to 5 with 2.
3, 3, 4, and 5 double bonds, respectively) and prostomial lipids by ar-
gentation thin-layer chromatography. The fatty acid standards with the
greatest degree of saturation interact least with the silver nitrate on the
TLC plate and therefore migrate furthest. The prostomial lipid extract
shows a spot with an identical Rf value to the eicosatrienoic acids. This
spot, when scraped off a washed plate, shows sperm maturation factor
(SMF) activity in vitro

umn injection. A linear thermal gradient program from
90-300C at 20C-min ' was used with helium as the
carrier gas (25 cm s~ ' ).

Results

Analysis of prostomial lipid extract and
20 fatty acids by TLC

TLC on activated silica gel plates, using the upper
phase of: ethyl acetate:2,2,4-trimethylpentane:acetic
acid:water (45:25: 10:50 v/v) as a solvent, allows the sepa-
ration of C20 fatty acids from their metabolites. The bio-
logical activity associated with prostomial lipid samples
is associated with a region of the TLC plate that is identi-
cal to the position where free fatty acids are found (Rf
0.78-0.82). This chromatographic separation cannot
distinguish between fatty acids with varying degrees of
unsaturation.

Argentation TLC (using the same solvent system and
TLC plates impregnated with 5% AgNO 3 ) allows fatty
acids of the same carbon number to be separated accord-
ing to the number of double bonds in the molecule.
Figure 1 illustrates the results of this separation. SMF ac-
tivity was recovered from a region of the plate which cor-
responds to the position of the C20:3 acids (8,11,1 4-eico-
satrienoic acid and 1 1,14,17-eicosatrienoic acid). This

technique is unable to separate these two isomers be-
cause they are identical in their degree of unsaturation.

Sperm activation by C20 fatty acids

In vitro bioassay of eicosanoic, 1 1-eicosenoic, 11,14-
eicosadienoic, 8,1 1,14-eicosatrienoic acid, 1 1,14,17-ei-
cosatrienoic, 5,8,1 1,1 4-eicosatetraenoic (arachidonic),
and 5,8,1 1,14, 1 7-eicosapentaenoic acids for the ability
to induce sperm activation showed that both 8,11,1 4-ei-
cosatrienoic acid and arachidonic acid displayed biologi-
cal activity. The results are summarized in Table I. Con-
centration ranges for biological activity of 8,1 1,14-eico-
satrienoic acid and arachidonic acid are based on nine
replicate experiments producing mean minimum con-
centrations required for a response of 4.47 X 10 5 At and
2.28 X 10~ 4 M, respectively. These data indicate that
8,1 1,14-eicosatrienoic acid is about five times more ac-
tive in this system than in arachidonic acid.

Studies of cyclooxygenase and lipoxygenase pathways

The preparation of prostomial extract in the presence
of inhibitors of cyclooxygenase activity (aspirin, indo-
methacin, tolazoline) or lipoxygenase (butylated hy-
droxytoluene) did not effect the SMF activity of the ex-
tract. Aspirin at concentrations of 5 mM and 10 mA/
caused sperm lysis (the reasons for this are not clear, but
this effect is unlikely to be related to the cyclooxygenase
inhibitory property of the aspirin). These results suggest
that there is no conversion of parent fatty acid to a bio-
logically active metabolite via either the cyclooxygenase
or lipoxygenase pathway. However, polychaete enzymes
metabolizing fatty acids may differ from vertebrate
cyclooxygenases and lipoxygenases, and substances used
as inhibitors may not effect enzyme activity. Quercetin,
another lipoxygenase inhibitor, could not be used in this

Table I

Sperm activation by C20 fatly acids

Threshold

concentration

for activation

Fatty acid

Activity (Mean S.E.; n =

9)

A eicosanoic

-

B 1 1 -eicosenoic

-

C 1 1.14-eicosadienoic

-

D 8.1 1.14-eicosatrienoic

+ 4.47 1.46 X 10' 5

M

E 11,14,17-eicosatrienoic

-

F 5,8,11,1 4-eicosatetraenoic

+ 2.28 1.78 x 1(T 4

M

G 5,8, 11,14,1 7-eicosapentaenoic

-

H TFSW control

FATTY ACID ACTIVATION OF SPERMATOZOA

x \Q-*M
X \Q-*M

Table II

I'lic c/lccts i>/'cvclt>o\yKi'nu.ti' und lipoxygenase on \pcrm

aclivalion hy (. '- tally iiculx

Threshold concentration
Activity for activation

a. Incubation with

cyclooxygenase
Arachidonic acid

incubated with bovine

lung cyclooxygenase

Arachidonic acid + I.25X10' 5 M

Bovine lung homogenate

(cyclooxygenase)
TFSW

b. Incubation with

lipoxygenase
Arachidonic acid

incubated with

soybean lipoxygenase + 2.5

Arachidonic acid + 4.0

Soybean lipoxygenase
TFSW

study because of a non-specific effect on spermatozoa,
which will be reported elsewhere.

Incubation of arachidonic acid with bovine lung ho-
mogenate (cyclooxygenase) or soybean lipoxygenase was
carried out to examine whether there was (a) a reduction,
(b) enhancement, or (c) the same level of sperm activa-
tion after converting the fatty acid substrate to metabo-
lites. Table II shows that both incubation with bovine
lung homogenate and soybean lipoxygenase brought
about a reduction in the fatty acid incubate's ability to
activate spermatozoa. This suggests that the fatty acid
has been largely converted to cyclooxygenase and lipoxy-
genase metabolites, which cannot activate the spermato-
zoa. Thin layer chromatographic analysis of the incu-
bates confirm that most of the fatty acid is converted dur-
ing incubation (Fig. 2). Thin layer chromatography of
prostomial homogenate shows that most of the fatty acid
remains unmetabolized. This indicates that the fatty acid
is not normally converted to a metabolite by endogenous
polychaete enzymes, which may be outcompeted for
substrate during incubations with exogenous cyclooxy-
genase or lipoxygenase.

Incubation of prostomial homogenate with soybean li-
poxygenase results in a total loss of SMF activity. This
indicates the conversion of a fatty acid in the prostomial
homogenate to non-active metabolites, and also suggests
that it is this fatty acid component of the prostomial ho-
mogenate that causes sperm activation in vitro.

Incubations of 8, 11,14-eicosatrienoic acid and
prostomial extract with BSA

BSA (bovine serum albumin) was incubated with
8,1 1,14-eicosatrienoic acid and prostomial homogenate

to investigate the possible interference of BSA on the
ability of both the fatty acid and prostomial extract to
activate spermatozoa of Arenicola marina. It has long
been known that fatty acids interact strongly with serum
albumin (Goodman, 1958). Figure 3 shows that the abil-
ity of both 8,1 1,14-eicosatrienoic acid and prostomial
homogenate to activate spermatozoa is markedly re-
duced by the addition of BSA. This evidence lends fur-
ther support to the suggestion that it is a fatty acid com-
ponent of the prostomial homogenate that causes sperm
activation in vitro.

Further purification of SMF from
prostomial homogenate

Figure 4 shows the purification steps employed for the
purification of SMF from crude prostomial homogenate,
using the method developed by Jouvenaz et ai, (1970)
and Van Dorp ( 197 1 ) for the extraction of prostaglan-
dins from biological tissues. The figure also traces the bi-
ological activity through the purification steps. SMF ac-
tivity is finally recovered in an ethyl acetate fraction.

1.0

0.8

0.6

Rf value

0.4

0.2

0.0

1 : Arachidonic acid incubated with bovine lung

2 : Arachidonic acid

3 : Prostomial lipids

Figure 2. TLC analysis of prostomial lipids. arachidonic acid, and
arachidonic acid following incubation with cyclooxygenase. Arachi-
donic acid can be seen at an Rf value of about 0.78. The cyclooxygenase
products are clearly visible with Rf values lower than that of arachi-
donic acid itself. Prostomial lipid extract shows no spots which corre-
spond to cyclooxygenase products, and which may have arisen as a
result of action by endogenous cyclooxygenases.

M. G. BENTLEY ET AL

1.0-]

o x

|1
I 1

1

100 1000 10000 100000

BSA Concentration (ng/ml)

Figure 3. Minimum concentrations of (a) 8.1 1,14-eicosatnenoic
acid, and (b) prostomial homogcnatc required to bring about sperm
activation in the presence of bovine serum albumin (BSA). The fatty
acid concentration, or the concentration of prostomial extract required
to bring about sperm activation //; wm>. increases with the concentra-
tion of dissolved BSA. Data shown are the mean (SE) minimum con-
centrations required to bring about sperm activation in three replicated
experiments.

Prostaglandins remain in the pH 7.8 Tris buffer and
would be recovered only in an ethyl acetate fraction from
Tris buffer at pH 4.0. This indicates that SMF activity is
not recovered with prostaglandins but is recovered in the
fatty acid fraction.

A parallel approach to the purification of SMF has
been carried out using Sep-Pak cartridges and a succes-
sion of aqueous and organic solvents. Table III shows
that SMF activity is recovered in the same fractions as
the 8,1 1,14-eicosatrienoic acid standard.

Gas chromaiographic analysis n/'prnstumial lipids

The results of separation of methyl esters of prostomial
lipid extracts are shown in Figure 5. Three peaks with
retention times corresponding to methyl esters of
5,8,1 l,14-eicosatetraenoicacid{8.72 min), 8,1 1,14-eico-
satrienoic acid (8.81 min), 1 1.1 4,1 7-eicosatrienoic acid
(8.93 min), can be identified. This clearly indicates the
presence of 8,1 1,14-eicosatrienoic acid in prostomial
lipid extracts obtained from prostomia showing SMF ac-
tivity in vitro.

Discussion

The results obtained from thin layer chromatography
of prostomial total lipid extracts described above showed
that SMF activity co-migrated with 20-carbon fatty acid
standards. In particular, it is associated with eicosatrie-
noic acids (demonstrated by argentation TLC). The bio-
assay of C20 fatty acids show that only two of the fatty

acids tested brought about the activation (dissociation of
the morulae and the acquisition of motility) in vitro: ara-
chidonic and 8,1 1,14-eicosatrienoic acids. Arachidonic
acid, while capable of activating spermatozoa, does not
co-migrate with prostomial SMF in the argentation
TLC. Clearly, then, arachidonic acid and SMF are not
the same substance. All eicosatrienoic acids co-migrate
in this TLC system but only 8,1 1,14-eicosatrienoic acid
causes sperm activation in vitro. While 8,1 1,14-eicosa-
trienoic acid co-migrates with SMF, and has biological
activity identical to SMF, this is insufficient evidence to
propose that they are the same.

Arachidonic acid, 8,1 1,14-eicosatrienoic acid, and ei-
cosapentaenoic acid are all naturally occurring 20-car-
bon fatty acids that differ in the number of double bonds,
having 4, 3, and 5 double bonds, respectively. They are
all precursors for a range of pharmacologically active
molecules, the eicosanoids. Each of these three fatty

1.97g prostomia

Homogenize in 2ml dislilled water \-

bioassay

Add 10 ml ethanol

Centrifuge & wash precipitate
with a) 5ml ethanol

b) 10ml ethanol -diethyl ether (l:lv/v)

Pool supernatant

bioassay

Add 2. 5ml saline,
reduce volume to 2.5ml

Ad]ust to pH4 with citric acid
extract with 7ml petroleum ether

Extract aqueous phase with
3x2 volumes ethyl acetate

Reduce volume of organic
fraction to 5ml,

Add 1.5ml Tris buffer at pH7,8

bioassay of organic fraction
bioassay of aqueous fraction

bioassay of organic fraction
bioassay of aqueous fraction

bioassay of organic fraction
bioassay of aqueous fraction

-ve

+ve

+ve
-ve

+ ve
-ve

Ethyl acetate fraction stored
under helium at -20 C

Figure 4. Extraction procedure used for sequential purification of
sperm maturation factor (SMF) using organic solvents and aqueous
buffers (after Jouvenaz el ai. 1970). Following each purification step,
aqueous and organic phases were dried under helium, resuspended. and
tested for their ability to activate sperm //; vitro. The response is repre-
sented here as +ve or -ve, where +ve indicates the presence of SMF
activity evidenced by sperm morula breakdown and the presence of
free-swimming spermatozoa, and -ve indicates no activation of sper-
matozoa.

FATTY ACID ACTIVATION OF SPERMATOZOA

Table III
ofprostomialSMFon ODS silica cartridges

Eluent from
cartridge

Activity of

prostomium

extract

Activity of 8. 1 1,14-
eicosatrienoic acid

1. 20 ml 30%
ethanol

2. 20mlH : Odist.

3. 10 ml petroleum
etherchloroform
(65:35 v/v)

4. 10 ml methyl
formate

5. 10 ml 80%
ethanol

acids gives rise to a series of prostaglandins (PGs): arachi-
donic acid, which is the best known and probably the
most important, is converted to series 2 PGs; 8,1 1,14-
eicosatrienoic acid is converted to series 1 PGs. Eicosa-
pentaenoic acid, which is the most important C20 fatty
acid in marine organisms, gives rise to series 3 PGs.

The use of the principal enzymes involved in the me-
tabolism of the fatty acids to their respective prostaglan-

dins (cyclooxygenase) and other metabolites (lipoxygen-
ase) combined with the use of selective inhibitors permits
the possible pathways involved to be elucidated. Evi-
dence shown above, by using bovine lung homogenate
and soybean lipoxygenase, which both caused a marked
reduction of SMF activity of prostomial homogenate,
suggests strongly that a fatty acid present in prostomial
homogenate is responsible for the SMF activity. The use
of inhibitors of cyclooxygenase and lipoxygenase sug-
gests that there is no conversion of fatty acid in prosto-
mial homogenate to metabolite(s), which may have po-
tent biological activity as previously suggested (Bentley,
1986a).

Compared to other invertebrate groups, notably the
insects, and the Crustacea, little is known of the chemical
nature of polychaete hormones. To date, no hor-
mone from polychaete tissues has been completely puri-
fied or its structure elucidated. Grothe el al. (1987)
identified catecholamines in the nervous system of
Ophryotrocha puerilis, which may have an endocrine re-
lated function. Numerous vertebrate-like peptides have
been identified in the nervous system of polychaetes
(Dhainaut-Courtois el al.. 1985), but functions have yet
to be ascribed to these putative hormones. The possible
action of a fatty acid as a hormone may seem unlikely.

a- s -

Figure 5. Gas chromatograph of fatty acid methyl esters (FAMEs) of biologically active prostomial
lipids showing identified peaks corresponding to 5,8,1 1,14-acid (C20:4), 8,1 1,14-eicosatrienoic acid (C20:
3w6). and 1 1 , 14, 1 7-eicosatrienoic acid (C20:3w3) with retention times of 8.72 min, 8.8 1 min, and 8.93
min, respectively. These peaks correspond in absolute retention times, and relative distances to FAME
standards of the three acids.

M. G. BENTLEY ET AL.

but the certain presence in the prostomium ofArenicola
marina of a fatty acid that acts on distant target cells
(sperm morulae in the coelomic fluid) may well be an
example of such a "hormone." Its hormonal role is fur-
ther supported by the cyclical nature of its appearance
in prostomial extracts. Bentley (1985) showed that SMF
activity of prostomial extracts is maximal around the
breeding season of given populations and is non-existent
during the post-spawning period.

Fatty acids and prostaglandins are present in a wide
range of lower animals (Srivastava and Mustafa, 1984;
Stanley-Samuelson, 1987). These essential fatty acids
and their metabolites also have an effect on many aspects
of reproduction in marine invertebrates. The endocrine
control of oocyte maturation in asteroid echinoderms is
now well understood and involves the action of a peptide
gonad-stimulating substance (GSS), 1 -methyl adenine,
and an intracellular maturation-promoting factor
(MPF) (see Giese and Kanatani, 1987 for review). One-
methyl adenine acts on the oocytes to bring about matu-
ration. However, Meijer ct al. ( 1986) demonstrated that
arachidonic acid mimics the action of 1 -methyl adenine
on starfish oocytes in vitro. It is possible that 1 -methyl
adenine acts as a "second messenger" or that the arachi-
donic acid mimics some hitherto unidentified fatty acid.

A tri-hydroxy metabolite of arachidonic acid has been
identified as the hatching factor in the barnacle Semibal-
tinns (Balamis) balanoidcs (Clare ct al.. \ 982, 1985; Hol-
land el al., 1985). Prostaglandins also cause spawning of
the abalone, Haliolis nifi'scenx, and the mussel, Mytilus
edidis ( Morse et al.. 1977).

Pharmacologically active metabolites of arachidonic
and related fatty acids are characteristically short-lived
substances produced close to, or at, their site of action.
The parent fatty acids are often metabolized by the target
cells themselves. This may occur at the cell surface or
intracellularly. Typically this metabolism occurs as a re-
sult of the action of lipoxygenases or cyclooxygenase (PG
synthetase). The precise nature of the enzymes may vary
between phyla, and there is evidence that those found
in some invertebrates (e.g.. Lymnaea stagnalis) may be
different to those occurring in vertebrates (Clare et al..
1986). In the starfish oocyte, arachidonic acid is con-
verted to HETEs at the plasma membrane (Meijer et al.,
1986). It may be that 8,1 1 , 1 4-eicosatrienoic acid is me-
tabolized by the sperm morulae ofArenicola marina and
this will be investigated by the use of radiolabeled precur-
sors. The maturation of starfish oocytes by arachidonic
acid is inhibited in a dose-dependent manner by the pres-
ence of BSA, however, maturation induced by 1 -methyl
adenine (the natural inducer) is not. The activation of
spermatozoa of A. marina by prostomial extract or
8, 1 1 , 1 4-eicosatrienoic acid are both inhibited in a similar
dose-dependent manner by BSA. This may be further ev-

idence to suggest that the fatty acid from the prosto-
mium, causing sperm activation in A. marina, is a pri-
mary inducer rather than a "second messenger."

Purification procedures, followed by structural analy-
sis, must be performed to identify the chemical nature of
any endocrine substance with certainty. For example,
the barnacle hatching factor was identified by organic ex-
traction and subsequent GC-MS analysis (Holland et al.,
1985). One of the problems often encountered is obtain-
ing sufficient starting material for purification. The sepa-
ration procedures described in this paper show that SMF
of Arenicola marina has identical chromatographic
properties to 8,1 1,1 4-eicosatrienoic acid, and that
8,1 1,1 4-eicosatrienoic acid is present in the fatty acid
component of prostomial extract. The use of bonded-
phase C18 cartridges as a purification stage should per-
mit sufficient quantities of SMF to be extracted to com-
plete mass spectrometrical analysis.

Acknowledgments

The authors gratefully acknowledge the support of a
Royal Society European Programme Fellowship to
M.G.B., under the tenure of which this work was com-
menced; the award of support from the Royal Society
Browne Fund to S.C.; and the award of an SERC post-
graduate studentship to A. A. P. The authors also thank
Prof. F. D. Gunstone and Mr. K. Black (Department of
Chemistry, University of St. Andrews) for their assis-
tance with GC analysis.

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Development of Nerve Cells in Hydrozoan Planulae:

III. Some Interstitial Cells Traverse the

Ganglionic Pathway in the Endoderm

VICKI J. MARTIN

Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

Abstract. Hydrozoan planulae of Pennaria tiarella
possess migratory stem cells interstitial cells that are
capable of self renewal and can differentiate into either
ganglionic nerve cells or nematocytes. The commitment
and differentiation of a subpopulation of larval endoder-
mal interstitial cells to the neural pathway were exam-
ined using light immunocytochemistry and transmission
electron microscopy. Embryos of different ages, from 8
to 96 h, were tested for their ability to bind rabbit antise-
rum raised to the neuropeptide FMRFamide. A subpop-
ulation of interstitial cells in the anterior endoderm of
the planula begins to express a FMRFamide-like antigen
between 48 and 72 h postfertilization. Concurrent with
this endodermal interstitial cell expression, a subset of
ectodermal ganglionic cells with FMRFamide-like im-
munoreactivity appears in the anterior end of the plan-
ula. Ultrastructural examination of the interstitial cell
population in the anterior planular endoderm, at 48 h
in development, indicates that, based upon morphology,
there are at least three subsets of interstitial cells in this
region: undifferentiated interstitial cells, interstitial cells
traversing the nematocyte differentiation pathway, and
interstitial cells traversing the neural differentiation
pathway. The endodermal interstitial cells entering the
neural pathway form a Golgi complex, electron-dense
droplets, dense cored vesicles, and microtubules. Neurite
formation does not occur in the endoderm; rather, neu-
rites are only found in association with ectodermal gan-
glionic cells. Furthermore, planulae lack fully differenti-
ated endodermal neurons. This study demonstrates that,
during embryogenesis, some interstitial cells destined for
neural differentiation are committed in the endoderm
before their emigration to the ectoderm, begin to express

Received 5 June 1984; accepted 17 November 1989.

cytochemical and morphological features of neural
differentiation while in the endoderm, and migrate to the
ectoderm as neuroblasts.

Introduction

The hydrozoan planula larva is an especially good sys-
tem with which to examine the commitment and differ-
entiation of cells during development: the number of cell
types in the larva is small; their arrangement is simple;
and neither the variety nor the arrangement of larval
cells are very far from those of the adult (Martin and
Thomas, 1980; Martin el al., 1983; Thomas et al., 1987;
Martin. 1988a, b,c).

The hydrozoan planula contains a population of mi-
gratory undifferentiated cells: interstitial cells. Interstitial
cells are capable of self-renewal, and can differentiate
into either ganglionic nerve cells or nematocytes ( Martin
and Thomas, 198 la, b; Martin, 1988a). Interstitial cells
arise in the endoderm. later migrate into and populate
the ectoderm, and eventually differentiate into the two
classes of cells (Martin and Archer, 1986). Do the inter-
stitial cells ( 1 ) migrate as uncommitted cells and become
committed by some sort of positional cues upon arrival
in the ectoderm, or (2) are committed before they leave
the endoderm, and migrate into the ectoderm to com-
plete differentiation? The second alternative is correct for
nematocytes, (Martin and Archer, 1986), and in this pa-
per I show that it is also correct for neurons (i.e.. gangli-
onic cells).

This research describes a series of histological experi-
ments designed to determine whether interstitial cells in
a hydrozoan planula develop neuronal characteristics
(ganglionic features) before arriving at their final destina-
tion in the ectoderm. The numbers and locations of in-

10

NEURAL DIFFERENTIATION

11

terstitial cells and ganglionic cells in hydrozoan embryos
of different ages were determined by light microscopy
and transmission electron microscopy (TEM). The abil-
ity of these embryos to bind a rabbit antiserum raised to
the neuropeptide FMRFamide [such immunoreactivity
has been demonstrated in planular sensory cells ( Martin,
1988b)] was tested to determine whether the antigen is
expressed by ganglionic cells or interstitial cells differen-
tiating along the ganglionic pathway. Anti-FMRFamide
was used in this study because when it is applied to cni-
darians, the peptides bound are likely to be related to
pGlu-Gly-Arg-Phe-amide, which is present in large
amounts in nervous systems of adult anthozoans and
probably also in hydrozoans and scyphozoans (Graff and
Grimmelikhuijzen, 1988). The planular results show
that a subpopulation of interstitial cells in the anterior
endoderm of 48 h planulae expresses morphological and
cytochemical features of ganglionic cell differentiation.
Thus, at least some interstitial cells for the neural differ-
entiation pathway are committed in the endoderm and
actually traverse the ganglionic pathway in the endo-
derm.

Materials and Methods

Mature colonies of Pennaria tiarella were collected
from pier pilings in Morehead City, North Carolina.
Fronds from male and female colonies were placed to-
gether in the dark at 6:00 pm. At 9:00 pm the bowls were
returned to the light and, within an hour, early cleavage
embryos were found in the bottoms of the dishes. Em-
bryos were collected, placed in small finger bowls of fil-
tered seawater, and reared at 23C.

Embryos of seven different ages: 8-, 10-, 16-, 24-, 48-,
72-, and 96-h, were prepared for transmission electron
microscopy. Animals were fixed for 1 h in 2.5% glutaral-
dehyde, pH 7.4, in 0.2 M phosphate buffer. They were
subsequently postfixed for 1 h in 2% osmium tetroxide
(pH 7.2, in 1.25% sodium bicarbonate), dehydrated in
an ethanol series, infiltrated, and embedded in Spurr's
embedding medium. Serial thick and thin sections were
cut with a Porter-Blum MT-2B ultramicrotome. Thick
sections were mounted on gelatin-coated slides, stained
with 0.5%. toluidine blue in 1% sodium borate, and ex-
amined with a Zeiss research microscope. Thin sections
were placed on 1 50-mesh copper grids and stained with
3.5% uranyl acetate in ethanol followed by lead hydrox-
ide. Grids were examined and photographed with a Hi-
tachi H-600 transmission electron microscope.

Wholemounts and paraffin sections of the selected em-
bryonic stages were tested for their ability to bind a rabbit
antiserum raised to FMRFamide (Immuno Nuclear
Corporation). Twenty-four-hour planulae, treated for 2
h with 0.2% colchicine in seawater and subsequently al-

lowed to recover for 24 h, were also exposed to the
FMRFamide antiserum. Such colchicine treatment
eliminates the entire interstitial cell system i.e.. intersti-
tial cells, nematoblasts, nematocytes, and ganglionic
cells (Martin and Thomas, 1981b).

To visualize the binding of FMRFamide antiserum on
wholemounts, the procedure presented by Martin
( 1988b) was followed with some modifications. Animals
were fixed for 1 h in 10% formalin in seawater and subse-
quently washed 3 times, for 15mineach, in 10mA/ phos-
phate-buffered saline (PBS, pH 7.2). Incubation with the
FMRFamide antiserum was for 1-4 h, with the primary
antibody diluted 1:200 with 10 mM PBS, pH 7.2, con-
taining 0.1% sodium azide, 0.3% Triton X-100, and 2%
fetal calf serum. Incubations were carried out with plan-
ulae in lid-covered 96 well tissue culture plates placed on
a rotating shaker platform set at 60 rpm. At the end of
the first incubation period, the primary antibody was re-
moved, and animals were washed 3 times, for 15 min
each, in 10 mM PBS, pH 7.2. Incubation with the second
antibody was for 1 h in fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit immunoglobin
(Boehringer Mannheim). The FITC-tagged antibody was
diluted 1:120 in 10 mM PBS, pH 7.2, containing 0.1%
sodium azide, 0.3% Triton X-100, and 10% fetal calf se-
rum. The second incubations were also done in 96 well
plates rotated at 60 rpm. After the second incubation,
animals were washed for three 15-minute changes in 10
mM PBS, pH 7.2. Wholemounts were examined for
fluorescently labeled cells with a Zeiss microscope
equipped with epi fluorescence.

To visualize binding of FMRFamide antiserum to par-
affin sections of embryos, samples fixed in formalin were
dehydrated through an alcohol series, infiltrated and em-
bedded in paraffin, and serially sectioned at 8 nm. Nine
sections were mounted in the center of a glass slide, three
rows one above the other, and each row containing three
sections. Slides were rehydrated to distilled water, and
the sections were surrounded by an outer ring of vacuum
grease. Grease application was done in a moist chamber
to prevent the sections from drying. FMRFamide antise-
rum was placed in the grease-created wells, thus immers-
ing the sections. The slides were placed in a covered
moist chamber and rotated at 20-40 rpm for 1-4 h. PBS
rinses and incubation in the second antibody were car-
ried out in the moist chamber. After incubation, the
grease was removed from the slides; the sections were
covered with mineral oil and examined for fluorescently
labeled cells.

Binding specificity of the FMRFamide antiserum was
determined by preincubating a 1 :200 dilution of the anti-
serum with 10 Mg/ml synthetic FMRFamide (Peninsula
Lab) for 24 h at 4C before using it to stain the embryos.

12

V. J. MARTIN

Results

Mature planula (72-96 h postfertilization)

The mature planula consists of an ectoderm, an acellu-
lar mesoglea, and an endoderm (Fig. 1). The ectoderm
contains epithelial cells (epitheliomuscular, glandular,
and sensory), interstitial cells, and their derivatives
(nematoblasts, nematocytes. and ganglionic cells),
whereas, the endoderm has gastrodermal epithelial cells,
interstitial cells, and nematoblasts. Interstitial cells, nem-
atoblasts, nematocytes, and ganglionic cells are easily
identified in planular tissue at the light microscopic level
(Figs. 1-3). Interstitial cells small round cells measur-
ing 7.5 jiin in diameter contain a centrally located nu-
cleus with one to several nucleoli. They possess few cy-
toplasmic organelles and are scattered among the epithe-
lial cells in both the ectoderm and the endoderm along
the entire anterior-posterior axis of the planula (Fig. 2).
Nematoblasts (developing nematocytes) range from 10
to 12.5 /urn in diameter and have distinctive dark- or
light-staining capsules (Figs. 1, 2). Each capsule houses
a nematocyst thread that may possess barbs and spines.
Nematoblasts are located in both the ectoderm and the
endoderm and are mostly confined to the anterior and
middle two-thirds of the planular axis. Mature nemato-
cytes are found only at the ectodermal surfaces of planu-
lae and exhibit the same distribution pattern as that of
the nematoblasts. Ganglionic cells are 5 //m in diameter,
exhibit a spindle shape, and are positioned all along the
planular anterior-posterior axis at the base of the ecto-
derm just above the mesoglea (Fig. 3). The ganglionic
perikaryon, its long axis oriented parallel to the meso-
glea, contains a Golgi complex, microtubules, mitochon-
dria, electron-dense droplets, and dense cored vesicles.
Neurites project from either side of the cell bodies and
form an extensive ectodermal neural plexus above the
mesoglea (Figs. 3, 4). Such neurites are filled with micro-
tubules, mitochondria, electron-dense droplets, and
dense cored vesicles ( Fig. 4 ). Electron-dense droplets and
dense cored vesicles are found exclusively in differenti-
ating and full-differentiated nerves. The endoderm lacks
ganglionic cells and a neural plexus.

Interstitial cells and nematoblasts are migratory,
whereas, nematocytes and ganglionic cells are not (Mar-
tin and Archer, 1986). These migratory cells move as sin-
gle cells, and migration has been observed from the endo-
derm to the ectoderm and, once in the ectoderm, up and
down the planular axis.

FMRFamide-like immunoreactivity is detected in a
subpopulation of ganglionic cells in the planular ecto-
derm at 72 h postfertilization, just before metamorphosis
(Fig. 5). Such immunopositive nerve cells are located at
the base of the ectoderm above the mesoglea and are con-
fined to the anterior head and anterior sides of the plan-

Figure 1 . Longitudinal section of a 72 h planula showing ectoderm
( EC), mesoglea ( M ) and endoderm ( E ). Endodermal nematoblasts (sin-
gle arrow) and interstitial cells (double arrows) and an ectodermal gan-
glionic cell (triple arrows) are visible. X250.

Figure 2. Endodermal interstitial cells (arrows) in a 72 h planula.
Each cell contains a large nucleus with a prominent nucleolus and few
other cytoplasmic organelles. x620.

Figure 3. Ectodermal ganglionic cell (arrow) in a 72 h planula. The
cell body is oriented parallel to the mesoglea and neurites (double ar-
rows) extend from each side of the perikaryon. x620.

NEURAL DIFFERENTIATION

13

Figure 4. Ectodermal neurites of ganglionic cells. These neurites form a plexus just above the mesoglea
and are rich in microtubules, mitochondria, electron-dense droplets (single arrows) and dense cored vesi-
cles (double arrows), xl 9.000.

ula. Cell bodies of the immunopositive ganglionic cells
are stained, whereas their neurites (processes) are not.
Nematoblasts and nematocytes do not produce the

Figure 5. Wholemount of a 72 h planula. A subpopulation of gan-
glionic cells (arrow) in the ectoderm of the planula exhibits FMRF-
amide-like immunoreactivity. Such ectodermal ganglionic cells are
confined to the anterior head and anterior sides of the planula. X200.

FMRFamide-like peptide, as indicated by their lack of
staining. Furthermore, the majority of planular intersti-
tial cells do not stain with the antibody. There is, how-
ever, a small subset of interstitial cells in the anterior en-
doderm of 72-h planulae that does express a FMRFami-
de-like peptide. Such positive cells first produce the
neuropeptide at 48 h in development and are described
below (see Forty-eight hour planula).

Gastrulating embryo (8-10 h postfertilization)

Embryos gastrulate between 8-10 h postfertilization
resulting in the formation of an immature. 10-h planula.
This young planula consists of an ectoderm, an acellular
mesoglea, and an endoderm (Figs. 6, 7). The ectoderm
contains epithelial cells (dark-staining epitheliomuscular
cells and light-staining glandular cells) and is devoid of
interstitial cells, nematoblasts, nematocytes, and gangli-
onic cells. The endoderm consists of an outer epithelial
layer of gastrodermal cells surrounding a central core of
tightly packed interstitial cells (Figs. 6, 7). This core of
interstitial cells extends the entire length of the planular

14

V. J. MARTIN

Figure 6. Cross section of a 10-h planula. The embryo consists ot'an ectoderm (EC), an acellular meso-
glea (M), and an endoderm (E). The endoderm is composed of an outer columnar epithelial layer (G)
surrounding a central core of lightly staining interstitial cells (arrows). The ectoderm contains epithelio-
muscular cells (dark-staining cells) and glandular cells (light-staining cells), but is devoid of interstitial cells,
nematoblasts, nematocytes, and ganglionic cells. X320.

Figure 7. Endodermal region of a 10-h planula. Clusters of interstitial cells (arrows) occupy the central
endoderm. EC, ectoderm; G, epithelial layer of endoderm: M, mesoglea. 320.

anterior-posterior axis. These lightly staining, oval-
shaped interstitial cells possess a large, centrally located
nucleus with one to several nucleoli. Dark-staining gran-
ules occupy the cytoplasm of these young interstitial
cells, however, such granules disappear as the cells ma-
ture. Interstitial cells of late planulae possess few granules
(see Fig. 2).

Interstitial cells traverse the nematocyte differentia-
tion pathway in the endoderm (see Figs. 1, 2, 8). Such
cells are distinguished by the appearance of either a dark-
or light-staining nematocyst capsule. The capsule en-
larges to an extent that it displaces the nucleus to one side
of the cell. A few endodermal nematoblasts, confined to
the anterior and middle two-thirds of the endoderm.
have been observed in the 10-h planula. Interstitial cells
traversing the neural differentiation pathway have not
been seen in the immature planula.

Interstitial cells and nematoblasts emigrate as single

cells from the endoderm to the ectoderm. Interstitial cells
migrate out from all locations along the planular endo-
dermal axis, whereas outward nematoblast migration is
confined to the anterior and middle endodermal regions.
Interstitial cells and nematoblasts first appear in the
planular ectoderm at 14 h postfertilization (Martin and
Archer, 1986). Their ectodermal distribution corre-
sponds to their above-mentioned migration patterns.

Immature planulae (10 h) do not express a FMRF-
amide-like antigen as indicated by their lack of staining.

Yininxphimtla (24 h postfertilization)

By 24 h, the planular ectoderm contains epithelial cells
(epitheliomuscular, glandular, and sensory), interstitial
cells, nematoblasts, a few nematocytes, and ganglionic
cells; the endoderm has gastrodermal epithelial cells, in-
terstitial cells, and nematoblasts. Both interstitial cells

NEURAL DIFFERENTIATION

15

Figure 8. Longitudinal section of a 24-h planula. Differentiating
nematohlasts (single arrows) are visible in both the endoderm (E) and
the ectoderm (EC). A young ganglionic cell (double arrows) is seen at
the base of the ectoderm above the mesoglea (M). Its neurites are not
yet fully formed. Triple arrows, endodermal interstitial cells. X250.

Figure 9. Ectodermal ganglionic cell (arrow) in a 24 h planula. Note
its spindle shape and extending neurites. X620.

and ganglionic cells occupy the entire anterior-posterior
axis of the planula, whereas, nematoblasts and nemato-
cytes are confined to the anterior and middle regions of
the animal (Figs. 8, 9). In the ectoderm, ganglionic cells
and nematoblasts are positioned in close proximity to
the mesoglea, and interstitial cells are located slightly
above these cells (i.e., toward the outer ectodermal sur-
face). In the endoderm, interstitial cells and nemato-
blasts may be found in the central core or out closer to
the mesoglea. As planulae mature (24-96 h) the numbers
of ectodermal and endodermal interstitial cells, ectoder-
mal and endodermal nematoblasts, ectodermal nemato-
cytes, and ectodermal ganglionic cells increase.

At 24 h, the nervous system begins to form (Martin,
1988a, b). This neural system is entirely ectodermal and
consists of ganglionic cells (interstitial cell derivatives)
and sensory cells (epithelial derivatives). Ganglionic cells
form a neural plexus composed of cell bodies and their

neurites; this plexus extends the entire length of the plan-
ula and is located just above the mesoglea (Fig. 9). These
ganglionic cells have originated from interstitial cells that
have migrated from the endoderm to the base of the ecto-
derm. Once in this ectodermal position, they elaborated
morphological features characteristic of ganglionic cell
differentiation. Interstitial cells traversing the ganglionic
pathway in the endoderm have not been observed at this
stage. Sensory cells first arise in the anterior end of the
planula (later in development they appear all along the
length of the planula) and extend from the free surface
of the planula to the ganglionic plexus where they insert
neurites into the plexus (Martin, 1988b).

Twenty-four hour ganglionic cells do not produce a
FMRFamide-like peptide, however, the sensory cells do
(Martin, 1988b). The FMRFamide-like peptide is first
observed in the apices of sensory cells and only later in
their mid to basal regions. FMRFamide-like positive sen-
sory cells are observed throughout the remaining larval
period (Figs. 10, 11). Interstitial cells and nematoblasts
lack immunostaining at this stage, as does the entire en-
doderm.

Forty-eight hour planula

The distribution of interstitial cells and their progeny
in the 48-h planula is similar to that observed in the 24-
h planula. By 48 h, the numbers of these cells have dra-
matically increased in both germ layers.

At 48 h, a subpopulation of interstitial cells in the ante-
rior endoderm begins to express a FMRFamide-like anti-
gen(Figs. 10, 1 1). These positive-staining interstitial cells
are found exclusively in the anterior-most endodermal
region of the planula and are present in the central endo-
dermal core and in the outer endodermal periphery
(Figs. 10, 11). Depending upon the plane of section, the
FMRFamide-positive interstitial cells exhibit either a
mesenchymal shape or an oval morphology. Interstitial
cells located in the mid to posterior endodermal regions
do not express the FMRFamide-like peptide, as indi-
cated by an absence of staining (Fig. 12). Just after the
endodermal appearance of these FMRFamide-like posi-
tive interstitial cells, a few immunopositive ganglionic
cells are detected in the ectoderm above the mesoglea
confined to the anterior head and anterior sides of the
planula. Their cell bodies are stained whereas their neu-
rites are not. This is a full day after ganglionic cells first
appear in the planular ectoderm. Nematoblasts and
nematocytes do not stain for the FMRFamide-like pep-
tide at this stage.

Between 48 and 72 h, the number of immunopositive
endodermal interstitial cells and immunopositive ecto-
dermal ganglionic cells increase. Their distribution is
limited to the anterior end of the planula.

16

V. J. MARTIN

Figure 10. Longitudinal paraffin section of a 48-h planula. A suhpopulation of interstitial cells (arrows)
in the anterior endoderm of the planula begins to express a FMRFamide-like antigen at this stage in devel-
opment. A. anterior; E. endoderm; EC, ectoderm; M, mesoglea; P, posterior. 250.

Figure II. Longitudinal paraffin section of a 48-h planula. This section is taken from a deeper region
of the same planula shown in Figure 10. A subset of interstitial cells in the anterior endoderm expressing a
FMRFamide-like antigen is visible, as are FMRFamide-positive sensory cells (double arrows). A. anterior;
E, endoderm; EC. ectoderm; M. mesoglea; P, posterior. >250.

NEURAL DIFFERENTIATION

17

Ultrastructural examination of interstitial cells in the
anterior endoderm of 48-h planulae indicates that, based
upon morphology, at least three subsets of interstitial
cells are found in this region: undifferentiated interstitial
cells, interstitial cells traversing the nematocyte differen-
tiation pathway (nematoblasts), and interstitial cells
traversing the ganglionic differentiation pathway (neuro-
blasts) (Figs. 14-19). All three subpopulations can be
found in the central endodermal core and at the periph-
ery of the endoderm. These three subsets are also founc 1
in older planulae (72 h) in the same endodermal posi
tion. Undifferentiated interstitial cells are characterized
by a centrally located nucleus with a nucleolus, and a
cytoplasm containing free ribosomes, a few mitochon-
dria, and a few segments of rough endoplasmic reticulum
(Fig. 14). These interstitial cells, as of yet, show no spe-
cific organelles indicative of a particular differentiation
pathway. Interstitial cells committed to the nematocyte
differentiation pathway have a cytoplasm rich in rough
endoplasmic reticulum and form a distinctive nemato-
cyst capsule (Fig. 15). This capsule is in close proximity
to the nucleus and often displaces it to one side of the
cell. Interstitial cells undergoing neural differentiation
(ganglionic cell pathway) form a Golgi complex, elec-
tron-dense droplets, dense cored vesicles, and microtu-
bules (Figs. 16-19). These electron-dense droplets and
dense cored vesicles occupy the cell bodies of the devel-
oping endodermal ganglionic cells and are morphologi-
cally identical to the droplets and vesicles found in the
ectodermal ganglionic cell bodies and neurites (see Figs. 4,
16, 17, 18, and 19). These developing endodermal neuro-
blasts do not form neurites in the endoderm, as neurites have
only been observed in the ectoderm of the planula.

Colchicine-treated embryos

Embryos treated with colchicine and subsequently al-
lowed to recover for one to two days lack all interstitial cells
and their differentiated progeny (ganglionic cells, nemato-
blasts, neuroblasts, and nematocytes) (Martin and Thomas,
1 98 1 a). When such epithelial planulae are exposed to FMR-
Famide antiserum, they show no immunostaining (Fig. 13).
There are no immunopositive interstitial cells, immunoposi-
tive neuroblasts, or immunopositive ganglionic cells.

Discussion

Research presented here, as well as past work (Martin
and Archer, 1986), indicates that at least some larval in-

terstitial cells are committed within the endoderm to the
differentiation of either nerve cells or nematocytes.
These restricted cells enter a differentiation pathway in
the endoderm, and most probably migrate as nemato-
blasts or neuroblasts to a position in the ectoderm where
differentiation is completed. This process probably ac-
counts for the spatial distribution of the interstitial cell
system in the larval ectoderm.

With regard to ganglionic cell formation, this study
demonstrates a subpopulation of anterior endodermal
interstitial cells that shows early signs of neural cyto-
chemical differentiation by expressing a FMRFamide-
like antigen. Concurrent with the appearance of these
immunopositive interstitial cells, TEM indicates that a
subset of interstitial cells in the same anterior endoder-
mal region develops morphological features indicative of
neural differentiation: formation of a Golgi complex,
electron-dense droplets, dense cored vesicles, and micro-
tubules. This subset of interstitial cells probably includes
the FMRFamide-positive interstitial cells.

Furthermore, a subset of FMRFamide-positive gangli-
onic cells appears in the anterior ectoderm between 48-
72 h. Because this occurs just after the endodermal ap-
pearance of the immunopositive interstitial cells, and be-
cause both populations are confined to the same anterior
head region, the immunopositive interstitial cells have
probably migrated to the base of the ectoderm where
they differentiated into ganglionic cells. Alternatively,
the interstitial cells traversing the neural pathway in the
endoderm might never migrate to the ectoderm but sim-
ply remain and complete their differentiation, or die, in
the endoderm. The alternative is unlikely because the
planular endoderm lacks fully differentiated ganglionic
cells [i.e.. they do not form neurites in the endoderm;
neurite formation constitutes the last step in ganglionic
cell differentiation (Martin, 1988a)], and TEM studies
reveal no signs of degenerating cells in the endoderm at
any stage of planular development.

The movements of interstitial cells, nematoblasts, and
neuroblasts in planulae appear to be coordinated, as evi-
denced by their final placement within the ectoderm. In-
terstitial cells, which divide and possibly remain as stem
cells, migrate out from all regions of the endoderm and
distribute themselves along the whole planular axis in the
ectoderm. Developing nematoblasts emigrate from the
endoderm in a specific region of the planula (anterior to
mid endoderm) and concentrate in an ectodermal area
extending from the anterior end of the planula to the

Figure 12. Paraffin section of the posterior (P) region of a 48 h planula. The posterior endoderm (E) is
devoid of FMRFamide-positive interstitial cells. X250.

Figure 13. Paraffin section of a mature "recovered" colchicine-treated planula. Such epithelial planu-
lae lack FMRFamide-like activity as indicated by the absence of staining. X250.

18

V J. MARTIN

WP'i

-

NEURAL DIFFERENTIATION

19

Figure 18. Golgi region of a "neural" endodermal interstitial cell in a 48 h planula. Several mitochon-
dria and microtubules are seen in close proximity to the Golgi. x37,400.

Figure 19. Electron-dense droplets (arrows) in the Golgi region of a developing endodermal ganglionic
cell. Such droplets are characteristic of neural differentiation. X20.400.

mid-region of the planula. Interstitial cells destined to
form ganglionic cells migrate out from all regions of the
central endoderm and are evenly distributed along the
planular anterior-posterior axis in the ectoderm. Since
the interstitial cells and their progeny exhibit a rather

precise positioning within the ectoderm, some mecha-
nism of directed migration may be operating in the plan-
ula. The FMRFamide findings support the notion of di-
rected migration. FMRFamide-positive endodermal in-
terstitial cells and FMRFamide-positive ectodermal

Figure 14. Endodermal interstitial cell in the anterior region of a 48-h planula. This undifferentiated
cell contains a centrally located nucleus (N), a few segments of rough endoplasmic reticulum (arrow), a
few mitochondria, and numerous free ribosomes. Although not visible in this plane of section, the intersti-
tial cell also contains a prominent nucleolus. This interstitial cell has migrated from its site of origin in the
central endoderm to the outer endoderm (E) and is in close proximity to the mesoglea (M). x 14.400.

Figure 15. Developing nematoblast in theanteriorendodermofa48-h planula. Interstitial cells travers-
ing the nematocyte differentiation pathway are characterized by the appearance of large amounts of rough
endoplasmic reticulum (arrow) and by the formation of a nematocyst capsule (C). Such cells eventually
emigrate to the ectoderm. N, nucleus. X 10,800.

Figure 16. Interstitial cell traversing the neural differentiation pathway in the anterior endoderm of a
48-h planula. Such interstitial cells committed to the ganglionic pathway form small electron-dense drop-
lets (arrows) and dense cored vesicles (see Fig. 17), develop a Golgi (see Fig. 18), and accumulate microtu-
bules in their cytoplasm (see Fig. 18). These cells do not develop neurites in the endoderm. N, nucleus.
X9.600.

Figure 17. Developing ganglionic cell in the endoderm of a 48-h planula. The cytoplasm of the differ-
entiating interstitial cell becomes filled with electron-dense droplets (single arrow) and dense cored vesicles
(double arrows). Similar droplets and vesicles are abundant in the cell bodies and the neurites of ectodermal
ganglionic cells (see Fig. 4). N. nucleus. X2 1,600.

20

V. J. MARTIN

ganglionic cells are confined to the same anterior head
region of the late planula. As stated previously these posi-
tive interstitial cells probably emigrated to the ectoderm
and formed the positive ganglionic cells. The fact that
the FMRFamide-positi ve ganglionic cells are confined to
a specific region in the ectoderm and not distributed at
random suggests directed migration.

Acknowledgments

This research was supported by National Science
Foundation Grants DCB-8702212, Career Advance-
ment Award DCB-871 1245, and DCB-8942149.

Literature Cited

Graff, D., and C. J. P. Grimmelikhuij/en. 1988. Isolation of <Glu-
Gly-Leu-Arg-Trp-NH : (Antho-RWamide II). a novel neuropeptide
from sea anemones. f-'EBS I. ell 239: 137-140.

Martin, V. 1988a. Development of nerve cells in hydrozoan planulae:
I. Differentiation of ganglionic cells. Biol. Bull. 174: 3 19-329.

Martin, V. 1988b. Development of nerve cells in hydrozoan planu-
lae: II. Examination of sensory cell differentiation using electron
microscopy and immunocytochemistry. Biol. Bull. 175: 65-78.

Martin, V. 1988c. Development of the interstitial cell system in a ma-
rine hydrozoan polyp. Am /.ool 28: 95A.

Martin, V., and VV. Archer. 1986. Migration of interstitial cells and
their derivatives in a hydrozoan planula. Dev Biol 116:486-496.

Martin, \ ., and M. Thomas. 1980. Nerve elements in the planula
larva of the hydrozoan Pennaria liarella. J Morphol. 166: 27-36.

Martin, V., and M. Thomas. 198la. The origin of the nervous system
in Pennaria tiarella as revealed by treatment with colchicine. Biol.
Hull 160:303-310.

Martin, V., and M. Thomas. 1981b. Elimination of the interstitial
cells in the planula larva of the marine hydrozoan Pennaria liarella
J Exp. '/.ool. 217:303-323.

Martin, V., F.-S. Chia, and R. Koss. 1983. A fine-structural study of
metamorphosis of the hydrozoan Mitrocomella polydiaiiemala .1
Morphol. 176:261-287.

Thomas, M., G. Freeman, and V. Martin. 1987. The embryonic ori-
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Reference: Bio/. Bull 178: 21-24. (February. 1990)

Putative Immunological Influence Upon Amphibian

Forelimb Regeneration. II. Effects of X-Irradiation

on Regeneration and Allograft Rejection

RAYMOND E. SICARD AND MARY F. LOMBARD

Department of Pediatrics, Rhode Island Hospital. Providence, Rhode Island 02903,
and Department of Biology. Regis College, Weston, Massachusetts 02193

Abstract. Influence of the immune system on epimor-
phic regeneration of amphibian limbs has been suggested
but not proved. The present investigation explored this
hypothesis by examining the effects of x-irradiation on
forelimb regeneration and rejection of skin allografts.
Two kRad x-irradiation was provided either to a single
limb or as whole-body irradiation to intact newts (with 1
limb shielded). Complete suppression of regeneration
was observed when limbs to be amputated were irradi-
ated directly. In addition, irradiated limbs displayed se-
vere and protracted inflammation, with total resorption
of the affected limbs in 85% of the cases. Moreover,
delays in both the rate of forelimb regeneration and
allograft rejection were found in animals receiving
whole-body irradiation. However, in these cases neither
forelimb regeneration nor allograft rejection were sup-
pressed. These observations diffuse the challenge raised
by irradiation studies to the notion of possible immuno-
logical influence on epimorphic regeneration. Moreover,
the delays observed in both regeneration rate and allo-
graft rejection following whole-body irradiation are con-
sistent with possible interaction between the immune
system and the regenerating limb. Nevertheless, confir-
mation that such interaction occurs and is integral to epi-
morphic regeneration must await further investigations.

Introduction

Epimorphic regeneration of lost appendages in verte-
brates is accomplished by a mass of dedifferentiated tis-
sues the regeneration blastema. This mass of tissue is
derived at the site of amputation (Butler, 1935: Brunst

Received 30 December 1988: accepted 30 November 1989.

and Cheremetieva, 1936; Butler and O'Brien, 1942). Al-
though it has been well-established that the blastema is
not produced by an accumulation of blood cells, an in-
fluence of the immune system on the progress of regener-
ation has been suggested (Prehn, 1970). This hypothesis
arose because of perceived similarities between sarcomas
and the regeneration blastema. In essence, this hypothe-
sis considers that immunostimulation might promote
blastemal growth just as mild immunostimulation pro-
motes the growth of certain sarcomas (Prehn, 1970,
1972; Prehn and Lappe, 1971).

The validity of this hypothesis has yet to be either con-
firmed or refuted conclusively. Nevertheless, several
studies have demonstrated effects on the progress of re-
generation of substances that might have altered immu-
nological status (Sicard, 1981; Schotte and Sicard, 1982;
and Sicard and Laffbnd, 1983). These experiments em-
ployed chemical interventions and assessed their impact
on forelimb regeneration but not on immunological
function. More recent investigations have examined pa-
rameters of immunological status (Sicard and Lombard,
1989). One of these studies disclosed modified respon-
siveness to T-cell mitogens during epimorphic regenera-
tion (diminished responsiveness during dedifferentiation
and blastema formation; augmented responsiveness dur-
ing proliferative stages) but not following nonamputa-
tional trauma. Another study observed reciprocal effects
of graft rejection and forelimb regeneration. That is, re-
generation rate could be accelerated or delayed when
combined with allograft challenge, depending upon the
temporal relationship between both operations. Sim-
ilarly, the rate of allograft rejection could be altered by
events of epimorphic regeneration.

Although the results of these previous investigations

21

22

R. E. SICARD AND M. F. LOMBARD

are consistent with immunological influence on epimor-
phic regeneration, they cannot yet prove that such influ-
ence occurs. On the other hand, the studies that estab-
lished that the blastema is of local origin (Butler, 1935;
Brunst and Cheremetieva, 1936; Butler and O'Brien,
1942) might present a formidable challenge to this no-
tion. If whole-body irradiation rendered the subjects im-
munoincompetent, their subsequent regeneration of
forelimbs occurred without immunological influence.
However, if the animals retained immunocompetence,
then regeneration occurred against a background of po-
tential immunological influence. The absence of specific
mention of increased morbidity or mortality in irradi-
ated animals by earlier investigators leads us to the con-
clusion that immunocompetence was at least adequate
to meet challenges of infection in these animals. Never-
theless, this requires verification.

The present investigation was undertaken to confront
this potential challenge to the hypothesis that the im-
mune system might influence the initiation and progress
of epimorphic regeneration. This investigation em-
ployed x-irradiation as the means of intervention and as-
sessed its effects both on forelimb regeneration and im-
mune status (skin allograft rejection). Documentation of
an impaired immune response (i.e., delayed graft rejec-
tion) in the absence of an effect on forelimb regeneration
would challenge or refute the immunostimulation hy-
pothesis as it applies to epimorphic regeneration. On the
other hand, demonstration of intact immunocompe-
tence (i.e., essentially normal allograft rejection) in irra-
diated newts regenerating amputated forelimbs removes
the challenge presented by these earlier studies. How-
ever, it cannot establish that the immune system plays a
role in epimorphic regeneration.

Table I
Effect of irradiation on forelimb regeneration

Materials and Methods

Animals

Adult newts (Notophthalmus viridescens), obtained
from Tennessee, were maintained in glass bowls in Holt-
freter's solution at 2 1 2C. Two control groups were
established: (1) regenerating controls and (2) allograft
controls. These groups received no treatment other than
amputation or a skin graft and marked the "normal" rate
of regeneration or graft rejection. Irradiations and surgi-
cal manipulations were performed on newts anesthetized
in 0. 1% aqueous methane tricaine sulfonate (MS-222).

Irradiation

Initial treatment consisted of x-irradiation. Experi-
mental animals were divided into two groups, one receiv-
ing whole-body irradiation, except for a shielded limb,
and a second group in which only one limb was irradi-

Treatment

Suppressed

or aborted' Mid-bud :

Palette

Digital

None 0/15

2 kRad whole body

(shielded limb) 3/10

2 kRad irradiated

limb 13/13** >76**

23 4 33 4 42 4
22 2 47 12* 53 10*

1 Number of cases/total number within group.
: Mean number ot days 1 S.D.
*P<0.01;**P<0.001.

ated. Total irradiation was either 2 kRad (whole-body
and limb) or 2.2 kRad (whole body only). Irradiation was
provided using a Picker-Gemini 320 kV industrial x-ray
unit equipped with an aluminum filter. Output intensi-
ties of 1 25 or 1 60 kV were employed for 17.5 min to yield
2 and 2.2 kRad irradiation, respectively. Animals were
positioned 1 5 cm from the source and radiation was ad-
ministered dorsally. Shielding was provided by a 6 mm
thickness of lead plate.

Skin grafts

Subsequently, one group of nontreated newts and two
groups of newts receiving whole-body irradiation re-
ceived skin allografts. These animals were used to assess
effects of irradiation in cellular immunity. Reciprocal al-
lografts consisted of small pieces of skin, approximately
2 mnr, implanted into wound sites created by removal
of skin used as grafts for other animals. Thus each newt
served as both a donor and a recipient. In addition, a
small group of control and irradiated animals received
autografts.

Amputations

The remaining control and irradiated animals were
used to evaluate effects on regeneration. All regeneration
groups were subjected to unilateral forelimb amputation
through the distal stylopodium. In all instances, any por-
tions of humerus extending beyond the wound surface
were carefully trimmed.

Results and Discussion

Effects on regeneration

Regeneration occurred among all control animals and
progressed to the early digital stage in approximately 42
days (Table I). Whole-body irradiation suppressed regen-
eration of shielded limbs in 3 of 10 newts and signifi-
cantly (P < 0.01 ) slowed the rate of regeneration among

REGENERATION AND GRAFT REJECTION

23

Table II

Effect of irradiation (ingraft

Treatment

Retained
skin graft'

Interrupted
circulation 2

Loss of
pigment

Rejection

Autografts:

None

3/3

>76

2kRad

3/3

>76

Allografts:

None

3/14

12 1

188

27+7

2 kRad

1/12

186*

24 6

33 10

>2 kRad

0/10

19 7*

30 6*

36 + 6*

1 Number of cases/total number within group.
: Mean number of days I S.D.
*F<0.05.

the remainder (Table I). Thus, there was an adverse effect
on forelimb regeneration of whole-body irradiation.

In contrast to the results of the shielded-limb group,
irradiation of limbs caused total suppression of regenera-
tion in all cases. This is in complete accord with observa-
tions from other laboratories (reviewed in Wallace,
1981). Moreover, irradiation of the limbs led to severe
and persistent inflammation and ultimately to resorp-
tion of the limbs in 85% of the cases. These events ap-
peared strikingly reminiscent of those described by
Schotte and Butler (1941) in larval Ambystoma follow-
ing limb denervation and by Butler (1933) in larval Am-
bystoma following irradiation. These investigators as-
cribed the resorption phenomenon to a failure of de-
differentiation to stop and the progressive stages of
regeneration to commence. They inferred that apparent
resorption of the stump occurred because of an inability
to retain an appendage of dedifferentiated tissues. How-
ever, the aggravated initial inflammation followed by
loss of pigmentation, and subsequent resorption of soft
tissues observed in this study resembled more the rejec-
tion of a foreign graft (Cohen, 1966). The extent to which
this similarity can be pursued and its implications are the
objects of additional studies.

Effects on allograft rejection

Skin autografts were tolerated by those few control
and irradiated animals used for that purpose (Table II).
In addition, rejection of skin allografts occurred in 1 1 of
14 controls and 21 of 22 irradiated animals (Table II).
However, the rate of rejection appeared to be sensitive to
irradiation. In particular, irradiation above 2 kRad sig-
nificantly delayed (P < 0.05), but did not suppress, allo-
graft rejection. These results suggest that irradiation
affected the immunological status of the newts; however,
at the dosages used, this effect did not cripple the newts'
immune system.

Demonstration of delays in the rate of regeneration of
shielded limbs of otherwise whole-body irradiated ani-
mals suggest that one or more factors outside of the am-
putation site had been adversely affected by irradiation.
To this observation, the resorption of amputated irradi-
ated limbs presents an intriguing counterpoint. Further-
more, the persistence of inflammation and subsequent
erosion (resorption) of the stump seems reminiscent of
graft rejection. Therefore, it is tempting to suggest that
the factors extrinsic to the limb that were affected by irra-
diation are associated with the immune system. In fact,
the occurrence of both of these manifestations is consis-
tent with interactions between the immune system and
the amputated limb.

Speculations

Similarities between these data and previous observa-
tions of limb resorption following irradiation (Butler,
1 933) or limb denervation (Schotte and Butler, 1 94 1 ) in
larval Ambystoma prompt the speculation: Following
amputation, dedifferentiation of local tissues occurs. If
dedifferentiated cells are stabilized and activated (e.g., by
neurotrophic factors), they modulate immunological ex-
pression favoring blastema formation and possibly con-
tributing to promoting blastemal growth. However, if
stabilization and activation does not occur, presumptive
blastemal cells are eliminated by activated immunologi-
cal defenses (Prehn, 1970; Coleman el al. 1989). More-
over, the absence of a pool of accumulating blastema
cells (e.g.. following irradiation) might lead to limb re-
sorption in a futile attempt to establish such a pool.

Conclusions

The design of this investigation does not enable the
means through which x-irradiation induced the particu-
lar effects observed to be known with certainty; conse-
quently, alternative interpretations of our observations
might be equally tenable. Nevertheless, the results of this
investigation demonstrate that regeneration of shielded
forelimbs by newts otherwise receiving whole-body irra-
diation occurs in animals that are still immunocompe-
tent, at least in terms of allograft rejection. In addition,
these data suggest that x-irradiation can affect the expres-
sion of epimorphic regeneration through central, as well
as local, effects. Furthermore, this impairment of regen-
eration appears to occur in parallel to retardation of the
rate of allograft rejection. Consequently, a relationship
between immunological expression and epimorphic re-
generation is suggested. Moreover, these results remove
the potentially devastating challenge to this hypothesis
presented by earlier investigations of limb regeneration
in which x-irradiation was used.

24

R. E. SICARD AND M. F. LOMBARD

Acknowledgments

The technical assistance of Kara Anderson, Katherine
Costello, and Laurie Peluso is gratefully acknowledged.

Literature Cited

Brunst, V. V., and E. A. Cheremetieva. 1936. Sur la perte locale du
pouvoir regenerateur chez le triton et 1'axolotl causee par 1'rradia-
tion avec les rayons x. Arch. Zool. Expil. Gen. 78: 57-67.

Butler, E. G. 1933. The effects of x-radiation on the regeneration of
the forelimb of Amhlysloma larvae. ./ Exp /no/. 65: 27 1-316.

Butler, E. G. 1935. Studies on limb regeneration in x-rayed Amhly-
stoina\arvae.Amil. Rec 62: 295-307.

Butler, E. G., and J. P. O'Brien. 1942. Effects oflocalized x-radiation
on regeneration of the urodele limb. Anal Rec 84: 407-4 1 3.

Cohen, N. 1966. Tissue transplantation immunity in the adult newt,
Diemtctvliis viruleseen.s II. The rejection phase: first- and second-
set allograft rejections and lack of sexual dimorphism. J Exp. /.mil
163: 173-190.

Coleman, R., M. Lombard, H. Sicard, and N. Rencricca. 1989.
Fundamental Imiiiitnulnuy. Unit 8: Cancer and Transplantation.
Wm. C. Brown Publishers. Dubuque. IA. Pp. 427-504.

Prehn, R. T. 1970. Immunosurveillance, regeneration, and oncogen-
esis. Progr. Exp. Tumor Res 14: 1-24.

Prehn, R. T. 1972. The immune reaction as a stimulator of tumor
growth. Science 176: 1 70- 171.

Prehn, R. T., and M. Lappe. 1971. An immunostimulation theory of
tumor development. Transplant. Rev 7: 26-54.

Schotte, O. E., and E. G. Butler. 1941. Morphological effects of de-
nervation and amputation of limbs in urodele larvae. J. Exp. Zool.
87: 279-322.

Schotte, O. E., and R. E. Sicard. 1982. Cyclophosphamide-induced
leukopenia and suppression of limb regeneration in the adult newt,
Notophlhalniits viruiescens. J. Exp. Zool. 222: 199-202.

Sicard, R. E. 1981. The effects of putative immunological manipula-
tions upon the rate oflimb regeneration in adult newts, Notophthal-
niii.s viriilexcens. IRCSMed. Sci.i. 9: 692-693.

Sicard, R. E., and W. T. Laffond. 1983. Putative immunological in-
fluence upon amphibian forelimb regeneration. I. Effects of several
immunoactive agents on regeneration rate and gross morphology.
Exp. Cell Bin/ 51: 337-344.

Sicard, R. E., and M. F. Lombard. 1989. Epimorphic regeneration
and the immune system. Pp. 107-1 19 in Reccnl Trends in Regener-
ation Research. V. Kiortsis. S. Koussoulakos, and H. Wallace, eds.
Plenum Publ. Corp.. New York.

Wallace, II. 1981. Vertebrate Limb Regeneration Wiley and Sons,
Chichester.

Reference: Biol. Hull. 178: 25-32. (February, 1 WO)

Correlation of Abnormal Radular Secretion with Tissue

Degrowth During Stress Periods in Helisoma

trivolvis (Pulmonata, Basommatophora)

DAVID A. SMITH 1 AND W. D. RUSSELL-HUNTER 23

1 \Vabash College, Department of Biology, Crawfordsville, Indiana 47933: 2 Syracuse University,

Department of Biology, Syracuse, New York 13244-1270: and* Marine

Biological Laboratory, Woods Hole, Massachusetts 02543

Abstract. Laboratory experiments on starvation stress
in Helisoma trivolvis elucidate a relationship between
modifications of radular secretion and tissue degrowth
resulting from stress. Tissue losses in starved adults
ranged from 4.5% at 40 days to 27.7% at 160 days, with
negligible mortality (<2%). Modifications in radular se-
cretion that paralleled tissue loss involved not only ab-
normal secretion of individual teeth and of tooth rows,
but especially an increased "packing" of radular rows per
unit ribbon length. Radular length remained constant
during experimental trials, however the mean number of
tooth rows increased by almost 47% after 120 days of
food deprivation. Radular patterns reflecting degrowth
observed in these experiments were paralleled in radulae
taken from overwintered animals sampled from natural
populations. Rates of radular turnover averaged between
2.3% new growth per day (43 days to turnover) and 4.0%
new growth per day (25 days to turnover). Radular sam-
ples could provide for post hoc detection of recent peri-
ods of tissue degrowth in snails, just as evidence of longer
periods of tissue degrowth can be detected in the shells
of long-lived bivalves.

Introduction

Natural populations of aquatic molluscs can experi-
ence tissue loss during winter. This phenomenon in-
volves complex shifts in metabolism and has been called
"degrowth" (Russell-Hunter, 1985). Previous reports
have demonstrated that physiological stress, both short-
term and of longer duration, can temporarily affect radu-
lar secretion (Isarankura and Runham, 1968; Kerth,

Received 6 July 1 989; accepted 14 November 1989.

1971; Fujioka, 1985; Smith, 1987). The laboratory ex-
periments reported here were designed to clarify the rela-
tionship between abnormal radular secretion and con-
current tissue degrowth resulting from starvation stress.
Applied to field populations, these observations could
provide an independent (short-term) method for detect-
ing periods of starvation that had occurred shortly before
sampling. This would complement the long-term detec-
tion of stress-induced degrowth based on "oversized"
shellsfRussell-Hunterrffl/., 1984), or on modified catab-
olism (Russell-Hunter el al. 1983). The Ramshorn snail
of eastern North America, Helisoma trivolvis (Say,
1817), was particularly suitable for these experiments be-
cause it performs well in laboratory culture, and because
recent studies have documented not only the biometry
and mechanics of its radula (Smith, 1987, 1988, 1989),
but also its actuarial bioenergetics, including its capacity
for degrowth (Russell-Hunter and Eversole, 1976; Rus-
sell-Hunter et al, 1983, 1984).

Detailed analysis of radula-tooth biometry in Heli-
soma has shown significant levels of interpopulation
variation in this species (Smith, 1987, 1989). As with
similar studies on Lymnaeid pulmonates by Berrie
(1959) and Hunter (1975), in Helisoma there are no ob-
servable ecophenotypic effects on tooth shape. Despite
this constancy (within individuals, and within populations)
more general aspects of radular secretion, including the
number and density of tooth rows, can be modified by envi-
ronmental stress. Short exposures to near-freezing tempera-
tures will produce a zone of modified tooth rows on the rad-
ula ribbon (Isarankura and Runham, 1968; Kerth, 1971;
Fujioka, 1985; Smith, 1987), and longer exposures to the
stress of starvation will produce "bunching" or "packing" of
radular rows, as described below.

25

26

D. A. SMITH AND W. D. RUSSELL-HUNTER

Work on the bioenergetics of tissue degrowth in Heli-
sorna is also cognate to these experiments. Held in the
laboratory in a metabolic framework simulating that of
natural overwintering, a representative cohort of Hcli-
soma showed a 50% loss of tissue biomass (involving per-
haps 20% loss of protein) with only 10%> mortality over
132 days (Russell-Hunter and Eversole, 1976). Meta-
bolic shifts during the degrowth process were studied by
Russell-Hunter el al. ( 1983) using nearly concurrent as-
sessments of oxygen consumption and of nitrogenous
excretion. There was a clearly controlled differential ca-
tabolism of protein resources during degrowth. One of
the first quantitative reports of direct field evidence for
tissue degrowth during winter is for natural populations
ofHelisoma trivolvis and ofLymnaea palustris in central
New York state (Russell-Hunter ct al.. 1 984).

The existence of all these recent reports not only made
stocks of Hclisoma appropriate for these experiments,
but also made it likely that a history of recent degrowth
could be detected by detailed examination of the radula.
In field studies this might come to parallel evidence
(Clark, 1976; Mallet et al.. 1987; Peterson el al.. 1985)of
longer periods of degrowth and regrowth which can be
detected in the shells of long-lived bivalves.

Materials and Methods

Hclisoma trivolvis is one of the more common gastro-
pod molluscs of central New York state. This euryoecic,
pulmonate snail is found primarily in eutrophic environ-
ments including lakes, ponds, streams, farm ponds, and
drainage ditches. Mature adults used for laboratory anal-
ysis of tissue degrowth were taken from a small pond in
Ithaca, New York (7622.96'W, 4225.78'N). To study
the effects of overwintering on radula secretion, as well
as the patterns of radular regrowth during early spring,
snails were sampled in April and May, 1985, from four
additional field sites (Eaton Reservoir, 7542.27'W,
4251.10TSf; Meadowbrook Pond, 7607.08'W, 4301.59'N;
Otter Pond, 7632.83'W, 4309.52'N; and Silver Lake,
Remsen, 7508.19'W. 4320.97'N).

Methods used to quantify tissue degrowth were modi-
fied from those of Russell-Hunter and Eversole (1976),
and protocols for radula preparation are detailed by
Smith (1987). To initiate an investigation of tissue de-
growth, more than 400 adult snails were collected at the
Ithaca field site in October 1985. This sample reflects the
natural variation in a single generation of Helisoma as it
moves into winter conditions. All shells were measured
with dial calipers (0. 1 mm) for maximum shell diame-
ter (MD). On the basis of MD, individuals were then di-
vided into three size classes: <14.0, 14.1-16.9, and
>17.0 mm. Two individuals from each class were then
cultured together in translucent plastic beverage cups (n
= 6 per container) in approximately 400 ml of filtered

pond water. At this time, groups, each containing six
snails, were randomly designated (using Japanese icosa-
hedral dice) as "fed" or "starved" experimentals or as
baseline controls. Fed animals were provided fresh let-
tuce for the duration of the experiment; starved animals
were starved for 1 20 days and then fed lettuce for the last
40 days of the trial. The experiment was run in a B.O.D.
chamber at 8C. Cold fluorescent lights illuminated the
cultures on a 14L/IOD cycle. Cups were cleaned, and
provided with fresh, filtered, water each week.

Samples were taken at 0, 40, 80, 120, and 160 days
both for analysis of tissue degrowth and for radular prep-
arations. Of 264 animals used in this study, 72 were des-
ignated controls and 192 were experimentals. Of these
192, 144 were used for tissue analysis and 48 were used
for estimating radular degrowth. At 40-day intervals,
samples of 18 snails from each treatment were assessed
for shell and tissue dry weight. Individuals were oven
dried at 65C, treated with an excess of 8.5%. HNO, ( 1 2%
v/v nitric acid), washed, and then redried, giving two dry
weights, whole snail and tissue, and, by subtraction, a
value for dissolved calcium carbonate. Tissue degrowth
was then calculated as the difference between actual tis-
sue dry weight (TDW) and that predicted from initial tis-
sue-to-shell regressions established at the start of the trial
(TDWp).

To determine the effects of food deprivation on radu-
lar secretion, 6 specimens from each of the above treat-
ments were sampled every 40 days. Individuals were sac-
rificed in boiling water and removed from their shells.
The buccal mass of each individual was removed, soft-
ened in saturated KOH for 2-5 seconds, and transferred
to distilled water. Radulae were then removed with fine
forceps, placed onto clean glass slides, arranged, and cov-
ered with coverglasses. Preparations were then held in
distilled water for 24 hours, dehydrated in 70%> EtOH
and air dried. New coverglasses and mounting fluid were
then applied. Abnormal radular secretion was quantified
by first dividing each radula into three equal sectors on
the basis of overall length. The total number of tooth
rows per sector was then counted.

To study the natural patterns of return to normal rad-
ular secretion following overwinter stress, adult Hcli-
soma were collected in early spring at four field sites.
Sampling continued until evidence of abnormal secre-
tion (row-packing) was no longer present. Radular
growth rates were calculated as length of new growth as
a fraction of ribbon length.

Methods used to study radular turnover in the labora-
tory follow Isarankura and Runham (1968). Pond water
was cooled to approximately PC. Snails were placed in
this bath for 24 hours. Individuals were then returned to
room temperature (18C) and were provided fresh let-
tuce. Individuals were sacrificed daily until regions of
radular malformation were absent.

RADULAR CORRELATES OF DEGROWTH

27

Table I

Tissue tifgrowlli in Helisoma trivolvis

A. 40d 80d I20d I60d
Fed -1.9 1.63 -6.5 1.41 -8.7 1.50 -11.3+1.91
Unfed -2.2+1.54 -8.6+1.62 -13.1 + 1.69 -14.6+1.80

B. 40 d 80 d 12()d 160 d
Fed* -3.5 + 2.58 -1I.O2.46 -15.7 + 2.29 -19.0 3.05
Unfed** -4.5 2.57 -15.6 + 3.44 -23.8 2.05 -27.7 + 2.83

* ANOVA F,. 68 = 7.438, P < 0.00 1
** ANOVA F 3 . 6g = 1 3.878, P< 0.001

Data were subject to arcsin-square root transformation before
analysis.

A. Change in tissue dry weight in milligrams (as TDW-TDWp, n
= 18) over 160 days. B. Change in tissue dry weight as a percentage of
predicted tissue dry weight [((TDW-TDWp)/TDWp)- 100, n = 18],
over 1 60 days.

Results

At the start of the laboratory trial 72 control individu-
als had been sacrificed. For these, analysis showed that
tissue dry weight (mg) related to shell dry weight (mg)
as TDW = 0.254- SOW + 1.335 (r = 0.956, n = 72, P
< 0.001). With each set of known values of SOW, this
relationship was then used as a predictor of TDW. The
deviation of predicted (TDWp) from expected TDW for
each individual was used as an indicator of tissue growth
or of tissue degrowth. These values for each of four sam-
pling periods are set out in Table I. Two-hundred and
sixty-four individuals began the trial; four died (<2%)
and were replaced with parallel experimental animals.
Tissue degrowth clearly occurred by 80 days in both sets
of experimental animals, and this had nearly doubled by
160 days (Table I). Degrowth over 160 days of food de-
privation ranged from 4.5% tissue loss at 40 days to
27.7% tissue loss at 1 60 days. These values correspond to
2.2 mg below predicted tissue dry weight and 14.6 mg
below TDWp, respectively. Degrowth in animals belong-
ing to the fed treatment ranged from 3.5% at 40 days to
19.0% at 160 days. Levels of tissue loss in fed and unfed
treatments did not differ (P > 0.05) at 40 and at 80 days.
At 120 and at 160 days, however, treatments did show
significantly different mean levels of degrowth (t i:o days
= 2.414, n = 36, P < 0.05; t lw) days = 2.098, n = 36,

Tissue degrowth in the experimental snails was paral-
leled by abnormal radular secretion (Fig. 1 ). This was
manifest not only in malformations of individual radular
rows (including smaller lateral, marginal, and rachidian
teeth; irregular lateral, marginal, and rachidian teeth;
and missing marginal teeth), but also, and most consis-
tently, by an increased number of radular rows (packing)
per unit ribbon length. Radular length remained con-

stant during experimental trials, however the mean
number of tooth rows increased by almost 47% after 1 20
days of food deprivation (Table Ha). Observations
showed this increase was associated with the generative
(posterior) end of the radular ribbon. Although secretory
activity of the odontoblasts continued during the trial,
secretion by the membranoblasts (which produces
lengthening of the radula ribbon) proceeded at a much
reduced relative rate (Fig. 2). [The precise mechanism of
post-secretory radular transport remains uncertain. The
topic has been reviewed by Runham (1963), Mischor
and Miirkel (1984), and Mackenstedt and Markel
( 1987).] This differential activity of odontoblasts and of
membranoblasts resulted in an increased density of
tooth rows at the posterior end of the radula ribbon (Fig.
2). During the last 40 days of the trial (refeeding), radular
transport was restored and the proper pattern of radular se-
cretion was again established (Table lib). Once a normal pat-
tern of secretion was established, the region between the
tightly compressed rows (generated during stress) and the
normally deposited rows (generated during refeeding) pro-
vided a marker which could be used to quantify radular
turnover rates either in experimental or in natural popula-
tions.

Patterns of abnormal radular secretion observed in the
laboratory were paralleled in radulae taken from animals
sampled from five field sites (Fig. 3). Weekly sampling
in early spring allowed a unique opportunity to estimate
radular growth and turnover rates under natural condi-
tions. Values among five sites ranged between 3-4% new
growth per day. This figure corresponds to approxi-
mately 5-7 rows per day, and to 225-315 teeth per day.
Radulae from Meadowbrook Pond showed the slowest
turnover (2.3% growth/day, 43 days to turnover) while
radulae from Ithaca turned over most rapidly (4.0%/day,
25 days to turnover). Radulae from the other three sites
turned over in approximately 30 days (Eaton, 2.9%/day,
34 days turnover; Otter, 3.6%/day, 28 days turnover,
Remsen, 3.5%/day, 29 days turnover). These rate data
agree with laboratory trials, which showed ribbon turn-
over in 30 days at room temperature. Field rates also
agree with those determined by Isarankura and Runham
(1968) who reported average radular production of ap-
proximately 3.2 rows per day (minimum 0.5, maximum
7.5, average minimum 2.8, average maximum 4.2 rows/
day) for a variety of molluscs including Helix and Lym-
naea. At one field site (Ithaca) the progress of return to
normal radular secretion correlated with changes in tis-
sue regrowth (as tissue/shell) (r = 0.937, n = 5, P < 0.05)
indicating that radular growth is associated with in-
creases in early spring tissue biomass.

Discussion

There is no doubt that, for Helisoma trivolvis at least,
our experimental conditions closely match those of over-

28

D. A. SMITH AND W. D. RUSSELL-HUNTER

Figure I. Untouched photographs to show radular malformations in Ilcli^onui A. Normal radula. B.
Radula alter 7? days of food deprivation. In both A and B. the most recently secreted radular rows are at
the top of each photograph, and the rows in use are near the bottom. C. Enlarged view of rachidian and
lateral-tooth region from B. Note lateral tooth malformations and row packing. D. Enlarged view of margi-
nal-tooth region from B. Note that marginal teeth are absent from the region of degrowlh.

wintering in natural populations. Degrowth in three field
populations measured by Russell-Hunter el al. (1984)
showed average losses in tissue biomass of 24.7%, 28.3%,
and 41.3%. The maximum loss of 27.7%. over 160 days
in these experiments is appropriate.

The results of the present investigation confirm that
the physiological stress of starvation in Hclisnma results
not only in tissue degrowlh but also in concurrent
changes in radular secretion. The significance of this con-

currence is twofold, involving first, possible insight into
the fundamental control mechanisms of stress response
in molluscs, and second, the possibility (for applied stud-
ies) of post hoc detection, in natural populations, of ear-
lier periods of starvation or similar stress. Before review-
ing these two aspects, however, it is necessary to set out
certain strengths and weaknesses in laboratory starvation
experiments.

In general, quantitative studies of any kind of stress on

Table II

Modification <>l nuluhir iccrctmn in Helisoma tnvolvis

A.

Od

40 d

80 d

120d

16()d

Length (mm)*

2.7 0.04

2.9 + 0.16

2.7 0.17

2.7

0.19

2

4 0.29

Total rows**

138 + 1.9

169 9.4

192 9.8

202

13.7

194

27.1

Rows/mm***

52 0.9

59 + 1.3

70 +2.0

77 +

4.1

82

9.7

* ANOVA F 4 . 20 = 0.707.

P > 0.5. "ANOVA

F 4 . ;o = 3.072. P< 0.05.

***ANOVA F 4 :il = 6

724./><0.01.

B.

Od

40 d

80 d

120d

160d

Anterior

34.6 + 0.40

30.6 + 0.51

28.8 + 0.92

26.0

1.48

31

4+ 3.23

Middle

32.2 0.37

29.2 + 0.97

27.6 0.40

24.4

1.17

32

8+ 4.89

Posterior

33.2 + 0.49

40.6 + 0.87

43.4+ 1.03

49.2 +

2.25

36

,2 2.82

Row number

138 1.9

169 9.4

192 9.8

202 +

13.7

194

27.1

A. Basic statistics for abnormal radular secretion in unfed snails over 160 days (n = 5). B. Sector analysis as percent per sector based on total
number of rows (average total on last line).

RADULAR CORRELATES OF DEGROWTH

29

100

90

35

- 25

80

70

60

50

- 15

o

- 5

40

80
DAYS

120

160

Figure 2. Patterns of radular modification and tissue degrowth in
laboratory stocks of Helisoma trimlnx. Main plot (A) shows correla-
tion of tissue degrowth and abnormal radular secretion. Insert plot (B)
shows pattern of radular packing (see text for further explanation). Ver-
tical bars are 95% confidence limits ol each mean.

animals in laboratory culture must not involve high rates
of mortality. Our survivorship rate (>98%) in the experi-
mental groups is clearly satisfactory. Evidence of de-
growth in snails and in other shelled molluscs is based
on the permanence of the calcareous shell as a record of
previous tissue biomass. In a review of molluscan de-
growth studies, Russell-Hunter (1985) emphasizes an
important caveat, that ratio measurements of tissue-shell
relationships and, hence, predicted values of tissue bio-
mass should be obtained only from those species that de-
monstrably show no shell resorption. Helisoma trivolvis
has been well studied in this respect, and its shell does
not change in mass or in composition (Russell-Hunter
andEversole, 1976; Russell-Hunter et al., 1983, 1984).

A more immediate difficulty is that, with experimental
groups set up as in the present series, it is empirically
impossible to provide polar trophic conditions. Under
our experimental conditions, "fed" snails are not sati-
ated, while "unfed" snails are not totally starved (micro-
organisms are present in 7-day-old water and on shells).
Our controls represent unstressed snails, the fed snails
represent some nutritional stress, and the unfeds greater
stress. In similar experiments, which assessed the control
of differential catabolism (by measuring oxygen con-
sumption and nitrogenous excretion) during degrowth
(Russell-Hunter^ al., 1983), highly stressed snails estab-
lished an effective regime of metabolic compensation (by
reducing the proportion of protein catabolism) more
rapidly than less stressed snails. Unlike shell mass, tissue
biomass is not a static value (see Russell-Hunter and
Buckley. 1983, for discussion of this in the actuarial bio-
energetics of molluscan productivity). While any indi-
vidual organism remains alive, its tissue biomass contin-

ues to be in turnover. Thus, growth represents a positive-
value (and degrowth a negative value) for a combined
net rate that involves both inputs and outputs as rate
functions (Russell-Hunter and Buckley, 1983; Russell-
Hunter et al., 1983).

Tissue degrowth in our experiments was paralleled by
abnormal patterns of radular secretion. This was mani-
fest in several ways. Smaller lateral, marginal, and rachi-
dian teeth; irregular (malformed) lateral, marginal, and
rachidian teeth; and missing marginals were readily ap-
parent (Fig. Ic). Most consistently, tissue degrowth was
correlated with an increase in the number of radular
rows per unit ribbon length. At 40 days it was apparent
that either ( 1 ) production of subradular membrane by
the membranoblasts and transport by the inferior epithe-
lium had slowed, or (2) the production of radular teeth
by the odontoblasts had hastened. Regardless of the rela-
tive contributions of these alternative processes, the re-
sult is the same. An obvious zone (Figs. 1, 4) of denser
row-packing has been created. These observations, after
confirmation from field analysis, suggest that activity of
membranoblast and odontoblast cell lines is differen-
tially impaired during periods of food deprivation. The
nature of the control mechanism regulating these cells is
still uncertain so it is not possible to determine how food
deprivation influences the results documented here.
However, membranoblast activity is reduced to a greater
extent than that of the odontoblasts during periods of
sustained stress, and this differential secretory response
produces the characteristically packed rows. The fact
that there are no observable ecophenotypic effects on
tooth shape (thought to be under rigid genetic control in

1.0

o

o;
o

O

0.8

0.6

0.4

0.2

4.15

4.25 5.05
DATE

5.15

Figure 3. Return to normal radular secretion in spring in five natu-
ral populations of Helisoma, demonstrating recovery from radular
row-packing overwinter (and from presumptive overwinter tissue de-
growth). Vertical bars are 95% confidence limits of each mean.

30

D. A. SMITH AND W. D. RUSSELL-HUNTER

Eaton

B

1 mm

C.

Remsen

1 mm

Figure 4. Llnlouched photographs showing patterns of return to normal radular secretion in two natu-
ral stocks of lltiiMiiiui irivolvis. Note that the most recently secreted radular rows are at the top of each
photograph. Radulae from Eaton Reservoir (top, A-D. left to right) represent 29%, 40%, 61%, and 80%
regrowth (as fraction new rows of total rows). Radulae from Silver Lake. Remsen (bottom. E-H. left to
right) represent 17%, 36%', 53% and 76%' regrowth. Samples from both sites were taken at weekly intervals
beginning 4. 22. 85. Actual sizes are: Eaton (top, left to right), 3.1. 3.7, 3.2, and 3.8 mm, Remsen (bottom,
left to right). 3.7, 4.3. 3.8. 3.4 mm.

each stock or population; Smith, 1987, 1989) empha-
sizes the unique significance of row-packing as a predict-
able response to environmental stress.

In one respect, that of the time sequence of return to
normal tissue growth after stress, the radular record of

row-packing can be more useful than any assessments
based on shell-tissue ratios. In field studies of tissue de-
growth (Russell-Hunter el a/., 1984), one pond stock of
Hclisoma recovered from 41.3% average degrowth to
only 32.8% over three months in spring. In another stock

RADULAR CORRELATES OH DEGROWTH

31

(from a highly eutrophic lake), overwinter degrowth was
eliminated (47.1% net growth) in two spring months.
The data on radular recovery (corresponding to re-
growth) from the five sites sampled during this study not
only show that the process was complete within 25-43
days, but also indicated its temporal sequence in stages.
As noted above, there are two significant aspects to the
concurrence of abnormal radular secretion and of tissue
degrowth as consequences of starvation stress. The first
concerns a matter of fundamental biology in attempting
to deduce the control mechanisms involved and, ulti-
mately, the sequence of causality. All patterns of re-
sponse to environmental stress have evolved to increase
the fitness of individuals, and all basically require (i) re-
ceptors monitoring changes in the rate of abiotic and
physiological parameters, (ii) some system capable of in-
tegrating such inputs, and (iii) effector tissues that carry
out the response. In the case of the response to starvation
in gastropods, we have quantified for (iii) several kinds
of effects, we can deduce something of (ii), but we are
almost completely ignorant of (i) in specific terms. At the
very least we know that the simultaneous effects include
both abnormal radular secretion and general tissue de-
growth. The former involves both absolute and relative
reductions of the secretory activity of membranoblasts.
The latter involves not only highly reduced levels of gen-
eral catabolic activity but also a metabolic shift towards
relatively higher turnover of nonprotein carbon. As has
been noted (Russell-Hunter, 1985), such controlled
differential catabolism can be considered an appropriate
parsimony in the net flow through of amino acids (rather
than as the defense of a static protein biomass).

There are obvious elements of adaptive conservation
in the fact that odontoblast activity is less reduced than
membranoblast activity, and in the preservation (rela-
tively) of structural proteins in the tissues. Both differen-
tial processes are adaptive in their potential to accelerate
return to normal secretion and tissue regrowth when the
period of stress has ended. Parenthetically, it should be
noted that this capacity for controlled tissue degrowth
[increasing individual survivorship under certain envi-
ronmental conditions by a decrease in individual energy
content (Russell-Hunter, 1985; and references therein)]
compels reconsideration of certain fitness predictions
from simple models of age structure and energy parti-
tioning between growth and reproduction (see for exam-
ple, Williams, 1966; Tinkle and Hadley, 1975; Browne
and Russell-Hunter, 1978).

It seems likely that the ganglia of the snail's central
nervous system are involved in integration after the on-
set of starvation stress. It is unlikely that the integrating
system is linked neurally to the rate-controlling cells for
membranoblast secretion and those of differential pro-
tein catabolism, and barely possible that a specialized en-
docrine tissue if involved. It can be postulated that the

most likely link is through neurosecretory cells. Other
systems of integrated control in molluscs involve neu-
rosecretion. For example, sex change in Crcpidida (Rus-
sell-Hunter et ai, 197 1 ), and cyclic reproductive behav-
ior in high littoral snails (Price, 1979) involve neurose-
cretion. Despite the degree of integration of the response
to overwinter starvation, it may not be appropriate to
term this a diapause, since it is less obligate and more
plastic in these snails than in those nematodes and insect
larvae from similar habitats for which an innate and es-
sential seasonal diapause has been described. However,
there is integration of responses (probably involving neu-
rosecretion), and there can be no question either of ab-
normal radular secretion (row-packing) causing tissue
degrowth or even of tissue degrowth causing row-pack-
ing directly. Although the common cause of both sets
of responses appears to be the stress of starvation, these
statements belong within David Hume's ( 1 748) regular-
ity theory of causation, which remains appropriate for
the logical description of such biological sequences, de-
spite being currently unfashionable among many profes-
sional philosophers.

Conclusions from these experimental data have a sec-
ond significance to applied biology: the possibility of a
retrospective detection, in the field, of earlier periods of
stress affecting natural populations. Just as the trunks of
long-lived forest trees can record in their rings the histori-
cal sequence of drought years and of minor forest fires,
so the shells of long-lived bivalve molluscs (Clark, 1976;
Mallet et ai, 1987; Peterson et al., 1985) can record, in
their growth rings, a history of severe winters. Radular
records of degrowth periods as zones of modified tooth-
row secretion may provide a history of more recent envi-
ronmental stress. This may be of applied value in some
gastropod stocks by using comparative spring samples of
radulae from known populations to assess relative levels
of overwinter starvation, and thence to predict produc-
tivity for the rest of the year. In addition, similar radular
records could be useful in assessing the metabolic stress
of a transient period of pollution (such as an oil spill) on
populations of freshwater or marine littoral gastropods,
even if no records had been obtained before the popula-
tions were stressed.

Acknowledgments

Work was supported by grants from the Senate Re-
search Committee of Syracuse University (D.A.S. and
W.D.R-H.) and the Theodore Roosevelt Memorial Fund
(D.A.S.). Preparation of this manuscript was supported
by the Treves and Carscallen Funds of Wabash College.
This is contribution #102 of the Upstate Freshwater In-
stitute.

32

D. A. SMITH AND W. D. RUSSELL-HUNTER

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Reference: Biol. Bull 178: 33-45. (February. 1990)

A Decapod Hemocyte Classification Scheme
Integrating Morphology, Cytochemistry, and Function

JO ELLEN HOSE, GARY G. MARTIN, AND ALISON SUE GERARD
Department of Biology. Occidental College, Los Angeles. California 90041

Abstract. We have examined the hemocytes of three
decapod crustaceans (Homarus americanus, Panulirns
interruptus, and Loxorhynchus grandis) and propose a
classification of these cells based on morphology, cyto-
chemistry, and studies of cell functions. In all species,
hyaline cells and granulocytes were identified. Although
we have retained the widely used names for these cells,
we show that traditional morphological features alone do
not accurately differentiate between these categories.
Historically, the term hyaline cell refers to hemocytes
that contain no or only a few cytoplasmic granules,
whereas granulocytes contain abundant granules. How-
ever, the size and number of granules in hyaline cells
vary greatly between species and therefore are not useful
criteria for identifying these cells. Since morphological
identification alone is inadequate and misleading, espe-
cially with regard to hyaline cells, a combination of mor-
phological, cytochemical and functional methods is nec-
essary to identify decapod hemocytes. Features of hya-
line cells include: a higher nucleocytoplasmic ratio than
that of granulocytes, the presence of abundant small
(~50 nm), round, electron-dense deposits in the cyto-
plasm, and their accumulation of trypan blue dye prior
to cytolysis. Granulocytes do not take up trypan blue or
lyse during a 5-min incubation, and they contain pro-
phenoloxidase and hydrolases. Hyaline cells are involved
in the initiation of hemolymph coagulation whereas
granulocytes are involved in defense against foreign ma-
terial by phagocytosis and encapsulation. We propose
that these criteria be applied to other crustacean species
and expect that they will facilitate our understanding of
the physiological roles of their hemocytes.

Introduction

In crustaceans, circulating hemocytes are thought to
be involved in hardening of the exoskeleton, prevention

Received 3 April 1 989; accepted 30 November 1989.

of blood loss and the confinement of invasive organisms
by clot formation, recognition of non-self, phagocytosis,
and encapsulation (Bauchau, 1981; Ratnerand Vinson,
1983). Although recent research has expanded the vari-
ous physiological roles played by crustacean hemocytes,
extention of this information from one species to an-
other is difficult because of the lack of a unified classifi-
cation scheme for the hemocytes of all Crustacea. Prior
hemocyte classification systems rely on tinctorial proper-
ties of the cells, which are often subtle or subjective, and
seldom apply to other species ( Martin and Graves, 1985).
Using the penaeid shrimp Sicyonia ingentis as a proto-
type for decapod crustaceans, a hemocyte classification
system was developed, which relates cellular morphology
at the light and electron microscope levels, cytochemis-
try, and three essential functions: clotting, phagocytosis,
and encapsulation (Martin et a!., 1987; Hose etal., 1987;
Omori et al., 1989; Hose and Martin, 1989). The choice
of this species proved serendipitous because the three
types of hemocytes are morphologically distinct and clot-
ting occurs by explosive cytolysis (Tail's type C coagula-
tion; Tait, 1911), making identification of the clotting
cell type relatively easy. At the electron microscope level,
the cells that initiate clotting are readily identified by sev-
eral features typical of hyaline cells (small size, a high
nucleocytoplasmic ratio, and scarcity of cytoplasmic
granules) and by the presence of numerous, small (~50
nm diameter), electron-dense deposits in the cytoplasm.
In addition, the hyaline cells selectively stain with Sudan
black B. as does coagulogen extracted from cell-free
hemolymph of Paniilirus interruptus and Astacus lepto-
clactyhts (Durliat, 1985). During lysis of these cells, the
deposits appear to extend through breaks in the plasma
membrane and hydrate to produce the clot (Omori et
al.. 1989). The granulocytes are larger cells with a lower
nucleocytoplasmic ratio and contain numerous small
(0.4 ^m diam.) or large (0.8 ^m diam.) granules. Granu-
locytes (small and large granule hemocytes) show no

33

34

J. E. HOSE ET AL

morphological changes during coagulation and are capa-
ble of phagocytosis of bacteria and encapsulation of fun-
gal hyphae. Phagocytosis is accomplished primarily by
small granule hemocytes (Hose and Martin, 1989); they
contain many vesicles and occasional granules that stain
for acid hydrolases (acid phosphatase, /3-glucuronidase,
and nonspecific esterase) (Hose et al., 1987). Encapsula-
tion is initiated by large granule hemocytes and, to a
lesser extent, by small granule hemocytes (Hose and
Martin, 1989). Prophenoloxidase (PPO), an enzyme in-
volved with melanization of encapsulated material and
possibly the recognition of non-self items (Soderhall.
1982), is most abundant in large granule hemocytes and
to a lesser degree in some small granule cells. In contrast,
hyaline cells, which do not phagocytize bacteria or assist
in capsule formation, do not contain PPO and only
rarely acid phosphatase (Hose el al.. 1987). Thus the di-
vision of shrimp hemocytes into two functional groups,
hyaline (or clotting) cells and granulocytes, is supported
by morphology, cytochemistry, and function.

This paper extends the results of the shrimp studies to
other decapods and attempts to develop a unified hemo-
cyte classification system for the diverse assemblage of
crustaceans. This diversity is exemplified by the exis-
tence of multiple coagulation mechanisms. In contrast
to clotting via explosive cytolysis as in the shrimp (type
C according to Tait, 1911), other decapods exhibit type-
A coagulation which is distinguished by the formation of
a dense hemocyte network which seals oft' the injury and
plasma coagulation is not apparent or type-B coagula-
tion in which hemocyte aggregation is followed by
plasma coagulation (Tait, 191 1). In the present study, we
examined the hemocytes in one species with type-A co-
agulation (a crab, Loxorhynchus grandis), one species
with type-B coagulation (the Maine lobster, Homarm
anicriainus), and one species with type-C coagulation
(the spiny lobster, Pamdiriis interniptus). Light and elec-
tron microscopic features of hemocytes from these three
decapods are compared to those identified in the shrimp
and correlated with a suite of cytochemical characteris-
tics (Sudan black B, acid phosphatase, and PPO) and a
group of essential physiological functions (clotting,
phagocytosis, and encapsulation). The methods pre-
sented here should facilitate study of decapod hemocytes
by providing a framework for practical hemocyte classi-
fication.

Materials and Methods

Animals

Spiny lobsters (P. interruptus] and sheep crabs (L.
grandis) were collected in less than 1 m of water at King
Harbor Marina, Los Angeles, California. Maine lobsters
(H. americtiHiis) were purchased commercially. Crusta-

ceans were maintained in flow-through aquaria at 18C
and only intermolt animals were studied.

Microscopic examination of hemocytes

Freshly fixed hemocytes were examined by light mi-
croscopy (LM) (brightfield and phase contrast optics) to
determine cell size, cell shape, granule size, and differen-
tial hemocyte counts. An aliquot of hemolymph (usually
0.2 cc) was withdrawn from the ventral sinus or heart
into a 1 cc syringe containing 0.4 cc of fixative (2.5% glu-
taraldehyde in 0. 1 A/sodium cacodylatepH 7. 8 contain-
ing 12% glucose).

Excess fixative was added to a second 0.2 cc hemo-
lymph aliquot, and the cells were processed for examina-
tion by electron microscopy. The cells were fixed for 2 h
at room temperature and pelleted (10,000 X g for 1.5
min). Following a 10-min wash in 0.1 M sodium caco-
dylate (pH 7.8) containing 24% sucrose, the cells were
post-fixed in 1% OsO 4 in 0.1 M sodium cacodylate for 1
h at room temperature. Each sample was stained en bloc
for 1 h with 3% uranyl acetate in 0. 1 M sodium acetate,
dehydrated in a graded series of ethanol, and infiltrated
and embedded in Spurr's (1969) low viscosity plastic.
Thin sections (90 nm) were cut on a Porter Blum MT2B
ultramicrotome, stained with lead citrate and exam-
ined in a Hitachi HU 1 1 A transmission electron micro-
scope (TEM).

Nucleocytoplasmic ratios were determined by divid-
ing the area of the nucleus by the area of the cell. For
hyaline and small granule hemocytes, both areas were
clearly identified in light micrographs of immediately
fixed cells and measured using a digitizing tablet and Sig-
ma-Scan computer software (Jandel Scientific). Be-
cause the nucleus is difficult to visualize in phase contrast
images of large granule hemocytes, measurements were
made from thick plastic sections. To ensure that cells
were sectioned through their greatest axis, only large
granule hemocytes showing typical length and width
measurements were used. There was no difference in size
measurements of fixed cells examined in wet mounts by
phase optics and cells embedded in plastic and sectioned.

Identification of cell-type initiating coagulation

Two previously used approaches helped to identify the
type of hemocyte initiating coagulation of the hemo-
lymph: ( 1 ) visual examination of hemocyte types accu-
mulating trypan blue, an event we have previously
shown to be a direct precursor to cytolysis and ensuing
clot formation and (2) ultrastructural examination of he-
mocytes fixed at stages during clot formation (Omori et
al.. 1989). For the first technique, 0.1 cc of freshly drawn
hemolymph was gently mixed on a glass slide with 0. 1 cc
of a 1.2% solution of trypan blue in seawater. Within 1-
2 min, certain hemocytes accumulate the blue color in

DECAPOD HEMOCYTE CLASSIFICATION

35

both the cytoplasm and nucleus. By 5 min these cells lyse
and the cytoplasm is lost, but the blue staining nuclei
remain. Individual cells may be identified and observed
as they accumulate the dye and lyse. After 5 min, the
number of blue stained nuclei and the cells remaining
intact and colorless were counted. Six hundred cells were
evaluated for each species.

The second method provided ultrastructural informa-
tion on the type of hemocyte that initiates coagulation as
well as changes in these cells during cytolysis. Aliquots
of hemolymph (0. 1 cc) and seawater (0. 1 ml) were mixed
for times ranging from 1 5 s to 5 min and then fixed by the
addition of an excess amount of gluaraldehyde fixative and
prepared for TEM examination as described above.

Phagocytosis of bacteria by hemocytes

In vitro phagocytosis experiments were performed as
described by Hose and Martin (1989). A glass micro-
scope coverslip was placed into each of two sterile plastic
Petri dishes and each covered with 20 ml of shrimp cul-
ture medium (SCM, Brody and Chang, in press). Ap-
proximately 0.3 cc of hemolymph was added over each
coverslip, and hemocytes were allowed to settle and at-
tach to the coverslips for 15 min. Approximately
100,000 cells of a Gram-negative marine bacterium (Cy-
top/iaga sp.; Occidental College Isolate 1 ) were added to
one of the dishes. Cultures were incubated at 12C for 3
h. Coverslips were fixed in methanol for 5 min and
stained with May Grunwald-Giemsa. Differential counts
of approximately 200 hemocytes were performed and the
numbers of phagocytic cells (hemocytes containing at least 1
bacterium within a vacuole) were recorded. Dead hemocytes
were differentiated from viable cells by the presence of
nuclear degeneration (karyolysis, pycnosis).

Fungal encapsulation

The method of Hose and Martin (1989) was used to
determine the types of hemocyte that attached to fungal
hyphae and initiated capsule formation. Approximately
1 ml of hemolymph was added to 1 2 ml SCM in a 1 5 ml
plastic centrifuge tube. Small cubes (0.5 mm 3 ) of Sabou-
raud-dextrose agar containing primarily hyphae of Fu-
sarium solani (University of Arizona strain 1623C) were
added to the tube; the culture was incubated at 1 2C. Af-
ter 1. 2, and 5 min, a cube was removed and washed
gently in SCM to remove nonadherent hemocytes. The
cell types attached to the fungus were identified using phase
contrast microscopy (total of 200 cells for each species).

Hemocyte cytochemistry

Hemolymph (0.2 cc) was withdrawn into 0.2 cc of
12.5% unbuffered citrate anticoagulant (which prevents
lysis of hyaline cells), spread on three glass microscope

slides, and allowed to air dry. Constituents of hemocyte
granules and cytoplasm were visualized using methods
given in Hose et al. (1987). Smears were prepared from
six individuals of each species. Where possible, 200 cells
per slide were evaluated using brightfield microscopy
( 1000X); each hemocyte was categorized and individual
cellular reactions were recorded.

Lipids and lipoproteins were demonstrated using a
commercial Sudan Black B kit (Sigma Chemical Co. Kit
#380). Glutaraldehyde-fixed hemocytes were processed
according to provided directions except that the nuclear
counterstain was not used. Cytoplasmic staining was
differentiated from staining of granule or plasma mem-
branes and was termed a positive reaction (Hose et al.,
1987). Occasionally entire granules were stained by Su-
dan Black B; these are noted in the results.

Prophenoloxidase (PPO) activity was evaluated in
smears fixed in 2.5% glutaraldehyde in 0.1 M phosphate
buffer (pH 7.4) for 1 h at 4C. The smears were rinsed
three times in phosphate buffer ( 1 5 min each ), then incu-
bated in 0.1% L-DOPA in phosphate buffer for 16 h at
room temperature. Black staining of the granules was in-
terpreted as a positive reaction (Hose el al., 1987).

Acid phosphatase in glutaraldehyde-fixed hemocytes
was visualized using a commercial research kit (Sigma
Chemical Co. Kit #386). Naphthoi AS-B1 phosphate was
used as the substrate, yielding a red-violet reaction prod-
uct (naphthol AS-Bl-fast garnet GBC complex). Al-
though the location and abundance of acid phosphatase
was species-specific, the rating system previously used for
the shrimp (Hose el al., 1 987) was acceptable for use with
the lobsters. In the shrimp we recognized the following
categories: rare (0 to 3 positive foci per cell), few (4 to 10
foci per cell), intermediate ( 1 1 to 30 foci per cell), and
many (>30 foci per cell). Because L. grandis contained
more acid phosphatase foci than the shrimp, the rating
system was slightly modified for evaluation of crab he-
mocytes. A distribution of the number of positive foci
per cell was constructed and the limit for the "rare" cate-
gory placed between the groups containing rare and few
foci. Thus while the rare category consisted of less than
four foci for shrimp and the lobsters, it was enlarged to
include less than six foci for the crab. A rare response was
interpreted as negative. Remaining limits were identical
for all four species. Two hundred cells were evaluated for
each test; each hemocyte was identified and individual
cellular reactions were recorded.

Results

Description of hemocyte types

Using morphological criteria previously developed for
the shrimp (Martin et al., 1987), three basic cell types
were observed in each of the decapods studied; one type

36

Comparative hemocytc morphology

J. E. HOSE ET AL
Table I

Large granule cells

Small granule cells

Hyaline cells

Homarus americanus

Cell size (length x width)
Granule diameter
Number ot granules
Nucleocytoplasmic ratio

Panulirus interruptus
Cell size (length width)
Granule diameter
Number ot granules
Nucleocytoplasmic ratio

Loxorhynchus grandis
Cell size (length width)
Granule diameter
Number of granules
Nucleocytoplasmic ratio

23.42.2 x 12. 3 1.3(11)

1.3 0.1 (30)
72. 7 4.6 (30)
20.3 1.8(10)

22.6 l.3x 14.1 0.5(12)

1 .4 0.1 (30)
34.0 + 2.4(30)

I8.3 1.3(8)

20.4 0.5 < 13. 7 0.6 (1 9)

1.4 + 0.1 (30)
28.0 4. 1 (30)
17.5+ 1.5(1 1)

20.9 0.6 x 13.9 0.3 (18)

0.9 + 0.1 (30)

27. 3 5.6 (30)

27.5 2.4(10)

1 9. 7 0.9 x 10.0 0.3 (18)
0.8 0.1 (33)
10.8 4.3 (30)

24.6 2.1 (16)

I 7.9 0.5 X 10.6 0.4 (15)

1.0 + 0.1 (30)

8.0 1.6(30)

23.5 1.0(11)

14.1 + 0.8X 11.20.6(16)

0.9 0.1 (30)
13.9 + 3.0(30)
40.0 2.5 (13)

14.2 + 0.5 X 10.6 0.2 (18)

1.2 0.1 (30)
5.6 + 0.8(30)

40.2 1.5(16)

14.5 1.0 x 9.1 0.3(12)
1.0 + 0.1 (30)

38.3 5.6 (30)
36.0+ 1.5(16)

Measurements are mean standard error (number ot measurements). Cell sizes are presented in length width. Cell and granule sizes are in
/jm. Number ot'granules is the number per sectioned cell. Nucleocytoplasmic ratios are percentages.

T-tests showed significantly smaller hyaline hemocyte size for all three species compared to either small or large granule hemocytes (P < 0.05).
Nucleocytoplasmic ratios for hyaline hemocytes of all three species were significantly larger than those of small or large granule hemocytes (P
< 0.05).

of hyaline cell and two subgroups of granulocytes (small
and large granule hemocytes) (Table I).

Hyaline Cells (Figs. 1A-6A). Hyaline cells were the
most morphologically diverse type of hemocyte. When
examined by phase contrast microscopy, they were gen-
erally ovoid in shape, smaller than granulocytes and with
a higher nucleocytoplasmic ratio (Table I), and either
contained few large granules (P. interruptus) or numer-
ous smaller granules (//. americanus and L. grandis).
Most hyaline cells of //. americanus measured 21X11
Atm although smaller hemocytes (from 12 ^m in the larg-
est dimension) were occasionally observed. The smaller
cells may represent immature hyaline cells because they
were continuous in size with the hyaline cell and proba-
bly correspond to the prohyalocyte category recognized
by Cornick and Stewart (1978). Hyaline hemocytes of
H. americanus had numerous (14/section) small, ovoid
granules, 0.9 jum long, the contents of which appeared
homogeneous and electron dense at the EM level. The
cytoplasm contained Golgi bodies, abundant rough en-
doplasmic reticulum (RER), a circumferential band of
microtubules, a few vesicles, mitochondria, and small
(~50 nm diam.), round, electron-dense deposits (Fig. 7).
Hyaline cells of the crab (L. grandis) resembled those of
the Homarus, except they contained more granules (40/
section) that, at the EM level, were ovoid, homogeneous,
and electron dense. Hyaline hemocytes of the spiny lob-
ster (P. interruptus) were distinctive in that only a few
(6/section) large (1.2 ^m diam.) granules were present.
Ultrastructurally the granules had a punctate pattern.

Otherwise, features of the cytoplasm were similar to
those described for the other species.

Granulocytes. Granulocytes could be easily differen-
tiated into two groups using phase contrast microscopy:
small (Figs. 1B-6B) and large (Figs. 1C-6C) granule he-
mocytes. Small granule hemocytes contained few to
many, round, dark, small (usually <1.0^m diam.) gran-
ules and a relatively small, centrally located nucleus,
whereas the cytoplasm of large granule hemocytes was
packed with larger (1.3-2.0 nm diameter), refractile
granules that obscured the eccentrically placed nucleus
(Table I). However, it was sometimes difficult using
TEM to distinguish between small granule hemocytes
containing numerous granules and large granule hemo-
cytes because the sectioned granules appeared similar in
size. These cells may be part of a single line of maturation
in which the number and size ofgranules in small gran-
ule hemocytes increase until the cell is recognized as a
large granule hemocyte. To distinguish between small
and large granule hemocytes, we relied on ( 1 ) the loca-
tion of the nucleus (centrally or eccentrically placed) and
(2) the presence of only large granules (> 1.2 ^m diam.) in
large granule hemocytes while small granule hemocytes
may contain both large and small granules. Both types of
granules were often surrounded by a clear (artifactual?)
space (see Figs. 6B and C), and in P. interruptus, granules
in the large granule hemocytes often did not section
cleanly but appeared fractured (see Fig. 4C), unlike those
present in small granule cells. The cytoplasm of granulo-
cytes, both small and large granule hemocytes, contained

DECAPOD HEMOCYTE CLASSIFICATION

37

Figures 1-3. Light micrographs of hemocytes from Panulirux interruptus (Fig. 1), Htmiarus ameri-
canus (Fig. 2), and Loxorhynchus grandis (Fig, 3) showing hyaline cells (column A), small granule (column
B), and large granule (column C) types. Note the small size of the hyaline cells compared to the granulo-
cytes. The large granule cells are highly refractile and it is difficult to observe the nucleus. All figures at
2600X; scale bar =

Golgi bodies, RER, vesicles, mitochondria, ribosomes,
and microfilaments scattered between the granules (see
Fig. 7). Microtubules, typically in a band adjacent to the
plasma membrane, were commonly seen.

Hemocyte differential counts

Differential counts were performed using phase con-
trast LM and TEM (Table II). Although individual
paired counts are similar, we consider the TEM counts
more accurate for comparison because of the inherent
greater resolution. P. interruptus had the highest percent-
age of hyaline cells at 56%, whereas L. grandis and //.
americanus were considerably lower at 2 1 % and 27%, re-
spectively. Large granule granulocytes constituted be-

tween 10% and 13% of the total with small granulocytes
comprising about 65%. in H. americanus and L. grandis
and 31%. in P. interruptus.

Clotting

Patterns of coagulation. The species studied represent
the three coagulation patterns described by Tail (1911).
In L. grandis (Tail category A), the bulk of the clot con-
sisted of long cellular aggregations linked by strands of
clot material. The clot produced by H. americanus (Tail
category B) contained isolated islands of coagulated
hemolymph with intervening areas of packed hemo-
cytes, whereas in P. interruptus (Tail category C), cell ag-

Figures 4-6. Transmission electron micrographs of hemocytes from Panulints inierrupiu* (Fig. 4).
Homarus americanus (Fig. 5), and Loxorhynchusgrandis(F\g. 6) showing hyaline (column A), small gran-
ule (column B) and large granule (column C) types. Granules are present in hyaline cells although not
abundant. Small granule hemocytes are characterized by small granules in a relatively large amount of
cytoplasm and in large granule hemocyles the granules till much ol the cytoplasm. All figures at 5500X;

scale bar = 5 ^m.

38

Figure 7. Transmission electron micrograph of a hyaline hemocyte from Homarus americanux show-
ing cytoplasmic deposits (arrows), RER (R), a granule (G). vesicles (V), nucleus (N). and edge of Golgi
body (GB). 70,000x; scale bar = 0.5 ^m.

Figure 8. Light micrograph of a large granule hemocyte from Pamdinis inlemiptux 2 min after mixing
equal volumes of hemolymph and seawater containing trypan blue. Note the cell is intact and has spread
on the glass substrate. Nucleus (N); filopodia(F). 2000X; scale bar = 10 ^m.

Figure 9. Light micrograph of two hyaline cells from P. inlcrrupius from the same preparation as cells
in Figure 8. These cells have not attached to the substrate and they have lysed, leaving a light (blue-stained)
nucleus (N), blebs (B), and thin rim of residual cytoplasm (arrows). 2000 X; scale bar = 10 ^m.

Figure 10. Light micrograph of hemocytes from P. inlvrruplus during early clot formation (2 min
after mixing with seawater). Note the clusters of hemocytes between the large circular areas of coagulated
hemolymph (C). Large granule hemocytes (L); small granule hemocytes (S); and hyaline cell (H). 700X;
scale bar = 25 ^m.

Figure 11. Light micrograph of hemocytes from Loxorhynchus ,i,'*w/<//.v during early clot formation (2
min after mixing with seawater). Note the large aggregation of hemocytes. Obvious areas of coagulated
hemolymph are not present. 700X; scale bar = 25 ^m.

39

40

J. E. HOSE ET AL.

Table II

Comparative hemocyle differential counts using light tiiul tranxmixxiim
electron microscopy

Large granule Small granule

cells cells Hyaline cells

Homarus

americanus

Light 16.4 + 2.7 60.2 3.6 22.4 2.4

microscopy ( 10.0-26.5; 9) (41.5-76.2; 9) ( 1 1.9-34.2; 9)

Electron 10.3 1.6 62.4 2.1 27.3 2.8

microscopy (7. 8-19. 5; 7) (53.8-71.3; 7) ( 15.2-35.9; 7)
Panulirus

intern/plus

Light 9.8 2.6 29.2 + 3.6 61.0 + 3.4

microscopy (4.0 + 21.0; 6) (22.0-45.5:6) (50.0-72.0; 6)

Electron 12.6+1.7 31. 1 1.7 56.3 1.7

microscopy (6. 3-18. 7; 8) (26.6-40.2; 8) (48.5-62.8; 8)
Loxorhynchus

grandis

Light 14.1+2.3 67. 8 5. 3 18.1 3.8

microscopy (6.0-2 1.0; 6) (44.0-79.0; 6) ( 1 1.0-35.0; 6)

Electron 10.4+0.7 68.81.6 20.81.6

microscopy (8.0-13.0:6) (62.2-72.0:6) (15.4-25.6:6)

Values are mean + standard error (range; number of measurements).

gregates were small and widely separated by clotted he-
molymph.

Trypan blue experiments. Previous work with deca-
pods has shown hemocyte lysis to be the initiating step
in hemolymph coagulation. The method used here to
identify lysing hemocytes simulates coagulation result-
ing from a seawater influx such as a break in the exoskel-
eton. The addition of trypan blue (which does not alter
the process of coagulation) facilitates early identification
of lysing hemocytes. Cell categorization at the LM level
is impossible following lysis; thus TEM was used to de-
scribe successive steps in coagulation (see next section).
When hemolymph was mixed with seawater containing
trypan blue, two populations of cells were observed. In
one group, the cells contained distinct granules, spread
on the glass slide, extended filopodia(Fig. 8), and did not
accumulate trypan blue during the observation period.
This group was composed of small and large granule he-
mocytes. The second group of cells remained ovoid and
did not attach to or spread on glass slides; the cytoplasm
and nucleus of these cells appeared blue. By 1 min, mul-
tiple blebs were apparent beneath the plasma membrane.
Cytolysis occurred by 2 min in L. grandis and by 5 min

for the lobsters, leaving a blue-staining nucleus and frag-
ments of cytoplasm (Fig. 9). Visual observations of indi-
vidual cells confirmed that the lysing cells were hyaline
cells in all three species (Table III). In addition, the per-
centage of lysing cells in each species corresponded to
the percentage of hyaline cells obtained from differential
counts. Using the trypan blue method, the smallest pro-
portion of clotting cells was found in L. grandis (19%),
with //. americanus intermediate (34%) and P. interrup-
tus highest (50-70%). We suggest that the coagulation
patterns reported by Tail (191 1) are not three distinct
categories but rather represent a continuum relating to
the percentage of hyaline cells. Species with the highest
hyaline cell percentage exhibit type C coagulation; lysis
of these cells results in large expanses of coagulated he-
molymph with small intervening clusters of granulo-
cytes. As the percentage of hyaline cells decreases, so
does the amount of coagulated hemolymph. Therefore,
in animals with type A coagulation, like the crab, the pre-
dominant feature is the aggregated granulocytes which
are, in fact, connected by strands of coagulated hemo-
lymph.

The trypan blue method was particularly useful with
the crab because the hyaline cells are not abundant, lyse
faster than in the other species, and the granulocytes rap-
idly attach to one another to produce large aggregations.
Thus lysed cells in unstained preparations were difficult
to identify within the mass of cells (primarily granulo-
cytes) that appear unaffected and intact.

Morphology of coagulation. When equal volumes of
hemolymph and seawater were mixed, coagulation be-
gan immediately and produced a strong clot within a few
minutes. Clots produced in P. interrupt us were com-
posed primarily of coagulated hemolymph with hemo-
cytes scattered throughout the matrix (Fig. 10). In H.
americanus. less coagulated hemolymph was present
and tightly compacted clusters of granulocytes were con-
spicuous. In L. grandis, the bulk of the clot was produced
by aggregated cells (Fig. 1 1 ) and only strands of coagu-
lated hemolymph.

In all three species, changes in cell morphology oc-
curred primarily in the hyaline cells. Early changes in-
volving loss of plasma membrane integrity were identical
to those observed in the preceding trypan blue experi-
ments. Electron microscopy revealed that the centers of
the hyaline cell blebs appeared empty and were sur-
rounded by round, electron-dense deposits (Fig. 12).
Granules in these cells, which were homogeneous in sec-
tioned material and dispersed throughout the cytoplasm.

Figure 1 2. Transmission electron micrograph of a hyaline cell from L. grandis 2 min after mixing with
seawater showing the formation of blebs (B). Note the homogeneous granules (G) aggregated around the
nucleus (N) and the small electron-dense deposits (arrows) throughout the cytoplasm. 12,000> ; scale bar
= 2.5 fim.

DECAPOD HEMOCYTE CLASSIFICATION
Table III

41

Comparative liemocyte lunctiun.t

Large granule cells
(% positive)

Small granule cells
(% positive)

Hyaline cells
(% positive)

Homarus americanus

Clotting:
% accumulate trypan blue

Phagocytosis:
% phagocytic
%dead(+ bacteria)
%dead(- bactena)

Encapsulation:

% of adherent cells
Painilirus interrupt us

Clotting:

% accumulate trypan blue

Phagocytosis:
% phagocytic
% dead (+ bacteria)
%dead(- bacteria)

Encapsulation:

% of adherent cells
Loxorhynchus grandis

Clotting:

% accumulate trypan blue

Phagocytosis:
% phagocytic
%dead(+ bacteria)
%dead(- bacteria)

Encapsulation:
% of adherent cells

0.0

39.3
9.3
0.0

63.3

32.0
0.0
0.0

68.4

0.3

70.0
4.8
16.7

67.2

1.2

93.1
11.5

0.0

21.2

83.1
16.2
0.0

25.3

0.7

95.9
6.1

1.7

28.7

100.0

0.0
83.0
29.7

0.0

100.0

0.0
31.6

1.6

6.3

100.0

0.0
95.0
24.3

4.1

Mean percentage of each category which accumulates trypan blue, is phagocytic, or initiates encapsulation by adhering to fungal hyphae. In the
clotting experiments. >100 hemocytes in each category were evaluated from each of 5 animals. In the phagocytosis experiments, >100 hemocytes
in each category were evaluated from a single animal. Percentages of dead hemocytes were compared in the presence ( + ) and absence ( - ) of bacteria.
In the encapsulation experiments, > 100 hemocytes in each category were evaluated from each of 5 animals.

became concentrated around the nuclear envelope. As
the plasma membrane over the blebs ruptured, cyto-
plasm containing the deposits and disrupted organelles
was released. Surrounding the degenerating hyaline cell,
long strands were formed in the hemolymph, which ap-
parently hydrated into typical clot material. Concur-
rently, granules developed a scalloped margin, their con-
tents became grainy, and adjacent granules sometimes
fused. Granules released their constituents either by exo-
cytosis or lysis into the cytoplasm.

As hyaline cells of P. interniptus lysed, spheres of coag-
ulated hemolymph developed around each cell (Fig. 10).
The spheres expanded and fused with adjacent spheres
to produce a continuous hemolymph clot with clusters
of granulocytes scattered between roughly spherical ar-
eas of coagulated hemolymph. Hemolymph clots of Ho-
marus contained fewer areas of coagulated hemolymph
and larger intervening granulocyte clusters. In contrast,
the clotted hemolymph of L. grandis was composed of
masses of aggregated granulocytes (Fig. 1 1 ) often adher-
ing to long strands of clot material. The granulocytes in

all three species did not lyse during the 1-h time period
examined in this study, although they extended filo-
podia. Large and small granule hemocytes rarely dis-
played exocytosis of granules.

Phagocytosis of bacteria

Phagocytosis of the Gram-negative bacterium Cyio-
phaga sp. was performed by most small granule hemo-
cytes, some large granule hemocytes, and none of the hy-
aline hemocytes (Table III). The percentage of phago-
cytic small granule hemocytes ranged from 83% to 96%,
whereas only 30% to 67% of the large granule cells were
phagocytic. In contrast to incubation in seawater, where
cytolysis of hyaline cells was observed, most hyaline cells
and granulocytes remained viable when incubated in
SCM (shrimp culture medium) for the 2-h duration of
the phagocytosis experiments. However, enhanced au-
tolysis was observed when hyaline cells and granulocytes
were cultured in the presence of bacteria (Table III). Hya-
line cells that did not lyse during the experiments did not

42

J. E. HOSE ET AL.

Table IV
Comparative hemocyte cytochemistry

Large
granule cells

Small
granule
cells

Hyaline cells

Hninuni.'iiimi'riainii.'i

Acid phosphatase

48.3 8.5

23.6 8.0

0.5 0.5

(25.0-80.0)

(6.7-59.5)

(0.0-2.9)

Prophenoloxidase

86.6 5. 2

10.8 1.3

0.0 0.0

(71.4-100.0)

(5.0-14.3)

(0.0-0.0)

Sudan Black B

0.0 0.0

0.4 0.4

99.4 0.6

(0.0-0.0)

(0.0-2.5)

(96.1-100.0)

Piiiiulirit.t inti'mi/Hits

Acid phosphatase

72.1 6.4

69.4 7.2

4.9 1.7

(44.4-90.0)

(4 1.0-9 1.5l

(0.0-1 1.1)

Prophenoloxidase

96.8 3.4

35.2 4.9

0.2 0.2

(91.7-100.0)

(20.7-55.7)

(0.0-1.0)

Sudan Black B

0.0 0.0

(i.o 0.0

99.6 + 0.2

(0.0)

(0.0)

(98.8-100.0)

Loxorhynchus

grandi \

Acid phosphatase

88. 7 5. 8

76.6 7. 7

11.4 2. 8

(61.5-100.0)

(51.9-96.5)

(1.9-22.6)

Prophenoloxidase

100.0 0.0

53.4 4.2

1.0 1.0

(100.0)

(41 8-66.7)

(0.0-5.9)

Sudan Black B

0.0 0.0

0.4 0.2

100.0 J no

(0.0)

(0.0-1.3)

(100.0)

Percentage of positive hemocytes standard error. Minimum and
maximum values are in parenthesis. Twenty large granule. 50 small
granule, and 50 hyaline hemocytes were examined from each of 6 ani-
mals.

attach to the glass and spread as did the granulocytes, but
often adhered to the granulocytes and remained ovoid.
Total phagocytosis rates (denned as the number of
phagocytic cells divided by the total number of surviving
hemocytes) were 79% and 88%, respectively, for //.
americanus and L. grand is (the two species with no sur-
vival of hyaline hemocytes) and 54% for P. internipius
which had higher hyaline cell survival.

Encapsulation of fungal hyphac

LM observations of initial hemocyte contact with fun-
gal hyphae showed that approximately two-thirds of ad-
herent cells were large granule hemocytes, between 20%
and 30% were small granule hemocytes and only small
percentages were hyaline hemocytes (Table III). For all
three species, percentages of adherent large granule cells
were enriched 7 to 15 times over those found in hemo-
lymph.

Hemocyte cytochemistry

Hemocyte smears were stained to identify sites of lipid
(with Sudan black B), or acid phosphatase, or PPO activ-
ity (Table IV). Sudan black B, which stains lipids and

lipoproteins, produced diffuse cytoplasmic staining in all
hyaline cells of each species. However, the staining inten-
sity was less than that previously reported for deposit-
containing hyaline cells of the penaeid shrimp in which
the entire cytoplasm is darkly stained in a clumpy pat-
tern (Hose el al. 1987). In the present species, the light
grey, homogeneous staining of the cytoplasm was diffi-
cult to detect without prior experience with the stain.
The most distinctive feature in Sudan black-stained cells
was that the nucleus in hyaline cells was obsurred by the
stain, similar to that observed in the shrimp (Hose el al.,
1987). Granules in the hyaline cells of all three species
did not accumulate the stain, although membranes
around the larger granules in some hyaline hemocytes of
//. americanus displayed intense staining. The cyto-
plasm of the granulocytes remained unstained by Sudan
black B and the nucleus was always visible. Except for
intense staining of granule membranes in large granule
hemocytes of//, americanus, staining reactions of granu-
locytes were identical to those reported for shrimp hemo-
cytes.

Reaction sites demonstrating acid phosphatase were
rare in hyaline cells and more abundant in granulocytes
(Table IV). For each species, ranges of the percentages of
positive cells did not overlap between the two categories.
Most small granule hemocytes had few to an intermedi-
ate number of foci. As observed in the shrimp (Hose el
al., 1987), not all large granule hemocytes contained re-
action sites, but positive cells had numerous foci. In L.
grandis, acid phosphatase was primarily located in the
granules, although some vesicles and tubules (most likely
RER) stained positive as well. In the lobsters, only a few
granules contained acid phosphatase and most of the re-
action sites were located in vesicles and tubules of RER.

PPO activity was restricted to granulocytes (only one
out of approximately 300 hyaline hemocytes of the crab
and spiny lobster appeared positive). From 11% (//.
americanus) to 53% (L. grandis) of small granule hemo-
cytes had positively stained granules, whereas most
(>87%) large granule hemocytes contained numerous
dark-staining granules. The cytoplasm of large granule
cells also contained PPO while staining in small granule
hemocytes was confined to the granules.

Discussion

Our results suggest that hemocytes of decapod crusta-
ceans are composed of two major groups, hyaline cells
and granulocytes, which have distinct functional and cy-
tochemical differences. Most investigators have histori-
cally recognized these two categories and separated them
using morphological criteria (see Martin and Graves,
1985, for review). However, our work demonstrates that
the morphological features traditionally used to identify
these categories do not reliably correlate with cellular

DECAPOD HEMOCYTE CLASSIFICATION

43

functions. Although granulocytes of the three species
studied in this paper, the penaeid shrimp (Sicyonia in-
gentis) used to develop the system, and several other spe-
cies described in the literature (Bauchau, 1981) are
morphologically almost indistinguishable, hyaline he-
mocytes constitute a heterogeneous group. Our observa-
tions may explain much of the confusion in the literature
regarding hemocyte morphology and function. Such dis-
crepancies have prevented information obtained on a
particular species to be readily interpreted with regard to
other decapods. In some cases, functional studies have
not identified cell types involved in hemolymph coagula-
tion and phagocytosis. For example, both Schapiro cl al.
(1977) and Goldenberg et al. (1986) presented quantita-
tive data on phagocytosis of bacteria by H. americaniis
hemocytes, but neither group could identify the phago-
cytic hemocytes. In other cases, morphological identifi-
cation did not correspond to functional roles. For in-
stance, Soderhall cl al. (1986) refer to the hyaline cell
as the main phagocytic hemocyte in the crab Carcinus
maemis whereas in the crayfish Pacifastacus leniiisculus
phagocytosis is performed by both hyaline cells and
semigranular cells. Such lack of consistency in ascribing
similar functions to apparently similar hemocyte types
stems from difficulties of using a classification system
based on traditional morphological interpretations of hy-
aline and granular hemocytes (i.e., granule number and
size). Our data show that, for the four species investigated
thus far, function is correlated with other morphological
features such as cell size, nucleocytoplasmic ratio, and
the presence of cytoplasmic deposits.

Historically, a second area of confusion is the identity
of the type of hemocyte that initiates coagulation. Re-
sponsible cells have been described as either "explosive
corpuscles" and "hyaline cells" (Wood and Visentin,
1967;Woodrta/., 1971; Ravindranath, 1980) or granu-
locytes (Toney, 1958; Hearing and Vernick, 1967;
Mengeot et al., 1977; Madaras et al., 1981). Our studies
provide an answer for the apparent confusion regarding
the identity of clotting hemocytes. Hyaline cells lyse and
initiate coagulation in all species; however in different
species, these cells exhibit variations in the abundance
and size of granules. For example, the granules of Loxo-
rhynchus grandis are so abundant that the hyaline cells
are easily confused with granulocytes while in Panulirus
interruptiis, the granules in large granule and hyaline he-
mocytes are approximately the same size. Hyaline hemo-
cytes do have in common numerous, 50-nm diameter
cytoplasmic deposits. These deposits can be detected us-
ing TEM, a technique rarely included in previous classi-
fication schemes, and by their propensity to stain with
Sudan Black B.

We avoided the use of the term "hyaline cell" in our
previous publications (Martin and Graves, 1985; Martin
et al., 1987; Hose et al.. 1987; Hose and Martin, 1989;

Omori et al., 1989) in an attempt to avoid bias in devel-
oping a classification scheme and instead referred to de-
posit cells (with and without granules), small granule and
large granule hemocytes. We now consider that shrimp
deposit cells are equivalent to hyaline cells. Therefore,
in an attempt to simplify the classification of crustacean
hemocytes, we suggest the following categories of hemo-
cytes: hyaline, small and large granule hemocytes. It is
very important to recognize that morphology alone is in-
sufficient for assigning any cell to one of these categories;
instead the following criteria for hemocyte identification
are suggested.

Hyaline cells have a nucleocytoplasmic ratios of >0.35
and lyse during clot formation. Because lysis is rapid,
identification of these cells, especially in species with rel-
atively low numbers of hyaline cells, is facilitated by mix-
ing a trypan blue-seawater solution with hemolymph.
Hyaline cells turn blue prior to lysis, thereby allowing
morphological identification of the cell and observation
of changes in cell morphology during coagulation. At the
TEM level, these cells contain tiny cytoplasmic deposits
that appear to be involved with the clotting process be-
cause they are only present in the hyaline cells and their
release from the lysing cell precedes hemolymph coagu-
lation. In addition, hyaline cells in the species we have
studied (this paper and Hose et al., 1987), selectively
stain with Sudan Black B. Although this is a general stain
for lipid. it has also been shown to stain coagulogen iso-
lated electrophoretically (Durliat, 1985). Coagulogen
may be contained within hyaline hemocytes or perhaps
produced but not stored in high levels by these hemo-
cytes. In crustacean hyaline cells, the cytoplasmic depos-
its are sudanophilic, with the most intense staining ob-
served from the clustered deposits present in shrimp
(Hose et al.. 1987). Although the test is useful, interspe-
cific variations in the intensity of Sudan Black B staining
are subtle and require careful interpretation. The less
subjective criterion for a positive reaction is the obscu-
rance of the nucleus by the stain.

Our results suggest that coagulation in decapods in-
volves a common mechanism; the release of cytoplasmic
material through breaks in the plasma membrane, possi-
bly including the granules. The identity of the materials
released is not clear. It has been suggested that ( 1 ) coagu-
logen, the clotting protein, is found in the plasma and
activated by chemicals released from hemocytes and (2)
coagulogen and its activators are released from cells (see
Omori et al., 1989). Ghidalia el al. (1981) reviewed this
topic and demonstrated the presence of coagulogen in
the plasma of decapods representing Tail's (1911) three
patterns of coagulation. Although the presence of coagu-
logen in plasma could result from lysis of hyaline hemo-
cytes during cell separation, these investigators used an
anticoagulant (1:9, hemolymph: 10% sodium citrate, v:v)
which we have shown to be effective in preventing hya-

44

J. E. HOSE ET AL.

line cell lysis. They conclude that differences between the
three coagulation patterns are probably due to the man-
ner in which the clot-initiating materials are released.
From the present study we show that decapods placed in
Tail's category C (characterized by rapid gelation of the
plasma) have twice the percentage of hyaline cells as in
species where hemocyte aggregation occurs followed by
slight gelation of the plasma (Tail's category A). What
remains unclear is Ihe localizalion of Ihe clolling prolein
coagulogen (in cells, plasma, or both) and an idenlifica-
lion of Ihe material released from Ihe hyaline cells lhal
iniliales coagulalion. The most abundanl cyloplasmic
malerial released during coagulalion is Ihe eleclron-
dense deposils. These deposils were idenlified in Ihe hya-
line cells of all decapods we examined using TEM and
appear similar lo published micrographs of coagulocyles
in some insecls (Ralcliffe and Rowley, 1979). Clearly a
specific labelling lechnique for Ihese deposils and coagu-
logen is needed, as in Bohn ct al.'s ( 198 1 ) immunocylo-
chemical study of insecl coagulogen.

Granulocyles, Ihe second major category of decapod
hemocyles, have a nucleocyloplasmic ralio of <0.35 and
Ihey do nol accumulate Irypan blue or lyse rapidly in
cullure. They are identified by the presence of numerous
cytoplasmic granules, positive staining reactions for acid
phosphatase and PPO, and in vitro phagocytosis of bacle-
ria and allachmenl to fungal hyphae. The two subdivi-
sions of granulocyles may be dislinguished by ( 1 ) cenlral
location of nuclei in small granule hemocyles and eccen-
Iric location of nuclei in large granule hemocytes, (2) Ihe
presence of only large granules in large granule hemo-
cytes whereas in small granule hemocyles there is a mix-
lure of granules wilh varying sizes, and (3) Ihe refraclile
nalure of granules only in large granule hemocyles when
examined by phase conlrasl microscopy.

The functional roles of granulocyles correlale well
wilh observed cylochemical fealures. Granulocyles are
Ihe primary defensive cells of Ihe hemolymph and Ihe
Iwo sublypes perform overlapping funclions. Small
granule hemocyles are Ihe main cells involved in phago-
cylosis and conlain many lysosomes. while large granule
cells, which mosl frequently iniliale encapsulation of
fungi, show more intense staining for PPO (Hose and
Martin, 1989).

The funclional and cytochemical crileria for recogniz-
ing two categories of hemocytes (hyaline cells and granu-
locyles) are further supported by observalions of hemo-
cyte maturation within Ihe hemalopoielic lissue of the
shrimp ( Martin etui., 1987). In this species, we observed
mitosis only in agranular hyaline cells and small granule
hemocyles. Clusters of hyaline cells and granulocyles
were segregaled within Ihe hemalopoielic lissue (Martin
el ul., 1987). We propose that the Iwo hemocyle catego-
ries represent two cell lines. Cell size is significantly
smaller in hyaline cells and is disconlinuous belween hy-

aline cells and small granule hemocyles. The nucleocy-
loplasmic ratios of hyaline cells of shrimp and the three
species considered here are significanlly higher lhan
Ihose of granulocyles. The ralios of Iwo granulocyle cate-
gories overlap and decrease in large granule hemocyles
coincidenl wilh increases in granule number and size
(Table I). Granulocyles Ihus appear as a conlinuum of
differenlialion from the less mature small granule hemo-
cyles lo the large granule hemocyles.

To summarize, a combinalion of morphological, cylo-
chemical and funclional melhods musl be used lo iden-
lify decapod hemocyles, because Iradilional morpholog-
ical fealures are inadequate and misleading, especially
with regard to hyaline cells. Further sludies by invesliga-
lors ulilizing other decapods are necessary to lest the use-
fulness of Ihis classificalion scheme and lo offer improve-
menls by developing more specific crileria.

Acknowledgments

We want to thank Heidi Parker and Laura Targgart
for collecling and mainlaining Ihe cruslaceans; Sidne
Omori, Calhy Corazine, Celesle Chong, and Erin Camp-
bell for lechnical support; and Dr. Don Lightner and Le-
ona Mohney for supplying the cullures ofFusariwn so-
luni. The projecl was supported by NSF granl DCB-
85021 50 loGM and JEH.

Literature Cited

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C'r//v. Vol. 2. Academic Press. New York.
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of hemolymph clotting in the insect LeuL'opliaca medarae (Rlatta-

ria). J Cmnp. /Virv/o/ I43B: 169-184.
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canux) hemocytes: classification, differential counts and associated

agglutmin activity. J. Invcnchr. 1'alhol. 31: 194-203.
Durliat, M. 1985. Clotting processes in Crustacea Decapoda. Biol.

KIT 60:473-498.
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ard. 1981. Coagulation in decapod Crustacea. / Comp I'/iyxiol.

142:473-478.
Goldenberg, P. Z., A. H. Greenberg, and J. M. Gerrard. 1986.

Activation of lobster hemocyles: cytoarchitcctural aspects. J. Invcr-

ichr I'athol 47: 143-154.
Hearing, V. J., and S. H. Vernick. 1967. Fine structure of the blood

cells of the lobster. Homarus americanus. Cites. Sci. 8: 170-186.
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1987. Cytochemical features of shrimp hemocytes. Biol Bull.

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cytes in the ridgeback prawn Sicyoniu ingenue Burkenroad 1938. J.

Invcnchr Pallwl. 53: 335-346.
Madaras, F., M. Y. Chew, and J. D. Parkin. 1981. Purification and

characterization of the sand crab (Ovalipes bipustulalus) coagulo-
gen (nbnnogen). Thromb. Haemosl. 45: 77-81.

Martin, G. G., and B. L. Graves. 1985. Fine structure and classifica-
tion of shrimp hemocytes. J. Moiphol 185: 339-348.

DECAPOD HEMOCYTE CLASSIFICATION

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Martin, G.G..J. E. Hose, and J.J. Kim. 1987. Structure of hemato-
poietic nodules in the ridgeback prawn Sicynnia mgentis: light and
electron microscopic observations. ./ Morphol 192: 193-204.

Mengeot, J. C., A. G. Bauchau, M. B. DeBrouwer, and E. Passelecq-
Gerin. 1977. Isolement des granules des hemocytes de Homarus
vulgaris. Examens electrophoretiques du contenu proteique des
granules. Coinp. Kiochem Phy\iol 58(A): 343-403.

Omori, S. A., G. G. Martin, and .J. E. Hose. 1989. Morphology of
hemocyte lysis and clotting in the ridgehack prawn, Sicyonia m-
genlis. Cell Tissue Res. 255: 1 17-123.

Ratcliffe, N. A., and A. F. Rowley. 1979. Role of hemocytes in de-
fense against biological agents. Pp 332-414 in Insect Hemocytes:
Development, Form, Functions and Techniques. A. P. Gupta, ed.
Cambridge University Press. Cambridge.

Ratner, S., and S. B. Vinson. 1983. Phagocytosis and encapsulation:
cellular immune responses in Arthropoda. Am. Zoo/. 23: 185-194.

Ratindranath. M. 11. 1980. Haemocytes in haemolymph coagulation
of arthropods. Biol. Rev 55: 139-170.

Shapiro, H. C., J. F. Steenbergen, and /. A. Fitzgerald. 1977.
Hemocytes and phagocytosis in the amencan lobster. Homarus

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Soderhall, K. 1982. Prophenoloxidase activating system and melani-
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Co/up Immunol 6: 60 1 -6 1 I .

Soderhall, K., V. J. Smith, and M. W. Johansson. 1986. Exocytosis
and uptake of bacteria by isolated haemocyte populations of tun
crustaceans evidence for cellular co-operation in the defense reac-
tions of arthropods. Cell Tissue Rex. 245: 43-49.

Spurrs, A. 1969. A low viscosity epoxy embedding medium for elec-
tron microscopy. J. Ultraxtnicl. Res. 26:31-43.

Tail, J. 1911. Types of crustacean blood coagulation. J. Mar Biol.
Assoc. U. A'. 9: 191-198.

Toney, M. E. 1958. Morphology of the blood cells of some Crustacea.
Growth 22: 35-50.

Wood, P. J., and L. P. Visentin. 1967. Histological and histochemical
observations of the hemolymph cells in the crayfish, Orconectes vir-
ilis J Morphol. 123:559-568.

Wood, P.J., J. Podlewski, and T. E. Shenk. 1971. Cytochemical ob-
servations of hemolymph cells during coagulation in the crayfish.
Ommeaes virilis. J Morphol 134: 479-488.

Reference: Bio/ Bull 178: 46-54. (February, 1990)

Respiratory Responses of the Blue Crab Callinectes
sapidus to Long-Term Hypoxia

PETER L. DEFUR 1 *, CHARLOTTE P. MANGUM 2 , AND JOHN E. REESE 2

^ Department of Biology, George Mason University, Fairfax, Virginia 22030 and 2 Department of
Biologv. College of William and Mary, Williamsburg, I'irginia 23185

Abstract. Blue crabs (Callinectes sapidus) were held in
hypoxic (50-55 mm Hg) water for 7-25 days. Post-
branchial blood PO 2 fell by about 80% within 24 h and
then remained unchanged. Postbranchial blood total
CO : increased within 24 h and remained elevated for the
duration of the experiment. There was no change in post-
branchial blood pH, osmolality, or Cl. Lactate, urate,
and Ca all raise the O : affinity of blue crab hemocya-
nin; by 25 days, blood lactate and urate had risen slightly,
but Ca +: had increased dramatically. Hemocyanin con-
centration had also increased by 25 days. At both 7 and
25 days there was an intrinsic increase in hemocyanin-O 2
affinity and a change in subunit composition. The highly
adaptive homotropic change is believed to be due to an
attendant shift in the proportions of two of the three vari-
able monomeric hemocyanin subunits. Thus, both het-
erotropic and homotropic adaptations enhance blood
oxygenation at the gill during long-term hypoxia.

Introduction

The respiratory response to long term hypoxia, de-
fined here as exposure for three or more days, has been
examined in six species of aquatic crustaceans: three
crayfish (McMahon el a/., 1974; Dejours and Armand,
1980; Wilkes and McMahon, 1982a, b), a lobster (Mc-
Mahon et a/., 1978), a crab (Burnett and Johansen,
1981), and a prawn (Hagerman and Uglow, 1985). In
all cases, the initial response was hyperventilation, which
resulted in a respiratory alkalosis. Subsequently, how-
ever, the response in different species became diverse.

Received 1 November 1988; accepted 30 November 1989.
* Present address: Environmental Defense Fund. 1 108 E. Main St..
Richmond. VA 232 19.

Blood pH either returned in full (Wilkes and McMahon,
1982a) or in large part (Butler et ai, 1978; McMahon et
ai, 1978) to the normoxic level, or remained decidedly
alkalotic for as long as 3-8 days (Dejours and Armand,
1980; Burnett and Johansen, 1981).

Crustacean hemocyanins (Hcs) typically have very
large normal Bohr shifts; the quantity A log Pso/ApH is
commonly near -1 (Mangum, 1980). Thus, the alkalo-
sis, which had also been observed during acute hypoxia
(Truchot, 1975; Burnett, 1979), would have the impor-
tant respiratory consequence of raising blood O : affinity.
The increases were observed, but were attributed by pre-
vious workers to the rise in blood pH. We now know
that at least three other allosteric effectors, viz.. L-lactate
(Truchot, 1980; Booth et ai. 1982), Ca +: (Mangum,
1985) and urate (Morris et ai. 1985; Lallier et ai. 1987),
also may increase HcO 2 affinity during acute hypoxia.
The levels of these effectors in the blood during pro-
longed exposure, however, are not known.

Intrinsic changes in O 2 affinity of He in response to
prolonged changes in environmental factors have re-
cently been observed in both crayfish (Rutledge, 1981)
and crabs (Mauro and Mangum, 1982; Mason et ai,
1983; Mangum and Rainer, 1988). In the blue crab, Cal-
linectes sapidus Rathbun, salinity-induced changes are
accompanied by shifts in the concentrations of two of the
5-6 subunits of the He polymers (Mason et ai. 1983).
The changes in one of the two subunits fully explains
the attendant shift in O : affinity (Mangum and Rainer,
1988).

Although an intrinsic molecular change would not be
expected to occur during acute hypoxia, it might occur
during long-term hypoxia. In the shrimp Crangon cran-
gon. He levels increase sharply during prolonged hypoxia

46

HYPOXIA IN BLUE CRABS

47

(Hagerman, 1 986); a similar increase in the blue crab ap-
pears to hasten intrinsic molecular adaptation to a salin-
ity change (Mason el a/., 1983). Therefore, we have ex-
amined the possibility that a change in net synthesis or
degradation during hypoxia produces additional or re-
placement molecules that differ from those in normoxic
animals.

The blue crab inhabits many bodies of water that are
not invariably normoxic (Carpenter and Cargo, 1957;
May, 1973; Carlo, 1979; Harpers al., 1981; Turner and
Allen, 1982). Lethal levels (PO 2 < about 50 mmHg) of-
ten kill animals that cannot escape from pots (Carpenter
and Cargo. 1957). Free-ranging animals may even
emerge into air (Loesch, 1960; Officer el al., 1984), de-
spite limited tolerance of it. In the Chesapeake Bay sys-
tem, sublethal O : levels, still well below normoxia, are
so widespread that crabs must encounter them for long
periods. Water PO 2 in the range 50-100 mmHg is char-
acteristic of the Chesapeake Bay for several months dur-
ing the spring and fall (Officer el al., 1984; Seliger el al,
1985). In the summer, cyclical destratification in the
channels produces sublethal hypoxia throughout the wa-
ter column for several weeks at a time (Webb and D'Elia,
1980). Processes ranging from tidal flushing of the
marshes, to seiching of the water in the channels, pro-
duce sublethal hypoxia in extensive areas of shallow wa-
ter as well (Carpenter and Cargo, 1957; Axelrad el al.,
1976; Kemp and Boynton, 1980; Taft el al, 1980; Ma-
lone el al, 1986; MacKiernan, 1987).

We have determined the response of blood respiratory
and osmotic variables of the blue crab Callinectes sapi-
dus Rathbun to sublethal hypoxia. The treatments in-
clude acute exposure, more prolonged exposures similar
to those employed in previous studies, and still more pro-
longed exposures designed to elicit intrinsic changes in
the He molecule. We have measured all of the known
physiological effectors of HcO 2 binding, both hetero- and
homotropic.

Materials and Methods

Animals

Large (ca. 1 20-220 gm wet wt.), male, intermolt crabs
were obtained from commercial watermen or collected
by the first author near the Rhode River or the mouth of
the Patuxent River in Maryland. They were returned to
Fairfax. Virginia, and maintained in open containers
(100-200 1) of natural, aerated water (500-530 mOsM,
2 1 -23C) for 4-7 days prior to the experimental hypoxia.
Water osmolality was monitored frequently and distilled
water added as needed; water pH was also monitored and
kept above 7.9 by the addition of NaHCO,. Crabs were
fed thawed smelt twice a week throughout the control

and experimental periods, but not within 24 h of sam-
pling.

Design

The experimental protocol consisted of taking blood
from the same crabs before, during, and following expo-
sure to hypoxia. Insofar as possible, the design of paired
observations on the same individuals was maintained,
and the data were analyzed accordingly. The significance
of changes in blood pH. PO : , total CO 2 , lactate, Cl, and
osmolality was tested according to Student's /-test for un-
grouped (paired) data as the mean of the differences of
each value from the control for the same individual, the
null hypothesis being that there was none. Because the
same individuals were sampled repetitively, the blood
samples were necessarily small (0.5 ml), thus insufficient
volumes of many samples remained for the other mea-
surements. The paired observations design could not be
maintained for the analysis of Ca +2 , urate, and He con-
centrations; these values were analyzed by Student's t-
test for grouped data (two samples). For measurements
such as O 2 binding, which require a total of more than
0.5 ml of material, samples were pooled; the results were
analyzed by regression. In these measurements, pooled
samples for 1 and 7-day exposure were made up of
blood from the same individuals, whereas that for 25-day
exposure was composed of blood from different animals.

Hypoxia

Nitrogen gas was bubbled into the water to reduce the
PO 2 from 140-155 to approximately 55 mm Hg in 3-4
h, and then the bubbling was stopped. Thereafter a slow,
steady air flow was maintained, and N 2 was bubbled into
the water only as needed to offset the air.

N 2 flow was regulated by a metering system that bal-
anced N 2 against rising O 2 . The system consisted of an
O 2 electrode and meter (Instrumentation Laboratories
Models 1703B and 113, respectively), the output of
which provided the signal for a logic circuit that con-
trolled an electric gas valve. The circuit was set to evalu-
ate the output of the meter every 5 min and to open the
valve if the PO 2 had increased above the set point of 50
mmHg. Thus, once the initial PO 2 of 50 mmHg was
reached, further changes were confined to the ranges 50-
55 mmHg and occurred slowly. The continuous airflow
stirred the water and ensured that O 2 uptake by the crabs
did not reduce water PO 2 below 50 mmHg.

Blood sampling

Postbranchial hemolymph samples for the determina-
tion of in vivo respiratory variables were withdrawn
through holes in the carapace dorsolateral to the heart.

48

P. L. DEFUR ET AL.

The holes had been drilled four or more days prior to
the control period, and covered with latex rubber affixed
with cyanoacrylate cement. On occasion, prebranchial
hemolymph was also withdrawn from the base of one
of the legs for the measurement of Cl, osmolality, and
lactate.

Blood samples were withdrawn into iced syringes and
immediately placed on ice to slow clotting. After deter-
mination of blood gas and acid-base variables, these sam-
ples were frozen for the remaining analyses. The samples
for HcO : binding were kept cool and, with the exception
noted below, unfrozen; O : binding measurements on
blood from normoxic and hypoxic animals were made
within a few days.

In vivo variables

Hemolymph pH was measured with a thermostatted
glass capillary electrode (Radiometer G299A) and meter
( Radiometer PHM 84). PO 2 was measured with a polaro-
graphic electrode (Radiometer E5046) and acid-base an-
alyzer (PHM 72). Total CO : was determined in 50 n\
samples with a Corning Model 965 CO 2 analyzer. Lac-
tate was measured enzymatically (Sigma Procedure No.
826), with the modifications for He-containing blood de-
veloped by Graham el al. ( 1983). Osmolality was deter-
mined with a vapor pressure osmometer ( Wescor Model
5100C).

Ca 1 " activity was determined with a Radiometer elec-
trode and PHM 84 meter, following 1:99 dilution with
0.05 Tris Maleate buffer, pH 7.6 (Mangum and Lyk-
keboe. 1979). Chloride was measured by electrometric
titration (Corning Model 920).

We determined urate as the quinoneimine produced
by digestion with uricase (Sigma Procedure No. 685), af-
ter first verifying that 100% of the urate added to test
samples of blood could be recovered. Because He ab-
sorbs at 685 nm, the absorbance was measured in repli-
cate, once with and once without the analytical reagents,
and the interference of He was subtracted.

HcO : binding and He concentration

Hemolymph was declotted with a tissue grinder, cen-
trifuged, and then dialyzed at 4C for 24-28 h against a
saline made up according to Mason el al. (1983). HcO :
binding was determined by the cell respiration method,
in which the deviation from a constant rate of O? deple-
tion is used to estimate fractional oxygenation at the
measured PO : (Mangum and Lykkeboe. 1979).

Before determining He concentration in the blood, we
eliminated the effect of light scattering, dissociating the
native polymers to monomeric subunits by dilution ( 1:
39) with Tris HC1 containing 50 mM EDTA. Absor-
bance of He was measured at 338 nA/with a Milton Roy

Spectronic 501 spectrophotometer; the concentration
was calculated using the extinction coefficient for por-
tunid He reported by Nickerson and Van Holde ( 197 1 ).

Electrophoresis

Alkaline dissociation electrophoresis (Hames and
Rickwood, 1981) of He monomers on polyacrylamide
gel slabs was performed as described by Mangum and
Rainer (1988). In the present case, aliquots of the three
pools of blood used to compare O 2 binding in normoxic
and hypoxic animals were run on the same gels, which
were scanned with a Gelman Instruments Model 3372
integrating densitometer (modified for transparent me-
dia). Changes were estimated by comparing peaks of the
variable subunits with that of an invariant subunit.

Results

The experiment was performed three times. The first
hypoxic exposure period was 7 days, and samples were
taken at - 1 (control), 1, 4, and 7 days. The second and
third exposure periods were 25 and 23 days, and samples
were taken at -1, 7, 9, or 16, and 23 or 25 days. In the
first experiment, the crabs were also sampled one day af-
ter the return to normoxic water.

Behavior and mortality

When ambient PO 2 fell to 50 mm Hg, most of the
crabs became active and moved slowly around the
aquarium, as reported by Lowery and Tate ( 1 986); some
crabs tried to climb out of the water. Elevated activity
ceased within a few hours, and the animals became qui-
escent for the duration of the hypoxic exposure. They
frequently buried in the sand lining the bottom of the
aquarium.

Mortality was low. There was none during the first ex-
periment and only 20% during the longer exposures. In
our experience, this level would be low under normoxic
conditions.

Hemolymph variables

Many of the data for normoxic animals are unexcep-
tional (Table I, day - 1 ), but pH and PO : are high relative
to those in the literature for this species (e.g., Weiland
and Mangum, 1975; Mangum el al.. 1985). Our value
for blood urate in normoxic animals is also considerably
lower than that reported by Morris el al. (1986) for the
crayfish Austropotamobius pallipes (0.35 mM}. but it is
similar to the figure (0.08 mM) found in the portunid
crab Carcinus niaenas (Lallier et al.. 1987). The low
urate levels in the portunid bloods may explain the ab-
sence (Mangum, 1983) or small size (Truchot, 1975) of

HYPOXIA IN BLUE CRABS

49

Table 1

Respiratory variables* in the heinolvmpli of blue erabs exposed lo moderate hypoxic? for 7-25 days 3 (day - 1 = control)

No.
animals

PaO,
(mm Hg)

pHa

CaCO 2

(mM)

Lactate(mM)

Ca +2 Urate
(mM) (mM)

[He]

(g/ 100 ml)

Duration 4
(days) 7 23-25

7

23-25

7 23-25

7 23-25

7 23-25

23-25 23-25

23-25

Day

-1 8 9-11

9x

70

7.81 7.71

2.3 2.3

0.01 0.94

6.73 0.05

3.11

(control)

3

8

0.03 0.03

0.2 0.4

0.01 0.09

0.75 0.02

0.41

1 8

18

7.79

5.4

0.08

4

0.02

0.3

0.05

4 8

15

7.80

5.1

0.08

4

0.04

0.3

0.06

7 8 4-8

13

22

7.83

4.6 3.6

0.09

4.73 0.03

1.26

+ 2

2

0.03

0.4 0.2

0.05

0.75 + 0.00

0.11

Recovery 8

87

7.77

3.3

0.00

10

0.02

0.2

0.00

9 6

19

7.81

2.6

0.80

3

0.06

0.2

0.09

16 4

21

3

23-25 5-11

21

7.76

3.2

1.79

10.1 0.14

4.40

4

0.04

0.3

0.50

0.5 0.02

0.19

1 Top no. = mean, bottom no. = S.E.

: -50-55 mm Hg, 21-2.VC, 500-530 mOsM.

3 Symbols: PaO 2 = postbranchial blood PO 2 , pH = postbranchial blood pH. CaCO 2 = postbranchial blood total CO : .

4 Two columns under the first five headings represent different exposure periods, as indicated.

changes in HcO : affinity following dialysis of normoxic
serum against a physiological saline.

Within 24 h of the onset of the 7-day hypoxic exposure
in the first experiment, postbranchial blood PO ; (PaO : )
fell by 80% and total CO, (CaCO 2 ) more than doubled
while pH remained unchanged (Table I). The subse-
quent changes in these three variables are not significant
(P > .05). The apparent increase in lactate is not signifi-
cant (P > .05) if the difference at each sampling period is
tested against zero. If the particular sampling period is
disregarded and the maximum increase for each individ-
ual is tested against the null hypothesis, however, then
the mean increase (0.18 .06 mM) is significantly
greater than zero (P < .01 ). More important, this change
is very small, indicating a highly aerobic condition.
Within 24 h of return to normoxic water, control levels
of postbranchial PO 2 were restored, although total CO 2
remained slightly elevated (P < .02).

In the second and third experiments, the same and
longer periods of exposure (23-25 days) to hypoxia re-
sulted in similar patterns of PO 2 , pH, and total CO : (Ta-
ble I). Once again blood lactate increased slightly, al-
though in this case significantly (P = .05), regardless of

how the data are grouped. At 25 but not 7 days, blood
Ca +2 rose by a large amount (P = 0.025), blood urate rose
significantly (P < .001 ), and He concentration increased
by almost half (P < . 05).

In none of the three experiments, did blood Cl or os-
molality change, nor were there any coherent trends in
these variables. The mean values (S.E., n = 61) for all
periods are 355 (4) mA/Cl and 756 (5) mOsm.

HcO } binding and subunit composition

At the end of the 7-day period, a change in HcO 2
affinity had clearly occurred (Fig. 1, upper panel). The
95% confidence intervals around the regression lines de-
scribing the data for -1 and 7 days in Figure 1 do not
overlap at any point (Table II). The slopes of regression
lines (-0.97 0.21 95% C.I. for normoxic and -1.1 1
0.1 1 for 7 days, hypoxic animals), and thus the Bohr
shifts, do not differ significantly.

The relationship between cooperativity (n) and pH of
the decapod Hcs is usually quite complex, often reaching
a maximum in the middle of the physiological pH range
and showing lower values at the extremes. No very sensi-

50

P. L. DEFUR ET AL

60 I

40

20

(torr)

7.0

7.2

7.4

7.6

7.8

8.0

8.2

PH

"so

o

o

7.0

7.2

7.4

7.6

7.8

8.0 8.2

Figure 1. Oxygen binding by stripped He of normoxic (O), 7 days
hypoxic (). and 25 day hypoxic (A) blue crabs. 25C, 0.05 M Tns
maleate buffered saline containing 494 mM NaCl, 1 6 mM KC1. 23 mA/
CaCI : . 23 mA/ MgCl, 25 mA/(Na) : SO 4 and 2 mM NaHCO,. Upper
panel shows oxygen affinity (PsuK and lower panel shows the coopera-
tivityat PCK = P w (n,,,).

live procedure for data analysis is available. In the lower
panel of Figure 1, cooperativity seems to decrease at 7
days, but the mean values are not significantly different
(P = .10). A Mann-Whitney U test also did not distin-
guish a significant change.

There was a clear change in the subunit composition
of the Hcs (Fig. 2). Specifically, subunits 3, 5, and 6 de-
creased in concentration relative to subunit 4, which has
remained invariant in samples taken, by now, from more
than 500 individuals (Mangum, unpubl. obs.; Rainer,
1988).

After 25 days of hypoxia, HcO : affinity increased fur-
ther (Fig. 1). The 95% confidence interval around a re-
gression line describing O : affinity does not overlap those
for control or 7-day hypoxic animals in any part of the
pH range (Table II). The slope of the regression line de-

scribing the 25 day data (-1.00 0.17 95% C.I.) does
not differ from the other two. In this case, the mean value
for cooperativity (1.95 0.06 S.E.) of the He from 25
day hypoxic animals (Fig. 1) differs significantly (P= .02)
from that for control and 7 day hypoxic animals (2.41
0.14).

All three variable subunits decreased further in con-
centration relative to subunit 4 (Fig. 2). Indeed the pres-
ence of subunit 3, which is sometimes completely absent
(Mason et ai. 1983), is dubious. By the end of 25 days of
hypoxia, the concentration of subunit 6 had dropped
from the highest in the control period to rank fourth; no.
5 had dropped from second to third; and no. 3 had
dropped from clearly present to undetectable, or nearly
so. The two weak bands appearing between peaks 1 and
2 of the He from hypoxic crabs (Fig. 2B, C) are usually
not present: they are not copper containing and have no
influence on oxygen binding (Mangum and Rainer,
1988).

Although the effects of elevated Ca +2 (and L-lactate)
on C. sapidus He are well known (e.g.. Booth et a/., 1982;
Mangum, 1983; Mason el al., 1983; Johnson el ul.,
1 984), those of urate are not. Therefore we used the small
amount of (frozen) blood remaining after the measure-
ment of extrinsic co-factors to examine urate sensitivity.
Figure 3 shows that small quantities of urate clearly raise
O : affinity of the He of animals exposed to hypoxia for 25
days, with its altered subunit composition. The positions
(but not slopes) of regression lines describing the data for
0. 0.55. and 2.35 mM urate all differ at P = 0.05. Al-
though the data suggest little further difference beyond
1.17 mA/. the small number of observations permitted
by the volume of material available mandates some cau-
tion on this point.

We emphasize that, unlike the measurements in Fig-
ure 1 , those in Figure 3 were made on Hcs that had been
frozen for several months. As mentioned earlier (Man-
gum, 1983), freezing does not usually influence Pso (see
also Morris, 1988), at least if the He retains its native

Table II

Ninety-five percent confidence intervals around semilogarithmic

(log Y) regression lines fit in P 50 data in Figure I

PH

Control
(r = 0.946)

7-day hypoxia
(r 2 = 0.994)

25-day hypoxia
(r : = 0.965)

7.0

57.2-60.5

49.7-53.5

31.0-33.7

7.2

36.6-38.6

30.5-32.7

19.6-21.2

7.4

23.4-24.6

18.7-20.0

12.4-13.4

7.6

15.0-15.7

11.6-12.2

7.86-8.46

7.8

9.54-10.1

7.01-7.48

4.96-5.35

8.0

6.09-6.44

4.29-4.59

3.13-3.39

8.2

3.88-4.12

2.62-2.81

1.97-2.15

HYPOXIA IN BLUE CRABS

51

Figure 2. Densitometer scan of slab gels, showing native suhunits
(numbered peaks) of blue crab He separated by charge (subunit 1 is at
the anodal end). The He applied to the gels was from the same samples
from which data were collected for Figure 1. A. Normoxic. B. 7-day
hypoxic. C. 25-day hypoxic. Subunit 3 in C is dubious.

optical properties. The control values in the two figures
are essentially identical (95% confidence intervals
around regression lines broadly overlap). Because freez-
ing frequently influences cooperativity, however (Man-
gum, 1983; S. Morris, pers. comm.), we did not analyze
the cooperativity of the thawed samples. Morris el al.
(1986) found no effect of urate on cooperativity.

Discussion

Blood pH.PO : . and CO :

In view of the unanimity of previous reports of blood
alkalosis accompanying hypoxia of virtually any dura-
tion in crustaceans, we were surprised to find none in the
present experiments. Hyperventilation and alkalosis are
not always precisely correlated; in the crayfish O. rusti-
cus, ventilation returns to control levels while blood pH

is still elevated (Wilkes and McMahon, 1982a). But all
reports agree that blood pH rises at some point. In fact,
in severely hypoxic C. maenas, Lallier et al. (1987) re-
ported a pH increase of more than 0.3 units accompany-
ing an increase in lactate of 25 mA/, despite no change
in base. In other investigations of C. sapidus, we have
either found (Pease et al., 1986), or not found (Mangum
and Weiland, 1975, and unpubl. obs.), a hypoxic alkalo-
sis. The response in this species is apparently highly la-
bile, for reasons that are presently unclear. The increases
in lactate observed here seem too small to offset a respira-
tory alkalosis brought about by vigorous hyperventila-
tion (Pease and deFur, 1987). Further increases in pH
and PO 2 might have been precluded because ventilation
was already high. Elevated ventilation during the control
period could have arisen from sensory stimulation (Mc-
Donald^/ al., 1977) and been unrelated to ambient PO 2 .

Extrinsic modulation of HcO : affinity

In many crustaceans L-lactate is a physiologically im-
portant modulator of HcO ; affinity, both during exercise
and hypoxia (Booth et al., 1982; Graham et al.. 1983)
and very severe environmental hypoxia (Lowery and
Tate, 1986; Lallier et al.. 1987). The small increases ob-
served here would raise HcO 2 affinity at physiological pH
by less than 1 mmHg. The increase in urate would raise
HcCK affinity by a similarly small amount. In contrast,
Ca +2 may be an important effector after 23-25 (but not
7) days, by which time the increase in Ca +: would raise
HcO : affinity by more than 5 mmHg.

40

20

Pso
(torr)

7.1

7.3

7.5

7.7

7.9

8.1

8.3

PH

Figure 3. Effect of urate on O 2 affinity of stripped He from hypoxic
animals. Conditions as in Figure I . (O) no urale: ()0.57 mA/;(A) 1.15
mM: and (D) 2.35 mA/.

52

P. L. DE FUR ET AL.

These conclusions, inferred from the data in Figure 3
and those of Mason et at. (1983) and Johnson el at.
(1984). assume that the two organic effectors act com-
pletely independently and cumulatively, which is not en-
tirely true (S. Morris, pers. comm.). The interaction of
Ca +2 with the organic effectors is included in the present
data for HcO ; affinity because the actions of urate and
lactate were determined in the presence of Ca +: . The in-
teraction of urate and lactate would further diminish
their effects, albeit by a small amount.

Intrinsic adaptation ofHcO? affinity

Estuarine (and also normoxic) blue crabs transferred
to high salinity in the laboratory show a rapid decrease
in He concentrations. Concomitantly, HcCK affinity in-
creases and the levels of subunits 3 and 5 decrease,
closely resembling the He in animals freshly caught at a
seaside location. Subunit 6 remains unchanged. In sea-
side (and normoxic) animals transferred to low salinity,
the He concentration rapidly increases while the HcO :
affinity decreases (Mason et ul., 1983). The intrinsic
change opposes the effects of salinity-induced changes in
extrinsic co-factors.

In initial samples freshly taken at the estuarine and
seaside localities, the vast majority of animals exhibited
the molecular phenotype associated with the comparable
acclimation salinity in the laboratory (C. P. Mangum
andG. Godette, unpubl. obs.; Rainer et a/.. 1985). How-
ever, subunit 6 was variable in both samples, implicating
another environmental or physiological effector unre-
lated to salinity per se. Moreover, when the field study
was enlarged, no clinal variation of the three subunits
was obvious along a salinity gradient between the two
localities (Rainer, 1988); these findings also suggest a
confounding variable.

Under the same ionic conditions, the O : affinities of
blue crab Hcs composed of different combinations of the
variable chains indicate that the levels of subunits 3 and
6 both influence oxygen binding; the effects of variation
in subunit 5 are not entirely clear (Mangum and Rainer,
1988). Changes in subunit 3 (alone) can fully explain the
difference between seaside and estuarine animals. How-
ever, changes in subunit 6 (alone), smaller than those ob-
served here, significantly alter HcO : affinity by almost
20% at physiological pH. Although there is no difference
between an He with low levels of only subunit 3, and one
with low levels of both 3 and 5. an He with low levels of
5 alone has not been examined.

The present results suggest that blue crab He is intrin-
sically adaptable to prolonged hypoxia as well as to salin-
ity. The adaptation may be expedited by an increase in
He concentration, which is clear at 25 days, and it is ac-

companied by changes in the same three subunits al-
ready known to be variable. A decrease in concentration
of subunits 3 and 6 during hypoxia has the same effect
as that of decreasing either alone or in combination; i.e.,
increasing O 2 affinity (Mangum and Rainer, 1988). The
present findings suggest that, while subunits 3 and 5
respond to a change in both salinity and oxygen, subunit
6 responds only to oxygen. The changes in subunits 3
and 5 as a result of hypoxia were much smaller than the
salinity-induced changes, but the changes in P 5I) were
about the same in the two groups, at physiological pH.
The smaller changes in the amounts of subunits 3 and 5
in hypoxia may be due to lower levels at the onset of
hypoxia (for comparison see fig. 1 in Mangum and
Rainer, 1988). The oxygen-induced change in subunit 6,
however, was much larger than observed by Mangum
and Rainer (1988). A greater change in subunit 6 may
offset a smaller change in subunit 3, and the intrinsic ad-
aptation of HcO : affinity to hypoxia may involve the
change in subunit composition. Moreover, we suggest
that the variation of subunit 6 in nature is related to hyp-
oxia, which does not vary along a salinity gradient in a
simple fashion.

Finally, the increase observed here in He concentra-
tion occurs widely in hypoxic crustaceans. In different
species its magnitude may be much greater, it may occur
in a far shorter period, and it may occur at a much lower
temperature (Hagerman and Oksama, 1985; Hagerman
and Uglow. 1985; Hagerman, 1986). It will be interesting
to learn whether intrinsic molecular adaptability is sim-
ilarly widespread.

Acknowledgments

Supported by NSF Grant DCB 84-14856 (Regulatory
Biology) to CPM. This work is a result of research spon-
sored in part by NOA A Office of Sea Grant, U. S. Depart-
ment of Commerce, under Grant No. NA85AA-D-
SGO 1 6 to the Virginia Graduate Marine Science Consor-
tium and Virginia Sea Grant College Program. The
U. S. Government is authorized to produce and distrib-
ute reprints for governmental purposes notwithstanding
any copyright notation that may appear hereon.

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207: 983-985.

Weiland, A. L., and C. P. Mangum. 1975. The influence of environ-
mental salinity on hemocyanin function in the blue crab, Calli-
nectes sapidus. J .v/>. /.not 193: 265-274.

VVilkcs, P. R. H., and B. R. McMahon. 1982a. Effect of maintained
hypoxic exposure on the crayfish Orconectes rusticus. I. Ventila-
tory, acid-base and cardiovascular adjustments. J. Exp. Bio/ 98:
119-137.

Wilkes, P. R. H., and B. R. McMahon. 1982b. Effect of maintained
hypoxic exposure on the crayfish Ora.mee.les rusticus. II. Modula-
tion of haemocyanin oxygen affinity. J Exp. Biot 98: 1 39-149.

Reference: Biol. Bull 178: 55-64. (February. 1440)

The Horseshoe Crab Tachypleus tridentatus has Two
Kinds of Hemocytes: Granulocytes and Plasmatocytes

PER PLOUG JAKOBSEN AND PETER SUHR-JESSEN

Department ofAnatomv& Cytology, University of Odense, Campusvej 55, 5230 OdenseM, Denmark

Abstract. For the first time, the fine structure of the he-
mocytes from the horseshoe crab Tachypleus tridentatus
is investigated by transmission electron microscopy and
light microscopy serial sectioning. Two morphologically
distinct, ellipsoidal, and mononucleate hemocytes
granulocytes (amebocytes) and plasmatocytes are re-
vealed. Granulocytes constitute about 97% of the hemo-
cytes. They have a marginal band of microtubules, a het-
erochromatic nucleus, distended but poorly developed
RER, few free ribosomes, few mitochondria, and many
large secretory granules. The majority of these granules
have a uniform content and are mature. Structured gran-
ules located in the proximity of Golgi complexes may be
immature transitional stages leading to the mature uni-
form granules. Upon stimulation with endotoxin from
gram negative bacteria, the mature granules become
transitory structured before exocytosis. In contrast, the
immature granules are not exocytosed. Plasmatocytes
constitute about 3% of the hemocytes. They differ from
granulocytes by having an euchromatic nucleus, a well-
developed RER of flattened or tubular cisternae, many
free ribosomes, many mitochondria, but only few, if any,
large secretory granules. Apparently, plasmatocytes are
not affected by endotoxin. The relationship and possible
functions of granulocytes and plasmatocytes are dis-
cussed and compared with those of the horseshoe crab,
Limulus polyphemus.

Introduction

Horseshoe crabs are "living fossils." which have un-
dergone little morphological evolution during the last
360 million years; they can be traced back more than 500
million years (Sekiguchi and Sugita, 1980; Shishikura el

Received 14 June 1 984; accepted 28 November 1984.

al., 1982;Mikkelsen, 1988). If this stability is reflected in
their physiology, studies of their immune defense system
may shed light on when and how the different parts of it
evolved in horseshoe crabs and possibly also in higher
and more recent phyla.

Inoculation of gram negative bacteria or their endo-
toxins into the hemolymph of horseshoe crabs cause fatal
intravascular coagulation (Bang, 1956). This involves
exocytosis of the large secretory granules from the he-
mocytes. These granules contain coagulogen and all
other proteins necessary for the coagulation (Levin and
Bang, 1964; Ornberg and Reese, 1981; Iwanaga el al.,
1986; Suhr-Jessen et al., 1989). Hemocyte (amebocyte)
lysates can be made from all four extant species of horse-
shoe crabs, and are now extensively used to detect min-
ute quantities of endotoxin (Shishikura el al., 1 983; Wat-
son et al., 1987).

The horseshoe crab best characterized is Limulus poly-
phemus. Until recently, only one hemocyte, the granulo-
cyte, had been identified in this species (Dumont et al.,
1966; Levin and Bang, 1 968; Copeland and Levin, 1985;
Tablin and Levin. 1988). However, a second hemocyte,
the plasmatocyte, has been identified independently by
light microscopical observations of live cells, by light mi-
croscopical serial sectioning of fixed cells, and by trans-
mission electron microscopy alone and combined with
immuno-gold labeling (Suhr-Jessen eta/., 1989). Inaddi-
tion, cyanocytes and cyanoblasts have been reported to
be present in the sinusoids around the compound eyes
(Fahrenbach, 1970). Early light microscopical studies
suggested that Tachypleus tridentatus had two kinds of
granulocytes (Shishikura et al.. 1977; Shishikura and
Sekiguchi, 1979).

The aim of the present study is to characterize the
fine structure of T. tridentatus hemocytes the cellu-
lar part of the immune defense system in the pres-

55

56

P P. JAKOBSEN AND P. SUHR-JESSEN

'

RER
v

X
^

,

M~

\

:.;'

jJHtj

3p>

9

PER

RER

5 jjm

-

* "^ g

PM

3

250 nm

50 nm

Figure 1. I'm/iv/'/i'ii^ initcntaius granulocyte with its heterochromatic nucleus (N), and many large
secretory granules (GR). Mitochondria (M). Rough endoplasmic reticulum (RER). Marginal band (arrow-
heads).

T.\Cin'l'U-:i'S IRIDI.M III .S III MOO IIS

57

500 nm

'

500 nm

Figure 5. The distended rough endoplasmic reticulum (RER) from a Tacliyi>lcn.\ iridematus granulo-
cyte. Golgi complex (G).

Figure 6. The flattened or tubular RER from a T. Iridentatux plasmatocyte. Many free ribosomes are
present (arrowheads). Golgi complex (G); nucleus (N).

Figure 1. T. Iridcnlalus granulocyte with a structured (immature) large granule (IG) located in close
proximity to a Golgi complex (G). Centrioles (C): mitochondria (M); uniform (mature) granule (MG);
nucleus (N).

ence and absence of endotoxin. We show that the gen-
eral circulation of T. tridenlatm contains plasmato-
cytes and a single class of granulocytes. Furthermore,

a temporal relationship is described for the formation
to final secretion of the large secretory granules in the
granulocytes.

Figure 2. T. tridenlalm plasmatocyte with its euchromatic nucleus (N), well-developed RER. and
many mitochondria (M). Marginal band (arrowheads).
Figures 3, 4. Longitudinal and transverse sections of marginal bands of microtubules (arrowheads) in

T. indcnlalus hemocytes. Plasma membrane (PM).

58

P. P. JAKOBSEN AND P. SUHR-JESSEN

12a

Figures 8-10. Differently structured immature large granules from Tachyplen\ tridcnlatus granulo-
cytes. Insert: close-up of the about 17-nm tubular structures in transverse and longitudinal section (bar
equals 100 nm). Apparently, a coated pit (CP) and a coated vesicle (CV) are present. Golgi complexes (G).

TACHYI'LKUS TRIDENTATUS HEMOCYTES

59

Materials and Methods

Six adult T. tridentatus females (males were not avail-
able) (prosomal width: 30-33 cm) were collected in the
Tonkin Gulf, China, and kept in seawater (3.0% NaCl)
at 15C at The Danish Aquaculture Institute, H0rsholm,
for up to nine months. Throughout this period, hemo-
lymph samplings from all animals gave similar results.
Hemolymph was drawn by cardiac puncture at the etha-
nol-cleaned prosoma-opistosoma junction. Access to the
heart was made by a 19-gauge needle alone or combined
with a 5-ml syringe containing fixative or, as part of a
total bleed of the animal, through a large cut by a sterile
(LPS-free) scalpel. The three methods gave similar re-
sults. Hemolymph was floating directly into 5% glutaral-
dehyde in 0.1 M sodium cacodylate buffer, pH 7.4, to
give a final glutaraldehyde percentage of no less than 4.
Samples were also incubated for 5 to 300 s with 10 4
10"" g E. coli endotoxin (Sigma no. L 3755)/ml hemo-
lymph prior to fixation. The fixed samples were pro-
cessed as described (Willumsen el ai, 1987). Transmis-
sion electron microscopy sections (about 50 nm) were
mounted on pioloform F-50 coated Cu- or Ni-grids, con-
trasted with lead citrate, examined in a Jeol JEM-100CX
electron microscope at 80 kV, and photographed using
Agfa-Gevaert 23D56 film. Light microscopy serial sec-
tions (about 1 .0 /urn) were stained with toluidin blue, ex-
amined in a Zeiss microscope (numerical aparture: 1 .30)
at 400X using immersion oil, and photographed using
Kodak panatomic X film. To eliminate inaccuracies due
to minor differences in the thickness of the sections, a
plasmatocyte was always compared with a nearby granu-
locyte starting and ending at almost the same section
numbers. In the two cells, the number of cuts through
mitochondria rather than the actual number of mito-
chondria was determined. Assuming that the mitochon-
dria are randomly oriented and approximately of the
same size in the two cells, any consistent deviation from
1 in the PL/GR ratio reflects differences in numbers of
mitochondria.

Results

General morphology of the hemocytes

T. tridentatm hemocytes are spheroid, and about 1 5-
20 jum at their longest axis (Figs. 1,2, 15). A marginal
band of microtubules run parallel to the longitudinal axis
of the cells at least one microtubule diameter beneath

the plasma membrane (Figs. 1-4). The almost parallel
arrangement of the microtubules, combined with the
electron-dense material seen between them, suggest that
they are connected (Figs. 3, 4). Each hemocyte has a sin-
gle, non-lobated nucleus containing one or a few
nucleoli. The cells also contain rough endoplasmic re-
ticulum (RER), free ribosomes, mitochondria with la-
mellar cristae, and Golgi complexes with 3-6 layers of
cisternae the cis-ones being more distended than the
trans-ones (Figs. 1-2, 5-7). The paired centrioles form
an obtuse angle to each other (Fig. 7). No sign of mitosis
was seen in any of the examined hemocytes. Digestive
vacuoles and apparently coated pits and coated vesicles
are also present (Figs. 9, 14). No cytoplasmic crystals
were observed.

Granulocytes

About 97% of the hemocytes are granulocytes. They
have a heterochromatic nucleus, a poorly developed but
distended RER, few free ribosomes, and few mitochon-
dria (Figs. 1, 5). However, their most prominent feature
is the many large secretory granules with diameters
around 1-2 ^m (see below).

Large secretory granules

The majority of the large secretory granules in granu-
locytes have a uniform content (Fig. 1). However, one
class of granules, with structures ranging from amor-
phous to highly organized tubules with diameters around
1 7 nm, are seen in close proximity to Golgi complexes
(Figs. 7-10). When hemocytes are stimulated with endo-
toxin a second class of structured granules containing tu-
bules with diameters around 10 nm become transitorily
present (Fig. 1 1). A reverse relationship seems to exist
between the numbers of structured granules of the sec-
ond class and the uniform granules. After this, exocytosis
occurs (Fig. 1 2). In contrast, structured granules of the
first class are usually not exocytosed following stimula-
tion with endotoxin (Figs. 13, 14). Following exocytosis,
the granulocytes gain numerous pseudopodia, and the
organelles collect in the center of the cell surrounded by
microtubules (Figs. 13, 14).

Plasmatocytes

Plasmatocytes constitute about 3% of the hemocytes.
This conclusion is reached by examining duplicate sam-

Kigure 1 1 . Granulocyte from T. tridentatus incubated with 10 4 g endotoxin per ml hemolymph for
30 s. A stimulated large secretory granule (SG) is in close connection with the plasma membrane (PM). Its
tubular structures (arrows) have a diameter around 10 nm, while 17-nm tubular structures (arrowheads)
are present in the immature granule (IG) located in close proximity to a Golgi complex (G).

Figure 12. Successive stages in exocytosis of the large secretory granules from T tridcnlatus granulo-
cytes. Bar length: 500 nm.

60

P. P. JAKOBSEN AND P. SUHR-JESSEN

-

- ' :

*

*v*c

%%*&&

'

Figure 13. Granulocyte from Tachypleus tridentatus incubated with 10 4 g endotoxin per ml hemo-
lymph for 300 s. The large secretory granules are exocytosed (arrow), except the immature ones (1G). and
pseudopodia (P) are projected. Nucleus (N).

pies from each of six animals. From all samples, at
least 10 sections, each containing more than 100 he-
mocytes, were examined by light microscopy; at least
10 sections were examined by transmission electron
microscopy. The plasmatocyte has an euchromatic
nucleus, a well-developed system of flattened or tubu-
lar cisternae of RER, and many free ribosomes (Figs.
2, 6). Mitochondria are approximately three times as

frequent as in granulocytes (Table I). Plasmatocytes
contain few, if any, large secretory granules. These
observations are confirmed by LM serial sections of
12 different plasmatocytes (Fig. 15): two plasmato-
cytes contained zero, five contained one, three con-
tained two, and two contained three large granules.
Plasmatocytes are not affected by endotoxin stimula-
tion.

lACini'LEVS TRIOENTATL'S HEMOCYTES

61

Figure 14. Granulocyte from Tachyplcus tridentalus incubated with 10 * g endotoxin per ml hemo-
lymph for 300 s. After exocytosis, the remaining organelles collect in the middle of the cell surrounded by
microtubules (arrowheads) as observed also in Limn/us polyphemus (Tablin and Levin. 1988). Digestive
vacuoles (DV). Immature granule (IG); nucleus (N).

Discussion

Two major groups ofhemocytes

We reveal one granular and one almost agranular type
of hemocyte in the general circulation of T. tridentatus
(Figs. 1, 2, 15). In agreement with the terminology from
other arthropods, including other chelicerates. these he-
mocytes are named granulocytes and plasmatocytes, re-
spectively (Gupta, 1979; Sherman, 1981; Gupta, 1985;
Suhr-Jessen et al., 1989). Their main differences are
summarized in Table II. The plasmatocyte has not pre-
viously been observed in T. tridentatus, but it makes up
about 3% of the hemocytes in all samples from the six
animals studied.

The plasmatocyte is not a cyanoblast or a cyanocyte
(Fahrenbach, 1970), because plasmatocytes have the
same size, are present in the general circulation of all ani-

mals studied at all times, and do not contain cytoplasmic
crystals.

The plasmatocyte is not a granulocyte that exocy-
tosed during sampling, because the two cells differ in
amounts of hetrochromatin, RER, free ribosomes, and
mitochondria (Table II). In other systems, such dra-
matic changes usually takes hours. Furthermore, the
plasmatocyte has the smooth ellipsoidal shape with a
marginal band characteristic of the unstimulated gran-
ulocyte in contrast to the pseudopodial form following
exocytosis (Figs. 1, 13; Dumont etai, 1966; Armstrong,
1980; Armstrong and Rickles, 1982; Armstrong, 1985;
Tablin and Levin, 1988). However, it cannot be ex-
cluded that plasmatocytes are granulocytes, which have
undergone spontaneous exocytosis so early prior to
hemolymph sampling that the marginal band of micro-
tubules have reformed. Because the production of gran-

62

P. P. JAKOBSEN AND P. SUHR-JESSEN

. .

N

Figure 15. Serial sections of a plasmatocyte from Tachyplciis trulcntaliix. The nucleus ( N) is uniformly
euchromatic: a single large granule (GR) and many mitochondria (M) are present. The neighboring granu-
locytes contain many large secretory granules, but few mitochondria.

ulocytes is not continuous (Cohen, 1 985), this latter in-
terpretation implies either: ( 1 ) that approximately 3%
of the hemocytes in all T. tridcntatus examined are con-
stantly recovering from spontaneous exocytosis, and
that the transition from plasmatocyte to granulocyte is
so fast that intermediate stages are at least one order of
magnitude less frequent than plasmatocytes; or (2) that
approximately 3% of the hemocytes in each animal re-
cover from a single burst of exocytosis long before the
first sampling, and that this recycling is blocked at the
plasmatocyte stage. In L. polyphcmits. the indepen-
dence of granulocytes and plasmatocytes is further sup-
ported by the detection of coagulogen only in granulo-
cytes (Suhr-Jessen et al.. 1989).

Large secretory granules

The large structured granules seen in the proximity of
Golgi complexes in granulocytes are apparently not

affected by endotoxin (Figs. 7-10, 13, 14). This supports
the interpretation that this class of structured granules is
an immature stage leading to the mature uniform secre-

Table I

iMin nl the ahiindnncc of mitochondria in plasmatocytes (PL)
Tachypleus tndentatus

Serial
section

Number of mitochondria in

Ratio
PL/GR

Plasmatocvte

Granulocyte

#1

#:

#3

154
276
138

48
86
51

3.25
3.21
2.71

Total

568

185

= 3

Each serial section number refers to one plasmatocyte and one gran-
ulocyte. Eighteen to 23 serial sections were required to completely sec-
tion a cell.

I'RIDLM'AI'L'S HEMOCYTES

63

Table II

.1 comparison o/l he main differences between plasmatocytes ami
graiiiilocyte.s in Tachypleus tridcntatus

Plasmatocyte

Granulocyte

Nucleus

Euchromatic

Heterochromatic

RER

Flattened and well

Distended hut

developed

poorly developed

Free ribosomes

Many

Few

Large secretory

Few if any

Many

granules

Mitochondria

Many

Few

Frequency

3%

97%

tory granules, as suggested for L. polyphemus (Copeland
and Levin, 1985;Suhr-Jessen eta/.. 1989). Following en-
dotoxin stimulation, the content of the mature uniform
granules become transitorily structured before exo-
cytosis (Fig. 11). This resembles the situation in rat
mast cells and human platelets (Bloom, 1974; Morgen-
stern et ai, 1987). The different responses to endotoxin
suggest that immature and mature secretory granules
contain different membrane proteins.

Immune defense

Granulocytes and plasmatocytes from the Asian T. tri-
dentatus are cytologically indistinguishable from those
in the American L. polyphemus (Dumont el ai, 1966;
Shishikura et ai, 1977; Gupta, 1979; Nemhauser et ai,
1 980; Ornberg and Reese, 1981; Shishikura el ai. 1982;
Armstrong, 1985; Copeland and Levin, 1985; Tablin
and Levin, 1988; Suhr-Jessen et ai. 1989). This suggests
that the cellular part of their immune defense systems
has remained unchanged for more than 140 million
years (Shishikura et ai. 1982).

Do granulocytes also participate in the endocytic part
of the immune defense system, as debated by Armstrong
and Levin (1979)? Although digestive vacuoles are pres-
ent (Fig. 14), we have not observed the formation of large
endocytic vacuoles, neither in plasmatocytes nor in gran-
ulocytes, but both cells form micropinocytotic (coated)
vesicles (Fig. 9). It is tempting to speculate that granulo-
cytes and plasmatocytes may operate together, and with
the humoral part of the immune defense system, to rec-
ognize and destroy invading microorganisms.

Although the cellular part of the immune defense sys-
tem in horseshoe crabs has been studied extensively, nei-
ther the hemocyte stem cell nor its location, regulation of
maturation, differentiation, or proliferation is elucidated
(Cohen, 1985). The fate of the granulocytes after exo-
cytosis is also unknown. The gathering of the organelles
in the middle of the granulocyte after exocytosis might
be the first step in a recovery process (Fig. 14).

The present study describes a hitherto overlooked
hemocyte, the plasmatocyte, in the general circulation
of T. tridentatiis. It also extends previous studies of the
temporal relationship between maturation and struc-
ture of the large secretory granules in granulocytes.
Both results prompt several questions pertinent to the
molecular biology, structure, and function of hemo-
cytes in horseshoe crabs in particular, and to the evolu-
tion and cell biology of the immune defense system in
animals in general.

Acknowledgments

We thank Tom Mikkelsen for providing the horseshoe
crabs and Ulla Hauschildt for technical assistence with
the transmission electron microscopy and light micros-
copy preparations. Support from Knud H0jgaards Foun-
dation (to PPJ) and a student fellowship from the Carls-
berg Foundation (to PPJ) is gratefully acknowledged.

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Reference: Biol. Hull 178: 65-73. (February. 1990)

A! Adenosine Receptor Modulation of Adenylyl Cyclase
of a Deep-living Teleost Fish, Antimora rostrata

JOSEPH F. SIEBENALLER 1 AND THOMAS F. MURRAY 2

^Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803,
and 'College oj Pharmacy, Oregon State University, Corvallis, Oregon 97331

Abstract. Low temperatures and high hydrostatic pres-
sures are typical of the deep sea. The effects of these pa-
rameters on transmembrane signal transduction were
determined through a study of the A, adenosine recep-
tor-inhibitory guanine nucleotide binding protein-ade-
nylyl cyclase system in brain membranes of the bathyal
teleost fish. Antimora rostrata (Moridae). The compo-
nents of this system were analyzed at 5C and 1 atm, and
the role of the A, receptor in the modulation of adenylyl
cyclase was determined. The A, selective radioligand N h -
['H]cyclohexyladenosine bound saturably, reversibly,
and with high affinity. The K^ of N 6 -['H]cyclohexyladen-
osine estimated from kinetic measurements was 1.11
nAf; the Kj determined from equilibrium binding was
4.86 nAf. [ 32 P]ADP-ribosylation of brain membranes by
pertussis toxin labeled substrates with apparent molecu-
lar masses of 39,000 to 4 1 ,000 Da. Basal adenylyl cyclase
activity was inhibited in a concentration-dependent
manner by the A! adenosine receptor agonist N 6 -cyclo-
pentyladenosine (IC 50 = 5.08 nM). The inhibition of ad-
enylyl cyclase activity was dependent upon GTP. Basal
adenylyl cyclase activity was unaffected by 272 atm of
pressure. The efficacy of 100 /iA/ N 6 -cyclopentyladeno-
sine as an inhibitor of adenylyl cyclase was the same at
atmospheric pressure and at 272 atm. The inhibition of
adenylyl cyclase by the agonist 5'-N-ethylcarboxami-
doadenosine (100 pM) at 272 atm was twice that ob-
served at atmospheric pressure. Although consideration

Received 1 September 1989; accepted 30 November 1989.

Abbreviations: ATP. adenosine triphosphate; cAMP. cyclic adeno-
sine monophosphate; [ 3 H]CHA, N 6 -['H]cyclohexyladenosine; CPA.
N''-cyclopentyladenosine; G protein, guanine nucleotide binding pro-
tein; GTP, guanosine triphosphate; NAD, nicotinamide adenine dinu-
cleotide. NECA, 5'-N-ethylcarboxamidoadenosine; R-PIA. N^-phenyl-
isopropyladenosine, R(-) isomer. S-PIA. N 6 -phenylisopropyladeno-
sine, S( + ) isomer.

of the effects of low temperature and high hydrostatic
pressure on acyl chain order suggest that deep-sea condi-
tions will perturb membrane function, signal transduc-
tion by the A, receptor system of the bathyal fish A. ros-
trata is not disrupted by deep-sea conditions.

Introduction

The low temperatures and high hydrostatic pressures
of the deep ocean may disrupt the biochemical and phys-
iological functions of organisms colonizing this habitat
(Siebenaller and Somero, 1978, 1989). Membrane-asso-
ciated systems are likely to be particularly sensitive be-
cause of the ordering effects of these environmental vari-
ables on the organization of acyl chains of lipids (Chong
and Cossins, 1983; Hochachka and Somero, 1984).
Comparisons of homologous cytoplasmic proteins from
deep- and shallow-living teleost fishes have established
the importance of adaptation to deep-sea temperatures
and pressures (Siebenaller and Somero, 1989). Studies of
membranes and associated systems in deep-sea organ-
isms indicate that these systems also adapt (e.g.. Cossins
and Macdonald, 1984, 1986; DeLong and Yayanos,
1985, 1 987; Gibbs and Somero, 1989).

To further understand the effects of deep-sea condi-
tions, and to identify potential adaptations of transmem-
brane signal transduction, we studied the A, adenosine
receptor and its associated effector elements in brain tis-
sue of a deep-living cold-adapted marine teleost fish.
Antimora rostrata. The objectives of this study were: (1)
to determine whether the A, receptor of a typical deep-
sea species is coupled to adenylyl cyclase, and (2) to as-
certain whether this coupling is functional under the
conditions of low temperatures (0-6C) and high hydro-
static pressures (85-250 atm) experienced by A. rostrata.
To this end, we undertook a molecular dissection of this

65

66

J. F. S1EBENALLER AND T. F. MURRAY

signal transduction system, characterized ligand binding
to the A, receptor, identified the associated GTP-binding
proteins, and determined basal adenylyl cyclase activity
and the role of A, receptor agonists and GTP-binding
proteins in modulation of cAMP accumulation.

A. rostrata is a benthopelagic morid commonly found
in the Atlantic and South Pacific Oceans at bathyal
depths of 850 to 2500 m (Iwamoto, 1975; Wenner and
Musick, 1977). [Pressure increases 1 atm (=101.3 kPa)
for every 10 m of depth in the ocean.] A. rostrata is re-
placed by the congener. 1. micmlepis in the North Pacific
(Small, 1 98 1 ). Many adaptations to hydrostatic pressure
and low temperature have been documented for these
species (e.g., Hochachka, 1975; Siebenaller and Somero,
1979; Somero and Siebenaller, 1979; Cossins and Mac-
donald, 1984, 1986; Avrova, 1984; Yancey and Siebe-
naller, 1987; Hennessey and Siebenaller, 1987; Gibbs
and Somero, 1989).

A, adenosine receptor modulation of adenylyl cyclase
was selected for two reasons as a model with which to
examine pressure and temperature effects on transmem-
brane signal transduction. First, our previous studies had
documented the widespread distribution of this receptor
among the classes of chordates, including deep-occurring
teleosts (Siebenaller and Murray, 1986, 1988), and we
had also identified potentially adaptive differences in li-
gand binding among species (Murray and Siebenaller,
1987; Siebenaller and Murray, 1988). Agonist occupa-
tion of the A, adenosine receptor inhibits cAMP accu-
mulation in mammalian central nervous tissue prepara-
tions (reviews by Wolff tv a/., 1981; Londos et a/.. 1983;
Snyder, 1985; Williams, 1987). The A, adenosine recep-
tor is coupled to adenylyl cyclase [ATP pyrophosphate-
lyase (cyclizing); EC 4.6.1.1] by an inhibitory guanine
nucleotide binding protein (G, protein). Agonist occu-
pation of the other subclass of adenosine receptor cou-
pled to adenylyl cyclase, the A : receptor, stimulates ad-
enylyl cyclase activity. These receptors are further distin-
guished on the basis of the rank order potencies of
adenosine analogs (Daly, 1983a, b; Stone, 1985; Wil-
liams, 1987).

At a measurement temperature of 22C, the binding
affinities, specificities, and pharmacological profiles of
the A i adenosine receptors in teleost fishes are similar to
those of mammals (Siebenaller and Murray, 1986; Mur-
ray and Siebenaller, 1987). The binding of agonists to the
high affinity state of mammalian A, receptors is dis-
rupted by the low temperatures typical of body tempera-
tures of many cold-adapted fishes (e.g.. Bruns et a/.,
1980; Trost and Schwabe, 1981; Murphy and Snyder,
1982;Lohserfa/.. 1984; Siebenaller and Murray, 1988).
However, agonist recognition and binding properties of
the A, adenosine receptors are retained in evolutionary
adaptation to different body temperatures. For instance.

for eight vertebrate species with body temperatures of 1 -
40C, Kj values for the agonist N 6 -['H]cyclohexyladeno-
sine <[ 3 H]CHA) measured at 5C varied 30-fold; how-
ever, the binding affinities vary only four-fold when com-
pared at temperatures similar to the species' body tem-
peratures (Siebenaller and Murray, 1988).

Materials and Methods
Specimens

Demersal adult Antimora rostrata (Moridae) were col-
lected by otter trawl at their depths of typical abundance
(850-2500 m) off the coast of Newfoundland, Canada,
on a cruise of the R/V Gyre in May 1986. Brain tissue
was dissected, frozen in liquid nitrogen at sea, and trans-
ported to the laboratory where tissues were maintained
at -80C until used. For the [ 3: P]ADP-ribosylation ex-
periments described below, brain tissue from the mac-
rourids, Macrourus hergla.\, Coryphaenoides rupestris,
and C. armatus, taken off the coast of Newfoundland,
the scorpaenids, Sebastolobus alascanus and 5. altivelis,
taken on a cruise of the R/V H 'ecoma off the coast of
Oregon, and the salmonid, Oncorhynclnts mykiss
(Salmo gairdneri), raised at the Food Toxicology and
Nutrition Laboratory of Oregon State University, were
also used. The macrourid species were chosen because
they represent a primarily deep-sea family. The Sebasto-
lobus species have been employed in a variety of pressure
adaptation studies (Siebenaller and Somero, 1989), and
O. mykiss is a pelagic freshwater species.

Reagents

[Adenylate- 3: P]-nicotinamide adenine dinucleotide
([ 3: P]NAD, 31.31 Ci/mmol), [ 3 H]CHA (34.4 Ci/mmol),
[-- : P]ATP (800 Ci/mmol) and [ 3 H]cAMP (30.5 Ci/
mol) were from DuPont NEN (Wilmington, Delaware).
The R- and S-diastereomers of N 6 -phenylisopropyladen-
osine (PIA), 5'-N-ethylcarboxamidoadenosine (NECA),
and papaverine were obtained from Research Biochemi-
cals. Inc. (Wayland, Massachusetts). Pertussis toxin was
from List Biological Laboratories (Campbell, Califor-
nia). Electrophoresis reagents and molecular weight
standards were from Bio-Rad (Richmond, California).
Adenosine deaminase (Sigma, Type VI), N 6 -cycIopentyI-
adenosine (CPA), 2-chloroadenosine, and all other
chemicals used were from Sigma Chemical Co. (St.
Louis, Missouri). Water was processed through a four-
bowl Milli-Q purification system (Millipore, Bedford,
Massachusetts).

Preparation of brain membranes

Antimora rostrata brain membranes for assays of li-
gand binding were prepared following the procedures de-
scribed by Murray and Siebenaller ( 1987).

ADENYLVL CYCLASE OF A DEEP-SEA FISH

67

For adenylyl cyclase assays, brain tissue was disrupted
with a Dounce (pestle A) in 100 volumes of 10 mM
HEPES, pH 7.6 at 5C, and centrifuged at 27,000 X g(0-
4C) for 10 min. The pellet was resuspended in buffer,
centrifuged at 27,000 X g for 10 min, resuspended in
buffer, and 7.5 units/ml of adenosine deaminase were
added. The homogenate was incubated at 18C for 30
min, chilled on ice, centrifuged at 27,000 X g, and the
pellet resuspended in buffer and 7.5 units/ml adenosine
deaminase. Fifty microliters of this homogenate were
used in the adenylyl cyclase assays.

For ADP-ribosylation experiments, membranes were
homogenized with a Dounce (pestle A) in 40 volumes of
50 mM Tris-HCl, pH 7.6 at 5C. The homogenate was
centrifuged at 27,000 X g for 10 min. The pellet was re-
suspended in 40 volumes of Tris-HCl buffer. Fifty micro-
liters of this were used for the ribosylation experiments.

Protein was determined by the method of Lowry et al.
(1951) following solubilization of the samples in 0.5 M
NaOH. Bovine serum albumin (Sigma Chemical Co.)
was used as the standard.

Time course of agonist association and dissociation

Aliquots of brain membranes were incubated with
2.85 nM [ 3 H]CHA in 50 mM Tris-HCl, pH 7.6 at the
incubation temperature of 5C. Nonspecific binding was
determined simultaneously in the presence of 60 nM R-
PIA. For the dissociation experiments, samples were first
incubated at 5C with 2.85 nM [ 3 H]CHA for 240 min to
allow binding to reach equilibrium. R-PIA (60 nAI) was
added in a negligible volume (1% of the total) to initiate
the dissociation reaction. Samples were started at timed
intervals, and all incubations were terminated simulta-
neously by filtration over No. 32 glass fiber filter strips
(Schleicher and Schuell Inc., Keene, New Hampshire)
using a cell harvester (Brandel Instruments, Gaithers-
burg, Maryland). The data were analyzed as described
below.

Equilibrium binding assay for membrane bound
A, adenosine receptors

The rapid filtration assay described by Bruns et al.
(1980) and Murray and Cheney (1982) was used with
minor modifications to determine the specific binding of
the A,-selective agonist [ 3 H]CHA to A. rostrata brain
membranes. Assays were conducted in Tris-HCl, pH 7.6,
at the incubation temperature of 5C. The procedures de-
scribed in Siebenaller and Murray (1988) were followed.
Brain membrane protein (0.4- 1 .2 mg) was added to each
assay tube.

[ 32 F\ADP-ribosylation

Pertussis toxin-catalyzed [ 3: P]ADP-ribosylation of
GTP binding proteins followed the procedures described

in Ribeiro-Neto et al. ( 1 985 ) and Greenberg et al. (1987).
The 100-jul incubation mixture contained 100 mM Tris-
HCl, pH 7.5, at the incubation temperature of 5C, 25
mM dithiothreitol, 2 mM ATP, 0. 1 mM GTP, 5
[ 32 P]-NAD, 1.5 ng soybean trypsin inhibitor, 15 ^
tracin, 2 ^g pertussis toxin, and 37-92 ng membrane pro-
tein. After 3 h, the reaction was stopped by adding 50 /ul
of stop solution (3% sodium dodecyl sulfate, 42% glyc-
erol, 1 5% 2-mercaptoethanol, 200 mM Tris-HCl, pH
6.8, at 20C) and boiled for 5 min. The denatured sam-
ples were subjected to sodium dodecyl sulfate polyacryl-
amide electrophoresis in a 1.5-mm thick 12.5% acryl-
amidegel following Laemmli(1970). The gel was stained
with 0.25% Serva Blue R (Serva Fine Biochemicals,
Westbury, New York) in 25% 2-propanol, 10% acetic
acid, destained and dried. The dried gels were exposed to
Kodak (Rochester, New York) X-Omat AR film. Du-
Pont Cronex Lightning Plus intensifying screens were
used.

Adenylyl cyclase assays

The standard adenylyl cyclase assay contained in a to-
tal volume of 150 //I, 10 to 20 jtg of A. rostrata brain
membrane protein, 50 mM HEPES, pH 7.6 at the assay
temperature of 5C, 50^Af 2-deoxy-ATP, approximately
1 to 1.5 X 10" cpm [- 32 P]ATP, 10 juMGTP, 6.25 mM
Mg acetate, 100 mM NaCl, 7.5 units creatine kinase, 5
mM phosphocreatine, 1.5 /*g soybean trypsin inhibitor,
1 5 Mg bacitracin, and other constituents as indicated be-
low. Assays were conducted in triplicate in a refrigerated
water bath for 2 h. The reaction was stopped by adding
250 fi\ of 2% sodium dodecyl sulfate, 45 mM ATP, and
1.3 mA/cAMP. The samples were boiled for 3 min and
600 ^1 of water were added. [ 3: P]cAMP generated in the
assays was determined according to Salomon et al.
(1974).

For assays of the effects of hydrostatic pressure on ad-
enylyl cyclase activity and inhibition, samples were
transferred to polyethylene tubing. The tubing was
trimmed to exclude air bubbles and sealed using a pipet
heat sealer. [ 3 H]cAMP (approximately 20,000 cpm) was
used as an internal standard to monitor the recovery of
sample through the sealing and incubation, and through
the subsequent column chromatography steps isolating
the [ 32 P]cAMP from the [ 3: P]ATP following Salomon et
al. (1974). The pK a of HEPES, the buffer used in these
experiments, is relatively insensitive to pressure (Bern-
hardt et al., 1988). Samples were incubated in high pres-
sure vessels maintained at 5C in a refrigerated circulat-
ing water bath. The high pressure apparatus is described
in Hennessey and Siebenaller (1985). Samples were incu-
bated for 1 20 min. The time required to seal and pressur-
ize a group of four samples and the time required to re-

68

J. F. SIEBENALLER AND T. F. MURRAY

move the samples was less than 6% of the incubation
time at elevated pressure. Samples sealed and incubated
at atmospheric pressure have adenylyl cyclase activities
identical to samples that are incubated in test tubes.

Data analysis

The kinetic parameters for the time course of associa-
tion and dissociation of specific [ 3 H]CHA binding were
estimated using the equations of Weiland and Molinoff
(1981). Rate constants were calculated by least squares
linear regression.

The association data were analyzed as a pseudo-first
order reaction described by the equation:

In

[B,J

-[B])

k ,)t = k ohs t

where [B C J is the amount of [ 3 H]CH A bound at equilib-
rium, [B] is the amount bound at time t, [L] is the con-
centration of [ 3 H]CHA. The pseudo-first order rate con-
stant, k ohs is determined from the slope of plots of In
[B eq ]/([B e J - [B]) versus time (Weiland and Molinoff,
1981; Kitabgi r/<//.. 1977).

The first order dissociation constant (k ,) was deter-
mined from a plot of the equation:

In [B]/[B ] = k ,t

where [B ] is the concentration of bound [ 3 H]CHA at
time 0. The ratio of the rate constants (k ,/k, ,) provides
an estimate of the equilibrium dissociation constant (KJ
for [ 3 H]CHA binding.

Saturation isotherms were analyzed using LUNDON-
1 (Lundon Software, Inc., Cleveland, Ohio) iterative
curve fitting routines (Lundeen and Gordon, 1985).

Concentration-response data for inhibition of ade-
nylyl cyclase were analyzed by fitting a three parameter
logistic equation to the data. The equation used was:

Y =

E-I

1 +

X

+ 1

(1C.,,,)

where Y is the rate of adenylyl cyclase activity (pmol
cAMP min ' mg protein ') in the presence of a given
concentration of adenosine analog (X); E is the activity
of adenylyl cyclase in the absence of adenosine analog; I
is the activity in the presence of a maximally inhibiting
concentration of adenosine analog; and IC 5( , is the con-
centration of adenosine analog that produces a half-max-
imal inhibition of the adenylyl cyclase activity. The con-
centration-response data were analyzed with an un-
weighted nonlinear regression analysis program using
FITFUN, a computer modeling program on the
PROPHET II Computer System.

120 150 180 210 240

TIME (min)

Figure 1. Time course of association ol specific [ 3 H]CHA binding
loAnttmura rosiraui brain membranes at ?C. [ 3 H]CHA(2.85 nM) was
incubated at 5C with 0.52 mg membrane protein per tube prepared as
described in Materials and Methods. Nonspecific binding was mea-
sured in the presence of 60 fiM R-PIA and was constant throughout
the association reaction. Inset shows the pseudo-first order replot of
pHlCHA association data. A single representative experiment is
shown.

Results

Time course of association and dissociation

At 5C, the agonist fHJCHA bound specifically and
reversibly to the A. rostrata brain membranes. By 120
min, the specific binding of [ 3 H]CHA was 87% of that
observed at 240 min (Fig. 1). Nonspecific binding was
constant throughout the association reaction. The k obs
calculated from the plot of In [B e J/([B c J - [B]) against
time was 0.0484 0.007 1 9 min ' (Fig. 1 , inset). Exami-
nation of the data in the inset suggest that a multiple pa-
rameter fit might provide a better fit. However, we ap-
proximated k ohs by a single parameter because we did not
have sufficient data to fit a multiple parameter model.
The dissociation rate constant (k ,) was determined di-
rectly by following the dissociation of specifically bound
[- H]CHA by the addition of 60 pM R-PIA. The [- H]-
CHA was readily dissociated on addition of excess R-PIA
(Fig. 2). The k_, calculated from the plot of In [B]/[B (1 ]
versus time was 0.0 136 0.0006 min" 1 (Fig. 2, inset). K^
of [ 3 H]CHA was estimated from the ratio of k i/k 4 i.
This kinetically derived estimate of the equilibrium dis-
sociation constant, K^ , was 1.11 nM.

Equilibrium saturation analysis

The specific binding of [ 3 H]CH A at 5C to A. rostrata
brain membranes was saturable (Fig. 3). Specific bind-
ing, denned as total binding minus nonspecific binding

ADENYLYL CYCLASE OF A DEEP-SEA FISH

69

B

92.5k
66.2k

120

TIME (min)

Figure 2. Time course of R-PIA-induced dissociation of specific
[ 3 H]CHA binding in Antimora rostrata brain membranes. Membranes
(0.52 mg protein) were first incubated at 5C with 2.85 nM ['H]CHA
for 240 min to allow binding to reach equilibrium. Sixty pM R-PIA
was added in a negligible volume (1% of the total incubation volume)
to initiate the dissociation reaction. The nonspecific binding, which has
been subtracted from each experimental point, was determined in the
presence of 60 pM R-PIA. Inset depicts the first-order replot of dissocia-
tion data and represents the best least-squares regression line. Data
shown are from a single experiment.

determined in the presence of 30 nAf R-PIA, saturated
over the range of 0.096 to approximately 16 nA/. Non-
specific binding increased linearly as a function of ['HI-
GH A concentration. Analysis of the [ 3 H]CH A saturation
isotherm, using equations based on mass action princi-
ples, indicated that the data were adequately described
by interaction with a single high affinity state of the re-
ceptor. Computer modeling of replicate experiments

5 10

PH]CHA (nM)

Figure 3. Equilibrium saturation binding of ['H]CHA \oAntimuru
rostrata brain membranes at 5C. Membranes were incubated 1 50 min
with 1 1 concentrations of free ['H]CHA ranging from 0.096 to 15.7
nA/in this experiment. Specific binding is indicated by the filled circles.
Nonspecific binding, shown by the open triangles, was determined in
the presence of 30 iiM R-PIA.

MR

1234567 1234567

Figure 4. Autoradiogram of teleost fish brain membrane prepara-
tions [ 32 P]ADP-nbosylated by pertussis toxin at 5C. Preparations were
incubated for 2 h with [ 32 P]NAD with (A) or without (B) pertussis
toxin. The samples were denatured and subjected to sodium dodecyl
sulfatepolyacrylamideelectrophoresisin 1 2.5% acrylamide gels follow-
ing Laemmli ( 1 970). The gels were dried and exposed to x-ray film.
I. Antimora rostrata, 1. Scbasiolohux alascanus. 3. 5. altivelis,
4. Macrurus hcrglax. 5. CoryphaemMfx rupestris, 6. C. armatus,
1. Oncorhvnchux mvkiss.

yielded an average Kd value of 4.86 0.95 nM with a
density of 25.6 2.02 fmol mg membrane protein '.
The Kj value obtained from the saturation isotherm is
in relatively good agreement with the kinetically derived
K<, value of 1 . 1 1 nM.

[ 3: P]ADP-ribosylation

Figure 4 shows an autoradiogram of a sodium dodecyl
sulfate Laemmli gel used to resolve brain membrane
preparations incubated with [ 32 P]NAD in the presence
and absence of activated pertussis toxin. [ 3: P]ADP-ribo-
sylation did not occur in the absence of pertussis toxin.
Substrates of approximately 39,000 to 41,000 Da appar-
ent molecular mass were specifically labeled in the brain
membrane preparations from each of the seven teleost
species surveyed. In the autoradiogram some prepara-
tions clearly have two labeled substrates, e.g., Macrourus
berglaxand Coryphoenoides rupestris (Fig. 4). The ribo-
sylation reaction was performed at 5C to maintain
membrane viscosity close to the body temperatures of
these species. At this temperature, pertussis toxin-cata-
lyzed ribosylation of membranes from cold-adapted
deep- and shallow-living marine teleosts and the fresh-
water species, O. mykiss. results in labeling of compo-
nents similar to those of warm-adapted species. The rela-
tive molecular masses of the components ribosylated are
in the range corresponding to the molecular masses re-
ported for the alpha subunits of the GTP-binding pro-
teins G, and G , an "other" G protein of unknown func-
tion, in other species (4 1 ,000 Da and 39,000 Da, respec-
tively; Oilman, 1987;PfeufferandHelmreich, 1988).

Adenylyl cyclose

The time courses for basal and 0. 1 \iM forskolin-stim-
ulated adenylyl cyclase activity in A. rostrata brain mem-

70

J. F. S1EBENALLER AND T F. MURRAY

120

Time (min)

Figure 5. Time course of the adcnylyl cyclase reaction at 5C in
brain membranes from Anlimora roMruui. Open circles: basal adenylyl
cyclase activity. Filled circles: forskolin (0.1 p.M) stimulated activity.
Reaction mixture contained 50 m,U HHPES, pH 7.6, at 5C, 4 m.U
magnesium acetate, 50 n.M 2-deoxy ATP. 100 M cAMP. 200 ,,A/ pa-
paverine,6.3 mg creatine phosphate, 2 mg creatine phosphokinase, 100
m-A/NaCl, 10 jiA/ GTP, and 2.5 units/ml adenosinedeaminase.

branes are shown in Figure 5. At 5C, both the basal and
forskolin-stimulated rates were linear for at least 120
min. An incubation time of 1 20 min was used for all sub-
sequent experiments.

The inhibition of basal adenylyl cyclase activity by N h -
cyclopentyladenosine (CPA) was used as a biochemical
measure of the extent of coupling of A, receptors to ad-
enylyl cyclase via a guanine nucleotide regulatory pro-
tein. CPA is a highly selective A , adenosine receptor ago-
nist and was used to eliminate potential interactions with
the A 2 adenosine receptor (Bruns el ai. 1986; Williams
ft ai, 1986) or with the P-site on the catalytic subunit of
adenylyl cyclase (Londos el ai. 1983; Blair et ai. 1989;
Johnson et ai. 1989). The dependence of CPA-induced
inhibition of adenylyl cyclase activity on GTP concen-
tration is depicted in Figure 6. Basal adenylyl cyclase ac-
tivity in the absence and presence of 100 ^A/ CPA is
shown. CPA had little effect on adenylyl cyclase activity
in the absence of added GTP; however, at GTP concen-
trations of 1 to 100 nM. the A i selective agonist inhibited
activity. A representative concentration response curve
in Figure 7 depicts the inhibition of basal adenylyl cy-
clase activity in A. rostrata brain membranes by various
concentrations of CPA. The IC ?I) value for inhibition of
adenylyl cyclase was 5.08 2.65 fiM. The maximal inhi-
bition of basal activity by CPA ranged from 7 to 17%.
Other adenosine analog agonists, such as R-PIA, 2-chlo-
roadenosine, and NECA displayed similar efficacies as
inhibitors of adenylyl cyclase activity (data not shown).
The concentration-dependent inhibition of adenylyl cy-
clase activity is consistent with the involvement of A, re-
ceptors in the inhibitory modulation of the enzyme.

CD

E

10 .

E

Q.

O

10 10

GTP (M)

Figure 6. Effect of GTP concentration on Anlimora rostrata brain
membrane basal adenylyl cyclase activity in the absence (open circles)
and presence (filled circles) of 100 ^A/CPA at 5C.

As shown in Figure 8, the basal adenylyl cyclase activ-
ity of the deep-living fish A. rostrata was unaffected by
272 atm of pressure, which is comparable to the pres-
sures experienced by the species at the lower end of its
depth range. At atmospheric pressure, basal adenylyl cy-
clase activity is 3.3 0.07 pmol min ' mg membrane
protein"'. Basal activity is unchanged at 272 atm (3.3
0.11 pmol min ' mg '). The efficacy of the adenosine
receptor agonists CPA ( 100 /uM) and NECA (100 pM)
as inhibitors of adenylyl cyclase were compared at atmo-
spheric pressure and 272 atm. As shown in Figure 8,
the increased pressure doubled the mean percentage in-
hibition of basal activity by NECA over that observed at
1 atm ( 14 8% at atmospheric pressure, 29 2% at 272
atm) but had no effect on the inhibition by CPA (9 5%
at atmospheric pressure and 8 5% at 272 atm).

cr>

E

\

c

1

o

E

CL

<

o

CPA IC 50 = 5.08 2.65

2.9-

2.7-

i
25

50
CPA

75

100

Figure 7. Inhibition of Antimora roslrala brain membrane basal
adenylyl cyclase activity by the A, adenosine receptor agonist CPA at
5C.

ADENYLYL CYCLASE OF A DEEP-SEA FISH

71

1.0--

_D
0)

cr

0.5--

1 atm

272 atm

Pressure

Figure 8. The effects of hydrostatic pressure on Anlimora mslrata
basal adenylyl cydase activity (open bar) and inhibition of basal ade-
nylyl cyclase activity by the adenosine analogs CPA (100 //A/; filled
bar) and NECA (100 /i.U. 1 hatched bar). Membranes were incubated at
atmosphenc pressure or 212 atm pressure for 2 h at 5C. All values are
standardized to the I atm basal adenylyl cyclase activity. The 1 atm
and 272 atm basal activities were 3.3 pmol mm ' mg protein' '. The 1
atm data are the mean of three replicates: the 272 atm values are the
mean of six replicates. The average standard errors are 1 1.7% of the
values of the mean.

Discussion

Binding of the agonist [ 3 H]CHA to the A, receptor in
A. rostrata brain membranes at 5C is saturable and
readily reversible (Figs. 1 . 2. 3). At 5C, the rate constants
determined for A. rostrata from association-dissociation
experiments are lower than those reported for two scor-
paenid fishes, Sebastolobus alascanus and 5. altivelis, at
a measurement temperature of 22C (Murray and Siebe-
naller, 1987). The k obs fortheA rostrata binding reaction
is only 2 1 to 25% of the values obtained for the Sebasto-
lobus species ai 22C. Thek_! value for A. rostrata is only
40 to 65% of the 22C values. At 22C the binding reac-
tion in Sebastolobus membranes is complete in 30 min.
In contrast, at 5C, the reaction in A. rostrata mem-
branes takes more than four times as long to reach equi-
librium (Fig. 1). At 5C, which approximates the body
temperatures of these three species, the K^ values are
similar (Siebenaller and Murray, 1988).

At 5C, the rank order potencies of agonists is compat-
ible with that expected for the A, receptor (Fig. 9; Siebe-
naller and Murray, 1988). The K, values indicate dis-
crimination of the R- and S-diastereomers of PIA (4.5
and 1 1 5.9 nM, respectively). The rank order potency se-
ries expected for A, receptors is R-PIA > 2-chloroadeno-
sine > NECA > S-P1A. and for A : receptors NECA > 2-
chloroadenosine > R-PIA > S-PIA (Daly, 1983 a, b;
Stone, 1985; Williams, 1987). The rank order potencies,
the discrimination between R-PIA and S-PIA, and the
Kj of [ 3 H]CHA values are characteristic of an A, adeno-
sine receptor.

The substrates specifically ["P]ADP-ribosylated by
pertussis toxin in A. rostrata and six other teleost species
(Fig. 4) have apparent molecular masses characteristic of
the class of alpha subunits from the guanine nucleotide
binding regulatory proteins G, and G (Oilman, 1987;
PfeufTer and Helmreich, 1988). The GTP-dependence of
CPA-induced inhibition of cAMP accumulation (Fig. 6)
demonstrates a role for these G proteins in the coupling
of the A, receptor to negative modulation of adenylyl
cyclase activity in A. rostrata brain membranes. In the
presence of GTP, CPA inhibited basal adenylyl cyclase
activity with an IC 5I) of 5 .08 2.65 \iM ( Fig. 7 ). The max-
imal inhibition ranged from 7 to 17%. This degree of in-
hibition is similar to the maximal inhibition of adenylyl
cyclase by CPA in embryonic chick heart membranes
( Blair rt al. 1989).

The A, adenosine receptor of the deep-living teleost,
Anlimora rostrata, is capable of modulating the activity
of adenylyl cyclase under the conditions of low tempera-
ture and high hydrostatic pressure, which characterize
the bathyal habitat (Fig. 8). Experiments currently un-
derway, using brain tissues from other species, indicate
that the A, adenosine receptor-G,-adenylate cyclase sys-
tem can be markedly perturbed by hydrostatic pressures
less than the 272 atm used in the present study. For in-
stance, in shallower-occurring fishes, basal adenylyl cy-
clase activity is inhibited 1 1 to 25%> by 136 atm pressure
(Siebenaller and Murray, work in progress). In contrast,
A. rostrata brain tissue adenylyl cyclase is unaffected by
272 atm pressure, the highest pressure tested. The effi-
cacy of agonists at the A] adenosine receptor is not les-
sened by increased pressure (Fig. 8). CPA-induced inhi-
bition of adenylyl cyclase was unaltered, and the efficacy
of NECA increased. Thus, basal adenylyl cyclase activ-
ity, as well as signal transduction by the A, receptor sys-

100--

o

cr

i

o
o

O

m

-10 -9 -8 -7 -6 -5 -4
LOG DISPLACER CONCENTRATION (M)

Figure 9. Inhibition of specific [ 3 H]CHA binding in Aniimora nn-
Iraui brain membranes by adenosine analogs: R-PIA (open circle),
NECA (open triangle), 2-chloroadenosine (filled circle). S-PIA (filled
triangle). Eleven concentrations of each analog were incubated with
membranes and 7.9 nA/ [ 3 H]CHA for 1 50 mm at 5C.

72

J. F. SIEBENALLER AND T. F. MURRAY

tern, are functional under the conditions of pressure and
temperature at which A. rostrata occurs.

Consideration of the effects of low temperature and
high hydrostatic pressure on membrane viscosity (Cos-
sins and Macdonald, 1989) suggests that any of the com-
ponents of the A, receptor-G, protein-adenylyl cyclase
complex may be susceptible to perturbation in organ-
isms colonizing the deep sea. For A. rostrata, the func-
tion of this transmembrane signaling complex is main-
tained at low temperature and high hydrostatic pressure.
The pressure insensitivity of this membrane-associated
system in A. rostrata is analogous to the pressure adapta-
tions observed for cytoplasmic proteins (Siebenaller and
Somero, 1 989). The K m values of NAD-dependent dehy-
drogenases of deep-living species are relatively insensi-
tive to perturbation by pressure. In contrast, homologous
enzymes from shallow-living, cold-adapted species are
perturbed by pressures as low as 68 atm. By having pres-
sure-resistant enzymes, function is preserved over the
range of depths that may be experienced by an individual
during ontogeny or diel vertical migrations, or by a spe-
cies maintaining populations over a broad depth gradi-
ent (Siebenaller, 1987).

Gibbs and Somero (1989) hypothesized, based on
their study of Na + /K + -ATPase in teleost gill tissue, that
clear adaptations of membrane-associated systems to
pressure may only be apparent in species occurring at
depths greater than 2000 m. Although our data do not
directly test this hypothesis, the pressure insensitivity of
the A | receptor and effector system in A. rostrata. which
commonly occurs to depths of 2500 m, are compatible
with their suggestion. This pressure resistance in A. ros-
trata is a standard with which to compare the effects of
environmental parameters on transmembrane signal
transduction in other deep- and shallow-occurring spe-
cies.

Acknowledgments

This research was supported by NSF grant DCB-
8710155, and ONR contracts N00014-88-K-0426,
N0014-88-K.-0432, and ONR grants N00014-89-J-1865
and N00014-89-J-1869. Shiptime on the R/V \Vcconui
off the coast of Oregon was supported by NSF grant
DCB-8710155. Shiptime on the R/V Gyre off the coast
of Newfoundland was supported by NSF grant DMB-
8502857 to Dr. A. F. Riggs. We thank Drs. A. Riggs and
R. Noble for their help in obtaining specimens and Drs.
P. Franklin and M. Leid for their help with the ADP-
ribosylation experiments.

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Control of Cnida Discharge: III. Spirocysts are
Regulated by Three Classes of Chemoreceptors

GLYNE U. THORINGTON AND DAVID A. HESSINGER 1

Department of Physiology and Pharmacology. School of Medicine,
I.onui Linda University. Loma Linda, California 92350

Abstract. Spirocysts are two to three times more abun-
dant than nematocysts in the feeding tentacles of aconti-
ate sea anemones. Despite their prevalence, little experi-
mental work has been done on the discharge of Spirocysts
because of the difficulty in detecting and counting them
after they have discharged. To circumvent this problem,
we have developed a simple, reliable, enzyme-linked lee-
tin sorbent assay (ELLSA) for quantifying discharged
spirocysts. With this method, we have shown that the dis-
charge of spirocysts, like that of mastigophore nemato-
cysts, is chemosensitized in a dose-dependent manner by
three classes of low molecular weight substances, typified
by N-acetylneuraminic acid (NAN A), glycine, and cer-
tain heterocyclic amino compounds, such as proline and
histamine. We also show that spirocysts exhibit consider-
able agonist-specific variation in the dose-responses of
discharge, suggesting the existence of multiple popula-
tions of spirocyst-bearing cnidocyte/supporting cell
complexes (CSCCs). Our findings call into question
commonly held views regarding the respective roles of
spirocysts and mastigophore nematocysts in the reten-
tion of captured prey.

Introduction

The cnidom of the feeding tentacles of acontiate sea
anemones, including Aiptasia patlida. consists of three
types of cnidae: spirocysts; microbasic p-mastigophore
nematocysts, and basitrichous isorhiza nematocysts
(Hand. 1955) in approximate ratios of 3:1:0.3, respec-
tively (Bigger, 1982: Watson and Mariscal, 1983). Cni-
dae function primarily in the capture of prey (Ewer,
1947), in aggression (Purcell, 1977; Bigger, 1982), in de-

Received 17 July 1989; accepted 30 November 1989.
' To whom all correspondence should be addressed.

fense( Francis, 1973), and in the attachment to appropri-
ate substrates (Mariscal, 1972).

Spirocysts are adherent cnidae found only in zoan-
tharian anthozoans (Mariscal et al.. 1978; Mariscal,
1 984). An undischarged spirocyst consists of a single-lay-
ered capsule containing a long, spirally coiled, inverted
tubule of uniform diameter (Mariscal, 1974). The tubule
lacks spines, but bears hollow rods that dissociate upon
discharge to form a web of fine, adhesive microfibrillae
(Mariscal el al.. 1977).

Unlike nematocysts, discharged spirocysts are difficult
to see under the light microscope due to their non-refrac-
tile, transparent capsules (Weill, 1934). Because the tu-
bules of discharged spirocysts entangle extensively (Ste-
phenson. 1929; Skaer and Picken, 1965; Picken and
Skaer, 1966; Mariscal, 1974; Mariscal et al., 1977), it is
difficult to visually distinguish individual tubules. Thus,
it is tedious and time-consuming to visually count spiro-
cysts discharged onto test probes.

To circumvent this difficulty, we developed a simple,
sensitive, and reproducible assay to quantify spirocysts
discharged onto test probes. The method is based on the
recent discovery that the everted tubules of spirocysts
have a high affinity for free and conjugated N-acetylated
sugars such as occur on mucins, asialomucins, and mu-
copolysaccharides ( Watson and Hessinger, in prep. ). The
terminal sugars of the unbranched oligosaccharide
chains of bovine submaxillary asialomucin are N-acetyl-
galactosamine. This saccharide binds specifically to the
lectin from I 'icia villosa. Subsequent to binding asialo-
mucin to discharged spirocysts, we determine the num-
ber of discharged spirocysts adhering to gelatin-coated
test probes by measuring the amount of asialomucin
bound to probes using a peroxidase conjugate to the
I 'icia lectin.

We describe a relatively rapid enzyme-linked, lectin

74

CHFMORECEPTORS SENSITIZE CNIDOCVTES TO DISCHARGE SPIROCYSTS

75

sorbent assay (ELLSA) to determine the number of spi-
rocysts discharged onto test probes. Using the ELLSA,
we show that three classes of agonists sensitize spirocytes
to discharge their spirocysts in response to triggering me-
chanical stimuli. The dose-response curves of spirocyst
discharge to the agonists indicate that multiple popula-
tions of discharging spirocysts exist, each characterized
by different sensitivities to the agonists.

Materials and Methods

Sea anemone maintenance

Monoclonal sea anemones (Aiplasia pallida, Carolina
strain) were fed and maintained individually in glass
finger bowls containing natural seawater at 24 1C
as previously described (Thorington and Hessinger,
1988a).

Experimental animals and test solutions

Prior to each experiment, animals of the same size
were starved for 72 h and kept under defined conditions
and lighting (Thorington and Hessinger, 1 988a). Test so-
lutions of chemosensitizing agonists (N-acetylneur-
aminic acid, glycine, proline and histamine; Sigma, St.
Louis, Missouri) were prepared in natural, filtered (Type
1, Whatman) seawater adjusted to pH 7.6 with 1 N HC1
or NaOH. Animals were permitted to adapt to changes
of medium for 10 min before cnidocyte responsiveness
was measured.

Assays of cnidocyte responsiveness

Three methods were used to measure the discharge of
cnidae: (1) cnida-mediated adhesive force; (2) micro-
scopic enumeration of discharged microbasic p-mastigo-
phores and spirocysts; and (3) an indirect, solid-state en-
zyme-linked lectin sorbent assay (ELLSA) of discharged
spirocysts.

Cnida-mediated adhesive force. Cnida-mediated ad-
hesive force was measured as previously described
(Thorington and Hessinger, 1988a). In principle, this
technique involves using a small, gelatin-coated nylon
bead attached to a strain gauge via a stainless steal wire
shaft. The gel-coated bead is made to contact the tip of a
tentacle on an anemone in a finger bowl containing a
solution of chemosensitizing agent in seawater. The dis-
charge of cnidae initiated by contact of the probe with
the tentacle results in the tubules of the everting cnidae
either adhering to or penetrating the gelatin surface.
Withdrawing the probe from the tentacle causes the dis-
charged cnidae to exert an opposite and downward force
on the probe, which is measured from a gravimetrically
calibrated force-transducer connected to a strip-chart re-
corder. The adhesive force, measured in hybrid units of
mg-force (mgf), is the force required to break the cnida-

mediated attachment between the probe and the tenta-
cle. It is an aggregate measure of several contributions,
including the different kinds of discharged cnidae and
the inherent "stickiness" of the tentacle surface, and is
proportional to the total number of cnidae discharged
onto the probe (Geibel el ai. 1988).

Enumeration of discharged mastigophores and spiro-
cysts. Following the measurement of adhesive force, the
same gel-coated probes were used to visually count the
number of adhering mastigophore nematocysts by meth-
ods previously described (Geibel et ai, 1988).

Discharged spirocysts were visually counted by the
same procedures used for discharge mastigophores. Even
with phase contrast optics, however, fully discharged
spirocysts were extremely difficult to see and time-con-
suming to count. To expedite counting of discharged
spirocysts adhering to test probes, we developed a fast
and reliable micro-assay termed an ELLSA.

Indirect, solid-state enzyme-linked lectin sorbant assay
(ELLSA)

This assay for quantifying discharged spirocysts is
based upon the observation that the everted tubules of
discharged spirocysts bind conjugated N-acetylated sug-
ars with high affinity (Watson and Hessinger, in prep.).
In brief, the assay involves first dipping the gel-coated
tips of spirocyst-bearing probes into a solution of asialo-
mucin, then into a solution of I 'icia villosa lectin/peroxi-
dase conjugate, followed by colorimetric measurement
of bound peroxidase activity. Some of the N-acetylgalac-
tosaminyl residues on the asialomucin molecule bind to
the adhesive "glue" of the everted tubules while the re-
maining terminal sugars bind the lectin/peroxidase.

Buffers. The following buffers were prepared: Buffer A
(0.69 M NaCl and 0.25 M phosphate, pH 7.6); Buffer B
(0. 1 5 M NaCl and 0.0 1 A/ phosphate, pH 6.0 containing
0.02% Tween 20); Buffer C (0.15 M NaCl and 0.01 M
phosphate, pH 6.0); and Buffer D (0.5 M sodium citrate-
HC1 pH 5.3).

Asialomucin solution. Asialomucin (12 //g/ml; A-
0789, Sigma) in filtered seawater was divided into 10 ml
aliquots and stored frozen. For assays, a solution of asia-
lomucin (10.8 Mg/ml) was prepared by adding nine parts
of the stock solution to one part of Buffer D.

Leclin/enzyme conjugate. Horseradish peroxidase
conjugated to I 'icia villosa lectin (E-Y Laboratories, San
Mateo, California) was diluted to a final concentration
of 1.5 Mg/ m ' m Buffer A. Aliquots of lectin/enzyme con-
jugate were protected from light and frozen ( 20C) un-
til used. Mannose ( 50 mA/) was added immediately prior
to using the lectin conjugate to minimize nonspecific in-
teractions between the lectin and the gelatin on test
probes.

Enzyme substrate. Hydrogen peroxide (30%; Sigma)

76

G. U. THORINGTON AND D. A. HESSINGER

was daily diluted to 3% ( v/v) with distilled water and then
to 0.3% with Buffer C. The final substrate solution was
prepared immediately before use by adding 6 ml of 0.3%
HoO : to 0.05 ml of 1% o-dianisidine (Sigma) in meth-
anol.

Assay procedure. The wells of flat-bottomed, 96-well
microtiter plates (Dynatech) were each rinsed with 200
jul of Buffer B, emptied, and then air dried for 30 min.
Test probes were secured to a plastic holder that permit-
ted individual probes to be immersed in the contents of
separate wells without coming into contact with the sides
or bottom of the wells. All incubations were performed
at room temperature. Probes were incubated in the asia-
lomucin solution for 30 min, then rinsed by immersing
in individual wells containing Butter C for 2 min, and
finally air-dried for 5 min.

Mucin-treated probes were incubated in separate wells
containing 200 ^1 of lectin/enzyme conjugate for 60 min
in the dark. Following a 2-min rinse in Buffer C, they
were transferred to wells containing 200 n\ of enzyme
substrate where they were incubated for 60 min. Follow-
ing the incubation, the probes were removed and 50 ^1
40% sodium azide in Butter C was added to each of the
wells to stop peroxidase activity.

The absorbance of each well was measured at 492 nm
using a microtiter well spectrophotometer (Model EL
308, Biotek Instruments, Cambridge, Massachusetts).
The mean values of controls, consisting of gel-coated
probes, which had not been touched to sea anemone ten-
tacles, were subtracted from the values of individual ex-
perimental probes. Probes sputter-coated with gold for 4
min at 1 5 M using a Polaron E 5 100 sputter coaler and
then dipped into asialomucin (10.8 Mg/ml) in Buffer D
were used as external standards to assess reactivity of re-
agents and to normalize data from test probes to the stan-
dard curve, when necessary. The "gold" standard gave
absorbances of 0.07 (0.003 S.E.M.) O.D. at 492 nm.
For experimental probes, the absorbance at 492 nm is
linearly and directly proportional to the number of dis-
charged spirocysts. Measurements of absorbance are di-
rectly converted to the number of discharged adherent
spirocysts on a probe by extrapolation from the standard
curve.

Results

Optimal dilution of ELLSA reagents

Checkerboard titrations of asialomucin and of lectin
peroxidase were performed in microtiter plate wells. The
dilutions ranged from 1:3 to 1:81 for asialomucin and 1:
8 to 1:5832 for lectin peroxidase. Test probes were gold-
coated insect pins (See Materials and Methods) with
heads of 0.8 mm diameter. Negative controls were
probes treated with 0.01 M phosphate buffered saline,
pH 6.0. The dilutions chosen were those giving the great-

16

E '2

UJ

O

m
cc

8

m

NUMBER OF SPIROCYSTS X 10' 2

Figure I. Standard curve for ELLSA determination of discharged
spirocysts. The number of discharged spirocysts counted on test probes
is plotted against absorbance at 490 nm. Solid circles represent mean
values obtained from tentacles chemosensitized by N-acetylneur-
aminic acid and open circles from histamine-sensitized animals. Each
point is the mean of separate absorbancy readings (n = 24) and direct
countings (n = ll)(R = 0.99).

est difference between controls and experimentals. The
optimal dilution for asialomucin was 1:27, which is
equivalent to 10.8 ng/m\; and for the lectin peroxidase it
was 1.5

Standard cun-e

A standard curve was constructed by plotting visually
counted spirocysts per probe as a function of absorbance
at 490 nm. Visual counts of spirocysts discharged onto
probes from animals that were chemosensitized by vari-
ous concentrations of either NANA or histamine were
performed under phase contrast optics. Direct counts
and absorbancy readings were obtained using replicate
probes from the same animals. For absorbancy readings,
a total of four separate experiments were performed and
averaged. Each experiment consisted of six replicate
probes. A linear and direct relationship existed between
the absorbance and the visually counted discharged spi-
rocysts (Fig. 1).

Adhesive force measurements

Dose-response curves, expressing the mean adhesive
force for all tested chemosensitizers, are biphasic. The
curves for glycine, histamine, proline, and NANA ex-
hibit a sigmoidal region of sensitization at low concentra-
tions of sensitizer, a maximum response or effect (E max )
at higher concentrations (EC 100 ), and a region of appar-
ent desensitization occurring at still higher concentra-

CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS

Table I
Dose-responseparameters of agonist-sensitized cmida discharge and adhesive force measurements from MptasiapaSida tentacles

77

Spirocysts

Mastigophores

Adhesive force

Agonists

E

(no.)

EC.oo

Ko.5 (A

r)

E ma x (I

,0.)

EC- UK)

1

M) 5 ("

/)

E max (mgf) EC IOO Ko. 5 (M)

Glvcine

107

15

5 X 10-"

5.4 x 10 l2

160

24

10"

1.2 x

10 -"

0.23

8.7 0.7 10" 2.0 x 10 8 0.2

Histamine

12.0 0.9 2.7X1Q- 7 1.4X10 "0.1

Peak 1

138

18

2.7 X 10 *

9.7 x 10 "'

1.0

1 10

23

10-"

1.6 x

10'

Peak 2

159

23

2.7 X 10"

l.Ox 10 '

0.3

201

40

io- 7

1.9 X

10 *

0.3

Proline

10.7 1.0 10-" 3.6x10 "0.4

Peak 1

128

6

2.7 x 10 *

3.2 x 10 "

0.2

70

15

10 "

5.4 x

10 "

0.3

Peak 2

93

7

2.7 x 10"

1.7 X IO- 7

0.3

86

4

10"

5.0 x

io- 7

NANA

157

9

10 5

8.1 x

10"

0.5

14.0+1.0 l.SxlO 5 3.6xlO~ 7 0.5

Peak 1

233

15

io- 8

5.0 x IO- 9

Peak 2

396

9

io- 7

8.0 x to- 8

0.4

Peak 3

172

46

10~ s

3.2 x 10"

1.0

Emax (no.) represents the maximal number of cnidae discharged onto single test probes at optimal sensitization. ECioo is the molar concentration
of agonist producing a maximal effect. This value was obtained by visual inspection of dose-response curves. K<, 5 (M) represents the molar concen-
tration of agonist producing the half-maximal effects. E max (mgf) represents the maximal cnida-mediated adhesive force at optimal sensitization.
Both E,^ and Ko 5 values are determined from least-square double reciprocal plot of the sensitized region of the dose-response curve. Values
represent the response to agonists alone (/ c . controls subtracted) and are means standard error of the mean.

tions (Figs. 2A, 3 A, 4A, and 5 A, respectively). The dose-
response curves differ with regard to the specific dose-
response parameters (Table I): E max , the maximum
effect; Ko. 5 , the dose at which a half-maximum effect oc-
curs; and ECiou. the dose at which the maximum effect
occurs.

Dose-responses of mastigophore and spirocyst discharge

Glvcine. The dose-response curves representing the
discharge of mastigophores (Fig. 2B) and spirocysts ( Fig.
2C) to glycine are biphasic. The dose-response of the dis-
charge of spirocysts to glycine consists of a single modal
dose-response similar to that obtained from adhesive
force measurements and from the discharge of mastigo-
phores (Fig. 2A, B). However, there are significant
differences in the dose-responses of these two types of
cnidae. The response of spirocysts sensitized by glycine
is shifted significantly to the left of the glycine-sensitized
mastigophore response, indicating that responding spiro-
cytes are approximately 10,000 times more sensitive to
glycine than are the responding mastigophore-bearing
cnidocytes (Table I).

Before chemosensitization, the mean number of dis-
charged spirocysts on control probes was 116; after sensi-
tization, the number rose to 2 1 4. This is equivalent to an
average increase of 86%. Because insignificant spirocyst
discharge occurs at higher concentrations, and because
the dose-response for adhesive force and for discharged
mastigophores coincide, it appears that the discharged
mastigophores are the major contributors to glycine-in-
duced adhesive force.

Histamine. The dose-responses of discharging spiro-

cysts and mastigophores sensitized by histamine are bi-
modal, each displaying two biphasic peaks (Fig. 3) that
are complementary and non-overlapping. The two peaks
of discharging mastigophores each appear to be about
ten times more sensitive to histamine than the corre-
sponding two peaks of discharging spirocysts.

Proline. The dose-response curves of cnida discharge
to proline (Fig. 4) are similar to those obtained for hista-
mine. Both the mastigophore and spirocyst response pro-
files are bimodal, but unlike histamine, they are comple-
mentary and coincidental, rather than non-overlapping.
The discharge of spirocysts is less sensitive to proline
than to histamine (Table I).

N-acetylneuraminic acid (NANA). The pattern of dis-
charge elicited by NANA for spirocysts is trimodal, but
for mastigophores it is modal. This is in contrast to the
responses elicited by the tested "amino" agonists in
which agonist-induced patterns were similar for both
spirocysts and mastigophores. Each of the three biphasic
spirocyst responses is fairly narrow (Fig. 5C), in compari-
son to the mastigophore response (Fig. 5B), which spans
a range of NANA concentrations of five to six orders of
magnitude.

Effect of target hardness on retention of cnidae

To determine whether the hardness of the target con-
tributes to the number of cnidae retained on target
probes, we varied the concentrations of the gelatin used
(5-50%; w/v) to coat target probes. We sensitized all
anemones at !0~ 5 M NANA to assure that the number
of discharging cnidae remained constant. Thus, the
number of discharged cnidae retained on probes mea-

78

G. LI. THORINGTON AND D. A. HESSINGER

log Glycine Cone. (M)

Figure 2. Dose-responses of glycine on discharge of cnidae. A.
Effect of glycine on cnida-mediated adhesive force. Values express the
mean of four separate experiments. Each experiment consists of eight
replicate probes for each concentration: each probe and each tentacle
is used only once (n = 32). B. Effect of glycine on the number of dis-
charged mastigophores (n = 8). C. Effect of glycine on the number of
discharged spirocysts (n = 24). The number of spirocysts was deter-
mined by the ELLSA assay. Vertical bars represent the standard error
of the mean at 95% confidence limit.

showing maxima at 40% and steep declines at 50%. The
adhesion curve for spirocysts (Fig. 6B), on the other
hand, is sigmoidal, reaching a maximum at 30% and
then plateauing at harder coatings of gelatin.

At concentrations of gelatin below 20% the numbers
of retained mastigophores predominated by as much as
2.5-fold (Fig. 6A, D). Approximately equal numbers of
mastigophores and spirocysts were retained on probes

o

1
0.

g 2.0

*
o

1

E

sured the adhesion of the discharging cnidae to target
surfaces of differing degrees of hardness.

We find that the retention of discharged mastigo-
phores and spirocysts onto test probes of differing de-
grees of hardness is minimal at soft gelatin coatings of 5%
(Fig. 6A, B). The adhesion curves with respect to gelatin
concentration for retained mastigophores (Fig. 6 A) and
for adhesive force measurements (Fig. 6C) are biphasic.

log Histamine Cone. (M)

Figure 3. Dose-responses of histamine on discharge of cnidae. A.
Effect of histamine on cnida-mediated adhesive force. Values express
the mean of four experiments. Each experiment consists of eight repli-
cate probes for each concentration; each probe and each tentacle is used
only once (n = 32). B. Effect of histamine on the number of discharged
mastigophores (n = 8). C. Effect of histamine on the number of dis-
charged spirocysts (n = 24). The number of spirocysts was determined
by the ELLSA assay on Figure 2C. Vertical bars represent the standard
error of the mean at 95% confidence limit.

CHFMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS

79

45
2.0

o
01

2.5

log Proline Cone. (M)

Figure 4. Dose-responses of proline on discharge of cnidae. A.
Effect of proline on cnida-medialed adhesive force. Values express the
mean of four experiments. Each experiment consists of eight replicate
probes for each concentration; each probe and each tentacle is used
only once (n = 32). B. Effect of proline on the number of discharged
mastigophoresfn = 7).C. Effect of proline on the number of discharged
spirocysts (n = 24). The number ot spirocysts was determined as in
preceding figures. Vertical bars represent the standard error of the mean
at 95% confidence limit.

dae occur: the spirocysts, the microbasic p-mastigo-
phores, and the basitrichous isorhizas ( Hand, 1955). Re-
cently, using cnida-mediated measurements of adhesive
force in A. pallida. three different classes of chemorecep-
tors were identified that sensitize cnidocytes to discharge
their cnidae in response to triggering mechanical stimuli
(Thorington and Hessinger, 1988a, b). Although the dis-
charge of the microbasic p-mastigophores is under the

t

3

E
z

o

I

o

I

coated with 20, 30 and 40% gelatin (Fig. 6D). However,
the spirocysts predominated by about 3-fold at 50% gela-
tin (Fig. 6B, D).

Discussion

In the feeding tentacles of the sea anemone Aiptasia
pallida, as in all acontiate anemones, three types of cni-

loq NANA Cone. (M)

Figure 5. Dose-responses of N-acetylneuraminic acid (NANA) on
discharge of cnidae. A. Effect of NANA on cnida-mediated adhesive
force. Values express the mean of four experiments. Each experiment
consists of eight replicate probes for each concentration; each probe
and each tentacle is used only once (n = 32). B. Effect of NANA on the
number of discharged mastigophores (n = 1 1 ). C. Effect of NANA on
the number of discharged spirocysts (n = 24). The number of spirocysts
was determined as in preceding figures. Vertical bars represent the stan-
dard error of the mean at 95%> confidence limit.

80

G. U. THORINGTON AND D A. HESSINGER

03

LLJ
CC

O

Q.

o
g

co

o

o

X

o
o

CC
Q.
CO

O

Z

3.5

2.5

1.5

0.5

3.5

2.5

0.5

10' 5 M NANA
D CONTROL

A 1CT 5 M NANA
A CONTROL

10 5 M NANA
O CONTROL

10' 5 M NANA
CONTROL

80

70

60

D>

o

CC

o

LL
LLJ

CO
LLJ

I
O

50

40

CD

i

a

2.0

1.0

10

30

50

10

30

50

% GELATIN (wt/vol)

% GELATIN (wt/vol)

Figure 6. Dose-responses of retained discharged cnidae and of measured adhesive force using targets
coated with varying concentrations of gelatin. A. Effect of target hardness on the number of mastigophores
retained onto probes (n = 5). B. Effect of target hardness on the number of spirocysts retained onto probes
(n = 5). C. Effect of target hardness on the measured adhesive force (n = 5). D. Ratio of retained mastigo-
phores to retained spirocysts. All experiments were carried out either in 10 " M N-acetylneuraminic acid
or in seawater (controls). Data points are the mean standard error of the mean.

influence of at least two classes of sensitizing agonists,
namely glycine and N-acetylated sugars (Geibel et a/.,
1988), it is unknown whether such chemosensitizers.
along with a third class of sensitizers, typified by hetero-
cyclic amino compounds, also elicit similar responses
from spirocytes.

Spirocysts have been described ultrastructurally
(Mariscal and McLean, 1976; Mariscal et al., 1976,
1977), but few experimental studies have been per-
formed on spirocytes. The qualitative effects of remote
mechanical stimuli (Conklin and Mariscal, 1 976) and of
food extracts (Williams, 1968) on the discharge of spiro-

CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS

81

cysts have been reported. Until now, the local, chemical
control of spirocyst discharge and the purported primary
role of spirocysts in retaining captured prey has not been
quantitatively or experimentally verified. This lack of in-
formation is due in large part to the difficulty of detecting
discharged spirocysts because they possess a highly trans-
parent and non-refractile capsule. The counting of dis-
charged spirocysts by optical methods is further compli-
cated by the fact that the everted tubules entangle exten-
sively. While the visibility of the capsules of discharged
spirocysts is enhanced with phase contrast optics, the
counting of these cnida is, nonetheless, tedious and time-
consuming.

A rapid and sensitive assay of discharged spirocysts

To circumvent these problems, we developed a sensi-
tive indirect, solid-state, enzyme-linked lectin sorbant
assay (ELLSA) to detect discharged spirocysts. The assay
is highly reproducible and is significantly faster than vi-
sually counting discharged spirocysts using phase con-
trast optics. The potential applications of this procedure
include enumerating discharged spirocysts on experi-
mental targets as well as detecting and characterizing the
adhesive substance of spirocysts. In the present report we
use this assay to study the effects on spirocyst discharge
of two classes of substances known to sensitize the dis-
charge of mastigophores (Geibel el al., 1988), in addition
to a third class of sensitizer known to sensitize cnida-me-
diated adhesive force (Thorington and Hessinger,
1988b).

Sensitiiation ofspirocytes to discharge spirocysts

We have found that the three known classes of sensitiz-
ers as typified by glycine, NANA, and the heterocyclic
amino compounds, histamine and proline, all sensitize
spirocyst- and mastigophore-bearing CSCCs, albeit in
very different and specific ways. In spite of the variability
in sensitivity, magnitude, and pattern of spirocyte re-
sponsiveness induced by these agonists, each of the dose-
response profiles consists of one or more biphasic peaks.
Each biphasic peak reveals a region of sensitization
reaching a maximal effect (E max ), followed by a region of
desensitization at higher concentrations. The dose-re-
sponse parameters (Table I) indicate that the discharge
of spirocysts is most sensitive to glycine, followed by his-
tamine, proline, and then NANA, while the discharge of
mastigophores is most sensitive to histamine, followed
by proline, NANA, and glycine. The differences in the
sensitivity ofspirocytes and nematocytes to glycine were
the most pronounced.

In addition to differences in sensitivity to agonists, the
dose-response patterns also exhibited differences. In con-
trast to the modal (i.e. biphasic) dose-responses exhibited
by measurements of adhesive force (Thorington and

Hessinger, 1988a, b; Geibel el al.. 1988; Figs. 2A, 3 A,
4A, 5A), we observe that dose-responses of the discharge
of spirocysts to glycine is modal, while the dose-re-
sponses to proline and histamine are both bimodal, and
the response to NANA is trimodal. These contrast to the
dose-responses of discharging mastigophores, which for
glycine and NANA are modal, while for proline and his-
tamine are bimodal. Although the dose-responses of
mastigophore and spirocyst discharge are not coinciden-
tal for any of the tested agonists, except possibly proline,
the fact that all of the adhesive force dose-response
curves are coincidental with those of the mastigophores
implies that discharging mastigophores contribute sig-
nificantly more to adhesive force than do discharging
spirocysts.

Are all of the receptors effecting multimodal responses
(i.e.. NANA) associated directly with the cnidocytes or
possibly located on remote sites where they exert indirect
control over cnidocyte responsiveness, such as via the
nervous system or by initiating changes in behavior that
affect the availability of cnidae to discharge? By using
mucin-labelled colloidal gold, we find that 99.4% of the
labelled gold binds to supporting cells adjacent to spiro-
cytes and nematocytes (Watson and Hessinger, 1988),
while no label binds to tentacle sensory cells. We con-
clude that the receptors to the multimodal agonist,
NANA, are entirely located on supporting cells of CSCCs
and not on remote sensory sites.

A salient feature of modal dose-responses is that the
response is "turned off" at concentrations of agonist ex-
ceeding those needed to evoke a maximum response.
Where multimodal responses are exhibited, high concen-
trations of agonist turn off the response of CSCCs having
dose-response maxima below that concentration. The
existence of bimodal and, particularly, trimodal dose-re-
sponses provides for discharge of cnidae over a wide
range of agonist concentrations while ensuring that only
a portion of the available CSCCs are sensitized at any
one time and dose. Thus, the total number of discharging
cnidae never reaches the total number present. This
effectively conserves cnidae by preventing both excessive
discharge against living prey and nonproductive dis-
charge against killed prey.

Multiple populations ofcnidocyte/supporting cell
complexes (CSCCs)

The display of bimodal and trimodal dose-responses
implies the existence of multiple populations of spiro-
cytes distinguished by different sensitivities (i.e., KO 5 val-
ues) to a given agonist. That multiple populations of
CSCCs exist is indicated by the fact that there are CSCCs,
termed type C CSCCs, that discharge their cnidae in re-
sponse to tactile stimuli in the absence of added agonist
(Figs. 2, 3, 4, 5), in addition to CSCCs, termed type B

82

G. U. THORINGTON AND D. A. HESSINGER

CSCCs, that require chemosensitization by agonists be-
fore they can be triggered to discharge by static (i.e., non-
vibrating) targets. Furthermore, mastigophore-bearing
CSCCs triggered by targets vibrating at specific frequen-
cies (Watson and Hessinger, 1989) are termed type A
CSCCs. Although we do not yet know if vibration-sensi-
tive, spirocyst-bearing type A CSCCs exist, there obvi-
ously exist different populations of spirocyst- and nema-
tocyst-containing CSCCs distinguished by differences in
their sensitivities and specificities to agonists and by the
ways they are triggered by mechanical stimuli to dis-
charge their cnidae.

the primary contributors to measured adhesive force. On
the other hand, when targets are too hard for the dis-
charging mastigophores to penetrate, then the spirocysts
predominate as the retained cnida and, collectively, they
provide the major contribution to adhesive force. Thus,
the correlation between measured adhesive force and the
number of discharging mastigophores on both dose-re-
sponsive curves and on adhesion curves suggests that dis-
charged mastigophores contribute significantly more to
adhesive force than do discharged spirocysts under con-
ditions in which the target is penetrable to discharging
mastigophores.

Roles of discharged mastigophores and spirocysts
in the capture of prey

In the light of our current findings, the commonly ac-
cepted roles of the spirocysts and the mastigophores in
the capture and adherence of prey must be re-evaluated
and modified. We consider these matters from the per-
spectives of two questions addressed by this report: (i)
which of the two kinds of discharged cnida contribute
most to cnida-mediated adhesive force; and (ii) which
physical types of target retain the two kinds of cnida.

To adequately address the first question, we must rec-
ognize that the cnida-mediated components of adhesive
force measurements reflect both the number and the
kinds of cnidae discharging onto targets. A quantitative
analysis of the contributions and magnitudes of these in-
dividual factors to adhesive force is beyond the scope of
the present discussion, but we can make preliminary
qualitative assessments based upon the findings pre-
sented here. We have seen for several agonists that the
dose-responses for adhesive force measurements and for
the number of discharged mastigophores coincide and
more closely resemble each other than do the dose-re-
sponses for discharging spirocysts ( Figs. 2-5 ). This is also
seen by comparing EC 100 values for the discharge of cni-
dae with those for adhesive force (Table I). To assess the
second question, we performed measurements of adhe-
sive force and cnidae discharge in which the hardness of
the gelatin-coating on the target probes was varied. With
gelatin coatings below 20%, the number of discharged
mastigophores retained on the target probes predomi-
nated over spirocysts (Fig. 6A, D), presumably because
proportionally fewer discharging spirocysts can adhere
to the "softer" targets. Equal and maximal numbers of
mastigophores and spirocysts are retained on probes
coated with 20, 30, and 40% gelatin (Fig. 6D). Above
40% gelatin, however, the spirocysts predominate (Fig.
6B. D), presumably because mastigophores are incapa-
ble of penetrating these "harder" targets. Thus, when the
targets are too soft for the discharging spirocysts to ad-
here, the penetrant nematocysts predominate as the kind
of cnida retained on the target and, collectively, they are

Conclusions

In this paper, we show that the discharge of spirocysts
is chemosensitized by the same agonists that sensitize the
discharge of mastigophores. That is not to say that there
may not also exist agonists that sensitize only the dis-
charge of mastigophores or of spirocysts. However, the
dose-responses of these two kinds of cnidae differ both
qualitatively and quantitatively.

The dose-responses of discharging mastigophores are
either modal (e.g.. to glycine and NANA) or bimodal
(e.g.. to proline and histamine), and are coincidental to
dose-responses obtained from measuring adhesive force
under the same conditions. We have presented strong ev-
idence that the similarity between the dose-response
curves of adhesive force measurements and the discharge
of mastigophores is due to the discharged mastigophores
contributing significantly more to cnida-mediated ad-
herence onto 30% gelatin-coated targets than the dis-
charged spirocysts.

It seems appropriate, therefore, to modify the pur-
ported roles of penetrant microbasic p-mastigophores
and adhesive spirocysts in the capture of prey. Spirocysts
have been generally regarded as the primary means by
which adhesion of prey to the tentacle occurs (Williams,
1968; Doumenc, 1971; McFarlane and Shelton, 1975;
Mariscal, 1984). Mastigophores have been regarded ^
primarily penetrating and envenomating prey while, by
implication, not contributing significantly to prey adhe-
sion unless the tubules wrap around bristles or projec-
tions on prey (Mariscal, 1984). Our findings, however,
indicate that mastigophores play a significant, and some-
times primary, role in the adhesion of prey, depending
most likely upon the hardness of the prey surface. In-
deed, it appears that mastigophores and spirocysts may
be complimentary in their relative contributions to prey
adhesion so that the contribution of mastigophores to
adhesive force predominates with soft-surfaced targets,
which they can penetrate. The contribution of spiroi , ^
to adhesive force predominates when the target surface
is hard enough for the spirocysts to adhere and too hard
for the mastigophores to penetrate. Thus, in addition to

CHEMORECEPTORS SENSITIZE CNIDOCYTES TO DISCHARGE SPIROCYSTS

83

penetrating and immobilizing prey, discharging mastigo-
phores contribute significantly, even predominantly, to
the adhesion of prey, provided they are able to penetrate
the surface of the prey.

Acknowledgments

Funded in part by NSF grant DCB-8609859 to D.A.H.

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CONTENTS

DEVELOPMENT AND REPRODUCTION

Bentley, M. G., S. Clark, and A. A. Pacey

The role of arachidonic acid and eicosatrienoic acids
in the activation of spermatozoa in Arenicola manna
L. (Annelida: Polychaeta) .

Martin, VickiJ.

Development of nerve cells in hydrozoan planulae:
III. Some interstitial cells traverse the ganglionic
pathway in the endoderm

Sicard, Raymond E., and Mary F. Lombard

Putative immunological influence upon amphibian
forelimb regeneration. II. Effectsof x-irradiation on
regeneration and allograft rejection

ECOLOGY AND EVOLUTION

Smith, David A., and W. D. Russell-Hunter

Correlation of abnormal radular secretion with tis-
sue degrowth during stress periods in Helisoma tn-
volvis (Pulmonata, Basommatophora) .... 25

GENERAL BIOLOGY

Hose, Jo Ellen, Gary G. Martin, and Alison Sue
Gerard

A decapod hemocyte classification scheme integra-
ing morphology, cytochemistry, and function

PHYSIOLOGY

33

deFur, Peter L., Charlotte P. Mangum, and John E.
Reese

Respiratory responses of the blue crab Callinectes
sapidus to long-term hypoxia 46

Jakobsen, Per Ploug, and Peter Suhr-Jessen

The horseshoe crab Tachypleus tridentatus has two
kinds of hemocytes: granulocytes and plasmatocytes

Siebenaller, Joseph F., and Thomas F. Murray
A, adenosine receptor modulation of adenylyl cy-
clase of a deep-living teleost fish, Antimora rostrata . .

Thorington, Glyne U., and David A. Hessinger
Control of cnida discharge: III. Spirocysts are regu-
lated by three classes of chemoreceptors 74

55

65

Volume 178

THE

Number 2

BIOLOGICAL
BULLETIN

APR 2 5 1990

APRIL, 1990

Published by the Marine Biological Laboratory

THE

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PUBLISHED BY
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APR 2 5 1990

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The Sperm Transfer System in Kinbergonuphis simoni

(Polychaeta:Onuphidae)

HWEY-LIAN HSIEH 1 AND JOSEPH L. SIMON

Department of Biology, University of South Florida, Tampa. Florida 33620

Abstract. Tube dwelling Kinbergonuphis simoni (San-
tos, Day and Rice) achieves a 98.9% fertilization effi-
ciency by means of a sperm transfer system involving
spermatophores and seminal receptacles. The spermato-
phores are mushroom-shaped structures released as
clumps. The seminal receptacles are paired sac-like or-
gans embedded in the dorsal epidermis of female genital
segments. Males release spermatophores into the envi-
ronment, and females pick them up with their ventral
palps and first pair of parapodia. Stored sperm remain
viable for fertilization for at least one month. Spermato-
phore release and egg laying are independent of the pres-
ence of the opposite sex. Advantages associated with this
system are discussed, and include asynchronous repro-
duction, a long breeding season, reduced sperm loss, and
reduced exposure to risks. This sperm transfer mode is
the first reported in the family Onuphidae and is pro-
posed for other small, tube-dwelling onuphids.

Introduction

Sperm transfer in polychaetes occurs in two main
modes: non-aggregate transfer, in which sperm are free
swimming and not packed together before reaching eggs;
and aggregate transfer, in which sperm are packed to-
gether by varying complex structures before reaching
eggs.

Of the non-aggregate transfer modes, three different
types have been recorded: broadcast spawning (Clark,
1961; Schroeder and Hermans, 1975), copulation (Just.
1914; Gray, 1969; Schroeder and Hermans, 1975; West-
heide, 1984), and pseudocopulation (Reish, 1957; Petti-
bone, 1963; Daly, 1973). Three types of aggregate trans-

Received 17 October 1989: accepted 29 January 1990.
' Present address: Institute of Zoology, Academia Sinica, Taipei, Tai-
wan, 11529R. O. C.

fer have been recognized: indirect hypodermic impreg-
nation, free transfer of spermatophores, and free transfer
of spermatozeugmata. Among these three types, sperma-
tozeugmata transfer (Austin, 1963; Eckelbarger, 1974)
has not been elucidated with certainty, and will not be
discussed further here.

In hypodermic impregnation, males actively place
spermatophores on the body surface of females. Sperm
may then be collected into seminal receptacles, or may
penetrate through the epidermis into the coelom of fe-
males (Ax, 1968;Jouin, 1970; Westheide, 1984). In free
transfer, the spermatophores are released into the envi-
ronment and later picked up by females. Seminal recep-
tacles are often noted. Free spermatophore transfer has
been well demonstrated in members of the spionid genus
Polydora (Rice, 1978a, 1987a), and has been strongly
suggested to occur in serpulids and sabellids (Daly and
Golding, 1977; Picard, 1980). The members of these
three Families are tube dwellers.

Life history characteristics and habitat choice have
been considered strong selective forces for the mode of
sperm transfer (Rice, 1978a; Clark, 1981; Mann, 1984;
Westheide, 1984). For example, sessile or tube dwelling
life styles limit direct bodily contact, or decrease the mo-
bility of individuals so that encounters between sexes
are infrequent or impossible; thus, neither copulation,
pseudocopulation, nor indirect hypodermic impregna-
tion would be favored. Broadcast spawning or free trans-
fer of spermatophores may be the only alternative for
such species. However, broadcast spawning requires
large numbers of gametes and synchronous reproduction
in the population. The disadvantages of broadcast
spawning have been reported (e.g., in corals, Harrison et
ai. 1984;Shlesingerand Loya, 1985; and in sea urchins,
Pennington, 1985). In contrast, free spermatophore
transfer with sperm storage, as found in the spionid Poly-

85

86

H. HSIEH AND J. L. SIMON

tiora. has been proposed as an efficient low risk mode of
sexual reproduction (Rice, 1978a). Liberation of sper-
matophores into the sea also has been considered as an
adaptive character in sessile tubicolous pogonophorans
(Fliigel, 1977) and vermetid gastropods (Hadneld and
Hopper, 1980). Recently, a high efficiency of fertilization
has been recorded in bivalves with similar free spermato-
phore transfer ( 6 Foighil, 1985).

Reproduction in the Onuphidae has been reviewed in
general, and developmental patterns have been studied
in a few species (Blake, 1975; Fauchald, 1983; Paxton,
1986; Hsieh and Simon, 1987). However, no studies
have been done on sperm transfer modes in this group.
The characteristics of life style and life history of Kinhcr-
gonnpliis simoni are similar to those of many Po/ydora
species. Both are dioecious tube dwellers and are small in
size. Females produce few, large yolky eggs, brood their
young in the tubes, and have an extended breeding sea-
son (Rice, 1978a, b; Hsieh and Simon, 1987; Hsieh and
Simon, unpub. data).

The goals of this study are to address: ( 1 ) the mode of
sperm transfer in Kinbergonuphis simoni: (2) the fertil-
ization efficiency of this mode; and (3) the possibility that
the mode represents convergent evolution between spio-
nids and onuphids.

Materials and Methods

Seminal receptacles

Worms were collected from an intertidal sandy flat in
Upper Tampa Bay, Florida, and brought alive into the
laboratory in January 1985. The presence of seminal re-
ceptacles was determined as follows: Two treatments
control and isolation were set up. Five replicates were
used as controls. In each replicate, a pair of male and
female worms were reared in a plastic dish surrounded
by mesh cloth to keep adults and juveniles from escap-
ing. In the isolation experiment, seven females were sep-
arately incubated in the same way. Three of the seven
females were brooding when experiments began. The fe-
males were tapped out of their tubes and thus separated
from their young. These young were at embryonic or seg-
mented stages. Seawater, which had been sealed in jars
for four months, was filtered through Whatman No. 1
filter paper before being added to the aquaria. Salinity
and temperature were maintained at 22%o and 20C, re-
spectively. The presence of larvae and juveniles was
noted at one- or two-week intervals. This study was con-
ducted for three months.

Spermatophores

Mature worms were collected in March 1988 to deter-
mine the occurrence of spermatophores. In the labora-

tory, males were reared in mesh-enclosed dishes with and
without females. Salinity was kept at 22-24%, and tem-
perature at 20-22C. Observations on behavior and sper-
matophore production were made at intervals of 2 to 3
h during daylight hours for three weeks. In all laboratory
experiments, the worms were fed ground alfafa.

Seminal receptacles and spermatophores: morphology

Mature females collected in May 1985, were prepared
for paraffin sections after being fixed in Bouin's fixative.
Subsequently, they were cut into 7- to 10-jjm sections
and stained in Ehrlich's hematoxylin and eosin (Knud-
sen, 1966). Spermatophores were prepared for SEM
studies following the procedures of Hsieh and Simon
(1987).

Fertilization efficiency

Worms were collected at the study site monthly in
1982, and from June to October in 1985, to determine
the fertilization efficiency. Worms were relaxed in 0. 1 5%
propylene phenoxytol and fixed in 10% formalin in the
field. Broods were examined in the laboratory. Unfertil-
ized eggs could be recognized by a white coloration and
a clear space appearing at one end (Fig. 1). Only the
broods at early developmental stages (blastula to 5-set-
iger stages) were used to avoid underestimating the num-
ber of unfertilized eggs due to disintegration. Fertiliza-
tion efficiency was expressed as the percentage of eggs
fertilized of all eggs spawned.

Results

Seminal receptacles

Table I shows that, over three months, paired females
produced one to three normally developing broods. Iso-
lated females also laid eggs, suggesting that spawning was
not induced by the presence of males. In some isolated
females (No. 3, 4, and 6), eggs were present in maternal
tubes, but no development was observed. In four of the
seven isolated females, each produced only one viable
brood, indicating that females did store sperm, but that
the amount was insufficient for subsequent broods. In
isolated females, to 7 juveniles were produced, while in
control pairs 9 to 64 offspring were produced (Table I).

Spermatophores

Spermatophores were released from male tube open-
ings as clumps, which stuck to the bottom of the culture
dishes or to pieces of debris. Occasionally, in clean dishes
where no food particles were present, spermatophores
were trapped in the water surface film. Freshly released
spermatophores were white, almost transparent, very

POLYCHAETE SPERM TRANSFER
Table I

( 'out/wrist i ol breeding sum's* between isolated /enuile.\ anil paired male* and females of Kinbergonuphis simoni reareil
in the laboratory from January to March,

87

Date (1985)

Number of

Number of

viable broods

juveniles

Treatment Jan 27 Feb 3

10 17

24 Mar 3 9

produced

produced

Control pairs

1 J. 3

*

* * J.6. *

2

9

2 J.27 *

* J.4

* * J, 28

3

64

3

* *

J, 17

1

17

4 J. 18 *

* J, 16.*

* * J.8

3

42

5 J. 5 *

* *

J.7

2

12

Isolated females

1 J.2

1

2

2

* *

J,4

1

4

3

* *

*

4 *

* *

*

5 brooder J. 3

1

3

6 brooder

* *

*

7 brooder J. 7

1

7

Larvae or eggs present in maternal tubes; J = juveniles present in dishes: numerals following J = brood sizes.

sticky, and easily broken when handled. Motile sperm
were observed inside the intact spermatophores. The
thin mucous sheets to which the spermatophores were
attached were quickly broken down by ciliates or rotifers;
however, the spermatophores themselves remained in-
tact for more than 48 h.

Spermatophores produced at one time by individual
males could form more than one clump. The number of
spermatophores in each clump varied, ranging from 33
to 160 (mean 1 S.E. = 84.90 12.66, n = 10). The
average number of spermatophores produced by an indi-
vidual male at one time was 80.25 1 8.26 (n = 4). Sper-
matophores were present in all of the culture dishes, with
or without females, indicating that production of sper-
matophores was not influenced by the presence of fe-
males or other males.

Seminal receptacles and spermatophores: morphology

Seminal receptacles are found in the genital segments
of females, which run roughly from the 80th segment to
the 100th segment. They are located dorsal and posterior
to the nephridiopores, near the intersegmental junctions
(Figs. 2, 3). Seminal receptacles are paired, blind, sac-like
organs embedded in the body wall (Fig. 4). Each sac is
about 40 ^m long, and possesses a single 6 /urn wide open-
ing to the exterior. The wall of the sac is composed of
columnar cells, except at the blind end where cuboidal
cells predominate (Fig. 5). Some sacs are branched into
two to four lobes (Fig. 6), and the number of lobes varies
among and within females.

Each spermatophore is mushroom shaped, with a stalk
and a spherical portion (Figs. 7, 8) containing sperm.
Heads of individual spermatophores are about 40 ^m in
diameter, with the stalk about 135 ^m in length. The
spherical heads are covered by two layers, the outer char-
acterized by a granular appearance, and the inner one
with symmetrically arranged bands (Fig. 9a-c). Not ev-
ery spermatophore produced is equipped with both lay-
ers, some occasionally lacking the outer layer (see arrows
in Fig. 7). The sperm from broken spermatophores are
morphologically identical to mature sperm seen in the
coeloms of males (Fig. 10; also see Fig. 20c in Hsieh,
1984).

Transfer of spermatophores

Although the direct release of spermatophores from
male gonoducts was not observed, expulsion of sper-
matophores from the tube openings of males was ob-
served several times. When spermatophores were placed
around the tube openings of female worms, these fe-
males usually within one minute would extend their
anterior body portions out of the tubes, searching. Upon
locating the spermatophores, they would pick them up
with their first pair of parapodia and ventral palps, and
then immediately withdraw to their tubes. In one in-
stance, some spermatophores were literally carried out of
a female's tube by larvae when the female and larvae
were disturbed by routine observations. Upon examina-
tion under SEM, these spermatophores did not contain
the outer granular layer (see Fig. 9c), suggesting that fe-

88

H. HS1EH AND J. L. SIMON

.' A

*"* ifc/

Figure 1. Unfertilized eggs and a developing embryo within a tube of Kinbergonuphis sinioni Dl
= developing larva with 5 setigers; Ue = unfertilized eggs; Sg = sand grams.

Figure 2. Dorsal view (SEM) of a female A'm/vn,><>m</)/i/s \imoni showing the relative positions of
seminal receptacles (arrows), nephridiopores (Np) and intersegmental junctions (Ij). A = anterior end of
the worm; G = gill.

POLVCHAETE SPERM TRANSFER

89

males might manipulate spermatophores and mechani-
cally break down the layers.

Fertilization efficiency

Fifty-one broods and 982 spawned eggs were exam-
ined. Out of 982 eggs, only 1 1 were unfertilized; this rep-
resents a fertilization efficiency of 98.9%.

Discussion

Sperm transfer in Kinbergonuphis simoni involves
spermatophores and seminal receptacles. The seminal
receptacles are embedded in the dorsal body wall, with
external openings close to the nephridiopores. The ne-
phridiopores serve as gonopores, while the adults' tubes
serve as brood chambers. A similar spatial arrangement
has also been found in the spionids (Soderstrom, 1920;
Simon, 1967; Rice, 1987a), in the serpulids Spirorbis
spirorbis (L.) (Daly, 1978), and in the capitellids (Eckel-
barger and Grassle, 1987). A comparable situation also
occurs in the galeommatacean bivalve Mysella tumida,
where eggs are spawned, sperm are stored, and young are
brooded in the same place: the suprabranchial chamber
(O Foighil, 1985). The close proximity between openings
of the sperm storage organs, gonoducts and brood cham-
bers leads to a high fertilization efficiency. Efficiency has
been shown to be 98.9% in A", simoni (this study), 98.8%
in S. spirorbis (Da\y, 1978), 100% in Capite//a(Ecke\bar-
ger and Grassle, 1987), and 99.9% in a galeommatacean
bivalve (6 Foighil, 1985).

In Kinbergonuphis simoni the production of sper-
matophores can occur in the absence of females, and the
release of eggs also can occur without the presence of
males. Such phenomena have also been observed in spio-
nids (S. Rice, University of Tampa, pers. comm.). More-
over, in A. simoni, Polydora ligni, and Pseudopolydora
paucibranchiata (Myohara, 1980), females isolated from
males can continuously produce viable offspring until
the stored sperm are depleted. Two cases in A. simoni
(Hsieh, unpub. data) also showed that, after their male
mates had died, each remaining female (isolated from
other males) continued to produce 4 more viable broods

over periods of 30 to 40 days. These features reveal that
sperm release and egg laying can take place separately,
and that sperm are stored alive until needed. Thus, the
pressure of breeding synchrony required in broadcast
spawning is relieved. Consequently, asynchronous, pro-
longed, or even continuous reproduction can take place
in the population over time. This prediction is consistent
with the breeding pattern found in the field (Hsieh and
Simon, unpub. data). Asynchrony and extended breed-
ing may have advantages since the risk of reproductive
failure can be spread over several reproductive events,
and the chance that at least some offspring survive is
higher (Stearns, 1976).

Sperm loss due to dilution by seawater has been as-
sessed experimentally in broadcast spawning sea urchins
(Pennington, 1985). With successful spermatophore
transfer, females are provided with sperm packed in such
a localized dense form that sperm dilution does not oc-
cur. Furthermore, in Kinbergonuphis simoni, spermato-
phores can remain intact for one to two days after being
released into seawater. The reduction of sperm loss may
be one of the advantages of having spermatophores.

The morphology of spermatophores in polychaetes is
varied and species specific (e.g.. in the spionids, Rich-
ards. 1970;Greve, 1974; Rice, 1978a;andinanonuphid.
this study). Despite the morphological differences, the
method of spermatophore transfer in polychaetes ap-
pears similar. Spermatophores are released from the
male tubes and subsequently are picked up by females.
As proposed by Rice ( 1978a), this demonstrates that in-
dividuals do not have to leave their tubes or burrows for
sperm transfer, and implies that the risks of being ex-
posed to predation or disturbance are minimized when
compared to other alternatives (e.g.. copulation, pseudo-
copulation, or indirect hypodermic impregnation).

Table II summarizes the modes of sperm transfer
known in polychaetes. Both the spionids and the
onuphid have seminal receptacles in females, and sper-
matophores in males, for free transfer. In both families,
worms are tube dwellers, tend to aggregate, and have
similar life histories, suggesting similar selective pres-
sures. In the Onuphidae, the predominant developmen-
tal pattern is lecithotrophic. Small-sized onuphids brood

Figure 3. Sagittal section of a female Kinbergonuphis simoni showing the position of seminal recepta-
cles (arrows). A = anterior end of the worm; D = dorsal; V = ventral; Ve = vitellogenic egg; Se = septa;
Yo = young oocytes.

Figure 4. Cross section of a female Kinbergonuphis simoni showing paired seminal receptacles (ar-
rows). G = gut; V = ventral nerve cord.

Figure 5. Individual seminal receptacle in female Kinbergonuphis simoni showing stored sperm (S).
Co = columnar cell; Cu = cuboidal cell;O = opening of seminal receptacle.

Figure 6. Branched seminal receptacles in female Kinhergtmiiphis simoni. (a) Bi-lobed. (b) Four-lobed.
S = stored sperm.

90

H HSIEH AND J. L. SIMON

Figure 7. A clump of spermatophores produced by a male Kinbergonuphis xiinoni. A = a piece of
ground altafa: Sp = individual spermatophore: St = stalk of spermatophore; Arrows = spermatophores
without outer layers.

POLYCHAETE SPERM TRANSFER
Table II

Summary of sperm transfer modes in polychaete Families

91

Family

Adult life

Mode of sperm transfer

Reference

A) Non-aggregate transfer

Nereidae Eunicidae Benthic motile forms Broadcast spawning, mature individuals undergo swarming Clark, 1961; Schroeder and

Syllidae spawning with epitoke or stolon formation

Pisonidae Interstitial forms Copulation, females have seminal receptacles, males have

Saccocirridae copulatory organs

Nereidae Polynoidae Benthic motile forms Pseudocopulation with pair formation or aggregations

Phyllodotidae

Hermans, 1975
Gray. 1969; Westheide, 1984

Reish, 1957; Daly, 1973;
Pettibone. 1963

B) Aggregate transfer

Dinophilidae
Protodrilidae
Hesionidae

Arenicolidae

Spionidae
Onuphidae

Interstitial forms Hypodermic impregnation associated with spermatophores. Ax, 1968;Jouin, 1970;

seminal receptacles are present Westheide. 1984, 1988

Benthic hurrowers Free spermatophore transfer, the presence of seminal Okuda, 1946

receptacles is unknown

Benthic tube dwellers Free spermatophore transfer, seminal receptacles are present Soderstrom, 1920; Simon. 1967;

Rice, 1978a, 1987a

Benthic tube dwellers Free spermatophore transfer, seminal receptacles are present This study

C) Sperm transfer modes uncertain

Alciopidae

Syllidae

Terebellidae

Pectinariidae

Capitellidae

Pelagic forms

Benthic motile forms

Serpulidae Sabellidae Benthic tube dwellers

Copulation or hypodermic impregnation has been suggested;

naked sperm masses are embedded in epidermis; seminal

receptacles are present
Copulation has been suggested; spermatophore-like

structures are present in seminal receptacles
Benthic tube dwellers Pseudocopulation with pair formation or free transfer of

aggregated sperm has been suggested; males become errant

during spawning period; sperm morulae and free sperm

are produced; the presence of seminal receptacles is

unknown
Free transfer of aggregated sperm has been suggested; sperm

packets (spermatozeugmata) are produced; the presence of

seminal receptacles is unknown
Copulation, pseudocopulation or free spermatophore

transfer has been suggested; seminal receptacles are

present
Free spermatophore transfer has been suggested; seminal

receptacles are present

Benthic tube dwellers

Benthic burrowers or
tube dwellers

Pettibone, 1963; Rice, 1987b;
Rice and Eckelbarger, 1989

Goodrich, 1930
Eckelbarger, 1974

Austin, 1963, 1965

Hartman, 1947; Reish, 1974;
Eckelbarger and Grassle. 1987

Daly and Golding, 1977; Picard,
1980

larvae in maternal tubes (Fauchald, 1983; Hsieh and Si-
mon, 1987). These reproductive characteristics lead us
to predict that the sperm transfer mode seen in Kinbergo-
nuphis simoni and spionids may commonly operate in
other small onuphids as well. This proposal may also ex-
tend to other tube dwelling or burrowing polychaetes, if
they share similar characters with onuphids and spionids
in their life histories. Certainly, extensive studies cover-

ing tube dwellers and burrowers from different taxa are
needed to test this hypothesis.

Most spionid spermatophores are morphologically
adapted for flotation or enhanced suspension in the wa-
ter (Rice. 1978a). In contrast, spermatophores of Ki/ihcr-
gonuphis simoni are sticky and often deposited on the
bottom of culture dishes, or tangled with objects such
as detritus or food particles, suggesting that the dispersal

Figure 8. Individual spermatophore of Kinbergonuphis simoni. H = head portion; St = stalk.

Figure 9. Covering layers of spermatophores in Kinbergonuphis simoni. (a) Outer layer with granular
appearance, (b) Inner layer exposed after the outer layer being partially eroded. O = outer layer, (c) Inner
layer showing symmetrical bands.

Figure 10. A broken spermatophore of Kinbergonuphis xiinoni showing escaped sperm (S).

92

H. HSIEH AND J. L. SIMON

ability of spermatophores is low. As a result, the ex-
change of spermatophores between individuals in distant
populations is greatly reduced. Any changes in the tim-
ing of release, quantity and quality of spermatophores,
or in the processes of spermatophore transfer and sperm
storage, may alter the efficiency of fertilization. In turn,
different methods of fertilization can serve as a barrier
between populations and act as a reproductive isolation
mechanism. In A", simoni, preliminary analyses of isoen-
zymes and morphometric measurements of populations
from Tampa Bay, Ft. Myers, and the Indian River indi-
cate that these populations differ (K. Fauchald, Smith-
sonian Institution, pers. comm.), suggesting that some
form of divergence has taken place. Comparative studies
of sperm transfer modes and sperm storage mechanisms
among distantly distributed populations would be useful.

Acknowledgments

We thank Drs. B. Cowell, E. McCoy, S. Bell, and F.
Fried! for their advice during this study. We also thank
Drs. S. Rice, K. Fauchald, C.-P. Chen, and two anony-
mous reviewers for their discussions and comments. Spe-
cial thanks are due C.-P. Chen for his help in preparing
histological sections.

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Rice, S. A. 1987a. Sperm storage organs in spionid polychaetes, im-
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POLYCHAETE SPERM TRANSFER

93

Rice, S. A. I987b. Reproductive biology, systematics and evolution
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Rice, S. A., and K. J. Eckelbarger. 1989. An ultrastructural investiga-
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Schroedcr, P. O., and C. O. Hermans. 1975. Annelida: Polychaeta.
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Soderstrom, A. 1920. Studien uber die polychaetenfamilie Spioni-
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Weslheide, \V. 1984. The concept of reproduction in polychaetes
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Reference: Biol. Bull 178: 94-100. (April. 1990)

Structure and Function of a Special Tissue in the

Female Genital Ducts of the Chinese

Freshwater Crab Eriocheir sinensis

TAI-HUNG LEE* AND FUMIO YAMAZAKI

Laboratory of Embryology and Genetics, Faculty of Fisheries,
Hokkaido University, Hakodate, Hokkaido 041, Japan

Abstract. The histological anatomy of the genital ducts
of adult females of Eriocheir sinensis was studied before
and after copulation, and during and after egg-laying. A
strongly basophilic valve-like tissue was discovered at the
junction of the spermatheca and the oviduct. This tissue
prevents communication between the spermatheca and
the oviduct except during oviposition. At this time, it
functions as a valve, allowing ripe eggs out of the oviduct
and preventing sperm from entering the oviduct during
and after egg-laying. These findings suggest that the ac-
tual site of gamete contact in E. sinensis is within the
spermatheca, instead of in the lumen of the ovary or in
the oviduct. The presence of the valve-like tissue assures
that the ripe eggs collected from the ovary during egg-
laying are unfertilized. This observation is of great im-
portance for obtaining unfertilized ripe eggs in studies
of artificial fertilization (//; vitro) and hybridization. The
valve-like tissue has not been described in other brachy-
urans, and this genital duct should be classified as new
for the Brachyura.

Introduction

The goal of this study was to define the actual site of
fertilization in the Chinese freshwater crab. Eriocheir si-
nensis, in preparation for artificial fertilization (//; vitro).
This crab is widely distributed in fresh and brackish wa-
ters in southeastern China and has great economic value
in the country.

The female reproductive system of the Brachyura,
with the exception of two superfamilies, consists of a se-
ries of ducts leading from the ovary to the exterior of the

Received 15 May 1989; accepted 19 January 1990.
* To whom all correspondence should he addressed.

animal. These ducts are composed of four regions: ovi-
duct, spermatheca, vagina, and vulva (Hartnoll, 1968).
During copulation, the male transfers its spermato-
phores into the spermatheca of the female; therefore, fer-
tilization in the Brachyura is generally accepted as being
internal. But what is the actual site of this fertilization?
And what is meant by "internal fertilization" in the Bra-
chyura? These two questions have not yet been answered
conclusively.

Early reports were contradictory. Binford (1913) sug-
gested that in Menippe mercenaria, fertilization occurred
in the lumen of the ovary, because spermatozoa were
found on the surface of the ripe eggs, and many could
develop into embryos. Spalding (1942), Cheung (1966),
and Goudeau ( 1982) reported that fertilization of Card-
mix maenas occurs in the lumen of the ovary or within
the oviduct. On the other hand, studies ofPortunus san-
giiinolentus (by Ryan, 1 967 ) and Libinia emarginata (by
Hinsch, 1971) suggested that the spermatozoan contacts
the membrane of the ripe egg internally, and that the re-
maining processes in fertilization occur outside the body of
the female. This suggestion agrees with that of Yonge ( 1 937).

In E. sinensis, we found a valve-like tissue within the
spermathecal wall which is connected to the oviduct. Ex-
cept during oviposition, this valve-like tissue prevents
communication between the oviduct and the sperma-
theca. During egg-laying, the tissue functions as a valve,
freeing eggs and preventing spermatozoa from entering
the oviduct. Thus, we will comment on the expression
"internal fertilization" as it pertains to E. sinensis and
other brachyurans.

Materials and Methods

Specimens of the Chinese freshwater crab, Eriocheir
sinensis. were obtained from Yang Qin Lake in Jiangsu

94

GENITAL DUCT TISSUE IN E SINENSIS

95

mu

op-

Fif>urv I. Schematic illustration of the longitudinal section of the
genital ducts of the female Eriocheir sinensis. Abbreviations: (co) co-
lumnar epithelium; (en) endocuticle; (ex) exocuticle; (ep) epicuticle;
(In) hinge; (in) inner wall; (mu) muscles; (op) operculum; (ou) outer
wall; (ova) ovary; (ovi) oviduct; (spa) spermatheca; (vag) vagina; (val)
valve-like tissue; (vu) vulva. Bar = 1 mm.

Province, China, in November 1986 during the crab's
spawning migration season. The maximum carapace
widths of the specimens used in this study were 7-8 cm.
Some specimens were used immediately after they ar-
rived at the laboratory (Faculty of Fisheries, Hokkaido
University, Hakodate, Japan); these were at the germinal
vesicle stage. The remaining specimens were maintained
for about two months in individual compartments of a
well-aerated, closed circulating seawater system (28%o S,
20C). Female genital ducts, in various stages before
and after copulation, during egg-laying, and one day and
four days after egg-laying were excised carefully and fixed
in Bouin's solution for histological observations. Sections
(8-10 jmi) were made by the standard paraffin method and
stained with Delafield's haematoxylin and eosin.

Results

Structure of the female genital ducts and the
valve-like tix\ne

In adults of Eriocheir sinensis, we found that the fe-
male genital ducts have four regions: oviduct, sperma-
theca, vagina, and vulva (Fig. 1 ).

The oviduct, a short, tube-like passage connecting the
ovary and the spermatheca, is about 4. 1 mm long. The
opening of the oviduct leading to the spermatheca is on
the epithelium of the spermatheca, just above the outer
wall of the vagina. The undulant wall of the oviduct is
composed of a columnar epithelium. The cavity of the
oviduct is full of basophilic material in colloidal form.

The spermatheca is ovoid, about 19 mm high and 8

mm wide. It consists of a single crumpled layer of colum-
nar epithelium; its cavity is filled with a basophilic colloi-
dal substance. The portion of the spermathecal cavity
nearest the vagina sharply tapers downward, its narrow
end continuous with the cavity of the vagina. The vagina,
about 3 mm long, is formed by two cuticular walls; one
face (inner wall) is invaginated into a concavity of the
other (outer wall) (Fig. 2). Muscles run diagonally from
the inner wall to the sternum. The opening of the vagina
(i.e., the vulva) is on the sternite of the sixth thoracic seg-
ment. The vulva is characterized by the presence of an
operculum the continuation of the inner wall of the va-
gina. The operculum can be opened and closed by con-
tracting and relaxing the muscles of the inner wall during
copulation or egg-laying.

A strongly basophilic tissue (hereafter referred to as the
valve-like tissue) can be found at the opening of the sper-
matheca leading to the oviduct (Fig. 1 ; Fig. 3A); it is about
1 . 1 mm high and 0.4 mm wide. Because the border of the
valve-like tissue is connected to the epithelium of the open-
ing, it prevents communication between the oviduct and
the spermatheca. The tissue is composed of a mass of cells
in which no nuclear division is observed; thus, it appears to
originate from epithelial cells somewhere nearby.

Under light microscopy, the tissue appeared to be a
syncytium because no cell membranes were observed.
Most of the nuclei are nearly oval, with a long axis of
about 5.6 ^m and a short axis of about 3.5 urn; the direc-
tions of the long axes are random (Fig. 3B). In contrast,
the nuclei in the middle of this tissue are somewhat con-
densed and spindle-shaped, with a long axis of about 7.3
jum and a short axis of about 2.0 ^m; all the long axes of
these nuclei point toward the cavity of the spermatheca
(Fig. 3C). The nuclei within the middle part of that sur-
face of the tissue facing the spermathecal cavity appeared
to be pycnotic (Fig. 3D). This observation suggests that
old nuclei are displaced through the middle part of the
tissue into the cavity of the spermatheca.

ou

en

vag

Figure 2. Schematic illustration of a transverse section of the va-
gina showing its concave shape. Abbreviations: (en) endocuticle; (ep)
epicuticle; (in) inner wall; (mu) muscles; (ou) outer wall; (vag) vagina.

96

T. LEE AND F. YAMAZAKI

' B

^^HHHH|
:>" ^

Figure 3. Transverse sections from the middle of the valve-like tissue and oviduct before copulation. A:
Whole figure of the valve-like tissue. B: Most of the nuclei of the tissue are oval and the directions of long axes
are at random. C: The nuclei in the middle of the tissue are somewhat condensed and spindle-shaped, all the
long axes of these nuclei point toward the cavity of the spermatheca. D: The nuclei within the middle part of
that surface of the tissue facing the spermathecal cavity appear to he pycnotic. Abbreviations: (co) columnar
epithelium; (ovi) oviduct; (spa) spermatheca: (val) valve-like tissue. Bar (A) = 200 pm. Bar (B, CD) = 20 fim.

Structural changes in the valve-like tissue
and the oviduct

Five hours after copulation. Spermatophores and free
spermatozoa introduced during copulation swell the
spermatheca to about three times its pre-copulatory size.
The valve-like tissue is a bit flattened due to the pressure
of the seminal fluid (Fig. 4A). Besides these, no other
changes in the oviduct were observed.

During egg-laying. Changes occur in both the oviduct
and the valve-like tissue. The undulant surface of the ovi-
duct straightens, and its circumference expands to some

degree. The valve-like tissue is perforated by the ex-
truded eggs in the middle with its broken parts prolonged
toward the cavity of the spermatheca (Fig. 4B, C, Fig.
5A). Figures 4D and E and 5B show that near the end of
egg-laying, eggs are extruded continually. The split parts
of the tissue are closely attached to each other when there
are no eggs passing through.

One day after egg-laying. A new thin layer of valve-
like tissue appears on the border that is connected to the
epithelium of the spermatheca. The split parts of the
valve-like tissue have already fused together. However,
no nuclear-division is observed in this tissue. The old tis-

$$:$&'&:.

GENITAL DUCT TISSUE IN E. SINENSIS

iiiiiiiiiiiiii ' mmam

97

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Figure 4. Transverse sections from the valve-like tissue and oviduct showing the structural changes in
different stages. A: Five hours after copulation. B and C: During egg-laying (from the same spermatheca).
D and E: Near the end of egg-laying (from the same spermatheca). F: One day after egg-laying. G: Four
days after egg-laying. Abbreviations: (eg) egg; (mu) muscles; (nt) new valve-like tissue; (ot) old valve-like
tissue; (ovi) oviduct; (sp) spermatozoa; (spa) spermatheca; (val) valve-like tissue. Bar (A, C, E, F, G) = 200
Mm. Bar(B, D)

98

T. LEE AND F. YAMAZAKI

OVI

CO

sp

-Fig.4B ovi
-Fig.4C

Fig.4D

Fig.4E

Figure 5. Schematic illustration of the longitudinal sections of the valve-like tissue. A: Based on obser-
vation of continuous transverse sections of the same spermatheca as shown in Figure 4B and C. B: Based
on observation of continuous transverse sections of the same spermatheca as shown in Figure 4D and E.
Abbreviations: (co) columnar epithelium; (eg) egg; (ovi) oviduct; (sp) spermatozoa; (spa) spermatheca; (val)
valve-like tissue.

sue is being discharged into the cavity of the sperma-
theca. The surface of the oviduct changes again from be-
ing straight to undulant (Fig. 4F).

Four days after egg-laying. The old valve-like tissue is
almost discharged and a new valve-like tissue is formed
(Fig. 4G).

No spermatozoa were found inside the oviduct or the
ovary during the different stages discussed above. More-
over, none of the ripe eggs removed from the egg-laying
ovary developed into embryos.

Discussion

In the present study, no spermatozoa were found in
either the oviduct or the ovary in Erioclieir sinensis be-
fore, during, or after egg-laying. Moreover, the ripe eggs
removed from the egg-laying ovary did not develop into
embryos. Therefore, the spermatozoa in the sperma-
theca never entered the oviduct or ovary. This phenome-
non can be explained by the presence of the valve-like
tissue. This tissue not only prevents the sperm from en-
tering the oviduct or the ovary before egg-laying, but it
also functions as a valve. It allows ripe eggs out of the
oviduct during egg-laying and prevents the sperm from
entering the oviduct, both near the end of egg-laying and
after egg-laying, by closing once the positive pressure of
the seminal fluid acts upon it ( Fig. 6 ). Therefore, the only
site where the eggs and sperm come into contact is in the
spermatheca.

In E. sinensis, egg-laying continues for approximately
1 5-30 min. About 300,000-500,000 eggs or more can be
found in one brood. The capacity of the spermatheca is
no more than 100 eggs, and the opening of the vagina
will only allow the passage of two or three eggs at one
time. Thus, we estimate that the time an egg takes from
entering the spermatheca to release from the vagina is no

more than 1 s (unpub. data). Accordingly, there is only
enough time for the sperm to attach to or penetrate the
surface of the outer membrane of the ripe egg in the sper-
matheca, so the remaining events of fertilization must
then occur externally. This conclusion is similar to the
suggestions made by Yonge (1937), Ryan (1967), and
Hinsch(l971).

Because fertilization is a series of phenomena that gen-
erally involves the contact of sperm and egg, penetration,
and karyogamy, the term "internal fertilization" is ap-
parently not appropriate forE. sinensis. However, in this
study, we could not determine whether any interaction
(e.g., acrosome reaction) occurred between the sperm
and the egg within the spermatheca. If such an interac-
tion does occur, then fertilization in this crab cannot be
external. On the other hand, if the sperm simply attaches
to the egg membrane and has no further interaction with
it within the spermatheca, then the term "external fertil-
ization" is applicable. Hence, further research is required
to determine whether this crab performs "external fertil-
ization."

In early studies of the brachyurans by Binford (1913;
Menippe nicrcencina), Ryan (1967; Port nuns sanguino-
lentns), and Hartnoll (1968; Carcinus maenas, Ifyas
coarctatus and Hyas araneus), the structure of the ovi-
duct and its opening into the spermatheca were de-
scribed as being similar to one another. Unlike E. si-
nensis. the oviducts of these crabs do not function as a
passage from the ovary into the cavity of the sperma-
theca; rather, they are simply a convoluted cord of cells
with a blind end extended toward the stratified epithe-
lium of the spermatheca, except during ovulation (Fig.
7A). Some time before either ovulation or egg-laying,
this cord of cells forms a passage between the ovary and
the cavity of the spermatheca; but no valve-like tissue
preventing the sperm from entering the oviduct was ob-
served (Fig. 7B).

GENITAL DUCT TISSUE IN E SINENS1S

99

OVI

CO

val

spa

spa

A B

Figure 6. Schematic illustration of the transverse sections of the valve-like tissue showing the function
of freeing eggs and preventing sperm from entering the oviduct. A: Valve-like tissue opens when the eggs
are extruded. B: Valve-like tissue closes when the positive pressure of the seminal fluid acts upon it. Abbre-
viations: (co) columnar epithelium; (eg) egg; (ovi) oviduct; (spa) spermatheca; (val) valve-like tissue. Arrows
indicate the directions of positive pressure.

Our view of the relation between the structural
changes in the oviduct and the site of fertilization differs
from those of previous investigators. Ryan ( 1967) found
a small amount of sperm in the open oviduct of P. san-
guinolentus. With no further explanation, he concluded
that the spermatheca was the site of sperm-egg contact,
and that the rest of the fertilization process occurred ex-
ternally. He thought that there was insufficient time for
the sperm to penetrate the egg within the body of the
female crab. Diesel (1989) also reported sperm-egg con-
tact within the spermatheca of /. phalangium, but did
not report whether any sperm were present in the oviduct
or ovary immediately before or after spawning. In studies
of M. mercenaria (by Binford, 1913), and C. macnas (by
Spalding, 1942; Cheung, 1966; Goudeau, 1982), the au-
thors believed that fertilization occurred in the lumen of
the ovary or within the oviduct because: ( 1 ) sperm were
found in the lumen of the ovary; and (2) some of the eggs
removed from that ovary could develop into embryos.
We cannot deny that sperm might be naturally pressed
into the oviduct and the lumen of the ovary when the
blind-ended oviduct opens. However, the evidence cited

ovi

se

Figure 7. Schematic illustration of the transverse sections of the
oviduct and spermatheca described in early studies by Binford (1913),
Ryan (1967). and Hartnoll (1968). A: Non-spawning stage. B: Some
time before or during ovulation and egg-laying. Abbreviations: (ovi)
oviduct: (se) stratified epithelium; (spa) spermatheca.

by Binford (1913) and other investigators is too weak to
support their conclusions. During their dissection and
removal, the genital organs may have experienced nega-
tive pressure inside the ovary, drawing the sperm into the
oviduct or the ovary. This artificial phenomenon might
have misled investigators. This may also account for the
presence of the sperm in the oviduct of P. sanguinolentus
(by Ryan, 1967). Further experiments are needed to clar-
ify the site of fertilization in the crabs that have no appa-
ratus to prevent sperm from entering the oviduct.

There are two known types of oviducal openings into
spermatheca: one is that reported by Binford (1913),
Ryan (1967), and Hartnoll (1968), and the other is the
one we describe in the present study. Besides E. sinensis,
we also found the same valve-like tissue and patent ovi-
duct in Eriocheir japonicus and Hemigrapsus sangui-
neus (unpub. data). In Pachygrapsus crassipes, Chiba
and Honma (1971) discovered an oviduct of the same
structure; unfortunately, they did not mention whether
there was a valve-like tissue. What does the distribution
of these two types of openings in the Brachyura mean?
What is their taxonomic significance? Does the valve-like
tissue have functions other than preventing sperm from
entering the oviduct? From where are the cells that re-
build the split valve-like tissue?

The presence of the valve-like tissue in crabs helps en-
sure that the ripe eggs removed from the egg-laying ovary
are all unfertilized. This finding is of importance for ob-
taining unfertilized ripe eggs in studies of artificial fertil-
ization (in vitro) (Lee and Yamazaki, 1989) in E. si-
nensis. Furthermore, crabs with this valve-like tissue
would be good laboratory animals for studies on fertiliza-
tion, hybridization, and embryology in the Brachyura.

Acknowledgments

The authors thank Dr. Akira Goto of Laboratory of
Embryology and Genetics, Faculty of Fisheries at Hok-

100

T. LEE AND F. YAMAZAK1

kaido University, for his constructive criticism, and John
Goodier for reading the manuscript. We also thank our
friends Chun-Min Liu and Yaichiro Kamataki for their
assistance in collecting the specimens, and Paul Endo for
his helpful suggestions. Finally we wish to express our
hearty thanks to Mrs. Enid Mok Lee for her continuous
assistance.

Literature Cited

Binford, R. 1913. The germ-cells and the process of fertilization in
the crab, Menippe mercenaria. J Morpli 24: 147-201.

Cheung, T. S. 1966. The development of egg-membranes and egg at-
tachment in the shore crab, Carciiiu.^ luacnas. and some related
decapods./ Mar. Biol .-l.vvoc. V K 46: 373-400.

Chiba, A., and Y. llonma. 1971. Studies on gonad maturity in some
marine invertebrates II. Structure of the reproductive organ of the
lined shore crab. A'/'/)/'"" .VH/MW Gakkaishi. 37: 699-706. (in Japa-
nese, with English abstract)

Diesel, R. 1989. Structure and function of the reproductive system of
the symbiotic spider crab Imitinis plialanxiwn (Decapoda: Maji-

dae): observations on sperm transfer, sperm storage, and spawning.
J. Crust. Biol. 9:266-277.

Goudeau, M. 1982. Fertilization in a crab: I. Early event in the ovary,
and cytological aspects of the acrosome reaction and gamete con-
tacts. 7V.v.vwCV//14:97-ll 1.

Hartnoll, R. G. 1968. Morphology of the genital ducts in female
crabs. / Limn. Soc. (Zool). 47: 279-300.

Hinsch.G. \V. 1971. Penetration of the oocyte envelope by spermato-
zoa in the spider crab. J L'llrastrucl. Res 35: 86-97.

Lee, T. II.. and F. Yamazaki. 1989. Cytological observations on the
fertilization in the Chinese freshwater crab Erioclieir .v/wmv.v by
artificial insemination (in vitro) and incubation. Aquaculture 76:
347_36().

Ryan, K. P. 1967. The structure and function of the reproductive sys-
tem of the crab. Purtunus saiiguinolentu.i(Hert>sl) (Brachyura: Por-
tunidae). II. The female system. Pmc. Symp. Crustacea, Mar liiol
,-l.v.sw , India. Jan. 12-15. 1965. Ernakulam. Pt II: 522-544.

Spalding, J. F. 1942. The nature and formation of the spermatophore
and sperm plug in Carcumt nuicnas. Q J Microsc. Sci. 83: 399-
422.

e, C . M. 1937. The nature and significance of the membranes
surrounding the developing eggs Homarus vulgaris and other Deca-
poda. Pruc. tool. Sue. Loud. A. 107: 499-5 1 7.

Reference: Biol. Bui! 178: 101-1 10. (April, I WO)

Sperm Attachment and Acrosome Reaction

on the Egg Surface of the Polychaete,

Tylorrhynchus heterochaetus*

MASANORI SATO 2 AND KENZI OSANAI

Marine Biological Station, Toliokit University, Asarnitshi, Aomori, 039-34, Japan

Abstract. Sperm binding to the egg envelope (chorion)
was examined in fixed eggs and isolated chorions of the
polychaete, Tylorrhynchus heterochaetus. Sperm bind-
ing included two successive steps: attachment (acroso-
mal outer surface-chorion binding) before the acrosome
reaction and adhesion (acrosomal process-chorion bind-
ing) after the acrosome reaction. The attachment be-
tween sperm head-tip and the outermost layer of the cho-
rion was observed in Ca-free seawater, in which the acro-
some reaction did not occur. The surface of the chorion
was stained with phosphotungstic acid (PTA). Sperm did
not attach to pronase-treated eggs, in which the PTA-
positive layer disappeared. When isolated chorions were
soaked in distilled water for several hours, they lost the
capacity for sperm attachment, and the PTA-positive
layer thinned. The acrosome reaction was induced by
material that was dissolved from the chorions into dis-
tilled water. This suggests that both the receptor for
sperm attachment and the inducer of the acrosome reac-
tion are involved in the PTA-positive layer.

Introduction

In many animals, ripe unfertilized eggs have one or
more extracellular coats (envelopes). During fertiliza-
tion, egg envelopes play a key role in sperm binding, in
the induction of the sperm acrosome reaction, and in the
exclusion of supernumerary sperm (see Epel and Vac-

Received 16 December 1988; accepted 18 December 1989.

1 Contribution No. 558 from the Marine Biological Station. Tohoku
University.

2 Present address: Department of Biology. Faculty of Science, Kago-
shima University, Korimoto, Kagoshima 890, Japan.

quier, 1978;Lopo, 1983;MonroyandRosati, 1983;Jaffe
and Gould, 1985).

Previous studies on sperm-egg binding have suggested
that two types of binding exist (see Epel and Vacquier,
1 978): ( 1 ) binding between the outer surface of unreacted
sperm heads and egg envelopes before the acrosome re-
action (referred to as attachment in the present paper)
in mice (Saling and Storey, 1979; Bleil and Wassarman,
1983; Wassarman et a/., 1985; Soldani and Rosati,
1987), ascidians (DeSantis et a/., 1980; Rosati, 1985), a
horseshoe crab (Brown, 1976; Barnum and Brown,
1983), polychaetes (Anderson and Eckberg, 1983; Osa-
nai, 1983; Sato and Osanai, 1983, 1986), an abalone
(Lewis et ai, 1982) and a sea urchin (Aketa, 1973, 1975);
and (2) binding between acrosomal processes of reacted
sperm and egg envelopes after the acrosome reaction (re-
ferred to as adhesion in the present paper) in ascidians
(DeSantis et ai, 1980; Rosati, 1985), a horseshoe crab
(Brown, 1976), polychaetes (Osanai, 1983; Sato and Osa-
nai, 1983, 1986), sea urchins (Summers and Hylander,
1975; Vacquier, 1980), a sand dollar (Summers and Hy-
lander, 1974), bivalves (Hylander and Summers, 1977;
Brandriff et a!., 1978), and a crustacean (Clark et ai,
1981). Studying sperm-egg binding can be difficult be-
cause, during normal fertilization in most organisms, the
acrosome reaction usually follows sperm attachment too
quickly to be examined. Using phase-contrast micros-
copy, Osanai (1983) observed that sperm remained at-
tached to isolated egg envelopes (chorions) without the
acrosome reaction in Ca-free solution, and that sperm
adhered to the chorion with its acrosome reacted in Ca-
containing solution in the polychaete Tylorrhynchus het-
erochaetus.

In the present study, we use electron microscopy to

101

102

M. SATO AND K. OSANAI

b .

c

Figure I. Sperm binding to the fixed Tylorrhynchus eggs. Two
hours after insemination. 260. (a) Tylorrliynchu.i sperm were bound
to the egg in artificial ordinary seawater. (b) Tylurrliym'lius sperm were
bound to the egg in artificial Ca-free seawater. (c) Sperm of the sea star
I \icniM peel 1 11 1 tern were not bound to the egg in artificial ordinary sea-
water.

confirm these sperm bindings, and demonstrate that fac-
tors for the reception of initial sperm attachment and for
the induction of sperm acrosome reaction are distributed
in the outermost layer of the egg envelope all over the egg
surface.

Materials and Methods

Preparation oj gametes

Mature worms of the nereidid polychaete Tylorrhyn-
c/ius heterochaetus were collected in Natori, Miyagi Pre-
fecture, Japan. They were placed in 30% seawater (salin-
ity: about 10), and refrigerated at 0-5C. Gametes were
obtained by compressing or cutting the body with for-
ceps. Unfertilized eggs were washed several times in 30%
seawater; sperm were diluted in ordinary seawater (cf.
Osanai, 1978).

Experimental media

Natural seawater filtered with a paper filter (Toyo-
roshi No. 2) or Herbst's artificial seawater (ordinary and
Ca-free seawater) modified by Motomura (1938) were
used. Ca-free seawater was prepared by substituting
NaClforCaCl 2 .

Isolation ofchorion from eggs

Egg envelopes (chorions) were isolated from unfertil-
ized eggs as described by Osanai ( 1 976, 1 983). The unfer-
tilized eggs were suspended in 30% seawater and then
gently homogenized with a teflon homogenizer. The ho-
mogenate was centrifuged at 300 X g for 5 min. After
removing the supernatant, the sedimented chorions were
resuspended in fresh 30% seawater and centrifuged
again. Transparent chorions were obtained by repeating
this procedure several times.

Preparation of fixed eggs

The sperm-egg binding process was examined using
fixed unfertilized eggs as described in sea urchins by Kato
and Sugiyama (1978). The eggs were prefixed in 1%. glu-
taraldehyde in 30% seawater for 0.5-2 h. After rinsing in
30%' seawater several times, the eggs were inseminated.
In other cases, unfertilized eggs were pretreated with
0.1% pronase ( Kaken Chemical Co.) in 30% seawater for
20 min prior to prefixation.

Insemination

Tylorrhynchus eggs are fertilizable in media over a
wide range of salinity. In this study, sperm were added to
isolated chorions and fixed eggs in 30%- or 100%> seawa-
ter. When insemination occurred in Ca-free seawater,
the chorions and eggs had been rinsed several times in
Ca-free seawater, so that the concentration of contami-
nating Ca at insemination might be less than 1/1000 of
that in ordinary seawater. Egg or chorion suspensions ( 1 -
5 X 10 2 /ml) were inseminated with sperm suspension
(final concentration: 10 4 dilution of dry sperm, 5 X 10 6 -
1 X 10 7 /ml) at room temperature (10-20C).

SPERM-EGG BINDING IN TYLORRHYNCHUS

103

AV

Figure 2. Attachment ot'unreacted sperm to the fixed eggs in artificial Ca-free seawater. One minute
after insemination. X31.500. (a) A spermatozoon attached to the first layer ( I ) of chorion by its head-tip
without any acrosomal change. (2, 3. 4) The second, third, and fourth layer of the chorion, respectively.
AV: acrosomal vesicle, (b) A spermatozoon with its acrosomal vesicle open at the head-tip. The outer
membrane of sperm head was attached to the first layer of chorion.

Test ofacrosome reaction-inducing activity in material
dissolving from cliorions

Isolated chorions were placed in distilled water (DW)
(2% V/V) for 0.5-5 h. The chorion suspension was fil-
tered through filter paper. The filtrate was diluted with
ordinary seawater to 30% seawater and used as chorion
extract.

Sperm suspension was added to the chorion extract
(final sperm concentration: 1-5 X 10 7 /ml). The test solu-
tion was fixed with 1-2% glutaraldehyde 15-40 min

later. Sperm acrosome reaction was checked by phase-
contrast microscopy (X 1000).

Electron microscopy

Specimens were fixed in 2% glutaraldehyde in 70-90%
seawater for several days at 0-4C. After rinsing, they
were postfixed in 1% OsO 4 in 70-90% seawater for 1 h at
0-4C. The specimens were dehydrated in ethanol and
embedded in Epon 812. Thin sections were stained with
uranyl acetate and lead citrate or with 1 0% phosphotung-

104

M. SATO AND K. OSANAI

I

Figure 3. Adhesion of reacted sperm to the fixed eggs in artificial ordinary seawater (a, b, c). One
minute after insemination. The acrosomal process (AP) penetrated the first and second layers of chorion
and adhered to the third layer. The outer membrane of acrosome was in contact with the fibrous compo-
nent of the first laver of chorion (arrows). 35,000.

stic acid (PTA), and then examined with a transmission
electron microscope.

Results

Sperm binding to fixed eggs

In both ordinary seawater and Ca-free seawater, Tylor-
rhyncliux sperm were bound to the surface of fixed eggs
at their head-tip (Fig. la, b). When the fixed eggs were
inseminated with sperm of the sea star Asterina pectini-
fera and the sea urchin Strongylocentrotus nuclus, the
sperm were not bound to the eggs (Fig. Ic).

The chorion (1-1.5 ^m thick) consists of four layers
(Fig. 2; see also Sato and Osanai, 1 983). The first (outer-
most) layer is composed of a row of small packed spheres
wrapped in fibrous matter. The second layer is composed
of less electron-dense material. The third layer is a thin
electron-dense layer. The fourth layer (innermost and
thickest) is composed of densely packed material with
many cavities opening toward the inner surface.

The ultrastructure of the sperm-egg binding was exam-
ined with specimens fixed 1 min after insemination. In
Ca-free seawater, most sperm were attached to the first
layer of chorion by their head-tip without any acrosomal
change, though the acrosomal vesicle had opened in a
few sperm (Fig. 2). The opening of the acrosomal vesicle
was sometimes observed in free sperm suspended in sea-
water, and the usual morphological change associated

with a true acrosome reaction did not occur. Thus, open-
ing of the vesicle may be either a spontaneous phenome-
non or an artifact of fixation. In any case, the outer sur-
face of the sperm head-tip made contact with the fibrous
component of the first layer.

When live eggs were inseminated in Ca-free seawater,
sperm temporarily attached to the egg surface, but soon
detached. The sperm-egg attachment without a sperm
acrosome reaction seems to be less stable in live eggs than
in fixed eggs.

In ordinary seawater, most sperm bound to the cho-
rion underwent the acrosome reaction and formed a lob-
ular acrosomal process (Fig. 3). The acrosomal process
penetrated the first and second layers of the chorion and
adhered to the third layer. At the same time, the outer
membrane of the acrosome (the lateral side of the opened
acrosomal vesicle) continued to contact the fibrous com-
ponent of the first layer of the chorion. Some sperm that
had not undergone the acrosome reaction were attached
to the first layer of the chorion.

Sperm binding to isolated chorions

In intact unfertilized eggs, the first layer of the chorion
was stained by PTA (Fig. 4a, see also. Sato and Osanai,
1983). The outer surface of the isolated chorion (mor-
phologically similar to the third layer) was stained with
PTA (Fig. 4b), suggesting that the first and second layers
had been deformed or had collapsed onto the third layer.

SPERM-KGG BINDING IN TYLORRHYNCHVS

105

1234

Figure 4. (a) Ultrastructure of the surface of an intact unfertilized
egg. A section stained with phosphotungustic acid (PTA). The first layer
(1) of the chorion was stained intensively. (2, 3, 4) The second, third,
and fourth layer of the chorion, respectively. > 40.800. (b) Ultrastruc-
ture of the isolated chorion. A section stained with PTA. The outer
surface (arrow), which was morphologically similar to the third layer,
was stained. X45.600. (c) Sperm binding to the isolated chorions in
artificial ordinary seawater. Two minutes after insemination. X140.

Sperm were bound to the isolated chorion in both or-
dinary and Ca-free seawater, and the bindings of sperm
were kept for a long time (Fig. 4c). Because the isolated

Table I

Percentage of occurrence of acrosome reaction in \pcnn hound to
isolated chorion in presence or absence of Co?*

Percentage of acrosome reaction

Expt. No.

Ordinary seawater

Ca-free seawater

1

83.0

0.4

2

64.4

3

61.3

0.2

4

11.6

Isolated chonons were fixed 1-10 min after insemination. Occur-
rence of acrosome reaction was checked by phase contrast microscopy
in 200-300 spermatozoa on 3-6 chorions.

chorion was transparent, the acrosome reaction of the
attached sperm could be checked by phase-contrast mi-
croscopy. In ordinary seawater, many sperm underwent
the acrosome reaction (Table I, Fig. 5). These sperm were
also examined by electron microscopy. The lobular acro-
somal process adhered to the outer surface of the chorion
and did not penetrate it (Fig. 6a). In Ca-free seawater, the

KigureS. Phase contrast micrographs of sperm binding to the trans-
parent isolated chorions. x 1 900. (a) Control. An intact free-swimming
spermatozoon, (b, c) Spermatozoa undergoing acrosome reaction and
adhering to the chorion in artificial ordinary' seawater. Ten minutes
after insemination. Arrows indicate the development of acrosomal pro-
cess, (d) Spermatozoa attached to the chorion without acrosome reac-
tion in artificial Ca-free seawater. An arrow indicates an intact acroso-
mal vesicle. Ten minutes after insemination.

106

M. SATO AND K. OSANAI

Figure 6. Fleet ron mierographs of sperm binding to the isolated
chorion. One minute after insemination. 24.800. (a) A spermatozoon
adhering to the outer surface of chorion with the spread acrosomal pro-
cess (AP) in artificial ordinary seawater. (h) A spermatozoon attached
to the outer surface of the chorion without acrosome reaction in artifi-
cial Ca-free seawater. AV: Acrosomal vesicle.

sperm bound to the isolated chorion did not undergo the
acrosome reaction (Table I. Fig. 6b). The outer acroso-
mal membrane of sperm head-tip was attached to the
chorion surface. No spermatozoon was bound to the in-
ner surface of the chorion (the fourth layer) in both ordi-
nary and Ca-free seawater.

Effect of pronase treatment of eggs on sperm-egg binding

We tried to remove the egg-surface component that
binds sperm and induces the acrosome reaction. Unfer-
tilized eggs were pretreated with pronase and then fixed.
After rinsing, the eggs were inseminated in 30% seawater.

Sperm attachment was blocked or greatly reduced (Table
II, Fig. 7).

The pronase-treated eggs were examined by electron mi-
croscopy. The first and the second layers were removed from
the chorion in the pronase-treated eggs (Fig. 8). The outer
surface of the chorion did not stain with PTA.

Acrosome reaction-inducing activity in chorion extract

Chorion extracts were prepared by soaking chorions
in DW. Sperm were added to the chorion extract diluted
with natural seawater. Many of the free swimming sperm
underwent acrosome reaction (Fig. 9, Table III). The
sperm did not undergo acrosome reactions in control
media (30% seawater).

After isolated chorions were treated with DW, they
were mixed with sperm in 30% seawater. Few sperm were
bound to the chorions (Fig. 10). These chorions were ex-
amined by electron microscopy. The PTA-positive layer
at the outer surface of the chorions became thinner after
the DW-treatment (Fig. 1 1). No morphological change
was observed in other parts of chorion.

Discussion

Osanai (1983) used light microscopy to examine
sperm binding to the isolated chorion in Tylorrhyncluts
heteroclmetm. He showed that sperm binding includes
two steps: sperm attachment before the acrosome reac-
tion and sperm adhesion after the acrosome reaction. He
also showed that the progression from sperm attachment
to adhesion requires external calcium ions. We used elec-
tron and light microscopy to observe sperm-chorion
binding using fixed eggs and isolated chorions. Our re-
sults confirm the validity of Osanai's (1983) report.

Figure 7. Inhibition of sperm binding by pronase-pretreatment of eggs. The eggs were inseminated in
30% natural seawater after glutaraldehyde-fixation, and observed 10 min after insemination. X 140. (a) The
eggs pretreated with pronase for 20 min. Sperm did not bind to the eggs, (b) The eggs without pronase-
pretreatment. Many sperm hound to the eggs.

SPERM-EGG BINDING IN TYLORRHYNC/ll'S

Table 1 1
i 'I v/xvm binding by pronase-prelreatrnenl o/eggx

107

Eggs

No. of sperm hound on
the egg contour*

Pronase-treated
Untreated

3.20.7(n = 20)
66.5 3.6 (n = 12)

! Average SD (No. of eggs examined).

Sperm attachment and adhesion were demonstrated ul-
trastructually in Ca-free seawater and ordinary seawater,
respectively. Both were also photographed 1 min after
insemination during normal fertilization (Sato and Osa-
nai. 1983). However, in normal fertilization, the sperm
attachment step is rather inseparable, because it is fol-
lowed quickly by the acrosome reaction. We could sepa-
rate the sperm attachment step in Ca-free medium, in
which the acrosome reaction was prevented.

Sperm attachment occurred at only the head-tip of

Figure 9. Induction of acrosome reaction by the chorion extract.
*2, 100. (a) Unreacted sperm in control medium (30% seawater). (b. c)
Sperm undergoing acrosome reaction in the chonon extract.

conspecific sperm. This appears to be a species-specific
and site-specific reaction for the first sperm-egg recogni-
tion. Specific attachment between sperm and egg enve-
lope before the acrosome reaction is also known in an
ascidian (Rosati and De Santis, 1978) and in mammals
(Wassarman et al. 1985). Sperm attachment was inde-

Figure 8. infrastructure of the pronase-treated eggs. The eggs were
treated with pronase for 20 min. X24.000. (a) A section stained with
uranyl acetate and lead citrate. Most parts of the first and second layers
of the chorion disappeared with a few spherical components of the first
layer remaining on the surface (arrow), (b) A section stained with phos-
photungstic acid. The outer surface of the chorion was not stained as
compared with the untreated egg and the isolated chorion (Fig. 4).

Table III

osonii' reaction-inducing activity of the chorion extract

Percentage of

acrosome

Duration of

reaction*

incubation in

Expt. No.

distilled water (h)

Extract

Control**

1

0.5

82.4

2

1.5

3.7

2.7

3

5

77.4

1.9

4

5

19.1

7.6

* Occurrence of acrosome reaction was checked in 100- 1 50 sperma-
tozoa by phase contrast microscopy.
** 30% natural seawater.

108

M. SATO AND K. OSANAI

Figure 10. Decrease of sperm binding by soaking of the isolated
chorions in distilled water. The isolated chorions were inseminated in
30 <r f natural seawater just after preparation (a) or after distilled water-
treatment of the chorions for 90 min (3 times). Many sperm bound to
the chorion in the former (a), but not in the latter (b) 30 min after
insemination. 130.

(see Epel, 1978). Sperm aggregation can be induced even
in a medium of low Ca 2+ concentration, in which the
acrosome reaction is prevented (Dan. 1954). Aggrega-
tion-inducing and acrosome reaction-inducing factors
were separated from jelly of a starfish (Uno and Hoshi,
1978). Uno and Hoshi (1978) considered that sperm ag-
gregation might reflect the initial sperm-egg interaction,
i.e., the sperm attachment to the jelly surface.

In intact Tylorrhynchits eggs, the PTA-positive fibrous
substance coats the outer surface of chorion. Our results
show that both the receptor for sperm attachment and
the inducer for the acrosome reaction are associated with
the PTA-positive layer. It is unknown whether the com-
ponents of the PTA-positive layer function differently or
whether a single factor has both functions. Because the
PTA-positive layer corresponds to a periodic acid-Schiff
(PAS)-positive one observed by light microscopy (Sato
and Osanai, 1983) and is digested by pronase, it seems
to contain polysaccharide and protein. The glycoprotein
ZP3 of mouse zona pellucida is responsible for both
sperm attachment and the induction of the acrosome re-
action (Bleil and Wassarman, 1983). Other reports

pendent of external calcium ions in Tylorrhynchus het-
erochaetus, while it was calcium-dependent in mouse
eggs ( Sal ing i>/ a/.. 1 978; Saling and Storey. 1979; Soldani
and Rosati, 1987). Why sperm attachment to live Tylorr-
hynchus eggs is less stable than to fixed eggs or isolated
chorions, in Ca-free seawater, is unknown. The outer-
most layer of the chorion of live eggs may be less tightly
fastened to the chorion proper. Alternatively, attached
sperm may be detached by a factor secreted from live
eggs, as in normally fertilized eggs (Osanai, 1976). How-
ever, it is unknown whether unfertilized eggs secrete the
sperm-detaching factor in Ca-free solution.

The acrosome reaction in Tylorrhynchus heterochae-
tus evidently requires external calcium ions as in sea ur-
chins (Dan, 1954; Collins and Epel, 1977). The jelly, the
outermost layer of the sea urchin egg envelope, induces
the acrosome reaction and sperm aggregation behavior

Figure 1 1. Ultrastructural alteration of the isolated chorion by the
distilled-water treatment. Sections were stained with phosphotungstic
acid(PTA). -36.700. (a) An isolated chorion just after preparation, (b)
An isolated chorion after distilled-water treatment for 60 mm. The
PTA-positive layer at the outer surface of the chorion became thinner.

SPERM-EGG BINDING IN TYLORRHYM'I/LS

109

showed that a sugar or glycoprotein on an egg envelope
plays an important role in sperm attachment or induc-
tion of the acrosome reaction in the eggs of a mouse
(Shur and Hall, 1982a, b). an ascidian (Rosati and De-
Santis, 1980; Pinto ct a/.. 1981), a horseshoe crab (Bar-
num and Brown, 1983), a sea urchin (Segall and Len-
narz, 1979; Yoshida and Aketa, 1983), a seastar (Uno
and Hoshi. 1978), and a bivalve (Tumboh-Oeri and
Koide. 1982).

The acrosomal process of the fertilizing spermatozoon
fuses with a microvillus projecting from the egg through
the chorion in T. heterochaetus (Sato and Osanai, 1983).
However, no morphological difference of the PTA-posi-
tive layer was observed between regions around micro-
villi and the other regions. In contrast with T. hetero-
chaetus. the acrosome reaction-inducing activity was lo-
calized to a limited number of specialized sites above egg
microvilli in the nereidid polychaete Ncanlhes japonica
(Sato and Osanai, 1986).

Sperm seem to initiate the acrosome reaction just after
attaching to the chorion in the presence of Ca. Their
acrosomal processes usually penetrate the first and sec-
ond layers of the chorion and adhere to the third layer.
Such sperm can fuse with an egg microvillus to fertilize
the egg (Sato and Osanai, 1983). The initial attachment
between the sperm head-tip and the outermost layer of
chorion may be important for keeping the sperm ori-
ented for successful fertilization during the acrosome re-
action. Holding the sperm erect on the chorion surface
should lead to proper penetration and adhesion of the
acrosomal process.

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Reference: Biol. Hull 178: 111-117. (April. I WO)

A Photoperiod Determined Life-Cycle
in an Oligochaete Worm

BERND SCHIERWATER 1 AND CARL HAUENSCHILD

Zoologisches Institut der Technischen Universilaet, Pockelsstr. Wa,
3300 Braunschweig, West Germany

Abstract. For one common cosmopolitan naidid
worm, Stylaria lacustris, we studied the effects of differ-
ent environmental factors upon ( 1 ) the alternation of re-
productive modes, (2) the rates of population increase,
and (3) the combination of each of ( 1 ) and (2 ). While age,
temperature, population density, or rate of feeding did
not affect the mode of reproduction, photoperiod had a
dominant effect. Under long-day conditions (LD > 12:
12), all worms reproduced exclusively by paratomic fis-
sion, theoretically ad infinitum. When transferred to sh-
ort-day conditions (LD < 12:12) the worms ceased vege-
tative reproduction, and within 2 to 4 weeks developed
the hermaphroditic genital apparatus and a clitellum. Af-
ter an additional two weeks, the first cocoons were pro-
duced. The switch to the bisexual mode of reproduction
was cum grano sails irreversible. These findings are con-
sistent with observations of field samplings, and allow
one to predict the annual life-cycle strategy of S. lacus-
tris. This is the first example of a photoperiod deter-
mined life-cycle within the oligochaete worms.

The vegetative mode of reproduction led to extremely
high rates of population increase, whereas with the bisex-
ual mode of reproduction the number of individuals was
roughly stable. However, because 5. lacustris could not
withstand temperatures of 5C or lower, the switch to
sexual reproduction and the formation of diapausing co-
coons appear to be the only mechanism of overwinter-
ing. Nevertheless, some 'asexual' clones never switch to
sexual reproduction, whereas a loss of the asexual vegeta-
tive mode of reproduction did not occur. In contrast to
some general predictions from life-history theories, the
reproductive strategy of 5. lacustris is highly prepro-

Received 13 November 1989; accepted 2 January 1990.
1 Present address: Yale University. Department of Biology. P.O. Box
6666, New Haven, CT 065 1 1 .

grammed and cannot respond to sudden and unexpected
environmental changes.

Introduction

With the pollution of our environment, present day
ecology demands investigation of the biological mecha-
nisms that regulate the distribution and dynamics of
populations in a given environment (e.g., Brinkhurst and
Jamieson, 1971; McElhone, 1978; Brinkhurst and Cook,
1980; Tauber et al., 1986; Zaslwaski, 1988; Klerks and
Levington, 1989). The rapid increase in theories on the
evolution of life histories demands extended experimen-
tal work and empirical data (cf. Stearns, 1976, 1980; Rez-
nick, 1985; Hoekstra, 1987; Michod and Levin, 1988;
Hauenschild, 1989;Nunney, 1989).

The study of particular oligochaete worms can be
highly fruitful to our understanding of both the ecologi-
cal and the evolutionary implications of animal life-cy-
cles for two reasons: ( 1 ) the oligochaete worms in general
are regarded as perhaps the most important group con-
cerned with the retrieval of organic matter in freshwaters
(e.g., Brinkhurst and Jamieson, 1971; Dumnicka and
Pasternak, 1978; Brinkhurst and Cook, 1980). (2) Those
oligochaete worms that are capable of reproducing both
by a bisexual and by a vegetative mode of reproduction,
in particular the Aeolosomatidae and Naididae, allow in-
traspecific comparisons of the consequences of sexual vs.
asexual life-history tactics; such systems allowing experi-
mental work are badly needed but are difficult to find (cf.
Bell, 1980; Townsend and Calow, 1981; Calow, 1983;
Reznick, 1985; Hoekstra, 1987; Abugov, 1988; Schier-
water, 1989; Hadrys et al., 1990). Unfortunately, the
number of well understood life-cycles that include an 'al-
ternation of reproductive modes,' is surprisingly low (cf.,
Giese, 1959; Giese and Pearse, 1959; Kinne, 1970;
Brinkhurst and Cook, 1980; Townsend and Calow,
1981; Holm, 1988).

in

112

B. SCHIERWATER AND C. HAUENSCHILD

Records of the reproductive ecology of most oligo-
chaetes are limited to notes on the presence of sexually
mature specimens in field populations, but almost no
conclusions can be drawn from these scattered notes;
thus, little is known about the mechanisms affecting the
mode of reproduction and hence their annual life-cycles
(e.g., Vershinin and Semernoi, 1977; McElhone, 1978,
1982; Mill, 1978; Brinkhurst and Cook, 1980; Pascar-
Gluzman, 1981; Wetzel, 1982).

In this study we will present the annual life-cycle
model as well as quantitative data on the consequences
of sexual vs. asexual reproduction for the cosmopolitan
naidid Stylaria lacustris Linnaeus 1 758. Its life-cycle has
not been described, though growth rates of vegetative
worms have been studied (Streit, 1978; McElhone, 1982;
Finogenova, 1984) and several brief notes about sexual
worms are available (Kamlyuck and Kovaltchuk, 1972;
McElhone, 1978, 1982; Wetzel, 1982). McElhone (1982)
suggested that food supply, food quality, and water qual-
ity may affect the 'alternation of reproductive modes' in
S. lacustris. In this study we will demonstrate that the life
cycle of S. lacustris is strictly and exclusively determined
by the photoperiod (day-length). We shall discuss this
first finding of a photoperiod-determined life-cycle in an
oligochaete worm in an ecological context regarding the
evolution of the life-history strategy.

Materials and Methods

Animals

Stylaria lacustris is one of the most common and
widely distributed oligochaete species, found in Europe,
Asia, Africa, and North America (e.g., Brinkhurst and
Jamieson, 1971; Vershinin and Semernoi, 1977; McEl-
hone, 1978;Pascar-Gluzman, 1981; Wetzel, 1982). In 5.
lacustris, the vegetative mode of reproduction follows
the type of paratomic fission of animal chains of between
two to three individuals (Stephenson, 1 930). The biology
of sexual reproduction has not been described.

Worms were counted as 'sexual' if either gonads and/
or a clitellum were visible. All other worms were called
'vegetative' independent of the formation of tomits.

Field samples

All animal material of Stylaria lacustris was collected
from the field at different times and transferred into the
laboratory for culturing under defined laboratory condi-
tions. Worms were collected in W. Germany from a
pond at Weddel. Braunschweig, in July 1985, Sept. and
Oct. 1987, June and July 1988, and from the river II-
menau at Uelzen in August 1986, '87, '88. Right after
sampling, as many worms as possible were isolated and
checked within 24 h for their reproductive status (i.e.,

presence or absence of a clitellum, gonads, tomits) by
means of a dissection microscope at 20X.

Laboratory studies

Under defined laboratory conditions, we investigated
whether the following environmental factors influence
the 'alternation of reproductive modes': temperature,
feeding, population density, and photoperiod.

Culturing. Culture dishes (400 cm 3 'deep freeze' plastic
containers) were kept in thermoregulated rooms or in
chambers providing temperature constancy to 1C, as
controlled by mini-max-thermometers; normal photo-
period setting was LD = 16:8, unless otherwise stated. S.
lacustris was cultured either in filtered and heated (2 h at
80C) water of its natural environment, or in carbonic-
acid-free natural mineral water ('VitteF or 'Volvic').
Worms were fed on the green algae Haematococcus la-
custris and Goniuin sociale ad lib. The air bubbled cul-
ture dishes were washed, and water and food were re-
newed twice a week.

Acclimation time to any new experimental condition
was 24 h. The highest changes in temperature were 5C
per day. Temperature changes of 10C were done step-
wise within 4 days. For long-period observations on the
reproductive activity under different feeding, tempera-
ture, and photoperiod conditions, acclimation time was
14 days, unless otherwise stated.

Animals were observed through a binocular micro-
scope ('Zeiss' 475052-9901 ) with variable magnifications
from 8x to 50x. One ocular was equipped with a //m-
scale for in vivo measurements of one-dimensional dis-
tances.

Photoperiod settings. Experiments on the effects of
photoperiod on the mode of reproduction were run at 20
1 C, unless otherwise stated. The following LD settings
were used: LD = 6: 1 8, 1 2: 1 2, 1 6:8, 1 8:6, and 24:0. Light
intensities were 500-2000 Ix during light periods and
= 0.05 Ix in the dark, respectively. The light intensities
were measured with a lightmeter (Gossen, Mavolux 6C
18493), and because of the use of fluorescent lights (Os-
ram L40W/22-1), the light intensity values have to be
taken with care. During all experiments and observa-
tions on the effects of temperature, feeding rate, popula-
tion density and age. light-dark rhythm was held con-
stant at LD = 16:8.

Mean doubling times. For observations on mean dou-
bling times (mdt), 30 worms each were placed in plastic
chambers of =400 cm 3 , and the number of worms per
chamber was counted once a week. After each counting,
the total number of worms was reduced to a maximum
of 50 worms per chamber. Two populations (one from
Weddel pond and one from the river Ilmenau) were fol-
lowed over 8 weeks (2 weeks acclimation plus 6 weeks
of registration) at 10, 15, 20, and 25C. The mdt's were

PHOTOPERIOD1SM IN AN OL1GOCHAETE

113

u

T>

O

CL

03
L

X

(D

to

100

50

A) LD = 6ilB T=20 t

. B) LD = 6,18 T=20 C

A) LO = 12,12 T=20 t

* B) LO = 12,12 T=20 C

14 21 28

t I me (d)

35 42

Figure 1 . Examples for the time course of switching from the vege-
tative to the bisexual mode of reproduction induced by the photoperiod
in Sty/aria lacusiris. Vegetatively reproducing worms from the field
were exposed to short-day conditions of either LD = 1 2: 1 2 or LD = 6:
18 at day in the figure; A = Weddel 1985, B = Ilmenau 1986 popula-
tion; the points for LD = 16:8 are not shown, for all of them would
lie along the abscissa (= 0% sexual reproduction); N > 1000 for each
population.

calculated from the initial population size (N 1 ), the final
population size (N2) and the time (t) in days between the
countings: mdt = log 2 t/(log N2 - log N 1 ).

Statistics. The non-parametric Mann-Whitney-U test
(two-tailed) was used to compare the means of two inde-
pendent samples, and the Jonckheere test was used to
look for monotonous trends in three or more indepen-
dent samples (Lienert, 1976). The number of statistical
replicates (i.e.. number of cultures tested under the same
experimental conditions) is given as n in the text,
whereas N means the total number of worms checked for
their reproductive mode during one experiment.

The alternation of reproductive mode worked only in
one direction, i.e., only vegetative worms switched to
sexual reproduction. Once this switch had occurred, it
was cum grano salis irreversible. Ninety-one clitellate
worms had been transferred back from LD = 6: 1 8 to LD
= 16:8 (T = 20C) and watched for 42 continuous days.
Only three worms reverted to vegetative asexual repro-
duction by forming tomits; in these three worms the cli-
tellum of the parent individual was retained, whereas the
daughter individuals showed normal asexual shape.

Bisexual reproduction excluded fission, i.e., clitellate
worms never formed tomits. Therefore, once a popula-
tion became sexual, the number of worms per chamber
became constant or even declined slightly, because some
worms always died after changing the photoperiod. Un-
der laboratory conditions, the number of cocoons pro-
duced per sexual worm was low at all tested tempera-
tures. Rates of cocoon production ranged from 0.25 to
2.0 cocoons per sexual worm until death. The highest
rates of cocoons produced per sexual worm, within 3
weeks after the clitellum became visible, were 0.5 (T
= 15C), 0.7 (T = 20C), and 0.8 (T = 25C). Generally
the first cocoons were produced at 16.3 5.61 days
(range 7-3 1 days; N = 650, n = 13) after clitellum forma-
tion. Bisexual reproduction thus led to a very limited rate
of production of reproductive units. Cocoons were lem-
on-shaped and preferably placed in the corners of the
culture dishes. Mean SD cocoon length was 0.8 0.07
mm (n = 78), and each cocoon contained a maximum
of three embryos.

Asexual clones. Two culture populations of 5. lacustris
that came from the Weddel pond and had been cultured
exclusively vegetatively (T = 20, LD = 16:8) over 14 or
23 months, respectively, failed to show a photoperiodic

Results

Photoperiod

The mode of reproduction was strictly determined by
the photoperiod. Under long-day conditions (LD > 12:
12) the worms reproduced exclusively vegetatively by
paratomic fission. Exposure to short-day conditions ( LD
< 12) induced a quantitative switch from paratomic fis-
sion to sexual reproduction within 10 to 30 days (Fig.
1), i.e.. the hermaphroditic genital apparatus has been
developed. The formation of the clitellum always oc-
curred after the gonads became visible (in some cases up
to 25 days later). The reaction time to the short-day con-
dition, i.e.. for the switch in the mode of reproduction,
was affected by the temperature. One population was di-
vided into three portions and each portion cultured at
either 15, 20, or 25C. With increasing temperature the
time for switching from the vegetative to the sexual mode
of reproduction was significantly shortened (P < 0.001,
Jonckheere, N = 3 X 38; see Fig. 2).

c

O

O

L
D.

O

L

100

50

OOP D LO = 6,18 T=15 C

-.-. C) LO = 6.18 T=20 C

- C) LO = 6.18 T=25 C

4 21 28
t I ma (d)

35 42

Figure 2. Example of the effect of temperature on the time course
of switching from the vegetative to the bisexual mode of reproduction
in Sly/aria lacusiris; one vegetative population (C = Weddel 1988) of
1 14 worms was divided into 3 portions, and each group of 38 worms
was exposed to one of three different temperatures under short-day con-
ditions (LD = 6: 1 8) at day 0.

114

B. SCHIERWATER AND C. HAUENSCHILD

Table I

The mean doubling limes (nidi) ofvciu'lativcly reproducing Stylaria
lacustns at different experimental temperatures

range

T

mdt

ra

N

n

[d]

min

max

10

277

7

11.1 4.04

9.3

16.9

15

657

12

6.9 2.47

4.8

10.9

20

733

12

5.1 2.83

2.7

13.2

25

854

12

5.0 3.49

2.5

14.4

The means SD and the ranges over a 6-week observation period of
each two cultures are given. At 10C, one culture died after the first
week of observation period, hence here n = 7.

reaction. Samples of each population were tested at both
LD = 1 2: 1 2 and LD = 6: 1 8 each at 1 5C and 20"C over
13 weeks. From 238 worms, only 8 worms, i.e., =3%,
became sexually mature.

Mean doubling times for vegetative populations. The
differences in mdt between the Weddel and the Ilmenau
populations were not significant (U-test) and hence the
groups were pooled in Table I. The mdt decreased sig-
nificantly with increasing temperature (P < 0.05, Jonck-
heere). However, the differences between 20C and 25C
were not significant (U-test).

Other environmental factors

No S. lacustris worm reproduced sexually when the
light period was > 12 h per day. Thus, the mode of repro-
duction was independent of temperature, feeding and
population density, and age (see Table II).

Temperature. Different populations of 5. lacustris
were exposed to temperatures between 5 and 30C for 14
weeks. Temperatures of 5C and 30C were not tolerated,
and all experimental animals died within 6 days (N = 2
X 63, n = 2 X 3). In the zone of thermal-tolerance, not
one sexually reproducing individual was found (N
> 1000) during 14 weeks of observation. One culture at
10C was cultured for another 9 weeks and checked 3
times per week. On 10 October 1988 (after 21 weeks in
controlled conditions), two worms were found that had a
well developed genital apparatus and a clitellum. Neither
worm was found after two weeks; their fate is unknown.
Cocoons were not found.

Feeding. Feeding rates reduced to 3 days feeding per
week over 10 weeks never led to a sexual worm. All
worms kept reproducing vegetatively (N > 600, n = 3).
Starvation experiments, resulting in LD 5(I values of 18
3.75 days (range for total population extinction: 20-
31 days), also never led to sexually reproducing worms
(N = 100, n = 3; T = 20C, LD = 16:8).

Population density. Different population densities of

0.1-1.5 worms cm 2 bottom area of culture dish were
tested over 10 weeks at T = 20C. No sexual worms were
found (N> 1000, n = 3).

Field samples

Field samples of S. lacustris were taken at different
times of the year. Only in one sample, collected in Octo-
ber 1987, were both vegetative as well as sexual worms
found. In all other samples collected between June and
September exclusively aclitellate worms were found (see
Table III). The only exception was observed on 19 Au-
gust 1988. Two clitellate worms were detected in a field
sample from the Weddel pond collected on 1 7 July 1988.
The sample (including plant material) had been stored
in a plastic beaker in our laboratory for four weeks at
natural daylight. Other worms found in the sample were
aclitellate (N = 108) on 19-21 August.

Discussion

Although studies of the seasonal development of or-
ganisms have always occupied an important place in ex-
perimental biology, the leading role of the signaling fac-
tors in determining seasonal phenomena have been
largely unknown. For 5". lacustris, the results of this study
unmistakably demonstrate that the life-cycle is strictly
determined by the photoperiod as the relevant external
signaling factor. Since the outstanding discoveries of
Garner and Allard (1920) and Rowan (1926) on photo-
periodic phenomena in plants and animals, many im-
portant contributions have derived from studies in par-
ticular on polychaetes and insects (for overview see
Giese, 1959; Kinne. 1970; Segal, 1970; Hauenschild.
1975; Tauber et a/., 1986; Zaslawski, 1988), but none
from the phylogenetically closely related oligochaetes.

The alternation of reproductive modes

Stylaria lacustris apparently measures the day-length
(proximate factor) to prepare for the sharp temperature
decline (ultimate factor) during winter. Under long-day
(summer) conditions, the worms reproduced exclusively
asexually by paratomic fission, theoretically ad infini-
tum. In the 1960's, Hauenschild (unpubl.) cultured a
population of 5. lacustris for more than six continuous
years in the laboratory at LD = 16:8 and T = 20C, and
he did not find a single sexually mature worm during this
period. In the short-day (autumn conditions), S. lacustris
reproduces only once and then the worms die. Hence, S.
lacustris can best be termed as a 'continuous asexual and
monotelic bisexual breeder' (using the terminology as re-
viewed by Mill, 1978).

The two findings of sexual worms of unknown origin
under long-day conditions can hardly weaken the results.
However, the two observed asexual populations that al-

PHOTOPER1ODISM IN AN OLIGOCHAETE 115

Table 1 1

The effects of age and different environmental factors on the switch from vegetative to bisexual reproduction in Stylaria lacustris

Age

Temperature

Pop. dens.

Feeding rate

Short -day

N^(n)

>1000(5)

> 1000 (5)

> 1000 (3)

>600(3)

>1000(5)

time[d]

98

98

70

70

21-36

sexual p ]

>95

N veg = initial number of vegetative worms exposed to the conditions listed.

(n) = Number of different populations tested.

time = Observation time.

Short-day = LD < 12:12. The photopenod (short-day) is the only factor found to determine the switch from the vegetative to the bisexual mode
of reproduction. Under long-day conditions the worms never became sexually mature (as followed continuously over more than 20 generations),
independent of temperature, population density (pop. dens.) and feeding rate.

most did not switch to sexual reproduction under short-
day conditions are noteworthy. It is unknown whether
an irreversible genetically based loss of sexuality or some
kind of 'permanent modification' had occurred in these
populations. The latter was first observed by Hauen-
schild (1956, 1957) in the anthomedusae of Eleutheria
dichotoma from the Mediterranean. Here, a small per-
centage of primary medusae directly budded off from the
polyp was regularly found to be asexual. In the field a loss
of the sexual mode of reproduction is known from the
sedentary polychaete Ctenodrilus serratus. In the North
Sea, C. serratus reproduces exclusively asexually by par-
atomic fission, whereas in the Mediterranean Sea sexu-
ally mature (hermaphroditic) worms are known. In S.
lacustris, asexual clones were only found in populations
that had been cultured vegetatively for a long time in the
laboratory (here, more than 14 or 23 months, respec-
tively). Whether field samples also include a small per-
centage of asexual clones cannot be answered, because
some worms always died when changing the photope-
nod from long- to short-day. In the field, the asexual
clones would go extinct whenever the temperature
dropped below 5C, i.e.. normally during the winter in

the Palaearctic area. Only the 'normal' clones showing
the photoperiodic reaction can survive. However, in bio-
topes showing annual temperature fluctuations between
only 10C and 25C, a loss of the bisexual mode of repro-
duction in field populations of S. lacustris seems likely,
analogous to the polychaete C. serratus. If those habitats
are found this can be easily tested by exposing popula-
tion samples to short-day conditions.

The annual life-cycle

From the findings of this study, the life-cycle of S. la-
custris can be roughly described as shown in Figure 3.
The prediction is rough in the sense that the tested LD
scalings were broad and the within-population genetic
variation is unknown. The predicted life-cycle from this
study allows 5. lacustris to start sexual reproduction and
hence production of diapausing cocoons prior to. and in
anticipation of, the cold winter period, which is critical
for the worms' survival (cf. Denlinger et al.. 1978; In-
grisch, 1984; Zaslawski, 1988). At 52N. lat. this would
be from October to November, corresponding to the nat-
ural habitats in north Germany, where ponds usually do

Table III

Proportions of reproductive modes infield samples of Stylana lacustris

Date

Place

N

Sex

[N]

Veg
[N]

{ }
[N]

cut

[%]

Aclit
[%]

14July 1985

pond Weddel

26

11

15

100

10 Aug. 1986

river Ilmenau

161

68

93

100

30 Aug. 1987

river Ilmenau

107

57

50

100

20 Sept. 1987

pond Weddel

4

1

3

100

16 Oct. 1987

pond Weddel

48

41

7

85

15

12 June 1988

pond Weddel

96

53

43

100

Samples were taken from different plants (e.g.. of the genera Ceratophyllum, Cham, Elodea. Nasturtium) from a pond at Weddel (near Braun-
schweig) or from the river Ilmenau near Uelzen (Niedersachsen. W. Germany); Oct. 16. 1987 is the only sample collected under natural short-day
conditions: { | refers to individuals in which no kind of actual reproduction was obvious; Clit and Aclit refer to clitellate and aclitellate individuals,
respectively.

116

B. SCHIERWATER AND C. HAUENSCHILD

O)

JB
X

o>

T3

23456789
month

10

Figure 3. The annual lite-cycle of Sly/aria lacuxtri.i. The external
signaling factor daylength is given for 52N. lat. The laboratory studies
and field samples suggest that the switch from asexual paratomic fission
to bisexual reproduction occures in mid September at this latitude.
About three weeks later the first worms become sexually mature, and
the first diapausing cocoons are produced during October. Further ex-
planations are given in the text.

not freeze before December. The genetic variation
within and between populations would be the predicted
basis for adapting 5". lacustris to different annual cycles
with respect to temperature and photoperiod (e.g.. Sauer,
1977; Sauer el a/.. 1986; Groeters and Dingle, 1987).
This could be tested by collecting 5. lacustris at different
latitudes with similar annual temperature cycles, or at
different altitudes with similar annual photoperiod cy-
cles, and measuring the threshold for the photoperiodic
reaction (c;/. Hairston and Olds, 1984, 1986).

'All or nothing' life-history strategy

By using the vegetative mode of reproduction, S. la-
custris can double its number at least every 5 days at 20
to 25C (Table I). Data on the rates of population in-
crease by vegetatively reproducing populations of S. la-
custris given in the literature range from mean doubling
times of 3.6 to 12 days between 15C and 20C (Streit,
1978; McElhone, 1982; Finogenova, 1984). The data
agree with Streit (1978), who calculated mdt of 3.6 (T
= 19C) and McElhone (1982), who estimated values of
4-6 days (T == 20C). In the latter case it is not clear
whether these are mean values or maximal values. Dur-
ing summer all efforts are invested in vegetative repro-
duction, leading to the most rapid population increases,
regardless of the actual physical environmental condi-
tions (r-strategy); during one season (April through Sep-
tember) one single worm can theoretically give rise to a
population of 3.4 billion worms. During autumn all
effort is invested in sexual reproduction, i.e., the number
of cocoons produced for overwintering is maximized, re-
gardless of whether the winter temperatures go below 5C

or not. An unexpected and abnormal temperature de-
cline to <5C during summer or early autumn would
lead to the total extinction of populations. Furthermore,
the reproductive strategy cannot respond to unexpected
changes in other environmental factors, like food supply
and population density. Therefore, the life-cycle strategy
of S. lacustris is an 'all or nothing' strategy maximizing
reproductive output as far as possible (cf. Hirshfield and
Tinkle, 1975; Pianka, 1976; Stearns, 1976). This is con-
sistent with a high degree of adaptation to predictable
annual cycles of environmental conditions; thus the life
history strategy of S. lacustris does not fulfill some pre-
dictions from life history theories. In a fluctuating and
unpredictable environment (such as small freshwater
ponds), we would expect S. lacustris (a) to reach early
sexual maturity, instead of postponing it as far as possible
to the end of the season or (b) to show reproductive flex-
ibility in order to minimize the risk of total failure in a
bad year (cf. Stearns, 1976, 1980; Glesener and Tilman,
1978; Mill, 1978; Sauer, 1984; Groetersand Dingle,
1987).

The high abundances and the wide distribution of 5.
lacustris, however, indicate that the photoperiodic life-
cycle strategy can also be very successful in oligochaetes.
It seems unlikely that S. lacustris should be the only oli-
gochaete worm that has been found to synchronize its
life-cycle with the seasons of the year by measuring day-
length, and it might be reasonable to look for photoperi-
odic reactions in other oligochaete worms that perform
an alternation of reproductive modes.

Acknowledgments

We thank Hansi Fauter, Heike Fee Hadrys, and Dr.
D. Teschner(all Braunschweig). We are indebted to Prof.
L. W. Buss, Dr. N. W. Blackstone, and Matt Dick (all
Yale) for their critical comments and corrections on ear-
lier versions of the manuscript. The work was supported
by a grant from 'GradFoeg des Landes Niedersachsen,'
W. Germany.

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Visualization of the Transparent, Gelatinous House of

the Pelagic Tunicate Oikopleura vanhoeffeni

Using Sepia Ink

PER R. FLOOD 1 . DON DEIBEL 2 , AND CLAUDE C. MORRIS :

1 Institute oj 'Anatomy, University of Bergen, Arstadveien 19, 5009 Bergen, Norway and

^'Marine Science Research Laboratory, Ocean Sciences Centre, Memorial University

of Newfoundland, St. John's, Newfoundland, Canada, A1C 5S7

Abstract. Appendicularian tunicates of the genus Oi-
kopleura feed using an external, acellular, transparent
structure known as the house. Previously, dilute particu-
late dyes have been used to visualize the internal struc-
ture of this house. However, because of toxicity, large
particle size, and flocculation, many of these dyes have
been of limited practical and scientific use. We report on
a new marker, the ink from the cephalopod Sepia offi-
cinal is. that solves many of these problems.

Specimens of Oikopleura vanhoeffeni relished Sepia
ink, having dark black stomachs and producing many
dark fecal pellets over several days. When O. vanhoeffeni
expanded houses in dilute ink, the internal walls, septae,
and filters were shown in great detail, whereas high con-
centrations of ink showed delicate patterns of lines on
the internal walls.

We present documentary photographs of previously
unillustrated or undescribed morphologies: the escape
slot; the incurrent funnels; two dimples caused by inser-
tion of suspensory filaments on the upper wall of the pos-
terior chamber, a large, posterior keel; both the open and
closed positions of the exit valve; and the complex pat-
tern of lines on the inner walls. However, the external
walls of the house had no affinity for the dye and could
only be seen by dark field illumination.

We believe that Sepia ink can be used to visualize
functionally important transparent structures of other
gelatinous zooplankton and can be a colloidal marker in
feeding experiments of a wide range of filter feeders.

Received 25 September 1989; accepted 30 January 1990.

Introduction

Oikopleurid appendicularians are suspension feeding
zooplankters that are surrounded by a transparent, acel-
lular, gelatinous "house," which they secrete. The house
contains a complex system of fine filters that are used
by the animals to concentrate and remove food particles
from suspension. Using its muscular tail as a pump, the
animal draws water into the house through a pair of
coarse, bilateral, incurrent filters. The water is then
pumped through the tail chamber into bilateral passage-
ways leading to the lateral edge of expansive food-con-
centrating filters. Here much of the water is pushed
through a mesh with 0.22 nm pore size (Deibel et a/..
1985). Particles are retained between the food concen-
trating filter screens, resulting in a concentrated food sus-
pension that is drawn into a medial food-collecting
tube leading to the animal's mouth. This food suspen-
sion is 100 to 1000 times more concentrated than are
particles in the environment surrounding the animal (Jor-
gensen, 1984; Flood, in prep.). A third filter inside the
pharynx of the animal traps the food particles for inges-
tion. The filtered water exiting the food concentrating
filter leaves the house through a narrow exit spout and
valve, producing a jet that propels the house and animal
slowly through the sea.

The existence of the house has been known since the
work of Fol (1872) and Lohmann (1899). However,
many details of its structure remained unknown until re-
cent improvements in microscopical techniques and spe-
cial staining procedures made further progress possible.
Dilute particulate dyes have been used to visualize the
internal walls, chambers, and filters of the house (All-
dredge. 1977; Flood, 1978, 1983; Deibel et a/.. 1985;

118

VISUALIZATION OF OIKOPLEURA HOUSES USING SEPIA INK

119

Deihel, 1986; Fenaux, 1986). When added to seawater,
dye particles are retained by the filters within the house
in the same way as are naturally occurring particles. Loh-
mann (1899) and Alldredge (1977) used dilute suspen-
sions of carmine particles to visualize both the incurrent
and food-concentrating filters of many oikopleurids.
However, the animals may not feed normally when car-
mine is present (Alldredge, 1977). We have found that
freshly prepared dilutions of carmine and extreme care
are required to prevent the animals from leaving their
houses. In addition, carmine particles settle rapidly and
stain only the incurrent and food-concentrating filters.

Fenaux ( 1986) used dilute India ink to stain the incur-
rent and food concentrating filters, and the internal walls
and septae of houses of Oikopleura dioica. One of us
(P.R.F.) has used a similar technique since 1978. If India
ink is added to seawater before the animal expands a new
house, all internal walls and septae of the house are
stained (Flood, in prep.). However, if the ink is added
after the house has been expanded, only the food-con-
centrating filter is stained. This approach requires freshly
prepared ink solutions and great care to prevent the ani-
mal from leaving its house. The carbon particles that
make up India ink tend to aggregate in seawater and set-
tle rapidly as do particles in carmine suspensions.

Deibel ( 1986) used several types of particles to mark
specific parts of the house of Oikopleura vanhoeffeni
differentially. These particles included finely ground
charcoal, starch, latex beads, and the unicellular green
alga I\ochrysis galbana. Charcoal particles adhered spe-
cifically to an intermediate, coarse screen between the
two walls of the food-concentrating filter. The alga, on
the other hand, stuck to the upper and lower walls of the
food-concentrating filter. Starch granules did not adhere
to any of the filters of the house, but stained the pharyn-
geal filter within the trunk of the animal. This suggests
that physical and chemical properties of both the marker
and the house structures in question affect the staining
result.

We recently found another marker that may be used
to visualize structural details of oikopleurid houses that,
in the past, have been difficult or impossible to docu-
ment. Information about these structures is needed to
understand the behavioral and functional details of the
feeding process on which the ecology of these animals
depends. The new marker may be used not only to visu-
alize feeding structures and quantify particle clearance
rates of pelagic tunicates, but also to observe transparent
structures of other marine plankton.

Materials and Methods

Individuals of Oikopleura vanhoeffeni. in their houses,
were collected in 500-ml glass jars by SCUBA diving in

Logy Bay, Newfoundland, during May and June 1989.
Animals were maintained in these jars for up to 10 days
in laboratory tanks containing circulating seawater at 1
to 5C, about 1C above ambient sea surface tempera-
ture.

Ink was collected from freshly dead specimens of the
cuttlefish Sepia officinal/sal the Plymouth Marine Labo-
ratory, Devon, U.K. The ink duct of each animal was
clamped with a hemostat while the ink sac was removed.
Once excised, a loop of the duct was placed in a collec-
tion vial and the duct cut to allow the ink to drain into
the vial. This ink was diluted immediately with 10 parts
of distilled water and 1 0.000 units of pennicillin G added
per ml of solution to help prevent bacterial decomposi-
tion. Diluted ink remains liquid for several years, but un-
diluted ink coagulates after several weeks at room tem-
perature. The ink was dispersed with gentle agitation in
seawater to a dilution of cu. 10 ? just before use.

Solid Sepia ink is available commercially, but must
be ground before use. It also contains phenol or other
chemical preservatives that may be noxious or toxic to
marine animals, and therefore it was not used in these
experiments.

Houses were examined using a Wild M420 macro-
scope with bright or dark-field illumination. The light
source used for routine observation was a 100W halogen
lamp, whereas two modified Sunpak GX14 electronic
flashes and a Wild MRS 5S/-5 1 photoautomat were used
for still photography. We used Kodak Ektachrome 100
and 400 ASA film for slides, and Kodak Tmax at 100 to
3200 ASA for prints. By using 0.5- and 2.0-times acces-
sory lenses on the macroscope, final magnification of the
photographs ranged from 1 .25- to 25-times.

Results and Comments

Liquid Sepia ink was easily miscible in seawater, and,
contrary to other dyes that have been used to visualize
the house of Appendicularia. it stayed evenly dispersed
in solution for up to 14 days.

Transmission electron microscopy (TEM) revealed
that this ink consists of uniformly spherical melanin
granules with diameters ranging between 56 and 161 nm
(arithmetric mean = 102 nm. Standard deviation = 21
nm. Flood, pers. obs. by TEM).

In spite of this low particle size, some of the ink parti-
cles were easily concentrated and ingested by Oi-
koploeura vanhoeffeni. In fact, these animals seemed to
relish the Sepia ink, having full stomachs and producing
abundant opaque, black fecal pellets (Fig. 1) that ap-
peared to be composed entirely of ink.

However, much of the ink passed through the house
of Oikopleura vanhoeffeni, without being witheld by the
food-concentrating filters. Some of these particles ad-

120

P. R. FLOOD ET AL

EW

:

ExV

Figure I . Lateral view of a live Oikupleura vanhoeffeni heating its tail inside a house faintly stained by
Sepia ink. Bright field macrograph at seven times magnification. [The nomenclature used is adopted from
Flood ( 1 983) and is largely a direct translation of Lohmann's German names (Lohmann. 1956).]

In addition to numerous details of the inside walls (/") of the house, like the prominent exit spout
(ExS) and valve (E\ "I "), a keel (A ), cushion chambers lateral (l.CC) and antero-medial (aCC) to the inlet
openings, inlet funnels (//"). roof dimples (rd), and a roof hump (rli), numerous internal details can be
seen. The animals trunk ( 7>), tail ( Ta), and escape chamber (EsCh) as well as the trunk chamber ( TrCh),
tail chamber (TaCh). supply passage (SP). and suspension of the food-concentrating niters (FCF) in the
posterior chamber (PCh) are faintly outlined. Numerous fecal pellets (/"/*) stained completely black by
Sepia ink are seen along the floor of the posterior chamber. The external wall (/:'H') is only visible above
the hump in the inside roof.

1mm

hered to the internal walls and septae of the house and
made them easily visible.

By varying the concentration of Sepia ink from experi-
ment to experiment, the intensity of staining could be
controlled to reveal different features of the house. When
a specimen of Oikopleura vanhoeffeni expanded its
house in seawater containing very dilute Sepia ink, the
internal walls, septae, and niters were shown in great de-
tail (Fig. 1 ), whereas heavy staining made the house less
transparent and revealed delicate patterns of lines and
fields on the internal walls and septae throughout the
house (Figs. 2, 3).

The outer wall of the house, however, had no affinity
for the ink and was rarely seen at all in bright field illumi-
nation (Fig. 1). However, in most cases its presence was
revealed by adhering detritus particles. This was particu-
larly true for the prominent bow of the house (Fig. 2). In

dark field illumination, on the other hand, the external
walls and their variable thickness in distinct parts of the
house became more evident (Fig. 3).

The difference in volume between the internal water-
filled spaces and the total house could be estimated from
such pictures. If the internal transverse diameter of the
house was considered to be unity, the external transverse
diameter was generally close to 1.2, the internal longitu-
dinal diameter about 1.3. and the outside longitudinal
diameter about 1 .7. Considering the house to be an ellip-
tical rotatory body, this makes the total volume approxi-
mately 1.5 times as large as the internal water-filled
spaces. We do not know if the spaces between the inside
and outside walls are filled with a compact (gel-like) sub-
stance or if they are water-filled chambers unaccessible
to the Sepia particles.

By varying the staining intensity of the house, we dis-

VISUALIZATION OF O1KOPLEURA HOUSES USING SEPIA INK

121

ExS

EsP

.

/

4

1mm

Figure 2. Top view of live Oikupk'iira vanhoeffeni inside its house. Bright field macrograph at seven
times magnification after strong staining with Sepia ink.

Intricate patterns of Sepia ink are seen on many walls, as for example near the escape passage (EsP) and
supply passages (SP). Note also the attachment (arrows) of the animal trunk (7>) to the walls separating
the trunk chamber (TrCli) from the escape chamber (EsC'h). The inlet openings (1O), inlet niters (//'/), and
the inlet funnels (IFu) are visible on both sides of the house. Note the prominent bow (B) made visible
only by adhering detritus particles. Otherwise, same labeling as in Figure I.

covered many structural details of which we were pre-
viously unaware or had insufficient knowledge. Here we
will only describe some of the most prominent features
and comment briefly on their functional significance.

1 I ) The escape port in the anterior chamber (Figs. 2,
5B). The animal forces its way through this preformed
weak part when it leaves the house, thereby tearing it
open to a wide escape slot. This escape port is covered by
the massive bow of the house (Fig. 2), and somehow a
preformed channel must exist through this bow material
towards the external house wall. Otherwise the animal
could not force its way out of the house as easily, fre-
quently, and uniformly as it does (cp. Fenaux, 1985).

(2) The incurrent funnels leading into the house (Figs.
1-3). In the only existing description of the house ofOi-
koplewa vanhoeffeni (Deibel, 1986. Fig. 1), these have
been given quite a different shape from what we have
been able to photograph.

(3) The attachment of the anterior walls of the incur-
rent funnels to the lateral part of the trunk (Fig. 2). These
walls seem to meet the trunk exactly where the Langer-
hans bristle is located. Through this sensory organ the
animal may monitor accordingly the inflation and con-
dition of the house (Bone and Ryan, 1979). Perhaps the
entire house in this context may be regarded as a tactile
sensory structure.

A rather rigid suspension of the trunk of the animal
within the house is needed for the tail to perform its
pumping action. This prevents the "tail from wagging
the dog" as may be observed just before the animal leaves
its house, when the trunk has detached from some of its
anchoring points. Stimulation of the Langerhans recep-
tor may then initiate the vigorous jerk and swimming
movement that enables the animal to detach completely
from the house and force its way through the escape slot.

(4) The shape of prominent chambers and lateral flaps

122

P. R. FLOOD ET AL

Figure .V Inhabitated house of Oikopleura vanhocffeni as seen in dark held illumination from a point
above, behind, and to the side of the house. (The axes of the house as it normally moves through the sea
are indicated in the lower right hand corner.) Magnified seven times.

Note the prominent patterning of the internal walls (/IT) corresponding to the attachment sites of the
filter ridges of the food-concentrating filters (arrows) along the periphery of the supply passages (SP), and
the presence of a prominent semitransparent jelly-like substance (G') covering the posterior side and the
anterior bow-like pole (/J) of the house. The outer limit of this jelly-like substance represent the true exter-
nal walls (All) of the house. The orientation of the house as it moves through the water is indicated by
axes in the lower right hand corner of the figure. For other abbreviations refer to Figure 1 .

in the anterior part of the house, medial and lateral to
the incnrrent openings (Fis>.s. 1-3). These chambers are
probably rilled by water flowing from the tail chamber
through a hole in its distal floor (Flood, in prep.). It is
also possible that the anterior chambers communicate
with the upper compartment of the posterior chamber
and may be tilled by water via this route, as suggested by
Fenaux (1986). A positive pressure in the anterior cham-
bers surrounding the incurrent funnels is needed to resist
the collapsing force generated by the negative pressure
within these passageways as water is drawn into the
house. The lateral flaps may serve as vertical stabilizers
to control the orientation of the house as it moves
through the sea or as flaps to prevent the immediate re-

clogging of the incurrent niters after they have been back-
washed (Flood, in press).

(5) Two large dimples in the inner house wall of the
upper compartment of the posterior chamber (Figs. 1. 3).
These probably represent the anchoring sites of suspen-
sory filaments originating somewhere along the anterior
edge of the food-concentrating filters.

(6) A medial hump in the inner house wall above the
trunk oj the animal (Fig. I). The external house wall had
its highest optical density and could be faintly seen even
in bright field illumination above this hump. Although
of unknown functional significance, this hump is also
found in houses of Oikopleura dioica and O. labrado-
riensis( Flood, pers. obs.).

VISUALIZATION OF OIKOPLEURA HOUSES USING SMI. -I INK

123

(7) A large "keel" at the hack of the house just above
the exit valve (Figs. 1. 3). This keel may serve as a rudder
to inhibit rolling and to facilitate looping motions as the
house is propelled through the water. A looping motion,
which has been described for other oikopleurans by All-
dredge (1976), allows the animal to stay within and ex-
ploit a patch of nanoplankton more efficiently than by a
linear motion. This keel was discovered by Deibel
(1986), but due to poor visibility, even in dark field illu-
mination, his description is incomplete (Compare his
Fig. 1 to our Fig. 1 ).

(8) A posterior exit spout and valve below the longitu-
dinal midline of the house (Figs. I, 3). Strong staining by
Sepia ink allowed us to observe the opening and closing
action of this pressure sensitive valve. In its closed posi-
tion its upper and lower lips were inverted (Fig. 4A). One
to five seconds after the pumping action of the tail started
and increased the pressure inside the posterior chamber
and exit spout, the lips everted and exposed a medial oval
opening with a strongly birefringent and elastic rim (Fig.
4B). This central exit opening was evident even when the
animal pumped slowly. However, when the tail pumped
at maximum efficiency, the exit spout became much
longer, and four additional exit openings were exposed
peripheral to the central one. The tissue surrounding the
exit valves was then stretched to such a degree that it left
very little contrast in our photographs (Fig. 4C). Fenaux
(1986), studying Oikopleura dioica houses, found the
four peripheral openings to open before the central one.

The propulsive thrust generated by the jet of water
leaving the house was directed somewhat below the cen-
ter of the house, resulting in a tendency to turn the front
of the house upward. When combined with the slightly
upward-pointing bow and the directional control of the
keel and lateral flaps (see above), this thrust will result in
a slow upward movement of the house, or even a vertical
looping motion as sometimes seen in the field (cp. All-
dredge, 1976).

The more intense staining resulting from higher con-
centrations of Sepia ink revealed delicate patterns of
lines and fields on most internal walls of the house (Figs.
2, 5). In some areas, complex patterns of straight or
curved lines were visible (Fig. 5A). These may corre-
spond to decorated filaments, corrugated surfaces, or
small pockets. In other areas, faint patterns of polygonal
fields were apparent (Fig. 5B). Although each polygon
was quite large, their pattern reminded us of the oi-
koplast cell pattern on the trunk of the animal. These
cells are responsible for the production of the house
(Lohmann, 1933/1956); perhaps Sepia ink might be
used to map the areas of the house made by individual
cells. This represents a major problem yet to be properly
elucidated for all appendicularians.

Discussion

The usefulness of the Sepia ink for visualizing distinct
parts of Oikopleura houses probably depends on three
or four factors: ( 1 ) Sepia ink forms stable solutions in
seawater and does not aggregate and sediment like most
other particulate dyes. Such flocculent particles seem to
interfere with the house expansion process of animals
kept in captivity. (2) The particle size of Sepia ink is
small enough to allow a significant proportion of parti-
cles to pass the food-concentrating filters to stain the
walls of the posterior chamber, the exit spout, and possi-
bly the anterior chambers of the house. (3) The animals
seem to relish the Sepia ink as a food source and do not
find it noxious or toxic like many other dyes. (4) The
physico-chemical properties of the Sepia ink particles
may be particularly favorable to stain the internal walls
and septae of the house.

These excellent properties of Sepia ink may make it
useful in the study of other gelatinous zooplankters.

The reason why Sepia ink, like all other particulate
dyes we have used, failed to stain the external walls of the
house remains obscure. It may depend on special physi-
co-chemical properties of this layer, but a more likely ex-
planation may be that the dye particles are prevented
from having direct physical contact with it. The walls
surrounding the water-filled spaces inside the house are
probably not entirely waterproof. Due to the higher hy-
drostatic pressure inside the house, water will seep slowly
out through the walls, leaving its particles behind as a
decoration on the internal walls, and producing a thin
halo of particle-free water just outside the house. Such a
halo may be enough to prevent the proper staining of the
external walls.

The pore size of the food-concentrating filters of Oi-
kopleura vanhoeffeni 1.0 X 0.22 ^m according to
Deibel et al. (1985) was significantly larger than was
the particle size of Sepia ink (0. 1 0.02 ^m according to
Flood, unpub. res.). In spite of this, the animals used in
this study easily concentrated and ingested the dye, and
incorporated it into fecal pellets. This may depend on a
selection of the largest particles in the ink, on a selection
of aggregated particles, or on an ability to retain smaller
particles than hitherto believed. In fact, the carbon bud-
gets of oikopleurans seem to be such that ingested parti-
cles > 0.2 ^m in diameter rarely account for more than
30% of the energy expenditure for growth, respiration,
and house production (Paffenhofer, 1976;Gorsky, 1980;
King, 198 1 ). It seems likely that the animals may obtain
much of their nurishment from particles < 0.2 ^m in
diameter, or from dissolved organic matter. We foresee
the use of monodisperse Sepia ink particles in future
feeding experiments on appendicularians and other filter
feeding marine animals.

124

P. R. FLOOD ET AL

PExO

CExO

4C

PExO

EW

Figure 4. Bright field macrographic details of the exit spout and valve of a heavily Sepia-ink stained
house of Oikopleura vanhoeffeni at 20 times magnification.

(A) In its closed state. (B) in its half open state, and (C) in its full open position. Unfortunately the exit
openings themselves [one central (CExO) and four peripheral ( PE\O)\ didn't give sufficient contrast to be
seen in picture C. The external wall (EM ") of the house is seen next to the exit spout.

Figure 5. Bright field macrographic details of an Oikopleura vanhoeffeni house heavily stained by Sepia
ink at 23 times magnification.

In (A), parallel ruffles ( TChR) and numerous pockets ( TCliP) are seen in the roof of the tail chamber. In
( B), polygonal fields ( /)/) resembling cell outlines are seen next to the escape passage ( EsP) of the house.

VISUALIZATION OF OIKOPLEURA HOUSES USING SEPIA INK

125

Acknowledgments

Many thanks to the members of the Diving Unit (G.
Chaisson, Divemaster) of the Ocean Sciences Centre,
Memorial University, for assisting D.D. with the collec-
tion of Oikoplcwa vankocffcni. and to Mr. Edward
Downton for designing and fabricating laboratory equip-
ment. This work is a result of a Bergen-Memorial Uni-
versities Exchange Fellowship to P.R.F., and we thank
Drs. Bodil Larsen and R. L. Haedrich, Memorial Uni-
versity, for making this visit possible. This work was sup-
ported by a grant from the Norwegian Research Council
for Science and the Humanities to P.R.F., and by Oper-
ating and Equipment Grants from the Natural Sciences
and Engineering Research Council of Canada to D.D.
This is Ocean Sciences Centre contribution number 6 1 .

Note added in proof: We have used commercial ink from
Sepia recently available from Sigma Chemical Co. (St.
Louis, Missouri). Oikoplcwa vanhoeffeni took up this
ink similarly to that we collected from Sepia ourselves.

Literature Cited

Alldredge, A. L. 1976. Field behavior and adaptive strategies of ap-
pendicularians (Chordata: Tunicata). Mar. Binl. 38: 29-39.

Alldredge, A. I.. 1977. House morphology and mechanisms of feed-
ing in the Oikopleuridae (Tunicata. Appendicularia). J. /.<iol
(Loml.)lSl: 175-178.

Bone, Q., and K. P. Ryan. 1979. The Langerhans receptor of Oi-

koplcwa (Tunicata: Larvacca). ./ Mar. Biol. Assoc. U. K. 59: 69-
75.

Deibel, t>. 1986. Feeding mechanism and house of the appendicular-
ian Oikoplcwa vanhocllcni. Mar. Biol. 93: 429-436.

Deibel, D., M.-L. Dickson, and C . V. I.. Powell. 1985. Ultrastructure
ot the mucous feeding tiller of the house of the appendicularian
Oikoplcwa vanhocltcni. Mar. i.col I'rogr. Ser 27: 79-86.

Kenaux, R. 1985. Rythm of secretion of oikopleurid's houses. Bull
Mar Sci. 37: 498-503.

Fenaux, R. 1986. The house ofOikopleura dioica (Tunicata. Appen-
dicularia): structure and functions. Zoomorphology 106: 224-23 1 .

Flood, P. R. 1978. Filter characteristics of appendiculanan food
catching nets. Expericntia 34: 173-1 75.

Flood, P. R. 1983. The gelationous house ofOikopleura dioica (Ap-
pendicularia, Tunicata); its architecture and water filtration mecha-
nism. Ann. Meet. Western Soc. Naturalists. Burnaby. B. C. Canada.
Dec. 1983 (Abstract).

Fol, H. 1872. Etudes sur des Appendiculaires de Detroit de Messine.
Mem. Soc. Phys. Hist. Nat. Geneve 21(2): 445-499.

Gorsky, G. 1980. Optimisation des cultures d'appendiculaires. Ap-
proche du metabolisme de O dioica. Ph. D. thesis, Univ. P. & M
Curie, Paris VI. 110pp.

Jorgensen, C. B. 1984. Effect of grazing: metazoan suspension feed-
ers. Pp. 445-464 in Heterotrophic Activity in the Sea. }. E. Hobbie
and P. J. leB. Williams, eds. New York, Plenum Press.

King, K. R. 1981. The quantitative natural history of Oikoplcwa di-
oica (Urochordata. Larvacea) in the laboratory and in enclosed wa-
ter collumns. Ph. D. thesis. Univ. Washington, Seattle.

Lohmann, H. 1899. Das Gehause der Appendicularien. sein Bau.
seine Funktion und seine Entstehung. Schr. Nantrwiss. I 'er. Schles-
H-ix-Holstein 11:347-407.

Lohmann, H. 1933/1956. Appendiculariae. Handb. Zool. 5,11: 15-
202.

PafTenhofer, G. A. 1976. On the biology of the Appendiculana of the
southeastern North Sea. lO/li Enr. Symp. Afar. Biol.. Oslcml, Bel-
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Reference: Biol. Bull. 178: 126-136. (April. 1490)

The Morphology and Mechanics of Octopus Suckers

WILLIAM M. KIER AND ANDREW M. SMITH*

Department of Biology, Coker Hall, CB# 3280, The University of North Carolina,
Chapel Hill. North Carolina 27599-3280

Abstract. The functional morphology of the suckers of
several benthic octopus species was studied using histol-
ogy and cinematography. The suckers consist of a tightly
packed three-dimensional array of musculature. Three
major muscle orientations are found in the wall of the
sucker: ( 1 ) radial muscles that traverse the wall; (2) circu-
lar muscles that are oriented circumferentially around
the sucker, including a major and minor sphincter mus-
cle; and (3) meridional muscles that are oriented perpen-
dicular to the circular and radial muscles. The connec-
tive tissue of the sucker includes inner and outer fibrous
connective tissue layers and an array of crossed connec-
tive tissue fibers embedded in the musculature of the
sucker.

Attachment is achieved by reducing the pressure in-
side the sucker cavity. We propose the following mecha-
nism to explain this pressure reduction. Contraction of
the radial muscles thins the wall and thus increases the
enclosed volume of the sucker. If the sucker is sealed to
the substratum, however, the cohesiveness of water re-
sists this expansion. Thus, contractile activity of the ra-
dial muscles reduces the pressure of the enclosed water.
The radial muscles are antagonized by the circular and
meridional muscles so that the three-dimensional array
of muscle functions as a muscular-hydrostat. The
crossed connective tissue fibers of the sucker may store
elastic energy, providing a mechanism for maintaining
attachment over extended periods.

Introduction

Octopus suckers perform a remarkable variety of func-
tions. Packard (1988) listed six distinct roles of the suck-
ers of benthic octopuses including: (1) locomotion; (2)
anchoring the body and holding prey; (3) sampling, col-

Received 16 October 1989; accepted 30 January 1990.
* Order of authorship is merely alphabetical.

lecting, and manipulating small objects; (4) chemotactile
recognition; (5) displays; and (6) cleaning maneuvers.
These diverse roles demand that the suckers be flexible
and dexterous yet capable of generating large forces (see
Dilly c/ ai, 1964). Previous research has focussed on the
chemotactile ability of the suckers (see Wells, 1978), on
the sensory receptors of the suckers (Graziadei, 1962;
Graziadei and Gagne, 1976a, b). and on their morphol-
ogy (see below). Our understanding of how the sucker
generates the movements that allow it to manipulate and
forcefully grip objects is incomplete.

The morphology of octopus suckers has been de-
scribed previously. Nixon and Dilly (1977) described the
surface features of octopus and squid suckers from
different genera. The sucker musculature has been de-
scribed by Girod (1884), Guerin (1908), Nachtigall
(1974), Niemiec (1885), and Tittel (1961, 1964), but the
proposed mechanisms of action are incorrect both in
their analysis of the function of the musculature and in
understanding the ability of water to sustain sub-ambi-
ent pressures. Previous studies also overlooked impor-
tant features of the connective tissue.

The suckers are muscular-hydrostats as defined by
Kier and Smith (1985) (see also Smith and Kier, 1989).
The musculature is arranged in a tightly packed, three-
dimensional array that provides the skeletal support and
the force for movement. This type of system produces
movements that are localized and remarkably complex,
allowing precise changes in shape by bending, contracting,
or stretching at any point. In this paper we describe the
muscle arrangements in the suckers of several octopus spe-
cies and discuss the function of these arrangements.

Materials and Methods

Experimental animals

Specimens of Eledone cirrosa were supplied by The
Laboratory of the Marine Biological Association of the

126

SUCKER FUNCTIONAL MORPHOLOGY

127

United Kingdom, Plymouth. Specimens of Octopus jou-
bini and Octopus maya were supplied by The Marine
Biomedical Institute of the University of Texas Medical
Branch at Galveston, Texas. Specimens of the Octopus
bimaculoides/bimaculatus complex (see Pickford and
McConnaughey, 1949) were supplied by Pacific Bio-Ma-
rine, Venice, California, and Chuck Winkler Enterprises,
San Pedro, California. Observations of sucker behavior
and kinematics were made primarily on O. bimacu-
loides/bimaculatus and O. maya. A detailed morpholog-
ical analysis of the suckers was performed on specimens
of E. cirmsa, O. joubini, and O. bimaeulatus/bimacu-
loides.

Histology

Blocks of arm tissue that included several suckers were
obtained from freshly killed animals that were anesthe-
tized in 1% ethanol in seawater. The tissue was fixed in
Bouin-Dubosq fixative (Humason, 1979) or in 10% for-
malin in seawater for 24-48 h. In some cases, blocks of
tissue were obtained from specimens that had been fixed
whole in 10% formalin in seawater after anesthesia. The
tissue was dehydrated in ethanol and embedded in par-
affin (MP 56C). The blocks were sectioned serially at 5-
10 /urn on a rotary microtome. Serial sections were made
in three mutually perpendicular planes. The sections
were stained using one of the following techniques: ( 1 )
Mallory's triple stain as outlined by Pantin (1946); (2)
Milligan trichrome stain; (3) Picro-Ponceau with iron
hematoxylin; or (4) Mowry's colloidal iron method. The
procedures followed for stains 2-4 above are outlined by
Humason (1979). Sections were examined with bright-
field, phase contrast, and polarized light microscopy.

Computer-assisted three-dimensional reconstruction

The extrinsic musculature of the suckers of one speci-
men of E. cirrosa was examined using a computer
program for three-dimensional reconstruction (PC3D
Three-Dimensional Reconstruction Software, Jandel
Scientific, Corte Madera, California). Serial frontal sec-
tions (see description of section planes below) 10 nm
thick were used for the reconstructions. The outlines of
the major muscle groups of every fourth section were
traced using a camera lucida on a compound micro-
scope. Alignment of the series of tracings was performed
according to the visual best-fit method (Gaunt and
Gaunt, 1978; Young etai, 1985). The tracings were then
digitized with a Numonics 2210 digitizing tablet. The
PC3D software, running on a CompuAdd 286/12 AT
microcomputer, stacked the outlines of specified muscle
bundles from each section, producing a three-dimen-
sional representation of the muscular morphology that
could be viewed in any orientation. The reconstructions

shown in Figure 8 were plotted on a Hewlett-Packard HP
7475A plotter.

Cinematography

A specimen of 0. maya was filmed walking on a glass
aquarium wall with a Canon Scoopic 1 6mm movie cam-
era filming at 48 frames/s using Eastman Ektachrome
Video News Film. The film was viewed frame by frame
on an L-W International film analyzer, and calipers were
used to measure the diameter of the sucker and the diam-
eter of the opening to the acetabulum. The measurement
error was <5%. Measurements were made from one 100-
ft roll of film, choosing every sucker (total of 26 suckers)
that attached or released and whose outlines were dis-
tinct enough to measure. Suckers attached to the glass
could be distinguished because they remained stationary
relative to the movement of the arm.

Results

Gross morphology of the suckers

The gross morphology of the suckers of different octo-
pus species has been described previously (Girod, 1 884;
Guerin, 1908; Niemiec, 1885; Nixon and Dilly, 1977;
Packard, 1988), and a brief summary of observations on
the species we examined is provided here. The sucker
consists of two general regions: the acetabulum and in-
fundibulum (Girod, 1884) (Fig. 1). The infundibulum is
the exposed portion of the sucker that is applied to the
substratum during attachment. The acetabulum is a
more or less spherical cavity that opens to the infundibu-
lum through a constricted orifice (Fig. 1). The surface
of the infundibulum bears a series of radial grooves and
ridges while the surface of the acetabulum is smooth. The
sucker is covered by a chitinous cuticle or sucker lining
(see below) that is particularly well-developed on the in-
fundibulum. The sucker lining is shed periodically and
renewed continuously (Girod, 1884; Naef, 1921-1923;
Nixon and Dilly, 1977; Packard, 1988). The infundibu-
lum is encircled by a rim covered with a deeply folded,
loose epithelium. The suckers are attached to the arms
by a short muscular base that is covered by a continua-
tion of the dermis and epidermis of the arms. A single
row of suckers is present on the arms of E. cirrosa and
two rows of suckers are present on the arms of the Octo-
pus species.

Sucker microanatomy

For the purposes of this discussion, we refer to trans-
verse and frontal sectional planes. Transverse sectional
planes are defined as sections perpendicular to the long
axis of the arm. Frontal sections are parallel to the plane
defined by the opening of the sucker.

Intrinsic sucker musculature. Although we did not

128

W. M. K.IER AND A. M. SMITH

Figure I . Schematic diagram of the microanatomy ot'the sucker of
Octopus in transverse section. A. acetabulurn; AR. acetahular roof;
AW. acetabular wall: C, circular muscle; CC, crossed connective tissue
fibers; D, dermis; E, extrinsic muscle; EC, extrinsic circular muscle; EP,
epithelium; IN, infundihulum; 1C, inner connective tissue layer; M,
meridional muscle; OC. outer connective tissue layer; R, radial muscle;
SI. primary sphincter muscle; S2, secondary sphincter muscle.

make a systematic study of a wide range of sucker sizes
and sucker locations on the arms, the general arrange-
ment of the muscle and connective tissue of the suckers
is the same for the different species and sucker sizes we
examined. Several minor differences between genera
were observed and are noted below. The acetabular and
infundibular portions of the sucker consist primarily of a
tightly packed, three-dimensional array of muscle fibers.
The muscle fibers can be categorized by orientation into
three major groups: radial muscle fibers that extend
across the wall of the sucker more or less perpendicular
to the inner surface; circular muscle fibers that are ori-
ented circumferentially around the sucker and parallel
to the frontal plane; and meridional muscle fibers that
are oriented perpendicular to the radial and circular
muscle fibers (Fig. 1 ).

The acetabular portion consists of a wall region and a
domed roof. The acetabular wall includes radial, circu-
lar, and meridional muscle fibers. The acetabular roof
includes radial and meridional muscle fibers but lacks
circular muscle fibers. Radial muscle fibers extend be-
tween their origins and insertions on an inner fibrous
connective tissue layer lining the acetabulum and an
outer fibrous connective tissue layer encapsulating the
sucker (Figs. 1-3). As the radial fibers project toward the
outer surface, they interdigitate with bundles of meridio-
nal muscle fibers. In the acetabular wall, the radial mus-

cle fibers also interdigitate with circular muscle bundles
(Fig. 2). The circular muscle bundles extend around the
perimeter of the acetabular wall.

The location of the circular and meridional muscle
bundles in the acetabular wall of the suckers of Eledone
cirrosa is different from that of the Octopus species exam-
ined in this study. In E. cirrosa, the meridional muscle
bundles are located peripheral to the circular muscle
bundles. In the Octopus species, however, the arrange-
ment is reversed; a distinct series of circular muscle bun-
dles are located peripheral to the meridional muscle bun-
dles (Compare Figs. 1 and 2).

In addition to the circular muscle bundles of the ace-
tabular wall, a mass of circular muscle forms a sphincter
located adjacent to the inner surface at the level of the
narrow orifice that connects the infundibulum to the ac-
etabulum (Figs. 1, 2). A secondary sphincter is also evi-
dent near the junction between the outer surfaces of the
walls of the acetabulum and infundibulum and has a
cross-sectional area that is approximately 1 0% of the area
of the primary sphincter.

The meridional muscle fibers project from a point
near the apex of the acetabular roof toward the sphincter
muscles as an array of flat bundles that lie between the
radial muscle fibers. When the outer surface of the ace-
tabular roof is viewed in a grazing frontal section, the
meridional muscle fiber bundles appear to be arranged in
a stellate pattern (Fig. 4). Many of the meridional muscle
fibers insert on the outer connective tissue layer at the
level of the sphincter muscles. Some meridional muscle
fibers extend into the wall of the infundibulum.

The arrangement of muscle fibers in the wall of the
infundibulum is similar to that of the acetabular wall de-
scribed above. Radial muscle fibers extend across the
wall from their origins and insertions on the inner and
outer connective tissue layers of the infundibulum. The
radial muscles pass between a series of flat bundles of
circular muscle fibers located adjacent to the inner sur-
face of the infundibular wall (Fig. 5). Meridional muscle
fibers are also present in the infundibular wall (Fig. 1,
Fig. 5). Many originate on the outer connective tissue
layer at the level of the sphincter muscles and extend to-
ward their insertion at the margin of the infundibulum
while others appear to be extensions of the meridional
fibers of the acetabular wall. The bundles of meridional
fibers are flat and are interwoven between the radial mus-
cle fibers.

Sucker connective tissue. The two major components
of the connective tissues of the sucker are an array of
crossed connective tissue fibers embedded in the muscu-
lature of the acetabular roof, and the inner and outer
connective tissue capsules. Thin layers of connective tis-
sue also surround the circular and meridional muscle
bundles of the sucker. It is likely that the connective tis-
sue fibers observed in the sucker are collagenous because

SUCKER FUNCTIONAL MORPHOLOGY

129

they appear birefringent when viewed with polarized
light microscopy and show staining characteristics typi-
cal of collagen.

The inner and outer connective tissue capsules are
compact layers of fibers that enclose the sucker muscula-
ture. The layers appear to be arranged as a crossed-fiber
array when viewed in transverse sections that graze the
inner or outer surface of the wall (Fig. 6). At the level of
the sphincter muscles, fibers of the outer connective tis-
sue layer penetrate into the musculature of the sucker
wall (Fig. 5). These fibers branch repeatedly and extend
to the primary sphincter muscle, dividing it into fasci-
cles. The extension of the outer connective tissue capsule
that encloses the infundibulum is thinner than that of the
acetabulum. The inner connective tissue capsule extends
from the acetabulum to the infundibulum without any
appreciable change in thickness.

In addition to the connective tissue layers encasing the
sucker, crossed connective tissue fibers are present in the
musculature of the roof of the acetabulum (Figs. 1, 3).
These fibers extend between the inner and outer connec-
tive tissue capsule at oblique angles to the radial muscle
fibers. They are reminiscent of the "intermuscular" con-
nective tissue fibers described by Gosline and Shadwick
(1983a, b) and Bone et cil. (1981) in the mantle of squid
and cuttlefish and those described by Kier (1989) and
Kieret al. (1989) in the fins of squid and cuttlefish. How-
ever, the angle they make with the radial fibers is not con-
stant (Fig. 3). These connective tissue fibers do not occur
in the acetabular wall. The boundary between the acetab-
ular roof and the acetabular walls includes a particularly
robust band of intermuscular connective tissue fibers,
and the wall is thinner at this point (Fig. 1 ).

Sucker epithelium. Several distinct zones of epithe-
lium are present on the sucker (see also Girod, 1884;
Guerin, 1908; Nixon and Dilly, 1977; Packard, 1988).
The epithelium lining the infundibulum consists of tall
columnar cells resting on a basal lamina and the inner
connective tissue capsule. These cells secrete a tough,
chitinous cuticle (Hunt and Nixon, 1981). The surface
of the cuticle bears numerous tiny denticles or pegs, each
secreted by a single columnar epithelial cell (see Nixon
and Dilly, 1977). The epithelial cells lining the radial
grooves of the infundibulum are cuboidal, and the cuti-
cle lining the grooves lacks denticles. The cells of the epi-
thelium lining the acetabulum are cuboidal. In addition,
the denticles are rudimentary or absent from the cuticle
lining the acetabulum. The transition between the epi-
thelial surfaces of the infundibulum and acetabulum oc-
curs at the level of the primary sphincter muscle (Figs. 1 ,
2, 5). Another transition is observed in the groove that
separates the rim and the infundibulum. The epithelial
cells in the groove are cuboidal and the cuticle is thin and
lacks denticles. The epithelium covering the pillows and
folds of the rim is columnar and the underlying dermis

is loose and folded. An additional differentiation of the
epithelium was observed in a zone surrounding the
sucker rim. Cells in this zone showed intense staining by
Mowry's colloidal iron stain (Humason, 1979) for acid
mucopolysaccharides(Fig. 7).

Girod ( 1 884) described the infundibulum of the suck-
ers of Octopus vulgaris as being covered by numerous
small "hillocks" of tall columnar epithelial cells and cuti-
cle with denticles. He describes the epithelium between
the hillocks as being flattened. Although small hillocks
are visible on the surface of the infundibulum or on shed
sucker linings of the species we examined, no differenti-
ation of the epithelium was observed between the hill-
ocks. A flattened epithelium was only observed in the
radial grooves.

Extrinsic sucker musculature. The suckers are at-
tached to the arms by a series of extrinsic muscle bundles
(see also Guerin, 1908). A group of major extrinsic mus-
cle bundles is associated with each sucker and originates
on the connective tissue sheath surrounding the arm
musculature (Kier, 1 988) and extends orally to converge
on the sucker (Fig. 8). These bundles insert on the outer
connective tissue capsule of the sucker at the level of the
sphincter muscle (Figs. 1, 2). The extrinsic muscle bun-
dles are, in turn, surrounded along much of their length
by a sheet of circumferential muscle fibers (Fig. 8). In
addition to the major extrinsic muscle bundles illus-
trated in Figure 8, a medial group of smaller diameter
extrinsic muscle bundles was observed in the region en-
closed by the major extrinsic bundles. Although many
are oriented parallel to the major bundles, some follow
oblique courses, crossing from one side to the other.

Kinematics

Octopus suckers are capable of a wide range of move-
ments. The animals explore their environment with their
arms, holding their suckers extended and splayed out.
The muscular base that attaches the sucker to the arm
can elongate to twice its resting length, extending the
suckers away from the arm. Sometimes individual suck-
ers were observed to probe through small openings such
as a screen, then extend fully and tilt up and down or
side to side. If the sucker is stimulated lightly, it either
extends to attach to the stimulus or withdraws, always
orienting so that the infundibulum faces the object.
When the octopus is active, the infundibuli of the suckers
are flattened. Sucker "footprints" in wax show that the
entire infundibulum is pressed firmly against the substra-
tum during attachment. When the animal is at rest, the
infundibuli are cone-shaped.

An octopus can grip nearly any size object with its
suckers. They seem to prefer large flat surfaces but can
easily grip irregular objects and objects smaller than their
suckers. When manipulating threads or thin sheets, the

130

W. M. K.IER AND A. M. SMITH

Figure 2. Photomicrograph of a transverse section of a sucker from Eledone cirrosa in the region of
the primary and secondary sphincter muscles (SI, S2) and the acetabular wall. The radial muscles (R)
extend from the inner connective tissue capsule (1C) to the outer connective tissue capsule (OC). Inter-
woven between the radial muscle fibers are meridional muscles (M) and circular muscles (C). An extrinsic
muscle (E) inserts on the outer connective tissue capsule adjacent to the secondary sphincter muscle. The
photomicrograph was made using bnghtfield microscopy of a 15 ^m-thick paraffin section stained with
Milligan tnchrome. The scale bar equals 0.5 mm.

Figure 3. Photomicrograph of a transverse section of a sucker of Octopus bimaculoides/bimaculatus
in the region of the acetabular roof. The crossed connective tissue fibers (CC) extend across the roof from
the inner (1C) to the outer (OC) connective tissue capsule at oblique angles to the radial muscle fibers.
Meridional muscle fibers (M) are also visible adjacent to the outer surface of the acetabular roof. The
intersection of the meridional bundles at the axis of radial symmetry is apparent in the top of the micro-

SUCKER FUNCTIONAL MORPHOLOGY

131

suckers sometimes fold so that the two halves of the in-
fundibulum grasp the object like a mittened hand (see
also Packard, 1988). A sucker can grip a strand of fishing
line and pull on it with surprising force. When it does
this, the crease of the fold is usually parallel to the long
axis of the arm. Suckers are often observed to fold over
the corner of an object without noticeably weakening the
force of attachment. A striking example of this occurs
when a sucker is attached to the end of a cylinder with a
smaller diameter than that of the sucker. Here the perim-
eter of the infundibulum folds around the side of the cyl-
inder while the remainder of the infundibulum presses
flat against the end.

The movies allowed us to distinguish and quantify
changes in the sucker's dimensions during suction, par-
ticularly the diameter of the large sphincter. We consid-
ered the diameter of the orifice leading to the acetabulum
to be the same as the diameter of the inner surface of the
sphincter. We measured the diameter of the rim and the
diameter of the orifice when the sucker was attached (x)
and when it was relaxed (x ). When gripping, the rim
diameter increased from its resting state (x = 1 .26xo 94 ;
r = 0.82) as does the orifice diameter (x = 1.48x 087 ; r
= 0.80). The movies also showed that the roof of the ace-
tabulum does not press against the substratum during at-
tachment, contrary to the mechanism reported by Pack-
ard (1988).

Discussion

Principles of forming a suction attachment

Suckers attach to the substratum by forming a seal at
the rim and reducing the pressure in the acetabular cav-
ity. This decrease in pressure has been measured and can
account for all of the attachment force of octopus suckers
(A. M. Smith, in prep). The acetabular cavity is filled
with water, and the ability of water to withstand this de-
crease in pressure is critical to sucker function. The dis-
tinction between water-filled and air-filled suckers has
not been emphasized in previous studies of sucker func-
tion (see Denny, 1988).

A sucker filled with air has different mechanical re-
quirements from one that is filled with water. An air-
filled sucker must significantly increase its enclosed vol-
ume to decrease the pressure in the cavity. Starting from
0. 1 MPa ambient pressure ( 1 atm), doubling the volume

would halve the pressure to 0.05 MPa, increasing the vol-
ume ten times would only reduce the pressure to 0.01
MPa. To create a vacuum, the cavity must be reduced to
a negligible volume before attachment. The lowest possi-
ble pressure inside such a sucker would be a vacuum (0
MPa). At normal ambient pressure (0. 1 MPa), the force
holding this sucker and the substratum together would
be 0.1 MPa multiplied by the area exposed to the
vacuum.

Octopus suckers operate in water rather than air,
which leads to two important functional consequences:
first, the sucker can decrease pressure without detectably
expanding, and second, the pressures generated will not
necessarily be limited to a vacuum. Water is essentially
inexpansible at physiological stresses because of its cohe-
sive strength. Therefore, water resists the activity of the
muscles that expand the enclosed volume. Thus, if more
water does not leak into the sucker, the muscles involved
in generating suction contract isometrically, reducing
the water's pressure. As long as the water adheres to all
surfaces, the sub-ambi