E X O S K E L E T A L A B R A S I O N AS AN INDICATOR OF
R E P R O D U C T I V E READINESS IN T H E SPIDER CRAB
LIBINIA EMARGINATA
J. S. B. Ahl, H. Laufer, A. J. Ahl, a n d P. Takac
A B S T R A C T
Patterns o f exoskeletal abrasion on the carapace o f long-clawed males of the spider crab
Libinia e m a r g i n a t a appeared to follow specific sequences throughout the year. T h e degree o f
abrasion coincided with the enlargement o f the reproductive system and increased levels o f
methyl farnesoate in the h e m o l y m p h . The patterns o f abrasion apparently m a r k e d the time
elapsed since the last molt, and w h e n the abrasion extended b e y o n d the midcarapace, it appeared
to indicate increased reproductive readiness.
In the spider crab Libinia emarginata, reproductively active males are recognized by
their claw morphology and the condition of
the exoskeleton on the dorsum o f the carapace (cephalothorax). Dominant males are
large-bodied, have long claws (the length of
the propodus exceeds that o f the carapace),
and the exoskeleton is abraded to the extent
that much of the epicuticle and setae have
been removed exposing the exocuticle of
the dorsal carapace ( H o m o l a e t al., 1991;
Sagi et al., 1993). These abraded males aggressively compete for receptive females
(Sagi et al., 1994), have reproductive systems that may constitute as much as 2 . 5 6.0% o f their body wet-weight, and have
high hemolymph levels o f methyl farnesoate (MF), which is a putative reproductive
hormone (Laufer et al., 1987).
The claw morphology is the result o f differential growth during postlarval development that is especially accelerated during
the final morphometric molt to the longclawed form (Aldrich, 1974). Newly molted
long-clawed males appear velvety due to
the dense covering of scales and serrulate
setae, which are collectively referred to as
the epicuticle by H o m o l a e t al. (1991).
These males, unlike their abraded counterparts, do not mate, have small reproductive
systems (�2% wet body weight), and have
low hemolymph levels o f M F (Sagi et al.,
1994). After the morphometric molt, unabraded long-clawed males must undergo a
gradual transformation, externally and internally, before becoming reproductively
active.
In this study, we describe a patterned disappearance of the epicuticle from the cara-
pace, w h i c h s e e m s to coincide with increases in t h e s i z e o f t h e r e p r o d u c t i v e s y s t e m , as
w e l l a s i n c r e a s e s in t h e l e v e l s o f M F p r e s e n t
in the h e m o l y m p h . W i t h the c o m p l e t i o n o f
all o f t h e s e p r o c e s s e s , the a n i m a l s r e a c h full
behavioral
ductives.
competence
as
primary
repro-
M A T E R I A L S AND M E T H O D S
A n i m a l s . - S p e c i m e n s o f Libinia e m a r g i n a t a were collected from L o n g Island S o u n d o f f Connecticut by the
staff o f the N e w E n g l a n d Utilities Millstone P o w e r
Plant Environmental Laboratory, Waterford, Connecticut, at 2 - w e e k intervals from April through D e c e m b e r
1993 and 1994. In the first year o f study, the crabs
were caught with an otter trawl, but in the following
year, they were obtained with baited lobster pots. The
animals were transported to the University o f Connecticut, Storrs, where large-bodied long-clawed males
were immediately evaluated after being chilled on ice
until quiescent. The lengths of the claws and carapaces
were m e a s u r e d to the nearest 0.1 mum with a vernier
caliper. The crabs were weighed to the nearest 0.01 g,
after which a sample o f h e m o l y m p h was taken.
L o n g - c l a w e d males (N = 330) were separated into
groups according to the pattern o f abrasion on the carapace. Identification o f the abraded regions o f the exoskeleton was according to the terminology o f Felgenhauer (1992).
G o n a d o s o m a t i c Index ( G S I ) . - T h e GSI is the wet
weight o f the reproductive system (testes, vas deferens,
and accessory glands) expressed as a percentage o f the
body weight.
Hemolymph Levels of Methyl Farne.roate ( M F ) . - P r i o r
to dissection, 2-ml samples o f h e m o l y m p h were taken
from the base o f the walking legs, using a 5-ml syringe
with an 18-gauge needle. The samples were transferred
to 15-ml borosilicate tubes containing ice-cold acetonitrile (5 ml) a n d 4 % N a C l (2 ml). The samples were
vigorously mixed, and then stored at - 2 0 ° C if they
were not extracted immediately. Prior to extraction, an
aliquot ( I S - 2 5 ng) o f cis-trans MF, a nonbiologically
active isomer, was added to each tube as an internal
standard (Sagi et al., 1993). T h e samples were extracted twice with 500 u.1 o f hexane. The extractions were
pooled, then stored in tightly capped 1.5-ml amber
glass vials at -20°C. Before analysis by High Performance Liquid Chromatography (HPLC), each sample
volume was reduced by evaporation under nitrogen to
approximately 100-250 pl. All of the sample was then
injected into a Waters HPLC system (model 501 pump
and Waters 486 absorbance detector set at 214 nm).
Cis-trans and all-trans MF, the biological isomer, were
separated using normal phase with a 5-wm MicrosorbMV column (Rainin Instrument Co.), and running solvent of 1% diethyl ether in hexane (flow rate of 1.5
ml/min, 1,000-1,500 psi). Elution times for cis-trans
and all-trans MF were determined by running a mixture of the isomers as an external standard. Peak areas
were calculated with Maxima 820 software. The
amount of MF present in each sample was determined
by comparing the peak area of the internal standard
(cis-trans MF), which represented a known amount, to
the peak area of the all-trans MF.
Data Analysis.-Significant differences between mean
GSIs and between mean MF levels in the hemolymph
were determined using Student's t-test (Sokal and
Rohlf, 1969) and Sigma Plot® software.
ed. This pattern occurred over the hepatic
region and extended posteriorly to the mesobranchial and urogastric regions, and always included the " C h e e k s , " " P a t e , " and
" S p i n e " patterns. Completely (C) abraded
crabs were those animals lacking epicuticle
over much o f the carapace (greater than
one-half exposure).
Males exhibiting abrasion patterns were
present in all field collections (Table 1).
However, recently molted unabraded individuals (U) (without " C h e e k s " ) were conspicuously absent during the late spring to
early fall months (June-October). In addition, completely abraded males (C) were
scarce in the early spring (April-May) and
late fall (December) collections.
RESULTS
The mean GSIs of male crabs at each
stage o f the abrading process are shown in
Fig. 2. The GSI was significantly larger (P
� 0.05) in those animals which exhibited
an abrading pattern on the dorsum of the
carapace: Pate (1.56 ± 0.098%), Spine
(1.71 ± 0.08%), Half (2.12 ± 0.08%), and
Complete (2.02 ± 0.080%). The Unabraded
and Cheeks animals had GSIs that were less
than 1 % o f their body weight (0.49 ±
0.044% and 0.76 ± 0.073%, respectively).
Pattern o f Abrasion
L o n g - c l a w e d large c a r a p a c e ( 6 5 - 1 0 0
m m - C L ) males were separated into six categories based upon the degree and pattern
of abrasion present on the carapace (Fig. 1 ).
Recently molted animals were completely
covered with a dense velvety epicuticle, and
were called unabraded (U). The first and
smallest area o f abrasion appeared on the
suborbital portion of the carapace immediately lateral and inferior to the sulcus of the
eyestalk. The exposed area was oval in
shape and light colored, giving the animal
the appearance o f having " c h e e k s . " This
pattern was called " C h e e k s " (Ch). This initial pattern o f abrasion was always present
even when subsequent abrasion occurred on
other parts o f the carapace. The next area
to be affected was located immediately posterior to the rostrum and sulci o f the eyestalks on the protogastric region on the dorsum of the carapace. This pattern was
somewhat circular giving the animal the appearance o f being " b a l d . " This pattern was
n a m e d " P a t e " (P). In a n o t h e r pattern,
which was always observed as an extension
o f the " P a t e , " abrading followed the line
of the middorsal spines extending from the
protogastric region to the posterior edge of
the c a r a p a c e , a n d w a s r e f e r r e d to as
" S p i n e " (S). Crabs with abrasion extending
over one-third to one-half o f the dorsal carapace were identified as " H a l f " (H) abrad-
Gonadosomatic Indices (GSIs) and
Abrasion Pattern
M F H e m o l y m p h Levels and Abrasion
Patterns
The mean ( ± S E ) hemolymph levels o f
M F show an increasing trend as the animals
become more abraded (Fig. 3). The Unabraded and Cheeks animals have significantly lower (P :5 0.05) M F levels (0.474
± 0.110 ng/ml and 0.949 ± 0.258 ng/ml,
respectively) compared to those animals
with the most abrasion, such as the Half
(3.038 ± 0 . 8 9 3 ng/ml) and Complete
(3.334 ± 0 . 9 1 8 ng/ml). Animals with the
Pate and Spine patterns had M F levels that
were intermediate (1.241 ± 0.215 ng/ml
and 1.482 ± 0.139 ng/ml, respectively) to
those o f the Cheeks, Half, and Complete
patterns, but were significantly greater than
that o f the Unabraded Pattern.
DISCUSSION
Male L. e m a r g i n a t a undergo a differentional molt to a long-clawed form, prior to
becoming primary reproductives (Homola
Fig. 1. Patterns o f epicuticlar abrasion on the carapace o f the spider crab Libinia e m a r g i n a t a . a, Unabraded;
b, Cheeks; c, Pate; d, Spine; e, Half; f, Completely abraded. E a c h scale = 10 m m .
Table 1. N u m b e r o f long-clawed large-carapace male
Libinia e m a r g i n a t a collected each month that were undergoing abrading.
et al., 1991; Sagi et al., 1993). When they
are newly molted, they are covered with a
thick velvety epicuticle, which becomes
worn off (abraded) over time. We have
found that abrading follows a specific sequence o f patterns that also coincides with
the enlargement of the reproductive system
and increased levels of M F in the hemolymph. This suggests that the patterns mark
time that has elapsed since the last ecdysis
and are not random. This is further substantiated by the conspicuous absence from
the sampling population o f crabs with specific abrading patterns at certain times of
the year.
Recently molted unabraded male crabs
were entirely absent from the collections
during August through October, but were
commonly present during N o v e m b e r and
December. This suggests that there is an annual abrading cycle which follows the annual molting event that takes place during
the late summer and early fall (Hinsch,
1972). Molting crabs are gregarious, forming aggregations of hundreds o f individuals
(DeGoursey and Stewart, 1985) that molt
somewhat synchronously. Thus, newly
molted long-clawed males, most likely belonging to the same cohort, would exhibit
early stages of abrading (Cheeks and Pate)
the following spring. They become more
abraded as summer progresses. If molting
were to take place year-round, one would
find newly molted, unabraded long-clawed
males continuously represented in the population, but that was not the case.
Spider crabs in Long Island Sound are
k n o w n to migrate from deep water (100 m)
where they overwinter to shallow water ( 5 8 m) where they aggregate for mating and
molting (DeGoursey and Auster, 1992).
Since the mating aggregations are com-
Fig. 2. Gonadosomatic indices (mean and SE) of
long-clawed male Libinia emarginata with different
patterns of epicuticular abrasion. U, Unabraded; Ch,
Cheeks; P, Pate; S, Spine; H, Half; C, Completely
abraded.
prised o f females and large males, it is possible that the scarcity o f abraded males in
the spring is due to their participation in
these mating aggregations, which segregates them from the population of crabs that
were collected.
It is thought that male spider crabs exhibiting the enlarged or elongated claws are
in t e r m i n a l a n e c d y s i s ( H a r t n o l l , 1963;
Hinsch, 1972). This has been tested and
verified in L. emarginata by the lack o f
molting in these males after eyestalk ablation (Takac et al., unpublished), which is a
well-known technique utilized to initiate
molting in Crustacea (Zeleny, 1905). In terminally molted animals, the epicuticle covering the exoskeleton is gradually worn
Fig. 3. H e m o l y m p h levels (mean and SE) of methyl
farnesoate present in long-clawed male Libinia emarg i n a t a at different stages of abrading. U, Unabraded;
Ch, Cheeks; P, Pate; S, Spine; H, Half; C, Completely
abraded.
away. Since the epicuticle is secreted by the
epidermis only during premolt, it is not replaced if it is removed or d a m a g e d (Halcrow, 1988) unless the animal undergoes
another molt cycle.
The loss of the epicuticle in terminally
molted animals is probably due to mechanical abrasion that occurs during grooming
and social behavior. Although grooming behavior is poorly developed in brachyuran
decapods (Bauer, 1981), the Cheeks and
Pate patterns could be produced by repeated
rubbing and picking motions with the chelae, which we have observed in animals
maintained in aquaria. The other abrading
patterns that occur on the dorsum of the
carapace may be the consequence o f aggregative or podding behavior, which is characterized by the active intermingling o f
many crabs piling one on top of the other
(Carlisle, 1957; DeGoursey and Auster,
1992; Stevens et al., 1992, 1994).
In conclusion, an abraded carapace in the
adult male spider crab L. e m a r g i n a t a m a y
be evidence of anecdysis as well as reproductive readiness. The progression of
abrading patterns discussed here indicates
the passage of time since the last molt, and
seems to be an external indicator that males
are undergoing physiological transitions.
Abrading can therefore be used to identify
those spider crab males that are switching
their developmental program from growth
(molting) to anecdysis and active reproduction.
ACKNOWLEDGEMENTS
The research reported here was supported in part by
grants from the Sea Grant College Program, National
Oceanographic and Atmospheric Administration
(NOAA). We thank D. Dodge and the personnel of the
Environmental Control Laboratory of the Millstone
Nuclear Power Station, Waterford, Connecticut, for
generously providing living specimens of L. emarginata.
LITERATURE C I T E D
Aldrich, J. C. 1974. Allometric studies on energy relationships in the spider crab Libinia e m a r g i n a t a
( L e a c h ) . - B i o l o g i c a l Bulletin 147: 2 5 7 - 2 7 3 .
Bauer, R. T. 1981. G r o o m i n g behavior and m o r p h o l ogy in the decapod Crustacea.�Journal o f Crustacean Biology 1: 153-173.
Carlisle, D. B. 1957. On the hormonal inhibition o f
m o u l t i n g in d e c a p o d Crustacea. II. T h e terminal
a n e c d y s i s in c r a b s . - J o u r n a l o f the M a r i n e Biology
Association o f the United K i n g d o m 36: 2 9 1 - 3 0 7 .
D e G o u r s e y , R. E., and P. J. Auster. 1992. A mating
aggregation o f the spider crab, Libinia e m a r g i n a t a . - J o u r n a l o f Northwest Atlantic Fisheries Science
13: 7 7 - 8 2 .
, a n d L. L. Stewart. 1985. Spider crab podding
b e h a v i o r and mass m o l t i n g . - U n d e r w a t e r Naturalist, Bulletin o f the A m e r i c a n Littoral Society 15: 1 3 16.
Felgenhauer, B. E. 1992. External a n a t o m y and integu m e n t a r y structures.�In: F. W. Harrison, treatise
ed., Microscopical a n a t o m y o f invertebrates. F. W.
Harrison and A. G. Humes, eds. Vol. 10. D e c a p o d
Crustacea. Pp. 7�43. Wiley-Liss, Inc., N e w York,
N e w York.
H a l c r o w , K . 1988. A b s e n c e of epicuticle from the repair cuticle p r o d u c e d by four malacostran crustac e a n s . - J o u r n a l o f Crustacean Biology 9: 346�354.
Hartnoll, R. G. 1963. The biology of the M a n x spider
c r a b s . - P r o c e e d i n g s of the Zoological Society o f
L o n d o n 141: 4 2 3 - 4 9 6 .
Hinsch, G. W. 1972. Some factors controlling reproduction in the spider crab, Libinia emarginata.�Biological Bulletin 143: 3 5 8 - 3 6 6 .
H o m o l a , E., A. Sagi, and H. Laufer. 1991. Relationship of c l a w form and exoskeleton condition to reproductive system size a n d methyl farnesoate in the
male spider crab, Libinia emarginata.�Invertebrate
R e p r o d u c t i o n and D e v e l o p m e n t 20: 2 1 9 - 2 2 5 .
Laufer, H., D. Borst, F. C. Baker, C. Carrasco, M. Sinkus, C. C. Reuter, L. W. Tsai, a n d D. S. Schooley.
1987. Identification o f a juvenile hormone-like c o m p o u n d in a c r u s t a c e a n . - S c i e n c e 235: 2 0 2 - 2 0 5 .
Sagi, A., E. H o m o l a , and H. Laufer. 1993. Distinctive
reproductive types o f male spider crabs Libinia
e m a r g i n a t a differ in circulating and synthesizing
methyl farnesoate.�Biological Bulletin 185: 1 6 8 173.
, J. S. B. Ahl, H. Danaee, and H. Laufer. 1994.
Methyl farnesoate levels in male spider crabs exhibiting active reproductive b e h a v i o r . - H o r m o n e s and
B e h a v i o r 28: 2 6 1 - 2 7 2 .
Sokal, R. R., and F. J. Rohlf. 1969. Introduction to
b i o s t a t i s t i c s . - W . H. F r e e m a n � Co., San Francisco,
California. Pp. 1-368.
Stevens, B. G., W. E. Donaldson, and J. A. Haaga.
1992. First observations o f podding behavior for the
Pacific lyre crab Hyas lyratus (Decapoda: Majidae).�Journal o f Crustacean Biology 12: 193�195.
, J. A. Haaga, and W. E. Donaldson. 1994. Aggregative mating in Tanner crabs, Chionoecetes
b a i r d i . - C a n a d i a n Journal of Fisheries and Aquatic
Sciences 51: 1 2 7 3 - 1 2 8 0 .
Zeleny, C. 1905. C o m p e n s a t o r y r e g u l a t i o n . - J o u r n a l
o f Experimental Zoology 2: 1-102.
RECEIVED: 2 0 O c t o b e r 1995.
ACCEPTED: 28 F e b r u a r y 1996.
Addresses: (JSBA, HL, A J A ) D e p a r t m e n t o f Molecular and Cell Biology, U - 1 2 5 , University o f Connecticut, Storrs, Connecticut 06269, U.S.A.; (PT) Institute
o f Z o o l o g y and Ecozoology, Bratislava, Slovakia.