D E V E L O P M E N T OF N E U R O N S EXHIBITING FMRFAMIDERELATED I M M U N O R E A C T I V I T Y IN T H E C E N T R A L
N E R V O U S S Y S T E M O F L A R V A E OF THE SPIDER CRAB
HYAS ARANEUS L. (DECAPODA: MAJIDAE)
S t e f f e n H a r z s c h a n d R a l p h R. D a w i r s
A B S T R A C T
The ontogeny of neuropeptide-expressing neurons during larval development of the crustacean central nervous system is as yet poorly understood. Recently, h o m o l o g u e s o f the molluscan
neuropeptide F M R F a m i d e have b e e n isolated from the central nervous system o f adult crustaceans. In order to identify sets o f neurons which might express F M R F a m i d e - r e l a t e d neuropeptides and to follow their d e v e l o p m e n t , whole m o u n t preparations o f the brain and ventral
n e r v e cord o f laboratory-reared spider crab larvae (Hyas a r a n e u s ) were labeled with an antibody
against the tetrapeptide F M R F a m i d e . The distribution and morphology of F M R F a m i d e - l i k e
immunoreactive neurons, their p r i m a r y neurites, and neuropils were m a p p e d throughout develo p m e n t of the stages zoea I a n d zoea 2. Ontogenetic alterations in the staining pattern were
recorded and the results c o m p a r e d to the distribution o f F M R F a m i d e - l i k e immunoreactivity in
other crustaceans.
The tetrapeptide F M R F a m i d e (Phe-MetArg-Phe-NH,) and FMRFamide-related
peptides (FaRPs) form a large neuropeptide
family which is widely distributed among
invertebrates and vertebrates (Price and
Greenberg, 1989; Greenberg and Price,
1992; Walker, 1992). An increasing amount
o f literature on the structural diversity and
neurohormonal action o f invertebrate FaRPs and other peptides in coelenterates (review by G r i m m e l i k h u i j z e n e t al., 1992),
molluscs (review by M u n e o k a and Kobayashi, 1992), plathyelminths (review by Fairweather and Halton, 1991), insects (review
by Nassel, 1993; Nassel et al., 1994), the
chelicerate Limulus (Gaus et al., 1993;
Groome, 1993), and crustaceans (review by
Keller, 1992) highlights a growing appreciation of the importance o f these substances. In crustaceans, the FaRPs identified
so far are 7 - 9 amino acids in length and
have the tetrapeptide core FLRFamide in
common. The two octapeptides which have
been isolated from lobster pericardial organs (F, and F.) (Trimmer et al., 1987) differ by single amino extensions from two rec e n t l y identified crayfish h e p t a p e p t i d e s
(NF, and DF,) (Mercier et al., 1993), but
all four peptides share a c o m m o n C-terminal hexapeptide amide core (RNFLRFamide). On the other hand, the only completely sequenced brachyuran F a R P (Krajniak, 1991) is a nonapeptide which has a
serine for asparagine substitution within the
macruran hexapeptide core. Immunocytochemical studies using antibodies against
FMRFamide-like substances have revealed
that FaRP-containing neurons are widely
distributed throughout the crustacean central nervous system, including the stomatogastric nervous system ( M a r d e r e t al.,
1987; Coleman et al., 1992), ventral nerve
cord ( D i r c k s e n e t al., 1987; Arbiser and
Beltz, 1991; Elofsson, 1992), eyestalk ganglia (Jacobs and Van Herp, 1984; Mangerich et al., 1987), and brain (Callaway et al.,
1987; Mangerich and Keller, 1988; Schmidt
and Ache, 1994). However, the highest peptide concentrations have been detected in
the pericardial organs, and, therefore, these
substances are thought to act as circulating
hormones (Kobierski et al., 1987; Mercier
et al., 1991). The release of crustacean
FaRPs has been reported to exert a cardioexcitatory effect (Mercier and Russenes,
1992; Skerrett et al., 1995), to enhance synaptic transmission in abdominal extensor
muscles (Mercier et al., 1990; Friedrich et
al., 1994; Skerrett et al., 1995) and the dactyl opener muscle (Worden et al., 1995),
and to modulate muscle receptor organs
(Pasztor and Golas, 1993). Furthermore,
FaRPs are among the peptides which modulate the motor pattern in the stomatogastric
system (Hooper and Marder, 1984; Katz
and Harris-Warrick, 1990).
However, apart from this wealth o f literature on the distribution of FaRPs in the
adult crustacean nervous system, virtually
nothing is known about the embryonic and
larval development o f neurons containing
FMRFamide-like peptides. In general, the
ontogeny of crustacean neurotransmitter
systems is poorly understood. Current investigations in this field outline only the
morphogenesis of neuronal structures
which are immunoreactive to antibodies
against the biogenic amine serotonin, and
against another peptide, proctolin (Beltz et
al., 1990; Helluy et al., 1993; Harzsch and
Dawirs, 1995). Nevertheless, the brachyuran larval central nervous system is a particularly attractive model to study its development and metamorphic respecification
( " N e u r o m e t a m o r p h o s i s " ; Harris, 1990),
since the larvae undergo a striking double
metamorphosis which is characterized by
fundamental alterations in morphology and
behavioral patterns. Thus, several aspects o f
neurogenesis in spider crab larvae have already been described which are related to
the metamorphic events (Harzsch and Dawirs, 1993, 1994, 1995). The scope o f the
present study is to identify F M R F a m i d e like immunoreactive structures in the larval
brain and ventral nerve cord and to map
their distribution and morphology through
early larval development. Ontogenetic alterations in the staining pattern are recorded
and the results compared to the distribution
o f FMRFamide-like immunoreactivity in
other crustaceans.
MATERIALS AND METHODS
Z�rtYtf.—Ovigerou.s females of Hyas araneus L. were
dredged from the "Tiefe Rinnc" south of the Island of
Helgoland (North Sea, German Bight) and kept in
flow-through systems with natural sea water in the laboratory. Newly hatched larvae were reared at a constant temperature of 12°C, a light-dark cycle of 10:14
h and fed on brine shrimp nauplii, Artemiu sp. The
rearing procedure has been described in detail by Anger and Nair (1979) and Anger and Dawirs (1981).
Larval development is constant under these conditions
and an exact determination of the larval stage, age, and
even molt cycle is possible (Anger, 1983). The larval
stages and their respective duration at the given temperature are: zoea I (I 1 days), zoea 2 (14 days, metamorphosis I ), megalopa (29 days, metamorphosis 2),
crab 1. A description of the larval morphology has
been provided by Christiansen (1973).
Immunocytochemistry.-FMRFamide-like immunoreactive neurons were labeled with a polyclonal antiserum to the sequence Arg-Phe-amide in free-floating
w h o l e mounts o f the larval brain and ventral nerve
cord (VNC). The antiserum was obtained by immunizing rabbits with synthetic F M R F a m i d e which was
coupled via glutaraldehyde to b o v i n e thyroglobulin
(Grimmelikhuijzen and Spencer, 1984). Incubation of
the a n t i s e r u m w i t h s e p h a r o s e - b o u n d F M R F a m i d e ,
F L R F a m i d e , or R F a m i d e abolished all staining. The
antiserum has been shown to be m o s t sensitive to the
C-terminal sequence - R F a m i d e (-Arg-Phe-NH2; Grimm e l i k h u i j z e n , 1985). It has b e e n u s e d to label
F M R F a m i d e immunoreactive structures in the m e d u s a
Polyorchi.s penicíllatus Agassiz (see G r i m m e l i k h u i j z e n
and Spencer, 1984), in several species o f H y d r a (Grimmelikhuijzen, 1985), and in a n u m b e r o f polychaete
species (Windoffer, 1992). Specifity controls o f the
a n t i - F M R F a m i d e antiserum included omission of the
first antibody and preincubation o f the antiserum with
1 0 - ' M F M R F a m i d e (16 h, 4°C). In these controls,
neuronal structures were not labeled.
The larvae were fixed for 16 h in 4 % paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4, PB) at
4°C. T h e i r carapaces were punctured previously to improve penetration of the fixative. T h e brain and V N C
were then r e m o v e d using microtweezers. T h e optic
lobes and abdominal ganglia 2 - 6 had to be omitted
from this study b e c a u s e it was impossible to dissect
them d u e to their small size. S p e c i m e n s were w a s h e d
in several changes of buffer for 2 h and afterward
preincubated in 0.1 M phosphate buffered saline (PBS)
containing 1% normal goat serum ( N G S ) and 0 . 1 5 %
Triton X-100 ( P B S - T X ) for 2 h at 21°C. T h e antiF M R F a m i d e antibody (1:1,000 in P B S - T X ) was applied for 16 h at 4°C. T h e n the specimens were incubated in a goat anti-rabbit bridge antibody (1:50, Sigma) for 3 h at 21 °C and subsequently for another 3 h
in the P A P antibody c o m p l e x (1:500, Sigma), both in
0 . M PBS and 1% NGS. After 15 min, preincubation
in 0 . 0 1 3 % d i a m i n o b e n z i d i n e ( D A B , Sigma), the tissues w e r e treated with 0 . 0 1 3 % D A B and a reagent
containing hydrogen peroxide, cobalt chloride, and
nickel chloride ( A m e r s h a m International, United Kingd o m , R P N 20) for 7 - 9 min to reveal the peroxidase
label. Finally, w h o l e - m o u n t preparations were dehydrated, mounted in Eukitt (Riedel-de Haen, AG, 30926
Seelze, G e r m a n y ) and covered with a coverslip. Preparations were o b s e r v e d with a Polyvar microscope
(Reichert-Jung) and labeled neurons and neuropils
d r a w n using a c a m e r a lucida. In the larval stages investigated, zoea 1 and zoea 2, the pattern o f F M R F a m ide-like immunoreactivity is reproducible between different specimens and is therefore illustrated in semischematic drawings. These are based on the analysis
o f at least 10 specimens for each stage. The n o m e n clature o f central nervous structures has been slightly
modified from that previously used by H a r z s c h and
Dawirs (1993) for larvae o f C a r c i n u s m a e n a s (L.) (Decapoda; Brachyura). It is in line with the c o m m o n nomenclature for h o m o l o g o u s brain structures in d e c a p o d
C r u s t a c e a proposed by S a n d e m a n et al. (1992).
RESULTS
Brain
Immunocytochemistry
for F M R F a m i d e -
r e l a t e d p e p t i d e s in t h e C N S
labels
cell
bodies,
their
of crab larvae
primary
neurites,
and an e x t e n s i v e n e u r o p i l a r n e t w o r k .
FMRFamide-like immunoreactive projections from the lateral protocerebrum enter
the medial protocerebrum via the protocerebral tracts (PT, Fig. 1). The number of fibers within these tracts increases from 2 in
zoea 1 to at least 5 in zoea 2. These fibers
terminate in the anterior immunoreactive
protocerebral neuropil (APN) where they
spread out and form a fine, dense network
(Figs. 1, 2A). Several ventrally situated
commissural fibers join these paired neuropils. The posterior immunoreactive protocerebral neuropils (PPN) consist o f a
weakly labeled anterior portion and a posterior lobe which is labeled more strongly.
A pair o f commissural tracts joins the
paired PPNs. These commissures anteriorly
and posteriorly flank the central body which
is not immunolabeled itself. The olfactory
lobes (OL), which are part of the deutocerebrum, are strongly immunoreactive and
the labeling reveals a glomerular organization o f the neuropil (Figs. 1, 2A). The olfactory lobes enlarge from zoea 1 to zoea
2 and the number of glomeruli seems to increase. In contrast to the protocerebral and
deutocerebral neuropils, the tritocerebral
immunoreactive fibers do not form distinct
neuropil bodies but consist of a scattered
meshwork. The paired tritocerebral neuropils (TCN) are joined by a commissural fiber bundle. Several projections extend anteriorly into the protocerebrum. Two more
pairs o f labeled fiber bundles leave the tritocerebrum posteriorly via the esophageal
connectives (OC) and enter the commissural ganglia (CG, Figs. 1, 2A) which flank
the esophageal foramen. They contribute to
the spherical networks in the commissural
ganglia and then extend farther posteriorly
into the subesophageal ganglia.
The brain (excluding the optic lobes) o f
a late zoea 1 contains about 30 labeled neurons which are antigenic to the antibody,
while in zoea 2 shortly before metamorphosis about 60 neurons are immunoreactive.
Thus, the number o f labeled neurons in the
brain doubles during development of the
zoea. Many of these cells are located in the
anterior medial cell cluster (AMC, Figs. 1,
2A). Most other cells are scattered in the
ventral cell cortex. The axonal projections
can be traced only in a few cases. There is,
for example, a pair of large neurons, the
neurites of which can be followed until they
enter the posterior immunoreactive protocerebral neuropil (PPN). Furthermore, the
processes o f some tritocerebral neurons can
be traced until they scatter in the tritocerebral neuropils (TCN, Fig. 1). Close to the
medial border of the olfactory lobes (OL)
there is a conspicuous cluster o f small, dorsally situated neurons. In some preparations
o f the brain o f zoea 2, a bundle of fibers
could be seen to leave this cluster and penetrate into the olfactory neuropil.
Ventral Nerve Cord
At least three small labeled fibers leave
the commissural ganglia (CG) posteriorly
via the esophageal connectives (OC) and
enter the subesophageal ganglia (SOG, Fig.
1). T h e r e is a b r o a d d o u b l e b a n d o f
FMRFamide-like immunoreactive material
which reaches dorsally throughout the entire length of the nerve cord on each side
from the first subesophageal ganglion (SOG
1) to the last thoracic ganglion (TG 5, Figs.
1, 2B). The staining density and the number
o f single longitudinal fibers which can be
distinguished in these bands (at least five in
zoea 2) increases throughout the development of zoea 1 toward zoea 2. Segmental
thickenings along the bands indicate the location of the six fused subesophageal ganglia (Fig. 1). Furthermore, segmental commissures connect the hemiganglia across
the midline. In the third subesophageal ganglion, a small paired side-branch of the medial bands leaves the nerve cord via the respective nerve root. The thoracic commissures are difficult to recognize in zoea 1,
but they become clearly discernible in zoea
—
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>
Fig. 1A, B. Z o e a 1 and Z o e a 2, c a m e r a lucida drawings o f F M R F a m i d e - l i k e immunoreactive cell bodies,
fibers, and neuropil o f brain and ventral nerve cord o f Hyas araneu.r. A G 1, abdominal ganglion 1; A M C ,
anterior medial cluster; APN, anterior protocerebral immunoreactive neuropil; CG, commissural ganglion; 0 ,
e s o p h a g e a l foramen; OC, esophageal connective; OL, olfactory lobe; POC, postesophageal commissure; PPN,
posterior protocerebral immunoreactive neuropil; PT, protocerebral tract; S, sternal artery; S O G I - 6 , subesophageal ganglia 1-6; T C N , tritocerebral neuropil; T G 1-5, thoracic ganglia 1-5. Scale bar = 50 fLm.
2. The neuropil glomeruli of the thoracic
ganglia do not show any immunoreactivity
in zoea 1 (Figs. 1, 2B). However, in zoea 2
the neuropil has acquired a pattern of fine,
yet dense labeling (Figs. 1, 2C). Furthermore, the thoracic neuropil seems to be
subdivided into a double row of 5 - 7 glomerulus-like structures. The double band o f
labeled fibers leaves the fifth thoracic ganglion posteriorly to merge with the neuropil
of the first abdominal ganglion (Figs. 1, 2B,
AG 1).
The number of immunolabeled cells in
the ventral nerve cord (excluding the abdominal ganglia) increases from 2 0 - 2 5 in
late zoea 1 to about 50 in zoea 2 shortly
before metamorphosis. Since this twofold
rise in numbers resembles the situation in
the brain, the two parts o f the central nervous system which have been studied here
seem to share similar dynamics in neurogenesis. In zoea 1, all but one of the labeled
cells are situated in the six subesophageal
ganglia (Fig. 1 A). The most remarkable of
these cells is a bilateral pair of large neurons in the first subesophageal ganglion
(Fig. 2D). Their neurites ascend dorsally
and join the fibers in the longitudinal immunoreactive band. In some preparations,
these processes seemed to travel posteriorly
and then leave the nerve cord via the nerve
roots o f the third subesophageal ganglion.
In zoea 2, a cluster o f 3 or 4 smaller cells
has been added in a similar anterior position, the fibers of which share the same projection pattern with the large cells (Fig.
1B). Furthermore, there are segmentally repeated clusters of 2 - 6 cells, situated medially in the ventral cell cortex o f subesophageal ganglia 3 - 6 (Fig. 1, top of Fig. 2C).
Their neurites ascend vertically and then
take a sharp lateral bend to join the longitudinal neuropil bands. The position o f
these clusters is unchanged during the transition from zoea 1 to zoea 2, but new cells
are added in the more anterior clusters. The
somata of another type o f segmentally arranged, FMRFamide-like immunoreactive
cells in the subesophageal ganglia are located ventrolaterally with their processes
traveling medially into the central neuropil
(Fig. 1 ). Again, new cells of this type are
added during larval development. The thoracic ganglia (TG 1-5) do not bear immunolabeled cells in zoea 1 with the exception
o f an unpaired, large neuron at the midline
between the fifth thoracic hemiganglia (Fig.
2E). However, 10 more cells are immunoreactive in zoea 2 (Fig. 1 B). They are located in the ventral cell cortex o f thoracic
ganglia 2 and 3 and their fibers course medially to join the central neuropil. In abdominal ganglion 1 (AG 1), apart from a
pair o f small cells, another large, unpaired
cell can be found in a position similar to
the cell in thoracic ganglion 5 (Fig. 1).
DISCUSSION
Development of the Larval System o f
Peptidergic Neurons
Using immunocytochemical methods, we
have shown that already at hatching an
elaborate system o f FMRFamide-like immunoreactive neurons and fibers is present
in zoeae of the spider crab Hyas araneus.
T h e most prominent immunoreactive neuropils are the anterior and posterior protocerebral neuropils, the olfactory lobes, and
the neuropil of the six subesophageal ganglia, whereas the five thoracic ganglia initially comprise less labeled fibers. However,
there are significant alterations in the staining pattern through larval development, the
most striking of which is a twofold increase
in the number of immunolabeled neurons.
Using the method o f in vivo incorporation
o f BrdU (5-Bromo-2'-Deoxyuridine) to lab e l m i t o t i c cells, H a r z s c h a n d D a w i r s
( 1994) have shown that the proliferative ac-
Fig. 2. Whole mounts o f central nervous system o f Hyas a r a n e u s processed for FMRFamide-like immunohistochemistry. A: Zoea 1, brain. APN, anterior protocerebral immunoreactive neuropil; CG, conunissural ganglion;
OL, olfactory lobe; PPN, posterior protocerebral immunoreactive neuropil; TCN, tritocerebral neuropil. B: Z o e a 1,
ventral nerve cord. A G 1, abdominal ganglion 1; S, sternal artery; S O G 1-6, subesophageal ganglia 1-6; T G I 5, thoracic ganglia 1-5. C: Z o e a 2, part of ventral nerve cord. T G 1-5, thoracic ganglia 1-5, arrow indicating
glomerular structure of neuropil. D: Zoea 1, pair o f labeled cells at anterior margin o f first subesophageal ganglion,
broken line indicating ganglion midline. E: Zoea 1, unpaired labeled cell between fifth thoracic hemiganglia. S,
sternal artery, broken line indicating ganglion midline. Scale bars A, B, C = 40 �Lm; D, E = 10 wm.
tion of segmental neuroblasts adds large
numbers o f new neurons to the embryonic
nervous system during the zoeal stages. The
present study may serve as an example to
point out the proliferation of an identified
class of neurons which are specified by
their neurotransmitter expression. Furthermore, during development most F M R F a m ide-like immunoreactive neuropils enlarge,
their staining intensity increases, and new
labeled fibers are added. This is especially
true for the five thoracic ganglia which at
hatching still lack any immunoreactivity,
but show a strong, glomerular pattern of
r e a c t i v i t y for F M R F a m i d e - l i k e p e p t i d e s
shortly before metamorphosis. It must be
recognized that in the zoeal stages only the
appendages which are related to the subesophageal ganglia 1-5 operate, while the
appendages of the more caudal segments
are still under development. Hence, a developmentally advanced system o f peptidergic neurons is present in the subesophageal ganglia, while in the thoracic ganglia
this system is required only shortly before
metamorphosis, in the course of which the
thoracic legs start their locomotive function.
The release of crustacean F M R F a m i d e related peptides (FaRPs) in vivo has been
shown to exert a cardioexcitatory effect by
increasing both the rate and amplitude of
contractions (Krajniak, 1991; Mercier and
Russenes, 1992; Skerrett et al., 1995). Furthermore, FaRPs seem to play hormonal
roles in modulating neuromuscular transmission, for example, by increasing the
transmitter release presynaptically (Mercier
et al., 1990; Friedrich et al., 1994; Worden
et al., 1995) and enhancing the amplitude
o f excitatory junctional potentials (Pasztor
and Golas, 1993; Skerrett et al., 1995) of
phasic extensor muscles. The modulatory
action o f FaRPs on neurons of a central pattern generator has been demonstrated in the
crustacean stomatogastric ganglion (STG),
which receives a distinct input o f peptidergic fibers (Marder et al., 1987; Coleman,
1992). Hence, bath application of F M R F amide to the STG increases the frequency
o f the pyloric motor output (Hooper and
Marder, 1984). Taken together, there is reason to argue that a minimum number o f
neurons that express FaRPs is a necessary
prerequisite for suitable motor behavior in
crustaceans. In this respect, the progressive
development of the larval thoracic peptidergic system probably correlates with the
maturation of an adequate motor innervation o f the thoracic legs, which do not take
up their final function before metamorphosis.
Immunocytochemistry against the biogenic amine serotonin in spider crab larvae
has yielded a developmental pattern which
is comparable to the innervation pattern of
FMRFamide-like immunoreactive neurons
(Harzsch and Dawirs, 1995). There is a significant anterior-posterior gradient in the
expression of serotonin during larval development, and, therefore, the neuropilar
immunolabeling is more distinct in the
more advanced anterior ganglia than in the
posterior ones. Nevertheless, during subsequent metamorphic development the serotonergic innervation also increases in these
ganglia. However, the overall structure o f
the serotonergic system seems to be closer
to the adult state at hatching than the
FMRFamide-like immunoreactive system.
Thus, the number o f serotonin-immunoreactive cells in the ventral nerve cord increases only by 10% from hatching to crab
1 (Harzsch and Dawirs, 1995), compared to
a 100% increase in the number o f peptidergic neurons from hatching to late zoea
2. In the brain the relation is a 60% increase
of serotonergic neurons from hatching to
crab 1 against a 100% increase of peptidergic cells from hatching to late zoea 2.
F u r t h e r m o r e , during larval d e v e l o p m e n t
there is no comparable part of the serotonergic system which progresses as rapidly
as the peptidergic fiber system within the
thoracic ganglia during one larval stage
only. These data suggest that the development o f the aminergic system is rather more
advanced at hatching as compared to the
peptidergic system. Beltz et al. (1990) and
Helluy et al. (1993) described the appearance of neurons which are immunoreactive
to serotonin and the pentapeptide proctolin
in the developing nervous system of the
American lobster and reached similar conclusions. These authors reported that at
hatching only 10% and in the final larval
stage roughly 30% of the adult set of peptidergic neurons are immunoreactive, while
the complete set of serotonergic neurons is
already in place at hatching.
Comparison to other Crustaceans
Although detailed information on the distribution of FMRFamide-like immunoreactive neurons in the brain and ventral nerve
cord is available only for the Astacidea,
there are several remarkable similarities to
the staining pattern in brachyuran larvae.
The arrangement of numerous immunoreactive cells in the anterior cell cluster (AMC)
has been described in the lobster H o m a r u s
americanus H. Milne Edwards (see Kobierski et al., 1987) as well as the crayfish Orconectes limosus (Rafinesque) (see Mangerich and Keller, 1988). Furthermore, the
cluster of small labeled cells close to the
olfactory lobes (OL) seems to be a c o m m o n
feature of these species and Hyas a r a n e u s
( " m e d i a l c l u s t e r " in K o b i e r s k i et al.
(1987), "anterior olfactory cells" in Mangerich and Keller (1988)), and we therefore
propose that it is homologous within the
decapods. Both studies on the astacidean
species, as well as our study on brachyuran
larvae, demonstrate a glomerular structure
of the neuropil in the olfactory lobes. This
type o f organization appears to be widespread in decapods (Schmidt and Ache,
1992; Mellon and Alones, 1993; Sandeman
et al., 1993) and therefore seems to be essential for olfactory processing. The neurites o f serotonin-containing olfactory interneurons also have been shown to participate
in the formation of this glomerular structure
both in adult (Sandeman et al., 1988; Sandeman and Sandeman, 1994) and larval
crayfish and lobsters (Helluy et al., 1993).
Interestingly, larvae of the spider crab do
not display any serotonin-immunoreactivity
in the olfactory neuropil (Harzsch and Dawirs, 1995), although this is the case in
adult crabs (Johansson, 1991). However,
our study on peptidergic immunoreactivity
clearly demonstrates that a glomerular organization is already present at hatching.
Since considerable interest has been shown
in the deutocerebral development in crustaceans (Helluy et al., 1995), studies on the
development of additional neurotransmitter
systems may shed further light on this topic.
In the ventral nerve cord, the only cluster
of FMRFamide-like immunoreactive cells
which can be readily identified within different species is the group o f small and
larger immunoreactive cells in the anterior
lateral part of the first subesophageal ganglion. These cells are present in adult Orconectes limosus (see Mangerich and Keller, 1988) and another crayfish P r o c a m b a rus clarkli (Girard) (see M e r c i e r e t al.,
1991), as well as our larval preparations,
and, therefore, may be a c o m m o n feature of
decapods. However, the detection of other
homologous neurons in the more caudal
subesophageal ganglia and the thoracic ganglia is difficult, due to varying experimental
methods and differing modes o f illustrating
the results in the studies cited. Since comparing larval with adult animals is not easy,
f u t u r e studies on the a d u l t p a t t e r n o f
FMRFamide-like immunoreactivity in spider crabs could produce interesting results.
ACKNOWLEDGEMENTS
The authors thank Dr. K. Anger for generously providing rearing and laboratory facilities at the Marine
Station of the Biologische Anstalt Helgoland, and Mr.
S. Pfannschmidt for technical assistance. We are especially thankful to Dr. J. Harms (Helgoland) who supported our laboratory work in a most reliable way. We
are also indebted to Prof. W. Westheide and Mrs. M.
Miiller (Osnabruck) for kindly providing the antiFMRFamide antiserum, and to Dr. R. Windoffer (Hamburg) for his helpful advice. Mrs. Fay Misselbrook is
gratefully acknowledged for correcting the English
draft.
LITERATURE C I T E D
Anger, K. 1983. M o u l t cycle and m o r p h o g e n e s i s in
H y a s a r a n e u s larvae (Decapoda, Majidae), reared in
the laboratory.�Helgoländer w i s s e n s c h a f t l i c h e
M e e r e s u n t e r s u c h u n g e n 36: 285�302.
���, and R. R. Dawirs. 1981. Influence o f starvation on the larval d e v e l o p m e n t o f H y a s a r a n e u s
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RECEIVED: 21 F e b r u a r y 1995.
ACCEPTED: 27 April 1995.
Address: D e p a r t m e n t o f N e u r o a n a t o m y , Faculty o f
Biology, University of Bielefeld, Postbox 10 01 31, D33501 Bielefeld, Germany.