Table Of ContentReference: Btol. Bull. 180: 65-71. (February.
Putative Molt-Inhibiting Hormone in Larvae
of the Shore Crab Carcinus maenas L.:
An
Immunocytochemical Approach
S. G. WEBSTER' AND H. DIRCKSEN*
SchoolofBiologicalSciences, University CollegeofNorth Wales, Bangor. GwyneddLL572UW, UK,
and*Institutfiir Zoophysiologie. Universitdt Bonn, EndenicherAllee 11-13.
D-5300 Bonn 1. Germany.
Abstract. Immunocytochemical investigations of the 1989 for recent reviews). Despite recent advances in our
eyestalk ofCarcinus maenaszoeal larval stages, usingan knowledge concerning mechanisms of molt control in
antiserum directed against putative Carcinus molt-inhib- adult decapod crustaceans, little is known about the reg-
iting hormone (M1H), revealed immunopositive neuronal ulation ofmolting in larval crustaceans. This deficiency
structures. Thesestructuresincluded perikaryaassociated has been reiterated in a recent review by Christiansen
with the medulla terminalis X-organ, parts ofthe sinus (1988).
gland tract, and the neurohemal organ the sinus gland. Evidence for molt regulation by MIH in crustacean
Apart from an increase in volume ofthe sinus gland be- larvae has, until recently, been obtained byeyestalk abla-
tween zoeal stage I and II, no striking changes in the to- tion experiments (for references see Charmantier et ai,
pography or morphology ofthe MIH neurosecretory sys- 1988;Christiansen 1988), which havegiven equivocal re-
tem were observed. Immunopositive structures were sults, suggesting that in some instances, the larval molt is
found in similar locations to those seen in adult crabs. notregulated by MIH until shortly before metamorphosis.
Our results suggest that the control of molting by MIH However, with regard to morphological correlatesofneu-
in crustacean larvae may be similar to the currently ac- rosecretory structures in larval eyestalks, several reports
cepted model of molt control in adult decapod crusta- (Orlamunder, 1942; Pyle, 1943; Hubschman. 1953; Dahl,
ceans. 1957; Matsumoto, 1958; Little, 1969; Zielhorst and Van
Herp, 1976; Bellon-Humbert eta/., 1978;Gorgels-Kallen
Introduction and Meij, 1985) detail the ontogeny oflarval neurosecre-
tory systems in a wide variety of crustaceans. With the
Acurrent modelofmoltcontrol indecapodcrustaceans exception ofstudies by Gorgels-Kallen and Meij (1985),
involves regulation of ecdysteroid synthesis by a molt- Beltz and Kravitz (1987), and Beltz et a/., (1990), there
inhibiting hormone (MIH), released by neurosecretory arenootherstudiesinwhich neurosecretorysystemscon-
neurons in the eyestalk. Much evidence has now accu- taining immunocytochemically defined neuropeptides
mulated suggesting that increased synthesis and liters of have been described in crustacean larvae.
circulatingecdysteroidsnecessary forinduction ofpremolt Recently, we have characterized a neuropeptide from
aredirectly repressed bythis neuropeptide, thus inhibiting the sinus gland ofCarcinus maenas, which, by virtue of
proecdysis and molting. Nevertheless, alternative hy- its ability to repressecdysteroidogenesis by Y-organscul-
potheses have implicated processes such as metabolism tured in vitro, could be described as a putative MIH
and excretion of ecdysteroids in molt regulation (see (Webster, 1986; Webster and Keller, 1986). It should be
Skinner, 1985; Webster and Keller, 1988; Watson el ai. stressed that the precise significance and function ofthis
neuropeptide as a molt-inhibitor in vivo has not yet been
Received 23 May 1990;accepted 6 November 1990. elucidated, and until suitable in vivo bioassays are devel-
1 Towhom correspondence shouldbesent. oped, the status of MIH must remain "putative." Re-
65
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> xr 'v'^-it',4.X^f0^e,1g-"'^
>- x^~v ^. J.w^n^f-c'/*^f'.^v^
Figure 1. CharacteristicstructuresofMIH-immunoreactive(IR) neuronsin prezoeal(a,c),stageIzoea
(h. d-f. left eye), and stage II zoea (g-l) eyes ofCarcinus maenas larvae. Phase contrast micrographs of
immunostained semithin (1 nm) transverse sections. (Orientation ofdorsal parts oflarvae to the tops of
micrographs.)
66
MOLT-INHIBITING HORMONE IN CRAB LARVAE 67
cently, we demonstrated that the neurosecretory system ing to Rice and Ingle (1975). Under these maintenance
produced putative MIH in the eyestalk ganglia ofseveral conditions, survival wasgood (80%), and instardurations
adult brachyuran crustaceans (Dircksen el at.. 1988). Be- were approximately: Z I: 7, Z II: 5, Z III: 6, Z IV: 7, M:
causethese studies provide compellingevidence tosuggest 8, days. Samples of larvae were taken at the middle of
that MIH isasecretable neuropeptide, and in viewofour each instar, which wasconsidered tobeduringintermoult.
earlier observations on the nature and mode ofaction of
this neuropeptide on ecdysteroid synthesis in Carcinus Tissueprocessingand immunocytochemistry
(Webster and Keller. 1986; Lachaise el al. 1989), it
seemed opportune to examine the larval eyestalk neu- Fixations were carried out in a mixture of2% parafor-
rosecretory system immunocytochemically, using anti- maldehyde,M2% glutaraldehyde. and 0.1% saturated picric
bodies raised against Carcinus MIH. Evidence presented acid in 0.1 sodMium cacodylate bumffeMr, pH 7.4, supple-
here suggests that a functional MIH-like neurosecretory men4tCed with 0.5 sucrose and 5 CaCl2 for 2-4 h
system exists in all larval stages ofCarcinus. at according to Dircksen et al. (1987). Tissues were
washed extensively in the same buffer, dehydrated, and
Materials and Methods embedded in low viscosity resin (Spurr, 1969). Semithin
frontal cross-sections ( 1 ^m) through the whole animal
Laboratory rearing oflarvae were cut on a LKB Ultrotome III or a Reichert Ultracut
E, and processed forimmunocytochemistry usinga rabbit
Ovigerous Carcinus maenas L. females were collected antiserum (code R1TB) directed against HPLC-purified
from the Menai Strait, North Wales, between May and MIH ofMCarcinus (Dircksen el al., 1988), diluted 1:4000
July, and maintained in the laboratory until larvae were in0.01 phosphatebufferedsaline(PBS)and PAPstain-
released. Only positively phototropic, rapidly swimming ing techniques (Dircksen et al., 1987). Micrographs were
larvae were collected. Rearing techniques were initially taken with a Zeiss Axioskop using phase contrast optics
based upon thoseofRiceand Ingle(1975), butwere found and documented on Agfapan 25 film.
to be inadequate. Successful rearing to first crab with a
high survival wasachieved usinga mixed diet of(A) phy- Results
toplankton (Tetraselmis clniii), (B) rotifers (Brachionus
plicatilis), (C) barnacle nauplii (Ehninius modeslus), and Despite several attempts to improvethe penetration of
(D) brine shrimp nauplii (Anemia salina). During each fixative into the eyestalks (for example, by piercing the
larval stage, prey ratios were supplied as follows: Zoea I exoskeleton behind the eyestalks, using other fixatives or
fixation times), adequate fixation ofmegalopae and first
III (C):l, (D):l. Zoea IV, Megalopa and First crab (D):l. crab stages was impossible. Thus, by necessity, this study
With theexception ofphytoplankton (culture density ca. is restricted to the zoeal stages of Carcinus, and in later
106cellsmr': 1 part = 15 ml),thetotalpreyconcentration zoeal stages problems with fixation and tissue shrinkage
was around 25-50 items per ml. Larvae were reared in were encountered. A sometimes confusing feature ofthe
50-ml plastic containers in constantly aerated, filtered zoeal eyestalk wasthe presenceofapigmented perineural
seawater (33%o) under ambient temperature (15-18C) sheath (Fig. 2c, 2f), which could have been identified as
and photoperiod (L 15-18 h: D9-6 h). Maximum density an immunopositive structure. Thisproblem wasresolved
oflarvae was 1 per 5 ml. Water and food were changed by using normal bright field optics, under which immu-
everytwo days, at which time instars were staged accord- nopositive material appears brownish, or by higher mag-
fa)MIH-IRaxonprofileswithinthesinusgland(centerofrectangle)ofaprezoea.Noteommatidialpnmordia.
brain (*) and yolk droplets(arrowhead), (b) MIH-IR axon profiles within the sinusgland (rectangle) ofa
stage I zoea. Note dense pigmentation at the base oftheommatidia, and well-developed neuropilesofthe
lamina ganglionaris (LG). medulla externa (ME), and the brain (*). (c, d) Higher magnifications ofsinus
glandscorrespondingtorectanglesina.b.(e)Cross-sectionedMIH-IRaxons(insetenlargedfromtherectangle).
(f)Two MIH-IR perikarya in an anteriordorsalcell groupofthelefteyestalkganglia(inset enlarged from
therectangle), (g) MIH-IR axon profilesinthesinusgland(rectangle)ofastageII zoeaadjacent tothe ME
andlargehemolymphspaces.Notestalkformationoftheeyeatthisstage,(i)Cross-sectionedMIH-IRaxons
in the medulla terminalis. (k)Threeclustered MIH-IR penkarya in an anteriordorsal position ofthe pre-
sumptive X-organ cell group. Note well-developed ganglia and neuropiles in the eye. (h,j, I) Higher mag-
nificationsofrectanglesoutlined ing,i,k.Noteaxonprofilesandputativeterminalsabuttingonthesurface
ofthe sinus gland (h) and dark PAP reaction products restricted to the cytoplasm ofthe penkarya (1) of
MIH-IR neurons.
Scalebars: 50^m in a.b, e. f,g, i. k. 10/jm inc.d, h.j, 1,and insetsine, f.
^r
,c^ 1-
I ,I'M
m
^
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& ~'^wft"
M]$&.
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.-
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-;-- :
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\ '-r\--:--' V- " '^&?Jss*r / , ,>: x v . > , \ "..''.M' -' ,_
'
w&
V . .^
Figure2. CharacteristicstructuresofMIH-immunoreactive(IR)neuronsinstageIIIzoea(a-f,lefteye)
andstageIVzoea(g-1,righteye)eyesofCarcimismacnaslarvae.Phasecontrastmicrographsofimmunostained
semithin(1 ^m)transversesections.(Orientationofdorsalpartsofthelarvaetothetopsofthemicrographs.)
68
MOLT-INHIB1TING HORMONE IN CRAB LARVAE 69
nification (Fig. 2f) when the black pigment granulescould been demonstrated in all zoeal instarsofCarcinuslarvae.
be clearly resolved by phase contrast optics. Surprisingly, larval immunopositive structures were to-
M1H immunoreactivity was found in all zoeal stages pographically and morphologicallysimilartothose found
examined, including the so-called prezoeal stage, which, in the adultcrab. However, very few(maximum 4) MIH-
in view ofits brevity (ca. 30 min), and association with immunoreactive perikarya were observed in any larval
hatching, could well be described as an embryonic molt. stage, compared to the adult crab where there are 32-36
(Fig. la, c). Ingeneral, MIH immunoreactivitywasfound MIH-immunoreactive perikarya (Dircksen et ai, 1988).
instructuressimilartothose found in theadult, including Itislikelythattheincreasein numberofimmunopositive
perikarya in a position similar to the X-organ in adults, cells during larval to juvenile/adult development is due
an X-organ sinus gland tract, and a sinus gland (Figs. 1, to increased MIH gene expression rather than by cell di-
2). In several preparations the sinusgland appeared to be vision because neuroblasts are generally considered to be
A
in close proximity to a large hemolymph vessel (Figs. Ih, too highly differentiated to undergo further division.
MIH
2a, d). By serially sectioning through the entire eyestalk, striking similarity of the larval immunopositive
a maximum offour immunopositive perikarya ofabout structures to those ofthe adult concerns the morphology
8-10 ^m in diameter were observed in all zoeal stages ofthe X-organ sinus gland tract. In the adult, MIH im-
localized in a cluster ofneuroblasts in an anterior dorsal munoreactive axons form a peripheral tract around the
position ofthe eyestalk, with large nuclei and scarce cy- central axon bundle containing crustacean hyperglycemic
toplasm (Figs. If, k, 1. 2c, f, i). Axonal projections were hormone (CHH) immunopositive axons(Dircksen etai,
CHH
found in the medulla terminalis of the well-developed 1988). Although wedid notdetermine in thepresent
eyestalk ganglia in a typical circular arrangement offour study, the similarity in the arrangement ofthe four MIH-
cross-sectioned axons (Figs. Ij. 2e, k), reminiscent ofthe immunoreactive axonsaround acentral tract was clearly
axonal arrangement in the adult crab. This pattern was suggestive ofthe adult morphology.
found in all zoeal stages. Despite exhaustive investigation, Several studies have reported thegeneral development of
the only discernable change in the morphology of the neural systemsin thecrustacean eyestalk. Cellscorrespond-
neurosecretory structures was the size ofthe sinus gland, ingto the X-organ have been found in the first larval stages
whichappearedtoincrease in volumebetween zoea I and ofall species examined (Birgits. Orlamiinder, 1942; Horn-
II, when the eye became stalked and mobile. Indeed, it arm. Pinnotheres. Pyle, 1943: Crangon. Dahl, 1957; Pota-
was frequently difficult to observe the sinusgland in zoea mon. Matsumoto, 1958; Palaemonetes, Hubschman, 1963;
I due to its small size, but in zoea II, the sinus gland was Palaemon. Little, 1969, Bellon-Humbert et ai, 1978; As-
often the most striking immunopositive structure (Fig. tacus, Zielhorst and Van Herp, 1976, Gorgels-Kallen and
Ib, d, g, h). In control incubations, preabsorbtion ofthe Meij, 1985). With regard to the development ofthe sinus
antiserum with 2 nmolesofMIH perjulofcrudeantiserum gland, for freshwater crustaceans, which hatch at an ad-
completely abolished immunostaining, thus proving the vanced developmental stage, the sinus gland is present in
specificity ofthe immunocytochemical detection (results the first larval stage(Matsumoto, 1958;Gorgels-Kallen and
not shown). Meij, 1985). In marine crustaceans, which hatch at a rela-
tively early stage ofdevelopment, and which often undergo
Discussion a lengthy planktonic existence prior to a dramatic meta-
In the present study, the location ofperikarya, axons, morphosis, all studies suggest that the sinus gland develops
andsinusgland terminals immunopositive for MIH have (orcan first beobserved) late in larval life,ataboutthetime
(a) MIH-IR axon profiles in the sinusgland (rectangle) adjacent to the large hemolymph vessel (*) ofthe
eyestalk. (b)Section slightlyanteriorto(a)showingthesinusgland(arrowhead)andcross-sectioned MIH-
IR axons (rectangle) in the medulla terminalis. (c) Four MIH-IR penkarya (rectangle) are found in an
anteriordorsal positionofthe presumptiveX-organcellgroup, (d,e. I") Highermagnificationsofrectangles
outlinedin a. b,c. MIH-IR putativeaxonterminalsadjacenttothe hemolymph vessel(*) are found in the
sinusgland (d). Note also cross-sectioned MIH-IR axons (e) in the medulla terminalis and strong immu-
noreactivityofthreeperikarya(f,arrowheads).Arrowsin(f)pointtodarkpigmentsusuallyfoundinperineural
sheaths ofeyestalk ganglia, (g) MIH-IR axon profiles in the sinus gland (rectangle) adjacent to the large
hemolymph vessel (*) ofthe eyestalk. (h) Cross-sectioned axons ofthe presumptive X-organ sinus gland
(XO-SG)tractinthemedullaterminalis. (i)Two MIH-IR perikarya in thepresumptive X-organcellgroup
in a dorsal anterior position ofthe proximal eyestalk ganglia, (j, k, 1) Higher magnification ofrectangles
outlined in g, h, i, MIH-IR axon profilesand putative axon terminals abuttingon thesurface ofthe sinus
gland,(*) indicates hemolymph vessel, (j), MIH-IRaxonsin the XO-SGtract(k)and twostrongly immu-
nopositive XOperikarya(I). Note unstained axonsin thecenterofthe XO-SG tract(k).
Scalebars: 50^m in a-c,g-i. 10j/m in d-f,j-l.
70 S. G. WEBSTER AND H. DIRCKSON
of metamorphosis (stage V Palaemonetes, Hubschman, periments in crab larvae. A further problem, which re-
1963, Palaemon, Bellon-Humbert et a/., 1978; stage III mains unresolved, concerns the increase in number of
Homarns. Pyle 1943). Apartfrom areportbyJaques(1975) immunoreactive perikarya between the last zoeal stage
demonstratingthepresenceofasinusgland in stage I Squilla and the adult. It is possible that this transition occurs
mantis larvae, this paper reports the first demonstration of during metamorphosis (a phenomenon wecould notelu-
a sinusgland in first stage larvae ofa marine decapod crus- cidatedue todifficulties in achievingadequate fixation of
tacean, andis undoubtedlyduetothegreat resolvingpower megalopae and first crab stages). If the MIH secretory
ofimmunocytochemical techniques compared to conven- system became syntheticallyactive atthistime, and stored
tional histochemical staining methods. To our knowledge, MIH was released, then previous observations regarding
the only other reports using immunocytochemical tech- the failure to accelerate molting in zoeal larvae, and the
niquesto identify larval neurosecretory structuresare those appearance ofthe sinus gland as a structure stainable by
byGorgels-Kallen and Meij (1985),demonstratingthe neu- conventional histochemical methods prior to metamor-
CHH
rosecretory structures containing immunoreactivity phosis, couldbe reconciled with the model ofmoltcontrol
inAstaaisleptodactylnslarvae, and Beltzand Kravitz(1987) suggested by Freeman et al. (1983).
and Beltz et al. (1990), demonstrating proctolin-like im-
munoreactivity in theCNS oflarval Homarnsamericanus.
While immunocytochemical evidence indicates that Acknowledgments
Carcinus zoeae possess a M1H neurosecretory system, We are grateful to Mr. M. Budd, School ofOcean Sci-
which may participate in the control of larval molting, ences, Menai Bridge, UK, forculturingthe phytoplankton
experimentsinvolvingeyestalk ablation in several species and rotifersused inthisstudy, and formuch useful advice
ofcrustacean larvae(see specific examplesin Charmantier concerning larval rearingtechniques. This work was sup-
et al.. 1988; Christiansen, 1988) have demonstrated that, ported by a Royal Society University Research Fellowship
ingeneral,eyestalkablation isonlyeffectiveinaccelerating (S.G.W.). Financial support from the British Council for
proecdysis and molting when performed during the last
travel to Bangor (H.D.) is gratefully acknowledged.
instar before metamorphosis. Although the deficiencies
of these experiments have been commented upon by
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868.
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MOLT-INHIBITING HORMONE IN CRAB LARVAE 71
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