Reference: 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 ^ b .y^^k'iAvis <v\J !p"V'*f(frrA ' ' C :> ->./ . v.s'v"' $?. r ^ ~> >'-^*^V^_*,.. ./'^y. : -r A > ' ' * -^' /<^;i' '/&'''*'IW- 't?' /\ ''*"^.^'T-,CA4 '^: W-- '<& ^ i--V ; ^ ] d > 3 i ! . VMi * $& ; . . *? :\. I HI U::^S ^.A| , - -,^ ,> ll:;.:'-. l'. , "'.' ;;ic^ K N .x'- 1 i i - K $-,; / \, . ;. f .* '# <-^Y\^^ -' ' ^Uw^ ' ; i i NI v. ?" 'j;SsrS.:/-o'ci > 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 ^ '.-! *1- ' ' '-"" , ' /IYIS^ m & ~'^wft" M]$&. v .- s .V-i,-.-. >..\ g ^mm^'-^^\ . ^-: ' ' $ ..'--'' :;'- \.fo ', ,' ,' '' ;' yj$! - f-' v.. ./ -;-- : ; m';f^*w^T[,^U-^,,-; /.:- .c. ,-:- Mr- $-^>T,S$B^",,$-.P-,$../,'-^.&' ^-'^/.t!'.'*> . " \ '-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 Freeman and Costlow (1980), particularly with regard to Literature Cited difficulties in determining the precise duration ofinstars andthetimeofinitiationofproecdysisin rapidly moulting Bellon-llumbert, C, M. J. P. Thijssen, and F. Van Herp. larvae, it has been suggested (Freeman et al., 1983) that 1978. Development, location and relocation ofsensory and neu- the larval molt cycle is not regulated by MIH until meta- rosecretorysitesin theeyestalksduringthelarvaland postlarval life ofPalaemon.terrains(Pennant).J. Mar. Biol. Assoc. U.K. 58: 851- morphosis. However, studies demonstrating that larval 868. ecdysteroid liters cycle in a molt-stage-dependent manner Beltz,B.S.,andE.A.Kravitz.1987. Physiologicalidentification,mor- in much the same way asadults (Changand Bruce, 1981; phological analysisanddevelopmentofidentifiedserotonin-proctolin Spindler and Anger, 1986), and a report by Snyder and containingneurons inthelobsterventral nervecord.J. Neurosci. 7: 533-546. Chang(1986), demonstrating that increases in proecdysial ecdysteroid titer induced by eyestalk removal ofStage II Beltoz,faB.ppSe.,arMa.ncPeonotfess,erSo.toMn.inHealnlduyp,raocntdolEi.nA.imKmruanvoirtze.ac1t9i9v0i.tiePsatitnetrhnes Homarnszoeaecan be repressed by the injection ofadult developing nervous system ofthe American lobster. / Neurobiol. sinus gland extracts, strongly support the hypothesis that 21:521-542. larval molting (or at least, initiation ofproecdysis) is reg- C'hang,E.S.,andM.J.Bruce. 1981. Ecdysteroidlitersoflarvallobsters. ulated by MIH, and the results presented here would also Com/1 Biochein. Physiol. 70A: 239-241. Charmantier, G., M. Charmantier-Daures, and D. E. Aiken. support this hypothesis. However, it should be stressed 1988. Larval development and metamorphosis ofthe American that no firm inferences as to the function ofthe immu- lobsterHomarusamericanus(Crustacea,Decapoda):effectofeyestalk noreactive MIH can yet be made;itis notknown whether ablation and juvenile hormone injection. Gen. Comp. Endocrinol. larval MIH-immunoreactive material is identical to that 70: 319-333. in adults, although the antiserum used displays a very Christiansen, M. E. 1988. Hormonal processesindecapodcrustacean high specificity in immunodot assays (Dircksen el al.. Dahll,arEva.e.19S5y7m.p.EZmoborl.yoSloocg.yLoofudX.-o5r9g:a4n6s-i6n8.Crangonallmanni. Nature 1988), RIA, and ELISA (Webster, unpub.), or whether it 179:482. is released during the zoeal stages. Although in vivo ex- Dircksen, H., C. A. Zahnow, G. Gaus, R. Keller, K. R. Rao, and J. P. periments involving injection ofMIH or sinus gland ex- Riehm. 1987. The ultrastructure ofnerveendingscontaining pig- tracts into zoeal larvae and subsequent monitoring of ment dispersing hormone (PDH) in crustacean sinus glands: iden- proecdysisorinstarlengthwould undoubtedlystrengthen t2i5f0i:ca3ti7o7n-3b8y7.an antiserum against synthetic PDH. Cell Tiss. Res. hypothesesconcerning larval molt control, the small size Dircksen,H.,S.G.Webster,andR.Keller.1988. Immunocytochemical ofmost crab zoeae argues against the success ofsuch ex- demonstration of the neurosecretory systems containing putative MOLT-INHIBITING HORMONE IN CRAB LARVAE 71 molt-inhihitinghormoneand hyperglycemichormoneintheeyestalk Portumdae)rearedinthelaboratory. Bull Bril Mus. (Not. Hist.)28: ofhrachyuran crustaceans. CellTiss. Res 251: 3-12. 103-119. Freeman,J. A.,andJ. D.Costlow. 1980. The moltcycle and itshor- Skinner,D.M. 1985. Interactingfactorsinthecontrolofthecrustacean monalcontrolinRhithmpanopeusliarnsilarvae.Dev Biol 74:479- moltcycle.Am. Zool 25: 275-284. 485. Snyder, M.J., and E. S.Chang. 1986. Effectsofsinusgland extracts Freeman,J.A.,T.L.West,andJ.D.Costkm. 1983. Postlarvalgrowth on larval molting and ecdysteroid liters ofthe American lobster. injuvenile Rlulhropanopeus harnsii. Biol Bull 165:409-415. Homants amcricanus. Biol Bull 170: 244-254. Gorgels-Kallen,J. L.,andJ.T. A. Meij. 1985. Immunocytochemical Spindler, K. D., and K. Anger. 1986. Ecdysteroid levels during the study ofthe hyperglycemic hormone (CHH)-producing system in larval development ofthe spider crab Hya.i araneus. Gen. Camp. the eyestalk ofthe crayfish Astacus leptodactylusduring larval and Endoa-moL 64: 122-128. postlarval development. J Morphol. 185: 155-163. Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium Hubschman,J.II. 1963. Developmentandfunctionofneurosecretory forelectron microscopy. J. Ultrastruc. Res. 26: 31-43. sites in the eyestalks oflarval Palaemoneles (Decapoda, Natantia). Watson,R.D.,E.Spaziani,andW.E.Bollenbacher. 1989. Regulation Biol. Bull. 125:96-113. ofecdysonesynthesisin insectsandcrustaceans: acomparison. Pp. Jaques, F. 1975. Decouverte de la glande du sinus chez la larve de 188-203 in Ecdysone. From Chemistry10ModeofAction, J. Kool- Si/iii/lu mantis (stade I) (Crustace, Stomatopodes). Ultrastructure. man. ed. GeorgThieme Verlag. Stuttgart. C. R Acad. Sa. (D) (Paris) 280: 1575-1577. Webster, S. G. 1986. Neurohormonal control ofecdysteroid biosyn- Lachaise, F., M. Hubert, S.G. Webster, and R. Lafont. 1988. Effect thesisby Carcinus maenas Y-organs in vitroand preliminary char- ofmolt-inhibitinghormoneonketodiolconversionbycrabY-organs. acterization ofthe putative moult-inhibiting hormone (MIH). Gen. J Insect Physiol. 34: 557-562. Comp Endocrinol. 61: 237-247. Little,G.1969. Thelarvaldevelopmentoftheshrimp.Palaemonmac- Webster.S.G.,and R.Keller. 1986. Purification,characterizationand rodaaylusRathbun,rearedinthelaboratory,andtheeffectofeyestalk amino acid composition ofthe putative moult-inhibiting hormone extirpation on development. Crustaceana 17: 69-87. (MIH)ofCarcinin >iiiicna\(Crustacea, Decapoda)./ Comp. Physiol Matsumoto, K. 1958. Morphological studieson theneurosecretion in B 156: 617-624. crabs. Biol J. Okayama L'nir. (Japan)4: 103-176. Webster, S. G., and R. Keller. 1988. Physiology and biochemistry of Orlamunder, J. 1942. Zur Entwicklung und Formbildung des Birgiix crustacean neurohormonai peptides. Pp. 173-196 inNeurohormones lalro L. mil besonderer Beriacksichtigung des X-Organs. Z Wiss. inInvertebrates, M.C.ThorndykeandG.J.Goldsworthy.eds.Cam- Zool. 155:280-316. bridge LJniversity Press. Pyle,R.W. 1943. Thehistogenesisandcyclicphenomenaofthesinus Zielhorst, A. J. A. G., and F. Van llerp. 1976. Developpement du gland and X-organ in Crustacea. Biol. Bull 85: 87-102. systeme neurosecreteur du pedoncle oculaire des larves d' Astacus Rice,A. L.,and R. W.Ingle. 1975. ThelarvaldevelopmentofCarcinus leptodactylussalinus Nordmann (Crustacea, Decapoda, Reptantia): maenax(L.)andC mediterraneusCzerniavsky(Crustacea.Brachyura. Microscopiephotonique. C R Acad. Sci(D)(Paris)283: 1755-1758.