Fisheries Science 62(6) , 909-913 (1996)

Immunohistochemical Localization of in the Cephalic Ganglion of the Land Crab Chiromantes haematocheir

Yoshiharu Honma,*1 Keiko Takano,*1 Akira Chiba,*2 and Shunya Oka*2 *1Faculty of Science , Niigata University, Ikarashi, Niigata 950-21, Japan *2School of Dentistry at Niigata , Nippon Dental University, Hamaura, Niigata 951, Japan (Received March 27, 1996)

To determine the distribution of Y (NPY)-like immunoreactivity in the brain (cephal ic ganglion) of the land crab Chiromantes haematocheir, an immunohistochemical study was carried out using the streptavidin-biotin technique, with special regard to a possible relationship with the mollus can cardioexcitatory tetra- (FMRFamide), , and urotensins I and ‡U. NPY-like im munoreactivity was found to occur widely in the crab brain, including several cell groups in the protocerebrum and deutocerebrum, while NPY-positive fibers were seen in many portions of the neuro piles. In most of the NPY-positive cells, FMRFamide-like immunoreactivity was demonstrated, suggest ing colocalization of both . Although an immunopositive oxytocin-like substance was detected sporadically in the crab brain, no urotensins were detected. These results suggested that NPY-like pep tide, in conjunction with FMRFamide-like peptide in the brain of C. haematocheir, functions as a neu rotransmitter or neuromodulator during the process of integration. Key words: immunohistochemistry, urotensin, oxytocin, , FMRFamide, cephal ic ganglion, Chiromantes haematocheir (Crustacea), neuropeptides

The land crab Chiromantes haematocheir (de Haan), a Biological Station, Niigata University, located on the very common decapod crustacean on the Japan coast, is northwestern coast of Sado Island in the Sea of famous for its peculiar spawning behavior, linked with Japan, from late May to late September, 1994. The cara semilunar periodicity.1-4) The reproductive cycle and fine pace width of animals ranged from 30 to 40 mm. structure of the spermatozoa of this species have been After removing the chelipeds and ambulatory legs, the previously clarified.1,5) In spite of being a commercially carapace was lifted off and the entire cephalo-thorax, in unimportant but experimentally useful species, detailed cluding the abdomen, was immersed in Bouin's solution research on the internal organs, including the nervous sys (without acetic acid) prepared with seawater. Following tem, is scarce. this, the cephalic ganglion (brain) was removed, and Recently, an immunohistochemical examination of the returned to fresh Bouin's solution where it remained over distribution of neuropeptide Y (NPY) in the abdominal night. The fixed blocks were subsequently dehydrated ganglion of the sea hare, Aplysia kurodai, a very common through a graded alcohol series, cleared, embedded in opisthobranch mollusc on the Japan coast, with respect to paraffin or paraplast, and cut serially at 10ƒÊm thickness, the coexistence of FMRFamide was made .6) Subsequently, chiefly in the horizontal direction. In addition, to deter Too and roll7) demonstrated FMRFamide-like im mine the running direction of the nerve fibers, thick sec munoreactivities in the central nervous system of the sea tions, 20-25 ,ƒÊm, were cut. Adjacent sections were mount scallop Placopecten magellanicus. ed on slides alternately left to right so as to produce mirror The brain structures of several species of decapod crusta images of each other. ceans have been studied in detail by Tsvilenneva and Tito va,81Tsvilenneva et al. 9)and Sandeman et al.10)In addition, Immunohistochemitry immunoreactivity of several neuropeptides in decapod Deparaffinized, dehydrated sections were incubated in crustaceans has been reported by several researchers ,1-18) 0.5% H202 for 20 min to suppress endogenous peroxidase but no studies have focused on the Japanese crab. The activity. After a short rinse in buffer (phosphate-buffered present work complements these earlier studies. saline, PBS), pH 7.6, the sections were incubated in 10% normal goat serum (Nichirei, Tokyo) in PBS for 15 min at Materials and Methods room temperature to reduce background staining. The sec tions were then processed using a commercial kit (SBA-OP Animals and Tissue Preparations kit, Nichirei, Tokyo) for the streptavidin-biotin peroxi A number of land crabs Chiromantes haematocheir, in dase technique. cluding both young and adults, were collected from the One of the correlative (mirror image) sections was incu rocky beach on and near the campus of the Sado Marine bated for 2 hrs with rabbit anti-synthetic porcine NPY an- 910 Honma et al. tiserum (working dilution 1:3000; UCB Bioproduct, Belgi dase (PAP), and treated with 4 chloronaphthal/H202 for um) and the other with FMRFamide (1:3000; Cambridge 10 min. Finally, after rinsing in PBS, the sections were Res. Biochem., U.K.) for 24 hrs at room temperature. mounted with glycerine PBS and observed under a light Both sections were then washed in PBS, incubated for 15 microscope. min with a secondary antibody (biotinylated, goat anti-rab Immunohistochemical procedures for urotensins I and bit IgG, 1:50), washed again in PBS and treated for 15 min 11 have been given elsewhere.20) For primary antiserum, with streptavidin-biotin peroxidase complexes (1:100) at polyclonal antioxytocin (working dilution 1:15,000; UCB room temperature. Finally, the sections were immersed in Bioproduct, Belgium) was used, the sections being incubat a fresh solution of 3,3'-diaminobenzidine 4 HCl (20 mg/ ed overnight at room temperature. To test the specificity 100 ml) and H202 (0.01%) in Tris buffer (pH 7.6) for 10 of the immunoreaction, the primary antiserum was 15 min at room temperature to reveal the reaction replaced by 1) normal rabbit serum, 2) synthetic oxytocin

product. (1 and 10ƒÊM, Penninsula Lab., USA) and 3) synthetic Blocking studies were performed by preabsorbing each isotocin (I and 10ƒÊM, UCB Bioproduct, Belgium), respec antiserum with the homologous antigen (1ƒÊM, 10ƒÊM) of tively. In all the sections resulting from these three proce each NPY or FMRFamide, and vice versa. In addition, dures immunostaining was absent. Therefore, the tissue APP (avian ) and PYY (peptide antigen(s) were considered to comprise immunohistochemi YY) were used to supplement these studies. The correlative cally oxytocin and isotocin-like substances. sections were processed immunohistochemically using Several sections were counterstained with hematoxylin preabsorbed and nonpreabsorbed antisera. eosin double stain to provide information on the general For simultaneous demonstration of NPY and FMRF histology of the ganglion. amide immunoreactivities in the same sections, double im munoenzymatic labelling was applied following the recom Results mendation of Girod et al.19) I After routine immunoreactivi ty and visualization by a primary antibody, the sections Following the examination of the serial sections, the ana were immersed in 0.1 M glycine HCl buffer (pH 2.2) for 1 tomical layout and immunoreactive structures of the 2 hrs and washed by stirring, the buffer solution being cephalic ganglion were diagrammatically illustrated and changed three times. Following this, the sections were summarized (Figs. 1 and 2C). rinsed in PBS, reacted successively against another prima No urotensin I or ‡U immunoreactivity was clearly seen. ry antibody, a second antibody and peroxidase-antiperoxi Oxytocin-like immunoreactive varicose fibers occurred spo-

Fig. 1. Diagrammatic illustration of the cephalic ganglion of the land-crab Chiromantes haematocheir, showing neuropeptide immunoreactive struc tures. A, dorsal view; B, ventral view. MN, median nerves; CB, central body; PT, protocerebral tract; OMNv, oculomotor nerve; A,Nv, antenna I nerve; A11Nv, antenna ‡Unerve; TNv, tegumentary nerve; OL, olfactory lobe; EC, esophageal connectives; AMPN, anterior medial protocerebral neuropile; TNv, tegumentary neuropile; I, anterior medial cells; ‡U, dorsal lateral cells; ‡V. ventral lateral cells; ‡W, olfactory lobe cells; V,

posterior lateral cells; •œ•œ, NPY and FMRFamide immunoreactive neuropiles;•œ•œ, NPY and FMRFamide immunoreactive cells;•E•E•E, NPY and FMRFamide immunoreactive fibers. Neuropeptides in the Land Crab Brain all

Fig. 2. Photomicrographs of immunoreactive structures of the land-crab Chiromantes haemat ocheir. A: Oxytocin-like immunoreactive varicose fibers in the proximal part of the protocerebral tract . (•~400) B: Oxytocin-like immunoreactive varicosities in the anterior optic neuropile near the central body. (•~400) C: Horizontal section of the cephali c ganglion showing the NPY-like im munoreactive structures consisting of cell clusters and fibers. (•~50) D: Enlarged view of left side portion of Fig . 2 C, showing broad distribution of dense immunoreactive substance. (•~100) E: NPY immunoreactive cells in the anterior medial cell clusters (i ndicated by numbers 1-3). (•~200) F : Correlative FMRFamide immunoreactive cells as in Fig. 2 E: (•~200) G: NPY immunoreactive cells and fibers i f n the ventral lateral cells and ol actory lobe cells. (•~200) H: Correlative FMRFamide immunoreactive fibers as in Fig . 2 G: (•~200) I: NPY immunoreactive fibers in the pro tocerebral tract. (•~200) J: Correlative FMRFamide immunoreactive fibers as in Fig . 2 I. (•~200) 912 Honma et al.

radically in the proximal part of the protocerebral tract Chiba et al., *3 however, reported that neither of the uro (Fig. 2A), the esophageal connectives and the anterior op tensin-I or ‡U-like substances have been demonstrated in tic neuropile, just lateral to the central body (Fig. 2B), the brain of the land crab. It is difficult to explain such a whereas no oxytocin-positive cell bodies were demonstrat discrepancy between the land crab and littoral crab, while ed. the role and significance of oxytocin-like substances in the NPY-like immunoreactivity was demonstrated in every crab brain are as yet unknown, although their apparent cell cluster and numerous fibers of the braaan (Figs. 2C,D). demonstration indicates either their existence or that of In and near the olfactory lobe, small positive cells (neur another substance, close to the oxytocin antigen. Van Deij ons) were exclusively dominant in the ‡W cell cluster (olfac nen et al.12) reported oxytocin-positive scattered fibers in tory lobe cells), whereas the ‡V cell cluster (ventral lateral the optic ganglia of the crayfish, although its biochemical cells) were characterized by large cell size. Both large and nature remained unknown. small cells coexisted and mingled with each other in the V NPY-immunoreactivity was seen widely in the somata cell cluster (posterior lateral cells). Immunoreactive cells of every cell cluster, the neuropile fibers of every were detected in the I cell cluster (anterior medial cells) cerebrum, and the olfactory globular tract and central

(Fig. 2E) located on the upper part of the brain, the ‡U cell body of Chiromantes haematocheir. The demonstration cluster (dorsal lateral cells), the ‡V cell cluster (Fig. 2G). of colocalization of NPY and FMRFamide substances in olfactory lobe cells and posterior lateral cells near the the same cells was of interest for a comparison with the ner olfactory lobe. However, no large cells showed any vous tissues of many other phylogenetically distant immunoreaction with NPY antiserum (Figs. 2C,D). animals. Both substances, colocalized or cross-reacted in Immunopositive fibers were found in the protocerebrum the ganglional cells and fibers, have been identified in the (Fig. 21), deutocerebrum and tritocerebrum, although a sea hare,) hagfish,23) goldfish 21) and rat,26) to mention a different spotted pattern of immunoreactivity was demon few. Therefore, cells containing both NPY-and strated in the olfactory lobe. Thick fibers with varicosities FMRFamide-related substances seemed to be widely dis were apparent in the olfactory globular tract, and a posi tributed throughout the lower and higher animals. As stat tive reaction was found in the central body (one of the neu ed previously for the sea hare,) the present demonstrations ropiles). of both NPY and FMRFamide in the land crab brain are FMRFamide-like immunoreactivity was found in similarly considered to be involved in the neurotransmis almost all of the NPY-like immunoreactive cells. From sion and/or neuromodulation in the brain of the land the use of double immunoenzymatic labeling and the crab. On the other hand, after cross preabsorption experi correlative sections, the almost complete coexistence of ments on the cephalic and thoracic nervous system of the NPY-like and FMRFamide-like substances in the cells and locust Locusta migratoria, Remy et al.26) were of the opin thick fibers was determined (Figs. 2E-J). ion that NPY and FMRFamide antisera recognize two The preabsorption test demonstrated negative im distinct antigenic sites belonging either to a large polypep munoreactivity in the cases of NPY antiserum affected tide, or to two distinct neuropeptides. with NPY (i.e., homologous antigen) and FMRFamide-an The architecture, microscopic architecture and anatomi tiserum with FMRFamide. On the contrary, immunostain cal nomenclature of decapod crustaceans have been thor ing by NPY antiserum preabsorbed with FMRFamide and oughly described and proposed by Tsvilenneva and Tito FMRFamide antiserum with NPY resulted in positive im va8) and Sandeman et al.10) According to them, the main munoreactivities in the same cells. These results indicated masses of ganglional cells consisted of 5 parts: two med that the NPY antiserum could recognize NPY, but not ian-(anterior and posterior) and 3 pairs of lateral-cells. FMRFamide-like antigen, and FMRFamide antiserum, The same was confirmed in the present study. The main FMRFamide, but not NPY-like antigen. In addition, im tracts, nerves, connectives, and neuropiles (such as pro- munostaining by NPY antiserum preabsorbed with APP or PYY brought about negative immunoreactivities in the same cells, while FMRFamide antiserum with APP was positive (Table 1). Table 1. Cross-reactivity of antisera toward NPY, APP, PYY and Discussion FMRFamide

From the phylogenetic point of view, Oka et al.20-22)and Chiba et al. *3 examined urotensin-like immunoreactivity in several vertebrates and marine invertebrates in conjunc tion with NPY, SP (), and FMRFamide. They detected UI-immunoreactivities in the nerve cord of the lancelet Branchiostoma floridae and the cerebral ganglia of the littoral crab Gaetice depressus, squid Todarodes pacificus, and sea hare Aplysia kurodai, and further ob served the contact of NPY positive fibers with Ul-im munoreactive neural structures in the lancelet and sea APP, avian pancreatic polypeptide; FMRFamide, Phe-Met-Arg-Phe-NH2 (mol luscan cardioexcitatory tetrapeptide); NPY, neuropeptide Y; PYY, polypeptide hare. YY; +, positive immunoreactivity; -, negative immunoreactivity; /, not tested.

*3 Abst . Metg. Japan. Soc. Fisheries Sci., October, 1995, p. 227 (in Japanese). Neuropeptides in the Land Crab Brain 913 tocerebral, oculomotor nerve,antenna I and 11 nerves, teg o- chem. Physiol., 21, 394-400 (1985). mentary nerve, median nerve, esophageal connectives, 10) D. Sandeman, R. Sandeman, C. Derby, and M. Schmidt: Morpho anterior medial protocerebral neuropiles, and central logy of the brain of crayfish, crabs, and spiny lobsters:a common body= neuropile) described by them were also found in C. nomenclature for homologous structures. Biol. Bull., 183, 304-326 haematocheir. However, they did not give a detailed (1992). 11) M. Fingerman, M. M. Hanumante, G. K. Kulkarni, R. Ikeda, and description and classification of the cellular properties of L. L. Vacca: Localization of substance P-like , leucine- immunoreactivities. -like,methionine-enkephalin-like, and FMRFamide-like immuno

Schmidt and Ache17,18)considered the FMRFamide-im reactivity in the eyestalk of the fiddler crab, Uca pugilator . Cell munoreactive descending neurons and afferent branches in Tiss. Res., 241, 473-477 (1985). the brain and thoracic ganglia of the spiny lobster Panuli 12) J. E. van Deijnen, F. Vek, and F. van Herp: An immunohistochemi rus argus, as chemosensory elements. Mercier et al.15) cal study of the optic ganglia of the crayfish, Astacus leptodactylus demonstrated FMRFamide-like immunoreactivity in the (Nordmann 1842) with antisera against biologically active peptides of vertebrates and invertebrates. Cell Tiss. Res., 240, 175-183 crayfish Procambarus clarki, that acted as neuro-hor (1985). mones. Charmantier-Daures et al.13) recognized NPY-like 13) M. Charmantier-Daures, J-M. Danger, P. Netchitailo, G. Pelletier, immunoreactivity in the eyestalk of the lobster Homarus and H. Vaudry: Mise en evidence immunocytochimique de sub gammarus. Using 15 antisera, including anti-FMRFamide, stances apparentees au facteur natriuretique auriculaire et au neuro raised against invertebrate and vertebrate biologically ac peptide Y dans les pedoncules oculaires de Homarus gammarus tive neuro and gastrointestinal peptides, van Deijnen et (Crustacea, Decapoda). C. R. Acad. Sc., Paris, 305, 479-483 (1987). 14) A. J. Mercier, I. Orchard, and V. TeBrugge: FMRFamide-like im al.12)distinguished 30 cell groups in the optic ganglia of the munoreactivity in the crayfish nervous system. J. exp. Biol., 156, crayfish Astacus leptodactylus. Gaus et al.28) succeeded in 519-538 (1991). the isolation of five FMRFamide-like neuropeptides from 15) A. J. Mercier and R. T. Rusenes: Modulation of crayfish hearts by the brain and circumoesophageal rings of the horseshoe FMRFamide-related peptides. Biol. Bull., 182, 333-340 (1992). crab Limulus polyphenus. They considered that one of 16) U. Audehm, A. Trube, and H. Dircksen: Patterns and projections these may be involved in cardioregulation. Using the shore of crustacean cardioactive-peptide-immunoreactive neurons of the terminal ganglion of crayfish. Cell Tiss. Res., 272, 473-485 (1993). crab Carcinus maenas, Dircksen and Keller27)prepared a di 17) M. Schmidt and B. W. Ache: FMRFamide-like immunoreactivity in agrammatic illustration of the central nervous system and presumptive chemosensory afferents of the spiny lobster, Panulirus demonstrated the existence of a novel cardioactive peptide argus. Brain Res., 653, 315-324 (1994a). (CCAP). Remy et al.26)also discussed the diverse functions 18) M. Schmidt and B. W. Ache: Descending neurons with substance of these two peptides. On the basis of present knowledge, P/FMRF amide-like immunoreactivity target the somata of olfacto it is surmised that NPY and FMRFamide or their related ry interneurons in the brain of the spiny obster, Panulinus argus. substances may act as neurotransmitters or neuromodula Cell Tiss. Res., 278, 337-352 (1994b). 19) C. Girod, N. Durand, and M. Raccurt: Immunostaining of a cell tors in a variety of physiological processes in the crab type in the islets of Langerhans of the monkey Macaca irus by an brain. tibodies against S-100 protein. Cell Tiss. Res., 244, 11-16 (1987). 20) S. Oka, A. Chiba, and Y. Honma: Immunohistochemical distribu References tion of urotensins I and ‡U in the central nervous system of the Senegal bichir, Polypterussenegalus. Zool. Sci., 12, 311-315 (1995). 1) Y. Honma, Y. Umegawa, and A, Chiba: Studies on gonad maturity 21) S. Oka, A. Chiba, and Y. Honma: Urotensin-like immunoreactivity in some marine invertebrates-XI. Maturation and spawning behav in the nervous system of lower vertebrates and invertebrates. Zool. ior of the land-crab, Sesarma haematocheir (de Haan), on the coast Sci., 9, 1274 (1992). of Sado Island in the Sea of Japan. Ann. Rep. Sado Mar. Biol. 22) S. Oka, A. Chiba, and Y. Honma: Peptidergic innervation in the Star., Niigata Univ., 11, 113-123 (1981). caudal neurosecretory system of some fishes as revealed by double 2) T. Kitami and Y. Honma: Notes on the behavior of the land-crab, immunostaining. Zool. Sci., 10 (Suppl.), 138 (1993). Sesarma haematocheir (de Haan), on the coast of Sado Island in the 23) A. Chiba and Y. Honma: FMRFamide immunoreactive structures in the brain of the brown hagfish, Paramyxine atami: relationship Sea of Japan. Res. Crustacea, 11, 113-123 (1981). 3) M. Saigusa: Adaptive significance of a semiluna rhythm in the ter with neuropeptide Y-immunoreactive structures. Histochemistry, 98, 33-38 (1992). restrialstrial, Sesarma. Biol. Bull., 160, 311-321 (1981). 4) M. Saigusa: Larval release rhythm coinciding with solar day and 24) L. E. Muske, G. J. Dockray, K. S. Stell, and W. K. Stell: Segrega tion of FMRFamide-like immunoreactive efferent fibers from NPY tidal cycles in the terrestrial crab, Sesarma-harmony with the semilu immunoreactive amacrine cells in goldfish retina. Cell Tiss. Res., nar timing and its adaptive significance. Biol. Bull., 162, 311-321 247, 299-307 (1987). (1982). 25) B. M. Chronwall, J. A. Olschowka, and T. L. O'Donohue: Histo 5) Y. Honma, M. Ogiwara, and A. Chiba: Studies on gonad maturity chemical localization of FMRFamide-like immunoreactivity in the in some marine invertebrates-XII. Light and electron microscope rat brain. Peptides, 5, 569-584 (1984). studies on spermatozoa of the land-crab, Sesarma haematocheir (de 26) C. Remy, J. Guy, G. Pelletier, and H. H. Boer: Immunohistological Haan). Rep. Sado Mar. Biol. Star., Niigata Univ., 22, 13-21 (1992). demonstration of a substance related to neuropeptide Y and 6) Y. Honma, T. Kawahara, and A. Chiba: Immunohistochemical FMRFamide in the cephalic and thoracic nervous systems of the localization of neuropeptide Y-like substance in the abdominal gan locust Locusta migratoria. Cell Tiss. Res., 254, 189-195 (1988). glion of the sea hare, Aplysia kurodai: relationship with FMRF 27) H. Dircksen and R. Keller: Immunocytochemical localization of amide-like substance. Fisheries Sci., 60, 53-58 (1994). 7) C. K. L. Too and R. P. Croll: Detection of FMRFamide-like im CCAP, a novel crustacean cardioactive peptide, in the nervous sys tem of the shore crab, Carcinus maenas L. Cell Tiss. Res., 254, munoreactivities in the sea scallop Placopecten magellanicus by im 347-360 (1988). muno-histochemistry and western blot analysis. Cell Tiss. Res., 28) G. Gaus, K. E. Doble, D. B. Price, M. J. Greenberg, T. D. Lee, and 281, 295-304 (1995). B.-A. Battelle: The sequences of five neuropeptides isolated from 8) V. A. Tsvilenneva and V. A. Titova: On the brain structures of dec Limulus using antisera to FMRFamide. Biol. Bull., 184, 322-329 apods. Zool. Jb. Anat., 113, 217-266 (1985). 9) V. A. Tsvilenneva, V. A. Titova, and T. V. Kvashina: Brain topo (1993)chem. peptide(Crustacea, Physiol., Y nomenclature dans Decapoda). for stancesapparentees 21,leshomologous au pedoncules394-400 facteur C. 519-538 R. (1991).15) Acad.(1985).10) natriuretiqueA. structures. J.D. oculaires MercierSandeman, andR. Sc.,Sandeman, R. C. T. Derby, Rusenes: Modulationand Paris, Biol. de auriculaire M. Homarus of 305, Bull., Schmidt:crayfish 479-483 (1985).13) 183, etM. Charmantier-Daures, gammarus auJ-M. hearts Morphology Danger, 304-326ofP. neuro(1987).14)Netchitailo, theM.G.brainA.Fingerman, Pelletier, (1992).11) byM. J.of M.H. Mercier, FMRFamide-relatedcrayfish,peptides. I.Hanumante, Vaudry:Biol. crabs, Orchard,G. MiseBull.,andandK. and182,en Kulkarni, spiny 333-340argus. V. R. evidenceBrain lobsters:aTeBrugge: Ikeda,(1992).16) FMRFamide-like immunocytochimiqueandcommonRes., L.653,immunoreactivity U.de L.315-324 Vacca:Audehm, in sub LocalizationA.(1994a).18) theTrube, ofand crayfish M.substanceH. nervousSchmidt P-like,Dircksen: -like,system.andleucine-enkephalinPatterns J. andmethionine-enkephalin-like, (Nordmann B.exp. W. projectionsandBiol.,1842) Ache: crustacean156,with DescendingFMRFamide-likecardioactive-peptide-immunoreactiveantisera neuronsimmunoreactivity againstneuronswithin biologicallyofof substance active the the amide-likepeptidesganglion eyestalk terminalimmunoreactivity vertebratesof oftarget crayfish. theP/FMRFandthe Cell ofsomata fiddler invertebrates.Tiss. of Res.,crab,olfactory Cell272, tibodies 473-485Ucainterneurons againstTiss. (1993).17)in pugilator.S-100Res., Cellthe protein.240,Res.,M. brainCell Schmidt 241,Tiss. 175-183Tiss. and473-477of Res., theB. (1985).12)244, W. spiny Ache:11-16 FMRFamide-like lobster,J.(1987).20)immunoreactivity PanulinusE. vanin argus.S. Deijnen,presumptiveTiss.chemosensory F.Oka,Res.,afferentsVek,A. Cell278, andof Chiba, 337-352immunoreactiveandthe F. vanamacrine (1994b).19)Y.spiny Herp:Honma: lobster,cellsAn Immunohistochemical C.Panulirus inimmunohistochemical Girod,distribution study N.goldfish ofof Durand, retina. urotensinsthe andCelloptic I M.Tiss. ganglia and Raccurt:Res., ofImmunostaining 299-307 ‡Uthe of247, (1987).25) crayfish, ina Astacus with B.thecellleptodactylusM. neuropeptide incentral Chronwall,type J.nervoustheY-immunoreactive FMRFamide A. systemislets structures.in Olschowka, of andHistochemistry,the ofLangerhans 33-38 T.Senegal of thecephalicL. (1992).24) bichir,theO'Donohue: and Polypterussenegalus.Histochemicalmonkey Zool.L. thoraciclocalization MacacaSci.,E. nervous12,Muske, ofirus 311-315G.98, systems FMRFamide-likeby (1995).21)J. ofimmunoreactivityDockray,an K. theS.in S. Oka, Locustaimmunostaining.Stell, locustA. theZool.and migratoria.Chiba, brain. Sci.,ratand CellPeptides,W. 10 5,Tiss.Y.K. Honma: Res.,569-584(Suppl.),Stell: Urotensin-like Segregation254,138 (1984).26)immunoreactivity 189-195of (1993).23) the FMRFamide-like (1988).27) C. nervousimmunoreactive Remy, A.systemefferentJ.H.Chiba fibers Guy, DircksenofG.fromand Pelletier,lower and Y. andNPYvertebrates in Honma:R. H.and FMRFamide Keller:H. invertebrates. Boer:immunoreactive ImmunocytochemicalZool.Immunohistologicalstructures 9,localization 1274 theofSci.,of (1992).22)brain a aCCAP, novelofinS. crustacean substance the Oka, cardioactivebrown A.related peptide, Chiba,hagfish, indemonstration Paramyxinetoand the atami:Y. nervousneuropeptide relationship Honma: systemPeptidergicY ofinnervation and thein shore the neurosecretorycrab, caudal systemCarcinus maenasof L. some Cellfishes Tiss.as Res.,revealed 254,by (1988).28) double 347-360 G.Gaus, K. E. Doble, D.B. Price, M.J. Greenberg, T.D. Lee, and Battelle: B.-A.Thesequences of fiveneuropeptides isolatedfromusing Limulusantisera to FMRFamide. Biol.Bull., 184,322-329 (1993). graphy of the shore crab, Hemigrapsus sanguineus. J. Evol. Bi