Immunolocalization of allatostatin-like neuropeptides and their putative receptor in eyestalks of the tiger prawn, Penaeus monodon

Allatostatin (AST)-like immunoreactivity (IR) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using four anti-AST antibodies. Depending on the antisera, AST-like immunoreactivity was detected in neuronal bodies of the lamina ganglionalis, cell bodies anterior to the medulla externa and cell bodies on the anterior and posterior of the medulla terminalis. Neuronal processes in neuropiles of the medulla externa, medulla terminalis, sinus gland and nerve fibers in the optic nerve were also recognized. No IR in cell bodies or in nerve fibers was found in the medulla interna. Strong AST-like immunoreactivity was found in hundreds of cells of the X organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X organ cells of the medulla terminalis in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk.     

Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon

FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.     

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.     

Localization of substance P-like,leucine-enkephalin-like,methionine-enkephalin-like,and FMRFamide-like immunoreactivity in the eyestalk of the fiddler crab,Uca pugilator

Summary The occurrence and distribution of substance P (SP)-like, methionine-(Met)- and leucine-(Leu)-enkephalinlike, and FMRFamide-like immunoreactivities were determined in the neuroendocrine complex of the eyestalk of the fiddler crab, Uca pugilator, by immunocytochemistry. SP-like immunoreactivity was found in the optic peduncle, sinus gland, medulla externa, medulla interna, lamina ganglionaris, and retinular cells. Met-enkephalin-like and Leuenkephalin-like immunoreactivity was observed in most of the retinular cells, optic peduncle, sinus gland, medulla terminalis, and lamina ganglionaris. However, Met-enkephalin-like, but no Leu-enkephalin-like, immunoreactivity was seen in the medulla terminalis X-organ. FMRFamide-like immunoreactivity could be seen in all parts of the eyestalk except in the sinus gland, lamina ganglionaris, and retinular cells. FMRF-amide-like activity was especially strong in the three chiasmatic regions connecting the optic ganglia. The possibility that these four peptides may function as neuroregulators in the fiddler crab is discussed.This investigation was supported in part by Grant No. PCM-8300064 from the National Science Foundation to MF and Biomedical Research Support Grant No. 2 SO7RRO5373 SUB from the University of Kansas Medical Center to LLV     

中华绒螯蟹窦腺神经末梢及X-器官神经分泌细胞的类型

在电子显微镜下观察了性未成熟的中华绒螯蟹黄蟹的窦腺及X－器官的超微结构。X－器官位于眼柄神经节终髓的腹外侧,与窦腺位置斜相对,窦腺主要由神经分泌细胞的末梢和胶质细胞组成。神经末梢含有大量的膜结构包围的颗粒、线粒体、粗面内质网和许多电子透明的小泡,末梢外周有时可见指状突起。依据颗粒的大小、形状、电子致密度以及胞质特征,可区分出6种类型的窦腺神经末梢及7种X－器官神经分泌细胞。观察了末梢中神经分泌颗粒的胞吐作用方式的释放过程,并且尝试对窦腺不同末梢中的颗粒及X－器官神经分泌细胞中的颗粒作了比较,发现二者之间具有较好的对应性,即电子致密度无大的变化,形态特征相似,只是大小稍有增加。     

Immunocytochemical localization of a substance in the eyestalk of the prawn,Palaemon serratus,reactive with an anti-FMRF-amide rabbit serum

Summary By use of a specific antiserum against the molluscan cardio-excitatory tetrapeptide FMRF-amide in combination with the PAP-method it was possible to obtain positive immunocytochemical reactions in several neurosecretory regions of the eyestalk of the prawn Palaemon serratus. FMRF-amide-like material was found in perikarya and nerve fibers of the medulla terminalis and in neurons in the lamina ganglionaris. The immunoreactivity observed in the glandular tissue located at the basal insertion of the eyestalk muscles must be ascribed to a non-specific reaction. The identification of immunopositive nerve fibers, ending on a nerve bundle in the medulla terminalis, and the fact that immunoreactive material was absent in the neurohemal sinus gland seem to indicate a neurotransmitter/neuromodulator function.     

Localization of crustacean hyperglycemic hormone (CHH) in the X-Organ sinus gland complex in the eyestalk of the crayfish,Astacus leptodactylus (Nordmann, 1842)

The eyestalk of Astacus leptodactylus is investigated immunocytochemically by light, fluorescence, and electron microscopy, using an antiserum raised against purified crustacean hyperglycemic hormone (CHH). CHH can be visualized in a group of neurosecretory perikarya on the medualla terminalis (medulla terminalis ganglionic X-organ: MTGX), in fibers forming part of the MTGX-sinus gland tractus, and in a considerable part of the axon terminals composing the sinus gland. Immunocytochemical combined with ultrastructural investigations led to the identification of the CHH-producing cells and the CHH-containing neurosecretory granule type.     

Two-dimensional gel electrophoresis of neurosecretory polypeptides in crustacean eyestalk

Summary A knowledge of the precise location of neurosecretory cell bodies is a prerequisite for studying the synthesis and subsequent processing of neurosecretory polypeptides stored in axon terminals comprising the sinus gland of the crustacean eyestalk. Structural data establish that the X organ in the medulla terminalis ganglion (mtXo) of the crayfish eyestalk represents 90–95% of the cell bodies actively synthesizing neurosecretory vesicles stored in the neurohemal sinus gland (Fig. 4). These cell bodies transport rather than accumulate neurosecretory vesicles as judged by light and electron microscopy suggesting that neurohormone precursors, but not subsequently stored products, might be found there. Two-dimensional electrophoresis of sinus gland and mtXo homogenates support this hypothesis. In crayfish, lobster and blue crab, stained two-dimensional gels display a number of sinus gland-specific polypeptides whose high concentrations and low molecular weights are consistent with stored neurosecretory material (Table 1). These neuropeptides are not detected in mtXo homogenates or in non-neurosecretory neural tissue with Coomassie Blue staining. By decreasing the porosity of the second dimension, the two-dimensional gel technique has proven useful in determining the molecular weights of a variety of neurosecretory polypeptides stored in the sinus gland. The crayfish and lobster store several polypeptides of ca. 7,000 Dalton. The blue crab stores two 7,000, two 13,000 and three 20,000 Dalton sinus gland polypeptides detected in stained gels.Following a 4 h incubation in³H-labelled amino acids, predominantly labelled 19,000–21,000 Dalton polypeptides are detected in crayfish mtXo homogenates by 2-D gel autoradiography (Fig. 12). Concomitantly, three labelled polypeptides (4,000–10,000 Dalton) appear in the sinus gland (Fig. 13), suggesting that they are cleaved from 19,000–21,000 Dalton molecules. This study is the first to examine neurosecretory precursors and their putative cleavage products in the Crustacea.Abbreviations mtXo medulla terminalis X organ - NEPHGE non-equilibrium pH gradient electrophoresis - PAF paraldehyde fuchsin - SDS sodium dodecylsulfate     

A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod crustacea

The eyestalk of the astacideans Orconects limosus, Nephrops norvegicus, and Homarus gammarus, and the palinuran Palinurus vulgaris, was examined with an antiserum raised against purified crustacean hyperglycemic hormone (CHH) of the astacidean species Astacus leptodactylus. A distinct immunopositive reaction occurs in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), in the MTGX-sinus gland tractus, and in a considerable part of the sinus gland. The immunoreactive sites in the eyestalk of the investigated species correspond to the site of production, storage, and release of the CHH. Preliminary investigations with this antiserum also indicate that a positive immunoreaction can be obtained in the eyestalk of other decapod crustaceans, for example, of the brachyuran Macropipus puber and the caridean Palaemon serratus.     

Occurrence of L- and D-crustacean hyperglycemic hormone isoforms in the eyestalk X-organ/sinus gland complex during the ontogeny of the crayfish Astacus leptodactylus.

We studied the ontogeny of the eyestalk structure and of the L-CHH and d-Phe3-CHH synthesis in the X-organ/sinus gland (XO/SG) complex by light microscopy and immunocytochemistry in the freshwater crustacean Astacus leptodactylus. The optic ganglia start to differentiate in embryos at EI 190 microm (EI: eye index; close to 410 microm at hatching). At EI 270 microm, the three medullae (externa, interna, and terminalis) and the lamina ganglionaris are present and are organized as in the adult eyestalk. The L-CHH was localized in perikarya of neuroendocrine cells, in their tracts, and in SG from the metanauplius stage to the adult. The d-Phe3-CHH was visualized in XO perikarya, in their tracts and in SG of embryos from EI 350 microm and in all later studied stages. Co-localization of both CHH stereoisomers always occurred in the d-Phe3-CHH-producing cells. These results show that the synthesis of CHH enantiomers starts during the embryonic life in A. leptodactylus, and that the d-isomer is synthesized later than its L-counterpart. We discuss the post-translational isomerization as a way to generate hormonal diversity and the putative relation between d-Phe3-CHH synthesis and the ability to osmoregulate, occurring late during the embryonic life of Astacus leptodactylus.     

Immunohistochemical Identification of Hyperglycemic Hormone- and Molt-Inhibiting Hormone-Producing Cells in the Eyestalk of the Kuruma Prawn, Penaeus japonicus

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.     

Appearance and innervation of CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus examined after tracing with Lucifer Yellow

Summary By injection of the fluorescent dye Lucifer Yellow into individual Crustacean Hyperglycemic Hormone (CHH)-producing cells, the shape of these neurosecretory cells in the eyestalk of the crayfish Astacus leptodactylus can be traced. A highly fluorescent perikaryon gives rise to an axon that can be followed by the fluorescent label to the neurohemal region, the sinus gland. The proximal part of that axon sends out extensive branches into the neuropil of the medulla terminalis. Electron-microscopic investigations reveal synaptic input to these axonal ramifications.     

Immunocytochemical localization of CCAP,a novel crustacean cardioactive peptide,in the nervous system of the shore crab,Carcinus maenas L.

Summary Polyclonal antibodies were raised in rabbits against synthetic crustacean cardioactive peptide (CCAP) conjugated to bovine thyroglobulin, and were used to map CCAP-immunoreactive structures in the central nervous system of Carcinus maenas. As expected, the neurohemal pericardial organs (PO) displayed abundant immunoreactivity in nerve fibers and terminals. In addition, immunoreactive neurons were demonstrated in other parts of the nervous system. At least some of them do not appear to terminate in neurohemal structures and may have a non-endocrine, as yet unknown function. Immunoreactive perikarya with a diameter of 25–30 m occur in the brain. They project into the optic and antennary neuropil, and into the eyestalk. One cell was found in the medulla terminalis of the eyestalk and in the connective ganglion, respectively. From the latter, axonal branches could be traced into the brain and the thoracic ganglia (TG). In the TG, small-diameter perikarya give rise to extensive networks of varicose fibers. Some of the perikarya occur in a characteristic paired arrangement with larger CCAP-immunoreactive somata (diameter 40–50 m). These pairs of one small and one large cell occur in all mouthpart and leg segments of the TG, except the abdominal ganglia (AG), where only large cells were found. The main projections of the large neurons comprise one or more fibers in each of the seven segmental nerves (SN), leading to neurosecretory terminals in the PO. The fibers in the SN are joined by branches of an ascending axonal tract from the large perikarya in the AG. The large-type perikarya are considered to be the principal source of CCAP in the PO. The optic ganglia in the eyestalk, except the medulla terminalis, the neurohemal sinus gland and the stomatogastric nervous system are devoid of CCAP-immunoreactivity.In axon terminals of the PO, CCAP is not colocalized with other PO-neuropeptides, i.e. proctolin-, FMRFamide-like, and Leu-enkephalin-like immunoreactive materials. Electron-microscopic immunocytochemistry revealed a distinct CCAP-containing granule type in specific axon profiles and terminals in the PO.The architecture of CCAP-immunoreactive neurons is discussed with respect to previous morphological studies on the origin and pathways of fibers terminating in the PO.Dedicated to Professor K.E. Wohlfarth-Bottermann, Bonn, on the occasion of his 65^th birthday     

Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.     

Structural re-evaluation of the neurosecretory system in the crayfish eyestalk

Summary The topography of the neurosecretory system in the decapod eyestalk has not been precisely delineated with light microscopy. Cobalt iontophoresis and electron microscopy have proved useful in clarifying the microstructure of this system. The sinus gland (sg) of the crayfish eyestalk consists of aggregated axon terminals which end at or near the blood space, lontophoresing cobalt back through the cut base of the sinus glands reveals proximal cell bodies in the eyestalk only in the X organ (Xo) region. Electron microscopy demonstrates that axons from about 115 neurosecretory cell bodies in the Xo form the Xo-sg tract. Intermingled with these Xo somata are smaller non-neurosecretory cell bodies which do not send axons into the sinus gland. One of these exhibits catecholamine fluorescence. Backfilling also reveals a second group of fibres which run from the brain along the optic tract and into the sinus gland. These brain-sg fibres are smaller in diameter than Xo-sg axons and lack neurosecretory vesicles. From these fibres collaterals extend into the eyestalk neuropil, especially in the proximity of the visual elements. The possible function of these non-neurosecretory processes within the sinus gland is discussed.This work was supported by a National Research Council of Canada grant     

Molt-inhibiting hormone immunoreactive neurons in the eyestalk neuroendocrine system of the blue crab, Callinectes sapidus

The production of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated by a neuropeptide, molt-inhibiting hormone. It is generally agreed that molt-inhibiting hormone is produced and released by the eyestalk neuroendocrine system. In the present study, immunocytochemical methods were used to detect molt-inhibiting hormone immunoreactive neurons in eyestalk ganglia of the blue crab, Callinectes sapidus. The primary antiserum used was generated against molt-inhibiting hormone of the green shore crab, Carcinus maenas. A preliminary Western blot analysis indicated the antiserum binds molt-inhibiting hormone of Callinectes sapidus. Using confocal and conventional immunofluorescence microscopy, molt-inhibiting hormone immunoreactivity was visualized in whole mounts and thin sections of Callinectes sapidus eyestalk ganglia. Immunoreactivity was detected in 15-25 neurosecretory cell bodies in the medulla terminalis X-organ, their associated axons and collateral branches, and their axon terminals in the neurohemal sinus gland. The cellular organization of molt-inhibiting hormone immunoreactive neurons in blue crabs is generally similar to that reported for other crab species. The combined results suggest the cellular structure of the molt-inhibiting hormone neuroendocrine system is highly conserved among brachyurans.     

Ontogeny of neuroendocrine centers in the eyestalk of Homarus gammarus embryos: an anatomical and hormonal approach

Summary

The ontogeny of the eyestalk neuroendocrine centers of the European lobster, Homarus gammarus, throughout embryonic development has been studied using light and electron microscopy, and the localization of specific neuroendocrine substances has been identified by immunocytochemistry. The procephalic lobes, which are the prospective eyestalks, develop progressively during embryonic development. In the nauplius stage two neuron masses are well defined. The visual structure originates from one of them and the neuroendocrine structure from the other. The four definitive optic ganglia are present at the mid-metanauplius stage and retain their appearance and location in larvae and adults. The organ of Bellonci, an internal sensory structure, appears at the mid-metanauplius stage and is mainly characterized by onion bodies. The medulla terminalis X-organ complex, an important neuroendocrine system, is present and already functional at the beginning of the embryonic metanauplius stage. Two neurohormones have been visualized immunocytochemically: the crustacean hyperglycemic hormone (CHH) and the gonad inhibiting hormone (GIH). Both neuropeptides are localized in the perikarya of neuroendocrine cells of the X-organ as well as in their tracts joining the presumptive sinus gland. However, the sinus gland has only been observed in the early larval stages just after hatching.     

Serotonin-containing Cells in the Nervous System and Other Tissues During Ontogeny of a Lancelet,Branchiostoma floridae

Abstract Serotonin-containing cells are described by immunohistochemistry throughout lancelet ontogeny. Such cells are first detected in the 2-day larva: these are (1) enterochromaffin cells in the inner epithelium of the gut and (2) anterior serotonergic neurons at the rostral end of the nerve cord. In the 6-day larva, relatively low levels of serotonin appear in ventro-lateral perikarya and cell processes of intraspinal serotonergic neurons scattered along the nerve cord. In the 18-day (early metamorphic) larva, antero-lateral serotonergic neurons are detected near the rostral end of the nerve cord as two small, bilateral clusters of perikarya with axons that descend the nerve cord; at later developmental stages, these axons extend almost to the posterior end of the body. In the 21-day (mid-metamorphic) larva, serotonin can no longer be detected in the anterior serotonergic neurons, but serotonin-containing cells are found subjacent to the inner epithelium of the digestive caecum and in the peribranchial epithelium covering the primary gill bars. In the discussion, we suggest that the anterior serotonergic neurons may play a role in larval photoreception and that the antero-lateral serotonergic neurons may be homologous to vertebrate hindbrain neurons with axons descending the spinal cord to modulate undulation (if this homology is valid, the anterior limit of the lancelet hindbrain would be roughly 100 μm behind the rostral tip of the nerve cord).     

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.     

Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii

Heptapeptide (YANAVQV-NH2 = T-) and octapeptide (YANAVQTV-NH2 = T+), the putative C-terminus of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, was synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization in swiss mice. Specificity of the antisera against both peptides was determined by indirect immunoperoxidase ELISA. The best response of antiserum against each peptide was used to determine the presence of the natural CHH in the eyestalk extract after separation by one step of RP-HPLC using dot-ELISA. The peptide immunoreactive substances were found in fraction 30 using anti-T- antiserum and in fraction 38 using anti-T+ antiserum. However, the CHH activity was found only in fractions 37-39. Immunocytochemical localization of peptide immunoreactive substances in the eyestalk of M. rosenbergii using the anti-T- antiserum did not show any specific staining. In contrast, the anti-T+ antiserum revealed specific staining on a group of 24 +/- 5 neurons in medulla terminalis ganglionic x-organ and their processes through the sinus gland. Similar results were also obtained using the eyestalk of another species, the giant tiger prawn Penaeus monodon, in which 34 +/- 4 neuronal cells were recognized. These results strongly indicate that the anti-T+ antibody can bind to the natural CHH while the anti-T- antibody can not; therefore, this isoform of CHH in M. rosenbergii should consist of 72 residues and threonine is predicted to be present at position 71.