Differential expression of neuropeptide F in the digestive organs of female freshwater prawn, Macrobrachium rosenbergii, during the ovarian cycle.

Neuropeptide F is a key hormone that controls feeding in invertebrates, including decapod crustaceans. We investigated the differential expression of Macrobrachium rosenbergii neuropeptide F (MrNPF) in the digestive organs of female prawns, M. rosenbergii, during the ovarian cycle. By using RT-qPCR, the expression of MrNPF mRNA in the esophagus (ESO), cardia (CD), and pylorus (PY) of the foregut (FG) gradually increased from stage II and peaked at stage III. In the midgut (MG), hindgut (HG), and hepatopancreas (HP), MrNPF mRNA increased from stage I, reaching a maximal level at stage II, and declined by about half at stages III and IV (P < 0.05). In the ESO, CD, and PY, strong MrNPF-immunoreactivities were seen in the epithelium, muscle, and lamina propria. Intense MrNPF-ir was found in the MG cells and the muscular layer. In the HG, MrNPF-ir was detected in the epithelium of the villi and gland regions, while MrNPF-ir was also more intense in the F-, R-, and B-cells in the HP. However, we found little colocalization between the MrNPF and PGP9.5/ChAT in digestive tissues, implying that most of the positive cells might not be neurons but could be digestive tract-associated endocrine cells that produce and secrete MrNPF to control digestive organ functions in feeding and utilizing feed. Taken together, our first findings indicated that MrNPF was differentially expressed in digestive organs in correlation with the ovarian cycle, suggesting an important link between MrNPF, the physiology of various digestive organs in feeding, and possibly ovarian maturation in female M. rosenbergii.

The presence and distribution of gamma-aminobutyric acid and dopamine during the developmental stages of the sea cucumber, Holothuria scabra, with emphasis on settlement organs.

In the sea cucumber, Holothuria scabra, the competent larvae require main settlement organs (SOs), including the ciliary bands (CiBs), tentacles (Ts), podia (PDs), and cues from neurotransmitters, including gamma-aminobutyric acid (GABA) and dopamine (DA), for successful settlement. In the present study, we investigated the spatial distribution of GABA and DA in the developmental stages of H. scabra, with special emphasis on SOs by detecting immunoreactivity (-ir) against these two neurotransmitters. Strong GABA-ir and DA-ir cells and fibers were specifically detected in several SO structures, including CiBs, CiB cells (CiBCs), and long cilia (LCi), of H. scabra larvae. Additionally, we found intense GABA-ir and DA-ir cells in the epithelial lining of bud-papillae (BP) and mesothelium (Me) in the stem (S) region of Ts in larvae and juveniles. Intense GABA-ir and DA-ir were observed in the epineural nerve plexus (ENP) and hyponeural nerve plexus (HNP) of Ts in H. scabra pentactula and juvenile stages. Staining for these two neurotransmitters was particularly intense in the PDs and their nerve fibers. We also found significant changes in the numbers of GABA-ir and DA-ir-positive cells and intensities in the CiBs, Ts, and PDs during the developmental stages. Taken together, we are the first to report on the existence and distribution of GABAergic and dopaminergic systems in structures associated with the settlement. Our findings provide new and important insights into the possible functions of these two neurotransmitters in regulating the settlement of this sea cucumber species.

Comparative neuroanatomical distribution and expression levels of neuropeptide F in the central nervous system of the female freshwater prawn, Macrobrachium rosenbergii, during the ovarian cycle.

Neuropeptide F (NPF) plays critical roles in controlling the feeding and reproduction of prawns. In the present study, we investigated changes in the expression levels of Macrobrachium rosenbergii neuropeptide F (MrNPF), and its neuroanatomical distribution in eyestalk (ES), brain (BR), subesophageal ganglion (SEG), thoracic ganglia (TG), and abdominal ganglia (AG), during the ovarian cycle of female prawn. By qRT-PCR, the amount of MrNPF transcripts exhibited a gradual increase in the ES, BR, and combined SEG and TG from stages I and II, to reach a maximum level at stage III, and slightly declined at stage IV, respectively. The highest to lowest expression levels were detected in combined SEG and TG, BR, ES, and AG, respectively. MrNPF immunolabeling was observed in several neuronal clusters, associated fibers, and neuropils of these central nervous system (CNS) tissues. MrNPF-ir was more intense in neurons and neuropils of SEG and TG than those found in other parts of the CNS. The number of MrNPF-ir neurons and intensity of MrNPF-ir were higher in the ES, BR, SEG, and TG at the late stages than those at the early stages of the ovarian cycle, while those in AG exhibited insignificant change. Taken together, there is a correlation between changes in the neuroanatomical distribution of MrNPF and stages of the ovarian cycle, implying that MrNPF may be an important neuropeptide that integrates sensory stimuli, including photo-, chemo-, and gustatory receptions, to control feeding and reproduction, particularly ovarian development, of this female prawn, M. rosenbergii.

Distribution of abalone egg-laying hormone-like peptide in the central nervous system and reproductive tract of the male mud crab, Scylla olivacea.

The mud crab, Scylla olivacea, is a high value economic marine animal in Thailand. However, collection of these crabs from natural habitat for local consumption and export has caused rapid population decline. Hence, aquaculture of this species is required and to this measure understanding of endocrine control of their reproduction must be understood. Egg laying hormone (ELH) is a neuropeptide synthesized by the bag cells (neurons) in the abdominal ganglia of Aplysia gastropods. It plays a critical role in controlling egg production and laying in gastropods, and its possible homolog (ELH-like peptide) was reported in the neural and ovarian tissues of prawns and recently in female reproductive tract of the blue swimming crab, Portunus pelagicus. In this study, we have studied the histology of the male reproductive tract in Scylla olivacea which are comprised of anterior testis, posterior testis, early proximal spermatic duct (ePSD), proximal spermatic duct (PSD), middle spermatic duct (MSD) and distal spermatic duct (DSD), by immunohistochemistry, detected an abalone ELH- immunoreactivity (aELH-ir) in epithelium of ducts in posterior testis and epithelium of all parts of spermatic duct. Furthermore, we could detect aELH-ir in neurons of cluster 9, 11, olfactory neuropil (ON) in the brain and in the small neurons located between the third and the fourth thoracic neuropils (T3-T4) and between the fourth and the fifth thoracic neuropils (T4-T5) of thoracic ganglia. Thus, the presence of aELH in male S. olivacea was designated the role of female egg laying behavior in the male mud crab.

The expression and distribution of a leptin receptor in the central nervous system, digestive organs, and gonads of the giant freshwater prawn, Macrobrachium rosenbergii.

In the present study, the presence and distribution of leptin receptor (LEP-R) in central nervous system, digestive organs, gonads of giant freshwater prawn, Macrobrachium rosenbergii, were investigated with Western blot and immunohistochemistry. By Western blot a LEP-R with a molecular weight (MW) of 100 kDa was detected in the brain, thoracic ganglia, abdominal ganglia, hepatopancreas, all parts of the gastrointestinal tract, ovaries, and testes. In hepatopancreas and foregut, another intense positive band was detected at molecular weight of 30 kDa, which could be an isotype of LEP-R. By immunohistochemistry, LEP-R-ir was detected in the neurons, and neuropils in the brain, thoracic ganglia, and abdominal ganglia. In the gastrointestinal tract, there was intense LEP-R-ir in the apical part of the epithelial cells of the foregut, midgut, and hindgut. In addition, LEP-R-ir was found in the Restzellen(R)cells and Fibrillenzellen(F) cells in the hepatopancreas. In the ovary, LEP-R-ir was detected in early stage of oocytes and mature oocytes. Intense LEP-R-ir was observed in spermatogonia and spermatocytes of the small and orange claw male prawns. In addition, LEP-R was seen in the high epithelium of spermatic ducts from all male morphotypes. In summary, the detection of the LEP-R-ir suggests the existence of a LEP-R in several organs of M. rosenbergii. Through binding with leptin peptide, LEP-R may be an important signaling molecule that has critical functions in modulating and controlling food intake, energy expenditure, and reproduction in this prawn.

Distribution and dynamic expression of serotonin and dopamine in the nervous system and ovary of Holothuria scabra during ovarian maturation.

In the present study, the distribution and dynamic expression of serotonin and dopamine in the nervous system and ovary of the sea cucumber, Holothuria scabra, during different ovarian stages were investigated. We found that serotonin-immunoreactivity was more intense in the neurons and neuropils of the outer ectoneural part, the inner hyponeural part, and the wall of hyponeural canal of radial nerve cord during the mature stages of ovarian cycle, whereas dopamine-immunoreactivity was detected at a higher intensity in these tissues during the early stages. Both neurotransmitters were detected in the ectoneural part of the nerve ring. In the ovary, serotonin intensity was more intense in the cytoplasm of late oocytes, while dopamine-immunoreactivity was more intense in the early stages. The changes in the levels serotonin in the radial nerve cord and oocytes are incremental towards the late stages of ovarian maturation. In contrast, dopamine levels in the nervous tissues and oocytes were more intense in early stages and became decremental towards the late stages. These findings suggest that serotonin and dopamine may have opposing effects on ovarian development in this sea cucumber species.

Transcriptomic discovery and comparative analysis of neuropeptide precursors in sea cucumbers (Holothuroidea).

Neuropeptides synthesized and released by neuronal cells play important roles in the regulation of many processes, e.g. growth, feeding, reproduction, and behavior. In the past decade, next-generation sequencing technologies have helped to facilitate the identification of multiple neuropeptide genes in a variety of taxa, including arthropods, molluscs and echinoderms. In this study, we extend these studies to Holothuria scabra, a sea cucumber species that is widely cultured for human consumption. In silico analysis of H. scabra neural and gonadal transcriptomes enabled the identification of 28 transcripts that encode a total of 26 bilaterian and echinoderm-specific neuropeptide precursors. Furthermore, publicly available sequence data from another sea cucumber, Holothuria glaberrima, allowed a more in-depth comparative investigation. Interestingly, two isoforms of a calcitonin-type peptide precursor (CTPP) were deduced from the H. scabra transcriptome - HscCTPP-long and HscCTPP-short, likely the result of alternative splicing. We also identified a sea cucumber relaxin-type peptide precursor, which is of interest because relaxin-type peptides have been shown to act as gonadotropic hormones in starfish. Two neuropeptides that appear to be holothurian-specific are GLRFA, and GN-19. In H. scabra, the expression of GLRFA was restricted to neural tissues, while GN-19 expression was additionally found in the longitudinal muscle and intestinal tissues. In conclusion, we have obtained new insights into the neuropeptide signaling systems of holothurians, which will facilitate physiological studies that may enable advances in the aquaculture of sea cucumbers.

Leptin-like immunoreactivity in the central nervous system, digestive organs, and gonads of the giant freshwater prawn, Macrobrachium rosenbergii.

Leptin, a highly conserved adipocyte-derived hormone, plays important roles in a variety of physiological processes, including the control of fat storage and metabolic status which are linked to food intake, energy homeostasis, and reproduction in all vertebrates. In the present study, we hypothesize that leptin is also present in various organs of the fresh water prawns, Macrobrachium rosenbergii. The existence and distribution of a leptin-like peptide in prawn tissues were verified by using Western blotting (WB) and immunohistochemical detection (ID) using primary antibody against human leptin. With WB, a leptin-like peptide, having a molecular weight of 15kDa, was detected in the brain, thoracic ganglia, abdominal ganglia, parts of the gastro-intestinal tract, hepatopancreas, adipocytes and gonads. By ID, leptin immunoreactivity (leptin-ir) was detected in the brain, thoracic ganglia and intersegmental commissural nerve fibers of abdominal ganglia. In the gastrointestinal tract, there was intense leptin-ir in the apical part of the epithelial cells of the cardiac and pyloric parts of the stomach. In the midgut and hindgut, the leptin-ir was detected in epithelial cells and basal cells located near the basal lamina of the epithelium. In addition, there was leptin-ir in the Restzellen cells in the hepatopancreas which produce digestive enzymes. In the ovary, the strong intensity of a leptin-ir was detected in the cytoplasm of middle to late stage oocytes, whereas no positive staining was detected in follicular cells. An intense leptin-ir was detected in spermatocytes and sustentacular cells in the seminiferous tubules in the testes of small and orange claw males. Taken together, the detection of the leptin-ir in several organs implicates the existence of a leptin-like peptide in various organs of the freshwater prawn; and like in vertebrates this peptide may be an important hormonal factor in controlling feeding and reproductive process.

Characterization and tissue distribution of neuropeptide F in the eyestalk and brain of the male giant freshwater prawn, Macrobrachium rosenbergii.

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, -II, -III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and -II precursor proteins, which show high similarity (91-95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and -II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.

Changes in the levels, expression, and possible roles of serotonin and dopamine during embryonic development in the giant freshwater prawn, Macrobrachium rosenbergii.

We investigated the changes in the levels of serotonin (5-HT) and dopamine (DA), and their possible roles during embryonic development of the freshwater prawn, Macrobrachium rosenbergii. The 5-HT and DA concentrations were quantified using high performance liquid chromatography with electrochemical detection (HPLC-ECD). The levels of 5-HT and DA gradually increased from early developing embryos to late developing embryos. The 5-HT concentrations gradually increased from the pale yellow egg to orange egg stages, and reaching a maximum at the black egg stage. DA concentrations were much lower in the early embryos than those of 5-HT (P<0.05), and gradually increased to reach the highest level at the black egg stage. Immunohistochemically, 5-HT was firstly detected in the early embryonic stages, whereas DA developed later than 5-HT. Functionally, 5-HT-treated female prawns at doses of 2.5×10(-5), 2.5×10(-6) and 2.5×10(-7)mol/prawn, produced embryos with significantly shortened lengths of early embryonic stages, whereas DA-treated prawns at all three doses, exerted its effects by significantly lengthening the period of mid-embryonic stage onwards. These results suggest significant involvement of 5-HT and DA in embryonic developmental processes of this species.

Spermatogenesis in the rock oyster, Saccostrea forskali (Gmelin, 1791).

Morphology of the differentiating spermatogenic cells of the rock oyster Saccostrea forskali (Bivalve: Ostreidae) was investigated by light and transmission electron microscopy. The testis is formed by several branching acini containing developing spermatogenic cells, classified into 7 stages based on nuclear characteristics, patterns of chromatin condensation and cytoplasmic contents. The spermatogonium is characterized by a euchromatic nucleus with a prominent nucleolus. The cytoplasm contains several round granulo-fibrillar dense bodies surrounded by numerous mitochondria. The round nucleus of the primary spermatocyte contains patches of electron-dense heterochromatin, numerous proacrosomal vesicles, ribosomes and mitochondria. The secondary spermatocytes contain a reticulated chromatin pattern and reduced number of proacrosomal vesicles. The early spermatids contain a small amount of euchromatin among dense patches of heterochromatin. A large single acrosomal vesicle is located in the posterior part of the cell. The middle spermatid is characterized by the migration of an acrosomal vesicle to the anterior part of the nucleus. The late spermatids contain highly condensed heterochromatin blocks and the acrosomal vesicle becomes cup-shaped and invaginated at the basal part. The spermatozoon contains a barrel-shaped head covered with the cup-like acrosome. At this stage, the subacrosomal space contains an axial rod in subacrosomal materials. Three to four transverse bands appear at the anterior region of the acrosome. The middle piece consists of spherical mitochondria surrounding the proximal and distal centrioles. The flagellum consists of 9+2 axonemal microtubule doublets surrounded by the plasma membrane. Our electron microscopic study of spermatogenesis in the S. forskali provides important new information on the mechanism of development of spermatogenesis of this species.

Spermatophore affects the egg-spawning and egg-carrying behavior in the female giant freshwater prawn, Macrobrachium rosenbergii.

In crustaceans, mating occurs during the ecdysis after female molting. During this period, a male transfers its spermatophore into a female which, in some species, stores the spermatophore for a long period prior to spawning and fertilization. However, in some species including the giant freshwater prawn, Macrobrachium rosenbergii, the male deposits its spermataphore onto the external surface of the thoracic segment of the female which affects the spawning time and maternal behavior. This study investigated the spawning behavior of the M. rosenbergii females, which was divided into pre-spawning, spawning, and post-spawning phases. It was revealed that mated female prawns with attached spermatophore exhibited an earlier spawning than unmated individuals, leading to assessment of the factors that may elicit this phenomenon. Four groups of female prawns were allocated to groups including mating females with spermatophore still attached, mating females with the spermatophore removed, artificially inseminated females with spermatophores, and an unmated control. There was a significant reduction in the time of egg-spawning in the presence of spermatophores, and the mating activity was also a contributing factor. Furthermore, over 90% of the mated and artificially inseminated females in which spermatophores were deposited carried the eggs in the abdominal brood chamber until completion of embryonic development while others discarded the eggs within 2 days post-spawning. This study implies that the spermatophore may contain ovulation-inducing factors which stimulate an earlier spawning and fostering of brooding behavior.

Distribution of serotonin and dopamine in the central nervous system of the female mud crab, Scylla olivacea (Herbst).

In crustaceans serotonin (5-HT) and dopamine (DA) are neurotransmitters that play roles in the modulation of numerous physiological functions, including reproduction. However, in the mud crab, Scylla olivacea, the distributions of 5-HT and DA in the CNS have not yet been investigated. The aim of our study was to map the distributions of these two neurotransmitters in the central nervous system (CNS) of the female of this crab during the late stage of ovarian development. We found 5-HT immunoreactivity (-ir) and DA-ir in many parts of the CNS, including the eyestalk, brain, and thoracic ganglia. In the eyestalk, 5-HT-ir was localized in the medulla terminalis (MT), hemi-ellipsoid body (HB), and protocerebral tract (PT), whereas DA-ir was present in neuronal cluster 1, the LG neuropils, and PT. In the brain, 5-HT-ir and DA-ir were detected in cells and fibers of neuronal clusters 6, 7, 8, 9, 10, 11, 14, and 15. In the ventral nerve cord, 5-HT-ir was present in neurons of the abdominal ganglia, whereas DA was only present in fibers. These spatial distributions of 5-HT and DA suggest that they may be involved in the neuromodulation of important physiological functions, including ovarian maturation, as shown in other non-crab decapods.

Alterations in the levels and distribution of octopamine in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei, and its possible role in ovarian development.

Octopamine (OA) is a major neurotransmitter that has not been studied in the Pacific white shrimp, Litopenaeus vannamei. Therefore, we investigated changes in OA levels, its distribution in regions of the central nervous system (CNS) and ovary during the ovarian maturation cycle, as well as its possible role in regulating ovarian maturation. OA exhibited the highest concentration in the brain and thoracic ganglia at ovarian stage II, and then declined to the lowest concentration at ovarian stages III and IV. In the cerebral ganglia, OA-immunoreactivity (OA-ir) was present in neurons of clusters 6, 17, the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic ganglia and abdominal ganglia, OA-ir was detected in several neuropils, neurons and fibers. The high level of intensity in OA immunostaining was observed in early developmental stage of oocyte by comparison with low level of OA-ir in late stages of oocyte development. Functionally, OA-injected female shrimps at doses of 2.5×10(-7) and 2.5×10(-6)mol/shrimp, showed significantly decreased gonado-somatic indices, oocyte diameters, and hemolymph vitellogenin levels, compared with control groups. This study showed changes of OA in the CNS and ovary reaching the highest level in early ovarian stages and declining in late stages, and it decreased hemolymph vitellogenin levels, suggesting significant involvement of OA in female reproduction in this species.

Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica.

Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Schistosoma mansoni, Moniezia benedeni, Avitellina centripunctata, Trichuris sp., Haemonchus placei and Setaria labiato-papillosa. Localization of CatL1 in each developmental stages of F. gigantica by immunoperoxidase technique, using these MoAbs as probes, indicated that CatL1 was present at high concentration in the caecal epithelium and caecal lumen of metacercariae, NEJ, 1, 3, 5-week-old juveniles and adult fluke. This finding indicated that CatL1 is a copiously expressed parasite protein that is released into the ES, thus CatL1 and its MoAb could be a good candidate for immunodiagnosis of fasciolosis in ruminant and human.

Immunohistochemical detection of GnRH-like peptides in the neural ganglia and testis of Haliotis asinina.

Gonadotropin releasing hormone (GnRH) is a peptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the distribution pattern of two isoforms of GnRH-like peptides in the neural ganglia and testis of reproductively mature male abalone, H. asinina, by immunohistochemistry and whole mount immunofluorescence. We found octopus (oct) GnRH and tunicate-I (t) GnRH-I immunoreactivities (ir) in type 1 neurosecretory cells (NS1) and they were expressed mostly within the ventral horn of the cerebral ganglion, whereas in pleuropedal ganglia they were localized primarily in the dorsal horn. Furthermore, tGnRH-I-ir were strongly detected in fibers at the caudal part of the cerebral ganglia and both ventral and dorsal horns of the pleuropedal ganglia. In the testis, only octGnRH-ir was found primarily in the granulated cell and central capillaries within the trabeculae. These results suggest that multiple GnRH-like peptides are present in the neural ganglia which could be the principal source of their production, whereas GnRH may also be synthesized locally in the testis and act as the paracrine control of testicular maturation.

Neuronal classification and distribution in the central nervous system of the female mud crab, Scylla olivacea.

The mud crab, Scylla olivacea, is one of the most economically valuable marine species in Southeast Asian countries. However, commercial cultivation is disadvantaged by reduced reproductive capacity in captivity. Therefore, an understanding of the general and detailed anatomy of central nervous system (CNS) is required before investigating the distribution and functions of neurotransmitters, neurohormones, and other biomolecules, involved with reproduction. We found that the anatomical structure of the brain is similar to other crabs. However, the ventral nerve cord (VNC) is unlike other caridian and dendrobrachiate decapods, as the subesophageal (SEG), thoracic and abdominal ganglia are fused, due to the reduction of abdominal segments and the tail. Neurons in clusters within the CNS varied in sizes, and we found that there were five distinct size classes (i.e., very small globuli, small, medium, large, and giant). Clusters in the brain and SEG contained mainly very small globuli and small-sized neurons, whereas, the VNC contained small-, medium-, large-, and giant-sized neurons. We postulate that the different sized neurons are involved in different functions.

The effects of serotonin, dopamine, gonadotropin-releasing hormones, and corazonin, on the androgenic gland of the giant freshwater prawn, Macrobrachium rosenbergii.

Neurotransmitters and neurohormones are agents that control gonad maturation in decapod crustaceans. Of these, serotonin (5-HT) and dopamine (DA) are neurotransmitters with known antagonist roles in female reproduction, whilst gonadotropin-releasing hormones (GnRHs) and corazonin (Crz) are neurohormones that exercise both positive and negative controls in some invertebrates. However, the effects of these agents on the androgenic gland (AG), which controls testicular maturation and male sex development in decapods, via insulin-like androgenic gland hormone (IAG), are unknown. Therefore, we set out to assay the effects of 5-HT, DA, l-GnRH-III, oct-GnRH and Crz, on the AG of small male Macrobrachium rosenbergii (Mr), using histological studies, a BrdU proliferative cell assay, immunofluorescence of Mr-IAG, and ELISA of Mr-IAG. The results showed stimulatory effects by 5-HT and l-GnRH-III through significant increases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). In contrast, DA and Crz caused inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). Moreover, the prawns treated with Crz died before day 16 of the experimental period. We propose that 5-HT and certain GnRHs can be now used to stimulate reproduction in male M. rosenbergii, as they induce increases in AG and testicular size, IAG production, and spermatogenesis. The mechanisms by which these occur are part of our on-going research.

Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease.

A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica.

Structure of the olfactory receptor organs, their GABAergic neural pathways, and modulation of mating behavior, in the giant freshwater prawn, Macrobrachium rosenbergii.

In the giant male prawn, Macrobrachium rosenbergii, the olfactory system is thought to be the main pathway for modulating sexual behavior through pheromone perception. In this report, we first used gross anatomical, histological, and SEM methods to describe the structures of the olfactory receptors (sensilla setae), their neural pathways, and possible role in modulating mating behavior. On the surfaces of antennule and antenna filaments there are four types of sensory receptors, viz single spike-like setae, single flagellum-like setae, multiple flagella-like setae, and aesthetascs (ASs). The ASs, which had previously been proposed to be odor receptor setae, are found only on the short filament of lateral antennule (slAn). Each AS on the slAn connects with olfactory receptor neurons (ORNs), whose axons form an outer central antennule nerve (ocAnNv), which then connects with the olfactory neutrophil (ON) of the brain. Thus, the slAn is the major olfactory organ that conveys sensory inputs from each AS to the ON within the deutocerebrum. GABA immunoreactivity was present in ASs, neurons of ORNs, inner central antennular, lateral tegumentary nerve, ocAnNv and the ON, inferring that GABA is the likely neurotransmitter in modulating olfaction. Disruption of the slAn by ablation or covering with Vaseline, resulted in significant reduction of mating behavior, indicating that this organ is crucial for sex pheromone perception. Identification of the active pheromones and further bioassays are now being performed.