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.

Variation of prostaglandin E2 concentrations in ovaries and its effects on ovarian maturation and oocyte proliferation in the giant fresh water prawn, Macrobrachium rosenbergii.

Prostaglandins (PGs) are important bioactive mediators for many physiological functions. In some decapod crustaceans, prostaglandin E2 (PGE2) has been detected in reproductive organs, and may play a role in the control of ovarian maturation. However, in the freshwater prawn, Macrobrachium rosenbergii, the presences of PGE2 and key enzymes for PGE2 biosynthesis, as well as its effects on ovarian maturation have not yet been investigated. In this study we reported the presence of PGE2, cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) in the ovarian tissues of M. rosenbergii, using immunohistochemistry. Intense immunoreactivities of PGE2 (PGE2-ir), COX1 (Cox1-ir) and PGES (PGES-ir) were detected in previtellogenic oocytes (Oc1 and Oc2), while the immunoreactivities were absent in the late vitellogenic oocytes (Oc4). This finding supports the hypothesis that the PGE2 biosynthesis occurs in the ovary of this prawn. To ascertain this finding we used LC-MS/MS to quantitate PGE2 concentrations during ovarian developmental cycle. The levels of PGE2 were significantly higher in the early ovarian stages (St I and II) than in the late stages (St III and IV). Moreover, we found that administration of PGE2 stimulated the ovarian maturation in this species by shortening the length of the ovarian cycle, increasing ovarian-somatic index, oocyte proliferation, and vitellogenin (Vg) level in the hemolymph.

Histological organization of the central nervous system and distribution of a gonadotropin-releasing hormone-like peptide in the blue crab, Portunus pelagicus.

We present a detailed histological description of the central nervous system (CNS: brain, subesophageal ganglion, thoracic ganglia, abdominal ganglia) of the blue crab, Portunus pelagicus. Because the presence of gonadotropin-releasing hormone (GnRH) in crustaceans has been disputed, we examine the presence and localization of a GnRH-like peptide in the CNS of the blue crab by using antibodies against lamprey GnRH (lGnRH)-III, octopus GnRH (octGnRH) and tunicate GnRH (tGnRH)-I. These antibodies showed no cross-reactivity with red-pigment-concentrating hormone, adipokinetic hormone, or corazonin. In the brain, strong lGnRH-III immunoreactivity (-ir) was detected in small (7-17 µm diameter) neurons of clusters 8, 9 and 10, in medium-sized (21-36 µm diameter) neurons of clusters 6, 7 and 11 and in the anterior and posterior median protocerebral neuropils, olfactory neuropil, median and lateral antenna I neuropils, tegumentary neuropil and antenna II neuropil. In the subesophageal ganglion, lGnRH-III-ir was detected in medium-sized neurons and in the subesophageal neuropil. In the thoracic and abdominal ganglia, lGnRH-III-ir was detected in medium-sized and small neurons and in the neuropils. OctGnRH-ir was observed in neurons of the same clusters with moderate staining, particularly in the deutocerebrum, whereas tGnRH-I-ir was only detected in medium-sized neurons of cluster 11 in the brain. Thus, anti-lGnRH-III shows greater immunoreactivity in the crab CNS than anti-octGnRH and anti-tGnRH-I. Moreover, our functional bioassay demonstrates that only lGnRH-III has significant stimulatory effects on ovarian growth and maturation. We therefore conclude that, although the true identity of the crab GnRH eludes us, crabs possess a putative GnRH hormone similar to lGnRH-III. The identification and characterization of this molecule is part of our ongoing research.

Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression.

Red pigment concentrating hormone (RPCH) is a member of the chromatophorotropic hormones and, in crustaceans, it is synthesized in the eyestalk. We have isolated a full-length cDNA for a RPCH preprohormone gene (Scyol-RPCH) from the eyestalks of female mud crabs, Scylla olivacea. The open reading frame consists of 642 nucleotides, and encodes a deduced 108 amino acid precursor protein, which includes a signal peptide, the RPCH (pQLNFSPGWamide), and an associated peptide. We show that the mud crab RPCH peptide exhibits 100% identity with 15 other decapods. Expression of Scyol-RPCH within adult mud crab takes place in the eyestalk, brain, and ventral nerve cord, comprising subesophageal ganglion, thoracic ganglion, and abdominal ganglion. In situ hybridization demonstrates specific expression within neuronal clusters 1, 2, 3, and 4 of the eyestalk X-organ, clusters 6, 8, 9, 10, and 17 of the brain, and in neuronal clusters of the ventral nerve cord. We found that administration of 5-HT up-regulates RPCH gene expression in the eyestalk, suggesting that RPCH may play a role as a downstream hormone of 5-HT.

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.

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus.

The crustacean X-organ-sinus gland (XO-SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO-SG, raises new questions concerning the established mode of MIH action.

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii.

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

Existence and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei.

We used antibodies against octopus gonadotropin-releasing hormone (octGnRH) and tunicate GnRH (tGnRH-I) in order to investigate the existence and distribution of GnRH-like peptides in the central nervous system (CNS) and in the ovary during various stages of the ovarian cycle of the white shrimp, Litopenaeus vannamei. OctGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in several regions of the supraesophageal ganglion (brain), subesophageal ganglion (SEG), thoracic ganglia, and abdominal ganglia. In the brain, both octGnRH immunoreactivity (ir) and tGnRH-I-ir were detected in neurons of clusters 6, 11, 17, and associated fibers, and the anterior medial protocerebral, posterior medial protocerebral, olfactory, and tegumentary neuropils. In the SEG and thoracic ganglia, octGnRH-immunoreactive and tGnRH-I-immunoreactive neurons and fibers were present in dorsolateral and ventromedial cell clusters and in surrounding fibers. Only immunoreactive fibers were detected in the abdominal ganglia. In the ovary, both octGnRH and tGnRH-I were detected at medium intensity in the cytoplasm of early step oocytes (Oc2) and, at high intensity, in Oc3. Furthermore, octGnRH-ir and tGnRH-I-ir were intense in follicular cells surrounding Oc2 and Oc3. The presence of GnRH-ir in the CNS and ovary indicates that GnRH-like peptides occur in the white shrimp, and that GnRHs are involved in the reproductive process, especially ovarian maturation and the differentiation of oocytes, as reported in other species.

Distribution and changes of serotonin and dopamine levels in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei, during ovarian maturation cycle.

We investigated changes in serotonin (5-HT) and dopamine (DA) levels and in their distribution patterns in the central nervous system (CNS) and ovary during the ovarian maturation cycle in the Pacific white shrimp, Litopenaeus vannamei. The concentrations of these two neurotransmitters were determined by using high performance liquid chromatography with electrochemical detection. The 5-HT concentration exhibited a gradual increase in the brain and thoracic ganglia during early ovarian stages I, II, and III, reaching a maximum at the mature ovarian stage IV, whereas DA showed its highest concentration at ovarian stage II in the brain and thoracic ganglia and then declined to its lowest concentration at ovarian stage IV. In the ovaries, 5-HT was lowest at ovarian stage I and gradually increased to a peak at ovarian stage IV. Conversely, the concentration of DA was highest at ovarian stages I and II and lowest at ovarian stage IV. In the brain, 5-HT immunoreactivity (-ir) from stage IV and DA-ir from stage II were distributed extensively in neurons of clusters 6, 11, and 17, in fibers, and in the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic, and abdominal ganglia, both 5-HT-ir and DA-ir were detected in neuropils and surrounding neurons and fibers. 5-HT-ir and DA-ir were more intense in the thoracic ganglia than in other parts of the CNS. In the ovary, 5-HT-ir exhibited high intensity in late oocytes, whereas DA-ir was more intense in early oocytes. Thus, opposing changes occur in the levels of these two neurotransmitters and in their specific localizations in the CNS and ovary during ovarian maturation, indicating their important involvement in female reproduction.

Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone.

In molluscs, the neurotransmitter serotonin (5-HT) has been linked to a variety of biological roles including gamete maturation and spawning. The possible involvement of 5-HT in abalone gamete release was demonstrated by a dose-dependent increase in Haliotis rubra gonad contractile bioactivity following 5-HT stimulation. Physiological functions associated with 5-HT, are mediated through binding to 5-HT receptors. A cDNA encoding a putative 5-HT receptor consisting of 359 amino acids was isolated from the tropical abalone H. asinina, termed 5-HT(1 ha). The 5-HT(1 ha) shares G-protein-coupled receptor motifs with metazoan 5-HT receptors, including predicted transmembrane domains, active sites for protein kinase action, and N-linked glycosylation sites. However, the third intracellular loop of 5-HT(1 ha) is relatively short, and only six transmembrane domains are predicted, implying a truncated receptor. Phylogenetic analysis with known 5-HT receptor genes suggests that 5-HT(1 ha) belongs to the type 1 5-HT receptor family. Expression analysis by RT-PCR showed that 5-HT(1 ha) mRNA was present in all tissues examined, including the neural ganglia and gonad tissues. Immunocytochemistry revealed the presence of 5-HT(1 ha) specifically within the soma of neuronal cells located in the outer cortex of both cerebral and pleuropedal ganglia. In ovarian and testicular tissues, 5-HT(1 ha) immunoreactivity was observed in epithelial cells of the outer capsule and connective tissue of the trabeculae to which the gamete follicles adhere. Whether this receptor transcript is translated to a functional protein needs to be verified, but if so, it could play a role in reproduction.

Mitochondrial DNA sequence and gene organization in the [corrected] Australian blacklip [corrected] abalone Haliotis rubra (leach).

The complete mitochondrial DNA of the blacklip abalone Haliotis rubra (Gastropoda: Mollusca) was cloned and 16,907 base pairs were sequenced. The sequence represents an estimated 99.85% of the mitochondrial genome, and contains 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes found in other metazoan mtDNA. An AT tandem repeat and a possible C-rich domain within the putative control region could not be fully sequenced. The H. rubra mtDNA gene order is novel for mollusks, separated from the black chiton Katharina tunicata by the individual translocations of 3 tRNAs. Compared with other mtDNA regions, sequences from the ATP8, NAD2, NAD4L, NAD6, and 12S rRNA genes, as well as the control region, are the most variable among representatives from Mollusca, Arthropoda, and Rhynchonelliformea, with similar mtDNA arrangements to H. rubra. These sequences are being evaluated as genetic markers within commercially important Haliotis species, and some applications and considerations for their use are discussed.

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.

In silico Neuropeptidome of Female Macrobrachium rosenbergii Based on Transcriptome and Peptide Mining of Eyestalk, Central Nervous System and Ovary.

Macrobrachium rosenbergii is the most economically important of the cultured freshwater crustacean species, yet there is currently a deficiency in genomic and transcriptomic information for research requirements. In this study, we present an in silico analysis of neuropeptide genes within the female M. rosenbergii eyestalk, central nervous system, and ovary. We could confidently predict 37 preproneuropeptide transcripts, including those that encode bursicons, crustacean cardioactive peptide, crustacean hyperglycemic hormones, eclosion hormone, pigment-dispersing hormones, diuretic hormones, neuropeptide F, neuroparsins, SIFamide, and sulfakinin. These transcripts are most prominent within the eyestalk and central nervous system. Transcript tissue distribution as determined by reverse transcription-polymerase chain reaction revealed the presence of selected neuropeptide genes of interest mainly in the nervous tissues while others were additionally present in the non-nervous tissues. Liquid chromatography-mass spectrometry analysis of eyestalk peptides confirmed the presence of the crustacean hyperglycemic hormone precursor. This data set provides a strong foundation for further studies into the functional roles of neuropeptides in M. rosenbergii, and will be especially helpful for developing methods to improve crustacean aquaculture.

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.

Changes of phosphatidylcholine and fatty acids in germ cells during testicular maturation in three developmental male morphotypes of Macrobrachium rosenbergii revealed by imaging mass spectrometry.

Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity.

Identification of genes associated with reproduction in the Mud Crab (Scylla olivacea) and their differential expression following serotonin stimulation.

The central nervous system (CNS) is often intimately involved in reproduction control and is therefore a target organ for transcriptomic investigations to identify reproduction-associated genes. In this study, 454 transcriptome sequencing was performed on pooled brain and ventral nerve cord of the female mud crab (Scylla olivacea) following serotonin injection (5 µg/g BW). A total of 197,468 sequence reads was obtained with an average length of 828 bp. Approximately 38.7% of 2,183 isotigs matched with significant similarity (E value < 1e-4) to sequences within the Genbank non-redundant (nr) database, with most significant matches being to crustacean and insect sequences. Approximately 32 putative neuropeptide genes were identified from nonmatching blast sequences. In addition, we identified full-length transcripts for crustacean reproductive-related genes, namely farnesoic acid o-methyltransferase (FAMeT), estrogen sulfotransferase (ESULT) and prostaglandin F synthase (PGFS). Following serotonin injection, which would normally initiate reproductive processes, we found up-regulation of FAMeT, ESULT and PGFS expression in the female CNS and ovary. Our data here provides an invaluable new resource for understanding the molecular role of the CNS on reproduction in S. olivacea.

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.

Expression of the male reproduction-related gene in spermatic ducts of the blue swimming crab, Portunus pelagicus, and transfer of modified protein to the sperm acrosome.

Expression of a sex-specific gene in Macrobrachium rosenbergii (Mr-Mrr), encoding a male reproduction-related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp-Mrr) and Scylla serrata (Ss-Mrr). We then investigated expression of Pp-Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp-Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp-Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti-Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp-Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs.

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.

Ultrastructure of differentiating oocytes and vitellogenesis in the giant freshwater prawn, Macrobrachium rosenbergii (De Man).

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone-shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc(1)) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc(2)) show nuclear heterochromatin with a "clock face" pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc(2). Early vitellogenic oocytes (Oc(3)) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc(4)) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc(3) and Oc(4), but there may be some undetected exogenous Vg production.