Napamanee Kornthong

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.

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.

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.

Composition and Localization of Lipids in Penaeus merguiensis Ovaries during the Ovarian Maturation Cycle as Revealed by Imaging Mass Spectrometry

Ovary maturation, oocyte differentiation, and embryonic development in shrimp are highly dependent on nutritional lipids taken up by female broodstocks. These lipids are important as energy sources as well as for cell signaling. In this study, we report on the compositions of major lipids, i.e. phosphatidylcholines (PCs), triacylglycerols (TAGs), and fatty acids (FAs), in the ovaries of the banana shrimp, Penaeus merguiensis, during ovarian maturation. Thin-layer chromatography analysis showed that the total PC and TAG signal intensities increased during ovarian maturation. Further, by using gas chromatography, we found that (1) FAs 14∶0, 16∶1, 18∶1, 18∶2, 20∶1, and 22∶6 proportionally increased as ovarian development progressed to more mature stages; (2) FAs 16∶0, 18∶0, 20∶4, and 20∶5 proportionally decreased; and (3) FAs 15∶0, 17∶0, and 20∶2 remained unchanged. By using imaging mass spectrometry, we found that PC 16∶0/16∶1 and TAG 18∶1/18∶2/22∶6 were detected in oocytes stages 1 and 2. PCs 16∶1/20∶4, 16∶0/22∶6, 18∶3/22∶6, 18∶1/22∶6, 20∶5/22∶6, and 22∶6/22∶6 and TAGs 16∶0/16∶1/18∶3, 16∶0/18∶1/18∶3, 16∶0/18∶1/18∶1, and 16∶0/18∶2/22∶6 were present in all stages of oocytes. In contrast, the PC- and TAG-associated FAs 20∶4, 20∶5, and 22∶6 showed high signal intensities in stage 3 and 4 oocytes. These FAs may act as nutrition sources as well as signaling molecules for developing embryos and the hatching process. Knowledge of lipid compositions and localization could be helpful for formulating the diet for female broodstocks to promote fecundity and larval production.

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.

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.

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. Microsc. Res. Tech. 77:189–200, 2014.

Transcriptomic analysis of the autophagy machinery in crustaceans

BackgroundThe giant freshwater prawn, Macrobrachium rosenbergii, is a decapod crustacean that is commercially important as a food source. Farming of commercial crustaceans requires an efficient management strategy because the animals are easily subjected to stress and diseases during the culture. Autophagy, a stress response process, is well-documented and conserved in most animals, yet it is poorly studied in crustaceans.ResultsIn this study, we have performed an in silico search for transcripts encoding autophagy-related (Atg) proteins within various tissue transcriptomes of M. rosenbergii. Basic Local Alignment Search Tool (BLAST) search using previously known Atg proteins as queries revealed 41 transcripts encoding homologous M. rosenbergii Atg proteins. Among these Atg proteins, we selected commonly used autophagy markers, including Beclin 1, vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), p62/sequestosome 1 (SQSTM1), and lysosomal-associated membrane protein 1 (Lamp-1) for further sequence analyses using comparative alignment and protein structural prediction. We found that crustacean autophagy marker proteins contain conserved motifs typical of other animal Atg proteins. Western blotting using commercial antibodies raised against human Atg marker proteins indicated their presence in various M. rosenbergii tissues, while immunohistochemistry localized Atg marker proteins within ovarian tissue, specifically late stage oocytes.ConclusionsThis study demonstrates that the molecular components of autophagic process are conserved in crustaceans, which is comparable to autophagic process in mammals. Furthermore, it provides a foundation for further studies of autophagy in crustaceans that may lead to more understanding of the reproduction- and stress-related autophagy, which will enable the efficient aquaculture practices.

Identification and expression of prostaglandin E synthase (PGES) gene in the central nervous system and ovary during ovarian maturation of the female mud crab, Scylla olivacea

Prostaglandins have important physiological roles in marine invertebrates, including larval development and reproduction. The prostaglandin E concentration fluctuates during the ovarian development of crustaceans. The biosynthetic pathway of prostaglandin, however, has not been well studied in portunid crabs, including in the mud crab, Scylla olivacea. In this study, the aim was to investigate the presence of prostaglandin E synthase (PGES), enzyme that catalyzes the terminal conversion in the prostaglandin E2 (PGE2) biosynthesis, and its gene expression in the central nervous system (CNS) and ovary during ovarian maturation of S. olivacea. cDNA sequence encoding PGES was cloned from the S. olivacea ovary. The PGES transcript of S. olivacea (Scyol-PGES) consists of 1258 nucleotides, which encodes for 420 amino acid PGES protein precursor. Investigation of gene expression by RT-PCR indicated that Scyol-PGES was detected in all organs studied. Based on in situ hybridization, Scyol-PGES was detected in the I to III stages for oocyte development of Stage 3 of ovarian development, and in the CNS, including the various neuronal clusters of the brain. In the ventral nerve cord, the Scyol-PGES gene was expressed in the neurons within the subesophageal, thoracic and abdominal ganglia. The Scyol-PGES gene expression as indicated by relative abundance of mRNA in the Stage 4 of ovarian development was greater than that at Stages 1 to 3 of ovarian development. This is the first report on PGES in the mud crab, S. olivacea, and its gene expression suggested the involvement of PGES in the ovarian development of this species.