Saowaros Suwansa-ard

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.

Steroids and genes related to steroid biosynthesis in the female giant freshwater prawn, Macrobrachium rosenbergii

The giant freshwater prawn, Macrobrachium rosenbergii, is important to many Asian countries due to its high economic value as an aquaculture product. With demand increasing, there is requirement for a better understanding of the biosynthetic components that regulate its growth and reproduction, including steroids, in order to help increase production. Vertebrate-type steroids and their receptors were identified in crustaceans and implicated in reproduction. In this study, we presented the sex steroids estradiol and progesterone by LC-MS/MS in female M. rosenbergii, and reveal steroidogenic-related genes by in silico analysis of de novo assembled transcriptomes. Comparative analysis with other species was performed to confirm their putative role, as well as tissue-specific and quantitative gene expression. We reveal 29 transcripts that encode for steroidogenic-related proteins, including steroidogenic enzymes, a nuclear steroid hormone receptors, and a steroidogenic factor. Moreover, we identified for the first time the presence of steroidogenic factor 1, StAR-related lipid transfer protein, estradiol receptor- and progesterone-like protein in M. rosenbergii. Those targeted for gene expression analysis (3 beta-hydroxysteroid dehydrogenase, 17 beta-hydroxysteroid dehydrogenase, estrogen sulfotransferase and progesterone receptor-like) showed widespread expression within many tissues, and at relatively high levels in the central nervous system (CNS) during ovarian maturation. In summary, we provide further evidence for the existence of steroidogenic pathways in crustaceans, which may be useful for advancing prawn aquaculture.

The neuropeptidome of the Crown-of-Thorns Starfish, Acanthaster planci

Outbreaks of Crown-of-Thorns Starfish (COTS; Acanthaster planci) are a major cause of destruction of coral communities on the Australian Great Barrier Reef. While factors relating to population explosions and the social interactions of COTS have been well studied, little is known about the neural mechanisms underlying COTS physiology and behaviour. One of the major classes of chemical messengers that regulate physiological and behavioural processes in animals is the neuropeptides. Here, we have analysed COTS genome and transcriptome sequence data to identify neuropeptide precursor proteins in this species. A total of 48 neuropeptide precursors were identified, including homologs of neuropeptides that are evolutionarily conserved throughout the Bilateria, and others that are novel. Proteomic mass spectrometry was employed to confirm the presence of neuropeptides in extracts of radial nerve cords. These transcriptomic and proteomic resources provide a foundation for functional studies that will enable a better understanding of COTS physiology and behaviour, and may facilitate development of novel population biocontrol methods. SIGNIFICANCE The Crown-of-Thorns Starfish (COTS) is one of the primary factors leading to coral loss on the Great Barrier Reef, Australia. Our combined gene and proteomic findings of this study reveal the COTS neuropeptidome, including both echinoderm-like neuropeptides and novel putative neuropeptides. This represents the most comprehensive neuropeptidome for an echinoderm, contributing to the evolving knowledge of the COTS molecular neurobiology that may assist towards the development of biocontrol methods.

Gonadotropin-releasing hormone and adipokinetic hormone/corazonin-related peptide in the female prawn.

Crustacean neuropeptides (NPs) play important roles in the regulation of most physiological activities, including growth, molting and reproduction. In this study, we have performed an in silico analysis of female prawn (Macrobrachium rosenbergii) neural transcriptomes to identify NPs not previously identified. We predict that approximately 1309 proteins are destined for the secretory pathway, many of which are likely post-translationally processed to generate active peptides. Within this neural secretome, we identified a gene transcript that encoded a precursor protein with striking similarity to a gonadotropin-releasing hormone (GnRH). We additionally identified another GnRH NP superfamily member, the adipokinetic hormone/corazonin-related peptide (ACP). M. rosenbergii GnRH and ACP were widespread throughout the nervous tissues, implicating them as potential neuromodulators. Furthermore, GnRH was found in non-neural tissues, including the stomach, gut, heart, testis and ovary, in the latter most prominently within secondary oocytes. The GnRH/corazonin receptor-like gene is specific to the ovary, whereas the receptor-like gene expression is more widespread. Administration of GnRH had no effect on ovarian development and maturation, nor any effect on total hemolymph lipid levels, while ACP administration decreased oocyte proliferation (at high dose) and stimulated a significant increase in total hemolymph lipids. In conclusion, our targeted analysis of the M. rosenbergii neural secretome has revealed the decapod GnRH and ACP genes. We propose that ACP in crustaceans plays a role in the lipid metabolism and the inhibition of oocyte proliferation, while the role of the GnRH remains to be clearly defined, possibly through experiments involving gene silencing.

Neuropeptides encoded within a neural transcriptome of the giant triton snail Charonia tritonis, a Crown-of-Thorns Starfish predator

HIGHLIGHTSNeuropeptide repertoire identified for the endangered giant triton snail.Extensive transcriptome derived from giant triton snail cerebral ganglia.Reproduction neuropeptides include APGWamide, conopressin and egg laying hormone. ABSTRACT Neuropeptides represent a diverse class of signaling molecules originating from neural tissues. These chemical modulators orchestrate complex physiological events including those associated with growth and reproduction. De novo transcriptome sequencing of a cerebral ganglion library of the endangered giant triton snail (Charonia tritonis) was undertaken in an effort to identify key neuropeptides that control or influence its physiology. The giant triton snail is considered a primary predator of the corallivore Acanthaster planci (Crown‐of‐Thorns Starfish) that is responsible for a significant loss in coral cover on reefs in the Indo‐Pacific. The transcriptome library was assembled into contigs, and then bioinformatic analysis was used to identify a repertoire of 38 giant triton snail neuropeptide precursor genes, and various isoforms, that encode conserved molluscan neuropeptides. C. tritonis neuropeptides show overall precursor organisation consistent with those of other molluscs. These include those neuropeptides associated with mollusc reproduction such as the APGWamide, buccalin, conopressin, gonadotropin‐releasing hormone (GnRH), NKY and egg‐laying hormone. These data provide a foundation for further studies targeted towards the functional characterisation of neuropeptides to further understand aspects of the biology of the giant triton snail, such as elucidating its reproductive neuroendocrine pathway to allow the development of knowledge based captive breeding programs.

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.

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

HighlightsTranscriptome sequencing of Holothuria scabra.Identification of putative neuropeptides in H. scabra and H. graberrima transcriptomes.Comparative analysis of potential holothurian specific neuropeptides and their tissue expressions. ABSTRACT 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.

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 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.