Transcriptomic Analysis of the Kuruma PrawnMarsupenaeus japonicusReveals Possible Peripheral Regulation of the Ovary

Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawnMarsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-alpha and -beta, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicussinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP usingEscherichia coliexpression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates

Editorial: Crustacean reproductive physiology and its applications

[Extract] Crustacea constitute an important taxonomic group in aquatic ecosystems and form an important sector of aquaculture industry. Novelties in studies on crustacean reproductive physiology help shedding new lights on deeper understanding of the mechanisms of sex determination and differentiation of crustaceans. The application of established and innovative techniques based on such knowledge will contribute significantly to progress in the crustacean aquaculture industry. The objective of this Special Issue was to provide a forum for researchers to report upon their cutting-edge research in Crustacean Reproductive Physiology and its Applications. This Research Topic comprises ten original research articles

Vasopressin-oxytocin–type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians

Vasopressin/oxytocin (VP/OT)–related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the “platytocin” system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an “antidiuretic hormone” and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses

甲殻類の生殖制御と栄養～インスリンシグナル経路からの新規アプローチ～

application/pdf本研究では、クルマエビを対象にしたトランスクリプトーム解析により、ILP-1、ILP-2、ゴナデュリン (GON) というインスリン様ペプチドを見出した。成体を用いた解析では、ILP1は卵巣、ILP2は精巣、GONは輸精管というように、生殖腺において遺伝子発現の性的二型や部位特異的発現がみられた。一方、ポストラーバ変態後74日までは、遺伝子発現における明確な雌雄差は確認できなかった。ILP1と2は、卵巣や肝膵臓での卵黄タンパク質の発現に影響を及ぼさなかった。In this study, three novel insulin-like peptides [ILP-1, ILP-2, and gonadulin (GON)] were found by transcriptome analysis in the kuruma prawn. Tissue-specific expression analyses on adults revealed sexual dimorphism and site-specific expression of these genes in the gonads: ILP1 in the ovary, ILP2 in the testis, and GON in the vas deferens. In contrast, no clear sex differences in gene expression were identified at least until 74 days after post-larval metamorphosis. Synthesized ILP1 and ILP2 had no effect on vitellogenin gene expression in the incubated ovary or hepatopancreas fragments.2018年度～2021年度科学研究費補助金(基盤研究(C))研究成果報告書18K0581

Preparation of two recombinant crustacean hyperglycemic hormones from the giant freshwater prawn Macrobrachium rosenbergii, and their hyperglycemic activities

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks, and regulates glucose levels in the hemolymph. In the giant freshwater prawn (Macrobrachium rosenbergii), two cDNAs encoding different CHH molecules were previously cloned by other workers. One of these (Mar-CHH-2) was expressed only in the eyestalks, whereas the other (Mar-CHH-L) was expressed in the heart, gills, antennal gland, and thoracic ganglion, but not in the eyestalks. However, their biological activities had not yet been characterized. Therefore, in this study, recombinant Mar-CHH-2 (rMar-CHH-2) and Mar-CHH-L (rMar-CHH-L) were produced using an E. coli expression system, by expression in bacterial cells and recovery in the insoluble fraction. Thereafter, rMar-CHH-2 and rMar-CHH-L were subjected to refolding and were subsequently purified by reversed-phase HPLC. The rMar-CHH-2 and rMar-CHH-L thus obtained exhibited the same disulfide bond arrangements as those of other CHHs reported previously, indicative of natural conformation. In in vivo bioassay, rMar-CHH-2 showed significant hyperglycemic activity, whereas rMar-CHH-L had no effect. These results indicate that Mar-CHH-L does not function as a CHH, but may have some other, unknown function

Cloning and expression of a cDNA encoding an insoluble matrix protein in the gastroliths of a crayfish, Procambarus clarkii

In the crayfish Procambarus clarkii, the gastroliths are formed as a paired structure in the stomach during the premolt period, and contain calcium carbonate and a small amount of an organic matrix. In this investigation, a cDNA encoding an insoluble matrix protein was isolated from P. clarkii. The open reading frame encoded 505 amino acid residues including two unique repeated sequences. The N-terminal half of the amino acid sequence, which included 10-amino-acid repeats, exhibited a high degree of similarity to that of involucrin, a protein synthesized in human keratinocytes. Northern blot analysis revealed that mRNA encoding the matrix protein is specifically expressed in the gastrolith discs during the premolt period in which the gastroliths formed. In the gastrolith discs, levels of expression of this mRNA correlated increases in weights of the gastroliths concomitant with their formation. Organ culture of the gastrolith discs suggested that expression of mRNA in the discs is induced by molting hormone, 20-hydroxyecdysone. These results reinforced the relationship between the matrix protein and formation of the gastroliths. Functional analysis showed that the protein inhibits calcium carbonate crystallization in a solution system, suggesting that the protein plays a role in the calcification of the gastroliths