Kuruma Prawn Marsupenaeus Japonicus Research Articles

Distribution and role of d-glutamate, a novel d-amino acid identified in animals, in the reproductive tissues of male kuruma prawn Marsupenaeus japonicus.

Some aquatic invertebrates contain free d-alanine. We previously showed copious amounts of free d-glutamate, a novel d-amino acid, in the tissue of the male reproductive organs of Marsupenaeus japonicus. Herein, we clarified the distribution and potential role of d-glutamate and d-alanine in male reproductive tissues, namely the testis, vas deferens and seminal receptacle at different growth stages of M. japonicus. The percentage of d-glutamate to total glutamate was over 50% in these tissues. In particular, the content of d-glutamate was the most abundant in the vas deferens, the ratio of d-glutamate to total glutamate was approximately 80%. In contrast, d-alanine content was the lowest in the vas deferens among these tissues. d-Glutamate content was the highest when the prawn weighed 12g, indicating that d-glutamate is actively synthesized in the younger stage. Our findings suggest that d-glutamate plays an important role in the reproductive functions of M. japonicus.

Dual roles of crustacean female sex hormone during juvenile stage in the kuruma prawn Marsupenaeus japonicus

The crustacean female sex hormone (CFSH) has been identified as a female-specific hormone that plays a crucial role in female phenotype developments in the blue crab Callinectes sapidus. To date, its homologous genes have been reported in various decapod species. Additionally, unlike the blue crab, several species have two different CFSH subtypes. The kuruma prawn Marsupenaeus japonicus is a representative example species of this phenomenon, having two CFSH subtypes identified from the eyestalk (MajCFSH) and ovary (MajCFSH-ov). Eyestalk-type MajCFSH is expressed predominantly in the eyestalk at the same level in both sexes, indicating no female-specificity. Here, we conducted gene knockdown analysis of eyestalk-type MajCFSH using sexually immature juveniles of kuruma prawn (average body length: ∼10 mm) to elucidate its physiological functions. As a result, MajCFSH-knockdown did not affect the development of sex-specific characteristics such as external reproductive organs, while it induced apparent growth suppression in male juveniles, implying that MajCFSH may play a male-biased juvenile growth role. Moreover, MajCFSH-knockdown female and male juveniles changed their body color to become brighter, indicating that MajCFSH has the ability to change body color by dispersing the pigment granules in the chromatophore. Overall, our present study improved our understanding of the physiological roles of CFSH using kuruma prawn.

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus.

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.

Update on the source of phenoloxidase activity in the hemolymph of kuruma prawn Marsupenaeus japonicus

Crustacean phenoloxidase (PO) and hemocyanin (Hc) are classified as type 3 copper proteins. PO catalyzes the oxidation of mono- and di-phenol compounds, which is the rate-limiting step of melanization, while Hc generally functions as a dioxygen-transporting protein in the hemolymph of arthropods. To date, many studies have shown PO activity in Hc, which is inspired by their structural similarity. Here, the source of PO activity in crustaceans was re-examined by purifying Hc and PO exclusively from the hemolymph of kuruma prawn. The conventional procedure for the preparation of arthropod Hc, which includes precipitation of Hc by ultracentrifugation and subsequent purification by size exclusion chromatography, was not able to completely remove hemolymph-type PO from Hc. In contrast, fractionation with 50% saturation of ammonium sulfate and subsequent hydrophobic chromatography yielded sufficiently pure Hc, which contained no detectable PO protein and virtually no PO enzymatic activity. These results indicate that the main source of PO activity in the hemolymph of kuruma prawn is hemolymph-type PO and that the improved purification method of Hc is preferable for evaluating the PO activity of Hc.

Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction.

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity.

Transcriptomic Analysis of the Kuruma Prawn Marsupenaeus japonicus Reveals 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 prawn Marsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-α and -β, 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 japonicus sinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP using Escherichia coli expression 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.

Impacts of Methyl Farnesoate and 20-Hydroxyecdysone on Larval Mortality and Metamorphosis in the Kuruma Prawn Marsupenaeus japonicus.

Physiological functions of juvenile hormone (JH) and molting hormone have been demonstrated in insects. JH, molting hormone and their mimics (insect growth regulators, IGRs) show endocrine-disrupting effects not only on target pest insects but also on other arthropod species such as crustaceans. However, little is known about the endocrine-disrupting effects of IGRs on benthic crustaceans. In this study, laboratory experiments were conducted to investigate effects of representative innate JH in crustaceans (methyl farnesoate, MF) and molting hormone (20-hydroxyecdysone, 20E, active form of ecdysteroid) on larval stages of the kuruma prawn Marsupenaeus japonicus, which is a decapod crustacean living in warm seawater. Larval development of kuruma prawn progresses in the order of nauplius, zoea, mysis, and then post-larvae with molting and metamorphosis, but it is unknown whether both MF and 20E have crucial roles in metamorphosis and molting of this species. Treatments of either MF or 20E on shrimp larvae were attempted at each developmental stage and those effects were validated. In terms of EC50 values between mortality and metamorphosis, there were apparent differences in the transition from nauplius to zoea (MF: 7.67 and 0.12 μM; 20E: 3.84 and 0.06 μM in survival and metamorphic rates, respectively). In contrast, EC50 values in MF and 20E treatments showed high consistency in the transitions between zoea to mysis (EC50 values for survival; MF: 1.25 and 20E: 0.22 μM), and mysis to post-larvae (EC50 values for survival; MF: 0.65 and 20E: 0.46 μM). These data suggest that nauplius has strong resistance against exposure to MF and 20E. Moreover, both chemicals induced high mortality triggered by the disruption of molting associated with metamorphosis. To our knowledge, this is the first experimental evidence that investigates in vivo physiological functions of MF and 20E in the larval stages of kuruma prawn, shedding light on not only ecotoxicological impacts of IGRs released into nature, but also endocrine mechanisms underlying larval development with metamorphosis in benthic decapod crustaceans.

Tissue distribution and biochemical characteristics of receptors for sinus gland peptide VII as a crustacean hyperglycemic hormone and vitellogenesis-inhibiting hormone of the kuruma prawn, Marsupenaeus japonicus

Crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH) belong to the CHH family, a neuropeptide superfamily conserved in ecdysozoans. To date, no receptor for the CHH family peptides has been identified in crustaceans. Here, we used a CHH family isoform, Mj-sinus gland peptide (SGP)-VII, as a representative of CHH and VIH in order to determine its target tissues and obtain biochemical information regarding its receptor in the kuruma prawn Marsupenaeus japonicus (Crustacea, Decapoda). An in vitro binding assay using a radiolabeled recombinant Mj-SGP-VII and tissue membranes showed that ligand-receptor binding was specific and dissociable. Six tissues, including the hepatopancreas, gill, heart, skeletal muscle, hindgut, and ovary, were identified as the main targets for Mj-SGP-VII. Scatchard analysis of these six tissues determined the dissociation constant and maximum binding capacity values as Kd = 0.86–3.6 nM and Bmax = 102–915 fmol/mg protein, respectively. Of these six tissues, the hepatopancreas, heart, and ovary showed changes in the levels of ligand-binding after the elimination of endogenous ligands by eyestalk ablation. In the hepatopancreas, an increase in the amount of ligand-binding was observed after eyestalk ablation, independent of gender, which appears to be associated with hypoglycemia caused by the treatment. The change observed in the hepatopancreas was due to the increase in the ligand-binding capacity, but not in the ligand-binding affinity, of the receptors. Furthermore, chemical cross-linking analysis demonstrated the presence of target tissue-specific receptors for Mj-SGP-VII with molecular masses of 34–62 kDa. Collectively, the present data provided important information on tissue distribution, temporal changes in expression level, and molecular mass, for the identification and characterization of receptors for CHH family peptides in crustaceans.

CDNA cloning and in situ localization of a crustacean female sex hormone-like molecule in the kuruma prawn Marsupenaeus japonicus

Recently, a crustacean female sex hormone (CFSH) was purified from the female eyestalk of the blue crab Callinectes sapidus. This report suggested that CFSH plays an essential role in the development of female secondary sexual characteristics. In order to broaden current knowledge of CFSH, here we report the cloning of the cDNA encoding a putative crustacean female sex hormone in Marsupenaeus japonicus (Maj-CFSH). The deduced Maj-CFSH precursor showed identical composition with other CFSHs, including a signal peptide, a CFSH-precursor-related peptide (CPRP), a dibasic processing signal and a mature Maj-CFSH. Although the amino acid sequence identities between Maj-CFSH and C. sapidus CFSH was low (38.9%), positions of eight cysteine residues and the N-glycosylation motif were found to be conserved between the two sequences. Tissue distribution showed that Maj-CFSH was expressed in both female and male eyestalks with roughly the same level. Analysis of in situ hybridization showed that Maj-CFSH was expressed in neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), medulla interna ganglionic X-organ (MIGX), as well as neural cells around the medulla externa in both female and male eyestalks. Our results suggest that Maj-CFSH is not a female-specific molecule and, therefore, is a CFSH homolog, which may have various, as yet undetermined functions in males, as well as females of M. japonicus.

Effects of maternal influences and timing of spawning on intraspecific variations in larval qualities of the Kuruma prawn Marsupenaeus japonicus

Decreases in intraspecific variations in offspring qualities resulting from reductions in female body size in harvested populations can reduce the ability of these species to adapt to variations in environmental conditions. This, in turn, may result in greater fluctuations in recruitment of resources. In species in which offspring quality differs by female parent size, preserving differences and preventing limitations in female size among populations would be important. The effects of maternal factors on intraspecific variations in larval quality of the Kuruma prawn (Marsupenaeus japonicus) were analyzed by determining the relationships between larval variability (body size and resistance to starvation) and maternal variability (maternal body size, spawning timing, and maternal body condition) in the laboratory from June to October 2015. In addition, temporal variations in parental body size of fished M. japonicus stock were examined by field investigation for ten years between 1996 and 2010 in Bungo-suido Channel, Japan. Body size and starvation resistance of hatched larvae varied by spawning timing (spawning date) and corresponded with seasonal availability of food for larvae in the field. Larger females produced larger larvae with stronger starvation resistance. The mean size of harvested female prawns has decreased significantly over time. Stocks of M. japonicus with reduced mean female size would show reductions in intraspecific variations in quality and quantity of larvae at the population level, resulting from observed maternal influences and size-dependent female reproductive potential. If fishing activity reduces body size, marine reserves can be used as a possible management option.

Growth-related changes in shell-length of short-neck clam Ruditapes philippinarum ingested by kuruma prawn Marsupenaeus japonicus

Growth-related changes in shell-length of short-neck clam Ruditapes philippinarum ingested by kuruma prawn Marsupenaeus japonicus

Initiation and Termination of Spawning Season of Female Kuruma Prawn Marsupenaeus japonicus in Western Enshu-nada, Japan

Initiation and Termination of Spawning Season of Female Kuruma Prawn Marsupenaeus japonicus in Western Enshu-nada, Japan

Effects of high-salinity seawater acclimation on the levels of d-alanine in the muscle and hepatopancreas of kuruma prawn, Marsupenaeus japonicus

Changes in d- and l-alanine contents were determined in the muscle and hepatopancreas of kuruma prawn Marsupenaeus japonicus, during acclimation from seawater containing 100% salinity to artificial seawater containing 150% salinity. In the hepatopancreas, contents of both amino acids increased by approximately threefold. The activity of alanine racemase, which catalyzes the interconversion of d- and l-alanine, also increased in the high-salinity seawater. In addition, the expression of the gene encoding alanine racemase increased in the hepatopancreas with an increase in the alanine racemase activity. These data indicate that the biosynthesis of d- and l-alanine is controlled by the gene expression level of alanine racemase, and d-alanine in the hepatopancreas functions as a major osmolyte for isosmotic regulation. In contrast, the content of d-alanine and alanine racemase activity did not change in the muscle during hyper-osmotic acclimation. Therefore, we suggest that d-alanine, which exists in the several tissues of M. japonicus, is considered to be utilized in some different physiological phenomena in different tissues.

Manipulation of Spawning by Suppressing Maturation under Low Temperature Treatment in Kuruma Prawn Marsupenaeus japonicus

Manipulation of Spawning by Suppressing Maturation under Low Temperature Treatment in Kuruma Prawn Marsupenaeus japonicus

Evaluation of Maturity of the Kuruma Prawn Marsupenaeus japonicus by Ovary Shadow Ratio

Evaluation of Maturity of the Kuruma Prawn Marsupenaeus japonicus by Ovary Shadow Ratio

Isolation of crustacean hyperglycemic hormone-family peptides with vitellogenesis-inhibiting activity from the shiba shrimp Metapenaeus joyneri

Vitellogenesis-inhibiting hormone (VIH) in Crustacea belongs to the crustacean hyperglycemic hormone (CHH)-family. In order to characterize multiple VIH molecules in the shiba shrimp Metapenaeus joyneri, four CHH-family peptides designated as Mej-SGP-I, -II, -III, and -IV were purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. The vitellogenesis-inhibiting activities of these peptides were examined using ex vivo incubation of ovarian fragments of the kuruma prawn Marsupenaeus japonicus. All of the four peptides significantly reduced vitellogenin mRNA levels in the ovarian fragments. Among them, the inhibitory activity of Mej-SGP-III which was the most abundant CHH-family peptide in the sinus gland was apparent. Therefore, a cDNA encoding the Mej-SGP-III precursor was cloned by RT-PCR coupled with 5′- and 3′-RACE. The mature Mej-SGP-III was found to consist of 72 amino acid residues containing six conserved cysteine residues and possess an amidated C-terminus. The mature Mej-SGP-III showed the highest sequence identity to Pej-SGP-V (one of six CHH/VIHs in M. japonicus) and Liv-SGP-G (one of six VIHs in Litopenaeus vannamei). These results indicate that Mej-SGP-III is one of the hormones regulating vitellogenesis in M. joyneri.

Occurrence of the kuruma prawn Marsupenaeus japonicus (Spence Bate, 1888) in the Celtic Sea, English Channel, and North-West France

The most northern occurrence in European waters of the Japanese or kuruma prawn Marsupenaeus (Penaeus) japonicus Bate, 1888 is reported from the Celtic Sea. Two specimens were captured during the winter and spring of 2007 from depths ~40 to 60m over mobile sediments by trawling. Other northern European records are reviewed.

X 線 CT 三次元イメージによるクルマエビの筋肉構造

独特の食感を有するボイルしたクルマエビについて，これを X 線 CT で観察したところ，腹部は 3 つの筋群（前斜筋，中央筋および横断筋）が絡み合って形成された特徴的な構造を有していることを見出した。すなわち，中央筋が前部前斜筋と後部前斜筋を束ね，それらを横断筋が保持した複雑な構造を有しており，このことが独特の食感に起因すると推察した。

The ex vivo effects of eyestalk peptides on ovarian vitellogenin gene expression in the kuruma prawn Marsupenaeus japonicus

Seven major peptides belonging to the crustacean hyperglycemic hormone family were purified from the sinus gland located in the eyestalk of the kuruma prawn Marsupenaeus japonicus, and their effects on vitellogenin gene expression were examined using the ex vivo ovary incubation system. Six molecular species of crustacean hyperglycemic hormone, Pej-SGP-I, -II, -III, -V, VI, and VII, displayed significant inhibitory effects on vg expression with almost the same efficacies, whereas Pej-SGP-IV (known as molt-inhibiting hormone) did not. Two chromatophorotropic peptides, red pigment-concentrating hormone and pigment-dispersing hormone, which were also present in the sinus glands, did not have a clear effect on the gene expression levels in this incubation system. These results suggest that the six crustacean hyperglycemic hormones are potentially capable of acting as vitellogenesis-inhibiting hormones in M. japonicus.

Toxicity of chlorothalonil to marine organisms

We evaluated the acute toxicity of the antifouling biocide 2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile (chlorothalonil, TPN) to an alga (Skeletonema costatum), crustacea (Tigriopus japonicus, kuruma prawn Marsupenaeus japonicus), and a teleost fish (red sea bream Pagrus major) commonly found in the coastal waters of Japan. We also performed acute toxicity and early-life-stage tests for TPN using mummichog Fundulus heteroclitus and used the results to predict the chronic toxicity of TPN to red sea bream. The acute toxicity values of TPN (based on measured concentrations) for S. costatum, T. japonicus, M. japonicus, red sea bream, and mummichog were 0.95, 16, 290, 35, and 61 μg/l, respectively. The early-life-stage test with mummichog embryos revealed that hatchability and growth were the most sensitive indicators, and the lowest- and no-observed-effect concentrations, based on the measured concentrations, were 32 and 11 μg/l, respectively. We estimated the chronic toxicity value for red sea bream to be 11 μg/l based on these toxicity values. Considering the current measured concentrations of TPN in coastal waters of Japan, its environmental impact would appear to be limited.