Comparative transcriptomic characterization of the ovary in the spawning process of the mud crab Scylla paramamosain.

Oviposition is induced upon mating in most insects. Spawning is a physiological process that is fundamental for the reproduction of Scylla paramamosain. However, the molecular mechanisms underlying the spawning process in this species are poorly understood. Herein, comprehensive ovary transcriptomic analysis was conducted at the germinal vesicle breakdown stage (GVBD), spawning stage, 0.5 h post-spawning stage, and 24 h post-spawning stage of S. paramamosain for gene discovery. A total of 67,230 unigenes were generated, and 27,975 (41.61%) unigenes were annotated. Meanwhile, the differentially expressed genes (DEGs) between the different groups were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was subsequently conducted. These results suggested that octopamine (OA) and tyramine (TA) could induce oviposition, while dopamine (DA) and serotonin (5-hydroxytryptamine [5-HT]) inhibit oviposition. The 20-hydroxyecdysone (20E) and methyl farnesoate (MF) signal pathways might be positively associated with oviposition. Furthermore, numerous transcripts that encode neuropeptides and their G-protein-coupled receptors (GPCRs), such as CNMamide, RYamide, ecdysis-triggering hormone (ETH), GPA2/GPB5 receptor, and Moody receptor, appear to be differentially expressed during the spawning process. Eleven unigenes were selected for qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our work is the first spawning-related investigation of S. paramamosain focusing on the ovary at the whole transcriptome level. These findings assist in improving our understanding of spawning regulation in S. paramamosain and provide information for oviposition studies in other crustaceans.

Identification of transient receptor potential channel genes from the swimming crab, Portunus Trituberculatus, and their expression profiles under acute temperature stress.

BACKGROUND: Temperature is an important environment factor that is critical to the survival and growth of crustaceans. However, the mechanisms by which crustaceans detect changes in temperature are still unclear. The transient receptor potential (TRP) channels are non-selective cation channels well known for properties in temperature sensation. However, comprehensive understandings on TRP channels as well as their temperature sensing functions are still lacking in crustaceans. RESULTS: In this study, a total of 26 TRP genes were identified in the swimming crab, Portunus trituberculatus, which can be classified into TRPA, TRPC, TRPP, TRPM, TRPML, TRPN and TRPV. Tissue expression analysis revealed a wide distribution of these TRP genes in P. trituberculatus, and antennules, neural tissues, and ovaries were the most commonly expressed tissues. To investigate the responsiveness of TRP genes to the temperature change, 18 TRPs were selected to detect their expression after high and low temperature stress. The results showed that 12 TRPs showed induced gene expression in both high and low temperature groups, while 3 were down-regulated in the low temperature group, and 3 showed no change in expression in either group. CONCLUSIONS: This study characterized the TRP family genes in P. trituberculatus, and explored their involvement in response to temperature stress. Our results will enhance overall understanding of crustacean TRP channels and their possible functions.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis.

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Putative Role of CFSH in the Eyestalk-AG-Testicular Endocrine Axis of the Swimming Crab Portunus trituberculatus.

It has been shown in recent studies that the crustacean female sex hormone (CFSH) plays a crucial role in the development of secondary sexual characteristics in Decapoda crustaceans. However, research on the function of CFSH in the eyestalk-AG-testicular endocrine axis has been inadequate. We cloned and identified a homolog of CFSH, PtCFSH, in this study. RT-PCR showed that PtCFSH was mainly expressed in the eyestalk. A long-term injection of dsPtCFSH and recombinant PtCFSH (rPtCFSH) in vivo showed opposite effects on spermatogenesis-related gene expression and histological features in the testis of P. trituberculatus, and was accompanied by changes in AG morphological characteristics and PtIAG expression. In addition, the phosphorylated-MAPK levels and the expression of several IIS pathway genes in the testis was changed accordingly in two treatments, suggesting that PtCFSH may regulate the testicular development via IAG. The hypothesis was further validated by a mixed injection of both dsPtCFSH and dsPtIAG in vivo. The following in vitro studies confirmed the negatively effects of PtCFSH on AG, and revealed that the PtCFSH can also act directly on the testis. Treatment with rPtCFSH reduced the cAMP and cGMP levels as well as the nitric oxide synthetase activity. These findings provide vital clues to the mechanisms of CFSH action in both the eyestalk-AG-testis endocrinal axis and its direct effects on the testis.

Molecular Characterization of the Cytochrome P450 Epoxidase (CYP15) in the Swimming Crab Portunus trituberculatus and Its Putative Roles in Methyl Farnesoate Metabolism.

CYP15, which encodes a microsomal cytochrome P450 enzyme, could be involved in juvenile hormone biosynthesis in insects. In this study, a full-length cDNA of CYP15 was cloned from the swimming crab Portunus trituberculatus. This PtCYP15 amino acid sequence contains six conserved domains, which is a typical feature of the cytochrome P450 family. Phylogenetic tree analysis results showed that PtCYP15 clusters in a single branch of crustacean species, suggesting that CYP15 may be more widely present in crustaceans. The PtCYP15 mRNA has a broad pattern of tissue expression in P. trituberculatus, including high levels of expression in the hepatopancreas of both sexes and in the ovary of female crabs. During ovarian development stages, PtCYP15 mRNA is highly expressed in stages I and II and less so in stages III and IV in the hepatopancreas and the ovary of the female crabs. These expression profiles are opposite those of methyl farnesoate in hemolymph, suggesting that PtCYP15 might be involved in methyl farnesoate metabolism. In vitro studies show that only methyl farnesoate upregulated vitellogenin expression in the hepatopancreas, suggesting that methyl farnesoate might be the equivalent of juvenile hormone III in crustaceans. Methyl farnesoate treatment increased levels of PtCYP15 in explants of the hepatopancreas and ovary, while juvenile hormone III treatment reduced levels of PtCYP15 mRNA in ovary explants, suggesting that PtCYP15 might be involved in degrading methyl farnesoate. Furthermore, PtCYP15 mRNA expression levels were inhibited by adding juvenile hormone III to ovary explants. These findings provide foundational information for future research on methyl farnesoate metabolism in crustaceans.

Molecular identification and putative role of insulin growth factor binding protein-related protein (IGFBP-rp) in the swimming crab Portunus trituberculatus.

The insulin-like growth factor/insulin-like polypeptide (IGF/ILP) signaling is vital for growth, physiological metabolism, development, and reproduction. Insulin-like growth factor-binding protein (IGFBP) is involved in the insulin signaling pathway in both vertebrates and invertebrates and is critical for various physiology functions. Herein, we cloned and characterized the full-length cDNA of IGFBP-rp in the swimming crab, Portunus trituberculatus (PtIGFBP-rp). The deduced amino acid sequence of PtIGFBP-rp was found to contain three key domains (insulin-like binding (IB) domain, the kazale-type serine protease inhibitor (KAZAL) domain, and the immunoglobulin-like C2 (IGc2) domain). Results showed that PtIGFBP-rp shared the same expression pattern as P. trituberculatus insulin androgenic gland hormone (PtIAG) transcripts during the embryonic larval, juvenile crab stage and the androgenic gland (AG) developmental cycle. Moreover, PtIGFBP-rp transcripts were also present in high abundance in hepatopancreas, muscle, and androgenic glands. The regulatory relationship between PtIGFBP-rp and PtIAG was investigated by RNA interference and co-localization assays, which showed a co-localization relationship and feedback regulation between them. Bilateral eye stalk ablation (ESA) increased the expression of PtIGFBP-rp in the AG at 7 d after surgery. These results demonstrate the involvement of PtIGFBP-rp in the signaling regulatory network of IAG in P. trituberculatus.

Identification and characterization of expression profiles of neuropeptides and their GPCRs in the swimming crab, Portunus trituberculatus.

Neuropeptides and their G protein-coupled receptors (GPCRs) regulate multiple physiological processes. Currently, little is known about the identity of native neuropeptides and their receptors in Portunus trituberculatus. This study employed RNA-sequencing and reverse transcription-polymerase chain reaction (RT-PCR) techniques to identify neuropeptides and their receptors that might be involved in regulation of reproductive processes of P. trituberculatus. In the central nervous system transcriptome data, 47 neuropeptide transcripts were identified. In further analyses, the tissue expression profile of 32 putative neuropeptide-encoding transcripts was estimated. Results showed that the 32 transcripts were expressed in the central nervous system and 23 of them were expressed in the ovary. A total of 47 GPCR-encoding transcripts belonging to two classes were identified, including 39 encoding GPCR-A family and eight encoding GPCR-B family. In addition, we assessed the tissue expression profile of 33 GPCRs (27 GPCR-As and six GPCR-Bs) transcripts. These GPCRs were found to be widely expressed in different tissues. Similar to the expression profiles of neuropeptides, 20 of these putative GPCR-encoding transcripts were also detected in the ovary. This is the first study to establish the identify of neuropeptides and their GPCRs in P. trituberculatus, and provide information for further investigations into the effect of neuropeptides on the physiology and behavior of decapod crustaceans.

Molecular Characterization of the Insulin-Like Androgenic Gland Hormone in the Swimming Crab, Portunus trituberculatus, and Its Involvement in the Insulin Signaling System.

The insulin-like androgenic gland hormone (IAG) is mainly produced in the androgenic gland (AG) of the male crustaceans and is a crucial regulator in male sexual differentiation. In the current study, the full-length cDNA of IAG in the swimming crab, Portunus trituberculatus (Pt-IAG), was cloned and characterized. Similar to other reported IAGs, the deduced amino acid sequence of Pt-IAG consists of signal peptide, B chain, C peptide, and A chain, containing six conserved cysteines that form two interchain disulfide bonds and one intra-B chain disulfide bond. Tissue distribution analysis suggested that the Pt-IAG cDNA was highly expressed in the AG and was slightly expressed in several other tissues. A short-term silencing of PtIAG with double-stranded RNA was found to reduce the transcript levels of insulin receptor (Pt-IR) and insulin-like growth factor-binding protein (Pt-IGFBP), suggesting the Pt-IAG might perform its biological function through the insulin family-based signaling system. Bilateral eyestalk ablation (ESA) induced the expression of Pt-IAG in the AG at 4 and 7 days after surgery, while the transcript levels of Pt-IR in the AG and testis and Pt-IGFBP in the muscle, testis, and thoracalia ganglia were significantly decreased from 1 day after surgery. The results suggested that the Pt-IR and Pt-IGFBP might also be the targets of eyestalk neuropeptides and responded to the ESA independent of IAG regulation.

Molecular characterization of the Sex-lethal gene in mud crab Scylla paramamosain and its potential role in sexual development.

Sex-lethal (Sxl) gene operates as a master switch of sexual differentiation in Drosophila melanogaster. In this study, we cloned and characterized the full-length cDNA of Sxl in mud crab (Scylla paramamosain) (SpSxl). The deduced amino acid sequence of SpSxl contained two RNA-binding motifs (RRM), namely RRM1 and RRM2. The SpSxl mRNA levels were abundant in the androgenic glands of the male crab, implying its potential role in male sexual development. This hypothesis was supported by the RNAi experiment, revealing that the injection of SpSxl dsRNA in vivo caused an increase in the expression of SpIAG, which is a key gene of male sexual differentiation in crustaceans. The interference result of SpTra-2 suggested that SpSxl and SpTra-2 may be involved in sex-differentiation by direct or indirect regulation of SpIAG gene. The eye stalk ablation (ESA) experiments further confirmed that SpSxl could not regulate SpIAG through eyestalk neuropeptide, implying other regulation pathways. In addition, treatment with SpSxl dsRNA had no effects on SpDmrt expression, suggesting that sex determination in S. paramamosain is different from that in D. melanogaster.

Role of Kruppel homolog 1 (Kr-h1) in methyl farnesoate-mediated vitellogenesis in the swimming crab Portunus trituberculatus.

Similar to the role of juvenile hormone (JH) in insects, methyl farnesoate (MF), the unepoxidized form of JH III, regulates many developmental processes in crustaceans, such as molting and reproduction. We have previously showed that the JH receptor, Methoprene-tolerant (Met), which is also a candidate receptor for MF, might be involved in the MF-mediated vitellogenesis in the swimming crab Portunus trituberculatus. In this study, the role of Kruppel homolog 1 (Kr-h1), a transcription factor downstream Met in JH signaling, was further investigated. The deduced protein of Pt-Kr-h1 contained seven repeats of zinc finger motifs, similar to Kr-h1s from other crustacean species, but differing from the eight zinc finger motifs found in insect Kr-h1s. MF treatment in vitro induced the expression of Pt-Kr-h1 in hepatopancreas but not ovary, which is similar to the MF-responsive pattern of Pt-Met as previously reported. Moreover, the expression of Pt-Kr-h1 decreased significantly after treating with Pt-Met dsRNA, strongly indicating that the Pt-Kr-h1 might be involved in the Met-mediated MF signaling pathway. RNAi of Pt-Met and Pt-Kr-h1 both led to a decrease in vitellogenin (Vg) expression, and the reduction cannot be rescued by adding MF, suggesting the regulation of vitellogenesis by MF may act through Met and Kr-h1. These results would help to enhance the current understanding of the regulatory mechanism of MF signaling, and provide a vital resource for further research into the evolution of hormonal pathways in arthropods.

Identification and characterization of a novel calreticulin involved in the immune response of the Zhikong scallop, Chlamys farreri.

Calreticulin (CRT) is a multifunctional calcium-binding chaperone shared among vertebrates and invertebrates. In this study, a novel CRT (CfCRT) was identified in the Zhikong scallop Chlamys farreri by rapid amplification of cDNA ends. The full-length cDNA was composed of 1345 bp, which included a 1158 bp open reading frame, a 25 bp 5'-untranslated region (UTR) and a 162 bp 3'-UTR. The predicted molecular mass of CfCRT was 44.8 kDa. CfCRT contained three highly conserved domains (N-, P- and C-domains) essential to the function of CRT. BLAST analysis revealed significant sequence similarity (73%-92%) with CRT proteins from other mollusks. The mRNA transcripts of CfCRT were present in all the tested tissues of Zhikong scallops, with the higher expression level in the hemocytes and mantle. After stimulation by Vibrio anguillarum, the mRNA transcript of CfCRT in hemocytes was significantly upregulated. Recombinant plasmid pBCRT was successfully expressed in Escherichia coli BL21 (DE3). The recombinant (r)CfCRT protein could bind to the surface of several bacteria including the Gram-negative bacteria V. anguillarum, E. coli, and the Gram-positive bacterium Staphylococcus aureus. Moreover, rCfCRT was able to suppress their growth significantly. These results indicate that CfCRT might act as an immune effector in Zhikong scallop innate immunity.

Cloning of two carboxylesterase cDNAs from the swimming crab Portunus trituberculatus: Molecular evidences for their putative roles in methyl farnesotae degradation.

The sesquiterpenoid methyl farnesoate (MF) is the unepoxidized form of insect juvenile hormone (JH) III, and is considered an equivalent of JH in crustaceans. Degradation of MF is similar to that of JH which occurs through ester hydrolysis by specific carboxylesterases (CXEs). In this study, the full-length cDNAs of two JH esterase-like CXEs were cloned from the swimming crab, Portunus trituberculatus. The predicted amino acid sequences of the two PtCXEs contain the conserved motifs including catalytic triad and oxyanion hole, which are the hallmark of the CXE family proteins. The phylogenetic analysis showed that the two PtCXEs may belong to the hormone/semiochemical processing group of CXE family, indicating their possible roles on metabolism of hormones. Transcripts of both PtCXEs were most abundant in hepatopancreas and the PtCXE2 was also highly expressed in ovary. The mRNA levels of two PtCXEs in hepatopancreas were induced by in vivo MF treatment and eyestalk ablation, further indicating their potential in degrading MF. However, during the ovarian maturation, expression of the two PtCXEs increased significantly in the early-vitellogenic stage, prior to the remarkable rise in hemolymph MF titer reported by our previous studies. Taken together, our results suggest that the two PtCXEs can potentially serve as the MF esterases, but their catalytic activity may not be restricted to MF.

Molecular characterization of methoprene-tolerant gene (Met) in the swimming crab Portunus trituberculatus: Its putative role in methyl farnesoate-mediated vitellogenin transcriptional activation.

Methoprene-tolerant (Met) belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of nuclear transcriptional regulators, and is the leading candidate receptor for the insect Juvenile hormone (JH) and the crustacean methyl farnesoate (MF). In the present study, a full-length cDNA of Met was cloned from the swimming crab, Portunus trituberculatus (PtMet). The PtMet amino acid sequence was found to contain domains characteristic of the bHLH-PAS family proteins and to have several conserved amino acid residues specifically responsible for JH or MF binding. In addition to its extensive tissue expression, PtMet mRNA is highly expressed in the female hepatopancreas, the primary site of vitellogenin (Vg) synthesis in P. trituberculatus. PtMet expression was significantly increased in the early-vitellogenic stage of ovarian development, which indicated that likely involvement in vitellogenesis regulation. In vitro, MF induced up-regulation of PtMet and PtVg expression in the hepatopancreas, but not in the ovary. In vivo, MF induced PtMet and PtVg expression in the hepatopancreas,and only PtMet expression was induced in the ovary. Taken together, these results suggest that Met can potentially serve as a MF receptor in crustaceans. In addition, the binding of Met to MF might transcriptionally regulate Vg synthesis in the hepatopancreas.

Identification of a new calreticulin homolog from Yesso scallop (Patinopecten yessoensis) and its role in innate immunity.

Calreticulin (CRT) is a multifunctional and highly conserved Ca2+-binding protein shared among vertebrates and invertebrates. In this study, we cloned and characterized a CRT gene, PyCRT, from Yesso scallop, Patinopecten yessoensis. The full-length cDNA of PyCRT was 1830 bp, including a 1242 bp open reading frame (ORF), a 29 bp 5'-untranslated region and a 559 bp 3'-untranslated region. PyCRT was consisted of three distinct structural and functional domains (N-, P- and C-domains), a signal peptide and an endoplasmic reticulum (ER) retrieval signal sequence (HDEL). Tissue specific expression analysis showed that PyCRT was distributed widely in Yesso scallop, and was highly expressed in the mantle and hemocytes. After Vibrio anguillarum challenge, the expression of PyCRT in hemocytes had a significant increase and reached the maximum level at 12 h post-infection. We also demonstrated for the first time in mollusc that the recombinant PyCRT (rPyCRT) could bind to the Gram-negative bacterium V. anguillarum, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. Our results suggested that the CRT gene from Yesso scallop possessed immune-related regulatory functions in the innate immune system in scallops.

Role of Halloween genes in ecdysteroids biosynthesis of the swimming crab (Portunus trituberculatus): Implications from RNA interference and eyestalk ablation.

Molting, including metamorphosis molting in arthropods are controlled by the ecdysteroids that are synthesized and secreted by the crustacean Y-organ (YO) or the insect prothoracic gland (PG). The Halloween genes encoding the enzymes mainly involved in the biosynthesis of ecdysteroids are well studied in insects but not in crustaceans. Given the importance of Halloween genes in ecdysteroids biosynthesis, we have previously reported the cDNA cloning of disembodied (Dib) in P. trituberculatus. Here, cDNA sequences of another two Halloween genes, Spook (Spo) and Shadow (Sad), were further identified and characterized. The predicted amino acid sequences for these two Halloween genes of Portunus trituberculatus were compared to those of several other arthropods, and several typical domains of the cytochrome P450 mono-oxygenase (CYP) were identified. Similar to the tissue distribution of Dib, the Spo and Sad also showed high specificity to the YO. RNA interference (RNAi) of these 3 genes indicated they all play essential role in ecdysteroids biosynthesis. To investigate the relationships of the Halloween genes to the eyestalk neuropeptides such as molt-inhibiting hormone (MIH), effects of eyestalk ablation (ESA) on the expression of Dib, Spo and Sad were detected. Expression of Dib and Sad, but not Spo, was significantly induced by ESA. The result indicated that the inhibition of MIH in ecdysteroids biosynthesis may be partly through the transcriptional regulation of certain Halloween genes, such as Dib and Sad, while the Spo might not be the target for MIH signal.

The nuclear receptor E75 from the swimming crab, Portunus trituberculatus: cDNA cloning, transcriptional analysis, and putative roles on expression of ecdysteroid-related genes.

The nuclear receptor E75 is an early-responsive gene in 20-hydroxyecdysone (20E) signaling pathway, and is found to play essential roles in many aspects of arthropods development. In this study, a cDNA encoding the E75 nuclear receptor of the swimming crab, Portunus trituberculatus was cloned using RT-PCR and RACE. The PtE75 cDNA was 3211bp in length, and encodes a protein of 795 amino acids. The DBD region of the predicted amino acid sequence for PtE75 was highly conserved with other arthropoda E75s, while its LBD region was more similar to decapod E75s. Tissue distribution analysis showed that PtE75 transcript was widespread among tissues and relatively abundant in Y-organ, epidermis, eyestalk, and muscles. PtE75 exhibited tissue-specific expression patterns in these four tissues, which may depend on the distinct action of 20E on different tissues. The expression of PtE75 in Y-organ and epidermis were induced by eyestalk ablation (ESA), indicating its responsiveness to the increasing hemolymph 20E titer. To identify potential targets for ecdysteroid control the in Y-organ, epidermis, and eyestalk, the expression change of an ecdysteroid synthesis gene PtSad in Y-organ, a chitin degradation gene PtChi1 in epidermis, and the molt-inhibiting hormone gene PtMIH in eyestalk were investigated after silencing of PtE75 mRNA. The double stranded RNA (dsRNA) of PtE75 caused a loss in PtChi and PtMIH expression, while an increase in PtSad expression. The results suggested that the ecdysteroids may act through E75, and play a stimulatory role on chitin degradation in epidermis and MIH synthesis in eyestalk, and an negative feedback effect on ecdysteroid synthesis in Y-organ.

The potential role of juvenile hormone acid methyltransferase in methyl farnesoate (MF) biosynthesis in the swimming crab, Portunus trituberculatus.

Juvenile hormone (JH) and methyl farnesoate (MF) play essential roles in the development and reproduction of insects and crustaceans respectively. Juvenile hormone acid methyltransferase (JHAMT) catalyzes the methyl esterification in insect JH biosynthesis, while the corresponding step in crustacean MF biosynthesis was long thought to be catalyzed by farnesoic acid O-methyltransferase (FAMeT). However, the new discovery of JHAMT orthologs in crustaceans indicates that JHAMT may also play essential role in the MF biosynthesis in crustaceans. Here we cloned and characterized the full-length cDNA encoding JHAMT in the swimming crab Portunus trituberculatus (PtJHAMT). Sequence and structure analysis of PtJHAMT revealed that it was composed of a 6-stranded ß sheet with 9 α helices, and contained a signature Sadenosyl-L-methionine (SAM) binding motif, which is the hallmark in all SAM dependent methyltransferases (SAM-MTs). Several active sites that are critical for the interaction of SAM and JH/FA substrate were also conserved in PtJHAMT. The gene expression of PtJHAMT was highly specific to the mandibular organ, which is the sole site of MF synthesis. PtJHAMT expression significantly increased in the late-vitellogenic stage and mature stage, which suggests a possible role of PtJHAMT in modulating ovarian development. The role of PtJHAMT and PtFAMeT in MF biosynthesis was further investigated by RNA interfering (RNAi). Injection of PtJHAMT and PtFAMeT dsRNA both led to a decrease in hemolymph MF titers. Injection of PtHMGR dsRNA caused the decrease in PtJHAMT expression, but had no effect on mRNA level of PtFAMeT. Together these results suggested that JHAMT and FAMeT are both involved in the MF biosynthesis in crustaceans, while the JHAMT is highly specific to FA substrate, and FAMeT may have more catalytic functions.

Complete mitochondrial genome of the giant croaker Nibea japonica (Perciformes, Sciaenidae) and phylogenetic analysis of the Sciaenidae.

The giant croaker Nibea japonica (Perciformes, Sciaenidae) is an important economic fish distributing in the East China Sea, South China Sea, and Japan southern coast. In this study, the complete mitochondrial genome of N. japonica was firstly determined. It is 16 496 bp-length and consists of 22 tRNA genes, 13 protein-coding genes, two rRNA genes, and a control region. Except for eight tRNA and ND6 genes, all other mitochondrial genes are encoded on the heavy strand. Phylogenetic analysis revealed that N. japonica, A. amoyensis, and other seven fish first clustered into the Argyrosominae clade. It is consistent with the taxonomic status. Then, the Argyrosominae, Pseudosciaeninae, and Sciaeniae formed the sister group, while the Johniinae became a separate clade, which is inconsistent with the previous phenotypic report. It is suggested that the researches of single gene and taxionomic might lose some significant evolutionary characters. This study will contribute to phyogenetic analysis of the Sciaenidae and the natural resources conservation.

Complete mitochondrial genome sequence for the cuneate drum Nibea miichthioides (Perciformes, Sciaenidae) and its phylogeny.

The cuneate drum, Nibea miichthioides, is a native sciaenid species with relevant commercial importance in China. However, the wild resource represented by this species has been severely damaged owing to overfishing and water pollution. For conserving and recovering the species through appropriate measures, genetic studies on the population are necessary. The complete mitochondrial genome of N. miichthioides is a supplement to the mitogenome database for giant croakers and can be used to address taxonomic problems and phylogenetic relationships in Sciaenidae. In this study, we sequenced and characterized the complete mitochondrial genome of N. miichthioides. The full length of the complete mitochondrial DNA was 16,490 bp. It contained 13 protein-coding genes, 22 tRNA genes, two rRNA genes and a control region. All 21 tRNA genes can fold into a typical cloverleaf structure except for tRNASer (AGY), which lacks a dihydrouridine arm. The phylogenetic analysis using the complete mitochondrial genome revealed that N. miichthioides, A. amoyensis, N. japonica and A. japonicus might be grouped in Argyrosomus, but not belonged to Nibea of Argyrosominae, which was highly consistent with the proposal of Talwar. This investigation provides an effective molecular tool for genetic research on and protection of this endangered species.

Hemolymph Levels of Methyl Farnesoate During Ovarian Development of the Swimming Crab Portunus trituberculatus, and Its Relation to Transcript Levels of HMG-CoA Reductase and Farnesoic Acid O-Methyltransferase.

Methyl farnesoate (MF) is a sesquiterpene compound and the crustacean homolog of insect juvenile hormones. MF has multiple physiological functions involving the regulation of molting, reproduction, metamorphogenesis, behavior, and osmoregulation. In this study, the hemolymph levels of MF during ovarian development of Portunus trituberculatus were measured by gas chromatography-mass spectrometry (GC-MS). The results showed that the hemolymph level of MF in P. trituberculatus was low during stages I and II, increased considerably in stage III, and remained high in stage IV. Correlation of MF level with ovarian maturation indicates the putative stimulatory role of MF in this physiological process. As 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyltransferase (FAMeT) are two essential enzymes in MF biosynthesis, their transcript levels during ovarian development were detected by quantitative real-time PCR (qPCR). Transcript levels of HMGR and FAMeT exhibited variation trends similar to that of the level of MF in hemolymph. This might indicate that high expression of HMGR and FAMeT could result in an increase in the production of MF, which ultimately affects ovarian development.