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The doublesex/mab-3 related transcription factor (Dmrt) gene family plays a conserved sexual regulatory role in different phyla, thus providing an important clue for studying the sex mechanism of crustaceans. In this study, cDNAs of three Dmrt homologs were cloned and characterized in the swimming crab, Portunus trituberculatus (Miers, 1876). Sequence analysis indicated the obtained Dmrts might be orthologs of the invertebrate Dmrt1 (iDmrt1) described in the spiny lobster, Sagmariasus verreauxi (H. Milne-Edwards, 1851). Real-time quantitative PCR revealed that the PtiDmrt1 transcripts were highly expressed in testis, muscle, Y-organ, and gill, and in the C2 stage during the developmental process. In addition, RNAi of PtiDmrt1 showed that the gene expression of insulin-like androgenic gland hormone (IAG) decreased remarkably at 24 h after injection, but increased to a higher level than in the control group at 48 h post injection. The results suggested that the PtiDmrt1 is a potential regulator of IAG, although the underlying mechanism may be complicated.

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.

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.

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.

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.

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.

The hydrophilic modification of a polyvinylidene fluoride (PVDF) microfiltration membrane via pretreatment with argon plasma and direct surface-initiated atom transfer radical polymerization (ATRP) was studied. Both modified and unmodified PVDF membranes were characterized by Fourier transform infrared spectroscopy (FTIR), water contact angle, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and pore size distribution measurements. FTIR and XPS spectra confirmed that sulfobetaine methacrylate (SBMA) had been grafted onto the membrane surface. The initial contact angle decreased from 87.0° to 29.8° and a water drop penetrated into the modified membrane completely in 8 s. The pore size distribution of the modified membrane exhibited a smaller mean value than that of the original membrane. The antifouling properties of the modified PVDF membrane were evaluated by a filtration test using bovine serum albumin (BSA) solution. The results showed that the initial flux of the modified membrane increased from 2140.1 L/m 2 h to 2812.7 L/m 2 h and the equilibrium flux of BSA solution increased from 31 L/m 2 h to 53 L/m 2 h.

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 β 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.

The oxygen mass transfer characteristics of a membrane contactor with a hydrophobic polypropylene hollow fiber membrane are experimentally studied herein. The examination focuses on influences of oxygen intake pressure (0.12, 0.13, and 0.14 MPa) and water flux (from 100 L/h to 320 L/h) on oxygen mass transfer coefficient. Experimental results indicate that the inflection point of oxygen mass transfer coefficient shows up when water flux increases under specific intake gas pressures. The mass transfer coefficient goes up remarkably along with rise of oxygen intake pressure. A novel mass transfer model based on the concept of the liquid film renewal time (△tj) and the solubility equilibrium time (△t⁎) is proposed. In this model, the total mass transfer rate is influenced both by liquid film renewal rate between fluid bulk region and liquid film at the gas–liquid interface, and by oxygen diffusion rate in the twice interface renewal process. The model interprets the experimental mass transfer phenomena well.

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.

In this study, the membrane compaction of polysulfone (PS), poly(ether sulfone) (PES) and poly(vinylidene fluoride) (PVDF) flat sheet membranes with finger-like porous structure during pressure-driven filtration process was investigated. The parameters of membrane compression index (sc), membrane rebound index (sr) and membrane base resistance (RT0) were proposed to explore the microfiltration membrane compressibility and its effects to performance degradation. The results revealed that the PS, PES and PVDF membranes all showed the maximum compression index and low rebound index in the initial pressure-driven filtration process. (sc − sr) decreased after three supercharging–decompression filtration cycles. sc and sr tended to be equal and stabilized at 0.41 ± 0.06, 0.35 ± 0.06 and 0.25 ± 0.04 for the PVDF, PES and PS membranes respectively. It unveiled a reduction of the residual deformation and the stability of the membrane structure. The elastic deformation exerted a more important effect on the flux of PVDF membrane than that of PES and PS membrane. The study also indicated that the compression deformation resulted in smaller pore size and larger membrane base resistance after continuing pressure-driven filtration process, which lead to the decay of membrane flux.

Methyl farnesoate (MF) is a sesquiterpene compound and the crustacean homolog of insect juvenile hormones. MF has multiple physiological functions involv- ing the regulation of molting, reproduction, metamorpho- genesis, behavior, and osmoregulation. In this study, the hemolymph levels of MF during ovarian development of Portunus trituberculatus were measured by gas chromatog- raphy-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-methyl- glutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyltransferase (FAMeT) are two essential enzymes in MF biosynthesis, their transcript levels during ovarian de- velopment 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 hemo- lymph. 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.

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.

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.

To elucidate the role of the retinoid X receptor (RXR) in moulting and ovarian development of crustaceans, the full-length cDNA of RXR (PtRXR) in Portunus trituberculatus (Miers, 1876) was cloned by nested reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of PtRXR was determined to be 1365 bp in length and contained an open reading frame (ORF) of 1140 bp encoding a 379-amino-acid residue protein. The deduced amino-acid sequence of PtRXR shared high identities with other known RXRs. Phylogenetic analysis showed that PtRXR was clustered among crustacean RXRs and located closer to the vertebrate RXRs than the insect ultraspiracle (USP, an orthologue of RXR). Quantitative real-time PCR (qRT-PCR) was used to analyse the tissue distribution of PtRXR and its expression patterns during the moulting cycle and the second ovarian development of P. trituberculatus. The results showed that PtRXR was widely distributed in the tested tissues. PtRXR mRNA levels were significantly high in ovary and Y-organs (YO) of intermoult crabs. The mRNA levels of PtRXR in YO and mandibular organs (MO) decreased significantly from intermoult to premoult. In addition, PtRXR was expressed at each stage of the second ovarian development in ovary, hepatopancreas, YO and MO, and the expression levels reached maximal values when the ovary reached the final stage of maturation. These results indicate that PtRXR might have an important role in regulating the moulting and ovarian development of P. trituberculatus.

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.

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.

In the present study, a genomic DNA of MIH (GenBank: #EU869539) was cloned from the swimming crab, Portunus trituberculatus (Miers, 1876). The genome DNA, consisting of 2865 bp, is comprised of three exons interrupted by two introns. Multiple sequence alignments revealed that in the 5 upstream region of MIH, sequences with high similarity to arthropod initiator, TATA box, CREB (cyclic AMP response element binding) protein were the common structure. The signal peptide in the genomic DNA was encoded by exon1 and exon2, which was interrupted by 242 bp-intron (intron1), located between gln12 and arg13. The mature peptide was encoded by exon2 and exon3, which was interrupted by 313 bp-intron (intron2), at the position between 2nd and 3rd nucleotides of the codon encoding arg41. Pot-MIH was expressed only in the eyestalk ganglia, ovaries, testes, posterior spermatic duct, bristle in ejaculatory duct, cranial ganglia, and thoracic ganglia, as determined in various tissues by semi-quantitative RT-PCR. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using the Escherichia coli (Migula, 1895) expression system. Two constructs were designed to yield either a mature MIH fusion protein with and without histidine (His) tag at the carboxyl terminus. The rMIH protein was detected by SDS-PAGE and Western blot analysis, indicating that the antibody prepared by two rMIH proteins has high specificity.

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.

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.

Two full-length cDNA (Pt-CHH1 and Pt-CHH2) sequences encoding crustacean hyperglycemic hormone (CHH) were cloned from tissues of the swimming crab (Portunus trituberculatus) using RACE. Pt-CHH1 was cloned from eyestalk, whereas Pt-CHH2 was cloned from thoracic ganglia. Sequence and structure analyses of Pt-CHH1 and Pt-CHH2 suggest that they may be generated from alternative splicing. Tissue distribution showed that transcript of Pt-CHH1 was only detected in eyestalk, while transcript of Pt-CHH2 was observed in several extra-eyestalk tissues. The transcript levels of Pt-CHH1 and Pt-CHH2 during molting and ovarian development were determined using qPCR. In molting process, level of Pt-CHH1 in eyestalk increased from stage A (postmolt), and to significant higher at stage C (intermolt), then decreased during premolt (D0–D4). In ovarian development, level of Pt-CHH1 in eyestalk decreased from previtellogenic stage (II), and to significant lower at mature stage (IV). The expression patterns of Pt-CHH2 in thoracic ganglia and Y-organ were distinct from that of Pt-CHH1 in eyestalk. The combined results suggest that Pt-CHH1 may be involved in inhibition of molting and ovarian development, whereas Pt-CHH2 may have other physiological functions.

To study the regulatory role of farnesoic acid O-methyl transferase(FAMeT)in molting process of crustaceans,the full-length FAMeT cDNA(GenBank accession number:KC192659)of Portunus trituberculatus is cloned by using reverse transcript PCR(RT-PCR)and rapid-amplification of cDNA ends(RACE).FAMeT cDNA contains a 201 bp 5′-untranslated region(5′-UTR),a 318 bp 3′-untranslated region(3′-UTR)and a 825 bp opening reading frame,which encodes 274 amino acid residues.Alignment of deduced amino acid sequence with FAMeT amino acid sequences of other crustaceans revealed that it shares the highest identity with P.pelagicus among the identities ranging from 75% to 97%.The amino acid residues consist of two copies of CF(CPAMD8/FAMeT)domain,which are the hallmark domain of FAMeT and are present in all crustacean FAMeTs.Quantitative real-time PCR(qRT-PCR)was used to quantify the relative expression level of FAMeT in different tissues and at molting stages in P.trituberculatus.The results showed that FAMeT was expressed in various tissues.The mRNA level of FAMeT was the highest in taoracic ganglia,followed by gill and mandibular organ(MO).During the molting process,the expression of FAMeT in MO increased to the maximum at D1 stage,then gradually decreased to the minimum at D4 stage.The results suggest that FAMeT plays an important role in molting regulation in P.trituberculatus.