Huiyang Huang

An insulin-like androgenic gland hormone gene in the mud crab, Scylla paramamosain, extensively expressed and involved in the processes of growth and female reproduction.

Insulin-like androgenic gland hormone (IAG) produced by androgenic gland (AG) in male crustaceans is regarded as a key regulator of sex differentiation. As a member of the insulin/insulin-like growth factor family, IAG is also likely involved in regulating somatic growth. In this study, a full-length cDNA of IAG (termed Sp-IAG) was isolated from the mud crab, Scylla paramamosain. Genomic DNA of Sp-IAG was also cloned, analysis of which reveals that Sp-IAG gene is organized in a 4 exon/3 intron manner. RNA in situ hybridization analysis detected positive signals in both type I and type II AG cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that Sp-IAG was expressed not only in AG, but also in many other tissues. Sp-IAG expression levels in ovaries were examined at different stages of ovarian development (stages I to V); it was found that the expression was maintained at low levels during undeveloped stage (stage I) to late vitellogenic stage (stage IV) and then increased significantly at mature stage (stage V), suggesting that Sp-IAG may participate in inhibiting oocyte growth and vitellogenesis. The expression pattern of Sp-IAG during the molting cycle of the first stage crabs (C1) was also determined. Sp-IAG expression level continuously decreased from 0 h C1 (postmolt) crabs to 96 h C1 (premolt) crabs, and then increased significantly in the newly molted second stage crabs (C2, postmolt). The combined results suggested for the first time that IAG is involved in regulating ovarian development and somatic growth in crustaceans.

Changes in progesterone levels and distribution of progesterone receptor during vitellogenesis in the female mud crab (Scylla paramamosain)

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.

Immune responses of prophenoloxidase in the mud crab Scylla paramamosain against Vibrio alginolyticus infection: in vivo and in vitro gene silencing evidence.

Phenoloxidase (PO) plays an important role in arthropod melanization. In the present study, a proPO gene was obtained from the mud crab Scylla paramamosain, then we localized the proPO mRNA in hemocytes and detected the expression of proPO after bacterial challenge. In vivo and in vitro gene silencing mediated by dsRNA was also used to investigate the function of proPO in innate immune. The full-length of the proPO cDNA was 2600 bp and the predicted ORF encoded a protein of 673 amino acids with a predicted molecular mass of 77.3 kDa. The deduced amino acid and the main functional domain of proPO shared a high similarity to the mud crab Scylla serrata. In situ hybridization assay showed that the proPO mRNA was localized in the granular and semi-granular cells. The expression level of proPO in hemocytes showed a clear time-dependent pattern during the 96 h course after stimulated by Vibrio alginolyticus. In this study, high expression levels were observed at 3, 12, 24 and 48 h, respectively and the highest expression level was observed at 12 h, and this suggested that proPO was induced by bacteria and involved in immune response. In vivo proPO and GFP dsRNA treatment experiments showed that, proPO mRNA transcript was reduced to 39%, but the PO activity showed no significant difference (P > 0.05). Results indicated that the expression of proPO could be inhibited by dsRNA, and the enzyme activity may be influenced by incomplete knockdown of proPO, or hemocyanin, and other proPO isoforms as well. In vitro proPO-silenced experiments showed that the levels of proPO were decreased by 36%, 64% and 77% at 8, 16 and 32 h, respectively. Meanwhile, the quantity of bacteria was significantly larger in proPO dsRNA treatment than that in control at 3 h, calculated by 4,6-diamino-2-phenylindole staining (P < 0.01). These data demonstrated that the proPO gene plays an important role in the control of systemic bacterial infections and could help us to elucidate the defense role of the proPO-activating system in crabs. In addition, in vitro gene silencing operation mediated by dsRNA was expected to be a new tool for investigating the function of genes in crustaceans in the case of lacking cell line.

The mechanism of regulation of ovarian maturation by red pigment concentrating hormone in the mud crab Scylla paramamosain

In this study a full-length cDNA (Sp-RPCH) was cloned from the eyestalk ganglia of the mud crab Scylla paramamosain. Sp-RPCH is 660 base pairs in length and its open reading frame encodes a precursor that is predicted to be processed into a 25-residue signal peptide, a mature red pigment concentrating hormone (RPCH, an octapeptide), and a 75-residue precursor-related peptide. Phylogenetic analysis indicates that it clusters with other crustacean RPCHs and belongs to the adipokinetic hormone/RPCH peptide superfamily. Sp-RPCH gene expression was detected, using an end-point polymerase chain reaction (PCR), not only in the eyestalk ganglia but also in the brain and thoracic ganglia. Quantified using a real-time PCR, Sp-RPCH gene expression levels in the three tissues fluctuated along a cycle of ovarian maturation, with the levels progressively increased from stages I to IV, after which the expression levels decreased (although they remained significantly higher than stage I levels) when the ovary reached the mature stage (stage V). It was demonstrated using a patch clamp analysis that synthetic RPCH was able to evoke a Ca(2+) current in dissociated brain neurons and synthetic RPCH significantly increased the mean oocyte diameter of the ovarian tissues co-cultured with the eyestalk ganglia, brain, or thoracic ganglia; the stimulatory effect of RPCH was absent when the nervous tissues were not included in the ovarian incubation. Animals administrated with RPCH had significantly higher levels of gonad-somatic index, hepatopancreas-somatic index, and vitellogenin gene expression, when compared to control animals receiving a saline injection. The combined results clearly show that RPCH is involved in ovarian maturation in the mud crab; the stimulatory effects of RPCH are likely mediated by its actions on the release from the nervous tissues of factor(s) that directly regulate vitellogenesis in the ovary and hepatopancreas.

Two beta-pigment-dispersing hormone (β-PDH) isoforms in the mud crab, Scylla paramamosain: Implication for regulation of ovarian maturation and a photoperiod-related daily rhythmicity

In crustaceans, the neuropeptide pigment dispersing hormone (PDH), including α- and β-PDH, is mainly involved in color changes related to the dispersion of integumental pigments and shielding pigments in the compound eye. In this study, we cloned two β-PDH isoforms in the mud crab Scylla paramamosain (termed Sp-β-PDH-I and II, respectively). The tissue distribution analysis in the females showed that Sp-β-PDH-I was mainly expressed in the eyestalk and to a much lesser extent in the brain, thoracic ganglion and ovary; however, Sp-β-PDH-II was exclusively distributed in the eyestalk. From there, we detected Sp-β-PDHs expression levels in the eyestalks (for Sp-β-PDH-I and II) and ovaries (for Sp-β-PDH-I) at different stages of ovarian development. The expression of Sp-β-PDH-I was consistent between the eyestalk and ovary; it maintained high levels from the pre-vitellogenic stage to the vitellogenic stage and then decreased significantly during the mature stage. By contrast, Sp-β-PDH-II expression levels were high only during the vitellogenic stage and significantly lower during the pre-vitellogenic and mature stages. Additionally daily expression analysis of the first stage crabs during the 24-h period showed that the expression level of Sp-β-PDH-II had an obvious daily rhythmicity and bright light could inhibit Sp-β-PDHs expressions. Moreover, photoresponses of Sp-β-PDHs further indicated that the daily rhythmicity was closely regulated by photoperiods. The combined results suggested for the first time that PDH is involved in regulating ovarian maturation in crustaceans and that a photoperiod-related daily rhythmicity of PDH exists in the juvenile crabs.

Profiles of gonadotropins and steroid hormone-like substances in the hemolymph of mud crab Scylla paramamosain during the reproduction cycle

To elucidate the role of gonadotropins-like substances in mud crab Scylla paramamosain, hemolymph samples were measured for concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH) and steroid hormones by enzyme-linked immunosorbent assay (ELISA). Hormonal concentration data were analyzed in association with the stages of gonadal development. ELISA has shown that in the female crab, the level of FSH reaches its peak in the early stage of ovary development, while estradiol and LH peaked during the late maturing stage of the ovary. In the male crab, testosterone and FSH culminated during the spermatid stage, and the level of LH peaked during the sperm stage. These results indicated that substances resembling the vertebrate FSH and LH are present in the hemolymph of S. paramamosain, and they may be involved in the development of the gonad.

Insights into insulin-like peptide system in invertebrates from studies on IGF binding domain-containing proteins in the female mud crab, Scylla paramamosain

Insulin-like peptides (ILPs) have been proved to exist extensively in invertebrates and play critical roles in regulating growth, metabolism and reproduction. ILP signaling system has been well defined in insects, with all key components homologous with vertebrate IGF signaling; however, counterparts of IGF binding proteins (IGFBPs) in vertebrates are not included in this system because of lacking sufficient researches in the related aspect. The present study firstly reports the identification of three kinds of invertebrate IGF binding (IB) domain-containing protein genes from the mud crab Scylla paramamosain. Gene expression analysis suggested that they might be closely involved in ovarian development, but with separate roles. Subsequent bioinformatics analysis and in vitro experiments indicated that they are likely to serve as endogenous ILP-specific binding proteins in invertebrates. More importantly, based on the current evidence we inferred that in invertebrate, ILP system might take the place of IGF system in vertebrate species.

Two transcripts and the expression profiles of cyclin A in ovary of the mud crab, Scylla paramamosain

Two transcripts of cyclin A (SPCyA) were identified and characterized from ovaries of the mud crab, Scylla paramamosain. The sequences of the two transcripts contained the same open reading frame and 5′ untranslated region (UTR), but differed in 3′ UTR. The polyadenylation signals “AATAAA” were found in the different sites, which might generate alternative polyadenalation and thus the different length of the transcripts. The dendrogram, based on comparison of cyclin A proteins in different species, agreed with the classic taxonomic structure. Reverse transcription-PCR (RT-PCR) showed that the SPCyA mRNA was expressed at the highest level in the ovary. Real-time RT-PCR revealed that the amount of SPCyA mRNA in the ovary increased from the first stage to the fourth stage, then declined at the fifth stage (p < 0.05). It implies that SPCyA is closely related to oogonial proliferation (mitosis) and oocyte meiosis.

Molecular cloning, expression profiles and subcellular localization of cyclin B in ovary of the mud crab, Scylla paramamosain

A full-length cDNA of cyclin B was isolated from ovary of the mud crab (Scylla paramamosain) in this study. This transcript encodes a polypeptide of 401 amino acids, which is highly homologous to cyclin B protein family. Reverse transcription-PCR (RT-PCR) showed that cyclin B mRNA was expressed at highest levels in ovary of the mud crab. During the ovarian maturation process, real-time RT-PCR revealed that the abundance of cyclin B mRNA increased from the second stage (early-developing stage) to the fourth stage (nearly-ripe stage) and reached the peak level at the fifth stage (ripe stage). This result indicates the identified cyclin B gene might be related to the cell proliferation in ovary, both mitotically and meiotically. Immunohistochemistry showed that cyclin B protein was localized in the cytoplasm of prophase oocytes at the second stage while enriched in the nuclei of pro-metaphase oocytes at the fourth stage. It suggests the tested cyclin B protein might play different roles in ovary at the two stages.

Implication for the regulation of catabolism drawn from the single insulin-like growth factor binding domain protein (SIBD) gene in the mud crab, Scylla paramamosain.

Insulin-like growth factor (IGF) signaling system holds a central position in regulating growth and metabolism in vertebrates. As critical components of this system, the IGF-binding proteins (IGFBPs) play important roles in regulating the biological activities of IGFs. Recently, the single IGF-binding domain protein (SIBD) was identified in invertebrates and its sequence was highly homologous with the N-terminal domain of IGFBP. In view of the possible role as counterparts of vertebrate IGFBPs, SIBDs have attracted the ever-increasing attention. This study reports the identification of a 1284bp SIBD gene (Sp-SIBD) from a member of commercially important family of Portunidae. The tissue distribution analysis showed that Sp-SIBD was mainly expressed in the nervous tissues and hepatopancreas. RNA in situ hybridization analysis showed that the positive signals were predominantly distributed in the secretory cells of the hepatopancreas. Subsequently, we examined the effects of various stresses, including hyperosmotic stress, hyperthermia, activated stress and fasting, on glucose levels in the hemolymph and Sp-SIBD expressions in the hepatopancreas. Interestingly, we found that Sp-SIBD expression was strongly up-regulated in response to these catabolic circumstances. Given the previous findings of insulin-like peptides (ILPs) in invertebrates, we speculate that invertebrate ILPs and SIBDs promise to serve as a pair of counterparts of IGFs and IGFBPs from vertebrate species respectively. In this context, the combined results suggested, by analogy with IGFBP 1 from vertebrates, for the first time that SIBD might play a key physiological role by sequestering ILPs to inhibit energy-expensive growth until conditions are more favorable.