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A new antimicrobial peptide named SCY2 with 65.08% identity in amino acid sequence to the known scygonadin (SCY1) was first characterized in Scylla paramamosain based on its cloned full-length cDNA and genomic DNA sequences. The SCY2 gene was dominantly expressed in the ejaculatory duct of male crabs and its mRNA transcripts were discerned mainly in the glandular epithelium of the inner wall and the secretion inside the ejaculatory duct. Although the SCY2 gene could not be induced with the challenge of the bacteria and fungi tested, its induction reached the highest level at the peak period of mating in mature male crabs either in June or November, suggesting its induction was likely related to seasonal reproduction changes. Moreover, it was interesting to note that, from analysis of its transcripts and protein, SCY2 was significantly expressed only in the ejaculatory duct of pre-copulatory males before mating, however it was clearly detected in the spermatheca of post-copulatory females after mating accompanied by the decreased level of SCY2 expression in the ejaculatory duct. These results suggested that the SCY2 was probably transferred from the male during mating action with the female for the purpose of protecting fertilization. The recombinant SCY2 was more active against the Gram-positive than the Gram-negative bacteria tested. It was further observed that the SCY2 transcripts were significantly increased with addition of exogenous progesterone in tissue cultures whereas the several other hormones tested had no any effect on SCY2 expression, indicating that there might be a relationship between the SCY2 expression and the induction of hormones in vivo. In summary, this study demonstrated that one role of SCY2 was likely to be involved in crab reproduction and it exerted its reproductive immune function through the mating action and the maintenance of inner sterility in the spermatheca of the female, thus leading to successful fertilization of S. paramamosain., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The macrophage migration inhibitory factor (MIF) family, consisting of MIF and D-dopachrome tautomerase (DDT) in vertebrates, is evolutionarily ancient and has been found across Kingdoms including vertebrates, invertebrates, plants and bacteria. The mammalian MIF family are chemokines at the top of the inflammatory cascade in combating infections. They also possess enzymatic activities, e.g. DDT catalysis results in the production of 5,6-dihydroxyindole (DHI), a precursor of eumelanin. MIF-like genes are widely distributed, but DDT-like genes have only been described in vertebrates and a nematode. In this report, we cloned a DDT-like gene, for the first time in arthropods, and a second MIF in mud crab. The mud crab MIF family have a three exon/two intron structure as seen in vertebrates. The identification of a DDT-like gene in mud crab and other arthropods suggests that the separation of MIF and DDT preceded the divergence of protostomes and deuterostomes. The MIF family is differentially expressed in tissues of adults and during embryonic development and early life. The high level expression of the MIF family in immune tissues, such as intestine and hepatopancreas, suggests an important role in mud crab innate immunity. Mud crab DDT is highly expressed in early embryos, in megalops and crablets and this coincides with the requirement for melanisation in egg chorion tanning and cuticular hardening in arthropods, suggesting a potential novel role of DDT in melanogenesis via its tautomerase activity to produce DHI in mud crab. The clarification of the presence of both MIF and DDT in this report paves the way for further investigation of their functional roles in immunity and in melanogenesis in mud crab and other arthropods., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Vertebrate gamma-interferon inducible lysosomal thiol reductase (GILT) is an IFN-γ-inducible protein and is involved in MHCII-restricted antigen processing and cross-presentation of MHCI-restricted antigens in adaptive immunity. Outside of the endocytic MHC pathway, GILT regulates the cellular redox state, inhibits T cell activation, neutralizes extracellular pathogens and is also a host factor of some bacterial pathogens. In this report, we isolated and characterized three divergent GILT-like genes, GILT1, GILT2 and GILT3, which share only 30.9-40.4% identities in a crustacean mud crab Scylla paramamosain. Whilst the crab GILT1 and GILT3 possess four and five exons, respectively, the GILT2 is intronless, suggesting that GILT2 may arise from a recent retroposition event. The invertebrate GILT-like genes have diverse gene organizations and may be evolved in a species/lineage-specific manner as suggested by phylogenetic tree analysis. The amino acid sequences equivalent to human mature GILT are well conserved, including the GILT signature and nine of the ten cysteine residues that potentially form 5 disulfide bonds in human GILT, across the animal kingdom. However, most invertebrate GILT-like molecules lack the human-type N-terminal propeptide, as well as the human-type C-terminal with a conserved cysteine residue, suggesting differences in post translational processing and mode of action. All the three GILT-like genes are highly expressed in the hepatopancreas and up-regulated by pathogenic bacterial infection suggesting a role in immune defense against bacterial diseases. This study may provide the basis for further investigation of the expanding functions of GILT-like molecules in immunity and other physiological processes in mud crabs and other animals., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The antimicrobial peptide scygonadin (Scy) was first isolated from the gonad of Scylla serrata and its gene is predominantly expressed in the ejaculatory duct of adult males. Thus, its function was predicted to be associated with reproductive immunity, but this is still unclear and needs further investigation. In our study, the expression pattern of Scy at different developmental stages of both male and female S. paramamosain was investigated, so that the potential function of this peptide could be examined. Using real-time quantitative PCR, Scy mRNA transcripts were demonstrated obviously in the vulnerable embryos and larvae-zoea I but very weakly detected in the larvae-zoea III, megalops and juveniles. The gene expression pattern showed a decreasing trend during the early developmental stages. The Scy gene had low expression in the ejaculatory duct of small and medium crabs (100g and 200g in weight) whose gonads were underdeveloped. However, the level of Scy expression was significantly increased in large crabs (300g in weight), which had normally become sexually mature at this size. It was further observed that the numbers of Scy mRNA transcripts in sexually mature crabs were significantly more abundant than in immature ones. In addition, the Scy gene was significantly expressed in the ejaculatory duct of mature male crabs during the mating period (April and May) and reached their highest expression in May. Using immunohistochemistry, the Scy protein was strongly detected in the testis and seminal vesicle of small crabs. However, in large crabs, Scy protein was intensively present in more tissues than in small crabs, including the ejaculatory duct, posterior ejaculatory duct, gill and muscle of males, and also in the spermatheca, gill and muscle of females. It is also interesting to note that Scy mRNA transcripts were detected in other crab species and showed similar expression pattern to those in S. paramamosain. This study extended our knowledge concerning the antimicrobial peptide scygonadin, which has its function principally in the ejaculatory duct of males but which may also play a role at different developmental stages of S. paramamosain from embryogenesis to maturation, and is also widely distributed in other crabs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Scygonadin (Scy) is an important antimicrobial peptide which was first isolated from the seminal plasma of Scylla serrata (now renamed as Scylla paramamosain). Elucidation of the Scy expression pattern in tissues will help in understanding its potential function associated with the reproductive immunity. In our study, Scy mRNA transcripts and its protein were found widely distributed in mature male and female crabs. Scy mRNA transcripts were significantly demonstrated in the ejaculatory duct and hemocytes of males but were much less expressed in the other tissues tested. In addition, Scy mRNA transcripts were discerned in a number of cells in the glandular epithelium of the inner wall and in the secretion inside the ejaculatory duct using the in situ hybridization method. In females, Scy mRNA transcripts were obviously demonstrated in the hemocytes and gills but weakly detected in other tissues tested. The copy number of scygonadin mRNA transcripts in the ejaculatory duct of males was greatly higher than those in other tissues, in particular, was over 60,000 fold that in the hemocytes of females. Using immunohistochemistry, the Scy protein was found at higher levels in male tissues than in female ones, particularly in the reproductive duct of males. It was also interesting to note that Scy gene expression was not significantly induced with lipopolysaccharide challenge. However, it was highly expressed in the ejaculatory duct and the seminal vesicle of pre-copulatory males and in the spermathecae of post-copulatory females under mating conditions. The results suggested that Scy, as an important antimicrobial component, probably performed more functions in males, and was likely to be involved in a function associated with crab fertilization and reproduction in both males and females during mating., (Copyright © 2011 Elsevier Ltd. All rights reserved.)