Three new piscidins from orange-spotted grouper (Epinephelus coioides): Phylogeny, expression and functional characterization.

The present study reports the identification, and characterization of three new putative piscidin paralogues, ecPis-2, ecPis-3 and ecPis-4, from orange-spotted grouper (Epinephelus coioides). The cDNA of the three piscidins with the 207, 216, and 231 nt open reading frame encoded respectively a 68-, 71-, and 76-amino acid preprotein consisting of the predicted signal peptide, and putative mature peptide and prodomain. The phylogenetic analysis indicated that multiple piscidin paralogues in one fish species are highly diversified, the analysis suggested that the piscidins should be a family belonging to the superfamily of ancient cationic, linear, and amphipathic host defence peptides widespread across invertebrate and vertebrate taxa comprising insect cecropins and ceratotoxins, and the amphibian dermaseptins. The synthetic putative mature peptides, ecPis-2S, ecPis-3S and ecPis-4S, had strong activities against bacterial and fungal species. EcPis-3S exhibited powerful activity against the infective stage of Cryptocaryon irritans, theronts. The full length ecPis-2 and ecPis-4 by removal of signal peptide, ecPis-2L and ecPis-4L respectively, had potency against bacterial, fungal and parasitic species. The peptide ecPis-2S was proved to exist in spleen of orange-spotted grouper by HPLC followed by ESI-LCMS analysis. Basal transcriptions of ecPis-2, ecPis-3 and ecPis-4 were detected not only in the potential sites of pathogen entry such as gills, skin and intestine, but also in tissues such as head kidney, trunk kidney, blood cells, and spleen with highly abundant immune cells, however different paralogues expressed constitutively with different levels in the tissues. In addition, the expression of ecPis-2, ecPis-3 and ecPis-4 was upregulated in orange-spotted grouper challenged by Vibrio Parahaemolyticus, in different tissues at different time point after bacteria injection. These results support ecPis-2, ecPis-3 and ecPis-4 being the important immune-related genes in orange-spotted grouper innate immune system and playing multifunctional and complementary roles following their structural and functional diversification, and expression pattern difference. Finally, this study facilitates the evaluation of ecPis-2S, 2L, ecPis-3S, and ecPis-4S, -4L as potential templates of therapeutic agents against pathogens.

Characterization and expression analysis of two novel CCR6 chemokine receptors and their three potential ligands CCL20Ls of grouper (Epinephelus coioides) post Cryptocaryon irritans infection.

CCR6 have been demonstrated playing an important role in immune cells homing to mucosal tissues, mediating antigen presentation and immune response in mammals. CCR6 in lower vertebrate leukocyte homing has not yet been revealed. Cryptocaryon irritans is believed to be a good pathogen model for skin and gill mucosal immunity. In this study, we identified two CCR6s and their three possible ligands CCL20 like cDNA sequences, designated as grouper EcCCR6A, EcCCR6B, EcCCL20L1, EcCCL20L2 and EcCCL20L3. It is interesting to find that EcCCR6A has a longer second extracellular loop than EcCCR6B, which is more similar to mammalian CCR6. Tissue distribution analysis showed that EcCCR6A pronouncedly dominates in gill and brain while EcCCR6B dominates in head kidney, trunk kidney and thymus. Three chemokine ligands have their own distinct expression pattern in health grouper tissues. EcCCL20L1 dominates in spleen and head kidney, EcCCL20L2 dominates in gill and thymus, whereas EcCCL20L3 dominates in skin and brain. The expression patterns of these chemokines and chemokine receptors were detected in C. irritans infected grouper and the results showed that EcCCR6A, EcCCR6B and EcCCL20L1 were significantly up-regulated in the skin of C. irritans infected fish, which indicated these two chemokine receptors and their ligand may play important role in immune cells' homing to skin mucosal immune tissues under pathogen caused inflammation.

Integrative description of a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Shennongjia, Central China.

A new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Xiangxi River, Shennongjia Forestry District, Hubei Province, China, is described on the basis of an integrative approach, involving morphology, and molecular systematics. The new species Dugesia saccaria A-T. Wang & Sluys, sp. nov. is characterized by the following features: a dumb-bell-shaped, muscularized hump located just anterior to the knee-shaped bend in the bursal canal; a ventrally displaced ejaculatory duct, which, however, opens terminally through the dorsal portion of the blunt tip of the penis papilla; a ventrally located seminal vesicle, giving rise to a vertically running duct that eventually curves downwards to communicate with the ejaculatory duct via a small diaphragm; oviducts opening asymmetrically into the dorsal portion of the common atrium and at the knee-shaped part of the bursal canal. The phylogenetic position of the new species was determined using four molecular markers (18S rDNA; ITS-1; 28S rDNA; COI), which suggested that it groups with other species of Dugesia from the Australasian and Oriental biogeographical regions.

Molecular cloning of orange-spotted grouper (Epinephelus coioides) TLR21 and expression analysis post Cryptocaryon irritans infection.

TLR21, a non-mammalian Toll like receptor, was recently identified in chicken as a pattern recognition receptor of unmethyl-CpG ODN, functionally similar to that of mammalian TLR9. Its role in fish immune defense and whether it is involved in anti-parasite immunity has not yet been proven. In this study, we identified a cDNA sequence encoding orange-spotted grouper Toll-like receptor 21 (EcTLR21), the open reading frame (ORF) was 2937 bp encoding a putative polypeptide of 979 amino acid residues. Some conserved motifs in mammalian TLR9 were also conserved in grouper and other fish species' TLR9 and TLR21. Tissue distribution analysis indicated that EcTLR21 is broadly expressed in all the tissue we tested except muscle. High expression levels were found in the head kidney, trunk kidney, spleen and heart. Post Cryptocaryon irritans infection, TLR21 and TLR9 transcripts were induced at the local infection sites (skin and gill), while suppressed in systemic immune organs (spleen and head kidney), indicating that these two receptors may play a role in host anti-parasitic immune responses.

Orange-spotted grouper (Epinephelus coioides) TLR2, MyD88 and IL-1ß involved in anti-Cryptocaryon irritans response.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish, and can have a devastating effect on aquacultured fish populations. The role of TLR signaling pathways in anti-parasitic immune responses is poorly understood in fish. In this paper, we first cloned Epinephelus coioides MyD88 full-length cDNA (EcMyD88) and its respective gene. The open reading frame (ORF) of cDNA is 873bp encoding 291 amino acid residues. Similar to other species, the EcMyD88 gene contains of five conserved exons and four diverse introns. The constitutive expression of EcMyD88 was detected in the gill, trunk kidney, head kidney, spleen, and heart in high concentrations, while the skin, brain, liver, and muscles contained much lower titers, indicating that EcMyD88 may play a crucial role in host innate immunologic surveillance. To identify the potential role of TLR pathways in fish anti-C. irritans immune responses, we chose three important molecules involved in anti-parasite responses, TLR2, MyD88 and IL-1ß to indicate TLR pathway's signal-in, signal transduction, and signal-out functions, respectively. The expression profile of these three genes was detected in grouper infected by C. irritans. Results showed these molecules each experience significant changes within the skin, gill (two infected mucosal sites), head kidney and spleen (two systematic immune organs) after C. irritans infection. These findings indicate the TLR signaling pathway may play an important role in host defense against C. irritans.

Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans.

Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and α-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit α, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while ß-tubulin, V-type ATPase α subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, ß-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75 kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit α, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development.

Molecular cloning of NCCRP-1 gene from orange-spotted grouper (Epinephelus coioides) and characterization of NCCRP-1(+) cells post Cryptocaryon irritans infection.

Nonspecific cytotoxic cells (NCCs) are an important cytotoxic cell population in the innate teleost immune system. The receptor designated "NCC receptor protein 1" (NCCRP-1) has been reported to be involved in the recognition and activation of NCCs. In this study, the full-length cDNA of Epinephelus coioides NCCRP-1 (ecnccrp-1) was cloned. The open reading frame (ORF) of ecnccrp-1 is 699 bp, encoding a 232 amino acid protein that includes proline-rich motifs at the N-terminus and is related to the F-box associated family. Although a bioinformatics analysis showed that EcNCCRP-1 had no signal peptide or transmembrane helices, a polyclonal antibody directed against recombinant EcNCCRP-1 efficiently labeled a membrane protein in the head kidney, detected with Western blot analysis, which indicated that the protein localized to the cell surface. RT-PCR showed that the constitutive expression of ecnccrp-1 was higher in the lymphoid organs, such as the trunk kidney, spleen, head kidney, and thymus, and lower in brain, heart, fat, liver, muscle, and skin. After infection with Cryptocaryon irritans, the transcription of ecnccrp-1 was analyzed at the infected sites (skin and gills) and in the systemic immune organs (head kidney and spleen). At the infected sites, especially the skin, ecnccrp-1 expression was upregulated at 6h post infection, reaching peak expression on day 3 post the primary infection. However, the expression patterns differed in the systemic immune organs. In the spleen, ecnccrp-1 was gradually increased in the early infection period and decreased sharply on day 3 post the primary infection, whereas in the head kidney, the transcription of ecnccrp-1 was depressed during almost the whole course of infection. An immunohistochemical analysis showed that EcNCCRP-1(+) cells accumulated at the sites of infection with C. irritans. These results suggested that NCCs were involved in the process of C. irritans infection in E. coioides.