Comparative transcriptome analysis reveals immunoregulation mechanism of lncRNA-mRNA in gill and skin of large yellow croaker (Larimichthys crocea) in response to Cryptocaryon irritans infection.

BACKGROUND: Cryptocaryonosis caused by Cryptocaryon irritans is one of the major diseases of large yellow croaker (Larimichthys crocea), which lead to massive economic losses annually to the aquaculture industry of L. crocea. Although there have been some studies on the pathogenesis for cryptocaryonosis, little is known about the innate defense mechanism of different immune organs of large yellow croaker. RESULTS: In order to analyze the roles of long non-coding RNAs and genes specifically expressed between immune organs during the infection of C. irritans, in this study, by comparing transcriptome data from different tissues of L. crocea, we identified tissue-specific transcripts in the gills and skin, including 507 DE lncRNAs and 1592 DEGs identified in the gills, and 110 DE lncRNAs and 1160 DEGs identified in the skin. Furthermore, we constructed transcriptome co-expression profiles of L. crocea gill and skin, including 7,503 long noncoding RNAs (lncRNAs) and 23,172 protein-coding genes. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the DEGs and the target genes of the DE lncRNAs in the gill were specifically enriched in several pathways related to immune such as HIF-1 signaling pathway. The target genes of DE lncRNAs and DEGs in the skin are specifically enriched in the complement and coagulation cascade pathways. Protein-protein interaction (PPI) network analysis identified 3 hub genes including NFKBIA, TNFAIP3 and CEBPB, and 5 important DE lncRNAs including MSTRG.24134.4, MSTRG.3038.5, MSTRG.27019.3, MSTRG.26559.1, and MSTRG.10983.1. The expression patterns of 6 randomly selected differentially expressed immune-related genes were validated using the quantitative real-time PCR method. CONCLUSIONS: In short, our study is helpful to explore the potential interplay between lncRNAs and protein coding genes in different tissues of L. crocea post C. irritans and the molecular mechanism of pathogenesis for cryptocaryonosis. HIGHLIGHTS: Skin and gills are important sources of pro-inflammatory molecules, and their gene expression patterns are tissue-specific after C. irritans infection. 15 DEGs and 5 DE lncRNAs were identified as hub regulatory elements after C. irritans infection The HIF-1 signaling pathway and the complement and coagulation cascade pathway may be key tissue-specific regulatory pathways in gills and skin, respectively.

Characterization of the complete mitochondrial genome of Miamiensis avidus causing flatfish scuticociliatosis.

Miamiensis avidus is a parasitic pathogen that causes the disease scuticociliatosis in teleost fish species. It is a ciliate and a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. The complete mt-genome of M. avidus was linear and 38,695 bp in length with 47 genes, including 40 protein-coding genes, two ribosomal RNA (rRNA) genes, and five transfer RNA (tRNA) genes. Of these, 20 genes typically belong to the clusters of orthologous groups, playing roles in energy production and conversion, translation, ribosomal structure and biogenesis, and defense mechanisms. This is the first report of sequencing and characterization of the mt-genome of M. avidus, which was observed to be linear and possessing the typical ciliate mitochondrial genome organization and phylogenetic relationships. Remarkable differences were observed between M. avidus and other ciliates in the mitochondrially encoded rRNAs, extensive gene loss in ribosomal genes and tRNAs, terminal repeat sequences, and stop codon usage. A comparative and phylogenetic analysis of M. avidus and Uronema marinum of the order Hymenostomatida, which is most closely related to the order Philasterida, signified the promise of the mitogenome data of M. avidus as a valuable genetic marker in species detection and taxonomic research. The present study has potential applications in epidemiological studies and host-parasite interaction investigations facilitating disease control.

Transcriptome analysis of grass carp (Ctenopharyngodon idella) and Holland's spinibarbel (Spinibarbus hollandi) infected with Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a protozoan ciliate that causes white spot disease (also known as ichthyophthiriasis) in freshwater fish. Holland's spinibarbel (Spinibarbus hollandi) was less susceptible to white spot disease than grass carp (Ctenopharyngodon Idella). In this study, grass carp and Holland's spinibarbel are infected by I. multifiliis and the amount of infection is 10,000 theronts per fish. All grass carp died within 12 days after infection, and the survival rate of Holland's spinibarbel was more than 80%. In order to study the difference in sensitivity of these two fish species to I. multifiliis, transcriptome analysis was conducted using gill, skin, liver, spleen and head kidney of Holland's spinibarbel and grass carp at 48 h post-infection with I. multifiliis. A total of 489,296,696 clean reads were obtained by sequencing. A total of 105 significantly up-regulated immune-related genes were obtained by Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in grass carp. Cluster of differentiation 40 (CD40), cluster of differentiation 80 (CD 80), tumor necrosis factor-alpha (TNF-α), toll-like receptor 4 (TLR-4), interleukin 1 beta (IL-1ß) and other inflammatory-related genes in grass carp were enriched in the cytokine-cytokine receptor interaction pathway and toll-like receptor pathway. In Holland's spinibarbel, a total of 46 significantly up-regulated immune-related genes were obtained by GO classification and KEGG pathway enrichment analysis. Immune-related genes, such as Immunoglobin heavy chain (IgH), cathepsin S (CTSS), complement C1q A chain (C1qA), complement component 3 (C3) and complement component (C9) were enriched in phagosome pathway, lysosome pathway and complement and coagulation concatenation pathway. C3 was significantly up-regulated in gill and head kidney. Fluorescence in situ hybridization (FISH) showed that the C3 gene was highly expressed in gill tissue of Holland's spinibarbel infected with I. multifiliis. A small amount of C3 gene was expressed in the gill arch of grass carp after infected with I. multifiliis. In conclusion, the severe inflammatory response in vivo after infecting grass carp with I. multifiliis might be the main cause of the death of grass carp. The extrahepatic expression of the gene of Holland's spinibarbel might play an important role in the immune defense against I. multifiliis.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Transcriptome analysis in Takifugu rubripes and Dicentrarchus labrax gills during Cryptocaryon irritans infection.

Takifugu rubripes and Dicentrarchus labrax are important commercial fish in China that are under serious threat from Cryptocaryon irritans. C. irritans is a ciliated obligate parasite that causes marine white spot disease and leads to heavy economic losses. We analysed the transcriptome in the gills of T. rubripes and D. labrax to compare differentially expressed genes (DEGs) and pathways during infection with C. irritans. In total, we identified 6,901 and 35,736 DEGs from T. rubripes and D. labrax, respectively. All DEGs were annotated into GO terms; 6,901 DEGs from T. rubripes were assigned into 991 sub-categories, and 35,736 DEGs from D. labrax were assigned into 8,517 sub-categories. We mapped DEGs to the KEGG database and obtained 153 and 350 KEGG signalling pathways from T. rubripes and D. labrax, respectively. Immune-related categories included Toll-like receptors, MAPK, lysosome, C-type lectin receptor and NOD-like receptor signalling pathways were significantly enriched pathways. In immune-related signalling pathways, we found that AP-1, P38, IL-1ß, HSP90 and PLA were significantly up-regulated DEGs in T. rubripes, but P38 and PLA were significantly down-regulated in D. labrax. In this study, transcriptome was used to analyse the difference between scaly and non-scaly fish infection by C. irritans, which not only provided a theoretical basis for the infection mechanism of C. irritans, but also laid a foundation for effectively inhibiting the occurrence of this disease. Our work provides further insight into the immune response of host resistance to C. irritans.

Dose effects of a DNA vaccine encoding immobilization antigen on immune response of channel catfish against Ichthyophthirius multifiliis.

Channel catfish (Ictalurus punctatus) vaccinated with pcDNA3.1-IAg52b plasmid DNA vaccine encoding immobilization antigen genes of Ichthyophthirius multifiliis (Ich) produced anti-Ich antibodies and were partially protected (20% survival) in a previous study. Here we evaluated whether a higher dose or two doses of pcDNA3.1-IAg52b vaccine could provide better protection for catfish against Ich. Fish were distributed into 6 groups and vaccinated using following schemes: 1.10 µg pcDNA3.1-IAg52b fish-1, 2.20 µg pcDNA3.1-IAg52b fish-1, 3. two doses of 10 µg pcDNA3.1-IAg52b fish-1 with 7 days between doses, 4.20 µg pcDNA3.1 fish-1 (mock-vaccinated control), 5.15,000 live theronts fish-1 (positive control), and 6. non-vaccinated and non-challenge control. Parasite infection levels, serum anti-Ich antibody levels, fish mortality and immune-related gene expression were determined during the trial. Fish vaccinated with a single dose of 20 µg pcDNA3.1-IAg52b fish-1 or two doses of 10 µg fish-1 had higher anti-Ich antibody levels than fish receiving a single dose of 10 µg fish-1. Survival was significantly higher in fish receiving 20 µg vaccine fish-1 (35.6%) or 2 doses of 10 µg fish-1 (48.9%) than fish injected with a single dose of 10 µg fish-1 (15.6%) or mock-vaccinated control (0%). Fish vaccinated at the dose 20 µg fish-1 had higher expression of vaccine DNA in muscle than fish vaccinated with 10 µg fish-1. Fish vaccinated with the DNA vaccine showed higher up-regulation than mock-vaccinated control in the expression of IgM, CD4, MHC I and TcR-α genes during most of time points after vaccination. Further studies are needed to improve efficacy of DNA vaccines by using multiple antigens in the DNA vaccines.

Transcriptomic analysis of immunity in rainbow trout (Oncorhynchus mykiss) gills infected by Ichthyophthirius multifiliis.

The parasite Ichthyophthirius multifiliis infecting skin, fins and gills of a wide range of freshwater fish species, including rainbow trout, is known to induce a protective immune response in the host. Although a number of studies have reported activation of several immune genes in infected fish host, the immune response picture is still considered incomplete. In order to address this issue, a comparative transcriptomic analysis was performed on infected versus uninfected rainbow trout gills and it showed that a total of 3352 (7.2%) out of 46,585 identified gene sequences were significantly regulated after parasite infection. Of differentially expressed gene sequences, 1796 genes were up-regulated and 1556 genes were down-regulated. These were classified into 61 Gene Ontology (GO) terms and mapped to 282 reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Infection of I. multifiliis induced a clear differential expression of immune genes, related to both innate and adaptive immunity. A total of 268 (6.86%) regulated gene sequences were known to take part in 16 immune-related pathways. These involved pathways related to the innate immunity such as the Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration. Elevated transcription of genes encoding the TLR 8 gene and chemokines (CCL4, CCL19, CCL28, CXCL8, CXCL11, CXCL13, CXCL14) was recorded indicating their roles in recognition of I. multifiliis and subsequent induction of the inflammatory response, respectively. A number of upregulated genes in infected gills were associated with antigen processing/presentation and T and B cell receptor signaling (including B cell marker CD22 involved in B cell development). Overall the analysis supports the notion that I. multifiliis induces a massive and varied innate response upon which a range of adaptive immune responses are established which may contribute to the long lasting protection of immunized rainbow trout.

Quantitative shotgun proteomics distinguishes wound-healing biomarker signatures in common carp skin mucus in response to Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a ciliated protozoan parasite recognized as one of the most pathogenic diseases of wild and cultured freshwater fish. Fish skin mucus plays a significant role against invading pathogens. However, the protein-based modulation against infection with I. multifiliis, of host fish at this barrier is unknown. Thus, we investigated the skin mucus proteome of common carp using a shotgun proteomic approach at days 1 and 9 after I. multifiliis exposure. We identified 25 differentially expressed proteins in infected carp skin mucus. Upregulated proteins were mainly involved in metabolism, whereas downregulated proteins were mainly structural. This is the first proteomic analysis of infected common carp skin mucus, and it provides novel information about proteome alteration caused by I. multifiliis. Furthermore, we identified novel proteins with yet unknown function in common carp following penetrating injuries such as olfactomedin 4, lumican, dermatopontin, papilin and I cytoskeletal 18. This analysis, therefore, represents a key for the search for potential biomarkers, which can help in a better understanding and monitoring of interactions between carp and I. multifiliis. This proteomic study not only provides information on the protein-level pathways involved in fish-ciliate interactions but also could represent a complementary system for studying tissue repair.

Molecular identification and functional characterization of IRAK-3 from a teleost fish, the orange-spotted grouper (Epinephelus coioides).

Interleukin-1 receptor-associated kinase-3 (IRAK-3) is a unique IRAK family member, which negatively regulates the TLR-mediated immune response in mammals. However, the function of IRAK-3 remains to be elucidated in fish. In the present study, an IRAK-3 cDNA sequence (EcIRAK-3) with an ORF of 1776 bp encoding 591 amino acids was identified in the orange-spotted grouper (Epinephelus coioides). Sequence analysis indicated that EcIRAK-3 shared the conserved structure characteristics and functional sites of vertebrate IRAK-3, and has a high sequence identity and phylogenetic relationship with that of other fish species. The genomic EcIRAK-3 ORF contained 13 exons and 12 introns, which was similar to that of most other fish species. In healthy grouper, EcIRAK-3 was ubiquitously expressed in seven tested tissues with the highest expression in the gills. Following Cryptocaryon irritans infection, the EcIRAK-3 transcript was up-regulated in the gills during the course of the experiment, but down-regulated in the spleen at an earlier point in time. EcIRAK-3 was localized in both the cytoplasm and nucleus in a condensed form, and its cellular distribution was affected by the death domain and ProST domain. In addition, EcIRAK-3 significantly increased MyD88-mediated NF-κB activity, and its function was ProST domain and kinase domain dependent. Taken together, the results obtained here have contributed to the understanding of the function of IRAK-3 in fish.

Identification and functional analysis of grouper (Epinephelus coioides) B-cell linker protein BLNK.

B-cell linker protein (BLNK) is an adaptor protein that plays a crucial role in the B cell antigen receptor (BCR) signal pathway. To investigate the function of BLNK in teleost fish, we cloned a BLNK ortholog gene from the orange-spotted grouper (Epinephelus coioides). Homology analysis showed that the grouper BLNK (EcBLNK) had a 34%-77% amino acid identity in comparison to other vertebrates and shared the highest amino acid identity with BLNK from the Asian seabass Lates calcarifer. EcBLNK comprises an N-terminal SAM domain and a C-terminal B-cell linker SH2 domain. Ten tyrosine residues were well conserved between teleost fish and mammals. Tissue distribution analysis showed that EcBLNK was expressed mainly in immune organs and expression was at the highest level in head kidney. Co-localization of EcBLNK and EcCD79a was observed in transfected HEK293T cells. Overexpression of EcBLNK did not activate nuclear factor kappa-light-chain-enhancer of activated B cells. The protein level of EcBLNK in grouper head kidney leukocytes was increased by stimulation with lipopolysaccharide. In groupers infected with Cryptocaryon irritans, EcBLNK was regulated in the infected sites and the systemic organ which suggests that EcBLNK was activated in the immune response to parasite infection.

Characterization and expression patterns of ERK1 and ERK2 from Epinephelus coioides against Cryptocaryon irritans infection.

Mitogen-activated protein kinases (MAPKs), a group of serine-threonine protein kinases, play a crucial role in immunoreaction response to extra environmental stresses. In this study, two novel MAPKs, Ec-ERK1 and Ec-ERK2, were identified from Epinephelus coioides. Both Ec-ERK1 and Ec-ERK2 sequences contain a highly conserved Thr-Glu-Tyr (TEY) motif, an HRD domain, and an ATP binding loop containing GXGXXG. An analysis of phylogenetic relationships demonstrated that ERK amino acid sequences were conserved between different species indicating that the functions may be similar. Ec-ERK1 and Ec-ERK2 mRNA can be detected in all thirteen tissues examined, but the expression level is different in these tissues. The expression patterns of these two genes in E. coioides were also detected against Cryptocaryon irritans infection, which is capable of killing large numbers of fish in a short time and has a serious impact on aquaculture. The expression was up-regulated in most of the tissues examined, with the highest expressions of Ec-ERK1 (3.9 times) occurring in the head kidney and Ec-ERK2 (3.5 times) occurring in the spleen. There was no significant correlation between the expression of Ec-ERK1/Ec-ERK2 and the expression of nuclear factor kappaB (NF-kB). The results indicated the sequences and the characters of Ec-ERK1/ERK2 were conserved, Ec-ERK1/ERK2 showed tissue-specific expression patterns in healthy grouper, and their expressions were significantly varied post C. irritans infection, suggesting Ec-ERK1/ERK2 may play important roles in these tissues during pathogen-caused inflammation.

ParTIES: a toolbox for Paramecium interspersed DNA elimination studies.

MOTIVATION: Developmental DNA elimination occurs in a wide variety of multicellular organisms, but ciliates are the only single-celled eukaryotes in which this phenomenon has been reported. Despite considerable interest in ciliates as models for DNA elimination, no standard methods for identification and characterization of the eliminated sequences are currently available. RESULTS: We present the Paramecium Toolbox for Interspersed DNA Elimination Studies (ParTIES), designed for Paramecium species, that (i) identifies eliminated sequences, (ii) measures their presence in a sequencing sample and (iii) detects rare elimination polymorphisms. AVAILABILITY AND IMPLEMENTATION: ParTIES is multi-threaded Perl software available at https://github.com/oarnaiz/ParTIES. ParTIES is distributed under the GNU General Public Licence v3.

Transcriptomic profiling of Tibetan highland fish (Gymnocypris przewalskii) in response to the infection of parasite ciliate Ichthyophthirius multifiliis.

Gymnocypris przewalskii is a native cyprinid in the Lake Qinghai of the Qinghai-Tibetan Plateau. G. przewalskii is highly susceptible to the infection of a parasite, Ichthyophthirius multifiliis, in the artificial propagation and breeding. To better understand the host immune reaction to I. multifiliis infection, we characterize the gene expression profiles in the spleen of healthy and I. multifiliis infected G. przewalskii by RNA-seq. Totally, the transcriptomic analysis produces 463,031,110 high quality reads, which are assembled to 213,538 genes with N50 of 1918 bp and the average length of 1205 bp. Of assembled genes, 90.52% are annotated by public databases. The expression analysis shows 744 genes are significantly changed by the infection of I. multifiliis, which are validated by qRT-PCR with the correlation coefficient of 0.896. The differentially expressed genes are classified into 689 GO terms and 230 KEGG pathways, highlighting the promoted innate immunity in I. multifiliis infected G. przewalskii at 2 days post infection. Our results pinpoint that the up-regulated genes are enriched in TLR signaling pathway, inflammatory response and activation of immune cell migration. On the contrary, complement genes are down-regulated, indicating the evasion of host complement cascades by I. multifiliis. The repressed genes are also enriched in the pathways related to metabolism and endocrine, suggesting the metabolic disturbance in I. multifiliis treated G. przewalskii. In summary, the present study profiles the gene expression signature of G. przewalskii in the responses to I. multifiliis infection, and improves our understanding on molecular mechanisms of host-parasite interaction in G. przewalskii, which focuses the crucial function of TLRs, cytokines and complement components in the host defense against I. multifiliis.

Transcriptomic variation of locally-infected skin of Epinephelus coioides reveals the mucosal immune mechanism against Cryptocaryon irritans.

Fish skin is the largest immunologically active mucosal organ, providing first-line defense against external pathogens. However, the skin-associated immune mechanisms of fish are still unclear. Cryptocaryon irritans is an obligate ectoparasitic ciliated protozoan that infects almost all marine fish, and is believed to be an excellent pathogen model to study fish mucosal immunity. In this study, a de novo transcriptome assembly of Epinephelus coioides skin post C. irritans tail-infection was performed for the first time using the Illumina HiSeq™ 2500 system. Comparative analyses of infected skin (group Isk) and uninfected skin (group Nsk) from the same challenged fish and control skin (group C) from uninfected control fish were conducted. As a result, a total of 91,082 unigenes with an average length of 2880 base pairs were obtained and among them, 38,704 and 48,617 unigenes were annotated based on homology with matches in the non-redundant and zebrafish database, respectively. Pairwise comparison resulted in 10,115 differentially-expressed genes (DEGs) in the Isk/C group comparison (4,983 up-regulated and 5,132 down-regulated), 2,275 DEGs in the Isk/Nsk group comparison (1,319 up-regulated and 956 down-regulated) and 4,566 DEGs in the Nsk/C group comparison (1,534 up-regulated and 3,032 down-regulated). Seven immune-related categories including 91 differentially-expressed immune genes (86 up-regulated and 5 down-regulated) were scrutinized. Both DEGs and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and immune-related gene expression analysis were used, and both analyses showed that the genes were more significantly altered in the locally-infected skin than in the uninfected skin of the same challenged fish. This suggests the skin's local immune response is important for host defense against this ectoparasite infection. Innate immune molecules, including hepcidin, C-type lectin, transferrin, transferrin receptor protein, serum amyloid A, cathepsin and complement components were significantly up-regulated (fold-change ranged from 3.3 to 12,944) in infected skin compared with control skin. The up-regulation of chemokines and chemokine receptors and activation of the leukocyte transendothelial migration pathway suggested that leucocytes intensively migrated to the local infected sites to mount a local immune defense. Toll-like receptors (TLRs) 1, 2, 5 and 5S were most significantly up-regulated in the infected skin, suggesting that these TLRs may be involved in parasite pathogen-associated molecular pattern (PAMPs) recognition. Up-regulation of the dendritic cell markers CD209 and CD83 and other antigen presentation pathway molecules provided evidence for skin local antigen presentation. Up-regulation of the T cell markers CD4 and CD48, B cell markers CD22 and CD81 and B cell receptor signaling kinase Lyn, showed the presence and population expansion of T/B cells at locally-infected sites, which suggested possible activation of a local specific immune response in the skin. Our results will facilitate in-depth understanding of local immune defense mechanisms in fish skin against ectoparasite infection.

Characterization and expression analysis of six interleukin-17 receptor genes in grouper (Epinephelus coioides) after Cryptocaryon irritans infection.

Interleukin-17 receptors (IL17Rs) mediate the activation of several downstream signal pathways to induce inflammatory response and contribute to the pathology of many autoimmune diseases. In this study, six IL17Rs (IL17RA1, RA2, RB, RC, RD and RE) were cloned and characterized from Epinephelus coioides, an orange-spotted grouper. Multiple sequence alignment and structural analysis revealed that all members of IL17Rs were low in sequence identity with each other. But their structures were conservative in grouper, which contain signal peptide, extracellular FNIII domain (IL17RA1/RA2/RB) or IL-17_R_N domain (IL17RC/RD/RE), transmembrane domain and SEFIR domain in their intracellular region. The analysis of tissue distribution showed these six genes were ubiquitously and differentially expressed in all major types of tissues. What's more, it is interesting to find their high expression in immune tissues (liver, gill, skin and thymus). IL17RA1 and IL17RA2 were significantly down-regulated at all time-points in gill and spleen after Cryptocaryon irritans infection, however, there was no significant change in other grouper IL17Rs. It suggests that the C. irritans may escape from the host immunity or the host prevents serious inflammation by inhibiting the expression of ILl7Rs.

Grouper (Epinephelus coioides) TCR signaling pathway was involved in response against Cryptocaryon irritans infection.

T cell activation is a complicated process accompanying with the activation of T cell receptor (TCR) signaling pathway, which is not well described in teleost fish. The initiation of this pathway depends on the interaction of membrane TCR co-receptors (e.g. CD4/8, CD3 and CD45) and a series of cytoplasmic protein tyrosine kinases (e.g. Lck, Fyn and ZAP70). Cyptocaryon irritans is a ciliate pathogen of marine fish white spot disease causing huge economic lost in marine aquaculture. This parasite can infect fish gill and skin and is considered to be a good pathogen model for fish gill and skin mucosal immunity. Our previous studies showed the locally mucosal antibody response was important for fish defense against this parasite. While how TCR signaling pathway involved in T cell activation to help B cell activation in C. irritans infected fish is still not known. In the present study, we cloned a grouper TCR co-receptor gene EcCD3ε (537 bp) and its three kinase genes, including EcLck (1512 bp), EcFyn (1605 bp) and EcZAP70 (1893 bp). Homology analysis showed that they all shared the highest identity with corresponding genes from Takifugu rubripes (EcCD3ε 41%, EcLck 88%, EcFyn 98% and EcZAP70 93%), and their conserved motifs involved in the signaling transduction were analyzed. The tissue distribution analysis showed these four genes were high expressed in thymus, and it is interesting to find their comparative high expression in skin, gill and midgut mucosal immune tissues. In C. irritans infected grouper, the expression of three TCR co-receptors (EcCD4-1, EcCD3ε and EcCD45) and three kinases (EcLck, EcFyn and EcZAP70) was tested in skin, gill, head kidney and spleen at 0, 12 h, 24 h, 2 d, 3 d, 5 d and 7 d. All six genes were significantly up-regulated in skin at most tested time points, which indicate the possibility of skin local T cell activation to support the local antibody response. Compared to three TCR co-receptors, significantly up-regulation of three kinases were seen in the spleen, and the spleen fold changes of these three kinases were much higher than head kidney, which indicates spleen maybe the major systematic immune organs for T cell activation in C. irritans infected fish.

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.

Transcriptome analysis of the Larimichthys crocea liver in response to Cryptocaryon irritans.

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics.

Transcriptome and analysis on the complement and coagulation cascades pathway of large yellow croaker (Larimichthys crocea) to ciliate ectoparasite Cryptocaryon irritans infection.

Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A = 42; C:A = 28; C:B = 42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed.

Characterization and functional analysis of a tandem-repeat galectin-9 in large yellow croaker Larimichthys crocea.

Galectins are a family of endogenous lectins with ß-galactosides affinity, playing significant roles in the innate immunity of vertebrates and invertebrates. In this report, a new galectin-9 cDNA was identified and characterized in large yellow croaker Larimichthys crocea (designated as LcGal-9). The complete cDNA sequence of LcGal-9 was 1795 bp, with an open reading frame (ORF) of 1032 bp encoding 343 amino acids. The putative LcGal-9 protein contained two carbohydrate recognition domains (CRDs) connected by a linker peptide, with each carrying two conserved ß-galactoside binding motifs H-NPR and WG-EE-, and it possessed neither a signal peptide nor a transmembrane domain. LcGal-9 protein shared 43-74% identity with galectin-9 sequences from other species. The qRT-PCR analysis revealed that LcGal-9 mRNA was constitutively expressed in all tissues examined, predominately expressed in liver, spleen, gill, kidney, head-kidney and intestine. Western blot analysis showed that LcGal-9 protein was highly expressed in liver, spleen, intestine, kidney, head-kidney, skin, gill, and heart, but not detected in muscle and plasma. LcGal-9 mRNA transcripts were induced by poly I:C in the liver (from 6 h to 48 h), spleen (at 12 h) and head-kidney (at 12 h and 24 h). In contrast, Vibrio parahaemolyticus caused a significant down-regulation in these three tissues, except for in spleen of 48 h and head-kidney of 3 h. Post-infection with Cryptocaryon irritans, the transcripts were dramatically up-regulated in gill, skin, spleen and head-kidney during initial infection period, while significant down-regulation afterward was also observed both in spleen and head-kidney. The recombinant LcGal-9 (named as rLcGal-9) purified from Escherichia coli BL21 (DE3) demonstrated hemagglutination against human, rabbit and L. crocea in a Ca(2+)-independent manner, which was inhibited by α-Lactose and LPS. The results of bacterial agglutination assays showed that rLcGal-9 was able to agglutinate Gram-negative bacteria V. alginolyticus and Aeromonas hydrophila in a Ca(2+)-independent manner. By immunohistochemistry assay, significant increases of LcGal-9 protein appeared in the spleen stimulated with poly I:C (for 12 h) and V. parahaemolyticus (for 48 h) compared with the control. Based on the collective data, LcGal-9 might play an important role in innate immune responses, especially defense against Gram-negative bacteria in L. crocea.