Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.     

Rapid adaptation of molecular resources from zebrafish and medaka to develop an estuarine/marine model

Many estuary and coastal waters are highly threatened by heavy anthropogenic pollutants. Oryzias melastigma, also called O. dancena, a marine medaka that showed sensitive response to hypoxia and estrogenic endocrine disruptors in previous studies, is becoming a sentinel species for marine ecotoxicology studies. However, the lack of strong molecular foundation and knowledge of early developmental stages hampers its practical applications. Combining our research strength on zebrafish embryos, this study revealed both morphological and molecular (at mRNA and protein levels) development of embryos of this emergent model. Whole mount immunostaining technique specific for O. melastigma was successfully developed based on zebrafish standard protocols. We demonstrated that 17 out of 61 primary antibodies, which were previously tested in zebrafish, showed specific immunoreactivity with O. melastigma. These antibodies clearly illustrated the embryonic development of target tissues (principally neurons) in this medaka. Additionally, partial cDNA fragments of 11 organ-specific marker genes were isolated according to genomic resources of zebrafish, Japanese medaka and other fishes. Of the 11 genes, 8 are widely used as organ markers and their expression patterns were remarkably similar to their homologues in zebrafish and Japanese medaka. The expression profiles of the remaining 3 genes in fish are reported for the first time. These molecular markers (17 antibodies and 11 mRNA probes) can be used as responsive indicators in environmental toxicity evaluation. Moreover, this study brought forward and demonstrated the advantage of transferring techniques and resources from one model to another to hasten the research of interest.     

The genome of the marine medaka Oryzias melastigma

Marine medaka (Oryzias melastigma) is considered to be a useful fish model for marine and estuarine ecotoxicology studies and has good potential for field‐based population genomics because of its geographical distribution in Asian estuarine and coastal areas. In this study, we present the first whole‐genome draft of O. melastigma. The genome assembly consists of 8,602 scaffolds (N50 = 23.737 Mb) and a total genome length of 779.4 Mb. A total of 23,528 genes were predicted, and 12,670 gene families shared with three teleost species (Japanese medaka, mangrove killifish and zebrafish) were identified. Genome analyses revealed that the O. melastigma genome is highly heterozygous and contains a large number of repeat sequences. This assembly represents a useful genomic resource for fish scientists.     

Expression profile of immune-related genes in Lates calcarifer infected by Cryptocaryon irritans

Cryptocaryon irritans causes Cyptocaryonosis or white spot disease in a wide range of marine fish including Lates calcarifer (Asian seabass). However, the immune response of this fish to the parasite is still poorly understood. In this study, quantitative polymerase chain reaction (qPCR) was performed to assess the expression profile of immune-related genes in L. calcarifer infected by C. irritans. A total of 21 immune-related genes encoding various functions in the fish immune system were utilized for the qPCR analysis. The experiment was initiated with the infection of juvenile fish by exposure to theronts from 200 C. irritans cysts, and non-infected juvenile fish were used as controls. Spleen, liver, gills and kidney tissues were harvested at three days post-infection from control and infected fish. In addition, organs were also harvested on day-10 post-infection from fish that had been allowed to recover from day-4 up to day-10 post-infection. L. calcarifer exhibited pathological changes on day-3 post-infection with the characteristic presence of white spots on the entire fish body, excessive mucus production and formation of a flap over the fish eye. High quality total RNA was extracted from all tissues and qPCR was performed. The qPCR analysis on the cohort of 21 immune-related genes of the various organs harvested on day-3 post-infection demonstrated that most genes were induced significantly (p < 0.05) in all tissues, particularly liver (11/21 genes) and kidney (11/21). The expression profile demonstrated that induction of the MHC Class IIα gene was the highest compared to the other genes followed by serum amyloid A, CC chemokine and hepcidin-2 precursor genes. In fish that were allowed to recover from the C. irritans infection (10 days post-infection), expression of the immune-related genes was down-regulated to levels similar to the control fish. These results provide insights into the interaction between C. irritans and L. calcarifer and suggest that the innate immune system plays an important role in early defence against parasite infection allowing the fish to eventually recover from the infection.     

Antinematode Activity of Violacein and the Role of the Insulin/IGF-1 Pathway in Controlling Violacein Sensitivity in Caenorhabditis elegans

The purple pigment violacein is well known for its numerous biological activities including antibacterial, antiviral, antiprotozoan, and antitumor effects. In the current study we identify violacein as the antinematode agent produced by the marine bacterium Microbulbifer sp. D250, thereby extending the target range of this small molecule. Heterologous expression of the violacein biosynthetic pathway in E. coli and experiments using pure violacein demonstrated that this secondary metabolite facilitates bacterial accumulation in the nematode intestine, which is accompanied by tissue damage and apoptosis. Nematodes such as Caenorhabditis elegans utilise a well-defined innate immune system to defend against pathogens. Using C. elegans as a model we demonstrate the DAF-2/DAF-16 insulin/IGF-1 signalling (IIS) component of the innate immune pathway modulates sensitivity to violacein-mediated killing. Further analysis shows that resistance to violacein can occur due to a loss of DAF-2 function and/or an increased function of DAF-16 controlled genes involved in antimicrobial production (spp-1) and detoxification (sod-3). These data suggest that violacein is a novel candidate antinematode agent and that the IIS pathway is also involved in the defence against metabolites from non-pathogenic bacteria.     

Effects of dietary vitamin D3 administration on innate immune parameters of seabream (Sparus aurata L.)

The present study assesses the in vivo effect of vitamin D₃ or cholecalciferol on some innate immune parameters of the gilthead seabream (Sparus aurata L.). Cholecalciferol was orally administered to seabream specimens in a commercial pellet food supplemented with 0 (control); 3750; 18,750 or 37,500 U kg^?1 and fish were sampled after 1, 2 and 4 weeks of treatment. Serum and head– kidney leucocytes were obtained and humoral (peroxidase and complement activity) and cellular (leucocyte peroxidase content, phagocytic, respiratory burst and natural cytotoxic activities) innate immune parameters were measured. Diet supplementation with 37,500 U kg^?1 cholecalciferol for 2 or 4 weeks resulted in a significant increase in phagocytic ability or serum peroxidase content, respectively, whereas the 3750 and 18,750 U kg^?1 supplemented diets led to significant increases in the phagocytic capacity of leucocytes at week 2 compared with the values found in control fish. Natural cytotoxic activity was increased in leucocytes from fish fed for 1 week with 3750 U kg^?1 cholecalciferol. No significant differences were observed in complement activity or in respiratory burst activity in the assayed conditions. These results suggested that dietary vitamin D₃ administration has an effect on the innate immune parameters of gilthead seabream. The immunostimulant effect was greater on the cellular innate immune parameters assayed, suggesting that similar receptors to those present in mammals are involved in the action of this vitamin in the fish immune system.     

Identification of immunostimulatory DNA-induced genes by suppression subtractive hybridization

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80-90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODN-stimulated bone marrow-derived macrophages: NF-kappaB p105, IRF-1, PA28beta, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms.     

Ontogeny and tissue-specific expression of immune-relevant genes in Catla catla (Hamilton)

India is the second largest fish producing country in the world with production of 12.6 million tonnes (mt) in 2017–18, and Indian major carps (IMCs) contribute bulk of this fish production. Catla, Catla catla is the fastest growing species among IMCs. However, the survival rate of catla during larval rearing is normally lower than the other IMCs i.e rohu Labeo rohita, and mrigal Cirrhinus mrigala. Continuous efforts are devoted for the identification of nutritional and environmental requirements of fish larvae in order to reduce hatchery mortalities. However, very little information is available regarding physiology of the immune system, especially during the late larval and juvenile stages. Hence, understanding the ontogenetic development of immune-relevant genes in the larval stages of catla will serve as the markers for the development of immune competence and thereby, will be beneficial in developing effective immune intervention strategies. In the present study, expression profiles of some of the important innate (IL-1β, IL-10, iNOS and C3) and adaptive immune (RAG-1, Ikaros, IgM and IgZ) genes during ontogenetic developmental stages and in different tissues of catla were investigated using quantitative real-time PCR. The results revealed that immune genes IL-1β, C3, IgM and IgZ were detected in the unfertilized eggs indicating their maternal inheritance. Immune genes, IL-1β, IL-10 and iNOS were expressed significantly during initial larval developmental stages whereas C3, RAG-1, Ikaros, IgM and IgZ showed significant expression during advanced stages of larval development in catla i.e. from 23 days post hatch (dph). Study of tissue distribution pattern of the genes indicated that innate immune genes were ubiquitously expressed in different tissues with varying degree of expression, whereas adaptive immune genes were predominantly expressed in lymphoid organs of catla. The information thus generated will improve knowledge on the maturation of the immune system in catla and will aid in deciding the appropriate age for vaccination in this teleost species.     

Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus

The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5′-untranslated region (UTR) of 17 bp, a 3′-UTR of 76 bp, and a stop codon TAA in 3′-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being challenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the immune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene discovery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expression studies in kelp grouper.