Infertility control of transgenic fluorescent zebrafish with targeted mutagenesis of the dnd1 gene by CRISPR/Cas9 genome editing.

Transgenic technology and selective breeding have great potential for the genetic breeding in both edible fish and ornamental fish. The development of infertility control technologies in transgenic fish and farmed fish is the critical issue to prevent the gene flow with wild relatives. In this study, we report the genome editing of the dead end (dnd1) gene in the zebrafish model, using the CRISPR/Cas9 technology to achieve a loss-of-function mutation in both wild-type zebrafish and transgenic fluorescent zebrafish to develop complete infertility control technology of farmed fish and transgenic fish. We effectively performed targeted mutagenesis in the dnd1 gene of zebrafish with a single gRNA, which resulted in a small deletion (-7 bp) or insertion (+41 bp) in exon 2, leading to a null mutation. Heterozygotes and homozygotes of dnd1-knockout zebrafish were both selected by genotyping in the F 1 and F 2 generations. Based on a comparison of histological sections of the gonads between wild-type, heterozygous, and homozygous dnd1 zebrafish mutants, the dnd1 homozygous mutation (aa) resulted in the loss of germ cells. Still, there was no difference between the wild-type (AA) and dnd1 heterozygous (Aa) zebrafish. The homozygous dnd1 mutants of adult zebrafish and transgenic fluorescent zebrafish became all male, which had normal courtship behavior to induce wild-type female zebrafish spawning. However, they both had no sperm to fertilize the spawned eggs from wild-type females. Thus, all the unfertilized eggs died within 10 h. The targeted mutagenesis of the dnd1 gene using the CRISPR/Cas9 technology is stably heritable by crossing of fertile heterozygous mutants to obtain sterile homozygous mutants. It can be applied in the infertility control of transgenic fluorescent fish and genetically improved farmed fish by selective breeding to promote ecologically responsible aquaculture.

Maintenance of Genetic Diversity of Black Sea Bream despite Unmonitored and Large-Scale Hatchery Releases.

Stock enhancement, used for replenishing depleted wild finfish populations, is an aggressive approach. Stock enhancement projects in Taiwan involve black sea bream (Acanthopagrus schlegelii), a major commercial species. During 2004-2015, even management agencies conducted stock enhancement projects, leading to numerous private releases that have not been recorded. Stock enhancement by a private hatchery without accurate genetic records may lead to a genetic structure change in wild populations. Using allele frequencies at nine microsatellite loci, we studied the genetic effects of stock enhancement in 19 samples collected from populations in the hatcheries and the wild. In 458 individuals from nine hatchery samples, most populations showed weak but significant genetic differences and complex clusters in structure analysis, indicating dramatic stock change within and among hatcheries. The 10 wild populations (n = 773) also had a complex genetic composition and were genetically different among sampling sites and times. However, a simple and clear cluster in structure analysis was found for only one sampling site, which had no release history. Thus, stock enhancement with complex genetic sources helps maintain genetic diversity but dramatically changes the genetic structure within and among wild populations, especially when stock enhancement is successful.

Multigenerational study of life history traits, bioaccumulation, and molecular responses of Pseudodiaptomus annandalei to cadmium.

Metal pollution provide a substantial challenge for environmental health. This study investigated the multigeneration effects of cadmium on populations of the copepod species Pseudodiaptomus annandalei, exposed to a sublethal concentration, 40 µg/L of cadmium (Cd), over 10 generations. At the end of each generation, copepod individuals were collected to estimate fecundity, bioaccumulation, and real time qPCR quantification of selected differentially expressed genes to evaluate Cd effects and sex-specific responses of copepods across multiple generations. Our results revealed a sex-specific accumulation of Cd integrating 10 successive generations. The concentration of Cd was significantly higher (p < 0.05) in males than in females. We also observed a generational increase in Cd accumulation. Fecundity increased, with the exception of the first generation, possibly as a compensation for a decrease of copepod population size under Cd exposure. Protein expression of copepods exposed to Cd occurred in a sex-specific manner. Hemerythrin was mostly up-regulated in both copepod sexes exposed to Cd with males having the highest expression levels, while heat shock protein 70 was mostly up-regulated in males and down-regulated in female copepods, both exposed to Cd. Although copepods are known to develop adaptive mechanisms to tolerate toxic chemicals, continuous exposure to metals could lead to the bioaccumulation of metals in their offspring through maternal transfer and direct uptake from the medium over several generations. As a consequence, increased metal concentrations in copepods could result in physiological damage, reducing their fitness, and possibly compromise copepod population structures. This study showed that mortality, life history traits and molecular responses of a copepod species provided important toxicological endpoints and bio-markers for environmental risk assessments. Environmental pressure resulting from continuous exposure to persistent pollutants like Cd, could have evolutionary significance. The tendency for copepods to selectively adapt to a toxic environment through modifications, could increase their chance of survival over a long term.

Identification of Genes Related to Cold Tolerance and Novel Genetic Markers for Molecular Breeding in Taiwan Tilapia (Oreochromis spp.) via Transcriptome Analysis.

Taiwan tilapia is one of the primary species used in aquaculture practices in Taiwan. However, as a tropical fish, it is sensitive to cold temperatures that can lead to high mortality rates during winter months. Genetic and broodstock management strategies using marker-assisted selection and breeding are the best tools currently available to improve seed varieties for tilapia species. The purpose of this study was to develop molecular markers for cold stress-related genes using digital gene expression analysis of next-generation transcriptome sequencing in Taiwan tilapia (Oreochromis spp.). We constructed and sequenced cDNA libraries from the brain, gill, liver, and muscle tissues of cold-tolerance (CT) and cold-sensitivity (CS) strains. Approximately 35,214,833,100 nucleotides of raw sequencing reads were generated, and these were assembled into 128,147 unigenes possessing a total length of 185,382,926 bp and an average length of 1446 bp. A total of 25,844 unigenes were annotated using five protein databases and Venny analysis, and 38,377 simple sequence repeats (SSRs) and 65,527 single nucleotide polymorphisms (SNPs) were identified. Furthermore, from the 38-cold tolerance-related genes that were identified using differential gene expression analysis in the four tissues, 13 microsatellites and 37 single nucleotide polymorphism markers were identified. The results of the genotype analysis revealed that the selected markers could be used for population genetics. In addition to the diversity assessment, one of the SNP markers was determined to be significantly related to cold-tolerance traits and could be used as a molecular marker to assist in the selection and verification of cold-tolerant populations. The specific genetic markers explored in this study can be used for the identification of genetic polymorphisms and cold tolerance traits in Taiwan tilapia, and they can also be used to further explore the physiological and biochemical molecular regulation pathways of fish that are involved in their tolerance to environmental temperature stress.

Fish-specific TLR18 in Nile tilapia (Oreochromis niloticus) recruits MyD88 and TRIF to induce expression of effectors in NF-κB and IFN pathways in melanomacrophages.

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.

Progranulin A Promotes Compensatory Hepatocyte Proliferation via HGF/c-Met Signaling after Partial Hepatectomy in Zebrafish.

Compensatory hepatocyte proliferation and other liver regenerative processes are activated to sustain normal physiological function after liver injury. A major mitogen for liver regeneration is hepatocyte growth factor (HGF), and a previous study indicated that progranulin could modulate c-met, the receptor for HGF, to initiate hepatic outgrowth from hepatoblasts during embryonic development. However, a role for progranulin in compensatory hepatocyte proliferation has not been shown previously. Therefore, this study was undertaken to clarify whether progranulin plays a regulatory role during liver regeneration. To this end, we established a partial hepatectomy regeneration model in adult zebrafish that express a liver-specific fluorescent reporter. Using this model, we found that loss of progranulin A (GrnA) function by intraperitoneal-injection of a Vivo-Morpholino impaired and delayed liver regeneration after partial hepatectomy. Furthermore, transcriptome analysis and confirmatory quantitative real-time PCR suggested that cell cycle progression and cell proliferation was not as active in the morphants as controls, which may have been the result of comparative downregulation of the HGF/c-met axis by 36 h after partial hepatectomy. Finally, liver-specific overexpression of GrnA in transgenic zebrafish caused more abundant cell proliferation after partial hepatectomy compared to wild types. Thus, we conclude that GrnA positively regulates HGF/c-met signaling to promote hepatocyte proliferation during liver regeneration.

Development of EST-Molecular Markers from RNA Sequencing for Genetic Management and Identification of Growth Traits in Potato Grouper (Epinephelus tukula).

The accuracy and efficiency of marker-assisted selection (MAS) has been proven for economically critical aquaculture species. The potato grouper (Epinephelus tukula), a novel cultured grouper species in Taiwan, shows large potential in aquaculture because of its fast growth rate among other groupers. Because of the lack of genetic information for the potato grouper, the first transcriptome and expressed sequence tag (EST)-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed. Initially, the transcriptome was obtained from seven cDNA libraries by using the Illumina platform. De novo transcriptome of the potato grouper yielded 51.34 Gb and 111,490 unigenes. The EST-derived SSR and SNP markers were applied in genetic management, in parentage analysis, and to discover the functional markers of economic traits. The F1 juveniles were identified as siblings from one pair of parents (80 broodstocks). Fast- and slow-growth individuals were analyzed using functional molecular markers and through their association with growth performance. The results revealed that two SNPs were correlated with growth traits. The transcriptome database obtained in this study and its derived SSR and SNP markers may be applied not only for MAS but also to maintain functional gene diversity in the novel cultured grouper.

Development of Disease-Resistance-Associated Microsatellite DNA Markers for Selective Breeding of Tilapia (Oreochromis spp.) Farmed in Taiwan.

There are numerous means to improve the tilapia aquaculture industry, and one is to develop disease resistance through selective breeding using molecular markers. In this study, 11 disease-resistance-associated microsatellite markers including 3 markers linked to hamp2, 4 linked to hamp1, 1 linked to pgrn2, 2 linked to pgrn1, and 1 linked to piscidin 4 (TP4) genes were established for tilapia strains farmed in Taiwan after challenge with Streptococcus inae. The correlation analysis of genotypes and survival revealed a total of 55 genotypes related to survival by the chi-square and Z-test. Although fewer markers were found in B and N2 strains compared with A strain, they performed well in terms of disease resistance. It suggested that this may be due to the low potency of some genotypes and the combinatorial arrangement between them. Therefore, a predictive model was built by the genotypes of the parental generation and the mortality rate of different combinations was calculated. The results show the same trend of predicted mortality in the offspring of three new disease-resistant strains as in the challenge experiment. The present findings is a nonkilling method without requiring the selection by challenge with bacteria or viruses and might increase the possibility of utilization of selective breeding using SSR markers in farms.

Differential gene expression profile of male and female copepods in response to cadmium exposure.

In this study, the whole transcriptome and sex-specific differential gene expression of the copepod Pseudodiaptomus annandalei exposed to cadmium (Cd) were investigated. P. annandalei were exposed to 40 µg/L Cd from the naupliar stage to male and female adults. High-throughput transcriptome sequencing (RNA-seq) was performed with copepod samples using an Illumina Hiseq™ 2000 platform. TransDecoder analysis found 32,625 putative open reading frame contigs. At p-values of <0.001, a total of 4756 differentially expressed genes (DEGs) (2216 up-regulated and 2540 down-regulated genes) were found in male copepods. Whereas a total of 2879 DEGs (2007 up-regulated and 872 down-regulated genes) were found in female copepods. A few selected cellular stress response genes, involved in xenobiotic metabolism, energy metabolism, growth, and development as a result of Cd exposure in the copepods were discussed. The study showed that most of these processes were changed in a sex-specific manner, accounting for the different sensitivities of male and female copepods. Results suggest and reinforce that sex is an important factor to be considered in ecotoxicogenomics.

Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.

Granulin peptide GRN-41 of Mozambique tilapia is a novel antimicrobial peptide against Vibrio species.

In our previous study, the novel GRN-41 peptide generated from alternative splicing of the Mozambique tilapia PGRN1 gene was identified to be a potent peptide that protected against V. vulnificus in the transgenic zebrafish model by modulating innate immune-related genes. In this study, the anti-bacterial activities of synthetic Mozambique tilapia GRN-41 peptide (OmGRN-41) against various bacterial pathogens were investigated. The results showed that OmGRN-41 had bactericidal activity against Vibrio species, including V. vulnificus, V. alginolyticus, and V. harveyi, but exhibited bacteriostatic activity against V. parahaemolyticus. OmGRN-41 maintained bactericidal activity (64 µM) against V. vulnificus at pH 2 to pH 10 or after heat treatment for 1 h at high temperatures between 40 °C and 100 °C. TEM observations revealed that the outer membrane of V. vulnificus was disrupted by OmGRN-41, leading to morphological rupture and loss of cytoplasmic contents. Additionally, little hemolytic activity against tilapia and sheep erythrocytes was detected after treatment with 128 µM OmGRN-41. OmGRN-41 can effectively enhance the survival of Nile tilapia infected by V. vulnificus. Our results suggest that the OmGRN-41 is a novel antimicrobial peptide possessing bactericidal activity, especially against Vibrio species. These results indicate that OmGRN-41 can be applied in human Vibriosis treatment and has the potential to defend against Vibrio spp. infection in critical aquaculture organisms.

A potent tilapia secreted granulin peptide enhances the survival of transgenic zebrafish infected by Vibrio vulnificus via modulation of innate immunity.

Progranulin (PGRN) is a multi-functional growth factor that mediates cell proliferation, survival, migration, tumorigenesis, wound healing, development, and anti-inflammation activity. A novel alternatively spliced transcript from the short-form PGRN1 gene encoding a novel, secreted GRN peptide composed of 20-a.a. signal peptide and 41-a.a. GRN named GRN-41 was identified to be abundantly expressed in immune-related organs including spleen, head kidney, and intestine of Mozambique tilapia. The expression of GRN-41 and PGRN1 were further induced in the spleen of tilapia challenged with Vibrio vulnificus at 3 h post infection (hpi) and 6 hpi, respectively. In this study, we established three transgenic zebrafish lines expressing the secreted GRN-41, GRN-A and PGRN1 of Mozambique tilapia specifically in muscle. The relative percent of survival (RPS) was enhanced in adult transgenic zebrafish expressing tilapia GRN-41 (68%), GRN-A (32%) and PGRN1 (36%) compared with control transgenic zebrafish expressing AcGFP after challenge with V. vulnificus. It indicates tilapia GRN-41 is a potent peptide against V. vulnificus infection. The secreted tilapia GRN-41 can induce the expression of innate immune response-related genes, such as TNFa, TNFb, IL-8, IL-1ß, IL-6, IL-26, IL-21, IL-10, complement C3, lysozyme (Lyz) and the hepatic antimicrobial peptide hepcidin (HAMP), in adult transgenic zebrafish without V. vulnificus infection. The tilapia GRN-41 peptide can enhance the innate immune response by further elevating TNFb, IL-1ß, IL-8, IL-6, and HAMP expression in early responsive time to the V. vulnificus challenge in transgenic zebrafish. Our results suggest that the novel GRN-41 peptide generated from alternative splicing of the tilapia PGRN1 gene is a potent peptide that defends against V. vulnificus in the transgenic zebrafish model by modulation of innate immunity.

Inducible liver-specific overexpression of gankyrin in zebrafish results in spontaneous intrahepatic cholangiocarcinoma and hepatocellular carcinoma formation.

Liver cancer is the second leading cause of death worldwide. As such, establishing animal models of the disease is important for both basic and translational studies that move toward developing new therapies. Gankyrin is a critical oncoprotein in the genetic control of liver pathology. In order to evaluate the oncogenic role of gankyrin without cancer cell inoculation and drug treatment, we overexpressed gankyrin under the control of the fabp10a promoter. A Tet-Off system was used to drive expression in hepatocytes. At seven to twelve months of age, gankyrin transgenic fish spontaneously incurred persistent hepatocyte damage, steatosis, cholestasis, cholangitis, fibrosis and hepatic tumors. The tumors were both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). ICC is the second most frequent primary liver cancer in human patients and the first to develop in this tumor model. We further investigated the role of complement C3, a central molecule of the complement system, and found the expression levels of both in mRNA and protein are decreased during tumorigenesis. Together, these findings suggest that gankyrin can promote malignant transformation of liver cells in the context of persistent liver injury. This transformation may be related to compensatory proliferation and the inflammatory microenvironment. The observed decrease in complement C3 may allow transforming cells to escape coordinated induction of the immune response. Herein, we demonstrate an excellent zebrafish model for liver cancers that will be useful for studying the molecular mechanisms of tumorgenesis.

Complete Genome Sequence of Streptococcus iniae 89353, a Virulent Strain Isolated from Diseased Tilapia in Taiwan.

Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis.

Transcriptome analysis of the effect of polyunsaturated fatty acids against Vibrio vulnificus infection in Oreochromis niloticus.

Vibrio vulnificus infection causes severe economic losses in Oreochromis niloticus aquaculture by inducing pro-inflammatory cytokines, that lead to inflammation and mortality. Omega-3 fatty acids, such as Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA), have been reported for their anti-inflammatory and antibacterial abilities in murine and zebrafish models. However, the anti-inflammatory and antibacterial functions of DHA and EPA in commercial aquaculture organisms such as Oreochromis niloticus remain unknown. The present study demonstrates antibacterial function and transcriptional modulation of inflammation-associated genes by DHA and EPA in Vibrio vulnificus infection in Oreochromis niloticus fish models. The administration of EPA or DHA improved the Oreochromis niloticus survival rate against Vibrio vulnificus infection. The induction of proinflammatory cytokines, Interleukin (IL)-1ß, IL-6, Tumor necrosis factor (TNF)-α, and Toll-like receptor (TLR)-2 by Vibrio vulnificus was suppressed in fish that were administered DHA. Bacterial membrane disruption and the killing of Vibrio vulnificus by EPA and DHA was observed using SEM, TEM, and cytoplasm leakage studies. In silico analysis of the transcription profile in Ingenuity Pathway Analysis software showed that DHA may enhance anti-Vibrio vulnificus activity in Oreochromis niloticus via the activation of peroxisome proliferator-activated receptor α (PPARα) to inhibit nuclear factor kappa B and suppress hepatocyte nuclear factor 4 α (HNF4α). In summary, the results of the present study demonstrated that DHA and EPA reduce the severity of Vibrio vulnificus infection and increase the survival rate of Oreochromis niloticus.

Lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in white shrimp Litopenaeus vannamei.

Lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and ß-1,3-glucan (ßG), known as pathogen-associated molecular patterns (PAMPs), and subsequently trigger innate immunity. Several seaweed polysaccharides and seaweed extracts increase immune parameters and resistance to pathogens. Here, we constructed the expression vector pET28b-LvLGBP and transferred it into Escherichia coli BL21 (DE3) for protein expression and to produce the recombinant protein LGBP (rLvLGBP) in white shrimp Litopenaeus vannamei. We examined the binding of rLvLGBP with seaweed-derived polysaccharides including alginate, carrageenan, fucoidan, laminarin, Gracilaria tenuistipitata extract (GTE), and Sargassum duplicatum extract (SDE), and examined the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and each polysaccharide. We also examined the binding of rLvLGBP with LPS and ßG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and ßG (rLvLGBP-ßG). An ELISA binding assay indicated that rLvLGBP binds to LPS, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE with dissociation constants of 0.1138-0.1770 µM. Furthermore, our results also indicated that the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE significantly increased by 328%, 172%, 200%, 213%, 197%, 194%, 191%, and 197%, respectively compared to controls (cacodylate buffer). We conclude that LvLGBP functions as a PRP, recognizes and binds to LPS, ßG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.

Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV.

Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

BACKGROUND: Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. RESULTS: Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1ß, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. CONCLUSIONS: Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.

Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity-related complement pathway in Epinephelus coioides.

BACKGROUND: Orange-spotted grouper (Epinephelus coioides) with protogynous hermaphroditic features are one of the most economically important aquaculture species in Taiwan. However, larvae stage grouper are susceptible to infection by the bacterial pathogen Vibrio alginolyticus. To better understand the molecular mechanisms of the immune response to V. alginolyticus in Epinephelus coioides larvae, we used high-throughput deep sequencing technology to study the effect of infection on gene expression. RESULTS: A total of 114,851,002 reads were assembled, consisting of 9,687,355,560 nucleotides; these were further assembled into 209,082 contigs with a mean length of 372 bp. Gene ontology (GO) analysis of the transcriptome revealed 12 cellular component subcategories, 16 molecular function subcategories, and 42 biological process subcategories (P value <0.05). A total of 32664 Epinephelus coioides genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG); 1504 differentially expressed genes (DEGs) were subsequently identified, in 12 categories (P value <0.05). Vibrio infection affected the expression of genes involved in complementation, coagulation cascades, pathogen (Staphylococcus aureus) infection, phagosome activity, antigen processing, and the antigen presentation pathway. CONCLUSION: We conclude that the complement pathway of innate immunity and the hepicidin antimicrobial peptide may play important roles in the defense of Epinephelus coioides larvae against V. alginolyticus, and the immune response may activate at 4 h after bacterial infection. These results implicate the complement pathway signal pathway in immunity during V. alginolyticus infection at early developmental stages, enhancing our understanding of the mechanisms underlying the immune response to Vibrio infection in Epinephelus coioides.