Betaine Alleviates High-Fat Diet Induced Excessive Lipid Deposition in Gibel Carp Hepatopancreas and L8824 Cells by Enhancing VLDL Secretion through HNF4α/MTTP Pathway.

Betaine, a methyl donor, plays a crucial role in lipid metabolism. Previous studies have shown that appropriate betaine supplementation in a high-fat diet reduces triglycerides (TG) of serum and hepatopancreas in fish. However, the underlying mechanism remains unclear. This study examined whether betaine can enhance the secretion of very low-density lipoprotein (VLDL) and sought to identify the specific mechanisms through which this enhancement occurs. A lipid accumulation model was established in gibel carp and L8824 cells using a high-fat diet and oleic acid, respectively. Different doses of betaine (1, 4, and 16 g/kg in the diet; 400 µmol in cell culture) were administered, and measurements were taken for lipid deposition, gene expression of HNF4α, MTTP, and ApoB, as well as the regulation of Mttp and Apob promoters by HNF4α. The results showed that betaine supplementation mitigated lipid droplet accumulation, TG levels, and VLDL production induced by the high-fat diet in gibel carp hepatopancreas and L8824 cells. Moreover, betaine not only increased VLDL content in the cell culture supernatant but also reversed the inhibitory effects of the high-fat diet on protein expression of MTTP, ApoB, and HNF4α in both gibel carp hepatopancreas and L8824 cells. Additionally, HNF4α exhibits transactivating activity on the promoter of Mttp in gibel carp. These findings suggest that betaine supplementation exerts its effects through the HNF4α/MTTP/ApoB pathway, promoting the assembly and secretion of VLDL and effectively reducing lipid accumulation in the hepatopancreas of farmed gibel carp fed a high-fat diet.

Full-Length RNA Sequencing Provides Insights into Goldfish Evolution under Artificial Selection.

Goldfish Carassius auratus is an ideal model for exploring fish morphology evolution. Although genes underlying several ornamental traits have been identified, little is known about the effects of artificial selection on embryo gene expression. In the present study, hybrid transcriptome sequencing was conducted to reveal gene expression profiles of Celestial-Eye (CE) and Ryukin (RK) goldfish embryos. Full-length transcriptome sequencing on the PacBio platform identified 54,218 and 54,106 transcript isoforms in CE and RK goldfish, respectively. Of particular note was that thousands of alternative splicing (AS) and alternative polyadenylation (APA) events were identified in both goldfish breeds, and most of them were inter-breed specific. RT-PCR and Sanger sequencing showed that most of the predicted AS and APA were correct. Moreover, abundant long non-coding RNA and fusion genes were detected, and again most of them were inter-breed specific. Through RNA-seq, we detected thousands of differentially expressed genes (DEGs) in each embryonic stage between the two goldfish breeds. KEGG enrichment analysis on DEGs showed extensive differences between CE and RK goldfish in gene expression. Taken together, our results demonstrated that artificial selection has led to far-reaching influences on goldfish gene expression, which probably laid the genetic basis for hundreds of goldfish variations.

The Impact of Rearing Salinity on Flesh Texture, Taste, and Fatty Acid Composition in Largemouth Bass Micropterus salmoides.

It is of great significance for the aquaculture industry to determine how rearing salinity impacts fish flesh quality. In the present study, largemouth bass was cultured in different salinities (0%, 0.3%, 0.9%) for 10 weeks, and the effect on flesh texture, flavor compounds, taste, and fatty acid composition was evaluated. We show that rearing salinity not only increased flesh water-holding capacity, but also enhanced muscle hardness, chewiness, gumminess, and adhesiveness, which was consistent with the finding in the shear value test. Morphology analysis further revealed that the effect of salinity on flesh texture was probably related to changes in myofibril diameter and density. As for the taste of the flesh, water salinity improved the contents of both sweet and umami amino acids, and reduced the contents of bitter amino acid. Meanwhile, the content of IMP, the dominant flavor nucleotide in largemouth bass muscle, was significantly higher in the 0.9% group. Interestingly, electronic-tongue analysis demonstrated that the positive effect of salinity on flavor compounds enhanced the umami taste and taste richness of flesh. Moreover, rearing salinity improved the contents of C20: 5n-3 (EPA) and C22: 6n-3 (DHA) in back muscle. Therefore, rearing largemouth bass in adequate salinity may be a practical approach to improving flesh quality.

Seawater Culture Increases Omega-3 Long-Chain Polyunsaturated Fatty Acids (N-3 LC-PUFA) Levels in Japanese Sea Bass (Lateolabrax japonicus), Probably by Upregulating Elovl5.

The fatty acid compositions of the fish muscle and liver are substantially affected by rearing environment. However, the mechanisms underlying this effect have not been thoroughly described. In this study, we investigated the effects of different culture patterns, i.e., marine cage culture and freshwater pond culture, on long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis in an aquaculturally important fish, the Japanese sea bass (Lateolabrax japonicus). Fish were obtained from two commercial farms in the Guangdong province, one of which raises Japanese sea bass in freshwater, while the other cultures sea bass in marine cages. Fish were fed the same commercial diet. We found that omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) levels in the livers and muscles of the marine cage cultured fish were significantly higher than those in the livers and muscles of the freshwater pond cultured fish. Quantitative real-time PCRs indicated that fatty acid desaturase 2 (FADS2) transcript abundance was significantly lower in the livers of the marine cage reared fish as compared to the freshwater pond reared fish, but that fatty acid elongase 5 (Elovl5) transcript abundance was significantly higher. Consistent with this, two of the 28 CpG loci in the FADS2 promoter region were heavily methylated in the marine cage cultured fish, but were only slightly methylated in freshwater pond cultured fish (n = 5 per group). Although the Elovl5 promoter was less methylated in the marine cage reared fish as compared to the freshwater pond reared fish, this difference was not significant. Thus, our results might indicate that Elovl5, not FADS2, plays an important role in the enhancing LC-PUFA synthesis in marine cage cultures.

Draft genome and SNPs associated with carotenoid accumulation in adductor muscles of bay scallop (Argopecten irradians).

Carotenoids are commonly deposited in the gonads of marine bivalves but rarely in their adductor muscles. An orange-adductor variant was identified in our breeding program for the bay scallop Argopecten irradians. In the present study, bay scallop genome survey sequencing was conducted, followed by genotyping by sequencing (GBS)-based case-control association analysis in a selfing family that exhibited segregation in adductor color. K-mer analysis (K=17) revealed that the bay scallop genome is about 990 Mb in length. De novo assembly produced 217,310 scaffold sequences, which provided 72.1% coverage of the whole genome and covered 72,187 transcripts, thereby yielding the most informative sequence resource for bay scallop to date. The average carotenoid content of the orange-adductor progenies was significantly higher than that of the white-adductor progenies. Thus, 20 individuals of each subgroup were sampled for case-control analysis. As many as 15,224 heterozygous loci were identified in the parent, among which 9280 were genotyped in at least 10 individuals of each of the two sub-groups. Association analysis indicated that 126 SNPs were associated with carotenoid accumulation in the adductor muscle and that 88 of these were significantly enriched on 28 scaffolds (FDR controlled P < 0.05). The SNPs and genes located on these scaffolds can serve as valuable candidates for further research into the mechanisms by which marine bivalves accumulate carotenoids in their adductor muscles.

Comparative analysis of the survival and gene expression of pathogenic strains Vibrio harveyi after starvation.

This study aimed to evaluate the survival and gene expression of Vibrio harveyi under starvation conditions. The microcosms V. harveyi were incubated in sterilized seawater for 4 weeks at room temperature. Overall, the cell numeration declined rapidly about 103 CFU/ml during starvation, with a tiny rebound at day 21. Scanning electron microscopy revealed that rod-shaped cells became sphere with a rippled cell surface. By polymerase chain reaction (PCR) assay, nine genes, named luxR, toxR, vhhB, flaA, topA, fur, rpoS, mreB and ftsZ, were detected in the non-starved cells. In the starved cells, the expression levels of the detected genes declined substantially ranging from 0.005-fold to 0.028-fold compared to the non-starved cells performed by reverse transcription quantitative real-time PCR with 16S rRNA as the internal control. In the recovering cells, the expression levels of the detected genes, except luxR and mreB, were upregulated dramatically compared to the wild, especially topA (23.720-fold), fur (39.400-fold) and toxR (9.837-fold), validating that the expressions of both the metabolism and virulence genes were important for growth and survival of V. harveyi. The results may shed a new light on understanding of stress adaptation in bacteria.

Passive protection effect of anti-Vibrio anguillarum IgY-encapsulated feed on half-smooth tongue sole (Cynoglossus semilaevi) against V. anguillarum.

Vibrio anguillarum is one of the most harmful pathogens associated with hemorrhage septicemia syndrome in the half-smooth tongue sole (C. semilaevis) due to its high virulence. In this study, we attempted to treat half-smooth tongue sole with anti-V. anguillarum egg yolk powder to elicit a passive immunity directly against V. anguillarum infection. Anti-V. anguillarum IgY was ß-cyclodextrin encapsulated in egg yolk powders as feed, which could avoid antibody inactivation in the gastrointestinal tract of half-smooth tongue sole. The IgY had an inhibiting effect on the infection of V. anguillarum in vitro. The survival rate of half-smooth tongue sole fed with basal diet containing 15% anti-V. anguillarum egg yolk powder was 70% after 7 days post-V. anguillarum challenge (10(7) CFU), which was significantly higher than those fed without anti-V. anguillarum egg yolk powder. As well, the bacterial burden in blood, liver, spleen and kidney was significantly lower in half-smooth tongue sole fed with specific IgY than those fed with non-specific IgY. These results suggested that pathogen-specific IgY may provide a valuable treatment for vibriosis infection and can be a promising food additive.

The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition.

Passive Immune-Protection of Litopenaeus vannamei against Vibrio harveyi and Vibrio parahaemolyticus Infections with Anti-Vibrio Egg Yolk (IgY)-Encapsulated Feed.

Vibrio spp. are major causes of mortality in white shrimp (Litopenaeus vannamei) which is lacking adaptive immunity. Passive immunization with a specific egg yolk antibody (IgY) is a potential method for the protection of shrimp against vibriosis. In this study, immune effects of the specific egg yolk powders (IgY) against both V. harveyi and V. parahaemolyticus on white shrimp were evaluated. The egg yolk powders against V. harveyi and V. parahaemolyticus for passive immunization of white shrimp were prepared, while a tube agglutination assay and an indirect enzyme-linked immunosorbent assay (ELISA) were used for detection of IgY titer. Anti-Vibrio egg yolk was encapsulated by ß-cyclodextrin, which could keep the activity of the antibody in the gastrointestinal tract of shrimp. The results showed that the anti-Vibrio egg powders had an inhibiting effect on V. harveyi and V. parahaemolyticus in vitro. Lower mortality of infected zoeae, mysis, and postlarva was observed in groups fed with anti-Vibrio egg powders, compared with those fed with normal egg powders. The bacterial load in postlarva fed with specific egg powders in seeding ponds was significantly lower than those fed with normal egg powders in seeding ponds. These results show that passive immunization by oral administration with specific egg yolk powders (IgY) may provide a valuable protection of vibrio infections in white shrimp.

RestrictionDigest: A powerful Perl module for simulating genomic restriction digests

Background: Reduced-representation sequencing technology is widely used in genotyping for its economical and efficient features. A popular way to construct the reduced-representation sequencing libraries is to digest the genomic DNA with restriction enzymes. A key factor of this method is to determine the restriction enzyme(s). But there are few computer programs which can evaluate the usability of restriction enzymes in reduced-representation sequencing. SimRAD is an R package which can simulate the digestion of DNA sequence by restriction enzymes and return enzyme loci number as well as fragment number. But for linkage mapping analysis, enzyme loci distribution is also an important factor to evaluate the enzyme. For phylogenetic studies, comparison of the enzyme performance across multiple genomes is important. It is strongly needed to develop a simulation tool to implement these functions. Results: Here, we introduce a Perl module named RestrictionDigest with more functions and improved performance. It can analyze multiple genomes at one run and generate concise comparison of enzyme performance across the genomes. It can simulate single-enzyme digestion, double-enzyme digestion and size selection process and generate comprehensive information of the simulation including enzyme loci number, fragment number, sequences of the fragments, positions of restriction sites on the genome, the coverage of digested fragments on different genome regions and detailed fragment length distribution. Conclusions: RestrictionDigest is an easy-to-use Perl module with flexible parameter settings. With the help of the information produced by the module, researchers can easily determine the most appropriate enzymes to construct the reduced-representation libraries to meet their experimental requirements.

SNP identification by transcriptome sequencing and candidate gene-based association analysis for heat tolerance in the bay scallop Argopecten irradians.

The northern bay scallop Argopecten irradians irradians (Lamarck) and the southern bay scallop Argopecten irradians concentricus (Say) were introduced into China in the 1980s and 1990s, and are now major aquaculture molluscs in China. Here, we report the transcriptome sequencing of the two subspecies and the subsequent association analysis on candidate gene on the trait of heat tolerance. In total, RNA from six tissues of 67 and 42 individuals of northern and southern bay scallops, respectively, were used and 55.5 and 34.9 million raw reads were generated, respectively. There were 82,267 unigenes produced in total, of which 32,595 were annotated. Altogether, 32,206 and 23,312 high-quality SNPs were identified for northern and southern bay scallops, respectively. For case-control analysis, two intercrossed populations were heat stress treated, and both heat-susceptible and heat-resistant individuals were collected. According to annotation and SNP allele frequency analysis, 476 unigenes were selected, and 399 pairs of primers were designed. Genotyping was conducted using the high-resolution melting method, and Fisher's exact test was performed for allele frequency comparison between the heat-susceptible and heat-resistant groups. SNP all-53308-760 T/C showed a significant difference in allele frequency between the heat-susceptible and heat-resistant groups. Notably, considerable difference in allele frequency at this locus was also observed between the sequenced natural populations. These results suggest that SNP all-53308-760 T/C may be related to the heat tolerance of the bay scallop. Moreover, quantitative expression analysis revealed that the expression level of all-53308 was negatively correlated with heat tolerance of the bay scallop.

Goldfish transposase Tgf2 presumably from recent horizontal transfer is active.

Hobo/Activator/Tam3 (hAT) superfamily transposons occur in plants and animals and play a role in genomic evolution. Certain hAT transposons are active and have been developed as incisive genetic tools. Active vertebrate elements are rarely discovered; however, Tgf2 transposon was recently discovered in goldfish (Carassius auratus). Here, we found that the endogenous Tgf2 element can transpose in goldfish genome. Seven different goldfish mRNA transcripts, encoding three lengths of Tgf2 transposase, were identified. Tgf2 transposase mRNA was detected in goldfish embryos, mainly in epithelial cells; levels were high in ovaries and mature eggs and in all adult tissues tested. Endogenous Tgf2 transposase mRNA is active in mature eggs and can mediate high rates of transposition (>30%) when injected with donor plasmids harboring a Tgf2 cis-element. When donor plasmid was coinjected with capped Tgf2 transposase mRNA, the insertion rate reached >90% at 1 yr. Nonautonomous copies of the Tgf2 transposon with large-fragment deletions and low levels of point mutations were also detected in common goldfish. Phylogenetic analysis indicates the taxonomic distribution of Tgf2 in goldfish is not due to vertical inheritance. We propose that the goldfish Tgf2 transposon originated by recent horizontal transfer and maintains a highly native activity.

[Cloning of goldfish hAT transposon Tgf2 and its structure].

The hAT transposon family, including hobo of Drosophila, Ac of maize (Zea mays L.) and Tam3 of snapdragon (Ceratophyllum demersum L.), is proposed to be involved in transposition between genomic DNAs in "cut and paste" patterns. In 1996, a transposon of Tol2, the first autonomous transposon in vertebrate, was identified from the genome of albino medaka fish (Oryzias latipes). Since then, a new transgenic and gene trap system based on Tol2 has been developed and widely used in zebrafish. In this study, we designed gene-specific primers based on the conserved regions of amino acid sequences between medaka Tol2 and maize Ac. Meanwhile, PCR was carried out in 19 fish species or strains including goldfish. Finally, another similar hAT transposon, termed as Tgf2, was identified in the genomes from different strains of goldfish. Goldfish Tgf2 is 4720 bp in length including 4 exons and shares an identity of 97% with medaka Tol2. Distinct differences were observed in the terminal inverted repeat (TIR) and subterminal repeat (STR) regions between Tgf2 and Tol2. In addition, the internal inverted repeat (IIR) region of Tgf2 (1453 bp-2091 bp) tended to form a "+" crossing structure, rather than a stem-loop structure in medaka Tol2. The regions, including TIR, STR, and IIR, were supposed to be closely related to the transposing activities of hAT transposon family members. From these sequence differences, we may expect a probably high activity of goldfish Tgf2 in transposition and it could be further used as a tool for transgenesis and gene trap in aquaculture fish in future.