DNA Methylation Profiling of Ovarian Tissue of Climbing Perch (Anabas testudienus) in Response to Monocrotophos Exposure.

Epigenetic modifications like DNA methylation can alter an organism's phenotype without changing its DNA sequence. Exposure to environmental toxicants has the potential to change the resilience of aquatic species. However, little information is available on the dynamics of DNA methylation in fish gonadal tissues in response to organophosphates. In the present work, reduced-representation bisulfite sequencing was performed to identify DNA methylation patterns in the ovarian tissues of Anabas testudienus exposed to organophosphates, specifically monocrotophos (MCP). Through sequencing, an average of 41,087 methylated cytosine sites were identified and distributed in different parts of genes, i.e., in transcription start sites (TSS), promoters, exons, etc. A total of 1058 and 1329 differentially methylated regions (DMRs) were detected as hyper-methylated and hypo-methylated in ovarian tissues, respectively. Utilizing whole-genome data of the climbing perch, the DMRs, and their associated overlapping genes revealed a total of 22 genes within exons, 45 genes at transcription start sites (TSS), and 218 genes in intergenic regions. Through gene ontology analysis, a total of 16 GO terms particularly involved in ovarian follicular development, response to oxidative stress, oocyte maturation, and multicellular organismal response to stress associated with reproductive biology were identified. After functional enrichment analysis, relevant DMGs such as steroid hormone biosynthesis (Cyp19a, 11-beta-HSD, 17-beta-HSD), hormone receptors (ar, esrrga), steroid metabolism (StAR), progesterone-mediated oocyte maturation (igf1ar, pgr), associated with ovarian development in climbing perch showed significant differential methylation patterns. The differentially methylated genes (DMGs) were subjected to analysis using real-time PCR, which demonstrated altered gene expression levels. This study revealed a molecular-level alteration in genes associated with ovarian development in response to chemical exposure. This work provides evidence for understanding the relationship between DNA methylation and gene regulation in response to chemicals that affect the reproductive fitness of aquatic animals.

Revealing Alteration in the Hepatic Glucose Metabolism of Genetically Improved Carp, Jayanti Rohu Labeo rohita Fed a High Carbohydrate Diet Using Transcriptome Sequencing.

Although feed cost is the greatest concern in aquaculture, the inclusion of carbohydrates in the fish diet, and their assimilation, are still not well understood in aquaculture species. We identified molecular events that occur due to the inclusion of high carbohydrate levels in the diets of genetically improved 'Jayanti rohu' Labeo rohita. To reveal transcriptional changes in the liver of rohu, a feeding experiment was conducted with three doses of gelatinized starch (20% (control), 40%, and 60%). Transcriptome sequencing revealed totals of 15,232 (4464 up- and 4343 down-regulated) and 15,360 (4478 up- and 4171 down-regulated) differentially expressed genes. Up-regulated transcripts associated with glucose metabolisms, such as hexokinase, PHK, glycogen synthase and PGK, were found in fish fed diets with high starch levels. Interestingly, a de novo lipogenesis mechanism was found to be enriched in the livers of treated fish due to up-regulated transcripts such as FAS, ACCα, and PPARγ. The insulin signaling pathways with enriched PPAR and mTOR were identified by Kyoto Encyclopedia of Genes and Genome (KEGG) as a result of high carbohydrates. This work revealed for the first time the atypical regulation transcripts associated with glucose metabolism and lipogenesis in the livers of Jayanti rohu due to the inclusion of high carbohydrate levels in the diet. This study also encourages the exploration of early nutritional programming for enhancing glucose efficiency in carp species, for sustainable and cost-effective aquaculture production.

An in vitro refolding method to produce oligomers of anti-CHIKV, E2-IgM Fc fusion subunit vaccine candidates expressed in E. coli.

Recombinant envelope protein-1 (E1) and E2 of Chikungunya virus (CHIKV) has been shown to elicit neutralizing antibodies and a balanced Th1/Th2 response in mice however with limited protection. Recently reported CHIK virus-like particles showed augmented immunity and protection in adult mice in comparison to E1 and E2, however exacerbated the disease in aged subjects. In order to improve the overall efficacy of protein based vaccines, novel strategies need to be adopted. The discovery of IgM Fc receptor (FcµR) and its role in humoral immune response led us to hypothesise that fusion of an antigen with Fc of IgM may enhance its immunogenicity by polymerizing it and FcµR mediated activation of B and other immune cells. We report in the current study, expression of E2 subunit of CHIKV in fusion with various IgM Fc domains/peptides in E. coli, their in-vitro refolding, characterization and immune response in C57BL/6 mice. Candidates fused with CH3-CH4 Fc fragment produced stable oligomers, whereas the one fused with peptides remained monomeric. The latter elicited a strong humoral and a balanced Th1/Th2 response in mice, whereas the polymeric candidate despite eliciting a strong humoral response, stimulated a biased Th1 response and exhibited higher virus neutralization in Vero cells.

Revealing liver specific microRNAs linked with carbohydrate metabolism of farmed carp, Labeo rohita (Hamilton, 1822).

The role of microRNA in gene regulation during developmental biology has been well depicted in several organisms. The present study was performed to investigate miRNAs role in the liver tissues during carbohydrate metabolism and their targets in the farmed carp rohu, Labeo rohita, which is economically important species in aquaculture. Using Illumina-HiSeq technology, a total of 22,612,316; 44,316,046 and 13,338,434 clean reads were obtained from three small-RNA libraries. We have identified 138 conserved and 161 novel miRNAs and studies revealed that miR-22, miR-122, miR-365, miR-200, and miR-146 are involved in carbohydrate metabolism. Further analysis depicted mature miRNA and their predicted target sites in genes that were involved in developmental biology, cellular activities, transportation, etc. This is the first report of the presence of miRNAs in liver tissue of rohu and their comparative profile linked with metabolism serves as a vital resource as a biomarker.

Liver-Specific microRNA Identification in Farmed Carp, Labeo bata (Hamilton, 1822), Fed with Starch Diet Using High-Throughput Sequencing.

The liver is an important central organ, which controls carbohydrate metabolism through maintaining glucose homeostasis by a tightly regulated system of genes or enzymes. The microRNAs are small non-coding RNAs playing an important role in the regulation of genes associated with developmental biology, physiology, metabolism, etc. Thus, in this study, we have intended to detect liver-specific microRNAs in farmed carp, Labeo bata, upon being fed a diet with different levels of carbohydrates. Here, we have conducted the experiment for 45 days using fingerlings of farmed carp fed with 20% (control), 40%, and 60% gelatinized starch levels. The liver tissues were collected from each treatment and processed for RNA isolation, small RNA library preparation, and high-throughput sequencing using Illumina NexSeq500. Through sequencing, 15,779,417 reads in 20% CHO, 13,959,039 in 40% CHO, and 13,661,950 in 60% CHO reads were generated for control and treated fishes using three small RNA libraries. We have investigated 445 novel and 231 conserved microRNAs in 20%, 40%, and 60% carbohydrate (CHO), respectively, through computational analysis. The differential expression analysis of miRNAs was carried out between different treatments compared with control and this study depicted 117 known and 114 novel miRNA genes involved in carbohydrate metabolic pathways. Further, target prediction and gene ontology analysis revealed that miRNAs were involved in several pathways such as signaling pathway, G protein pathway, complement receptor-mediated pathway, dopamine receptor signaling pathway, epidermal growth factor pathway, and notch signaling pathway. The predicted miRNA sites in targeted genes were associated with cellular activities, developmental biology, DNA binding, Golgi apparatus, extracellular region, catalytic activity, MAPK cascade, etc. Overall, we have generated a vital resource of liver-specific miRNAs involved in metabolic gene regulation. These studies further will help develop miRNA inhibitors to study their role during carbohydrate metabolism in farmed carp.