Complete mitochondrial genome of critically endangered catfish Hemibagrus punctatus (Jerdon, 1849) and comparative analysis for insights into the phylogeny of hemibagrids through mitogenomic approach.

BACKGROUND: Hemibagrus punctatus (Jerdon, 1849) is a critically endangered bagrid catfish endemic to the Western Ghats of India, whose population is declining due to anthropogenic activities. The current study aims to compare the mitogenome of H. punctatus with that of other Bagrid catfishes and provide insights into their evolutionary relationships. METHODS AND RESULTS: Samples were collected from Hemmige Karnataka, India. In the present study, the mitogenome of H. punctatus was successfully assembled, and its phylogenetic relationships with other Bagridae species were studied. The total genomic DNA of samples was extracted following the phenol-chloroform isoamyl alcohol method. Samples were sequenced, and the Illumina paired-end reads were assembled to a contig length of 16,517 bp. The mitochondrial genome was annotated using MitoFish and MitoAnnotator (Iwasaki et al., 2013). A robust phylogenetic analysis employing NJ (Maximum composite likelihood) and ASAP methods supports the classification of H. punctatus within the Bagridae family, which validates the taxonomic status of this species. In conclusion, this research enriches our understanding of H. punctatus mitogenome, shedding light on its evolutionary dynamics within the Bagridae family and contributing to the broader knowledge of mitochondrial genes in the context of evolutionary biology. CONCLUSIONS: The study's findings contribute to a better understanding of the mitogenome of H. punctatus and provide insights into the evolutionary relationships within other Hemibagrids.

Development of novel microsatellite markers for population differentiation and detection of natural selection in wild populations of butter catfish, Ompok bimaculatus (Bloch, 1794).

BACKGROUND: Butter catfish (Ompok bimaculatus) is a preferred species in South East Asia, with huge aquaculture potential. However, there is limited information about genetic stock composition due to insufficient markers. The goal of this study was to develop de novo microsatellite markers. METHODS AND RESULTS: For sequencing, genomic SMRT bell libraries (1.5 Kbp size) were prepared for O. bimaculatus. A total of 114 SSR containing sequences were used for primer designing. Polymorphic loci were validated by genotyping 83 individuals from four distant riverine populations, viz., Brahmaputra, Bichiya, Gomti and Kaveri. A total of 30 microsatellite loci were polymorphic, of which five were found to be associated with functional genes and eight (four positive and four negative) loci were found to be under selection pressure. A total of 115 alleles were detected in all loci and PIC ranged from 0.539 to 0.927 and pair-wise FST values from 0.1267 to 0.26002 (p < 0.001), with an overall FST value of 0.17047, indicating the presence of population sub-structure. Cross-species transferability of 29 loci (96.67%) was successful in congener species, Ompok pabda. CONCLUSION: The novel SSR markers developed in this study would facilitate stock characterization of natural populations, to be used in future selection breeding programs and planning conservation strategies in these species. Identified non-neutral markers will give insights into the effect of local adaptation on genetic differentiation in the natural population of this species.

Transcriptome Analysis Revealed Osmoregulation Related Regulatory Networks and Hub Genes in the Gills of Hilsa shad, Tenualosa ilisha, during the Migratory Osmotic Stress.

Tenualosa ilisha (Hilsa shad), an anadromous fish, usually inhabits coastal and estuarine waters, and migrates to freshwater for spawning. In this study, large-scale gill transcriptome analyses from three salinity regions, i.e., fresh, brackish and marine water, revealed 3277 differentially expressed genes (DEGs), out of which 232 were found to be common between marine vs freshwater and brackish vs freshwater. These genes were mapped into 54 KEGG Pathways, and the most significant of these were focal adhesion, adherens junction, tight junction, and PI3K-Akt signaling pathways. A total of 24 osmoregulatory genes were found to be differentially expressed in different habitats. The gene members of slc16 and slc2 families showed a dissimilar pattern of expressions, while two claudin genes (cldn11 & cldn10), transmembrane tm56b, and voltage-gated potassium channel gene kcna10 were downregulated in freshwater samples, as compared to that of brackish and marine environment. Protein-protein interaction (PPI) network analysis of 232 DEGs showed 101 genes to be involved in PPI, while fn1 gene was found to be interacting with the highest number of genes (36). Twenty-five hub genes belonged to 12 functional groups, with muscle structure development with seven genes, forming the major group. These results provided valuable information about the genes, potentially involved in the molecular mechanisms regulating water homeostasis in gills, during migration for spawning and low-salinity adaptation in Hilsa shad. These genes may form the basis for the bio-marker development for adaptation to the stress levied by major environmental changes, due to hatchery/culture conditions.

Transcriptome analysis revealed hub genes for muscle growth in Indian major carp, Catla catla (Hamilton, 1822).

Catla (Catla catla) is the fastest growing Indian major carp species and forms an important component of the freshwater aquaculture systems in the Indian sub-continent. The molecular mechanisms of growth of the species in response to seasonal water temperature variations hitherto are still unknown. In the current study, high-throughput transcriptome sequencing was used to study the differential gene expression pattern in catla muscle tissues between pre-winter and post-winter fingerling groups and fast-growing table size fish. Transcriptome analysis identified 1677 differentially expressed genes (DEGs) in three different growth stages and 236 common DEGs between fingerling at low temperature and table fish post-winter, including four genes under GH/IGF1 axis and 163 genes under signature for compensatory muscle growth. Molecular pathways for the mapped genes identified 42 KEGG pathways and the critical pathways under Environmental Information Processing identified were PI3K-Akt signaling, AMPK signaling pathway, Calcium signaling pathway and MAPK signaling pathway. In this study, 14 differentially expressed potential regulatory hub genes for growth were identified, for the first time and categorized into three major GO groups: unfolded protein binding, rNA processing and biogenesis and muscle development and differentiation. These regulatory hub genes, except acta1, were found to be upregulated in fast-growing table size and post-winter fingerling groups. The results provided valuable information about the key genes, with potential to be used as biomarkers of growth in breeding programs and contributed to our understanding of the molecular mechanisms and pathways regulating muscle growth, in response to temperature fluctuations and different growth stages in C. catla.

Discovery of alternatively spliced isoforms and long non-coding RNA in full length brain transcriptomes of anadromous Hilsa shad, Tenualosa ilisha (Hamilton, 1822).

BACKGROUND: Full length transcriptomes, achieved through long-read sequencing, along with the isoform analysis can reveal complexities in the gene expression profiles, as well as annotate the transcriptomes of non-model organisms. METHODS AND RESULT: Full length transcripts of brain transcriptome of Tenualosa ilisha, Hilsa shad, were generated through PacBio single molecule real-time sequencing and were characterized. A total of 8.30 Gb clean reads were generated, with PacBio RSII, which resulted in 57,651 high quality consensus transcripts. After removing redundant reads, a total of 19,220 high-quality non-redundant transcripts and 17,341 full length ORF transcripts were classified to 7522 putative ortholog groups. Genes involved in various neural pathways were identified. In addition, isoform clusters and lncRNAs were discovered, along with Hilsa specific transcripts with coding frames and 29,147 SSRs in 944 transcripts (1141 annotated). CONCLUSION: The present study provided, for the first time, a comprehensive view of the alternative isoforms of genes and transcriptome complexity in Hilsa shad brain and forms a rich resource for functional studies in brain of this anadromous fish.

Tissue specific alpha-2-Macroglobulin (A2M) splice isoform diversity in Hilsa shad, Tenualosa ilisha (Hamilton, 1822).

The present study, for the first time, reported twelve A2M isoforms in Tenualosa ilisha, through SMRT sequencing. Hilsa shad, T. ilisha, an anadromous fish, faces environmental stresses and is thus prone to diseases. Here, expression profiles of different A2M isoforms in four tissues were studied in T. ilisha, for the tissue specific diversity of A2M. Large scale high quality full length transcripts (>0.99% accuracy) were obtained from liver, ovary, testes and gill transcriptomes, through Iso-sequencing on PacBio RSII. A total of 12 isoforms, with complete putatative proteins, were detected in three tissues (7 isoforms in liver, 4 in ovary and 1 in testes). Complete structure of A2M mRNA was predicted from these isoforms, containing 4680 bp sequence, 35 exons and 1508 amino acids. With Homo sapiens A2M as reference, six functional domains (A2M_N,A2M_N2, A2M, Thiol-ester_cl, Complement and Receptor domain), along with a bait region, were predicted in A2M consensus protein. A total of 35 splice sites were identified in T. ilisha A2M consensus transcript, with highest frequency (55.7%) of GT-AG splice sites, as compared to that of Homo sapiens. Liver showed longest isoform (X1) consisting of all domains, while smallest (X10) was found in ovary with one Receptor domain. Present study predicted five putative markers (I-212, I-269, A-472, S-567 and Y-906) for EUS disease resistance in A2M protein, which were present in MG2 domains (A2M_N and A2M_N2), by comparing with that of resistant and susceptible/unknown response species. These markers classified fishes into two groups, resistant and susceptible response. Potential markers, predicted in T. ilisha, placed it to be EUS susceptible category. Putative markers reported in A2M protein may serve as molecular markers in diagnosis of EUS disease resistance/susceptibility in fishes and may have a potential for inclusion in the marker panel for pilot studies. Further, challenging studies are required to confirm the role of particular A2M isoforms and markers identified in immune protection against EUS disease.

Character based identification system for Elasmobranchs for conservation and forensic applications.

The term Elasmobranchs or Chondrichthyans refer to the group of marine organisms include sharks, skates, rays and chimeras. Due to high demand most of the Elasmobranch species depleted drastically in recent years. Being the apex predators in the marine environment most of the Chondrichthyan species are vulnerable with some risk of extinction. Accurate identification of species is prerequisite for conservation and management of these declining resources. In this context, character-based identification methods are of immense use which classify specimen to species using classification rules that compactly characterized species in terms of key diagnostic nucleotides in the selected gene sequences. In the present study, a total of 82 species of elasmobranches distributed in Indian waters is taken as the target group. A total of 20-30 mitochondrial CO1 sequences of each species were downloaded and modified. Using the BLOG2.0 software, species specific diagnostic nucleotides were identified for the selected group of species. Based on identified diagnostic nucleotide sites, species-specific probes with a length range of 23-30 bp were designed. These probes could be useful in detecting species of interest in customized microarrays (DNA chip). The technique is a powerful tool to obtain a significant amount of accurate information quickly and conveniently and could be used for forensic applications and conservation of elasmobranches in Indian waters.

First report on vertical transmission of a plasmid DNA in freshwater prawn, Macrobrachium rosenbergii.

Outbreak of WSSV disease is one of the major stumbling blocks in shrimp aquaculture. DNA vaccines have shown potential for mass scale vaccination owing to their stability, cost effectiveness and easy maintenance. Development of economically feasible delivery strategies remains to be a major challenge. This study demonstrates vertical transmission of a plasmid DNA in a decapod Macrobrachium rosenbergii for the first time. Females at three different maturation stages (immature, matured and berried) and mature males were injected with a plasmid DNA and allowed to spawn with untreated counterparts. Using specific primers the plasmid DNA could be amplified from the offspring of all groups except that of berried females. For this confirmation genomic DNA was isolated from 3 pools of 10 post larvae in each group. This presents an ideal strategy to protect young ones at zero stress.