Rajeev K. Singh

Hypoxia induced altered expression of heat shock protein genes (Hsc71, Hsp90α and Hsp10) in Indian Catfish, Clarias batrachus (Linnaeus, 1758) under oxidative stress

Heat shock proteins (Hsps) are typically associated with stress response and tolerance. The Indian catfish, Clarias batrachus, is a freshwater air-breathing hypoxia tolerant teleost and is potentially important catfish species for aquaculture and for its economic value as food. The present study aimed at determining the transcriptional response of three Hsps, CbHsc71, CbHsp90α and CbHsp10 in hypoxia tolerant Indian catfish, C. batrachus, under experimental and natural hypoxia. The expression profile of above three genes were studied under different periods of hypoxia, through qRT-PCR. Primers were designed from ESTs obtained through SSH libraries constructed from hypoxia treated fishes. The Hsp10 ESTs and deduced protein was in silico characterized for its ORF and for its physical and chemical properties, respectively, using GeneScan, blastp, scanprosite, superfamily and other softwares. A phylogenetic tree was constructed based on deduced amino acid sequences of Hsc71, Hsp90α, Hsp90β of Homo sapiens and other fishes along with CbHsp10 protein in MEGA4. The deduced protein sequences of CbHsp10 was found to have characteristic Hsp10 family signatures, and it is proposed for inclusion of methionine in the consensus sequences of Hsp10 family signature, after the “proline” residue. At transcription level, these genes were found to be differentially regulated under hypoxia stress, in different tissues of C. batrachus. The CbHsc71 and CbHsp90α were up-regulated after short and long-term hypoxia, whereas CbHsp10 was significantly down-regulated after short-term hypoxia. The differential expression of these Hsps may play a role in protection and survival under hypoxia induced oxidative stress in C. batrachus.

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN + I + G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN + I + G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN + I + G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN + I + G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN + I + G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN + I + G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN + I + G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN + I + G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN + I + G 0.022 to 0.025) shared the same lineage with T. putitora (TrN + I + G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.

Evaluation of Genetic Variation in the Clown Knifefish, Chitala chitala, Using Allozymes, RAPD, and Microsatellites

Twenty-seven enzyme systems, six random amplified polymorphic DNA (RAPD) primers, and two microsatellite loci were tested to determine intraspecific divergence in the natural population of the endangered Indian featherback fish, Chitala chitala, for the first time. The 262 samples of C. chitala were collected from six riverine locations in India: the Satluj, Ganga (Ghagra, Bhagirathi, and Brahmaputra), Mahanadi, and Narmada river systems. The analysis revealed population subdivisions, with an FST value from 0.1235 (95% confidence 0.0868–0.1621) for RAPD and a combined FST of 0.0344 (95% confidence 0.0340–0.0350) for microsatellite loci. An analysis of 38 allozyme loci did not reveal any polymorphism in the samples from any of the riverine localities; a possible explanation for this could be that the ancestors of Chitala could have faced a population reduction in prehistoric periods, as low allozyme variation is also reported for other species of Chitala from south Asia.

Complete mitochondrial genome sequences of two endangered Indian catfish species, Clarias batrachus and Pangasius pangasius.

Abstract Complete mitogenome sequences for Clarias batrachus (magur) and Pangasius pangasius (family Claridae and Pangasiidae, respectively) were generated, which were 16,511 and 16,476 bp, respectively. Both the mitogenomes contained 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs and 1 non-coding (control) region. The order of the genes was identical to that observed in most other vertebrates. The genome resource of complete mitogenome sequence of Indian catfish species generated during this study would be useful in phylogenetic studies.

Identification of candidate reference genes for quantitative expression analysis by real-time PCR for hypoxic stress in Indian catfish, Clarias batrachus (Linnaeus, 1758)

The selection of most appropriate reference gene(s) is a crucial step in studies quantifying gene expression by quantitative real-time PCR (qPCR). As a need reference gene(s) should be unaffected at transcription level by experimental conditions and/or tissue types. In this study, 11 candidate reference genes were tested in Clarias batrachus for their expression stability using the geNorm, NormFinder and BestKeeper statistical algorithms, and were compared in different tissues (brain, heart, liver, muscle, spleen and head kidney) and treatments (before and after hypoxia exposure). The results indicated that in brain, heart, liver, muscle, spleen and head kidney, respectively, 28S rRNA/TUB, 28S rRNA/TUB, RPL30/28S rRNA, RPL30/TUB, ELF-1A/28S rRNA and ELF-1A/TUB gene pairs were highly stable and were suitable as reference genes to study oxidative stress, while ACTB and B2M were the least stable genes in examined tissues under normoxic and/or hypoxic conditions. The observations suggested the consideration of tissue types and use of at least two reference genes, instead of one in accurate normalization of qPCR data.

Molecular characterization and expression analysis of PPP1R3C in hypoxia-tolerant Indian catfish, Clarias batrachus (Linnaeus, 1758) under hypoxia

Hypoxia is an important environmental stressor that leads to rapid adaptive changes in metabolic organization. However, the molecular mechanisms of hypoxia tolerance in fish remain largely unknown. The present work was focused on understanding the molecular mechanisms and signaling pathways that may lead to tolerance of Clarias batrachus to hypoxic stress. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is a new hypoxia-inducible factor (HIF) targeted gene and is regulated by HIF-1 under hypoxic conditions. Overexpression of PPP1R3C increases glycogen accumulation through activation of several enzymes and processes. In this study, for the first time, full length cDNA of PPP1R3C from C. batrachus was characterized and its expression pattern in the brain, liver, muscle and spleen under short (progressive hypoxia; PH, 1h, 6h and 12h) and long-term (natural) hypoxic conditions was investigated. The complete cDNA of PPP1R3C was of 1499 bp, encoding 285 amino acid residues. The identified protein had a protein phosphatase 1 binding motif and a carbohydrate binding domain, thought to be involved in the regulation of glycogen metabolism. Short-term hypoxia exposure caused significant increase in PPP1R3C transcripts in the liver (6h; 6.96 fold and 12h; 3.91 fold) and muscle (progressive hypoxia; 3.46 fold), while, after long-term hypoxia exposure, significant up-regulation in the liver (7.77 fold) and spleen (6.59 fold) tissues was observed. No significant differences were observed in the brain for any time periods. Thus PPP1R3C may play an important role in the tolerance of C. batrachus to hypoxia.

Complete sequence and characterization of mitochondrial genome in great snakehead, Channa marulius (Hamilton, 1822)

Abstract The complete nucleotide sequence of mitogenome of the great snakehead, Channa marulius (Channidae), was determined and found to be 16,569 base pairs in length. The content and arrangement of different genes on the mitogenome was found similar to other typical teleosts. The overall base composition of the L-strand was found to be T (19.1%), C (31.5%), A (34.8%) and G (14.6%). The control region was 915 nt long and without any repetitive region. The mitogenome sequence data would be useful for studying phylogenetic relationship of C. marulius with other perciform species.

Isolation and characterization of polymorphic microsatellite loci in yellowtail catfish, Pangasius pangasius (Hamilton, 1822).

A total of nine polymorphic microsatellite loci were obtained from a genomic library of Pangasius pangasius (order Siluriformes, family Pangasiidae). Samples from rivers Bhagirathi (n = 22) and Mahanadi (n = 20) were genotyped for each of the nine microsatellite loci to determine genetic variation. The mean number of alleles per locus was 5.22 in Bhagirathi and 5.78 in Mahanadi; and expected heterozygosity ranged from 0.567 (Bhagirathi) to 0.578 (Bhagirathi). Significant deviation (P < 0.003) from Hardy–Weinberg expectations was evident at three loci, Ppa2 (Bhagirathi), Ppa14 (Mahanadi) and Ppa28 (Bhagirathi and Mahanadi). The identified microsatellite loci were found to be promising for population genetics studies of P. pangasius.

Population genetic structure of Macrobrachium rosenbergii (Palaemonidae) from Indian waters using mitochondrial ATPase 6/8 gene.

Abstract Macrobrachium rosenbergii, giant freshwater prawn, is one of the most commercially important crustaceans. In the present study, primers for ATPase 6/8 region of mt-DNA were designed and successfully amplified (827 bp) in the species. The nucleotide variation in ATPase 6/8 gene revealed the population structuring in natural populations of M. rosenbergii in Indian waters. A total of 35 haplotypes were observed in 93 individuals collected from different locations. Low nucleotide diversity and high haplotype diversity were noticed for the ATPase 6/8 gene. Significant pairwise FST and, haplotype network indicated occurrence of distinct populations. Observed mismatch distribution and Tajima’s D test suggested demographical stability of giant freshwater prawn. The genetic stock structure revealed in this study will be helpful for conservation and management of stocks of M. rosenbergii in Indian waters.

Ion torrent next-generation sequencing reveals the complete mitochondrial genome of endangered mahseer Tor khudree (Sykes, 1839)

Abstract The complete mitochondrial genome of an endangered mahseer (Deccan mahseer), Tor khudree was sequenced using Ion torrent platform for the first time. The genome sequence was 16 573 bp in size, and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and 1 control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A: 31.9%, G: 15.6%, C: 27.68%, T: 24.76%, A + T content 56.6% and the G + C content 43.32%. The phylogenetic tree constructed using a maximum likelihood model showed sister relationship between T. khudree and Tor tambroides.