Identification of differentially expressed genes and SNPs linked to harvest body weight of genetically improved rohu carp, Labeo rohita.

In most of the aquaculture selection programs, harvest body weight has been a preferred performance trait for improvement. Molecular interplay of genes linked to higher body weight is not elucidated in major carp species. The genetically improved rohu carp with 18% average genetic gain per generation with respect to harvest body weight is a promising candidate for studying genes' underlying performance traits. In the present study, muscle transcriptome sequencing of two groups of individuals, with significant difference in breeding value, belonging to the tenth generation of rohu carp was performed using the Illumina HiSeq 2000 platform. A total of 178 million paired-end raw reads were generated to give rise to 173 million reads after quality control and trimming. The genome-guided transcriptome assembly and differential gene expression produced 11,86,119 transcripts and 451 upregulated and 181 downregulated differentially expressed genes (DEGs) between high-breeding value and low-breeding value (HB & LB) groups, respectively. Similarly, 39,158 high-quality coding SNPs were identified with the Ts/Tv ratio of 1.23. Out of a total of 17 qPCR-validated transcripts, eight were associated with cellular growth and proliferation and harbored 13 SNPs. The gene expression pattern was observed to be positively correlated with RNA-seq data for genes such as myogenic factor 6, titin isoform X11, IGF-1 like, acetyl-CoA, and thyroid receptor hormone beta. A total of 26 miRNA target interactions were also identified to be associated with significant DETs (p-value < 0.05). Genes such as Myo6, IGF-1-like, and acetyl-CoA linked to higher harvest body weight may serve as candidate genes in marker-assisted breeding and SNP array construction for genome-wide association studies and genomic selection.

A novel myxozoan parasite, Ellipsomyxa boleophthalmi sp. nov. (Myxozoa: Ceratomyxidae) in the brackishwater fish, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae) from India.

A novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0-10.7 × 6.0-7.8 µm. The two, spherical polar capsules measured 2.7 µm in diameter and enclosed 3-4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri.

Transcriptome analysis of Clarias magur brain and gonads suggests neuro-endocrine inhibition of milt release from captive GnRH-induced males.

Transcriptome analysis of Clarias magur brain and gonads at preparatory, mature, 6 and 16 h post-GnRH injection (hpi) stages yielded 9.5 GB data with 39,738 contigs. Sequences of 45 reproductive genes were identified for the first time in C. magur along with unique and differentially expressed genes. The expression of 20 genes was validated by qRT-PCR. Upregulation of Cyp11A1, Cyp17A1 and FTZF1 genes in the 16hpi testis accompanied by the 17ß-HSD3 expression indicates testosterone (T) synthesis in response to LH surge, while reduced expression of CYP11B1 suggests a high T: 11-KT ratio. It is evident by the gene expression analysis that the inhibitory neurotransmitter GABA, altered T: 11-KT, increased testicular bile acids, and oxytocin-like neuropeptide in the male brain, appear to be involved in arresting the pulsatile motion of testicular smooth muscles. The work generates important leads for an effective induced breeding strategy for silurid catfish.

Establishment of taxonomic status of Macrobrachium indicum Jayachandran amp; Joseph (Decapoda : Palaemonidae) through molecular characterization with a note on related species.

Macrobrachium indicum, a south Indian freshwater prawn described from Vellayani lake, Kerala is closely related to Koua river prawn, M. australe which in turn resembles M. ustulatum. Morphologically the three species pose difficulties in identification. A comparison of morphological characters of M. indicum, M. australe and M. ustulatum has been made in the paper. The COI sequence data for the species has been generated and made comparison with the existing COI barcode sequences for M. australe and showed clear difference between the species, thereby established the taxonomic validity of the species. Alignment of the present study sequences with reported sequences of M. australe revealed homologous region of 434 bp. Out of 434 nucleotides, the number of conserved and variable nucleotides were 313 and 121, respectively. Based on the P value (0.002) of Mann-Whitney U test, it was observed that the GC content at codon third base position between M. indicum and M. australe is significantly different. Because of the complexity of the species group a type material is highly necessary for confirmation in future studies and since holotype is irrecoverably lost, a neotype has been selected from the present collection and deposited in the CMFRI Museum at Kochi, Kerala with accession number: ED.2.2.1.8.

Multigene barcoding and phylogeny of selected Engraulidae species.

Anchovies (Engraulidae) are one of the ecologically important groups and often difficult to identify due to their small size and overlapping morphological characters. In the present study, reference DNA barcodes were generated for 82 individuals representing 13 species of Engraulidae family using mitochondrial Cytochrome c oxidase subunit I (COI) and 16S rRNA genes. The average genetic distance value of COI gene for conspecific, congeneric and confamilial is 0.25, 20.45 and 22.28%, respectively. Mitochondrial 16S rRNA showed an average divergence value of 0.60, 10.28 and 14.37% for within species, between species and within families, respectively. Comparison of the present study reference barcodes with the reported sequences revealed high frequency of misidentification of species and possible occurrence of cryptic species in this family. Phylogenetic tree reconstructed using different methodologies revealed monophyletic nature of genus Stolephorus and the evolutionary relationship within genus Stolephorus is defined as ([S. insularis: S. tamilensis] S. dubiosus (S. waitei [S. commersonnii: S. indicus])).

Molecular identification and phylogenetic assessment of species under genus Parapenaeopsis Alcock, 1901, from Indian waters.

Genus Parapenaeopsis constitute an important fishery resource in Indian waters. Parapenaeopsis stylifera, being morphologically very similar to Parapenaeopsis coromandelica, mainly differentiated using telsonic armature and it is subject of controversy regarding the number of armature for the species. The present study was aimed to resolve the taxonomic ambiguity between P. stylifera and P. coromandelica and to study phylogenetic relationship among five species under the genus Parapenaeopsis using partial sequences of mitochondrial 16S ribosomal DNA (rDNA) and cytochrome c oxidase subunit I (COI) genes. Sequence analysis revealed an average genetic divergence value ranging from 7.65 to 20.3% for COI gene and 2.4 to 12.0% for 16S rDNA gene between five species under study. The result shows that P. stylifera and P. coromandelica Alcock, 1906 are genetically a distinct species with average divergence value of 7.6% based on COI sequence and 2.4% based on the 16S rDNA. However, systematic relationship between the P. stylifera and P. coromandelica were closer than other species, thus formed separate clade in phylogenetic tree. The species specific molecular signatures developed in the present study will be helpful in effective fisheries management of the species in Indian waters.

Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus.

TLR5 is one of the important PRR (pathogen recognition receptors) and plays a fundamental role in pathogen recognition and activation of innate immune responses. It recognizes bacterial flagellin and stimulates the production of proinflammatory cytokines, through signalling via the adaptor protein MyD88. In this study, we characterized partial TLR5 (soluble form) gene from Pangasianodon hypophthalmus and analysed its expression profile upon challenge by Edwardsiella tarda. Bioinformatic analysis of gene sequence revealed a putative protein of 266 amino acids with four Leucine rich repeats. Quantitative expression analysis of TLR 5S showed its wide distribution in various organs and tissues. However, significant expression of TLR5S was observed in liver and spleen at 12 h (∼207.8 fold, p < 0.05). Significant upregulation was observed in kidney at 72 h.p.i. (50 folds, p < 0.05) indicating that the kidney provides longer protection almost till the activation of the adaptive immune system. This study enriches the knowledge of TLR5S in boosting the innate immunity against bacterial invasion in fish.

Genetic diversity and demographic history of the giant river catfish Sperata seenghala inferred from mitochondrial DNA markers.

The giant river catfish Sperata seenghala has huge demand in South Asian countries due to its low number of intramuscular bones and nutritive value. However, the culture practises for this fish have not been standardized and the current demand for this fish is being met by capture fisheries only. Unregulated and indiscriminate fishing would lead overexploitation of fish stocks subsequently stock depletion. Genetic diversity between populations would give insight about population structure and demography. In the present study, S. seenghala stocks from three rivers, namely Ganga, Brahmaputra and Mahanadi were characterized using cytochrome b gene and D-loop region. Moderate to high haplotype diversity and low nucleotide diversity values were observed in all populations. Analysis of molecular variance and pairwise FST values showed significant genetic differentiation among populations. Patterns of diversity, haplotype networks and mismatch distribution strongly suggest a historical influence on the genetic structure of S. seenghala populations. S. seenghala stocks from these three rivers are genetically distinct units and management measures should be formulated separately for each population.

Complete mitochondrial genome of threatened mahseer Tor tor (Hamilton 1822) and its phylogenetic relationship within Cyprinidae family.

The mahseers (Tor, Neolissochilus and Naziritor) are an important group of fishes endemic to Asia with the conservation status of most species evaluated as threatened. Conservation plans to revive these declining wild populations are hindered by unstable taxonomy. Molecular phylogeny studies with mitochondrial genome have been successfully used to reconstruct the phylogenetic tree and to resolve taxonomic ambiguity. In the present study, complete mitochondrial genome of Tor tor has been sequenced using ion torrent next-generation sequencing platform with coverage of more than 1000 x. Comparative mitogenome analysis shows higher divergence value at ND1 gene than COI gene. Further, occurrence of a distinct genetic lineage of T. tor is revealed. The phylogenetic relationship among mahseer group has been defined as Neolissochilus hexagonolepis ((T. sinensis (T. putitora, T. tor), (T. khudree, T. tambroides)).

DNA barcoding of marine ornamental fishes from India.

India has rich marine ornamental fish diversity with 400 fish species distributed in Gulf of Munnar/Palk Bay, Gulf of Kutch, and in reefs around Andaman & Nicobar and Lakshadweep Islands. Marine ornamental fish identification at the field level is very difficult because of their high diversity and profound changes in appearance during their developmental stages and camouflage. To facilitate ornamental fish trading with ease and in compliance with the biodiversity act, DNA barcoding technique could be used to accurately identify species. In this study, DNA barcodes were generated for 31 species of commercially important marine ornamental fishes from India. The average genetic distance (K2P model) within species, genus, and family was 0.446, 13.08, and 20.09%, respectively. Intraspecific variation has increased several folds (15-20 times) after including conspecific sequences from different geographical locations. The presence of allopatric lineages/cryptic species was observed in the Indo-pacific region. The NJ tree constructed based on K2P values showed distinct clusters shared by congeneric species specific to populations.

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

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.

DNA barcoding of elasmobranchs from Indian coast and its reliability in delineating geographically widespread specimens.

Identification of elasmobranchs by conventional taxonomy is difficult due to similarities in morphological characters. Species-specific molecular markers are good choice for identifying species irrespective of it's life stage. Recently, mitochondrial cytochrome c oxidase subunit I (COI) gene got global recognition as a barcode gene to discriminate all animals up-to species level. In this study, mitochondrial COI partial gene was used to develop DNA barcodes for 18 species of elasmobranchs (10 species of sharks and 8 species of rays). The COI barcodes clearly distinguished all the species with high interspecific distance values than intraspecific values. The average interspecific and intraspecific distance values are 8.6% and 0.3% for sharks, respectively and 12.4% and 0.63% for rays, respectively using K2P method. The Neighbor-Joining tree showed distinct clusters shared by the species of same genera. The COI barcodes were also used to estimate allopatric divergences for selected species across broad geographical locations and found that Sphyrna lewini, Aetobatus narinari and Neotrygon kuhlii have cryptic diversity.

DNA barcoding of gobiid fishes (Perciformes, Gobioidei).

Gobiids constitute a major proportion of fish population in both tropical and temperate freshwater as well as marine ecosystem. Due to their small size, cryptic ecology and ambiguous morphological characters, gobiids diversity was not documented completely. In this study, DNA barcodes were generated for 11 species of gobiids, collected from the Ashtamudi Lake, India. The mitochondrial COI gene was amplified using universal primers and the resulted 650 bp amplicon was sequenced. The COI barcodes clearly distinguished all the species with high inter-specific genetic distance values than intra-specific values based on K2P (Kimura 2 Parameter) model. The average genetic distance (K2P model) within species, genus and family was 1.2%, 22.2% and 25.3%, respectively. In addition to barcode-based species identification system, Nucleotide Diagnostic (ND) characters specific for species were identified. The Neighbor-Joining tree revealed distinct clusters shared by the species of same genera.

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.

Molecular phylogeny of elasmobranchs inferred from mitochondrial and nuclear markers.

The elasmobranchs (sharks, rays and skates) being the extant survivors of one of the earliest offshoots of the vertebrate evolutionary tree are good model organisms to study the primitive vertebrate conditions. They play a significant role in maintaining the ecological balance and have high economic value. Due to over-exploitation and illegal fishing worldwide, the elasmobranch stocks are being decimated at an alarming rate. Appropriate management measures are necessary for restoring depleted elasmobranch stocks. One approach for restoring stocks is implementation of conservation measures and these measures can be formulated effectively by knowing the evolutionary relationship among the elasmobranchs. In this study, a total of 30 species were chosen for molecular phylogeny studies using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA gene and nuclear Internal Transcribed Spacer 2. Among different genes, the combined dataset of COI and 12S rRNA resulted in a well resolved tree topology with significant bootstrap/posterior probabilities values. The results supported the reciprocal monophyly of sharks and batoids. Within Galeomorphii, Heterodontiformes (bullhead sharks) formed as a sister group to Lamniformes (mackerel sharks): Orectolobiformes (carpet sharks) and to Carcharhiniformes (ground sharks). Within batoids, the Myliobatiformes formed a monophyly group while Pristiformes (sawfishes) and Rhinobatiformes (guitar fishes) formed a sister group to all other batoids.