High Genetic Differentiation and Genetic Diversity in Endangered Mahseer Tor khudree (Sykes, 1839) as Revealed from Concatenated ATPase 6/8 and Cyt b Mitochondrial Genes.

The Deccan mahseer, Tor khudree (Sykes 1839), belonging to family Cyprinidae is an important food and a game fish distributed in peninsular India. Due to overfishing and habitat destruction, the species is declared endangered and placed on the IUCN red list. Therefore, a well-designed conservation program may be essential to get this species protected in its natural habitat. We used a total of 152 samples from four rivers of peninsular India to assess the genetic diversity and structure of the mahseer using concatenated sequences of two mitochondrial genes, ATPase 6/8 (790 bp) and Cyt b (1000 bp). High haplotypic diversity was seen with 44 haplotypes. Individual gene wise haplotypes included 10 and 21 haplotypes for ATPase6/8 and Cyt b, respectively. AMOVA revealed most of the genetic variations (71.02%) to be within the populations. Significant genetic differentiation was observed between all population pairs, with FST values ranging from 0.121 to 0.372, with minimum between Tunga and Tungabhadra population and maximum between Tunga and Periyar population. Haplotype network showed one ancestral haplotype (TKACH04). Significant negative Fu's F and unimodal mismatch distribution suggested recent demographic expansion. The results of the present study would serve as a useful resource for further research on population genetics and conservation programs of the species.

Effect of dietary vitamin E supplement on the reproductive performance and gene expression profiling in Ompok bimaculatus during breeding period.

This study aimed to evaluate the effect of vitamin E on the reproductive responses and the expression of reproductive genes in butter catfish (Ompok bimaculatus). Diets with different levels of vitamin E supplementation (0, 100, and 150 mg/kg) were provided to groups of fish for 60 days. After rearing for 60 days fish were induced with synthetic hormone, that is, Ovatide to study the breeding performance. Ovatide was administrated intramuscularly at the rate of 0.5 and 0.25 mL/kg of body weight for females and males, respectively, in all the treatments. Breeding performance result shows that supplements of vitamin E positively influenced the GSI%, fertilization rate, fecundity, and hatching rate. To understand the relationship between vitamin E and the breeding performance of the fish at the molecular level, a gene expression study was conducted. This study employed real-time PCR for the selected genes critical for reproductive function. In the brain, testis, and ovary dietary vitamin E supplementation of 100 mg/kg significantly enhanced the mRNA transcription of FSHR, Brdt, ESR1, 17beta2, and LHR. Hence, it can be said that 100 mg/kg of vitamin E supplementation in the diet during the reproductive period of O. bimaculatus could improve breeding performance and the mRNA expression of reproductive hormone receptor genes in both males and females of O. bimaculatus.

Photoperiod manipulation improves growth performance, haematological indices and economic returns of Indian butter catfish, Ompok bimaculatus (Bloch, 1794).

Photoperiod manipulation is emerging as an effective approach for regulating physiological functions in fish. This study aimed to assess the impact of photoperiod on the growth performance, haematological responses, and economic returns of the endangered and highly valued Indian butter catfish, Ompok bimaculatus. Fish with an average body weight of 28.60 ± 4.78 g were randomly placed in six FRP tanks, each measuring 120 × 45 × 60 cm3. Each tank contained 20 fish exposed to a light intensity of 1500 lx under different photoperiods [24:0 light: dark (L: D), 15 L: 9D, 12 L: 12D, 9 L: 15D, 0 L: 24D and a natural photoperiod (control)], and fed at a daily rate of 2% of their body weight twice daily for 60 days. The fish in the 15 L: 9D photoperiod exhibited the highest final weight (g), percentage weight gain, specific growth rate (SGR) and survival rate, while the lowest was displayed in 24 L: 0D photoperiod group. The feed conversion ratio (FCR) was at its lowest in the catfish subjected to the 15 L: 9D photoperiod. Regarding haematological parameters, the 15 L: 9D photoperiod group showed higher total erythrocyte count, total leukocyte count, haemoglobin levels, and haematocrit values compared to the other groups. Conversely, the 0 L: 24D group, which experienced prolonged darkness, exhibited the lowest values in these parameters. Moreover, the 24 L: 0D, 9 L: 15D, and 0 L: 24D groups displayed a lower mean corpuscular volume (MCV) but higher mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) when compared to the control group. The economic analysis revealed that O. bimaculatus reared in a moderate photoperiod (15 L: 9D) displayed better growth, feed utilization, and overall health. This finding suggests that adopting a 15 L: 9D photoperiod can lead to enhanced production and improved economic returns for farmers culturing this high-value catfish in the future.

Effect of photoperiod on serum biochemistry, electrolytic balance, acute phase response and histopathology of butter catfish, Ompok bimaculatus (Bloch, 1794).

The present study was executed to evaluate the effect of photoperiod on serum biochemical parameters (glucose, cortisol, ALT, AST and LDH), electrolytic balance (Sodium and potassium), acute phase response (CRP) and histopathology (liver, kidney and skin) of an endangered high valued catfish, Ompok bimaculatus. Catfish (21.00 ± 1.53 cm and 30.00 ± 2.31 g) from the acclimatized stock were randomly distributed to six 120 × 45 × 60 cm3 FRP tanks (n = 20 fish per tank) and exposed to 1500 lx light intensity under different photoperiods [24:0 light: dark (L: D), 15L: 9D, 12L: 12D, 9L: 15D, 0L: 24D and a natural photoperiod (control)], and fed at a daily rate of 2% of bodyweight, twice a day for 60 days. Serum glucose, cortisol and enzymes including aspartate transaminase (AST), lactate dehydrogenase (LDH), alanine transaminase (ALT), and acute phase reactant, such as C-reactive protein (CRP) increased significantly (P < 0.05) in continuous light (24L: 0D), continuous dark (0L: 24D) and short day (9L: 15D) photoperiods, whereas in 15L: 9D and 12L:12D photoperiods, those were in decreasing trend. Serum electrolytes, i.e. potassium level was elevated and sodium level was declined in 24L: 0D, 0L: 24D and 9L: 15D photoperiod groups. Moreover, significant histological alterations in the liver, kidney and skin tissue were also evidenced in the experimented catfish. Typical polygonal hepatocytes with normal blood vessels in liver and normal organization of kidney were seen in catfish of 15L: 9D group. Histological analysis of other groups displayed nuclear degeneration, karyorrhexis, karyolysis, melanomacrophages, nuclear hypertrophy, sinusoid dilation and vacuolar degeneration in liver and hyaline droplets accumulation, granular degeneration, fragmentation of glomerulus and focal necrosis of epithelial cells in kidney. Additionally, general structure of the skin was observed in control group as well as in 15L: 9D group. Contrarily, in 24L: 0D group increased number of mucous cells and vacuoles was observed in the skin of butter catfish. In 9L: 15D and 0L: 24D photoperiods, O. bimaculatus exhibited ruptured epithelial cells, enlarged alarm cells, fat cells, necrotic cells and vacuoles in the skin tissue. The present study depicted that 15L: 9D photoperiod can induce better health of catfish, O. bimaculatus, which, in turn, can help farmers to increase the production of this high valued catfish in future.

Revelation of candidate genes and molecular mechanism of reproductive seasonality in female rohu (Labeo rohita Ham.) by RNA sequencing.

BACKGROUND: Carp fish, rohu (Labeo rohita Ham.) is important freshwater aquaculture species of South-East Asia having seasonal reproductive rhythm. There is no holistic study at transcriptome level revealing key candidate genes involved in such circannual rhythm regulated by biological clock genes (BCGs). Seasonality manifestation has two contrasting phases of reproduction, i.e., post-spawning resting and initiation of gonadal activity appropriate for revealing the associated candidate genes. It can be deciphered by RNA sequencing of tissues involved in BPGL (Brain-Pituitary-Gonad-Liver) axis controlling seasonality. How far such BCGs of this fish are evolutionarily conserved across different phyla is unknown. Such study can be of further use to enhance fish productivity as seasonality restricts seed production beyond monsoon season. RESULT: A total of ~ 150 Gb of transcriptomic data of four tissues viz., BPGL were generated using Illumina TruSeq. De-novo assembled BPGL tissues revealed 75,554 differentially expressed transcripts, 115,534 SSRs, 65,584 SNPs, 514 pathways, 5379 transcription factors, 187 mature miRNA which regulates candidate genes represented by 1576 differentially expressed transcripts are available in the form of web-genomic resources. Findings were validated by qPCR. This is the first report in carp fish having 32 BCGs, found widely conserved in fish, amphibian, reptile, birds, prototheria, marsupials and placental mammals. This is due to universal mechanism of rhythmicity in response to environment and earth rotation having adaptive and reproductive significance. CONCLUSION: This study elucidates evolutionary conserved mechanism of photo-periodism sensing, neuroendocrine secretion, metabolism and yolk synthesis in liver, gonadal maturation, muscular growth with sensory and auditory perception in this fish. Study reveals fish as a good model for research on biological clock besides its relevance in reproductive efficiency enhancement.

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.

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.

Dynamics of expression of antibacterial and antioxidant defence genes in Indian major carp, Labeo rohita in response to Aeromonas hydrophila infection.

Cells produce large number of antioxidant molecules to prevent reactive oxygen species-induced self-damage during microbial assault while generating simultaneously number of antimicrobial molecules to target the pathogen. The present study was aimed at looking into molecules involved in antibacterial and self-protection mechanism of a host Labeo rohita when challenged with a pathogenic bacterium Aeromonas hydrophila. Expression profiles of few of the important host antibacterial genes viz., inducible nitric oxide synthase (iNOS), lysozyme G (LysoG), apolipoprotein A-I (ApoA-I) and hepcidin, and self-defence anti-oxidant genes viz., manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidases (GPx3) were examined in skin and muscle tissues of bacteria challenged fish. Transcription levels of iNOS, LysoG, ApoA-I, hepcidin, catalase, GPx3 and MnSOD were significantly upregulated (P < 0.05) in both tissues at different time points post-bacterial challenge. Increased expression of antibacterial genes in the muscle and skin clearly explains strong defensive mechanism activated in fish tissues in terms of both oxygen-dependent (iNOS) and independent (lysozyme) ways of microbe reduction, and bacterial lysis via production of antimicrobial molecules (ApoA-I and hepcidin) in the host. Simultaneous upregulation of MnSOD, GPx3 and catalase genes explains their involvement in patrolling the cells with regulated production of reactive oxygen species and keeping at a safe level to protect the host's own cells from oxidative damage.

Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus.

Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

Understanding feeding competition under laboratory conditions: Rohu (Labeo rohita) versus Amazon sailfin catfish (Pterygoplichthys spp.).

Competitive interactions between species is widely prevalent within the animal world. In this manuscript, we attempted to understand feeding competitions between the Amazon sailfin catfish, an invasive species introduced globally, and rohu, a keystone species native to several countries within southeast Asia. We used two different size classes of each species, large-size having total length (TL, from snout tip to caudal fin) of 15-20 cm and fingerling having TL<6 cm, and feeding duration was used as a proxy to understand competition. Our results demonstrated that feeding durations of large-size rohu were either similar or significantly (P<0.05) higher in presence of catfish when compared to trials in presence of conspecifics, indicating that large-size rohu is not a weak competitor. However, feeding durations of fingerling rohu was significantly (P<0.05) reduced in presence of both large-size and fingerling catfish, when compared to trials in presence of conspecifics. Moreover, fingerling rohu also displayed freeze (alarm) behavior in presence of the catfish. Interestingly, presence of rohu had no significant (P>0.05) impact on feeding durations of catfish. Overall, the study demonstrated that invasive catfish may behaviorally outcompete fingerling rohu, thus, threatening the sustenance of a species that is native to several freshwaters around the globe.