Influence of stocking density and environmental factors on the expression of insulin-like growth factors in cage-reared butter catfish (Ompok bimaculatus, Bloch 1794) within a large reservoir ecosystem.

In vertebrates, insulin-like growth like factors (IGFs) play an important role in growth and other physiological processes. The GH-IGF axis is considered a valuable tool to monitor fish growth performance. Herein, we report the molecular characterization of igf-1, igf-2, and ß-actin transcripts and relative expression of igf-1 and igf-2 in the liver and muscle tissue of cage-reared butter catfish, Ompok bimaculatus, in response to different stocking densities (T1, 15 fingerlings m-3; T2, 25 fingerlings m-3; and T3, 35 fingerlings m-3) over 180 days of culture duration. The length of the partial amplified transcript sequence of Obigf-1, Obigf-2, and Obß-actin was 325, 438, and 924 bp, respectively. Phylogenetically, Obigf-1 and Obigf-2 were closely clustered with catfishes, viz., Clarias magur, Bagarius yarrelli, and Silurus asotus. The expression of igf-1 was significantly downregulated in the liver at higher densities after 120 days as biomass in the cages increased, while igf-2 expression did not change with the stocking densities over the culture period. Cortisol concentration was significantly elevated in T3 groups post 150 days of the culture period and correlated negatively with the expression of igf-1 (p < 0.05) and igf-2 (p > 0.05). Environmental parameters, pH, TDS, hardness, conductivity, and alkalinity showed a significant positive correlation with hepatic IGF expression. Our study indicates that the liver-derived igf-1 plays a more important role in the regulation of growth in response to culture density in the species studied, and thus, igf-1 can be used effectively as a biomarker for growth. Furthermore, this study will help in planning a proper harvest schedule and optimize the culture practices of O. bimaculatus in an open water cage system.

Exposure to different temperature regimes at early life stages affects hatching, developmental morphology, larval growth, and muscle cellularity in rainbow trout, Oncorhynchus mykiss.

In this study, the effects of temperature on hatching, yolk-sac absorption, larval metamorphosis, post-metamorphic growth, developmental morphology, and muscle cellularity were assessed in rainbow trout, during its early development (until 52 days post-hatching, dph). From the eyed-ova stage, embryos were exposed to either low (8 ± 1 °C, LT-8) or high (16 ± 1 °C, HT-16) temperatures until hatching. Following hatching, half of the sac-fry from LT-8 group were shifted to higher temperature (16 ± 1 °C, LHT-16), and half from HT-16 group were shifted to medium temperature (13 ± 1 °C, HMT-13), for larval rearing. Incubating the eyed-ova at 16 °C preceded the hatching by 6 days, synchronized hatching duration, and minimized hatchlings' size-variation. However, it yielded smaller and morphologically less developed individuals compared to those incubated continuously at 8 ± 1 °C. Post-hatch shifting of sac-fry to high and medium temperatures, respectively, from the initial low and high regimes differentially affected the length and weight of fish. The effect on length was immediate and temporary, but on weight, it appeared to be permanent. Red muscle hypertrophy was observed to be high in HT-16 and HMT-13 individuals (high-temperature incubated groups). White muscle hypertrophy was high in HT-16 and LHT-16 individuals (high post-hatch rearing temperature groups). The effect of early-life temperature regimes on developmental morphology was found to be strong at 22 dph (82.5%) and comparatively weak at 52 dph (65%). The post-hatch rearing temperature caused an immediate but temporary effect on fin development, mainly pectoral, caudal, and anal fin (seen only at 22 dph, not at 52 dph). Contrarily, incubation temperature affected fin position, in a delayed but persistent manner (subtle at 22 dph, but stronger at 52 dph). Overall, this study provides new insights on temperature-dependent changes in developmental morphology, muscle cellularity, and larval growth in rainbow trout and shows that incubation temperature affects ontogeny profoundly than post-hatch thermal regimes.

Molecular characterization of four innate immune genes in Tor putitora and their comparative transcriptional abundance during wild- and captive-bred ontogenetic developmental stages.

The current study was designed to characterize immune genes and compare their expression during ontogenetic developmental stages in progenies of wild-collected and captive-matured T. putitora. The full-length cDNA sequences of Tptlr2, Tpmyd88, Tpcd36, and Tpil8 were 2814, 1545, 1807, and 653 bp in length, with ORFs of 2379 bp, 855 bp, 1410 bp, and 297 bp, encoding for putative peptides of 793, 284, 469 and 98 amino acids, respectively. The predicted peptide sequences of the genes had high sequence homology and structural conservation with other teleost fishes, especially cyprinids. The expression of Tptlr2 was relatively low in both wild- and captive-bred offsprings during the early embryonic stages, but significantly increased later in development. The mRNA abundance of the Tpmyd88 gene was significantly low until the blastula stage, then increased notably from the gastrula stage to the advanced fry stage. The Tpcd36 expression elevated during later developmental stages, peaking at advanced fry stage in both. On the other hand, expression of Tpil8 was relatively low until the blastula stage and showed a moderate increase from the organogenesis stage onwards in wild-bred offsprings, whereas a significant upregulation was seen in the captive-bred offsprings from the organogenesis stage until the advanced fry stage, with its maximum expression at the pre-metamorphosis stage. Overall, the findings suggest the crucial role of Tpmyd88, Tptlr2, Tpcd36, and Tpil8 in inducing innate immunity in embryonic and larval stages of T. putitora. Further, the considerably higher expression of the immune genes in the embryonic and larval stages of captive-bred offsprings may indicate a stronger immune system.

Concurrent changes in thermal tolerance thresholds and cellular heat stress response reveals novel molecular signatures and markers of high temperature acclimation in rainbow trout.

The objective of this study was to better understand the molecular mechanisms which regulate acclimatory responses and thermal safety margins of rainbow trout (Oncorhynchus mykiss) at temperatures above physiological optimum. For this, we investigated the time course of changes in critical thermal tolerance thresholds and associated hepatic and renal transcript abundance of molecular markers related to cellular stress response, during high temperature acclimation. The experimental fish were initially acclimated to 17 °C and later exposed to a gradually raised elevated temperature regime (22 °C) for a period of 30 days. CTmax, CTmin and mRNA expression of candidate markers were examined before the thermal challenge (T0) and over the time-course (days) of high temperature exposure (T1, T3, T7, T15 and T30). With respect to organismal response, CTmax was significantly elevated at T3, but the degree of gain in heat tolerance was not persistent. Contrarily, we observed a gradual loss in cold tolerance with highest CTmin estimate at T30. Based on the time-course of mRNA expression, the studied markers could be categorized into those which were persistently elevated (hsp70a, hsp70b, hspa5, hsp90a, hsp90b, stip1 and serpinh1 in kidney and hsp90b in liver); those which concurred with changes in CTmin (hspbp1, hsp90b, stip1, gr1, hif1a, hyou1, tnfa and tlr5 in kidney); and those which concurred with changes in CTmax (hsp90a, serpinh1, tlr5 and lmo2 in liver). Apparently, transcriptional changes in kidney and liver reflected CTmin and CTmax trend, respectively. Expression profile of stip1 and tlr5 suggest that they are potential novel markers which could reflect thermal limits in rainbow trout. Hepatic metabolic markers were either initially elevated (alt, glud, g6pase1) or down-regulated at different time-points (ast2, gls1, fas, cpt1b, mtor), linked to gluconeogenesis and metabolic depression, respectively. Whereas, growth-axis markers showed no significant differences. Overall, this time-course analysis has revealed potential associations in organismal and tissue-specific cellular response to high temperature acclimation in a thermally sensitive coldwater ectotherm.

Molecular characterization of non-specific immune genes of endangered golden mahseer (Tor putitora) and their expression during embryonic and larval development.

The present study was undertaken to characterize and analyze the expression of non-specific immune genes to get an insight into the early immune status of endangered golden mahseer. In this study, the full-length mRNA sequence of IFNγ, TNFα, C3, and IL10 was 927, 1409, 5125 and 1177 bp with an ORF of 558, 765, 4938, and 540 bp, encoding a putative protein of 185, 254, 1645, and 179 amino acid residues, respectively. The deduced amino acid sequences of these genes shared highly conserved structures with those from other cyprinids. Ontogenic real-time qPCR results indicated that expression of IFNγ and TNFα was lower until the morula stage and increased from blastula stage and found maximum at the organogenesis stage. Expression of the C3 gene was lower until the gastrula stage, followed by a linear increase from organogenesis to the pre-metamorphosis stage. The expression of IL10 was significantly lower during early developmental stages (till gastrula stage) and reached maximum at organogenesis. The level of IL1ß was found maximum in unfertilized eggs and remained elevated till the morula stage. TLR4 expression remained lower during the initial developmental stages and reached the maximum at the organogenesis stage. The expression level of defensin1 was substantially low until the organogenesis stage. In comparison, hepcidin1 was found considerably high until the blastula stage and remained significantly lower during later stages of development. Overall, the data generated improves knowledge on the immune status of endangered golden mahseer during embryonic and larval development, which may help develop effective immunomodulatory interventions during nursery rearing of golden mahseer to produce fry with better fitness.

Dietary soy lecithin augments antioxidative defense and thermal tolerance but fails to modulate non-specific immune genes in endangered golden mahseer (Tor putitora) fry.

A 70-day experiment was carried out to assess the effect of different levels (0, 1 and 2%) of soy lecithin in the diet on growth, survival, antioxidant defense markers, immune gene expression and thermal tolerance limits of golden mahseer, Tor putitora fry. Percentage weight gain, specific growth rate (SGR %) and survival of mahseer fed lecithin supplemented diets were not significantly different from those of the control group. Also, the mRNA expression levels of different immune related genes such as tnfα, il-1ß, il-10, complement-3, interferon-gamma (ifnγ) and tlr4 were unaffected by dietary lecithin supplementation. Nevertheless, superoxide dismutase (SOD) activity was significantly greater in the lecithin-fed groups than the control fish. The glutathione-S-transferase (GST) activity was exceptionally high in the 2% lecithin supplemented group compared to the rest two groups. This increase in antioxidant status with dietary lecithin supplementation, however, was not reflected in the whole body malonaldehyde (MDA) levels, as it did not vary significantly among the dietary groups. Importantly, dietary inclusion of soy lecithin significantly increased upper thermal tolerance limits as evidenced by higher CTmax and LTmax values. Likewise, golden mahseer fry fed with lecithin supplemented diets (both 1 and 2%) registered significantly lower critical and lethal thermal minimum (CTmin and LTmin) values than the control group, indicating higher cold tolerance capacity. Our results thus demonstrate that the dietary inclusion of soy lecithin could enhance the upper and lower thermal tolerance limits and antioxidant status of golden mahseer fry and failed to enhance immune related gene expression.

Temperature and oxygen related ecophysiological traits of snow trout (Schizothorax richardsonii) are sensitive to seasonal changes in a Himalayan stream environment.

In this study, we investigated the seasonal changes in key eco-physiological traits of a wild population of snow trout, Schizothorax richardsonii from river Gola in the Indian Himalayan region over one year. Live specimens (5.8-31.4 g) were electro-fished from their natural habitat during representative months of four seasons with notable differences in water temperature, oxygen concentration and saturation. After 24-72 h of captive-acclimation, the fishes were examined for upper and lower critical thermal limits (CTmax and CTmin), incipient lethal oxygen thresholds (ILOC and ILOS), apparent routine and maximum oxygen consumption rates (MO2rout and MO2max), and blood haemoglobin-haematocrit. Across the seasons, mean CTmin and CTmax values ranged from ∼0 to 34.6 °C, suggesting a relatively wide acute thermal tolerance range for this predominantly cold-water fish. Changes in the habitat's thermal condition during winter to summer was reflected in the CTmin (∼0-2.4 °C) and CTmax (31.7-34.4 °C) estimates, while the highest thermal scope (CTmax-CTmin; 33.2 °C) was recorded in autumn. Concurrently, the incipient lethal hypoxia threshold observed in autumn (ILOS-2.6% and ILOC-0.19 mgO2/L) was significantly lower than the other three seasons, possibly linked to warm-acclimation. The reduction in blood haemoglobin-haematocrit levels during winter could limit the oxygen carrying capacity, with possible reciprocations in thermal tolerance and aerobic metabolism. Concerning body mass corrected oxygen consumption, the apparent MO2rout was found to increase in a temperature-dependent manner from 150.3 mgO2/kg/h at 12 °C to 315.2 mgO2/kg/h at 26 °C, with Q10 ranging from 1.6 to 2.2. Whereas, changes in MO2max was not temperature sensitive (Q10 of 0.7-1.3), except during spring-summer (Q10-2), with lowest and highest measurements in spring and autumn (934 and 1514 mgO2/kg/h), respectively. Collectively, these data form the first information report on the seasonal plasticity in thermal and respiratory physiology of a Schizothoracine fish species, bearing significance for their conservation, aquaculture and habitat monitoring.

Molecular characterisation and transcriptional regulation of muscle growth regulatory factors myogenin and myogenic factor 6 in the Trans-Himalayan cyprinid fish Schizothorax richardsonii.

In vertebrates, myogenic regulatory factors (MRFs) play an important role in muscle growth through the processes of cell determination and differentiation. Herein, we report the molecular characterisation of two MRFs, myogenin (myog) and myogenic factor 6 (myf6) in Indian snow trout Schizothorax richardsonii. The full length mRNA sequence of Srmyog and Srmyf6 was 1437 and 1296 bp, with an ORF of 762 and 720 bp, encoding a putative protein of 253 and 239 amino acids, respectively. In silico analysis and predicted tertiary protein structure revealed that both these nuclear localized MRFs contained the highly conserved basic helix loop helix motif, to potentially aid in dimerization with functional partners and DNA binding. Phylogenetically, the deduced protein sequences of Srmyog and Srmyf6 closely clustered with high altitude dwelling cyprinids, suggesting common ancestry. Tissue-wise, transcripts of Srmyog and Srmyf6 were abundant in skeletal muscle, affirming their muscle-specific role. Moreover, Srmyog was highly expressed in females as compared to males, whereas Srmyf6 expression was higher in older-bigger individuals as compared to younger-smaller fish. Nutritional status (fed-starved-refed) and diet composition (protein and lipid levels) had no significant influence on Srmyog expression. But, Srmyf6 expression was elevated in fishes re-fed after 3 weeks of starvation and in those fed low protein diet. Only rearing temperature was found to coherently influence Srmyog and Srmyf6 transcripts, with highest expression at 18 °C, which favoured growth. Overall, molecular characterisation of the structure and regulation of these MRFs is the first step taken towards deciphering slow growth in this important Himalayan cyprinid.