Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

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.

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.

Comparison of glucose and lipid metabolic gene expressions between fat and lean lines of rainbow trout after a glucose load.

Two experimental rainbow trout lines developed through divergent selection for low (Lean 'L' line) or high (Fat 'F' line) muscle fat content were used as models to study the genetic determinism of fat depots. Previous nutritional studies suggested that the F line had a better capability to use glucose than the L line during feeding trials. Based on that, we put forward the hypothesis that F line has a greater metabolic ability to clear a glucose load effectively, compared to L line. In order to test this hypothesis, 250 mg/kg glucose was intraperitoneally injected to the two rainbow trout lines fasted for 48 h. Hyperglycemia was observed after glucose treatment in both lines without affecting the phosphorylation of AMPK (cellular energy sensor) and Akt-TOR (insulin signaling) components. Liver glucokinase and glucose-6-phosphate dehydrogenase expression levels were increased by glucose, whereas mRNA levels of ß-oxidation enzymes (CPT1a, CPT1b, HOAD and ACO) were down-regulated in the white skeletal muscle of both lines. Regarding the genotype effect, concordant with normoglycemia at 12 h after glucose treatment, higher muscle glycogen was found in F line compared to L line which exhibited hyperglycemia. Moreover, mRNA levels of hepatic glycolytic enzymes (GK, 6PFK and PK), gluconeogenic enzyme PEPCK and muscle fatty acid oxidation enzymes (CPT1a, CPT1b and HOAD) were concurrently higher in the F line. Overall, these findings suggest that F line may have a better ability to maintain glucose homeostasis than L line.

Insulin regulates lipid and glucose metabolism similarly in two lines of rainbow trout divergently selected for muscle fat content.

Two experimental rainbow trout lines were developed through divergent selection for low (Lean 'L' line) or high (Fat 'F' line) muscle fat content. Previous nutritional studies suggested that these lines differed in their regulation of lipid and glucose metabolism. Since insulin acts as an anabolic hormone by regulating lipid and glucose metabolism, we put forward the hypothesis that F line might have a stronger sensitivity to insulin than L line. In order to test this hypothesis, bovine insulin was injected into rainbow trout of the two lines fasted for 48 h. As expected, insulin induced hypoglycemia and activated Akt-TOR signaling both in the liver and muscle of the two lines. We demonstrate that this was coupled with increased expression of insulin dependent glucose transporter (GLUT4) and transcription factors of fatty acid anabolism (LXR and SREBP1c) in the muscle and liver, respectively, and lower mRNA levels of fatty acid oxidation enzymes (CPT1a, CPT1b and HOAD) in the white muscle of both lines. Regarding the genotype effect, TOR signaling response to insulin was stronger in F line as reflected by the higher phosphorylation of S6 protein and elevated mRNA levels of lipogenic enzyme (FAS) in the liver of F line. This observation was concordant with the higher plasma concentrations of free fatty acids and triglycerides in F line. Moreover, mRNA levels of hepatic gluconeogenic enzymes (G6Pase2, FBPase and PEPCK) and muscle fatty acid oxidation enzymes (CPT1a, CPT1b, HOAD and ACO) were higher in the F line. However, very few insulin-genotype interactions were detected, indicating that insulin induced similar changes in lipid and glucose metabolism in both lines.

Glucose metabolic gene expression in growth hormone transgenic coho salmon.

Salmonids are generally known to be glucose intolerant. However, previous studies have shown that growth hormone (GH) transgenic coho salmon display modified nutritional regulation of glycolysis and lipogenesis compared to non-transgenic fish, suggesting the potential for better use of glucose in GH transgenic fish. To examine this in detail, GH transgenic and non-transgenic coho salmon were subjected to glucose tolerance test and subsequent metabolic assessments. After intra-peritoneal injection of 250mg/kg glucose, we analysed post-injection kinetics of glycaemia and expression of several key target genes highly involved in glucose homeostasis in muscle and liver tissues. Our data show no significant differences in plasma glucose levels during peak hyperglycaemia (3-6h after injection), demonstrating a similar glucose tolerance between transgenic and non transgenic. However, and unrelated to the hyperglycaemic episode, GH transgenic fish return to a slightly lower basal glycaemia values 24h after injection. Correspondingly, GH transgenic fish exhibited higher mRNA levels of glucokinase (GK) and glucose-6-phosphate dehydrogenase (G6PDH) in liver, and glucose transporter (GLUT4) in muscle. These data suggest that these metabolic actors may be involved in different glucose use in GH transgenic fish, which would be expected to influence the glucose challenge response. Overall, our data demonstrate that GH transgenic coho salmon may be a pertinent animal model for further study of glucose metabolism in carnivorous fish.

Metabolism and fatty acid profile in fat and lean rainbow trout lines fed with vegetable oil: effect of carbohydrates.

The present study investigated the effect of dietary carbohydrates on metabolism, with special focus on fatty acid bioconversion and flesh lipid composition in two rainbow trout lines divergently selected for muscle lipid content and fed with vegetable oils. These lines were chosen based on previously demonstrated potential differences in LC-PUFA synthesis and carbohydrate utilization. Applying a factorial study design, juvenile trout from the lean (L) and the fat (F) line were fed vegetable oil based diets with or without gelatinised starch (17.1%) for 12 weeks. Blood, liver, muscle, intestine and adipose tissue were sampled after the last meal. Feed intake and growth was higher in the L line than the F line, irrespective of the diet. Moderate postprandial hyperglycemia, strong induction of hepatic glucokinase and repressed glucose-6-phosphatase transcripts confirmed the metabolic response of both lines to carbohydrate intake. Further at the transcriptional level, dietary carbohydrate in the presence of n-3 LC-PUFA deficient vegetable oils enhanced intestinal chylomicron assembly, disturbed hepatic lipid metabolism and importantly elicited a higher response of key desaturase and elongase enzymes in the liver and intestine that endorsed our hypothesis. PPARγ was identified as the factor mediating this dietary regulation of fatty acid bioconversion enzymes in the liver. However, these molecular changes were not sufficient to modify the fatty acid composition of muscle or liver. Concerning the genotype effect, there was no evidence of substantial genotypic difference in lipid metabolism, LC-PUFA synthesis and flesh fatty acid profile when fed with vegetable oils. The minor reduction in plasma glucose and triglyceride levels in the F line was linked to potentially higher glucose and lipid uptake in the muscle. Overall, these data emphasize the importance of dietary macro-nutrient interface in evolving fish nutrition strategies.

Selection for high muscle fat in rainbow trout induces potentially higher chylomicron synthesis and PUFA biosynthesis in the intestine.

Two lines of rainbow trout divergently selected for muscle fat content, fat line (F) and lean line (L) were used to investigate the effect of genetic selection on digestion, intestinal nutrient transport and fatty acid bioconversion, in relation to dietary starch intake. This study involved a digestibility trial for 2 weeks using Cr(2)O(3) as inert marker, followed by a feeding trial for 4 weeks. For the entire duration, juvenile trout from the two lines were fed diets with or without gelatinized starch. Blood, pyloric ceca, midgut and hindgut were sampled at 24 h after the last meal. Transcripts of the proteins involved in nutrient transport and fatty acid bioconversion were abundant in the proximal intestine. GLUT2 transcripts were slightly higher in the F line ceca than in the L line. Dietary starch intake did not enhance the transcription of intestinal glucose transporters, SGLT1 and GLUT2; but it was associated with the higher expression of ApoA1 and PepT1 in the midgut. Significantly, the F line exhibited higher intestinal mRNA levels of MTP, ApoA4, Elovl2, Elovl5 and D6D than the L line, linked to chylomicron assembly and fatty acid bioconversion. Apparent digestibility coefficients of protein, lipid and starch were high in both lines, but not significantly different between them. In conclusion, we found a higher potential of chylomicron synthesis and fatty acid bioconversion in the intestine of F line, but no adaptive transcriptional response of glucose transporters to dietary starch and no genotypic differences in nutrient digestibility.

Regulation of metabolism by dietary carbohydrates in two lines of rainbow trout divergently selected for muscle fat content.

Previous studies in two rainbow trout lines divergently selected for lean (L) or fat (F) muscle suggested that they differ in their ability to metabolise glucose. In this context, we investigated whether genetic selection for high muscle fat content led to a better capacity to metabolise dietary carbohydrates. Juvenile trout from the two lines were fed diets with or without gelatinised starch (17.1%) for 10 weeks, after which blood, liver, muscle and adipose tissues were sampled. Growth rate, feed efficiency and protein utilisation were lower in the F line than in the L line. In both lines, intake of carbohydrates was associated with a moderate post-prandial hyperglycaemia, a protein sparing effect, an enhancement of nutrient (TOR-S6) signalling cascade and a decrease of energy-sensing enzyme (AMPK). Gene expression of hepatic glycolytic enzymes was higher in the F line fed carbohydrates compared with the L line, but concurrently transcripts for the gluconeogenic enzymes was also higher in the F line, possibly impairing glucose homeostasis. However, the F line showed a higher gene expression of hepatic enzymes involved in lipogenesis and fatty acid bioconversion, in particular with an increased dietary carbohydrate intake. Enhanced lipogenic potential coupled with higher liver glycogen content in the F line suggests better glucose storage ability than the L line. Overall, the present study demonstrates the changes in hepatic intermediary metabolism resulting from genetic selection for high muscle fat content and dietary carbohydrate intake without, however, any interaction for an improved growth or glucose utilisation in the peripheral tissues.