Higher intake of ß-glucan impairs reproduction in a female teleost, Tor putitora (Hamilton, 1822).

Although the immuno-modulatory and stress-relieving properties of ß-glucan is well elucidated in humans and other animal models, including fish, its role as a dietary supplement on reproduction is extremely scarce. Therefore, in this study, adult female fish were fed one of four test diets having 0 (control), 0.5, 1, and 1.5% ß-D-glucan for 130 days and its effect on reproductive performance, ovarian and liver histology, sex hormones, and transcript abundance of selected reproduction-related genes was assessed. Low dietary intake of ß-glucan improved fertilization and hatching rates (p<0.05). The relative fecundity and percentage of spawning females were higher (non-significant) in 0.5% ß-glucan-fed groups. Surprisingly, even after 130 days, spawning did not occur in 1.5% ß-glucan-fed individuals. Irrespective of ß-glucan intake, all the brooders recorded similar plasma 17ß-estradiol and maturation-inducing hormone (p>0.05). Higher intake of ß-glucan (1.5%) upregulated aromatase genes without a parallel increase in 17ß-estradiol. However, plasma vitellogenin increased with increasing ß-glucan up to 1.0% then declined at 1.5% (p<0.05). The fish that received control, 0.5, and 1.5% ß-glucan recorded similar vitellogenin levels in their plasma. Significantly higher plasma cortisol was evidenced in 1.5% ß-glucan fed brooders (p<0.05). Histologically, higher follicular atresia and leaking of yolk material was evidenced in 1.5% ß-glucan-fed group. Liver histology revealed the highest nutrient/lipid accumulation in fish that received 1.0% and 1.5% ß-glucan. This study demonstrated the stimulatory effect of ß-glucan intake at a lower dose (0.5%) on reproduction. However, higher intake (1.5%) could perturb normal reproductive function in a fish model and caused an increased number of atretic follicles leading to spawning/reproductive failure.

Expression of genes encoding non-specific immunity, anti-oxidative status and aquaporins in ß-glucan-fed golden mahseer (Tor putitora) juveniles under ammonia stress.

The study investigated the effects of dietary administration of ß-glucan on aquaporins and antioxidative & immune gene expression in endangered golden mahseer, Tor putitora juveniles, exposed to ammonia stress. For that, fish were fed experimental diets having 0 (control/basal), 0.25, 0.5, and 0.75% ß-d-glucan for five weeks and then exposed to ammonia (10 mgL-1 total ammonia nitrogen) for 96 h. Administration of ß-glucan differentially influenced the mRNA expression of aquaporins, anti-oxidative, and immune genes in ammonia-exposed fish. For instance, the transcript abundance of catalase and glutathione-s-transferase in gill varied significantly among the treatment groups, with the lowest levels in 0.75% ß-glucan fed groups. At the same time, their hepatic mRNA expression was similar. Congruently, transcript abundance of inducible nitric oxide synthase considerably decreased in the ß-glucan fed ammonia-challenged fish. Conversely, the relative mRNA expression of various immune genes viz., major histocompatibility complex, immunoglobulin light chain, interleukin 1-beta, toll-like receptors (tlr4 and tlr5) and complement component 3 remained largely unchanged in ammonia-exposed mahseer juveniles that were fed with graded levels of ß-glucan. On the other hand, a significantly lower transcript level of aquaporins 1a and 3a was noticed in the gill of glucan-fed fish compared to ammonia-exposed fish that received the basal diet. However, branchial aquaporin 3b remained unaltered. Altogether, this study showed that dietary intake of 0.75% ß-glucan improved resistance to ammonia stress to a certain degree, probably through activating anti-oxidative system and reducing brachial ammonia uptake.

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.

Quantitative proteomic analysis of GnRH agonist treated GBM cell line LN229 revealed regulatory proteins inhibiting cancer cell proliferation.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptor, a rhodopsin-like G-protein coupled receptor (GPCR) family member involved in GnRH signaling, is reported to be expressed in several tumors including glioblastoma multiforme (GBM), one of the most malignant and aggressive forms of primary brain tumors. However, the molecular targets associated with GnRH receptor are not well studied in GBM or in other cancers. The present study aims at investigating the effect of GnRH agonist (Gosarelin acetate) on cell proliferation and associated signaling pathways in GBM cell line, LN229. METHODS: LN229 cells were treated with different concentrations of GnRH agonist (10-10 M to 10-5 M) and the effect on cell proliferation was analyzed by cell count method. Further, total protein was extracted from control and GnRH agonist treated cells (with maximum reduction in cell proliferation) followed by trypsin digestion, labeling with iTRAQ reagents and LC-MS/MS analysis to identify differentially expressed proteins. Bioinformatic analysis was performed for annotation of proteins for the associated molecular function, altered pathways and network analysis using STRING database. RESULTS: The treatment with different concentrations of GnRH agonist showed a reduction in cell proliferation with a maximum reduction of 48.2% observed at 10-6 M. Quantitative proteomic analysis after GnRH agonist treatment (10-6 M) led to the identification of a total of 29 differentially expressed proteins with 1.3-fold change (23 upregulated, such as, kininogen-1 (KNG1), alpha-2-HS-glycoprotein (AHSG), alpha-fetoprotein (AFP), and 6 downregulated, such as integrator complex subunit 11 (CPSF3L), protein FRG1 (FRG1). Some of them are known [KNG1, AHSG, AFP] while others such as inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), ITIH4, and LIM domain-containing protein 1 (LIMD1) are novel to GnRH signaling pathway. Protein-protein interaction analysis showed a direct interaction of KNG1, a hub molecule, with GnRH, GnRH receptor, EGFR and other interactors including ITIH2, ITIH4 and AHSG. Overexpression of KNG1 after GnRH agonist treatment was validated using Western blot analysis, while a significant inhibition of EGFR was observed after GnRH agonist treatment. CONCLUSIONS: The study suggests a possible link of GnRH signaling with EGFR signaling pathways likely via KNG1. KNG1 inhibitors may be investigated independently or in combination with GnRH agonist for therapeutic applications.

Transgenerational effects of ß-glucan on thermal tolerance, growth performance, and immune gene expression of endangered cyprinid Tor putitora progeny.

Nutritional programming signifies a process in which broodstock feeding approaches have long-term effects on the subsequent progeny. The present study aimed to elucidate whether supplementing golden mahseer, Tor putitora broodstock diets with ß-glucan affects progeny growth performance, survival, thermal tolerance, and non-specific immunity. Initially, the growth performance of progeny produced from brooders fed with different levels of ß-glucan was non-significant. However, on the 15th and 35th DPH, the maximum weight was observed in fry obtained from the brooders fed with 0.5% followed by 1.0% ß-glucan. Furthermore, on 50th DPH, significantly higher weight was registered in the fry from the 0.5% ß-glucan fed group while 1.0% ß-glucan group had no transgenerational effect on growth. The condition factor of fry obtained from golden mahseer brooders fed with a 0.5% ß-glucan diet was greater than the control and 1.0% ß-glucan fed group. On the other hand, we did not find any significant transgenerational influence of ß-glucan on the survival of the progeny. The thermal tolerance of fry produced from brooders fed with ß-glucan was significantly modulated at both end-points (CTmax and CTmin). Expression of interleukin-1ß was significantly up-regulated in fry obtained from ß-glucan fed brooders. In contrast, the expression level of tumor necrosis factor-α was significantly higher only in fry produced from 1.0% ß-glucan fed brooders. The expression of immunoglobulin light chain and serum amyloid A gene was significantly higher in fry produced from 0.5% ß-glucan fed brooders. Overall results suggest that the dietary provisioning of ß-glucan in golden mahseer brooders can be a strategy to produce healthy and robust fry in captivity for stock enhancement and conservation programs.

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.

Dietary ß-glucan influences the expression of testicular aquaporins, antioxidative defence genes and sperm quality traits in endangered golden mahseer, Tor putitora (Hamilton, 1822).

The effect of dietary ß-glucan on seminal plasma composition, sperm characteristics, expression of aquaporins, and antioxidative defence genes of golden mahseer was evaluated. For that, four experimental diets containing 0 (control), 0.5, 1, and 1.5% ß-glucan were fed to male golden mahseer brooders for 130 days. Feeding of 0.5% ß-glucan was found to improve sperm characteristics, viz. sperm count, motility, viability, and morphology with no effect on gonadosomatic index and seminal plasma energy resources. The marked down-regulation in the transcript abundance of testicular aqp3a noticed in 1.5% ß-glucan fed brooders corresponds to their poor sperm quality. Further, the mRNA expression of genes encoding antioxidant enzymes, namely gst and sod1, was lowest in 0.5% ß-glucan fed brooders. In contrast, control and higher ß-glucan (1 and 1.5%) groups displayed relatively higher expression levels of testicular gst and sod1. On the other hand, the higher seminal plasma total antioxidant capacity observed in 0.5 and 1% ß-glucan fed brooders indicated increased scavenging ability of reactive oxygen species. Overall, supplementation of 0.5% ß-glucan improved sperm quality and antioxidative potential, but the higher inclusion (1.5%) negatively affected sperm characteristics. Collectively, dietary ß-glucan (0.5%) can be a practical approach to developing quality broodstock of golden mahseer.

ß-glucan modulates non-specific immune gene expression, thermal tolerance and elicits disease resistance in endangered Tor putitora fry challenged with Aeromonas salmonicida.

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of ß-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of ß-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of ß-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1ß were significantly upregulated, while ifn-γ and il-10 were unaffected by ß-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, ß-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of ß-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CTmax and lower CTmin than the control group. After feeding ß-glucan for eight weeks, the fish were bath challenged with a bacterial pathogen, Aeromonas salmonicida. The challenge study results revealed that ß-glucan intake improved most of the studied immune parameters, resulting in lower mortality. Overall, dietary inclusion of ß-glucan (0.5-1.0%) was efficient in improving the immune responses, thermal tolerance, and disease resistance of T. putitora fry.

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.