Dietary arginine attenuates hypoxia- induced HIF expression, metabolic responses and oxidative stress in Indian Major Carp, Cirrhinus mrigala.

Hypoxia is a common stressor in aquaculture systems, which causes severe physiological disturbances, ultimately leading to mortality or reduced productivity. Arginine, as a precursor of NO, has a role in enhancing oxygen delivery. Thus, an experiment was conducted to evaluate the effect of dietary arginine (Arg) in Cirrhinus mrigala exposed to hypoxia. The fish were fed with different levels of arginine for 60 days and exposed for 72 h to a sublethal level of hypoxia (0.50 ± 0.16 mg/L dissolved oxygen [DO]). The six treatment groups with three replicates were N0 (0% Arg + Normoxia), H0 (0% Arg + Hypoxia), N0.7 (0.70% Arg + Normoxia), H0.7 (0.70% Arg + Hypoxia), N1.4 (1.40% Arg + Normoxia), H1.4 (1.40% Arg + Hypoxia). Eighteen experimental units with twelve animals (5.8 ± 0.18 g) each were used for the trial.The results indicated that supplementation of arginine at 0.7 and 1.4% enhanced the hypoxia tolerance time, although the high dose (1.4%) did not yield any further increments. The exposure to hypoxia up-regulated Hypoxia Inducible Factor (HIF)-1α mRNA expression and supplementation of arginine significantly decreased hypoxia induced up-regulation of HIF at 1.4%. Arginine supplementation partially or completely normalised the hypoxia induced changes in the metabolic enzymes of C. mrigala. The fish exposed to hypoxic conditions exhibited significantly higher (P < 0.05) lipid peroxidation levels than those maintained under normoxic conditions, while arginine feeding significant in reducing lipid peroxidation. Antioxidant enzyme activities were significantly higher (P < 0.05) in hypoxia-exposed carp, indicating increased oxidative stress during the hypoxic exposure, that was improved in Arg-supplemented groups. However, arginine did not modulate erythrocyte countsalthough itreduced the erythrocyte fragility. We conclude arginine supplementation is effective in ameliorating hypoxia induced metabolic alterations and improving antioxidant defences in fish.

Nested PCR assay for specific and sensitive detection of Alternaria carthami.

Alternaria leaf spot (ALS) caused by Alternaria carthami Chowdhary is one of the major threats to the cultivation of safflower in the world. The pathogen is seed borne and requires early detection for restricting its transmission and proliferation. A PCR-based diagnostic assay was developed for easy, quick and reliable detection of A. carthami in infected seeds and leaf samples of safflower. A primer set, AcSPF and AcSPR was designed using ribosomal internal transcribed spacer regions of A. carthami that consistently produced a distinct amplicon of 340 bp with DNA extracted from thirty A. carthami isolates. The specificity of the primer was confirmed using strains of 26 other strains of Alternaria and four other fungal pathogens of safflower. The sensitivity of detection was further enhanced from concentration of 100 pg by simple PCR to as low as 10 pg fungal DNA by a nested PCR assay using ITS and AcSPF and AcSPR primers. The primer pair also facilitated detection of A. carthami in infected seeds and leaf samples. The study provides an accurate and sensitive diagnostic tool for detection of A. carthami.

Dietary GABA enhances hypoxia tolerance of a bottom-dwelling carp, Cirrhinus mrigala by modulating HIF-1α, thyroid hormones and metabolic responses.

The Indian major carp, mrigal (Cirrhinus mrigala), is a bottom-dwelling fish that can survive hypoxic episodes in its natural environment. We hypothesise that it can better survive hypoxic conditions by altering metabolic responses through GABA (Gamma-aminobutyric acid) supplementation. In the first experiment, the hypoxia tolerance time of the fishes was evaluated under extreme anoxic conditions after feeding with GABA, which showed that GABA had improved survival time under hypoxia. To study the response of dietary GABA in hypoxia-exposed fish, the branchial HIF-1α expression levels, serum thyroid hormone levels and hepatic metabolic responses were assessed in the subsequent experiment. The treatment groups were fed for 60 days with experimental diets containing 4 levels of GABA (0.00% G, 0.50% G, 0.75% G and 1.0%G) and were subjected to 72-h hypoxia exposure (0.5 ± 0.02 mg L-1 dissolved oxygen (DO)) whereas a control group was maintained under normoxic conditions (6.0 ± 0.21 mg L-1 DO). The five treatment groups with three replicates were C0 (0% G + normoxia), H0 (0% G + hypoxia), H0.5 (0.50% G + hypoxia), H0.75 (0.75% G + hypoxia) and H1.0 (1.00% G + hypoxia). The results indicated that GABA supplementation triggered downregulation of HIF 1 alpha expression. When compared with the control group, decreased thyroxine (T4) and triiodothyronine (T3) levels were observed in the GABA-fed hypoxic groups. However, TSH (thyroid stimulating hormone) level remained unchanged in all the treatments. The LDH (lactate dehydrogenase) level in hypoxia-exposed groups was decreased by GABA supplementation. Our study demonstrated that GABA supplementation restores acute hypoxia-induced HIF-1α expression, thyroid hormone levels and LDH activities. On the other hand, it enhanced the citrate synthase (CS) activities at 0.5-1.00%, which showed a sharp decline in hypoxia. Hypoxia caused increase in the serum metabolites such as glucose, lactate, cholesterol and triglycerides. However, GABA supplementation was partially effective in reducing glucose and lactate level while triglycerides and cholesterol values remained unchanged. Overall, our results suggested a potential role of GABA in suppressing metabolism during hypoxia exposure, which can increase the chances of survival of the species Cirrhinus mrigala during hypoxia.

Population structure and virulence analysis of Alternaria carthami isolates of India using ISSR and SSR markers.

Alternaria leaf spot caused by Alternaria carthami is one of the most devastating diseases of safflower. Diversity among 95 isolates of A. carthami was determined using virulence assays, enzyme assays, dominant (ISSR) and co-dominant (SSR) markers. Collections and isolations were made from three major safflower producing states of India. The virulence assays categorised the population into four groups based on level of virulence. Estimation of activities of cell wall degrading enzymes (CWDE) yielded concurrent results to virulence assays with maximum CWDE activities in most virulent group. Eighteen ISSR primers were used and 23 polymorphic microsatellite markers were developed to assess the genetic diversity and determine the population structure of A. carthami. Analysis of ISSR profiles revealed high genetic diversity (Nei's Genetic diversity index; h = 0.36). Microsatellite markers produced a total of 56 alleles with an average of 2.43 alleles per microsatellite marker and Nei's genetic diversity index as h = 0.43. Unweighted Neighbor-joining and population structure analysis using both the marker systems differently arranged the isolates into three clusters. Distance analysis of the marker profiles provided no evidence for geographical clustering of isolates, indicating that isolates are randomly spread across India, signifying high potential of the fungus to adapt to diverse regions. Microsatellite markers clustered the isolates in consonance to the virulence groups in the dendrogram. This implies that the fungus has a high potential to adapt to resistant cultivars or fungicides. The information can aid in the breeding and deployment of A. carthami resistant varieties, and in early blight disease management in all safflower growing regions of the world.

Regulation of compensatory growth by molecular mechanism in Labeo rohita juveniles under different feeding regimes.

A study was carried out to assess the regulation of compensatory growth under different restriction feeding regimes in Labeo rohita juveniles by the interaction of various feed intake and growth regulating genes. A 60 day feeding trial was conducted with five treatment groups, Control (3% body weight, bw), T1 (alternate days), T2 (0.5% bw), T3 (1% bw) and T4 (2% bw) and feeding was done for first 30 days of the trial. For next 30 days, all the treatment groups were fed at a rate of 3% bw as in the control group. There was significant (p < 0.05) difference in the weight gain among the treatment groups with lowest FCR and highest PER was found in T2 group. Ghrelin gene mRNA levels were upregulated during first 30th days of the trial with highest expression levels in the T2 group. The expression levels of leptin gene mRNA were found significantly different (p < 0.05) among the treatments, which was down-regulated during initial 30 days and upregulated as the experiment progress towards 60th day. The IGF-1 mRNA expression levels were upregulated more in liver compared to the muscle tissue. The results of the study suggest that increased ghrelin levels and decreased leptin levels lead to hyperphagia during the onset of refeeding, which further triggers the compensatory growth in L. rohita. The present study describes the molecular mechanism behind the compensatory growth following a different feed restriction regime in L. rohita which is regulated due to the interaction of different energy homeostasis and growth regulating genes.

Functional analysis of diacylglycerol O-acyl transferase 2 gene to decipher its role in virulence of Botrytis cinerea.

Gray mold disease inflicted by Botrytis cinerea is a serious menace responsible for significant economic loss worldwide. Due to its polyphagous nature, the pathogen has enthused inquisitiveness in researchers to unravel its complexity. Agrobacterium tumefaciens-mediated transformation was used to generate insertional mutants of Botrytis cinerea. A mutant (BCM-55) with disruption in a gene (BcDGAT2) that encodes for diacylglycerol O-acyl transferase 2 (DGAT2), showed enervated virulence on various hosts' tissues. Enzyme DGAT2 is crucial in the final step of synthesis of triacylglycerol (TAG) that plays an important role in homeostasis of membrane and cellular processes. However, the role of DGAT2 has never been reported in a phytopathogenic fungus. In this study, BCM-55 was characterized to ascertain the role of DGAT2 in virulence of B. cinerea. The insertional mutant was defective in spore production and lacked sclerotia formation as a consequence of lower accumulation of TAG. A significant delay in spore germination in BCM-55 was accompanied with a low penetration potential. Hyphae of the mutant formed swollen endings with considerable impairment in penetration. Deletion of BcDGAT2 also led to increased sensitivity towards cell wall and membrane-disturbing agents. Furthermore, BCM-55 was deficient in the production of oxalic acid and showed lower activity of a cell wall-degrading enzyme, polygalacturonase. The role of BcDGAT2 in virulence was further confirmed by targeted deletion and complementation of the gene. The results insinuate a crucial role of BcDGAT2 in penetration and consequently virulence of B. cinerea. The study provides novel insights into plant-pathogen interactions that can be exploited to develop suitable disease management strategies.

Arbuscular Mycorrhiza Augments Arsenic Tolerance in Wheat (Triticum aestivum L.) by Strengthening Antioxidant Defense System and Thiol Metabolism.

Arbuscular mycorrhiza (AM) can help plants to tolerate arsenic (As) toxicity. However, plant responses are found to vary with the host plant and the AM fungal species. The present study compares the efficacy of two AM fungi Rhizoglomus intraradices (M1) and Glomus etunicatum (M2) in amelioration of As stress in wheat (Triticum aestivum L. var. HD-2967). Mycorrhizal (M) and non-mycorrhizal (NM) wheat plants were subjected to four levels of As (0, 25, 50, and 100 mg As kg-1 soil). Although As additions had variable effects on the percentage of root colonized by the two fungal inoculants, each mycobiont conferred benefits to the host plant. Mycorrhizal plants continued to display better growth than NM plants. Formation of AM helped the host plant to overcome As-induced P deficiency and maintained favorable P:As ratio. Inoculation of AMF had variable effects on the distribution of As in plant tissues. While As translocation factor decreased in low As (25 mg kg-1 soil), it increased under high As (50 and 100 mg As kg-1 soil). Further As translocation to grain was reduced (As grain:shoot ratio) in M plants compared with NM plants. Arsenic-induced oxidative stress (generation of H2O2 and lipid peroxidation) in plants reduced significantly by AMF inoculation. The alleviation potential of AM was more evident with increase in severity of As stress. Colonization of AMF resulted in higher activities of the antioxidant enzymes (superoxide dismutase, catalase, and guaiacol peroxidase). It increased the concentrations of the antioxidant molecules (carotenoids, proline, and α-tocopherol) than their NM counterparts at high As addition level. Comparatively higher activities of enzymes of glutathione-ascorbate cycle in M plants led to higher ascorbate:dehydroascorbate (AsA:DHA) and glutathione:glutathione disulphide (GSH:GSSG) ratios. Inoculation by AMF also augmented the glyoxalase system by increasing the activities of both glyoxalase I and glyoxalase II enzymes. Mycorrhizal colonization increased concentrations of cysteine, glutathione, non-protein thiols, and activity of glutathione-S-transferase that facilitated sequestration of As into non-toxic complexes. The study reveals multifarious role of AMF in alleviation of As toxicity.

Terpenoids in plant and arbuscular mycorrhiza-reinforced defence against herbivorous insects.

BACKGROUND: Plants, though sessile, employ various strategies to defend themselves against herbivorous insects and convey signals of an impending herbivore attack to other plant(s). Strategies include the production of volatiles that include terpenoids and the formation of symbiotic associations with fungi, such as arbuscular mycorrhiza (AM). This constitutes a two-pronged above-ground/below-ground attack-defence strategy against insect herbivores. SCOPE: Terpenoids represent an important constituent of herbivore-induced plant volatiles that deter herbivores and/or attract their predators. Terpenoids serve as airborne signals that can induce defence responses in systemic undamaged parts of the plant and also prime defence responses in neighbouring plants. Colonization of roots by AM fungi is known to influence secondary metabolism in plants; this includes alteration of the concentration and composition of terpenoids, which can boost both direct and indirect plant defence against herbivorous insects. Enhanced nutrient uptake facilitated by AM, changes in plant morphology and physiology and increased transcription levels of certain genes involved in the terpenoid biosynthesis pathway result in alterations in plant terpenoid profiles. The common mycorrhizal networks of external hyphae have added a dimension to the two-pronged plant defence strategy. These act as conduits to transfer defence signals and terpenoids. CONCLUSION: Improved understanding of the roles of terpenoids in plant and AM defences against herbivory and of interplant signalling in natural communities has significant implications for sustainable management of pests in agricultural ecosystems.

Pulsatile Clavicular Swelling as the Sole Presentation of Asymptomatic Renal Cell Carcinoma with an Isolated Metastasis to the Clavicle: A Case Report.

Renal Cell Carcinoma (RCC) is notorious for its unpredictable dissemination patterns involving both lymphatic and haematogenous route without a clear-cut preponderance for any. Unlike other intra-abdominal malignancies like colorectal carcinoma, renal cell carcinoma is often known to metastasize to distant sites even after a confident and unsuspecting curative resection or often as the primary presentation constantly adding and agitating our presentation patterns for this sneaky tumour. Presented below is a case of such an unsuspecting female patient. She was referred from the orthopaedic OPD to surgery OPD with a right clavicular swelling which was histopathologically examined and diagnosed as a metastatic renal cell carcinoma following which she was diagnosed with right renal cell carcinoma.