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Species diversification from major to minor carps for their sturdiness and initial higher growth, and also a quest for antibiotic-free aqua farming in the subcontinent, mandates search for and evaluation of alternatives. An experiment was performed to investigate the potential of fructooligosaccharide (FOS) and Bacillus subtilis (BS) (alone or as synbiotics) in promoting growth and immunity against infections in Labeo fimbriatus fingerlings. Six iso-nitrogenous and iso-lipidic diets containing combinations of two levels of FOS (0% and 0.5%) and three levels of BS (0, 10 4 , 10 6  CFU/g feed) were fed to fish for 60 days. At the end of the feeding trial, twenty-four fish from each group were injected intra-peritoneally with pathogenic strain of Aeromonas hydrophila O:18 to test the immunoprotective efficacy of the supplements against bacterial infection. BS, but not FOS, significantly improved (P < 0.05) growth and feed utilisation attributes like percentage weight gain (PWG), specific growth rate (SGR) and feed conversion ratio (FCR). There were interactive effects of FOS and BS on PWG, SGR and FCR; however, the effects were not additive in nature. These beneficial effects of BS, alone or in combination with FOS, were corroborated by increased protease activity, microvilli density and diameter and number of goblet cells. Overall beneficial effects of FOS and BS included improved erythrocyte (RBC), hemoglobin (Hb), total protein and globulin levels. Total leucocyte (WBC) count and immunological parameters like respiratory burst activity of leucocytes (NBT reduction), lysozyme activity, albumin: globulin ratio and post-challenge survival were significantly improved by both FOS and BS, and their dietary combination yielded the highest improvement in these parameters. Synergistic effects of FOS and BS as dietary supplements indicate that a combination of 10 6  CFU/g BS and 0.5% FOS is optimal to improve growth, feed utilisation, immune functions, and disease resistance in L. fimbriatus fingerlings., (© 2023. The Author(s).)

Understanding changes in biometric indices and metabolism in fish exposed to feed deprivation may be useful in aquaculture. The present study elucidates the effect of feed deprivation on physio-biochemical responses, such as changes in biometric indices, nutrient mobilization patterns, and enzyme activities in rohu ( Labeo rohita ). Experimental fish (av. wt. 3.41 ± 0.07 g) were deprived of feed and sampled at intervals of 0, 15, 30, 45, and 60 days to measure weight, length, body composition, and the activities of enzymes involved in digestion, metabolism, and antioxidation. A decrease in body weight, condition factor (CF), hepatosomatic index (HSI), and gastrosomatic index (GSI) was observed during the initial stage of feed deprivation (15 to 30 days) but remained unchanged thereafter. The total carbohydrate and lipid content also decreased rapidly up until 30 days, then stabilized. However, the reduction in tissue protein content (% wet weight) continued gradually with the duration of feed deprivation from 12.85 ± 0.36 at 0 days to 10.04 ± 0.67 at 15 days, 8.79 ± 0.59 at 30 days, 6.95 ± 0.69 at 45 days, and 6.16 ± 0.8 at 60 days, which was lower, compared to the other two body constituents. Amylase, protease. and lipase activities significantly reduced up until 30 days, but then stabilized. Although G6PDH enzyme activity decreased, gluconeogenic (LDH, AST, and ALT) and antioxidative (SOD and catalase) enzyme activities increased during initial feed deprivation (up to 30 days). A tissue-specific difference in amino acid metabolism with a major role of ALT in liver and AST in muscle was observed. This study revealed that rohu fingerlings adapted well to feed deprivation up until 30 days, beyond which there was an overall deterioration in the metabolic functions.

We investigated the metabolic and cellular stress responses in an endemic catfish Horabagrus brachysoma acclimated to ambient (26°C), 31, 33 and 36°C for 30 days. After acclimation, fish were sampled to investigate changes in the levels of blood glucose, tissue glycogen and ascorbic acid, activities of enzymes involved in glycolysis (LDH), citric acid cycle (MDH), gluconeogenesis (FBPase and G6Pase), pentose phosphate pathway (G6PDH), protein metabolism (AST and ALT), phosphate metabolism (ACP and ALP) and energy metabolism (ATPase), and HSP70 levels in various tissues. Acclimation to higher temperatures (33 and 36°C) significantly increased activities of LDH, MDH, ALP, ACP, AST, ALT and ATPase and blood glucose levels, whereas decreased the G6PDH enzyme activity and, tissue glycogen and ascorbic acid. Results indicated an overall increase in the carbohydrate, protein and lipid metabolism implying increased metabolic demands for maintaining homeostasis in fish acclimated to higher temperatures (33 and 36°C). We observed tissue specific response of HSP70 in H. brachysoma, with significant increase in gill and liver at 33 and 36°C, and in brain and muscle at 36°C, enabling cellular protection at higher acclimation temperatures. In conclusion, H. brachysoma adjusted metabolic and cellular responses to withstand increased temperatures, however, these responses suggest that the fish was under stress at 33°C or higher temperature., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Endosulfan is one of the most hazardous organochlorines pesticides responsible for environmental pollution, as it is very persistent and shows bio-magnification. This study evaluated the impact of acute endosulfan toxicity on metabolic enzymes, lysozyme activities, heat shock protein (Hsp) 70 expression, and histopathology in Tilapia (Oreochromis mossambicus). Among the indicators that were induced in dose dependent manner were the enzymes of amino acid metabolism (serum alanine aminotransferase and aspartate aminotransferase), carbohydrate metabolism (serum lactate dehydrogenase), pentose phosphate pathway (Glucose-6-phosphate dehydrogenase) as well as lysozyme and Hsp70 in liver and gill, while liver and gill Isocitrate dehydrogenase (TCA cycle enzyme) and marker of general energetics (Total adenosine triphosphatase) were inhibited. Histopathological alterations in gill were clubbing of secondary gill lamellae, marked hyperplasia, complete loss of secondary lamellae and atrophy of primary gill filaments. Whereas in liver, swollen hepatocyte, and degeneration with loss of cellular boundaries were distinctly noticed. Overall results clearly demonstrated the unbalanced metabolism and damage of the vital organs like liver and gill in Tilapia due to acute endosulfan exposure., (© 2014 Wiley Periodicals, Inc.)

This study aimed to determine the cellular alterations in the gill of Labeo rohita exposed to lethal temperature maxima (LTM ax ) and lethal temperature minima (LTM in ) by means of transmission electron microscopy (TEM). Acclimation of advanced fingerlings of L. rohita was carried out at 26°C for 30 days. Acclimated fish were subjected to a constant rate of increase or decrease in temperature (0.3°C/min) until the LTM ax and LTM in values were reached. Dissected gills were processed for TEM, both at the end of acclimation period at ambient temperature (26°C) and at lethal temperatures. Results indicated that at ambient temperature, the gill tissues appeared normal. However, significant changes were observed at lethal temperatures. The gill tissues at lethal temperature maxima showed severely damaged lamellae, with more vacuolated space. At lethal temperature minima, gill tissues showed increased density of mitochondria. Our prima-facie report indicated that L. rohita exposed to lethal temperatures exhibited marked ultrastructural changes in the gills., (© 2013 Blackwell Verlag GmbH.)

The epidermal secretion of fish contains various non-specific immune substances that act as the first line of defense against invading pathogens. The present study investigated the level of mucosal antibodies, the activities of hemagglutinin and protease, and other enzymes in the skin mucus of farm reared olive flounder (Paralichthys olivaceus) for 1 year, in order to gain an insight into the relationship between these mucosal immune substances and their seasonal variation. These levels varied significantly during different months of sample collection. The present study showed a positive correlation between water temperature and the level of mucosal antibodies, and an inverse relationship between the level of mucosal antibodies and the activity of mucosal hemagglutinin and protease, but no relationship between lysozyme activity and other innate immune substances. This relationship is thought to be a compensatory response in olive flounder to protect itself against pathogenic microorganisms which are inherently present in the aquatic environment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Every organism responds to heat stress by synthesizing a group of evolutionarily conserved proteins called the heat-shock proteins (HSPs) that, by acting as molecular chaperones, protect the cell against the aggregation of denatured proteins and play a significant role in adaptation to temperature. The present study aimed to investigate the critical thermal maxima (CTMax) and the expression of HSP70 in different tissues (gill, brain, muscle and liver) of an endemic catfish Horabagrus brachysoma acclimated at either 20 or 30°C for 30 days. To understand the HSP70 response, fish acclimated to the two temperatures were exposed to preset temperatures (26, 30, 34, 36 and 38°C for 20°C acclimated fish and 32, 34, 36, 38 and 40°C for 30°C acclimated fish) for 2 h, followed by 1 h recovery at their respective acclimation temperatures. The HSP70 levels in the gill, brain, muscle and liver tissues were determined by Western blotting of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A significant (P < 0.05) increase in the CTMax values was observed for fish acclimated at 30°C (41.86 ± 0.39°C) than those acclimated at 20°C (39.13 ± 0.18°C). HSP70 was detected in all the tissues with the highest level in the liver followed by intermediate levels in muscle and brain, and lowest level in gill tissue, irrespective of the acclimation temperatures (20 or 30°C). The HSP70 levels were significantly higher (P < 0.05) in the tissues of fish acclimated at 30°C than those acclimated at 20°C. The mean induction temperature of HSP70 in all the tissues of fish acclimated at either 20 or 30°C was 30 and 34°C, respectively. The optimum temperature for HSP70 induction in all the tissues of fish acclimated at 20°C was 36°C, whereas for fish acclimated at 30°C was 36°C for gill and 38°C for brain, muscle and liver. Decreased levels of HSP70 were noted in all the tissues of fish when exposed to temperatures that exceeded the optimum temperatures for HSP70 inductions. Overall results indicated that acclimation temperature influences both temperature tolerance and induction of HSP70 in H. brachysoma.

A 2 × 2 × 2 factorial experiment was conducted to delineate the main effect of water soaking of plant ingredients, phytase, cellulase, and their interactions on the growth and digestive enzyme activities of Labeo rohita fingerlings. Two basal diets were prepared using water-soaked (S) or unsoaked (US) plant-based ingredients. Feed of US ingredients was supplemented with phytase (U kg(-1)) and cellulase (%) at the level of 0, 0 (C(us)); 500, 0 (T(1)); 0, 0.2 (T(2)); 500, 0.2 (T(3)), and feed of S ingredients at 0, 0 (C(s)); 500, 0 (T(4)); 0, 0.2 (T(5)), and 500, 0.2 (T(6)), respectively. Three hundred and sixty fingerlings were randomly distributed into eight treatments, each with three replicates. Soaking of the ingredients for 24 h significantly reduced the tannin content. However, feeding of S diets did not improve the fish growth. Highest performance was recorded in the T(3) group. A significant interaction between dietary phytase and cellulase was observed for apparent net protein utilization. Tissue crude protein, ether extract, and ash content of the fingerlings were observed highest in the T(3) group. Activities of amylase, protease, and lipase were recorded highest in the T(3) group. Results suggested that soaking of plant-based ingredients reduces tannin content; however, growth and digestive enzyme activities of group fed soaked diet were not improved, possibly due to leaching of soluble nutrients. Probably, a shorter duration soaking may be effective in reducing tannin content and avoiding nutrients leaching.

Two experiments, one short-term and one long-term, were conducted to elucidate the acute and chronic effects, respectively, of endosulfan exposure on the haemato-immunological and histopathological responses of Channa puncatatus. In the short-term study, fish were exposed to sublethal endosulfan (8.1 μg l(-1)) for 12, 24, 36, 48, 72 and 96 h. In the long-term study, fish were fed with normal diet and simultaneously either exposed to endosulfan (1.2 μg l(-1)) for 90 days or not. Results showed that the ascorbic acid levels in both the liver and the muscle decreased significantly (P < 0.05) by acute and chronic endosulfan exposure. The haemoglobin (Hb) level reduced significantly (P < 0.05) by 15.5% within 12 h of acute endosulfan exposure, further decreased by 25.8% after 24 h of exposure, however, thereafter the values increased and at the end of 72 h returned to normal levels. Almost similar trend was observed for the erythrocyte (RBC) count. The WBC count and the nitroblue tetrazolium (NBT) value showed a general increasing trend with increase in the duration of acute endosulfan exposure. The chronic exposure of C. punctatus to endosulfan significantly (P < 0.05) lowered the Hb level, RBC and WBC counts, NBT reduction value and the plasma parameters such as plasma protein, albumin (A) and globulin (G) compared with that of the control (except for A/G ratio). Endosulfan exposure also severely altered the liver histological structure. Overall results indicated that both short-term acute and long-term chronic endosulfan exposure had a significant impact on the haemato-immunological parameters and tissue histopathology of C. punctatus.

Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.

The biochemical and stress responses of two Indian major carps, rohu Labeo rohita and mrigal Cirrhinus mrigala were studied after acclimating them to four preset temperatures (26, 31, 33 and 36 degrees C) for 30 days. The blood glucose and liver glycogen levels showed an inverse trend in both the species and were significantly different in L. rohita at higher temperatures. The decrease in the liver glycogen level of C. mrigala, however, was not significant. Plasma cortisol levels increased significantly whereas the ascorbic acid content in the brain and kidney of both the species decreased significantly with increasing temperatures. Total lipid content in the liver of both the species decreased significantly with increasing acclimation temperatures. The phospholipid concentration decreased in L. rohita with increasing acclimation temperatures, and in C. mrigala the values decreased up to 33 degrees C and increased at 36 degrees C. In C. mrigala, the cholesterol level decreased up to 33 degrees C and then increased at 36 degrees C, but the absolute value was lower in comparison to L. rohita. The cholesterol levels, however, were not significantly different in L. rohita. Triglycerides and free fatty acids concentrations decreased significantly with increasing acclimation temperatures in both the species. The present study indicates species-specific metabolic responses of L. rohita and C. mrigala to thermal acclimation.

The rates of antibiotic susceptibility and resistance were investigated in Streptococcus iniae and Streptococcus parauberis isolates obtained from diseased olive flounders (Paralichthys olivaceus) collected from fish farms in Jeju Island, Korea. Isolates of S. iniae (n=65) were susceptible to cefotaxime, erythromycin, ofloxacin, penicillin, tetracycline and vancomycin, as demonstrated by the minimum inhibitory concentration (MIC) test. Isolates of S. parauberis (n=86) were highly resistant to erythromycin (58% of the 86 isolates tested) and tetracycline (63% of the 86 isolates tested). Fifty-four isolates of tetracycline-resistant S. parauberis contained the tet(M/O/S) genes, of which 39 and 12 isolates contained the tet(M) and tet(S) genes, respectively, whereas 3 isolates contained both the tet(M) and tet(S) genes. Among the erythromycin-resistant isolates of S. parauberis (n=50) only 14 contained the erm(B) gene. These results suggest that the tet(S) and erm(B) genes of S. parauberis are involved in the acquisition of high-level resistance to erythromycin and tetracycline. Our findings reveal a high rate of antibiotic resistance among strains of S. parauberis and emphasize the need to develop an appropriate vaccine to reduce the use of antibiotics.

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis, respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges-Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.

A feeding trial of 120 days was conducted to study the effect of graded levels of dietary phosphorus on haematology, serum protein concentrations and HSP70 expression in fingerlings of the Indian major carp, Catla (Catla catla). Eight isonitrogenous and isoenergetic purified diets were formulated to contain graded levels of dietary phosphorus (dP), i.e., T(1), 0.1%; T(2), 0.3%; T(3), 0.5%; T(4), 0.7%; T(5), 0.9%; T(6), 1.1%; T(7), 1.3%; or T(8), 1.5%. Four hundred and eighty fish (average weight 4.23 +/- 0.016 g) were equally distributed into 24 tanks forming eight treatments with three replicates each. The fish were fed daily at the rate of 3.5% body weight in two instalments. At the end of feeding trial fish were sampled to study total RBC and WBC count, haemoglobin, serum lysozyme activity, serum total protein, albumin (A), globulin (G) concentration and HSP70 expression. Total RBC count, haemoglobin concentration and serum lysozyme activity did not vary significantly in response to different dietary phosphorus concentrations. Total WBC count was found to be significantly (P < 0.05) higher in T(1 )relative to all other treatments. Serum albumin and A/G ratio was found to be significantly lower in fish of T(1) and T(2) in relation to T(7) group (P < 0.05). Serum globulin and total protein levels remained unaffected by variations in dietary phosphorus. HSP70 expression was observed in T(1) group (0.1% dP) in gills and brain tissue, but not in liver and muscle tissues. No HSP70 expression was observed in fish of T(4) (0.7% dP) and T(8) (1.5% dP) treatments. These prima facie results suggest that dietary phosphorus had only minor influence on the haemato-biochemical parameters studied; however dietary phosphorus deficiency caused organ specific induction of HSP70 in catla fingerlings.

Apart from increased temperature, thermal effluents discharged through cooling systems of nuclear power plants may often contain chlorine (used against bio-fouling), which may affect the immune status of fish. Therefore, a 28-day trial was undertaken to delineate the effect of high temperature and a persistent sub-lethal chlorine exposure on immunomodulation in Cyprinus carpio advanced fingerlings. Fish were acclimated to four different temperatures (26, 31, 33 and 36 degrees C) and maintained for 30 days in two different groups. One group was exposed to persistent chlorine (0.1mgL(-1)) and was compared with their respective temperature control groups (without chlorine exposure). Expression of heat shock proteins (hsp 70) was tested in muscle after 28 days using Western blotting. Haematological parameters (erythrocyte count, leucocyte count, haemoglobin), serum parameters (total protein, albumin, globulin, A/G ratio) and respiratory burst activity were tested to assess immuno-competence of C. carpio in response to temperature and chlorine exposure. Results indicated that hsp 70 was induced at 36 degrees C in temperature control groups but not in their respective temperatures in the presence of chlorine. Haematological parameters such as haemoglobin, erythrocyte and leucocyte counts appeared depressed in chlorine treated groups as compared to their respective temperature control groups. Serum protein and globulin were affected due to chlorine exposure at different acclimation temperatures. A decrease in NBT activity was recorded in chlorine treated groups as compared to their respective temperature control groups. Overall results indicate that increasing acclimation temperatures alters the immune status of C. carpio advanced fingerlings and persistent sub-lethal exposure to chlorine augments this temperature induced immunosuppression.