Environmental impacts and effects on greenhouse gas emissions in shrimp feed production system for aquaculture - A case study in India.

Attributional life cycle assessment study examines the environmental impact of raw materials, machinery, and unit operations. In the present work, an attributional life cycle assessment (LCA) was employed to assess the environmental and greenhouse gas impacts of a shrimp feed production system. A commercial shrimp feed mill in Tamil Nadu, India, provided inventory data for one-ton shrimp feed (functional unit) for a Cradle-to-Gate evaluation using environmental impact methodologies, specifically Impact 2002+ in SimaPro® (V9.3.0.3) software. The results showed that human health (0.003357 DALY), ecosystem quality (2720.518 PDF × m2 × yr), climate change (2031.696 kg CO2 eq), and resources (71019.42 MJ primary) were the most significantly impacted. The human health category was found to be the most prominent after normalization and weighting (0.47 pt), and strategies were suggested accordingly. The GWP20 and GWP100 measures for long-term climate change were calculated to be 8.7 and 7.33 kg CO2 eq, respectively. Cast iron used in machinery production (GWP 20-15.40%, GWP100-134.5%) and electricity use (GWP 20-6.13%, GWP 100-6.9%) accounted for sizable portions of the burden. Feed production is estimated to contribute 0.2% of global CO2 emissions within the proposed global context. These findings are significant regarding economically and environmentally sustainable shrimp feed production worldwide.

Isolation of Aeromonas salmonicida subspecies salmonicida from aquaculture environment in India: Polyphasic identification, virulence characterization, and antibiotic susceptibility.

This study reports the polyphasic identification, characterization of virulence potential, and antibiotic susceptibility of Aeromonas salmonicida subspecies salmonicida COFCAU_AS, isolated from an aquaculture system in India. The physiological, biochemical, 16s rRNA gene sequencing and PAAS PCR test identified the strain as Aeromonas salmonicida. The MIY PCR tests established the subspecies as 'salmonicida'. The in vitro tests showed the isolated bacterium as haemolytic with casein, lipid, starch, and gelatin hydrolysis activity, indicating its pathogenic attributes. It also showed the ability to produce slime and biofilm, and additionally, it possessed an A-layer surface protein. In vivo pathogenicity test was performed to determine the LD50 dose of the bacterium in Labeo rohita fingerlings (14.42 ± 1.01 g), which was found to be 106.9 cells fish-1. The bacteria-challenged fingerlings showed skin lesions, erythema at the base of the fins, dropsy, and ulcer. Almost identical clinical signs and mortalities were observed when the same LD50 dose was injected into other Indian major carp species, L. catla and Cirrhinus mrigala. Out of the twelve virulent genes screened, the presence of nine genes viz., aerA, act, ast, alt, hlyA, vapA, exsA, fstA, and lip were detected, whereas ascV, ascC, and ela genes were absent. The A. salmonicida subsp. salmonicida COFCAU_AS was resistant to antibiotics such as penicillin G, rifampicin, ampicillin, and vancomycin while highly sensitive to amoxiclav, nalidixic acid, chloramphenicol, ciprofloxacin, and tetracycline. In summary, we have isolated a virulent A. salmonicida subsp. salmonicida from a tropical aquaculture pond which can cause significant mortality and morbidity in Indian major carp species.

Deciphering the Probiotic Potential of Bacillus amyloliquefaciens COFCAU_P1 Isolated from the Intestine of Labeo rohita Through In Vitro and Genetic Assessment.

In this study, a bacterial strain COFCAU_P1, isolated from the digestive tract of a freshwater teleost rohu (Labeo rohita), was identified as Bacillus amyloliquefaciens using 16S rRNA gene sequence analysis combined with amplification of species-specific BamHI and barnase genes. The probiotic potential of the strain was evaluated using an array of in vitro tests along with safety and genetic analyses. The isolate showed potent antimicrobial response against several fish pathogenic bacteria, survived a wide pH range (2-9), and was resistant up to 10% bile salt concentration. With regard to the in vitro adhesion properties, the strain showed significantly high in vitro adhesion to mucus, auto and co-aggregation capacity, and cell surface hydrophobicity. The strain was non-haemolytic, able to produce extracellular enzymes, viz., proteinase, amylase, lipase, and cellulase, and showed significant free radical scavenging activity. A challenge study in rohu revealed the strain COFCAU_P1 as non-pathogenic. The presence of putative probiotic marker genes including 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase, arginine/ornithine antiporter ArcD, choloylglycine hydrolase, LuxS, and E1 ß-subunit of the pyruvate dehydrogenase complex was confirmed by PCR, suggesting the molecular basis of the probiotic-specific functional attributes of the isolate. In conclusion, the in vitro and genetic approaches enabled the identification of a potential probiotic from autochthonous source with a potential of its utilization in the aquaculture industry.

Pseudomonas aeruginosa FARP72 Offers Protection Against Aeromonas hydrophila Infection in Labeo rohita.

Use of probiotics as the biocontrol agent for disease prevention in aquaculture is gaining importance as an alternative to the indiscriminate use of antibiotics and other chemotherapeutics. In view of this trend, the probiotic properties of a potent antagonistic bacterium, Pseudomonas aeruginosa FARP72, was characterized in terms of safety, antagonistic activities, in vitro immunomodulation, and in vivo disease resistance. Immunomodulatory activity was ascertained by measuring the production of intracellular superoxide anion, nitric oxide, total leukocyte peroxidase content, and the leukocyte proliferation in head kidney leukocytes. The bacterium isolated from the skin mucus of freshwater catfish Clarias batrachus was harmless to Labeo rohita. It showed inhibitory activity against Aeromonas caviae, A. hydrophila, Edwardsiella tarda, Pseudomonas putida, and Streptococcus agalactiae as revealed by cross and parallel streaking methods. Significantly higher superoxide anion and nitric oxide production, peroxidase content, and proliferative responses of leucocytes delineated the strains' ability to interact with immune cells to activate the immune system in vitro. Significant growth inhibition of A. hydrophila from 1.55 × 105 CFU/mL was observed when co-cultured with P. aeruginosa FARP72 in phosphate-buffered saline (PBS) at levels ranging from 2.61 × 107 to 2.61 × 109 CFU/mL in 10 days. P. aeruginosa FARP72 increased the survival rate of rohu fingerlings against pathogenic A. hydrophila challenge in biocontrol study in vivo as determined by cohabitation challenge. These results suggest that P. aeruginosa FARP72 is a potential probiotic strain and can be used in aquaculture to improve the health status and disease resistance of fish.

Modulation of systemic and mucosal immune responses of Catla catla (Hamilton, 1822) experimentally challenged with gill monogeneans.

The present study investigated the modulation of systemic and mucosal immunity of catla (Catla catla) experimentally challenged with gill monogeneans. Fingerlings of catla (24.2 ±â€¯1.5 g) without any parasites were randomly distributed into two groups each comprising quadruplicate tanks (10 fish per tank). The fish in the infected group were challenged with gill monogeneans by cohabitation method. The control group fish were not exposed to parasites. Serum and mucus were collected from both control and infected fish and different immunological parameters were measured at weekly interval for four weeks. Monogeneans on the fish gills were also quantified from infected fish at weekly interval. Different systemic immunological parameters viz. myeloperoxidase content, alkaline phosphatase and hemolytic activity of serum showed a significant increase (p < .05) in infected group. However, respiratory burst activity, total serum protein and anti-protease activity did not show any significant enhancement (p < .05) in infected group compared to control. In case of mucosal parameters, myeloperoxidase content, alkaline phosphatase activity, total protein content and anti-protease activity of mucus showed significant enhancement (p < .05) in infected group compared to control. Parasite prevalence was 100% in the infected group in all the sampling weeks. The mean intensity of parasites was progressively increased and the highest number of monogeneans per gill (45 ±â€¯4.56) was observed during 4th week post challenge. Control group had mean intensity and prevalence of zero. In conclusion, there was the participation of immune system components against monogenean infection in catla. However, the progressive increase in monogenean intensity indicates some immune evasion mechanism available with the monogenean to co-exist with the host in the face of host's immune responses.

Paraprobiotic preparation from Bacillus amyloliquefaciens FPTB16 modulates immune response and immune relevant gene expression in Catla catla (Hamilton, 1822).

The present study evaluated the paraprobiotic effect of heat-inactivated Bacillus amyloliquefaciens FPTB16 on immunological response and immune relevant gene expression in catla (Catla catla). Heat inactivation of viable cells of B. amyloliquefaciens was done at 60 °C for 2 h. For preparation of paraprobiotic supplemented diet, the heat-inactivated bacteria were added to the basal diet (control) at three different inclusion levels i.e., 107, 108 and 109 cells g-1 diet. Fish (25.98 ± 2.57 g) were fed with these diets and various immune responses and immune relevant gene expressions were measured after 4 weeks of feeding. Biochemical parameters were also measured along with the immunological responses. Immunological parameters viz. oxygen radical production, serum lysozyme activity and total serum protein content showed significant enhancement (p < 0.05) in fish fed with 108 and 109 cells g-1 diet. Significant enhancement in myeloperoxidase activity was observed in all the dietary groups compared to control. Alkaline phosphatase activity showed significant enhancement (p < 0.05) in fish fed with 107 and 108 cells g-1 diet. Biochemical parameters viz. GPT, GOT and glucose content did not show any significant difference in any of the dietary groups. Immune relevant genes viz. IL-1ß, TNF-α, C3 and iNOS showed significantly higher expression in either liver or head-kidney tissues in most of the cases. However, IFN-γ expression showed a down-regulation pattern in both the tissues. In conclusion, the dietary supplementation of heat-inactivated B. amyloliquefaciens enhanced the immunity of catla, particularly at 108 cells g-1 diet. The results collectively suggest the paraprobiotic applicability of B. amyloliquefaciens in aquaculture.

Biofloc technology application in indoor culture of Labeo rohita (Hamilton, 1822) fingerlings: The effects on inorganic nitrogen control, growth and immunity.

The present study was undertaken to investigate the effect of biofloc-based aquaculture system on inorganic nitrogen control, and growth and immunological responses of rohu (Labeo rohita). Fingerlings of rohu were cultured in indoor biofloc tanks in light-limited condition for 16 weeks with daily addition of molasses as carbon source to maintain the C/N ratio at 15. Different water quality parameters were measured at weekly interval. Growth, survival and welfare indices including stress and immunological status were measured after the end of the experimental period. Factor analysis using water quality variables indicated ammonia immobilization by heterotrophic bacteria, rather than nitrification, as the dominant mechanism influencing ammonia removal in the biofloc system. Ammonia immobilization was concomitant with microbial biomass production, as also revealed through factor analysis. The biofloc culture system enhanced the growth of rohu significantly than the control fish. As a measure of stress, the serum glucose content did not differ significantly between the fish cultured in biofloc and control system. Among the immunological parameters, respiratory burst and alkaline phosphatase activity showed significant enhancement in fish grown in biofloc system than the control. However, myeloperoxidase activity and total serum protein content did not show any statistically significant difference between biofloc and control system. The enhanced growth and welfare of the cultured fish may be attributed to in situ maintenance of water quality within the biofloc system and presence of microbial floc.

Dietary Bacillus subtilis FPTB13 and chitin, single or combined, modulate systemic and cutaneous mucosal immunity and resistance of catla, Catla catla (Hamilton) against edwardsiellosis.

Effects of dietary administration of Bacillus subtilis FPTB13 and chitin, single or combined, on the systemic immunity, mucosal immunity and resistance of catla (Catla catla) against Edwardsiella tarda infection were investigated. The probiotic attributes of B. subtilis was tested by conducting antagonism study, safety in catla, in vitro immunomodulation and dietary immunomodulation. Results of these studies indicated the probiotic potential of the strain. From the preliminary dietary immunomodulation study, a dose of 10(9) B. subtilis cells g(-1) was selected for inclusion into diets for subsequent experiments. Experimental diets were prepared by adding B. subtilis (10(9) cells g(-1)), chitin (2%) and their combination to the basal diet. Different systemic and mucosal immunological parameters viz. oxygen radical production, myeloperoxidase content, lysozyme activity, total protein content and alkaline phosphatase activity showed significant enhancement (p<0.05) after 2 weeks of feeding with the combined diet. B. subtilis and chitin alone also significantly elevated most of the immune responses. All the diets significantly increased the resistance of catla against E. tarda challenge. The highest post-challenge survival was observed in combined group (i.e. 63.33%). In conclusion, B. subtilis and chitin, alone or combined, had a health ameliorating effect in catla. The results also collectively suggest the usefulness of applying a combined probiotic and immunostimulant supplemented diet to achieve greater benefits.

Inactivated Probiotic Bacteria Stimulate Cellular Immune Responses of Catla, Catla catla (Hamilton) In Vitro.

In the present study, we investigated the in vitro immunostimulatory effects of inactivated form of two potential probiotics, Bacillus amyloliquefaciens FPTB16 and B. subtilis FPTB13 in catla (Catla catla). Catla head kidney leukocytes (HK) were incubated with the bacteria alive or inactivated with heat shock (2 h at 60 °C), UV light (for 2.5 h) and formalin (1.0%, v/v) treatment (for 24 h at 4 °C) at different concentrations (10(7), 10(8) and 10(9) cells ml(-1)). After incubation, different cellular immune parameters such as respiratory burst activity, nitric oxide production, leukocyte peroxidase content and proliferative response were analyzed. The inactivated probiotic preparations stimulated all the cellular immune parameters of catla HK leukocytes in vitro. Among the different inactivated preparations, heat-treated form exhibited the best result. The lowest dose (10(7) cells ml(-1)) of both the strains showed the maximum stimulation. The results collectively suggest the efficacy of inactivated preparations to be used as immunostimulant in aquaculture.

Effect of Edwardsiella tarda immunization on systemic immune response, mucosal immune response and protection in catla (Catla catla).

The effect of immunization on systemic and cutaneous mucosal immune responses of fish and their possible relation with protection has not been fully assessed. In this study, healthy catla (Catla catla) were immunized against Edwardsiella tarda using two antigenic preparations namely, whole cell bacterin (B) and bacterin mixed with Freund's complete adjuvant in a 1:1 (v/v) ratio (B+A) followed by a booster dose after 3 weeks of first injection. Different systemic and cutaneous mucosal immune responses were measured at weekly interval upto 8th week post vaccination (pv). Fish were challenged 8 weeks pv with live E. tarda to study vaccine induced protection. The result showed that although there were strong systemic as well as mucosal immune responses, particularly after booster dose, the challenge produced low to moderate protection in terms of relative percent survival (RPS). The maximum RPS (50 %) was recorded in the adjuvanted bacterin group after 8 weeks pv. Low to moderate protection after challenge, which may be attributed to the intracellular nature of E. tarda and/or use of crude antigenic preparation, accounts for new strategy to be developed for immunization programme against such intracellular pathogen. The results collectively suggest possible involvement of systemic as well as mucosal immune responses in inducing protective immunity in catla.

Effects of potential probiotic Bacillus amyloliquefaciens [corrected] FPTB16 on systemic and cutaneous mucosal immune responses and disease resistance of catla (Catla catla).

Effects of dietary administration of Bacillus amyloliquefaciens FPTB16 on systemic and mucosal immunity and disease resistance of catla (Catla catla) against Edwardsiella tarda infection were evaluated in the present study. The laboratory maintained B. amyloliquefaciens was used to study antagonistic activity against fish pathogenic bacteria by agar well diffusion assay. Healthy catla were challenged by this bacterium for determination of its safety. For preparation of probiotic supplemented diet, the bacteria were added to the basal diet (control) at three different inclusion levels i.e., 1 × 10(9), 1 × 10(8) and 1 × 10(7) CFU/g diet. Fish (weight 25-30 g) were fed with these diets and various immune parameters and disease resistance study were conducted at 4 weeks and 8 weeks post-feeding. The bacterial antagonism study showed inhibition zone against E. tarda, Aeromonas hydrophila, Vibrio parahaemolyticus and V. harveyi. B. amyloliquefaciens was harmless to catla as neither mortalities nor morbidities were observed after the challenge. Study of different systemic and mucosal immunological parameters viz. superoxide anion production and nitric oxide production, myeloperoxidase content, lysozyme activity and total protein content showed significant enhancement (p < 0.05) in fish fed with 10(8) and 10(9) CFU/g B. amyloliquefaciens at both time points with the highest values observed in case of 10(9) CFU/g. For fish fed with 10(7) CFU/g B. amyloliquefaciens, all the parameters showed significant enhancement (p < 0.05) at both time points except the lysozyme activity of serum at 8 weeks. Diet containing 10(8) and 10(9) CFU/g B. amyloliquefaciens significantly enhanced (p < 0.05) the resistance of catla against bacterial challenge at both time points. These results collectively suggest that B. amyloliquefaciens is a potential probiotic species and can be used in aquaculture to improve health status and disease resistance with an optimal dietary supplementation of 10(9) CFU/g.

In vitro immunopathogenesis of Edwardsiella tarda in catla Catla catla (Hamilton).

Immunopathogenesis of Edwardsiella tarda in Catla (Catla catla) was evaluated by in vitro interaction of catla head kidney (HK) phagocytes with the bacterium. Interactions of E. tarda with catla HK phagocytes were studied by light microscopy as well as by phagocytosis, intracellular replication, superoxide anion and nitric oxide production assays. Both opsonised and non-opsonized bacteria could adhere to, survive and replicate within phagocytes. Furthermore, there was no significant alteration in superoxide anion production by phagocytes when infected with E. tarda, indicating that they were able to avoid and/or resist reactive oxygen species mediated killing by the phagocytes. Enhancement of nitric oxide production indicated its possible involvement as inflammation mediator to facilitate pathogenesis. The results suggest the fact that E. tarda strain has the ability to resist phagocyte mediated killing, thereby proliferating within the host and causing infection.

Low pH, dissolved oxygen and high temperature induces Thelohanellus rohita (myxozoan) infestation in tropical fish, Labeo rohita (Hamilton).

In Labeo rohita, myxozoan infection is very common and here occurrence and distribution of myxozoan gill parasites were studied with an objective to evaluate the relationship between myxozoan infections with different abiotic factors. All ponds were infected with myxozoan infection. Sampling of water and fish was done fortnightly and soil sample on monthly basis for about 6 months. L. rohita is infected with the one myxozoan species Thelohanellus rohita. The first, second and third gill arches showed higher distribution of myxozoans than the fourth one. Posterior hemibranch of second gill arch was the most preferred site for parasite attachment. The mean intensity of parasite was significantly higher (p < 0.05) in the size class of 8-9 cm in comparison to the other size classes. There were profound variability in the prevalence, abundance and mean intensity of gill myxosoporeans from three ponds. During the start of the sampling, myxozoans were present in all the ponds but their intensity varied in different ponds. These may be due to the variability in the abiotic factors of individual ponds. Most importantly acidic pH, lower DO and higher temperature promote myxozoan infestation and their propagation. Low soil pH is also seen to enhance their propagation. Our data clearly highlighted that prevalence, intensity and abundance of T. rohita strongly influenced by the above environmental parameters and suggested that its life cycle is probably effected by their change; our hypothesis must be regarded as speculative as long as further detail study is not carried out.

Effects of water borne iron on spawn of Indian major carps (Catla catla (Ham.), Labeo rohita (Ham.) and Cirrhinus mrigala (Ham.)).

Effects of water-borne iron on Indian major carps spawn were evaluated in the present study. Ferrous sulphate was used to prepare different test iron concentrations. Mrigal had the lowest 96 h LC(50) value of 0.30 ± 0.06 mg L(-1) while rohu had the highest value of 0.73 ± 0.06 mg L(-1) of iron. Accumulation of iron in mrigal spawn was highest whereas it was lowest in catla. Abnormal behaviour and reduced growth were observed in chronic toxicity. Application factors were calculated to establish acceptable ranges and safe levels.

Immune responses and protection in catla (Catla catla) vaccinated against epizootic ulcerative syndrome.

Three different antigenic preparations from the epizootic ulcerative syndrome (EUS) pathogen Aphanomyces invadans were evaluated as vaccine candidate in catla (Catla catla). Anti-catla enzyme immunoconjugate was prepared after isolating catla immunoglobulin and raising hyperimmune sera against it, in rabbit. Three antigens namely, fungal extract (FE), fungal extract mixed with Freund's incomplete adjuvant (FIA) in a 1:1 (v/v) ratio (FE + A) and extra cellular product (ECP) were prepared and three groups of catla were vaccinated intramuscularly with all these antigens (200 µg/fish). Different cellular and humoral immune responses were measured for the entire vaccinated and control group on 0th, 5th, 15th and 25th day post vaccination. Thirty days after the vaccination, the fish were challenged with an A. invadans zoospore dose of 1 × 10(5) ml(-1) and mortality and relative percent of survival (RPS) were recorded. Study of cellular immunological parameters including antigen-specific leukocyte proliferation, antigen-specific nitric oxide production and superoxide anion production showed significantly higher (p < 0.05) values, in general, on 5th and 15th day post vaccination than the 0th day. Among all the antigenic groups, FE + A showed most significant response compared to the other groups. Among the humoral immune responses, lysozyme activity showed almost similar trend like cellular parameters. Anti-Aphanomyces antibody production was measured by enzyme-linked immunosorbent assay (ELISA) and it was increased with increasing days of vaccination in all the vaccinated groups with the highest observed on 25th day. Among the antigens, FE + A showed the highest antibody production following vaccination. The result of the homologous pathogen challenge study showed reduction in mortality in all the vaccinated groups. However, this reduction was not statistically significant (p > 0.05). Increased immune responses and protection have important implications with regard to the control of EUS by vaccination.

Effect of geometric and process variables on the performance of inclined plate settlers in treating aquacultural waste.

Inclined plate settlers are used in treating wastewater due to their low space requirement and high removal rates. The prediction of sedimentation efficiency of these settlers is essential for their performance evaluation. In the present study, the technique of dimensional analysis was applied to predict the sedimentation efficiency of these inclined plate settlers. The effect of various geometric parameters namely, distance between plates (w(p)), plate angle (alpha), length of plate (l(p)), plate roughness (epsilon(p)), number of plates (n(p)) and particle diameter (d(s)) on the dynamic conditions, influencing the sedimentation process was studied. From the study it was established that neither the Reynolds criterion nor the Froude criterion was singularly valid to simulate the sedimentation efficiency (E) for different values of w(p) and flow velocity (v(f)). Considering the prevalent scale effect, simulation equations were developed to predict E at different dynamic conditions. The optimum dynamic condition producing the maximum E is also discussed.