Immuno-stimulatory effect and toxicology studies of salt pan bacteria as probiotics to combat shrimp diseases in aquaculture.

The shrimp aquaculture industry has experienced serious economic losses due to diseases caused by Vibrio species. The application of antibiotics to combat diseases has led to environmental hazards, antibiotic-resistance in pathogens and accumulation of antibiotics in tissues. This study explores the use of probiotics as an alternative to antibiotics. A probiotic consortium SFSK4 (comprising salt pan bacteria Bacillus licheniformis TSK71, Bacillus amyloliquefaciens SK27, Bacillus subtilis SK07, Pseudomonas sp. ABSK55) was used as a water additive during shrimp culture. It significantly increased shrimp (Litopenaeus vannamei) immunity i.e. total hemocyte count, phagocytosis, total plasma protein, respiratory burst and bactericidal activity as compared to the control. It also stimulated the phenoloxidase activity by two-fold. Proteomic analysis revealed the differential expression of 50 immune proteins (39 up-regulated and 11 down-regulated) in SFSK4 treated shrimps. Four major immune modulation proteins viz. Caspase2, GTPase activating protein, Hemocyanin and Glucan pattern-recognition lipoprotein involved in cell mediated immune response were identified in SFSK4 treated shrimp hemolymph. SFSK4 decreased shrimp mortality by more than 50% against pathogens. Toxicology studies revealed that administration of the highest dose of probiotic (1012 CFU/mL) showed no adverse effect on shrimp survival (LC50 analysis) and neither exhibited cytotoxicity. Genotoxicity study confirmed that the probiotic did not cause DNA damage in shrimps. The findings suggest that the probiotic SFSK4 is an eco-friendly water additive to enhance shrimp immunity against diseases in aquaculture, which could help curtail environmental hazards as an effective alternative to antibiotics.

DNA damage and biochemical responses in estuarine bivalve Donax incarnatus (Gmelin, 1791) exposed to sub-lethal concentrations of an organophosphate pesticide monocrotophos.

Monocrotophos (MCP) is a highly toxic and broad-spectrum pesticide extensively used for agricultural and household purposes. The present study was aimed to evaluate the genotoxicity and alterations in the biochemical and physiological conditions induced by monocrotophos in a non-target organism, an estuarine bivalve, Donax incarnatus. The bivalves were exposed to three sub-lethal concentrations (6.8, 13.7, and 27.45 ppm) of MCP for a period of 72 h. DNA damage was assessed using the comet assay. Oxidative stress was analyzed using catalase, glutathione peroxidase, and superoxide dismutase. Neurotoxicity was evaluated using the acetylcholinesterase assay (AChE) and the physiological condition was assessed using the condition index (CI). A significant concentration-dependent increase of DNA damage was observed as well as a decline in the activities of the antioxidant enzymes. However, a decrease in DNA damage was observed with advancing time. A significant decrease of AChE activity and CI was observed in the bivalves exposed to MCP. Positive correlations were also observed between DNA damage and the antioxidant enzymes whereas negative correlations were observed between AChE and the antioxidant enzymes indicating MCP toxicity mediated by oxidative stress.

Tellurite biotransformation and detoxification by Shewanella baltica with simultaneous synthesis of tellurium nanorods exhibiting photo-catalytic and anti-biofilm activity.

Tellurite reducing bacterial strain was isolated from Zuari estuary, Goa India which could tolerate 5.5 mM potassium tellurite with a minimum inhibitory concentration of 6 mM. This strain was designated as GUSDZ9 and was identified as Shewanella baltica (accession number: MF350629) based on 16S rRNA gene sequencing and BLAST analysis. The Diethyl-dithiocarbamate based colorimetric analysis clearly demonstrated a complete reduction of 2 mM tellurite to elemental tellurium during the late stationary phase. Te Nanoparticles (TeNPs) biosynthesis which initiated at early log phase (i.e. 4 h) was evidently monitored through colour change and a peak due to surface plasmon resonance at 210 nm using UV-Vis spectroscopic analysis. X-ray crystallographic studies and transmission electron microscopy revealed unique nano-rods with a diameter ranging from 8 to 75 nm. Energy dispersive X-ray analysis further confirmed the presence of pure tellurium. The biogenic TeNPs at 10 and 5 µg/mL evidently demonstrated 90% degradation of methylene blue dye and anti-biofilm activity against potential Gram-positive and Gram-negative human pathogens respectively. The alkaline comet assay revealed time and dose-dependent genotoxicity at concentrations higher than 15 µg/mL of TeNPs. This study clearly demonstrated the potential of Shewanella baltica strain GUSDZ9 in bioremediation of toxic tellurite through bio-reduction into elemental tellurium and involvement of biogenic TeNPs in the photo-catalytic reduction of methylene blue and anti-biofilm activity. This is the first report of its kind on the synthesis of biogenic TeNPs from Shewanella baltica demonstrating photo-catalytic, anti-biofilm activity as well as genotoxicity.

Bioaccumulation of trace metals and total petroleum and genotoxicity responses in an edible fish population as indicators of marine pollution.

The present study reports the genetic damage and the concentrations of trace metals and total petroleum hydrocarbons prevailing in natural populations of an edible fish, Arius arius in different seasons along the coast of Goa, India as an indicator of the pollution status of coastal water. Fish were collected from a suspected polluted site and a reference site in the pre-monsoon, monsoon and post-monsoon seasons. Physico-chemical parameters as well as the concentrations of total petroleum hydrocarbons (TPH) and trace metals in the water and sediment as well as the tissues of fish collected from these sites were recorded. The genotoxicity status of the fish was assessed employing the micronucleus test and comet assay. A positive correlation (p<0.001) was observed between the tail DNA and micronuclei in all the fish collected. Multiple regression analysis revealed that tissue and environmental pollutant concentrations and genotoxicity were positively associated and higher in the tissues of the fish collected from the polluted site. Pollution indicators and genotoxicity tests, combined with other physiological or biochemical parameters represent an essential integrated approach for efficient monitoring of aquatic ecosystems in Goa.

Evaluation of DNA damage induced by gamma radiation in gill and muscle tissues of Cyprinus carpio and their relative sensitivity.

The effect of radiation on the aquatic environment is of major concern in recent years. Limited data is available on the genotoxicity of gamma radiation on different tissues of aquatic organisms. Hence, the present investigation was carried out to study the DNA damage induced by gamma radiation in the gill and muscle tissues and their relative sensitivity using the comet assay in the freshwater teleost fish, common carp (Cyprinus carpio). The comet assay was optimized and validated in common carp using cyclophosphamide (CP), a reference genotoxic agent. The fish were exposed (acute) to various doses of gamma radiation (2, 4, 6, 8 and 10Gy) and samplings (gill and muscle tissue) were done at regular intervals (24, 48 and 72h) to assess the DNA damage. A significant increase in DNA damage was observed as indicated by an increase in % tail DNA for all doses of gamma radiation in both tissues. We also observed a dose-related increase and a time-dependent decrease of DNA damage. In comparison, DNA damage showed different sensitivity among the tissues at different doses. This shows that a particular dose may have different effects on different tissues which could be due to physiological factors of the particular tissue. Our study also suggests that the gills and muscle of fish are sensitive and reliable tissues for evaluating the genotoxic effects of reference and environmental agents, using the comet assay.

Microplastics in decapod crustaceans: Accumulation, toxicity and impacts, a review.

The presence of microplastics in the aquatic environment poses a serious threat not only to aquatic organisms but also to human beings that consume them. The uptake and effects of microplastics have been studied in almost all groups of aquatic organisms. This review details the different aspects of microplastics exposure in an ecologically and economically important group of crustaceans, the Decapods. A majority of Decapod crustaceans such as prawns, shrimp, crabs, lobsters and crayfish are consumed as seafood and play important roles in food chains and food webs. Numerous studies are available on the accumulation of microplastics in tissues such as the gills, hepatopancreas and gastrointestinal tract in these organisms. Experimental studies have also highlighted the toxic effects of microplastics such as oxidative stress, immunotoxicity and reproductive and developmental toxicity in them. This review also summarizes the ecological impacts and implications in human beings as well as lacunae with regard to microplastic uptake in Decapods.

The Backwater Clam (Meretrix casta) as a bioindicator species for monitoring the pollution of an estuarine environment by genotoxic agents.

The coast of Goa receives anthropogenic stress through its major rivers, which carry mining wastes, including iron and manganese ores from upstream mining sites, and petroleum hydrocarbons from shipping activities. These contaminants show seasonal variation in concentration and may be bioaccumulated by fauna inhabiting these waters. These fauna, including the bivalve molluscs, are particularly at risk due to these insults. In the present study, the use of the backwater clam, Meretrix casta (Chemnitz), as a bioindicator species was evaluated, comparing two sites (Vasco and Palolem) on the Goan coast. DNA damage was assessed in the gill cells using the micronucleus and comet assays; physiological condition was determined from the condition index. These values were tested for correlations with the concentrations of total petroleum hydrocarbons and trace metals in the whole soft tissues and with the physico-chemical parameters of water from these sites. Specimens collected from Vasco showed high incidence of micronuclei and % tail DNA and a low condition index ratio compared to those from Palolem, which correlates with the higher level of pollutants in the bivalves the former site. We believe that M. casta is a suitable species for biomonitoring studies of this type.

The effect of gamma radiation on the Common carp (Cyprinus carpio): In vivo genotoxicity assessment with the micronucleus and comet assays.

Radioactive wastes may be leached into freshwater, either accidentally or in industrial effluents. We have studied gamma radiation-induced DNA damage in the freshwater fish Cyprinus carpio. Fish were irradiated with 2-10Gy gamma radiation and genotoxic effects in blood cells were studied with the micronucleus (MN) and comet assays. Micronuclei and a dose-dependent increase in comet-tail DNA were seen in dose- and time-dependent studies. The highest % tail DNA was observed at 24h, declining until 72h, which may indicate the repair of radiation-induced DNA single-strand breaks after gamma radiation. However, double-stranded DNA damage may not have been repaired, as indicated by increased micronuclei at later periods. A positive correlation was observed between the comet and micronucleus assay results. This study confirms the mutagenic/genotoxic potential of gamma radiation in the Common carp, as well as the possible combined use of the micronucleus and comet assays for in vivo laboratory studies with fresh-water fish for screening the genotoxic potential of radioactive pollution.