Bioaccumulation of TBT and Its Cellular Toxic Effects on the Freshwater Prawn Macrobrachium rosenbergii.

To test the toxic effects of tributyltin (TBT), Macrobrachium rosenbergii were exposed to three concentrations of TBT viz. 10 ng/L, 100 ng/L and 1000 ng/L for 90 days. The bioaccumulation of TBT level varied in hepatopancreas based upon dose dependent manner. Histopathological results revealed the reduction in basement membrane thickness, disruption of the hepatopancreatic tubules and abnormal lumen in hepatopancreas of TBT treated prawns. The ultrastructure of the control prawn showed normal architecture of cellular organelles with prominent nuclei in hepatocytes. On the other hand, many vacuoles, irregular arrangements of microvilli, swollen mitochondria, distorted rough endoplasmic reticulum cisternaes and abnormal nucleus were seen in the TBT treated group. Further, the biochemical and vitellogenin content were altered remarkably due to TBT exposure. It directly indicated that TBT had conspicuously inhibited the vitellogenesis. Therefore, it was inferred that the administration of TBT has considerably affected the hepatopancreatic functions in M. rosenbergii.

Screening, partial purification of antivibriosis metabolite sterol-glycosides from Rhodococcus sp. against aquaculture associated pathogens.

The present study probed the antimicrobial potential of a rare mangrove associated actinomycetes against an array of aquatic bacterial pathogens causing disease outbreak in fin and shellfish. Antibacterial activity results implied that the mangrove associated actinomycetes RAS7 exhibited striking inhibitory activity against the tested aquatic bacterial pathogens. Identification of strain RAS7 through polyphasic and 16S rRNA sequencing affirmed that the strain belongs to Rhodococcus sp. Optimization of culture conditions for antibacterial activity by Rhodococcus sp. inferred that it grew well and exerted notable antagonistic activity in medium supplied with 1% galactose and peptone as carbon and nitrogen sources. Similarly, the strain grown in 0.1% tyrosine, 1% NaCl, pH 7.5 and temperature 35 °C recorded maximum bioactivity against the test pathogens. The crude ethyl acetate extract of Rhodococcus sp. at 200  µg/ml recorded markedly pronounced growth inhibitory activity ranged between 14 and 29 mm. The cytotoxic effect of crude extract against brine shrimp Artemia salina nauplii registered LC50 value of 134.294 µg/ml after 24 h of exposure. The secondary metabolite was separated using Ethyl acetate: Methanol (7:3) as solvent system through TLC. The TLC autobiogram mapped the active spot in TLC with Rf value of 0.84. Analysis of chemical constituents and FT-IR spectral analysis substantiated that the active principle in bioassay guided fraction was sterol-glycosides.

Environmentally benign antifouling activity and toxic properties of bioactive metabolites from mangrove Excoecaria agallocha L.

This study was aimed to investigate the antifouling (AF) potentials and toxic properties of methanol extract from leaves of mangrove Excoecaria agallocha. Antimicrofouling activity results inferred that this extract strongly inhibited fouling bacterial and microalgal growth. This extract had also inhibited the settlement of brown mussel Perna indica and larvae of barnacle Balanus amphitrite. Further, EC50 < LC50 and therapeutic ratio > 1 together propagated non-toxic nature of the extract. Mollusk foot adherence assay result showed complete inhibition of foot spreading and loss of attachment of common rocky fouler Patella vulgata to the substrata. Field assay results affirmed that this extract effectively deterred settlement of biofoulers. Purification and GC-MS analysis of bioassay-guided active spot evidenced presence of three major compounds (> 85%) responsible for the promising AF activity. The identified lead compounds subjected to an estimation (BIOWIN™) program developed by United States Environmental Protection Agency (USEPA) predicts that they are biodegradable in nature. Graphical abstract.

Characterization of haloalkalophilic organic solvent tolerant protease for chitin extraction from shrimp shell waste.

Halophilic organic solvent tolerant protease (HOSP) producing Paracoccus saliphilus APCMST-CS5 was isolated from the marine sediment samples and identified through 16S rRNA sequence analysis. P. saliphilus APCMST-CS5 registered maximum HOSP production of 1,321.70U/ml in the medium contained the most significant parameters such as shrimp shell powder (SSP), CaCl2, NaCl, and sardinella powder (SP), obtained through Placket-Burman and Response Surface Methods. HOSP was further purified to 22.68 fold purity with 29.71 U/mg specific activity and its molecular weight was 39kDa. The HOSP was stable at 60°C, 9.0 pH, 3.0M NaCl concentration and it also showed maximum activity at other tested parameters. Interestingly the purified HOSP showed better antibiofilm ability against tested pathogens. Also, the HOSP effectively deproteinized (85.64%) shrimp shell chitin which in turn maximum and exhibited higher antioxidant activity. The commercial and experimental shrimp shell chitin showed similar peak pattern in FTIR and 13C CP/MAS NMR spectral analysis.

Biosynthesis, purification and characterization of ß-1,4-xylanase from a novel mangrove associated actinobacterium Streptomyces olivaceus (MSU3) and its applications.

The present study was undertaken to evaluate the xylanolytic properties of an actinobacterium Streptomyces olivaceus (MSU3) isolated from the sediment sample of mangrove environment. It showed highest xylanase activity on initial screening in Breg's mineral salts medium supplemented with 0.5% xylan. Further the organism expressed maximum xylanase production at optimized culture conditions of pH 7, temperature 30 °C with 2.5% of inoculum size at 72 h of incubation, the nutrient sources like sucrose (2%) and yeast extract (3%) have observed as best carbon and nitrogen sources respectively. In purification, the xylanase resulted 4.27fold increase with the yield of 15.57% at the final step using sephadex G-75 chromatography. The molecular weight of the purified xylanase was observed as 42 kDa on 10% SDS-PAGE and further identified by MALDI-TOF-MS analysis. It belongs to GH43 family encoded 485 amino acid residues and the xylanase gene isolated using PCR amplification was partially sequenced. It showed 98% sequence similarity to the xylanase gene of S. olivaceus. The maximum activity of purified xylanase was observed at pH 8, temperature 40 °C and also the production medium substituted with Fe3+ metal ion, 2.0% xylan and 1.5% NaCl along with Km and Vmax values of 8.16 mg/ml and 250.01 µg/min/mg, respectively. In xylanolytic hydrolysis of pretreated agro-wastes, especially the sugarcane juice substituted medium yielded maximum (52.19%) reducing sugar, followed by bioethanol production (4.19  g/L) at 72 h of incubation. Based on the results, it could be confirmed that the selected isolate is a potent strain and it can able to produce xylanase through fermentation process and also it can able to convert the pretreated agro-wastes into economically important byproduct like bioethanol.

In vitro antioxidant activities of an exopolysaccharide from a salt pan bacterium Halolactibacillus miurensis.

Exopolysaccharide (EPS) designated as HMEPS was isolated from Halolactibacillus miurensis and purified through gel filtration chromatography. EPS extracted from the supernatant yielded a 56.1% total carbohydrate content. The ash and moisture content were 12.1% and 1.5% respectively. Galactose and glucose were found as main monosaccharides of the HMEPS through HPLC analysis. FT-IR spectra of the HMEPS revealed its composition with hydroxyl, alkenes, amide and carboxyl as functional groups and aliphatic amine and alkynes at the fingerprint region. In vitro antioxidant activity was investigated against hydroxyl, DPPH, superoxide free radicals and the scavenging activity against all were found to be dose dependent proportionately. HMEPS showed higher reducing ability against superoxide radical and potency in chelating the ferrous ions. 10mg/ml of HMEPS was found equivalent to 2.7units of ascorbic acid through the total antioxidant assay. Phylogenetic relation of H. miurensis SEEN MKU3 (GenBank number KT803852) was plotted with MEGA 5.0.

Pharmacological importance of sulphated polysaccharide carrageenan from red seaweed Kappaphycus alvarezii in comparison with commercial carrageenan.

Pharmacological properties of native carrageenan (κ) extracted from Kappaphycus alvarezii and commercial carrageenan (Sigma-Aldrich) were evaluated using in vitro antioxidant, anticancer and antidiabetic studies. Phytochemical analysis of native and commercial carrageenans showed the presence of alkaloids, saponins, steroids, gums & mucilages and carbohydrate. Both native and commercial carrageenans exhibited better antioxidant activities such as total antioxidant capacity (87±0.47 and 82.6±0.47µg A.A/g), hydroxyl radical scavenging activity (61.4±0.27 and 58.66±0.31µg/ml), nitric oxide radical scavenging activity (80.42±0.22 and 73.66±0.22µg/ml), DPPH radical scavenging activity (56.26±0.20 and 53.67±0.082µg/ml) and reducing power assay (46.57±0.32 and 42.54±0.27µg/ml) at the maximum concentration of 100µg/ml carrageenans. These results indicated that native carrageenan from K. alvarezii possessed better antioxidant potential in comparison with commercial carrageenan. Anticancer activities of both carrageenans showed excellent inhibition on the growth of breast, colon, liver and osteosarcoma cell lines at the maximum concentration of 150µg/ml. Native carrageenan exhibited an excellent anticancer activity on colon carcinoma cell lines (67.66±0.168%) with the IC50 value of 73.87µg/ml and commercial carrageenan possessed a potent inhibition on the growth of breast cancer cell lines (67.33±0.077%) with the IC50 value of 123.8µg/ml. These results clearly indicated the beneficial effect of native and commercial carrageenans as anticancer agents being a free radical scavenger. Anti-diabetic property of both carrageenans showed inhibition effect on α- glucosidase enzyme. The inhibitory effect depends on concentration of carrageenans and it was recorded that maximum (74.49±1.05 and 67.42±0.63) inhibitory effect of α- glucosidase enzyme at 500µg/ml concentration.

In vitro-Scientific evaluation on antimicrobial, antioxidant, cytotoxic properties and phytochemical constituents of traditional coastal medicinal plants.

In the present study, evaluation of antimicrobial, antioxidant, phytochemical constituents and toxicological properties of six coastal medicinal plants (CMP's) such as Ipomea biloba, Cantharanthus roseus, Cymbopogon citratus, Vitex negundo, Thespesia populnea and Pandanus tectorius was done. The maximum antimicrobial activity was recorded by methanolic extracts of V. negundo and T. populnea against bacterial and fungal pathogens. Similarly, methanolic extracts of V. negundo and T. populnea evidenced highest antioxidant properties. The extract of T. populnea showed the maximum cytotoxicity against Artemia salina with the LC50 value of 478.11µg/ml. The hemolytic property of CMP's extracts was V. negundo (8.91%), T. populnea (21%) and C. citratus (64%) also the hemolytic index did not show any hemolysis of human erythrocytes. Furthermore, the TLC separation of V. negundo and T. populnea extracts exhibited the presence of Flavonoids (Rf-0.74) and Terpenoids (Rf-0.64). The present findings propose the promising clinical applications of selected CMP's.

Production, purification and characterization of halophilic organic solvent tolerant protease from marine crustacean shell wastes and its efficacy on deproteinization.

The quantum of marine fish wastes produced by fish processing industries has necessitated to search new methods for its disposal. Hence, this study is focused on production and purification of halophilic organic solvent tolerant protease (HOSP) from marine Alcaligenes faecalis APCMST-MKW6 using marine shell wastes as substrate. The candidate bacterium was isolated from the marine sediment of Manakudi coast and identified as A. faecalis APCMST-MKW6. The purified protease showed 16.39-fold purity, 70.34 U/mg specific activity with 21.67 % yield. The molecular weight of the purified alkaline protease was 49 kDa. This purified protease registered maximum activity at pH 9 and it was stable between pH 8-9 after 1.30 h of incubation. The optimum temperature registered was 60 °C and it was stable between 50 and 60 °C even after 1.30 h of incubation. This enzyme also showed maximum activity at 20 % NaCl concentration. Further, manganese chloride, magnesium chloride, calcium chloride and barium chloride influenced this enzyme activity remarkably and it was also found to be enhanced by many of the tested surfactants and solvents. The candidate bacterium effectively deproteinized the shrimp shell waste compared to the other tested crustaceans shell wastes and also attained maximum antioxidant activity.

Deproteinization potential and antioxidant property of haloalkalophilic organic solvent tolerant protease from marine Bacillus sp. APCMST-RS3 using marine shell wastes.

The current increase in the vast amount of marine crustacean shell waste produced by the fish processing industries has led to the need to find new methods for its disposal. Hence, the present study was carried out via marine shell wastes as substrate for protease production. The maximum production (4000.65 U/ml) from Bacillus sp. APCMST-RS3 was noticed in 3:1% shrimp and oyster shell powder (SOSP) as substrate. Purified protease showed 53.22% and 22.66% enzyme yield; 3.48 and 8.49 fold purity with 40 kDa molecular weight; whereas, its Km and Vmax values were 0.6666 g/l, 1111.11 U/ml. This enzyme showed optimum activity at pH 9 and 60 °C temperature. Also, it retained maximum protease activity in the presence of NaCl (2.5 M), surfactants (Tween 20, 40, 60, 80 and SDS) and metal ions (MnCl2, CaCl2, HgCl2 and BaCl2) and solvents. The candidate bacterium effectively deproteinized (84.35%) shrimp shell and its antioxidant potentials.

Dietary effect of Sargassum wightii fucoidan to enhance growth, prophenoloxidase gene expression of Penaeus monodon and immune resistance to Vibrio parahaemolyticus.

The polysaccharide fucoidan from brown seaweed Sargassum wightii was extracted and it was incorporated with pellet diets at three concentrations (0.1, 0.2 & 0.3%). The fucoidan incorporated diets were fed to shrimp Penaeus monodon for 60 days and the growth performance was assessed. The weight gain and SGR of control group was 6.83 g and 9.72%, respectively, but the weight gain and SGR of various concentrations (0.1-0.3%) of fucoidan incorporated diets fed groups of shrimp was increased from 7.30 to 8.20 g and 9.83 to 10.03%, respectively. After 60 days of feeding experiment, the relative quantification of prophenoloxidase gene of experimental groups over control group was analysed by RT-PCR and it was ranged between 2.13 and 7.95 fold increase within 33.52-34.61 threshold cycles, respectively at 0.1-0.3% concentrations of fucoidan. After 60 days of feeding experiment, the P. monodon were challenged with shrimp pathogen Vibrio parahaemolyticus and the mortality percentage was recorded daily up to 21 days. The reduction in mortality percentage of experimental groups over control group was recorded from 44.56 to 72.79%, respectively in 0.1-0.3% of fucoidan incorporated diets fed groups. During challenge experiment, all the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity, superoxide dismutase activity, phagocytic activity, bactericidal activity and bacterial clearance ability of experimental groups were significantly (P < 0.05) increased than control group. The V. parahaemolyticus load was enumerated from the infected shrimp at every 10 days intervals during challenge experiment. In control group, the Vibrio load was increased in hepatopancreas and muscle tissues from 10th to 21st days of challenge test. But in the experimental groups, the Vibrio load in both the tissues decreased positively from 10th to 21st days of challenge duration. It is concluded that the S. wightii fucoidan had enhanced the innate immunity and increased resistance to V. parahaemolyticus infection in P. monodon.

Antifouling and toxic properties of the bioactive metabolites from the seagrasses Syringodium isoetifolium and Cymodocea serrulata.

The present study documents the antifouling and toxic properties of seagrasses Syringodium isoetifolium and Cymodocea serrulata. For that, the seagrasses S. isoetifolium and C. serrulata were extracted individually using organic solvents viz. dichloromethane, acetone and methanol. Amongst the extracts, the maximum antimicrofouling and antimacrofouling activities were exhibited by methanol extracts of both the seagrasses. The Minimal Inhibitory Concentration (MIC) of methanolic extracts of seagrasses was ranged from 1.0 to 10µg/ml against test biofilm bacteria and microalgal strains. Similarly, 100% fouling inhibition of limpet Patella vulgata was found at 6.0mg/ml of methanolic extracts of seagrasses. The mussel Perna indica showed 50% of byssal production and attachment inhibition at 21.51±2.03, 17.82±1.07µg/ml and the anticrustaecean activity for 50% mortality of Artemia salina was recorded at 732.14±9.21 and 394.16±5.16µg/ml respectively for methanolic extracts of S. isoetifolium and C. serrulata. The minimal inhibitory and higher lethal concentrations of active methanol extracts shows it׳s less toxic nature. Based on the prolific results, methanol extracts of S. isoetifolium and C. serrulata were subjected to purification using silica gel column and thin layer chromatography. Then the active compounds of the bioassay guided fractions were partially characterized using gas chromatography coupled with mass spectroscopy (GC-MS) and keyed out that fatty acids (C16 to C24) were the major components which responsible for the antifouling properties of the candidate seagrasses.

Influence of probiotic bacterium Bacillus cereus isolated from the gut of wild shrimp Penaeus monodon in turn as a potent growth promoter and immune enhancer in P. monodon.

A probiotic bacterium isolated from the gut of wild shrimp Penaeus monodon rendered maximum antagonistic activity against shrimp pathogens and was capable of producing extracellular enzymes. The probiotic bacterium was identified as Bacillus cereus through 16S rRNA sequencing. The lyophilized B. cereus was supplemented with shrimp basal diet at four different concentrations (0.1­0.4%/100 g feed) in D1­D4 diets. The viability of probiotic bacterium in the test diets was evaluated during the study period at various time intervals. The viability ranged from 50.24 ± 1.42 to 180.34 ± 1.30 CFU/g in D1 to D3 diets on the 30th day, whereas it was slightly declined from 45.23 ± 1.30 to 169.13 ± 1.18 CFU/g during the 90th day of storage. A control diet (C), devoid of probiotic supplementation was also simultaneously prepared. During experimentation, P. monodon postlarvae (PL-15) were cultured in individual one tonne capacity FRP tanks in triplicates provided with equal amount of substratum (clay soil) and fed with these respective diets at ad libitum for 90 days. Survival was high (82.0 ± 1.60%) in D4 diet fed shrimp as against a low survival of 65.0 ± 1.33% displayed by control diet fed shrimp. Overall growth responses inferred that a maximum production of 10.45 ± 0.275 g, SGR of 4.40 ± 0.179% and a better FCR of 1.27 ± 0.081 were obtained in D4 diet fed shrimp. However, the water quality parameters showed nonsignificant (P > 0.05) variations among the control and the probiotic treated groups. The tested immunological parameters such as Total haemocyte count, phenoloxidase activity, respiratory burst activity, lysozyme activity, plasma protein concentration and bactericidal activity were higher in D4 diet fed P. monodon, when compared to that of other diets fed shrimp. It is therefore suggested that lyophilized probiotic B. cereus at a concentration of 0.4%/100 g feed was efficient in stimulating the growth and immunity in shrimp.

Optimization of bacteriocin production by Lactobacillus sp. MSU3IR against shrimp bacterial pathogens.

BACKGROUND: Aquaculture is one amongst the growing and major food producing sectors. Shrimp culture is one of the subsectors of aquaculture that attracts more attention because of the economic interest. However, the shrimp culture systems have been facing severe consequences and economical losses due to disease outbreaks. Risk of disease outbreak can be combated with the application of probiotics. For economically viable production of such probiotic products, the present study provides information on the optimization and partial purification of bacteriocin produced by a goat milk isolate Lactobacillus sp. MSU3IR against the shrimp bacterial pathogens. RESULTS: Bacteriocin production was estimated as a measure of bactericidal activity (arbitrary Unit/ml) over the test strains. The optimum culture conditions and media components for maximum bacteriocin production by Lactobacillus sp. MSU3IR were: pH: 5.0, temperature: 30°C, carbon source: lactose; nitrogen source: ammonium acetate; NaCl: 3.0% and surfactant: Tween 80. MRS medium was found to extend better bacteriocin production than other tested media. Upon partial purification of bacteriocin, the SDS-PAGE analysis had manifested the presence of two peptide bands with the molecular weight of 39.26 and 6.38 kDa, respectively. CONCLUSION: The present results provide baseline trend for the statistical optimization, scale up process and efficient production of bacteriocin by the candidate bacterial strain Lactobacillus sp. MSU3IR which could be used to replace the usage of conventional chemotherapeutics in shrimp culture systems.

Sodium alginate from Sargassum wightii retards mortalities in Penaeus monodon postlarvae challenged with white spot syndrome virus.

Sodium alginate extracted from brown seaweed Sargassum wightii (16.35 ± 1.42%, mean [±SD] yield from 5 extractions) was prepared as a powder or beads and used to enrich Artemia nauplii at concentrations of 100, 200, 300 and 400 mg l-1. The alginate-enriched nauplii were fed to Penaeus monodon shrimp postlarvae (PL) stage 15 (PL15, i.e. 15 d old) for 20 d. Mean weight gain and specific growth rate over this period were 0.24 g and 15.8%, respectively, in PL groups not fed alginate, and 0.20-0.28 g and 14.7-16.5%, respectively, in PL groups fed alginate. Amongst PL35 then challenged with white spot syndrome virus (WSSV) by immersion, all PL not fed alginate died within 9 d. However, amongst PL fed the 4 concentrations of alginate powder or beads, mortality rates reduced with increasing alginate concentration, and between 25 and 32% PL remained alive when the bioassay was terminated on Day 21. Amongst alginate-fed PL groups compared with the control group, mortality was reduced by 26.5 to 58.4%. Nested PCR detection of WSSV revealed sodium alginate concentration-dependent reductions in infection loads. The data indicate that sodium alginate extracted from brown seaweed and fed to P. monodon can retard progression of WSSV disease.

The effect of fucoidan from brown seaweed Sargassum wightii on WSSV resistance and immune activity in shrimp Penaeus monodon (Fab).

The polysaccharide-fucoidan was extracted from brown seaweed Sargassum wightii and characterized through FT-IR and (13)C &(1)H NMR analysis. The extracted fucoidan was supplemented with pellet diets at three different concentrations (0.1, 0.2 and 0.3%). The fucoidan supplemented diets were fed to Penaeus monodon for 45 days, then challenged with WSSV and the mortality percentage was recorded daily up to 21 days. During the challenge test, the control group showed 100% mortality within 10 days, but in the experimental groups, the mortality percentage (51-72% within 21 days) was decreased considerably (P < 0.05) with respect to the concentrations of fucoidan. The reduction in mortality percentage of experimental groups over control group was ranged from 50.81 to 68.06%. During challenge experiment, the immunological parameters such as THC, prophenoloxidase activity, respiratory burst activity, superoxide dismutase activity and phagocytic activity were measured before injection of WSSV (0 day) and after the injection of WSSV on 10th and 21st days, respectively. All the immunological parameters of experimental groups were significantly (P < 0.05) increased than control group. RT-PCR analysis confirmed the considerable reduction of WSSV DNA copy numbers with respect to the concentration of fucoidan. It was concluded that P. monodon fed with fucoidan of S. wightii supplemented diet had enhanced the innate immunity and increased resistance against WSSV infection.

Antipyretic, wound healing and antimicrobial activity of processed shell of the marine mollusc Cypraea moneta

Objective: Some of the products derived from marine organisms have been recommended in alternative system of medicine especially Siddha medicine for several treatments. Among the marine molluscs, Cypraea moneta shell has been used as siddha medicine from ancient days. But no systematic study has been done on its efficacy as antipyretic, wound healing and as antimicrobial agent. In the present study, the protective action of processed shell powder of C. moneta was evaluated by us in an animal model for the above treatments. Methods: C. moneta shell powder was prepared by standard method described in Siddha medicine. Then the antipyretic, wound healing as well as antimicrobial effect of the processed powder was tested in Wister albino rats. Results: By the intravenous injection of yeast cell suspension into albino rats, the antipyretic effect of the shell powder given orally was studied by various concentrations of 0, 10, 20 and 30 mg/ml. The body temperature of the albino rat became normal within a short duration (3h). The wound healing effect of the shell powder was very effective. In the thigh region 2 cm wound was made and the different dosages of shell powder (C -Control, SD - Single dose, DD -Double dosage and TD -Triple dose/day) were applied externally as ointment. The scar was produced in eighth day onwards in DD and TD. Antimicrobial activity was studied in three different oppurtunistic human pathogens such as Micrococcus sp., Proteus vulgaris andSalmonella abory in different concentrations (2, 3, 4 and 5% w/v) of C. moneta shell powder extract. Among these, Proteus vulgaris showed the maximum zone of inhibition (15mm size) against 5% w/v concentration, followed by Micrococcus sp. (12mm) and S. abory (10mm) against the same concentration. Conclusions: The present observation suggested that, processed C. moneta shell powder can be used as an alternative medicine, and it has antipyretic, wound healing as well as antimicrobial properties.