Binding sites of mosquitocidal toxins of Pseudomonas fluorescens and Bacillus subtilis on pupae and larvae of Culex quinquefasciatus.

Two of the potential bacterial isolates, viz., Pseudomonas fluorescens (VCRC B-426) and Bacillus subtilis (VCRC B-471) whose toxins kill the mosquito pupae/larvae have been identified at our center. As the mode of action of these bacteria are not known, an attempt was made to find out the binding sites of the toxic proteins through immunological methods. Antibodies were raised in BALB/c mice and egg yolk system of chicken layers against the mosquitocidal proteins. The antibodies showed specific binding on to the cephalic and thoracic cuticle of the pupae as well as the paddles of the larvae, indicating the binding of the mosquitocidal proteins.

Bacillus sphaericus in the adults of Culex quinquefasciatus mosquitoes emerged from treated larvae and its effect on development of the filarial parasite, Wuchereria bancrofti.

Bacillus sphaericus is a bio-control agent effective against Culex quinquefasciatus, the vector of bancroftian filariasis. Apart from its larvicidal effect, there are reports of reduced infection of filarial parasites in mosquitoes exposed to it. In the present study, adults of Cx. quinquefasciatus emerged from B. sphaericus treated larvae were fed on blood samples positive for microfilariae of Wuchereria bancrofti and examined at various time intervals to assess the infection level. The rate of infection was reduced from 95% on day 1 post-feeding to 75% on day 13, when fed with blood sample containing 41 mf/20 µl. The mean parasite burden was also reduced from 4.9 per mosquito on day 1 to 2.15 on day 13. When fed with another sample (30 mf/20 µl), the infection was reduced from 100% on day 1 to 80% on day 13. Reduction in parasite burden was 4.0 to 1.75. Abnormally developed second-stage larvae of the parasite were seen in treated mosquitoes. Thus, the results indicated adverse effect of B. sphaericus treatment on infection and development of the filarial parasite in mosquitoes. The possible reason for the parasite regulation was studied through the assessment of the carryover of the bacterium as well as its toxins to the surviving mosquitoes. The presence of B. sphaericus was determined through plating of homogenate of survived mosquitoes on NYSM agar. Toxic protein was detected through immunoblotting. The bacterium as well as its 41.9-kDa toxic protein was found to be transmitted from larvae to adults and affected the parasite development, directly by the toxin or indirectly by eliciting humoral immune response of the mosquito.

Invasion of toxic marine cyanobacteria in to the tsunami affected coastal villages of southern India.

This documentation explores the facts about the invasion of marine cyanobacteria in to the tsunami affected coastal villages of Nagapattinam district of Tamilnadu and Karaikkal district of Pondicherry Union Territory (UT) in southern India. Water samples were collected from eight tsunami-hit coastal villages in different open water sources. The collected samples were processed for detecting marine cyanobacterial growth. Totally 110 water samples were processed, three samples were positive for the toxic cyanobacteria, Lyngbya sp., and nine for nontoxic species such as Epithemia sp.,, Johannesbaptistia pellucida, Oscillatoria princeps, Phormidium fragile, Synechocystis sp. Besides posing a public health risk because of the toxic cyanobacteria, the bloom formation by the cyanobacterial species such as Anabaena, Microcystis, Lyngbya, Plectonema, Phormidium contaminated the water bodies and deteriorated the water quality in the tsunami affected villages. The study revealed that another kind of public health risk from the invasion of toxic cyanobacteria to the costal ecosystem during the tsunami. It is necessary, in this context, that the surveillance mechanism, which is geared up during or after natural disasters, should have a provision to monitor the transportation of toxic elements/organisms from marine system to coastal/inland ecosystems and to control such organisms.

Coconut water as a cheap source for the production of delta endotoxin of Bacillus thuringiensis var. israelensis, a mosquito control agent.

Bacillus thuringiensis var. israelensis (B. t. i.) is being widely used in mosquito control programs. However, the large-scale production of this bacillus is expensive due to the high cost of the production medium. In this study, we attempted to develop a cost-effective medium, based on a locally available raw material namely coconut water which is available in plenty as waste product from coconut oil industry. The yield of cell mass, sporulation and mosquito larvicidal activity were studied by growing this bacterium in this waste product and in comparison with the conventional medium (NYSM). Cell mass yield of 3.1g/L, spore count of 3.4x10(11)spores/mL and mosquito larvicidal activity (LC(50)) of 14.85ng/mL (against early fourth-instar larvae of Aedes aegypti) were obtained with a 30h old culture of this bacterium grown in coconut water. This is almost similar to that obtained with NYSM medium. Hence, coconut water-based culture medium is economical for the production of B. t. i.

Production & purification of a fibrinolytic enzyme (thrombinase) from Bacillus sphaericus.

BACKGROUND & OBJECTIVE: Treatment of thromboembolic vascular disease has relied on anticoagulants. However, recognition that lysis of preformed fibrin could be accomplished in vivo by a process involving the conversion of inactive plasminogen to active plasmin enzyme led to an alternative enzyme-based approach. The drugs used for this therapy are called the fibrinolytic enzymes. In this study we attempted the production, purification and characterization of fibrinolytic enzyme from Bacillus sphaericus. METHODS: The seed was prepared in nutrient yeast salt medium (NYSM) in shake flask and organism was produced in 100 l pilot fermentor. Biomass was separated by centrifugation and crude protein was prepared by ammonium sulphate precipitation. Purification was done by ion exchange chromatography using Q sepharose followed by gel filtration chromatography using Sephacryl S- 300. Molecular weight was determined through HPLC. Fibrinolytic activity was assayed by fibrin plate method. RESULTS: The production method yielded 64 mg/l of the crude enzyme and after purification it was 6.3 mg/l. The molecular weight of the compound was 18.6 kDa. INTERPRETATION & CONCLUSION: The enzyme exhibited similar fibrinolytic activity as that of streptokinase, on fibrin plates that were devoid of plasminogen, suggesting that its fibrinolytic action is independent of plasminogen and it is not a plasminogen activator.

Muscoid fly populations in tsunami-devastated villages of southern India.

Several coastal villages of southern India were affected by the 26 December 2004 tsunami, and 10,749 people were killed. Investigation carried out in the affected villages during fourth, fifth, and sixth weeks posttsunami showed that the fly density was in the range of 12-91.8 flies per sweep net. In total, 3,259 flies belonging to eight species, namely, Musca domestica L., Musca vicina Macquart, Musca sorbens Wiedemann, Calliphora erythrocephala Robineau-Desvody, Sarcophaga ruficornis F., Chrysomyia sp. Robineau-Desvody, Chlorops sp., and Fannia sp. Robineau-Desvody, were recorded. M. domestica was the predominant species constituting 78.2% of the total flies collected. Density of flies was the highest in temporary shelters constructed for the victims, followed by centralized kitchens and devastated human settlements. Lack of waste control at centralized kitchens nearer to the shelters might be the reason for the high fly density in relief shelters. Under these circumstances, outbreak of fly-borne diseases is likely to be aggravated. Therefore, it is suggested that the ongoing space spraying be supplemented with effective waste control measures to reduce the high density of flies.

Development of lymphatic filarial parasite Wuchereria bancrofti (Spirurida: Onchocercidae) in mosquito species (Diptera: Culicidae) fed artificially on microfilaremic blood.

The efficiency of laboratory colonies of mosquitoes such as Anopheles stephensi Liston, Aedes aegypti (L.) Liverpool strain, Ae. aegypti wild type, Aedes albopictus (Skuse), Culex tritaeniorhynchus Giles, Culex sitiens Wiedemann, and Armigeres subalbatus Coquillett in supporting the development of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) microfilariae to infective larvae was investigated. The mosquitoes were fed on heparinized microfilaremic human blood by using a membrane-feeding unit with Parafilm as membrane. The rate of infection, parasite development, and parasite burden were compared with that in the known vector mosquito Culex quinquefasciatus Say. Cx. quinquefasciatus showed the highest percentage of infection, followed by Ae. aegypti Liverpool strain and An. stephensi. The rate of development of the parasite was more or less similar in all the three species, and infective larvae were found on day 13. When the larvae were harvested on day 17, Cx. quinquefasciatus yielded the highest numbers, followed by Ae. aegypti Liverpool strain and An. stephensi. The percentage of infection was low, and the development was slow in Cx. tritaeniorhynchus compared with the other susceptible species. The parasite developed to second-stage larvae only by day 22 and to infective larvae by day 28. When 2-wk-old Cx. tritaeniorhynchus were fed on microfilaremic blood, they could develop the parasite to infective larvae by day 13 postfeeding. All other species of mosquitoes tested were found to be refractory to parasite development. It is shown that Cx. quinquefasciatus is the most suitable mosquito host for the production of infective larvae. However, Ae. aegypti Liverpool strain, which is commonly used for Brugia malayi filarial parasite, also can be used for generation of W. bancrofti infective larvae to circumvent the problem of maintaining two mosquito species.

Optimization of media composition for the production of cyclosporin A by Tolypocladium species.

BACKGROUND & OBJECTIVE: Cyclosporins are produced by certain species of the filamentous fungi, belonging to the genus Tolypocladium. While there are numerous reports on the use of cyclosporins in clinical studies, reports on the various aspects of their production have been very limited. Therefore, this study was carried to optimize the medium composition for the production of cyclosporin A, produced by a strain of the filamentous fungus, Tolypocladium species by static fermentation. METHODS: The effect of different nutrients on the production of cyclosporin A, produced by Tolypocladium species in stationary culture was studied by growing the fungus for 21 days at 25 +/- 2 degrees C under different media composition. Cyclosporin A was extracted by homogenizing the fungal cells with methanol and the cyclosporin A level was analyzed by high performance liquid chromatography (HPLC). RESULTS: Among the six different media studied for the production of cyclosporin A, medium 'f' containing glucose (8%), casein acid hydrolysate (3%), malt extract (2%), peptone (1%) and DL- alpha-amino butyric acid (0.5%) favoured the maximum production (2.22 +/- 0.02 g/l medium or 5.85 +/- 0.35 g/kg biomass). INTERPRETATION & CONCLUSION: This study showed that by optimizing the composition of fermentation media enhanced production of cyclosporin A was obtained. Since the strain Tolypocladium (VCRC F21 NRRL No.18950) produces a high level of cyclosporin A in the identified fermentation medium, it could be exploited for industrial production.

Metastatic breast carcinoma discovered in a dentigerous cyst - a case report.

This paper reports a patient with a history of breast cancer, who presented with altered sensation to the right lower lip and chin. An orthopantomogram showed a probable dentigerous cyst associated with an unerupted lower wisdom tooth, which was closely related to the inferior dental canal. The tooth and cyst were enucleated under general anaesthesia. The subsequent histopathology report concluded that the cyst contained metastatic adenocarcinoma from a primary breast tumour.

Toxicity of a mosquitocidal metabolite of Pseudomonas fluorescens on larvae & pupae of the house fly, Musca domestica.

BACKGROUND & OBJECTIVES: Biological control through the use of parasitoids and pathogens is one of the alternatives to the use of chemical pesticides for control of insects of public health importance. At the Vector Control Research Centre, a liquid formulation developed using the metabolite of a Pseudomonas fluorescens strain was found to be lethal to larvae as well as pupae of vector mosquitoes. The lethal fraction of the metabolite is a protein with a molecular mass of 44 kDa and toxicity studies showed that it is safe to mammals. In the present study, this formulation was evaluated against immatures of the common house fly, Musca domestica, to find out whether it could be developed into a potential biocontrol tool. METHODS: Early second instar larvae of house fly were introduced into rearing medium incorporated with the formulation at concentrations of 1, 5, 10, 15, 20 and 25 per cent, which were equivalent to respectively 1.13, 5.63, 11.25, 16.88, 22.50 and 28.13 microg of the toxic protein/ g of rearing medium. Mortality was monitored until the emergence of adult house fly. Net mortality of larvae and pupae were calculated and the LC50 and LC90 values were determined through probit regression analysis. RESULTS: Larval mortality was obtained from day 3 to 6 post-treatment. Net mortality of larvae was higher at the concentration of 20 than at 25 per cent. However, it was higher at 25 per cent on day 5 and continued to day 6 when there was no larval mortality at other concentrations. The net mortality of pupae was higher than that of larvae at all the concentrations except at 20 per cent. The LC50 and LC90 values calculated from the net mortality of larvae and pupae together, from day 1 to 12 post-treatment, were respectively, 8.25 and 51.79 microg protein/g of the fly rearing medium. INTERPRETATION & CONCLUSION: The formulation prepared from the exotoxin of P. fluorescens was toxic to the house fly. Pupae were more susceptible than larvae and the activity of the toxin might have been through cuticular absorption. The results are indicative of the possibility of development of the mosquitocidal metabolite for house fly control through appropriate field evaluations.

Occurrence and diversity of mosquitocidal strains of Bacillus thuringiensis.

Ever since the discovery of the first Bacillus thuringiensis strain capable of killing mosquito larvae, namely, B. thuringiensis var israelensis, there are several reports from different parts of the world about the occurrence of mosquitocidal strains belonging to different subspecies/serotypes numbering thirty-six. The main sources of these wild type strains are soils/sediments, plants, animal feces, sick/moribund insects and waters. The toxicity of the strains within a subspecies/serotype varied widely. Some of the strains exhibited toxicity to mosquitoes as well as lepidopterans and dipterans (including mosquitoes) as well as plant parasitic nematodes.

A report on the demonstration of microfilariae of Brugia malayi in the brain of an experimental animal host, Mastomys natalensis.

The brain tissues of microfilaraemic animals, Mastomys natalensis, which were earlier inoculated (s.c.) with Brugia malayi infective larvae (100 each) were examined for the occurrence of Mf. This was done by staining squash preparations of the brain tissues which were cleared off from the vascular piamater. Animals with blood Mf count of 50 >/per 20 cu. mm were found to harbour Mf in the brain tissues. The Mf count in the brain varied from 5-86/81 cu. mm (sum of Mf detected in 3 tissue pieces, each of 27 cu. mm collected from 3 parts of the brain, viz., the cerebral hemispheres, cerebellum and medulla oblongata). Teh presence of Mf in the brain was confirmed by its detection in 20-micrometers-thick cryosections of the tissue. Also, fine needle aspirates of cerebral hemispheres of an animal showed live Mf.

Development of Wuchereria bancrofti in Culex quinquefasciatus that survived the exposure of sub-lethal dose of Bacillus sphaericus as larvae.

Development of Wuchereria bancrofti in Culex quinquefasciatus emerged from the larvae that survived the exposure of sub-lethal dose of Bacillus sphaericus was examined in the laboratory. Third instar larvae of Cx. quinquefasciatus were treated with B. sphaericus at a sub-lethal dose of 11.35 microg/250 ml. The female mosquitoes that emerged from the survived larvae were fed on microfilaraemic human blood and parasite development was monitored in the fed mosquitoes. Both treated and untreated mosquitoes could ingest microfilaria (mF) equally as there was no significant difference in mF density between them. But, density of developmental stages of the parasite in treated group was significantly lower. Since, there was no mortality of mosquitoes, the lower density of the developmental stages could be attributed to the loss of parasites in the treated mosquitoes. Consequently, the proportion of mosquitoes with infective larvae (L3) and number of L3 were also significantly lower in treated females. Delay in parasite development was also noticed in treated mosquitoes. The present study indicates that B. sphaericus, when applied at sub-lethal dose kills larvae, and in addition, inhibits development of the filarial parasite and consequently reduces L3 yield in adult mosquitoes that emerged from the survived larvae.

Efficacy of a floating sustained release formulation of Bacillus thuringiensis ssp. israelensis in controlling Culex quinquefasciatus larvae in polluted water habitats.

Larvicidal efficacy and residual activity of a floating sustained release formulation of Bacillus thuringiensis ssp. israelensis (Bti) was tested in polluted water habitats against Culex quinquefasciatus, the vector of bancroftian filariasis, breeding in cesspools and cesspits. A total of 25 habitats having 65 m(2) water surface area with C. quinquefasciatus immatures were treated with the formulation at the rate of 15 kg/ha. For comparison, in another area, 18 habitats with a water surface area of 70 m(2) were kept untreated. Evaluation was continued up to 179 days with three rounds of Bti application. The mean number of egg rafts did not differ significantly between treated and untreated habitats during the trial period indicating same level of recruitment. However, abundance of larvae and pupae was significantly lower in Bti treated habitats compared to that in untreated ones. The activity of the formulation, with 2 weeks delay in onset compared to treatment, caused >80% reduction in pupal abundance for a period of 30-34 days. Considering this, it is suggested that monthly application of Bti formulation will considerably reduce the recruitment of pupae of C. quinquefasciatus.

Field evaluation of a formulation of Pseudomonas fluorescens against Culex quinquefasciatus larvae and pupae.

VCRC B426, 0.09% emulsifiable concentrate (EC) formulation developed from a metabolite of Pseudomonas fluorescens was tested for efficacy against Culex quinquefasciatus larvae and pupae. At application rates of 100, 200, 300 ml/m2, the formulation caused 100% elimination of larvae and pupae at day 1 after treatments and >80% reduction in pupal density for periods of 7, 12 and 11 days in cesspits and 5, 9 and 10 days in U-shaped drains. In both the habitats, the efficacy of the formulation against pupae was 1.7 times more at 200 ml/m2 than at 100 ml/m2. An increase in dosage to 300 ml/m2 did not improve the efficacy in cesspits but a marginal increase was observed in drains.

Metabolites of fungi & actinomycetes active against mosquito larvae.

Extracellular secondary metabolites from 350 fungi and 94 actinomycetes were screened for larvicidal activity against Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. Of them, 133 fungal metabolites and 35 from actinomycetes were active. Two from Streptomyces sp. and one from Paecilomyces sp. were highly active with LC50 value of 1-3 microliters/ml. The metabolites were more toxic to C. quinquefasciatus than to A. stephensi or Ae. aegypti larvae.

Extra cellular hydrolytic enzyme secretion in Bacillus thuringiensis H14 & B. sphaericus & their significance in media design.

Level of extracellular proteolytic and amylolytic enzyme production was determined to assess the ability of these strains in utilizing complex carbon and nitrogen sources. Protease secretion reached maximum at around 12th h of growth in case of all the three B. thuringiensis strains and declined sharply thereafter. In B. sphaericus strains the protease level gradually increased and reached maximum at around 24th h of growth. Amylase activity was not detectable in the culture supernatants of B. sphaericus strains whereas all the three B. thuringiensis strains tested showed significant amount of amylase activity.

Method for storing Lagenidium (Oomycetes: Lagenidiales).

Techniques for storing the mosquito pathogenic fungus, Lagenidium, were evaluated. A technique, which involves storage of fungal mycelia in sterile distilled water of pH 6-7 with 0.0025 M glucose at 30-35 degrees C, was found to be useful. When stored in this manner the fungus retained it's larvicidal activity for 190 days.

Utility of cheap carbon & nitrogen sources for the production of a mosquito-pathogenic fungus, Lagenidium.

Attempts were made to produce Lagenidium in culture media containing corn starch, barley flour, wheat flour, jaggery, rice bran, rice flour or tapioca starch as the major carbon source and groundnut cake as nitrogen source in comparison with peptone-yeast extract-glucose-sunflower oil (PYGSF) agar medium. The number of zoospores produced by the mycelia grown in PYGSF agar medium was 20 x 10(4)/10 cm2 while that grown in the other media was 20-25 x 10(4)/10 cm2. With respect to the larvicidal activity, the mycelia grown for 7 days in the medium containing jaggery, tapioca starch, rice flour or rice bran caused 88-96 per cent mortality while that from other media, including PYGSF agar medium, caused 70-75 per cent mortality. Twelve batches of the fungus were produced using rice-groundnut cake-sunflower oil (RGNCSF) agar medium with consistent zoospore yield and larvicidal activity.

Isolation of mosquito-pathogenic Bacillus sphaericus & B. thuringiensis from the root surface of hydrophytes.

Attempts were made to isolate B. sphaericus and B. thuringiensis active against mosquito larvae from the root surface of hydrophytes. Out of 139 samples processed, 86 B. sphaericus and 23 B. thuringiensis isolates were obtained. Sixty two of the B. sphaericus isolates belonged to the serotype H5a5b, 2 to H6 and 22 isolates did not agglutinate with any of the 6 antisera tested. Twenty of the B. thuringiensis isolates belonged to the H14 serotype, 1 each to the H10 and H17 serotype(s) and 1 to an unknown serotype. Fifty nine of the B. sphaericus and 20 of the B. thuringiensis isolates fall under highly toxic category with the LC50 dose of 1-50 ng/ml for Culex quinquefasciatus third instar larvae.