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.

Transferrin in the mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae).

Transferrin is a defence protein known to be up-regulated upon infection of parasites/pathogens in Aedes aegypti mosquito. However, no information is available on its up-regulation in Culex quinquefasciatus, the vector of bancroftian filarial parasite. In the present study, enhancement of transferrin in C. quinquefasciatus infected with Wuchereria bancrofti is demonstrated through amplification of the specific mosquito transcript, its sequencing, cloning, and expression. By using two oligonucleotide primers, a 950-bp polymerase chain reaction product was obtained from the first strand cDNA made from RNA of C. quinquefasciatus infected with W. bancrofti. A 707-bp sequence encoding the mature portion of transferrin was confirmed by sequencing the product. This is the first report of transferrin expression in C. quinquefasciatus. The deduced amino acid sequence shared 85% homology with A. aegypti transferrin precursor molecule. Western blot analysis of haemolymph proteins of infected C. quinquefasciatus with antibodies raised against recombinant transferrin protein showed binding to a 66-kDa protein, confirming its identity as transferrin. Hence, this molecule also could be added to the list of immune molecules of C. pipiens group, such as the defensin, gambicin, and cecropin, which are already known.

In vitro screening of medicinal plant extracts for macrofilaricidal activity.

Methanolic extracts of 20 medicinal plants were screened at 1-10 mg/ml for in vitro macrofilaricidal activity by worm motility assay against adult Setaria digitata, the cattle filarial worm. Four plant extracts showed macrofilaricidal activity by worm motility at concentrations below 4 mg/ml and an incubation period of 100 min. Complete inhibition of worm motility and subsequent mortality was observed at 3, 2, 1 and 1 mg/ml, respectively, for Centratherum anthelminticum, Cedrus deodara, Sphaeranthus indicus and Ricinus communis. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was carried out at 1 mg ml(-1) and 4-h incubation period, and the results showed that C. deodara, R. communis, S. indicus and C. anthelminticum exhibited 86.56, 72.39, 61.20 and 43.15% inhibition respectively in formazan formation compared to the control.

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.

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.

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.

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.

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.

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.

Changes in the haemocyte population of the mosquito, Culex quinquefasciatus, following infection with the filarial parasite, Wuchereria bancrofti.

The mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is the vector of the filarial parasite Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), which causes human bancroftian filariasis. Information on the mosquito humoral response against the filarial parasite during the process of its infection and development is important, as it decides the vector competence of the mosquito. Visible changes in the haemocyte population of mosquito, if any, will be an indicator of the possible humoral factors. The present study was aimed at investigating changes in the populations of various types of haemocytes of Cx. quinquefasciatus following infection with W. bancrofti. On day 2 post-feeding on microfilaraemic blood, the haemolymph perfusate of infected mosquitoes with L1 stage of the parasite showed 44.1% granulocytes, 42% prohaemocytes and 13.9% plasmatocytes, whereas that of the control mosquitoes fed on amicrofilaraemic blood showed 63.4% plasmatocytes, 22.2% prohaemocytes and 14.4% granulocytes. Differences in the population numbers of haemocyte types between the infected and control were significant (P > 0.05). However, the mosquitoes examined on day 6 post-feeding, when the parasite was in L2 stage, did not show any such changes. But, similar changes reappeared on day 12 in mosquitoes with L3 stage of the parasite. The observed haemocyte population changes indicate the possibility of some amount of humoral immune response, through the production of certain immune molecules, in Cx. quinquefasciatus infected with W. bancrofti. The nature and exact role of such a response on the filarial parasite development need further investigation.

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.

Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride.

Secondary metabolites produced by Trichoderma viride, a deuteromycetes fungus, under submerged culture condition were formulated and evaluated for oviposition attractancy against gravid females of Culex quinquefasciatus mosquito. At a concentration of 10 g ml-1 the formulation showed remarkable attractancy with an oviposition active index (OAI) of +0.52. When the oviposition attractancy of the formulation was compared with a known oviposition attractant, p-cresol, both at 10 g ml-1, the former was found to be more attractive to result in 70% egg laying than the later with 30% egg laying. Thin layer chromatography fractions of the secondary metabolites showed that a fraction with Rf value of 0.88 was highly active as oviposition attractant with an OAI of +0.65. Further work on identification of the active principle(s) of the microbial formulation might lead to an oviposition attractant useful in mosquito vector management.

Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride

Secondary metabolites produced by Trichoderma viride, a deuteromycetes fungus, under submerged culture condition were formulated and evaluated for oviposition attractancy against gravid females of Culex quinquefasciatus mosquito. At a concentration of 10 æg ml-1 the formulation showed remarkable attractancy with an oviposition active index (OAI) of +0.52. When the oviposition attractancy of the formulation was compared with a known oviposition attractant, p-cresol, both at 10 æg ml-1, the former was found to be more attractive to result in 70 percent egg laying than the later with 30 percent egg laying. Thin layer chromatography fractions of the secondary metabolites showed that a fraction with Rf value of 0.88 was highly active as oviposition attractant with an OAI of +0.65. Further work on identification of the active principle(s) of the microbial formulation might lead to an oviposition attractant useful in mosquito vector management