Fungi Beauveria bassiana (Balsamo) Metabolites for Controlling Malaria and Filaria in Tropical Countries

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1 Fungi Beauveria bassiana (Balsamo) Metabolites for Controlling Malaria and Filaria in Tropical Countries GAVENDRA SINGH AND SOAM PRAKASH Environmental and Advanced Parasitology and Vector Control Biotechnology Laboratories, Department of Zoology, Dayalbagh Educational Institute, Dayalbagh, Agra-8, India Abstract: - We are revelation here the biolarvicidal activity of Beauveria bassiana metabolites on larvae of Anopheles stephensi Liston and Culex quinquefasciatus Say in laboratory conditions. The effectiveness of metabolite of Beauveria bassiana against An. stephensi has been evaluated at statistically significant concentrations viz. ml/l, ml/l, ml/l, ml/l, and ml/l respectively. The probit equations were derived to ascertain regression. These metabolites were found to be more effective on An. stephensi comparatively Cx. quinquefasciatus larvae. Therefore, the present laboratory results can be a significant tool for controlling of malaria and filaria by reducing mosquito larvae with more validation and field trials. Key- Words: Malaria; Filaria, Anopheles stephensi, Culex quinquefasciatus, Larvae, Beauveria bassiana, Biocontrol, Climate change. Introduction India is struggling to cope with outbreaks of mosquito borne diseases. Overburden hospitals and clinics in several cities in the North of India are scrambling to treat thousands of patients admitted in the recent years with symptoms of high fever severe body aches. Most of them have been stricken with the mosquito borne diseases. Presently, Climate change is also expected to bring more natural disaster. Such change is inevitably affect heath particularly in tropical countries. Malaria and Filaria are common and life threatening diseases in many tropical and sub tropical areas. The synthetic larvicides have been extensively used to control of vector species of mosquitoes. These larvicides have caused physiological resistance and adverse effects on nontarget organisms in addition to high operational cost. Fungal metabolite could play a significant role in pathogenesis and in larvicidial activity that can help in controlling mosquito population. In India, extracellular metabolites from several fungi have been tested for larvicidal activity and found effective against Stegomyia aegypti, Anopheles stephensi, and Culex quinquefasciatus (Vijayan and Balaraman, 99). Moreover, extracellular metabolites of Trichophyton ajelloi, Chrysosporiom tropicum, C. lobatum, Lagenidium giganteum have been reported for their larvicidal activity against An. stephensi and Cx. quinquefasciatus and St. aegypti from our laboratory (Mohanty and Prakash,,, 7, 8, Priyanka et al.,, Priyanka and Prakash,, Vyas et al.,, 7). Beauveria is one of the most frequently isolated entomogenus fungul genera has cosmopolitan distribution (Scholte et al., ). The extracellular secondary metabolites produced by entomopathogenic fungi have become a focus of interest for insect pathologists (Zizka and Weiser, 99). A number of entomopathogenic fungi have been used effectively to control the mosquito vectors for last few decades. However, in a study the effects of extracellular metabolites on mosquito larvae appear to be very limited in comparison to use of spores and mycelia of the fungi to control mosquito larvae (Priyanka, ). Many of the biological control agents, such as Bacillus thuringiensis and Bacillus sphaericus have shown their valuable efficacy against mosquitoes globally. However, simultaneously the resistance has also been started emerging in these larvicides (Singh and Prakash, 9a, Adak et al., 99, Rao et al., 99). In present study metabolites produced by B. bassiana were evaluated for all instars of An. stephensi, and Cx. quinquefasciatus and the results are reported here. ISSN: 79-8 ISBN:

2 Problem Formulation. Fungal strain and culture media The fungal strain B. bassiana was obtained from Microbial Type Culture Collection and Gene Bank (MTCC No.98) Institute of Microbial Technology, Chandigarh, India. The fungal colonies of were maintained on recommended autoclaved complete media (FeSo -.g, KCl-.g, KH PO -. g, MgSO. 7H O-.g, ZnSO -., Hydrolysed Casein-.g, Glucose-g, Peptone-g, Agar-g, Deionized water-ml). Culture filtrates were obtained by filtering of fungal culture through Whatman no. filter paper after day of incubation. These metabolites were used on larval instars of An. stephensi and Cx. quinquefasciatus.. Mosquito collection and rearing Larvae were collected from various localities including urban, rural and semi-urban regions of Agra (7 N, 78 E) in India. A colony of An. stephensi and Cx. quinquefasciatus were maintained in the laboratory at temperature ± C relative humidity of 7±% and photoperiod of :h (L:D). All the instars of An. stephensi and Cx. quinquefasciatus were maintained in separate containers. Larvae were placed in deionized water at ph 7.. To counteract evaporation, water was added daily using sterilized glassware. Larvae were fed on % glucose in deionized water.. Larval bioassay Larval bioassay have been conducted as per the guidelines of World Health Organization () a laboratory and field manual. Twenty five An. stephensi and Cx. quinquefasciatus larvae of first, second, third, and fourth instars were used for all bioassays. The tests were carried out in ml of deionized water in Schott Duran beakers. The bioassays were conducted separately for each instars. These entire tests were carried out in three replicates at 7± C to validate results. The mortality and survival were determined after h of the exposure without offering meal to larvae. Controls were set up in deionized water and run simultaneously with treatment beakers. Control and treatment larvae were kept in the same environmental conditions.. Statistics and analysis These bioassays studies were analyzed using the probit analysis (Finney, 97). Control mortality was corrected by Abbott s formula (Abbott, 9). The relation between probit of kill and log concentrations were established by the probit equation. The probit regression lines were drawn for each of the larval instars. Problem Solutions During bioassay statistically significant concentrations ml/l, ml/l, ml/l, ml/l, and ml/l were applied on An. stephensi and Cx. quinquefasciatus larvae. The different percent of mortalities were found in all instars of An. stephensi (Table ). The mortality rates on first instars (R =.8) and on second instars (R =.7) were found (Fig.). Similarly, the mortality rates on third instars (R =.8) and fourth instars (R =.87), and significant mortalities were found on all instars of An. stephensi (Fig. Moreover, a comparison of the percent mortality among the instars of Cx. quinquefasciatus showed that on first instars (R =.), second instars (R =.7). Third instars (R =.9) and fourth instars (R =.) (Fig., ). y =.x +.9 R² =.8 8 First instar y =.77x +.89 R² =.7 Second instar....8 Fig. Probit regression line depicting relationship metabolites of B. bassiana of h exposure for the first and second instars of An. stephensi. y =.899x +.9 R² =.8 7 Third instar y =.7x +. R² =.87 Fourth instar....8 Fig. Probit regression line depicting relationship metabolites of B. bassiana of h exposure for the third and fourth instars of An. stephensi. ISSN: 79-9 ISBN:

3 y =.7x +.7 R² =. 8 7 y =.99x +.9 R² =.7 First instar....8 Fig. Probit regression line depicting relationship metabolites of B. bassiana of h exposure for the first and second instars of Cx. quinquefasciatus. y =.x +.9 R² =.9 Third instar y = -.7x +.79 R² = Log concentration (mg/l) Fourth instar Fig. Probit regression line depicting relationship metabolites of B. bassiana of h exposure for the third and fourth instars of Cx. quinquefasciatus. Conclusion The entomopathogenic fungus B. bassiana is being considered as a biocontrol agent for the adult mosquito of An. stephensi and Cx. quinquefasciatus. The fungal biopesticide approach for control of female mosquitoes in contact with fungal spores from treated surfaces they rest to digest a blood meal. Infection with the entomopathogenic fungus B. bassiana dramatically reduces survival of An. stephensi mosquitoes by day (Thomas and Read, 7). However, a problem associated with using conidia is that they have no residual effect. They germinate in mosquito habitats even when not in contact with larvae. This limitation, along with the high doses needed is serious drawbacks for mosquito larvae control (Scholte et al., ). As per the previous studies most of researches are applying zoospores, conidia for the adult mosquito control (Kannan et al., 8, Achonduh and Tondje, 8, Mohanty and Prakash,, 8). Recently, World Health Organization reveals at midpoint between and, the current gains need to sustained, particularly in malaria where there has been little or no movement (World Health Statics 9). The present situation needs searches for effective mosquito pathogens that can be used in mosquito control operations. Moreover, the extracellular metabolites of Trichophyton ajelloi the efficacy of An. stephensi was similar to Cx. quinquefasciatus except in the case of LC 9 of the first instar larvae. The LC 9 values of first instar larvae was higher than that of the second instar larvae (Mohanty and Prakash, ). In present study the mortality rates of B. bassiana metabolites on An. stephensi first instars (R =.8), second instars (R =.7), third instars (R =.8) and fourth instars (R =.87) were found significantly effective comparatively Cx. quinquefasciatus larvae. The result of the present investigation could be applied for malaria control in semi-arid zone. Preliminary laboratory based bioassays revealed that mosquitoes were readily infected by exposure to entomopathogenic fungi isolates caused % mortality of adult Anopheles and Culex species in 7- days depending on dose, formulation and fungal strain (Scholte et al.,, Blanford et al., ). Moreover, few biopesticides products have been widely used, in spite of their potential (Thomas and Reid, 7). Indeed on global scale penetration of biocontrol technology into the pesticide market has been minimal, less than % of global pest control scale (US $b) comprises biocontrol. The 7% of this small proportion are biopesticide products that that are based on the crystal toxin forming bacterium Bacillus thuringiensis (Georgis, 997, Fravel, ). Premilary studies indicate viable spores can be recovered from treated after three months but that the percentage infection of mosquitoes exposed to these surfaces at this is very low. Moreover, studies on the demand for malaria control interventions indicate corrections between willingness to and socioeconomic status and potentiality very low threshold costs for driving net benefit from control technologies (Onwujekwe et al.,, Meltzer, ). Viruses, fungi, and protozoa are used in only a few niche markets and thus have largely failed as microbial insecticides, and will continue to fail until more efficacious mass production methods are developed. Despite these failures, research on insect pathogens lead to the development of transgenic insect-tolerent Bt crops arguably the most important advance in pest control technology ISSN: 79- ISBN:

4 of the latter of the th century (Federici, ). In this concern the cost of research and production at large always remain unconcining, however, when it get started in production in mass by companies, the cost generally get down to a workable price. The fungi can be isolated from environment. This could be good interface between community and laboratory results. The utility of B. bassiana larvicides can be good when we have recorded resistance in bacterial Bacillus sphaericus and chemical larvicides Gokilaht -S EC a synthetic pyrethroid (Singh and Prakash, 9a,b) and in Bti also. Recently World Health Organization (9) found in report world health statistic that the Japanese encephalitis and malaria are very difficult to identify without specialized laboratory tests that are often not available in developing countries. Moreover, in many setting, case of some diseases malaria is a common example, this are identified through clinical sign and symptoms alone. In this concern present study of B. bassiana metabolites can be a promising larvicidal potential against early larval instars of An. stephensi and Cx. quinquefasciatus also. The use of fungal metabolites at the larval stages of development of mosquitoes is one of the techniques which afford a cheap, easy to use and environmental friendly method of mosquito control. Therefore, the present laboratory results could be suggested we need to use fungal metabolites as larvicides which can be an alternative way to control larval population of malaria and filarial vectors. Acknowledgements: We thank Prof. V.G. Das, Director, Dayalbagh Educational Institute, for his encouragements. We are grateful for Department of Science and Technology for the financial support (DST, C-/ Soam Prakash) to conduct the research and to DST - FIST program (-8) for providing laboratories facilities. G. Singh is indebted to University Grants Commission (UGC), New Delhi, for an award of senior research fellowship. References: []Abbott WS, A method of computing the effectiveness of an insecticide, Journal of Economic Entomology, Vol 8, 9, pp.. [] Achonduh OF, Tondje PR, First report of pathogenicity of Beauveria bassiana RBL to the malaria vector, Anopheles gambiae s.i. (Diptera: Culicidae) in Cameroon, African Journal of Biotechnology, Vol.7, 8, pp.9 9. [] Adak T, Mittal PK, Raghvendra K, Subbarao, KS, Ansari MA, Resistance to Bacillus sphaericus in Culex quinquefasciatus Say 8, Current Science, Vol. 9, 99, pp [] Blanford S, Fungal pathogen reduces potential for malaria transmission, Science, Vol. 8,, pp.8. [] Federici, BA, Bacteria as biological control agent for insects: economics, engineering, and environment safety, Nobel Biotechnology for Biocontrol Agent Enhancement and Management, ISBN , pp., 7. [] Finney D J, Probit Analysis. rd edition. Cambridge University Press, Cambridge, United Kingdom, 97. [7] Fravel DR, Commercialization and implementation of biocontrol. Annual Review of Phytopatol, Vol.,, pp [8] Georgis R, Microbial insecticides: Novelity or necessity? (ed Evans HF) - Britis Crop Protection Council Monograph No 8, 997. [9] Kannan SK, Murugun K, Kumar AN,Ramasubramanian,N,Mathiyazhagan P, Adulticide effect of fungal pathogen, Metarhizium anisopliae on malaria vector Anopheles stephensi (Diptera: Culicidae), African Journal of Biotechnology, Vol. 7, 8, pp [] Meltzer MI et al., The house hold-level economics of using permethrin-treated bed nets to prevent malaria in children less than five years of age, American Journal of Tropical Medicine and Hygiene, Vol. 8,, pp. 9. []Mohanty SS, Prakash S,. Laboratory evaluation of Trichophyton ajelloi, a fungal pathogen of Anopheles stephensi and Culex quinquefasciatus, Journal of the American Mosquito Control Assocociation, Vol., pp. 7. []Mohanty SS, Prakash S, Efficacy of Chrysosporium lobatum against larvae of malaria vector, Anopheles stephensi in the laboratory, Current Science, Vol. 8,, pp []Mohanty SS, Prakash S, Extracellular metabolites of Trichophyton ajelloi against Anopheles stephensi and Culex quinquefasciatus larvae, Current Science, Vol. 8,, pp.. ISSN: 79- ISBN:

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