Advances in Microbiology, 2012, 2, 216-226 doi:10.4236/aim.2012.23026 Published Online September 2012 (http://www.SciRP.org/journal/aim) Distribution of cry and cyt Genes among Indigenous Bacillus thuringiensis Isolates with Mosquitocidal Activity Ayyasamy Mahalakshmi 1 , Kabilan Sujatha 1 , Poornima Kani 2 , Rajaiah Shenbagarathai 2* 1 UGC-NRCBS, School of Biological Sciences, Madurai Kamaraj University, Madurai, India 2 PG and Research Department of Zoology and Biotechnology, Lady Doak College, Madurai, India Email: * shenbagarathai@rediffmail.com Received June 11, 2012; revised July 5, 2012; accepted July 15, 2012 ABSTRACT Bacillus thuringiensis strains isolated from Madurai, TamilNadu, India were evaluated for their mosquitocidal activity, as well as cry and cyt genes diversity. It revealed that 99% of the parasporal crystal morphology of these isolates was spherical in nature and a variable percentage (0% - 100%) of toxicity was observed against Culex quinquefasciatus and Aedes aegypti. PCR analysis revealed that 53% of the isolates were positive for the various cry and cyt genes tested, whereas 47% did not produce any PCR product for the cry gene analyzed. Diverse pattern of cry and cyt genes distribu- tion was observed even in the isolates from the same sample. B. thuringienis subsp. LDC-9 showed three-fold higher toxicity against Culex quinquefasciatus than that of B. thuringiensis var israelensis which might be used as a potential strain to control mosquitoes in near future after field evaluation. Keywords: Bacillus thuringiensis; Crystal Endotoxin; Culex quinquefasciatus; Aedes aegypti 1. Introduction The search for native strains to control dipteran species have an impact on the control of mosquitoes worldwide, as vector borne diseases are major public health prob- lems and their prevalence has dramatically increased worldwide [1]. Chemical insecticides have been proved to be very effective in vector control programme, but their adverse environmental effects, insecticidal resis- tance and resurgence have prompted the search for alter- native strategies for insect pest control [2]. Among the various alternatives, B. thuringiensis and B. sphaericus are the most potent and successful group of organisms for effective control of pests among the microorganisms [3]. The environmental safety of Bt. based products em- ployed in pest control methods are well documented [4]. B. thuringiensis is a gram positive organism that synthe- sizes crystalline inclusions (δ-endotoxins) during sporu- lation. These toxins are highly specific, completely de- gradable and harmless to humans, vertebrates and plants. Hence, researchers across the world are interested for screening new strains with increased levels of insecti- cidal toxicity with a broader spectrum of activity [5]. The various screening programmes resulted in the number of B. thuringiensis strains not only active against Lepidop- tera, Diptera, Coleoptera but also against Hymenoptera, Phthiraptera or Mallophaga, Acari, Nematheliminthes, Platyhelminthes and Sarcomastigophora [6-8]. The insecticidal activity of B. thuringiensis strains against Dipterans is attributed to the presence of Cry and Cyt proteins [9]. Cry toxins are activated by host prote- ases, which interact with specific receptors located on the host cell surface, resulted in the formation of a pre-pore oligomeric structure that is insertion competent. In con- trast, Cyt toxins directly interact with membrane lipids and insert into the membrane [10]. The mosquitocidal activity of a B. thuringiensis strain is due to the additive effect of each toxin and a complex synergistic interaction among them. B. thuringiensis subsp. israelensis produces four Cry toxins such as Cry4Aa, Cry4Ba, Cry10Aa, and Cry11Aa, Cyt1Aa and Cyt2Ba respectively [11,12]. The presence of the Cyt toxin synergizes and delays or pre- vents the development of resistance to Cry toxins by functioning as a Cry-membrane bound receptor [10]. However, information on the diversity and distribution of cry and cyt genes among mosquitocidal B. thuringiensis isolates from southern part of India is negligible [13,14]. Hence, the present study was envisaged to analyze the distribution of the cry and cyt genes of indigenous mos- quitocidal B. thuringiensis isolates. 2. Materials and Methods 2.1. Sampling Procedure Triplicate samples were collected with an internal di- * Corresponding author. Copyright © 2012 SciRes. AiM