Exploration of Biocontrol Agents
Bti and Bs are the most commonly used larvicide for mosquito control. Bti has potent toxicity toward Anopheles and Aedes mosquito, but low toxicity against Culex. In contrast, Bs is the most toxic agent for Culex, but not for Aedes. Difference in activity spectra of both bacteria is due to difference in the mosquito-larvicidal proteins produced in the bacterial cells. Furthermore, variety of insecticidal proteins specific to different insects are produced in different B. thuringiensis strains. Therefore, microbial agents containing novel mosquito larvicidal proteins as well as those toxic against major insect pests should be explored. Thailand's rich biodiversity thus offers potential for this exploration.
Molecular Mechanism of Mosquito Larvicidal Toxins
We are currently studying structure-function relationships and molecular mechanism of two mosquito larvicidal toxins; Cyt toxin from Bacillus thuringiensis and binary toxin from Bacillus sphaericus. Cyt toxins (Cytolytic-endotoxins) are a group of proteins produced by some strains of Bt. These proteins have lethal activity against larvae of Dipteran insects (mosquito and black fly) in vivo. Current evidence indicates that Cyt toxins kill mosquito larvae by forming pores on the cell membrane in the larval gut. However, the detailed mechanism of this process is not clearly understood. The pore -forming mechanism and pore architecture of Cyt toxin integrated into biological membrane are under investigation in our laboratory. Another mosquito larvicidal toxin we are studying is "binary toxin". This toxin consists of two components, 42 kDa (BinA) and 51 kDa (BinB). Both proteins function together to kill mosquito larvae. BinB acts as specificity determinant by binding to a specific receptor presented on the gut cell membrane. The toxic component (BinA) then binds to BinB and the complex translocate into the cell and exert its toxicity through an unknown mechanism. We are now studying the molecular mechanism and structure-function relationships of both components. Information obtained from these investigations will be useful for engineering the protein to improve toxin potency,developing synergism with other toxins to broaden the host range, preventing or delaying the emergence of resistance or designing new immunotoxins.
Resistance Mechanism in Mosquito Larvae
Over 100,000-fold resistance to binary toxin in Culex has been found in Thailand and around the world when Bs is used continuously. Cross-resistance among different strains of Bs is also observed. The larger component of binary toxin, 51-kDa protein (BinB), binds to the mosquito larval midgut. Alteration in binding might result in loss of activity of the toxin. Currently, we are investigating the receptors (alpha -glucosidases) for the 51-kDa proteins from susceptible and resistant mosquito larvae collected in Thailand. We have found differences in binding between susceptible and resistance mosquitoes. Mutations in the alpha-glucosidase gene might therefore be responsible for the binary toxin resistance. However, more information at molecular level of the receptors is required to test this hypothesis.
Development of a host cell for production of insecticidal proteins
A gram-positive bacterium Bacillus subtilis has many beneficial features, including high capacity of protein secretion and non-pathogenicity, which allows its exploitation as a host for recombinant protein production. It offers an alternative system for protein production with cheaper cost. There are two major reasons hampering the use of B. subtilis as a cell factory; structural instability of the expression plasmid and degradation of secreted recombinant proteins by native extracellular proteases. Therefore, the expression system in B. subtilis for heterologous secretory proteins is currently setting up. The stable expression vector and B. subtilis stains with proteases deficient will be constructed.
Production of VIP3Aa for effective control of insect pests
Bacillus thuringiensis is the most extensively used biopesticide worldwide. For decades, the insecticidal activity of this bacterium is believed to associate with its ability to synthesize a group of crystal proteins, referred to as Cry and Cyt proteins. However, a group of proteins named vegetative insecticidal proteins (Vips) have been recently discovered and their essential roles in insecticidal activity of the bacterium have been proved. One of the the most active Vip protein is Vip3Aa, which is produced during vegetative stage and highly toxic against several insect species, including beet armyworm (Spodoptera exigua) and S. litura. The protein is reportedly much more toxic to S. exigua and S. litura than Cry proteins. However, application of the protein to control the insect pests is limited to an inadequate amount due to the lack of production technology. The effective expression system in B. thuringiensis is being developed in our laboratory. Our aim is to improv its expression level, stability and synergism with other insecticidal proteins.