Among disease carriers, mosquitoes are recognized as one of the most important vectors of human diseases. They are capable of transmitting serious, possibly even fatal diseases, such as mosquito-borne encephalitis, dengue, yellow fever, filariasis, and malaria. Transmission of disease occurs when an infected mosquito takes a blood meal. Even though uninfected mosquitoes do not transmit diseases, their bites can result in allergic reactions, which can produce significant discomfort and itching. Each type of disease is transmitted by different species of mosquito. Anopheline mosquito is the main malaria vector. Encephalitis is carried by Culex spp., while dengue and yellow fever are transmitted by Aedes spp. The reason why each species carries different diseases might be because of molecular incompatibilities between the mosquito and the disease agent.

Each year, approximately 300 million people in the developing countries are affected by malaria and over 2 million are killed. Dengue and encephalitis also affect thousands of people in urban areas. There is a close relationship between disease outbreak and the number of mosquito carriers in the area. In other words, the distributionof the disease is mainly determined by the distribution of each mosquito species. Therefore, to reduce the risk of disease, the mosquito population has to be controlled.

The most widely used biological agents for controlling mosquito larvae are Bacillus thuringiensis subsp. israelensis (Bti), and Bacillus sphaericus (Bs). These bacteria produce proteinaceous toxins that specifically kill certain species of mosquito larvae. The toxin is formed as a "crystal" in the bacterial cells. After ingestion by the mosquito larvae, the toxins are dissolved as a protoxin and activated by the larva proteases. The "active form" of the toxin will then bind to the midgut membrane and destroy the midgut cells, leading to starvation and death of the larvae. However, application of both bacteria is limited by the "longevity" of these biocontrol agents. The degradation of microbial proteins in the field is also a main limitation. In addition, toxin resistance has been observed in Culex spp. when B. sphaericus was used. It is thought that the use of a single protein toxin for a long time contributed to the emergence of resistance. Therefore, application of bacteria containing various proteins might be a more effective "broad-range biopesticide" and could overcome the resistance in the mosquito larvae.

The main objective of our group is to improve efficacy and safety of microbial agents for controlling mosquitoes and major insect pests.

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.

Collaboration with Local Scientists

  1. Institute of Molecular Biosciences, Mahidol University
  2. Faculty of Science, Mahidol University
  3. Department of Entomology, Kasetsart University
  4. Department of Agriculture, Ministry of Agriculture and Cooperatives
  5. National Institute of Health, Ministry of Public Health

Collaboration with International Scientists

  1. Department of Pharmaceutical Science, University of Maryland, USA
  2. Lawrence Berkeley National Laboratory, University of California, USA
  3. School of Biological Sciences, Washington State University, USA


Boonhiang Promdonkoy, Ph.D.
(Biochemistry, University of Cambridge, UK)

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Mongkon Audtho, Ph.D.
(Biochemistry, The Ohio state University, USA)

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Sumarin Soonsanga, Ph.D.
(Microbiology, Cornell University, USA)

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Chatchanun Trakulnaleamsai, M.Sc.
(Biochemistry, Chulalongkorn University)

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Amporn Rungrod, M.Sc.
(Biotechnology, KMUTT)

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Chalermphon Rattanalapho, B. Sc.
(Science and Technology, Thammasat University)

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