Cloning and expression of the gene for trypsin modulating oostatic factor and isolation of a new trypsin inhibitor from calopogonium mucunoides seeds to control the larvae of aedes albopictus skuse

Abstract

Mosquitoes are one of the major insect vectors that transmit many infectious diseases. Many discases like dengue fever, malaria, ctc. are transmitted by mosquitoes. Conventional control methods using different classes of chemical insecticides resulted in development of resistance in mosquitoes. In addition, these chemicals cause severe environmental contamination and health hazards. Hence newer strategies with target specificity need to be developed. In this study, two methods for the control of Aedes albopictus larvae is described. In the first method, wild-type or mutated Trypsin Modulating Oostatic Factor (TMOF) peptide was expressed in Escherichia coli and used for the control of Aedes albopictus larvae. In addition, the impact of the TMOF treatment on the larval gut microbiota is also examined. The second method utilises a new trypsin inhibitor isolated from the seeds of Calopogonium mucunoides to control the larvae of Aedes albopictus mosquito. Trypsin Modulating Oostatic Factor (TMOF) is a naturally occurring decapeptide which inhibits trypsin synthesis in mosquito. As trypsin is required for digestion of protein, the mosquito and mosquito larvae dies on treatment with TMOF. In this study, the gene coding for TMOF was cloned and expressed in Escherichia coli. Mutant TMOF was also developed and expressed in Escherichia coli. Bacterial cells expressing TMOF/mutant TMOF were lysed and used to prepare crude TMOF/mutant TMOF preparation. Second instar Aedes albopictus larvae were treated with the crude TMOF/mutant TMOF and significant mortality was observed within 48 hours of treatment. The LCso (48 hours) for crude TMOF was 242.1 = 6.04 ug/mL and that for crude mutant TMOF was 236.54 + 6.49 pg/mL. Crude TMOF/mutant TMOF were purified by nickel affinity chromatography. The purified TMOF/mutant TMOF was found to have an LCso (48 hrs) of 2.13 + 0.02 pg/mL (56.89 + 1.42 nM) and 1.91= 0.02 pg/mL (50.93 +1.50 nM) respectively to Aedes albopictus larvae. The yield of purified TMOF/mutant TMOF was 3.12 and 2.98 pg per milligram of bacterial pellet. The purified mutant TMOF is 1.12 times better than the purified wild type TMOF as a larvicide to the larvae of Aedes albopictus. The crude TMOF and crude mutant TMOF were also toxic to Culex quinquefasciatus larvae but not to non-target organisms like Drosophila melanogaster, Luprops tristis, Aplocheilus lineatus. Analysis of the gut microbiota of Aedes albopictus larvae on treatment with recombinant TMOF/mutant TMOF revealed that there is a reduction in the gut bacterial population compared to untreated larvae. The impact of gut microbiota towards the TMOF toxicity need to be investigated. As the mechanism of action of TMOF/mutant TMOF is receptor mediated and the receptor is only reported from mosquitoes, using recombinant TMOF/mutant TMOF as a larvicide is an ecofriendly method of mosquito control. For the isolation of a new trypsin inhibitor, a total of 45plant extracts were screened by trypsin inhibition assay. Using proteinase K the plant extracts with proteinaceous inhibitor was found and used to test Aedes albopictus larvae. The extract from the seeds of Calopogonium mucunoides showed the highest trypsin inhibition (87.99 + 0.38%) and percentage mortality (100 £ 0.00%) towards A. albopictus larvae. Thus, the seed extract from Calopogonium mucunoides was used for the purification of the trypsin inhibitor by ammonium sulphate fractionation, size exclusion chromatography, ion exchange chromatography and trypsin affinity chromatography. On toxicity analysis the LCso (48 hours) for Aedes albopictus larvae for crude, 0-30% ammonium sulphate fraction and size exclusion fraction were 0.50 = 0.01, 0.48 + 0.01, and 0.32 + 0.00 pg/mL, respectively. The purified Calopogonium mucunoides trypsin inhibitor (CmTI) had a subunit molecular weight of 22 kDa. The yield of purified CmTI was 0.1pg per gram of seed. For obtaining in large quantities, the CmTI gene can be cloned and expressed in a suitable host like bacteria or yeast, and heat killed cells expressing CmTI can be used as a mosquito larvicide.