BASIC RESEARCH ON INSECTICIDAL PROTEINS PRODUCED BY SYMBIONTS OF NEUROPTERA INSECTS

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The aim of this study is to isolate symbionts producing insecticidal proteins from Neuroptera insects, test for their toxicities against insects and elucidate their mode of actions. Enterobacter aerogenes and Bacillus cereus have been isolated from larvae of Myrmeleon bore. E. aerogenes was found to produce GroEL-like protein as one of active substances. On the other hand, B. cereus is known to secret phospholipase C as one of insecticidal toxins. GroEL like protein and phospholipase C were immunologically detected from the regurgitant of the antlion. Active principles, Toxins 1 and 2 were isolated as insecticidal proteins other than phospholipase C from the culture broth of B. cereus by testing their toxicity by injection. Amino acid sequencing of these proteins suggested that Toxins 1 and 2 are homologous to zinc methalloprotease inh2A and sphingomyelinase C, respectively. The genes for these toxins have been cloned and sequenced to deduce their primary structures.Since the insectici … More dal proteins produced by the symbionts exerted their toxicity rapidly, these proteins could attack nervous systems of insects. To prepare an assay system for the proteins, structural properties and sensitivity to neonicotinoid insecticides of nicotinic acetylcholine receptors (nAChRs) were also investigated by deploying homomer-forming chicken α7 nAChR subunit and two-electrode voltage-clamp electrophysiology as well as site-directed mutagenesis. It has been shown that G189 in loop F of the α7 nAChR is located close to the nitro group of imidacloprid a neonicotinoid in the binding site. On the basis of the crystal structure of acetylcholine binding protein from a snail Lymnaea stagnate that is highly homologous to the N-terminal agonist binding domain of the α7 subunit, Q79 in loop D of the α7 nAChR was found to be located in the proximity of G189 in loop F. Thus Q79 was replaced by either acidic or basic residues and the mutant receptors were functionally expressed in Xenopus oocytes to evaluate role of the residue in its interactions with neonicotinoids in the nAChR. The neonicotinoid sensitivity of the α7 nAChR was markedly reduced by the Q79E mutation, whereas it was enhanced by the Q79K and Q79R mutations, suggesting the data were the result of electronic interactions of the nitro group of neonicotinoids with the added residues. These neonicotinoid-resistant and sensitive nAChRs can also be used for testing the insecticidal proteins and comparing their mode of actions with that of neonicotinoids. Less