Receptors of peptide hormones in the gut of rice brown planthopper (Nilaparvata lugens) as targets for novel insecticides

Abstract

The rice brown planthopper, Nilaparvata lugens, is a pest of rice where it can be responsible for single season crop losses of up to 38% (IRRI statistic). Due to the absence of an effective Bacillus thuringiensis (Bt) toxin against these insect pests current control methods are reliant on synthetic insecticides. However, these broad acting insecticides can be harmful to beneficial insects, and resistance to these synthetic insecticides amongst N.lugens populations is now common. Therefore, other novel approaches are needed for the control of this rice pest species, which is addressed in this thesis. This project aims to characterise proteins expressed in the N.lugens gut that could act as potential 'targets' for the design of insecticidal proteins. G protein coupled receptor (GPCR) proteins were identified as 'target' proteins and N.lugens candidates were identified and characterised. A cDNA predicted to encode a diuretic hormone receptor (DHR) in Nilaparvata lugens was cloned using RNA extracted from gut tissue as a template in a PCR based strategy. The coding sequence (639 amino acids, 69.7 kDa) has seven predicted transmembrane domains and is a member of the calcitonin/secretin/corticotropin releasing factor family of G-protein coupled receptors. The protein has greatest similarity to Acheta domesticus DHR (Q16983) with 51% sequence identity. N.lugens DHR has a large N-terminal extracellular domain (amino acids 1-260) putatively involved in hormone binding. This domain was expressed as a recombinant protein in E.coli, purified under denaturing conditions, refolded and used to raise a polyclonal antibody in rabbit. Purified anti-DHR IgG bound specifically to a putative DHR polypeptide extracted from N.lugens gut tissues on western blots. Immunolocalisation experiments using dissected guts showed that anti-DHR antibody bound specifically to the Malpighian tubules. The N- terminal hormone-binding domain is located on the cell surface and is exposed to the haemolymph in vivo. Anti-DHR antibodies delivered to insects via artificial diet showed no binding to gut or tubule tissue, and had no effect on survival. Ingested antibodies were not detected in the haemolymph. Another GPCR protein with high similarity to insect allatostatin receptors (ALSTR) was isolated by PCR using highly degenerate primers, from N.lugens gut specific cDNA. The coding sequence (383 amino acids, M, 42.3 kDa) has seven predicted transmembrane domains and is a member of the SST/galanin/opoid receptor family of G-protein coupled receptors. The putative N.lugens ALSTR has greatest similarity to Periplaneta americana ALSTR (AF336364) with 69% overall sequence identity. Isolation of the full-length N.lugens ALSTR is the first step necessary for the design of novel insecticidal proteins.