The plasma membrane V-ATPase of Manduca sexta larval midgut is an electrogenic proton pump located in goblet cell apical membranes (GCAM); it energizes, by the voltage component of its proton motive force, an electrophoretic K+/nH+ antiport and thus K+ secretion (Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage, indicating net active K+ transport, was found to be more than 100 mV during intermoult stages but was abolished during moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton transport in GCAM vesicles were found to be reduced to 10-15% of the intermoult level. Immunocytochemistry of midgut cryosections as well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM demonstrated that loss of ATPase activity paralleled the disappearance of specific subunits. The subunits missing were those considered to compose the peripheral V1 sector, whereas the membrane integral V0 subunits remained in the GCAM of moulting larvae. The results provide, for the first time, evidence that a V-ATPase activity can be controlled in vivo by the loss of the peripheral V1 domain.

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Mitochondrial NADH dehydrogenase from bovine heart was photolabelled with the inhibitor [3H]dihydrorotenone. A constituent of the hydrophobic domain of the enzyme of M r 33 000 was the major site of labelling. The identity of this protein with the mitochondrially encoded ND‐1 gene product was established by immunoblotting and immunoprecipitation with an antiserum raised to the expected C‐terminal sequence of the human ND‐1 gene product.

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The nicotinic acetylcholine receptor pharmacological profile of sulfoxaflor in aphids is consistent with that of imidacloprid. Additionally, the insecticidal activity of sulfoxaflor and the current commercialised neonicotinoids is affected by the point mutation in FRC Myzus persicae. Therefore, it is suggested that sulfoxalfor be considered a neonicotinoid, and that this be taken into account when recommending insecticide rotation partnering for effective resistance management programmes.

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

A photoaffinity-labelling analogue of the respiratory inhibitor rotenone was synthesized from the naturally occurring rotenoid amorphigenin. The analogue inhibits NADH-ubiquinone oxidoreductase activity at concentrations comparable with those of rotenone. Photolysis of the radiolabelled analogue bound to isolated NADH-ubiquinone oxidoreductase resulted in preferential incorporation of radioactivity into a polypeptide of Mr 33 000, particularly at low concentrations of the inhibitor. Preparations of the enzyme differ in a parallel fashion in the content of this polypeptide, the degree of photolabelling by the analogue and their sensitivity to rotenone, providing further evidence that the 33 000-Mr protein forms part of the rotenone-binding site.

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