Distribution of cholinesterases in insects.

The study of toxicology and other related fields has been largely based on in vitro techniques. These methods have provided quantitative information on the effects of inhibitors on enzymes, but none on the localized effects of inhibitors on selected sites of action within the animal. Histochemical study of frozen sections does provide data on the site of action of toxicants. The utility of histochemistry in conjunction with in vitro methods is discussed.The substrates acetylthiocholine and phenyl thioacetate were utilized in demonstrating cholinesterase. Neither substrate penetrated well into freshly dissected nerve cord preparations, but both compounds were hydrolysed by sectioned tissue. The leaving group of phenyl thioacetate was demonstrated to be benzenethiol. In general, acetylthiocholine was hydrolysed slightly more rapidly by insect cholinesterases. A unique cholinesterase was found in motor end-plates of cricket muscle, which hydrolyses acetylthiocholine and which was inhibited by physostigmine. No other insect muscle preparation showed this activity. Topical application of insecticides showed that a vital site of action in flies is the peripheral area of the thoracic ganglia and that in crickets the brain and nerve cord are involved at knock-down. Kinetic data indicate that acetylthiocholine has a greater affinity than does phenyl thioacetate for a variety of enzyme sources. Ultrastructural evidence shows that cholinesterases that hydrolyse acetylthiocholine are membrane-bound. Phenyl thioacetate was found to be useful as a model in designing new insecticides.