A Study of the Effects of Azadirachtin on Specific Tissues in the Locust, Schistocerca gregaria

Azadirachtin was isolated from the seeds of the neem tree (Azadirachta indica) by solvent extraction and flash column chromatography. The recrystallised material was characterised by chromatography, NMR and melting point, and was found to have a purity of >95%. The yield, from Sri Lankan neem seeds, was 0.065% w/w. The pure compound was used to prepare the reduced derivative (22,23) dihydroazadirachtin and (22,23-3H2)- dihydroazadirachtin was also commercially prepared for tracer and metabolic studies. The radiolabelled tracer was used to follow tissue uptake, metabolism and excretion in the locust Schistocerca gregaria. It was found that an injected dose of the tracer was removed with great speed from the haemolymph, apparently by carrier-mediated specific mechanisms, into many of the locust tissues. Unlabelled analogues, injected in large excess, inhibited the clearance of the tracer to different extents and the results suggested that azadirachtin and its dihydro derivative have different affinities for the uptake mechanism. Radio-labelled dihydroazadirachtin applied topically to the locusts was shown to penetrate the insect to only a limited extent. A large fraction of the tracer was absorbed into the fat body as well as into gut, Malpighian tubules and nervous tissue. Binding of the dihydroazadirachtin was persistent and not easily displaced. There was no evidence of active excretion of dihydroazadirachtin by the Malpighian tubules. Metabolism of the dihydroazadirachtin was slow, and largely restricted to fat body and crop. Attempts, in vivo and in vitro, to show an effect of azadirachtin on Malpighian tubule function in a) water secretion and b) clearance of the sulphonic acid dye, amaranth, were unsuccessful. 3H-glycine and 35S-cysteine were used as tracers to examine the effects of azadirachtin on the incorporation of these amino acids into the protein and peptides in various tissues both in vivo and in vitro. The tissues examined were fat body, ovary, gut, brain and corpus cardiacum. It was found that the terpenoid eliminated the stimulatory effects of crude neurohormonal extracts from corpus cardiacum on protein synthesis in fat body and ovary, but had no effect on basal, unstimulated synthesis. On the other hand, the azadirachtin did not inhibit the stimulation of protein synthesis in the fat body due to application of pure juvenile hormone, suggesting that the effects on protein synthesis were differential. Azadirachtin appeared to have a direct inhibitory effect on protein biosynthesis in the mid-gut, and this could partly account for observed secondary antifeedant effects. Incorporation of 35S-cysteine into the polypeptides, including glutathione, synthesised in both brain and corpus cardiacum was strongly inhibited by azadirachtin. This suggested that many of the biological effects of the terpenoid are due to interference with neuroendocrine processes, most of which are mediated by peptide hormones