Abstract. Electrophoretic patterns of esterases from larvae of Tribolium castaneum fed on a diet which included hormetic concentrations of azadirachtin (viz. 0.001, 0.01 and 0.1 ppm) for 10 d were studied. The results showed a dose-dependent variation in the multiple molecular forms of the esterases. The variations, however, were not limited to the synthesis of new isoforms or the deletion of existing ones; there was also variation in their relative abundance. Key words. Esterases; azadirachtin; hormetic concentrations; hormesis.Potentially toxic substances at subinhibitory concentrations may also have stimulatory effects ~,2. This phenomenon is called hormesis a. The hormetic effect of azadirachtin, a tetranortriterpenoid from Azadirachta indica, was reported for the first time in Tribolium castaneum by us. In this report it was shown that at 0.001 ppm to 0.1 ppm the larvae weighed more than the controls 3. Azadirachtin, a potent insect growth inhibitor, is known to affect hormonal balance in insects 4 and hence its beneficial effects may be a consequence of favourable shift in hormonal balance, at subinhibitory concentrations. Esterases, in insects, have been implicated in reproductive behaviour 5, pheromone and hormone metabolism 6,7, digestion ~'9, neurotransmission I~ and the action of and resistance to insecticides particularly organophosphates (OPs) 1~--~3. The mechanism of hormesis is still unknown, although several hypotheses have been proposed ~. Hormesis has been suggested to be the consequence of transient or sustained overcorrections in response to low levels of inhibitory stimulus 1,14. The present study, therefore, is an attempt at understanding, at the molecular level, the involvement of esterases in the hormetic action of azadirachtin on Tribolium castaneum.
Materials and methodsA laboratory culture of Tribolium castaneum was maintained on a diet comprising white flour: white cornmeal: dried brewer's yeast (10:10:1.5) at 27+1~ and 60% RH. To obtain freshly hatched first instar larvae, eggs were obtained by infesting freshly sieved (mesh no. 40) diet with adults of both sexes. The eggs thus obtained were incubated at 30 ~ to give freshly hatched first instar larvae. Diet with different concentrations of azadirachtin was prepared by adding a known amount of azadirachtin (98% pure, a gift from Prof. H. Rembold, Max Planck Institut fiir Biochemie, Germany) from a stock solution.The carrier solvent used was AR grade acetone. Diet soaked in acetone alone served as the control. Treated and control diets were kept at 30 ~ for 48 h to allow complete evaporation of the solvent. Five freshly-hatched first instar larvae per replicate per concentration were allowed to feed on diet containing 0.001, 0.01 and 0.1 ppm azadirachtin or on the control diet for 10 d. For each treatment there were three replicate groups. This resulted in 15 ten-day-old larvae (III instar ~8 mg) per treatment after 10 d. These were separately homogenized in 3 ml of 40 mM phosphate buffer, pH 7.0. The homogenate was centrifuged at 10,0...
