General discussion—Session V

General Discussion- Session V TOXIC EFFECTS AND CAUSES OF DEATH IN INSECTS MEETER: As the toxicity of different agents' for different species is measured by LD50 determinations, it is important to know the causes of death. Some mammals, following exposure to organophosphorus compounds, may die primarily from a central respiratory paralysis, while others may die from a peripheral paralysis-both forms complicated to varying degrees by bronchoconstriction. What is known of the cause(s) of death in insects? HOLLINGWORTH: I think it would be fair to say that for no insect-insecticide combination is the ultimate cause of death known. Multiple lesions occur. It is reasonable to assume that, in insects, the primary site of attack by carbamates and organo- phosphorus compounds is cholinergic and that the central effects are probably the main ones. Respira- tory effects may well be of limited importance in insects, which generally rely on the passive diffusion of oxygen to the tissues via the tracheae, with pump- ing of air by body movement under some circum- stances. HOBBIGER: What are the criteria for "death" in insects? Does the difficulty of defining death give rise to variations in the LD50 values determined in different laboratories? WEIDEN: The criteria vary for different insects and different types of compound. One standard pro- cedure is to prod the insect to see if it will move. Mealworms can be dropped on a hot plate. Boll weevils are difficult to test because they " play dead". Experience is essential in order to know when to make a reading. In most published data on insect LD50 values, the time of reading after treatment is stated, and frequently the LD50 is given for more than one time period. Percentage knock-down is also frequently given. One reason for using the housefly for the determination of LD50 values is that experience has shown that the data obtained by different laboratories compare well. As with higher animals, of course, the strain must be specified. HOLAN: The best method for determining the death of intoxicated insects is the measurement of nerve impulses at chemoreceptors. When the nerve acti- vity of a randomly selected insect is zero, the insects can be considered dead. When testing slow-acting insecticides in the housefly. conventional obser- vations or nerve impulse measurements may show significant differences in 24-hour mortality. After 48 hours these differences are not significant. HOLMSTEDT: In mammalian pharmacology, physo- stigmine has been used to map out and prove cholin- ergic functions. In the early 1920s acetylcholine was finally shown to be the cholinergic transmitter. Has any entomologist ever tried to isolate a choline ester from insects, thus proving the occurrence of cholinergic mechanisms in the species? BOOTH: Both choline acetyltransferase and cholin- esterase have been isolated from several insects and purified in our laboratory. The presence of acetyl- choline has also been demonstrated. The literature contains a wealth of information on the abundance of cholinesterase, acetylcholine, and choline acetyl- transferase in insect tissues. There is little doubt that choline esters are present in insects or that a cholinergic system exists in insects. In fact, the titres of choline esters in insect tissues are usually higher by several orders of magnitude than those in mammalian tissues. AUGUSTINSSON: There is good evidence that acetyl- choline is present in the head (brain) of various insects. Acetylcholine is probably the only choline ester that is present. Our work since 1955 has demonstrated that acetylcholine and no other choline ester-including propionylcholine-is pre- sent in honey-bees. HOBBIGER: I should like to point out that the presence of high concentrations of acetylcholine, acetylcholinesterase, and choline acetyltransferase is not sufficient for postulating synaptic transmission of a cholinergic nature; all three are present in high concentrations in the human placenta, although the latter does not contain neuronal synapses. DAHM: Reviews by Chadwick (1963) and by Small- man & Mansingh (1969), cited in my paper, contain references to the isolation of acetylcholine. A speci- fic example of the identification of acetylcholine - 2292632 GENERAL DISCUSSION-SESSION V in cockroach blood occurs, I believe, in a report by Waller & Lewis (Journal of Insect Physiology, about 1960). HOLLINGWORTH: I believe that virtually all the elements of the cholinergic system have been detected in insects. The junctions between the cercal nerve and the giant fibres in the sixth abdominal ganglion of the American cockroach have been examined by several workers, including Dr Narahashi and Dr Shankland at Purdue. There is very strong evidence that these junctions have the usual cholinergic apparatus. NARAHASHI: Both acetylcholine and the choline acetyltransferase system have been demonstrated in insect nervous systems. From studies of the sixth abdominal ganglion of the cockroach, we obtained evidence that transmission across certain synapses between the cercal nerve fibres and the giant nerve fibres is mediated by acetylcholine. The effect of the latter was potentiated by physostigmine. How- ever, the neuromuscular junction of the cockroach is not cholinergic; we found that treatment with several anticholinesterase agents had no effect whatsoever. Although inhibition of cholinesterases is probably the principal mechanism responsible for the toxic action of organophosphorus compounds and carba- mates, it might be appropriate to point out that some of these compounds exert a direct action on the postsynaptic membrane. Such direct action is more important in mammalian toxicology, since it could cause serious side-effects. In insects, direct action may play a less important role in toxicity. Apart from the question of whether cholinesterase inhibition is mainly responsible for toxicity, the determination of 150 values seems to be very useful for the purpose of testing new organophosphorus compounds or carbamates. However, in discussing the mode of action, we should remember that a good correlation does not demonstrate a causal relationship. As pointed out by Dr Dahm, there are many examples of a lack of correlation between cholinesterase inhibition and the development of symptoms of poisoning. We have little knowledge of the nature of the transmitter substance in insects. I do not think that the nervous system of insects is more compli- cated than that of vertebrates; the reason for our poor knowledge of the transmitter substance is that very little effort has been put into studies of insect neurophysiology in comparison with those of verte- brate and general neurophysiology. The small size of insect tissues is not a great handicap in such studies; with highly refined techniques it is possible to identify a transmitter substance in the insect nervous system. To accomplish this, several tech- niques must be used, including (1) modern electro- physiological techniques, such as intracellular micro- electrode recording and iontophoretic application of suspected transmitter substances to the synaptic areas; (2) biochemical techniques, such as the microbioassay of transmitter substances; and (3) histochemical techniques, especially at the electron microscopic level. The identification of gamma- aminobutylic acid as the inhibitory transmitter sub- stance in crayfish neurones by Otsuka and Kravitz is a good example of such a study. We should have two goals for the purpose of designing new and better insecticides-first, to overcome the immediate problems of contamination of the environment and resistance in insects, and second, a long-term programme aimed at, say, 10 years from now. To meet the immediate prob- lems, the further development of organophosphorus compounds and carbamates, using 150 values as a measure of activity, would be a powerful approach. As a long-term goal, we should try to identify the transmitter substances in insects, since we should then be in a better position to develop new com- pounds having specific affinity for the insect ner- vous system and other physiological and biochemical factors. 230