Bull. Org. mond. Sant 1971, 44, 215-219 Bull. WldHlth Org.

Toxic Effects Produced in Insects by Organophosphorus Compounds*

P. A. DAHM 1

This paper reviews the toxicity of organophosphorus compounds in relation to cholin- esterase inhibition in insects. It covers anticholinesterase effects on different stages of the life cycle and the relationship between cholinesterase inhibition and lethality. Other effects of organophosphorus compoun ds, which may account for anomalies in insecticidal action, are also considered.

The lethality of modern insecticides has en- Smaliman & Mansingh (1969). The system consists couraged the acceptance of their use in spite of of three components: acetylcholine (ACh), the the fact that we have relatively little information synaptic transmitter; acetylcholinesterase (AChE), on their mode of action in insects. Only a decade the ACh-hydrolysing enzyme; and choline acetyl- ago, Heath (1961) expressed his concern at our lack transferase, the ACh-synthesizing enzyme. All of knowledge of the toxicity of organophosphorus three components are present in insects in higher compounds by stating that " almost every aspect titre than in most vertebrates. Maximum amounts of the action of phosphorus compounds on insects of AChE activity are associated with highly active is debatable. In part this is because many of the insects (Metcalf et al., 1955). Much evidence indicates techniques applied to the study of mammals are that the system functions in synaptic transmission not applicable to the study of insects, but it is in insects essentially as it does in vertebrates, but mainly because few workers have tried to tackle full proof is lacking. Neuromuscular transmission the problems presented. Because of the shortage in insects, unlike that in vertebrates, seems not to be of data it has to be assumed at present that phos- mediated by a cholinergic system. phorus compounds act in similar ways on insects There is only a limited amount of information of different species. It will be surprising if this relating the toxic effects of organophosphorus in- assumption is not proved to be an oversimplifi- secticides with the cholinergic system in various cation as more work is done." This paper examines stages ofinsect development. In the embryonic stage, the action oforganophosphorus compounds oncholin- this is because of uncertainty over the appearance ergic mechanisms, their effects on other enzymes, of the cholinergic components. Choline acetyltrans- and their miscellaneous effects. ferase seems to be one of the first detectable com- ponents. ACh appears simultaneously or sometimes EFFECTS ON CHOLINERGIC MECHANISMS after a short delay (Smallman & Mansingh, 1969). A hypothesis of a cholinergic system in insects The insensitivity of insect eggs to anticholinesterase has been developed to explain the action of organo- agents during early development indicates that phosphorus insecticides. Despite earlier uncertain- AChE may not be essential-or may not be present- ties (Chadwick, 1963), the evidence for a cholinergic during early embryogenesis (David, 1959; Meh- system in insects is now unequivocal according to rotra & Smallman, 1957; Smith & Wagenknecht, 1956). Nevertheless, ChE inhibition is the best- * Journal Paper No. J-6855 of the Iowa Agriculture and supported hypothesis for the toxicity of organo- Home Economics Experiment Station, Ames, Iowa, USA. insect embryogenesis. Projects No. 1351 and 1844. The preparation of this paper phosphorus compounds during was supported by US Public Health Service Research If ChE is not present during early embryonic develop- Grant ES-00205 from the Division of Environmental Health ment, other esterases may play important roles Sciences and North Central Regional Project NC-96. in explaining the toxicity of organophosphorus 1 Professor, Department of Zoology and Entomology, Iowa State University, Ames, Iowa, USA. compounds during this time, although the impor-

2629 -215 - 216 P. A. DAHM tance of these other esterases in the insect embryo vivo with mammals. It has been suggested that a is not clear. Research with organophosphorus ChE-reactivating factor exists in living flies (Mengle compounds and insect embryos has not revealed & O'Brien, 1960). an exploitable target for insecticidal action. Even The selectively toxic action of a few organo- though some organophosphorus compounds have phosphorus compounds is partly attributable to been shown to have ovicidal activity, this factor differences in cholinesterases (O'Brien, 1961; Win- is seldom evaluated because field studies usually teringham, 1969). The greater toxicity of the diiso- do not differentiate between insecticidal effects on propyl homologue of parathion to the housefly eggs and effects on other stages in the life cycle than to the honey-bee, Apis mellifera, is a result of (Smith & Salkeld, 1966). the greater sensitivity of housefly ChE to diiso- Studies with organophosphorus insecticides have propyl paraoxon and the higher levels of this com- shown that muscular movements of insects during pound in the housefly than in the honey-bee after hatching from the egg are controlled by a cholinergic treatment. The lower levels of diisopropyl paraoxon system (Mehrotra & Smallman, 1957). AChE in the honey-bee are caused by a slower rate of activity increases five-fold from the egg to the larval formation (Camp et al., 1969a, 1969b). The low rate stage (Casida, 1956). The levels of the cholinergic of detoxification of famphur t in the large milkweed components also increase between the first and the bug, Oncopeltus fasciatus, is balanced by the de- last larval instars. The cholinergic system does not creased sensitivity of its ChE to inhibition by fam- change significantly during the larva-pupa moult, oxon (O'Brien et al., 1965). but does increase greatly during pharate adult There are also reports of acetylcholinesterases development. The cholinergic components reach in arthropods that are relatively insensitive to in- their maximum levels shortly before adult emergence hibition by organophosphorus insecticides. For (Smaliman & Fisher, 1958). example, resistance to these insecticides in Biarra The ChE inhibition produced in vitro and in vivo and Ridgelands (Australia) strains of the tick by organophosphorus insecticides is not always Boophilus microplus seemingly is due to such a pro- consonant with the performance of the same com- perty of the enzyme (Wharton & Roulston, 1970). pounds in the field. Neither is it correct to assume A few organophosphorus insecticides have been that a high degree of ChE inhibition must be pro- used as systemic poisons in mammals to kill arthro- duced by a compound if it is to be effective in killing pod parasites. The insecticides or their metabolites insects. There are numerous examples where low are lethal because of ChE inhibition in the para- levels of ChE inhibition have been observed during sites. ChE inhibition was " complete " in the central the early symptoms of poisoning and, sometimes, nervous system of northern cattle grubs, Hypoderma even at death (Chadwick, 1963). In a recent study bovis, removed from heifers treated with Ruelene, t (Goszczynska & Styczynska, 1968), the amounts of virtually complete in grubs from heifers treated with malathion and diazinon required to produce 50 % trichlorfon, and usually complete in heifers treated inhibition of housefly, Musca domestica, brain AChE with coumaphos (Steward, 1969). Following the in vitro were 1.5 and 8 times, respectively, the amounts administration of cythioate,t famphur,t and fen- required for the same effect in vivo. The AChE inhi- thion to female rabbits, yellow-fever mosquitos, bition in flies in a state of "knock-down " was Aedes aegypti, and Rocky Mountain wood ticks, independent of the insecticide dosage. Even more Dermacentor alndersoni, were allowed to feed on extreme is a report of fatal poisonings of lepidopte- the rabbits. Mortality of the ectoparasites com- rous larvae by organophosphorus insecticides where menced when their ChE was 32% inhibited, and no inhibition of the central nervous system occurred was complete at 680% inhibition. The mortality of (Sundukov, 1969). the ectoparasites correlated more closely with de- Spontaneous recovery of housefly brain AChE, pression of their ChE activity than it did with inhibited by several organophosphorus compounds depression of the ChE activity of the host (Smith (DFP,t TEPP, parathion, paraoxon, malathion, & Goulding, 1970). and dimethoate) applied to the tip of the abdomen, has also been observed. This is in contrast to results obtained in vitro with houseflies and in EFFECTS ON OTHER ENZYMES t Names against which this symbol appears are identified Insects contain numerous hydrolytic enzymes in the Glossary on pages 445-446 with differing substrate spectra. The fact that a TOXIC EFFECTS OF OP COMPOUNDS IN INSECTS 217 few of them are inhibited by organophosphorus com- MISCELLANEOUS EFFECTS pounds suggests another mode of action of these Symptoms of poisoning by organophosphorus compounds. However, the inhibition of esterases insecticides in insects are difficult to interpret in does not seem to play as critical a role in the poi- relation to species, insecticide, dose, and interval -soning process as does the inhibition of AChE after poisoning (Chadwick & Hill, 1947; Chamberlain (Afsharpour & O'Brien, 1963; Brown, 1963; & Hoskins, 1951; O'Brien, 1956). Successive symp- Chadwick, 1963). As an example, 4 of 6 carboxylic toms of poisoning by DFP t and TEPP in the esterases separated by zone electrophoretic analysis American cockroach are hyperactivity and hyper- of the gastric secretion of American cockroaches, excitability, exaggerated tonus, ataxia, clonic and Periplaneta americana, were inhibited by dichlorvos. tonic convulsions, paralysis, and death (Chadwick A purified, heat-stable enzyme (designated E6), & Hill, 1947). About 50-75% of the AChE in the prepared from the original group of 6, lost 86% nerve cord of the American cockroach was inhibited of its capacity to hydrolyse tripalmitin in the pre- at the hyperactive stage of poisoning with parathion. sence of 10-3M dichlorvos, indicating that the enzyme At the same stage of poisoning with TEPP or para- was considerably less sensitive than AChE to thion in the honey-bee, at least 50% of the AChE inhibition (Cook et al., 1969). Conversely, two in the brain was inhibited and at least 90% was electrophoretically separated bands of housefly inhibited at the prostrate stage (Brown, 1963). esterases that are strong in organophosphate- Electrophysiological effects in the American resistant strains of the housefly were inhibited com- cockroach caused by TEPP, parathion, demeton, pletely by cyclic methylene-o-phenylene phenyl and demeton-methyl were an increase in spon- phosphate (" saligenin cyclic phenyl phosphate ", taneous activity and the prolongation of synaptic compound I, R = C6H5), but were little affected after-discharge, followed by a burst of discharges by the insecticidal methyl derivative (compound and block in the nerve cord. The changes in nervous I, R = CH3) (Eto, 1969). Although the principal activity paralleled AChE inactivation (Narahashi & Yamasaki, 1960). A review of changes in nervous activity with organophosphorus insecticide poison- ing (Brown, 1963) indicates that treatment of QQ OR the last abdominal ganglion of the American cock- 0' roach with DFP t causes a multiplication of dis- charges as they pass the synapse, followed by I an alternation of after-discharge and complete block. The block seems to be caused by ganglionic action of certain cyclic methylene-o-phenylene depolarization brought about by increased excitatory phosphates and phosphorothioates is the inhi- post-synaptic potentials. The alternating facili- bition of AChE by phosphorylation, it is possible tation-and-block effect exerted on the ganglia by that the alkylating activity of these cyclic esters may TEPP takes place at concentrations of the toxicant have some effects on the enzyme (Eto & Ohkawa, that produce about 95 % inhibition of AChE in the 1970). nerve cord. The effects of organophosphorus insecticides A study of the action of diazoxon on ChE activity on other enzymes and a few metabolic products also and nerve conduction in ganglia of the American have been investigated. Malathion has some inhi- cockroach showed that the metathoracic ganglion bitory effect on succinoxidase, glycolysis, and is about three times as susceptible as the sixth Krebs-cycle intermediates. Substantial concen- abdominal ganglion to diazoxon (Burt et al., 1966). trations (10-3M) of malathion or parathion are Severe functional abnormalities occur in the ner- necessary to inhibit cytochrome oxidase. Parathion vous system, although much of the ChE in the decreases dehydrogenase activity (Brown, 1963). ganglion is still uninhibited and some ChE inhibition Cytochrome oxidase, succinic dehydrogenase, and occurs before the function of the ganglion is affected. overall glycolysis were inhibited by trichlorfon These workers warn against using ChE inhibition in in houseflies (Pant, 1958). In none of these studies various body regions as an indication of the toxicity was the inhibition of other enzymes as specifically of organophosphorus insecticides. pronounced as was the inhibition of AChE by the Some organophosphorus insecticides, several same insecticides. other chemicals, and certain forms of stress cause 218 P. A. DAHM the release of pharmacologically active substances grity of the heart and circulation. If these functions that affect the nervous system of insects (Sternburg are disrupted by organophosphorus insecticides, et al., 1959; Sternburg, 1963). For example, the it seems unlikely that such interference is of primary nerve cord of the American cockroach perfused importance in the lethal effects of these chemicals. with TEPP releases a neurotoxin that induces exci- The following examples of variations in the effects tation and block of nervous activity (Sternburg of organophosphorus insecticides on insect heart- et al., 1959). The blood of TEPP-poisoned American beat are included in a review by Brown (1963). cockroaches contains a toxin that excites the nerve Injected parathion increased the rate of heart-beat cord of an untreated cockroach (Colhoun, 1958). in the American cockroach and in Anopheles Experimental procedures seem to exclude unhydro- quadrimaculatus, a mosquito vector of malaria; lysed ACh as the cause of the excitation (Sternburg, the isolated heart of the cockroach was stimulated 1960). Release of the active substances appears by paraoxon, but not by parathion; parathion and to be associated with excessive central nervous related compounds had no effect on the heart-beat system activity, whether initiated by disruption of of the adult large milkweed bug or of larvae of the ACh-AChE system by organophosphorus insec- the greater wax moth, Galleria mellonella. ticides, by physical stimulation (e.g., electrical), or Although the cockroach heart has been designated by excessive afferent bombardment of synaptic re- a neurogenic heart, this characterization stems pri- gions (e.g., by DDT). Stress in chemical poisoning marily from its elaborate innervation and an in- presumably originates at the primary target of the creased heart rate in response to ACh (McCann, insecticide, and the abnormal release of pharmaco- 1970). However, the action of pharmacologically logically active substances seems to be a secondary active compounds, including AChE-inhibiting car- effect. When these substances are released in greater bamates, on the heart of the American cockroach than normal amounts, they cause abnormal beha- suggests that a more appropriate classification would viour or even paralysis and death (Flattum & Stern- be a myogenic heart with extensive nervous control burg, 1970). These active substances have not been (Miller & Metcalf, 1968). identified but their presence suggests the possibility Lack of evidence makes the effect of organo- that certain substances or systems that might be phosphorus insecticides on the excretory function induced by organophosphorus and other chemicals of malpighian tubules a moot question. There might be exploited in the design of insecticides. is, however, a significant water loss to the gut in Organophosphorus insecticides do have an effect many cases of organophosphorus-insecticide poi- upon oxygen consumption, heart and blood, soning, and this could have drastic metabolic excretion, and water loss, but not always in a way consequences (Chadwick, 1963). A loss of body that can be uniquely ascribed to this class of insecti- fluids occurred in silkworms, Bombyx mori, and cides. The reports are conflicting and therefore larvae of Dendrolimus pini poisoned with parathion difficult to evaluate. For example, German cock- and in German cockroaches poisoned with TEPP. roaches, Blattella germanica, injected with parathion In German cockroaches poisoned with parathion- thion or TEPP showed an increase in oxygen con- methyl, however, there was little loss of water sumption during hyperactivity before paralysis (Brown, 1963). (Harvey & Brown, 1951); American cockroaches The histological effects of parathion that have injected with malathion did not (O'Brien, 1956). been observed in several insect species (Brown, The insect heart and blood presumably transport 1963) are not inevitably found in insects killed by organophosphorus insecticides and their active parathion or other organophosphorus insecticides. metabolic products to the sites of action. The heart At least one study has been made of an antidote in various invertebrates is under a cholinergic for poisoning by organophosphorus compounds system of control according to Chadwick (1963), in insects (Mengle & O'Brien, 1960). Injections of but to refer to " the invertebrate heart " in comparison pralidoxime iodide at rates of 50 ,g/g and 1 000 with the vertebrate heart is an oversimplification. ,ug/g had no effect on the mortality of houseflies Among the invertebrates, there are hearts that topically treated with parathion, parathion-methyl, physiologically and pharmacologically are of seve- paraoxon, malathion, DFP, t and TEPP. The ral different kinds. Some are not sensitive to ACh symptoms of poisoning were delayed about 1 hour or the ChE inhibitors. There is little indication that with parathion, malathion, and DFP.t No sub- insects are dependent for continued life on the inte- stantial changes were induced by pralidoxime TOXIC EFFECTS OF OP COMPOUNDS IN INSECTS 219 iodide (500 ug/g) in AChE inhibition in vivo after before the organophosphorus compound, simulta- poisoning by malathion and parathion. This was neously with it, or near the period of maximum equally true whether the oxime was injected 2 hours AChE inhibition.

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