Penetration of insecticides through the foregut of the honeybee (Apis mellifera L.)

The rates of penetration of ¹⁴C-labeled insecticides (parathion, carbaryl, and dieldrin) through the foregut of the honeybee (Apis mellifera L.) were measured in vitro and in vivo. Uptake of the insecticides from the lumen of the foregut into foregut tissue was directly proportional to insecticide lipophilicity, but penetration through the foregut was not. Of the three insecticides studied, parathion appeared to possess the optimal physicochemical characteristics required for penetration. The uptake of carbaryl and release of dieldrin by the foregut tissues may limit their respective penetration rates. Insecticide penetration was found to be inversely proportional to the sucrose concentration in the lumen of the foregut in both in vitro and in vivo studies. The oral toxicity of carbaryl showed a similar dependence on the sucrose concentration of the solution in which the insecticide was fed. The data presented indicate that the honeybee foregut is permeable to lipophilic compounds and strongly suggest that this permeability may contribute substantially to the toxicity of orally ingested insecticides in this insect.     

The mechanisms of carbaryl resistance in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

The physiological mechanisms of resistance to carbaryl were investigated in a carbaryl-resistant strain of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Piperonyl butoxide greatly reduced the resistance level from 90- to 6-fold, indicating that microsomal cytochrome P-450-dependent monooxygenases may play a major role in resistance. This finding is consistent with metabolic data in which the oxidative metabolism of carbaryl by midgut homogenates was five times more active in the resistant strain than in the susceptible strain. In addition, the resistant strain showed increased activities of microsomal hydroxylation and epoxidation compared to the susceptible strain. Cuticular penetration studies using [¹⁴C]carbaryl revealed that 55% of the applied radioactivity remained on the cuticle of resistant larvae while 32% remained on susceptible larvae 24 hr after topical treatment. The resistance appeared to be unrelated to target site insensitivity. It is concluded that the high level of resistance to carbaryl in this insect was mainly due to enhanced oxidative metabolism of the insecticide (via hydroxylation and epoxidation) with reduced cuticular penetration playing a very minor role, if any.     

Accumulation of malathion residues in grain stored on malathion-treated wood surfaces

Laboratory studies were conducted to determine whether insecticides applied to storage structures are able to move past a single layer of grain kernels to give adequate control of stored-grain insects. The extent of uptake of insecticides was assessed on three layers of wheat in contact with wood surfaces treated with malathion 836 g litre^?1 e.c. at 1–0 g a.i. m^?2 using chemical and biological methods at predetermined time intervals. There was a progressive uptake of malathion beyond the layer directly in contact with the treated surfaces and the uptake depended on the duration of storage. After 8 weeks a sufficient amount of malathion accumulated on the top layer of grain kernels to cause 100% mortality of adult Tribolium castaneum (Herbst).     

The biochemical basis of malathion resistance in the sheep blowfly,Lucilia cuprina

The in vivo and in vitro metabolism of [¹⁴C]malathion was studied in susceptible (LS) and malathion resistant (RM) strains of the sheep blowfly, Lucilia cuprina (Wiedemann). No difference was found between strains in the penetration, excretion, storage, or inhibitory potency of the insecticide. However, RM degraded malathion to its α- and β-monocarboxylic acid metabolites more rapidly than LS, both in vivo and in vitro. This enhanced degradation of [¹⁴C]malathion occurred in vitro in both mitochondrial and microsomal fractions of resistant flies. Kinetic analysis revealed that these fractions degraded malathion by discrete mechanisms. The enzymes from the mitochondria of both strains had the same K_m, whereas the microsomal enzyme from the RM strain had a fivefold higher K_m than that from the LS strain. Studies of esterase activities and the effect of enzyme inhibitors showed that both the mitochondrial and microsomal resistance mechanisms were the result of enhanced carboxylesterase activity. It was concluded that increased carboxylesterase detoxification of malathion adequately explained the high level of malathion resistance in RM if rate-limiting factors such as cuticular penetration were taken into account.     

Malathion resistance of stored-product insects in Israel

A study was conducted to determine the extent of resistance to malathion in field populations of insects collected from nine granaries located in different regions of Israel. The results showed that the maximum resistance factor calcuated from the LC_so ^s of the different insect species tested was:Tribolium castaneum (Herbst), x 538.0;Oryzaephilus surinamensis (L.), x 8.0;Sitophilus oryzae (L.), x 1.2; andRhyzopertha dominica (F.), x 9.0. There were significant differences between the resistance level among strains collected from different locations in Israel. By using triphenyl phosphate (TPP), an inhibitor of carboxyesterase, it was shown that, in the case ofT. castaneum andR. dominica, the resistance is a malathion-specific type and that in the case ofO. surinamensis it is partially non-specific to malathion. The significance of these findings in selecting new insecticides to replace malathion as a grain protectant was considered.     

Penetration and metabolism of [14C]carbaryl in larvae of the gypsy moth,Lymantria dispar (L.)

The absorption and metabolism of topical doses of carbaryl by larvae of the gypsy moth, Lymantria dispar (L.), were determined using ¹⁴C-ring-labeled material. Carbaryl penetration followed four distinct phases of linear absorption, i.e., 0–5 min, 5–60 min, 1–3 hr, and 3–12 hr; the absorption rates for the four phases were 2.8, 0.57, 0.07, and 0.02% per minute, respectively. Ninety percent of the total metabolism of carbaryl occurred within the first 3 hr; over the next 9 hr, metabolism was exceedingly slow with a linear rate of ca. 0.8% per hour. Carbaryl was always the major radiocarbon in the larvae and the feces, amounting to ca. 16 and 9%, respectively, at 12 hr. At 12 hr the metabolite composition was 5-hydroxy carbaryl >N-hydroxy carbaryl > 1-naphthol > 4-hydroxy carbaryl > 5,6-dihydro-5,6-dihydroxy carbaryl. Small amounts of an additional product were detected but not identified. Much of the excretion of carbaryl and metabolites occurred without conjugation. The amounts of carbaryl metabolized by second, third, fourth, and fifth instars were directly correlated with the insecticide tolerances and mixed-function oxidase activity of the various larval stages. The synergistic action of 2,6-dichlorobenzyl-2-propynyl ether and piperonyl butoxide was also correlated with inhibition of the oxidative pathway.     

Penetration and metabolism of topically applied permethrin and cypermethrin in pyrethroid-tolerant Wiseana cervinata larvae

The penetration, excretion, and metabolism of topically applied [¹⁴C]permethrin and [¹⁴C]cypermethrin have been examined in larvae of the porina moth Wiseana cervinata to determine the factors which affect body levels of unchanged pyrethroids. Metabolism was by hydrolysis and to a lesser extent oxidation and the primary metabolites were quickly conjugated to water-soluble products. Little excretion occurred and body levels of unchanged pyrethroids were dependent on the interaction of penetration and metabolism. cis-Cypermethrin was more resistant to metabolism than trans-cypermethrin and cis- and trans-permethrin. trans-Permethrin most readily penetrated into larvae. The body levels of unchanged permethrin were enhanced by pretreatment of larvae with the metabolic inhibitors carbaryl or piperonyl butoxide. Tolerance of the pasture pest porina to the synthetic pyrethroids is discussed in relation to these findings.     

Organophosphorus insecticides affect normal polyamine metabolism in amphibian embryogenesis

The objective of the present study was to evaluate the concentration- and time-dependent effects of the organophosphorus insecticides malathion and azinphos-methyl on polyamine metabolism, and relate them to normal and altered embryonic development of the common toad Rhinella arenarum. Control embryos showed that the higher polyamines spermidine and spermine acquired importance with respect to the diamine putrescine as embryonic development progressed. The activity of ornithine decarboxylase significantly decreased in complete operculum embryos. Continuous exposure to malathion caused a decrease in polyamine levels during embryonic development. However, there was an increase in putrescine levels in complete operculum embryos exposed to a sublethal concentration of the insecticide. Embryos exposed to malathion displayed a decrease in fresh weight and size, along with an increase in the number of malformed individuals. R. arenarum embryos exposed to a lethal concentration of azinphos-methyl showed an increase in putrescine levels and a decrease in spermidine and spermine levels, accompanied by an increase in ornithine decarboxylase activity. In conclusion, as the embryonic development of the toad R. arenarum progresses, polyamine metabolism shifts to higher polyamine levels with a more preponderant contribution of spermidine and spermine with respect to putrescine and involves a dramatic change in ornithine decarboxylase activity, one of the key regulatory enzymes of the pathway. Organophosphorus insecticides are capable of altering polyamine metabolism, slowing embryo development in parallel with a reduction in spermidine and spermine levels. An increase in the oxidative degradation of polyamines might be involved in the toxic action of organophosphorus insecticides and might also be related to other effects such as teratogenesis.     

Combinations of pirimiphos-methyl and carbaryl for stored grain protection

In laboratory experiments, whole wheat was treated with pirimiphos-methyl or carbaryl or combinations of these two insecticides; the treated grain was then adjusted to a 12% moisture content and stored at 25°C for bioassay at various intervals over a period of 39 weeks. Pirimiphos-methyl at 5.1 mg kg^?1 effectively controlled Sitophilus granarius (L.) and Tribolium confusum Jacquelin du Val but was ineffective against Rhyzopertha dominica (F.) CRD 118, a strain showing malathion resistance. Conversely, carbaryl at 6.5 mg kg^?1 (but not at 3.1 mg kg^?1) was effective against R. dominica, but ineffective against the other two species. A combination of pirimiphosmethyl + carbaryl, at 1.8 + 5.1 mg kg^?1, controlled S. granarius and R dominica but not T. confusum, whilst a 4.2 + 3.4 mg kg^?1 combination was relatively more effective against T. confusum but less so against R. dominica. In a separate experiment, whole wheat was treated with carbaryl at 2.5, 5.0 and 7.5 mg kg^?1 (nominal rates). Samples were stored and, at various times after the treatments, were bioassayed with R. dominica CRD 2, at 20, 25, 30 and 35°C. The results were comparable with those for the CRD 118 strain, but efficacy was reduced at higher temperatures. The combination of pirimiphos-methyl at 4–5 mg kg^?1 and carbaryl at 5–6 mg kg^?1 is suggested as a potentially useful grain protectant where R. dominica is a problem and long term storage is required. These results are discussed in relation to the protection of stored grain in Australia.     

Age-dependent variations in the response of several species of diptera to insecticidal chemicals

The toxicity of carbaryl to three species of fleshflies Sarcophaga bullata Parker, S. crassipalpis Macquart, and S. argyrostoma (Robineau-Desvoidy) varied considerably with age and sex. In contrast, the susceptibility of a blowfly, Phormia regina (Meigen) to carbaryl decreased with age and that of two muscid flies, Musca autumnalis DeGeer and Stomoxys calcitrans (L.), remained relatively constant. The synergistic activity of piperonyl butoxide varied inversely with the innate toxicity of carbaryl to each species suggesting that the observed age- and sex-dependent variations in carbaryl toxicity result mainly from differences in detoxifying capability. This was supported by in vitro measurements of oxidative microsomal metabolism. It was further established that differences in the rates of penetration and excretion of carbaryl and in the susceptibility of the head cholinesterase to carbaryl inhibition were of little importance in determining the susceptibility of the flies to this insecticide.     

Changes in malathion resistance with age in Anopheles stephensi from Pakistan

Resistance to malathion in Anopheles stephensi from Pakistan was measured at intervals during the first week of adult life. LT₅₀ values for homozygous resistant females decreased four-fold during the first 7 days of adulthood. A decrease in resistance with age also occurred in heterozygotes; the LT₅₀ values of males and females fell sevenfold during the first 5 days of adulthood. The sensitivity to malathion of a susceptible strain increased with age. A biochemical basis for the declining resistance levels was investigated. Resistant and susceptible adults were homogenized at intervals during the first week of adulthood and soluble extracts were incubated with [¹⁴C]malathion. The rate of malathion metabolism to mono- and dicarboxylic acids was faster in resistant than in susceptible mosquitoes. The rate of malathion metabolism decreased with age in both strains. A decrease in carboxylesterase activity with age in resistant and susceptible mosquitoes is thus responsible for the increasing sensitivity to malathion. Implications for the monitoring of resistance in the field by diagnostic dosages and for the future use of malathion in mosquito control are discussed.     

Detoxication capacity in the honey bee,Apis mellifera L

Various detoxifying enzymes, including microsomal oxidases, glutathione S-transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase, were assayed in adult worker bees (Apis mellifera L.) using midguts as the enzyme source. A cell-free system was used for all enzyme assays, except that microsomal oxidases required intact midgut because of the inhibitor encountered. Midgut microsomal preparations contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain the low microsomal oxidase activity in microsomes. All enzymes studied were active, suggesting that the high susceptibility of honey bees to insecticides is not due to low detoxication capacity. Sublethal exposure of honey bees to various insecticides had no effect on these enzyme activities, with the exception of permethrin which significantly stimulated the glutathione S-transferase, and malathion, which significantly inhibited the α-naphthylacetate esterase and carboxylesterase.     

Effect of insecticides on the short-circuit current and resistance of isolated frog skin

The effects of aldrin, carbaryl, α- and γ-chlordane, dichlorodiphenyldichloroethane (DDD), dichlorodiphenyltrichloroethane (DDT), dieldrin, endrin, heptachlor, lindane, methoxychlor, and nonachlor on the short-circuit current and resistance of the isolated intact frog skin were studied. The short-circuit current is approximately equivalent to the rate of active transport of sodium, while the resistance indicates the summed ionic permeability of the skin. At a concentration of 2 × 10^?4M, only carbaryl, DDD, dieldrin, and lindane produced significant (P<0.05, paired t test) changes in the short-circuit current. Nonachlor produced a decrease (P=0.12) in the short-circuit current and also increased the resistance (P=0.07). DDD, dieldrin, and carbaryl caused significant increases in short-circuit current while at the same time the resistance was significantly decreased. Lindane increased both the short-circuit current and the resistance. It was concluded that the frog skin probably contains effective permeability barriers that prevent externally applied insecticides from reaching the site of active sodium transport. It appeared likely that most of the insecticides produced the observed effects on the frog skin by altering the sodium permeability of the outer barrier.     

Role of a microsomal carboxylesterase in reducing the action of malathion in eggs of Triatoma infestans

A microsomal malathion carboxylesterase present in Triatoma infestans eggs was active from the first day of embryonic development. This microsomal egg malathionase (MEM) showed a unique band in polyacrilamide gel electrophoresis (PAGE) when malathion was used as substrate. In vivo metabolism of [¹⁴C]malathion during all embryonic development showed a 10% degradation due to carboxylesterases. The in vitro evaluation of the same metabolic pathway catalyzed by the microsomal fraction of T. infestans eggs showed partial inhibition by paraoxon. α- and β-malathion monoacids were identified as the main metabolites of the in vivo and in vitro metabolic pathways. The carboxylesterase band that appeared in PAGE (MEM) from the first day of embryonic development could be the main cause of malathion tolerance in T. infestans eggs.     

Why does DDT toxicity change after a blood meal in adult female Culex pipiens?

The toxicity of topically applied DDT to adult female anautogenous mosquitoes (Culex pipiens L.) showed dramatic variations in blood-fed insects. It decreased very rapidly about twofold to a minimum at 24 hr after a blood meal, then increased within 72 hr back to values typical of non-blood fed insects. A comparison of the metabolism of [¹⁴C]DDT in vivo revealed an increase in DDT dehydrochlorination to DDE at 72 hr after a blood meal, but this increase was not responsible for the variations in DDT toxicity at 24 hr. Changes in penetration rates were not observed and changes in the distribution of DDT could likewise not be related to the short period of decreased toxicity of DDT. Fenvalerate and trans-permethrin, two pyrethroid insecticides which are believed to have a mode of action similar to that of DDT, were also significantly less toxic 24 hr after a blood meal. By contrast, the cyclodiene insecticide aldrin and the carbamate insecticide propoxur were not less toxic 24 hr after a blood meal. The results suggest that after a blood meal an unidentified and transient change in C. pipiens specifically decreases DDT/pyrethroid toxicity. A hypothesis concerning this transient change is advanced. The results illustrate the difficulties in explaining physiological changes in insecticide toxicity.     

Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster

Cytochrome P450-dependent monooxygenases are important in the activation and detoxification of numerous insecticides. In this study, a Drosophila melanogaster Cyp6d4 null mutant was used to determine the role of this P450 in insecticide metabolism. This null mutant was generated by imprecise excision of a mobile P element located upstream to the P450 gene Cyp6d4. Comparative analysis between the non-functional mutant and relevant control strains shows that Cyp6d4 does not appear to be involved in the metabolism of chlorfenapyr, cypermethrin, diazinon, imidacloprid, malathion, oxamyl, parathion, or pyrethrum extract, even though these insecticides are known to be activated or detoxified by P450-monooxygenases. No obvious abnormalities in development were seen in the Cyp6d4 null mutant, indicating that Cyp6d4 is not critical for the metabolism of vital endogenous substrates.     

Intestinal metabolism and absorption of carbaryl studied with a portal fistula

These data were obtained by use of a total and continuous portal vein fistula which virtually eliminated vascular redistribution of compounds absorbed from the gastrointestinal tract to nondigestive tissues (i.e., liver). The method allows direct measurement of the compounds absorbed, which is especially important in metabolism studies of ingested toxic compounds. These studies demonstrated that in vivo metabolism did occur within the intestine during the process of absorption of the pesticide carbaryl (naphthyl N-methylcarbamate) and naphthol, the hydrolysis product of the pesticide. Portal absorption of naphthol from a liquid diet (46 ± 4% of dose/120 min) was slower than from Ringer medium (75 ± 1%/120 min); portal absorption accounted for 82 ± 8 and 83 ± 4%, respectively, of the ¹⁴C absorbed from the intestine. The proportion of hydrophilic ¹⁴C-metabolites (water soluble) in portal blood varied from 6 to 89% and was a function of the substrate, dose vehicle (liquid diet vs Ringer), and time of portal fluid collection. Metabolism in the small intestine before absorption was confirmed for both substrates. The principal lipophilic constituent in portal fluid was the unmetabolized substrate for both carbaryl and naphthol; the principal ampholyte metabolite was naphthyl glucuronide. Although these in vivo data are qualitatively similar to evidence from previous in vitro studies, this in vivo evidence demonstrated that the extent of metabolism (and possibly detoxication) was considerably less than would be predicted from in vitro studies and indicates that the hazard of ingestion of carbaryl and other lipophilic toxic agents may be greater than realized.     

Comparative ester hydrolysis of carbaryl and ethiofencarb in four mammalian species

The comparative ester hydrolysis and selective toxicity of carbamate insecticides were studied in four mammalian species. Hydrolysis rates of carbaryl and ethiofencarb (Croneton) were examined in the rat, mouse, guinea pig, and gerbil. Respiratory ¹⁴CO₂ resulting from the hydrolysis of orally administered [carbonyl-¹⁴C]carbamates (0.2 mg/kg) was taken as measure of in vivo hydrolytic capabilities. Ester hydrolysis was found to be greater for ethiofencarb than for carbaryl in all species tested, although the relative order of hydrolysis among species was the same with both compounds. After 24 hr, gerbils had hydrolyzed 91% of the ethiofencarb and 65% of the carbaryl. Guinea pigs hydrolyzed somewhat less of the compounds, 65 and 58%, but considerably more than rats and mice, about 40 and 25%. Comparing hydrolysis capabilities to acute toxicity data revealed that those species exhibiting the greatest hydrolysis were equally or more susceptible to carbamate poisoning than those having lesser hydrolytic capabilities. While ester hydrolysis destroys the anticholinesterase activity of carbamates, it is clear from these findings that factors other than hydrolysis are largely responsible for the variation in toxicity of the carbamates to different mammalian species.     

The in vitro degradation of [14C]malathion by enzymatic extracts from resistant and susceptible strains of Drosophila melanogaster

Enzyme preparations from Drosophila melanogaster flies degraded [¹⁴C]malathion to α- and β-malathion monoacids and, hence, were considered to contain malathion carboxylesterase (ME) activity. Although ME- activity was stable during preincubation in the absence of malathion, it decreased dramatically during the course of the reaction, and could not be completely recovered by Sephadex G-25 chromatography. Furthermore, the protein fraction after chromatography still contained ¹⁴C, suggesting that the enzyme had become inhibited by a bound, ¹⁴C-labeled derivative. Extracts from a resistant (malathion-selected), an intermediate control, and the susceptible Canton S strains of D. melanogaster differed in the lability of ME activity during the reaction. This difference was partly attributed to the production of small amounts of malaoxon (2–8%) by the extracts from the more resistant strains. No consistent strain differences were found when the rate of malathion degradation was measured during the first minute of reaction, either with or without a microsomal oxidase inhibitor (metyrapone) present. These results, together with the cross-resistance of the malathion-selected strain to other insecticides and the lack of a synergistic effect of two carboxylesterase inhibitors (triphenyl phosphate and S,S,S-tributylphosphorotrithioate) suggested that malathion carboxylesterase does not contribute significantly to the observed differences in malathion resistance between strains.     

Dual role of esterases in insecticide resistance in the green rice leafhopper

The role of esterases as related to insecticide resistance was studied in an organophosphorus (OP)-resistant strain of the green rice leafhopper. As judged by p-nitrophenyl acetate hydrolysis, 21, 5, and 74% of the esterase activity was located in nuclei/mitochondria, microsomes, and the soluble fraction, respectively. All the fractions were active in hydrolyzing malathion, paraoxon, and fenvalerate. Hydrolysis of malathion and fenvalerate increased with time while that of paraoxon reached a plateau within 15 min. Since a considerable amount of p-nitrophenol was detected in the paraoxon reaction at 0°C and at zero time, the formation of p-nitrophenol may be due to phosphorylation of the esterases rather than phosphorotriesterase action. The results suggest a dual role for esterases in resistance mechanisms; a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides, both of which would protect the intrinsic target, acetylcholinesterase, from inhibition. Chromatofocusing of the soluble fraction resolved five esterase peaks, I–V. These esterases were active toward the three general substrates as well as for the three insecticides tested, except for Peak I in which the overall activity was too low. Thin-layer agar gel electrophoresis showed that the chromatofocusing peaks I–V corresponded to the electrophoretic bands E₁–E₅, some of which were previously shown to be associated with OP resistance. The dual role of these esterases may explain the cross-resistance between malathion and other OP insecticides as well as synergism between OP and carbamate insecticides.