Mechanisms of DDT resistance in larvae of the mosquito Aedes aegypti L

Larvae of eight strains of Aedes aegypti were exposed to DDT and compared for resistance, DDT uptake, in-vivo breakdown of DDT and residual unmetabolised DDT. Resistance varied widely between strains, three being fully susceptible, two almost immune and three of intermediate resistance. Breakdown of DDT by dehydrochlorination to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (pp'-DDE) occurred in all strains and was greater in the five resistant types, but there was no significant correlation between the extent of breakdown in the resistant strains and the level of resistance. Moreover the overall difference between susceptible and resistant strains disappeared when they were compared at a low, almost sublethal, concentration of DDT. Larvae of resistant strains carried a greater absolute quantity of unmetabolised DDT in the body and were able to tolerate levels of DDT that were lethal to susceptible larvae. However the two most resistant strains (T8 and B51) contained significantly less DDT plus pp'-DDE than strains of intermediate resistance (T30 and BSJ) from which they had been derived. Addition of the synergist chlorfenethol to DDT increased its knockdown effect on all resistant strains, suggesting that dehydrochlorination was a factor in resistance. Three strains, two DDT-resistant and one DDT-susceptible, were tested with 1,1-bis(4-ethoxyphenyl)-2,2-dimethylpropane (I), an insecticide that cannot be dehydrochlorinated. All the strains were relatively tolerant to it although the DDT-susceptible strains were less tolerant. Addition of the synergist sesamex decreased the level of tolerance to I in all strains which suggested that microsomal oxidation made some contribution to it. It is concluded that three factors contribute to larval DDT resistance in A. aegypti; (a) increased metabolism to pp'-DDE; (b) increased tolerance to unmetabolised internal DDT; and (c) reduced content of DDT+pp'-DDE (only in the most resistant strains and due either to reduced absorption or increased excretion). These factors are discussed in relation to known larval resistance genes R^DDT1 and y.     

Mechanism of DDT resistance in larvae of the mosquito Aedes aegypti L.; The effect of DDT selection

Selection with pp'-DDT was applied to fourth-stage larvae of Aedes aegypti along four lines, starting with larvae of the F₂ generation from crosses between a susceptible strain and each of four resistant strains (two of Trinidad origin and two of Bangkok origin). Larval resistance increased substantially along each line but there was little or no change in the percentage breakdown of DDT to 1,1-dichloro-2,2-bis(4-chloro-phenyl)ethylene (pp'-DDE) in vivo and in no line were these two variables significantly correlated. Percentage breakdown was generally higher at 10 mg than at 50 mg litre^?1. DDT uptake (defined as content of DDT+pp'-DDE) was generally higher after exposure to 50 mg than to 10 mg litre^?1. It increased significantly with selection in the TE×NS line; it remained unchanged in the T8 × NS line; and in the other two lines (BSJ × NS and B51 × NS), it increased initially but dropped as selection progressed, the reduction being highly significant in the second of these lines. The amount of internal residual (unmetabolised) DDT tolerated by larvae of the TE × NS line also increased significantly with resistance. The levels in the other lines followed the pattern of uptake, remaining steady in T8 × NS despite the increase in survival, rising at first and then declining in the two Bangkok lines. Thus selection produced a higher tolerance to internal unmetabolised DDT in the two Trinidad lines but led ultimately to a lower content of DDT+pp'-DDE in the two Bangkok lines. The reasons for this behaviour are discussed.     

Accumulation and distribution of pp′-DDT and related compounds in an apple orchard. II. Residues in trees and herbage

DDT residues in or on the roots and leaves of the herbage and the roots, bark, leaves and fruit of the trees are given for an apple orchard sprayed annually (1953–1969). The distribution of DDT in both the grass and the grass roots was in circular areas of residues, with maximum values at each trunk and decreasing radially to each alley. Of the spray applied at the green cluster stage 80% was deposited on the grass sward and very little, if any, directly on the soil surface. The pp′-DDT content of the grass fell rapidly with successive mowings (from which the cuttings remained in situ) from 400 μg/g at spraying to 2 μg/g after nine months. 33 g/ha pp′-DDT was found in the herbage roots (0.87% of the total residues in the soil). The residues in the bark (87.5 g/ha) were much lower than expected after 13 years spray application. There were increased amounts of pp′-DDE, pp′-TDE and pp′-TDEE relative to pp′-DDT, indicating some breakdown on the bark, but the chief losses were attributed to volatilisation and to removal by wind and rain. The residue content of root bark varied from 3 μg/g near the emerging trunk to 0.05 μg/g at a depth of 90 cm. The pp′-DDT content of leaves at leaf fall rose from <1 ng/g after a single spring spray to 8.33 μg/g following an additional spray in late June. There was a large loss of DDT from the canopy between the June spray and leaf fall (440–480 g/ha down to 25 g/ha), attributed to volatilisation. The amount of pp′-DDT on the fruit, after a single spray, was 3 ng/g fresh weight (80.9 mg/ha out of a total of 1.0–1.5 kg/ha used).     

Residues of DDT in cottonseed after spraying with DDT and torbidan

DDT at 1.12 and 2.24 kg/ha a.i. and Torbidan at 5 and 10 litre formulation/ha (1 and 2 kg DDT/ha) were sprayed five times on cotton over a period of 15 weeks. Seeds from the first pick of the crop were found to contain as residues pp′-DDT and pp′-DDE [1,1-dichloro-2,2-di- (4-chlorophenyl)ethylene]. The highest residue level (0.783 parts/10⁶) was found in seeds from Torbidan 10 litre/ha treatment.     

Residues of organochlorine insecticides in some fishes and birds in the gezira of Sudan

Twenty-eight specimens representing five types of fish and five types of birds from the Gezira Research Farm, Wad Medani, Sudan, were analysed for residues of organochlorine insecticides. All fish muscle samples were found to contain pp′-DDE, pp′-TDE and pp′-DDT in total concentrations ranging from 0.27 to 16.0 mg/kg. In addition to DDT-type residues, dieldrin (HEOD) was also found in the majority of bird samples. Concentrations in bird breast muscle ranged from 0.07 to 5.5 mg/kg. When bird liver samples were examined they were found to contain higher residues than the breast muscle in nearly all cases.     

Pesticide residues in soil and foodstuff I. Chlorinated pesticides in cattle feed and milk produced in orchard and non-orchard areas

Milk, hay and silage produced in orchard and non-orchard areas have been analysed for their content of chlorinated pesticides. The residue levels in milk produced in orchard areas were about double those in milk produced in non-orchard areas. The levels in hay were much higher (30% up to sevenfold) in the hay produced in orchard areas and the levels in silage from the two areas showed small differences. Milk collected after cattle had been grazing in an orchard for 3 to 4 days contained 10% more pp′-DDT and 2-, 3-, 7- and 15-fold morepp′-DDE, pp′-TDE and heptachlor epoxide, gamma-BHC and pp′-TDE olefin [1-chloro-2,2-bis(p-chlorophenyl)ethylene], respectively, than did the samples collected one day before the cattle grazed in the orchard.     

Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica L

The mechanisms of resistance and cross resistance to the juvenoids methoprene and R-20458 in the house fly, Musca domestica, were examined. Radiolabeled methoprene was found to be metabolized faster in resistant and cross-resistant house fly larvae than in susceptible larvae, and methoprene and R-20458 penetrated more slowly into larvae of the resistant strain. In vivo and in vitro metabolism of methoprene was largely by oxidative pathways followed by conjugation in all strains examined, and little or no ester change of methoprene was noted in vitro. In vitro oxidative metabolism of methoprene, R-20458, juvenile hormone I, and several model substrates was higher in resistant and cross-resistant larvae than in susceptible larvae. Juvenoid functionalities susceptible to metabolic attack by resistant strains are indicated.     

Comparative excretion of DDT analogues into milk of indian buffalo,Bubalus bubalis L. following oral administration

Four groups of Indian buffaloes were fed daily with 25 mg of p,p′-DDT p,p′-TDE p,p′-DDE or o,p′-DDT for 100 days. Milk was analysed for organochlorine residues during this period and also for 100 days after pesticide administration had been discontinued. For the period showing ‘plateau level’ residues, 17.2% of p,p′-DDE, 17% of p,p′-TDE, 14% of p,p′-DDT as p,p′-DDT (3.5%); p,p′-TDE (10.5%); 3.2% of p,p′-DDT as o,p′-DDT (1.3%) and o,p'-TDE (1.9%) of their administered amounts were excreted in the milk. Since these compounds were excreted at different rates, the residue levels in the milk expected from a given feed would depend on their concentration and proportional distribution in the feed. The maximum tolerable content of DDT analogues in feed was derived to be 0.1 mg kg^?1 (dry weight basis) by using the maximum accumulation coefficient and incorporation of the necessary safety margin. It is concluded that Indian buffaloes fed with rations contaminated with a total of DDT analogues below this limit will yield milk of acceptable quality. Following the termination of feeding with contaminated rations, the elimination of p,p′-DDE in the milk took the longest time and that of o,p′-DDT the shortest. These results suggest that the time required for the initial high residue concentration to decline to less than the legal limit would be determined by the relative amounts of DDE, TDE and DDT in the milk, after elimination of the potent source of contamination.     

Residues of some chlorinated hydrocarbon pesticides in milk and milk products after their addition to the feed of cows

Lactating cows fed 0.05 part/million aldrin, 0.2 part/million γ-BHC and 2.0 parts/million pp'-DDT in their concentrate ration gave detectable residues of dieldrin, γ-BHC, pp'-DDE, pp'-TDE and pp'-DDT in the milk and also aldrin in the butter and cheese. The levels found after 28 days of feeding were not significantly greater than those after 14 days. Subsequent increase of the pesticide content of the feed increased the residue content of the milk, butter and cheese. Only a small percentage of pesticide residue was found in the buttermilk, separated milk and the whey.     

Inhibition of acetyl-coenzyme a carboxylase by coenzyme a conjugates of grass-selective herbicides

A total of 146 samples of different kinds of cheeses produced in Spain were analysed in order to ascertain the specific contamination pattern. The organochlorine compounds studied were those most commonly investigated in previous surveys: α-HCH, β-HCH, γ-HCH (lindane), γ-HCH, chlordane, aldrin, dieldrin, endrin, heptachlor, heptachlor epoxide, and the isomers and metabolites of DDT. α-HCH, β-HCH, γ-HCH, chlordane, p,p′, DDT, and p,p′-DDE were found in more than 76% of samples; p,p′-DDE and γ-HCH were the most frequently detected, with frequencies of 100 and 97.9% respectively. γ-HCH, aldrin, dieldrin, heptachlor, heptachlor epoxide, o,p′-DDT, p,p′-DDD and o,p′-DDD were observed at lower frequencies. No residues of endrin were detected in any sample. Insecticides exceeding the maximum residue limits (MRLs) were chlordane, β-HCH, α-HCH and γ-HCH, with 42, 20, eight and six samples respectively. Mean residues of organochlorines found were as follows (μ kg^?1 butterfat): α-HCH = 46.3; β-HCH = 46.5; γ-HCH = 54.2; δ-HCH = 16.9; aldrin = 16.7; dieldrin = 9.7; heptachlor = 15.9; heptachlor epoxide = 14.8; chlordane = 50.2; o,p′-DDT = 5.1; p,p′-DDT = 12.4; o,p′-DDT = 19.6; p,p′-DDD = 46.7; o,p′-DDE = 6.9; p,p′-DDE = 40.7 (.DDT = 55.0). Estimated dietary intakes (EDIs) from cheese consumption were compared to acceptable daily intakes (ADIs) for the pesticides where residues exceeded the MRL. EDIs calculated were in all cases below ADIs, and, therefore, based on the ADIs, there is no health risk involved in the consumption of cheese from Spain arising from organochlorine residues.     

Malathion carboxylesterase enzymes in Anopheles arabiensis from Sudan

Malathion resistance in Anopheles arabiensis from Sudan is monofactorially inherited and is expressed in the adults but not in the larvae. The resistance is suppressed by the esterase synergist, triphenylphosphate. Semipurification of the soluble esterase enzymes by Sephadex G-25 and Sephacryl S-200 gel filtration revealed no difference between the enzymes of the resistant and susceptible strains with α- or β-naphthylacetate (NA) with a fixed substrate concentration in either the adults or larvae. However, with the malathion-specific assay a second peak of activity was observed in the adult resistant strain which was not present in either the larvae of this strain or the larvae and adults of the susceptible strain. A corresponding threefold difference in the K_m value for α-NA was also observed in the resistant adults over the range of this second peak, but there was no change in the K_m with β-NA.     

Pyrethroid resistance and esterase activity in three strains of the cotton bollworm, Helicoverpa armigera (Hübner)

Microplate assay technique for estimation of esterase activity in a single insect was used in combination with dose mortality bioassays to detect pyrethroid resistance in three strains of Helicoverpa armigera and to study the frequency of pyrethroid resistant individuals within the population of the same strain at two larval stages, third and fifth instar. The third and fifth instar larvae of the field strains i.e., Nagpur strain and Delhi strain that displayed high degree of resistance towards deltamethrin, had higher esterase activity compared to a susceptible laboratory strain. The frequency distribution of individuals with elevated esterase activity above 1.00 absorbance unit was correlated with the resistance level of the strains. The frequency of resistant individuals in the third instar larvae of Nagpur strain and Delhi strain were 29% and 23%, respectively compared to 4% in the susceptible strain. The resistant individuals in the resistant strains have markedly increased in the fifth instar larvae with a frequency distribution of 63% and 90% in Delhi strain and Nagpur strain, respectively, while only 14% of individuals was found to have elevated esterase activity in the susceptible strain. The results demonstrated the role of esterase in pyrethroid resistance in H. armigera. Microplate assay proved to be a rapid and reliable biochemical technique for monitoring of pyrethroid resistance in H. armigera.     

Resistance monitoring in codling moth: a need for standardization

BACKGROUND: A wide range of methods has been used for monitoring resistance in Cydia pomonella L. However, the results from specific tests are not always concordant and they generate doubts over the extent to which the data represent the field situation. Therefore, the variability in the expression of resistance mechanisms and in resistance ratios between developmental stages and their reliability in a field situation were studied. RESULTS: Neonate larvae showed the highest insecticide resistance, except to spinosad. Resistance ratios were from 2 to 9.5, depending on the insecticide and the strain tested, when compared with the susceptible strain. Spinosad exhibited the highest resistance ratio for diapausing larvae (4.4, 12.2 and 4.4 for Raz, Rdfb and RΔ strains, respectively). Enzymatic ratios were higher in adults than in neonates or diapausing larvae, and the highest enzymatic ratios were observed for mixed‐function oxidases (7.3–36.7 for adults of resistant strains). CONCLUSIONS: The sensitivity of different instars depends on the insecticides used, and there is a differential expression of resistance mechanisms between stages. For the population considered in this study, tests on neonates provided the most consistent results to the field situation, but adult response to chlorpyrifos‐ethyl and azinphos‐methyl in the laboratory was also consistent with the field results. Copyright © 2008 Society of Chemical Industry     

Reproductive and developmental defects in a malathion-resistant,laboratory-selected population of Drosophila melanogaster

We have examined the chromosomal basis for reproductive and developmental defects that are associated with malathion resistance in a laboratory-selected population of Drosophila melanogaster. Strains homozygous for second or third chromosomes from this population were more resistant to malathion and had greater mixed-function oxidase activity, decreased fertility, and lower egg production when compared with first chromosome or susceptible strains. Some of the strains carrying resistant third chromosomes were developmentally delayed and required a significantly longer time to pupate. Delayed pupation was not associated with increased in vitro degradation of ecdysone by larvae having increased mixed-function oxidase activity, nor could it be reversed by feeding larvae ecdysone. Differences in mixed function oxidase activity among strains homozygous for second or third chromosomes were strongly correlated with malathion resistance but not with fitness. Although both second and third chromosome strains had high mixed-function oxidase activity, only fly extracts from the third chromosome strains oxidatively degraded [³H]juvenile hormone in vitro to a significant extent. A deficit of vitellogenic oocytes and increased egg laying by females in response to topically applied juvenile hormone-I supported the hypothesis that juvenile hormone titer was lower than normal in these strains. The results indicate that different polygenic systems control malathion resistance and associated fitness defects in this selected population of D. melanogaster. Although these systems are partly independent, they overlap due to pleiotropic effects of third chromosomal genes controlling mixed-function oxidase activity on female reproduction.     

Entomopathogenic nematodes for the management of Agrotis ipsilon: effect of instar, nematode species and nematode production method

BACKGROUND: Previous laboratory studies have indicated the potential of some entomopathogenic nematode (EPN) species for the control of larvae of the black cutworm (BCW). To determine the most promising EPN species and the most susceptible BCW stages, a more in‐depth evaluation of seven EPN species against different BCW instars was carried out, the efficacies of in vitro‐ and in vivo‐produced EPNs were compared and the suitability of BCW instars for EPN reproduction was examined. RESULTS: Heterorhabditis megidis was the most virulent species, irrespective of larval stage in small arenas, followed most often by H. bacteriophora. In pots with grass, Steinernema carpocapsae tended to be the most virulent species, followed by H. bacteriophora, H. megidis and S. riobrave. Fourth and/or fifth instars were the most susceptible stages to most EPN species, and pupae the least susceptible. Furthermore, H. bacteriophora, H. megidis and S. carpocapsae successfully reproduced in fifth and sixth instars and pupae. In vivo‐produced H. megidis and S. carpocapsae controlled fifth instars better than the corresponding in vitro‐produced products; production method did not affect H. bacteriophora and S. riobrave efficacy. CONCLUSIONS: Several in vitro‐produced commercial EPN strains were highly virulent to BCW and warrant further testing under field conditions, along with some in vivo‐produced strains. Copyright © 2011 Society of Chemical Industry     

Differential efficacy of three commonly used pyrethroids against laboratory and field‐collected larvae and adults of Helicoverpa zea (Lepidoptera: Noctuidae) and significance for pyrethroid resistance management

BACKGROUND: Helicoverpa zea (Boddie) pyrethroid resistance monitoring programs typically utilize cypermethrin in the adult vial test. Here we investigated if differences in insect growth stage and pyrethroid structure affect resistance ratios and discuss implications for pyrethroid resistance management. RESULTS: Vial bioassays with cypermethrin, esfenvalerate and bifenthrin were conducted on H. zea third instars and male moths from a susceptible laboratory colony and the F1 generation of a pyrethroid‐resistant field population. In the susceptible population, both growth stages were most sensitive to bifenthrin and adults were more sensitive to esfenvalerate than cypermethrin. LC₅₀ resistance ratios for the larvae and adults of the resistant population were approximately two times higher for bifenthrin than cypermethrin or esfenvalerate. CONCLUSION: For the resistant population, vial assays using either growth stage gave similar resistance ratios for each of the three pyrethroids, respectively, proving the adult vial test accurately reflects larval resistance. However, as resistance ratios varied considerably depending on the pyrethroid used, resistance ratio values obtained with one pyrethroid may not be predictive of resistance ratios for other pyrethroids. Our results suggest that carefully chosen pyrethroid structures diagnostic for specific mechanisms of resistance could improve regional monitoring programs. Copyright © 2009 Society of Chemical Industry     

Comparative toxicity,absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms

Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3–4 ×) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in vivo metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryl- and alkyltransferases) present in the soluble cell fractions were of lesser importance. O-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in vivo and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.     

Comparative metabolism of six ethoxychlor analogs and DDT in DDT-resistant and susceptible strains of the house fly Musca domestica L

The metabolic fate of six ³H-ring-substituted ethoxychlor analogs with altered aliphatic moieties and [¹⁴C]p,p′-DDT was investigated in susceptible and DDT-resistant strains of the house fly Musca domestica Linnaeus. The chloroalkane analogs, dichloroethane, chloropropane, and dichloropropane were primarily metabolized to the corresponding dehydrochlorinated products. This pathway was relatively more prominent in the resistant strain than in the susceptible strain. Biotransformation and detoxication of the isobutane, nitropropane, and neopentane derivatives was through microsomal oxidation (O-deethylation) of aryl ethoxy degradophores, and oxidation of the aliphatic moieties to produce the corresponding benzophenones, with no substantial differences between the resistant and susceptible strain. There was a strong correlation between the Taft ($\text{σ}{}^1$) values for the altered aliphatic moieties of chloroalkane analogs and their rate of dehydrochlorination in both the strains. These results suggest the importance of altered aliphatic moieties in developing resistance-proof DDT derivatives.     

Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides

Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro. The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants (k_i) differed by approximately 1.2- to 6-fold for a range of organophosphorous compounds and 17- to 1570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates.     

Pyrethroid synergists suppress esterase-mediated resistance in Indian strains of the cotton bollworm, Helicoverpa armigera (Hübner)

The role of esterase in pyrethroid resistance was studied in the final larval instar of different strains of the cotton bollworm, Helicoverpa armigera. The resistant strains viz., Nagpur strain and the Delhi strain were found to have elevated midgut esterase activity in comparison to the susceptible strain. Nagpur strain and Delhi strain have 2.24 and 1.73-fold higher esterase activity, respectively, than that of the susceptible strain. The Native PAGE displayed important differences in the midgut esterase isozyme pattern between the susceptible and the pyrethroid-resistant strains. Out of the 10 esterase isozyme observed, susceptible strain lacked three bands, E2, E6 and E10 that were found in the resistant strains. The potency of the synergists piperonyl butoxide (PBO) and dihydrodillapiole (DDA) as esterase inhibitor were also studied both in vitro and in vivo. The in vitro results clearly show that both PBO and DDA inhibited esterase activity in the two resistant strains, while there was almost no esterase inhibition in the homogenate of the susceptible strain. The in vivo inhibition studies (topical application of PBO and DDA followed by biochemical analysis) illustrated that PBO- and DDA-esterase binding is rather slow and non permanent process. Esterase inhibition did not occur immediately after the synergist treatment but at 4 and 8 h post treatment in case of PBO and DDA, respectively. Native PAGE revealed that the in vivo esterase inhibition caused by both PBO and DDA was due to the binding of the synergist with the E6 isozyme which was not present in the susceptible strain.