The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

Insecticide resistance status of Aedes aegypti (L.) from Colombia

BACKGROUND: To evaluate the insecticide susceptibility status of Aedes aegypti (L.) in Colombia, and as part of the National Network of Insecticide Resistance Surveillance, 12 mosquito populations were assessed for resistance to pyrethroids, organophosphates and DDT. Bioassays were performed using WHO and CDC methodologies. The underlying resistance mechanisms were investigated through biochemical assays and RT‐PCR. RESULTS: All mosquito populations were susceptible to malathion, deltamethrin and cyfluthrin, and highly resistant to DDT and etofenprox. Resistance to lambda‐cyhalothrin, permethrin and fenitrothion ranged from moderate to high in some populations from Chocó and Putumayo states. In Antioquia state, the Santa Fe population was resistant to fenitrothion. Biochemical assays showed high levels of both cytochrome P450 monooxygenases (CYP) and non‐specific esterases (NSE) in some of the fenitrothion‐ and pyrethroid‐resistant populations. All populations showed high levels of glutathione‐S‐transferase (GST) activity. GSTe2 gene was found overexpressed in DDT‐resistant populations compared with Rockefeller susceptible strain. CONCLUSIONS: Differences in insecticide resistance status were observed between insecticides and localities. Although the biochemical assay results suggest that CYP and NSE could play an important role in the pyrethroid and fenitrothion resistance detected, other mechanisms remain to be investigated, including knockdown resistance. Resistance to DDT was high in all populations, and GST activity is probably the main enzymatic mechanism associated with this resistance. The results of this study provide baseline data on insecticide resistance in Colombian A. aegypti populations, and will allow comparison of changes in susceptibility status in this vector over time. Copyright © 2011 Society of Chemical Industry     

Elucidating resistance in cotton genotypes to whitefly, <Emphasis Type="Italic">Bemisia tabaci,</Emphasis> by population buildup studies

Plant resistance has become an important component of integrated pest management (IPM) for management of whitefly, Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), an important pest of cotton in India. The present studies were undertaken to standardize the plant stage and identify resistant cotton genotypes against whitefly. Nine plant stages of F846, a susceptible cotton genotype, were exposed to whitefly for 25 days under no-choice conditions. The population buildup (eggs, nymphs, pupae and adults) was recorded. The 12-, 14- and 16-leaf stages were suitable for plant resistance studies against whitefly, and the 14-leaf stage was taken for further studies. Ten cotton genotypes of Gossypium hirsutum and two of G. arboreum were covered with split cages in which five pairs of B. tabaci (F₁) were released. The population buildup was recorded to categorize genotypes as resistant, moderately resistant, moderately susceptible or susceptible. The experiment was repeated with F₂ and F₃ generation whiteflies. Based on overall average score of three experiments, LD694 was rated as resistant; LK861, Supriya, RS2013, CNH911 and PA183 as moderately resistant; IS-376/4/1/20/72, NHH44, TxMaroon2-78, Bt 6304 and RS2098 as moderately susceptible; and F846 as susceptible. LD694 was found to be resistant in three consecutive generations of whitefly.     

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.     

Insecticide resistance in the tropical bedbug Cimex hemipterus

Insecticide resistance in the bedbug Cimex hemipterus was investigated using 4211 bedbugs collected from three districts of Sri Lanka. Insecticide bioassays were carried out with discriminating dosages of deltamethrin, permethrin, DDT, malathion, and propoxur. Activity levels of insecticide metabolizing enzymes and the insecticide target site acetylcholinesterase were monitored using biochemical assays. Percentage survivals after DDT, malathion, and propoxur exposure were 41-88%, 18-64%, and 11-41%, respectively. For deltamethrin and permethrin, KT₅₀/KT₉₀ (time to knock-down 50%/90% of the population) values were 0.5-24/1.0-58 and 1.3-10/2.5-47 h, respectively. Both elevated esterase and malathion carboxylesterase mechanisms were present in bedbug populations. Monooxygenase levels were heterogeneous. Organophosphate and carbamate target site acetylcholinesterase, was insensitive in 29-44% of the populations. High DDT resistance was probably due to glutathione S-transferases. Malathion carboxylesterases are mainly responsible for high malathion resistance. High tolerance to both DDT and pyrethroids suggests the presence of ‘kdr’ type resistance mechanism in one population.     

General esterase,malathion carboxylesterase,and malathion resistance in Culex tarsalis

The role of esterases in malathion resistance in Culex tarsalis has been investigated. When larvae of a resistant and a sensitive strain were placed in water containing [¹⁴C]malathion, malathion penetrated to give initially similar internal levels. With resistant mosquitoes, after 15 min the internal malathion concentration decreased to low levels while the monoacid degradation products accumulated in the larvae and were excreted into the surrounding water, whereas in susceptible larvae the internal malathion level stayed high and was lethal. It is suggested that the decrease in internal malathion and the resulting resistance were caused by an active malathion carboxylesterase in the resistant strain. A specific assay for malathion carboxylesterase with [¹⁴C]malathion showed 55 times more activity in resistant than in susceptible larvae, whereas when general esterase activity was assayed with α-naphthyl acetate only 1.7 times the activity was found. Analyses by starch gel electrophoresis showed a peak of malathion carboxylesterase, 60-fold higher from resistant than from susceptible larvae, in a gel zone which did not stain for general esterase activity. General esterases that did not hydrolyze malathion showed different electrophoretic patterns in the two populations, which are likely due to the nonisogenic character of the strains. These results show that use of a specific assay and the demonstration of degradation of malathion in vivo are essential for assessment of the contribution of esterase activity to the malathion-resistant phenotype in mosquito populations.     

Toxicity of ddt and malathion to various larval stages of Mamestra brassicae L.

The toxicity of DDT and malathion to the larvae of Mamestra brassicae was determined following several methods of application. The toxicity (LD₅₀), expressed as μg insecticide per g of insect, did not change significantly between larval instars (a) when either insecticide was injected into fourth to sixth instars; (b) when DDT was applied in the food of fifth and sixth instars; or (c) when malathion was applied topically to second to sixth instars. Significant changes in toxicity were found between successive instars when DDT was applied topically, but there was no clear trend. When malathion was applied in the food, the fifth instars were more susceptible than the sixth instars; it was found that the former consumed a toxic dose of malathion at a greater rate, and that probably malathion was degraded in the gut at a slower rate. In a contact test, the first to third instars were far more susceptible than the later instars to malathion; with DDT this trend was much less marked. Uptake studies with [¹⁴C]malathion showed that differences in the contact toxicity of malathion between instars could be explained, at least partly, by the decline in uptake per unit weight with increasing larval size.     

A field trial with permethrin against bodylice,pediculm humanus humanus in Egypt, 1976

Populations of bodylice, Pediculus humanus humanus L., in Egypt have become resistant to DDT and BHC, but not to malathion. A single dust application of permethrin (1 % a.i.; cis: trans-ratio 25:75) rendered 98.9% of the treated population louse-free. Thirty-seven days after dusting 91.3% and at the end of the 98-day observation period 64.6% of the population were free of infestation; during the same period the proportion of infested individuals rose by 12.3% in a similar untreated village. Permethrin is ovicidal against bodylice, affects the population of the human flea, PulexirritansL., and ishighly effective against the rat flea, Xenopsyllacheopis(Rothsch.).     

Non-target-site-based resistance to ALS-inhibiting herbicides in six Bromus rigidus populations from Western Australian cropping fields

BACKGROUND: Bromus rigidus is a common weed species that has increased in cropping fields owing to limited control options. During a random field survey in Western Australia, six B. rigidus populations that had survived in‐crop weed control programmes were collected. The study aimed to determine the resistance profile of these six populations. RESULTS: Based on dose–response studies, all six B. rigidus populations had a low‐level resistance to sulfosulfuron and sulfometuron (both sulfonylurea herbicides) while remaining susceptible to herbicides with other modes of action. ALS in vitro activity assays revealed no differences in enzyme sensitivity between susceptible and resistant populations, while the use of malathion (a cytochrome P450 inhibitor) in combination with sulfosulfuron caused the resistant populations to behave like the susceptible population. CONCLUSION: This study established that these six B. rigidus populations have a low‐level resistance to the ALS‐inhibiting sulfonylurea herbicides, but are able to be controlled by other herbicide modes of action. The low‐level, malathion‐reversible resistance, together with a sensitive ALS, strongly suggest that a non‐target‐site enhanced metabolism is the mechanism of resistance. Copyright © 2012 Society of Chemical Industry     

Preference of whitefly, Bemisia tabaci, towards black gram genotypes: Role of morphological leaf characteristics

Considering that certain morphological leaf characteristics and foliar pubescence may affect insect feeding preference on plant genotypes, the present studies were undertaken to study the variation in leaf morphology and to further evaluate the influence of leaf characteristics in imparting resistance to black gram (Vigna mungo (L.) Hepper) genotypes against Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Evaluation of nine black gram genotypes against B. tabaci in multiple-choice test revealed that moderately resistant genotypes KU 99-20 and NDU 5-7 recorded significantly lowest whitefly population (egg, nymph and adult) as compared to the susceptible genotypes IPU 02-043, KU 7-602, KU 7-605, KU 7-618 and Mash 1-1 and highly susceptible genotypes KU 7-504 and KU 7-505. Evidence for morphological leaf characteristics was gathered using data from leaf area meter, fluorescent, electron microscopy as well as direct visual observations. Correlations between morphological leaf characteristics of black gram genotypes and the whitefly population were examined. Leaf area, lamina thickness and trichome length were significantly and positively correlated with whitefly eggs, nymphs and adults whereas trichome density and angle were negatively correlated. Thus, black gram genotypes with narrow, thin and highly pubescent leaves having short, but erect trichomes should be selected for developing black gram varieties resistant to whitefly.     

Cross-resistance to pyrethroids and other insecticides in Aedes aegypti

Two substrains of Aedes aegypti, already resistant to DDT and pyrethroids, were further selected using either DDT or permethrin by mass exposure of the females only. DDT selection over 14 generations raised the resistance to DDT so far that no accurate LC₅₀ values could be determined. Selection with permethrin raised the tolerance to an irregular plateau 7–10 times the original. DDT selection in the adults raised the DDT resistance of the larvae, but this could be partly overcome using a dehydrochlorinase inhibitor. The resistance to pyrethroids was increased but tolerance of dieldrin, malathion and propoxur compounds was little changed. Permethrin selection of the adults raised resistance to pyrethroids more than DDT selection but also increased DDT resistance. Similar patterns were found for the larval insects. A strain from Demerara in Guyana showed both DDT and pyrethroid resistance, including strong resistance to pyrethrins together with dieldrin and propoxur. It was concluded that two major independent resistance mechanisms existed in the selected strains, a dehydrochlorinase affecting DDT alone, and an unknown mechanism, probably nerve insensitivity (kdr) affecting both DDT and pyrethroids.     

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.     

Oxidative cleavage of a carboxyester bond as a mechanism of resistance to malaoxon in houseflies

The synergistic effect of triphenyl phosphate (a carboxyesterase inhibitor), sesamex (inhibitor of microsomal oxidation) and O,O-diethyl O-phenyl phosphorothioate on the toxicity of malathion and malaoxon for one susceptible and two resistant strains of housefly was studies. It was found that in the resistant strain G (characterized by high carboxyesterase activity) both malathion and malaoxon were synergized by triphenyl phosphate, but only malaoxon (and not malathion) by sesamex. The other resistant strain E 1, moderately tolerant for malathion but highly resistant to malaoxon, differed from strain G in that triphenyl phosphate had no effect; its response to sesamex was similar to that of strain G. The third synergist, O,O-diethyl O-phenyl phosphorothioate, combined the properties of triphenyl phosphate and sesamex. It was found to be the best of the three compounds used.Biochemical in vitro studies showed that both resistant strains could degrade malaoxon oxidatively at a rate at least 10 × higher than that of the susceptible strain. This oxidation could be inhibited by very low concentrations of the thiono analogue; a malaoxon to malathion ratio of 10:1 gave an inhibition of about 70% at a malaoxon concentration of 5 × 10^?6M. The product of this oxidation is malaoxon β-monocarboxylic acid. This metabolite was also found 1 hr after application of malaoxon in vivo.The results mentioned in this paper indicate that houseflies may become resistant to malaoxon by an increased rate of oxidative carboxyester bond cleavage.     

一些农药对温室白粉虱的毒力及其抗药性测定

用11种农药对温室自粉虱(Trialeurodes vaporariorum Westw.)进行了室内毒力测定。结果表明:拟除虫菊酯类杀虫剂、灭螨猛对温室白粉虱各虫态毒力均高,并且随虫龄的增加,白粉虱的抗药性增强。稻丰散、乐果、氧化乐果对温室白粉虱若虫毒力尚可,但对其它虫态效果差。马拉硫磷、二嗪农、敌敌畏、洗衣粉除对一龄若虫有一定效果外,对其它虫态毒力极低。北京地区温室白粉虱对某些药剂已出现明显的抗药性。     

An Example of Cross-resistance to Pyrethroids in DDT-resistant Aedes aegypti

Comparisons of the susceptibility of several strains of adult Aedes aegypti were made. Mosquitoes from Bangkok and Jakarta were found to be highly resistant to DDT and resistant to pyrethroids relative to a laboratory strain. A strain from Singapore, where less DDT has been used, was susceptible to DDT and pyrethroids. Two strains from the Caribbean had LC₅₀ values to DDT 3 times that of the reference strain while the LC₅₀ values against bioresmethrin synergised with piperonyl butoxide were 1 1/2 times raised. Another two strains from central Africa were 2 times tolerant of DDT and 1 1/2 times tolerant of bioresmethrin plus piperonyl butoxide. Agents which block DDT-dehydrochlorinase, esterases and oxidases each caused small increases in the mortality of the Bangkok strain due to DDT and bioresmethrin as well as augmenting toxicity to the susceptible reference strain. It is tentatively suggested that resistance in the Bangkok strain is due to a combination of the actions of these and perhaps other resistance mechanisms.     

The biochemical nature of malathion resistance in Anopheles stephensi from Pakistan

Malathion resistance in Anopheles stephensi from Pakistan was synergized by triphenyl phosphate, primarily a carboxylesterase inhibitor. There was a slight degree of antagonism with piperonyl butoxide. The major metabolite of malathion in larvae of both the resistant and susceptible strains was malathion monocarboxylic acid. Resistant larvae produced about twice as much of this product as the susceptible larvae. This suggests that a qualitative or a quantitative change in a carboxylesterase enzyme may be the basis of malathion resistance in this strain. Analysis of general esterase levels to α- and β-naphthyl acetate showed that there was no quantitative change in the amount of carboxylesterase enzyme present in the resistant strain as compared to the susceptible.     

不同类型杀虫剂对温室白粉虱若虫的毒力研究

利用一龄若虫浸渍法测定了不同类型杀虫剂对温室白粉虱四季青种群的毒力,发现白粉虱对噻嗪酮、阿维菌素极其敏感,对吡虫啉和联苯菊酯次之,而对马拉硫磷和ＤＤＶＰ不敏感。     

Impact of sub-lethal residues of azinphos-methyl on the pheromone-communication systems of insecticide-susceptible and insecticide-resistant obliquebanded leafrollers Choristoneura rosaceana (Lepidoptera: Tortricidae)

The effects of sub-lethal residues of azinphos-methyl on pheromone production, calling, female attractiveness and the ability of males to locate sources of natural and synthetic pheromone were compared in azinphos-methyl-susceptible (susceptible) and azinphos-methyl-resistant (resistant) obliquebanded leafrollers, Choristoneura rosaceana (Harris). The amount of pheromone in susceptible females was reduced by 29-33% after exposure to azinphos-methyl; this treatment did not affect the pheromone content of resistant females. Azinphos-methyl-treated resistant females contained 39-43% less pheromone than azinphos-methyl-treated susceptible females. Resistant females that were not treated with azinphos-methyl contained 35-56% less pheromone than susceptible females that were not treated with insecticide. The incidence of calling was reduced by 67-100% in azinphos-methyl-treated susceptible females; the incidence of calling by resistant females was not affected by exposure to azinphos-methyl. The incidence of calling by azinphos-methyl-treated susceptible females was 58-100% lower than that of azinphos-methyl-treated resistant females. There was no difference in the incidence of calling between susceptible and resistant females that had not been treated with insecticide. In a flight tunnel, treatment with insecticide reduced the attractiveness of susceptible females by 38%; treatment with insecticide did not affect the attractiveness of resistant females. There was no difference in the proportion of males attracted to susceptible and resistant females that had, or had not been treated with insecticide. In an apple orchard, the attractiveness of susceptible and resistant females treated with azinphos-methyl was reduced by 84 and 12%, respectively. The proportion of males attracted to azinphos-methyl-treated susceptible females was 58% lower than the proportion attracted to azinphos-methyl-treated resistant females, whereas, if females were not treated with insecticide, the proportion attracted to resistant females was 57% lower than the proportion attracted to susceptible females. In a flight tunnel, azinphos-methyl did not affect the ability of susceptible or resistant males to locate a source of pheromone gland extract. Likewise, in an apple orchard, the insecticide treatment had no effect on the ability of susceptible or resistant males to locate a source of synthetic pheromone. In a flight tunnel, there was no difference in the proportion of azinphos-methyl-treated susceptible and resistant males locating a source of pheromone gland extract; however, in the orchard, 39% fewer azinphos-methyl-treated resistant males located a source of synthetic pheromone than azinphos-methyl-treated susceptible males. A similar proportion of susceptible and resistant males that had not been treated with insecticide located a source of pheromone gland extract in the flight tunnel, but in the orchard, the proportion of resistant males not treated with azinphos-methyl that located the source of synthetic pheromone was 32% lower than the proportion of susceptible males not treated with this insecticide. The implications of the differences in the effect of sub-lethal residues of azinphos-methyl on the pheromone communication system of susceptible and resistant moths are discussed in relation to the theory of the development of insecticide resistance, the detection of resistance in feral populations of moths using sex pheromone-baited traps, and the control of moths using sex pheromone-mediated mating disruption.     

A Field Method for Evaluating the Potential Durability of New Resistance Sources: Application to the Leptosphaeria maculans-Brassica napus Pathosystem

ABSTRACT To increase the longevity of new resistance genes by avoiding a rapid change in pathogen populations, we established a new field method to determine, before the release of a resistant cultivar, whether and how rapidly the pathogen population is capable of responding to the selective pressure we impose. This method was applied to the Leptosphaeria maculans-Brassica napus pathosystem. The potential durability of two new major resistance genes introgressed into B. napus from the Brassica B genome was tested separately for each gene under field conditions for 4 years. Successive inoculations with residues of the resistant lines mixed with susceptible contaminated plant material recovered at harvest the previous year were performed in autumn. The Jlm1 resistance gene originating from B. juncea conferred complete resistance on the B. napus-B. juncea recombinant lines MX and MXS to inoculation of the cotyledons with a large diversity of L. maculans isolates. It also gave a high level of stem canker resistance in the field against natural populations of the pathogen. A similar level of resistance was obtained in the B. napus-B. nigra addition line LA4+, containing B. nigra chromosome 4 in a B. napus background. In the second year of the field experiment (i.e., the first in which residues from the resistant lines were included in the inoculation material), both MX and LA4+ maintained a high level of resistance. In the third and fourth years of the field experiment, the resistance of MX and MXS exposed to inoculum produced from their own residues broke down, but against fungal populations from susceptible B. napus or resistant B. nigra material remained effective. In contrast, LA4+ remained highly resistant to all sources of inoculum for the 4-year experiment.