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Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae.

Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a gamma-aminobutyric acid (GABA) receptor subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Alanine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, this same alanine residue is replaced either by a serine, or, in some resistant strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of the green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other alleles with the following amino acid replacements: A302-->Glycine (allele G), A302-->SerineTCG (allele S) and A302-->SerineAGT (allele S'). Evidence from direct nucleotide sequencing and Single Stranded Conformational Polymorphism (SSCP) analysis shows that at least three of these different Rdl alleles (i.e. A, G and S) are commonly present in individual aphids or aphid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutation confirm the presence of two independent Rdl-like loci in M. persicae. One locus carries the susceptible alanine (A) and/or resistant glycine (G) allele while the other carries the two serine alleles (S or S'). Whereas resistance levels are correlated with the glycine replacement, the S allele was present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacements at the first (A/G) but not the second (S/S') locus. Phylogenetic analysis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen through a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit diversity are discussed.

Alanine↗

The genomics of insecticide resistance.

Genomic technologies are revealing several mechanisms of insecticide resistance involving enhanced detoxification or reduced target-site sensitivity that had previously defied molecular analyses. Genome projects are also revealing some potentially far-reaching consequences for pest-insect genomes of the rapid accumulation of multiple resistance mutations in very short periods of evolutionary time.

Animals↗

Insecticide resistance in malaria vectors: a new approach to an old subject.

The application of biochemical and molecular biological techniques to the study of insecticide resistance has revolutionized our understanding of the underlying genetic basis of resistance. Using the examples of glutathione S-transferase and esterase-based metabolic insecticide resistance, three different routes via which increased insecticide detoxication can be achieved are elucidated. An understanding of these molecular pathways opens up new avenues for manipulating mosquito populations to restore insecticide susceptibility to the vectors.

Animals↗

Current insecticide resistance patterns in mosquito vectors in Thailand.

Chemical pesticides are still commonly used in Thailand for control of agricultural pests and disease vectors. Organophosphates, carbamates and synthetic pyrethroids are commonly used for agricultural purposes, whereas synthetic pyrethroids have become more popular and predominate for public health use. The genetic selection of insecticide resistance (whether physiological, biochemical or behavioral) in pests and disease vectors has been extensively reported worldwide (Brown and Pal, 1971). The long-term intensive use of chemical pesticides to control insect pests and disease vectors is often cited as the reason behind the development of insecticide resistance in insect populations. Unfortunately, reliable information on vector resistance patterns to pesticides in Thailand is sparse because of a remarkable shortage of carefully controlled, systematic studies. This review gathers useful information on what is presently known about disease vector resistance to chemical pesticides in Thailand and provides some possible management strategies when serious insecticide resistance occurs.

Animals↗

Insect glutathione transferases and insecticide resistance.

Glutathione transferases (GSTs) are a diverse family of enzymes found ubiquitously in aerobic organisms. They play a central role in the detoxification of both endogenous and xenobiotic compounds and are also involved in intracellular transport, biosynthesis of hormones and protection against oxidative stress. Interest in insect GSTs has primarily focused on their role in insecticide resistance. GSTs can metabolize insecticides by facilitating their reductive dehydrochlorination or by conjugation reactions with reduced glutathione, to produce water-soluble metabolites that are more readily excreted. In addition, they contribute to the removal of toxic oxygen free radical species produced through the action of pesticides. Annotation of the Anopheles gambiae and Drosophila melanogaster genomes has revealed the full extent of this enzyme family in insects. This mini review describes the insect GST enzyme family, focusing specifically on their role in conferring insecticide resistance.

Animals↗

Relationship between amount of esterase and gene copy number in insecticide-resistant Myzus persicae (Sulzer).

Overproduction of the insecticide-degrading esterases, E4 and FE4, in peach-potato aphids, Myzus persicae (Sulzer), depends on both gene amplification and transcriptional control, the latter being associated with changes in DNA methylation. The structure and function of the aphid esterase genes have been studied but the determination of their copy number has proved difficult, a common problem with gene amplification. We have now used a combination of pulsed-field gel electrophoresis and quantitative competitive PCR to determine relative esterase gene copy numbers in aphid clones with different levels of insecticide resistance (R1, R2 and R3). There are approx. 4-fold increases between susceptible, R1, R2 and R3 aphids, reaching a maximum of approx. 80 times more genes in R3; this gives proportionate increases in esterase protein relative to susceptible aphids. Thus there is no overexpression of the amplified genes, in contrast with what was thought previously. For E4 genes, the loss of 5-methylcytosine is correlated with a loss of expression, greatly decreasing the amount of enzyme relative to the copy number.

5-Methylcytosine↗

Molecular mapping of insecticide resistance genes in the yellow fever mosquito (Aedes aegypti).

Several loci conferring insecticide resistance in the yellow fever mosquito (Aedes aegypti) have previously been mapped by simple recombinational mapping. Here we describe correlation of these resistance phenotypes with molecular gene probes for insecticide target sites by RFLP mapping. The para sodium channel gene homologue and the GABA receptor gene Resistance to dieldrin map to the same genome regions as the DDT/pyrethroid and cyclodiene resistance loci, respectively. Although the acetylcholinesterase (target site of organophosphorus and carbamate insecticides) gene Ace does not map to any known resistance locus, it maps very close to the sex-determining locus. We discuss the possibilities that, if identified, Ace-mediated resistance in A. aegypti will be sex linked or that, as suggested for anopheline mosquitoes, two independent Ace loci may exist, one of which is autosomal. These results support the importance of target site insensitivity as an insecticide resistance mechanism in mosquitoes.

Aedes↗

Subcellular distribution and characterization of esterase isozymes from insecticide-resistant and -susceptible strains of German cockroach (Dictyoptera: Blattellidae).

Esterases from insecticide-resistant and -susceptible strains of German cockroach, Blattella germanica (L.), were isolated and analyzed for differences in activity. The isozymes E5, E6, E7, and E8 of resistant strains indicated about two- to fivefold increases in activity compared with those of the susceptible strain. These isozymes constituted approximately 60% of the total activity in the resistant strains, whereas they contributed only about 40% activity in the susceptible strain. Subcellular distribution studies showed that most esterase activity is present in the 100,000 x g fraction of the homogenate (cytosolic). Only a small portion of activity was membrane bound. Molecular weights of the esterase isozymes calculated based on their retardation coefficients ranged between 48 and 81 kd.

Animals↗

Esterase B1 activity variation within and among insecticide resistant, susceptible and heterozygous strains of Culex quinquefasciatus (Diptera: Culicidae).

Amplification of the esterase B1 gene of Culex quinquefasciatus Say results in high titers of an esterase enzyme that confers resistance to organophosphate insecticides. Esterase activity of individuals was measured in samples from an organophosphate resistant strain (Tem-R), a susceptible strain (S-Lb), and their reciprocal F1 progeny. Within-strain variation, as measured by coefficients of variation, was fairly consistent between sexes within strains and among strains (average, 12%). On average, individuals from the Tem-R strain had about 120 times the esterase activity of individuals from the S-Lab strain. The mean esterase activities of the F1 strains were significantly higher than the average of the Tem-R and S-Lab strain mean esterase activities, suggesting enhanced expression of the amplified esterase B1 genes in F1 individuals. Reciprocal F1 strains did not differ significantly in esterase activity or resistance, indicating that maternal effects do not influence either of these measures in these strains. The levels of esterase activity of the strains are discussed in relation to their resistance.

Animals↗

Insecticide resistance genes confer a predation cost on mosquitoes, Culex pipiens.

Newly occurring adaptive genes, such as those providing insecticide resistance, display a fitness cost which is poorly understood. In order to detect subtle behavioural changes induced by the presence of resistance genes, we used natural predators and compared their differential predation on susceptible and resistant Culex pipiens mosquitoes, using strains with a similar genetic background. Resistance genes were either coding an overproduced detoxifying esterase (locus Ester), or an insensitive target (locus ace-1). Differential predation was measured between susceptible and resistant individuals, as well as among resistant mosquitoes. A backswimmer, a water measurer, a water boatman and a predaceous diving beetle were used as larval predators, and a pholcid spider as adult predator. Overall, the presence of a resistance gene increased the probability of predation: all resistance genes displayed predation costs relative to susceptible ones, at either the larval or adult stage, or both. Interestingly, predation preferences among the susceptible and the resistance genes were not ranked uniformly. Possible explanations for these results are given, and we suggest that predators, which are designed by natural selection to detect specific behavioural phenotypes, are useful tools to explore non-obvious differences between two classes of individuals, for example when they differ by the presence or absence of one recent gene, such as insecticide resistance genes.

Animals↗

Evaluation of insecticide resistance and biochemical mechanisms in a population of Culex quinquefasciatus (Diptera: Culicidae) from São Paulo, Brazil.

To establish an insecticidal resistance surveillance program, Culex quinquefasciatus mosquitoes from São Paulo, Brazil, were colonized (PIN95 strain) and analyzed for levels of resistance. The PIN95 strain showed low levels of resistance to organophosphates [malathion (3.3-fold), fenitrothion (11.2-fold)] and a carbamate [propoxur (3.0-fold)]. We also observed an increase of 7.4 and 9.9 in alpha and beta esterase activities, respectively, when compared with the reference IAL strain. An alteration in the sensitivity of acetylcholinesterase to insecticide inhibition was also found in the PIN95 mosquitoes. The resistant allele (Ace.1R), however, was found at low frequencies (0.12) and does not play an important role in the described insecticide resistance. One year later, Cx. quinquefasciatus mosquitoes were collected (PIN96 strain) at the same site and compared to the PIN95 strain. The esterase activity patterns observed for the PIN96 strain were similar to those of the PIN95 mosquitoes. However the occurrence of the Ace.1R allele was statistically higher in the PIN96 strain. The results show that esterase-based insecticide resistance was established in the PIN95 Cx. quinquefasciatus population and that an acethylcholinesterase based resistant mechanism has been selected for. A continuous monitoring of this phenomenon is fundamental for rational mosquito control and insecticide application programs.

Animals↗

Reduced response of insecticide-resistant aphids and attraction of parasitoids to aphid alarm pheromone; a potential fitness trade-off.

Response to the alarm pheromone, (E)-beta-farnesene, produced by many species of aphids, was assessed in laboratory bioassays using an aphid pest, Myzus persicae (Sulzer), and its primary endoparasitoid, Diaeretiella rapae (McIntosh). This was done in three separate studies, the first investigating responses of a large number of M. persicae clones carrying different combinations of metabolic (carboxylesterase) and target site (kdr) insecticide resistance mechanisms, and the other two investigating the responses of young virgin female adult parasitoids. In M. persicae, both insecticide resistance mechanisms were associated with reduced repellence suggesting that each has a pleiotropic effect on aphid behaviour. In contrast, D. rapae females were attracted to the alarm pheromone source. The implications of this apparent fitness trade-off for the evolution and dynamics of insecticide resistance, and the potential for using beneficial insects to combat resistance development are discussed.

Animals↗

Efficacy of Brahman breeding in the management of insecticide-resistant horn flies (Diptera: Muscidae) on beef cattle.

The efficacy of Brahman breeding used as an alternative tactic to manage insecticide-resistant populations of adult horn flies, Haematobia irritans irritans (L.), was determined. Concentration-mortality bioassays done at Booneville and Hope, AR, in 1988 and 1989, respectively, showed that horn fly populations were resistant to diazinon, pirimiphos methyl, tetrachlorvinphos, and methoxychlor. Data showed loss of field efficacy for coumaphos and delnav. Mean horn fly counts on Braham cows were significantly lower than on Angus cows for all sampling dates in 1989 and 1990. Mean fly counts on Brahman x Angus cows were approximately intermediate to the two purebred mean fly counts. Brahman breeding caused significant reductions in the number of organophosphate-resistant horn flies, which had been equal to or greater than that obtained from continued spraying with organophosphate insecticides. The Brahman x Hereford cows, which have one-eighth greater Brahman breeding than the Brangus cows, had fewer horn flies on 48 of 56 sampling dates in 1988-1990 and significantly fewer flies on 37 sampling dates. The effectiveness of Brahman breeding in causing lower numbers of insecticide-resistant horn flies significantly increased as the percentage of Brahman breeding increased.

Animals↗

Insecticide-resistant pollen beetles (Meligethes aeneus F) found in Danish oilseed rape (Brassica napus L) fields.

The pollen beetle is the most important pest in Danish oilseed rape fields. In 2001, we screened a broad range of pollen beetle populations for pyrethroid and dimethoate resistance. A standard dip-test was used to test insecticide resistance in 18 populations collected from oilseed winter and spring rape fields. The beetles were treated with four different insecticides: the pyrethroids tau-fluvalinate, lambda-cyhalothrin and esfenvalerate, and the organophosphate dimethoate. The results show that up to 99% of the pollen beetles survived Danish standard doses of pyrethroids and up to 36% of the beetles survived standard doses of dimethoate.

Animals↗

[Insecticide resistance of different populations of white-backed planthopper, Sogatella furcifera (Horváth) (Homoptera:Delphacidae)].

Resistance to some insecticides used commonly in different populations of the white-backed planthopper, Sogatella furcifera collected from Zhejiang, Guangxi, Yunnan and Hainan Province in China was measured. The specific activities of some resistance-associated enzymes, i.e. esterase, carboxylesterase, glutathione S-transferase and acetylcholinesterase from these populations were also measured. The resistant levels to malathion, methamidophos, isoprocarb and buprofezin were markedly higher in Yunnan and Hainan populations than in other populations, but considerably more heterogeneity in response to insecticides was found in Zhejiang and Guangxi populations. The specific activities of the enzymes were obviously higher in Zhejiang and Guangxi populations than in Yunnan and Hainan populations, and the distribution range of the enzyme activity was the widest in Zhejiang population, and the narrowest in Guangxi population. Finally, The relationship between the long-distance migration and the territorial difference in insecticide resistant level and activities of the enzymes in the populations of this planthopper was discussed.

Acetylcholinesterase↗

Comparative effect of fenitrothion treatment on intracellular protease activities in insecticide-resistant and susceptible strains of Musca domestica L.

In order to further elucidate the biochemical mechanisms responsible for insecticide resistance in insects, we have determined changes in the activity levels of a comprehensive range of proteolytic enzymes (cytoplasmic and lysosomal proteinases and peptidases, which play a key role in normal cell functioning) in fenitrothion-resistant (571ab) and susceptible (Cooper) strains of Musca domestica following in vivo exposure to the insecticide fenitrothion. Untreated insects of the resistant strain had significantly higher levels (20-100%) of activity for many protease types compared to the susceptible strain (whole body analysis). Exposure to fenitrothion resulted in further activity increases for most proteases at some point during the subsequent 24 h period in resistant strain insects; susceptible strain insects were also capable of similar increases in protease activities. We therefore suggest that it must be the combination of intrinsically higher protease levels (prior to pesticide exposure), together with the capacity to further increase protease activities following insecticide exposure, which is important in the mechanism by which proteases may confer survival advantages in insecticide resistant insects. We further speculate that this mechanism may involve increased supply of precursor amino acids from proteolytic degradation products to the intracellular pool, prior to de novo synthesis of detoxifying enzymes following insecticide exposure.

Animals↗