Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations

Two amino acid substitutions in acetylcholinesterase 1 (AChE1), G119S and F290V, are responsible for resistance to organophosphate and carbamate insecticides in Culex pipiens mosquitoes. These mutations generate very different levels of insensitivity to insecticide inhibitors. We described here a biochemical method that rapidly identifies AChE1 variants (susceptible, G119S and F290V, named S, R and V, respectively) present in individual mosquitoes. We investigated the frequency of AChE1 phenotypes in 41 field samples collected around the Mediterranean Sea. F290V substitution was found only in 15 samples and at low frequency, whereas G119S was highly spread in all samples. However, seven V distinct alleles were identified whereas only one R allele was present. The [V] enzymatic phenotype was never observed alone, and the V allele was always found associated with the susceptible and/or G119S AChE1 ([VS], [VR] or [VRS] phenotypes). Furthermore, we showed the presence of duplicated alleles, associating a susceptible and a V copy of the ace-1 gene, in most individuals analyzed for its presence. Evolutionary forces driving the large number of F290V ace-1 alleles and their low frequency in Mediterranean countries are discussed.     

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l⁻¹ and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1^R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1^R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1^V or the duplicated ace-1^D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.     

Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens

Gene duplication is thought to be the main potential source of material for the evolution of new gene functions. Several models have been proposed for the evolution of new functions through duplication, most based on ancient events (Myr). We provide molecular evidence for the occurrence of several (at least 3) independent duplications of the ace-1 locus in the mosquito Culex pipiens, selected in response to insecticide pressure that probably occurred very recently (<40 years ago). This locus encodes the main target of several insecticides, the acetylcholinesterase. The duplications described consist of 2 alleles of ace-1, 1 susceptible and 1 resistant to insecticide, located on the same chromosome. These events were detected in different parts of the world and probably resulted from distinct mechanisms. We propose that duplications were selected because they reduce the fitness cost associated with the resistant ace-1 allele through the generation of persistent, advantageous heterozygosis. The rate of duplication of ace-1 in C. pipiens is probably underestimated, but seems to be rather high.     

Evidence of introgression of the ace-1(R) mutation and of the ace-1 duplication in West African Anopheles gambiae s. s

Background

The role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s.

Methods and Results

To establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression.

Conclusions

These results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa.     

Detection and widespread distribution of sodium channel alleles characteristic of insecticide resistance in Culex pipiens complex mosquitoes in China

Culex pipiens complex mosquitoes are widely distributed throughout China and are known to be important disease vectors. Two pyrethroid resistance associated mutations have been identified in Cx. pipiens complex (Diptera: Culicidae), but there is little information on the diversity and distribution of kdr alleles in pyrethroid resistance in Cx. pipiens complex mosquitoes in China. In the present study, we report on a modified three tube allele-specific (AS)-PCR method for detecting the 1014F and 1014S alleles. The new technique was applied to identify the distribution of the two alleles in natural Cx. pipiens complex populations in China. The results confirmed that the new method is both sensitive and specific. The 1014F allele was found in all 14 of the field populations tested (frequency ranged from 6.8 to 76.2%) and the 1014S allele was found in almost two-thirds (frequency from 2.4 to 28.6%), indicating that the genotypes known to be associated with pyrethroid resistance are widespread in China. The resistance-associated alleles were more common in southern Chinese sampling sites than in northern sites. The coexistence of the two resistant mutations in individual mosquitoes was also observed in five of the field populations. Two alternative mutations within the L1014 codon were identified in Culex pipiens molestus Forskal, 1775, including a non-synonymous mutation resulting in a 1014C substitution.     

Contemporary evolution of resistance at the major insecticide target site gene Ace‐1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine–serine substitution at codon 119 of the Ace‐1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace‐1 119S haplotype, whereas 119G diversity was high overall but very low at non‐synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace‐1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace‐1 gene, whereas 119G alleles were unduplicated. Ace‐1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace‐1, emphasizing the need to integrate CNV analysis into genome scans for selection.     

我国草地贪夜蛾田间种群有机磷和氨基甲酸酯类杀虫剂靶标基因ace-1的基因型和突变频率

【目的】对入侵我国的草地贪夜蛾Spodoptera frugiperda有机磷和氨基甲酸酯类杀虫剂靶标基因ace-1的基因型进行分子检测,明确抗性基因频率,进而指导田间科学用药。【方法】采集中国12省份的草地贪夜蛾田间种群幼虫样本,提取单头样本的基因组DNA,利用特异性引物进行PCR扩增,获得ace-1基因片段。根据碱基、氨基酸序列比对和测序峰图分析,明确与有机磷和氨基甲酸酯类杀虫剂抗性相关的3个氨基酸突变位点A201S, G227A和F290V的基因型和抗性基因频率。【结果】通过DNA检测分析中国12省份草地贪夜蛾田间种群589头个体ace-1基因的基因型和突变频率发现,在A201S位点检测到137头个体为抗性杂合基因型,抗性基因频率为11.6%,未发现抗性纯合基因型个体;G227A位点589头个体均为敏感纯合基因型;F290V位点的抗性基因频率最高,达到57.1%,携带抗性基因的个体数量达到523头(占样本总数的88.8%)。【结论】结果表明入侵我国的草地贪夜蛾种群携带高频率的对有机磷和氨基甲酸酯类杀虫剂抗性基因。田间防治建议不用或少用有机磷和氨基甲酸酯类杀虫剂,同时进一步加强田间抗性监测工作。     

Forty years of erratic insecticide resistance evolution in the mosquito Culex pipiens

One view of adaptation is that it proceeds by the slow and steady accumulation of beneficial mutations with small effects. It is difficult to test this model, since in most cases the genetic basis of adaptation can only be studied a posteriori with traits that have evolved for a long period of time through an unknown sequence of steps. In this paper, we show how ace-1, a gene involved in resistance to organophosphorous insecticide in the mosquito Culex pipiens, has evolved during 40 years of an insecticide control program. Initially, a major resistance allele with strong deleterious side effects spread through the population. Later, a duplication combining a susceptible and a resistance ace-1 allele began to spread but did not replace the original resistance allele, as it is sublethal when homozygous. Last, a second duplication, (also sublethal when homozygous) began to spread because heterozygotes for the two duplications do not exhibit deleterious pleiotropic effects. Double overdominance now maintains these four alleles across treated and nontreated areas. Thus, ace-1 evolution does not proceed via the steady accumulation of beneficial mutations. Instead, resistance evolution has been an erratic combination of mutation, positive selection, and the rearrangement of existing variation leading to complex genetic architecture.     

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 γ-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?→ Serine^TCG (allele S) and A302?→?Serine^AGT (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.     

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.     

Global spread and genetic variants of the two CYP9M10 haplotype forms associated with insecticide resistance in Culex quinquefasciatus Say

Insecticide resistance develops as a genetic factor (allele) conferring lower susceptibility to insecticides proliferates within a target insect population under strong positive selection. Intriguingly, a resistance allele pre-existing in a population often bears a series of further adaptive allelic variants through new mutations. This phenomenon occasionally results in replacement of the predominating resistance allele by fitter new derivatives, and consequently, development of greater resistance at the population level. The overexpression of the cytochrome P450 gene CYP9M10 is associated with pyrethroid resistance in the southern house mosquito Culex quinquefasciatus. Previously, we have found two genealogically related overexpressing CYP9M10 haplotypes, which differ in gene copy number (duplicated and non-duplicated). The duplicated haplotype was derived from the non-duplicated overproducer probably recently. In the present study, we investigated allelic series of CYP9M10 involved in three C. quinquefasciatus laboratory colonies recently collected from three different localities. Duplicated and non-duplicated overproducing haplotypes coexisted in African and Asian colonies indicating a global distribution of both haplotype lineages. The duplicated haplotypes both in the Asian and African colonies were associated with higher expression levels and stronger resistance than non-duplicated overproducing haplotypes. There were slight variation in expression level among the non-duplicated overproducing haplotypes. The nucleotide sequences in coding and upstream regions among members of this group also showed a little diversity. Non-duplicated overproducing haplotypes with relatively higher expression were genealogically closer to the duplicated haplotypes than the other non-duplicated overproducing haplotypes, suggesting multiple cis-acting mutations before duplication.     

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using "optical tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.     

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47–0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.     

Fitness costs of insecticide resistance in natural breeding sites of the mosquito Culex pipiens

Genetic changes conferring adaptation to a new environment may induce a fitness cost in the previous environment. Although this prediction has been verified in laboratory conditions, few studies have tried to document this cost directly in natural populations. Here, we evaluated the pleiotropic effects of insecticide resistance on putative fitness components of the mosquito Culex pipiens. Experiments using different larval densities were performed during the summer in two natural breeding sites. Two loci that possess alleles conferring organophosphate (OP) resistance were considered: ace-1 coding for an acetylcholinesterase (AChE1, the OP target) and Ester, a 'super locus" including two closely linked loci coding for esterases A and B. Resistance ace-1 alleles coding for a modified AChE1 were associated with a longer development time and shorter wing length. The pleiotropic effects of two resistance alleles Ester1 and Ester4 coding for the overproduced esterases A1 and A4-B4, respectively, were more variable. Both A1 and A4-B4 reduced wing length, although only A1 was associated with a longer preimaginal stage. The fluctuating asymmetry (FA) of the wing did not respond to the presence or to the interaction of resistance alleles at the two loci at any of the density levels tested. Conversely, the FA of one wing section decreased when larval density increased. This may be the consequence of selection against less developmentally stable individuals. The results are discussed in relation to the local evolution of insecticide resistance genes.     

Comparison of Anopheles gambiae and Culex pipiens acetycholinesterase 1 biochemical properties

Selection of insensitive acetycholinesterase 1 (AChE1) has occurred in several mosquito species controlled with carbamate (CX) and organophosphate (OP) insecticides. In case of pyrethroid resistance, these insecticides represent an alternative for disease vector control program. Their heavy use in agriculture has selected resistant populations of Anopheles gambiae in West Africa. The evolution of resistance has to be studied to prevent, or at least slow down, the spread of resistant mosquito in wild populations. An. gambiae shares the same resistance mechanism to CX and OP insecticides as Culex pipiens, which was attributed to the G119S substitution in the AChE1 enzyme. By comparing resistant AChE1 from both species, we show here that similar resistance levels are obtained toward 10 insecticides of both classes. Moreover, similar AChE1 activity levels are recorded between either susceptible or resistant mosquitoes of both species. Enzymes belonging to both species seem thus to share identical properties. Consequently, we hypothesize that fitness cost associated with AChE1 insensitivity in C. pipiens mosquitoes should be similar in An. gambiae and thus be used in strategies to control resistant populations where malaria is prevalent.     

库蚊野生种群扩增等位基因重组现象分析

羧酸酯酶 (carboxylesterases)的过量产生是库蚊Culexpipiens对有机磷杀虫剂 (OP)产生抗性的主要机制。由est 3和est 2组成的酯酶超级基因座 (estersuper locus)的基因扩增是引起酯酶基因扩增的主要遗传学基础。通过淀粉电泳研究了采自广州、佛山、郑州的库蚊野生蚊虫种群 ,发现在这些种群中存在着扩增等位基因重组现象。该现象可能是蚊虫受到杀虫药剂的选择压力、等位基因多样性和等位基因型频率的影响。这将提供一个研究抗性进化的自然模型。     

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.     

Biotic interactions govern genetic adaptation to toxicants

The genetic recovery of resistant populations released from pesticide exposure is accelerated by the presence of environmental stressors. By contrast, the relevance of environmental stressors for the spread of resistance during pesticide exposure has not been studied. Moreover, the consequences of interactions between different stressors have not been considered. Here we show that stress through intraspecific competition accelerates microevolution, because it enhances fitness differences between adapted and non-adapted individuals. By contrast, stress through interspecific competition or predation reduces intraspecific competition and thereby delays microevolution. This was demonstrated in mosquito populations (Culex quinquefasciatus) that were exposed to the pesticide chlorpyrifos. Non-selective predation through harvesting and interspecific competition with Daphnia magna delayed the selection for individuals carrying the ace-1^R resistance allele. Under non-toxic conditions, susceptible individuals without ace-1^R prevailed. Likewise, predation delayed the reverse adaptation of the populations to a non-toxic environment, while the effect of interspecific competition was not significant. Applying a simulation model, we further identified how microevolution is generally determined by the type and degree of competition and predation. We infer that interactions with other species—especially strong in ecosystems with high biodiversity—can delay the development of pesticide resistance.     

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 γ-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 → Serine^TCG (allele S) and A302 → Serine^AGT (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. Received: 10 March 1998 / Accepted: 21 July 1998     

Analysis of the CCR5 gene coding region diversity in five South American populations reveals two new non-synonymous alleles in Amerindians and high CCR5*D32 frequency in Euro-Brazilians

The CC chemokine receptor 5 (CCR5) molecule is an important co-receptor for HIV. The effect of the CCR5*D32 allele in susceptibility to HIV infection and AIDS disease is well known. Other alleles than CCR5*D32 have not been analysed before, neither in Amerindians nor in the majority of the populations all over the world. We investigated the distribution of the CCR5 coding region alleles in South Brazil and noticed a high CCR5*D32 frequency in the Euro-Brazilian population of the Paraná State (9.3%), which is the highest thus far reported for Latin America. The D32 frequency is even higher among the Euro-Brazilian Mennonites (14.2%). This allele is uncommon in Afro-Brazilians (2.0%), rare in the Guarani Amerindians (0.4%) and absent in the Kaingang Amerindians and the Oriental-Brazilians. R223Q is common in the Oriental-Brazilians (7.7%) and R60S in the Afro-Brazilians (5.0%). A29S and L55Q present an impaired response to β-chemokines and occurred in Afro- and Euro-Brazilians with cumulative frequencies of 4.4% and 2.7%, respectively. Two new non-synonymous alleles were found in Amerindians: C323F (g.3729G > T) in Guarani (1.4%) and Y68C (g.2964A > G) in Kaingang (10.3%). The functional characteristics of these alleles should be defined and considered in epidemiological investigations about HIV-1 infection and AIDS incidence in Amerindian populations.