Distinct roles of two RDL GABA receptors in fipronil action in the diamondback moth (Plutella xylostella)

The phenylpyrazole insecticide fipronil blocks resistance to dieldrin (RDL) γ-aminobutyric acid (GABA) receptors in insects, thereby impairing inhibitory neurotransmission. Some insect species, such as the diamondback moth (Plutella xylostella), possess more than one Rdl gene. The involvement of multiple Rdls in fipronil toxicity and resistance remains largely unknown. In this study, we investigated the roles of two Rdl genes, PxRdl1 and PxRdl2, in P. xylostella fipronil action. In Xenopus oocytes, PxRDL2 receptors were 40 times less sensitive to fipronil than PxRDL1. PxRDL2 receptors were also less sensitive to GABA compared with PxRDL1. Knockout of the fipronil-sensitive PxRdl1 reduced the fipronil potency 10-fold, whereas knockout of the fipronil-resistant PxRdl2 enhanced the fipronil potency 4.4-fold. Furthermore, in two fipronil-resistant diamondback moth field populations, PxRdl2 expression was elevated 3.7- and 4.1-fold compared with a susceptible strain, whereas PxRdl1 expression was comparable among the resistant and susceptible strains. Collectively, our results indicate antagonistic effects of PxRDL1 and PxRDL2 on fipronil action in vivo and suggest that enhanced expression of fipronil-resistant PxRdl2 is potentially a new mechanism of fipronil resistance in insects.     

Sex-ratio distortion in Drosophila simulans: co-occurence of a meiotic drive and a suppressor of drive

A sex-ratio distortion factor was found at high frequency in D. simulans strains from Seychelles and New Caledonia. This factor is poorly or not expressed within those strains which are resistant to it. Its presence was detected by crossing females from New Caledonia or the Seychelles with males from a different geographic origin. Most of the F1 males obtained produced an excess of females (up to 99%) in their progeny. The two strains are infected with Wolbachia, but these micro-organisms are not involved in the sex-ratio distortion. The sex-ratio factor is shown to be an X-linked meiotic driver; nuclear resistance factor(s) act by suppressing the drive. It is likely that the same X-located driver invaded the two populations, which subsequently developed resistance factor(s) against it.     

Sequential evolution of a symbiont inferred from the host: Wolbachia and Drosophila simulans

This study aims to unravel the biogeography of a model symbiont/host system by exploiting the prediction that a symbiont will leave a signature of infection on the host. Specifically, a global sample of 1,442 Drosophila simulans from 33 countries and 64 sampling localities was employed to infer the phylogeography of the maternally inherited alpha-proteobacteria Wolbachia. Phylogenetic analyses, from three symbiont genes and 24 mtDNA genomes (excluding the A + T-rich region), showed that each of four Wolbachia strains infected D. simulans once. The global distribution and abundance of the Wolbachia strains and the three mtDNA haplogroups (D. simulans siI, siII and siIII) was then determined. Finally, network analyses of variable regions within siI (584 bp from seven additional lines) and siII (1,701 bp from 383 lines) facilitated a detailed biogeographic discussion. There is little variation in siIII and the haplogroup is restricted in its distribution. These data show how the history of an infection can be mapped by combining data from the symbiont and the host. They say little about the organismal history of the host because the mtDNA genome is a biased representation of the whole genome.     

Cis- and trans-acting genetic factors contribute to heterogeneity in the rate of crossing over between the Drosophila simulans clade species

In the genus Drosophila, variation in recombination rates has been found within and between species. Genetic variation for both cis‐ and trans‐acting factors has been shown to affect recombination rates within species, but little is known about the genetic factors that affect differences between species. Here, we estimate rates of crossing over for seven segments that tile across the euchromatic length of the X chromosome in the genetic backgrounds of three closely related Drosophila species. We first generated a set of Drosophila mauritiana lines each having two semidominant visible markers on the X chromosome and then introgressed these doubly marked segments into the genetic backgrounds of its sibling species, Drosophila simulans and Drosophila sechellia. Using these 21 lines (seven segments, three genetic backgrounds), we tested whether recombination rates within the doubly marked intervals differed depending on genetic background. We find significant heterogeneity among intervals and among species backgrounds. Our results suggest that a combination of both cis‐ and trans‐acting factors have evolved among the three D. simulans clade species and interact to affect recombination rate.     

Antagonistic responses to natural and sexual selection and the sex-specific evolution of cuticular hydrocarbons in Drosophila simulans

Natural and sexual selection are classically thought to oppose one another, and although there is evidence for this, direct experimental demonstrations of this antagonism are largely lacking. Here, we assessed the effects of sexual and natural selection on the evolution of cuticular hydrocarbons (CHCs), a character subject to both modes of selection, in Drosophila simulans. Natural selection and sexual selection were manipulated in a fully factorial design, and after 27 generations of experimental evolution, the responses of male and female CHCs were assessed. The effects of natural and sexual selection differed greatly across the sexes. The responses of female CHCs were generally small, but CHCs evolved predominantly in the direction of natural selection. For males, profiles evolved via sexual and natural selection, as well as through the interaction between the two, with some male CHC components only evolving in the direction of natural selection when sexual selection was relaxed. These results indicate sex‐specific responses to selection, and that sexual and natural selection act antagonistically for at least some combinations of CHCs.     

中国大陆拟果蝇(Drosophila simulans)的随机扩增多态性DNA分析

近几年发现了拟果蝇(Drosophila simulans)在中国大陆的广泛分布。用随机扩增多态性DNA(RAPD)方法研究了中国大陆38个不同地理群体的拟果蝇(D．slmulam)在DNA水平上的遗传多样性,初步讨论了拟果蝇在中国大陆的起源。以40种10bp长的寡聚核苷酸随机引物进行PCR扩增,根据遗传距离利用UPGMA法作出的相关聚类图显示：(1)38个地理群体按纬度以南京(NJ)为界明显地分为南北两大支系,北方支系以北京(BJ)为界又分为明显的两个亚支,一支为东北支系包括漠河、海拉尔、黑河、佳木斯、哈尔滨、长春、沈阳、丹东、延吉、图们等10个群体;另一支包括北京、大同、呼和浩特、银川、西宁、兰州、太原、石家庄、烟台、济南、徐州、连云港等12个群体。南方支系包括郑州、武汉、上海、南京、杭州、重庆、南昌、温州、长沙、贵阳、福州、昆明、厦门、广州、南宁、海南等16个群体。(2)各地理群体之间的遗传距离与地理分布有密切关系,基本按地理位置的相关性聚类在一起。根据了解到的事实,(1)拟果蝇在中国是一个外来种;(2)拟果蝇在中国大陆的入侵是最近30年左右的事情;(3)拟果蝇在全国的广泛分布是最近10多年的事情。所以拟果蝇各群体的聚类关系可能并不是地理分化的结果,而是由于建立各地方群体的祖先群体或个体本身具有不同的遗传组成,但是某些地理上相邻近的群体可能拥有共同的祖先群体或个体,从而造成了拟果蝇各地方群体随地理分布关系而聚类。推测有两种可能的原因会产生以上结果：一是中国大陆的拟果蝇有多个不同的来源;二是拟果蝇在扩张过程中经历了可产生奠基者效应或瓶颈效应的偶然事件。     

Cyclodiene resistance at the insect GABA receptor/chloride channel complex confers broad cross resistance to convulsants and experimental phenylpyrazole insecticides

This study investigated the pharmacological profile of cyclodiene resistance in Drosophila melanogaster and the mode of action of a phenylpyrazole insecticide, JKU 0422. Toxicological studies were performed with a sucrose bait assay containing the synergist piperonyl butoxide. The Maryland strain of D. melanogaster was resistant to dieldrin, lindane, picrotoxinin, TBPS, p-CN-TBOB, and JKU 0422. In contrast, this strain was susceptible to cypermethrin and the avermectins MK-243, abamectin, and abamectin 8,9-oxide. Neurophysiological studies showed that both TBPS and JKU 0422 reversed the inhibitory action of GABA in central nerve preparations from susceptible D. melanogaster. However, the response to these compounds was attenuated in nerve preparations from the resistant Maryland strain, which indicated that the resistance was expressed at the level of the nerve. Topical toxicity bioassays with JKU 0422 on susceptible (CSMA) and cyclodiene-resistant (LPP) strains of German cockroach revealed a resistance ratio of 553-fold for this compound. These studies demonstrate that cyclodiene resistance in D. melanogaster confers broad cross resistance toward compounds thought to block the GABA-gated chloride channel in a manner similar to the cyclodienes. Moreover, the cross resistance extends to JKU 0422, and resistance to this compound is also present in a strain of cyclodiene-resistant German cockroach. These toxicological results, along with the neurophysiological studies, confirm that JKU 0422 has a mode of action that is similar to the cyclodienes and TBPS. These findings suggest that the introduction and use of new chloride channel antagonists as insecticides should be managed carefully in order to prevent the rapid development of resistance in the field. © 1994 Wiley-Liss, Inc.     

Phenotypic plasticity of body pigmentation in Drosophila: influence of a developmental thermoperiodic regime in two sibling species

Abstract Variation of body pigmentation according to growth temperature was analysed in two European populations (Bordeaux and Cordoba) of the two sibling species Drosophila melanogaster (Meigen) and D. simulans (Sturtevant). Results obtained at constant temperatures were compared with those observed under alternating thermal regimes of various amplitudes and with a daily periodicity of 12–12 h. Abdominal pigmentation in segments 5, 6 and 7, and of their sum, was investigated in both species in females only. Thoracic trident pigmentation was studied in males and females but in Drosophila melanogaster only.
Thoracic pigmentation exhibited curvilinear convex reaction norms under constant conditions, with a darker pigmentation in the population (Bordeaux) living in a colder climate. No specific effects of alternating regimes were seen. For abdomen pigmentation, slightly different reaction norms were observed under constant conditions between Bordeaux and Cordoba strains. There were also slight differences between the two species. Alternating temperatures produced a significant decrease in average pigmentation, roughly proportional to the thermal amplitude.
Alternating stressing conditions induced also an increase in the phenotypic variability of abdominal pigmentation, especially in segments 6 and 7. Finally, because an increase of growth temperature results in a monotonically decreasing reaction norm of both abdomen pigmentation in females and a size-related trait, the wing/thorax ratio, we found a linear relationship between the two variables, with a very high overall correlation at constant temperatures ( r  = 0.99). The correlation was slightly less under alternating conditions, due to a broader dispersal of the experimental values around the regression lines.     

黑腹果蝇GABA受体GRD亚基和LCCH3 亚基的克隆、表达与鉴定

昆虫GABA受体（γ-aminobutyric acid receptor, GABAR）是杀虫剂的重要靶标之一。本研究以黑腹果蝇Drosophila melanogaster整体组织的cDNA作为模板, 采用RT-PCR技术扩增了黑腹果蝇GABA受体LCCH3亚基和GRD亚基的cDNA序列, 并克隆至pET-32a表达载体上, 测序结果表明获得的序列与基因库中已发表的序列一致性在99%以上, 无移码突变。在IPTG的诱导下, LCCH3基因成功在大肠杆菌Escherichia coli中表达, 而GRD基因未表达。通过包涵体洗涤、变性、Ni²⁺亲合层析纯化、稀释复性获得纯化的重组表达的LCCH3蛋白, 并用圆二色谱测定了目标蛋白的二级结构, 主要富含β结构。该研究结果为研究昆虫GABAR的结构和功能关系提供了重要的参考数据。     

Direct and correlated responses to selection for desiccation resistance: a comparison of Drosophila melanogaster and D. simulans

Replicate lines of Drosophila melanogaster and D. simulans originating from the same location in Australia were selected at two selection intensities (50%, 85% mortality) for increased resistance to desiccation, and scored for correlated responses to see if similar physiological changes were associated with the selection responses. Realized heritabilities were much higher in D. melanogaster. Selected lines of both species were more resistant than control lines to starvation and a toxic ethanol concentration. Both species also showed similar correlated responses for traits underlying the selection response: selected lines lost water at a slower rate and had reduced activity levels in a dry environment, but they did not differ in wet or dry body weight or in water content. For D. melanogaster, realized heritabilities for lines selected at 85% mortality were higher than for lines selected at 50% mortality, but there was no effect of selection intensity for D. simulans. Comparative studies of this nature may be useful in predicting the extent to which species can adapt to stress in the wild.     

Consequence of the presence of two different beta subunit isoforms in a GABA(A) receptor

The major isoforms of GABA(A) receptors are thought to be composed of two alpha, two beta and one gamma subunit(s). GABA(A) receptors containing two beta1 subunits respond differently to the anticonvulsive compound loreclezole and the general anaesthetic etomidate than receptors containing two beta2 subunits. Receptors containing beta2 subunits show a much larger allosteric stimulation by these agents than those containing beta1 subunits. We were interested to know how receptors containing both beta1 and beta2 subunits, in different positions respond to loreclezole and etomidate. To answer this question, subunits were fused at the DNA level to form dimeric and trimeric subunits. Concatenated receptors (alpha1-beta1-alpha1/gamma2-beta1, alpha1-beta2-alpha1/gamma2-beta1, alpha1-beta1-alpha1/gamma2-beta2 and alpha1-beta2-alpha1/gamma2-beta2) were expressed in Xenopus ooctyes and functionally compared in their response to the agonist GABA and to the positive allosteric modulators, loreclezole and etomidate. We have shown that (I) in the presence of both beta1 and beta2 subunits in the same pentamer (mixed receptors) direct gating by etomidate is similar to exclusively beta1 containing receptors; (II) In mixed receptors, stimulation by etomidate assumed characteristics intermediate to exclusively beta1 or beta2 containing receptors, but the values for the concentrations < 10 microM were always much closer to those observed in alpha1-beta1-alpha1/gamma2-beta1 receptors; and (III) mixed receptors show no positional effects.     

Resistance to dieldrin + fipronil assorts with chromosome inversion 2La in the malaria vector Anopheles gambiae

Cyclodiene insecticide resistance in malaria vector mosquitoes of the Anopheles gambiae species complex (Diptera: Culicidae) has been reported previously from several parts of Africa. We report resistance to dieldrin, a cyclodiene, in two laboratory strains of An. gambiae Giles sensu stricto code-named Ian P20 from Nigeria (1979) and CIG from Cote d'Ivoire (1997). Dieldrin resistance levels were high in adult female mosquitoes (40-75% survived exposure to 4% dieldrin for 1 h) and was closely linked with chromosomal paracentric inversion 2La. This inversion did not occur in Hardy-Weinberg proportions, but showed an excess of heterozygotes in both strains, which may account for the high levels of resistance. This linkage also suggests that dieldrin resistance in Ian P20 is dominant. After subsamples of strain Ian P20 were exposed for 1 h to dieldrin 4% or fipronil 2% (discriminating concentrations), the resultant mortality-rates (61% and 65%) were not significantly different. Most survivors after fipronil treatment also survived subsequent exposure to dieldrin (46/50=92%). This apparent cross-resistance between insecticides of two classes (cyclodiene and phenyl pyrazole) has implications for the management of insecticide resistance in wild populations of the An. gambiae complex.     

Molecular cloning,genomic structure,and genetic mapping of two Rdl‐orthologous genes of GABA receptors in the diamondback moth,Plutella xylostella

The Resistance to dieldrin (Rdl) gene encodes a subunit of the insect γ‐aminobutyric acid (GABA) receptor. Cyclodiene resistance in many insects is associated with replacement of a single amino acid (alanine at position 302) with either a serine or a glycine in the Rdl gene. Two Rdl‐orthologous genes of GABA receptors (PxGABARα1 and PxGABARα2) were cloned and sequenced from a susceptible strain (Roth) of Plutella xylostella. PxGABARα1 and PxGABARα2 showed 84% and 77% identity with the Rdl gene of Drosophila melanogaster at an amino acid level, respectively. The coding regions of PxGABARα1 and PxGABARα2 both comprise ten exons, with two alternative RNA‐splicing forms in exon 3 of both genes. At the orthologous position of alanine‐302 in D. melanogaster Rdl, PxGABARα1 has a conserved alanine at position 282. PxGABARα2 has a serine instead of an alanine at the equivalent position. With two informative DNA markers, both PxGABARα1 and PxGABARα2 were mapped onto the Z chromosome of P. xylostella. © 2010 Wiley Periodicals, Inc.     

Reduced neuronal sensitivity to dieldrin and picrotoxinin in a cyclodiene-resistant strain of Drosophila melanogaster (Meigen).

Toxicological and neurophysiological studies were performed to characterize the resistance mechanism in a cyclodiene-resistant strain of Drosophila melanogaster (Maryland strain). Dieldrin had an LC50 of 0.058 ppm against the larvae of susceptible D. melanogaster (Oregon-R wild type) when formulated in the rearing media. The LC50 of the resistant Maryland strain was 10.8 ppm, giving a resistance ratio (LC50-Maryland/LC50-susceptible) of 186-fold. Suction electrode recordings were made from peripheral nerves of the larval central nervous system to test whether reduced nerve sensitivity played any role in the observed resistance. In susceptible preparations (n = 5), inhibition of nerve firing by 1 mM gamma-aminobutyric acid (GABA) was effectively antagonized within 3-10 min by 10 microM dieldrin. In contrast, 30 min incubations with 10 microM dieldrin had no effect on preparations from cyclodiene-resistant individuals (n = 5). Similarly, 10 microM picrotoxinin blocked GABA-dependent inhibition in susceptible nerve preparations (n = 3). In recordings from resistant insects (n = 4), picrotoxinin displayed either weak antagonism of GABA or hyperexcitation indistinguishable from susceptible preparations. These results demonstrate that cyclodiene resistance in the Maryland strain of D. melanogaster 1) is expressed in immature stages, 2) is present at the level of the nerve, and 3) extends to picrotoxinin, albeit at a reduced level compared with dieldrin. The possible role of an altered GABA receptor in this resistance is discussed.     

Neuronal nicotinic acetylcholine receptors from Drosophila: two different types of alpha subunits coassemble within the same receptor complex

Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.