28 research outputs found
Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.
No full textBACKGROUND:Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE) genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos. PRINCIPAL FINDINGS:Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida): one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries. SIGNIFICANCE:The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies
Substrate specificity and promiscuity of horizontally transferred UDP-glycosyltransferases in the generalist herbivore Tetranychus urticae
No full textUridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the addition of UDP-sugars to small hydrophobic molecules, turning them into more water-soluble metabolites. While their role in detoxification is well documented for vertebrates, arthropod UGTs have only recently been linked to the detoxification and sequestration of plant toxins and insecticides. The two-spotted spider mite Tetranychus urticae is a generalist herbivore notorious for rapidly developing resistance to insecticides and acaricides. We identified a set of eight UGT genes that were overexpressed in mites upon long-term acclimation or adaptation to a new host plant and/or in mite strains highly resistant to acaricides. Functional expression revealed that they were all catalytically active and that the majority preferred UDP-glucose as activated donor for glycosylation of model substrates. A high-throughput substrate screening of both plant secondary metabolites and pesticides revealed patterns of both substrate specificity and promiscuity. We further selected nine enzyme-substrate combinations for more comprehensive analysis and determined steady-state kinetic parameters. Among others, plant metabolites such as capsaicin and several flavonoids were shown to be glycosylated. The acaricide abamectin was also glycosylated by two UGTs and one of these was also overexpressed in an abamectin resistant strain. Our study corroborates the potential role of T. urticae UGTs in detoxification of both synthetic and natural xenobiotic compounds and paves the way for rapid substrate screening of arthropod UGTs
Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay
No full textAbstract Background Mutations in the voltage-gated sodium channel at codon 1014 confer knock-down resistance (kdr) to pyrethroids in a wide range of insects. Anopheles gambiae exhibits two mutant alleles at codon 1014, serine and phenylalanine; and both are now widespread across Africa. Existing screening methods only allow for one resistant allele to be detected per assay. A new locked nucleic acid (LNA) qPCR assay was developed for the simultaneous detection of both mutant alleles and the wild type allele in a single assay. This tri-allelic detection assay was assessed as part of a study of the insecticide resistance in An. gambiae sensu stricto (s.s.) in the previously un-sampled area of Nord Ubangi, Democratic Republic of the Congo. Methods Samples from three sites were tested for insecticide susceptibility using WHO bioassays, with and without the synergist PBO preceding pyrethroid exposures, and were subsequently analysed for frequency and resistance-association of the Vgsc-1014 and Vgsc-N1575Y mutations. Results from the LNA-kdr 1014 assay were compared to results from standard TaqMan-kdr assays. Results Anopheles gambiae sensu lato (s.l.) was by far the predominant vector captured (84%), with only low frequencies of Anopheles funestus s.l. (9%) detected in Nord Ubangi. Molecular identification found An. gambiae s.s. to be the principal vector (99%) although Anopheles coluzzii was detected at very low frequency. Anopheles gambiae were susceptible to the carbamate insecticide bendiocarb, but resistant to DDT and to the pyrethroids permethrin and deltamethrin. Susceptibility to both pyrethroids was partially restored with prior exposure to PBO suggesting likely involvement of metabolic resistance. Anopheles gambiae s.s. was homozygous for kdr resistant alleles with both the L1014F and L1014S mutations present, and the N1575Y polymorphism was present at low frequency. The LNA-kdr assay simultaneously detected both resistant alleles and gave results entirely consistent with those from the two TaqMan-kdr assays. Conclusion This study provides rare data on insecticide resistance and mechanisms in Anopheles from the centre of Africa, with the first detection of N1575Y. Nord Ubangi populations of An. gambiae s.s. show insecticide resistance mediated by both metabolic mechanisms and Vgsc mutations. The LNA-kdr assay is particularly suitable for use in populations in which both 1014S and 1014F kdr alleles co-occur and provides robust results, with higher throughput and at a quarter of the cost of TaqMan assays
Rapid high throughput SYBR green assay for identifying the malaria vectors <i>Anopheles arabiensis</i>, <i>Anopheles coluzzii</i> and <i>Anopheles gambiae s</i>.<i>s</i>. <i>Giles</i>
No full textThe Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72°C for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.</div
MOESM3 of Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay
No full textAdditional file 3. Taqman genotype plots demonstrating mis-calling of genotype due non-specific binding of the mutant alleles. Mis-called genotypes are circled in black and the actual genotype indicated. A: Taqman kdr for L1014F detection. B: Taqman kdr for L1014S detection
MOESM2 of Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay
No full textAdditional file 2. Genotype frequencies for the VGSC L1014 mutations in Anopheles gambiae s.s. specimens from Uganda
MOESM4 of Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay
No full textAdditional file 4. Genotype frequencies for the VGSC N1575Y mutation in Anopheles gambiae s.l. specimens from DRC
MOESM1 of Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay
No full textAdditional file 1. Collection sites in the Gbadolite region of DRC. (A) Location of Gbadolite in DRC (shown in red) within Equateur province and the capital city, Kinshasa â
. (B) Fine-scale map of collection sites
Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus
No full textInternational audienceVector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions
Copy number variants underlie major selective sweeps in insecticide resistance genes in Anopheles arabiensis
No full textTo keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. The outdoor-biting malaria vector Anopheles arabiensis is of increasing concern for malaria transmission because it is apparently less susceptible to many indoor control interventions, yet knowledge of its mechanisms of resistance remains limited. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of globally high resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to the pyrethroid deltamethrin (commonly used in bednets) and PM (widespread use for indoor spraying), in An. arabiensis from 2 regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in metabolic resistance. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the carboxylesterase genes Coeae2g - Coeae6g. Using complementary data from another malaria vector, An. coluzzii, in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster (Cyp6aap_Dup33). Against this background of metabolic resistance, resistance caused by mutations in the insecticide target sites was very rare or absent. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with 3 An. arabiensis individuals from West Africa revealed a startling evolutionary diversity, with up to 5 independent origins of Vgsc-995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis
