Copy number variation (CNV) and insecticide resistance in mosquitoes: evolving knowledge or an evolving problem?

Copy number variation (CNV) in insect genomes is a rich source of potentially adaptive polymorphism which may help overcome the constraints of purifying selection on conserved genes and/or permit elevated transcription. Classic studies of amplified esterases and acetylcholinesterase duplication in Culex pipiens quantified evolutionary dynamics of CNV driven by insecticidal selection. A more complex and potentially medically impactful form of CNV is found in Anopheles gambiae, with both heterogeneous duplications and homogeneous amplifications strongly linked with insecticide resistance. Metabolic gene amplification, revealed by shotgun sequencing, appears common in Aedes aegypti, but poorly understood in other mosquito species. Many methodologies have been used to detect CNV in mosquitoes, but relatively few can detect both duplications and amplifications, and contrasting methods should be combined. Genome scans for CNV have been rare to date in mosquitoes, but offer immense potential to determine the overall role of CNV as a component of resistance mechanisms.sequencing, appears common in Aedes aegypti, but poorly understood in other mosquito species. Many methodologies have been used to detect CNV in mosquitoes, but relatively few can detect both duplications and amplifications, and contrasting methods should be combined. Genome scans for CNV have been rare to date in mosquitoes, but offer immense potential to determine the overall role of CNV as a component of resistance mechanisms

Development and deployment of an improved Anopheles gambiae s.l. field surveillance by adaptive spatial sampling design

Introduction: Accurate assessments of vector occurrence and abundance, particularly in widespread vector-borne diseases such as malaria, are crucial for the efficient deployment of disease surveillance and control interventions. Although previous studies have explored the benefits of adaptive sampling for identifying disease hotspots (mostly through simulations), limited research has been conducted on field surveillance of malaria vectors. Methods: We developed and implemented an adaptive spatial sampling design in southwestern Benin, specifically targeting potential and uncertain Anopheles gambiae hotspots, a major malaria vector in sub-Saharan Africa. The first phase of our proposed design involved delineating ecological zones and employing a proportional lattice with close pairs sampling design to maximize spatial coverage, representativeness of ecological zones, and account for spatial dependence in mosquito counts. In the second phase, we employed a spatial adaptive sampling design focusing on high-risk areas with the greatest uncertainty. Results: The adaptive spatial sampling design resulted in a reduced sample size from the first phase, leading to improved predictions for both out-of-sample and training data. Collections of Anopheles gambiae in high-risk and low-uncertainty areas were nearly tripled compared to those in high-risk and high-uncertainty areas. However, the overall model uncertainty increased. Discussion: While the adaptive sampling design allowed for increased collections of Anopheles gambiae mosquitoes with a reduced sample size, it also led to a general increase in uncertainty, highlighting the potential trade-offs in multi-criteria adaptive sampling designs. It is imperative that future research focuses on understanding these trade-offs to expedite effective malaria control and elimination efforts

Estimation of allele-specific Ace-1 duplication in insecticide-resistant Anopheles mosquitoes from West Africa

Background: Identification of variation in Ace-1 copy number and G119S mutation genotype from samples of Anopheles gambiae and Anopheles coluzzii across West Africa are important diagnostics of carbamate and organophosphate resistance at population and individual levels. The most widespread and economical method, PCR–RFLP, suffers from an inability to discriminate true heterozygotes from heterozygotes with duplication. Methods: In addition to PCR–RFLP, in this study three different molecular techniques were applied on the same mosquito specimens: TaqMan qPCR, qRTPCR and ddPCR. To group heterozygous individuals recorded from the PCR–RFLP analysis into different assumptive genotypes K-means clustering was applied on the Z-scores of data obtained from both the TaqMan and ddPCR methods. The qRTPCR analysis was used for absolute quantification of copy number variation. Results: The results indicate that most heterozygotes are duplicated and that G119S mutation must now be regarded as a complex genotype ranging from primarily single-copy susceptible Glycine homozygotes to balanced and imbalanced heterozygotes, and multiply-amplified resistant Serine allele homozygotes. Whilst qRTPCR-based gene copy analysis suffers from some imprecision, it clearly illustrates differences in copy number among genotype groups identified by TaqMan or ddPCR. Based on TaqMan method properties, and by coupling TaqMan and ddPCR methods simultaneously on the same type of mosquito specimens, it demonstrated that the TaqMan genotype assays associated with the K-means clustering algorithm could provide a useful semi-quantitative estimate method to investigate the level of allele-specific duplication in mosquito populations. Conclusions: Ace-1 gene duplication is evidently far more complex in An. gambiae and An. coluzzii than the better studied mosquito Culex quinquefasciatus, which consequently can no longer be considered an appropriate model for prediction of phenotypic consequences. These require urgent further evaluation in Anopheles. To maintain the sustained effectiveness carbamates and organophosphates as alternative products to pyrethroids for malaria vector control, monitoring of duplicated resistant alleles in natural populations is essential to guide the rational use of these insecticides

Field efficacy of pyrethroid treated plastic sheeting (durable lining) in combination with long lasting insecticidal nets against malaria vectors

BACKGROUND: Insecticide treated plastic sheeting (ITPS), sometimes known as durable lining, has potential as a long-lasting insecticidal surface for malaria vector control when used as lining for interior walls and ceilings inside the home. Against a backdrop of increasing long lasting net (LN) coverage, we examined the effect of combining permethrin-treated plastic sheeting (ITPS) with LNs in Burkina Faso. METHODS: A verandah trap experimental hut trial of ITPS with or without Olyset LN was conducted in the Vallée du Kou near Bobo-Dioulasso, where the two molecular forms of Anopheles gambiae s.s., S (frequency 65%) and M (frequency 35%), occur. The S form is mostly pyrethroid resistant (Fkdr = 92%) owing to the kdr mechanism, and the M form is mostly kdr susceptible (Fkdr = 7%). The treatment arms included ITPS, Olyset, ITPS plus Olyset, ITPS plus untreated net (with or without holes), and untreated control. RESULTS: ITPS was significantly inferior to Olyset LN in terms of mortality (37% vs 63%), blood feeding inhibition (20% vs 81%) and deterrence (0 vs 42%) effects, and hence altogether inferior as a means of personal protection (16% vs 89%). The addition of ITPS to Olyset did not improve mortality (62%), blood feeding inhibition (75%), deterrence (50%) or personal protection (88%) over that of Olyset used alone. Use of untreated nets - both holed and intact - with ITPS provided greater protection from blood-feeding. The intact net/ITPS combination killed more mosquitoes than ITPS on its own. CONCLUSIONS: Although ITPS has a potential role for community control of malaria, at low coverage it is unlikely to be as good as Olyset LNs for household protection. The combination of pyrethroid IRS and pyrethroid LN - as practiced in some countries - is unlikely to be additive except, perhaps, at high levels of IRS coverage. A combination of LN and ITPS treated with an alternative insecticide is likely to be more effective, particularly in areas of pyrethroid resistance

Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine