Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

Insecticides Susceptibility Status of Malaria Vectors in a High Malaria Endemic Tribal District Gadchiroli (Maharashtra) of India

Background and Objective: The current study was undertaken to determine insecticide susceptibility of malaria vectors in various villages of high malaria endemic PHCs of Gadchiroli district of Maharashtra. Methods: Adult malaria vectors were collected from the human dwellings/ cattle sheds of 156 villages of 18 malaria endemic PHCs. Susceptibility tests were carried out for different insecticides against An. culicifacies and An. fluviatilis mosquitoes as per the World Health Organization (WHO) procedure. Cone bioassays were also done to assess the quality and efficacy of indoor residual spray. Results:An. fluviatilis could be collected from 23 villages only and all the populations were fully susceptible to synthetic pyrethroid (deltamethrin) while being tolerant to organophosphorous (malathion). Susceptibility of An. culicifacies from 156 villages indicated that only 3 populations of An. culicifacies were resistant to deltamethrin while 57 populations were fully susceptible and other 96 populations were tolerant to deltamethrin. Resistance was recorded in 25 populations of An. culicifacies against malathion and 30 populations were tolerant to malathion insecticide. Remaining populations of An. fluviatilis and An. culicifacies were highly resistant to organochlorine. Results of cone bioassay revealed the mortality ranged from 32.5-51.1% on cemented and 27.5-43.3% on the mud wall sprayed with lambda cyhalothrin. Conclusion: The current study indicates that resistance has developed to synthetic pyrethroids in the major malaria vector An. culicifacies. Therefore, there is an urgent need for the evaluation of new insecticide molecules for better control of malaria vectors.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of insecticide resistance in the major malaria vector Anopheles coluzzii

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito

Wolbachia infections are a fascinating example of reproductive parasitism with strong potential to combat vector-borne diseases, due to their combined ability to spread in insect populations and block pathogen replication. Though the Wolbachia factors mediating the notable reproductive manipulation cytoplasmic incompatibility (CI) have now been identified as prophage WO genes cifA and cifB, the relative role of these genes is still intensely debated, with different models claiming that CI requires either both factors or cifB alone. Here we investigated whether cifA and cifB are sufficient to induce conditional sterility in the major malaria vector Anopheles gambiae, a species that appears to have limited susceptibility to invasion by Wolbachia. We report that CI can be fully recapitulated in these mosquitoes, and that cifB is sufficient to cause this reproductive manipulation. cifB-induced sterility is fully rescued by high levels of cifA expression in females. Surprisingly, however, when cifA is highly expressed in males alongside cifB, the CI phenotype is attenuated. cifB strongly impairs fertility also when expressed in the female germline, again mitigated by cifA. These data support a system whereby cifB and cifA must be fine-tuned to exercise CI and rescue, respectively, possibly explaining the limited success of Wolbachia at invading Anopheles. Our findings pave the way towards facilitating Wolbachia infections in anopheline vectors, for use in malaria control strategies.

High pyrethroid/DDT resistance in major malaria vector Anopheles coluzzii from Niger-Delta of Nigeria is probably driven by metabolic resistance mechanisms

Entomological surveillance of local malaria vector populations is an important component of vector control and resistance management. In this study, the resistance profile and its possible mechanisms was characterised in a field population of the major malaria vector Anopheles coluzzii from Port Harcourt, the capital of Rivers state, in the Niger-Delta Region of Nigeria. Larvae collected in Port-Harcourt, were reared to adulthood and used for WHO bioassays. The population exhibited high resistance to permethrin, deltamethrin and DDT with mortalities of 6.7% ± 2.4, 37.5% ± 3.2 and 6.3% ± 4.1, respectively, but were fully susceptible to bendiocarb and malathion. Synergist bioassays with piperonylbutoxide (PBO) partially recovered susceptibility, with mortalities increasing to 53% ± 4, indicating probable role of CYP450s in permethrin resistance (χ2 = 29.48, P < 0.0001). Transcriptional profiling revealed five major resistance-associated genes overexpressed in the field samples compared to the fully susceptible laboratory colony, Ngoussou. Highest fold change (FC) was observed with GSTe2 (FC = 3.3 in permethrin exposed and 6.2 in unexposed) and CYP6Z3 (FC = 1.4 in exposed and 4.6 in unexposed). TaqMan genotyping of 32 F0 females detected the 1014F and 1575Y knockdown resistance (kdr) mutations with frequencies of 0.84 and 0.1, respectively, while 1014S mutation was not detected. Sequencing of a fragment of the voltage-gated sodium channel, spanning exon 20 from 13 deltamethrin-resistant and 9 susceptible females revealed only 2 distinct haplotypes with a low haplotype diversity of 0.33. The findings of high pyrethroid resistance but with a significant degree of recovery after PBO synergist assay suggests the need to move to PBO-based nets. This could be complemented with carbamate- or organophosphate-based indoor residual spraying in this area.

Chemical composition and insecticidal activity of Aeollanthus pubescens Benth leaf essential oil on the malaria vector Anopheles gambiae (Diptera: Culicidae)

The use of synthetic insecticides is responsible for many cases of resistance in insects. Therefore, the use of natural molecules of ecological interest with insecticidal properties turns out to be an alternative approach to the use of synthetic insecticides. This study aims at investigating the larvicidal, adulticidal activity and the composition of the essential oil of Aeollanthus pubescens Benth on the major malaria vector Anopheles gambiae.The leaves of Aeollanthus pubescens were collected in the South of the Republic of Benin. Three reference strains of Anopheles gambiae s.s. such as Kisumu, Kiskdr and Acerkis were used. The chemical composition of the essential oil was analysed by gas chromatography coupled to mass spectrometry. Larvae were exposed to the essential oil extract for 24 h. Adult mosquitoes were exposed to the fragment nets coated with the essential oil for 3 min. Larval mortality and adult survivorship were monitored.Fourteen components were identified representing 98.31% of the total of oil. The major components were carvacrol (51.06 %), thymyle acetate (14.01 %) and γ-terpinene (10.60 %). The essential oil has remarkable larvicidal properties with LC50 of 29.26, 22.65, and 28.37 ppm respectively on Kisumu, Acerkis and Kiskdr strains. With the fragment net treated at 165 µg/cm2, the KDT50 of both Acerkis (1.71 s, p < 0.001) and Kiskdr (2.67 s, p < 0.001) individuals were significantly lower than that of Kisumu (3.77 s). The lifespan of the three mosquito strains decreased respectively to one day for Kisumu (p < 0.001), two days for Acerkis (p < 0.001) and three days for Kiskdr (p < 0.001) compared to their control.Our findings show that the Aeollanthus pubescens essential oil is an efficient larvicide and adulticide against malaria vector Anopheles gambiae. This bioinsecticidal activity is a promising discovery for the control of the resistant malaria-transmitting vectors.