Investigating molecular mechanisms of insecticide resistance in the Eastern Democratic Republic of the Congo.

BACKGROUND: Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. METHODS: Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. RESULTS: L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. CONCLUSIONS: This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme

Detection of insecticide resistance markers in Anopheles funestus from the Democratic Republic of the Congo using a targeted amplicon sequencing panel

Vector control strategies have been successful in reducing the number of malaria cases and deaths globally, but the spread of insecticide resistance represents a significant threat to disease control. Insecticide resistance has been reported across Anopheles (An.) vector populations, including species within the An. funestus group. These mosquitoes are responsible for intense malaria transmission across sub-Saharan Africa, including in the Democratic Republic of the Congo (DRC), a country contributing > 12% of global malaria infections and mortality events. To support the continuous efficacy of vector control strategies, it is essential to monitor insecticide resistance using molecular surveillance tools. In this study, we developed an amplicon sequencing (“Amp-seq”) approach targeting An. funestus, and using multiplex PCR, dual index barcoding, and next-generation sequencing for high throughput and low-cost applications. Using our Amp-seq approach, we screened 80 An. funestus field isolates from the DRC across a panel of nine genes with mutations linked to insecticide resistance (ace-1, CYP6P4, CYP6P9a, GSTe2, vgsc, and rdl) and mosquito speciation (cox-1, mtND5, and ITS2). Amongst the 18 non-synonymous mutations detected, was N485I, in the ace-1 gene associated with carbamate resistance. Overall, our panel represents an extendable and much-needed method for the molecular surveillance of insecticide resistance in An. funestus populations

Genomic and microscopic evidence of stable high density and maternally inherited <i>Wolbachia</i> infections in <i>Anopheles</i> mosquitoes

AbstractWolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has been detected within populations of Anopheles (An.) malaria vectors. In the An. gambiae complex, the primary vectors in Sub-Saharan Africa, Wolbachia strains are at low density and infection frequencies in wild populations. PCR-independent evidence is required to determine whether Wolbachia strains are true endosymbionts in Anopheles given most studies to date have used nested-PCR to identify strains. Here we report high-density strains found in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti and maternal transmission was observed. Genome sequencing of both strains obtained genome depths and coverages comparable to other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present indicating these strains possess the capacity to induce the phenotype cytoplasmic incompatibility which allows Wolbachia to spread through populations. The characteristics of these two strains suggest they are ideal candidates for Wolbachia biocontrol strategies in Anopheles.</jats:p

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Background: Wolbachia , a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the Anopheles (An.) genera, but has recently been found in An. gambiae s.l. populations.  As there are numerous Anopheles species that have the capacity to transmit malaria, we analysed a range of species to determine Wolbachia prevalence rates, characterise novel Wolbachia strains and determine any correlation between the presence of Plasmodium , Wolbachia  and the competing endosymbiotic bacterium Asaia . Methods: Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis of samples was undertaken using quantitative PCR, Sanger sequencing, Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial 16S rRNA gene.  Results : Novel Wolbachia strains were discovered in five species: An. coluzzii , An. gambiae s.s., An. arabiensis , An. moucheti and An. species ‘A’, increasing the number of Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with Wolbachia supergroup B strains.  We also provide evidence for resident strain variants within An . species ‘A’.  Wolbachia is the dominant member of the microbiome in An. moucheti and An. species ‘A’, but present at lower densities in An. coluzzii .  Interestingly, no evidence of Wolbachia/Asaia co-infections was seen and Asaia infection densities were also shown to be variable and location dependent.  Conclusions: The important discovery of novel Wolbachia strains in Anopheles provides greater insight into the prevalence of resident Wolbachia strains in diverse malaria vectors.  Novel Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other Anopheles mosquito species, which could be used for population replacement or suppression control strategies

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa.

Background:  Wolbachia, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the  Anopheles (An.) genera, but has recently been found in  An. gambiae s.l. populations in West Africa.  As there are numerous  Anopheles species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine  Wolbachia prevalence rates, characterise novel  Wolbachia strains and determine any correlation between the presence of  Plasmodium,  Wolbachia and the competing bacterium  Asaia. Methods:  Anopheles adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017.  Molecular analysis was undertaken using quantitative PCR, Sanger sequencing,  Wolbachia multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial  16S rRNA gene.  Results: Novel  Wolbachia strains were discovered in five species:  An. coluzzii,  An. gambiae s.s.,  An. arabiensis,  An. moucheti and  An. species A, increasing the number of  Anopheles species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with  Wolbachia supergroup B strains.  We also provide evidence for resident strain variants within  An. species A.  Wolbachia is the dominant member of the microbiome in  An. moucheti and  An. species A but present at lower densities in  An. coluzzii.  Interestingly, no evidence of  Wolbachia/Asaia co-infections was seen and  Asaia infection densities were shown to be variable and location dependent.  Conclusions: The important discovery of novel  Wolbachia strains in  Anopheles provides greater insight into the prevalence of resident  Wolbachia strains in diverse malaria vectors.  Novel  Wolbachia strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other  Anopheles mosquito species, which could be used for population replacement or suppression control strategies

Identification of main malaria vectors and their insecticide resistance profile in internally displaced and indigenous communities in Eastern Democratic Republic of the Congo (DRC)

Contains fulltext : 228539.pdf (publisher's version ) (Open Access

Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria