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.

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.

Background In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of the disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance. Methods Anopheles gambiae sensu lato mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. Five hundred and sixty-three mosquitoes underwent molecular characterization of vector species, presence of target site mutations (L1014F kdr , N1575Y and G119S Ace - 1 ), Plasmodium falciparum infection, and relative expression of three metabolic genes ( CYP6M2 , CYP6P3 and GSTD3 ). Results In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) of mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy–Weinberg equilibrium. The G119S Ace - 1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2 , CYP6P3 and GSTD3 , were found to be overexpressed in wild resistant and susceptible An. gambiae sensu stricto populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah. Conclusions Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as t...

Insecticide resistance is one of the primary threats to the recent gains in malaria control. This is especially true in Guinea, where long-lasting insecticidal nets are currently the primary vector control intervention. To better inform the national malaria control program on the current status of insecticide resistance in Guinea, resistance bioassays were conducted, using Anopheles gambiae s.l. Giles, in three sites. Molecular analyses were also done on An. gambiae s.l. to determine the species and find whether the target-site mutations kdr and Ace1R were present. Susceptibility tests revealed resistance to DDT and pyrethroids, although mosquitoes were susceptible to deltamethrin in two of the three sites tested. Mosquitoes were susceptible to bendiocarb, except in Kissidougou, Guinea. The kdr-west mutation was widespread and the frequency was 60% or more in all sites. However, the Ace1R mutation was present in low levels. Insecticide susceptibility should continue to be monitored in Guinea to ensure insecticide-based vector control methods remain effective.

Anopheles (An.) mosquitoes contain bacteria that can influence Plasmodium parasites. Wolbachia, a common insect endosymbiont, has historically been considered absent from Anopheles but has recently been found in An. gambiae populations. Here, we assessed a range of Anopheles species from five malaria-endemic countries for Wolbachia and Plasmodium infection. Strikingly, we found Wolbachia infections in 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 by this endosymbiont. Molecular analysis suggests the presence of phylogenetically diverse novel strains, while qPCR and 16S rRNA sequencing indicates that Wolbachia is the dominant member of the microbiota in An. moucheti and An. species ‘A’. We found no evidence of Wolbachia/Asaia co-infections, and presence of these endosymbionts did not have significant effects on malaria prevalence. We discuss the importance of novel Wolbachia strains in Anopheles and potential implications for disease control.