Mugenzi, L. J. | Search bioRxiv

bioRxiv2024

Contrasting patterns of Asaia sp association with Plasmodium falciparum between field collected Anopheles gambiae and Anopheles coluzzii from Cameroon

The presence of bacteria of the genus Asaia in mosquitoes makes them suitable candidates for malaria paratrangenic control. To better understand whether this bacterium could be used for malaria control, we investigated and quantified Asaia influence in An.Lgambiae sl populations naturally infected by the malaria parasite Plasmodium falciparum. Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during the dry and wet seasons. DNA was extracted from the whole individual mosquitoes and Asaia was quantified using real-time Q-PCR by amplification of 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in same mosquitoes. The density of Asaia was quantified in 864 mosquitoes. This included 439 An. gambiae collected from Bankeng and 425 An. coluzzii collected from Gounougou. The prevalence of Asaia in An. gambiae and An. coluzzii was 88.3% and 80.9% respectively, with An. gambiae were more heavily infected by Asaia than An. coluzzii. There was also a significant difference between the infection densities of Asaia per season in the two localities. We also had a significant difference between the infection densities of Asaia according to Plasmodium infection status in the two localities. However, no correlation was observed between the amount of Asaia and P. falciparum infection. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii. Further studies as experimental infection and innate immune response are required to better investigate the correlation between Anopheles mosquitoes, Asaia and Plasmodium. ImportanceMalaria control relies mainly on insecticide-based tools. However, the effectiveness of these tools is threatened by the widespread insecticide resistance in malaria vectors highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated and quantified Asaia influence in An.Lgambiae sl populations naturally infected by the malaria parasite Plasmodium falciparum. DNA was extracted from the whole individual mosquitoes collected from two localities, and then Asaia was quantified using real-time Q-PCR by amplification of 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in same mosquitoes and correlated Plasmodium infection them with the presence of Asaia infection. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii.

microbiology↗

bioRxiv2021

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted deep sequencing of candidate resistance genes and genomic loci detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNP was in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where CYP9K1 P450 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited a drastic reduction of genetic diversity in Uganda, in contrast to other locations, highlighting an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid mutation located between the meander and cysteine pocket of CYP9K1 was detected in all Ugandan mosquitoes. This study sheds further light on the complex evolution of metabolic resistance in a major malaria vector, by adding further resistance genes and variants that can be used to design field applicable markers to better track this resistance Africa-wide. Author SummaryMetabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used several DNA based approach to associate genomic differences between resistant and susceptible mosquitoes from several field and laboratory populations of the malaria vector Anopheles funestus. We followed-up our genomic analyses with functional validation of a candidate resistance gene in East Africa. This gene (CYP9K1) is a member of the cytochrome P450 gene-family that helps to metabolise, and thereby detoxify, pyrethroid insecticides. We show that this gene is a major driver of resistance to a specific sub-class of pyrethroid insecticides only, with moderate to no effects on other insecticides used against Anopheles funestus. We were able to link resistance in this gene to a mutation that changes the amino acid glycine to alanine that may impact how the protein-product of this gene binds to target insecticides. In addition to demonstrating the biochemical specificity of an evolutionary response, we have broadened the available pool of genes can be used to monitor the spread of insecticide resistance in this species.

genomics↗