Genetic surveillance of Plasmodium-Anopheles compatibility markers during Anopheles stephensi associated malaria outbreak.

Background: Despite a previous decline in malaria in Ethiopia, an outbreak in Dire Dawa in 2022 implicated the invasive vector Anopheles stephensi as responsible. The efficient transmission of Plasmodium by invasive An. stephensi raises questions about the molecular basis of compatibility between parasite and vector, and the origin of the Plasmodium being transmitted. The Plasmodium P47 gene is involved in parasite-vector interactions in the mosquito, and along with the corresponding mosquito P47 receptor (P47Rec), can be critical in the establishment of Plasmodium infections in anophelines.

Methods: Herein, we analyzed P47 and P47Rec sequences to determine the origin of Plasmodium detected in An. stephensi during the outbreak and evaluate markers of compatibility. This was completed using polymerase chain reactions and Sanger sequencing.

Results: Of 160 mosquitoes screened, 6.21% of the mosquitoes screened were positive for P. falciparum DNA and 4.37% were positive for P. vivax DNA. Analysis of geographically informative SNPs at positions 707 and 725 in Pfs47 revealed that these P. falciparum strains only exhibit the African haplotype. Minimum spanning network (MSN) analysis revealed connectivity between Pfs47 in Dire Dawa and Pfs47 sequences in Africa, further supporting that these Plasmodium strains are of African origin. We also evaluated the connectivity between Pv47 in this study and African and Asian Pv47 using MSN analysis. Pv47 in both continents displayed shared haplotypes, suggesting little differentiation between the African and Asian strains in P. vivax. Lastly, we identified a single amino acid change in the P47Rec within An. stephensi, which could act as a marker for the propensity of An. stephensi populations to outbreak.

Conclusions: Overall, these results provide evidence of African P. falciparum in invasive An. stephensi and identify P47Rec as a potential marker, which could be applied as a molecular diagnostic for propensity for an outbreak. The relatively high frequencies of Plasmodium parasites observed in An. stephensi may suggest that this mosquito species contributed to the malaria outbreak. Our findings lay the groundwork for further research into the interactions between the invasive mosquito species An. stephensi and African Plasmodium strains, with the goal of predicting future outbreaks.