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

Abstract

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.

Keywords: Anopheles stephensi; P47Rec; Pfs47; Pv47; Pvs47; Malaria outbreak; Vector-parasite interaction.

Fig. 1

Minimum spanning network of Pfs47 sequences from NCBI Genbank, MalariaGen database, and Dire Dawa. The number of sequences is represented by the size of circle, and single point mutations between haplotypes are represented by hash marks. Samples produced in this study are colored in bright pink. Countries where invasive An. stephensi has been detected are shown in purple, all other countries in Africa are shown in blue, countries in Asia are shown in green, countries in South America are shown in orange, and Papua New Guinea is shown in brown. Dire Dawa shares a major haplotype with other countries in Africa (Hap11 and Hap12) and has some unique haplotypes as well (Hap25, Hap17, Hap18, and Hap5). Hap13 is connected to a major African haplotype (Hap12) and the South American and Asian haplotypes (Hap14 and Hap8, respectively), and notably contains sequences from Sudan, India, and Papua New Guinea. Ghana shares a haplotype with the major Asian haplotype (Hap8). Thailand shares a haplotype with African sequences (Hap24)

Fig. 2

Minimum spanning network of Pv47 sequences from NCBI and Dire Dawa. The number of sequences is represented by the size of circle, and single point mutations between haplotypes are represented by hash marks. Samples produced in this study are shown in bright pink. Countries where invasive An. stephensi has been detected are shown in purple, all other countries in Africa are shown in blue, countries in Asia are shown in green, countries in South America are shown in orange, and Vanuatu is shown in brown. Hap3 represents most sequences from Africa and Asia, although Hap8 is also a major Asian haplotype that is also shared with the positive control from Ethiopia. South American sequences are present in Hap6 and Hap7. Samples from Dire Dawa share similarity with Hap3 but have two unique haplotypes (Hap11 and Hap4)

Fig. 3

Allele frequency of the amino acids histidine or glutamine at position 53 in different samples and reference genomes of An. stephensi. Invasive populations of An. stephensi are outlined in purple, native in gray, and reference in black. Allele frequency of H is denoted by red in the pie charts, and the frequency of Q is denoted by blue