The invasion of Anopheles stephensi in Africa warrants investigation of neighboring countries. In this study, genetic analysis was applied to determine the status of An. stephensi in southern Yemen. Cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) were sequenced in An. stephensi collected in Dar Sa'ad (Aden City), Tuban, Rodoom, Al Mukalla, and Sayhut, and phylogenetic analysis confirmed An. stephensi identity. Our analyses revealed that the ITS2 sequences were identical in all An. stephensi, while COI analysis revealed two haplotypes, one previously reported in northern Horn of Africa and one identified in this study for the first time. Overall, these findings revealed low levels of mitochondrial DNA diversity, which is consistent with a more recent population introduction in parts of southern Yemen relative to the Horn of Africa. Further, whole genomic analysis is needed to elucidate the original connection with invasive populations of An. stephensi in the Horn of Africa.

Anopheles stephensi invasion in Ethiopia poses a risk of increased malaria disease burden in the region. Thus, understanding the insecticide resistance profile and population structure of the recently detected An. stephensi population in Fiq, Ethiopia, is critical to inform vector control to stop the spread of this invasive malaria species in the country. Following entomological surveillance for An. stephensi in Fiq, Somali region, Ethiopia, we confirmed the presence of An. stephensi morphologically and molecularly in Fiq. Characterization of larval habitats and insecticide susceptibility tests revealed that Fiq An. stephensi is most often found in artificial containers and is resistant to most adult insecticides tested (organophosphates, carbamates, pyrethroids) except for pirimiphos-methyl and PBO-pyrethroids. However, the immature larval stage was susceptible to temephos. Further comparative genomic analyses with previous An. stephensi populations from Ethiopia using 1704 biallelic SNPs revealed genetic relatedness between Fiq An. stephensi and east-central Ethiopia An. stephensi populations, particularly Jigjiga An. stephensi. Our findings of the insecticide resistance profile, coupled with the likely source population of Fiq An. stephensi, can inform vector control strategies against this malaria vector in Fiq and Jigjiga to limit further spread out of these two locations to other parts of the country and continent.

Anopheles stephensi is a malaria vector that has been recently introduced into East Africa, where it threatens to increase malaria disease burden. The use of insecticides, especially pyrethroids, is still one of the primary malaria vector control strategies worldwide. The knockdown resistance (kdr) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel (vgsc) is one of the main molecular mechanisms of pyrethroid resistance in Anopheles. Extensive pyrethroid resistance in An. stephensi has been previously reported in Ethiopia. Thus, it is important to determine whether or not the kdr mutation is present in An. stephensi populations in Ethiopia to inform vector control strategies. In the present study, the kdr locus was analyzed in An. stephensi collected from ten urban sites (Awash Sebat Kilo, Bati, Dire Dawa, Degehabur, Erer Gota, Godey, Gewane, Jigjiga, Semera, and Kebridehar) situated in Somali, Afar, and Amhara regions, and Dire Dawa Administrative City, to evaluate the frequency and evolution of kdr mutations and the association of the mutation with permethrin resistance phenotypes. Permethrin is one of the pyrethroid insecticides used for vector control in eastern Ethiopia. DNA extractions were performed on adult mosquitoes from CDC light trap collections and those raised from larval and pupal collections. PCR and targeted sequencing were used to analyze the IIS6 transmembrane segment of the vgsc gene. Of 159 An. stephensi specimens analyzed from the population survey, nine (5.7%) carried the kdr mutation (L1014F). An. stephensi with kdr mutations were only observed from Bati, Degehabur, Dire Dawa, Gewane, and Semera. We further randomly selected twenty resistant and twenty susceptible An. stephensi mosquitoes from Dire Dawa post-exposure to permethrin and investigated the role of kdr in pyrethroid resistance by comparing the vgsc gene in the two populations. We found no kdr mutations in the permethrin-resistant mosquitoes. Population genetic analysis of the sequences, including neighboring introns, revealed limited evidence of non-neutral evolution (e.g., selection) at this locus. The low kdr mutation frequency detected and the lack of kdr mutation in the permethrin-resistant mosquitoes suggest the existence of other molecular mechanisms of pyrethroid resistance in eastern Ethiopian An. stephensi.

BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA to improve predictions of future spread. We investigated the genetic diversity of An. stephensi in eastern Ethiopia, where detection suggests a range expansion into this region, in order to understand the history of this invasive population. METHODS: We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Population genetic, phylogenetic, and minimum spanning network analyses were conducted for Ethiopian sequences. Molecular identification of blood meal sources was also performed using universal vertebrate CytB sequencing. RESULTS: Six An. stephensi COI-CytB haplotypes were observed, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. We observed population differentiation, with the highest differentiation between the northeastern sites compared to central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) and the southeastern sites. Phylogenetic and network analysis revealed that the HOA An. stephensi are more genetically similar to An. stephensi from southern Asia than from the Arabian Peninsula. Finally, molecular blood meal analysis revealed evidence of feeding on cows, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. CONCLUSION: We show that An. stephensi is genetically diverse in Ethiopia and with evidence of geographical structure. Variation in the level of diversity supports the hypothesis for a more recent introduction of An. stephensi into southeastern Ethiopia relative to the northeastern region. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the need for additional investigation into the potential for livestock movement to play a role in vector spread in this region.