This study complements the data on the distribution and abundance of invasive An. stephensi in Ethiopia. Entomological surveillance in 26 urban centers between 2021 and 2023 revealed that An. arabiensis was the predominant species and accounted for 79% of the collections, followed by An. stephensi, which contributed to 8% of the total catches. The relative abundance of adult An. stephensi was greater than An. arabiensis in Babile, Kebri Dehar, Danan, and Modjo. Among those sites, Modjo is located along the main ground transportation route or corridor that connects Ethiopia to Djibouti [36]. Generally, adult An. stephensi collections were low (0.15 catches/trap), and most of the immature collections were from artificial aquatic habitats.
Since the first detection of An. stephensi in eastern Ethiopia in 2016, new positive sites have been identified in subsequent surveys [36, 37, 57]. In line with this, we detected An. stephensi in western Ethiopia (Assosa) in an area bordering Sudan. We also found An. stephensi at all previously reported sites and at new sites along its purported invasion route. However, the site positivity of An. stephensi was relatively low (8/26 sampled sites) compared to that in other studies. One study that covered ten sites in eastern Ethiopia reported the existence of An. stephensi at all sites [36]. Similarly, sampling at 21 sites between 2018 and 2020 revealed 61.9% positivity for An. stephensi [37]. Another study conducted by the PMI Vector Link Ethiopia project showed the presence of An. stephensi in 16 urban settings, of which 9 (56.3%) sites were newly positive for An. stephensi [58]. This difference in collections might be attributed to the selection of study sites; most of the collection points in the previous studies were purposefully included to detect An. stephensi. The current study followed substantial random element steps in the selection of study sites and collection points within sites, which has the advantage of providing unbiased distribution estimates but reducing the probability of detection. In addition, some of our study sites were located far from major transportation corridors [59], which are considered the main invasion routes.
In line with the findings of other studies [36, 38], An. stephensi was more readily detectable as immature than as adults in most of positive sites. The highest proportion of An. stephensi collections (85.7%) were obtained as immatures (larvae and pupae) in aquatic habitats. A range of aquatic habitats were positive; for example, in Dubti, immature An. stephensi were detected in both artificial habitats (water tanks, barrels, buckets, tires) and natural habitats (ponds, streams, swamps, and marshes), in line with previous reports, An. stephensi can breed in various habitats with different salinity levels and water qualities [60]. In Modjo, Danan, Kebri Dehar, and Babile, immature An. stephensi were detected only in artificial habitats. This variation highlights how larval source management of An. stephensi, which has been recommended by the WHO [61] and is being implemented by the PMI VectorLink and others, will be more complex than simply targeting container habitats.
In the current study, An. arabiensis was the most abundant species at 20/26 sites, which is in line with the findings of other studies showing that this species is still the predominant malaria vector in the different ecoepidemiological settings of Ethiopia [19, 24, 62]. Even though An. arabiensis is considered less adapted to urban ecology [63], and our findings suggest that it is probably primarily responsible for malaria transmission in urban centers in Ethiopia. The other Anopheles species collected in this study were An. pharoensis, An. coustani, An. funestus, An. tenebrosus, and An. rufipes, which together accounted for 12.8% of the total adult Anopheles catches. Of these species, An. pharoensis and An. funestus were reported as secondary or suspected malaria vectors in Ethiopia [64]. We detected An. pharoensis infected with P. falciparum or P. vivax at one and five of our study sites, respectively. While An. coustani and An. funestus were detected with P. vivax sporozoites across four and three of the study sites, respectively.
The level of household exposure for An. stephensi was heterogeneous across the study sites, with household positivity for adults and immatures ranging from 18% in Metehara to 30% in Danan. The level of household exposure for adult An. stephensi was highest in the region where invasion was first reported (Danan) and lower in more central parts of the country (Awash Sebat Kilo and Metehara). A similar trend was observed for household exposure to the immature stage of An. stephensi.
Our findings revealed that An. stephensi prefers non-human vertebrate hosts for blood meal. The most prevalent blood meal among An. stephensi detected with sources of blood was cattle (69.2%), followed by goats (32.3%). This finding is consistent with previous studies in Ethiopia [37, 38] and India [65], which showed that most An. stephensi fed on livestock. One-third of An. stephensi were fed on unidentified blood meal sources, which might be due to a lack of host antibodies or primers for blood meal analysis. It is noteworthy that at some of the study sites, especially in eastern Ethiopia, the readily available animals were camels, where most of the tested An. stephensi were collected. Despite the relatively high non-human vertebrate host blood meal indices, 24.6% of An. stephensi were found with human blood, including mixed blood meal sources. The blood meal source of vectors might be affected by multiple factors, including host availability and proximity. This might explain why 76.9% of the An. stephensi and 56.4% of An. arabiensis were fed on a single blood meal source, either an animal or a human host.
Of the 194 screened An. stephensi, none were detected with Plasmodium parasites. This finding is similar to the findings of another study in which none of the tested An. stephensi was positive for Plasmodium [36]. However, another study conducted in 2019 in Awash Sebat Kilo reported an infection rate of 2.8% for P. vivax and 1.4% for P. falciparum from the homogenate of whole mosquitoes [38]. Findings from another study in which the head and thoraxes was used to detect Plasmodium by ELISA, revealed that the infection rate was 0.5% in Dire Dawa and 0.3% in Kebri Dehar for P. vivax [37]. The most recent study, from Dire Dawa, which implicated An. stephensi in an outbreak, detected a P. falciparum sporozoite rate of 1.2% [39]. Nonetheless, our study might be limited in its ability to elucidate An. stephensi sporozoite infection, as a large proportion of the adult catches (91/146) were from a single aquatic site via Prokopack aspirator.
There were some limitations to our study. The limited number of An. stephensi adults caught indicates the need for further studies into more efficient trapping methods. We employed both PCR and ELISA for detecting blood meal sources and Plasmodium infection in Anopheles mosquitoes. This might have limited the direct comparability of our findings across study sites. The collection round did not directly coincide with the malaria transmission season in Ethiopia, and some of sites were excluded because of civil unrest.