First report of Anopheles stephensi in Southern Ethiopia

Background Anopheles stephensi is an emerging exotic invasive urban vector of malaria in East Africa. The World Health Organization recently announced an initiative to take concerted actions to limit this vector’s expansion by strengthening surveillance and control in invaded and potentially receptive territories in Africa. This study sought to determine the geographic distribution of An. stephensi in southern Ethiopia. Methods A targeted entomological survey, both larvae and adult, was conducted in Hawassa city, Southern Ethiopia between November 2022 and February 2023. Anopheles Larvae were reared to adults for species identification. CDC light traps and BG Pro traps were used overnight both indoor and outdoor at selected houses to collect adult mosquitoes in the study area. Prokopack Aspirator was employed to sample indoor resting mosquitoes in the morning. Adults of An. stephensi was identified using morphological keys, and then confirmed by PCR. Results Larvae of An. stephensi were found in 28 (16.6%) of the 169 potential mosquito breeding sites surveyed. Out of 548 adult female Anopheles mosquitoes reared from larvae, 234 (42.7%) were identified to be An. stephensi morphologically. A total of 449 female anophelines were caught, of which 53 (12.0%) were An. stephensi. Other anopheline species collected in the study area included An. gambiae (s.l.), An. pharoensis, An. coustani, and An. demeilloni. Conclusion The study, for the first time, confirmed the presence of An. stephensi in southern Ethiopia. The presence of both larval and adult stages of this mosquito attest that this species established a sympatric colonization with native vector species such as An. gambiae (s.l.) in Southern Ethiopia. The findings warrant further investigation on the ecology, behavior, population genetics, and role of An. stephensi in malaria transmission in Ethiopia.


Background
Anopheles stephensi is the principal malaria vector of urban environments in Southeast Asia, the Middle East, and the Arabian Peninsula [1]. Since its initial discovery in Djibouti in 2012 [2], this vector species has been found spreading to eastern Ethiopia [3,4], Sudan [5], Somalia [1], and Kenya [6].
Its adaptation to urban environment [7] coupled with the rapid spread of An. stephensi in Eastern Africa poses serious challenges for the control and elimination of malaria in African's rapidly urbanizing nations. Malaria outbreak has been documented following its recent colonization of the Horn of Africa region [8] and a 40-fold increase in urban malaria incidence reported in Djibouti over the last decade [9].
According to geospatial modeling studies, many cities in sub-Saharan Africa have environmental conditions favorable for An. stephensi to proliferate, putting an additional 126 million Africans at risk of contracting malaria if the spread of An. stephensi is not curbed [10]. Another modeling study predicted that if a tailor-made An. stephensi interventions are not implemented, the number of malaria cases in Ethiopia might rise by 50% per a year [11].
Determining the geographic range of An. stephensi helps design tailored vector interventions. Given the importance of An. stephensi to transmit urban malaria and its potential effects on public health in Africa, the World Health Organization (WHO) recently launched an initiative to take coordinated actions to limit the vector's spread by improving An. stephensi surveillance and control in Africa [1]. While the spread of An. stephensi has recently been documented in several localities in Eastern, north eastern, and central Ethiopia [3,4], its occurrence and distribution in Southern Ethiopia is unknown. Thus, in response to WHO call, we conducted an entomological survey to detect and determine the geographic distribution of An. stephensi in Hawassa city, Southern Ethiopia.

Study sites
Entomological survey was conducted from November 2022 to February 2023 in Hawassa City in Southern Ethiopia (Fig. 1). Hawassa City is the largest city located in the middle of the Great Ethiopian Rift Valley and is the capita of Sidama Regional State. It is located at the elevation of 1,708 meters (5,604 ft) above sea level. The City's population was 502,980 in 2022 [12]. Being situated at the shore of Lake Hawassa, the city has seen rapid growth in recent years as it is destined to be one of Ethiopia's industrialhub in Southern Ethiopia. The City constantly sees new construction sites following its swift industrial boom in recent years. The average annual temperature and precipitation in the areas is about 21°C and 961 mm, respectively. A shorter rainy season occurs between March and May followed by a longer wet season between July and October. Malaria is seasonal in the area and the main malaria transmission period lies between September and December [12].
Malaria pro le review Laboratory con rmed malaria data was obtained from Hawassa City Health Department. The malaria morbidity data, which was sorted by age, sex, parasite species and residence for the previous nine years was collected and analyzed, and the annual transmission pattern was established.

Mosquito larval survey and rearing
Any water collections were surveyed for mosquito larvae and pupae between November 2022 and February 2023. To increase the likelihood of discovering An. stephensi, target sampling was conducted. Mosquito larvae and pupae of were dipped from likely larval breeding habitats including man-made water containers, freshwater pools, lake margins, discarded tires, plastic containers and concrete water collection tankers at construction sites. Dipping was done in accordance with larval search strategies recommended by WHO [13] . The larvae and pupae were brought in jars to the environmental lab at Hawassa University, where they were placed in trays and raised to adult in preparation for morphological identi cation. Each enamel tray containing larvae was labeled by habitat type from where the larvae were obtained in order to identify the species after adult emergence. The larvae were allowed to develop in the water that was drawn from the eld in order to maintain the same aquatic environment. The pupae were sorted and transferred with pipettes from the enamel trays to beakers with modest volumes of water, then kept inside cages. A dissecting microscope was used to identify emerging adults to the species using identi cation key [14]. All identi ed specimens were preserved individually in Eppendorf tubes for further analysis.

Adult mosquito survey
Three different types of traps were used to collect adult mosquitoes: CDC Light Traps (Model: John W. Hock CDC Light Trap 512, USA); Bioagents (BG-pro) Traps with lure; and Prokopack Aspirator (John W. Hock 1418, USA).
Collection with CDC Light Trap and BG-pro were made overnight from 18:00 to 06:00 h. A trap was suspended 1.5 meters above the ground in close proximity to a sleeping area where the people are protected by LLINs. Prokopack Aspirator was employed to sample indoor resting mosquitoes in the morning from 6:30 h to 8:00 h in 60 houses.
All collected adult mosquitoes were brought to the lab for species identi cation. Mosquitoes were killed by placing them in a refrigerator. The specimens were then sorted into culicines and Anopheles. Culicine were counted, recorded, and discarded. All anophelines were further sorted out to species using morphological key [14].

Molecular identi cation of An. stephensi
A subset of the morphologically identi ed An. stephensi specimens were molecularly analyzed in order to con rm the species. DNA was extracted from single leg using the Chelex method [15] with modi cation. Two methods were used to identify the species: (i) PCR endpoint assay using the internal transcribed spacer 2 (ITS2) locus; and (ii) sequencing portions of cytochrome c oxidase subunit 1 (cox1) and cytochrome B gene (cytb) loci. ITS2 endpoint assay was performed as previously described using the primers 5.8SB (5′-ATG CTT AAA TTT AGG GGG TAG TC-3′) and 28SC (5′-GTC TCG CGA CTG CAA CTG-3′) and the following modi cations: nal reagent concentrations and components were 0.5 μM for each primer; 1× DreamTaq Green Master Mix (Thermo Fisher Scienti c); and water for a total reaction volume of 17 μl. PCR reaction conditions were set as denaturation at 95 °C for 3 min, 35 cycles of 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and a nal step at 72 °C for 6 min. Anopheles stephensi specimens were identi ed by visualization of 522-bp band with gel electrophoresis; non-An. stephensi specimens do not amplify and no band was present [16]. Portions of the cox1 and cytb loci were also ampli ed for sequencing using previously detailed methods [17]. PCR products were puri ed and sequenced using Sanger technology by Genewiz Inc (South Plain eld, NJ). Sequences were cleaned and analyzed using CodonCode (CodonCode Corporation, Centerville, MA, USA). Next, cox1 and cytb sequences from An. stephensi were submitted as queries to the National Center for Biotechnology Information's (NCBI) Basic Local Alignment Search Tool (BLAST) [18] against the nucleotide collection in NCBI's GenBank. A threshold limit of 98% sequence similarity for cox1 was used to classify sequences into species [19].

Malaria morbidity
Over the last nine years, 128,946 malaria cases were reported in the study area. Plasmodium falciparum and P. vivax were prevalent in the area and nearly had identical proportions with 66,570 (51.6%) and 62,376 (48.4%), respectively. According to the data, malaria transmission was dropping for three years in a row, from 2014 to 2016. However, from 2017 to 2019, there was slight increment. Then, after a drop in year 2020, number of malaria cases skyrocketed in 2022 (Fig. 2).

Molecular identi cation of An. stephensi
Of the 50 morphologically identi ed An. stephensi specimens analyzed, ITS2 PCR results were obtained from all of them. We compared the morphological identi cation to the ITS2 PCR endpoint assay results.
With the PCR endpoint assay, 48 (96%) specimens were con rmed as An. stephensi. Only two of the 50 (4.0%) morphologically identi ed An. stephensi were not con rmed with the PCR endpoint assay. Sanger sequencing was performed for 30 con rmed and 2 not con rmed specimens. Of the 30 An. stephensi, 11 were found to be cox1 haplotype 2 (GenBank accession OQ865406) and cytb haplotype 2 (GenBank accession OQ863377), while 19 were cox1 haplotype 3 (GenBank accession OQ865407) and cytb haplotype 1 (GenBank accession OQ863376). The two specimens not con rmed by ITS2 PCR were identi ed as An. arabiensis by cox1 sequencing.

Discussion
The recent colonization of An. stephensi in Eastern Africa has called for additional molecular surveillance to delineate the expansion of this new species in Africa [3,4]. To the best of our knowledge, this is the rst report to con rm the presence of Anopheles stephensi in Southern Ethiopia. Recent increases in malaria transmission in this region coincided with the expansion of this species in Ethiopia.
Such rapid spread of An. stephensi to many areas of Ethiopia triggers the question of whether it is truly new arrival or whether it has been present for a while but gone unnoticed. Several hypotheses have been forwarded by the scholars, one of which is that An. stephensi was recently brought to Ethiopia from neighboring country Djibouti which Ethiopia uses as a major good importation route [10]. According to the present data, the Ethiopian isolate is most closely related to an isolate from Pakistan [4]. The second hypothesis involved An. stephensi being present in Ethiopia for a long period yet gone unnoticed. Provided the existing gaps in molecular vector surveillance, it is necessary to carefully re-examine the trend of entomological surveillance practices that entirely base on morphological keys for species identi cation. The morphological similarity of An. stephensi to other Anopheles species, such as An. arabiensis as documented in this study, plus the infrequent utilization and cost of molecular methods for identi cation, such as PCR and sequencing, could possibly explain why the species have been overlooked [4,20]. This study was carried out in Southern Ethiopia, which is far from the location where An. stephensi colonization was initially recorded in the country [4], suggesting that An. stephensi colonization is still ongoing. This nding underscores the critical need to expand molecular vector surveillance in all malaria-prone areas in order to map An. stephensi's geographic distribution in the country.
Water tanks at construction site, concrete water cisterns, and concrete water collection boxes for carwashes were the frequent habitats found to harbor An. stephensi in the study area. All these breeding sites were man-made, produced as a result of urbanization. Hawassa is Ethiopia's major cities, destined to be an industrial zone and rapidly expanding in recent years. Massive development projects are currently undergone, opening the door for the proliferation of new An. stephensi breeding environments. Building strong collaborations across many sectors, including as health, trade and industry, housing agencies, education, and municipalities, is critical to achieve successful vector control responses in such metropolitan contexts. Previous studies in several countries reported that arti cial containers are an appropriate breeding site for An. stephensi [3,4,21,22]. As the World Health Organization recommends, an integrated vector control strategy should be promoted in the area [1].
Discovering An. stephensi in Hawassa has important public health implications. A coincidental observation during the surveillance was the city's rising malaria cases after a decade-long decline. Plasmodium vivax and P. falciparum are both actively transmitted in the study area. Due to its vectorial capacity to spread both P. falciparum and P. vivax [7], the spread of An. stephensi might been contributing to increasing malaria transmission in the Southern Ethiopia. Studies have indicated that malaria incidence increased following the establishment of An. stephensi in eastern Ethiopia and Djibouti [8,9]. Further research is required to determine the relative contribution of An. stephensi to the city's rising malaria incidence because it coexists alongside native vector species such as An. arabiensis, An. coustani, and An. pharoensis. The survey result suggests the need for further study to elucidate the ecology, behavior, and population genetics of this invasive species. Better understanding on the extent of its distribution and its role in malaria transmission helps develop tailor-made vector intervention strategies in urban settings.

Conclusion
Anopheles stephensi coexists with native vector species in Southern Ethiopia. Further research is needed to determine its relative role in malaria transmission in the region. Heath authorities need to revise the existing vector control strategies to target Anopheles stephensi alongside the native malaria vector species.

Figure 1
Map of the study site and Anopheles stephensi larval habitat distribution