The introduction and rapid geographic range expansion of the Asian urban malaria vector Anopheles stephensi (Liston) in African cities has created concern among international health organizations and in the countries impacted (WHO 2022). Since it was first detected in Djibouti in 2012, An. stephensi has spread to Ethiopia, Somalia, Nigeria, Kenya, and Ghana (WHO 2022), with modeling predicting suitable environmental conditions throughout tropical African cities (Sinka et al. 2020, Whittaker et al. 2023). The World Health Organization (WHO) considers the spread of An. stephensi, an efficient and competent vector for both Plasmodium falciparum (Welch) and P. vivax (Grassi and Feletti), to be a major potential threat to malaria control and elimination in Africa (WHO 2022).

The WHO has launched a global initiative to stop the spread and to support an effective response to An. stephensi in Africa (WHO 2022). Since the alert, WHO has urged countries, especially those in and around the Horn of Africa, to take immediate action enhancing and expanding surveillance and control activities with the aim of eliminating this species from the invaded areas (WHO 2022). Larval source management to control An. stephensi is one of the key recommended activities and includes the treatment with WHO prequalified chemical or biological larvicides of those breeding sites that cannot be removed or modified immediately (WHO 2005). Evidence from Ethiopia indicates that An. stephensi, while resistant to adulticide insecticides such as pyrethroids, carbamates, organophosphates, and organochlorines (Balkew et al. 2020, 2021; Yared et al. 2020), are susceptible to the organophosphate temephos and microbial larvicide (Bacillus thuringiensis var. israelensis, de Barjac) (Teshome et al. 2023).

On March 6–14, 2023, we collected larvae of An. stephensi from Jigjiga city (9°21ʹ0.5″N; 42°48ʹ01.8″E) during the peak of the dry season to conduct larvicide efficacy studies. Jigjiga (population 763,509) is the capital of the Somali Region of Ethiopia and a rapidly growing urban center (Fig. 1). Although not endemic for malaria, Jigjiga has reported the presence of An. stephensi since 2018 (Balkew et al. 2021). For this study we identified sites with high potential to contain water with An. stephensi larvae. From all habitats surveyed (metal/plastic cisterns, 200 liter drums, buckets, truck tires) temporary water storage reservoirs located at houses under construction and/or reserved for production of cement blocks were found positive for Anopheles sp. larvae by regular dipping, supplemented with fish net collections (Fig. 1). Such construction pits consisted of a hole in the dirt, covered with orange plastic tarp (Fig 1) with a capacity of 8–40 m³ and used to store water for construction purposes. Prior experience of our team indicates that these sites are also positive for An. stephensi in the rainy season in other Ethiopian cities (Balkew et al. 2020, 2021).

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For our study, immature mosquito specimens (larvae and pupae) were collected with standard dippers and entomological aquatic nets (WHO 2011) and transported alive in plastic jars with water from their breeding sites to the insectary facility at the University of Jigjiga. All Anopheles larvae were separated, and a sample was reared to adults in cages (BugDorm-1Insect Rearing Cages, 30 × 30 × 30 cm) at the Entomology Laboratory of the University of Jigjiga. Although larvae were used in the bioassays, all Anopheles sp. pupae collected in the field were reared to adults in the laboratory inside 20 × 20 × 20 plastic cages (Bugdorm) to rule out the presence of other anopheline species cohabiting with An. stephensi. All adult female Anopheles mosquitoes were aspirated from cages and confirmed to be An. stephensi using the updated key to the females of Afrotropical Anopheles mosquitoes (Coetzee 2020). All emerged Anopheles adults aspirated from the cage were An. stephensi. All the adults visually identified as An. stephensi were molecularly confirmed to be this species using an allele-specific polymerase chain reaction and the sequencing of internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) loci (Carter et al. 2018).

For the bioassays, WHO guidelines for laboratory testing of mosquito larvicides were followed (WHO 2005). In 1 liter plastic containers, pooled batches of 25 larvae (late III to early IV instars) collected from the field were tested against spinosad (Natular® 30G), formulated as an extended-release granule and applied at a rate of 0.004 g per 1 liter of tap water. A total of 100 larvae were exposed to spinosad in 4 replicates with 25 larvae each. Four replicated containers water containing 25 larva each were used as controls. Larval mortality was recorded after 24 and 48 h. Dead larvae were those that could not be induced to move when they were probed with a needle in the cervical region, and moribund larvae were those incapable of rising to the surface or not showing the characteristic diving reaction when the water was disturbed or sank to the bottom (WHO 2005). The mortality of the test sample was calculated by summing the number of dead larvae across all exposure replicates expressed as a percentage of the total number of exposed larvae. The residual efficacy of the larvicide was not included in this study. All larvae were found dead at 24 h postexposure to spinosad at a concentration of 0.004 g/liter of water and remained dead at 48 h, indicating strong efficacy of spinosad against An. stephensi. No larval mortality was observed in the control.

Spinosad as an insecticide is composed of a mixture of spinosyn A and D as the active ingredient (AI) and derived from the soil actinomycete Saccharopolyspora spinosa (Mertz and Yao) under aerobic fermentation condition (Prabhu et al. 2011). Within 24 h, spinosad functions as both a contact and stomach poison for mosquito larvae, altering the function of the GABA-gated nicotinic channel and the nicotinic receptor, producing mainly involuntary muscle contractions, tremors, paralysis, and death (Prabhu et al. 2011). Natular G30 has previously been studied for its efficacy against An. arabiensis (Patton), An. gambiae (Giles), and An. funestus (Giles) in both permanent and semipermanent habitats in sub-Saharan Africa (Gimnig et al. 2020). In that study, 2 formulations of pinosad (emulsifiable concentrate and extended-release granules) were tested in semifield habitats and in a field trial in 2 villages, showing significant reductions in Anopheles sp. that resulted in significant reductions in larval occupancy, larval densities, and adult densities (Gimnig et al. 2020). Spinosad has not exhibited cross-resistance with other insecticides and can be alternated with all other classes of mosquito larvicides currently in use (WHO 2013).

Our findings are the first reporting of the efficacy of spinosad under laboratory conditions against larvae of the invasive An. stephensi in Africa. Our results also validate the results with different spinosad formulations against An. stephensi larvae in Asian countries (Kumar et al. 2011, Prabhu et al. 2011). The WHO guidelines for laboratory and field testing of mosquito larvicides indicate larval mortality should be recorded after 24 h exposure and 48 h for slow-acting larvicides (WHO 2005). In this study, the effects of spinosad were observed 24 h after exposure as an erratic diving or lack of mobility, and after 48 h all larvae were immobile at the bottom of the containers.

The preference of An. stephensi larvae for developing in construction pits and other artificial containers, particularly during the dry season, suggests possible control through the application of larvicides to these fixed habitats. In addition to treatment applied to water-holding containers during the dry season, the preventive use of granules to dry containers known to fill during the rainy season could also be considered (Aldridge et al. 2018, Britch et al. 2018, Golden et al. 2018). Further laboratory and field-based studies are necessary to determine the efficacy of spinosad larvicide against An. stephensi and other malaria vectors at different localities and under field settings. Exhaustive assessment of larval habitats and the identification of the cohabitants of this vector can also help in identifying effective tool(s) to control these vectors in an integrated approach.

We thank all the Jigjiga residents who allowed us to access their houses to collect mosquito larvae. Rapid Response Grant funding was provided by the Emory Global Health Institute to conduct this research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Emory Global Health Institute.