The study was conducted in Awash Subath Kilo town, Afar Region, Eastern Ethiopia, 214 km east of Addis Ababa. Characterized by its arid and hot climate, the region experiences an average annual temperature of 30°C and annual rainfall ranging between 1,300 and 1,874 mm. Malaria transmissions in this region is marked by unstability and seasonality, with Plasmodium falciparum and P. vivax being the predominant malaria parasite species.
Recent reports indicate that An.stephensi mosquitoes in this region have developed resistance to multiple classes of insecticides commonly employed in public health interventions 12,13. Consequently, there is a pressing need to explore effective larvicidal options for managing this invasive mosquito species. To assess the efficacy and residual activity of SumiLarv 2MR, various potential breeding habitats of An. stephensi were surveyed within Awash Subath Kilo town. Third and fourth mosquito larvae were sampled from these breeding sites using standard dippers to confirm their presence. The typical breeding sites are human-made containers, particularly water storage containers inside and outside residential houses, and large human-made cisterns. Subsequently, An.stephensi larvae collected from different artificial breeding sites were transported and to the experimental site and were directly used for the test.
Four standard plastic containers, domestic water storage in Ethiopia were used, each holding 100 L of water treated with a SumiLarv 2MR disc with 2g/disc of SumiLarv 2MR (pyriproxyfen; 40mg/disc), were set up alongside two plastic containers, each containing 100L of untreated water as a control. Similarly, four plastic containers, each filled with 250L of treated water with a SumiLarv 2MR disc, were prepared, with two plastic containers, each holding 250L of untreated water as control. Additionally, eight plastic containers, each containing 250L of water, were treated with half a SumiLarv 2MR disc to match the dosage of 1 disc per 500 L. Four plastic containers, each filled with 250 L of untreated water, were designated as controls for this treatment group (Table 1). All treated water was left to stand for 7 days before the introduction of An. stephensi larvae.
The efficacy and residual activity of SumiLarv 0.5G against An. stephensi larvae was carried out using six plastic containers, four containers each holding 100L of tap water and SumiLarv 0.5G, whereas two containers were untreated and used as controls. Similarly, four containers each holding 100L of tap water were used along sise two control containers were used to evaluate the activity of Abate 1SG against An. stephensi larvae following the recommended dosage (Table 1). In all water containers a untreated netting were placed over each container to prevent any emerging adults from escaping and to inhibit entry by wild free flying female mosquitoes ovipositing in the containers and to keep debris out. Moreover, wooden frames were put on each container over the netting for free flow of air and container lids were put over the wooden frame to avoid direct sunlight exposure(Supporting information, Plate S1).
Table 1
SumiLarv 2MR, SumiLarv 0.5G and Abate 1SG treatment schedule
Treatment | Dose rate | Replicate | Larvae Introduction |
T1 | SumiLarv 2MR 1 disc /100 L | 4 | Every two weeks |
T2 | SumiLarv 2MR 1 disc/250 L | 4 | Every two weeks |
T3 | SumiLarv 2MR 1 disc/500 L | 8 | Every two weeks |
T4 | SumiLarv 0.5G 0.2g/100 L | 4 | Every week |
T5 | Abate 1SG 10g/100L | 4 | Every week |
T6 | Control/ untreated | 12 | Every week/two weeks |
For SumiLarv 2MR and SumiLarv 0.5G residual activity tests, observation of adult emergence commenced immediately after the collection of pupae and continued for three days with pupal formation and, any instances of morphological abnormalities until all pupae either emerged as adults or were confirmed deceased. For Abate 1SG each day, assessments were made regarding survival or mortality of larvae. To mimic normal use conditions, 50% of the water in both treated and control containers was removed and replaced with water every seven days throughout the experiment. Prior to this water change, larvae that had not undergone pupation were removed and new larvae were introduced three days following the water change. This process was repeated every two weeks specifically for evaluations involving SumiLarv 2MR. Throughout the duration of the trial, adult mosquitoes that failed to extricate themselves from their pupal casings were classified as dead. The experiment spanned nine months, from April to December 2022, with all relevant timings logged. The calculation of the Adult Emergence Inhibition Rate (EI %) in response to exposure to SumiLarv 2MR was performed using the formula provided by the World Health Organization (WHO) in 2005.
$$\text{EI}\text{ }\left(\text{%}\right)\text{ =}\text{ }\text{100 - }\left( \frac{\text{T × 100}}{\text{C}} \right),$$
Where T and C are the % of adult mosquito emergence from treated and control sites, respectively.
Four plastic containers, each holding 100L of tap water, were treated with SumiLarv 0.5G to evaluate the residual efficacy of SumiLarv 0.5G. These containers were treated with a 0.2g/100L dose of SumiLarv 0.5G, as per the recommended dosage guidelines. In addition, two control containers, each also containing 100 L of water but without treatment were established in parallel to monitor natural survivorship and emergence patterns. The SumiLarv 0.5G granules were precisely weighed and then uniformly dispersed across each treatment container. Into each of these treated and control containers, batches of 20–25 larvae in their 3rd and 4th instar stages, from a field insectary of An. stephensi, were introduced. Throughout the experiment, a routine water replacement schedule of 50% was adhered every 7 days for both treated and control containers following the collection of pupae. Prior to each water replacement, any larvae that had failed to pupate were carefully removed and discarded. Subsequently, new larvae were introduced into the containers three days after the water exchange. This process was repeated on a weekly basis to ensure consistency in the experimental conditions.
During the trial period, any adult mosquitoes that remained trapped within their pupal exoskeletons were regarded as deceased. The trial spanned until mortality rates fell below 60% for two consecutive observations, with all timings diligently recorded. To quantify the inhibition of adult emergence, as described above (WHO 2005), was employed.
The efficacy and residual activity of Abate 1SG against An. stephensi larvae under semi-field conditions was assessed using plastic containers, each holding 100L of water, with four replicates treated with 10g per 100L of Abate 1SG granules, following the recommended dosage. In parallel, control groups were established with two replicates of plastic containers, each also holding 100L of water but without treatment. The granules were evenly distributed across the water surface of each container. Into each container, both treated and control, batches of 20–25 third and fourth instar An. stephensi larvae were introduced.
Mortality was assessed 24 hours post-treatment. Surviving larvae were carefully removed using a larval net, while pupae, if present, were collected daily with a pipette, counted, and then transferred into a 250ml beaker containing water from their respective treatment or control container. Pupae that pupated within one day post-larvae release were excluded from the analysis. Mortality and survival rates were recorded daily until all larvae had either emerged as adults or were dead. To mimic natural aquatic environments, 50% of the water in each container was replaced every seven days, with non-pupated larvae being discarded prior to the water change and new larvae introduced three days thereafter. Any adult mosquitoes unable to exit their pupal casings were considered dead. The duration of the trial was set until the mortality rate decreased to below 60% for two consecutive assessments, with all relevant timings noted.
Data analysis
The evaluation of adult mosquito emergence inhibition in treated containers took into account the mortality observed in control containers, using a correction factor for a more accurate assessment. The average percentage of adult emergence reduction for both SumiLarv 2MR and SumiLarv 0.5G treatments was compared through a one-way ANOVA analysis (utilizing SPSS version 20), to ascertain if there were statistically significant differences in the effectiveness of emergence inhibition between the two treatment groups.
Additionally, the mean larval mortality rates for mosquitoes in both treated and control containers were calculated for Abate 1SG. These mean percentage reductions in larval populations were then analyzed using a one-way ANOVA (via SPSS version 20) to assess the impact of Abate 1SG.. To provide a comprehensive understanding of the results, 95% confidence intervals (95% CI) for the mean larval mortality rates were computed.
The study also determined the duration of the residual larvicidal effects for each type of container used in the trial, defined as the number of days until a decrease in larvicidal efficacy was observed, specifically when larval mortality fell below 60%. Moreover, Sumilarv contents of the used SumiLarv 2MR were quantified by using high performance liquid chromatography (HPLC), based on in-house method modified from CIPAC method 715/MR/M/.