Background

Malaria remains a significant global health concern, primarily transmitted by infected Anopheles mosquitoes. In Africa, the Anopheles gambiae and An. funestus species complex/group are the primary vectors of malaria1. In Ethiopia, An. arabiensis, belonging to the An. gambiae complex, is the primary vector, accompanied by secondary vectors such as An. pharoensis, An. funestus, and An. nili2,3.

Anopheles stephensi, traditionally a principal malaria vector in the Arabian Penisula, has increasingly been detected in the Horn of Africa since its initial identification in Djibouti in 20124. Its presence has since been confirmed in Ethiopia’s Somali region5, with reports of its widespread distribution across eastern Ethiopia in 20196. Subsequently, it was also documented in central and Northwest Ethiopia7, highlighting the expanding range of the species within the country. Unlike other African malaria vectors, An. stephensi thrives not only in rural but also in urban environments, breeding in artificial water reservoirs such as domestic containers and garden reservoirs8. Exhibiting opportunistic feeding behavior on both humans and animals, predominantly outdoors, An. stephensi poses significant challenges for vector control efforts9. Furthermore, the development of resistance to many insecticides in vector control jeopardizes the efficacy of traditional control methods10.

Recent reports indicate that An. stephensi mosquitoes in this region have developed resistance to multiple classes of insecticides commonly employed in public health interventions11,12. Consequently, there is a pressing need to explore effective larvicidal options for managing this invasive mosquito species. Larvicides play a vital role in mosquito control strategies, targeting mosquito larvae and pupae13. Organophosphate larvicides like Abate 1SG (Temephos) disrupt the nervous system of immature larvae, while insect growth regulators (IGRs) such as SumiLarv 2MR and SumiLarv 0.5G interfere with insect development. Pyriproxyfen, the active ingredient in both SumiLarv formulations, acts as an IGR, mimicking juvenile hormone and inhibiting molting and reproduction. Prequalified by WHO as a larvicide that can be used in drinking water, SumiLarv 2MR provides slow-release larvicidal action, specifically targeting container-breeding mosquitoes, with a recommended dosage of one disc per 40–500 L of water. SumiLarv 0.5G, available in a granular form, is designed for open water sources and small and big containers, with residual activity lasting over one month. The use of IGRs like SumiLarv 2MR and SumiLarv 0.5G presents a promising alternative for mosquito control. Abate 1SG (Temephos) widely used for mosquito vector control.

Therefore, this study aimed to assess the efficacy and residual activity of SumiLarv 2MR and SumiLarv 0.5G compared to Abate 1SG against An. stephensi larvae under semi-field conditions. Such evidence-based evaluations are essential for guiding effective strategies in combating this invasive vector species in Ethiopia.

Materials and methods

The study was conducted in Awash Subath Kilo town, Afar Region, Eastern Ethiopia, located 214 km east of Addis Ababa. This region is characterized by an arid and hot climate, with an average annual temperature of 30 °C and annual rainfall ranging from 1300 to 1874 mm. Malaria transmissions in this region is unstable and seasonal, with Plasmodium falciparum and P. vivax being the predominant malaria parasite species.

To assess the efficacy and residual activity of SumiLarv 2MR, SumiLarv 0.5G, and Abate 1SG, various potential breeding habitats of An. stephensi were surveyed within Awash Subath Kilo town. Third and fourth instar mosquito larvae were sampled from these breeding sites using standard dippers to confirm their presence. Typical breeding sites included human-made containers, particularly water storage containers inside and outside residential houses, and large human-made cisterns. The collected An. stephensi larvae were then transported to the experimental site for testing.

Experimental setup

Plastic containers with capacities of 100L and 250L were used to assess the residual efficacy of SumiLarv 2MR. Specifically, four 100L containers each treated with one disc of SumiLarv 2MR, were compared to two untreated controls. Similarly, four 250L containers received one disc each, with two untreated controls. In addition, eight 250L containers were treated with a half-dose to match one disc per 500L, alongside four untreated controls. For SumiLarv 0.5G and Abate 1SG, four 100L containers were treated with each larvicide, with two untreated controls for each. Each container received 20–25 third and fourth instar An. stephensi larvae.

Untreated netting was placed over each container to prevent emerging adults from escaping, inhibit wild free-flying female mosquitoes from ovipositing, and keep debris out. Wooden frames were placed over the netting to allow airflow, and container lids were put over the wooden frame to avoid direct sunlight exposure (Supporting information, Fig. S1).

SumiLarv 2MR evaluation

The residual efficacy and activity of SumiLarv 2MR against An. stephensi larvae were evaluated using standard plastic containers used for domestic water storage in Ethiopia. Four replicate containers, each holding 100L of water treated with a SumiLarv 2MR disc (2 g/disc, containing 40 mg/disc pyriproxyfen), were used, while two containers with 100L of untreated water served as controls. Additionally, four containers, each filled with 250L of water and set up with a SumiLarv 2MR disc, and two containers with 250L of untreated water were used as controls. Furthermore, eight containers, each containing 250L of water, were treated with half a SumiLarv 2MR disc to match the dosage of one disc per 500L, with four containers of 250L of untreated water serving as controls for this treatment group (Table 1).

Table 1 SumiLarv 2MR, SumiLarv 0.5G and Abate 1SG treatment schedule.
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SumiLarv 0.5G evaluation

The efficacy and residual activity of SumiLarv 0.5G against An. stephensi larvae were evaluated using six replicates. Four containers, each holding 100L of water, were treated with precisely weighed and uniformly dispersed SumiLarv 0.5G granules, while two additional containers of the same size were left untreated to serve as controls.

Abate 1SG evaluation

The activity of Abate 1SG against An. stephensi was evaluated using four treated containers, each holding 100L of water and treated with 10 g per 100L of Abate 1SG granules following the recommended dosage, alongside two untreated control containers. The granules were evenly distributed across the water surface of each container.

Larvae and pupae management

Batches of 20–25 third and fourth instar larvae were introduced into both treated and control water containers. The larvae were provided with food made of ground-up dog biscuits every three days. Pupae were collected daily using a pipette, counted, and then transferred from each container into a 250 ml beaker, along with some treated water from their respective containers. The pupae were then placed in a paper cup covered with untreated netting secured with rubber bands (Supporting information, Fig. S2). Any larvae that pupated within one day after release were discarded, as it is crucial for the larvae to be exposed to pyriproxyfen for a few days before pupation to inhibit emergence.

Assessment of residual activity

For SumiLarv 2MR and SumiLarv 0.5G residual activity tests, observations 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, assessments were made daily regarding the survival or mortality of larvae. 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 250 ml beaker containing water from their respective treatment or control container. Mortality and survival rates were recorded daily until all larvae had either emerged as adults or died.

To mimic normal use conditions, 50% of the water in both treated and control containers was removed and replaced with water every two weeks for SumiLarv 2MR and SumiLarv 0.5G, and every week for Abate 1SG throughout the experiment (Table 1). 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. All treated water was left to stand for seven days before the introduction of An. stephensi larvae. Throughout 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. The trial duration was set until the mortality rate or inhibition rate decreased below 60% for two consecutive assessments. The calculation of the Adult Emergence Inhibition Rate (EI%) in response to exposure to SumiLarv 2MR was performed following a standard protocol.

$${\text{EI}} \, \left(\text{\%}\right)\text{ } = \, \text{100 - }\left( \frac{\text{T }\times \text{ 100}}{\text{C}} \right),$$

where T and C are the % of adult mosquito emergence from treated and control sites, respectively.

HPLC analysis

The contents SumiLarv in the used SumiLarv 2MR dics were quantified and compared with those in fresh SumiLarv 2MR discs using high performance liquid chromatography (HPLC). The analysis was conducted based on an in-house method, which was a modification of CIPAC method 715/MR/M/314 (The SOP used for HPLC analysis is included in the Supplementary Information).

Data analysis

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 analysed for Abate 1SG. These mean percentage reductions in larval populations were then analyzed using a one-way ANOVA (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.

Results

Efficacy and residual activity of SumiLarv 2MR against An. stephensi larvae under semi-field conditions

Anopheles stephensi pupae from 100L water containers treated with a single SumiLarv 2MR disc (at a dosage of 1 disc per 100L) exhibited a complete inhibition of adult emergence (Fig. 1). This 100% inhibition was consistently maintained throughout the nine-month study. In contrast, pupae from the control group, which were not exposed to the treatment, successfully emerged as adults, indicating no inhibition under control conditions.

Fig. 1
figure 1

SumiLarv 2MR with one disc used in a 100L treatment to inhibit the emergence of adult An. stephensi mosquitoes under semi-field conditions. Error bar indicates standard error.

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Similarly, in the 250L water containers treated with one SumiLarv 2MR disc, complete inhibition of adult emergence was observed, maintaining 100% efficacy up until week 29 ( Fig. 2). However, by week 35, the inhibition rate slightly decreased to 93%. Conversely, all pupae in the control group emerged as adults, showing no signs of inhibition.

Fig. 2
figure 2

SumiLarv 2MR with one disc used in a 250L treatment to inhibit the emergence of adult An. stephensi mosquitoes under semi-field conditions. Error bar indicates standard error.

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For the treatment involving 250L water containers with half a disc of SumiLarv 2MR disc, equivalent to a concentration of one disc per 500L, a consistent 100% inhibition of adult emergence was observed up to week 25 (Fig. 3). However, by the end of the seventh month (Week 27), and into the first (Week 33) and final week (Week 35) of the ninth month, inhibition rates declined to 98%, 67%, and 65% respectively. Despite this decline, all pupae in the control group emerged as adults, underscoring the absence of any inhibitory effect in the untreated containers (Fig. 3).

Fig. 3
figure 3

SumiLarv 2MR with half disc in a 250L (equivalent to 1 disc/500L) treatment to inhibit the emergence of adult An. stephensi mosquitoes under semi-field conditions. Error bar indicates standard error.

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Efficacy and residual activity of SumiLarv 0.5G against An. stephensi larvae under semi-field conditions

Anopheles stephensi pupae in 100L containers treated with SumiLarv 0.5G exhibitted a complete inhibition of adult emergence during the first three weeks of the study. However, in the fourth, fifth, and sixth weeks, the inhibition rate decreased to 87%, 89%, and 69% respectively (Fig. 4, Fig. S3). From the seventh to the eighth week, the inhibition rate fell sharply to less than 5%, representing a statistically significant decline (P < 0.05). By the ninth and tenth weeks, all pupae in the SumiLarv 0.5G treated containers developed into adults, indicating a total loss of inhibitory effect during this period, as shown in Fig. 4 and Fig. S3. The significant deop in emergence inhibition, begining in the seventh week, was accompannied by a corresponding rise in adult emergence (P = 0.0001). In contrast, the control group, which received no treatment, consistently demonstrated 100% pupal emergenc into adults throughout the study, with no observed inhibition. This stark comparison underscore the initial effectiveness of SumiLarv 0.5G in preventing adult mosquito emergence, which significantly diminishes over time.

Fig. 4
figure 4

Efficacy and residual activity of SumiLarv 2MR, Abate 1SG and SumiLarv 0.5G against An. stephensi in plastic containers under semi-field conditions.

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Efficacy and residual activity of Abate 1SG against An. stephensi larvae under semi-field conditions

The residual efficacy of Abate 1SG, applied at a concentration of 10g per 100L, remained at 100% during the first seven weeks of the trial, with a consistent mean larval mortality rate of 100% recorded 24 h post-exposure (Fig. 4, Fig. S4). However, from the eighth week onward, a marked decline in efficacy was observed, with mean larval mortality rates dropping to 69%, 25%, and 16% in the eighth, ninth, and tenth weeks, respectively (Fig. 4, Fig S2). By the eleventh and twelfth weeks, no larval mortality was recorded, and all larvae successfully underwent pupation and emerged as adults. Throughout the 12-week trial the control group experienced no larval mortality.

When compared to SumiLarv 0.5G, the residual activity duration of Abate 1SG did not differ significantly (P > 0.05), indicating that both products had similar mean durations of efficacy under the tested conditions. This comparison highlights the strong initial larvicidal effect of Abate 1SG, which, like SumiLarv 0.5G, diminishes over time, underscoring the need for periodic reapplication to maintain effective control over mosquito larvae populations.

SumiLarv 2MR demonstrated the longest residual efficacy, extending up to 35 weeks, significantly longer (P < 0.05) than Abate 1SG and SumiLarv 0.5G, which had residual efficacies lasting seventh and eighth weeks, respectively. These results underscore the superior long-term effectiveness of SumiLarv 2MR in preventing the emergence of An. stephensi adults under the tested conditions.

Pyriproxyfen residual content analysis in SumiLarv 2MR discs

After concluding the nine-month trial, the analysis of the SumiLarv 2MR discs showed that the remaining mean pyriproxyfen content in discs used in 100L, 250L, and halved discs in 250L water containers was 0.8%, 0.85%, and 0.92%, respectively (Fig. 5). This retention of pyriproxyfen demonstrates significant residual activity, with the discs retaining nearly 50% of their initial pyriproxyfen content (approximately 1%) by the end of the tral period. These findings underscore the sustained release and prolonged efficacy of SumiLarv 2MR, highlighting its potential for continued mosquito control effectiveness beyond the observed study period.

Fig. 5
figure 5

Pyriproxyfen residual content in used and fresh, unused SumiLarv 2MR discs. Error bar indicates standard error.

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Discussion

The study revealed a complete inhibition of An. stephensi emergence under semi-field conditions across three dosages of SumiLarv 2MR (1 disc/100L, 1 disc/250L, and 1 disc/500L), maintaining a consistent 100% inhibition over a six-month period. Furthermore, the residual activity persisted for more than 35 weeks (nine months), surpassing a 60% emergence inhibition threshold. In line with these findings, Ref.15 demonstrated 100% inhibition of adult emergence for over six months using SumiLarv 2MR discs placed in catch basins, while16 reported continued 100% effectiveness against Aedes-infested containers even after eight months of SumiLarv 2MR resin disc application in school settings. Similarly17 reported prolonged inhibition of adult emergence in Aedes mosquitoes reinforcing the robust and lasting impact of S SumiLarv 2MR. These results align with prior studies conducted in Cambodia18 where SumiLarv resin discs exhibited greater than 80% inhibition of adult emergence for up to 36 weeks under both laboratory and semi-field conditions.

Additionally, in a simulated field trial conducted in Thailand, more than 80% emergence inhibition of adult Ae. aegypti was observed throughout the 24-week study period. In a study where pyriproxyfen 0.5G-treated plastic containers exhibited complete inhibition against Ae. aegypti for 15 weeks19, with residual activity lasting up to 26 weeks under outdoor conditions. This disparity in results can be attributed to variations in factors such as water quality and mosquito species. Pyriproxyfen, functioning as an analog of juvenile hormone, acts as a growth regulator for insects, specifically targeting the pupal stage and impeding development into adult mosquitoes within treated areas.

Analysis of nine-month-old discs from the semi-field setting revealed that they maintained over 90% efficacy in 100L and 250L containers, and over 60% efficacy in 500L containers with weekly water changes of 50%. This persistence suggests sustained release of pyriproxyfen from the discs over an extended period. Comparatively, aged SumiLarv 2MR discs retained nearly 50% residual pyriproxyfen content, demonstrating that their extended efficacy remains comparable to that of fresh discs.

The sustained efficacy of SumiLarv 2MR, with residual activity lasting over 35 weeks post-treatment, underscores the effectiveness of this Matrix release formulation. This formulation ensures prolonged residual activity as the disc settles to the bottom of the water container, continuously releasing active ingredient to control mosquito larvae. Additionally, the SumiLarv 2MR disc boasts several key properties that distinguish it from other larvicides, notably its easily identify the application by size and color, and controlled release technology enabling SumiLarv 2MR to maintain a consistent pyriproxyfen concentration even after water replacement in containers, thereby mitigating the risk of larvicide overdose and reducing operational costs while preventing mosquito resistance16,20. Studies have shown that treating water storage containers in rural areas with SumiLarv significantly reduces the need for frequent treatments, leading to lower costs for anti-mosquito programs16,20.

Another pyriproxyfen product evaluated in this study is SumiLarv 0.5G. The study revealed that SumiLarv 0.5G effectively prevented mosquito emergence for three weeks, with residual activity lasting up to six weeks under semi-field conditions in plastic containers, where 50% water replaced weekly. These findings are consistent with previous research, which demonstrated that SumiLarv 0.5G effectively inhibited adult emergence for five-week period in An. gambiae s.s.21.

Moreover, several studies have shown the effectiveness of SumiLarv 0.5G against various mosquito species, including An. gambiae s.l. and Aedes mosquitoes, even at low concentrationsof the active ingredient21,22,23,24. SumiLarv 0.5G remained effective at lower doses, with low weekly emergence rates for up to six weeks. In Kenya, it was found that a concentration of 2 g SumiLarv 0.5G per 1000L effectively prevented mosquito re-infestation for three months13. Similarly, studies in Tanzania showed high efficacy of SumiLarv 0.5G in killing mosquito pupae for up to five weeks21.

The residual impact of SumiLarv 0.5G as observed in this study, is consistent with previous reports on its efficacy against An. stephensi and other mosquito species21,22,23,24. Furthermore, SumiLarv 0,5G granules at higher concentrations demonstrated nearly 100% inhibition of emergence over 90 days, even with water removal and addition, regardless of larval density25. Reports from Florida, USA have also confirmed that SumiLarv 0,5G can complete ly inhibit the emergence of Ae. aegypti for six weeks in outdoor plastic tubs22. The use of high doses of SumiLarv 0.5G may provide longer-lasting control of An. stephensi in various breeding sites, particularly concrete ponds and plastic containers.

On the other hand, Abate 1SG was found to cause 100% larval mortality for seven weeks when used at a dosage of 10 g/100L with 50% weekly water raplacement. This result is aligns with previous studies in Ethiopia as well as laboratory studies in India and southern Iran, which also showed An. stephensi larvae were susceptible to temephos at diagnostic doses11,26,27. However, it’s worth noting tha anotherr study reported a residual effect of 15 weeks for 1 mg a.i./L temephos in laboratory conditions19. Additionally, there was a report of complete Aedes larvae mortality for 12 weeks in plastic containers treated with temephos in Malaysia28. Nevertheless, studies in Malaysia have indicated reduced susceptibility of Aedes larvae to temephos due to resistance development29,30, emphasizing the need for alternative larvicides such as pyriproxyfen.

The variations in effectiveness observed in these studies are likely influenced by factors such as water volume, mosquito species, and pyriproxyfen formulations. However, it is evident that pyriproxyfen, particularly at lower concentrations, effectively prevented adult mosquitoes from emerging. These findings underscore the importance of innovative larvicides like SumiLarv 2MR in the fight against mosquito-borne diseases in the context of changing environmental conditions.

Conclusion

The results of this study emphasize the the long-lasting effetiveness of SumiLarv 2MR in combatting An. stephensi larvae, lasting for an impresive eight months. In comparison, SumiLarv 0.5G and Abate 1SG showed residual effectiveness for seven and five weeks respectively.It’s important to consider the specific uses of these larvicides, while SumiLarv 0.5G is suitable for a wide range of breeding sites such as ditches, pools, and containers, SumiLarv 2MR is formulated for clean domestic water storage vessels, making it particularly effective against An. stephensi larvae in such containers. The demonstrated effectiveness of SumiLarv 2MR as a long-lasting larvicide against An. stephensi larvae highlights its potential as an important tool in fighting this invasive mosquito species. By targeting clean water storage containers, SumiLarv 2MR offers an effective solution for controlling An. stephensi populations in areas where these containers are the main breeding sites.