Susceptibility of Aedes aegypti mosquitoes to pyrethroid insecticides and characterization of breeding habitats in selected districts of Mwanza, Tanzania

Abstract

Background

Tanzania has experienced outbreaks of dengue fever in major cities. The main vectors of the dengue virus in Tanzania are Aedes aegypti mosquitoes. The control of this mosquito vector is based on insecticide, and yet, the insecticide susceptibility of this species is not well known in many places in Tanzania. Conversely, the breeding habitats mostly preferred by this species are not well documented in the study area. Therefore, the present study aimed to determine the pyrethroid insecticide susceptibility status and breeding habitats preference of Ae. aegypti mosquito in the three sites from three districts in Mwanza, Tanzania. The assessment of Ae. aegypti mosquito 24-h percentage mortality was based on WHO criteria.

Results

A total of 850 Ae. aegypti were exposed to three pyrethroid insecticides. In Bwiru site, 100% mortality was observed for all three pyrethroids, indicating full susceptibility. At Igekemaja and Mwagagala villages, Ae. aegypti were resistant to all the three pyrethroid insecticides tested. In Igekemaja, there were variations in Ae. aegypti mortality rates induced by different insecticides, with mortality rates ranging from 72% for alphacypermethrin to 86% for deltamethrin. Although the mortality rates were lower than in Bwiru site, they were still substantial and statistically significant. The mortality rates in Mwagagala were lowest across the three insecticides, with mortality rates ranging from 60% for deltamethrin to 86% for alphacypermethrin. In Mwagagala, the Ae. aegypti mosquitoes were less susceptible to the insecticides tested. Aedes aegypti mosquito prefer breeding in abandoned old tires in urban area and in small containers and uncovered water storage containers in rural settings.

Conclusion

The study has revealed pyrethroid insecticide susceptibility status and breeding habitats of Ae. aegypti in the rural and urban settings in Mwanza, Tanzania. The study findings imply the need for public health interventions with focus on community education on mosquito control.

Background

Dengue virus is transmitted from one person to another through the bite of Aedes aegypti mosquito (Diallo et al. 2022; Kraemer et al. 2019). Dengue is a global epidemic, affecting large population of people who lives in countries where the disease is endemic (Kraemer et al. 2019). A recent study has reported the disease to occur in 195 countries globally between 1990 and 2017 (Zeng et al. 2021). The World Health Organization (WHO) has reported approximately 50 million cases of dengue (WHO, 2016a). In recent years, mosquito-borne arboviral diseases such as dengue, Zika, chikungunya and yellow fever have increased globally. The Word Health Organization (WHO) has documented a tenfold rise in reported cases between 2019 and 2000, reaching 5.2 million across 129 countries globally (WHO, 2016a). In 2023, over 5 million dengue cases and more than 5,000 related deaths were recorded across 80 countries. Africa is among the top four regions mostly affected by dengue, with 171,991 cases and 753 deaths in 2023 (Enitan et al. 2024). The incidence of the disease has increased in Africa since the 1980s, with many cases occurring in the East African countries (Diallo et al. 2022; Vairo et al. 2012). Tanzania has experienced several dengue outbreaks since 2014 to 2019 especially in the city of Dar es Salaam (Vairo et al. 2012, 2016). A previous study in northeast Tanzania has reported the dengue seroprevalence of 24.8% (Kajeguka et al. 2016). Information on dengue seroprevalence is lacking in other places of Tanzania where Aedes mosquito is present (Mweya et al. 2016). Although the first outbreaks of dengue fever in Africa date back to the early nineteenth century, its burden remains relatively unknown (Gainor, Harris and LaBeaud 2022). Factors contributing to this issue include its symptoms overlap with diseases like malaria, inadequate laboratory infrastructure for prompt detection, insufficient surveillance systems and limited vector control measures. Additionally, there is a significant lack of public awareness regarding the disease (Adesola et al. 2024).

Aedes aegypti mosquito lives in human habitats (Egid et al. 2022; Kahamba et al. 2020b). This mosquito prefers to bite outdoors during the day and in shady places, particularly early in the morning and late afternoon before sunset (Egid et al. 2022). This mosquito prefers to breed in small containers such as stagnant water, especially in household utensils, some plants leaf axils, roof gutters, car tires, buckets, cans and un-attended domestic wastes which can hold water for a long time such as coconut shells (Egid et al. 2022; Diallo et al. 2022; Surendran et al. 2021a). Few studies have reported on the distribution and diversity of mosquitoes and the role of Aedes mosquito in the transmission of arboviruses in Tanzania (Philbert & Msonga, 2020). Climate and environmental factors have major contribution in the spread of Aedes mosquitoes in many parts of Africa including Tanzania, which results in the arbovirus transmission (Mweya et al. 2016; Dong et al. 2022). The Ae. aegypti mosquitoes, the primary vectors of dengue virus, have been a significant concern in Tanzania, especially with the increasing incidence of dengue during dengue outbreaks (Philbert, Lyantagaye and Nkwengulila 2019; Mathias et al. 2017a; Philbert & Msonga, 2020). Despite the growing threat, the insecticide susceptibility status of Ae. aegypti in many parts of the country especially in Mwanza region, the second largest city in Tanzania, remains poorly understood. Since the control of mosquito vectors relies primary on the use of insecticides, addressing the lack of knowledge on insecticide resistance status is critical for effective vector control measures. Moreover, the ecological preference of Ae. aegypti such as its preferred breeding habitats, are not well known (Msellemu et al. 2020). The present study aimed to determine the pyrethroid insecticide susceptibility status and breeding habitats preference of Ae. aegypti mosquito in Mwanza, Tanzania.

Methods

Study area

The cross-sectional study conducted between March and November 2021, in three villages located in three different districts, namely Magu, Ilemela and Misungwi districts, in Mwanza region. The villages in the rural settings were Igekemaja (Magu district) and Mwagagala (Misungwi district) while the one in the urban setting was Bwiru site (Ilemela municipal council). Magu district is situated at 2°34.673 S and 33°07.170 E. The surrounding area is dominated with maize, rice and vegetables cultivation. Vegetables are irrigated with water from a seasonal river. Misungwi district is located at 3.0731° S and 32.9888° E. It is a rural district which covers an area of 2,122 km² and includes 27 wards, 78 villages and a population of 351,607 people (URT, 2022). The annual rainfall ranges from 0.5 mm to 58.8 mm, split in two rainy seasons (October to December and March to May) and a long dry season (June to September). Average altitude in the study area is 1,150 m. Majority of the residents in Magu and Misungwi districts are subsistence farmers who depend mainly on rainfall. They grow rice, cotton, maize, millets and legumes. They also keep livestock such as cattle, goats and sheep. Ilemela District is located at 2° 35′ 0″ S, 32° 55′ 0″ E and is one of the seven districts of the Mwanza Region in Tanzania. It is bordered to the north and west by Lake Victoria, to the east by Magu District and to the south by Nyamagana District. Part of the region's capital, the town of Mwanza, is within Ilemela District (Fig. 1). Dengue is an emerging concern in many regions including Mwanza due to the changing climate, increased urbanization and increased mobility of people. During the 2014, dengue outbreak, epidemiological evidence has revealed that out of the 961 confirmed cases reported from January to May 2014, only two cases were recorded in Mwanza, which is equivalent to 0.2% (Mweya et al. 2016). The three study sites within the Mwanza region is justified the rural and urban representation settings, allowing for a comparative analysis of health outcome influenced by different socioeconomic and environmental conditions. These sites are characterized by distinct agricultural practices which rely on seasonal rainfall and proximity to water bodies, making them potential for investigating insecticide susceptibility status and habitat characterization of the Ae. aegypti mosquito. Furthermore, the region’s emerging health concerns such as the spread of dengue during the 2014 outbreak make it justifiable study area for this study. The altitudes within the Mwanza region's districts vary slightly, with Magu District ranging from approximately 1,100 to 1,200 m above sea level, Ilemela district is between 1,140 and 1,200 m, and Misungwi district is around 1,150 m. These moderate elevations are characteristic of the Mwanza region, contributing to the region's climate and environmental conditions, which are important factors in understanding local health outcomes, particularly the transmission dynamics of vector-borne diseases like dengue.(https://en.wikipedia.org/wiki/Mwanza_Region).

Fig. 1

Map of Mwanza region showing study sites

WHO insecticide susceptibility bioassays

Aedes aegypti mosquito larvae were collected from different breeding sites. The water holding containers surveyed constituted by abandoned car tires, discarded domestic containers. Collected Aedes mosquito larvae were brought to the insectary for rearing. Emerged first filial generation (F1) of day three to five were tested to the three pyrethroid insecticides: permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%) using WHO guideline (WHO, 2016b). Four replicates, each with F1 unfed 20 female mosquitoes, were exposed to selected insecticides and another one replicate of F1 unfed 20 female adult mosquitoes were exposed oil-treated papers without insecticide (control) for 1 h. After 1 h, mosquitoes were transferred to the holding tubes and maintained on 10% glucose solution for 24 h. The assessment of 24-h percentage mortality rate of Ae. aegypti mosquito was based on WHO criteria. Mortality rate range 98–100% indicates susceptible mosquito population, less than 98% suggests possible resistance that need to be confirmed and below 90% indicates existence of resistance (WHO, 2018). These diagnostic doses were opted to provide information on assessment insecticide resistance status of local Ae. aegypti populations in Mwanza region, Tanzania, where such information is limited and could provide a better understanding of resistance patterns. However, similar doses for Ae. aegypti have been used by the previous studies conducted in Dar es Salaam and Ifakara, Tanzania, which provided a broader range of susceptibility for this mosquito species (Mathias et al. 2017b; Kahamba et al. 2020a).

Aedes mosquito breeding habitat characterization

In each site, Aedes breeding habitats were searched as previous described (Saleh et al. 2018; Toé et al. 2022). Standard dippers and pipettes were used to collected Aedes larvae in the breeding sites encountered. The use of either a standard dippers or pipettes depended on the type of the mosquito breeding ground encountered. All breeding sites encountered were recorded. Among the types of breeding sites encountered were the water storage containers, broken clay pots and abandoned old tires (Fig. 2 and 3). The breeding sites which contained larvae were record and larvae from each dipper were countered. In larval stages, usually distinguishing Aedes larvae from Mansonia, Anopheles and Culex is based on their morphological features. Aedes larvae can be distinguished by their short, stout siphon with a single pair of hair tufts and their angled resting position in the water. In contrast, Mansonia larvae lack a visible siphon as they attach to aquatic plants to breathe. Anopheles larvae also lack a siphon, resting parallel to the water surface, while Culex larvae have a long, slender siphon with multiple hair tufts and hang at a steeper angle. For this study, sample of larvae was reared in the insectary to adult for identification by using morphological taxonomic key (Rueda, 2004). The distance from the sites to the insectary varied from 1, 20 and 76 km from Bwiru, Magu and Mwagagala, respectively.

Fig. 2

Water storage container and broken clay pot as breeding sites for Ae. aegypti in rural settings

Fig. 3

Abandoned old tires as Ae. aegypti breeding sites in urban settings

There were few Anopheles gambiae sensu lato and Culex species found which were identified and discarded. The presence of Aedes larvae and the number of containers positive for Aedes mosquitoes were recorded.

Data analysis

The susceptibility status of an Ae. aegypti mosquito population to pyrethroid insecticides was assessed in three study areas, namely Bwiru (urban), Igekemaja (rural) and Mwagaga (rural)—according to the WHO criteria which is based on the 24-h post-exposure mortality rates. A total of 850 female Ae. aegypti mosquitoes were tested using three different pyrethroid insecticides: permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%). A mosquito population was considered susceptible if mortality rates were ≥ 98%, possible resistant if mortality ranged between 90 and 97%, and resistant if mortality was below 90% (WHO, 2018). Chi-square tests were used to compare mortality rates across sites for each insecticide, assessing whether observed differences were statistically significant.

The breeding sites productivity of Ae. aegypti was assessed by counting the average number of larvae at each container. This provided insight into the productivity and potential contribution of each habitat type to the overall mosquito population. Larval habitats were categorized into various container types across the study sites, and the average number of larvae per container was calculated to identify key productive breeding grounds. The Breteau Index (BI) was also calculated for each district, representing the proportion of inspected containers that contained Aedes larvae or pupae, thus indicating infestation levels (WHO, 2016b).

Results

Insecticide susceptibility status of Ae. aegypti

A total of 850 female Ae. aegypti were exposed to three pyrethroid insecticides, namely permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%), to reveal patterns of susceptibility status in different study sites. Table 1 highlights significant differences in insecticides performance between the study sites. The findings have indicated a strong correlation between susceptibility and geographic location. The presence of superscripts 'a' and 'b' provides a clear framework for understanding where the differences in mortality rates are statistically significant or not, emphasizing the varying effectiveness of the insecticides across different sites. At Bwiru site, Ae. aegypti were susceptible to all insecticide tested. At Igekemaja and Mwagagala sites, Ae. aegypti were resistant to all the three pyrethroid insecticides tested, although there was variation in 24-h post-exposure percentage mortality. At Igekemaja, the 24-h % mortality was 84%, 72% and 86%, when exposed to permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%), respectively. In Mwagagala, the 24-h % mortality was 79%, 86% and 60%, when exposed to permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%), respectively.

Table 1 Insecticide susceptibility status of Ae. aegypti in three sites

Aedes aegypti breeding habitats characterization

A total of 256 breeding sites of Ae. aegypti were characterized across three study sites: Bwiru, Igekemaja and Mwagagala. Mwagagala had the highest number of breeding habitats, accounting for 46.9% (n = 120) of the total, followed by Igekemaja with 40.2% (n = 103), while Bwiru had the least with 12.9% (n = 33). Open plastic bottles and broken clay pots were the most common types of breeding habitats in Igekemaja and Mwagagala, whereas abandoned old tires were more prevalent in Bwiru. A total of 3,592 mosquito larvae were collected. Among them, 3,577 (99.5%) were Ae. aegypti larvae were collected, with the highest average number of larvae per habitat being found in water storage containers in the rural sites of Igekemaja and Mwagagala, and abandoned tires in the urban site of Bwiru. The higher abundance of mosquito was observed at Bwiru (46 ± 24.0) (Table 2). Other mosquito genera, including An. gambiae s. l. (n = 2, 0.1%) and Culex (n = 13, 0.4%), were detected in old tires and broken clay pots, respectively.

Table 2 Aedes aegypti breeding habitat types encountered

The Breteau Index (BI) quantifies the proportion of inspected containers that contain Aedes larvae or pupae, expressed as a percentage. For Ilemela District, the BI is approximately 48.5%, indicating that nearly half of the inspected containers had larvae or pupae. Magu District has a BI of about 39.8%, suggesting a lower but still significant proportion of infested containers. In Misungwi District, the BI is the highest at around 60.8%, reflecting a substantial prevalencenumber of larvae and pupae among inspected containers. These indices highlight varying levels of mosquito infestation across the districts, with Misungwi experiencing the most significant problem (Table 2).

Discussion

In the present study, it has been revealed that Ae. aegypti mosquito is resistance to permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%) in Igekemaja and Mwagagala villages. These findings concur with a previous study conducted in Dar es Salaam, Tanzania, which reported that Ae. aegypti is resistant to permethrin (0.75%) and deltamethrin (0.05%) (Mathias et al. 2017). The increase over time in resistance to deltamethrin and permethrin among Ae. aegypti has been reported in Northern Nigeria (Mukhtar & Ibrahim, 2022) and in Cameroon (Montgomery et al. 2022). However, the resistance observed in the rural settings of Igekemaja and Mwagagala contrasts with findings from other rural areas, such as Ifakara and Muheza, where Ae. aegypti remained susceptible to these insecticides (Emidi et al. 2017; Kahamba et al. 2020).

The findings of the present study have revealed that Ae. aegypti was resistant to alphacypermethrin (0.05%) in Igekemaja and Mwagagala villages, similarly to the study conducted in Cameroon (Montgomery et al. 2022) and Cambodia (Maestre-Serrano et al. 2017). These findings contradict with previous studied conducted in Ifakara, Tanzania, which reported this species to be susceptible to pyrethroid tested (Kahamba et al. 2020a) The high Pyrethroids resistance among Ae. aegypti mosquito populations in rural settings can be attributed to the prolonged use of these insecticides for agriculture, veterinary and public health activities which may contribute to selection pressure (Emidi et al. 2017). . Interesting, Ae. aegypti was susceptible to permethrin (0.75%), alphacypermethrin (0.05%) and deltamethrin (0.05%) at Bwiru site which was an urban setting. This was in contrary to the study conducted in Dar es Salaam (Mathias et al. 2017). These findings call for close monitoring and management of insecticide resistance in both urban and rural areas.

This study identified key breeding habitats for Ae. aegypti in both urban and rural settings, with significant variation in larval density observed across different habitat type. However, there was variation in the number of habitats and larval density between the rural and urban settings, possibly due to the variations in environmental conditions and human activities which could have created mosquito breeding habitats. The most common type of breeding habitat encountered to harbor Ae. aegypti larvae was the open plastic bottles/containers and broken clay pots in Igekemaja and Mwagagala villages. The open plastic bottles/containers were also mentioned by previous studies as preferred breeding grounds for Ae. aegypti (Surendran et al. 2021). The water storage containers in the Igekemaja and Mwagagala have higher average number of larvae per habitat. The findings of this study concur with previous studies conducted elsewhere with similar settings in Tanzania, which identified discarded water holding containers as predominant breeding sites for Ae. aegypti mosquito (Kahamba et al. 2020b). The present findings are also comparable to a study conducted in Philippines (Edillo et al. 2012) and Sri Lanka (Surendran et al. 2021b). Contrary to the findings of this study, a previous study conducted in Dar es Salaam documented low productivity of Ae. aegypti in water storage containers (Mathias et al. 2017). The difference in habitat productivity could be due to the rural and urban environmental settings. The rural settings where the present study was conducted had limited water supply systems, whereby people use to store water for domestic purposes. At Bwiru, the breeding habitat type with higher average number of larvae per habitat was the abandoned old tires. This finding concurs with the previous studies conducted in Kilimanjaro and Ifakara in Tanzania and in Jaffna Peninsula, northern Sri Lanka, whereby the old tires were mentioned to be the preferred breeding ground for Ae. aegypti (Hertz et al. 2016; Surendran et al. 2021b; Kahamba et al. 2020).

The Breteau Index (BI) serves as a critical metric for understanding the prevalence of Aedes mosquitoes by measuring the percentage of inspected containers that harbor larvae or pupae. A previous study in Sri Lanka has proposed a BI value of 5 as the minimum threshold which does not require mosquito chemical control. If the BI value is between 5 and 20, and there are no reported cases, control should focus solely on reducing breeding sites without using chemical methods like fogging. In contrast, if there are reported dengue cases or if the BI exceeds 20, fogging is recommended (Udayanga et al. 2018). In Ilemela site, a BI of approximately 48.5% signifies that nearly half of the inspected containers were infested, suggesting a moderate level of mosquito activity and a considerable risk for mosquito-borne diseases. Conversely, Magu District, with a BI of 39.8%, demonstrates a relatively lower proportion of infested containers compared to Ilemela and Misungwi, but still presents a substantial concern for mosquito control efforts. Misungwi District, having the highest BI of around 60.8%, reveals the most severe infestation problem among the districts studied. This elevated BI reflects a high prevalence of mosquito larvae, underscoring the urgent need for intensified vector control measures in this area. Overall, these varying BI values highlight the diverse levels of mosquito infestation and the need for tailored intervention strategies to address the specific challenges in each district.

Conclusions

The present study has highlighted the notable differences in the insecticide susceptibility of Ae. aegypti in the urban and the rural settings, with full susceptibility in the urban area of Bwiru and resistance in the rural sites of Igekemaja and Mwagagala. This variation suggests that environmental factors and difference levels of insecticide exposure to this mosquito species could be driving the development of resistance in rural settings. These findings align with previous research indicating a widespread of pyrethroid resistance in Ae. aegypti, although localized differences, such as the susceptibility observed in Bwiru compared to Dar es Salaam, call for the need for monitoring of insecticide resistance. The findings about the key breeding habitats, such as the water storage containers in the rural areas and old tires in urban settings, highlights the importance of targeted environmental management and community engagement in vector control strategies to better control Ae. aegypti populations and mitigate the risk of mosquito-borne diseases. Knowledge obtained will likely help to provide guidance to the national vector control unit on Aedes and arboviral-borne emerging diseases management and preparedness in Tanzania.