The present study establishes insecticide susceptibility profiles of Aedes albopictus adult populations from 4 hot springs in Selangor, Malaysia, against 7 pyrethroids through an adult mosquito susceptibility bioassay. All Ae. albopictus populations were subjected to a 1-h exposure to each pyrethroid following the World Health Organization. The mortalities were recorded at 60 min of exposure to bifenthrin, 30 min for other pyrethroids, and 24 h posttreatment for all pyrethroids. Complete mortalities were observed upon exposures to the pyrethroids under 60 min and at 24 h posttreatment, excluding permethrin 0.25%, alpha-cypermethrin 0.05%, and bifenthrin 0.2%. These findings indicated that permethrin, deltamethrin, lambda-cyhalothrin, cyfluthrin, and etofenprox possess the recommended pyrethroid adulticide active ingredients that could be applied in vector control programs at these hot springs in the future. Nevertheless, the application of pyrethroids should be carefully monitored in rotation with other insecticide classes, including organophosphates and carbamates to avoid the development of insecticide resistance among mosquito vectors towards all insecticides. Although there were no reported cases of Aedes-borne pathogens at these hot springs to date, the current study results could still assist the Malaysian health authorities in determining approaches to control Aedes populations in these hot springs, if required in the future.
In recent decades, the spread of mosquito-borne arboviral diseases, namely, dengue, chikungunya, and Zika, have increased dramatically on a global scale. The mosquito-borne arboviral pathogens are transmitted through the bite of infected Aedes aegypti (L.) and Ae. albopictus (Skuse) mosquitoes (Peinado et al. 2022). High human morbidity and mortality rates due to these diseases have triggered public health concerns worldwide.
The control of mosquito-borne arboviral diseases depends on vector management due to the shortcomings of effective treatment or vaccines for the illnesses. Reducing human-mosquito interactions is also the essence of an effective control strategy. Nonetheless, chemical insecticides remain the primary vector control tools (Fansiri et al. 2022) despite the numerous mosquito control approaches, such as source reduction, habitat manipulation, and biological larvicide applications, delineated by the World Health Organization (WHO 2012).
Aedes aegypti and Ae. albopictus tend to blood feed on human hosts over other warm-blooded species (Kamgang et al. 2012, Pruszynski et al. 2020). Aedes aegypti is predominantly related to human population densities, while Ae. albopictus abundance coincides with vegetation (Tsai and Teng 2016, Dalpadado et al. 2022). Consequently, mosquito control operations are conducted in human habitation and public recreational sites typically surrounded by vegetation following the increased risk of mosquito-borne pathogen transmissions in these areas.
Hot springs are natural geothermal water environments that can be found worldwide. Humans visit hot springs to bathe in the hot water for therapeutic and stress-relieving purposes. Therefore, visitor traffic is commonly heavy, especially during weekends and holidays. Furthermore, ornamental plants and dense vegetation within the hot spring environments offer harborage for certain mosquito species, including Ae. albopictus.
Aedes albopictus reportedly prevail in outdoor vegetation (Dalpadado et al. 2022), leading to expectations of mosquito-borne infections in hot spring environments. In Malaysia, several hot springs scattered in some states, including Selangor, have become popular attractions among local and foreign tourists. The hot springs are located in suburban and rural areas and primarily surrounded by vegetation. In an unpublished study, Ae. albopictus was the most common mosquito species collected through ovitrapping performed in all 4 hot springs selected in the current study.
Mosquito vectors and humans co-occurrences create a critical necessity for effective mosquito control strategies around hot springs to prevent the pathogen transmissions by mosquitoes, particularly Aedes vectors. Selecting the precise insecticides to be employed in vector control operations at each hot spring is based on the susceptibility profiles of targeted mosquito species. Nonetheless, no susceptibility investigations involving mosquitoes from any of the hot springs in Malaysia have been performed. The current study aimed to determine the most suitable insecticide(s) to control the outdoor vector Ae. albopictus at selected hot springs in Selangor, Malaysia, according to their susceptibility profiles against the active ingredients (AIs) of different pyrethroids.
MATERIALS AND METHODS
Four hot springs popular among tourists were selected as the localities in this study: Selayang (SEL), Hulu Tamu Batang Kali (HTBK), Kuala Kubu Bharu (KKB), and Kerling (KERL). The hot springs are open to visitors daily. The study sites are located in the Gombak and Hulu Selangor districts in Selangor, Malaysia. The hot spring points in the areas are encircled by concrete pools, while their surroundings are skirted by forest vegetation. Table 1 summarizes the geographical descriptions of the sampling sites.
The Ae. albopictus reference strain
The present study utilized the Ae. albopictus laboratory strain (F80) as a reference strain, which was initially collected from Selangor, Malaysia, and reared in the insectarium of the Institute for Medical Research (IMR), Kuala Lumpur, Malaysia, for almost 20 yr. The Ae. albopictus laboratory strain has never been exposed to any insecticide since its colonization in the IMR insectarium.
The Ae. albopictus field population sampling
This study employed ovitrapping during Ae. albopictus field sampling (Lee 1992, Ministry of Health Malaysia 1997), where 50 ovitraps of 9.1 cm (height) × 6.8 cm (diam) were set in each area. Each ovitrap contained 10% hay infusion water (Wan-Norafikah et al. 2019) and an oviposition paddle. The ovitraps were placed randomly within the hot spring compound, including under the ornamental plants, near vegetation, and under the concrete benches. The ovitraps were deployed for 5 days to allow female adult mosquitoes to lay eggs before being retrieved and returned to the laboratory.
Rearing and identification of Ae. albopictus adults
The contents of the ovitraps retrieved in the present study were poured into different containers. Liver powder and small semicooked ox liver pieces were then provided to the hatched Ae. albopictus larvae in the containers. All larvae were reared to adulthood, which were identified to species based on the taxonomic and pictorial identification keys of Jeffery et al. 2012). Only Ae. albopictus adults (F0) were conserved in the current study, permitted to mate and blood feed to obtain the F1 progeny for use in adult mosquito susceptibility bioassay. The Ae. albopictus laboratory strain and hot spring populations were maintained in identical environments within the insectarium at 27 ± 2°C and 75 ± 10% RH.
In the adult mosquito susceptibility bioassay, impregnated papers of 7 pyrethroids: permethrin 0.25% and 0.75%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, cyfluthrin 0.15%, etofenprox 0.5%, alpha-cypermethrin 0.05%, and bifenthrin 0.2% were utilized. The impregnated papers were procured from the WHO Collaborating Centre, Vector Control Research Unit (VCRU), Universiti Sains Malaysia (USM), Penang, Malaysia, which were primarily produced by the VCRU at WHO-recommended diagnostic doses (WHO 1992, 1998, 2016; Table 2).
Adult mosquito susceptibility bioassay
The current study used the adult mosquito susceptibility bioassay outlined by the World Health Organization (WHO 2016). Twenty-five sucrose-fed 3–5-day-old adult female mosquitoes were held in a holding tube for 1 h to confirm their health. The mosquitoes were then transferred into an exposure tube lined with a pyrethroid-impregnated paper of a specific diagnostic dose and left exposed to the pyrethroid AI for 1 h. A silicone oil-impregnated paper produced by VCRU was also utilized in an exposure tube to serve as control. The bioassay in this study was performed in quadruplicate. Mosquito mortality rates were recorded every minute throughout the exposure period. Mosquitoes incapable of stably flying or resting were considered dead. Subsequently, the mosquitoes were transferred into holding paper cups and provided with 10% sucrose-soaked cotton balls. Cumulative mortality results were taken at 24 h postexposure.
The raw mortality data generated in this study were also assessed for normality with the Shapiro-Wilk evaluation. The mortality percentages were employed to reveal any significant differences between the Ae. albopictus populations via the one-way ANOVA and post hoc analyses, while the Pearson correlation assessment ascertained any significant associations between different pyrethroid AIs. A >0.4 (r > 0.4, P ≤ 0.05) correlation value (r) denoted significant cross-resistance, whereas an r of >0.8 showed strong cross-resistance between 2 pyrethroid AIs. The statistical analyses in this study were performed with IBM SPSS Statistics version 23.0 with a P = 0.05 significance.
The susceptibility of Ae. albopictus adult mosquitoes of the reference strain and those of the hot spring study localities against several pyrethroid adulticide AIs were evaluated through a 1 h exposure in adult bioassays. Based on the normality test conducted, all raw mortality data produced in this study were normally distributed (P > 0.05).
The 50% lethal time (LT50) values of the Ae. albopictus adult populations in the current study exposed to 0.25% and 0.75% permethrin ranged from 45.69 min to 85.48 min and 19.78 min to 23.86 min, respectively (Table 3). The resistance ratios for all field populations subjected to both permethrin concentrations were also below 1.00. Meanwhile, all Ae. albopictus adult populations exposed to deltamethrin 0.05%, lambda-cyhalothrin 0.05%, cyfluthrin 0.15%, and etofenprox 0.5% generated LT50 values within approximately similar ranges (16.90 min to 33.46 min). The resistance ratios of the mosquitoes exposed to the pyrethroid AIs were under 1.50.
Exposing the Ae. albopictus adult populations to alpha-cypermethrin 0.05% resulted in a higher LT50 range (23.48 min to 38.78 min) with resistance ratios of 1.02 or below. Conversely, the LT50 values (70.83 min to 237.83 min) for bifenthrin 0.2% were obtained only from samples collected in SEL, HTBK, and KERL hot springs, as only a few mosquitoes died during the exposure. The mortality value of Ae. albopictus of the reference strain and Kuala Kubu Bharu (KKB) hot spring exposed to the same pyrethroid AI did not reach the minimum of 5.00% (P = 0.05), thus, rendering their LT50 values indeterminable.
Within the 1-h exposure period, the cyfluthrin 0.15% exposure exhibited the fastest effects in causing 50.00% mortalities among the Ae. albopictus adult populations. In contrast, permethrin 0.25% and bifenthrin 0.2% recorded the slowest effects in exterminating 50.00% of each Ae. albopictus adult population within the same exposure period.
In this study, mortality percentages were calculated at 30 min of exposure to all pyrethroids, excluding bifenthrin 0.2%, which was determined at 60 min (Table 4). The Ae. albopictus adult populations subjected to permethrin 0.25% recorded not more than 10.00% mortality after 30 min. Nonetheless, the mortality increased between 76.00% and 96.00% upon exposure to permethrin 0.75%.
The Ae. albopictus populations subjected to deltamethrin 0.05%, lambda-cyhalothrin 0.05%, cyfluthrin 0.15%, and etofenprox 0.5% for 30 min documented 36.00% to 99.00% mortality. In contrast, the mortality percentages of all Ae. albopictus adult populations exposed to alpha-cypermethrin 0.05% were below 32.00% except for HTBK, at 85.00%, within a similar time frame. Nevertheless, the mortality percentage of the Ae. albopictus adult populations exposed to bifenthrin 0.2% were much lower, 2.00% to 27.00%.
The Ae. albopictus adult populations exposed to cyfluthrin 0.15% demonstrated the highest mortalities after 30 min of exposure, while the lowest were demonstrated among Ae. albopictus adult populations exposed to permethrin 0.25%. On the other hand, under 30.00% mortality of the populations evaluated was documented by bifenthrin 0.2% at 1-h exposure.
The Ae. albopictus reference strain adults demonstrated full susceptibility to almost all pyrethroid AIs after 24 h of recovery period, excluding the mosquitoes subjected to permethrin 0.25% exposure (51.00%) and bifenthrin 0.2% (67.00%; Table 5). The Ae. albopictus adults from SEL, HTBK, and KKB exhibited possible resistance against permethrin 0.25%, while the Ae. albopictus adults from KERL were resistant to permethrin 0.25%. All Ae. albopictus field populations revealed full susceptibility to permethrin 0.75%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, cyfluthrin 0.15%, and etofenprox 0.5%.
After the 24-h recovery period, only the Ae. albopictus adults from SEL demonstrated possible resistance against alpha-cypermethrin 0.05%, while the rest indicated susceptibility. On the other hand, only Ae. albopictus adults from SEL were fully susceptible to bifenthrin 0.2%, while Ae. albopictus adults from the other hot springs exhibited resistance against bifenthrin 0.2% 24 h postexposure (86.00% to 97.00%).
Based on the one-way ANOVA performed in the present study, significant differences (P ≤ 0.05) in mortality percentages at 30-min or 60-min and 24-h postexposure were documented in only a few pyrethroid exposures. Significant LT50 correlations were revealed between permethrin 0.75% and deltamethrin 0.05% (r = 0.909, P = 0.032), deltamethrin 0.05% and cyfluthrin 0.15% (r = 0.885, P = 0.046), and lambda-cyhalothrin 0.05% and cyfluthrin 0.15% (r = 0.965, P = 0.008), indicating intraclass cross-resistance between the pyrethroids involved.
According to WHO (2016), the mortality percentage of adult mosquitoes should be determined at 24-h postexposure to an insecticide AI. In the current study, Ae. albopictus populations subjected to adulticide pyrethroid AIs except for permethrin 0.25%, alpha-cypermethrin 0.05%, and bifenthrin 0.2% resulted in 100.00% mortalities at the end of 1-h exposure and 24-h posttreatment. Consequently, the shortest or longest time required by the populations to be affected by the pyrethroid could not be determined in this study. As a result, besides mortality percentages at 24-h postexposure, the current work recorded mortality percentages at 30-min exposure for all AIs except for bifenthrin 0.2%, where the Ae. albopictus adult populations did not demonstrate complete mortalities after 1 h.
As for permethrin 0.25% exposure, the mortality percentages were noted at 30-min exposure time so that these data were comparable with mortality percentages for permethrin 0.75% exposure. The mortality percentage of Ae. albopictus adult population from HTBK subjected to alpha-cypermethrin 0.05% was also logged at 30-min exposure, as the population exhibited complete mortality before the end of the exposure time.
The current study employed 0.25% and 0.75% permethrin. Initially, the higher concentration of permethrin impregnated papers was utilized as it was the only concentration available from VCRU. The permethrin 0.25% impregnated papers were later available following the WHO (2016) recommended diagnostic dose for Aedes mosquitoes. Nevertheless, WHO established a 0.4% diagnostic dose for permethrin in 2022, which was revealed after the completion of this study.
The results in the current study indicated that permethrin, deltamethrin, lambda-cyhalothrin, cyfluthrin, and etofenprox are suitable pyrethroid adulticide AIs and could be employed in vector control activities at the selected hot springs if required. The Ministry of Health Malaysia has also utilized some of the pyrethroids during vector control space treatments (Ong 2016). Nevertheless, the details on the application are currently unavailable for public access.
Although exposure to alpha-cypermethrin 0.05% resulted in various levels of susceptibility at 30 min of exposure, the mortality percentages recorded by the Ae. albopictus adult populations increased to over 97.00% at the end of 24-h posttreatment. The application of alpha-cypermethrin-based products could still be considered at selected hot springs if required. Nevertheless, alpha-cypermethrin-based substances necessitate a longer time to reach a high mortality compared to other pyrethroid AIs. Furthermore, the use of alpha-cypermethrin-based products in SEL and KERL should be closely monitored as the Ae. albopictus adult populations were no longer fully susceptible to the pyrethroid AI.
Employing the pyrethroids at the hot springs selected in this study should be conducted in rotation with other classes of insecticides, such as organophosphates, that are also utilized in the Malaysian vector control program to prevent or delay insecticide resistance among mosquito vectors. Alpha-cypermethrin has also been employed in indoor residual spraying (IRS) to combat malaria and visceral leishmaniasis in countries including India (Saurabh et al. 2020, Mishra et al. 2021), but information on its application in Malaysian vector control strategies is inadequate.
The Ae. albopictus adult populations exposed to bifenthrin 0.2% documented very low mortality percentages by the end of 1 h. Although the figures rose at 24-h posttreatment, the numbers were still less than the results obtained for alpha-cypermethrin 0.05%. If required in the future, bifenthrin could be utilized as an adulticide at the study sites. Nonetheless, careful considerations are necessary as the mosquitoes in this study demonstrated resistance to bifenthrin although only at diagnostic dosage.
Several reports in other countries have demonstrated the effectiveness of bifenthrin as a residual insecticide in barrier spray treatments on foliage and vegetation, thus providing an option for mosquito control (Fulcher et al. 2015, VanDusen et al. 2016). In Malaysia, employing bifenthrin as an adulticidal, larvicidal, and wall residual agent was significantly effective against laboratory-reared Ae. aegypti, Ae. albopictus, and Culex quinquefasciatus Say (Lee et al. 1997, Sulaiman et al. 2008). Nevertheless, information on the application of bifenthrin in vector control programs held by the Ministry of Health Malaysia and other local health authorities remains unspecified.
The significant intraclass cross-resistance between permethrin and deltamethrin, deltamethrin and cyfluthrin, and lambda-cyhalothrin and cyfluthrin suggested that similar metabolic enzymes or target-site mutation were involved in the resistance mechanisms against the pyrethroid AIs among Ae. albopictus adult populations assessed in this study.
The Ae. albopictus laboratory strain from IMR was the best option as a reference strain in the present study, considering that IMR is a research arm of the Ministry of Health Malaysia. Furthermore, access to susceptible Ae. albopictus strains from any well-established laboratories were not available.
Although the Ae. albopictus laboratory strain required longer than 60-min exposure time to 24-h postexposure to be affected by the insecticide AIs, the Ae. albopictus laboratory strain employed in the present study was completely susceptible to numerous other commonly available AIs (Elia-Amira et al. 2019, Lau et al. 2021, Wan-Norafikah et al. unpublished data). Nevertheless, continuous monitoring of its susceptibility against insecticide AIs and precautionary actions have been performed by IMR to maintain or enhance the sensitivity of the strain against the insecticide AIs and prevent further insecticide resistance development in the future.
Generally, the hot springs chosen in this study are equipped with similar essential facilities, such as concrete benches around the hot spring spots, resting gazebos, and washrooms. In terms of location, the KERL site is the only study area located marginally remote from any human dwelling. The other hot springs are situated in proximity to human housing areas, which are more prone to insecticide exposures during vector control operations. Nevertheless, no significant differences in susceptibility levels between the Ae. albopictus adult population from KERL and Ae. albopictus adult populations from other hot springs and laboratory strains against the same pyrethroid AIs were observed regardless of its unique location.
Close monitoring of the abundance and susceptibility levels of the mosquito vectors from the hot springs against insecticides is necessary because of the dense vegetation around the localities. The lush foliage, especially at KERL, could provide conducive breeding and resting habitats for numerous mosquito species, including Ae. albopictus. Consequently, possibilities of mosquito-borne infections spreading are increased in the areas assessed in this study.
The susceptibility status of Ae. albopictus adult populations in urban and rural neighborhoods and agricultural plantations against similar adulticide pyrethroid AIs has been reported by researchers globally. For instance, adult Ae. albopictus mosquitoes from the USA, Laos, and Papua New Guinea were susceptible to deltamethrin 0.05%, while its counterpart in Fanar, Lebanon, recorded susceptibility to permethrin 0.25% and lambda-cyhalothrin 0.03% (Marcombe et al. 2014, Tangena et al. 2018, Demok et al. 2019, Haddad et al. 2022). In another report, the Ae. albopictus adult populations from Andaman and Nicobar Islands in India demonstrated susceptibility to deltamethrin 0.05%, but resistance to permethrin 0.75%, lambda-cyhalothrin 0.05%, and cyfluthrin 0.15% (Sivan et al. 2015).
Two Ae. albopictus adult populations from Bangui, Africa, and several adult populations of the same species from Yaounde and Douala in Cameroon were reportedly resistant to deltamethrin 0.03%, 0.05%, alpha-cypermethrin 0.05%, and permethrin 0.75% (Ngoagouni et al. 2016, Kamgang et al. 2017, Yougang et al. 2022). Similarly, resistance against permethrin 0.75% was also recorded among 2 Italian Ae. albopictus populations (Pichler et al. 2018).
A study revealed that adulticide pyrethroid AIs required between 1 to 24 h to demonstrate their full effects on 14 Ae. albopictus adult populations collected from different districts in Sabah, Malaysia. Although the populations recorded under 90.00% mortalities at 60-min exposure to permethrin 0.75% and to etofenprox 0.50%, it improved to 100.00% mortality at 24-h posttreatment (Elia-Amira et al. 2019). Meanwhile, a majority of the 13 Ae. albopictus adult populations obtained from different districts in Sarawak were resistant to etofenprox 0.50%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, and permethrin 0.25% (Lau et al. 2021). Conversely, the populations were fully susceptible to cyfluthrin 0.15% (Lau et al. 2021). In another local study, 11 Ae. albopictus adult populations collected from dengue hotspots in Kuala Lumpur and Selangor documented resistance to deltamethrin 0.03% and permethrin 0.25% (Rasli et al. 2021).
Although no mosquito-borne disease case has been reported from the hot springs selected in this study, the susceptibility levels of Ae. albopictus populations should still be considered and utilized to delineate forthcoming plans for tackling Aedes-borne diseases. This is the first study to record the susceptibility status of mosquito vectors captured in the hot springs. Consequently, more studies involving mosquito vectors from commonly visited hot springs are needed.
The authors thank the Medical Entomology Unit, Institute for Medical Research, Ministry of Health Malaysia, for supplying the reference strain of mosquitoes. The technical assistance provided by the staff in the Laboratory Animal Care Unit, Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, throughout this study is also appreciated. This present study was funded under the Dana UiTM Cawangan Selangor Research Grant (DUCS 2.0) [600-UiTMSEL (PI. 5/4) (021/2020)] by the UiTM Selangor Branch.
Faculty of Medicine, Universiti Teknologi MARA (UiTM), Selangor Branch, Sungai Buloh Campus, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia.
HW ReNeU, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia.
Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Selangor Branch, Sungai Buloh Campus, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia.
Medical Entomology Unit, WHO Collaborating Centre for Vectors, Institute for Medical Research (IMR), Ministry of Health Malaysia, National Institute of Health, Block C, Jalan Setia Murni U13/52, Seksyen U13, Setia Alam, 40170 Shah Alam, Selangor, Malaysia.