Visceral leishmaniasis (VL), known as kala-azar in the Indian subcontinent, is a parasitic disease caused by the flagellated protozoan Leishmania that can be fatal if not treated promptly. The sandfly Phlebotomus argentipes is the only confirmed vector of VL in Southeast Asia, where it is controlled by indoor residual spraying (IRS), a synthetic insecticide. The use of DDT in VL control programmes has resulted in the development of resistance in sandflies, so DDT has been replaced by the insecticide alpha-cypermethrin. However, alpha-cypermethrin acts similarly to DDT, so the risk of resistance in sandflies increases under stress caused by repeated exposure to this insecticide. In this study, we assessed the susceptibility of wild mosquitoes and their F1 progeny using the CDC bottle bioassay.
We collected mosquitoes from 10 villages in Muzaffarpur district of Bihar, India. Eight villages continued to use high-potency cypermethrin for indoor spraying, one village stopped using high-potency cypermethrin for indoor spraying, and one village never used high-potency cypermethrin for indoor spraying. The collected mosquitoes were exposed to a pre-defined diagnostic dose for a defined time (3 μg/ml for 40 min), and the knockdown rate and mortality were recorded 24 h after exposure.
The kill rates of wild mosquitoes ranged from 91.19% to 99.47%, and those of their F1 generations ranged from 91.70% to 98.89%. Twenty-four hours after exposure, the mortality of wild mosquitoes ranged from 89.34% to 98.93%, and that of their F1 generation ranged from 90.16% to 98.33%.
The results of this study indicate that resistance may develop in P. argentipes, indicating the need for continued monitoring and vigilance to maintain control once eradication has been achieved.
Visceral leishmaniasis (VL), known as kala-azar in the Indian subcontinent, is a parasitic disease caused by the flagellated protozoan Leishmania and transmitted through the bite of infected female sand flies (Diptera: Myrmecophaga). Sand flies are the only confirmed vector of VL in Southeast Asia. India is close to achieving the goal of eliminating VL. However, to maintain low incidence rates after eradication, it is critical to reduce the vector population to prevent potential transmission.
Mosquito control in Southeast Asia is accomplished through indoor residual spraying (IRS) using synthetic insecticides. The secretive resting behaviour of the silverlegs makes it a suitable target for insecticide control through indoor residual spraying [1]. Indoor residual spraying of dichlorodiphenyltrichloroethane (DDT) under the National Malaria Control Programme in India has had significant spillover effects in controlling mosquito populations and significantly reducing VL cases [2]. This unplanned control of VL prompted the Indian VL Eradication Programme to adopt indoor residual spraying as the primary method of silverlegs control. In 2005, the governments of India, Bangladesh, and Nepal signed a memorandum of understanding with the goal of eliminating VL by 2015 [3]. Eradication efforts, involving a combination of vector control and rapid diagnosis and treatment of human cases, were aimed at entering the consolidation phase by 2015, a target subsequently revised to 2017 and then 2020.[4] The new global roadmap to eliminate neglected tropical diseases includes elimination of VL by 2030.[5]
As India enters the post-eradication phase of BCVD, it is imperative to ensure that significant resistance to beta-cypermethrin does not develop. The reason for the resistance is that both DDT and cypermethrin have the same mechanism of action, namely, they target the VGSC protein[21]. Thus, the risk of resistance development in sandflies may be increased by stress caused by regular exposure to highly potent cypermethrin. It is therefore imperative to monitor and identify potential sandfly populations resistant to this insecticide. In this context, the objective of this study was to monitor the susceptibility status of wild sandflies using diagnostic doses and exposure durations determined by Chaubey et al. [20] studied P. argentipes from different villages in Muzaffarpur district of Bihar, India, which continuously used indoor spraying systems treated with cypermethrin (continuous IPS villages). The susceptibility status of wild P. argentipes from villages that had stopped using cypermethrin-treated indoor spraying systems (former IPS villages) and those that had never used cypermethrin-treated indoor spraying systems (non-IPS villages) were compared using the CDC bottle bioassay.
Ten villages were selected for the study (Fig. 1; Table 1), of which eight had a history of continuous indoor spraying of synthetic pyrethroids (hypermethrin; designated as continuous hypermethrin villages) and had VL cases (at least one case) in the last 3 years. Of the remaining two villages in the study, one village that did not implement indoor spraying of beta-cypermethrin (non-indoor spraying village) was selected as the control village and the other village that had intermittent indoor spraying of beta-cypermethrin (intermittent indoor spraying village/former indoor spraying village) was selected as the control village. The selection of these villages was based on coordination with the Health Department and the Indoor Spraying Team and validation of the Indoor Spraying Micro Action Plan in Muzaffarpur District.
Geographical map of Muzaffarpur district showing the locations of villages included in the study (1–10). Study locations: 1, Manifulkaha; 2, Ramdas Majhauli; 3, Madhubani; 4, Anandpur Haruni; 5, Pandey; 6, Hirapur; 7, Madhopur Hazari; 8, Hamidpur; 9, Noonfara; 10, Simara. The map was prepared using QGIS software (version 3.30.3) and Open Assessment Shapefile.
The bottles for the exposure experiments were prepared according to the methods of Chaubey et al. [20] and Denlinger et al. [22]. Briefly, 500 mL glass bottles were prepared one day before the experiment and the inner wall of the bottles was coated with the indicated insecticide (the diagnostic dose of α-cypermethrin was 3 μg/mL) by applying an acetone solution of the insecticide (2.0 mL) to the bottom, walls and cap of the bottles. Each bottle was then dried on a mechanical roller for 30 min. During this time, slowly unscrew the cap to allow the acetone to evaporate. After 30 min of drying, remove the cap and rotate the bottle until all the acetone has evaporated. The bottles were then left open to dry overnight. For each replicate test, one bottle, used as a control, was coated with 2.0 mL of acetone. All bottles were reused throughout the experiments after appropriate cleaning according to the procedure described by Denlinger et al. and the World Health Organization [ 22 , 23 ].
On the day after insecticide preparation, 30–40 wild-caught mosquitoes (starved females) were removed from the cages in vials and gently blown into each vial. Approximately the same number of flies were used for each insecticide-coated bottle, including the control. Repeat this at least five to six times in each village. After 40 minutes of exposure to the insecticide, the number of flies knocked down was recorded. All flies were captured with a mechanical aspirator, placed in pint cardboard containers covered with fine mesh, and placed in a separate incubator under the same humidity and temperature conditions with the same food source (cotton balls soaked in 30% sugar solution) as the untreated colonies. Mortality was recorded 24 hours after exposure to the insecticide. All mosquitoes were dissected and examined to confirm species identity. The same procedure was performed with the F1 offspring flies. Knockdown and mortality rates were recorded 24 h after exposure. If mortality in the control bottles was < 5%, no mortality correction was made in the replicates. If mortality in the control bottle was ≥ 5% and ≤ 20%, mortality in the test bottles of that replicate was corrected using Abbott’s formula. If mortality in the control group exceeded 20%, the entire test group was discarded [24, 25, 26].
Mean mortality of wild-caught P. argentipes mosquitoes. Error bars represent standard errors of the mean. The intersection of the two red horizontal lines with the graph (90% and 98% mortality, respectively) indicates the mortality window in which resistance may develop.[25]
Mean mortality of F1 progeny of wild-caught P. argentipes. Error bars represent standard errors of the mean. The curves intersected by the two red horizontal lines (90% and 98% mortality, respectively) represent the range of mortality over which resistance may develop[25].
Mosquitoes in the control/non-IRS village (Manifulkaha) were found to be highly sensitive to the insecticides. The mean mortality (±SE) of wild-caught mosquitoes 24 h after knockdown and exposure was 99.47 ± 0.52% and 98.93 ± 0.65%, respectively, and the mean mortality of F1 offspring was 98.89 ± 1.11% and 98.33 ± 1.11%, respectively (Tables 2, 3).
The results of this study indicate that silver-legged sand flies may develop resistance to the synthetic pyrethroid (SP) α-cypermethrin in villages where the pyrethroid (SP) α-cypermethrin was used routinely. In contrast, silver-legged sand flies collected from villages not covered by the IRS/control programme were found to be highly susceptible. Monitoring the susceptibility of wild sand flies populations is important for monitoring the effectiveness of insecticides used, as this information may help in managing insecticide resistance. High levels of DDT resistance have been regularly reported in sand flies from endemic areas of Bihar due to historical selection pressure from the IRS using this insecticide [ 1 ].
We found P. argentipes to be highly sensitive to pyrethroids, and field trials in India, Bangladesh and Nepal showed that IRS had high entomological efficacy when used in combination with cypermethrin or deltamethrin [19, 26, 27, 28, 29]. Recently, Roy et al. [18] reported that P. argentipes had developed resistance to pyrethroids in Nepal. Our field susceptibility study showed that silverlegged sand flies collected from non-IRS exposed villages were highly susceptible, but flies collected from intermittent/former IRS and continuous IRS villages (mortality ranged from 90% to 97% except for sand flies from Anandpur-Haruni which had 89.34% mortality at 24 h post exposure) were likely resistant to highly effective cypermethrin [25]. One possible reason for the development of this resistance is the pressure exerted by indoor routine spraying (IRS) and case-based local spraying programs, which are standard procedures for managing kala-azar outbreaks in endemic areas/blocks/villages (Standard Operating Procedure for Outbreak Investigation and Management [30]. The results of this study provide early indications of the development of selective pressure against the highly effective cypermethrin. Unfortunately, historical susceptibility data for this region, obtained using the CDC bottle bioassay, are not available for comparison; all previous studies have monitored P. argentipes susceptibility using WHO insecticide-impregnated paper. The diagnostic doses of insecticides in the WHO test strips are the recommended identification concentrations of insecticides for use against malaria vectors (Anopheles gambiae), and the operational applicability of these concentrations to sandflies is unclear because sandflies fly less frequently than mosquitoes, and spend more time in contact with the substrate in the bioassay [23].
Synthetic pyrethroids have been used in VL endemic areas of Nepal since 1992, alternating with the SPs alpha-cypermethrin and lambda-cyhalothrin for sandfly control [31], and deltamethrin has also been used in Bangladesh since 2012 [32]. Phenotypic resistance has been detected in wild populations of silverlegged sandflies in areas where synthetic pyrethroids have been used for a long time [ 18 , 33 , 34 ]. A non-synonymous mutation (L1014F) has been detected in wild populations of the Indian sandfly and has been associated with resistance to DDT, suggesting that pyrethroid resistance arises at the molecular level, as both DDT and the pyrethroid (alpha-cypermethrin) target the same gene in the insect nervous system [17, 34]. Therefore, systematic assessment of cypermethrin susceptibility and monitoring of mosquito resistance are essential during the eradication and post-eradication periods.
A potential limitation of this study is that we used the CDC vial bioassay to measure susceptibility, but all comparisons used results from previous studies using the WHO bioassay kit. Results from the two bioassays may not be directly comparable because the CDC vial bioassay measures knockdown at the end of the diagnostic period, whereas the WHO kit bioassay measures mortality at 24 or 72 hours post-exposure (the latter for slow-acting compounds) [35]. Another potential limitation is the number of IRS villages in this study compared to one non-IRS and one non-IRS/former IRS village. We cannot assume that the level of mosquito vector susceptibility observed in individual villages in one district is representative of the level of susceptibility in other villages and districts in Bihar. As India enters the post-elimination phase of leukemia virus, it is imperative to prevent significant development of resistance. Rapid monitoring of resistance in sandfly populations from different districts, blocks and geographic areas is required. The data presented in this study are preliminary and should be verified by comparison with the identification concentrations published by the World Health Organization [35] to get a more specific idea of the susceptibility status of P. argentipes in these areas before modifying vector control programmes to maintain low sandfly populations and support leukemia virus elimination.
The mosquito P. argentipes, the vector of the leukosis virus, may begin to show early signs of resistance to the highly effective cypermethrin. Regular monitoring of insecticide resistance in wild populations of P. argentipes is necessary to maintain the epidemiological impact of vector control interventions. Rotation of insecticides with different modes of action and/or evaluation and registration of new insecticides is necessary and recommended to manage insecticide resistance and support the elimination of leukosis virus in India.
Post time: Feb-17-2025