Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Abstract

Thirty-two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda-cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda-cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda-cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities. Dengue/dengue hemorrhagic fever is one of the most serious and important resurgent tropical diseases worldwide (Gubler 1998). Dengue is the most common vector-borne viral disease globally, primarily affecting developing countries of tropical and subtropical regions, with an estimated 2.5 to 3 billion people at risk of infection and resulting in approximately 50 million dengue infections annually (Guzman et al. 2010). Aedes aegypti (L.) and Aedes albopictus (Skuse) are the primary vectors of dengue viruses in Southeast Asia, a region that historically represents the epicenter of transmission and disease occurrence (Gubler 1998). Both species are primarily day-biting mosquitoes that share many of the same behavioral attributes. Ae. aegypti is more prevalent near and inside human dwellings and is the principal vector in urban, more densely populated zones, whereas Ae. albopictus is more peridomestic and more closely associated with rural and less congested suburban areas. Historically, Ae. aegypti is often associated with the epidemic spread of dengue viruses; Ae. albopictus typically provides a secondary role in outbreak transmission and maintaining rural endemic cycles in humans. Both mosquito species also play a comparable dual role in the transmission of chikungunya virus, a disease that has recently emerged in a more frequent epidemic form in Asia, Indian Ocean countries, and southern Europe (Charrel et al. 2007). Effective control of these two mosquito species has proven extremely difficult with both vector and virus remaining entrenched and expanding in many disease endemic areas. Lacking an effective vaccine, vector control methods attacking both larval habitats and adult mosquito populations remains the primary method for reducing risk of dengue infection (Kongmee et al. 2004, Polsomboon et al. 2008). Unfortunately, control strategies aimed at elimination of preferred vector larval habitats through source reduction or periodic application of insecticides to water sources has often met with failure to sufficiently control these two species (Gubler 1998, Kongmee et al. 2004). Furthermore, control of adult mosquitoes using a variety of chemical means is fraught with complications including high cost, slow operational response, ineffective timing of application, low efficacy, and evolution of resistance to insecticides. However, the reduction of vector densities alone has often proven insufficient. A better understanding of the complex interplay of diverse factors, including site-specific social determinants, is critical for promoting a broader public health response leading to successful vector control management practices using routine larviciding and focal space spraying. Nevertheless, insecticides continue to play a crucial, if not indispensible, role in helping to reduce the risk of dengue transmission by complementary reduction of immature and adult vector mosquitoes (Grieco et al. 2007, Jirakanjanakit et al. 2007, Thanispong et al. 2008). Synthetic insecticides of various chemical classes have long been used in national public health vector control programs (Roberts and Andre 1994, Reiter and Gubler 1997), the vast majority of which today are pyrethroid-based formulations. In Thailand, combinations of pyrethroids are commonly used by home owners to control mosquitoes and other arthropod pests. Applied at relatively low concentrations, pyrethroids are considered to be effective and relatively safe compounds for controlling common house-frequenting mosquitoes, especially Ae. aegypti and Culex quinquefasciatus (Chareonviriyaphap et al. 1999, Somboon et al. 2003). Since 1994, deltamethrin has been used intensively in organized public health programs in Thailand to attempt interruption of dengue transmission following reports of outbreaks. Numerous permethrin and deltamethrin-based formulations (e.g., aerosols, coils, and gels) are commercially available to the general public but much less so for lambda-cyhalothrin (Paeporn et al. 1996, Chareonviriyaphap et al. 1999). The selection for resistance to pyrethroids by mosquitoes is largely attributed to frequent exposure to sub-lethal concentrations of commonly applied chemicals and has a direct bearing on the effective management and prevention of vector-borne diseases (Hemingway and Ranson 2000). The majority of published reports on pyrethroid resistance in Ae. aegypti and Ae. albopictus populations in Thailand have been restricted in geographical scope (Chadwick et al. 1977, Paeporn et al. 2004, Chareonviriyaphap et al. 1999, Yaicharoen et al. 2005, Sathantriphop et al. 2006, Jirakanjanakit et al. 2007, Thanispong et al. 2008). Without a better understanding of the temporal effects and outcomes of insecticide use on mosquito susceptibility, sustainable and successful vector control activities remain vulnerable. Routine monitoring of insecticide susceptibility integrated into control programs allow for more timely response and greater efficiency in controlling specific mosquito vectors. We report herein the susceptibility of Ae. aegypti collected across Thailand and Ae. albopictus collected in southern Thailand to three common synthetic pyrethroids used to control adult mosquitoes. Since a high level of permethrin resistance in Ae. aegypti was reported from Baan Suan, Nonthaburi Province (Sathantriphop et al. 2006), a decision to use a three-fold greater concentration (0.75%) was made in this study. Collection sites for Ae. aegypti were selected based on reported dengue cases in 2008. Ae. aegypti larvae and pupae were collected from containers located in and around houses in 32 separate sites (29 provinces) throughout Thailand in 2008 (Figure 1). Additionally, Ae. albopictus was collected as immature stages in rubber plantation areas from five collections located in four provinces of southern Thailand (Figure 1). Geo-coordinates of each site are provided in Table 1. Aedes aegypti and Aedes albopictus collection sites by province in Thailand. Field-collected larvae and pupae were reared in the insectary to the adult stage for morphological identification and colonization. Two fully susceptible populations of Ae. aegypti (USDA, USA) and Ae. albopictus (MOPH, Thailand) were used as a reference baseline for susceptibility to permethrin, deltamethrin, and lambda-cyhalothrin. The USDA laboratory population was provided by the Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL, U.S.A. This population has been continuously colonized for over 40 years. Aedes albopictus was originally collected from Nonthaburi Province and has been maintained in a colony at the MOPH for approximately ten years. All mosquito populations were carefully segregated to prevent contamination from cross-mating and reared under identical laboratory-controlled conditions at 25+5° C and 80+10%) in the Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand (Kongmee et al. 2004). Adult mosquitoes were allowed to mate and females permitted to blood feed on a live guinea pig beginning on the fourth day post-emergence. Moistened oviposition substrates were provided for females to deposit eggs. Following conditioning, eggs were rehydrated and allowed to hatch in pans reserved for each population. Subsequent susceptibility tests were restricted to F1-F3 generation female mosquitoes. Three synthetic pyrethroids (without addition of piperonyl butoxide or other synergists) were used in susceptibility testing. 1. Permethrin 92% pure active ingredient (a.i.) [IUPAC (3-phenoxybenzyl (1 RS, 3 RS, 1RS, 3 SR)-3 –(2, 2-dichlorovinyl)-2, 2-dimethylcyclopropanecarboxylate)] provided by Ladda Company, Bangkok, Thailand (June, 2008). 2. Deltamethrin 98% a.i. [IUPAC (S)-alpha-cyano-3-phenoxybenzyl (1R,3R)-3-(2,2-dibromovinyl)-2,2 dimethylcyclopropanecarboxylate] obtained from BASF (August, 2008), and 3. Lambda-cyhalothrin 98% a.i. [IUPAC (S)-alpha-cyano-3-phenoxybenzyl (Z)-(1R,3R)-3-(2-chloro-3,3,3-trifluropropenyl)-2,2-dimethylcyclopropanecarboxylate and (R)-alpha-cyano-3- phenoxybenzyl (Z)-(1S,3S)-3-(2-chloro-3,3,3-trifluropropenyl)-2,2-dimethyl cyclopropanecarboxylate] received from Syngenta Crop Protection, Thailand (June, 2008). Insecticide treated papers were produced at the Department of Entomology, Kasetsart University according to WHO specifications (WHO 1998). Test papers (Whatman® No. 1) 12 × 15 cm in size were individually impregnated with one of the following concentrations: 0.75% (equivalent 294 mg a.i./m2) permethrin, 0.05% (18.3 mg a.i./m2) deltamethrin, or 0.05% (19.4 mg a.i. /m2) lambda-cyhalothrin with silicon oil and acetone serving as diluents. Papers were treated at the rate of 2 ml of insecticide solution per sheet. Control papers were prepared in the same manner but impregnated with only 2 ml acetone solvent mixed with the non-volatile carrier silicon oil at 3.6 mg/cm2 of the carrier, i.e., 648 mg/paper or 0.66 ml/paper for silicon oil. Only three to five-day-old, non-blood fed adult female mosquitoes (the gender of interest as potential transmitter of dengue viruses) were used in tests. Test kits, procedures, data analysis and interpretation followed provided instructions (WHO 1998). Each test consisted of five clear plastic tubes lined inside with treated papers (two controls and three treatments). Twenty-five mosquitoes were introduced into each holding tube lined with clean (untreated) paper for 60 min to establish the vitality of mosquitoes before insecticide exposure. Dead and moribund mosquitoes were removed before beginning each test. Mosquitoes were exposed for 60 min with all tubes placed in the vertical (standing up) position for the duration of the test period. All tests took place during daylight hours (between 09:00 and 16:00) under identical environmentally controlled conditions (25+5° C and 80+10% RH). At the end of the test exposure, the number of knockdown (KD) mosquitoes was recorded. All mosquitoes (live and moribund) were carefully transferred to separate clean holding tubes and provided with 10% sugar solution applied to cotton wool. Mortality was recorded at 24 h post-exposure. Four test replicates (∼100 female mosquitoes total per test series) were conducted for each mosquito population and insecticide combination. Post-test KD and final (24 h) mortality were averaged among each test series. Analysis and interpretation of bioassay results were based on established criteria (WHO 1998). Mosquitoes were considered susceptible to insecticide if the percent mortality was >98%. Incipient insecticide resistance (also referred to as tolerance) is defined as final mortality ranging between 80 and 97%. Test populations were considered resistant if percent mortality was below 80%. Because only one concentration of each chemical was used in the assays, it was not possible to estimate varying percent lethal concentrations using a probit analysis. The summary findings of KD and mortality for Ae. aegypti and Ae. albopictus exposed to permethrin, deltamethrin, and lambda-cyhalothrin are provided in Table 2. For all tests, control mortalities for Ae. aegypti and Ae. albopictus were nil, thus indicating that colonized mosquitoes used in this study were healthy at time of testing. For all three compounds, knockdown response after 60 min was a highly accurate predictor of final recorded mortality at 24 h post-exposure. In many instances, the KD and mortality percentages were identical. The USDA Ae. aegypti control population was completely susceptible to all three chemicals along with 100% KD after 60 min. All 32 populations of Ae. aegypti had evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. In general, a higher level of physiological tolerance/resistance to permethrin was seen in northern populations compared to other geographical regions in Thailand. The highest resistance to permethrin was seen in populations from Lampang (43.54% mortality), followed closely by Chiang Mai (44.59% mortality) and Kamphaeng Phet (50.00% mortality). In all, five populations (55.6%) in northern Thailand were resistant. Physiological resistance was also observed in two populations from the eastern region, Chonburi and Chanthaburi, and in five populations (50% of tested populations) from the south [Surat Thani, Chumphon, Prachuap Khiri Khan, Namom (Songkhla), and Sadao (Songkhla)]. Incipient resistance to permethrin (80–97% mortality) was seen in all remaining populations and within all six regions. With deltamethrin, incipient resistance was seen in eleven of 32 populations (34.4%); six (60%) from the southern region, two populations each from eastern (67%) and northern (22.2%) regions, and one population from the northeastern region (12.5%). The remaining majority of Ae. aegypti tested were considered as susceptible to deltamethrin (98–100% mortality). All populations of Ae. aegypti were found completely susceptible (100% KD and mortality) to lambda-cyhalothrin. Among the five populations of Ae. albopictus tested, only one population (Songkhla-Sadao, 54% mortality) was considered permethrin-resistant, whereas the other four had evidence of moderate to light incipient resistance (84–96% mortality). In contrast, Ae. albopictus was found completely susceptible to deltamethrin and lambda-cyhalothrin. Preliminary studies using the recommended operational concentration of 0.25% permethrin for Ae. aegypti found that all samples were resistant to this dose and 0.5% (data not presented). Sathantriphop et al. (2006) reported using a 0.75% concentration of permethrin and finding high levels of resistance in Ae. aegypti from Baan Suan, Nonthaburi Province. Therefore, a decision was made to use a three-fold greater concentration (0.75%) for all tests used in this study. First to third generation field-collected populations of Ae. aegypti and Ae. albopictus were either tolerant or resistant to a three-fold higher concentration of permethrin above the normal recommended operational concentration. All 32 populations of Ae. aegypti had evidence of incipient resistance (62.5% of populations) or levels of survival deemed resistant (37.5%) to permethrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang in northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). Four populations of Ae. albopictus were found with incipient resistance (97 to 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) were susceptible (> 98% mortality) to 0.05% deltamethrin, with incipient resistance (observed 97 to 82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely (100%) susceptible to 0.05% dosage of lambda-cyhalothrin. All five populations of Ae. albopictus were completely susceptible to both deltamethrin and lambda-cyhalothrin. Geographically, collection sites in northern and southern provinces showed the highest percent resistance to permethrin by Ae. aegypti, 55.6% and 50% of populations tested, respectively. Deltamethrin tolerance (60% of populations) was more common in the south. In Thailand, synthetic pyrethroids, particularly permethrin and deltamethrin, were introduced in the last three decades and have been widely available for the control of mosquitoes and other insects (Chareonviriyaphap et al. 1999). Historically, pyrethroids have demonstrated great promise for effective mosquito control due to their low toxicity to humans and high potency at low doses quickly immobilizing and killing insects. However, the continuous use of particular insecticides for long periods of time has been associated with development of resistance in mosquitoes and thus reduced effectiveness controlling vectors (Hemingway and Ranson 2000). From the 1960s, investigations examining patterns of mosquito resistance to insecticides in Thailand began reporting varying and levels of tolerance/resistance to various synthetic chemicals used for control of adult and larval Ae. aegypti et al. et al. 1977, and Only recently have a studies made of Ae. albopictus et al. et al. 2005, Jirakanjanakit et al. 2007). the tolerance or resistance to permethrin deltamethrin has been in adult and larval Ae. aegypti in Thailand (Chareonviriyaphap et al. 1999, Somboon et al. Paeporn et al. 2004, Yaicharoen et al. 2005, et al. 2005, Sathantriphop et al. 2006, Jirakanjanakit et al. 2007, Thanispong et al. 2008). high levels of permethrin resistance were reported by Jirakanjanakit et al. mortality) and Thanispong et al. mortality) exposed to 0.25% not study showed relatively of resistance mortality) exposed to a three-fold higher concentration. Additionally, these in permethrin susceptibility in collection sites and levels of exposure to permethrin or is that insecticide i.e., and formulations permethrin, play a role for permethrin resistance in Ae. aegypti populations in Thailand as reported by Paeporn et al. and Thanispong et al. The use of treated with permethrin for disease control in many of Thailand and the use of for control of have the development of physiological resistance in this Incipient resistance to deltamethrin was detected in of 32 Ae. aegypti permethrin, the susceptibility to deltamethrin over time is the of use of this chemical and pyrethroid pyrethroids are also commercially available to the general public for use in for mosquitoes (Kongmee et al. 2004). of deltamethrin have been commonly used to dengue for many years. Incipient or resistance (WHO to permethrin was detected in populations of Ae. albopictus from the south. have been commonly used for mosquitoes Ae. in rubber and Since 2004, routine for control was of in areas the southern with or resistance also have of exposure to or use of permethrin or other of the populations of Ae. aegypti tested from the southern resistance to permethrin due to more exposure or to populations (60%) of Ae. aegypti also demonstrated incipient resistance to In contrast, all populations of Ae. albopictus remain completely susceptible to deltamethrin and lambda-cyhalothrin. However, this not the development to these two chemicals as a of the exposure to compounds et al. In addition to to these three chemicals over the varying of Aedes susceptibility seen between be to in and Permethrin is with other third generation pyrethroids that and over their and generation chemical Deltamethrin and lambda-cyhalothrin are more fourth generation pyrethroids with active greater and for effectiveness permethrin. The and also between permethrin on the one and deltamethrin and lambda-cyhalothrin on the other are in susceptible as or 2000). of is to by the in and thus a For insects susceptible to pyrethroid the is and are typically into two classes based on in specific and compounds (e.g., are associated with more of that is with their toxicity and less effects on 2 compounds and are approximately more pyrethroids for an that is with physiological response is that compounds have a for toxicity 2 a toxicity with in 2000). as a for selection of the higher associated with the in Thailand the potential toxicity of permethrin to levels potency of the other to this study was not including a component to the possible in permethrin or deltamethrin resistance. resistance to pyrethroids one or more including of the active The resistance results in reduced to A of mosquito including Ae. aegypti (Hemingway et al. have had that either a broader of pyrethroid or be to a of pyrethroids alone (Hemingway and Ranson 2000). studies a have been in a population of Ae. aegypti from that showed resistance to permethrin and and of pyrethroids typically one or more analysis had a of and with to the as evidence of site associated with Ae. aegypti in Thailand et al. Paeporn et al. selected for permethrin and deltamethrin resistance in separate laboratory populations of Ae. aegypti and detected and in addition to in in the population. Only were so that a also have been to play a relatively if in pyrethroid resistance in Ae. aegypti compared to resistance (Hemingway et al. resistance is the primary resistant populations in selection have from wide spread use of pyrethroids in organized space or the of these active in control the of resistance to after have the following with pyrethroids (Chadwick et al. monitoring of the resistance over one or this is selected and maintained in continue to play a crucial, if not indispensible, role in the control of most vector-borne This is particularly for transmission of dengue and chikungunya control by attacking both larval habitats and adult mosquitoes remains the principal method for reducing risk of dengue infection and Gubler the and in monitoring and is evidence to an in vector resistance in Thailand. is that investigations be to also on Ae. albopictus and the identification of the physiological and of resistance in the In this use of a three-fold in permethrin tolerance or resistance was in all populations of Ae. aegypti and Ae. albopictus tested, a wide geographical within Thailand. and of either direct or to a of pyrethroid chemicals and formulations have serious and for dengue control strategies using insecticides. and monitoring of mosquito vector populations remain the primary to timely and as to the effectiveness and use of insecticides under (Roberts and Andre further appropriate countermeasures (e.g., or of effective to control or mitigate the of resistance. a comprehensive for routine monitoring of susceptibility and of insecticide resistance to the identification of for resistance should be an of an insecticide aimed at effective integrated vector management This was by the Center of on and and of of Center of for and for Kasetsart University, Bangkok, Thailand and Thailand