First Report on Adulticide Susceptibility Status of Aedes albopictus , Culex quinquefasciatus , and Culex vishnui

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1 First Report on Adulticide Susceptibility Status of Aedes albopictus, Culex quinquefasciatus, and Culex vishnui from a Pig Farm in Tanjung Sepat, Selangor, Malaysia Author(s): Chee Dhang Chen, Van Lun Low, Koon Weng Lau, Han Lim Lee, Wasi Ahmad Nazni, Chong Chin Heo, Abdul Aziz Azidah, and Mohd Sofian- Azirun Source: Journal of the American Mosquito Control Association, 29(3): Published By: The American Mosquito Control Association DOI: URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Journal of the American Mosquito Control Association, 29(3): , 2013 Copyright E 2013 by The American Mosquito Control Association, Inc. FIRST REPORT ON ADULTICIDE SUSCEPTIBILITY STATUS OF AEDES ALBOPICTUS, CULEX QUINQUEFASCIATUS, ANDCULEX VISHNUI FROM A PIG FARM IN TANJUNG SEPAT, SELANGOR, MALAYSIA CHEE DHANG CHEN, 1 VAN LUN LOW, 1,4 KOON WENG LAU, 1 HAN LIM LEE, 2 WASI AHMAD NAZNI, 2 CHONG CHIN HEO, 3 ABDUL AZIZ AZIDAH 1 AND MOHD SOFIAN-AZIRUN 1 ABSTRACT. The present study aims to investigate the susceptibility status of Aedes albopictus, Culex quinquefasciatus, and Cx. vishnui collected from a pig farm in Tanjung Sepat, Selangor, toward 11 insecticides representing the classes of organochlorines, carbamates, organophosphates, and pyrethroids. The results of a World Health Organization adult mosquito bioassay revealed that Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui exhibited different susceptibility toward various insecticides. Overall, pyrethroids were able to induce rapid knockdown for all test mosquito species. The pyrethroids lambdacyhalothrin and etofenprox were able to cause high mortality (.80%) of all 3 species. The findings of the present study will benefit local authorities in selecting appropriate dosage of insecticides to be used in mosquito control in this area. KEY WORDS Malaysia Adulticide susceptibility, pig farm, Aedes albopictus, Culex quinquefasciatus, Culex vishnui, INTRODUCTION In Malaysia, Aedes albopictus (Skuse) has been implicated as a dengue vector (Lee and Rohani 2005). The dengue situations in Malaysia have reached an alarming level, with a total of 19,884 dengue cases and 36 deaths reported in 2011 (Ministry of Health Malaysia 2011). With regards to Culex species, Malaysian Culex vishnui (Theobald) and Cx. quinquefasciatus (Say) are known vectors of Japanese encephalitis and lymphatic filariasis, respectively (Vythilingam et al. 1995, 2005). A total of 283 human cases of viral encephalitis with 109 deaths have been reported in Malaysia from September 29, 1998, to December 1999 (Chua 2010). For lymphatic filariasis, an estimated 115 million and 13 million people have been infected with Wuchereria bancrofti (Cobbold) and Brugia spp., respectively, in endemic countries, including Malaysia (Michael and Bundy 1997, WHO 2010). Insecticides have played an important role in the control of mosquito vectors. Unfortunately, the ability of mosquito populations to develop resistance to every class of insecticide often leads to control failures. In Malaysia, numerous studies on insecticide resistance mainly focused on Ae. aegypti (L.), Ae. albopictus,andcx. quinquefasciatus (Lee et al. 1997, Chen et al. 2005, Nazni et al. 2005, Hidayati et al. 2011, Low et al. 2013b). However, report on the susceptibility status of Cx. vishnui 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia. 2 Medical Entomology Unit, WHO Collaborating Centre for Vectors, Institute for Medical Research, Jalan Pahang, 50588, Kuala Lumpur, Malaysia. 3 Faculty of Medicine, Universiti Teknologi Mara, Shah Alam, 40450, Selangor, Malaysia. 4 To whom correspondence should be addressed. against various insecticides has not been elucidated in this region. Indeed, there has been a lack of data on the insecticide susceptibility of Cx. vishnui from every part of the world. To the best of our knowledge, insecticide susceptibility status of this species has only been reported in India (Sundaram et al. 1989, Sharma et al. 2003), Cambodia (Kohn 1991), and China (Alout et al. 2007). Domestic pigs serve as amplifying hosts for viruses while mosquitoes serve as the important vectors prior to transmission to humans (Borah et al. 2013). Japanese encephalitis and Nipah viruses often affect pigs and pig-farm workers, as well as other pigfarming areas in Malaysia (Chua 2010). During the outbreaks, insecticide applications remain as the main control agents against mosquitoes. Despite the importance of insecticide application in vector control programs for many years, the evaluation of insecticide susceptibility status of mosquitoes in pigfarming areas has not been conducted. Given that the early detection and monitoring of resistance remain a vital part of insecticide resistance management, the present study is an attempt to 1) evaluate the susceptibility status of Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui obtained from a pig farm in Tanjung Sepat, Selangor, toward 11 insecticides belonging to the classes of organochlorines, carbamates, organophosphates, and pyrethroids, as part of an ongoing insecticide susceptibility investigation; and 2) to provide the 1st documented data on Cx. vishnui insecticide susceptibility status in Malaysia. MATERIALS AND METHODS Mosquitoes strain The present study was conducted at Tanjung Sepat, the largest pork producer in the state of Selangor, Malaysia. Investigation of the density 243

3 244 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 29,NO. 3 Fig. 1. Collection site of mosquito specimens in Tanjung Sepat, Selangor, Malaysia. of mosquitoes at a pig farm in Tanjung Sepat (2u N, 101u E) revealed that Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui were the dominant species (Sofian-Azirun et al., unpublished data). The public health implications of the occurrence of these 3 species therefore deserved specific attention in the assessment of their insecticide susceptibility status in the present study. The adult mosquitoes were collected at a pig farm located in Tanjung Sepat, Selangor, Malaysia, by using human landing catch (Fig. 1). Prior to the collection, collectors were briefed on the biosafety procedures and informed consent was obtained. This study was conducted from 0700 to 0700 h with 3 to 4 collectors stationed outdoors. All mosquitoes landing on human baits were caught using mm glass vials and subsequently plugged with cotton. Collected female mosquitoes were transported to the laboratory and identified according to the illustrated keys by Rattanarithikul and Panthusiri (1994) and Triplehorn and Johnson (2005). Identified female mosquitoes were subsequently used in adult susceptibility bioassays (Fig. 1). Insecticides A total of 11 insecticides belonging to organochlorines, carbamates, organophosphates, and pyrethroids were used in adult susceptibility bioassays. The World Health Organization (WHO) impregnated papers were purchased from WHOPES Collaborating Centre in Universiti Sains Malaysia, Penang. The diagnostic dosages and exposure times (%/h) were as follows: organocholorines (dichlorodiphenyltrichloroethans [DDT] 4.0%/4 h, dieldrin 4.0%/ 1 h), carbamates (propoxur 0.1%/2 h, bendiocarb 0.1%/1 h), organophosphates (malathion 5.0%/ 1 h, fenitrothion 1.0%/2 h), and pyrethroids (permethrin 0.75%/1 h, deltamethrin 0.05%/1 h, cyfluthrin 0.15%/1 h, lambdacyhalothrin 0.05%/ 1 h, etofenprox 0.5%/1 h). Insecticide susceptibility bioassays The bioassay procedure of WHO (1981) was performed against all adult female mosquitoes species, with minor modifications. A total of 15

4 SEPTEMBER 2013 ADULTICIDE SUSCEPTIBILITY OF AEDES AND CULEX IN MALAYSIA 245 sucrose-fed female mosquitoes were exposed to the diagnostic dosages of WHO-impregnated papers and the test was repeated 3 times. Control groups of mosquitoes were exposed to nontreated papers. The test tubes were covered with black cloth to make sure that the mosquitoes would be resting on the impregnated paper. For the 50% and 90% knockdown time (KT 50 and KT 90, respectively), a cumulative knockdown was recorded every minute for test insecticides within their respective exposure period, as recommended by WHO adult bioassay procedure. Mosquitoes were then transferred to holding tubes to observe for posttreatment effect. Cotton pads soaked in 10% sugar solution were provided during the 24-h recovery period. Mortality was recorded at 24 h posttreatment. Data analysis All data were subjected to probit analysis (Raymond 1985). The knockdown rates of mosquitoes within the exposure period were recorded every minute for the computation of KT 50 and KT 90. Based on the KT 50 value, a resistance ratio (RR) was calculated by dividing values for the field strain by those of the reference strain (Brown and Pal 1971). Calculated RR values.10 are indicative of high resistance, 5 10 are indicative of medium resistance, and,5 are indicative of low resistance (Mazzarri and Georghiou 1995). Mortality at 24 h posttreatment was also used to determine the susceptibility status: % mortality indicating susceptibility, 80 97% mortality suggesting the possibility of resistance that needs to be further confirmed, and,80% mortality suggesting resistance (WHO 2009). RESULTS The knockdown rates of Ae. albopictus, Cx. quinquefasciatus, andcx. vishnui obtained from a pig farm in Tanjung Sepat, Selangor are shown in Table 1, Table 2, and Table 3, respectively. All tested Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui exhibited rapid knockdown to cyfluthrin and deltamethrin, with KT 50 ranging from min and min, respectively. High resistance to carbamates and organophosphates was observed in Ae. albopictus, but KT 50 and KT 90 values could not be generated by probit analysis as the knockdown rates were,5%. Among the tested Cx. quinquefasciatus,,5% knockdown rates were observed in organochlorine bioassays (i.e., DDT and dieldrin). For Cx. vishnui,,5% knockdown rates were also recorded after exposure to fenitrothion. The knockdown percentage of Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui after exposure period of various insecticides (Table 4) showed that all 3 species tested with pyrethroids displayed 100% knockdown. Inversely, no knockdown was observed for Ae. albopictus and Cx. quinquefasciatus after exposure to the organophosphates and organochlorines, respectively. The percent mortality of all 3 species at 24 h posttreatment was used to determine susceptibility status (Table 5). Although knockdown was not observed in Ae. albopictus after exposure to the organophosphates, 20.00% mortality was observed at 24 h posttreatment with malathion and fenitrothion. For Cx. quinquefasciatus, 33.33% mortality was also recorded at 24 h posttreatment with DDT and dieldrin. Culex vishnui exhibited complete mortality due to all groups of insecticides at 24 h posttreatment, while Ae. albopictus exhibited complete mortality caused by organochlorines and pyrethroids. Conversely, various susceptibility trends toward all groups of insecticides were observed in Cx. quinquefasciatus. Culex quinquefasciatus exhibited a degree of recovery from exposure to pyrethroids and was resistant to cyfluthrin, deltamethrin, and permethrin, showing 26.67%, 33.33%, and 66.67% mortality, respectively. It was susceptible only to lambdacyhalothrin and etofenprox, bendiocarb, and fenitrothion, with 80.00%, 93.33%, 93.33%, and 100% mortality, respectively. Overall, pyrethroids conferred rapid knockdown effect toward Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui. The findings of this study also indicated that only lambdacyhalothrin and etofenprox were able to induce high mortality (.80%) against all 3 species at the pig farm in Tanjung Sepat. DISCUSSION In the present study, we found that Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui collected concurrently demonstrated a wide variation in susceptibility toward various classes of insecticides tested. Different activity behaviors (i.e., biting and resting behaviors), habitats, as well as environmental conditions, may allow the mosquitoes to be exposed to different insecticide treatments and consequently resulting in varied susceptibility (Liu et al. 2004, Surendran et al. 2012). The development of insecticide resistance to 4 major insecticide classes in Cx. quinquefasciatus has been well documented in many parts of the world, notably Malaysia (Chandre et al. 1997, Liu et al. 2004, Kasai et al. 2007, Pridgeon et al. 2008, Low et al. 2013a). Culex quinquefasciatus was more resistant to all groups of insecticides, compared to both Ae. albopictus and Cx. vishnui. This finding concurred with the study of Hidayati et al. (2005) where a laboratory strain of Cx. quinquefasciatus developed higher resistance levels toward organophosphate and pyrethroid

5 246 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 29,NO. 3 Table 1. Knockdown rates of Aedes albopictus obtained from a pig farm in Tanjung Sepat, Selangor. 1 Insecticide KT 50 (min) (95% CL) Laboratory strain Field strain KT 90 (min) (95% CL) Regression line KT 50 (min) (95% CL) KT 90 (min) (95% CL) Regression line DDT 4.0% ( ) ( ) y x ( ) ( ) y x Dieldrin 4.0% ( ) ( ) y x ( ) ( ) y x Propoxur 0.1% ( ) ( ) y x (ND) (ND) y x Bendiocarb 0.1% ( ) ( ) y x ND Malathion 5.0% ( ) ( ) y x ND Fenitrothion 1.0% NA ND Permethrin 0.75% ( ) ( ) y x ( ) ( ) y x Deltamethrin 0.05% ( ) ( ) y x ( ) ( ) y x Cyfluthrin 0.15% ( ) ( ) y x ( ) ( ) y x Lambdacyhalothrin 0.05% ( ) ( ) y x ( ) ( ) y x Etofenprox 0.5% ( ) ( ) y x ( ) ( ) y x Resistance ratio 1 ND, not determined by probit analysis as knockdown rate,5%, indicating high resistance; NA, not available; KT, knockdown time; CL, confidence limit.

6 SEPTEMBER 2013 ADULTICIDE SUSCEPTIBILITY OF AEDES AND CULEX IN MALAYSIA 247 Table 2. Knockdown rates of Culex quinquefasciatus obtained from a pig farm in Tanjung Sepat, Selangor. 1 Insecticide KT 50 (min) (95% CL) Laboratory strain Field strain KT 90 (min) (95% CL) Regression line KT 50 (min) (95% CL) DDT 4.0% ( ) ( ) y x ND Dieldrin 4.0% ( ) ( ) y x ND KT 90 (min) (95% CL) Regression line Propoxur 0.1% ( ) ( ) y x ( ) ( ,254.91) y x Bendiocarb 0.1% ( ) ( ) y x ( ) ( ) y x Malathion 5.0% ( ) ( ) y x ( ) ( ) y x Fenitrothion 1.0% ( ) ( ) y x ( ) ( ) y x Resistance ratio Permethrin 0.75% ( ) ( ) y x ( ) ( ) y x Deltamethrin 0.05% ( ) ( ) y x ( ) ( ) y x Cyfluthrin 0.15% ( ) ( ) y x ( ) ( ) y x Lambdacyhalothrin 0.05% ( ) ( ) y x ( ) ( ) y x Etofenprox 0.5% ( ) ( ) y x ( ) ( ) y x ND, not determined by probit analysis as knockdown rate,5%, indicating high resistance; KT, knockdown time; CL, confidence limit.

7 248 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 29,NO. 3 Table 3. Knockdown rates of Culex vishnui obtained from a pig farm in Tanjung Sepat, Selangor. 1 Field strain Insecticide KT 50 (min) (95% CL) KT 90 (min) (95% CL) Regression line DDT 4.0% ( ) ( ) y x Dieldrin 4.0% ( ) ( ) y x Propoxur 0.1% ( ) ( ) y x Bendiocarb 0.1% ( ) ( ) y x Malathion 5.0% ( ) ( ) y x Fenitrothion 1.0% ND Permethrin 0.75% ( ) ( ) y x Deltamethrin 0.05% ( ) ( ) y x Cyfluthrin 0.15% ( ) ( ) y x Lambdacyhalothrin 0.05% ( ) ( ) y x Etofenprox 0.5% ( ) ( ) y x ND, not determined by probit analysis as knockdown rate,5%, indicating high resistance; KT, knockdown time; CL, confidence limit. classes, as compared to laboratory strains of Ae. albopictus and Ae. aegypti. In addition, a similar finding has also been reported where Cx. quinquefasciatus exhibited very high levels of resistance to pyrethroids and organophosphates, compared to Ae. albopictus collected concurrently from the same study sites in Alabama and Florida (Liu et al. 2004). We noted that all species displayed a complete knockdown after exposure to pyrethroids, indicating that pyrethroids remain as the most effective control agents among all classes of insecticides for mosquito control in this area. However, a degree of recovery from pyrethroids exposure was demonstrated in Cx. quinquefasciatus, suggesting that Cx. quinquefasciatus from this study site was resistant to pyrethroids and such observation was in agreement with a previous report (Yap 2000). In Malaysia, pyrethroids are one of the most widely used household insecticides available in the form of aerosol, coils and mats, and liquid vaporizer, containing AI of d-allethrin, d-trans allethrin, transfluthrin, prallethrin, s-bioallethrin, deltamethrin, d-phenothrin, permethrin, and tetramethrin (Yap et al. 2000). Overuse of these pyrethroid-based insecticides might confer pyrethroid tolerance in this species. In addition, the behavior of Cx. quinquefasciatus to rest indoors (Tham 2000) increases higher frequency of exposure to pyrethroid-based insecticides that would subsequently lead to the development of insecticide resistance, due to selection pressure. Table 4. Knockdown percentage of Aedes albopictus, Culex quinquefasciatus, andcx. vishnui obtained from a pig farm in Tanjung Sepat, Selangor, after exposure period of various insecticides. Insecticide Ae. albopictus Cx. quinquefasciatus Cx. vishnui DDT 4.0% Dieldrin 4.0% Propoxur 0.1% Bendiocarb 0.1% Malathion 5.0% Fenitrothion 1.0% Permethrin 0.75% Deltamethrin 0.05% Cyfluthrin 0.15% Lambdacyhalothrin 0.05% Etofenprox 0.5%

8 SEPTEMBER 2013 ADULTICIDE SUSCEPTIBILITY OF AEDES AND CULEX IN MALAYSIA 249 Table 5. Percent mortality of Aedes albopictus, Culex quinquefasciatus, andcx. vishnui obtained from a pig farm in Tanjung Sepat, Selangor, at 24 h posttreatment of various insecticides. Insecticide Ae. albopictus Cx. quinquefasciatus Cx. vishnui DDT 4.0% Dieldrin 4.0% Propoxur 0.1% Bendiocarb 0.1% Malathion 5.0% Fenitrothion 1.0% Permethrin 0.75% Deltamethrin 0.05% Cyfluthrin 0.15% Lambdacyhalothrin 0.05% Etofenprox 0.5% With regards to organophosphate insecticides, Ae. albopictus exhibited 0% knockdown in malathion and fenitrothion bioassays, probably due to the overuse of organophosphates over the years, particularly the introduction of malathion, fenitrothion, and temephos by local authorities for mosquito control during the dengue outbreak in Tanjung Sepat areas. In addition, the low mortality rates of Ae. albopictus in carbamate bioassays (propoxur and bendiocarb) indicated a lack of effective control in this species. Both organophosphates and carbamates attack the acetylcholinesterase of insects leading to the development of cross-resistance when there is an excessive use of either class of insecticide (Hemingway et al. 2004). Moreover, when a resistant strain is being exposed to an insecticide continuously, the resistance might extend to other compounds of the same class of insecticides or to compounds with similar mode of action (Liu et al. 2004). Cross-resistance between organophosphates and carbamates could occur due to the target site insensitivity of acetylcholinesterase (Hemingway et al. 2004). However, no report of this mutation has been reported in Aedes mosquitoes so far. Hence, further investigation of acetylcholinesterase insensitivity in Ae. albopictus at a molecular level is proposed. In conclusion, different susceptibility toward various insecticides was observed in Ae. albopictus, Cx. quinquefasciatus, and Cx. vishnui in Tanjung Sepat. The present bioassay data suggest the use of lambdacyhalothrin and etofentox as the most effective insecticides for the control of the 3 species in this area. ACKNOWLEDGMENTS We wish to thank the University of Malaya Research Programme (RP003B-13SUS) for funding this research. We express our sincere thanks to Lay Ah Kwee for providing accommodation during the fieldwork in Tanjung Sepat, Selangor. REFERENCES CITED Alout H, Berthomieu A, Cui F, Tan Y, Berticat C, Qiao C, Weill M Different amino-acid substitutions confer insecticide resistance through acetylcholinesterase 1 insensitivity in Culex vishnui and Culex tritaeniorhynchus (Diptera: Culicidae) from China. J Med Entomol 44: Borah J, Dutta P, Khan SA, Mahanta J Epidemiological concordance of Japanese encephalitis virus infection among mosquito vectors, amplifying hosts and humans in India. Epidemiol Infect 141: Brown AW, Pal R Insecticide resistance in arthropods. Public Health Pap 38: Chandre F, Darriet F, Doannio JM, Rivière F, Pasteur N, Guillet P Distribution of organophosphate and carbamate resistance in Culex pipiens quinquefasciatus (Diptera: Culicidae) in West Africa. JMed Entomol 34: Chen CD, Nazni WA, Lee HL, Sofian-Azirun M Weekly variation on susceptibility status of Aedes mosquitoes against temephos in Selangor, Malaysia. Trop Biomed 22: Chua KB Epidemiology, surveillance and control of Nipah virus infections in Malaysia. Malays J Pathol 32: Hemingway J, Hawkes NJ, McCarroll L, Ranson H The molecular basis of insecticide resistance in mosquitoes. Insect Biochem Mol Biol 34: Hidayati H, Nazni WA, Lee HL, Sofian-Azirun M Insecticide resistance development in Aedes aegypti upon selection pressure with malathion. Trop Biomed 28: Hidayati H, Sofian-Azirun M, Nazni WA, Lee HL Insecticide resistance development in Culex quinquefasciatus (Say), Aedes aegypti (L.) and Aedes albopictus (Skuse) larvae against malathion, permethrin and temephos. Trop Biomed 22:45 52.

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