Aedes aegypti(Linnaeus) larvae from dengue outbreak areas in Selangor showing resistance to pyrethroids but susceptible to organophosphates

Acta Tropica 185 (2018) 115–126

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Aedes aegypti(Linnaeus) larvae from dengue outbreak areas in Selangor showing resistance to pyrethroids but susceptible to organophosphates

T

⁎

Cherng Shii Leong , Indra Vythilingam, Meng Li Wong, Wan-Yusoff Wan Sulaiman, Yee Ling Lau Department of Parasitology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia

A R T I C LE I N FO

A B S T R A C T

Keywords: Aedes aegypti Insecticides resistance Synergist WHO larva bioassays & biochemical assays

The resistance status of Selangor Aedes aegypti (Linnaeus) larvae against four major groups of insecticides (i.e., organochlorines, carbamates, organophosphates and pyrethroids) was investigated. Aedes aegypti were susceptible against temephos (organophosphate), although resistance (RR50 = 0.21–2.64) may be developing. The insecticides susceptibility status of Ae. aegypti larvae were found heterogeneous among the different study sites. Results showed that Ae. aegypti larvae from Klang, Sabak Bernam and Sepang were susceptible against all insecticides tested. However, other study sites exhibited low to high resistance against all pyrethroids (RR50 = 1.19–32.16). Overall, the application of synergists ethacrynic acid, S.S.S.- tributylphosphorotrithioate and piperonyl butoxide increased the toxicity of insecticides investigated. However, the application failed to increase the mortality to susceptible level (> 97%) for certain populations, therefore there are chances of alteration of target site resistance involved. Biochemical assays revealed that α-esterase, (Gombak, Kuala Langat, Kuala Selangor and Sabak Bernam strains) β-esterase (Klang and Sabak Bernam strains), acetylcholinesterase (Kuala Selangor and Sabak Bernam strains), glutathione-S-transferase (Kuala Selangor and Sabak Bernam strains) and mono-oxygenases (Gombak, Hulu Langat, Hulu Selangor and Kuala Langat strains) were elevated. Spearman rank-order correlation indicated a significant correlation between resistance ratios of: DDT and deltamethrin (r = 0.683, P = 0.042), cyfluthrin and deltamethrin (r = 0.867, P =0.002), cyflyuthrin and lambdacyhalothrin (r = 0.800, P =0.010), cyfluthrin and permethrin (r = 0.770, P =0.015) deltamethrin and permethrin (r = 0.803, P =0.088), propoxur and malathion (r = 0.867, P = 0.002), malathion and temephos (r = 0.800, P = 0.010), etofenprox and MFO enzyme (r = 0.667, P =0.050). The current study provides baseline information for vector control programs conducted by local authorities. The susceptibility status of Ae. aegypti should be monitored sporadically to ensure the effectiveness of current vector control strategy in Selangor.

1. Introduction Mosquitoes play an important role as vectors of parasites and pathogens, due to its blood-sucking characteristics (Benelli and Dunggan; 2018). Mosquitoes are threat to humans and animals due to its ability in transmitting destructive parasites and pathogens, including dengue, malaria, yellow fever, Zika virus, chikungunya, filariasis, encephalitis, heartworm and West Nile virus (Benelli, 2015; Mehlhorn, 2016). The battle against mosquito borne diseases is a huge challenge of public health importance (Benelli and Mehlhorn, 2016). Although malaria cases have declined in recent years, the other arboviruses such as dengue, Zika virus and chikungunya that are carried by Aedes mosquitoes have been frequently reported in many parts of the world (Attar, 2016; Benelli and Mehlhorn, 2016). To prevent outbreaks of mosquito borne diseases, control measures such as application of insecticides, either as space spraying, indoor residual spraying, long lasting insecticidal nets, bio-control agent and insect growth hormone ⁎

Corresponding author.

https://doi.org/10.1016/j.actatropica.2018.05.008 Received 16 January 2018; Received in revised form 2 May 2018; Accepted 6 May 2018 0001-706X/ © 2018 Published by Elsevier B.V.

were carried out (Benelli and Beier, 2017; Benelli and Romano, 2017 Benelli and Mehlhorn, 2016; Mehlhorn, 2016; Benelli, 2015). Integrated Vector Management (IVM) has been recommended in making use of the full range of vector control tools available to prevent outbreaks (WHO, 2012; Benelli and Beier, 2017). In recent years, One Health approach was introduced, a control strategy which emphasizes on cooperation among multiple disciplines to achieve the best health for humans, animals and the environment (Benelli and Beier, 2017; Benelli and Duggan, 2018). Dengue is a mosquito borne viral disease that causes serious public health problem in most of the tropical countries. The cases of dengue have increased 30 folds over the past five decades (WHO, 2017). There were 4.5 million dengue cases reported in 2016 and 3.9 billion people living in the areas with risk of dengue infection (WHO, 2017). In Malaysia, there were 101,357 cases of dengue with 237 deaths (Idengue MOH-Malaysia: unpublished recorded from http://idengue. remotesensing.gov.my/idengue/index.php). Selangor is one of the

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were provided for five – seven days old adult females Ae. aegypti using live white mouse (IACUC no.: 20150407/PARA/R/MBK). Bora-bora strain of Ae. aegypti was used as a reference susceptible strain. The reference strains have been reared in the insectary for 134 generations without exposure to insecticides.

states in Malaysia which reported the highest number of cases (51,652 cases) in the country (MOH, 2016). Aedes aegypti (Linnaeus) is the vector of dengue and it can transmit all four serotypes as well as other viral diseases like Chikungunya and Zika virus (Cheong et al., 1986; Benelli and Romano, 2017). Unfortunately, there are no drugs to treat dengue and the vaccine is only partially effective. Thus, vector control has been the hallmark of the dengue control programme in Malaysia. Application of larvicide like temephos (Abate 1% Sand Granules) which is an organophosphate has been used for many decades (Chen et al., 2005a) and carried out by house owners or during epidemics in which the health personnel will apply the larvicides to containers which cannot be disposed and where the water is required by the people. Besides community participation in source reduction, fogging or ultra-low volume (ULV) is carried out by the authorities when cases of dengue are reported. The insecticides used for fogging are mainly organophosphates (malathion & fenitrothion) and pyrethroids. Insecticides like pyrethroids are not only used in public health but also in agriculture and most of the aerosol insecticides used by house owners are also pyrethroids (Yap et al., 2000). The development of insecticides resistance has become a serious problem worldwide due to the excessive use of insecticides and also there is limited insecticides for use in public health. Aedes aegypti from different localities in the world have been documented to be resistant to several classes of insecticides. (Araújo et al., 2013; Garcia et al., 2009; Grisales et al., 2013; Lima et al., 2011; Marcombe et al., 2012; Ocampo et al., 2011; Prapanthadara et al., 2002). It is not known if the mosquitoes have become resistant to insecticides and that could be one of the reasons for the increase of dengue cases. On the other hand, because asymptomatic persons are more infectious to the virus (Duong et al., 2015), control measures instituted after the case has been reported is perhaps too late. Thus, a more proactive surveillance is needed to prevent the increase in the number of dengue cases and also the management and use of insecticides is important. Although numerous studies have been conducted in Malaysia with regards to insecticide resistance to Ae. aegypti (Chen et al., 2008; Hasan et al., 2016; Lee et al., 2008; Loke et al., 2012; Wan-Norafikah et al., 2010), no robust and comprehensive study has been carried out to compare the resistance/susceptibility status of the said species to all four classes of insecticides in dengue outbreak and non-outbreak areas in Selangor state. We therefore examined the susceptibility status of Ae. aegypti larvae to all four classes of insecticides using bioassays and biochemical methods. The efficacy of synergist on insecticides was also explored.

2.2. Insecticides tested All four major classes of the neurotoxic insecticides (technical grade) were used for the experiments. These were organochlorine: DDT (98%); carbamate: propoxur (99.8%); organophosphates: malathion (98.7%), temephos (97.5%); pyrethroids: cyfluthrin (99.8%), deltamethrin (99.6%), etofenprox (97.7%), lambdacyhalothrin (97.8%) and permethrin (98.1%). All insecticides were purchased from Sigma Aldrich (Germany). 2.3. Larval bioassays Larval bioassays were conducted following the larval susceptibility bioassay procedure (WHO, 1981, 2005). The test was conducted using disposable 300 ml paper cup containing 249 ml of distilled water with 1 ml of insecticide solution. Twenty to 25 late third to early fourthinstar larvae were randomly selected and transferred to the holding cup for 10–15 min before addition of the insecticide solution. The larvae were exposed for 24 h after which mortality was recorded. To determine the diagnostic dose, Bora-Bora strain was tested with five concentrations for each insecticide in nine replicates to obtain mortality that ranges from 5 to 95% to generate LC50 and LC99 values according to WHO guidelines (WHO, 1981, 2005). For each test, five controls were set using 1 ml of 10% ethanol. The diagnostic dose, which is defined as two times (2X) lethal concentration that kills 99% of the reference population tested (LC99 X 2). The mortality rate of F2 generation of field collected mosquito larvae were determined using the diagnostic dosage. Three tests, each performed on three different days, were carried out for each insecticide for all the nine field strains. Similarly, five to seven different concentrations for each insecticide were used to determine lethal concentration of the insecticide that kills 50% (LC50) and 99% (LC99) of test population. 2.4. Synergism tests All field strains of Ae. aegypti were subjected to synergism tests to evaluate their effectiveness against the detoxification of insecticides. Ethacrynic acid 99% (EA), Piperonyl butoxide 99% (PBO) and S.S.Stributlyphosphorotrithioate 97.2% (DEF) all from Sigma-Aldrich were used in this study. Larvae of reference strain were exposed to all three synergists at different concentrations, and maximum sub lethal concentration were determined. After a series of trial and error, the sublethal doses of larvae synergism test were 0.1, 0.01, and 5 mg/L for EA, DEF and PBO, respectively. Larvae synergism tests were conducted almost similar to larvae bioassays, with an additional step by mixing the synergist and insecticides in the ratio of 1:1 before exposing to larvae. Each of the synergist was added to all insecticides.

2. Materials and methods 2.1. Sample collection Selangor is the most populated and well-developed state in Peninsular Malaysia. Its central position contributed to rapid industrialization and is the main transportation hub for the country. Selangor contributes 23% of the total GDP of Malaysia (Department of Statistics, 2014) and comprises of nine districts. Aedes aegypti were collected from September 2015 to April 2016 using ovitraps and larval surveys from all nine districts in Selangor state. Fig. 1 shows the map of Selangor with ovitraps collection sites. Table 1 shows the districts and sites from where the study was conducted. The sampling sites were selected based on dengue outbreak and dengue free areas. Eggs collected from each site were hatched and maintained as a single colony. All emerged adult mosquitoes were identified to species by morphological characteristics (WHO, 2003). The Ae. aegypti colonies were maintained under standard insectary condition with 27 ± 2 °C, 75 ± 5% relative humidity, a 10 h : 14 h (light:dark) light cycles and provided with 10% sucrose solution (vitamin B complex). Blood meals

2.5. Biochemical assays The different enzyme levels in individual larvae were determined according to the WHO procedure, as described by Hemingway and Brogdon (1998). Briefly, fourth-instar larvae were (Bora-Bora and field strains) individually homogenized in 200 μL of distilled water (on ice). Twenty-five microliters of homogenate were used for the AChE (acetylcholinesterase) assay. The remaining homogenate was then centrifuged at 14,000 rpm at four degrees Celsius for one minute, and the supernatant was used as an enzyme source for all other biochemical assays. For each assay, blanks were included (only distilled water instead of mosquito homogenate). Ninety-four larvae per strain were 116

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Fig. 1. Map of Sealngor with ovitraps collection sites.

2.5.2. Non-specific esterase assay For each sample, 20 μL of supernatant obtained from the larval homogenate were added in duplicates to each well. This was followed by the addition of 200 μL of the substrate, 30 mM α-naphthyl acetate to one set of samples while 30 mM β-naphthyl acetate was added to the other. And the mixture was incubated at room temperature for 15 min., then 50 μL of fast blue stain was added to each well. After 5 min incubation, the microplate was read with 570 nm absorbance. The activity against each substrate was calculated from standard curves of absorbance for known concentrations of α-naphthol or β-naphthol. Enzyme activities were expressed as nmole of α-naphthol or β-naphthol/minute/mg protein.

analysed. These assays were performed in a 96-well microplate in duplicates and the absorbance (optical density [OD] values) was measured on the Infinite M200 Pro microtitre plate reader (Tecan Trading AG, Switzerland). Assay for each enzyme is briefly described below.

2.5.1. AChE assay Twentyfive μL of larval homogenate was mixed with 145 μL of triton phosphate buffer, followed by addition 10 μL of 0.01 M dithiobis 2-nitrobenzoic acid solution and 25 μL of 0.01 M acetylthiocholine iodide to initiate the reaction. Duplicate reactions were prepared for each sample. While one reaction was allowed to progress, the other was inhibited by adding 0.05 μL of 0.1 M propoxur. The OD of both reactions were measured (after incubation of 1 h) at 405 nm and the activity was expressed as percentage insensitive AChE activity after propoxur inhibition (Saelim et al., 2005).

2.5.3. GST (glutathione-S-transferase) assay From each larval homogenate, 10 μL of supernatant was added to duplicate wells. A 200 μl mixture of 63 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 10 mM reduced glutathione was added for each well. After 117

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the larval bioassays (Brown, 1958). Levels of resistance were calculated according to WHO (2016) standard where the calculated RR50 values < 1 were expressed as susceptible, RR50 values < 5 were expressed as low resistance, 5–10 were expressed as medium resistance and RR50 values > 10 were expressed as high resistance. If the control mortality was > 5%, but < 20% the percentage mortalities was corrected by Abbott’s (1925) formula. The mortality rate 98%–100% indicates susceptible, 80% - 97% suggests the possibility of resistance that need to be further confirmed and < 80% suggested resistance (WHO, 2016). Mortality rate and enzymes levels were subjected to Levene’s and Kolmogorov-Smirnov tests. Data not normally distributed were transformed to arcsine log to stabilize the variance. Two-sample t-test/MannWhitney non-parametric test was performed to compared the difference between field strains and Bora-bora strain. Statistical significance was assumed at P < 0.05. All data were analysed and interpreted using SPSS (IBM SPSS Statistics 19) software. All graph was generated using GraphPad Prism V.5.0 (GraphPad Software) and Microsoft Excel version 2016 (Microsoft Inc.).

Table 1 Description of study sites. District

Site collection

Coordination

Status

Landscape

Gombak

Batu Caves Cheras

Hulu Selangor

Batang Kali

Klang

Pulau Ketam

Kuala Langat

Banting

Kuala Selangor

Kuala Selangor Sabak Bernam Bandar Sunway Salak Tinggi

Dengueoutbreak Dengueoutbreak Non-dengueoutbreak Non dengueoutbreak Non-dengueoutbreak Dengue outbreak

Urban

Hulu Langat

3O25’80.90"N 101O 64’51.3"E 3°01'19.4"N 101°45'31.1"E 3°27'20.6"N 101°39'18.4"E 3°01'18.5"N 101°15'12.4"E 2°48'25.9"N 101°29'17.8"E 3°14'23.8"N 101°25'35.2"E 3°45'51.6"N 100°59'17.5"E 3°04'54.3"N 101°36'38.6"E 2°49'42.8"N 101°43'34.5"E

Non-dengue outbreak Dengue outbreak

Rural

Non-dengue outbreak

Sub-urban

Sabak Bernam Petaling Jaya Sepang

Urban Sub-urban Rural Sub-urban Urban

Urban

3. Results *Dengue-outbreak – Ovitrap was conducted after fogging and frequent dengue cases reported in the areas. *Non dengue-outbreak – Ovitrap was conducted with prior survey of no fogging activity conducted for at least 2 month.

The Bora-Bora strain of Ae. aegypti exhibited least toxicity to propoxur (LC50 0.41755 mg/L) and was most susceptible to deltamethrin (0.00006 mg/L). The diagnostic dosage of the different larvicides based on Bora-Bora strain are shown in Table 2. Larvae from all sites showed 100% mortality to porpoxur and malathion only (Table 3). For DDT, larvae from all sites showed more than 90% mortality with the exception of Gombak (88.44%) and Hulu Selangor (32.95%). This corroborates well with the RR50 value of these two strains which were 3.84 and 5.64 respectively. This shows that the Hulu Selangor strain was moderately resistant (Table 4). Larvae from six of the sites showed 100% mortality towards temephos while only the Hulu Langat, Petaling and Kuala Selangor strains the mortality ranged from 98.67% to 96.92%. However, the RR50 value of Petaling strain was 2.64 while all other strains had RR50 < 1 (Table 4). This shows that temephos was still susceptible to larvae from

20 min of incubation at room temperature, absorbance was determined at 340 nm. GST activity was calculated based on Beer’s Law (A = €cl) and reported as mmole of CDNB/minute/mg protein. Where A (OD value) was transformed to μmole of CDNB conjugates using the € (extinction coefficient) of 4.39 mM−1. The path length (the depth of the buffer solution in the microplate well,) was 0.6 cm. 2.5.4. MFO (Monooxygenases) assay For MFO activity 20 μL of supernatant was added to duplicate wells. This was followed by the addition of 80 μL of 0.625 M potassium phosphate buffer (pH 7.2), 200 μL of 3,3,5,5-tetramethylbenzidine (TMBZ) in methanol solution, and 25 μL of 3% hydrogen peroxide. It was incubated at room temperature for 2 h before microplate was read at 650 nm. The MFO activity was calculated based on the standard curve of absorbance for known concentration of cytochrome C (Brogdon and Janet, 1997). Enzyme activity is expressed as equivalent units of cytochrome P450/min/mg protein.

Table 2 Diagnostic dose (mg/L) of different larvicides based on Bora-Bora strain.

2.5.5. Protein assay To account for size variances among individuals, protein concentration was used as a standard correction factor for the analysis of all enzymes activities. A commercialized protein assay (Bio-Rad, USA) was used to obtain the bovine serum albumin standard curve. Protein concentration was calculated and transformed based on the bovine serum albumin standard curve. Ten microliters of larval homogenate solution were mixed with 300 μl of Bio-Rad dye reagent and incubated for 5 min. and was read at 570 nm.

Insecticides

n

LC50 (95% CL) (mg/L)

LC99 (95% CL) (mg/L)

Diagnostic dose (mg/L)

DDT

1203

1176

Malathion

1211

Temephos

1230

Cyfluthrin

1174

Deltamethrin

1245

Etofenprox

1372

Lambdacyhalothrin

1378

Permethrin

1423

5.60492 (3.0339713.95812) 5.82678 (2.07143463.92613) 0.31488 (0.231020.49175) 0.04121 (0.024730.08533) 0.00170 (0.001180.00289) 0.00145 (0.000540.01642) 0.08050 (0.065762.39374467.5) 0.01077 (0.004940.03337) 0.01631 (0.010300.03148)

11.210

Propoxur

0.15482 (0.128180.18591) 0.41755 (0.0218040.68622) 0.04457 (0.039970.04926) 0.00168 (0.001420.00199) 0.00019 (0.000170.00021) 0.00006 (0.000040.00008) 0.00441 (0.001130.03436) 0.00009 (0.00007 -0.00011) 0.00090 (0.000790.00103)

2.6. Statistical analysis The mortality rate (%) and resistance ratio (RR50) were used to express the Ae. aegypti susceptibility status. Mortality rate was used to evaluate the effectiveness of synergists against insecticides. On the other hand, resistance ratio provides information on the degree of resistance of field strains compared to reference strain. Larval bioassay data within the range 5–95% were subjected to probit analysis methods of Finney (1947) to obtain the LC50 and LC99 values for each insecticide. Data from bioassays were pooled for analysis. Resistance ratios (RR50) were calculated by dividing values for the field strain with the reference strain based on the LC50 obtained from 118

11.653

0.629

0.082

0.003

0.003

0.161

0.022

0.033

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Table 3 Mortality rate of Ae. aegypti larvae 24-hours after exposure to insecticides of diagnostic dosage and synergists. Insecticides + Synergists

Total exposed (n) DDT only DDT + PBO DDT + DEF DDT + EA DDT + 3 synergists Total exposed (n) Propoxur only Propoxur + PBO Propoxur + DEF Propoxur + EA Propoxur + 3 synergists Total exposed (n) Malathion only Malathion + PBO Malathion + DEF Malathion + EA Malathion + 3 synergists Total exposed (n) Temephos only Temephos + PBO Temephos + DEF Temephos + EA Temephos + 3 synergists Total exposed (n) Cyfluthrin only Cyfluthrin + PBO Cyfluthrin + DEF Cyfluthrin + EA Cyfluthrin + 3 synergists Total exposed (n) Deltamethrin only Deltamethrin + PBO Deltamethrin + DEF Deltamethrin + EA Deltamethrin + 3 synergists Total exposed (n) Etofenprox only Etofenprox + PBO Etofenprox + DEF Etofenprox + EA Etofenprox + 3 synergists Total exposed (n) Lambdacyhalothrin only Lambdacyhalothrin + PBO Lambdacyhalothrin + DEF Lambdacyhalothrin + EA Lambdacyhalothrin + 3 synergists Total exposed (n) Permethrin only Permethrin + PBO Permethrin + DEF Permethrin + EA Permethrin + 3 synergists

Mean % mortality ± SE Bora-Bora

Gombak

Hulu Langat

Hulu Selangor

Klang

1186 100 ± 100 ± 100 ± 100 ± 100 ± 1186 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1228 88.44 ± 1.82* 100 ± 0 96.89 ± 1.46 100 ± 0 100 ± 0 1234 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

1136 91.65 ± 2.94* 100 ± 0 100 ± 0 100 ± 0 100 ± 0 1158 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

1133 32.95 ± 3.68*a 89.38 ± 1.88*a 78.72 ± 2.85*a 95.11 ± 2.29a 80.22 ± 3.08*a 1143 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

1141 100 ± 100 ± 100 ± 100 ± 100 ± 1136 100 ± 100 ± 100 ± 100 ± 100 ±

1235 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1233 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1146 100 ± 100 ± 100 ± 100 ± 100 ±

1142 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1213 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1178 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1141 98.67 ± 0.94 100 ± 0 82.67 ± 2.49* 96.04 ± 1.48 100 ± 0

1147 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1156 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1211 64.33 ± 1.59* 100 ± 0a 85.42 ± 2.08*a 79.26 ± 1.34*a 100 ± 0a

1138 80.97 92.93 81.66 80.61 83.39

± ± ± ± ±

4.35* 1.61 2.39* 2.46* 2.38*

1146 11.03 39.00 13.56 44.44 34.58

± ± ± ± ±

1155 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1181 73.01 ± 1.86* 100 ± 0a 77.20 ± 1.48* 74.34 ± 2.11* 100 ± 0a

1132 60.35 82.42 65.89 73.68 74.87

± ± ± ± ±

3.38* 5.28*a 6.76* 4.50*a 4.11*a

1131 10.64 46.50 12.00 15.98 33.45

1150 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1168 89.78 ± 2.12 100 ± 0a 100 ± 0a 100 ± 0a 100 ± 0a

1137 94.32 ± 1.13* 96.49 ± 1.04* 89.08 ± 3.62* 100 ± 0 100 ± 0

1134 61.04 91.61 83.56 88.96 82.80

1151 100 ± 0

1913 75.56 ± 1.41*a

1141 91.91 ± 2.79

100 ± 0

100 ± 0a

95.16 ± 1.30

100 ± 0

a

0 0 0 0 0

0 0 0 0 0

*a

Sabak Bernam

0 0 0 0 0

1131 100 ± 100 ± 100 ± 100 ± 100 ± 1138 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1170 90.74 ± 0.69 100 ± 0 100 ± 0 100 ± 0 100 ± 0 1144 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

1144 97.33 ± 1.15 97.81 ± 0.96 100 ± 0 100 ± 0 100 ± 0 1190 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

1141 100 ± 100 ± 100 ± 100 ± 100 ± 1136 100 ± 100 ± 100 ± 100 ± 100 ±

1135 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1131 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1145 100 ± 100 ± 100 ± 100 ± 100 ±

1143 100 ± 100 ± 100 ± 100 ± 100 ±

1136 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1130 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1143 96.92 ± 1.28 97.33 ± 1.49 97.33 ± 1.49 96.89 ± 1.60 100 ± 0

1.61* 2.70*a 2.86* 4.59*a 3.81*a

1131 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1129 26.86 ± 2.23* 100 ± 0a 53.33 ± 4.42*a 75.78 ± 4.33*a 100 ± 0a

1133 51.69 54.67 53.28 50.37 58.58

± ± ± ± ±

± ± ± ± ±

2.22* 3.63*a 2.58* 3.06* 4.23*a

1143 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1128 44.22 ± 2.37*a 100 ± 0a 73.33 ± 3.20*a 62.22 ± 2.12*a 100 ± 0a

1163 26.45 29.50 37.33 83.92 89.85

± ± ± ± ±

± ± ± ± ±

2.91*a 2.26a 4.39*a 2.63a 2.29*a

1139 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1131 80.36 ± 1.46* 82.32 ± 2.56* 100 ± 0a 83.28 ± 2.35* 98.67 ± 0.94a

1140 90.78 ± 1.18 100 ± 0 100 ± 0 100 ± 0 100 ± 0

1133 29.95 ± 2.65*

1143 100 ± 0

1129 65.30 ± 3.12*

93.42 ± 3.00a

100 ± 0

92.44 ± 1.69*a

87.78 ± 2.97

96.00 ± 1.86

a

87.11 ± 1.74

100 ± 0

100 ± 0a

82.48 ± 2.62*

1204 100 ± 100 ± 100 ± 100 ± 100 ±

1192 100 ± 100 ± 100 ± 100 ± 100 ±

1148 96.18 ± 1.28 100 ± 0 91.73 ± 2.37 83.88 ± 3.85* 92.58 ± 2.36

0 0 0 0 0

Petaling

43.47 ± 4.60

*

100 ± 0

0 0 0 0 0

Kuala Selangor

*a

90.91 ± 3.13

100 ± 0

Kuala Langat

0 0 0 0 0

100 ± 0

0 0 0 0 0

*

70.41 ± 3.44

*

Sepang

0 0 0 0 0

1136 100 ± 100 ± 100 ± 100 ± 100 ± 1134 100 ± 100 ± 100 ± 100 ± 100 ±

1135 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1128 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1155 97.33 ± 1.15 95.16 ± 1.30 89.04 ± 1.93 100 ± 0 100 ± 0

1136 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1129 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

4.80* 2.83* 2.60* 3.42* 2.73*

1178 22.22 86.39 89.92 78.18 85.29

± ± ± ± ±

2.75* 3.12*a 2.54a 2.46*a 2.12*a

1131 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1127 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1.63* 2.08* 2.49* 4.31*a 2.23a

1153 24.44 85.33 66.46 78.34 80.97

± ± ± ± ±

2.35* 5.89*a 2.97*a 3.23*a 1.11*a

1143 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1129 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1143 93.33 ± 1.33 100 ± 0 95.03 ± 1.69 96.03 ± 1.33 100 ± 0

1139 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1128 100 ± 100 ± 100 ± 100 ± 100 ±

0 0 0 0 0

1137 70.36 ± 1.76*

1186 59.56 ± 2.94*

1143 100 ± 0

1127 100 ± 0

72.90 ± 2.74*

97.33 ± 0.94a

100 ± 0

100 ± 0

91.26 ± 2.38

a

100 ± 0

100 ± 0

90.88 ± 1.19

a

100 ± 0

100 ± 0

0 0 0 0 0

70.67 ± 1.49 a

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

100 ± 0

62.22 ± 5.13

93.33 ± 1.89

86.29 ± 2.36*a

100 ± 0

99.56 ± 0.44a

100 ± 0a

90.82 ± 2.59a

100 ± 0

100 ± 0

1134 91.57 93.81 91.61 87.32 91.59

1143 100 ± 100 ± 100 ± 100 ± 100 ±

1131 90.70 ± 2.50 100 ± 0 89.33 ± 1.76 65.33 ± 2.11* 100 ± 0

1139 42.67 53.78 55.66 76.46 88.48

1165 72.00 90.67 72.85 95.62 90.26

1143 100 ± 100 ± 100 ± 100 ± 100 ±

1131 100 ± 100 ± 100 ± 100 ± 100 ±

± ± ± ± ±

4.02 2.23 2.25 2.23* 2.71

0 0 0 0 0

± ± ± ± ±

3.19* 4.48* 6.42* 2.80*a 1.42a

± ± ± ± ±

2.21* 2.58a 3.21* 1.37a 2.32a

0 0 0 0 0

0 0 0 0 0

Mean % mortality followed by asterisk symbol denotes rates that were significantly different when compared with Bora-bora strain (P < 0.05, independent T−test). Mean followed by a superscript letter were significant different between synergist(s) threated versus non−synergist(s) threated, P < 0.05, Mann-Whitney U test.

119

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Table 4 Susceptibility of the Ae. aegypti larvae to various insecticides. Insecticide

Strain

n

LC50 (95% CL) (mg/L)

LC99 (95% CL) (mg/L)

Slope

x2(df)

RR50

DDT

Bora-Bora

1203

8.354 ± 0.091

0.79 (3) 11.30 (3)

–

1255

Hulu Langat

1146

4.594 ± 0.320

1.04 (3)

4.82

Hulu Selangor

1130

2.730 ± 0.060

1178

0.71 (3) 10.62 (3)

5.64

Klang Kuala Langat

1130 1208

2.16

Petaling

1167

8.251 ± 0.124

7.21 (3) 6.44 (3) 16.78 (3)

0.24

Kuala Selangor

Sabak Bernam

1250

9.002 ± 1.410

17.40 (3)

0.47

Sepang

1198

9.939 ± 0.137

11.89 (3)

0.26

Bora-Bora

1176

8.535 ± 0.771

11.82 (3)

–

Gombak

1235

10.383 ± 0.090

9.22 (3)

1.63

Hulu Langat

1134

9.637 ± 0.357

11.40 (3)

2.15

Hulu Selangor

1125

10.789 ± 0.322

16.88 (3)

2.12

Klang

1157

10.846 ± 0.330

9.89 (3)

0.66

Kuala Langat

1137

10.570 ± 0.469

21.71 (3)

0.25

Kuala Selangor

1178

11.163 ± 0.388

9.23 (3)

0.07

Petaling

1154

9.985 ± 0.296

3.96 (3)

3.10

Sabak Bernam

1229

10.292 ± 0.259

12.73 (3)

1.12

Sepang

1157

2.984 ± 0.064

3.29 (3)

2.69

Bora-Bora

1211

10.518 ± 0.352

1.55 (3)

–

Gombak

1253

12.334 ± 0.366

16.25 (3)

0.84

Hulu Langat

1243

11.809 ± 0.865

1144

11.039 ± 0.319

1.57 (3) 10.52 (3)

1.71

Hulu Selangor Klang

1245

11.543 ± 0.379

1.45 (3)

0.51

Kuala Langat

1131

11.699 ± 0.315

2.25 (3)

0.45

Kuala Selangor

1187

11.065 ± 0.400

4.22 (3)

0.65

Petaling

1233

10.471 ± 0.749

15.87 (3)

2.22

Sabak Bernam

1261

10.948 ± 0.344

1.57 (3)

0.59

Sepang

1167

11.077 ± 0.365

4.45 (3)

1.35

Bora-Bora

1230

10.715 ± 0.434

2.88 (3)

–

Gombak

1263

9.772 ± 0.832

8.23 (3)

0.38

Hulu Langat

1170

11.689 ± 1.258

5.17 (3)

0.43

Hulu Selangor

1129

10.021 ± 0.411

4.50 (3)

0.60

Klang

1162

10.472 ± 0.646

3.95 (3)

0.26

Kuala Langat

1135

5.60492 (3.03397-13.95812) 2.90911 (1.57590 - 29.21118) 6.31578 (4.31662-11.18140) 5.93164 (4.30124 - 9.64713) 0.72852 (0.14527 - 172.54671) 14.27176 (1.75692 - 2982.84299) 4.66156 (2.16930 - 28.09479) 10.37099 (1.46911 - 22748.65236) 5.52793 (0.72965 - 3081529.601) 9.89097 (1.16975 - 11820.30857) 5.82678 (2.07143-463.92613) 1.33782 (0.45234 - 41.37951) 4.57604 (2.27608-65.72541) 4.45991 (2.20196 - 186.53030) 2.84803 (1.22002 - 39.97898) 0.88398 (0.31580 - 5414.29368) 0.64951 (0.20942 - 15.21424) 6.45977 (4.95458 - 9.56722) 6.74790 (2.28082 - 533.40694) 4.60518 (3.68997 - 6.26600) 0.31488 (0.23102-0.49175) 0.66585 (0.20644- 46.84840) 0.24850 (0.21014 -0.31523) 1.41518 (0.34837-112.93681) 0.57982 (0.35655- 1.13705) 1.01909 (0.55904-2.29810) 0.62579 (0.38428-1.23725) 0.38919 (0.20978-9.38667) 1.0313 (0.21066-5518.11731) 1.14543 (0.71852-2.19390) 0.04121 (0.02473-0.08533) 0.26847 (0.02795 - 185.59243) 0.01576 (0.01405 - 0.01849) 0.04951 (0.02551 - 0.12992) 0.00618 (0.00401 - 0.01152) 0.11640 (0.04518 - 0.45052)

9.020 ± 0.135

Gombak

0.15482 (0.12818-0.18591) 0.59462 (0.42305 - 0.80232) 0.74569* (0.66852-0.83305) 0.87395* (0.78229 – 0.96309) 0.01222 (0.00595 - 0.02919) 0.03678 (0.01665 - 0.10266) 0.33465 (0.21873 - 0.46650) 0.15886 (0.04137 - 0.60838) 0.07203 (0.02274 - 0.32187) 0.04019 (0.01328 - 0.12832) 0.41755 (0.021804-0.68622) 0.68013 (0.03969 - 0.11284) 0.89864 (0.67758-1.40572) 0.88679 (0.52677 - 1.32921) 0.27382 (0.17599 - 0.41134) 0.10325 (0.04743 - 0.23894) 0.02732 (0.01622 - 0.04812) 1.29379 (1.18453-1.40352) 0.46913 (0.26301 - 0.84814) 1.12195 (1.01124 - 1.21150) 0.04457 (0.03997 - 0.04926) 0.03755 (0.01837-0.07813) 0.07612* (0.07054-0.08123) 0.03156 (0.01545-0.63627) 0.02275 (0.01911- 0.02699) 0.02024 (0.01637-0.02532) 0.02883 (0.02454-0.03403) 0.09891 (0.07175-0.15168) 0.02615 (0.00836- 0.06870) 0.06025 (0.05155-0.07078) 0.00168 (0.00142-0.00199) 0.00063 (0.00027 - 0.00200) 0.00072 (0.00069 - 0.00075) 0.00101 (0.00082 - 0.00124) 0.00043 (0.00037 - 0.00049) 0.00035 (0.00026 - 0.00048)

10.423 ± 0.306

4.77 (3)

0.21

Propoxur

Malathion

Temephos

10.014 ± 0.250 8.751 ± 0.147 10.373 ± 0.093

3.84

0.08

1.03

0.71

(continued on next page)

120

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Table 4 (continued) Insecticide

Cyfluthrin

Deltamethrin

Etofenprox

Lambda-cyhalothrin

Strain

n

LC50 (95% CL) (mg/L)

LC99 (95% CL) (mg/L)

Slope

x2(df)

RR50

Kuala Selangor

1192

14.65 (3)

0.65

1145

10.661 ± 1.069

0.081 (3)

2.64

Sabak Bernam

1226

10.476 ± 0.673

10.84 (3)

0.30

Sepang

1156

10.397 ± 0.547

9.14 (3)

0.90

Bora-Bora

1174

10.348 ± 0.876

2.74 (3)

–

Gombak

1258

8.695 ± 0.399

4.08 (3)

3.37

Hulu Langat

1211

9.899 ± 3.554

16.98 (3)

7.89

Hulu Selangor

1130

10.050 ± 0.685

8.21 (3)

30.05

Klang

1210

10.550 ± 0.552

6.72 (3)

0.16

Kuala Langat

1128

11.203 ± 0.715

24.13 (3)

24.74

Kuala Selangor

1157

10.094 ± 4.405

7.38 (3)

17.42

Petaling

1145

8.775 ± 0.256

18.51 (3)

32.16

Sabak Bernam

1229

10.780 ± 4.263

3.44 (3)

1.11

Sepang

1166

9.909 ± 0.458

3.09 (3)

0.37

Bora-Bora

1245

10.396 ± 0.674

5.90 (3)

–

Gombak

1255

9.116 ± 0.456

13.62 (3)

7.17

Hulu Langat

1147

9.943 ± 0.664

17.67 (3)

7.67

Hulu Selangor

1230

8.485 ± 0.589

3.86 (3)

9.67

Klang

1162

11.085 ± 0.418

8.24 (3)

0.67

Kuala Langat

1128

10.006 ± 0.609

5.17 (3)

6.50

Kuala Selangor

1206

10.538 ± 0.394

15.87 (3)

10.50

Petaling

1245

10.586 ± 0.688

3.56 (3)

10.67

Sabak Bernam

1236

10.633 ± 4.872

0.68 (3)

2.00

Sepang

1167

9.766 ± 0.405

4.98 (3)

0.83

Bora-Bora

1372

9.998 ± 0.242

36.15 (3)

–

Gombak

1269

11.141 ± 0.253

11.51 (3)

5.87

Hulu Langat

1138

10.643 ± 0.418

27.18 (3)

4.34

Hulu Selangor

1125

9.535 ± 0.205

12.95 (3)

9.64

Klang

1136

-11.156 ± 3.142

8.84

0.33

Kuala Langat

1136

9.342 ± 0.238

22.67 (3)

9.34

Kuala Selangor

1326

12.088 ± 0.203

14.37 (3)

4.90

Petaling

1236

11.287 ± 0.687

2.70 (3)

1.68

Sabak Bernam

1245

9.666 ± 0.353

18.26 (3)

1.59

Sepang

1142

9.773 ± 0.184

2.29 (3)

3.54

Bora-Bora

1378

10.309 ± 0.440

1.09 (3)

–

Gombak

1257

9.663 ± 0.304

9.59 (3)

5.56

Hulu Langat

1238

0.04613 (0.00923 - 97.22675) 0.01340 (0.01130 - 0.01711) 0.00610 (0.00228 - 0.19504) 0.02163 (0.00789 - 0.46839) 0.00170 (0.00118-0.00289) 0.41799 (0.12021-2.81811) 0.10585 (0.01464-2.659509) 0.07024 (0.02949- 1.08215) 0.00177 (0.00046 - 0.05691) 0.04348 (0.01506-423.58228) 0.06752 (0.02511-1.30090) 1.19962 (0.09741 - 3.41693) 0.02125 (0.01023-0.06014) 0.00912 (0.00381 - 0.03232) 0.00145 (0.00054-0.01642) 0.03282 (0.00453 - 4.22092) 0.00691 (0.00195 - 72.22754) 0.01861 (0.00974 - 0.05228) 0.00609 (0.00274 - 0.01869) 0.00742 (0.00452 - 0.01544) 0.03928 (0.00641 - 323.92784) 0.00674 (0.00471 - 0.01133) 0.00883 (0.00437-0.2405) 0.01756 (0.00633 - 0.07771) 0.80502 (0.06576-2.393744) 0.52865 (0.016850 - 19.62968) 0.50726 (0.09475-3.90320) 1.80965 (0.38277 - 10.09331) 0.18326 (0.03552-12.42996) 1.15045 (0.23817 - 4.27223) 0.82493 (0.026572 - 12.70036) 0.03227 (0.02570 - 0.04436) 0.20712 (0.04187 - 14.20724) 3.43937 (1.40752 - 12.31976) 0.01077 (0.00494-0.03337) 0.20603 (0.01922 - 623.98026) 0.01694 (0.00799 - 0.05301)

10.418 ± 0.405

Petaling

0.0011 (0.00042 - 0.00273) 0.00443* (0.00416 - 0.00469) 0.00050 (0.00031 - 0.00083) 0.00152 (0.00101 - 0.00256) 0.00019 (0.00017-0.00021) 0.00064 (0.00046 - 0.00092) 0.00150 (0.00050-0.00644) 0.00571 (0.00356-0.00821) 0.00003 (0.00001 - 0.00005) 0.00470 (0.00177-0.01109) 0.00331 (0.00185-0.00492) 0.00611 (0.00006 - 0.02859) 0.00021 (0.00016-0.00027) 0.00007 (0.00005 - 0.00009) 0.00006 (0.00004-0.00008) 0.00043 (0.00017 - 0.00158) 0.00046 (0.00021 - 0.00115) 0.00058 (0.00048 - 0.00069) 0.00004 (0.00003 - 0.00005) 0.00039* (0.00031 - 0.00042) 0.00063 (0.00023 - 0.00200) 0.00064* (0.00056 - 0.00073) 0.00012 (0.00009-0.00015) 0.00005 (0.00004 - 0.00008) 0.00441 (0.00113-0.03436) 0.02588 (0.01444 - 0.04785) 0.01916 (0.00405-0.13756) 0.04253 (0.01381 - 0.08993) 0.00145 (0.00069-0.00320) 0.04117 (0.00061 - 0.18208) 0.02163 (0.01264 - 0.03538) 0.00740 (0.00684 - 0.00799) 0.00702 (0.00233 - 0.02305) 0.01560 (0.01180 - 0.02084) 0.00009 (0.00007-0.00011) 0.00050 (0.00018 - 0.00172) 0.00021 (0.00017 - 0.00026)

9.451 ± 0.475

5.05 (3)

2.33

(continued on next page) 121

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Table 4 (continued) Insecticide

Permethrin

Strain

n

LC50 (95% CL) (mg/L)

LC99 (95% CL) (mg/L)

Slope

x2(df)

RR50

Hulu Selangor

1128

12.50 (3)

38.67

1129

11.840 ± 0.478

2.60 (3)

3.44

Kuala Langat

1128

11.203 ± 0.715

8.54 (3)

9.22

Kuala Selangor

1164

12.191 ± 0.331

2.96 (3)

9.00

Petaling

1322

11.004 ± 0.497

4.01 (3)

15.22

Sabak Bernam

1227

9.740 ± 0.515

13.07 (3)

4.44

Sepang

1238

10.808 ± 0.422

11.72 (3)

2.44

Bora-Bora

1423

10.707 ± 0.256

2.25 (3)

–

Gombak

1259

10.376 ± 2.849

6.76 (3)

1.87

Hulu Langat

1139

11.381 ± 0.717

23.94 (3)

1.19

Hulu Selangor

1354

11.229 ± 0.420

20.16 (3)

1.30

Klang

1355

10.866 ± 4.058

6.78 (3)

0.44

Kuala Langat

1129

11.730 ± 0.461

12.46 (3)

3.89

Kuala Selangor

1195

10.818 ± 0.560

19.50 (3)

7.80

Petaling

1128

10.146 ± 0.388

8.15 (3)

6.47

Sabak Bernam

1228

12.057 ± 0.802

4.61 (3)

0.44

Sepang

1137

0.04226 (0.01598 - 1.07557) 0.00862 (0.00522 - 0.01710) 0.02437 (0.00660 - 1.99993) 0.04898 (0.02780 - 0.10688) 0.02264 (0.01775 - 0.04160) 0.00907 (0.00295 - 0.26879) 0.01597 (0.00281 - 5.61853) 0.01631 (0.01030-0.03148) 0.31004 (0.05482-22.01905) 0.00750 (0.00303 - 11.80268) 0.03285 (0.00906 - 2.02573) 0.04622 (0.01544- 0.37133) 0.10752 (0.02900-7.47897) 0.04713 (0.01976 - 13.35656) 0.12193 (0.04221 - 3.16567) 0.00454 (0.00318 - 0.00738) 0.04228 (0.02125 - 0.10938)

11.504 ± 0.459

Klang

0.00348 (0.00209 - 0.00600) 0.00031 (0.00026 - 0.00037) 0.00083 (0.00049 - 0.00156) 0.00081* (0.00066 - 0.00099) 0.00137* (0.00118 - 0.00159) 0.00040 (0.00025 - 0.00067) 0.00022 (0.00010 - 0.00060) 0.00090 (0.00079-0.00103) 0.00168 (0.00084-0.00369) 0.00107 (0.00044 - 0.00209) 0.00117 (0.00068 - 0.00231) 0.00074 (0.00518- 0.01122) 0.00350 (0.00172-0.00722) 0.00702 (0.00373 - 0.01289) 0.00582 (0.00340 - 0.00917) 0.00040 (0.00035-0.00045) 0.00054 (0.00043 - 0.00069)

11.223 ± 0.358

4.08 (3)

0.60

* Asterisk symbol at the top show significantly higher when compared with Bora-Bora Strain (P < 0.05, independent T-test). Table 5 Correlation between resistance ratio of each insecticides of Ae. aegypti larvae. DDT

Propoxur

Malathion

Temephos

Cyfluthrin

Deltamethrin

Etofenprox

Lambdayhalothrin

Permethrin

DDT

–

Propoxur

r = 0.300 P = 0.433 r = 0.533 P = 0.139 r = 0.400 P = 0.286 r = 0.483 P = 0.187 r = 0.683 P = 0.042* r = 0.550 P = 0.125 r = 0.233 P = 0.546 r = 0.310 P = 0.417

r = 0.300 P = 0.433 –

r = 0.533 P = 0.139 r = 0.867 P = 0.002* –

r = 0.400 P = 0.187 r = 0.633 P = 0.067 r = 0.800 P = 0.010* –

r = 0.483 P = 0.187 r = 0.167 P = 0.668 r = 0.267 P = 0.488 r = 0.350 P = 0.356 –

r = 0.683 P = 0.042* r = 0.200 P = 0.606 r = 0.500 P = 0.170 r = 0.583 P = 0.099 r = 0.867 P = 0.002* –

r = 0.550 P = 0.125 r = -0.150 P = 0.700 r = -0.050 P = 0.099 r = -0.017 P = 0.966 r = 0.567 P = 0.112 r = 0.417 P = 0.265 –

r = 0.233 P = 0.546 r = -0.100 P = 0.798 r = -0.117 P = 0.765 r = 0.150 P = 0.700 r = 0.800 P = 0.010* r = 0.600 P = 0.088 r = 0.517 P = 0.154 –

r = 0.310 P = 0.417 r = -0.126 P = 0.748 r = 0.192 P = 0.620 r = 0.377 P = 0.318 r = 0.770 P = 0.015* r = 0.803 P = 0.009* r = 0.544 P = 0.130 r = 0.628 P = 0.070 –

Malathion Temephos Cyfluthrin Deltamethrin Etofenprox Lambdayhalothrin Permethrin

r = 0.867 P = 0.002* r = 0.633 P = 0.067 r = 0.167 P = 0.668 r = 0.200 P = 0.606 r = -0.150 P = 0.700 r = -0.100 P = 0.798 r = -0.126 P = 0.748

r = 0.800 P = 0.010* r = 0.267 P = 0.488 r = 0.500 P = 0.170 r = -0.050 P = 0.898 r = -0.117 P = 0.765 r = 0.192 P = 0.620

r = 0.350 P = 0.356 r = 0.583 P = 0.099 r = -0.017 P = 0.966 r = 0.150 P = 0.700 r = 0.377 P = 0.318

r = 0.867 P = 0.002* r = 0.567 P = 0.112 r = 0.800 P = 0.010* r = 0.770 P = 0.015*

r = 0.417 P = 0.265 r = 0.600 P = 0.088 r = 0.803 P = 0.009*

r = 0.517 P = 0.154 r = 0.544 P = 0.130

r = 0.628 P = 0.070

* Asterisk symbol at the top show significant correlation between resistance ratio of insecticides (P < 0.05; Spearman rank-order correlation).

deltamethrin (r = 0.683, P = 0.042), cyfluthrin and deltamethrin (r = 0.867, P =0.002), cyfluthrin and lambdacyhalothrin (r = 0.800, P =0.010), cyfluthrin and permethrin (r = 0.770, P =0.015), deltamethrin and permethrin (r = 0.803, P =0.088), propoxur and malathion (r = 0.867, P = 0.002), malathion and temephos (r = 0.800, P = 0.010) while there were no significant correlation with others insecticides (Table 5). To investigate the efficiency of synergists in improving the toxic effect of insecticides against mosquitoes, DEF, EA and PBO were added

all sites. Five insecticides belonging to the pyrethroid group were tested and of these the Klang, Sabak Bernam and Sepang strains showed 100% mortality to all five insecticides while the Gombak strain had 100% mortality to permethrin (Table 3). All other strains exhibited different degrees of resistance to pyrethroids with RR50 > 30 for Hulu Selangor strain against lambdcyhalothrin and cyfluthrin; the Petaling strain against cyfluthrin (Table 4). However, Spearman rank-order correlation indicated a significant correlation between resistance ratios of DDT and 122

Acta Tropica 185 (2018) 115–126

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Table 6 Mean ( ± SE) levels of insensitive acetylcholinesterase (AChE), glutathione-S-transferase (GST), non-specific esterase (α- and β-EST) and mono-oxygenase (MFO) activities of Ae. aegypti larvae in Selangor. Strain

N

AChE

Bora2 Gombak Hulu Langat Hulu Selangor Klang Kuala Langat Kuala Selangor Petaling Sabak Bernam Sepang

84 86 86 90 78 87 67 93 63 80

39.02 42.18 38.28 40.19 32.14 40.42 49.39 27.42 47.57 43.62

± ± ± ± ± ± ± ± ± ±

1.32 1.83 1.50 1.18 1.10 1.62 1.79* 0.61 1.67* 1.39

αEST

βEST

GST

8.17 ± 0.81 9.57 ± 0.57* 4.06 ± 0.32 8.24 ± 0.61 7.95 ± 0.55 9.88 ± 0.53* 12.08 ± 1.25* 2.66 ± 0.15 12.07 ± 1.00* 9.42 ± 0.68

8.56 ± 0.80 8.90 ± 0.60 9.02 ± 0.52 4.45 ± 0.60 10.17 ± 0.57* 9.02 ± 4.52 8.81 ± 0.64 2.56 ± 0.14 14.38 ± 0.98* 8.10 ± 0.59

0.17 0.15 0.05 0.14 0.20 0.17 0.23 0.06 0.33 0.21

MFO ± ± ± ± ± ± ± ± ± ±

0.01 0.01 0.00 0.01 0.01 0.01 0.02* 0.00 0.02* 0.01

0.21 0.46 0.36 0.44 0.22 0.57 0.21 0.16 0.29 0.27

± ± ± ± ± ± ± ± ± ±

0.00 0.01* 0.01* 0.01* 0.01 0.01* 0.00 0.00 0.01 0.00

* Asterisk symbol at the top show significantly higher when compared with Bora-Bora Strain (P < 0.05, Mann-Whitney test).

in combination with each insecticide. The mortality rate of field obtained Ae. aegypti larvae against different combination of insecticides and synergist are shown in Table 3. Generally, the synergists increased the mortality rate of different field strains and 100% mortality was achieved with combination treatment of synergist(s) and insecticide for some strains. Results show that PBO has significantly increased the mortality rate of almost all strains of Ae. aegypti against pyrethroids and 100% mortality were also achieved for several insecticides (DDT, temephos, cyfluthrin, deltamethrin, etofenprox, lambdacyhalothrin and permethrin). Results showed that synergist(s) increased the mortality of all strains of Ae. aegypti against all the tested insecticides. However, from the results, we observed that Hulu Selangor and Kuala Selangor strains showed low mortality rate (< 80%) against cyfluthrin and deltamethrin even when synergist(s) was added. To investigate the observed insecticide resistance of Selangor Ae. aegypti population due to the elevated enzyme levels, biochemical assays were conducted. About 730 larvae collected from the nine study sites were individually assayed for AChE, α-EST, β-EST GST and MFO enzymes activities and are shown in additional File (Figs S1-S5). All biochemical assays data were pooled and shown in Table 6. Results showed that Kuala Selangor and Sabak Bernam strain exhibited elevated level of altered AChE activity when compared with Bora-Bora strain. Significant elevation of α-EST levels was found in Gombak, Kuala Langat, Kuala Selangor and Sabak Bernam strain. Aedes aegypti larvae obtained from Klang and Sabak Bernam showed elevated level of β-EST compared to Bora-Bora strain. In glutathione-S-transferase assays, a significant increase in GST activity was detected in Kuala Selangor and Sabak Bernam strains. Of the nine field strains, four strains (Hulu Selangor, Gombak, Hulu Langat and Kuala Selangor) exhibited a significant increase in MFO activity. In addition, spearman rank-order correlation indicated there a significant correlation between resistance ratio of etofenprox and MFO enzyme (r = 0.667, P =0.050) (Fig. 2), however, no significant correlation was found with other insecticides

nor enzymes. 4. Discussion Although only temephos is used as a larvicide for Aedes larval control, we decided to test all four classes of insecticides since some of them are used for control of adult mosquitoes, be it for vectors of dengue or malaria. The scarcity of data on susceptibility of mosquito larvae against candidate insecticides is one of the factors that is limiting the achievement of the control programs. These control programs are usually conducted with insufficient information on the resistance selection risk posed by a given control agent in the field and perhaps the sample size used was also very small. The results of the current study provide a baseline dataset of the susceptibility of field strains of Ae. aegypti of Selangor against commonly used insecticides. Current study has demonstrated that larvae collected from Selangor are susceptible against carbamate and organophosphates. However, they showed different susceptiblility level against organochlorine and pyrethroids. Generally, all field strains were susceptible against malathion showing 100% mortality and RR50 values < 5 even though malathion has been used by the control programmes since 1970s (Tanrang and Vythilingam, 2004). During epidemics of dengue, control measures rely heavily on both thermal fogging and ULV to control and kill the infected Aedes mosquitoes rapidly, and organophosphates (especially malathion) has been the insecticide of choice (Lam and Tham, 1988). It is now being slowly replaced by other organophosphates and pyrethroids (Ong, 2016). Similarly, all field strains of Ae. aegypti larvae were susceptible against temephos, except the three strains which had a mortality > 96% but < 100%. However only the Petaling strain had an RR50 of 2.64. According to WHO, 2016, it is still considered susceptible as RR50 is < 5. Previous studies have reported that Malaysian Ae. aegypti larvae from Gombak (Chen et al., 2005b) and Shah Alam (Loke et al., 2010) showed mortality of [(5.33%–72.00%; at diagnostic dosage (DD) 0.012 mg/L)] and [(11 – 100%; DD 0.02 mg/L)] respectively implicating resistance against temephos. However, the diagnostic dosage being used in this study was higher (0.082 mg/L) compared to previous studies mentioned; therefore, it is not viable to compare with the previous studies. Furthermore, the guidelines of WHO (WHO, 2016) recommended that 1 mg/L (1%) temephos is the operational dosage for field usage. Chen et al. (2005b) obtained 100% mortality against resistant field strains of Ae. aegypti collected in Gombak when exposed to operational dosage of temephos. Temephos will still be an effective larvicide that could be used but monitoring for emerging resistance is required. The various strains of Ae. aegypti larvae showed different mortalities against DDT, but only the Hulu Selangor strain showed an RR50 > 5. This could be due to that district being a malarious district for many years. Although DDT indoor residual spraying for malaria has been banned since 1998 (Yap et al., 2000), the resistance phenotype may still

Fig. 2. Correlation between etofenprox and MFO activity of field Aedes aegypti. 123

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et al., 2003; Nauen, 2007). However, the current study shows the field strains tested were susceptible against temephos and malathion. Since DEF have significantly increased the mortality of certain field strains of Ae. aegypti larvae when exposed to pyrethroids perhaps elevated esterases may be involved in pyrethroid resistance. Insensitive AChE activity of field obtained Ae. aegypti larvae were minimal with only Sabak Bernam and Kuala Selangor strains showing significantly elevated insensitive AChE activity. Cuamba et al. (2010) suspected the hydrolysis of propoxur via others enzymes (such as ESTs) have decreased the level of insensitive AChE in field mosquitoes. Perhaps the concentration of propoxur for AChE inhibition was decreased as the biochemical assays were conducted using total enzyme extraction on mosquitoes (Koou et al., 2014). DDT resistance is associated with elevated GST level (Aïzoun et al., 2014; Rodríguez et al., 2007) and this was observed in both the Kuala Selangor and Sabak Bernam strains. This elevated GST level maybe due to the large-scale use of pyrethroids in public health activities as both DDT and pyrethroids are especially designed to target the voltage-gated sodium channel of arthropods (Hemingway et al., 2004). Multiple insecticides resistance implicated that more than one mechanisms are involved in insecticides resistance. As a matter of fact, evolution/mutation of multiple isolates is not a new phenomenon and is becoming a serious issue worldwide. Results of the current study do not support the hypothesis that enzyme activities are corresponding to insecticide susceptibility status of Ae. aegypti due to the lack of correlation between resistance level and enzyme activities. Yet, there are studies suggesting that enzyme activities are not axiomatically correlated with toxicological changes (Montella et al., 2012; Siegfried and Scott, 1992). This suggests that the occurrence of this incidence might be due to the evolution/mutation in voltage-gated sodium channel which is commonly found in pyrethroids resistant Ae. aegypti (Kasai et al., 2014; Stenhouse et al., 2013; Yanola et al., 2011). Therefore, we propose to conduct molecular work to further uncover the involvement of target site insensitivity mechanisms in Ae. aegypti. In summary, insecticide(s) resistance of Ae. aegypti larvae have been detected in the current study. Therefore, new strategy for vector control should be redesign with consideration of others aspect such as earlier prevention of outbreak (Lau et al., 2017), remodel the methods with data gather from research and modelling on prediction of outbreak (Benelli et al., 2018). For example, Lau et al. (2017) have reported that the data obtained from the combination usages of Gravid Ovipositing Sticky (GOS) traps and NS1 kit provided earlier detection of dengue outbreak in a premise in Malaysia. Thus, control measures can take action before the dengue outbreak. This control measures fits into One Health strategy, where all data collected in small scales can be used and applied in larger scales where disease outbreak can be preventing (Benelli et al., 2018).

remain in this mosquito population. Nazni et al. (2009) showed that the DDT resistance phenotype still persisted although the laboratory Ae. aegypti strain had been reared in the insectary for 1014 generations. However, it must be noted that DDT was only used in the malaria control programme in rural areas and was not used for dengue control programmes. Since DDT and pyrethroids share the same mode of action/target site, thus, this occurrence of resistance against DDT may be due to the application of pyrethroids based insecticides since 1996 until now (Selvi et al., 2010; Yap et al., 2000). It was shown that An. darlingi in South America was resistant against both DDT and pyrethroid insecticides, although application of DDT had been stopped for 17 years. Thus, it has been suggested that this cross resistance was due to the continuous usage of pyrethroid based insecticides in the study site, since both insecticides shared the same target site – voltage-gated sodium channels (Fonseca-González et al., 2009). Among all the insecticides tested in current study, field collected Ae. aegypti was most resistant against pyrethroids (except Klang, Sabak Bernam and Sepang strains). However, pyrethroids are not used as larvicides in Malaysia. The observed resistance is likely due to the usage of pyrethroid based adulticides, since pyrethroids is the class of insecticides used in household insecticides products and also used in treatment of bednets and fogging for control of malaria and dengue vectors, respectively (Nazni et al., 1998; Yap et al., 2000). Moreover, pyrethroids are one of the major class of insecticides used by pest control industry. It is suggested that these pyrethroid based insecticides products conferred the pyrethroid resistance detected in the current study. This type of observed resistance among the larval populations suggests that this phenotype acquired at the adult stage may be displayed by both mature and immature mosquitoes (Chavasse et al., 1997). However, it should be stressed that this resistance profile in larvae may not always be consistent in adult mosquitoes, and further adulticide bioassays are required for validation (Hemingway et al., 2002; Mebrahtu et al., 1997) and this would be conducted. Generally, the synergists have increased the mortality of field collected Ae. aegypti against insecticides and thus indicates that esterases, monooxygenase and glutathione-S-transferase play important role for DDT and pyrethroid resistance detected in the current study. It was found that PBO increased the toxicity of all pyrethroids compared to other synergists. PBO is known as MFO inhibitor, and MFO has been documented to play a role in pyrethroid resistance (Hasan et al., 2016; Kasai et al., 2014). Similarly, our study also suggests the involvement of MFO for the resistance of some field strains towards pyrethroids, where PBO have increased the mortality of some field collected Ae. aegypti to 100%. However, not all field strains have achieved susceptible level with the addition of PBO. Furthermore, there were six field strains (Gombak, Hulu Langat, Hulu Selangor, Kuala Langat, Kuala Selangor and Petaling) which showed resistance for two or more different pyrethroids and this could be due to the cross-resistance between the pyrethroid groups. Thus, this further suggests that involvement of more than one mechanism conferring insecticide resistance. Furthermore, cross resistance between propoxur and malathion, malathion and temephos, DDT and pyrethroid and within pyrethroids were detected in the current study. Cross resistance between DDT and pyrethroids has been commonly reported in Thailand (Prapanthadara et al., 2002), Colombia (Fonseca-González et al., 2009), Vietnam (Kawada et al., 2009) and also in Malaysia (Ishak et al., 2015). The mechanisms involved in this cross-resistance is mostly related to kdr mutation in voltage-gated sodium channel (Hemingway et al., 2002; Hemingway et al., 2004). Although cross resistance was detected for propoxurmalathion and malathion-temephos, however, all field strain Ae. aegypti larvae were susceptible against all three insecticides mentioned. This cross resistance emerges from the mutation of target site insensitivity of AChE (Liu et al., 2004). Biochemical assays have detected elevated levels of ESTs in most strains. Esterases detoxification is reported as one of the main reasons of resistance in Ae. aegypti populations due to organophosphates (Bisset

5. Conclusion Overall, dengue outbreak study sites have shown higher resistance compared to non-dengue outbreak areas. However, it was found that Sepang strain Ae. aegypti was susceptible against all tested insecticides. Results of current study showed that Ae. aegypti in Selangor are susceptible to organphosphates and carbamate. Temephos remains as an effective larvicide. It is important to note that Ae. aegypti have developed resistance to pyrethroids and is now one of the main class of insecticides used in vector control. The choice of alternative insecticides Bacillus thuringiensis israelensis (Bti) or insect growth regulator (IGR) should be recommended for future public health programs. The exact mechanism involved for DDT and pyrethroids resistance in current study remains to be explored to develop alternative measure to manage resistance in mosquitoes. The outcome of current study provides information on resistance and cross resistance pattern occurring in Selangor Ae. aegypti larvae. Thus, control programme should be aware of cross resistance to the same/related active ingredients application on 124

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the field. At the same time to ensure the success of the control programme, new developments to overcome resistance is needed and also novel strategies should be designed to prevent/minimize the spread and evolution of resistance. Perhaps detection of virus in mosquitoes will be more proactive strategy as control measures can be instituted before cases occur.

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