Susceptibility and Resistance of Field Populations of Anopheles sinensis (Diptera: Culicidae) Collected from Paju to 13 Insecticides.

OBJECTIVES: Over 20% of all malaria cases reported annually in the Republic of Korea (ROK) occur in Paju, Gyeonggi Province. Vector control for malaria management is essential, but the insecticide resistance of the vector, Anopheles mosquitoes, has been a major obstacle in implementing effective control. In this study, the insecticide resistance of the vector mosquitoes was evaluated and compared with that of vector mosquitoes collected from the same locality in 2001 and 2009.
METHODS: The insecticide resistance of Anopheles sinensis s.s. collected from Paju, Gyeonggi Province in the ROK was evaluated under laboratory conditions with a micro-application method using 13 insecticides currently used by local public health centers and pest control operators in the ROK.
RESULTS: Based on median lethal dose (LC50) values, An. sinensis s.s. were most susceptible to the insecticides bifenthrin, cyfluthrin, and etofenprox in that order, and least susceptible to permethrin. An. sinensis showed higher susceptibility to pyrethroids than organophosphates, except for fenthion and permethrin. In a comparative resistance test, the resistance ratios (RRs) of An. sinensis collected in 2012 (AS12) to the 13 insecticides were compared to the RRs of two strains of An. sinensis collected from the same locality in 2001 (AS01) and 2008 (AS08). With some exceptions, AS12 demonstrated higher resistance to all tested insecticides compared to AS01 and AS08, and less resistance to bifenthrin, cyfluthrin, and cypermethrin compared to AS01.
CONCLUSION: These results indicate that careful selection and rotation of these insecticides may result in continued satisfactory control of field populations of An. sinensis s.s. for effective malaria management in Paju.

Introduction

In the Republic of Korea (ROK, South Korea), Plasmodium vivax malaria has been endemic for centuries [1,2]. In the 1960s and 1970s, active and passive uses of pesticides were combined in an ambitious eradication project by the ROK government [1].In the mid-1970s, indigenous transmission of malaria was greatly reduced and the ROK was declared a “malaria free zone” by the World Health Organization (WHO) [3,4]. However, Plasmodium vivax re-emerged in Gyeonggi province in 1993. Subsequently, vivax malaria increased to 4412 cases in 2000, before declining to 864 cases in 2004, rising in 2005 [5,6], and again decreasing to 843 cases in 2011 [7]. Since 2001 over 20% of all reported vivax malaria cases have occurred Paju, Gyeonggi Province, which highlights the necessity of vector control in that locality. The vector mosquito is one of the most important factors in the transmission of vivax malaria and therefore must be effectively controlled. To protect public health, the susceptibility and resistance of vector populations to registered insecticides need to be monitored so that effective control measures can be implemented [8].

The principal measures to control mosquito populations include the use of various contact residual insecticides, e.g., organophosphates, carbamates, and pyrethroids. However, repeated use can result in the development of resistance to these insecticides [9-13]. Also, as insects become resistant, more insecticides may be applied, resulting in human and environmental health problems. Widespread insecticide resistance to commonly used and less expensive insecticides has been a major obstacle to implementing cost-effective and safe integrated mosquito management programs. In addition, the use of certain insecticides will likely be reduced in the near future in the USA by the U.S. Environmental Protection Agency (EPA) under the 1996 Food Quality and Protection Act [14].

These problems indicate the need to establish effective insecticide resistance management strategies, which include the collection of baseline data and determination of insecticide resistance trends. This study evaluated the susceptibility of An. sinensis collected in Paju in 2012 to 13 commonly used insecticides in the ROK and monitored changes in insecticide resistance compared to the same species collected in the same locality in 2001 and 2009.

Materials and Methods

Chemicals

Thirteen different insecticides purchased from Fluka (Buchs, Switzerland) were used in this study: bifenthrin (97.0% purity), cyfluthrin (93.0%), etofenprox (96.5%), fenthion (95.5%), cypermethrin (98.0%), λ-cyhalothrin (98.6%), α-cypermethrin (97.5%), deltamethrin (99.5%), dichlorvos (99.5%), chlorpyrifos (98.5%), fenitrothion (98.5%), profenofos (98.0%), and permethrin (95.5%). Triton X-100 was obtained from Shinoy Pure Chemicals (Osaka, Japan). All other chemicals used were of analytical grade and available commercially.

Mosquitoes

Engorged female mosquitoes were collected using black light traps (Yoshizawa type, FL 6w; Shinyoung Co., Seoul, Korea) and an aspirator at cow sheds in Tong Il Chon, Baegyeon, Paju, Gyeonggi Province from July to August, 2012. To induce oviposition, engorged females were placed individually in paper cups (350 mL) lined with filter papers and half filled with distilled water.

The eggs were allowed to hatch in larval rearing pans (15 × 15 × 4 cm). The larvae were provided with a mixture of Vivid-S (Sewhapet Co., Incheon, Korea) and Super Terramin (Sewhapet Co.) which was sprinkled over the surface of the water. Larvae were reared in an insectary at 25 × 1 °C, with 65 × 5% relative humidity and a photoperiod of 14 hours light:16 hours dark. The identification of field collected populations was confirmed by polymerase chain reaction (PCR) [15,16].

Mosquito identification

The identity of the Anopheles species was confirmed by PCR with genomic DNA extracted from the legs of individual adult mosquitoes. The PCR products were separated on a 2% agarose gel and visualized with Safe-Pinky DNA Gel staining solution (×10,000) (GenDEPOT, Barker, TX, USA). Fragment sizes were estimated by comparison to molecular weight standards provided by a 100-bp Ladder Molecular Weight DNA Marker (Bioneer, Seoul, ROK) (Table 1).

Table 1

Primers and PCR conditions for the identification of seven Anopheles spp.

Primer	Species	Sequence (5′→3′)	Diagnostic bands
Forward	Universal primer	TGT GAA CTG CAG GAC ACA TGA A	—
Reverse	Anopheles sinensis	ATT GTT GTC CAG CCC GCT AAC	500 bp
	Anopheles pulls	ATA TCA TGG CTT AAC ACC GCG T	260 bp
	Anopheles lesteri	TGC CTA GAA CTT CCG CCA ATC	300 bp
	Anopheles belenrae	CAT TTT TCA CGA CTG CGA CGG	190 bp
	Anopheles kleini	GCG TCC ATA CTG TCT CAA CGA	400 bp
	Anopheles sineroides	ACC GAG TGG CCT CAC TC	500 bp
	Anopheles koreicus	GTA TAC ACG CTT TGT ATG TGG GG	200 bp

Bioassay

A direct-contact mortality bioassay [4] was used to evaluate the toxicity of 15 larvicides to late third instars of An. sinensis s.s. from the field-collected colonies. Each larvicide was dissolved in methanol and then further diluted in distilled water containing Triton X-100 (20 μL/l). A total of 25 larvae from each colony were placed in paper cups (350 mL) containing test larvicide solutions (250 mL). The toxicity of each test larvicide was determined using four to six concentrations ranging from 1 ppm to 200 ppm. The control consisted of the methanol–Triton X-100 carrier solution in distilled water. Treated and control groups were held under the same conditions as used for colony maintenance.

Larvae were considered to be dead if they did not move when they were prodded with a fine wooden dowel 24 hours after treatment [17]. All treatments were replicated three times using 25 larvae/replicate. Because bioassays could not all be conducted simultaneously, treatments were blocked over time with a separate control treatment used for each block. Freshly prepared solutions were used for each block of bioassays [18].

Data analysis

Data were corrected for mortality using Abbott’s formula [19]. Mortality rates were analyzed using a probit analysis with SAS software (SAS, Cary, NC, USA). The resistance ratio (RR), defined as the ratio produced when the 50% mortality (LC50) values of the strain collected in 2012 (AS12) were divided by the LC50 values reported for mosquitoes tested in 2001 (AS01) and 2008 (AS08), was used as described by Shin et al [11].

The RRs were used to compare the susceptibility of larvae of field-collected An. sinensis collected and assayed in 2001, 2008, and 2012. RRs values of <10, 10–40, 40–160, and >160 were classified as low, moderate, high, and extremely high resistance, respectively [20]. The LC50 values of the treatments were considered to be significantly different from one another when their 95% confidence limits (CL) failed to overlap.

Results

The LC50 values demonstrated that the susceptibility of the larvae of An. sinensis s.s. collected from Paju in 2012 (AS12) was highest to bifenthrin, followed by cyfluthrin, etofenprox, fenthion, cypermethrin, λ-cyhalothrin, α-cypermethrin, deltamethrin, dichlorvos, chlorpyrifos, profenofos, fenitrothion, and permethrin, in that order (Table 2). AS12 showed the highest susceptibility to bifenthrin with LC50 found at 0.227 ppm, followed by cyfluthrin and etofenprox with LC50 at 0.446 ppm and 1.858 ppm, respectively, and demonstrated the lowest susceptibility to permethrin with LC50 at 12.485 ppm. AS12 exhibited a 50-fold lower susceptibility to permethrin than to bifenthrin. AS12 showed higher susceptibility to pyrethroids than organophosphates, except for fenthion and permethrin.

Table 2

Susceptibility of 13 insecticides against Anopheles sinensis s.s. using direct contact diffusion assay with 24-hour exposure

Insecticide	na	Slope (±SE)	LC50 (ppm)	95% CL	χ2	RSb
Bifenthrin	450	1.0 ± 0.09	0.227	0.166–0.312	5.35	1.0
Cyfluthrin	375	1.0 ± 0.12	0.446	0.315–0.692	4.69	2.0
Etofenprox	450	1.0 ± 0.90	1.858	1.360–2.331	6.53	8.2
Fenthion	300	1.3 ± 0.16	2.377	1.732–3.120	2.65	10.5
Cypermethrin	375	1.0 ± 1.12	2.419	1.748–3.217	1.83	10.7
λ-Cyhalothrin	300	1.2 ± 0.15	3.220	2.410–3.543	0.54	14.2
α-Cypermethrin	300	1.4 ± 0.16	3.323	2.561–3.689	2.70	14.6
Deltamethrin	375	1.4 ± 0.14	4.522	3.444–5.216	6.03	19.9
Dichlorvos	450	1.1 ± 0.13	4.850	3.907–5.224	3.57	21.4
Chlorpyrifos	375	1.2 ± 0.13	4.890	3.951–5.336	3.39	21.5
Profenofos	375	1.6 ± 0.14	5.829	4.916–7.361	3.89	25.7
Fenitrothion	450	1.3 ± 0.13	7.860	6.115–9.124	2.99	34.6
Permethrin	375	1.5 ± 0.16	12.485	9.799–13.599	0.45	55.0

Number of mosquitoes;

LC CL = confident limit; LC50 = median lethal dose; RS = relative susceptibility.

Comparative analysis of data for larvae collected from the same locality in 2001 (AS01) and 2008 (AS08) was carried out for the 13 insecticides [10, 21] (Table 3). Chang et al [10] showed that AS08 exhibited decreased pyrethroid resistance compared to AS01, except for permethrin and deltamethrin. The RR08-01 values of An. sinensis to pyrethroids ranged from 0.03 to 0.40 as follows: bifenthrin: 0.03; λ-cyhalothrin: 0.06; α-cypermethrin: 0.30; cypermethrin: 0.34; and cyfluthrin: 0.40. The RR08-01 values of deltamethrin and permethrin were 1.50 and 3.88 (low resistance level), respectively. However, AS12 showed higher pyrethroid resistance than AS08, with RR12-08 values ranging from 15.07 to 55.38 (moderate to high) as follows: α-cypermethrin: 55.38; λ-cyhalothrin: 40.25; bifenthrin: 25.22; and deltamethrin: 15.07. The RR12-08 values of An. sinensis to cypermethrin, cyfluthrin, and permethrin were less than 10-fold greater.

Table 3

Comparison of insecticide susceptibility of larvae of Anopheles sinensis s.s. among 2001 [21], 2008 [10], and 2012 strains collected in Paju, Republic of Korea

Insecticide	LC50 (ppm)
	AS01a	AS08	AS12	RR12-01b	RR12-08	RR08-01
Organophosphates
Fenthion	0.03	1.1	2.377	79.23	2.16	36.67
Dichlorvos	1.9	3.5	4.850	2.55	1.39	1.84
Chlorpyrifos	3.4	4.7	4.890	1.44	1.04	1.38
Profenofos	0.3	3.7	5.829	19.43	1.58	12.33
Fenitrothion	5.0	7.7	7.860	1.57	1.02	1.54
Pyrethroids
Bifenthrin	0.28	0.009	0.227	0.81	25.22	0.03
Cyfluthrin	0.5	0.2	0.446	0.89	2.23	0.40
Cypermethrin	4.7	1.6	2.419	0.51	1.51	0.34
λ-Cyhalothrin	1.3	0.08	3.220	2.48	40.25	0.06
α-Cypermethrin	0.2	0.06	3.323	16.62	55.38	0.30
Deltamethrin	0.2	0.3	4.522	22.61	15.07	1.50
Permethrin	0.8	3.1	12.485	15.61	4.03	3.88

AS01, colony of Shin et al, 2003 [21]; AS08, colony of Chang et al, 2009 [10]; and AS12, colony collected in 2012;

RR. LC50 = median lethal dose; RR = resistance ratio.

The resistance of An. sinensis to five organophosphates has continuously increased since 2001.

The RR08-01 of An. sinensis to organophosphates were low to moderate with values of 1.38 ppm to 36.67 ppm. AS08 showed moderate levels of resistance to fenthion and profenofos with RR values of 36.67 and 12.33, and low levels of resistance to dichlorvos, fenitrothion, and chlorpyrifos with RR values of 1.84 ppm, 1.54 ppm, and 1.38 ppm, respectively. When compared to AS01, the RR12-08 of An. sinensis to organophosphates were low with values ranging from 1.02 to 2.16, as follows: fenitrothion: 1.02; chlorpyrifos: 1.04; dichlorvos: 1.39; profenofos: 1.58; and fenthion: 2.16. Although the organophosphate resistance level of AS08 was low, resistance to organophosphates had not decreased. The RR12-01 of An. sinensis were low to high with values of 1.44 to 79.23. AS12 to fenthion demonstrated a high level of resistance with an RR value of 79.23, a moderate level of resistance to profenofos with an RR value of 19.43, and low levels of resistance to dichlorvos, fenitrothion, and chlorpyrifos with RR values of 2.55, 1.57, and 1.44, respectively.

Discussion

Insecticides have played a major role in the control of agricultural pests and vectors in the ROK, but their long and frequent use has resulted in significant insecticide resistance [8,10-13]. In our study, An. sinensis s.s. collected in 2012 showed high levels of pyrethroid resistance compared to samples gathered from the same locality in 2008.

These findings may be the result of increased use of pyrethroids for agricultural pest control. According to pest control operators in this area, they changed to pyrethroids in 2007 because organophosphates were failing to control agricultural pests. Because An. sinensis breeds mainly in paddy fields, it is under heavy selection pressure due to the agricultural application of insecticides [10,21]. Pyrethroids have also been used for thermal fogging, residual spraying, and as a repellent applied to clothing at low concentrations for medical pests. These uses may have resulted in the development of resistance to pyrethroids over 4 years. Although the use of organophosphates against agricultural pests has decreased in this area since 2007 and resistance to organophosphates is now low, constant use may maintain the same level of resistance of An. sinensis. An. sinensis collected in 2008 showed higher resistance to organophosphates than pyrethroids compared to the sample collected in 2001, because organophosphate insecticides have been used primarily to control agricultural pests from 2001 to 2007 [10].

Resistance monitoring is an effective component of resistance management as it provides current information on the response of An. sinensis populations to insecticides. Susceptibility tests need to be conducted over a broad area, as insecticide pressures and usage may very geographically. Insecticide failures in the ROK have probably occurred as a result of the development of field resistance [9-11,13,22]. Early detection of trends in the development of potential resistance can facilitate the use of synergists, rotation of insecticides and/or classes of insecticides, and alternative technologies that reduce dependence on and usage of chemical insecticides [13,23,24].

These results indicate that strategies which limit insecticide use and discourage it when no longer effective, and encourage the selective rotation of classes of insecticides provide increased vector control against field populations of the malaria vector, An. sinensis, in the ROK.