Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

doi:10.1016/j.pestbp.2006.06.009

Pesticide Biochemistry and Physiology

Volume 87, Issue 1, January 2007, Pages 54-61

https://doi.org/10.1016/j.pestbp.2006.06.009 Get rights and content

Abstract

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.

Introduction

The mosquito Anopheles sinensis is a major vector of malaria in Korea. In the1990s the national malaria eradication program, based mostly on chemical control of vector mosquitoes, resulted in the apparent disappearance of malaria in South Korea; however malaria has since resurged with more than 21,400 cases detected to date (Korea Center for Disease Control and Prevention, http://dis.cdc.go.kr/eng_statistics/statistics.asp). Since their introduction during the 1970s, insecticides including pyrethroids and organophosphates have been widely used for the control of medically important arthropod pests, including mosquitoes. Permethrin and DDVP (Dichlorvos) have been used in Korea as the principal active ingredients of both indoor and outdoor mosquito control products and have also been widely used for the control of mosquitoes and flies in animal farming. Intensive use of insecticides was quickly followed by development of insecticide resistance in a variety of mosquito species including An. sinensis.

It is extremely important to understand the mechanism of insecticide resistance in order to suppress and delay the development of resistance and establish a reliable resistance monitoring system. Consequently, many studies have been conducted on the molecular basis of insecticide resistance. One important mechanism of resistance to pyrethroids is characterized by a marked reduction in the intrinsic sensitivity of the insect nervous system to these compounds. This phenomenon was originally reported as knockdown resistance (kdr) in Musca domestica, and it was subsequently determined that a single mutation (leucine to phenylalanine, Leu1014Phe) in the S6 transmembrane segment of domain II in the sodium channel is associated with kdr to pyrethroids and DDT in both M. domestica [1] and the German cockroach, Blattella germanica [2]. Kdr-related mutations, identical or similar to the Leu-to-Phe mutation, have been also reported in Anopheles gambiae [3] and in a variety of pyrethroid-resistant arthropod species [4].

To establish a successful resistance management system, it is essential to develop more sensitive tools for the rapid estimation of resistance allele frequencies from field populations [5], [6]. Detection of the conserved mutations associated with insecticide resistance, such as the Leu-to-Phe mutation, has been achieved by various DNA-based genotyping techniques, including PCR amplification of specific allele (PASA) [7], bi-directional PCR amplification of specific allele (bi-PASA) [8], single stranded conformational polymorphism (SSCP) [9], minisequencing, and serial invasive signal amplification reaction (SISAR) [10]. Although these individual genotyping techniques are very useful in precise estimation of both frequency and genotype of resistance alleles, they usually require a great number of analysis and sample preparation, limiting their potential as a high throughput resistance monitoring tool. For the rapid monitoring of resistance in a large number of field populations of mosquito, necessary would be a genotyping technique based on pooled DNA samples that can be employed at the preliminary step of resistance monitoring prior to more elaborate individual genotyping.

In the present study, we demonstrate that the Leu-Phe sodium channel mutation is commonly found in most Korean populations of An. sinensis and describe a simple and accurate real-time PASA (rtPASA) protocol for the estimation of the kdr allele (Leu-Phe mutation) frequency on a population basis of An. sinensis. We also discuss the applicability of this protocol in large scale resistance monitoring and management.

Section snippets

Mosquitoes

Anopheles sinensis mosquitoes were collected using an aspirator or black-light trap. Blood-fed female mosquitoes were individually placed in a paper cup containing water and allowed to lay eggs under the conditions of 27 ± 2 °C temperature, 65 ± 5% relative humidity, and 12:12 photoperiod (light:dark). Hatched larvae were reared under the same conditions and used for RNA or DNA extraction. For individual genotyping, F1 larvae were obtained from 100 blood-fed females, combined and stored at −20 °C

Cloning of the IIS4-6 fragment of the para-homologous sodium channel gene

Two contiguous overlapping cDNA fragments (230-bp IIS4-IIS5 and 347-bp IIS5-IIIS3) of the para-homologous sodium channel gene were amplified by PCR using degenerate primer sets and sequenced. The assembled 531-bp cDNA fragments showed 97.2 and 96.6% sequence identity to the corresponding region of the sodium channel genes from An. gambiae and Aedes aegypti, respectively. A 1500-bp PCR product was generated using the sequence-specific primer set (Table 1) with genomic DNA as template. Two

Discussion

Sequence analysis of the IIS4-IIS6 segment of the sodium channel gene revealed that the Leu-Phe mutation known to confer kdr was present in all the regional populations of An. sinensis examined. However, the Met-Thr mutation known to be associated with the super-kdr trait was not found in the populations examined. In the para-homologous sodium channels present in a variety of insect species [4], the Leu residue is encoded by the codon CTT, in which a C to T substitution at the first nucleotide

Acknowledgments

This work was supported by Grant 02-PJ1-PG10-20405-0001 from the Korea Health Industry Development Institute. H.W. Kim and J.H. Baek were supported in part by Brain Korea 21 program.

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Pesticide Biochemistry and Physiology

Abstract

Introduction

Section snippets

Mosquitoes

Cloning of the IIS4-6 fragment of the para-homologous sodium channel gene

Discussion

Acknowledgments

References (17)

Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella

Pestic. Biochem. Physiol.

Estimation of knockdown resistance in diamondback moth using real-time PASA

Pest. Biochem. Physiol.

The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids

Insect Biochem. Mol. Biol.

The molecular biology of knockdown resistance to pyrethroid insecticides

Insect Biochem. Mol. Biol.

A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria

Mayo Clin. Proc.

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in an Iowa population of European corn borer (Lepidoptera: Crambidae)

J. Econ. Entomol.

DNA-based genotyping techniques for the detection of point mutations associated with insecticide resistance in Colorado potato beetle Leptinotarsa decemlineata

Pest Manag. Sci.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

Proc. Natl. Acad. Sci. USA

Cited by (39)

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Regional and seasonal detection of resistance mutation frequencies in field populations of Anopheles Hyrcanus Group and Culex pipiens complex in Korea

Insecticide susceptibility status of Phlebotomus argentipes and polymorphisms in voltage-gated sodium channel (vgsc) gene in Kala-azar endemic areas of West Bengal, India

Histopathological effects of cypermethrin and Bacillus thuringiensis var. israelensis on midgut of Chironomus calligraphus larvae (Diptera: Chironomidae)

Seasonal dynamics of insecticide resistance, multiple resistance, and morphometric variation in field populations of Culex pipiens