Elsevier

Acta Tropica

Volume 108, Issue 1, October 2008, Pages 54-57
Acta Tropica

Short communication
Development and application of a simple colorimetric assay reveals widespread distribution of sodium channel mutations in Thai populations of Aedes aegypti

https://doi.org/10.1016/j.actatropica.2008.08.004Get rights and content

Abstract

Dengue fever and its more serious complications dengue haemorrhagic fever and dengue shock syndrome are growing public health problems in tropical and subtropical countries. In the absence of a vaccine, most dengue control programmes rely heavily on the use of insecticides to target the Aedes mosquito vectors. As a limited number of insecticides are routinely used in control, monitoring for the presence of resistance is an essential component of dengue prevention programmes. The pyrethroid insecticides target the voltage-gated sodium channel on the insects’ neurons. Substitutions at residue 1016 of this protein have been associated with pyrethroid and DDT resistance in Aedes aegypti populations from Latin America and Asia. Here we report on the development of a simple colorimetric assay to detect these mutations in individual mosquitoes. Evaluation of this diagnostic assay on 180 Ae. aegypti individuals from Thailand revealed the presence of high frequencies of the Val1016Gly mutation throughout the country. The assay requires no specialised equipment and will enable monitoring for insecticide resistance associated alleles to be routinely incorporated into dengue surveillance operations.

Introduction

The mosquito Aedes aegypti Linn. is the major vector of dengue and dengue haemorrhagic fever worldwide. Currently the only method of preventing transmission of the virus that causes these diseases is to target the vector. Most vector control programmes rely extensively on a small number of classes of insecticides, and resistance to these insecticides is an increasing public health concern. Due to their rapid mode of action and low mammalian toxicity, pyrethroid insecticides have proved to be particularly useful in vector control. In the case of dengue vectors they are used currently as space sprays for adult mosquito control and, deployed as residues on window curtains or as container covers (Kroeger et al., 2006) and they are an important basis for novel approaches to dengue control for the future. However, resistance to pyrethroids is now widespread and has been reported in most regions where Ae. aegypti is established (Rodríguez et al., 2001, Rodríguez et al., 2002, Ponlawat et al., 2005, Brengues et al., 2003).

Both pyrethroids and the organochlorine insecticide, DDT, target the voltage-gated sodium channel on the insects’ neurons. Single amino acid substitutions in this ion channel have been associated with resistance to these insecticides. This form of resistance, known as knockdown resistance (kdr), has been observed in several insect species including the mosquitoes Anopheles gambiae (Martinez-Torres et al., 1998), Anopheles stephensi (Enayati et al., 2003), Culex quinquefasciatus (Martinez-Torres et al., 1999) and Aedes aegypti (Brengues et al., 2003). In Culex and Anopheles, kdr resistance is associated with the replacement of the leucine residue at position 1014 in the S6 segment of transmembrane domain II with either phenylalanine or serine. The codon usage of the Ae. aegypti sodium channel does not favour substitutions at residue 1014. Instead mutations at positions 923, 982, 1011 and 1016 have been detected in a study of 11 permethrin resistant Ae. aegypti populations collected from six different countries (Brengues et al., 2003) (Table 1). To date only substitutions at one of these four residues have been positively correlated with resistance to pyrethroid insecticides. Selection experiments with two different pyrethroids resulted in a dramatic increase in the frequency of the Ile1016 allele in Latin American strains of Ae. aegypti (Saavedra-Rodriguez et al., 2007). An alternative mutation at this codon, resulting in a Gly1016 replacement has been reported in Asian populations of Ae. aegypti (Brengues et al., 2003, Saavedra-Rodriguez et al., 2007).

Here we report on the development of a series of simple colorimetric assays to detect sodium channel mutations at residues 1011 and 1016 in Ae. aegypti. Using this technique, analysis of 180 individual mosquitoes collected from north, central and southern Thailand revealed that the Gly1016 mutation is widely distributed in this country. The Val1011 mutation was also detected but at lower frequencies.

Section snippets

Mosquito strains

The pyrethroid resistant Isla Mujeres strain originates from Mexico and was described in Saavedra-Rodriguez et al. (2007). The pyrethroid susceptible New Orleans strain was kindly provided by the centre for disease control (CDC), Atlanta, USA. Field material from Thailand (Fig. 1) was collected as larvae and reared to adults in the insectaries at Vector Borne Disease Control Unit, Phang nga, and subsequently at the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol

Results

A 457 bp fragment of genomic DNA was amplified from the S6 hydrophobic region of domain II of the voltage-gated sodium channel gene from Ae. aegypti from two laboratory strains (New Orleans susceptible strain and Isla Mujeres pyrethroid resistant strain from Mexico) and six field populations from Thailand. Sequence analysis revealed the three non-silent mutations reported earlier (Brengues et al., 2003), Gly1016, Ile1016 and Val1011 (Saavedra-Rodriguez et al., 2007). An intron, which varied from

Discussion

Our study shows that the HOLA assay is a reliable method to detect Ae. aegypti sodium channel mutations, permitting clear discrimination between homozygotes and heterozygotes. This assay can be performed on DNA extracted from individual dried mosquitoes and requires only a small DNA sample to obtain an accurate genotype. The reagents used are stable and easy to obtain and the methodology does not involve the use of radioisotopes or any specialised equipment. Hence it is an ideal assay to be

Acknowledgements

We would like to thank Mr Suvit Pengpit, Dr Audrey Lenhart and Dr Tom Scott for assistance with mosquito collections in Thailand and Dr Louise Kelly-Hope for the preparation of the map.

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