Ambiguous literature | kdr triple resistance mutation – Where has it really been found?

Editors note: This article is direct from our internal research diary

Words: Nancy M. Endersby-Harshman

The purpose of this article is to recommend very careful reading and analysis of the literature relating to sodium channel mutations in Aedes aegypti and equal care in writing about them.

I have uncovered some confusion in the mosquito literature about the kdr triple resistance mutation in the sodium channel of Ae. aegypti. The genotype in question consists of homozygous mutants for each of the sodium channel sites 1016, 1534 and 989 (numbered according to the sodium channel protein nomenclature of the housefly) in the one individual. These three sites, in particular combinations, have been shown to confer resistance to pyrethroid insecticides by functional analysis in Xenopus oocytes by Du et al. (2013). The kdr triple resistance mutation shows an extremely pyrethroid-resistant phenotype when expressed artificially in Xenopus oocytes (Hirata et al. 2014). Kawada et al. (2014) found two individual Ae. aegypti out of 240 that showed the triple resistance mutation in Myanmar. There is another verified example of a triple mutant from Saudi Arabia (Al Nazawi et al. 2017), but these mosquitoes were susceptible to deltamethrin (however, there were very few available to test). Although the genotype was created artificially in Xenopus oocytes by Hirata et al. (2014), it seems that the triple homozygous mutant is difficult to find in nature because of the linkage configuration of the 1016 and 1534 sites. Homozygous mutants at 1016 are very often linked to homozygous wildtype alleles at 1534 and vice versa. The 989 mutant generally follows the pattern of 1016. However, Hirata et al. (2014) caution that a single crossing over event could result in an individual with the triple mutation.

In a review of insecticide resistance in Ae. aegypti, Du et al. (2016) concluded that the triple mutant was also found in Yogyakarta, Indonesia, based on the study of Wuliandari et al. (2015) possibly due to some ambiguous wording on our part. Instead, Wuliandari et al. (2015) were referring to a single individual that was homozygous 1534C in the wMelPop line. It was not a homozygous mutant for 1016 or 989. A Thai reference (Yanola et al. 2011) also contains no evidence of a triple resistance mutant, but Du et al. (2016) use this work as an example of the same. I have also read the paper by Li et al. (2015) and I don’t believe they found the triple mutation in a single individual in China either though Du et al. (2016) cite their study as discovering one.

A more comprehensive review of insecticide resistance in Ae. aegypti and Ae. albopictus by Smith et al. (2016) only cite the Myanmar study as showing the triple mutant and have interpreted Wuliandari et al. (2015) as we intended. They and Du et al (2016), however, add more complications by citing Pang et al. (2015) as showing a double mutant which was homozygous at 1016 and 1534 (called inexplicably 1292 in this paper) from Singapore. Pang et al. (2015) did not screen the 989 site, but seem to have at least a very low frequency of the equally unusual double mutant. It may be that the genuine triple mutation could be found in Ae. aegypti in Singapore if the 989 site were screened as well. Al Nazawi et al. (2017) refer to another Indonesian study (Sayono et al. 2016).and mention that the triple mutation was found in some mosquitoes from Java, Indonesia. The study by Sayono et al. (2016) does appear to show the true triple homozygous mutant.

It is possible and common in some south east Asian populations to find individuals that are triple heterozygotes (heterozygous for each of 1016, 1534 and 989), but these individuals should not be referred to as triple resistance mutants. Triple heterozygotes probably occur by crossing the two different linkage types referred to above and do not arise from parents that are homozygous at both 1016 and 1534 in the one individual. We all must be very careful and unambiguous in how we refer to the triple kdr mutation so that no further mistakes invade or perpetuate in the literature. At this time, it seems that the only verified co-occurrence of triple homozygous mutations of 1016G/1534C/989P within an individual have been detected in Ae. aegypti populations in Myanmar (Kawada et al. 2014), Indonesia (Sayono et al. 2016) and Saudi Arabia (Al Nazawi et al. 2017). More studies of their resistance status when they occur in nature seem to be indicated.

References

Al Nazawi AM, Aqili J, Alzahrani M, McCall PJ, Weetman D (2017) Combined target site (kdr) mutations play a primary role in highly pyrethroid resistant phenotypes of Aedes aegypti from Saudi Arabia. Parasites & Vectors 10, 161.

Du Y, Nomura Y, Satar G, Hu Z, Nauen R, He SY, Zhorov BS, Dong K (2013) Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel. Proceedings of the National Academy of Sciences of the United States of America 110, 11785-11790.

Du Y, Nomura Y, Zhorov BS, Dong K (2016) Sodium channel mutations and pyrethroid resistance in Aedes aegypti. Insects 7, 1-11.

Hirata K, Komagata O, Itokawa K, Yamamoto A, Tomita T, Kasai S (2014) A single crossing-over event in voltage-sensitive Na+ channel genes may cause critical failure of dengue mosquito control by insecticides. PLoS Neglected Tropical Diseases 8, e3085.

Kawada H, Oo SZM, Thaung S, Kawashima E, Maung YNM, Thu HM, Thant KZ, Minakawa N (2014) Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar. PLoS Negl Trop Dis 8, e3032.

Li C-X, Kaufman PE, et al. (2015) Relationship between insecticide resistance and kdr mutations in the dengue vector Aedes aegypti in Southern China. Parasites & Vectors 8, 325.

Pang SC, Chiang LP, Tan CH, Vythilingam I, Lam-Phua SG, Ng LC (2015) Low efficacy of deltamethrin-treated net against Singapore Aedes aegypti is associated with kdr-type resistance. Tropical Biomedicine 32, 140-50.

Sayono S, Hidayati APN, Fahri S, Sumanto D, Dharmana E, Hadisaputro S, Asih PBS, Syafruddin D (2016) Distribution of voltage-gated sodium channel (Nav) alleles among the Aedes aegypti populations in Central Java Province and its association with resistance to pyrethroid insecticides. PLOS ONE 11, e0150577.

Smith LB, Kasai S, Scott JG (2016) Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases. Pesticide Biochemistry and Physiology 133, 1-12.

Wuliandari J, Lee S, White V, Tantowijoyo W, Hoffmann A, Endersby-Harshman N (2015) Association between three mutations, F1565C, V1023G and S996P, in the voltage-sensitive sodium channel gene and knockdown resistance in Aedes aegypti from Yogyakarta, Indonesia. Insects 6, 658-685.

Yanola J, Somboon P, Walton C, Nachaiwieng W, Somwang P, Prapanthadara LA (2011) High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand. Tropical Medicine & International Health 16, 501-9.