Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The voltage-gated sodium channel is the primary target of both pyrethroids and DDT, whose insecticidal effects are known to operate by altering the function of voltage-gated sodium channels in the nerve membranes of insects and preventing the repolarization phase of action potentials. The substitution of leucine to phenylalanine (L to F), the first mutation resulting from a single nucleotide polymorphism (SNP) in the insect sodium channel domain II segment 6 (IIS6), has been clearly associated with resistance to pyrethroids and DDT in many insect species (11-22). Subsequently, more than 20 sodium channel mutations have been reported in different insect species in comparison between resistant and susceptible ones. These data suggest that not only do mutations in the insect sodium channel play an important role in insecticide resistance, but also that L to P substitution is unlikely to be the sole mechanism governing kdr-mediated resistance. In our current study, we cloned and sequenced the full length of the sodium channel cDNAs from three different mosquito strains of Cx. Quinquefasciatus bearing different resistant phenotypes, ranging from susceptible (S-Lab), intermediary (parental HAmCqG0) to highly resistant (HAmCqG8, the 8th generation permethrin-selected offspring of HAmCqG0); conducted a systematic comparison of the sodium channel full length cDNA sequences and their deduce amino acid sequences among these three mosquito populations in order to identify nucleotide polymorphisms presented in the Culex soium channel; and investigated the correlation of the frequency of each mutation or codon resulting from each polymorphism at RNA level with the levels of permethrin resistance and evolution of the polymorphisms or mutations following permethrin selection. Three nonsynonymous (L1027F, A109S, and W1592R) and 6 synonymous (L897L, G936G, A1260A, D1264D, P1268P, and P1813P) mutations were found in the Culex mosquito sodium channel. A comparative framework was used to examine adaptive changes in the patterns of amino acid and codon usage in the 3 Culex mosquito strains under permethrin selection pressure, revealing the frequency of both nonsynonymous and synonymous mutations in the mosquito sodium channel underwent a rapid population expansion following permethrin selection. This finding suggests permethrin selection is the prevailing selective force in the evolution of the amino acid and codon usage of the mosquito sodium channel, thus affecting the sensitivity of the sodium channel to insecticides and enabling the mosquito to survive. This study provides the first evidence of the importance of synonymous mutations in insect response to insecticide selection. PARTICIPANTS: Qiang Xu, Ting Li, Lan Zhang, Lee Zhang, Nannan Liu TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This study for the first time demonstrated a correlation between the frequency of both nonsynonymous and synonymous mutations and levels of pyrethroid resistance in Culex mosquitoes. The findings of this study indicate that synonymous mutations may contribute to the insensitivity of sodium channels to pyrethroid insecticides, and that they are involved in the insect's response to permethrin selection. The information from the proposed research will allow us to apply extramural funding because it has a great impact on the mosquito management.
Publications
- N. Liu*. 2008. Recent Advances in Insect Physiology, Toxicology and Molecular Biology. Research Signpost, ISBN: 978-81-308-0242-8.
- Liu, N*. 2008. Insecticide resistance in mosquitoes: development and mechanisms. In: Recent Advances in Insect Physiology, Toxicology and Molecular Biology, Liu, N. (Ed.), Research Signpost, Kerala, India, pp. 75-91.
- Liu, N*. 2008. The mosquito sodium channel: adaptive changes of amino acid and codon basis in the evolution of pyrethroid resistance. International Drug Discovery Science and Technology, Beijing, China.
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Progress 10/01/05 to 09/30/08
Outputs
OUTPUTS: An extensive survey of permethrin resistance in Alabama designed to characterize the importance of the L-to-F kdr mutation in pyrethroid resistant Culex mosquitoes. Mosquitoes were collected from 19 counties, 17 in Alabama and 2 from neighboring counties in Florida and Tennessee. Culex quinquefasciatus from all the counties tested were found to have developed resistance to permethrin. Most of the mosquito populations tested showed a strong correlation between their level of resistance and the frequency of L-to-F mutation expression, suggesting the importance of target site insensitivity in the development of permethrin resistance. However, 4 populations with elevated levels of resistance showed no L-to-F mutation in their sodium channels. While it is possible that other mutations other than L-to-F are present in the sodium channel, resulting in permethrin resistance in these 4 field populations, resistance mechanisms other than target site insensitivity must also be considered. Target site insensitivity resulting from point mutations within the voltage-gated sodium channel of the insect nervous system is known to be of primary importance in the development of resistance to pyrethroid insecticides. Most previous research has focused on partial sodium channel sequences and nonsynonymous mutations; neither synonymous mutations nor evolutionally adaptive changes in the frequency of amino acid and codon usage of the insect sodium channel have been studied. Here, we report a systematic analysis of nucleotide polymorphisms through the entire sodium channel cDNAs among susceptible, intermediately resistant parental, and highly resistant offspring mosquitoes Culex quinquefasciatus and the dynamics of the synonymous and nonsynonymous nucleotide composition of the mosquito sodium channel under insecticide selection pressure. Three nonsynonymous and 7 synonymous mutations were found in the Culex mosquito sodium channel. We characterized adaptive changes in the patterns of amino acid and codon usage in the 5 Culex mosquito strains under permethrin selection pressure and found the frequency of both nonsynonymous and synonymous mutations in the mosquito sodium channel underwent a rapid population expansion following permethrin selection. We next to see if the prevalence of sodium channel mutation and mutation combination correlated with the sensitivity of mosquitoes to permethrin at different concentrations (or doses) and if a specific threshold of insecticide concentration presets at which a particular mutation or mutation combination occurs in a mosquito population or how the frequency of polymorphic mutation changes during the selection process. We found that the individuals caring homozygous susceptible alleles were gradually decreased following groups that increased their tolerance to permethrin treatments. In contrast, the individuals had homozygous polymorphic alleles were gradually increased following the groups' increasing in tolerance to permethrin treatments. These results strongly suggest the importance of these mutations in the development of resistance in mosquitoes. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This study, for the first time, demonstrated a correlation between the frequency of both nonsynonymous and synonymous mutations and levels of pyrethroid resistance in Culex mosquitoes. The finding has provided a framework for conducting the future functional studies on the roles of mutations in the mosquito sodiun channel sensitivity and insecticide resistance.
Publications
- Liu, N., Q. Xu, T. Li, L. He and L. Zhang. 2009. Permethrin resistance and target site insensitivity in the mosquito Culex quinquefasciatus in Alabama, U.S.A. J. Med. Entomol. 46, 1424-1429__________________________
- Xu, Q. and N. Liu. 2009. The development of pyrethroid resistance in the mosquito Culex quinquefasciatus. In: Recent Advances in Entomological Research: From Molecular Biology to Pest Management, Liu, T. X and Kang, L. (Eds.), Springer-High Education Press, China. 172-178____________
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: 1). Monitoring pyrethroid resistance in field populations of mosquitoes This year, we have further conducted mosquitoes from other 10 counties, including Tallapoosa County, Coosa County, Talladega County, Shelby County, Jefferson County, Blount County, Cullman County, Morgan County, and Limestone County of Alabama and Nashville of TN (as a reference). We conducted bioassays to examine the levels of resistance in the mosquito populations with permethrin. Each bioassay was repeated three times. Our results indicated that Cx. quinquefasciatus had highest levels of resistance with resistance ratios ranged from 125 to 1400 compared with other species and the laboratory susceptible S-Lab strain. Whereas, Cx. retuan showed the similar sensitivity to permethrin as that of the susceptible S-Lab strain. Monitoring A to T single nucleotide polymorphism (SNP) for the kdr allele in field populations of mosquitoes A substitution of leucine to phenylalanine (L to F) resulting from the A to
T single nucleotide change, termed the kdr mutation, in the sodium channel has been recognized as associated with pyrethroid resistance of mosquitoes. Comparison of resistance levels and the frequency of the A to T SNP for the kdr allele in each mosquito population is significant for determine the importance of the kdr mutation in resistance. We extracted RNAs from individual adult mosquitoes of each field mosquito population and detected the frequency and the homozygosity of the A to T SNP for the kdr allele using a SNaPshot Kit. Our study showed that the high frequency of the kdr allele (A/T or T) presented in all field populations of Cx. quinquefasciatus (Tables 1 and 2). Nevertheless, only the susceptible A allele was detected in all other mosquito populations. Our results revealed a strong correlation between the kdr allelic (A/T or T) expression and levels of permethrin resistance and susceptibility, suggesting that target site insensitivity is the major mechanism involved in
pyrethroid resistance in the field population of Cx. quinquefasciatus in Alabama. 3). Differential expression of genes in resistant Cx. quinquefasciatus in Alabama We have compared the gene expression profiles in resistant HAmAal mosquitoes from Huntsville, Alabama using a combination of subtractive PCR hybridization and cDNA microarray technique. By screening of 1500 cDNA clones from a mosquito subtractive library, we identified a set of genes with up-regulated expression in insecticide resistant HAmAal mosquitoes compared to those in susceptible mosquitoes. These genes are vital for cellular and molecular metabolism, signal transduction, vesicular and molecular transport, protein biosynthesis, ubiquitination, and neuronal survival, but most have not previously been implicated in insecticide resistance. Functional studies of resistance-associated up-regulated genes should shed new light on both the molecular basis of resistance and the regulatory pathways that lead to it.
PARTICIPANTS: Dr. Nannan Liu, Dr. Qiang Xu, Dr. Lee Zhang, Ms. Ting Li
Impacts
The project will provide the first information on pyrethroid resistance in mosquitoes in Alabama and improve our understanding of mechanisms governing resistance development, which, in turn, will be important for designing appropriate strategies for the mosquito management. The information from the proposed research will allow us to apply extramural funding because it has a great impact on the mosquito management.
Publications
- Liu, N., H. Liu, F. Zhu and L. Zhang. 2007. Differential Expression of Genes in Pyrethroid Resistant and Susceptible Mosquitoes, Culex quinquefasciatus (S.). Gene 394, 61-68. _____________________________________________________________________ _____________________________________
- Liu, N. 2007. Insecticide resistance in mosquitoes: development and mechanisms, Chapter 6 In N. Liu (ed.), Recent Advances in Insect Physiology, Toxicology and Molecular Biology, Research Signpost (In press).
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Progress 01/01/06 to 12/31/06
Outputs
1). Monitoring pyrethroid resistance in field populations of mosquitoes We have collected mosquitoes from Colbert, Winston, Cherokee, Jefferson, Lamar, and Montgomery counties as we proposed in the original proposal for year 1. We conducted bioassays to examine the levels of resistance in the mosquito populations with permethrin (a pyrethroid insecticide). Our results indicated that Cx. quinquefasciatus had highest levels of resistance with resistance ratios ranged from 125 to 1400 compared with other species and the laboratory susceptible S-Lab strain (Table 1). Whereas, Cx. retuans, Cx. nigripalpus, and Cx. salinarius showed the similar sensitivity to permethrin as that of the susceptible S-Lab strain. Although we will continually monitor mosquito resistance from other counties in the Alabama state in next two years as we proposed, our first year's data may suggest a common phenomenon of insecticide resistance in Cx. quinquefasciatus in Alabama. 2). Monitoring A to
T single nucleotide polymorphism (SNP) for the kdr allele in field populations of mosquitoes A substitution of leucine to phenylalanine (L to F) resulting from the A to T single nucleotide change, termed the kdr mutation, in the sodium channel has been recognized as associated with pyrethroid resistance of mosquitoes. Comparison of resistance levels and the frequency of the A to T SNP for the kdr allele in each mosquito population will be significant for determine the importance of the kdr mutation in resistance. We extracted RNAs from individual adult mosquitoes of each mosquito population and detected the frequency and the homozygosity of the A to T SNP for the kdr allele. Our study showed that the high frequency of the kdr allele (A/T or T) presented in all field populations of Cx. quinquefasciatus (Table 1). Nevertheless, only the susceptible A allele was detected in all other mosquito populations. Our results revealed a strong correlation between the kdr allelic (A/T or T)
expression and levels of insecticide resistance and susceptibility, suggesting that target site insensitivity is the major mechanism involved in the pyrethroid resistance in the field population of Cx. quinquefasciatus mosquitoes in Alabama. 3). Sodium channel gene expression in mosquito Aedes albopictus We have collected a mosquito strain of Aedes albopictus, HAmAalG0, from Huntsville, Alabama. Our bioassay result showed that HAmAalG0 had a normal susceptibility and low tolerance to permethrin compared to a susceptible strain, even though these pyrethroid insecticides have been used in the field for a long period of time in Alabama. This strain was reared in the laboratory and selected with permethrin for 5 generations. We detected no significant change in the level of resistance to permethrin in the selected mosquitoes compared with the parental strain HAmCqG0. We examined the allelic expression at the kdr site of the sodium channel gene in the Aedes mosquitoes. We have found that
every tested individual in HAmAalG0 and selected mosquitoes expressed A allele at the L-to-F kdr site encoding Leu, strongly corresponding to their susceptibility to insecticides.
Impacts
The project will provide the first information on pyrethroid resistance in mosquitoes in Alabama and improve our understanding of mechanisms governing resistance development, which, in turn, will be important for designing appropriate strategies for the mosquito management. The information from the proposed research will allow us to apply extramural funding because it has a great impact on the mosquito management.
Publications
- Liu, N., Q. Xu, and L. Zhang. 2006. Sodium Channel Gene Expression in Mosquitoes, Aedes albopictus (S.). Insect Sci. 13, 431-436. _____________________________________________________________________ ___
- Zhang, Q., Q. Xu, N. Liu. 2006. Sodium Channel Gene Expression in Mosquitoes Culex quinquefasciatus in Alabama State. J. Med. Entomol. (Submitted)
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