Presenter: Tara Thiemann PhD, Associate Professor, University of the Pacific with research contributions by graduate students Sumiko De La Vega and Bonnie Ryan
Culex tarsalis is an important vector of West Nile Virus and other arboviruses in the Western US. Once more common in rural areas, Culex tarsalis is now often found in residential areas and faces insecticide pressure both from vector control efforts and agricultural spraying. Until recently, few reports of insecticide resistance in Cx. tarsalis had been reported, but over the last few years that has changed.
To investigate this, Thiemann launched a research project with two main objectives. The first was to determine the prevalence of pyrethroid resistance among mosquitos in select study sites across five counties in Northern California. Once the onset of resistance could be verified, Thiemann wanted to better understand the mechanisms behind it, in particular, the presence of enzymatic resistance and target-site mutations.
Pyrethroids target the nervous system of the mosquito by binding to, and opening, voltage-gated sodium channels (VGSC) in synaptic cells. This binding activity holds those channels open, which in turn increases the influx of sodium into the neurons and starts a chain reaction that overexcites mosquito’s neurons. As a result, the mosquito uses all of its energy contracting muscles and sending action potentials, ultimately leading to death.
One way insecticide resistance develops is when mosquitos adapt and become able to produce more enzymes (esterases and oxidases in the case of pyrethroids) that are able to break down or inhibit the insecticide. A second mechanism is target site mutation. In the latter, a mutation in the insect’s DNA leads to an amino acid substitution that reduces the ability of the insecticide to bind to its target site. For Thiemann and her team, the mutation of interest was Knockdown Resistance (kdr) – mutations that prevent the insecticide from binding to the target site. Two common mutations in mosquitoes relate to changes in the gene that codes for voltage-gated sodium channels, substitutions involving the amino acid Leucine changing to Phenylalanine or Serine.
To first determine the prevalence of resistance, mixed age females were collected using CO2-baited traps. Between three and five populations were collected in each of the collaborating county districts in California: San Joaquin, Sac-Yolo, Placer, and Lake, and were evaluated with the CDC bottle bioassays. Mortality rates were recorded every 15 minutes for a two-hour period.
Among the susceptible (control) populations, results showed high and rapid mortality rates in bottles containing the pyrethroid Permethrin alone as well as in bottles containing Permethrin + PBO (a synergist that inhibits oxidases). While recorded mortality was also high for wild mosquitos in the Permethrin + PBO bottles, mortality rates in the Permethrin only bottles were typically quite low. The results not only confirmed the existence of resistant populations, but also, as Thiemann put it, that the challenge was “a bit more than we expected.” (see Summary of 17 Study Sites).
For the second objective of the study, researchers used enzyme (microplate) assays and molecular testing. In the microplate assays, mosquitoes were ground in a buffer and then added to the plates. A substrate and other reactants were then added to the plates to start a chemical reaction. The resulting product assumed a color varying in intensity depending on how much enzyme activity was in the sample. An instrument was then used to analyze the color to measure the amount of enzyme present.
The team measured for levels for a variety of detoxifying enzymes including oxidases, alpha-esterases, beta-esterases, glutathione-S-transferases (GSTs) and acetylcholinesterase (ACE), but the ensuing data proved difficult to analyze. While levels were elevated among the wild population samples, researchers learned that the control group also showed higher levels of the enzymes than they had anticipated. This made comparative analysis challenging. Thiemann states she is working with a statistician to determine if a different approach might be used to analyze the results.
Testing for target site mutations, however, proved far more definitive.
Thiemann and her team employed SYBR green qPCR molecular assays using three different primers that matched up to the three alleles they were looking for: Leucine, Phenylalanine, and Serine. The results showed a high prevalence of the Leucine to Phenylalanine kdr mutation as well as a lesser amount of the Leucine to Serine mutation (which are linked to insecticide resistance in these mosquito populations).
Intrigued by the results, Thiemann brought some Culex tarsalis samples “out of the freezer” from her grad school days (2006-2008) and was able to closely match them to a few capture sites from the 2018 study. Not only was the comparison conclusive, it was dramatic. Her analysis showed high prevalence of the kdr alleles, particularly the Leucine to Phenylalanine mutation. Moreover, the ten-year comparison showed that samples of kdr alleles had completely reversed from susceptible to a current high prevalence of resistance alleles. (see kdr Allele Prevalent Change Over Time).
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