Aquilegia, Vol. 30 No. 4, September-October 2006, Newsletter of the Colorado Native Plant Society

https://epublications.regis.edu/aquilegia/1117/thumbnail.jp

Using targeted next-generation sequencing to characterize genetic differences associated with insecticide resistance in Culex quinquefasciatus populations from the southern U.S.

Resistance to insecticides can hamper the control of mosquitoes such as Culex quinquefasciatus, known to vector arboviruses such as West Nile virus and others. The strong selective pressure exerted on a mosquito population by the use of insecticides can result in heritable genetic changes associated with resistance. We sought to characterize genetic differences between insecticide resistant and susceptible Culex quinquefasciatus mosquitoes using targeted DNA sequencing. To that end, we developed a panel of 122 genes known or hypothesized to be involved in insecticide resistance, and used an Ion Torrent PGM sequencer to sequence 125 unrelated individuals from seven populations in the southern U.S. whose resistance phenotypes to permethrin and malathion were known from previous CDC bottle bioassay testing. Data analysis consisted of discovering SNPs (Single Nucleotide Polymorphism) and genes with evidence of copy number variants (CNVs) statistically associated with resistance. Ten of the seventeen genes found to be present in higher copy numbers were experimentally validated with real-time PCR. Of those, six, including the gene with the knock-down resistance (kdr) mutation, showed evidence of a ≥ 1.5 fold increase compared to control DNA. The SNP analysis revealed 228 unique SNPs that had significant p-values for both a Fisher's Exact Test and the Cochran-Armitage Test for Trend. We calculated the population frequency for each of the 64 nonsynonymous SNPs in this group. Several genes not previously well characterized represent potential candidates for diagnostic assays when further validation is conducted

Zika Virus MB16-23 in Mosquitoes, Miami-Dade County, Florida, USA, 2016

We isolated a strain of Zika virus, MB16-23, from Aedes aegypti mosquitoes collected in Miami Beach, Florida, USA, on September 2, 2016. Phylogenetic analysis suggests that MB16-23 most likely originated from the Caribbean region

Characterization of <em>Culex pipiens</em> Complex (Diptera: Culicidae) Populations in Colorado, USA Using Microsatellites

<div><p>Mosquitoes such as those in the <em>Culex pipiens</em> complex are important vectors of disease. This study was conducted to genetically characterize <em>Cx. pipiens</em> complex populations in the state of Colorado, USA, and to determine the number of genetic clusters represented by the data. Thirteen populations located among four major river basins were sampled (n = 597 individuals) using a panel of 14 microsatellites. The lowest-elevation sites had the highest Expected Heterozygosity (H_E) values (range 0.54–0.65). AMOVA results indicated the presence of statistically significant amounts of variation within each level when populations were analyzed as one group or when they were grouped either by river basin or by their position on the east or west side of the Rocky Mountains. Most pairwise F_ST values were significant via permutation test (range 0–0.10), with the highest values from comparisons with Lamar, in southeast CO. A neighbor joining tree based on Cavalli–Sforza and Edwards’s chord distances was consistent with the geographic locations of populations, as well as with the AMOVA results. There was a significant isolation by distance effect, and the cluster analysis resolved five groups. Individuals were also assayed with an additional microsatellite marker, Cxpq78, proposed to be monomorphic in <em>Cx. pipiens</em> but polymorphic in the closely related but biologically distinct species <em>Cx. quinquefasciatus</em>. Low frequencies (≤3%) of <em>Cx. quinquefasciatus</em> alleles for this marker were noted, and mostly confined to populations along the Interstate 25 corridor. Pueblo was distinct in that it had 10% <em>Cx. quinquefasciatus</em> alleles, mostly of one allele size. The degree of population genetic structure observed in this study is in contrast with that of <em>Cx. tarsalis</em>, the other major vector of WNV in the western U.S., and likely reflects the two species’ different dispersal strategies.</p> </div

Analysis of Molecular Variance (AMOVA) results when populations are grouped by river basin.

<p>The amount of variation in each partition was significantly different from zero (<i>P</i><0.05) via permutation test. Corresponding fixation indices are given in the text.</p

Map of study area.

<p>Sample sites (n = 13) are shown, as well as the location of the Rocky Mountains (grey shading), major highways, and relevant waterways. Dashed lines indicate boundaries of the river basins in the state that were sampled in this study.</p

Microsatellite loci used or screened for population genetics analyses in this study.

<p>Dye, fluorescent label (D2 = black, D3 = green, D4 = blue); Size, range of allele sizes (in bp); H_E Expected Heterozygosity averaged across populations; N, number of alleles; Multiplex, which multiplex this locus is part of; Conc., concentration of each primer; Source, original source of locus: A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047602#pone.0047602-Molecular1" target="_blank">[6]</a>, B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047602#pone.0047602-Smith1" target="_blank">[8]</a>, C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047602#pone.0047602-Keyghobadi1" target="_blank">[7]</a>, D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047602#pone.0047602-Edillo1" target="_blank">[9]</a>.</p