Insecticide Resistance Bottle Bioassay Evaluation of Culex quinquefasciatus Mosquitoes From Coachella Valley, 2020

The objective of this study was to determine whether locally collected mosquitoes were resistant to adulticide products using the CDC bottle bioassay. The Coachella Valley Mosquito and Vector Control District evaluates adulticide resistance of field-collected mosquitoes to a susceptible colony strain on an annual basis. The results of these evaluations determine whether the products can efficiently reduce adult mosquito abundance and disrupt virus transmission.

Wild Culex quinquefasciatus mosquitoes were collected by placing gravid water bait (water infused with Brewer’s yeast and ground alfalfa pellets) in a plastic tub overnight and eggs were collected the next morning. Mosquitoes were collected from two neighborhoods in Indio, CA: Outdoor Resorts and North Indio. Three egg rafts were placed per larval rearing tray, and larvae were fed daily a mixture of liver powder, ground fish flakes, brewer’s yeast, and ground alfalfa pellets. Larvae were kept in rearing chambers at 55% relative humidity, 28°C, and 16:8 (L:D) h cycle. During the pupal stage, preliminary pupae were removed to increase the female:male ratio, and subsequent pupae were placed in a cage for emergence. Adults were 3–5 d old with mixed sex (60F:40M ratio) and fed 10% sugar water daily. The susceptible CQ1 colony strain (originally collected as adults from Merced, CA in the 1950s) is kept in colony under similar rearing conditions.

Two hundred fifty (250) milliliter glass Wheaton bottles were coated with 1 ml of the pesticide product diluted in acetone. Formulations of these products were calculated using the concentration of the active ingredient (a.i.) as indicated in the table. For the control, 1 ml of acetone solvent was added to the bottles. The inside of the bottles were fully coated before rolling in a fume hood to allow the solvent to evaporate. Once the solvent evaporated, the assays were conducted within 24 h.

For the assays, an average of 26 mosquitoes ranging from 11 to 41 mosquitoes were aspirated into each bottle. For each adulticide tested, four replicate bottles were used for each sample population and the susceptible colony mosquitoes. Mosquito mortality was assessed at 15 min after initial exposure and every 15 min thereafter until all mosquitoes in a bottle were dead or the 2 h count was completed. Mosquitoes were observed as knocked down or ‘dead’ if they could no longer have controlled flight or stand. Results of mosquito mortality were analyzed using WHO guidelines for mosquito mortality for bottle bioassays. We observed the mosquito mortality of the wild strain when the colony strain reached 100% mortality. Resistance was evident when fewer than 80% of the mosquitoes died at this diagnostic time.

The percent mortality at diagnostic dose are italicized in Table 1. This study documented evidence of resistance of local wild mosquitoes to adulticide products. Mosquitoes from North Indio and Outdoor Resorts showed similar rates of mortality in the bioassay with Zenivex E4 and Merus 3.0. Mortality of the two populations was different for the bioassay with Scourge 18 + 54 suggesting that a 3-mile distance, the distance between the two sites, may be enough to distinguish two mosquito populations.¹

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