Insecticide Resistance Bottle Bioassay Evaluation of Culex tarsalis 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 tarsalis mosquitoes were collected by placing carbon dioxide traps overnight and collecting the live adults the next morning. Mosquitoes were collected from two locations: Northshore (35.5327,-116.0353) and Westshore (33.4811,-116.0952). The live mixed-age females were used in the assays. For the susceptible colony mosquitoes (originally from Bakersfield, CA kept in colony since the 1950s), three egg rafts were placed per larval rearing tray. Larvae were fed daily a mixture of liver powder, ground fish flakes, brewer’s yeast, and ground alfalfa pellets and 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.

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 Table 1. 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 bottles were capped and the assays were conducted within 24 h.

For the assays, an average of 33 mosquitoes ranging from 12 to 55 mosquitoes were aspirated into each bottle. For each adulticide tested, four replicate bottles were used for each local 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. At the end of the study, the mosquitoes in each bottle were frozen, counted, and identified to species. For the collections from Northshore, the mosquitoes were comprised of 80–98% Cx. tarsalis, 1–20% Cx. erythrothorax, and 0–1% other species. For the collections from Westshore, the mosquitoes were comprised of 75–95% Cx. tarsalis, 5–24% Cx. erythrothorax, and 1% Cx. quinquefasciatus. 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 (Northshore) and Table 2 (Westshore). This study documented evidence of resistance of local wild mosquitoes to adulticide products. Some of the bottles were under-dosed, leading to the colony strain not reaching 100% mortality in several assays. The results were still included to demonstrate that the wild mosquitoes had lower mortality than the colony mosquitoes for many adulticide products.¹

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