EFFECT OF SEX AND AGE ON SURVIVAL OF ADULT CULEX TARSALIS FROM A SUSCEPTIBLE LABORATORY STRAIN EXPOSED TO PERMETHRIN IN THE CDC BOTTLE BIOASSAY

Culex tarsalis Coquillett (the western encephalitis mosquito) is a species of public health concern due to its role in the transmission of encephalitis-causing viruses affecting humans, birds, and other animal species (Reisen 2012). To help reduce the risk of mosquito-borne illnesses of humans and domestic animals, mosquito control programs often target adult mosquitoes using insecticides to reduce adult abundance (Richards et al. 2017). However, resistance of Cx. tarsalis to insecticides, including permethrin, is widespread, having been reported from Colorado and South Dakota (Strong et al. 2008, Vincent et al. 2018) and throughout California (Choi 2016; Hughes 2017; Hung et al. 2021, 2022; Tsecouras et al. 2023).

Public health agencies commonly evaluate adult mosquito resistance to insecticides using the Centers for Disease Control and Prevention (CDC) bottle bioassay, a rapid and efficient means to monitor population level changes in resistance phenotype (McAllister et al. 2020). In this assay, mosquitoes were exposed to a species-specific diagnostic dose of insecticide with those surviving exposure beyond a specified diagnostic time recorded as being resistant to the tested insecticide. The CDC bottle bioassay procedure states that adult mosquitoes can be utilized to test insecticide susceptibility regardless of sex, age, or physiological status (McAllister et al. 2020). The bioassay guidelines recommend that physiological status (fed, unfed, gravid) be recorded, recognizing that susceptibility to insecticides can vary between fed and unfed female mosquitoes (David and Bracey 1946, Hadaway and Barlow 1956, Allan 2011, Boubidi et al. 2016). In contrast, the related World Health Organization (WHO) bottle bioassay specifies the use of mosquitoes of a particular sex and age (3–5 day-old females; WHO 2022).

Variation in insecticide susceptibility by sex has been reported for Aedes aegypti (L.) (David and Bracey 1946) exposed to dichlorodiphenyltrichloroethane (DDT) or pyrethrins by spray mist and for Anopheles stephensi Liston exposed to DDT applied to the mosquito dorsum (Hadaway and Barlow 1956). In these tests, a greater susceptibility of males is perhaps related to their lower mean body weight relative to females (Hadaway and Barlow 1956). Insecticide susceptibility can also increase with mosquito age (David and Bracey 1946, Rajatileka et al. 2011, Machani et al. 2019), though other studies show no impact of age on insecticide susceptibility (Hadaway and Barlow 1956, Aïzoun et al. 2014, Saeung et al. 2024).

While mosquito control efforts generally target adult females as the blood-feeding sex, resistance testing procedures are greatly simplified when separation of the sexes is not required. In addition, evaluating insecticide resistance of mosquitoes from a field population often necessitates use of field mosquitoes of unknown, and likely varied, age in the bioassay. This contrasts with testing of a susceptible laboratory population where mosquito age is readily known, and often relatively young mosquitoes are utilized in the bioassay.

While performing permethrin resistance assays for Cx. tarsalis using the CDC bottle bioassay (Tsecouras et al. 2023), we were unable to locate published reports evaluating the effect of mosquito sex or age on the overall mortality rate. Since resistance of a field-collected mosquito population using the CDC bottle bioassay is a function of mosquito survival beyond a specified diagnostic time determined by the mortality rate of a susceptible strain, variation in mortality of the susceptible strain due to sex or age of mosquitoes tested could impact resistance determination of the field population.

The objective of this study was to determine if the mortality rate, diagnostic time (time to complete mortality), or determination of resistance varies by sex or age of adult Cx. tarsalis from a susceptible strain exposed to permethrin using the CDC bottle bioassay.

A permethrin-susceptible strain of Cx. tarsalis (Bakersfield lab strain; Hung et al. 2022) originally collected from California was acquired from the Coachella Valley Mosquito and Vector Control District (CVMVCD) and maintained at UC Riverside in an insectary room at 26°C, 50% RH, and 14:8 L:D with the light period bracketed by 1-h periods of dimmed light to stimulate sunrise and sunset, and otherwise following standard rearing protocols by Kauffman et al. (2017). Larvae were held within a 5.7-liter clear plastic container (rearing pan) and fed a blend of ground fish flakes, liver powder, Brewer’s yeast, and ground alfalfa pellets until reaching the pupal stage. Following the first appearance of pupae within the rearing pans, pupae were removed and placed into wax paper cups with water every 3 days to facilitate separation of adult mosquitoes by sex or age. Adult male mosquitoes emerge first (Walton and Eldridge 2020), so pupae removed from larval pans on earlier dates were primarily male. Pupal cups were then placed into labeled adult emergence cages (BioQuip®, Gardena, CA) provisioned with 10% sucrose in a 236-ml plastic deli container with a lid and cotton wick to provide a sugar food source for emerging adults. All pupae placed into a single adult emergence cage were therefore within 1–3 days of age.

Emergence cages were observed daily, and the date of first emergence was recorded. Adult mosquitoes were subsequently held within emergence cages for either 2–5 days (“young adults”) or 7–10 days (“old adults”) before using them for permethrin susceptibility testing.

The CDC bottle bioassay procedure was used to test for permethrin susceptibility (McAllister et al. 2020). Glass bottles (236 ml; model no. S-23397, ULINE, Ontario, CA) were treated with 40.85 μg permethrin dissolved in 1 ml of acetone to give a dosage of 0.19 μg/cm² of internal surface of the glass bottle in accordance with the CDC bottle bioassay procedures (McAllister et al. 2020). Control bottles were treated with 1 ml of acetone only. Bottles were hand rolled within a chemical fume hood until the acetone had evaporated to evenly coat the internal surface of the bottle with permethrin. Bottles were dried overnight in the fume hood before being used for testing procedures the following day. Bottle bioassays were performed in a well-lit insectary lab room at 24°C and 50% RH.

To perform an assay, approx. 25 adult Cx. tarsalis of the same sex and age were placed into each prepared bottle. Mosquitoes were aspirated from a labeled emergence cage using a Brigii Mini Vacuum (ASIN B07WN6D3TK, Brigii, Hong Kong, China) fixed with an aspirator tube made from a boba straw modified with a netting plug near the vacuum housing to prevent mosquitoes from reaching the vacuum. Aspirated mosquitoes were blown into a labeled glass bottle by reversing the airflow of the mini vacuum to begin the bioassay period. Each bottle had a “cap barrier” constructed from a plastic cap with a mesh netting top that contained a small hole big enough for the straw to be inserted to blow mosquitoes into the bottle but not large enough for mosquitoes to escape after removal of the straw.

On each testing date, a bioassay was performed using 3–5 permethrin-treated bottles and 1–2 control bottles depending upon the number of Cx. tarsalis available from rearing cages. Mortality of mosquitoes within each bottle was recorded every 5 min over the 30-min diagnostic test period indicated for Cx. tarsalis exposed to permethrin, using the CDC bottle bioassay (McAllister et al. 2020). Mosquitoes were recorded as dead if they could not stand upright or fly. At the end of the diagnostic test period, all bioassay bottles were placed into a lab freezer to kill any live mosquitoes prior to confirming the total number of mosquitoes in each bottle. The sex of adult mosquitoes within the glass bottles was easily determined during visual observation. For survival comparisons by mosquito sex, the few adults of the wrong sex that were inadvertently introduced into a bioassay bottle were excluded from analysis.

All data were analyzed in R (R Core Team 2022). Mortality at each observational time point was assessed as the number of Cx. tarsalis that were dead as a proportion of the total mosquitoes tested within each bioassay bottle. Abbott’s correction formula (Abbott 1925) was applied to any tested mosquito group with a control mortality of 3–10% (occurred on one assay date only for male mosquitoes) as per CDC guidelines for assessment of insecticide resistance variation among mosquito populations (McAllister et al. 2020). The mortality rate of mosquitoes by sex and age were visualized by Kaplan–Meier survival analysis using the R package “survival” version 3.3-1 (Therneau 2023), with further analyses for overall differences in mortality rate among tested groups by Cox proportional hazards model using the R package “coxph” (Therneau 2023) with data clustered by bioassay bottle and with statistical significance among test groups (females vs. males, young vs. old) confirmed by a Wald test. A chi-square test using the R package “chisquare” (Alberti 2024) was applied to assess differences in survival between the test groups at each observation timepoint in the CDC bottle bioassay.

Permethrin susceptibility by sex was evaluated using a total of 848 young adult mosquitoes, with 344 males and 215 females in permethrin-treated bottles and 187 males and 102 females in control bottles (Table 1). Permethrin susceptibility by female age was evaluated using a total of 763 adult female mosquitoes, with 248 young and 224 old females in permethrin-treated bottles and 146 young and 145 old females in control bottles (Table 1).

Mortality was slightly greater for males than for females during the first 5 min of the diagnostic period, (χ² = 4.14, P = 0.04), though mortality was not different by sex over the remainder of the diagnostic period. Mortality was greater for older females than for younger females during the 2nd observation period (5–10 min; χ² = 15.71, P = 7.38E-05), but was otherwise not different for the remainder of the diagnostic period. When evaluated over the entire diagnostic period, mortality rate varied slightly only by mosquito age (z ≤ 2.36, P = 0.02) not sex (z ≤ 1.64, P = 0.1).

All mosquitoes from each sex and age group for this susceptible mosquito strain died within the 30-min diagnostic time for this species, indicating similar overall susceptibility to permethrin according to bottle bioassay data interpretation (Fig. 1 and Table 2).

This study assessed whether the sex or age of adult mosquitoes from a permethrin-susceptible strain of Cx. tarsalis altered the mosquito survival rate when mosquitoes were exposed to a diagnostic dose of permethrin using the standard CDC bottle bioassay. This information is important since the CDC bottle bioassay method states that mosquitoes of either sex and any age may be used in these bioassays (McAllister et al. 2020), and because susceptible lab strains of mosquitoes are often used to identify the appropriate diagnostic period (exposure times for complete mortality) to evaluate resistance of field strains. However, data supporting a similar outcome when using either mosquito sex or varied adult age in this assay are lacking. If insecticide susceptibility varies with sex or age, then combining the sexes and various adult ages into a single assay could result in an error of interpretation, particularly if survival times vary substantially by sex or age.

While adult mosquitoes are easily sorted by sex in the laboratory for insecticide resistance testing, sorting requires additional time and extra handling that can increase mortality of field-collected mosquitoes prior to testing. Using mosquitoes of either sex in the bioassay greatly simplifies the pretesting preparation. Age of field-collected mosquitoes is typically unknown. To standardize age of field-collected mosquitoes, either immatures would need to be collected from a field site or the field population would need to be colonized and reared in the laboratory.

In the current study, all groups tested (either sex or age) died within the 30-min diagnostic period indicating susceptibility to permethrin. Thus, neither sex nor age had an impact on permethrin susceptibility for this susceptible strain of Cx. tarsalis as determined by the CDC bottle bioassay method (McAllister et al. 2020). Results in this study were similar to those obtained for young females from the same mosquito strain in a previous study (Tsecouras et al. 2023).

While all mosquitoes tested were susceptible to permethrin, differences in mortality by sex or age were observed during individual observation periods. Differences in mortality occurred during early observation periods, within 5 min for sex and between 5 and 10 min for age. Few mosquitoes survived for 15 min of permethrin exposure. This suggests that both sex and age can impact mortality resulting from permethrin exposure at least soon after exposure. However, when mortality rate was evaluated over the full diagnostic period, a significant effect of mosquito age but not sex suggests mortality is more affected by age than sex, perhaps due to increasing cuticle permeability or other physiological changes that occur with aging in mosquitoes (Machani et al. 2019). The effect of age on mortality following insecticide exposure was stronger for Anopheles gambiae (Giles) (Machani et al. 2019), though a greater age difference between tested groups was used in that study.

Reduced survival of older mosquitoes exposed to permethrin may have implications for public health. Older mosquitoes pose greater risk for pathogen transmission due to a greater opportunity for previous exposure to pathogens (Smith et al. 2004) and higher likelihood for pathogen transmission as a result of completing the pathogen extrinsic incubation period (Barker and Reisen 2019). Differences in the mortality rate by mosquito age suggest that control efforts targeting mosquitoes with permethrin or perhaps other related insecticides may have a greater impact on older female mosquitoes, perhaps achieving a greater reduction in pathogen transmission risk than might be anticipated if mosquito mortality was not impacted by mosquito age.

Sex-specific variation in insecticide susceptibility has been reported for other mosquito species (David and Bracey 1946, Hadaway and Barlow 1956), though these studies used different bioassay methods and thus are not directly comparable to the current study. With little difference in the overall mortality rate by sex in the current study, use of both sexes in the CDC bottle bioassay is appropriate to reduce both bioassay preparation time and handling of mosquitoes. Use of both sexes may also increase opportunity for early detection of insecticide resistance since the resistance phenotype may be sex specific and each sex could contribute resistance genes to later generations (McAllister et al. 2020).

Bakersfield lab strain mosquitoes were maintained at the Coachella Valley Mosquito and Vector Control District (CVMVCD) by Melissa Snelling. Funding was provided by the United States Department of Agriculture Hatch Act to AG and by a Moore Fellowship from the University of California to JT.