ABSTRACT

In several insect species, resistance to pyrethroids and DDT (dichlorodiphenyltrichloroethane) is linked to point mutations in the voltage-gated sodium channel (VGSC) gene. Pyrethroid-based insecticides prolong the opening of sodium channels, causing paralysis known as a “knockdown” effect before mortality occurs. Point mutations in the VGSC gene result in decreased pyrethroid binding and reduced sensitivity to the insecticide—this resistance mechanism is known as knockdown resistance (kdr) as insects do not die but recover from paralysis with time. In Culex mosquito species loss of target site sensitivity to pyrethroids is linked to a number of substitutions, one of which is leucine (L) to phenylalanine (F) at residue 1014 (L1014F) in the VGSC gene. Here we report the identification of kdr-associated pyrethroid resistance and developing resistance in Cx. quinquefasciatus field collections from Collier County, FL. Evaluation of position 1014 of the VGSC in Cx. quinquefasciatus collections from 7 locations in Collier County, FL, revealed a wide range of genotypes from one part of the district to the other. Centers for Disease Control and Prevention bottle bioassay, linear regression analysis, and cage trial evaluations suggest that the L1014F mutation plays a role, at least in part, to the pyrethroid resistance status of Cx. quinquefasciatus collected in Collier County, FL. Furthermore, we identified resistance attributed to both oxidase and esterase activity, indicating that multiple mechanisms are responsible for pyrethroid resistance in Collier County Cx. quinquefasciatus.

INTRODUCTION

Travel-related and local incidences of mosquito-borne diseases in the USA have increased in recent years. The southeastern USA is endemic with several mosquito-borne viruses transmitted by Culex mosquitoes, including West Nile virus and St. Louis encephalitis virus (Harwood et al. 1979). Current disease vector control strategies in the region are based primarily on the application of insecticides targeting both the juvenile and adult stages. Proper integrated pest management methods, including robust insecticide resistance monitoring, are paramount to reducing disease vector populations.

In several insect species, resistance to pyrethroids and DDT (dichlorodiphenyltrichloroethane) is linked to point mutations in the voltage-gated sodium channel (VGSC) gene. Sodium channels are a crucial component of the nerve-cell membrane and play a role in nerve-cell excitability. Pyrethroid-based insecticides prolong the opening of sodium channels, causing paralysis before mortality occurs, resulting in a “knockdown” effect. Point mutations in the VGSC gene result in decreased pyrethroid binding and reduced sensitivity to the insecticide—therefore, “knockdown” is observed but the insect recovers with time. This mechanism for resistance is commonly referred to as knockdown resistance (kdr). More than 50 nonsynonymous mutations in the VGSC have been identified in insect species (Dong et al. 2014). These mutations typically result in amino acid substitutions within the sodium channel. Pyrethroid resistance in mosquitoes has been attributed to several kdr mutations in Aedes aegypti (L.) (Brengues et al. 2003, Linss et al. 2014, Estep et al. 2018), Anopheles gambiae (Giles) (Martinez-Torres et al. 1999a, Ranson et al. 2000), and Culex species (Xu et al. 2006, Zhou et al. 2009, Chen et al. 2010, Yoshimizu et al. 2019). Adult mosquito survival rates after topical application and bottle bioassay have shown to correlate with high frequency of kdr mutations in Ae. aegypti (Aizoun et al. 2013, Estep et al. 2018).

The Centers for Disease Control and Prevention (CDC) bottle bioassay is the preferred method for insecticide resistance testing; however, these processes require additional vector control technicians and biologists dedicated to the collection and rearing of a large number of field-collected larvae—which can affect timely operational decision-making. The presence of allelic variation contributing to pyrethroid resistance has implications for a quick molecular diagnostic test for detection of resistance in field populations from already established operational traps. Several protocols for observing kdr mutations in mosquito populations have been created, including the use of standard polymerase chain reaction (PCR) (Martinez-Torres et al. 1999b), DNA melting analysis (Linss et al. 2014), and TaqMan assays (Chen et al. 2010). Despite our increasing knowledge of kdr mutations and associations with pyrethroid resistance, the operational significance of kdr genotyping as a screening tool for resistance by vector control agencies has yet to be determined.

In the USA, loss of target site sensitivity to pyrethroid-based insecticides in Culex mosquito species is commonly associated with a substitution of leucine (L) to phenylalanine (F) at residue 1014 (L1014F) in the VGSC gene. Another kdr mutation associated with the substitution of leucine to serine has also been implicated in pyrethroid resistance (Zhou et al. 2009). The L1014F mutation has been used to track pyrethroid resistance in Culex quinquefasciatus (Say) (Xu et al. 2005, 2006; Wondji et al. 2008; Zhou et al. 2009; Yoshimizu et al. 2019) and other Cx. pipiens (L.) complex mosquitoes (Scott et al. 2015, Yoshimizu et al. 2019). In Jacksonville, FL, the occurrence of a resistant genotypes in Cx. quinquefasciatus has shown to be as high as 65% (Zhou et al. 2009). Further, pyrethroid resistance linked to kdr mutations is common in Ae. aegypti populations throughout Florida, including 4 field collections examined in Collier County, FL (Estep et al. 2018). To further expand our knowledge of pyrethroid resistance and kdr allele frequency in Collier County mosquitoes, we performed kdr genotyping analysis on 7 Cx. quinquefasciatus field collections. Here we report the identification and operational significance of kdr-associated pyrethroid resistance in Cx. quinquefasciatus field collections from Collier County, FL. In addition, we assessed the role of oxidase, esterase, and glutathione transferase activity in contributing to pyrethroid resistance in Collier County Cx. quinquefasciatus mosquitoes.

MATERIALS AND METHODS

Mosquito collections and rearing

Between July 2017 to July 2018, 7 field collections of Cx. quinquefasciatus from Collier County, FL, were collected for this study (Fig. 1). For each field collection, approximately 100–500 larvae (F0) were collected from natural and artificial containers. Details regarding sampling locations can be found in Table 1 and Fig. 1. After identifying mosquito larvae species by morphology, mosquitoes were brought back to the insectary regulated at 28°C, 80% relative humidity, and a constant 14-h-light and 10-h-dark cycle.

Fig. 1

Sampling locations for Culex quinquefasciatus field collections in Collier County, FL. Seven locations were used for this study including: Cambier Park, Sugden Park, Naples Manor, Landfill, Big Cypress Elementary, Palmetto Elementary, and Sabal Palm Elementary.

Fig. 1

Sampling locations for Culex quinquefasciatus field collections in Collier County, FL. Seven locations were used for this study including: Cambier Park, Sugden Park, Naples Manor, Landfill, Big Cypress Elementary, Palmetto Elementary, and Sabal Palm Elementary.

Table 1

Sampling locations and genotyping data for Culex quinquefasciatus in Collier County, FL.1

Sampling locations and genotyping data for Culex quinquefasciatus in Collier County, FL.1
Sampling locations and genotyping data for Culex quinquefasciatus in Collier County, FL.1

kdr Genotyping analysis

For genotyping assays, newly emerged adults (male and female) were cold anesthetized, individually placed in sterile 1.5-ml microcentrifuge tubes, and stored at −80°C. Mosquitoes stored in 1.5-ml microcentrifuge tubes were used for DNA extraction. Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Catalog No. A1125; Promega, Madison, WI) according to manufacturer's protocol. Approximately 40–50 individual mosquitoes from each population were genotyped at residue 1014 of the VGSC using a TaqMan probe method, as previously described (Chen et al. 2010). Genotyping data were generated on Applied Biosystems® QuantStudio® 5 Real-Time PCR System (Thermofisher, Carlsbad, CA), and allelic calls were made using the QuantStudio Design and Analysis Software v1.4.1. In brief, 50 ng of DNA was used in a TaqMan assay reaction using probes labeled with VIC™ to detect wild-type L1014 allele and 6-FAM™ for detection of the L1014F allele, as previously described (Chen et al. 2010).

Genotype and allele frequencies along with 95% confidence intervals were calculated for each population (Table 1). The genotype frequencies were calculated by dividing the number of individuals with a given genotype by the total number of analyzed individuals. The equations below indicate as follows: f is the frequency of the genotypes, LL is the number of individuals homozygous for the L1014L genotype, LF is the number of heterozygous individuals, FF is the number of individuals homozygous for the L1014F genotype, and n is the total number of individuals tested.

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The allele frequencies were calculated using the following formulas, where f(L) is the frequency of the L1014L allele, f(F) is the frequency of the L1014F allele, LL is the number of individuals homozygous for the L1014L genotype, LF is the number of heterozygous individuals, and FF is the number of individuals homozygous for the L1014F genotype.

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Insecticide susceptibility tests

The susceptibility of Collier County Cx. quinquefasciatus to pyrethroids was evaluated using the CDC bottle bioassay protocol (Brogdon and McAllister 1998a, Brogdon and Chan 2010). Bottle bioassays were carried out using 3- to 4-day-old adult female mosquitoes from Cambier Park and Sabal Palm Elementary. Three assay bottles using approximately 20–25 female mosquitoes each were exposed to the CDC diagnostic dose of the technical-grade insecticides of either d-phenothrin (Sumithrin®) (22 μg/ml), pyrethrum (15 μg/ml), or naled (2.25 μg/ml) (CDC 2017); acetone was used as a control treatment. In additional experiments using formulated products, Anvil 10-10® (10% Sumithrin, 10% piperonyl butoxide [PBO]) (Clarke Inc., St. Charles, IL), Merus 2.0® (5% pyrethrins) (Clarke Inc.), and Dibrom® Concentrate (87.4% naled) (AMVAC Chemical Corp., New Port Beach, CA) were diluted in acetone to yield the equivalent CDC diagnostic dose of active ingredient. Knockdown was recorded every 15 min for 2 h. After 2 h, exposed mosquitoes were transferred to holding cages and provided a 20% sucrose solution. Mortality was then recorded at 24 h postexposure. The published CDC diagnostic time for technical-grade insecticides against the susceptible Cx. quinquefasciatus Sebring colony was used for classification of resistance status (CDC 2017). Diagnostic times for formulated products were not developed, and instead diagnostic times for technical-grade insecticides were used as reference. It is important to note that diagnostic times for the formulated products are likely shorter than for technical-grade insecticides. Collections were classified as resistant or susceptible using the World Health Organization guidelines (WHO 2013): 98–100% mortality at the recommended diagnostic time indicates susceptibility; 80–97% mortality at the recommended diagnostic time suggests developing resistance; and <80% mortality at the recommended diagnostic time suggests resistance. Percent mortality was determined using the following modified formula from Abbott (1925), and an average was produced among the 3 technical replicates:

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Percent recovery was determined using the following calculation, and an average was produced among the 3 technical replicates:

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Graphical analysis was produced using GraphPad Prism 8 (GraphPad Software, San Diego, CA).

Field evaluation of Cx. quinquefasciatus resistance status

Three- to 4-day-old adult female Cx. quinquefasciatus collected from Cambier Park and Sabal Palm were used in field tests using Anvil 10-10 and Merus 2.0. Anvil 10-10 was applied using a COLT-4 hand-fogger (London Fogger, Minneapolis, MN) at maximum application rate of 0.62 oz/acre (0.0036 lb AI/acre) while at a walking speed of 2 mph, according to manufacturer suggestions. Merus 2.0 was applied similarly at a maximum application rate of 0.78 oz/acre (0.0025 lb AI/acre), according to manufacturer suggestions. Both formulated products were diluted in mineral oil to obtain the flow rate needed to achieve the proper application rate. Three replicate test stations were utilized—which included slide impingers holding 2-mm Teflon-coated slides and a field cage holding 20–25 female Cx. quinquefasciatus mosquitoes collected from Cambier Park or Sabal Palm. Replicate test stations were arranged in a single sampling line (20 ft apart) perpendicular to wind direction. Applications were made 25 ft from test stations and perpendicular to wind direction to allow drift of the insecticide into the cages. The 2-mm slides and mosquito cages were collected 5 min postapplication, returned to the laboratory, and transferred to holding cages for evaluation. Knockdown was recorded every 15 min for 2 h, and mortality was recorded at 8 and 24 h posttreatment. Corrected percent mortality was calculated as described above. DropVision® (Leading Edge Associates, Fletcher, NC) was used to determine droplet size and density from the 2-mm slides. Applications were performed 3 times for each product. Graphical analysis was determined using GraphPad Prism 8.

Analysis of metabolic resistance

In order to assess the effect of metabolic resistance on the resistance status of Cambier Park and Sabal Palm Cx. quinquefasciatus mosquitoes, synergists were used according to the protocol described by the CDC (Brogdon and McAllister 1998b). Three technical replicates of approximately 20–25 mosquitoes were exposed to 1 of the 3 synergists: S.S.S-tributylphosphorotrithioate (DEF) (125 μg/bottle), which inhibits esterase activity; diethyl maleate (DEM) (80 μg/bottle), which inhibits glutathione transferase activity; and PBO (400 μg/bottle), which inhibits oxidase activity. After exposure for 1 h, mosquitoes were transferred to holding cages for recovery for another hour and then used in CDC bottle bioassays using technical-grade insecticides as described above. Graphical analysis and statistical significance using a 2-tailed Student's t-test was determined using GraphPad Prism 8.

RESULTS

Frequencies and distribution of L1014F allele in Collier County, FL

Voltage-gated sodium channel genotyping varied in genotype and allele frequencies among Cx. quinquefasciatus collections (Table 1). Cambier Park collections displayed the highest genotype and allele frequency of the L1014F conversion, with 25.64% (95% CI: 0.130–0.421) homozygous for the L1014F mutant allele (FF) and 53.85% (95% CI: 0.372–0.699) heterozygous (LF) for both alleles. The frequency of the L1014F allele (F) in Cambier Park Cx. quinquefasciatus was 52.56% (95% CI: 0.409–0.640). Homozygotes for the mutant allele were not detected in collections from Palmetto Elementary (95% CI: 0–0.090) or Sabal Palm Elementary (95% CI: 0–0.106); however, several heterozygous individuals were identified at 23.08% (95% CI: 0.111–0.393) and 12.12% (95% CI: 0.034–0.282), respectively. Furthermore, the frequency of the L1014F allele (F) from Palmetto Elementary and Sabal Palm Elementary was low, with 11.54% (95% CI: 0.054–0.208) and 6.06% (95% CI: 0.017–0.148), respectively. Frequency of the L1014F mutation was high in the “Old Naples” (Cambier Park) and “East Naples” (Sugden Park, Naples Manor, Eagle Lakes) localities, while decreasing in frequency in Cx. quinquefasciatus collected farther east in the county in areas such as “Golden Gate Estates” (Sabal Palm Elementary, Palmetto Elementary) (Table 1 and Fig. 1).

Insecticide susceptibility and identification of pyrethroid resistance

To assess the level of insecticide resistance in Collier County Cx. quinquefasciatus mosquitoes, field collections displaying the highest (Cambier Park: 52.56%) and the lowest (Sabal Palm: 6.06%) L1014F allele frequency were subjected to CDC bottle bioassay. Three common active ingredients in mosquito adulticide products were chosen for the assay: d-phenothrin (Sumithrin), pyrethrum, and naled. d-Phenothrin (Sumithrin) and pyrethrum were chosen because these materials serve as active ingredients in pyrethroid-based control materials recently adopted by Collier Mosquito Control District (CMCD). Further, naled is the active ingredient of an organophosphate-based insecticide, Dibrom Concentrate, also used by CMCD.

Cambier Park exhibited the highest level of pyrethroid resistance, with an average corrected percent mortality rate of nearly 20.66% for technical-grade pyrethrum (Fig. 2A) and 55.41% for Merus 2.0 (Fig. 2D), and 12.39% for technical-grade d-phenothrin (Sumithrin) (Fig. 2B) and 75.28% for Anvil 10-10 (Fig. 2E) at the CDC diagnostic time of 45 min. Even after exposure to the material for 2 h, a large proportion of Cambier Park Cx. quinquefasciatus remained active in the assay bottle. Furthermore, Cambier Park was susceptible to the organophosphate naled, with complete mortality at the CDC diagnostic time of 45 min for both the technical-grade naled (Fig. 2C) and Dibrom Concentrate (Fig. 2F). Sabal Palm Cx. quinquefasciatus appeared susceptible to d-phenothrin (Sumithrin) using Anvil 10-10 and developing resistance to pyrethrum using Merus 2.0, with an average knockdown of 98.33% (Fig. 2E) and 80.45% (Fig. 2D), respectively, at the CDC diagnostic time of 45 min. However, when CDC bottle bioassays were performed using technical-grade pyrethrum and d-phenothrin (Sumithrin), Sabal Palm Cx. quinquefasciatus displayed developing resistance to d-phenothrin (Sumithrin), with a mortality rate of 80.64% (Fig. 2B), and resistance to pyrethrum, with a mortality rate of 53.90% (Fig. 2A). Furthermore, Sabal Palm Cx. quinquefasciatus was susceptible to the organophosphate naled, with complete knockdown at the CDC diagnostic time of 45 min for technical-grade naled (Fig. 2C) and 96.4% knockdown for Dibrom Concentrate (Fig. 2F). Taken together, these data signify that Cx. quinquefasciatus mosquitoes collected from Cambier Park and Sabal Palm are resistant to pyrethroid-based insecticides but not to organophosphate-based materials. Furthermore, these data also signify the importance of utilizing technical-grade insecticides in a resistance monitoring program, as formulated products may mask resistance. It is important to note, that while the published CDC diagnostic time for Cx. quinquefasciatus was utilized as the basis for comparisons, a susceptible standard was not used in the assays. Further, diagnostic times were not determined for formulated product and instead CDC diagnostic times for technical-grade insecticides were used as reference.

Fig. 2

Centers for Disease Control and Prevention (CDC) bottle bioassays for Cambier Park (black solid line) and Sabal Palm (black dashed line) Culex quinquefasciatus mosquitoes. (A–C) CDC bottle bioassays using technical-grade insecticides: (A) 15 μg/ml pyrethrum, (B) 22 μg/ml d-phenothrin (Sumithrin®), and (C) 2.25 μg/ml naled. (D–F) CDC bottle bioassays using formulated products: (D) Merus 2.0®, (E) Anvil 10-10®, and (F) Dibrom Concentrate. Each formulated product was diluted in acetone to yield the equivalent CDC diagnostic dose of active ingredient per bottle. Solid vertical red line indicates published threshold for CDC diagnostic dose of the susceptible Cx. quinquefasciatus Sebring colony. Threshold times for formulated products (dashed vertical red lines) are unknown but provided for reference. Data represent 3 technical replicates and are shown as mean ± SEM.

Fig. 2

Centers for Disease Control and Prevention (CDC) bottle bioassays for Cambier Park (black solid line) and Sabal Palm (black dashed line) Culex quinquefasciatus mosquitoes. (A–C) CDC bottle bioassays using technical-grade insecticides: (A) 15 μg/ml pyrethrum, (B) 22 μg/ml d-phenothrin (Sumithrin®), and (C) 2.25 μg/ml naled. (D–F) CDC bottle bioassays using formulated products: (D) Merus 2.0®, (E) Anvil 10-10®, and (F) Dibrom Concentrate. Each formulated product was diluted in acetone to yield the equivalent CDC diagnostic dose of active ingredient per bottle. Solid vertical red line indicates published threshold for CDC diagnostic dose of the susceptible Cx. quinquefasciatus Sebring colony. Threshold times for formulated products (dashed vertical red lines) are unknown but provided for reference. Data represent 3 technical replicates and are shown as mean ± SEM.

Next, we asked whether the increased L1014F allele frequency in the Cambier Park Cx. quinquefasciatus collection has a significant impact on its pyrethroid resistance status. Phenotypic expression of kdr resistance can be determined by evaluating a population for recovery 24 h posttreatment. After completion of the CDC bottle bioassay with technical-grade d-phenothrin (Sumithrin) and pyrethrum, Cambier Park and Sabal Palm Cx. quinquefasciatus were transferred to holding cages and assessed for recovery 24 h postexposure. Cambier Park Cx. quinquefasciatus displayed a higher rate of recovery when exposed to technical-grade d-phenothrin (Sumithrin) and pyrethrum. With 2 h of exposure to pyrethrum and d-phenothrin (Sumithrin), Cambier Park Cx. quinquefasciatus reached a knockdown of 38.58% (Fig. 2A) and 28.85% (Fig. 2B), respectively. After a 24 h recovery period, percent mortality was reduced to 15.35% for pyrethrum (t = 3.43, df = 4, P = 0.0264) (Figs. 2A, 3A) and 8.58% for d-phenothrin (Sumithrin) (t = 5.10, df = 4, P = 0.0070) (Figs. 2B, 3A). This resulted in an average recovery rate of 67.78% for d-phenothrin (Sumithrin) and for 56.11% for pyrethrum in the Cambier Park Cx. quinquefasciatus. No significant recovery was observed in the Cx. quinquefasciatus collected from Sabal Palm for either d-phenothrin (Sumithrin) (t = 0.05, df = 4, P = 0.9612) or pyrethrum (t = 1.03, df = 4, P = 0.3612) (Fig. 3A). These results suggest that Cambier Park Cx. quinquefasciatus displays phenotypic characteristics of kdr-associated pyrethroid resistance.

Fig. 3

Phenotypic expression of knockdown resistance in Cx. quinquefasciatus mosquitoes in Collier County, FL. (A) Percent recovery at 2 h postexposure for Cambier Park and Sabal Palm. Data represent 3 technical replicates and are shown as mean ± SEM. A 2-tailed Student's t-test was performed to indicate statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001. (B) Linear regression analysis for the L1014F allele frequency against the percent recovery observed 24 h posttreatment for Cambier Park, Landfill, Big Cypress, and Sabal Palm Cx. quinquefasciatus mosquitoes. Goodness-of-fit statistical model (r2) indicates statistical significance.

Fig. 3

Phenotypic expression of knockdown resistance in Cx. quinquefasciatus mosquitoes in Collier County, FL. (A) Percent recovery at 2 h postexposure for Cambier Park and Sabal Palm. Data represent 3 technical replicates and are shown as mean ± SEM. A 2-tailed Student's t-test was performed to indicate statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001. (B) Linear regression analysis for the L1014F allele frequency against the percent recovery observed 24 h posttreatment for Cambier Park, Landfill, Big Cypress, and Sabal Palm Cx. quinquefasciatus mosquitoes. Goodness-of-fit statistical model (r2) indicates statistical significance.

To determine if there is a correlation between a sampling sites L1014F allele frequency and their postexposure recovery time, 4 of the collections (Cambier Park, Landfill, Big Cypress, Sabal Palm) were exposed to d-phenothrin (Sumithrin) or pyrethrum for 2 h using the CDC bottle bioassay. Mosquitoes were transferred to holding cages for 24 h and mortality was assessed. Recovery rates for each population were plotted against the frequencies of the L1014F allele. Using a linear regression model (GraphPad Software), we found that there is a significant correlation between the L1014F allele frequencies and recovery rates for pyrethrum (r2 = 0.9478, P = 0.0264) and for d-phenothrin (Sumithrin) (r2 = 0.9372, P = 0.0319) (Fig. 3B). These data indicate the L1014F mutation plays a role, at least in part, to the pyrethroid resistance status of Cx. quinquefasciatus collected in Collier County, FL.

Field evaluation of Cx. quinquefasciatus with high L1014F mutant allele frequency

Field cage trials were conducted in August 2018 in order to determine if resistance identified using laboratory techniques could be observed in real-world applications. Adult mosquitoes were exposed to Anvil 10-10 at an application rate of 0.62 oz/acre or Merus 2.0 at an application rate of 0.78 oz/acre. Applications selected represent maximum application rates per label requirements. Cambier Park Cx. quinquefasciatus adults displayed a reduced mortality rate of 51.62% with Merus 2.0 (Fig. 4A) and 52.22% with Anvil 10-10 (Fig. 4B) at 24 h postapplication, while 100% of the Sabal Palm Cx. quinquefasciatus were dead at 24 h posttreatment (P < 0.0001). Interestingly, despite the presence of the synergist PBO in Anvil 10-10′s formulation, Anvil 10-10 was not significantly more effective than Merus 2.0 at successfully knocking down Cambier Park Cx. quinquefasciatus. Recovery was not observed at 8 h or 24 h posttreatment, providing an example of formulated product masking resistance in the field—further stressing the importance of utilizing technical-grade insecticides in a resistance monitoring program. Furthermore, these results suggest that alternative mechanisms of pyrethroid resistance may play an important role in the pyrethroid resistance status of our field collections.

Fig. 4

Field cage trials using (A) 0.78 oz/acre of Merus 2.0® or (B) 0.62 oz/acre of Anvil 10-10® against Cambier Park and Sabal Palm Culex quinquefasciatus mosquitoes. Data represent 3 technical replicates and are shown as mean ± SEM.

Fig. 4

Field cage trials using (A) 0.78 oz/acre of Merus 2.0® or (B) 0.62 oz/acre of Anvil 10-10® against Cambier Park and Sabal Palm Culex quinquefasciatus mosquitoes. Data represent 3 technical replicates and are shown as mean ± SEM.

Analysis of alternative pyrethroid resistance mechanisms

To assess the role of metabolic resistance mechanisms in the resistance statuses of Cambier Park and Sabal Palm Cx. quinquefasciatus mosquitoes collected from each location were treated with synergists to inhibit oxidase (PBO), esterase (DEF), or glutathione transferase (DEM) activity. After exposure to the synergist PBO and DEF, Cambier Park Cx. quinquefasciatus mosquitoes displayed a partial reduction of the resistant phenotype observed with exposure to pyrethrum. Exposure of Cambier Park Cx. quinquefasciatus to PBO or DEF prior to CDC bottle bioassay using pyrethrum resulted in 85.22% (t = 5.546, df = 4, P = 0.0052) and 69.63% (t = 4.130, df = 4, P = 0.0145) mortality, respectively, while pyrethrum alone resulted in 20.65% mortality (Fig. 5A, 5B). Likewise, treatment with PBO and DEF resulted in an elimination of pyrethrum resistance in the Sabal Palm Cx. quinquefasciatus collection (t = 14.69, df = 4, P = 0.0001) (Fig. 5C, 5D). Treatment with DEM resulted in a partial reduction in the resistant phenotype observed in Sabal Palm Cx. quinquefasciatus (t = 2.953, df = 4, P = 0.0418) (Fig. 5C, 5D). Together these results suggest that oxidase and esterase activity play the primary role in the pyrethroid resistance status of Sabal Palm Cx. quinquefasciatus, while glutathione transferase activity may play a partial role. Furthermore, oxidase and esterase activity may play a partial role in Cambier Park Cx. quinquefasciatus resistance status.

Fig. 5

Centers for Disease Control and Prevention (CDC) bottle bioassay using 15 μg/ml pyrethrum in conjunction with exposure to 1 of 3 synergists: S.S.S-tributylphosphorotrithioate (DEF) (125 μg/bottle), diethyl maleate (DEM) (80 μg/bottle), and piperonyl butoxide (PBO) (400 μg/bottle), which inhibits oxidase activity. (A–B) Cambier Park Culex quinquefasciatus response to pyrethrum with synergist exposure and (C–D) Sabal Palm Cx. quinquefasciatus response to pyrethrum with synergist exposure. (A–D) Data represent 3 technical replicates and are shown as mean ± SEM. (A, C) Solid vertical red line indicates published threshold for CDC diagnostic dose of the susceptible Cx. quinquefasciatus Sebring colony. (B, D) A 2-tailed Student's t-test was performed to indicate statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

Fig. 5

Centers for Disease Control and Prevention (CDC) bottle bioassay using 15 μg/ml pyrethrum in conjunction with exposure to 1 of 3 synergists: S.S.S-tributylphosphorotrithioate (DEF) (125 μg/bottle), diethyl maleate (DEM) (80 μg/bottle), and piperonyl butoxide (PBO) (400 μg/bottle), which inhibits oxidase activity. (A–B) Cambier Park Culex quinquefasciatus response to pyrethrum with synergist exposure and (C–D) Sabal Palm Cx. quinquefasciatus response to pyrethrum with synergist exposure. (A–D) Data represent 3 technical replicates and are shown as mean ± SEM. (A, C) Solid vertical red line indicates published threshold for CDC diagnostic dose of the susceptible Cx. quinquefasciatus Sebring colony. (B, D) A 2-tailed Student's t-test was performed to indicate statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

DISCUSSION

Insecticide resistance represents one of the most important challenges faced by vector control agencies. Monitoring resistance status is essential for the selection of proper control methods. Additionally, vector control agencies need rapid and reliable methods for the detection and monitoring of insecticide resistance. It was recently reported that Ae. aegypti populations in Florida, including in Collier County, exhibit a high frequency of the kdr-resistant alleles (Estep et al. 2018). The identification of these resistance alleles in Ae. aegypti provide a mechanism for resistance to pyrethroid-based insecticides and may serve as a guide for vector control agencies in the rational use of specific control materials for Ae. aegypti. In order to expand CMCD's insecticide resistance map to include Cx. quinquefasciatus, we adopted an approach to assess pyrethroid resistance by examining the nucleotide diversity at position 1014 of the VGSC. We identified the presence of a point mutation resulting in the conversion of leucine (L) to phenylalanine (F) at position 1014 (L1014F) of the VGSC in several Cx. quinquefasciatus field collections located in the district. The frequency of the L1014F allele correlated significantly with recovery after CDC bottle bioassay performed using d-phenothrin (Sumithrin) and pyrethrum.

We found variation among sampling sites in frequencies of the VGSC gene L1014F allele, as well as variation in patterns of metabolic resistance. Cambier Park, located in the urban southwestern portion of the district, had the highest frequency of the L1014F allele, while Sabal Palm Elementary, located in the rural eastern portion of the district, displayed the lowest L1014F allele frequency. Pyrethroid resistance status and frequency of the L1014F mutation was highest in the urban areas of the district, while decreasing in resistance and frequency in Cx. quinquefasciatus collected in the more rural/newly developed locations. These results may relate to human population expansion and county growth, with older more urban areas of the district displaying a higher frequency of resistant alleles due to prolonged and increased likelihood of exposure to selective pressures, such as mosquito misting systems, private and commercial pest control applications, fertilizers and other materials associated with storm drain runoff. Although historical aerial treatment using pyrethroid-based control materials has been limited in Collier County, usage of pyrethroids by the local vector control agency (CMCD) cannot be ruled out as another means of selection. It has been shown that the L1014F mutation harbors a fitness cost to Cx. pipiens pallens (L.) in the absence of selective pressures (Chen et al. 2010), leading us to hypothesize that our Cx. quinquefasciatus mosquitoes are being exposed to continuous selective pressures. Further research is required to examine the temporal dynamics in relation to pyrethroid-based insecticide use and identify the selective pressures driving kdr-associated resistance in Collier County Cx. quinquefasciatus mosquitoes.

While the L1014F conversion and kdr-associated resistance was identified in Collier County Cx. quinquefasciatus, detoxification enzyme–based resistance appeared to play a more important role in their resistance levels in the field. Resistance attributed to oxidase and esterase activity was identified in both Cambier Park and Sabal Palm Cx. quinquefasciatus field collections, and most likely accounts for the pyrethroid resistance observed in field applications. Furthermore, recovery was not observed with the use of formulated product in CDC bottle bioassays or field applications, indicating masking of the kdr phenotype by formulated products. This identification signifies the importance of considering multiple modes of action in relation to pyrethroid resistance observed in the field.

Overall, this report provides insight into the use of kdr genotyping methods by vector control agencies to detect pyrethroid-resistant Cx. quinquefasciatus mosquitoes. Used in conjunction with CDC bottle bioassays and metabolic assays, kdr genotyping provides vector control agencies high-resolution resistance monitoring. While kdr genotyping may provide results for initial resistance monitoring by testing adults from already established operational traps, vector control agencies must not rule out the presence of other mechanisms of resistance, including oxidase, esterase, and glutathione S-transferases, in their districts' mosquito populations.

ACKNOWLEDGMENTS

The authors thank the Collier Mosquito Control District (CMCD) Board of Commissioners, Patrick Linn, and all the employees at CMCD who participated in sample collection and technical assistance, including Alexandria Watkins, D. Johnny Appazato, Nate Philips, Richie Ryan, and Derrick Klein. We also thank the CDC Southeastern Center of Excellence in Vector Borne Disease for providing funding for CMCD's research internship program. Further, we also thank the CDC Division of Vector-Borne Diseases for offering mosquito control districts with free Insecticide Resistance Kits and guidelines for evaluating insecticide resistance.

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