Habitat-Specific Host Selection Patterns of Culex quinquefasciatus and Culex nigripalpus in Florida

Culex nigripalpus Theobald and Cx. quinquefasciatus Say are considered primary amplification and/or bridge vectors of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) in Florida (Day et al. 1990, Turell et al. 2005, Vitek et al. 2008, Rochlin et al. 2019). This consideration is largely supported by vector competence studies (Sardelis et al. 2001, Rutledge et al. 2003, Colton and Nasci 2006), coincident distribution and abundance with human cases (Edman 1974, Day et al. 2015), and previously described host utilization patterns (Edman and Taylor 1968, Edman 1974, Molaei et al. 2007). While there has been considerable research on host feeding patterns of Cx. quinquefasciatus throughout other areas of the southern USA (Molaei et al. 2008, 2010; Mackay et al. 2010), there has been little focus on host usage of Cx. nigripalpus and Cx. quinquefasciatus in Florida. Understanding local host utilization patterns of both species is necessary to establish the potential roles of these species in arbovirus transmission cycles across diverse habitats (Rutledge et al. 2003, Kilpatrick et al. 2006, Day et al. 2015).

Historical annual surveillance of the mosquito population in Sarasota County, FL (C. Hancock, unpublished data), suggests that Cx. nigripalpus is in high relative abundance in rural habitats, moderate abundance in suburban habitats, and low numbers in urban areas, whereas the inverse pattern of abundance is found for Cx. quinquefasciatus (i.e., the relative abundance of Cx. quinquefasciatus is highest in urban areas). This is in line with previously published habitat associations of these species. Culex quinquefasciatus prefers the type of high-nutrient larval habitats more commonly found in urban areas (e.g., catch basins, wastewater treatment effluents, man-made containers, and bromeliad plants), whereas Cx. nigripalpus prefers a less nutrient-rich larval habitat and is more commonly found in freshly flooded habitats found in rural areas (e.g., roadside ditches and pastures) (Leisnham et al. 2014, Day et al. 2015, Watkins 2021). There have been few studies that have assessed the relationship between habitat and host feeding for these species, with little focus on the effects of anthropogenic habitat change on arbovirus ecology or disease risk (Steiger et al. 2016, Santos et al. 2019, Mann et al. 2020). Differences in host feeding patterns between different habitats has been limited to small-scale habitat descriptions (Patrican et al. 2007). In order to assess the potential impacts of anthropogenic habitats on mosquito-borne virus ecology and foraging behavior of these 2 mosquito vectors, more regional work involving habitat types is necessary.

Previous studies have indicated that Cx. quinquefasciatus shows a host preference for birds, while Cx. nigripalpus has been described as an opportunistic feeder (Edman 1974, Cohen et al. 2009, Mackay et al. 2010). There has also been evidence to suggest that Cx. nigripalpus exhibits a biphasic feeding pattern to support its role as a primary enzootic and epizootic vector of arboviruses in Florida (Edman and Taylor 1968). Seasonal shifts in host usage have been documented in other mosquito vectors, namely Culex pipiens L., and were correlated with arbovirus epidemics in humans (Kilpatrick et al. 2006). Seasonal feeding patterns in Cx. quinquefasciatus have not been well studied in Florida. The goal of this study was to investigate host utilization patterns of these 2 arbovirus vectors and how they may differ over time and in varying habitats. Understanding habitat-specific and temporal changes in host usage patterns is essential to determining the role(s) that mosquito vectors play in arbovirus transmission and spillover to humans.

Blood-engorged mosquitoes were collected from locations throughout Sarasota County, FL, over 3 years (May 2017–May 2020). Blood-engorged mosquitoes were mostly obtained via aspiration of resting shelters encountered during weekly surveillance efforts, and specimens encountered in Centers for Disease Control and Prevention light and gravid traps were also used for analysis when possible. Cold chain was maintained in processing collections. Individual, blood-engorged mosquitoes were separated by species and stored individually at −40°C until polymerase chain reaction (PCR) and sequencing could be performed. Mosquitoes were identified according to the features described by Darsie and Ward (2005).

In total, 140 collection sites were surveyed over the 3-year period. Collection sites were selected to represent urban (59 sites), suburban (51 sites), or rural (30 sites) habitats based on general observable features and later refined (Fig. 1). In general, urban habitats were areas with high human influence in the form of multiple family housing, small-lot neighborhoods, commercial properties, and were commonly coastal. Suburban sites were areas largely composed of focally distributed neighborhoods or medium-sized single-family home lots with little to no commercial properties. Rural areas were absent of human influence, agricultural lands, or contained ranch-style homes on large parcels of land. The classification of each trapping site was refined retrospectively according to the 2021 Florida Department of Environmental Protection Statewide Land Use and Land Cover geospatial data set (https://geodata.dep.state.fl.us). Under this scheme, “Urban and builtup” sites were classified under Level 1 land use values of 1,000 and 8,000; and “other” consisted of Level 1 values of 2,000, 3,000, 4,000, and 7,000 (Agriculture, Upland forests, Upland nonforested, and Barren land). Individual collection sites were then classified based on their within- and neighbor-polygon classification. For example, urban trapping sites were located within “Urban and builtup” polygons that were completely surrounded by “Urban and builtup” polygons; rural sites were located within “other” polygons completely surrounded by “other” polygons; and suburban sites were located within either an Urban or “other” polygon and surrounded by a mixture of habitat types, including wetland habitats. Comparison between the initial habitat assignment and the land use–based habitat assignment resulted in the reclassification of 7 trapping locations to match the land use–based habitat assignment.

Individual mosquitoes were homogenized in 250 μl of phosphate-buffered saline using disposable pestles. Homogenates were centrifuged and DNA extractions were performed on the total supernatant volume using the MagMAX CORE Nucleic Acid Purification Kit (ThermoFisher Scientific, Waltham, MA) on an automated extraction platform. Amplification of host DNA was conducted using up to 5 primer sets designed to amplify 16S rRNA, cytochrome oxidase I, or cytochrome b genes. Extracted DNA from samples was first screened by PCR with primers 16L1/H3056 (Hass et al. 1993) and/or RepCOI-F/RepCOI-R (Reeves et al. 2018). The RepCOI-F/RepCOI-R primer set was added to the screening process midstudy. Samples that did not produce an amplicon were then screened by an additional PCR protocol using primers L2513/H2714 (Kitano et al. 2007), which has been used to successfully amplify a range of host types with a slight bias towards amplifying sequences from amphibians and mammals (Burkett-Cadena et al. 2008). Finally, few samples producing no amplicon in previous screenings were tested using LO/H1 and/or L0/HO (Lee et al. 2008, Blosser et al. 2016).

In general, for each PCR, an aliquot of 2 μl of extracted DNA was used in a 20-μl reaction. Other reaction components consisted of 10 μl Platinum II Hot-Start Green PCR Master Mix (2×) (ThermoFisher Scientific), 0.4 μl of forward and reverse primers, and 7.2 μl molecular-grade water. Cycling conditions for all primer sets were 94°C for 2 min, 35 cycles of 98°C for 5 sec, and 60°C for 15 sec, then reactions were held at 4°C until processing. The PCR products were then loaded into and visualized on a 1.5% agarose gel. Amplified PCR products were sequenced in the forward direction by the Sanger method (Eurofins Genomics, Louisville, KY). No postsequencing editing of the resulting sequences was performed. The resulting nucleotide sequences were queried by a BLASTn search (basic local alignment and search tool) of National Center for Biotechnology Information's (NCBI) GenBank. Sequences to with ≥175 base pairs read and ≥95% similarity were considered confirmed to host species, as previously described (Blosser et al. 2016).

Individual samples of each species were stratified using multinomial variables for host (avian, mammalian, reptile/amphibian), collection month, and by collection habitat (Urban, Suburban, Rural). We tested the null hypothesis of equal host selection ratios with exact multinomial or binomial tests (R version 4.0.3, R Core Team 2018) and calculated 95% confidence intervals (CIs) around each proportion based on the standard normal distribution. Sampling effort was not homogeneous between habitats, and we could not verify the relative abundance of each species in each habitat by another less-biased method (e.g., light trapping). We therefore could not perform a similar statistical analysis testing the null hypothesis that mosquitoes have equal distribution between habitats. Instead, we tested the conditional independence between habitat and host group for each species based on a chi-square distribution. For post hoc analyses, we calculated the expected values based on i) equal selection of hosts in each habitat and ii) similar selection of hosts in each habitat using marginals (total counts) to calculate expected values. After testing the null hypothesis of conditional association under a chi-square distribution, we calculated standardized residuals (Agresti 2002) and/or estimated odds ratios (and 95% CIs) to identify specific differences in host selection between habitats. A time series was visualized to identify shifts in host selection, subdividing collection date by month.

From May 2017 to May 2020, 1,056 total blood-engorged mosquitoes were collected and tested to identify hosts. Both species were collected from resting shelters, no bloodfed Cx. nigripalpus were collected from gravid traps, and no bloodfed Cx. quinquefasciatus were collected from light traps. This resulted in the identification of host species from 920 of those blood meals (87.1% successfully sequenced). Culex nigripalpus blood meals accounted for 52.1% of the positively identified hosts, with a total of 479 identified and only 30 inconclusive (94.1% hosts successfully identified) (Table 1). Culex quinquefasciatus blood meals accounted for 47.9% and had a slightly lower success rate for positively identified hosts (73.7%), with 441 identified and 116 inconclusive (Table 1). All georeferenced data and host identifications are provided as a Supplemental Table; and additional data (raw sequences) are available upon reasonable request made to the corresponding author.

There were clear differences in host usage between Cx. nigripalpus and Cx. quinquefasciatus. Ectothermic hosts were only detected in Cx. nigripalpus and consisted of 1 from the American alligator (Alligator mississippiensis (Daudin)), 1 from the Cuban tree frog (Osteopilus septentrionalis (Duméril and Bibron)), and 87 blood meals from the brown anole (Anolis sagrei (Duméril and Bibron))—alone comprising 18.2% of the total hosts identified from Cx. nigripalpus. Secondly, there was a statistically significant difference in the number of avian versus mammalian hosts detected in each species (2-sided exact binomial tests of equal proportions, P < 0.001 for Cx. nigripalpus; P < 0.001 for Cx. quinquefasciatus). The estimated proportion of mammalian host usage for Cx. nigripalpus (versus avian) was 0.69 (95% CI = 0.64∼0.73), with a ratio of 2.2 mammalian hosts identified for every avian host. In contrast, the estimated proportion of mammalian host selection for Cx. quinquefasciatus was 0.27 (95% CI = 0.22∼0.32), with a ratio of 3.7 avian hosts identified for every mammalian host.

The top 5 avian hosts selected by Cx. nigripalpus were domestic chickens (Gallus domesticus (L.)) (34), eastern screech owls (Megascops asio (L.)) (16), northern cardinals (Cardinalis cardinalis (L.)) (12), mourning doves (Zenaida macroura (L.)) (9), and green herons (Butorides virescens (L.)) (6), comprising 16% of all identified meals and collectively 63% of avian meals (Table 2). The top 5 avian hosts selected by Cx. quinquefasciatus were domestic chickens (76), northern cardinals (64), Florida scrub jays (Aphelocoma coerulescens (Bosc)) (50), mourning doves (24), and northern mockingbirds (Mimus polyglottos (L.)) (24), comprising 54% of all meals and 68% of avian meals (Table 3). Thus, domestic chickens, northern cardinals, and mourning doves were commonly selected by both species of mosquito. The top 5 mammalian hosts of Cx. nigripalpus were eastern cottontail rabbits (Sylvilagus floridanus (J.A. Allen)) (65), domestic cows (Bos taurus (L.)) (59), white-tailed deer (Odocoileus virginianus (Zimmermann)) (37), common raccoons (Procyon lotor (L.)) (23), and domestic horses (Equus caballus (L.)) (19), comprising 76% of all mammalian meals and 42% of all meals (Table 2). The top 5 mammalian hosts of Cx. quinquefasciatus were common raccoons (29), Virginia opossums (Didelphis virginiana (Kerr)) (23), humans (11), domestic cats (Felis catus (L.)) (8), and black rats (Rattus rattus (L.)) (6), comprising 83% of all mammalian meals and 17% of total meals (Table 3).

There appeared to be differences in the abundance of bloodfed mosquitoes per habitat (Table 1 and Fig. 2). Specifically, there were 3 times more blood meals identified from Cx. quinquefasciatus obtained in urban habitats than in suburban or rural habitats, while Cx. nigripalpus were fairly evenly distributed between locations within the 3 habitat classifications (Table 1 and Fig. 2). We did not perform statistical analysis to test whether the distribution of a given species differed between habitats as we could not account for potential sampling bias (e.g., comparing relative abundance with a less-biased sampling method).

To test whether there was a difference in host usage in each habitat, we analyzed the conditional independence between host usage and habitat to account for potential sampling bias due to habitat. We found that host usage was conditionally dependent on habitat classification for Cx. nigripalpus, analyzing the 3 host classes (avian, mammalian, and reptile/amphibian) in the 3 habitat classifications (χ² = 65.7, df = 4, P < 0.001). As a post hoc test, to characterize the specific pattern of host usage in each habitat, we inspected the standardized residuals, assuming values >|3| are significantly different than expected (Agresti 2002). We found that mammalian hosts were used more frequently in rural habitats and less frequently in urban habitats than expected (based on the previously established proportion of mammal hosts of this species), while avian and/or reptile/amphibian hosts were used more often in urban habitats and less often in rural habitats. Culex nigripalpus host usage in suburban habitats was not significantly different from the general pattern of host usage for this species.

As only Cx. nigripalpus used ectothermic hosts in our study, and we were interested in comparing host selection between Cx. nigripalpus and Cx. quinquefasciatus, we repeated the analysis of conditional independence between habitat and host class using only mammalian and avian hosts for Cx. nigripalpus. Again, we found a significant difference in the distribution of observed hosts (avian versus mammalian) of Cx. nigripalpus in each habitat based on: i) the assumption that hosts would be used in equal ratios controlling for relative abundance in each habitat, χ² = 85.7, df = 2, P < 0.001; and ii) the assumption that hosts would be selected in similar ratios in each habitat (i.e., a higher proportion of mammalian blood meals, as previously noted), χ² = 37.1, df = 2, P < 0.001. The conditional odds ratios for Cx. nigripalpus selecting avian hosts in urban habitats was 3.92 (95% CI 2.27∼6.81) and in suburban habitats was 2.15× (95% CI 1.19∼3.87) Cx. nigripalpus selecting mammalian hosts in rural habitats. More simply stated, the mammalian to avian host ratio for Cx. nigripalpus was highest in rural habitats (6.05) and lowest in urban habitats (1.09).

A similar analysis of host selection by habitat for Cx. quinquefasciatus was troubled by the fact that only 6 blood meals were analyzed in the rural habitat (4 avian and 2 mammalian), and this limited the statistical power of the analysis. By combining suburban and rural habitat classifications, there was no difference in host usage between urban (79% avian) and nonurban (76% avian) habitats for Cx. quinquefasciatus under the assumption that hosts were selected in the same ratio in each habitat classification (χ² = 1.3, df = 1, P = 0.183). The avian-blood meal bias was evident in each habitat (conditional independence between habitat and host under the assumption that hosts were selected equally in each habitat (χ² = 143.2, df = 1, P < 0.001). In other words, Cx. quinquefasciatus used more avian hosts than mammalian hosts in every habitat.

Finally, we analyzed the seasonality of host selection by each species (Fig. 3). Approximately 90% of the blood meals from Cx. nigripalpus were encountered between June and October (Fig. 3A). While the majority of blood meals from June until August were from mammalian hosts (66–68%), a pattern of more catholic feeding was detected in September and October, with nearly equal proportions of avian, mammalian, and reptilian (Anolis sagrei) hosts (27–42%). For Cx. quinquefasciatus, 83% of the blood meals were encountered between January and August, and the proportion of avian hosts selected by Cx. quinquefasciatus decreased after this, with mammals comprising over half of the blood meals in October (N = 13; Fig. 3B). Having previously established the average proportion of mammalian hosts in Cx. quinquefasciatus was 0.21 (99% CI 0.16∼0.27), we estimate that significantly more mammalian hosts were used in January and September until December.

We detected clear differences in host usage between Cx. nigripalpus and Cx. quinquefasciatus. In general, Cx. nigripalpus used proportionally more mammalian hosts and Cx. quinquefasciatus used more avian hosts. Despite this pattern, Cx. nigripalpus used humans less often (2.2% of mammal hosts) than Cx. quinquefasciatus (11.8% of mammal hosts). Only Cx. nigripalpus used ectothermic hosts, the majority of which were anoles (Table 1). We also found a habitat-specific pattern of host usage for Cx. nigripalpus, but not for Cx. quinquefasciatus (Fig. 2). While Cx. quinquefasciatus used avian hosts and mammalian hosts at the same ratio in all habitats, significantly fewer mammalian hosts were observed in Cx. nigripalpus in urban habitats compared to suburban and rural habitats. For Cx. nigripalpus, utilization of both reptiles and birds for blood sources decreased in frequency as collections moved from urban to rural settings (Fig. 2). Finally, we noted a distinct seasonal pattern of host usage in Cx. quinquefasciatus—namely the increased proportion of mammalian feeding from September to January—while no such distinct pattern of host usage was observed in Cx. nigripalpus (Fig. 3).

Although Cx. nigripalpus primarily used mammals as hosts, more reptile feeding was observed in Cx. nigripalpus than in previous studies (Edman 1974). The increase in mammalian host usage in Cx. nigripalpus during summer months has been previously described at a lesser magnitude (Edman and Taylor 1968). However, reptiles accounted for roughly 20% of usage throughout winter and spring, with an increase to 40% in late fall (Fig. 3). The vast majority of reptilian hosts (97.8%) were sourced from the common brown anole (Anolis sagrei), an invasive anole that is abundant all year in Florida. As it has been shown in previous research that reptiles (including anoles) may play a role as overwintering reservoir hosts of eastern equine encephalitis virus (White et al. 2011), the rates of A. sagrei selection by Cx. nigripalpus in this study warrant further investigations into the potential of Cx. nigripalpus playing a larger role in the cycling and transmission of this virus.

Culex quinquefasciatus were collected more frequently in urban settings, which matches previously published habitat-associated abundance data, and therefore may accurately reflect host usage patterns in urban and suburban habitats (Patrican et al. 2007, Molaei et al. 2008, Mackay et al. 2010, Leisnham et al. 2014, Day et al. 2015, Santos et al. 2019, Mann et al. 2020). Culex quinquefasciatus had a significantly higher ratio of avian blood sources, regardless of habitat type. While Cx. quinquefasciatus is less prevalent in rural areas where enzootic foci of WNV are more likely to occur, the highly ornithophilic host usage pattern and known vector competence suggest it is involved in transmission of WNV in urban habitats in Florida (Day et al. 2015). The late-season shift in local Cx. quinquefasciatus from primarily avian hosts during winter and spring to increasing mammalian hosts from September to January (peaking in October) is a biphasic host feeding pattern that was previously described in Cx. nigripalpus (Edman and Taylor 1968). This shift of Cx. quinquefasciatus host usage from avian to mammal sources during the periods of highest transmission of WNV in Florida (Florida Department of Health 2021) (http://www.floridahealth.gov/diseases-and-conditions/mosquito-borne-diseases/surveillance.html), increased abundance in urban habitats, and moderate human feeding rates may drive spillover of WNV to humans and other mammals.

Both mosquito species are known to play a role in the transmission of arboviruses in Florida. The results of this study suggest that Cx. quinquefasciatus exhibits a more relevant host usage pattern for driving WNV enzootic and epizootic transmission cycles. Culex quinquefasciatus and other members of the Pipiens Complex are widely distributed and well-studied vectors of WNV in the eastern USA (Godsey et al. 2005, Mackay et al. 2010, Rochlin et al. 2019, Gorris et al. 2021). Culex nigripalpus is considered a major vector of SLEV and is a competent vector of WNV in Florida (Chamberlain et al. 1964, Dow et al. 1964, Day et al. 1990, Sardelis et al. 2001, Richards et al. 2011, Rochlin et al. 2019). However, because this mosquito species is limited to the southeastern USA and other subtropical and tropical locations (Carpenter and La Casse 1955), it is not well studied. This presents the need for further research into how host usage by Cx nigripalpus may affect its involvement in arbovirus transmission. To this end, our study contributes the range of hosts selected, demonstrating overrepresentation of specific classes of hosts for each mosquito species with respect to habitat and time of year.

Information on host usage is an important but seldom investigated component for understanding and mitigating arbovirus risk: it complements standard surveillance measures and offers insight into the temporal and spatial dynamics of virus activity to support further scientific research. We were primarily interested in demonstrating an approach to identify hosts of mosquitoes that is easy to implement and does not require extensive biomolecular knowledge—an ideal scenario for encouraging mosquito control districts to implement these procedures. In our study, no postsequencing quality control was performed prior to sequence identification (e.g., primer sequences were not trimmed, sequencing chromatographs were not inspected). Instead, the use of strict cutoff criteria for confirming host identification via Blastn search were adopted to reduce the possibility of ambiguous or inaccurate sequence identification (Blosser et al. 2016). This may have resulted in a loss of specificity of the identification, and we could not identify mixed meals. Nonetheless, we noted only a few equivocal identifications to the species level using this approach, and these were restricted to less commonly used hosts. For example, 11 identified sequences resulted in equivocal identifications between grackles (e.g., genus Quiscalus Vieillot) and robins (Turdus migratorius). This was negligible in our statistical analyses, as host classes (avian and mammalian) were used rather than specific species of hosts. However, species-level host identification data should be interpreted cautiously, even for the most common hosts, and particularly when extending the inference to known competent hosts of arboviruses. More bioinformatic processing of the resulting sequencings would likely have improved the confidence in the species-level identifications, as we used overall sequence similarity as a criterion for identifying a host.

An additional limitation of the study is presented in the lower sequencing success rate in blood samples from engorged Cx. quinquefasciatus. Many of these samples produced putative PCR products when processed with primer sets designed with a bias toward avian cytochrome b sequences. However, these samples failed to meet the cutoff criteria of 95% sequence similarity when comparing to the online sequence database (i.e., a Blastn search). These sequences would need additional testing with primers designed more specifically to amplify avian hosts to determine if certain avian species—perhaps cryptic species—were excluded or underrepresented by the primer sets used in this study. Additionally, the approach is dependent on the availability of voucher and/or reference sequences. In our study, for example, we repeated the previous Blastn searches for all sequences a final time in late 2021 and found several identifications of Cyanocitta cristata (L.) (blue jay) changed to Aphelocoma coerulescens (Florida scrub jay) after the addition of sequences to GenBank database in early 2021. Despite these limitations, we believe this method is highly adoptable for host identification, with caveats about the specificity of identification that can be reasonably expected. By employing multiple PCRs to analyze amplicon-negative sequences we were able to reach 87.1% blood meals identified, which seems to be an average success ratio compared to other similar studies. Although some primer sets were initially described for use in the study of reptiles, and others may have a known bias towards specific host classes, their overall success in amplifying products from a wide variety of hosts has been demonstrated in more than one study (Hass et al. 1993, Vidal et al. 2000, Burkett-Cadena et al. 2008, Blosser et al. 2016). Researchers and vector control organizations wishing to understand mosquito–host interactions to a species level must rely on sequencing multiple targets for confirmation and other follow-up experiments.

While there are many factors in determining the roles of individual mosquito vectors and their potential involvement in the transmission of arboviruses, research in host usage patterns is an integral facet in those determinations, particularly in a dynamic environment (Godsey et al. 2005, Kilpatrick et al. 2006, Molaei et al. 2007, Rochlin et al. 2019). As human development constantly reshapes the landscape, changing climate increases the range of temperate zones, and the consistent introduction of invasive animal and mosquito species occur, these patterns may shift over time (Steiger et al. 2016). These potential shifts produce the need for in-depth evaluations of understudied and occasional reevaluation of well-studied mosquito host usage patterns at the regional level. This study found that Cx. quinquefasciatus exhibits the more relevant host selection patterns and biphasic, seasonal usage of hosts necessary for driving local virus amplification and spillover than Cx. nigripalpus. We also conclude that Cx. nigripalpus host usage varies by habitat, is not highly seasonal, and includes a much larger proportion of reptile feeding than previously shown. While this study was designed to provide surface insight into the locally understudied ecology of these 2 mosquito species' host usage patterns, these results provide a basis for further research into more specific aims in determining relevance to arbovirus transmission potential, such as whether mosquitoes are feeding on highly competent hosts in specific habitats.

We extend our thanks to Nathan Burkett-Cadena and Bethany McGregor of the University of Florida's Medical Entomology Laboratory for their assistance with the sequencing protocols necessary for this work. We would also like to extend our gratitude for the collection and initial sorting of mosquitoes to contributing former and current staff of Sarasota County Mosquito Management Services: Madison Royer, Paul Hudson, Jennifer Serviss, Taylor Grennan, Wade Brennan, and Natalie Osborn.