Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator: An Update of Mosquito Species Records for Charlotte County, Florida

Nonnative mosquito species have been detected within the state of Florida over the last 20 years, with 5 species being detected within Florida's boundaries in just the past 7 years. These introductions may be attributed to a changing climate where increasing temperatures encourage mosquito migration from the South in addition to global connectivity in which mosquitoes can be inadvertently introduced through transports (Lounibos 2002, Franklinos et al. 2019). Recent ecological models have resulted in a heightened awareness across the state for nonnative detections as they are often invasive vector species (Campbell et al. 2021, Sloyer et al. 2022). During 2019–21, Charlotte County has documented 4 new species of adult mosquitoes, including Aedes pertinax (Grabham), Anopheles perplexens (Ludlow), Culex declarator (Dyar and Knab), and Cx. interrogator (Dyar and Knab). While public health implications of Ae. pertinax and An. perplexens are currently unknown in the USA, Cx. declarator has been implicated as a vector of the dog heartworm, Dirofilaria immitis (Leidy) (Labarthe et al. 1998, Darsie and Shroyer 2004), and Cx. interrogator is a known carrier of Culex flavivirus (Saiyasombat et al. 2010). Mosquito control public health agencies have indicated the need for implementing protocols to deal with detections of nonnative species, especially introduced invasives that are known to transmit pathogens of public health importance (Kondapaneni et al. 2021, Dye-Braumuller et al. 2022, Wilke et al. 2022). County-level species voucher records are a crucial stage of the process and should be taken with forethought to assist in the baseline collection of data for improving the detection of nonnative and invasive mosquito populations in any region surveyed by public health agencies (Darsie and Morris 2003, Riles and Connelly 2020).

Aside from An. perplexens, a native species in Florida, the other three hold nonnative status where their respective first detections in the state occurred from 2004 through 2016 (Darsie and Shroyer 2004, Shroyer et al. 2015, Shin et al. 2016). Anopheles perplexens is typically found in localized areas of the eastern USA, including northern and central Florida (Darsie and Ward 2005, Burkett-Cadena 2013). Aedes pertinax was originally limited to the Caribbean Islands (Darsie et al. 2010), and both Cx. declarator and Cx. interrogator are found in portions of southern Texas with ranges extending south through Mexico and Central America (Bram 1967, Darsie and Ward 2005). The possible range expansion demonstrates the need for heightened awareness across the state concerning identification of unknown mosquito species and their migration (Boehmler 2022, Short et al. 2022, Tyler-Julian et al. 2022). Standard microscopy methods using dichotomous identification keys is the forefront of determining species diversity locally and regionally; however, it does not always ensure accuracy due to reliance on specimen quality, similar characteristics among species within the same genus, and/or total absence of species in regional identification keys (Darsie and Morris 2003, Darsie and Ward 2005, Reeves et al. 2021, Riles 2021). For these reasons, the procedure of cytochrome c oxidase subunit I (COI) sequencing should be implemented in addition to visual identification methods to ensure accuracy when reporting the occurrence of unknown species introduced into any area that is being surveyed for public health, although it is important to have an accurate molecular reference collection available in addition to Barcode of Life Datasystems (BOLD) Identification Tool reference sequences. Here we report on the introduction of the aforementioned 4 species in Charlotte County, FL.

Charlotte County Division of Mosquito and Aquatic Weed Control (FL) performs county-wide surveillance at 17 routine static locations using the Centers for Disease Control and Prevention (CDC) miniature light traps (Model 512; John W. Hock Company, Gainesville, FL; hereafter referred to as CDC light traps). The CDC light traps were set with only incandescent light (Model No. CM-47, 6.3 V; John W. Hock Company) as an attractant, no other lures or attractants were used. Traps were set biweekly and operated for 24 h per collection period. Mosquitoes collected were brought to the laboratory and stored at −18°C for 1 h to ensure mortality. Mosquitoes were then sorted out from other insects and morphologically identified under a dissecting microscope (National Trinocular, 10–40× Stereo Zoom [420 series], Schertz, TX), using dichotomous identification keys (Darsie and Morris 2003, Darsie and Ward 2005), and recorded into the VectoStar (Skysoft Inc., Orlando, FL) database management system. Observed unknown or morphologically questionable species were placed into vials labeled with collection date, location, tentative species determination, and stored at −18°C for later visualization and/or DNA analysis. For DNA preparation, each specimen was placed in 95% ethanol for the DNA barcoding analysis comparing known COI gene sequences (Hebert et al. 2003).

Culex declarator was first collected and identified in Charlotte County on June 20, 2019, in a wooded residential lot in the western portion of the county (26°56′13.7328″N, 82°15′52.0272″W). In August 2019, 3 females were collected in the central portion of the county over 3 trap locations within semirural and suburban habitat designations (27°0′30.604″N, 82°5′16.288″W; 26°57′18.264″N, 81°59′16.748″W; 27°0′53.733″N, 82°3′38.044″W). The following year, 86 females were trapped at an additional 8 sites across the county.

Aedes pertinax was first recognized in Charlotte County on July 12, 2019, from a semirural trap site (26°55′14.9772″N, 82°0′53.2512″W) located in a patch of woods near an open grassy area. Thereafter, during the months of July and August, the species was documented at 15 out of all 17 trap sites with a mix of habitats ranging from rural to residential. The species often co-occurs with Ae. atlanticus Dyar and Knab and Ae. infirmatus (Dyar and Knab), all of which share similar morphologic characteristics.

In October 2021, specimens suspected to be Cx. interrogator (n = 10, F) were collected in Charlotte County from a rural oak hammock habitat in the eastern part of the county (26°59′7.278″N, 81°52′14.4876″W) and kept frozen according to analysis protocols until confirmation could be made. Subsequent collections (n = 5, F) were made in October through December at a semirural wooded site (26°57′18.264″N, 81°59′16.748″W) located just 7.5 mi southwest of the initial detection and a collection of 2 females was obtained from a 3rd site (26°56′15.015″N, 82°1′50.499″W) in November. All sites with these initial collections are located east of the Peace River.

A female mosquito tentatively identified as An. perplexens was collected on November 2021 at a wooded semirural site (26°57′18.2628″N, 81°59′16.7496″W). As of yet, no An. perplexens have been detected in neighboring Lee and Sarasota counties to the south and north, respectively. Initial collections are depicted in Table 1 and Fig. 1.

One representative adult specimen of each tentatively identified species was sent to the Florida Medical Entomology Laboratory (FMEL) Reeves Lab in Vero Beach, FL, for molecular analysis. From each of these specimens, one midleg was removed with flame-sterilized forceps and transferred to a 1.5-ml microcentrifuge tube. The remainder of each mosquito specimen was mounted on an insect pin and preserved as a voucher in the Reeves Lab reference collection. DNA was extracted from the detached mosquito legs using the Zymo Quick-DNA Miniprep Plus Kit (Genesee Scientific Corp., El Cajon, CA). After the 1st extraction buffer was added to the tube, the leg was macerated for approximately 3 min with a sterile plastic pestle. All subsequent steps followed the manufacturer's protocol. The barcoding region of the COI gene was amplified for each mosquito specimen by polymerase chain reaction (PCR) using either the LepF1 and LepR1 primers (Hebert et al. 2004) or the LCO1498 and HCO2198 primers (Folmer et al. 1994). Regardless of the primer pair used, each PCR was performed in a final volume of 25 μl that consisted of 10 μl 2× Apex Taq RED Master Mix (Genesee Scientific Corp., San Diego, CA), 2 μl of 10 μM forward primer, 2 μl of 10 μM reverse primer, 1 μl of extracted DNA, and 5 μl of sterile water. Thermal cycling conditions for each PCR were 94°C for 1 min, 5 cycles of 94°C for 30 sec, 45°C for 40 sec, and 72°C for 1 min, 35 cycles of 94°C for 30 sec, 51°C for 40 sec, and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. Seven microliters of each PCR product was electrophoresed on a 1.5% agarose gel for 45 min and visualized under a transilluminator to ensure amplification of a PCR product of the expected size. Amplicons were sent to Eurofins Genomics (Louisville, KY), where they were cleaned and sequenced using chain-termination sequencing (Sanger et al. 1977). The resulting DNA sequence chromatogram files were visualized in the bioinformatic software Geneious Prime 2020.1.2 Version 11.0.6 (Biomatters, Inc., San Diego, CA), and each sequence was examined and edited for quality.

Edited COI sequences were queried on the BOLD v. 4 Identification Engine for species-level identification by alignment to reference COI sequences (Ratsnasingham and Hebert 2007). Limitations to identifying mosquitoes through DNA barcoding include incomplete taxonomic coverage of reference sequences in publicly accessible databases and the existence of misidentified reference sequences and reference sequences that are not identified to species level (Beebe 2018). These issues are particularly relevant to An. perplexens (reference sequences absent from BOLD), and Cx. declarator and Cx. interrogator (misidentified reference sequences). To circumvent these limitations, COI sequences from the Charlotte County mosquito specimens were aligned to reference sequences curated by the Reeves Laboratory and derived from reliably identified specimens. Sequences from Charlotte County Ae. pertinax, An. perplexens, Cx. declarator, and Cx. interrogator were aligned to other Florida species from the Aedes subgenus Ochlerotatus, Anopheles subgenus Anopheles, and Culex subgenus Culex, respectively. From these alignments, neighbor-joining trees were constructed using the Geneious Tree Builder tool (Geneious Prime Version 11.0.6), with the Jukes–Cantor genetic distance model.

Based on the results of BOLD Identification Tool queries and neighbor-joining trees, DNA barcoding sequences confirmed the morphological identification of Ae. pertinax, An. perplexens, Cx. declarator, and Cx. interrogator in Charlotte County. The COI sequence from the specimen morphologically identified as Ae. pertinax was highly similar (>99%) to BOLD reference sequences labeled as Ae. pertinax and Ae. serratus (Theobald) collected on the Yucatan Peninsula. The latter species is morphologically similar to Ae. pertinax. In the neighbor-joining tree (Fig. 2), the Charlotte County Ae. pertinax sequences grouped together in a clade with sequences derived from other Florida Ae. pertinax specimens collected in Indian River, Miami-Dade, and Lee counties. This clade was distinct from those of all the other Aedes Protoculex Group species (Ae. atlanticus, Ae. dupreei Coquillett, Ae. tormentor Dyar and Knab) that occur in Florida, and similarity among sequences in this clade range from 98.7% to 100%. The COI sequence for the An. perplexens specimen did not match any BOLD reference sequences. Reference sequences labeled as An. perplexens have been uploaded to BOLD, but all are essentially identical to reference An. punctipennis Say sequences. Anopheles punctipennis is morphologically similar to An. perplexens. In the neighbor-joining tree (Fig. 3), the Charlotte County An. perplexens sequence formed a clade with other Florida specimens identified as An. perplexens from Brevard, Hillsborough, and Osceola counties. This sequence was 99.49–100% similar to other sequences in this clade. For Cx. declarator and Cx. interrogator, BOLD Identification Engine results were ambiguous, with Charlotte County COI sequences highly similar to reference sequences labeled as multiple Culex species. These results likely reflect the challenges with distinguishing several of the species of the subgenus Culex. In the Culex neighbor-joining tree (Fig. 4), sequences from the Charlotte County specimens of both species each formed respective clades with sequences from Florida Cx. declarator specimens from Indian River and Collier counties and Florida Cx. interrogator specimens from Osceola and Palm Beach counties, with each clade distinct from those of all other included Culex species.

Culex declarator, since the initial 2019 collections, has been observed in low numbers (n = 103) across multiple collection sites, possibly indicating establishment with a relatively even distribution. Currently no larvae have been collected for this species. The potential public health implications of Cx. declarator are unclear in Florida, although the species has been implicated as a vector for D. immitis due to their host preference of canine-baited traps (Labarthe et al. 1998, Darsie and Shroyer 2004). In 1998, Cx. declarator was first reported in Florida in Indian River County and did not become abundant until September 2003 (Darsie and Shroyer 2004). Prior to this, Cx. declarator was present in the USA only in portions of southern Texas (Breland 1954, Darsie and Ward 2005), though from there its geographic distribution extends south through Central America, the northern half of South America, and the Lesser Antilles (Bram 1967).

Aedes pertinax, since the initial detection in Charlotte County, FL, has been documented at nearly all surveillance sites (n = 703); currently no larvae have been collected for this species. The species had likely been collected prior to its detection but incorrectly identified as Ae. atlanticus due to similar morphologic characteristics at gross level as described by Shroyer et al. (2015). Shroyer et al. (2015) reported the first detections of Ae. pertinax in the USA through collections in Indian River County, FL, in 2011 and verified its presence through performing a morphometric genetic analysis. Prior to this, Ae. pertinax was known only from the Caribbean Islands (Darsie et al. 2010), which may serve as a corridor for mosquito introductions into the Florida Peninsula (Darsie and Shroyer 2004, Blosser and Burkett-Cadena 2017, Burkett-Cadena and Blosser 2017, Campbell et al. 2021). Voucher specimens were obtained from Sarasota County Mosquito Management Services to perform the initial verification of Ae. pertinax, which was further confirmed by DNA barcoding. Trained individuals need to have heightened awareness in Florida concerning the separation of morphologically similar mosquito species such as Ae. atlanticus, Ae. tormentor, and Ae. pertinax (Sither et al. 2013, Shroyer et al. 2015, Harrison et al. 2016). Refer to Shroyer et al. (2015) for morphological characters relevant to separating Ae. pertinax from other Florida Protoculex Group Aedes.

In 2013, Cx. interrogator larvae were collected in Broward County, FL (Shin et al. 2016). Prior to this detection, the US distribution of Cx. interrogator was limited to a few counties in Texas (Carpenter and LaCasse 1955, Darsie and Ward 2005) where the species reaches the northern limit of its distribution, occurring south through Mexico and Central America. Culex interrogator was previously understood to be absent from the Caribbean (Bram 1967) but has recently been reported for the first time from Cuba (Menzies et al. 2018) and Hispaniola (Sosa et al. 2020). Since its introduction into Florida, Cx. interrogator has migrated to multiple counties throughout the state (Riles and Connelly 2020). Collaboration with Lee County Mosquito Control District, a neighboring county to the south, helped to determine this was most likely Cx. interrogator via characteristic descriptions and photography of Lee County, FL, reference specimens. Once Cx. interrogator was determined, a specimen was sent to FMEL for COI sequencing verification where it was confirmed. Culex interrogator has been collected across 5 different sites (n = 20, F) in Charlotte County through the end of 2021; however, larvae of this species have not been collected in Charlotte County to date. Catch locations range from central to eastern portions of the county, with 4 sites designated as rural habitat and one a residential locality. The public health significance of Cx. interrogator in Florida is currently unknown, although pools in the Yucatan Peninsula of Mexico have been found infected with Culex flavivirus (Saiyasombat et al. 2010).

Anopheles perplexens is an uncommon mosquito that occurs in localized and disjunct areas of the eastern USA, including northern and central Florida (Darsie and Ward 2005, Burkett-Cadena 2013). Little is known concerning the medical importance of this particular species. To date, this has been the only confirmed An. perplexens collected in Charlotte County and the most recently discovered species in the county. This mosquito is morphologically similar to An. punctipennis and older, worn specimens may be confused for An. crucians Weidemann complex species.

Historically, routine trapping methodologies have remained unchanged in Charlotte County since CDC light traps were implemented several decades ago. Whereas many districts use lures and attractants such as CO₂ in addition to a light source, Charlotte County has only used the incandescent bulb that comes standard on a John W. Hock Company model 512 CDC light trap for routine 24-h site surveillance. That is not to say other trap types and attractants aren't currently being used for auxiliary collections, but to maintain baseline accuracy, all 17 routine static trap locations are still monitored in the original manner. Perhaps the biggest advantage of this method is a more manageably sized mosquito catch in which every single species is identified as opposed to aliquots where a new or rare species might be missed. Usual summertime trap catches using this method range from dozens up to over 2,000 mosquitoes in a single trapping period, with overall annual catches averaging 78 mosquitoes per catch period. In addition to manageable catch sizes, since the traps are run for 24 h, catches are not limited to mosquito species that are only active during crepuscular periods.

Due to operational constraints, localized collections of larvae for the purpose of identification are not often feasible and therefore have not been implemented as a standard form of species surveillance. While the nonnative species of Ae. pertinax, Cx. declarator, and Cx. interrogator have become commonplace in Charlotte County CDC light trap collections since initial detections, determination of establishment cannot be confirmed until respective larval collections are made. Because of this, current and future focus will be centered on obtaining an increased number of larval collections in an attempt to verify where the species are originating from.

The addition of 4 previously undetected species (Fig. 5) within the span of 3 years is quite significant. Documented species in Charlotte County have increased over this period by 10.5%, from 34 to 38 mosquito species. Culex declarator and Cx. interrogator occur in portions of southern Texas that share the same latitude as Charlotte County within the 26° parallel, so it is not surprising the county would be ecologically suitable for these species once they were introduced into Florida. Aedes pertinax was most likely established in the county prior to 2019, but because this species was not included in the Darsie and Morris (2003) or Darsie and Ward (2005), the primary resources used for morphological mosquito identification in Florida, specimens were considered anomalous to Ae. atlanticus at gross levels and perhaps misidentified. Anopheles perplexens is known to have sporadic distributions and is known to exist in the northern and central regions of Florida based on information from distribution maps of regional and national identification keys (Darsie and Ward 2005, Burkett-Cadena 2013) where peer-reviewed checklists of mosquito species suggest migration within northern Florida counties (Smith et al. 2020). For these reasons, along with changing weather patterns, it is important to remain vigilant when identifying mosquito species. Using references such as identification keys from multiple regions and recent peer-reviewed taxonomic literature along with communication with neighboring mosquito programs and molecular confirmation of morphological species identification can assist in recognizing when unknown or previously undetected species are first discovered.

Special thanks to Wade Brennan, Sarasota County Mosquito Management Services, and Kara Tyler-Julian, Lee County Mosquito Control District, for their help and communications with regards to newly encountered species. The authors would also like to show their gratitude to Roxanne Connelly for reviewing a previous draft of this manuscript.