Culex nigripalpus Distribution Expansion: First Record in Virginia, New County Records in North Carolina, and Revised United States Map

Culex nigripalpus was first described by Theobald in 1901 in St. Lucia, Lesser Antilles. It is classified as a Tropical or Neotropical species and is well documented throughout Central and South America as well as the Caribbean and the southeastern United States. Within the last 3 decades, this species has expanded northward and established populations in areas where it was initially an occasional transient. This is evidenced by collections in Kentucky (Minter et al. 2011, Dye 2016), Oklahoma (Bradt 2017), and North Carolina (this report) which are nigh on the northern limits of its current range. Recent discovery of the species in Suffolk, VA, at widely separated locations suggests the possibility of further range expansion into the Mid-Atlantic region. Likewise, new findings in Arkansas (Sames et al. 2021; D. Theuret, personal communication), Illinois (Kim and Stone 2018), and Missouri (Claborn et al. 2018) suggest a northern range expansion in the Midwest.

Within the US Mid-Atlantic states, Cx. nigripalpus bears remarkable similarity in adult morphology to Cx. salinarius Coq., Cx. restuans Theobald, and Cx. pipiens complex, especially when collected in mosquito traps where specimens are prone to damage. Culex pipiens complex in our study area includes Cx. pipiens L., Cx. quinquefasciatus Say, and their hybrids. We suspect misidentification, lack of mosquito surveillance or reporting thereof in many areas, as well as the lack of year-round surveillance—the majority of author collections in North Carolina and Virginia occurred in fall months (September–November)—could be underestimating the true geographical limits and prevalence of this species.

In addition to reporting this new northeastern limit of Cx. nigripalpus, this article presents an updated checklist of the mosquito species of Virginia and gives insight to its distribution expansion in North Carolina over the past few decades. A revised US distribution map for Cx. nigripalpus is also provided, as an extensive search of the available literature revealed numerous collections published in the 15 years since the widely referenced Darsie and Ward (2005).

During the months of April–October in 2017–20, mosquitoes were trapped weekly at more than 50 locations in the city of Suffolk, VA. From November to December, limited trapping took place at approximately 16 locations weekly due to decreased resources and unfavorable weather conditions. Sites included urban, suburban, and rural locations, as well as the Great Dismal Swamp National Wildlife Refuge. Primarily 3 trap types were used: 1) BG-Sentinel 2® (BGS2; Biogents AG, Regensburg, Germany) set with carbon dioxide (CO₂; from a gas cylinder with flow rate 200 ml/min) and a Biogents human-scent lure cartridge (Biogents AG); 2) Centers for Disease Control and Prevention light trap (CDCLT; BioQuip Products, Rancho Dominguez, CA) set using standard incandescent light bulb with CO₂ (as previously described); and 3) modified Reiter gravid traps (gravid trap; Reiter 1987) baited with a chicken manure, alfalfa, yeast, and water mixture. Modified collection bottle rotators (BioQuip Products) and fiber nursery pot resting boxes (Western Pulp Products Co., Corvallis, OR) were also occasionally used. Traps were routinely set at approximately 1300 h and picked up the following day at approximately 0700 h. Collection bags with live mosquitoes were retrieved and brought to the Suffolk Mosquito Control (SMC) laboratory where specimens were put in a freezer for 20 min to be sedated and subsequently identified by morphological characteristics using a Nikon SMZ1000 stereomicroscope (Nikon Instruments Inc., Melville, NC) and the most recent key for the Mid-Atlantic states (Harrison et al. 2016). Initial specimens were sent to coauthor (BAH) for confirmation; subsequent collections were stored in the SMC laboratory.

Out of the 50 routinely trapped locations, the 3 site descriptions where Cx. nigripalpus was first found in 2017 follow. Collection site 1 was in the Eclipse Community (36°54′38″N, 76°29′24″W) of Suffolk, VA, at the tip of the northwest peninsula where the Nansemond River meets the James River. The site is in a suburban neighborhood strewn with pockets of mostly loblolly pine (Pinus taeda L.) forest. The collection was made via a BGS2 set as previously described. This site has been a part of SMC routine disease and population surveillance since 2008 when the program was expanded to encompass citywide surveillance. A gravid trap is routinely set in this community as well, approximately 1.5 km to the north.

Collection site 2 was in the Sandy Bottom Community (36°52′20″N, 76°32′60″W) at the base of the northwest peninsula, located on an undeveloped road with an abandoned, dilapidated house 20 m away surrounded by agricultural land, loblolly pine forest, and scarce residential housing; part of SMC routine surveillance since 2013. The collection was from a CDCLT set as previously described.

Collection site 3 was in the Holland Historic District (36°40′57″N, 76°47′17″W), over 30 km south of the 1st 2 sites. This site is a small suburban outpost surrounded by agricultural land; SMC routine surveillance since 2007. The collection was from a CDCLT set as previously described. A gravid trap and BGS2 were also routinely placed at this site, 20 m to the south and east, respectively, due to historically heavy populations of Aedes albopictus (Skuse), Cx. restuans, and Cx. pipiens complex.

Apart from the 1st published record of Cx. nigripalpus in Union County in 1944 (Carpenter et al. 1945), a US Air Force report from Wayne County (McHugh et al. 1988), the recent report of the 1995 county-first records in Rowan County (Hartwig et al. 2018), and collections made 2017–18 in New Hanover and Wake counties (Day et al. 2020), there have been no other published reports of this species in North Carolina. Following US Army mosquito surveys and state entomological reports in the 1940s, little known effort was dedicated to investigating distribution data of species throughout the state. Most published studies and surveillance efforts were concentrated in the Piedmont and Coastal Plain regions. However, in 1994 the Public Health Pest Management (PHPM) Section of the North Carolina Department of Environment and Natural Resources (NC DENR) initiated a study of the western half of the state, including the Piedmont and Mountain regions.

Collections of larval mosquitoes were made using standard dippers (BioQuip Products); adult collection methods included human landing–biting collections, backpack and mouth aspiration, New Jersey light traps (NJLT; Mulhern 1942), and CDCLTs supplemented with dry ice as a CO₂ source. Collection sites included rural, suburban, and urban geographic areas; repeated sampling took place at those with high species diversity, typically that of public domain—local and state parks and ecological preserves. Larval samples were transported in Whirl-pak® bags (Nasco Sampling LLC, Madison, WI) and reared to adult or killed by heated water; adults were frozen via dry ice in an ice chest. Specimens were identified to species using various keys (Carpenter and LaCasse 1955, King et al. 1960, Darsie and Ward 1981, Slaff and Apperson 1989). Preserved specimens were deposited in the PHPM collection of the NC DENR or the Department of Entomology Insect Collection at North Carolina State University. Some of these collections were personally retained by coauthor (BAH) after budget cuts abolished the PHPM in 2011. These collections were then transferred to Brian Byrd at Western Carolina University after the passing of BAH in 2018. Additional unpublished records from other county jurisdiction programs in the state are reported and were collected through similar techniques. These collections were reported to and verified by PHPM staff including coauthors (BAH and PBW).

Throughout 2017, citywide, 455,989 adult female mosquitoes were collected and comprised 34 species (Table 1). On October 13, 2017, 1 Cx. nigripalpus female was collected from site 1, 2 females from site 2, and 1 female from site 3 (Fig. 1). Identifications of all specimens were confirmed by coauthor (BAH). In addition to the Cx. nigripalpus specimens, several other mosquito species were collected among the 3 trap sites (Table 2). Following these collections, adult trapping and larval dipping efforts increased in the surrounding areas. On December 6, 2017, another Cx. nigripalpus female was collected from site 1 (Table 2). These findings represent a new state record for Virginia and increases the number of reported mosquito species in the state to 58. Due to the 2 recent species records in the state of Virginia, Cx. coronator Dyar and Knab (Akaratovic and Kiser 2017) and Cx. nigripalpus (this report), we provide an updated checklist of mosquitoes for the state of Virginia (Table 1).

Following these initial discoveries, no specimens were collected the following year. However, 2 adult females were found in 2019: one from a BGS2 on May 21 and the other from a CDCLT on October 10 (Fig. 1, sites 4 and 5, respectively). Four more specimens were found in 2020, all adult females in BGS2 traps; 1 on November 10 and 3 on November 16, in similar habitats as previously described (Fig. 1, sites 6, 7, 8, respectively).

The following listed collections include 25 county-first records: 20 by coauthors (BAH and PBW) or associated personnel and 5 via personal communication (collectors listed in respective counties; confirmations by coauthor noted). In the case of Rowan County, these records were previously reported in conjunction with another study (Hartwig et al. 2018). In the case of New Hanover County, this report is the 1st known record in the county while many collections have been made in recent years (Day et al. 2020; M. Hemmen and J. Suggs, personal communication). Counties are listed in chronological order of 1st discovery.

Larval specimens (118 F, 76 M) were collected September–October 1991 with subsequent collections October–January in 1994–96 (R. Hickman, personal communication; confirmed by BAH). Larvae were collected from a variety of areas including wooded pools, sunny grassland pools and ditches, tidal, brackish, and even saline dredge pool habitats.

One female was collected on October 10, 1994, in a NJLT near the Coddle Creek Reservoir. Two more females were collected from the same site, 1 each in August and September 1995.

One specimen was collected on July 19, 1994; subsequently, a female was collected, September 2001, in a CDCLT near a stream monitoring station in the community of Etowah.

Three adult females were collected in a CDCLT on August 20, 1994, on State Road 1007 near Cane Creek; 2 more females were collected September 1, 1995.

One specimen was collected on July 20, 1994.

Several specimens, both larval and adult, were collected August–October 1995 at the Catawba College Ecological Preserve. Larvae were collected from shaded ditches or pools under young trees; adults were collected in CDCLT traps in nearby sunny, open field habitat (Hartwig et al. 2018).

Two larvae were collected from a horse pasture pool near Tanglewood Park on October 26, 1995. This species was collected very rarely until a decade later in 2015, at the same site. Fewer than 20 individuals each were collected in 2015, 2016, 2017, and 2019 by coauthor (RLH).

Two larvae were collected on September 8, 1995: one from a stump hole and the other from a temporary pool.

Three specimens were collected in September 1996.

Three adult females were collected from CDCLTs at the Pee Dee National Wildlife Refuge: 1 in September (C. Apperson, personal communication; confirmed by BAH) and 1 each in October and November 1996.

Five specimens were collected in September 1996. The species has since become well established in this county and grown to be one of the predominant species collected (M. Hemmen and J. Suggs, personal communication).

One specimen was collected in September 1996.

Two adult females were collected from CDCLTs near North Potts Creek (August 28, 2002) and Swearing Creek (September 12, 2002).

One adult female was collected September 26, 2002, from a CDCLT in a wooded area of a residential neighborhood in the town of Old Fort.

Six adult females were collected from a residential area of Rocky Mount, September–October 2017 (R. Collins, personal communication; confirmed by BAH).

Multiple adult specimens were collected in the fall of 2017 (E. McRoy, personal communication; confirmed by BAH).

Collections were made and confirmed by two of the coauthors (BAH and PBW) in the following counties between 1997 and 2002: Bladen, Duplin, Jackson, Jones, Macon, Onslow, Richmond, Sampson, and Scotland. With the passing of coauthor BAH, the exact dates and site descriptions within these counties are unable to be determined; however, these records (and those listed above with limited collection information—Columbus, New Hanover, Pender, and Transylvania) were stated in a local newsletter (Harrison and Whitt 2017).

Collections of Cx. nigripalpus over the past few decades in North Carolina and into Virginia show this species expanding northward in the Mid-Atlantic region of the USA. The 1st specimen of Cx. nigripalpus in North Carolina was discovered September 1, 1944, in a resting station at Camp Sutton in Monroe, Union County (Carpenter et al. 1945). Since this 1st publication, the species has been found in a number of counties throughout the state and appears to be well established in the southeastern corner; in New Hanover County it has grown to be one of the predominant species collected (Day et al. 2020; M. Hemmen and J. Suggs, personal communication). However, with the exception of 5 out of the 28 counties in North Carolina reporting collections—New Hanover, Rowan, Union, Wake, and Wayne (Carpenter et al. 1945, McHugh et al. 1988, Hartwig et al. 2018, Day et al. 2020)—the majority of these findings have not been published until now. The remaining 23 counties are reported for the 1st time here, improving the documented knowledge of the distribution of Cx. nigripalpus in North Carolina. Furthermore, collections in Suffolk, VA, are now the most northeastern records found to date in the USA. A county-level map of Virginia and North Carolina displays these recent findings (Fig. 2).

The northernmost published records of Cx. nigripalpus as of this report are in the Midwest region of the USA in Clark, IL (Kim and Stone 2018), Cass County, MO (Claborn et al. 2018), and Fayette County, KY (Dye 2016). Over the last 15 years, these northern outermost collections along with many other new findings throughout the southeastern USA have warranted an update to the widely recognized Darsie and Ward (2005) US map of Cx. nigripalpus. Our updated map corrects errors as described below with the 2005 map and fills in the new US distribution lines based on available species reports (Fig. 3).

Beginning with the western edge of the current distribution, in their earlier work, Darsie and Ward (1981) identified areas of collection in Oklahoma and Arizona that were referenced but omitted from the most recent Darsie and Ward (2005) map display. We have reincorporated these locations, as their sources appear to be verified. Additional records in Oklahoma have also been added (Parsons 1965, Bradt 2017, Bradt et al. 2018). Texas collections include records referenced but not displayed in Darsie and Ward (2005); primarily of note: El Paso and Val Verde counties (Hill et al. 1958).

Continuing eastward, new reports in Arkansas (Sames et al. 2021; D. Theuret, personal communication) and along the northern edge of the new limit line in Missouri (Claborn et al. 2018) and Illinois (Kim and Stone 2018) have extended the documented distribution of this species hundreds of miles into the northern Midwest. In Kentucky, the map now reflects both historic and recent collections: Jefferson County (Covell 1968), Caldwell County (Minter et al. 2011), and Fayette County (Dye 2016). For Tennessee, we have entered historical (Middlekauff and Carpenter 1944) as well as new findings in Chester (Cohen et al. 2009), Knox (Haddow et al. 2009), Blount, Fayette, Hardeman (Fryxell et al. 2014), and Davidson (Briggs 2021) counties.

Reports of Cx. nigripalpus along the northern edges of Louisiana (McHugh et al. 1988, Sames et al. 2021), Georgia (GMCA 2018), and northwestern South Carolina (Carpenter et al. 1945) further expand on the shown distribution in Darsie and Ward (2005). Publications in North Carolina over the last 3 decades (McHugh et al. 1988, Hartwig et al. 2018, Day et al. 2020) as well as those reported here, along with new findings in Virginia, extend the northeastern edge of the distribution for this species well into the northern Mid-Atlantic region. An extensive review of reports of this species shows it documented in 201 jurisdictions in 16 states of the continental USA (Table 3 and Fig. 3B).

In order to create a natural limit line, some areas of the map (Fig. 3A) have been filled in although there are no current collections to reference; however, jurisdictions with reported collections are identified and listed (Fig. 3B and Table 3). This is especially important to note for the portions of the limit line running through New Mexico, Kansas, and Indiana. These states are not included as part of the 16 listed as there are no known reports in these states currently. However, due to neighboring locations that have collected Cx. nigripalpus, we posit that it is highly likely the species exists in the areas between.

Identifying Cx. nigripalpus adults may be challenging initially in new areas of discovery, especially when collected in traps where specimens encounter a fan and are susceptible to damage. However, Harrison et al. (2016) lists multiple characteristics that clearly separate Cx. nigripalpus from Cx. pipiens complex, Cx. restuans, and Cx. salinarius, provided personnel performing identifications do not rush and overlook some features that require close scrutiny. Particularly of note, all of the Virginia specimens appear similar to Cx. salinarius, with sternal scales that are a light copper color, which is in contrast to Florida specimens whose sternal scales are pale-scaled (Nayar 1982). Therefore, it would seem essential to use the character “mesepimeron without middle patch of white scales” (Harrison et al. 2016) as the defining character, at least for the more northern specimens. This variation in morphological characters combined with the similarity to other species in the Culex genus, particularly Cx. salinarius, and the propensity for damage in adult surveillance traps may be resulting in misidentification and thus underrepresenting the true geographical range of this species.

Culex nigripalpus larvae are reasonably easier to identify than their adult stage and seem to tolerate diverse environmental conditions, including varying levels of salinity, sunlight, and organic matter. They have been found in both permanent and transient pools of water, natural and artificial containers, catch basins, effluent ponds, freshwater swamp, brackish water, and even salt-marsh habitat (Edman 1974, Nayar 1982, Allan et al. 2005, Rey et al. 2006, Smith et al. 2016). Adults have been collected in a variety of trap types, most commonly NJLT and CDCLT, but also Encephalitis Vector Survey, bait, and gravid traps as well as resting–ground aspiration (Nayar 1982, UF 1997, Lord and Day 2000, Zyzak et al. 2002); most recently the BGS2 (this report) and BG-Counter (Day et al. 2020). This diversity of larval habitats and both host- and oviposition-seeking adult trap collections highlights the opportunistic nature of this species and provides some explanation for its continued ability to expand its range so successfully.

Dispersal (mark–recapture) studies for Cx. nigripalpus have shown a flight range up to 5 km (Dow 1971, Nayar 1982, Turell et al. 2005). This species tends to appear most abundantly in the late summer and fall months throughout its range in the USA and its seasonal emergence therein shows a strong correlation with humidity and rainfall; adults typically emerge within 5–7 days following a heavy rain. Ideal conditions for abundant Cx. nigripalpus populations include an initial heavy rain followed by 1–3 wk of drought, ending with a 2nd heavy rainfall and humidity levels at or above 90% (Nayar 1982, Day and Curtis 1994, Tabachnick 2016, Wright 2017). In 2017, precipitation readings in Suffolk, VA, displayed a similar pattern prior to the 1st collections of Cx. nigripalpus. More than 100 mm of rain was recorded in 1 wk followed by 4 wk of dry conditions, and specimens were collected 2 days after a 16-mm recorded rainfall. It is worth noting, in this 1st year of Cx. nigripalpus collections in Virginia, New Hanover County, NC, attained their largest collections of the same species. Culex nigripalpus populations exploded in the fall and this species became the top collected in the county. It is also interesting to note, this same year (2017) Beaufort and Nash counties in North Carolina collected this species for the 1st time; both counties are approximately 100 km from the Suffolk, VA, southern border. While not considered a migratory species, increasing global temperatures and severe storm systems with heavy precipitation in the fall months when Cx. nigripalpus is at peak emergence, may be contributing to its range expansion.

Additionally, with the large diversity of larval habitats this species has been collected from, particularly artificial containers, the possibility of anthropogenic introduction cannot be ignored, especially in scenarios when single adults are collected at widely separated locations, as in the Virginia collections. One specimen in particular, collected in Suffolk, VA, in May 2019 in an urban area raised this concern as all other previous and subsequent collections in the state were in mostly rural or suburban habitats in fall months. This species should be considered quite opportunistic and capable of furthering its range around warm winters similar to other species with recent range expansions such as Cx. coronator (Akaratovic and Kiser 2017) and Mansonia titillans (Walker) (Moulis et al. 2015, Cartner et al. 2018).

Culex nigripalpus is documented feeding on an extremely diverse selection of hosts. Avian preferences include primarily passerine and gallinaceous birds. However, those of the orders Charadriiform, Ciconiiform, Cuculiformes, Pelecaniformes, and Strigiform (Edman 1974, Nayar 1982, Cohen et al. 2009, Mackay et al. 2010) have also been recorded. More than 11 mammalian species have been identified (Nayar 1982, Mackay et al. 2010) and various reptiles and amphibians are also reported (Day and Curtis 1994, Cohen et al. 2009). Host preferences of Cx. nigripalpus seem to be contingent on season and availability. This species is evidently quite opportunistic and while primarily an ornithophilic feeder in the winter, spring, and even early summer, it easily transitions to feeding on both avian and mammalian hosts in the late summer and fall. Thus, in areas where Cx. nigripalpus is present year-round, it may serve as both an amplification vector for virus in the spring as well as an enzootic vector in the fall (Turell et al. 2005).

The danger associated with the vector competency of Cx. nigripalpus should not be underestimated, particularly with regard to St. Louis encephalitis virus (SLE), but also West Nile virus (WNV), eastern equine encephalitis virus, and turkey malaria (Chamberlain et al. 1964, Forrester et al. 1980, Nayar 1982, Day and Stark 1996, Rutledge et al. 2003, Day and Shaman 2008). Although this species does not appear to be a large nuisance to humans, at least in Florida where it is widespread, this species is regarded as the most important vector for SLE and has been responsible for multiple epidemics since the 1950s (Vitek et al. 2008). Additionally, field transmission of WNV to sentinel chickens—an industry standard for assessing human risk—by Cx. nigripalpus was documented during a local outbreak in northern Florida in 2001 (Rutledge et al. 2003). With the vast range of habitats in which Cx. nigripalpus has been found, combined with its opportunistic feeding behavior, vector competency, and expanding northward distribution over the last few decades, the importance of continued monitoring and arboviral testing (when possible in areas with established populations) cannot be overemphasized.

The authors thank the following for personal communication of their data in North Carolina: Charles Apperson of North Carolina State University, Jeff Brown and Rick Hickman of Brunswick County, Robert Collins and Neal Watson of Nash County, Marie Hemmen and Jeff Suggs of New Hanover County, and Eugene McRoy of Beaufort County. For personal communication of data from the Arkansas Department of Health, the authors thank David Theuret. The authors are grateful to William Sames, US Army (retired) of Leakey, TX, for personal communication of collections in Arkansas and providing access to historical military records. For personal communication of data in Davidson County, TN, the authors thank Cierra Briggs, Entomology Department at Cornell University and Abelardo Moncayo, Tennessee Department of Health. For assistance with Suffolk, VA, field collections, we thank Ashley Byers, Amber Rymer, Cassidy McKelvie, and Kaitlyn Price of SMC. For North Carolina field collections, we thank NH Newton of PHPM DENR and TJ Wood of Mecklenburg County Environmental Health. We appreciate the time and effort of Brian Byrd of Western Carolina University for searching through pinned specimens of the deceased coauthor (BAH) and of James Pecor at the Walter Reed Biosystematics Unit, Smithsonian Institution for examining historical records. The authors are grateful to Charles Abadam, Mosquito Control Superintendent and the Department of Public Works in the City of Suffolk, VA, for coordination and funding support of this publication.

Of greatest significance is the posthumous coauthorship of Bruce A. Harrison to whom we are indebted and eternally grateful. Bruce was instrumental in alerting the Virginia authors to the imminent expansion of this species, offered expert taxonomic verification, linked extensive contacts, provided many records from his state (North Carolina) spanning decades, and contributed significantly to the analysis and revision of the manuscript even within days of his passing.