The subgenus Melanoconion of Culex is a diverse and taxonomically challenging group of mosquitoes (Diptera: Culicidae) endemic to the American tropics, with a few species occurring in temperate regions. With the introduction and establishment of Culex (Melanoconion) panocossa in southern Florida, the existing taxonomic resources for identifying species of Melanoconion in Florida need updating. Here we provide an up-to-date photographic key for identifying females of Culex (Melanoconion) spp. known to occur in Florida, USA, and summarize relevant literature on the biology of each species. Given the challenge of identifying members of subgenus Melanoconion, updated resources for identifying females of this medically important group are warranted. The keys, photographs, and summarized biological information in this report should facilitate research, surveillance, and decisions regarding control.
Rozeboom and Komp (1950) stated that “Few, if any, groups of mosquitoes present greater taxonomic confusion and difficulty than does the subgenus Melanoconion.” Melanoconion Theobald is a diverse and poorly known Culex subgenus with species distributed throughout much of the Americas. Diversity of Melanoconion is greatest in the neotropics, but several species occur in temperate areas of North and South America. A total of 167 species of Culex (Melanoconion) are currently recognized as valid species (Sá et al. 2020), with a number of species awaiting formal description (Hutchings et al. 2016). In the USA, diversity of Melanoconion is greatest in Florida, where 8 species are currently reported to occur (Table 1). Members of the subgenus are considered difficult to identify, especially as adult females, for which many species are morphologically similar, and descriptions are not available for all species (Torres-Gutierrez et al. 2018). For these reasons, species identification based on morphological features of adult females is problematic for operational mosquito control districts and researchers.
The subgenus Melanoconion is divided into 2 sections, the Spissipes Section and the Melanoconion Section, which differ substantially in their diversity and medical importance (Sá et al. 2020). The Spissipes Section is relatively small, containing just 23 species (Sallum and Forattini 1996, Sallum et al. 1997), but many members are important vectors of zoonotic Alphaviruses (Torres-Gutierrez and Sallum 2015), particularly Venezuelan equine encephalitis virus (VEEV) and Madariaga virus (formerly considered as South American strains of eastern equine encephalitis virus). Due to their importance in transmission of VEEV, host associations of a number of Spissipes Section members have been studied in some detail, and several species feed heavily upon rodents, the vertebrate hosts of VEEV (Davies 1978, Edman 1979, Christensen et al. 1996). The Melanoconion Section is more diverse, with 144 recognized species classified in 13 groups and 20 subgroups (Sirivanakarn 1983). Host associations of relatively few species of the Melanoconion Section are well documented, although many species feed mainly upon reptiles (Tempelis and Galindo 1975, Christensen et al. 1996, Burkett-Cadena et al. 2008, Reeves et al. 2019). A few species within the Melanoconion Section are generalists and feed upon birds, mammals, reptiles, and amphibians (Bingham et al. 2014, Blosser et al. 2016).
Considering the recent introduction and establishment of Culex (Melanoconion) panocossa Dyar in southern Florida (Blosser and Burkett-Cadena 2017), the existing taxonomic resources for identifying species of Melanoconion in Florida (Carpenter and LaCasse 1955, Knight and Haeger 1971, Darsie and Morris 2003, Darsie and Ward 2005) need revision. Here we provide an up-to-date photographic key for identifying females of Culex (Melanoconion) spp. known to occur in Florida, USA.
MATERIALS AND METHODS
Morphological keys were developed using conserved characters that are visible at magnification typical of standard stereomicroscopes (45×). The figures of this illustrated key consist of high-resolution focus-stacked color images of fresh, largely intact specimens that capture subtle variation in integumental shading that is typically used to identify females in this subgenus. We include additional characters in key couplets, particularly scale patterns of the dorsum of the head, features which are diverse within this group and useful for recognizing species in this challenging subgenus (Bangher 2020). Characters were combined from several sources to produce the key, including Carpenter and LaCasse (1955), Belkin et al. (1970), Knight and Haeger (1971), Sirivanakarn (1983), and Darsie and Ward (2005).
Images illustrating characters in this key were captured using a digital SLR camera (Canon 5DS R or Canon 6D; Canon USA, Melville, NY) with either a macro lens (Canon MP-E 65mm or 100mm L lens; Canon USA) and macro twin flash (Canon MT-24 EX [Canon USA] or Kuangren Macro Twin Lite KX-800 [Venus Optics, Hefei, China]), or a Canon 200mm L telephoto lens (Canon USA), with a 10× microscope objective (10× Mitutoyo Plan Apo Infinity-Corrected Objective; Edmund Optics, Barrington, NJ) mounted on the front. Sets of images for focus stacking were created using manual focusing or an automated focusing rail (Stackshot 3x; Cognisys Inc., Traverse City, MI). The distance between each step at which a photo was taken varied from 5 μm to 150 μm. Prior to photographing, adult specimens were affixed to standard glass microscope slides with human saliva, then positioned with standard entomological pins or minuten pins held by a pin vise. After a brief drying period (1–5 min), glass slides were held on a stage with small binder clips. Shallow depth of field images (aperture 4.0–5.6) were taken on manual settings (ISO = 100, shutter speed = 1/200 sec). The in-focus areas of the multiple resulting images, typically 15–25 (macro lenses) or 100–200 (10× microscope objective), were merged into a single composite image using image-stacking software (ZereneStacker [Zerene Systems, Richland, WA] or Helicon Focus [Helicon Soft Ltd., Kharkiv, Ukraine]). The images were cleaned and cropped in Adobe Photoshop version 21.0.2 (Adobe, San Jose, CA).
Key to the adult females of species of subgenus Melanoconion
Mesepimeron with large median patch of broad, pale scales (Fig. 2a) Cx. erraticus
Mesepimeron without scales, or with small narrow scales (Fig. 2b) 3
Pleural integument mostly pale yellowish, sharply contrasting with brown integument of scutum and dark brown spots on postpronotum, postspiracular area, and prealar knob (Fig. 3a) Cx. panocossa
Pleural integument and scutum brown (Fig. 3b) 4
Anterior median area of vertex with narrow decumbent scales, broad scales restricted to lateral portion of vertex (Fig. 4a); median mesepimeron integument distinctly pale with dark integument above and below (Fig. 4c) 5
Anterior median area of vertex of head mostly covered in broad, decumbent scales (Fig. 4b); mesepimeron without above distinct pattern of dark and pale integument, although large portions of the mesepimeron may have pale integumental areas (Fig. 4d) 6
Lateral patch of broad spatulate scales large, well evident in dorsal view of head (Fig. 5a); pale integumental area of mesepimeron not rounded posteriorly; mesomeron and lower mesokatepisternum pale (Fig. 5c); tarsi dark-scaled (Fig. 5e) Cx. atratus
Lateral patch of broad spatulate scales small, almost indistinct in dorsal view of head (Fig. 5b); pale integumental area of mesepimeron rounded posteriorly; mesomeron and lower mesokatepisternum not pale (Fig. 5d); hind tarsomeres with indistinct (Florida peninsula) or distinct (Lower Florida Keys) basal and apical bands of pale scales (Fig. 5f) Cx. cedecei
Thoracic pleural integument dark brown to black; mesepimeron with 1–11 small, scattered hairlike scales (unapparent, and best seen when illuminated from the side) (Fig. 7a); broad decumbent scales of head dark brown, often with bluish reflection (Fig. 7c) Cx. iolambdis
Thoracic pleural integument medium brown; mesepimeron with light area covering upper two-thirds (Fig. 8a); conspicuous patch of pale scales along posterior border of lower mesokatepisternum (Fig. 8a); broad decumbent scales of vertex pale gray or white (Fig. 8c) Cx. pilosus
Thoracic pleural integument light brown or reddish-brown; mesepimeron with light integumental area confined to rather narrow, pale border (Fig. 8b); lower mesokatepisternum with at most a few pale scales at posterior border (Fig. 8b); broad decumbent scales of vertex bluish-white (Fig. 8d) Cx. mulrennani
Florida has a greater diversity of Culex subgenus Melanoconion species than any other US state (8 nominal species). Within the state, the greatest diversity occurs in the southern third of the peninsula and the Florida Keys (Darsie and Morris 2003). Only 3 species (Cx. erraticus Dyar and Knab, Cx. peccator Dyar and Knab, and Cx. pilosus Dyar and Knab) occur in the panhandle and northern peninsula. The recognized northern limits of the geographic distribution of other Melanoconion spp. are rather ambiguous, in part because of challenges in morphological identification.
The species of Melanoconion that occur in Florida are divided among the taxonomic sections and groups of the subgenus, such that no 2 species occur in the same taxonomic group. In the Melanoconion Section, Cx. atratus Theobald belongs to the Atratus Group, Cx. erraticus to the Erraticus Group, Cx. iolambdis Dyar to the Bastagarius Group, Culex mulrennani Basham to the Inhibitator Group, Culex peccator to the Peccator Group, and Cx. pilosus to the Pilosus Group. In the Spissipes Section, Cx. cedecei Stone and Hair belongs to the Taeniopus Group, and Cx. panocossa to the Ocossa Group (Sirivanakarn 1983, Sallum and Forattini 1996). The diversity of groups in Florida facilitates morphological identification of species in this subgenus, which is exceedingly difficult for members within a group, based upon external morphology of the female (Sá et al. 2020). The distribution of Florida Melanoconion species among separate groups also aids in differentiating females by dissection of the cibarial armature (Williams and Savage 2009), which is often not possible within groups (Sirivanakarn 1983). When morphological identifications are not confident, or indicative of range expansion of a species, confirmation of species identifications should be performed using male terminalia (Carpenter and LaCasse 1955) or by molecular verification of DNA sequences (e.g., Williams and Savage 2011).
Variation within individuals or populations of a species can have significant consequences for developing accurate keys for morphological identification. Culex cedecei, for example, is often described as having narrow basal and apical white rings on hind tarsal segments 1 to 4 and segment 5 which is almost entirely white (Carpenter and LaCasse 1955, Darsie and Ward 2005); however, individuals from peninsular Florida rarely have recognizable pale-scaled bands on the hind tarsomeres. Specimens from the Lower Florida Keys (Boca Chica and Geiger Keys) typically have conspicuous tarsal bands (Fig. 5-f). This geographical variation in phenotypic form invariably leads to misidentification and underreporting. The keys provided here use characters that are geographically conserved among the Melanoconion spp. in Florida.
In Florida, “Cx. atratus” represents at least 2 cryptic species (Williams and Savage 2009, 2011), which are currently inseparable based on external morphology, but can be distinguished by the cibarial armature and molecular assays (Williams and Savage 2011). Only 1 of the 2 cryptic species is formally described (Williams and Savage 2009, 2011). Fourteen species of the Atratus Group are currently recognized (Sá et al. 2020). Interestingly, Sá et al. (2020) list, among other characters, that the mesepimeron of Cx. atratus s.s. is “entirely dark, without median pale area,” which is inconsistent with characters listed for this species in published keys that include the Florida Melanoconion species (Knight and Haeger 1971, Darsie and Morris 2003, Darsie and Ward 2005). Culex atratus s.s. was described by Theobald (1901) based on males and females from Jamaica. It is not currently known whether Cx. atratus B of Williams and Savage (2009, 2011) represents an undescribed species or a tropical member of the group that has expanded into Florida, perhaps from the Caribbean. According to Darsie and Ward (2005) and Darsie and Morris (2003), Cx. atratus lacks a patch of pale scales on the upper mesokatepisternum; however, we have observed considerable variation in this character and therefore not used it in this key. Culex atratus s.l., in Florida, are known to feed predominantly from reptiles, particularly Anolis spp. lizards (Reeves et al. 2019).
Culex cedecei has a complex taxonomic history and has been at times considered a synonym of Cx. opisthopus Komp and Cx. taeniopus Dyar and Knab, the Central American vector of VEEV. Knight and Haeger (1971) indicate that tarsi are “sometimes pale-ringed,” which perpetuates the challenge of morphological identification of this species, likely causing the distribution of the species to be underestimated. Keys by Darsie and Ward (2005) and Darsie and Morris (2003), which are used extensively by operational mosquito control districts in Florida, rely upon presence of pale-scaled bands on hind tarsomeres as definitive characters for this species. However, in their description Stone and Hair (1968) state that “most specimens of cedecei have the tarsomeres entirely dark or have only a trace of white flecks at the joints and only rarely is the entire fifth hind tarsomere pale.” The lack of pale bands in peninsular forms of this species has undoubtedly led to misidentification of Cx. cedecei in many areas of Florida where it occurs. Narrow decumbent scales over the majority of the vertex, and pale integumental spot with a distinctly rounded posterior edge on the mesepimeron, are a reliable combination of characters for this species.
Culex erraticus is widely recognized and occurs practically statewide (Smith et al. 2020, Riles and Connelly 2020). This common species is considered a vector of eastern equine encephalitis virus (Cupp et al. 2003, Bingham et al. 2016) and West Nile virus in the southern USA (Bolling et al. 2005, Cupp et al. 2007, Unlu et al. 2010). Adults can be quite variable in color and size, yet the large patch of white scales of the mesepimeron is a dependable character that is present in no other Florida Melanoconion species. Many species of the subgenus Culex have a similar patch of pale scales on the mesepimeron. Culex erraticus can be differentiated from species of subgenus Culex by the direction of the scales. In Cx. erraticus the scales are directed upward, whereas in subgenus Culex the scales are directed downward. Larvae are typically found in permanent freshwater wetlands with emergent vegetation (Burkett-Cadena 2013), and adult females are rather generalist in their host associations (Burkett-Cadena et al. 2008, Bingham et al. 2014).
Culex iolambdis is found mainly in coastal regions of southern Florida and is associated with mangrove habitats (Blosser et al. 2016). This species has been implicated in transmission of VEEV in Mexico (Scherer et al. 1964). Adults are typically quite small and darkly pigmented, with a nearly black, often shiny thoracic integument (Knight and Haeger 1971). The key character of adult female Cx. iolambdis is the 1–11 very small hairlike scales scattered about the central mesepimeron. These scales are best observed with incident light (from very low angle). Larvae are typically collected in association with the exposed roots of black mangroves (Avicennia germinansL.; Carpenter and LaCasse 1955), and adult females have relatively broad host associations, feeding from all terrestrial vertebrate classes (Blosser et al. 2016).
In the continental USA, Cx. mulrennani is known only from the Florida Keys and Florida Everglades. Adults are quite small. The pleuron has a light brown or reddish-brown coloration that is not shared by other Melanoconion that occur in southernmost Florida. The key character of pale border of the mesepimeron is not apparent in all specimens. Larvae are found in water deep in limestone solution holes (Basham 1948), and adults rest on the walls of these habitats (Reeves and Burkett-Cadena, unpublished observations). The species may be associated with the endangered pine rockland habitats of extreme southern Florida. Nothing is known about its host associations.
Culex panocossa, formerly known as Cx. aikenii (Aiken and Rowland), is found in freshwater wetlands of the southernmost Florida peninsula. Adults are recognizable by the pale yellowish coloration of the pleuron that contrasts with the darker brown integument of the scutum (Fig. 3a). The dorsal third of the pleuron has 2 small dark integumental spots on the postspiracular area and prealar knob (Fig. 3a). Although not used as a key character here, it should be noted that the vertex of the head has a median patch of narrow decumbent scales, consistent with other members of the Spissipes Section (Sallum and Forattini 1996). Larvae of Cx. panocossa are associated with water lettuce (Pistia stratiotes L.). Adult females are readily captured by carbon dioxide–baited light traps, feed mainly upon birds and mammals, and are confirmed vectors of VEEV in Panama (Galindo and Grayson 1971).
Culex peccator is considered to occur statewide, but is uncommon in the southern half of the state. This mosquito can be abundant in the freshwater swamps of the panhandle and northern peninsula, and feeds upon reptiles, amphibians, birds, and to a lesser extent mammals (Irby and Apperson 1988, Burkett-Cadena et al. 2008). Females are not easily recognizable, and generally lack conspicuous characters. The abdominal sternites are mostly pale and the mesepimeron has a rather large patch of broad pale scales (Burkett-Cadena 2013). Males, which are readily aspirated from their resting sites (hollow trees, undercut stream banks, animal burrows), have large conspicuous gonocoxites (Burkett-Cadena 2013). The presence of males in a sample may indicate the presence of conspecific females. Larvae are typically found in sluggish intermittent streams and creeks.
Culex pilosus is also found statewide. Larvae of Cx. pilosus inhabit floodwater pools that occur in virtually any habitat. Adults are rarely collected in light traps. Vertebrate hosts of adult females are lizards and other reptiles (Reeves et al. 2019). Identification of adult females of Cx. pilosus is challenging, as relatively few recognizable characters are available. The vertex of the head has pale decumbent scales, and the posterior border of the mesokatepisternum has a large patch of pale scales (Knight and Haeger 1971). The eyes of live adults are green. Knight and Haeger (1971) indicate that the light area of the mesepimeron covers the upper two-thirds of the sclerite; however, this character is not always evident. Identification of this species is reliably confirmed by the unique form of the cibarial armature (Williams and Savage 2009). Larvae and adult males of this species are easily distinguished from other species of Melanoconion in Florida, using characters of the larval siphon (Foote 1954) and male genitalia (Foote 1954).
Given the challenge of identifying members of subgenus Melanoconion, updated resources for identifying females of this medically important group are needed. The keys, photographs, and summarized biological information in this report is intended to facilitate research, surveillance, and decisions regarding control of Melanoconion in Florida.
The authors thank Carrie Cerminara and Lawrence Hribar for sharing locations where Cx. mulrennani were collected on Big Pine Key, and information regarding Cx. cedecei specimens from the Lower Florida Keys. Kristin Sloyer collected specimens of Cx. cedecei and Cx. panocossa. This study was supported by NIFA project FLA-FME-006106.