Introduction

With human-induced environmental change accelerating, current needs for biodiversity conservation far outweigh the available resources. Biodiversity conservation is reliant on up-to-date information on the abundance, diversity, and distribution of species to guide the implementation and deployment of conservation actions and resources (e.g., the purchasing of land and establishment of protected areas, drafting of legislation, development of species management plants, etc.)1. This critical information is generated by field surveys or biological monitoring, which requires considerable investment of resources. Vertebrates are often considered flagship, umbrella, keystone, or indicator species2,3, and, as such, these species or communities are the targets of field surveys and monitoring. Traditional methods of vertebrate survey and detection are effective, but tend to be time-consuming, expensive, and require extensive field and taxonomic expertise. Given these limitations, it is increasingly important to develop efficient and innovative ways to improve biodiversity survey and detection methods that leverage modern technologies in this critical era of biodiversity loss4.

Advances in molecular ecology and DNA sequencing technology have facilitated the implementation of environmentally derived DNA (eDNA) as a means of detecting vertebrates within complex environments by extracting DNA from water, air, soil, or other environmental samples such as spiderwebs or bulk arthropod samples5,6,7,8,9. Invertebrate-derived DNA (iDNA), i.e., DNA collected from invertebrates that feed on vertebrate blood, tissue, or excrement, builds on the eDNA approach and shows potential for use in biomonitoring applications10 and may enhance efficiency and cost-effectiveness in comparison to conventional surveys (e.g., direct observation, camera traps, drift fence trapping, etc.11,12). Invertebrate-derived DNA may enable researchers to glean insights that would be impossible through other sources of eDNA (e.g., water, air), though iDNA may be logistically more challenging to collect. Often, iDNA from an arthropod represents a noninvasive blood sample derived from an individual vertebrate animal. This nuance makes iDNA useful for tracking disease in vertebrate populations (i.e., xenosurveillance13) or, as a genetic sample that could be useful to conservation genetics, for example, in recognizing individuals or estimating abundance of vertebrate populations14. While other invertebrates (e.g., leeches, carrion flies) have been comparatively well-studied as sources of vertebrate DNA in iDNA-based biomonitoring, relatively few studies have assessed the potential of mosquitoes to detect vertebrate animals10,11,15,16. Their widespread geographic distribution, high abundance (in many terrestrial ecosystems), use of diverse vertebrate hosts, rapid blood meal digestion, and limited post-feeding flight ability make mosquitoes viable candidates for use in iDNA surveys of terrestrial vertebrates17,18,19. Further, mosquito host associations and molecular techniques for mosquito host identification have been studied for more than a century, albeit from a largely epidemiological context20. Despite these factors, mosquitoes have received comparatively little attention as candidate sources of iDNA and methodologies for conducting such surveys have not been fully explored.

Several recent studies investigated the potential of mosquito blood meals as iDNA for vertebrate detection in natural ecosystems. Kocher et al.21 collected hematophagous dipterans (sandflies and mosquitoes) to use as vertebrate samplers in the northern Amazon and concluded that mosquito blood meal analysis could be a helpful tool in biodiversity surveys but needed further investigation. Massey et al.22 collected carrion flies, sandflies, and mosquitoes in Mato Grosso, Brazil across urban and semi-urban habitats and compared the detection of terrestrial vertebrates from iDNA to detections from camera traps. Camera traps detected the highest species richness but were found to be biased towards carnivores and ungulates while the iDNA detections were more varied and included many vertebrate species and taxa (e.g., birds, bats, reptiles, amphibians) that were undetected by camera traps. Mosquitoes detected fewer hosts than other iDNA taxa, and were determined to be biased toward humans22. Danabalan et al.23 compared blood-fed mosquitoes, non-blood-fed mosquitoes and flies (Sarcophagidae, Calliphoridae, Muscidae) as sources of iDNA for detecting mammal diversity in Germany and found non-mosquito flies detected more mammals than mosquitoes, but the mosquito sampling methods (sweep netting) collected few blood-fed females and only 58 total blood meals were analyzed. Vieira et al.24 used 480 mosquito blood meals to detect host diversity and to screen for serological evidence of exposure to Ross River virus. Of these, 346 were identified representing 26 species, but this sample was dominated by human (73%) and cattle (9%) detections, likely reflecting the land-use of the study location, Brisbane, Australia. Additional research involving relatively small samples sizes concluded mosquitoes had potential as sources of iDNA25,26. Together, these studies suggest that mosquitoes can be useful sources of iDNA, albeit with limitations, and that methodologies for collecting and analyzing mosquito iDNA should be optimized.

Among the previous work exploring mosquitoes as a source of iDNA, only Danabalan et al.23 consistently identified collected mosquitoes to species. Host association is an important and idiosyncratic element of the biology of a mosquito species that is relevant to iDNA-based vertebrate detection using mosquitoes. Mosquitoes feed on all terrestrial vertebrate classes: Amphibia, Aves, Mammalia, Reptilia, and some species feed on fish and invertebrate annelids27,28. Among mosquito species, host associations are variable and understanding which mosquito species are associated with which vertebrate groups may be beneficial to interpreting the results of mosquito-based iDNA surveys or to designing and implementing surveys that target particular vertebrate groups or species. Mosquito host associations are nuanced, and go beyond simple classification of species as generalists or specialists. While a few mosquito species are relative generalists (i.e., species that feed on all terrestrial vertebrate classes without a strong association with particular classes), most specialize, to varying extents, on particular subsets of the vertebrate community within their ecosystems. Even among mosquito species that specialize on a particular vertebrate class, there may be especially strong associations with taxa within that class. For example, Culiseta melanura is strongly associated with avian hosts, especially passerine birds, while Culex erraticus, a relative generalist, often feeds from birds, but among birds, is strongly associated with wading birds29,30. Likewise, Culex cedecei feeds predominantly from mammals, but has a strong association with rodents31. Such nuanced host associations would be expected to make individual mosquito species more or less useful to a mosquito-based iDNA survey for vertebrates, depending on the objectives of the surveys (e.g., characterizing the vertebrate diversity of a site, detecting a particular target taxon of conservation or management importance). Thus, recognizing the mosquito species that have the greatest return on investment may be in important step in optimizing the utility and value of a mosquito-based iDNA survey, along with selecting appropriate sampling methods that maximize collection of the most useful species.

To better understand the feasibility of mosquito blood meal-based iDNA surveys of vertebrate diversity, we investigated mosquitoes as a means of detecting terrestrial vertebrate species at the DeLuca Preserve, in Osceola County, Florida and among mosquitoes, we assessed variation in the range of vertebrate hosts detected by individual mosquito species to explore how mosquitoes can be most effectively used in surveys for vertebrate animals. Mosquitoes were collected using methods expected to best target blood-fed female mosquitoes. Collected blood-engorged females were identified to species, and blood meals were preserved and screened for vertebrate DNA using DNA barcoding. The objectives of this work were: (1) to characterize the host associations of mosquito species and range of vertebrate animals detected by mosquito blood meal iDNA sampling at the DeLuca Preserve; and (2) to evaluate the utility of individual mosquito species for iDNA sampling based on the vertebrate “communities” detected by each mosquito species, diversity metrics (species richness, Shannon-Index, Gini-Simpson Index, estimated species richness, and sample completeness) of each community, and a measure of host detection efficiency (i.e., the number of vertebrate host species detected by mosquito species relative to the number of blood meals that were analyzed for that species).

Materials and methods

Study location

The study took place at the DeLuca Preserve, a ~ 10,900 ha conservation area maintained by the University of Florida located in Osceola County, Florida, USA. Natural and modified habitats at the DeLuca Preserve consisted of forests, wetlands, Florida scrub, citrus groves and pastureland. This land provides habitat for native and imperiled Florida wildlife such as the Florida panther (Puma concolor coryi), grasshopper sparrow (Ammodramus savannarum floridanus), red-cockaded woodpecker (Picoides borealis), and gopher tortoise (Gopherus polyphemus32). Additionally, the preserve was within a conservation focus area for the Everglades Headwaters National Wildlife Refuge and Conservation Area, situated between the Kissimmee Prairie Preserve and the Three Lakes Wildlife Management Area33.

Sampling sites

Within the preserve, mosquitoes were collected at sites in scrub, citrus grove, wetland, and forest habitats. Eight sampling sites were selected (Fig. 1), two in each of the habitat types, and mosquitoes were repeatedly sampled at each over an eight-month period. The forest areas sampled at the DeLuca Preserve were hardwood hammocks or mixed forest within mesic flatwood habitat. We sampled mosquitoes in a hardwood and sabal palmetto hammock and in a closed-canopy mixed woodland dominated by bald cypress, hardwoods, and pine, both embedded within a larger swath of mesic flatwoods. Scrub sites consisted of Florida scrub, a habitat found on dry, sandy ridges in Florida. The study habitat we classified as wetland consisted of a type of non-forested wetland habitat, depression marsh, a shallow, rounded, depression area that has sandy soil and herbaceous plants that grow in concentric bands, with the outermost bands being the driest, and the innermost being the wettest34. Citrus grove was the only human-modified habitat that was surveyed at the DeLuca Preserve. The groves consisted of citrus trees planted in rows on raised beds, often with tall grasses and other herbaceous vegetation growing in furrows between the rows, where water often pooled after rainfall35.

Fig. 1

Map of the eastern side of the DeLuca Preserve, Osceola County, Florida, USA, indicating the location of field sites where mosquitoes were collected (red squares). Blood-fed mosquitoes were collected from a total of eight sites, which represented four habitats, mesic flatwood forest, citrus grove, scrub, and depression marsh wetland. Two sites were located in each habitat type. At each site, one ten-minute aspiration was performed on each sampling day (n = 45) and five to seven resting shelters were deployed. Hashed black lines indicate the borders of the DeLuca Preserve. Inset (top right) indicates the location of the DeLuca Preserve in the state of Florida. Map created in ArcGIS Pro using the Statewide Land Use Land Cover shapefile hosted by the Florida Department of Environmental Protection.

Mosquito collection

Mosquitoes were collected in 2022 from January through August, across three seasons: winter (with collections taking place in January and February), spring (March, April, and May), and summer (June, July, August), to account for potential seasonal variation in mosquito abundance36, host association37,38,39 and vertebrate behavior (e.g., migration, seasonal activity, above ground activity). Mosquitoes were collected over nine sampling periods, three during each season. Each sampling period consisted of five consecutive days of sampling. Sampling took place in the morning to early afternoon (~ 09:00–13:00). The total number of field days of mosquito collection across the eight-month sampling period was 45, each with a duration of approximately four hours.

In order to maximize the number of collected mosquitoes and proportion of blood-engorged female mosquitoes collected, sampling efforts within each site focused on areas where mosquitoes were likely to rest after taking a blood meal from a host40,41. Two sampling methods were used to collect mosquitoes each day: resting shelters and a large-diameter aspirator. At each site, five to seven resting shelters were dispersed on the ground in dark areas shaded from morning sun exposure. The shelters were left in place throughout the duration of the study period. Mosquitoes were collected from each shelter daily during each sampling period. The shelters consisted of either a thick black trash bag fitted over a collapsible wire spiral frame or a similar commercially purchased collapsible black yard waste container. To collect mosquitoes from the shelters, a lid holding a collection cup was placed over the shelter entrance and the shelter was compressed several times, forcing the air inside the shelter, along with any mosquitoes, into the collection cup. Both construction of and collection from the shelters followed the methods described by Burkett-Cadena et al.42. The aspirator was created using an automotive radiator fan attached to a cylindrical wire field fencing frame covered in polyester tarp, based upon a model initially described by Nasci43 and modernized as described in Sloyer et al.44. The aspirator was powered by a 12-V rechargeable battery, and a mesh mosquito head net was used as a collection bag, attached to the inside of the aspirator. For each sampling day, each of the eight sites was aspirated once for approximately ten minutes. Aspirations at each site consisted of moving the running aspirator over likely mosquito resting microhabitats: leaf litter or undergrowth, buttressed tree trunks, recently disturbed areas of the ground, and herbaceous vegetation, depending on the habitat type. All collected mosquitoes were knocked down with carbon dioxide (dry ice), transferred into labeled vials, and transported in a cooler with dry ice to the Florida Medical Entomology Laboratory.

Mosquito identification and blood meal preservation

Field-collected mosquitoes were kept in a − 20 °C or − 80 °C freezer until processing. Mosquito specimens were examined under a stereoscope, separated by species, and counted. Species were morphologically identified using a dichotomous key to the mosquitoes of North America45,46. All blood-engorged females were separated from all other mosquitoes, counted, and given an identifying number. To preserve the host DNA, blood-engorged females were individually placed on Whatman Flinders Technology Associates (FTA) Classic Cards, and a sterile pipette tip was used to roll out the blood meal onto the card47,48. The blood meal was characterized on a scale of one to three based on the level of digestion48, either as blood-fed (BF) 1 (fresh, in early stages of digestion, and red in color), BF2 (partially digested, brown with some red coloration persisting), or BF3 (mostly digested and entirely brown or black in color). Cards with preserved blood meals were stored at room temperature.

DNA barcoding and sequencing

From each preserved blood meal, two 1 mm punches were taken from the associated Whatman FTA Card. Using the Hot Sodium Hydroxide and Tris (HotSHOT) method49,50, the DNA was extracted, then amplified by polymerase chain reaction (PCR) with primers targeting a fragment of the cytochrome c oxidase subunit I (COI) gene of vertebrates51,52. Three primer combinations were used in the hierarchical approach described in Reeves & Burkett-Cadena52: VertCOI_7194 (5′–CGM ATR AAY AAY ATR AGC TTC TGA Y–3′) + Mod_RepCOI_R (5′–TTC DGG RTG NCC RAA RAA TCA–3′), Mod_RepCOI_F (5′–TNT TYT CMA CYA ACC ACA AAG A–3′) + VertCOI_7216_R (5′–CAR AAG CTY ATG TTR TTY ATD CG–3′), and VertCOI_10096_F (5′–CHC AAT ACC AAA CNC CHY TNT TYG–3′) + Mod_RepCOI_R. The PCR products were visualized on an agarose gel and amplicons displaying a band of the expected size were sent to Eurofins Genomics (Louisville, KY) for chain termination sequencing53. The returned DNA sequence files were edited using Geneious Prime 2020.1.2 and submitted to the Barcode of Life Data System (BOLD) Version 4 Identification Engine for identification by comparison to reference sequences54. At the time of analysis, reference COI sequences were not available for Sylvilagus palustris (marsh rabbit) in publicly accessible sequence databases, and there appears to be geographic variation in Anolis carolinensis (green anole) COI sequences51. Thus, Sequences that were close matches (> 85% similarity) to Sylvilagus spp. (cottontail rabbits) and Anolis carolinensis (> 95% similarity) were compared to independently collected COI sequences from those species, derived from locally collected specimens51. Ten high-quality sequences were poor matches (94–97% similarity) to Setophaga and Geothlypis warblers. Because it was unclear what species these represented and how many species were represented, these ten sequences were excluded from the dataset of identified hosts, and those blood meals were considered unidentified. One high-quality sequence was a poor (94%) match to several passerine species that do not occur in Florida. Because this sequence was assumed to be derived from a passerine species not yet present in the BOLD database, and was distinct from all other blood meal sequences, it was included in the dataset as “Unidentified Passeriformes.” In two cases, COI sequences could not distinguish pairs of closely related species. Odocoileus virginianus (white-tailed deer) and Odocoileus hemionus (mule deer) have identical COI sequences and could not be separated. Because Odocoileus hemionus is not native to Florida and not known to occur in the wild, all Odocoileus sequences were attributed to Odocoileus virginianus. Similarly, Ardea herodias (great blue heron) and Ardea cocoi (cocoi heron) could not be separated, but all sequences matching this species pair were attributed to Ardea herodias based on geography.

Statistical analysis

All statistical analyses were run on R Version 4.3.155. We characterized mosquito host associations by compiling all host species detected by each mosquito species and by mosquitoes overall. Each assemblage of vertebrate species detected by a mosquito species was treated as a biological community. For each mosquito species and for all mosquito species combined, we constructed sample completeness profiles using the inext.4steps package56 to determine the upper bound of the proportion of total species in the assemblage (“assemblage” here referring to the range of vertebrate species the mosquito species feeds upon at the DeLuca Preserve) that were detected by the mosquito species, the proportion of individuals in the assemblage that are belong to detected vertebrate species, and the proportion of highly abundant species that were detected. To assess the diversity of the host communities detected by each mosquito species and by mosquitoes overall, vertebrate species count data were analyzed using the iNEXT package57. For each community, diversity metrics were calculated including species richness, Shannon index, Gini-Simpson index, and estimated species richness based on accumulation curves58. Accumulation curves were created using the iNEXT package in R for mosquito species with ten or more identified blood meals and for mosquitoes overall57. The curves were created to enable a robust comparison of the richness detected by each mosquito species based on asymptotic estimates of diversity. Accumulation curves were similarly created to assess the completeness of sampling (all mosquitoes species) of each vertebrate class individually to identify vertebrate classes that were well or poorly detected by the sampling effort.

Because mosquito host associations vary by species, we developed the metric “host detection efficiency” to evaluate and compare the ability of each mosquito species in our sample to contribute toward vertebrate diversity detection in our iDNA-based survey. Host detection efficiency was calculated as the square of host species richness detected by the mosquito species divided by the total number of blood meals analyzed for that species. The host detection efficiency represents a measure of the number of vertebrate host species detected by a mosquito species relative to the number of blood meals that were analyzed for the species. These values provide an indication of the return on investment for each mosquito species. Some mosquito species are narrowly associated with a small range of host species, and thus, may not be worth the expense and effort of including in a vertebrate diversity survey if the detection of as many vertebrate species as possible is the goal. Such species are expected to have host detection efficiency values closer to zero. Other mosquito species feed on a wider range of vertebrate species and are thus more useful to the goal of broad species detection than others. The larger the host detection efficiency value, the more efficient a mosquito species is at detecting the largest range of vertebrate species.

Results

A total of 54,637 mosquitoes were collected throughout the duration of the study, 3,508 (6.4%) of which were blood-fed. Of the blood-fed females, 2,051 (58.4%) resulted in species level vertebrate host identifications. For blood meals with digestion extent estimated as BF1 (n = 1,334), 90.0% resulted in positive identification of a vertebrate host species. For blood meals estimated as BF2 (n = 1,011), 64.8% resulted in positive host identifications, and for those categorized as BF3 (n = 1,163), 19.9% of blood meals resulted in a positive host identification.

Blood meals were collected from 21 mosquito species (Table 1) and included species from eight genera: Aedes, Anopheles, Coquillettidia, Culex, Culiseta, Mansonia, Psorophora, and Uranotaenia. Blood-fed Uranotaenia sapphirina were collected but excluded from analysis because this species is a specialist of annelid hosts in Florida28. Identifications for blood-fed females that were initially identified as Aedes atlanticus based on morphology and distribution45 were revised to Aedes atlanticus/tormentor because DNA barcoding analysis59 of specimens collected at the DeLuca Preserve for a separate project indicated that both morphologically similar species were present at the preserve (Table 3). Culex nigripalpus was the most abundant mosquito species in the blood meal sample, and was the species with the largest number (n = 1,024) of vertebrate host detections, representing 49.9% of all vertebrate detections from mosquito blood meals. Aedes infirmatus produced the second largest number of vertebrate detections, with 267 vertebrate detections (14.3%), followed by Culex pilosus with 262 detections (14.1%).

Host species representing four classes of terrestrial vertebrates were detected in mosquitoes collected at the DeLuca Preserve (Table 1). From the 2,051 total detections, 3.0% were amphibians (n = 61), 32.6% were birds (n = 668), 48.8% (n = 1,042) were mammals, and 15.7% (n = 322) were reptiles. In total, 86 vertebrate host species were detected. The full list of detected vertebrate species is available in Supplemental Table 1. Of these, seven were amphibians, 57 were birds, 14 were mammals, and eight were reptiles. All detected amphibian species were anurans (frogs). Detected bird species represented 12 orders, with Passeriformes (songbirds) being the most species rich (28 species). Detected mammal species represented eight orders and detected reptile species represented three, with squamates (lizards and snakes) the most species rich.

The five most-collected blood-fed mosquito species (Cx. nigripalpus, Cx. pilosus, Ae. infirmatus, Psorophora columbiae, and Cs. melanura) contributed 91.0% of all vertebrate detections. Among the other 16 mosquito species, fewer than 40 blood-fed individuals were collected from each, and of these, fewer than 10 blood-fed individuals were collected from 11 species throughout the duration of the study. In the overall sample of detected vertebrate hosts, a few host species were common while many were rare. Four host species were detected more than 100 times: Odocoileus virginianus (n = 788), Anolis carolinensis (n = 273), Meleagris gallopavo (wild turkey; n = 152), and Strix varia (barred owl; n = 113). Sixty-four of the 86 detected host species were detected fewer than ten times, and 24 were detected only once.

Mosquito host associations

Most mosquito species (Ae. atlanticus/tormentor, Ae. infirmatus, Aedes taeniorhynchus, Coquillettidia perturbans, Mansonia titillans, Psorophora ciliata, Ps. columbiae, Psorophora ferox) fed from a higher proportion of mammalian hosts than other vertebrate classes (Fig. 2). Culiseta melanura was the only mosquito species that fed primarily on birds (> 70% of identified bloodmeals), though the single identified Culex interrogator blood meal was derived from a bird. Culex pilosus fed primarily on reptiles, and both Culex territans and Uranotaenia lowii took more blood meals from amphibians than other host classes (Fig. 2). While no individual mosquito species fed evenly among all vertebrate classes, Cx. nigripalpus fed approximately evenly between birds and mammals, with 46% of blood meals from mammals and 48% from birds. Additionally, 6% of Cx. nigripalpus blood meals were derived from reptiles, and 0.1% from amphibians.

Fig. 2

Proportion of blood meals from each vertebrate class (amphibian, bird, mammal, reptile) for each mosquito species collected at the DeLuca Preserve, Florida, USA. Colors indicate vertebrate class (yellow = Amphibia, orange = Aves, red = Mammalia, dark purple = Reptilia). The sample size of identified blood meals for each mosquito is represented in parentheses after the species name.

For some mosquito species, a large proportion of the blood meal sample was derived from only one or two host species. For Ps. columbiae (n = 160), 91.3% of blood meals were derived from Odocoileus virginianus. Culex pilosus (n = 292) fed primarily on lizards of the genus Anolis, with 85% of blood meals derived from native Anolis carolinensis and non-native Anolis sagrei (brown anole).

Sample completeness

To compare the ability of mosquito species to contribute to detection of a broad range of host species in an iDNA survey, the assemblage of vertebrate host species detected by each mosquito species was treated as a biological community and sample completeness profiles and diversity metrics were calculated for each species and for all mosquito combined. Here, use of the word “community” refers to the assemblage of vertebrate species that are bitten by and detected or undetected by each mosquito species (or by all mosquito species combined) at the DeLuca Preserve during the sampling period. In the sample completeness profiles estimated for each vertebrate community sampled by a mosquito species, sample completeness increases with diversity order (q) for all mosquito species combined and for each mosquito species individually except for Ps. columbiae (Fig. 3). Thus, for all but Ps. columbiae, it is likely that there were undetected vertebrate species within each community. For the community sampled by all mosquito species combined, sample completeness for q = 0, 1 and 2 was 81.8%, 98.8%, and 100%, respectively (Table 2), indicating that at most 81.8% of the total vertebrate community fed upon by mosquitoes was detected, the species that were detected make up about 98.8% of the individuals in the community, and the detected species make up 100% of the individuals of the abundant species of the community. For all mosquito species combined, at least 1 – 81.8% = 18.2% of vertebrate species in the community were not detected in the sample. The undetected species make up an estimated 1 – 98.8% = 1.2% of the individuals in the community. Based on the q = 1 and q = 2 estimates of undetected diversity, essentially all abundant and highly abundant species in the community were detected.

Fig. 3

Profiles of sample completeness as a function of order q between 0 and 1 for vertebrate species detected by individual mosquito species and all mosquito species combined at the DeLuca Preserve, Osceola County, Florida, USA. Sample size of each species is indicated below species names. Mosquito species for which sample size was fewer than ten or for which number of host species detected was fewer than two are excluded.

For individual mosquito species, sample completeness estimates for q = 0, 1 and 2 for individual species varied, especially at q = 0 and q = 1. For q = 2, completeness estimates were near or at 100% (98.7–100%) except for Ae. atlanticus/tormentor (87.2%), indicating that essentially all abundant and highly abundant vertebrate species in the communities fed upon by each mosquito had been detected. For Ae. atlanticus/tormentor, about 12.8% of abundant species were undetected, though for this species, sample size was small (n = 10) and confidence bands were wide. Among individual mosquito species, sample completeness for q = 0 ranged from 26.2% to 100%. Sample completeness values at q = 0 indicate the upper bound of the proportion of species in the community that were detected by sampling. Because values for all species except Ae. atlanticus/tormentor were > 98.7% at q = 2 indicating that all or most of the abundant species were detected, it is likely that the vertebrate species that were undetected by all mosquito species other than Ae. atlanticus/tormentor were species that were rare in each community. The smallest sample completeness value at q = 0 was 26.2% in Cx. erraticus, implying that at least 73.8% of the vertebrate host species fed upon by Cx. erraticus at the DeLuca Preserve were not detected in the sample. At q = 0, the largest sample completeness value was 100% in Ps. columbiae, which fed predominantly from one species, Odocoileus virginianus. For q = 1, sample completeness ranged from 62.1% (Ae. atlanticus/tormentor) to 100% (Ps. columbiae), suggesting that for each species, the majority of individual vertebrate animals in each community belong to species that were detected by our sampling. For Ae. infirmatus, *Cx. nigrip