Human biting mosquitoes and implications for WNV transmission

doi:10.21203/rs.3.rs-1999780/v1

Background: West Nile virus (WNV), primarily vectored from Culex genus mosquitoes, is the most important mosquito-borne pathogen in North America, infecting thousands of humans and countless wildlife since its arrival in 1999. In locations with dedicated mosquito control programs, surveillance methods often rely on frequent testing of mosquitoes collected from a network of gravid traps (GTs) and CO₂-baited light traps (LTs). Traps targeting oviposition-seeking (e.g. GTs) and host-seeking (e.g. LTs) mosquitoes are vulnerable to trap bias, and captured specimens are often damaged, making morphological identification difficult.

Methods: This study leverages an alternative mosquito collection method, the human landing catch (HLC), as a means to compare sampling of potential WNV vectors to traditional trapping methods. Human collectors exposed one limb for 15 minutes at crepuscular periods (5:00-8:30am and 6:00-9:30pm daily, the time when Culex species are most actively host-seeking) at each of 55 sites in suburban Chicago, Illinois, for two summers (2018-2019).

Results: HLC collections resulted in 223 human seeking mosquitoes, of which 46 (20.6%) were Culex. Of the 46 collected Culex, 34 (73.9%) were Culex salinarius, a potential WNV vector species not thought to be highly abundant in the upper Midwestern United States. Per trapping effort, GTs and LTs collect greater than 7.5 times the number of individual Culex specimens than HLC efforts.

Conclusions: The less-commonly used HLC method provides important insight into the complement of human-biting mosquitoes in a region with consistent WNV epidemics. This study underscores the value of HLC collection methods as a complementary tool for surveillance to aid in WNV vector species characterization. However, given the added risk to the collector, novel mitigation methods or alternatives approaches must be explored to incorporate HLC collections safely and strategically into control programs.

Human Landing Catch

Culex salinarius

West Nile virus

zoonosis

vector-borne disease

spillover

West Nile virus (WNV) is a zoonotic mosquito-borne Flavivirus naturally maintained in a mosquito-bird-mosquito enzootic cycle [1, 2]. The risk of spillover to humans increases with greater exposure to several primary vector species of WNV within the Culex genus. Since the arrival of WNV to the United States in 1999, there have been 52,532 reported human infections, of which 2,456 (4.7%) died [3, 4]. More than two decades after its arrival, WNV remains the most important mosquito-borne pathogen in North America [5]. Now endemic, WNV persists throughout the United States, even in locations with operational area-wide vector control [6]. Effective mosquito control is augmented by a robust surveillance program based on traps gathering data on vector abundance and infection with WNV [7].

Gravid traps (GTs) and CO₂-baited light traps (LTs) are the most common tools for WNV surveillance used in the United States [8–10] and are extremely effective at collecting a large number of mosquito specimens per trapping effort; GTs tend to collect large numbers of Culex species mosquitoes while LTs trap a greater diversity of species. Mosquitoes trapped in GTs and LTs often consist of large collections spanning multiple mosquito genera, as well as a diversity of other non-Culicid insect species [11]. These large and diverse sample collections can make sorting and identifying WNV vector species labor-intensive. Adult mosquitoes collected from GTs and LTs are usually sorted by sex (often discarding male specimens) and are identified by key morphological features via a dissecting microscope. Mosquitoes collected from these types of traps pass through a bladed fan, and often get damaged, become desiccated (in dry climates), or moldy (in humid climates) if not collected within one or two days [12]. The methods of identification, in combination with high volumes of collected mosquitoes and difficulties in discerning subtle features across several key Culex species, are often prone to misidentification from human error [13, 14]. In addition to members of the Culex pipiens complex, Culex restuans, Culex salinarius, and even Culiseta inornata specimens can be misidentified as Culex pipiens or simply lumped into the term “Culex species”[15].

Deployment of a network of GTs and LTs, frequent mosquito collections, and rapid pathogen testing is considered the “status quo” for understanding mosquito abundance and WNV infection. When a combination of high abundance and high infection rates indicates increased human risk, mosquito control organizations will often then employ control efforts such as adulticide spraying or larvicide deposition [16, 17]. These control methods are noticeably effective in the immediate days following action. However, anecdotal evidence from Northern Illinois suggest that impacts on mosquito abundance from spray control methods may only be temporary, and only within a fairly small radius of the area of treatment, and populations recover rapidly, usually in less than 1 week [18, 19]. While the overall rapid reduction of all mosquitoes may be a goal for mosquito abatement agencies [20] and citizens [21], the key public health emphasis in relation to WNV surveillance and control should be to monitor and control species of mosquitoes involved in the enzootic transmission and spillover to humans. Spatially explicit, epidemiological and entomological forecasting efforts can be helpful in assisting abatement districts with efficient and targeted control. However, modeling human WNV illness is difficult and efforts to date have been found to vary in accuracy across varying spatial and temporal resolutions [6].

Given ongoing and persistent difficulties controlling mosquito populations, eliminating WNV from the environment, and the current limitations to predictive models of human WNV cases, we investigated whether adding information from human landing catch (HLC) collection methods to traditional collection methods in place to protect public health from arboviral threats, can improve targeted WNV surveillance and control efforts. A long-term WNV transmission research effort in the northwest suburbs of Chicago, IL, in close collaboration with the Northwest Mosquito Abatement District (NWMAD), has worked to pinpoint missing links between human WNV illness and mosquito infection at multiple spatial scales, including highly localized study sites ([6, 22, 23]). The goal of this study was to use HLC collection methods (considered the gold standard for assessing mosquito-human host interactions [24]) to evaluate the effectiveness in attracting Culex species mosquitoes targeting human bloodmeals at locations where there has previously been high incidence of human illness [25, 26]. We then compared the HLC data with traditional mosquito surveillance data using GT and LT data and determined key differences among collected mosquito diversity measures, and relative proportion of abundance. We hypothesized that HLC collection methods would provide a higher relative proportion of human-seeking mosquitoes, particularly among Culex species, thereby improving the understanding of potential vectors involved in WNV spillover to humans in the NWMAD. The insights into the interactions between humans and WNV vector species may lead to improved targeted surveillance for WNV vector species that seek human bloodmeals.

Ethics Statement

This project was approved by the Institutional Review Board (approval number 18908) of the University of Illinois at Urbana-Champaign, the Illinois Department of Public Health (IDPH), and the University of Illinois Biosafety Committee (approval number IBC-4307). All HLC participants were researchers and were informed and educated of the potential risks of the study prior to field collecting.

Study Sites

Mosquitoes collected via HLCs and human observations were conducted within the NWMAD, a ~ 240 mi² mosquito control agency area comprising the northwest suburbs of Chicago. Previous research had established 55 1-km wide hexagonal study locations within the NWMAD, providing representative focal study regions selected via stratification of high to low categorization values of previous human WNV risk (high to low) and prior modeling accuracy (high to low residual of predictions) [6]. Within each hexagonal region, a natural area (e.g. public park) was selected as a collection site, because they were easily accessible and allowed for collections during crepuscular periods (5–8:30 am and/or 6–9:30 pm) when Culex mosquitoes are most actively seeking blood meals [27, 28]. The sites also met the study criteria for locations where mosquito-human spillover likely occurs in the Midwest for two reasons: 1.) all locations were composed of heterogeneous landscapes consisting of a mix of domestic and peri-domestic built space, a body of water (e.g. pond, river), and natural green spaces rich with numerous avian species [29, 30], and 2.) humans were abundant and engaging in a variety of activities (e.g. resting to physical activity).

Human Landing Catches

Human landing catches (HLCs) were conducted between epidemiologic weeks 28–38 (early July – mid September) in the summers of 2018 and 2019. During these periods, each of the 55 1-km wide hexagonal study regions within the NWMAD were visited weekly for 15 minutes each. Additionally, the visiting order was rotated each week so that all sites were equally sampled at earlier and later times of the crepuscular period; a consistent number of sites (n = 27 to 28) were visited each day. Human collectors (n = 4) exposed any one to four limb(s) and collected landing mosquitoes via a mechanical aspirator (Fig. 2). Collectors were instructed to not apply any materials to their clothing or body that might be attractive or repellent to host-seeking mosquitoes (e.g. mosquito repellent, strong smelling deodorant, hair spray/gel, cologne, etc.). Mosquitoes were collected within 3–4 seconds after landing on the collector’s exposed skin. Over a 15-minute collection period, mechanically-aspirated mosquitoes were transferred to a pre-made paper cage and maintained live until transferred to -80°C freezer.

Within 3 days, all mosquitoes were removed from the freezer and morphologically identified by medical entomologists at the University of Illinois. Mosquitoes from the Culex genus were sent to the University of Maryland for morphological and molecular confirmatory species identification. Mosquitoes were shipped overnight on dry ice to the University of Maryland, College Park. Upon receipt, they were held at -80°C. Mosquitoes were dissected and abdomens retained as a voucher. Genomic DNA was extracted from heads and thoraces with a Qiagen DNeasy Blood and Tissue Kit (Cat. No. 69506, Qiagen Inc., Valencia CA, USA) according to standard protocol. Extracted DNA was amplified with a multiplexed polymerase chain reaction (PCR) targeting the 28S ribosomal subunit[31] to distinctly identify Culex pipiens (698 bp), Culex restuans (506 bp), and Culex salinarius (175 bp), using a reaction similar to that described in Rochlin et al. (2007), except the total reaction volume was 20 µl, and we used the MgCl₂ concentration standard in the Promega GoTaq buffer. A negative control, in which purified water replaced genomic DNA, was run with mosquito samples during each assay. PCR was run using a Bio-Rad T100 Thermal Cycler (Bio-Rad Life Sciences, Hercules, CA), with the following conditions: 96°C for 4 minutes, 35 cycles of 96°C for 15 seconds, 55°C for 30 seconds, 72°C for 90 seconds, then 72°C for 4 minutes. We visualized amplicons following gel electrophoresis on a 2% agarose gel at 120V for 60 minutes and specimens were identified using fragment lengths in parentheses above, using a 1kb ladder (Thermo Scientific Gene Ruler 1kb Plus, Waltham, MA) for comparison.

Human Observations

While HLC collections were conducted by a human collector, an accompanying researcher simultaneously recorded the number of unique human visitors within eyesight at the same location. Uelmen 2020 [33] provides additional data observing the unique activities, duration of activities, and apparent age and gender, to assess and quantify WNV risk and human behavior.

Using data from HLCs, as a proxy for mosquito biting rates, and human observations, as a proxy for human host availability, we derived two indices: the Nuisance Factor and Human WNV Added Risk. The majority of collected mosquitoes were non-Culex species and less likely to vector WNV and other regional mosquito-borne pathogens to humans. They are thus considered nuisances to the public, and the nuisance factor index is defined as:

$$Nuisance Factor=\frac{\frac{Human Observations}{Hour}*\frac{Nuisance Mosquitoes Collected}{Hour}}{100}$$

Conversely, the human WNV added risk factor was defined for mosquitoes collected from the Culex genus, by the following equation:

$$Human WNV Added Risk= \frac{\frac{Human Observations}{Hour}* \frac{Culex \text{s}\text{p}\text{e}\text{c}\text{i}\text{e}\text{s} \text{C}\text{o}\text{l}\text{l}\text{e}\text{c}\text{t}\text{e}\text{d}}{\text{H}\text{o}\text{u}\text{r}}}{100}$$

The denominator is used for ease of interpretation and visualization. All observed humans are assumed to be equally available to mosquitoes, regardless of activity and/or behavior.

Historic Mosquito Collections

Historical data from mosquito collections via GT and LT methods from four sources were examined, three from Chicago and surrounding suburbs, and another from Decatur, IL (Fig. 1). Trapping effort was standardized as combined trap night, a sum of the total number of nights GTs and LTs were set to collect mosquitoes. The relative abundance of Culex and non-Culex species mosquitoes were compared with human landing catch collections conducted in the NWMAD.

Public health agencies serving the city of Chicago and the surrounding suburbs in Cook and DuPage Counties, are among the best equipped (e.g. large annual budgets, number of personnel, and available equipment and tools) to combat mosquitoes and mosquito-borne diseases in the country. There are four dedicated abatement districts that serve the surrounding suburbs, as well as a vector control branch of the City of Chicago’s public health department that serves the greater metropolitan area. The Northwest Mosquito Abatement District (NWMAD) and the City of Chicago Vector Control provided surveillance records for presence of Culex salinarius in the greater Chicago area. In addition to these agency-provided data, collections conducted in the suburbs of southern Cook County (Hamer et al. unpublished) were included. The mosquito abatement district of Macon County, located in Central Illinois, provided surveillance records for the greater Decatur region. Like Cook and DuPage Counties, Macon mosquito abatement district (MMAD) maintains a long-standing, robust systematic surveillance program in Illinois and serves as an excellent comparator to the abundance and diversity of mosquitoes between the two regions.

Statistical Analyses

Descriptive analyses were conducted, using univariate tests for socioeconomic and demographic drivers of Culex collected via HLC methods. The mean of mosquito phenology and biting time of day were assessed by Tukey’s HSD. Mosquito abundance, diversity, and WNV illness risk were compared by trapping effort (ratio of trap nights to total mosquitoes by genus) and type (HLC, GT, LT, other) and assessed as multinomial distributions in a generalized linear regression. All statistical analyses were conducted in JMP (version 16.0.0, SAS Institute Inc., Cary, NC, 1989–2021) and R (version 4.1.2.).

Human Landing Catch Collections

Human landing catches resulted in a total of 223 mosquitoes, including 46 (20.6%) Culex specimens (Additional File 1: Figure S1). This resulted in a rate of 51.6 and 10.7 mosquitoes from all species and those from the Culex genus, respectively, per trap night. Mosquitoes from the Aedes genus were the most abundant (55.6%), followed by Culex (20.6%), Anopheles (19.7%), Coquillettidia (3.6%), and Psorophora (0.4%) (Fig. 3). The most abundant species collected were Aedes vexans (42.2%), Anopheles quadrimaculatus (18.8%), Culex salinarius (15.2%), and Aedes trivittatus (12.1%) (Additional File 1: Figure S2).

Mosquito landing rates significantly varied by epidemiological week by genus (p = 0.036). By species across collection season, Culex erraticus landing rates tended significantly later (mean epi-week of 36), and Culex restuans and Aedes trivittatus landing rates tended significantly earlier (mean epi weeks of 29 and 33, respectively) than all other species (Fig. 4).

Mosquito landing rates varied by time of day by genus and species (Fig. 5). Collectively, mosquitoes from Aedes and Psorophora landed significantly earlier in the day (peaking at 30 and 120 minutes before sunset, respectively, while Culex and Anopheles mosquitoes landed significantly later in the evening hours (peaking at 70 minutes after sunset for each). By individual species, only Culex restuans, Aedes vexans, and Psorophora ferox differed significantly in landing rates from the other species, peaking at 250 minutes after, 50 minutes after, and 120 minutes before sunset, respectively.

Human Activity and Risk

Human observations were recorded at every collection location, but nuisance mosquitoes were not present in twelve of the 55 locations and Culex mosquitoes were not present in thirty-three of the 55 locations (Fig. 6). A total of 2,821 individual humans were counted over 2,040 recording minutes (mean = 1.4 people per minute) during human landing catch collections. By recording location, human observations per 15 minute visit ranged from a maximum of 312 to a minimum of 1. The nuisance factor ranged from a maximum of 32.3 to a minimum of 0 and the human WNV added risk factor ranged from a maximum of 1.44 to a minimum of 0.

Historic Mosquito Abundance

A compilation of historical abundance records resulted in a total of 1,930,680 collected mosquitoes (all species) across the four historical data sources (Tables 1A, Additional File 1: S1). Of these, 454,223 (23.5%) were non-Culex species, and 1,476,457 (76.5%) were Culex species. Macon MAD provided the longest period of collections (17 years), but the NWMAD had the greatest number of mosquitoes collected (n = 1,169,168, 60.6%). Per trapping effort, standardized as mosquitoes collected per trap night, MMAD produced the most mosquitoes (37.1 per trap night) over the 17-year collection period.

Overall, Culex species mosquitoes were collected in greater numbers in GTs (n = 1,400,121, 94.8%) than in LTs (n = 75,777, 5.1%) (Table 1B). Per combined light and gravid trapping effort, MMAD collected more Culex species (32.8 per trap night) than NWMAD (17.3 per trap night). The ratio of Culex species mosquitoes collected from LT to GT in NWMAD and MMAD were similar (0.053 and 0.057, respectively), and from Southern Cook County traps it was 3-fold higher (0.193).

Human Landing Catch Comparisons to Historical Records

The NWMAD and MMAD collect high volumes of mosquitoes in GTs and LTs, including tens of thousands of potential WNV vectors every year. Human cases of WNV illness have occurred in Chicago, IL every year since 2002, despite rigorous mosquito control campaigns and dedicated abatement and local health districts working year-round [6]. Evaluating the results of collected mosquito species by varying trapping methods is not a precise method for determining abundances in a region, due to trap-specific biases, lack of a systematic collection regimen, and non-uniform distribution of equally placed trap types in the study region.

Based on the rich historic trapping data over the past decade, this study provides a reasonable baseline by which Culex species, and Culex salinarius in particular, are expected to be collected. Landing catches of collected Aedes, Culex, Anopheles, Coquillettidia, and Psorophora resulted 55.6%, 20.6%, 19.7%, 3.6%, and 0.4% proportion collected, respectively. Collections from NWMAD and MMAD resulted in proportions of Aedes, Anopheles, Coquillettidia, and Psorophora collections of 21.1% and 10.2%, 0.6% and 1.0%, 0.5% and 0.07%, and 0.01% and 0.2%, respectively. In contrast, proportions of Culex species collected at NWMAD and MMAD were 70.2% and 83.8% in GTs, and 3.7% and 4.8% in LTs, respectively. Overall, HLCs collect higher proportions of non-Culex mosquitoes than GTs or LTs. In a separate study, 785 Culex species mosquitoes from the North Shore and Northwest Mosquito Abatement Districts of Northern Cook County, IL were collected from GTs and LTs between 2017–2021 and submitted for confirmatory genetic species identification. Only 2 (0.25%) specimens were identified as Culex salinarius, compared to 73.9% of specimens from HLCs. Thus, the expected distribution of species collected will depend on the collection method.

Traditional trapping methods are ideal for the efficient collection and testing of potential WNV mosquito vectors. However, these methods are only effective in identifying potential vectors and controlling virus activity if testing is conducted frequently, as Culex populations are known to rebound rapidly after spraying [18]. Conversely, traditional trapping methods appear to collect a limited diversity of mosquito species [34, 35]. Explanations for this may be that other mosquito species may not have a biological preference or attraction to simulated baiting conditions (e.g. lights, CO2, lures, etc.), but are more likely the product of resource strain (e.g. limited human time to sort and identify mass quantities of mosquitoes). Human landing catch collections do not attract the same proportions of Culex species that GTs and LTs collect. Additionally, HLC collection methods have two major disadvantages: 1.) they are labor intensive and time-consuming, requiring a lot of human resources per captured mosquito, and 2.) the collector’s risks of acquiring mosquito-borne pathogens, many of which do not have specific treatments or cures, are increased. However, HLCs are better suited than GTs or LTs in estimating the community of female mosquitoes specifically seeking human hosts [24]. The potential benefits in a systematic and targeted design may pinpoint hotspots for the highest concentration of human-seeking mosquitoes. When used in a targeted-style approach, HLCs can narrow the geographic area of interest and provide a more efficient deployment for controlling potential human-seeking WNV mosquito vectors but approaches to safeguard the collector’s health must be further developed before widespread adoption of HLCs for monitoring can be considered [24]. Additionally, HLCs in this study were conducted in small natural areas, embedded within residential areas. Additional studies are needed to ascertain any influence on mosquito ecology within these two intersecting habitats [29]. Results from this 2-year study have made clear that Culex salinarius, a less commonly reported WNV vector species in the upper Midwestern United States, was not only present in the Chicago area, but was the most commonly collected WNV vector in HLC collections, accounting for 73.9% of all Culex species landings. Conversely, the two primary vectors of WNV in the Midwestern United States, Culex pipiens and Culex restuans, only comprised 4.3% and 2.2% of the total proportion of Culex species that landed on human collectors.

Implications for Human WNV Transmission

Culex salinarius Coquillett, commonly known as the unbanded saltmarsh mosquito, are among the most competent vectors of WNV in laboratory transmission studies [36–38]. Additionally, the virus has frequently been isolated from wild-caught specimens [39–43]. The etymology of the name salinarius pertains to salt but is slightly misleading given that the species appears to tolerate low to moderate levels of salt (3–11 ppt) in freshwater/brackish environments, particularly in ponds and other small bodies of water along coastlines [44–46]. Historically, this species distribution range stretches from Maine to Florida, and around the Gulf of Mexico to Texas [47]. Although this species has been collected as far north as Ontario, Canada, reports pertaining to their overall abundances in the upper Midwestern United States have remained low, although their presence has been reported from various locations in the Midwest [48–52]. Despite belonging to the same genus as Culex pipiens, these species do not contain high quantities of fat bodies and do not enter a reproductive diapause [53, 54]. Instead, overwintering is thought to occur in natural shelters and animal burrows, and females have been known to seek blood meals at the first signs of mild weather [47].

Perhaps the most startling difference between Culex salinarius and Culex pipiens is the host blood-meal preference; Culex pipiens highly prefers avian hosts, and in blood-meal analyses, mammalian hosts rarely exceed 15–20% [2, 55]. The host blood-meal preference for Culex salinarius exceeds 60% mammalian and between 35–40% avian [12, 56–58]. When factoring potential implications for WNV spillover in the Midwestern United States, Culex salinarius provides three potentially critical points of concern: 1.) this species is more commonly observed in its northern range, leading to new questions regarding the species’ northern geographic limit [59], and is either not commonly trapped via GT and LT methods, or not being correctly identified (via morphologic methods), 2.) efforts to mitigate potential WNV vectors are not designed for targeting Culex salinarius, given its tendency to breed in natural habitats, and 3.) the high mammalian host preference, aggressive biting nature, and high vector competence provide the key ingredients for a mosquito to potentially become a highly efficient bridge vector of WNV and other encephalitis viruses [56, 57, 60].

Upon discovering the high ratio of Culex salinarius landing rates, in comparison to any other Culex species in Chicago, additional data inquiries were made from prior research conducted in the region. Colleagues from the City of Chicago’s vector control program and a collaborative team working in various sites of Southern Cook County shared their abundance data for comparison (Tables 1, Additional File 1: S1). In summary, out of a total of 1,476,411 collected Culex specimens, only 1,665 (0.11%) Culex salinarius have been identified. Human landing catch efforts resulted in 34 Culex salinarius collections out of a total of 46 Culex specimens (73.9%). HLC efforts totaled 7.87 collections per trap night versus 0.022 collections per trap night for all other collection efforts. This equates to HLC collections producing over 364 times the amount of Culex salinarius than any other effort combined. One caveat to this finding, however, is potential for misclassification bias; HLC collections were speciated genetically, while other trapping data is generally based on high-throughput morphologic identification, which may fail to identify a species that is not expected in high numbers and is morphologically similar to a common species.

Interventions and Future Research

The surprising abundance of Culex salinarius in the human landing catch collections described here in Chicago is a testament to the effectiveness of the HLC collection method [61]. This study provides compelling evidence in support of alternative trapping methods as a useful tool to provide updates on the overall abundance and diversity of potential disease vectors that are targeting human hosts. As a standard public health measure, these type of environmental health “checkups” may be needed more frequently due to the rapidly changing forces of present day, such as new introductions of vectors and pathogens.

Public health and mosquito control should consider adding a supplemental plan to their current monitoring and mitigation strategies, occasionally conducting surveillance and/or targeting of natural breeding areas where Culex salinarius may reside (see Additional File 1: Text S1 for brief habitat analysis). While HLC collection methods are not practical for routine surveillance, sparing usage implemented at strategic time periods (e.g. for early season WNV “sentinel” use, at or around historic peak human transmission, in known “hot spot” human and/or bird transmission locations, etc.) could provide added utility and breadth to existing strategies, providing important information that may increase the efficiency and knowledge in targeting potential mosquito vectors. With the aid of human scented lures and/or CO₂, traps like BG-sentinels or CDC light traps may also be a viable alternative method for capturing large quantities of Culex salinarius and other human-host seeking mosquitoes in natural habitats [62–64].

While these incidental findings may have implications for the enzootic and zoonotic transmission potential of WNV, suggestions from this study remain as hypotheses and should be interpreted as a guide for improving targeted surveillance and WNV mitigation efforts. Onward research demands the frequent and repeated detection of WNV RNA in Culex salinarius females captured in their environment to substantiate the hypotheses provided in this manuscript. Additional HLC collections should be repeated and include a deeper evaluation of micro-scale factors that may influence a mosquito’s landing preferences, including preferred feeding locations on the human body (Additional File 1: Figure S3), microclimate (Additional File 1: Figure S4), and the addition of sampling residential areas.

In summary, this research provides an overview of the abundance and generic composition of mosquitoes collected in the Chicago and Decatur, IL regions. Traditional GT and LT collections are highly productive, particularly in collecting a broad array of Culex species. Human landing catch collection methods made clear that Culex salinarius is a potential WNV vector in frequent contact with humans in Chicago, IL. The potential of Culex salinarius as a WNV vector in the upper Midwestern United States has been underestimated for two reasons: 1.) commonly used traps only yielded very small fractions of identified Culex salinarius, and 2.) that the potential for misidentification of individual species in batch collections is high. However, individuals conducting HLC collection methods are subjected to increased risks of the very mosquito-borne pathogens intended for surveillance, and improvements to personal safety must be implemented before any consistent or systematic use. This study highlights the added utility of HLC collections as a tool for mosquito surveillance and public health officials to identify current and potential mosquito-borne disease risks.

WNV: West Nile virus; HLC: Human landing catch; CDC: Centers for Disease Control and Prevention; GT: Gravid trap; LT: Light trap; NWMAD: Northwest Mosquito Abatement District; IDPH: Illinois Department of Public Health; MMAD: Macon Mosquito Abatement District; PCR: Polymerase chain reaction

Acknowledgments

We thank the Macon Mosquito Abatement, especially Jason Probus and Sam Force, for supplying historic records to digitize for this study. We’d also like to thank Serena Schatz and Alvin Hernandez Reyes for their assistance with field work and data collection. The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Department of Health and Human Services.

Funding

This study was partially supported by Cooperative Agreement #U01 CK000505, funded by the National Science Foundation grants EF-0429124 and EF-0840403.

Availability of data and materials

The Chicago-area-HLC data used for analysis in this study is available in the Illinois HLC repository, https://github.com/juel15401/Chicago-area-HLC.git. Due to data privacy and sharing rights, please contact Dr. Gabe Hamer ([email protected]) for Southern Cook County mosquito data, Dr. John-Paul Mutebi ([email protected]) for 2005-2006 City of Chicago mosquito data, Dr. Patrick Irwin ([email protected]) for NWMAD mosquito data, or Jason Probus ([email protected]) for MMAD mosquito data.

Authors’ contributions

JAU conceptualized the study and use of HLC data. JAU, BL, and RLS designed the fieldwork component. PI, DB, AAB, JPM, GLM, and MF curated the historical mosquito identification, abundance and infection data from their respective institutions. CS and AM provided expert identification and critical HLC design and safety protocols. CS, AM, AAB, SJR, JPM, GLH, MF, and RLS provided supervision throughout the fieldwork and/analysis. JAU wrote the initial manuscript draft, conducted all analyses and evaluations, and created all the figures. All authors reviewed and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table 1. Mosquito collection data provided by each reporting location, indicating collections per trap night (A) and by trapping method (B). Detailed collection information by genus is available in supplemental materials.

A.

Location	Trapping Years	Trapping Nights All Methods	Total Mosquitoes Collected
			All species			*Culex* species			*Culex salinarius*
			Sum	per year	per trap night	Sum	per year	per trap night	Sum	per year	per trap night
Ultra-Fine-Scale NWMAD 55 Hexagons (HLC)	2018-2019	4.32*	223	111.5	51.62	46	23	10.65	34	17	7.87
NWMAD	2005-2016	67763	1580526	131710.5	23.32	1169168	97430.67	17.25	548	45.67	0.01
Southern Cook County, IL	2005-2012	N/A	5414	676.75	N/A	2222	277.75	N/A	22	2.75	N/A
City of Chicago	2005-2019	N/A							896	59.73	N/A
MMAD	2002-2018	9299	344517	20265.706	37.05	305021	17942.41	32.8	199	11.71	0.02
Total	54	77066.32	1930680	152764.46	111.99	1476457	115673.83	60.7	1699	136.86	7.9
*Trap night equivalent, converted from trap minutes

B.

Location	Trapping Years	Non-Culex species					*Culex* species
		Non-Culex species					All species					*Culex salinarius*
		Sum	LT	GT	HLC	Other	Sum	LT	GT	HLC	Other	Sum	LT	GT	HLC	Other
Ultra-Fine-Scale NWMAD 55 Hexagons (HLC)	2018-2019	177	0	0	177	0	46	0	0	46	0	34	0	0	34	0
NWMAD	2005-2016	411358	N/A				1169168	59127	1110041	N/A	N/A	548	548	N/A	N/A	N/A
Southern Cook County, IL	2005-2012	3192	N/A				2222	276	1433	0	513	22	18	4	N/A
City of Chicago	2005-2019	N/A										896	390	443	0	63
MMAD	2002-2018	39496	N/A				305021	16374	288647	N/A		199	196	3	N/A
Total	54	454223	0	0	177	0	1476457	75777	1400121	46	513	1699	1152	450	34	63
% Culex salinarius	--	--	--	--	--	--	0.12%	1.52%	0.03%	73.91%	12.28%

No competing interests reported.

Additionalfile1.docx
Additional file 1: Text S1. Appendix Table S1. Detailed mosquito collection information by genus, trap type, overall trapping effort, and submitting agency. Table S2. Univariate analysis suggests four independent variables may provide unique characteristics in determining the preferred habitat(s) of Culex salinarius within the NWMAD during the summers of 2018 and 2019. Further analysis is not warranted at this time, given the small number of specimens collected. Values in parenthesis indicate one standard error from the mean. No evidence of multicollinearity was present (Additional File 1: Table S3). Table S3. Correlogram testing for multicollinearity among four independent variables associated with Culex salinarius habitat preference within the HLC study regions of NWMAD. Figure S1. Cumulative HLC collections by mosquito genus for each of the 55 hexagons in the study region. Figure S2. Mosaic plot displaying frequency of genus and species collected by human landing catch by study location (HexID, n=55). For specific coordinates of each mosquito collection location within each HexID, contact the corresponding author. Figure S3. Mosaic plot of frequency of mosquito landing location on body of human collector by genus and species. The diagrams on the far right of the figure display the most common landing locations by nuisance (top) and Culex species (bottom) mosquitoes. Figure S4. Box plot distribution of average weather factors (temperature, humidity, and wind speed) during human landing catches by genus and species of collected mosquitoes. Temperature and wind speed were recorded by regionally reported value and by a handheld anemometer/thermometer combination device for specific, local values. Figure S5. Suitable larval habitats within the average female Culex salinarius flight range (2 km.²) for each collection from the City of Chicago, MMAD, and HLC study regions (55 1-km wide hexagons within the NWMAD).

Human biting mosquitoes and implications for WNV transmission

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Ethics Statement

Study Sites

Human Landing Catches

Human Observations

Historic Mosquito Collections

Statistical Analyses

Results

Human Landing Catch Collections

Human Activity and Risk

Historic Mosquito Abundance

Discussion

Human Landing Catch Comparisons to Historical Records

Implications for Human WNV Transmission

Interventions and Future Research

Conclusions

Abbreviations

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1