Optimal Collection Methods for Asian Longhorned Ticks (Ixodida: Ixodidae) in the Northeast United States

Abstract

The Asian longhorned tick, Haemaphysalis longicornis Neumann, is an invasive species in the United States. Since its earliest recorded presence in West Virginia in 2010, H. longicornis has been reported from 15 states. While its public health significance in the United States is unclear, globally it transmits pathogens that infect livestock and humans, causing economic losses and substantial morbidity. Management and control of H. longicornis requires knowledge of its biology, ecology, and distribution. Here, we address the need for effective collection methods for host-seeking H. longicornis as an important step for accurately assessing tick abundance and potential disease risk. The number of H. longicornis collected were compared across three collection methods (dragging, sweeping, CO₂ traps) and three tick check distances (5 m, 10 m, and 20 m) were compared for dragging and sweeping. Field collections were conducted from June through August 2019 in Westchester County, New York, and ticks were grouped by life stage to assess collection method efficiency. Results indicated that implementing shorter (5 m) tick check distance was ideal for adult and nymphal collections. The dragging method proved better than sweeping for adult collections; however, there was no significant difference between the methods for nymphal collections, at any tick check distance evaluated. CO₂ traps attracted H. longicornis, but additional research is necessary to devise an effective tick retaining method before the traps can be implemented in the field. The results are presented to inform and support H. longicornis surveillance and control programs across the nation.

The Asian longhorned tick, Haemaphysalis longicornis Neumann (Ixodida: Ixodidae), is a newly introduced invasive species in the United States and a potential vector of disease agents. As of January 2021, H. longicornis has been found in 15 eastern states, since it was first recorded from a deer in West Virginia in 2010 (Beard et al. 2018, CDC 2020, USDA 2021). An established population was first described parasitizing sheep in New Jersey in 2017 (Rainey et al. 2018). This tick species is native to Asia (China, Japan, Korea, and Russia) and is an established invasive species in Australia, New Zealand, and surrounding islands (Hoogstraal et al. 1968). Most of the research on H. longicornis originates from China, Korea, and New Zealand, out of concern for its impact on the spread of infection among humans and the effects on health and productivity of livestock.

Outside the United States, H. longicornis transmits pathogens that cause debilitating health problems among cattle, domestic animals, and humans. Infestation of this tick also leads to other consequences, causing economic loss in livestock and dairy production and physical discomfort (Hoogstraal et al. 1968, Heath 2016). Haemaphysalis longicornis can transmit bacterial and parasitic pathogens, such as Theileria orientalis (Heath 2016), Theileria sergenti, Theileria mutans and Coxiella burnetii (Hoogstraal et al. 1968), and viral pathogens, namely, Russian summer-spring encephalitis virus (Hoogstraal et al. 1968), and severe fever with thrombocytopenia syndrome virus (Luo et al. 2015). A recent study by Stanley et al. (2020) reported that colony-reared H. longicornis, derived from a U.S. population, can acquire Rickettsia rickettsii, the causative agent of Rocky Mountain Spotted Fever (RMSF), from an infected host and transmit the infection transstadially, transovarially, and to a naive animal host while feeding in the next life stage, in a laboratory setting. The role of H. longicornis in natural transmission of R. rickettsii remains unknown, but the above finding increases public health concerns. Moreover, a highly pathogenic strain of T. orientalis Ikeda, which is detrimental to cattle health and dairy production, has been detected among H. longicornis and cattle in Virginia (Hammer et al. 2015, Lawrence et al. 2016, Watts et al. 2016, Oakes et al. 2019, Thompson et al. 2020). This increases concerns for veterinary health and potential impact on cattle industry. Despite these possible threats, H. longicornis appears unable to serve as a vector for Borrelia burgdorferi sensu stricto (s.s.), the primary causative agent of Lyme disease in the United States (Breuner et al. 2019). In their study, Breuner et al. (2019) reported that B. burgdorferi s.s. acquired by larvae feeding on infected Mus musculus mice were lost during the molt to the nymphal stage. It is unclear whether this result translates to infection in white-footed mice (Peromyscus leucopus), one of the most effective natural reservoirs of Lyme disease spirochetes. However, it is encouraging to know that H. longicornis larvae are reluctant to feed on these rodent species under laboratory conditions (Breuner et al. 2019, Ronai et al. 2020).

Several aspects of the biology and ecology of H. longicornis make it a highly successful invader and an extremely difficult species to eradicate (Heath et al. 1987). If hosts are unavailable, it can survive for up to a year without feeding (Heath 1994). It can tolerate extremes of temperature and humidity; continuing development at temperatures as low as 12°C and as high as ~40°C and acclimatizing to extreme desiccation by modifying glycerol and protein content (Yano et al. 1987, Yu et al. 2014, Heath 2016). It reportedly spends most of its lifecycle in pastures and in herbaceous vegetation in forested areas (~80%), creating the need to apply chemical controls across vast areas of land, which is economically infeasible (Heath 1994). Moreover, the capacity of H. longicornis to reproduce parthenogenetically (without mating) ensures a constant regeneration of population. Haemaphysalis longicornis has three reproductive races: bisexual race (requires males and females to reproduce), parthenogenetic race (females reproduce without male fertilization), and aneuploid race (which reproduces both ways) (Suomalainen 1962, Oliver 1977, Cane 2010). Yet almost all H. longicornis adults collected in the United States are females, indicating the presence of the parthenogenetic race. These characteristics of H. longicornis biology are likely to ensure its further spread and establishment in other areas of the United States where suitable habitats are available (Rochlin 2018, Raghavan et al. 2019). Therefore, the extent of harm to human health and agriculture caused by this invasive tick depends partly on its climatic adaptation, habitat, and hosts, and our ability to develop appropriate monitoring and control solutions. Currently, the information about the biology, ecology, and distribution of H. longicornis populations in the United States is minimal.

Studies comparing collection methods for host-seeking ticks are limited, even more so for H. longicornis, and methods vary among published reports describing H. longicornis and its surveillance and control (Heath et al. 1987, Chong et al. 2013, Hammer et al. 2015, Tufts et al. 2020). To address the need to identify the optimal collection methods for H. longicornis in the United States, we compared the efficacy of three methods (dragging, sweeping, and CO₂ traps) and three tick check distances (5 m, 10 m, and 20 m) for dragging and sweeping. Check distance is a predetermined distance interval where fabrics are checked for tick specimens when dragging or sweeping. The check distance can impact the number of ticks collected during sampling (Borgmann-Winter and Allen 2019), but there is no data available yet for the optimal check distances for H. longicornis. In addition to comparing effectiveness of collection methods and tick check distances, we considered aspects of H. longicornis biology and ecology which may affect the efficacy of collection methods, with the aim of contributing to the foundation of knowledge regarding surveillance methods for this newly invasive tick.

Materials and Methods

Site Description

Haemaphysalis longicornis ticks were sampled from two locations, Armonk (41°12′74.8N, -73°73′02W) and Yonkers (40°96′01N, -73°89′2W), in Westchester County, New York State (Fig. 1A), where the tick population was recently established (Wormser et al. 2019). In Yonkers, collection sites were located on the southern end of a 42-km long public trail. The sites were approximately 5–12 m wide, covered with grasses and herbaceous vegetation (~10–45 cm tall) and surrounded by shrubs and vines such as multiflora rose (Rosa multiflora) and porcelain-berry (Ampelopsis brevipedunculata). Maple (Acer spp.) and oak (Quercus spp.) were predominant trees at the site. Vegetation and ground cover were similar on both sides of the trail with only minor differences. Most of the collection transects were shaded, with some portions exposed to direct sunlight. The trail passed through a residential area and was actively used by people for recreation. In Armonk, collection sites were covered in leaf litter from deciduous trees such as maple (Acer spp.), oak (Quercus spp.), American beech (Fagus grandifolia), and hickory (Carya spp.). Both locations provided habitat for wild animals such as white-tailed deer (Odocoileus virginianus), eastern chipmunk (Tamias striatus), gray squirrel (Sciurus carolinensis), and other animals that may serve as hosts for H. longicornis ticks.

Fig. 1.

Maps showing (A) the geographical location and sampling transects at collection sites located in (B) Yonkers and (C) Armonk, Westchester County, NY. In (A), sites are denoted with a red triangle and blue square for Yonkers and Armonk, respectively. In Yonkers (B), five 180 m transects were set with 20 m distance between each transect. In Armonk (C), three 60 m parallel transects were set in three locations with a 5 m distance maintained between each parallel transect.

Sampling

In Yonkers, five 180 m sampling transects were set up, with 2.5 consecutive transects on each side of the trail (Fig. 1B). Dragging and sweeping methods were rotated among those transects. For each 180 m transect, flag markers were placed at every 60 m interval and tick check distances (5, 10, and 20 m) were rotated, on different days, among those 60 m sections. A minimum of 20 m distance, lengthwise, was maintained between consecutive 180 m transects.

In Armonk, due to property boundary constraints and landscape features, three parallel 60 m transects were established (Fig. 1C). A buffer distance of 5 m was maintained between each parallel transects to avoid overlap during sampling. Transects were labeled with unique names (a, b, c) to aid in rotation of check distances among transects. Flag markers were placed at 0, 30, and 60 m to guide collectors.

Field collections were conducted from 12 June through 12 August 2019 between 0900 and 1900 h, on days without rain and <10 mph wind. Collection methods were cycled throughout the day to reduce the potential impact of temporal variation in tick activity. Temperature and relative humidity were recorded during each sampling event and ranged from 21.2–34.8°C and 30–83% RH, respectively.

Dragging

Dragging was performed using a 1 m² (1 m wide and 1 m long) double-sided corduroy fabric with a wooden dowel sewn into one edge and a 4-m nylon rope attached to the dowel (Bouseman 1990, Daniels et al. 1997). Two screw eyes were fitted at each end of the wooden dowel where the rope was attached, to make fabric easily detachable for cleaning.

During sampling, the fabric was pulled behind a collector, at a slow walking pace, making sure the drag cloth was parallel and in contact with the ground and vegetation (Fig. 2A). For each 60 m transect, the collector stopped at every 5, 10, or 20 m check distance, to scan the fabric for ticks. The check distances were rotated among 60 m transects as mentioned above. Ticks were collected using fine tipped forceps and stored in glass vials, labeled with collection date, location, collection method, and check distance. The number of adult and nymphal ticks were recorded, which was re-confirmed during species identification, in the laboratory. When all the ticks on one side of the cloth were collected, the cloth was gently flipped to the other side and the aforementioned processes were repeated. Given the small size and high numbers, larvae were removed from fabric using Scotch-Brite adhesive lint rollers (3M, St. Paul, MN). Each larva was later identified and counted. Collection vials and lint roller sheets were stored in a sealed plastic bag to provide secondary containment during transportation to the laboratory, where they were stored at −20°C until identification.

Fig. 2.

Photographs taken during field work that illustrate the three collection methods compared: (A) dragging, (B) sweeping, and (C) CO2 trap. For dragging (A), a 1 m2 white corduroy cloth was dragged behind a collector. For sweeping (B), a 0.5 m2 corduroy cloth attached to a PVC pipe handle was swept beside a collector, keeping the fabric parallel and in touch with the ground. For CO2 trap (C), styrofoam boxes with two holes on each side were filled with dry-ice and placed on a 1 m2 white fabric. Each trap was placed at least 10 m apart. Lids were kept off during collection to prevent holes from clogging due to condensation.

Sweeping

Sweeping is similar to flagging, another method employed in tick collection, with differences in fabric size, collection process, and construction—bent PVC pipe used for sweeping instead of straight wooden dowel (Ginsberg and Ewing 1989). Besides the varied construction design and fabric motion, a prominent difference between dragging and sweeping/flagging is the possible disturbance of vegetation (or lack thereof) caused by a collector. Unlike dragging where a collector walks in front of a fabric, disturbing the habitat before ticks encounter the fabric, sweeping/flagging are conducted in front or side of a collector, eliminating that potential human disturbance. Sweep construction and the action of fabric movement for this project were modified from those described by Carroll and Schmidtmann (1992) and Ginsberg and Ewing (1989), respectively. We used a 0.5 m² (0.5 m wide and 1 m long) double-sided corduroy fabric, which was attached to a PVC pipe handle. Similarly, the cloth was pulled in a straight line, at a walking pace, to the side of a collector, making sure to keep the fabric in contact with the ground (Fig. 2B). Fabric motion was modified to accurately calculate the area covered by sweeping. Rotation of check distances, sample collection, storage methods, and data recording were performed as described previously for dragging.

CO₂ Trapping

Several designs exist for using CO₂ baited traps in tick collection. Previously described designs can be expensive or require specialized construction, such as using large cylinders (Garcia 1962), fitting pipe dispensers to containers (Miles 1968), and constructing fiberglass lined or plywood boxes (Wilson et al. 1972, Falco and Fish 1991). To reduce the cost and labor associated with trap construction, we used styrofoam boxes (outer dimensions: 28 cm long × 23 cm wide × 16 cm tall with inner dimensions: 20. 5 × 15. 5 × 12 cm) for CO₂ reservoirs (Fig. 2C). Two small holes were drilled on each side of the box, allowing continued diffusion of CO₂ from pelleted dry ice to attract ticks (Wilson et al. 1972, Falco and Fish 1992). Traps were deployed without lids to prevent clogging of holes from condensation and to improve CO₂ dispersal.

Two to three traps were set out at once; boxes were placed 10 m apart, on 1 m² white corduroy cloths. Observations were conducted on four different dates in July and August. From initial observations, H. longicornis were attracted to the traps, but crawled away within 30 min. Therefore, for subsequent observations, the CO₂ traps were checked at every 5–10-min intervals. Collectors walked away from the traps between observation periods to minimize impact of observer proximity on tick behavior. Tick collection from the cloths was performed as described for dragging and sweeping, with an exception that the underside of the fabric was examined by gently lifting each corner, with the CO₂ boxes in place. The number of ticks were recorded, ticks were marked on their scutum using fine point DecoColor paint markers (Marvy Uchida, Torrance, CA) unique to each tick, and returned to their spot on the fabric. After one hour, ticks remaining on the fabric were collected, processed, and stored as previously described. In the laboratory, a few different methods were evaluated for improving the retention of H. longicornis around CO₂ reservoirs, including water and oil moats and tanglefoot brush-on glue (Sherpa 2019).

Tick Identification

In the laboratory, each individual tick specimen was sorted by life stage, sex, and identified to species using published identification guides (Durden and Keirans 1996, Egizi et al. 2019). Following identification, specimens were stored in 70% ethanol at room temperature.

Data Analysis

Statistical testing for differences among collection methods and tick check distances was performed using a negative binomial generalized linear mixed model in R using the following packages: lme4 (Bates et al. 2015), ggplot2 (Wickham 2016), tidyr (Wickham and Henry 2019), dplyr (Wickam et al. 2019), and ggthemes (Arnold 2019). Check distances and collection methods were used as fixed effects and transect was a random effect. Statistical significance was set at α = 0.05. For H. longicornis, statistical analysis was only possible for Yonkers due to the low number collected in Armonk (n = 13). We mostly collected I. scapularis nymphs in Armonk. Density of adults (DOA) and nymphs (DON) per 100 m² was calculated as $\left(\frac{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}H.longicornis\mathrm{l}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{d}}\right)\ast 100$ (CDC 2019). Mean numbers for methods and check distances were conducted for the total distances covered, whereas calculations of density took fabric size into account. Larvae were excluded from all the analyses because of their limited mobility when questing. Two extreme outliers (one from each dragging and sweeping methods) were removed from the nymphal data based on very high numbers. Extreme outliers were determined as numbers that were greater than two times the ‘outer fence’ value, which is calculated as third quartile plus three times the interquartile range {Q₃ + (3 * IQR)} (Norman and Streiner 2014). Data from CO₂ traps were not included in the analysis due to low number of ticks retained.

Results

Demographics and Phenology

A total of 3,785 H. longicornis were collected (159 adult females, 387 nymphs, and 3,239 larvae). In addition to H. longicornis, we collected three species of native ticks, namely, Ixodes scapularis (Say) (Ixodida: Ixodidae), Ixodes dentatus (Marx) (Ixodida: Ixodidae), and Dermacentor variabilis (Say) (Ixodida: Ixodidae) (Table 1). The highest number of H. longicornis was collected in Yonkers, whereas we collected predominantly I. scapularis in Armonk.

Table 1.

Numbers of ticks collected from June to August 2019, by species and life stage

Site	Tick species	Adult	Nymph	Larva	Total
Armonk	Ixodes scapularis	2	153	10,316	10,471
	Haemaphysalis longicornis	1	12	0	13
	Dermacentor variabilis	1	0	0	1
Yonkers	Haemaphysalis longicornis	158	375	3,239	3,772
	Ixodes scapularis	0	11	0	11
	Ixodes dentatus	0	1	0	1
	Dermacentor variabilis	1	0	0	1
Total		163	552	13,555	14,270

Larvae, nymphs, and adults of H. longicornis peaked at different points during the collection period (12 June 2019–12 August 2019). The nymphal life stage was the first to emerge, peaking in June, whereas adults were in greatest numbers in July and larvae in August (Fig. 3). Despite variation in life stages collected overtime, all three life stages co-occurred at several points during the summer. Nymphal numbers steadily declined over the summer.

Fig. 3.

The proportion of Haemaphysalis longicornis tick life stages sampled from Yonkers, NY, from June to August 2019 is demonstrated. Life stages were shaded from lightest to the darkest to reflect progression from nymph to adult to larva. Proportions were calculated as number of ticks collected per day per life stage divided by total ticks collected for each life stage across the entire season. Populations of nymphs, adults and larvae peaked in June, July, and August, respectively.

Comparison of Tick Collection Methods

Over the same sampling distances, the dragging method yielded an average of 2.2 times more adult H. longicornis than sweeping (z value = −2.744, P = 0.006; Fig. 4A). DOA for dragging and sweeping were 2.63 and 2.37 per 100 m², respectively. When compared per check distance, the differences in average numbers between dragging and sweeping were significant for 5 m (z ratio = 2.176, P = 0.03) and 20 m (z ratio = 3.116, P = 0.002). For the nymphal life stage, there was no significant difference between the two methods when mean numbers for the same distances were compared (Fig. 4B). DON per 100 m² for sweeping and dragging were 6.08 and 4.90, respectively. The CO₂ trap method proved ineffective without an efficient tick containment system around the CO₂ reservoirs. Additionally, the traps did not recover any larvae, indicating a bias toward life stages with greater mobility, favoring adults followed by nymphs.

Fig. 4.

The average difference in Haemaphysalis longicornis (A) adults and (B) nymphs collected by dragging versus sweeping methods across a 450 m distance. The number of H. longicornis adults collected using dragging versus sweeping was significantly different, but not for nymphs (P-value > 0.05).

Comparison of Tick Check Distances

Overall, the 5 m check distance yielded higher numbers of adult H. longicornis compared to longer intervals. On average 5 m check distance recovered 1.8-fold more adult ticks than 10 m and 2.5 times more than 20 m. Similarly, DOA was the highest for 5 m check distance (3.91 per 100 m²) followed by 10 m (2.13 per 100 m²) and 20 m (1.59 per 100 m²). When the means were compared per collection method, the differences were statistically significant between 5 m and 10 m (z ratio = 2.811, P = 0.01) for dragging, and between 5 m and 20 m (z ratio = 3.069, P = 0.006) and 10 m and 20 m (z ratio = 2.811, P = 0.01) for sweeping (Fig. 5A). The 5 m check distance also recovered a greater number of nymphs on average than longer intervals: 1.2 times more than 10 m and 1.6 times more than 20 m. Density of nymphs also followed the same pattern; nymphal density was reduced as the check distance increased. DON for 5 m, 10 m, and 20 m check distances were 7.29, 5.0, and 3.48 per 100 m², respectively. However, when these check distances were compared per collection method, we found no statistically significant differences (Fig. 5B).

Fig. 5.

A comparison of the average numbers of Haemaphysalis longicornis (A) adults and (B) nymphs collected across a 150 m distance using three check distances (5 m, 10 m, and 20 m). The figure is faceted by collection method. For adults (A), a significantly higher number of adults were recovered with a 5 m check distance (dragging) and 5 m and 10 m (sweeping; P-value < 0.05). For nymphs (B), the number of adults recovered with a 5 m check distance was trending higher (P = 0.06) than the 20 m distance, which is denoted by *.

Discussion

Effective collection methods are essential to determine tick density and seasonal activity, to evaluate the acarological risk of human tick exposure, and to obtain representative samples for monitoring the spread of pathogens. Standardized methods are also important to facilitate comparison between studies. The effectiveness of collection methods varies by tick species, life stage, and landscape features (Ginsberg and Ewing 1989, Falco and Fish 1992, Chong et al. 2013, Dantas-Torres et al. 2013). Studies comparing collection techniques for H. longicornis in the United States are lacking. To fill this knowledge gap, our study compared the effectiveness of collection methods that are commonly used for native ticks in the Northeast to monitor the newly introduced invasive tick, H. longicornis. We assessed efficacy of three methods (dragging, sweeping, and CO₂ traps) and three check distances (5, 10, and 20 m) for the collection of H. longicornis adults and nymphs.

Dragging, sweeping/flagging, and CO₂ baited traps are commonly used for native tick collection in the United States (CDC 2019). These methods differ in construction design, time and resources required, and field implementation. Globally, dragging method seems to be almost standardized with most studies using a 1 m² fabric that is pulled behind a collector. However, for sweeping/flagging methods, fabric size and motion of fabric in the field are widely variable (Ginsberg and Ewing 1989, Carroll and Schmidtmann 1992, Kinzer et al. 1990, Dantas-Torres et al. 2013, Rulison et al. 2013). The motion of fabric for sweeping method used in this project was modified from sweeping method described by Carroll and Schmidtmann (1992) and flagging method conducted by Ginsberg and Ewing (1989). The sweeping method however differed from flagging method described in recent studies (Dantas-Torres et al. 2013, Rulison et al. 2013). Sweeping in this project was conducted in a straight line on the side of a collector, instead of swinging the fabric in front of a collector in a scythe-like, chopping, or jabbing motion to penetrate thick vegetation, as described in above studies. This modification was necessary to accurately assess total area sampled with the sweep because the traditional sweeping/flagging methods, as described above, make it challenging to determine the actual area covered during sampling procedures. Therefore, it is likely that the comparative results for collection methods might have varied if a different fabric motion for sweeping, mentioned above, had been implemented. We used a smaller fabric size for sweep as it made the sweep light to carry and easy to maneuver.

When compared, dragging recovered a higher number and density of adult ticks than sweeping. However, there was no apparent difference in the efficacy of dragging and sweeping for the nymphal life stage. The result for adult life stage contrasted with Carroll and Schmidtmann (1992) and Chong et al. (2013). Carroll and Schmidtmann (1992) reported that the sweeping method was equally or more effective than dragging for collecting I. scapularis. Although I. scapularis was not the focus of our study, we compared the nymphal numbers collected in Armonk, and we did not detect significant differences between the collection methods as well as the check distances. Small sample size might have contributed to the indefinite results. The discrepancy between Carroll and Schmidtmann (1992) study and our results for adult H. longicornis is likely associated with the biological differences among the tick species, methodology, and the habitat types sampled. In our study, the size of the collection fabric for sweeping was half the size of that used for dragging; Carroll and Schmidtmann (1992) used 1-m square fabric for both methods. To eliminate the impact of fabric size, we calculated density of each life stage per 100 m² area. Similarly, Chong et al. (2013) reported no significant difference between dragging and sweeping when averages for nymph plus adult H. longicornis, collected from four different habitats, were compared. This variation is possibly contributed by difference in habitats sampled and motion of sweeping fabric, behavior difference between populations in the United States and South Korea, or the presence of different reproductive races (Arsnoe et al. 2015). South Korea has both parthenogenetic and bisexual reproductive races, whereas there has been no report of bisexual population in the United States (Hoogstraal et al. 1968). Further studies are required to confirm these possibilities.

Tick check distances vary among programs and projects, depending on the availability of resources, time, and personnel. Borgmann-Winter and Allen (2019) reported that estimated density of I. scapularis ticks decreased as the check distance increased. However, studies on effective check distance for H. longicornis is lacking. We found that shorter check distance (5 m) was most successful for collecting H. longicornis adults and nymphs, for both sweeping and dragging. Tick drop-off and behavior of H. longicornis may explain the superiority of 5 m, given the positive correlation between check distance and tick drop-off rate (Borgmann-Winter and Allen 2019). The 5 m check distance, which experienced the least amount of disturbance, recovered more ticks on average than longer check distances. Anecdotally, we observed that H. longicornis adults are more sensitive to disturbance than I. scapularis; H. longicornis dropped off at any external disturbance. Although 5 m check distance collected the most ticks, we recommend considering program goals and accessibility to resources, discussed below, to decide which check distance to implement. We also observed distinct differences in the abundance of H. longicornis at our two field sites, Armonk and Yonkers. We attribute that difference to geographical (Armonk is farther north) and habitat variations among these two sites, which could have led to variability in the introduction and dispersal of tick populations. In addition, unlike Armonk, which has a seemingly unlimited expansion of wooded areas where wild animals can roam freely, the trail in Yonkers provided a limited green corridor, at the center of a residential area. This might force the local H. longicornis hosts such as deer, squirrels, and chipmunks to congregate and spend more time in that green space, allowing constant food source for the maintenance of H. longicornis population. This, however, does not explain the lack of I. scapularis at the site (Yonkers), which also feeds on the same host animals. Therefore, additional, unexplained factors might also impact the growth and proliferation of H. longicornis and I. scapularis populations in these two locations.

CO₂ baited traps have been effective for collecting native ticks including Amblyomma americanum (Linnaeus) (Ixodida: Ixodidae) (Kinzer et al. 1990, Schulze et al. 1997), I. scapularis (Falco and Fish 1992), Amblyomma cajennense (Fabricius) (Ixodida: Ixodidae) (Guedes et al. 2012), and Ornithodoros parkeri (Cooley) (Ixodida: Argasidae) (Miles 1968). In this study, CO₂ traps were generally ineffective. Haemaphysalis longicornis (mostly adults) was attracted to the CO₂ traps but lost interest quickly and crawled away from the trap. We observed that most H. longicornis attracted to a trap exited the fabric around it within 10 min. However, some were retained near the reservoir for > 30 min, perhaps anesthetized by their closer proximity to CO₂ (Norval et al. 1989). We conclude the inefficacy of CO₂ traps likely reflects H. longicornis biology, rather than the tick density at our site, as reported for Ixodes ricinus (Linnaeus) (Ixodida: Ixodidae), where CO₂ traps outperformed dragging method at high-density sites, and were comparable at low tick density (Gray 1985). Collection of adults and nymphs around CO₂ traps, with open lids (to mitigate an issue of trap holes clogging from condensation), is evident that dispersion of a high concentration of CO₂ was attractive to H. longicornis. Norval et al. (1987) found that cattle and sheep odor plus high concentration of CO₂ stimulated unfed Amblyomma hebraeum (Koch) (Ixodida: Ixodidae) adults to emerge from soil and quest for hosts, however, that response was not observed when low concentration of CO₂ was dispensed. Such study on impact of varying amounts of CO₂ concentration on H. longicornis and its behavior is lacking. Therefore, in this study, it is unclear whether the high concentration of CO₂ led to rapid loss of interest as the ticks neared the trap or if low concentration would retain ticks for longer period. A few different methods were evaluated to improve the retention of H. longicornis around CO₂ reservoirs (Sherpa 2019). Among the solutions tried, oil moats and tanglefoot brush-on glue seemed to be most promising. However, ticks appeared to sense the retention substances and mostly evaded any contact. The design by Falco and Fish (1992) implemented a tick retaining method by using double-sided carpet tape, however, the design is impractical for most programs and projects. Trap construction required advanced carpentry skill and the traps were heavy and difficult to move in the field (Richard Falco, personal communication). CO₂ traps required significantly less collection time, so, with an effective way to retain specimens around the CO₂ reservoirs, this method may become an efficient H. longicornis monitoring tool.

In deciding which tick collection method to implement, programs need to consider the purpose of the surveillance, resources at their disposal, landscape/habitat type of collection sites, and compare the pros and cons of the methods. Based on the findings, we encourage tick surveillance and control programs that have access to resources (budget, time, and staff) and projects that are collecting specimen to compare tick density to employ a 5-m check distance for H. longicornis. However, if the program lacks personnel and the aim of the program is to check tick presence/absence, or just to collect ticks for pathogen testing, then implementing 10 m or 20 m check distances is advisable. Employing longer check distances will allow more landscape coverage in short time. Similarly, dragging is best utilized in open and low grass sites. We did not compare habitat differences, however, based on a published study, sweeping is recommended for sites with thick shrubs and hard to reach areas with brushy undergrowth (Carroll and Schmidtmann 1992). CO₂ traps might be an ideal choice for programs with budgets that limit the number of personnel and time for surveillance activity. However, as mentioned above, this method needs more research and an effective tick retention solution before it can be implemented as an efficient collection method for H. longicornis.

Following are some additional advantages and disadvantages of dragging and sweeping worth considering before deciding on which method to implement. The drag cloth is made of a larger fabric, so it is more readily kept in constant contact with the ground and the fabric spans a larger surface area than a sweep. However, it is difficult to implement this method in dense brush and vegetation as it requires a collector to walk in front of the fabric, increasing their tick exposure risk. Similarly, folding or flipping of drag fabric edges can go unnoticed until a collector stops to check. In contrast, sweeping allows a collector to avoid brambles and reduce potential exposure to ticks, any folding of fabric edges can be quickly fixed as the cloth is always visible to the collector, and scanning time is shortened due to smaller fabric size. Additionally, sweeping can be effectively used in narrow spaces and thick vegetation (Carroll and Schmidtmann 1992). However, because the fabric is small and light, even the smallest twig can flip the edges, which requires constant fixing/flattening of the fabric. This could however be fixed by adding weight to the sweep fabrics. Additional research is warranted to assess trade-offs between time, resources, and the effectiveness of collecting representative samples of H. longicornis from various commonly monitored habitats.

Altogether, our study fills the knowledge gap in comparing the effectiveness of collection methods and check distances for H. longicornis. The findings are crucial for estimating tick density and public health risk as well as for studying the ecology and biology of this newly invasive tick.