Conservation Wildflower Plantings Do Not Enhance On-Farm Abundance of Amblyomma americanum (Ixodida: Ixodidae)

Simple Summary Planting wildflowers is a commonly used tool to conserve pollinators. However, it is possible that wildflower plantings may inadvertently aid tick species, complicating both vector control and pollinator conservation programs. In this study, we tested whether conservation wildflower plantings enhanced the on-farm abundance of the lone star tick, Amblyomma americanum (L.). Over two years, A. americanum were sampled using dry ice traps in wildflower plots, weedy field margins, and forested areas. We found no more A. americanum in wildflower plots than in weedy field margins. Forested areas harbored the greatest number of A. americanum sampled. Overall, wildflower plots do not pose an increased risk of exposure to A. americanum on farms. Abstract Planting wildflowers is a commonly suggested measure to conserve pollinators. While beneficial for pollinators, plots of wildflowers may be inadvertently performing an ecosystem disservice by providing a suitable habitat for arthropod disease vectors like ticks. The lone star tick, Amblyomma americanum (L.), is a medically important tick species that might be able to utilize wildflower plantings as a suitable habitat. In this two-year study, ticks were sampled using dry ice baited traps from wildflower plots, weedy field margins, and forested areas to determine if wildflower plantings were increasing the on-farm abundance of A. americanum. Abiotic and biotic environmental variables were also measured to better understand which factors affect A. americanum abundance. We found no more A. americanum in wildflower plots than in weedy field margins. Forested areas harbored the greatest number of A. americanum sampled. The height of the vegetation in the sampled habitats was a significant factor in determining A. americanum abundance. Depending on the sampled habitat and life stage, this relationship can be positive or negative. The relationship with vegetation height may be related to the behavior of the white-tailed deer and the questing success of A. americanum. Overall, wildflower plots do not pose an increased risk of exposure to A. americanum on farms.


Introduction
The lone star tick, Amblyomma americanum (L.). (Ixodida: Ixodidae), is an aggressively-biting species that is a nuisance to humans and a pest of livestock [1]. It has been gaining attention as an important vector of human diseases, including erlichiosis, tuleramia, and Heartland virus [2,3]. populations [20,21], does the addition of plants aid A. americanum populations? The purpose of this study was to determine if on-farm wildflower plots can serve as quality habitat for A. americanum.

Study Area
Tick surveys were conducted at 10 farms in eastern Virginia and Maryland in 2018, and nine of the same farms in 2019. Nine of these farms had one wildflower plot installed during the spring of 2016; one farm had their wildflower plot seeded in the spring of 2015. Wildflower plot sizes ranged from 561-8600 m 2 , with an average size of 2360 m 2 . Three different wildflower mixes were used that were adapted to local soil conditions (Table 1). Wildflower plot establishment procedures were similar for each mix used. Generally, the site of the wildflower plot was tilled, packed, seeded, and packed once more-see Angelella and O'Rourke for further details of the fields seeded with the well-draining mix [31]. After establishment, the plots were mowed annually during the plant dormant season, between November and March. Table 1. Wildflower species used in well-draining soils (WD), well-draining replacement (WDr), and poorly draining soils (PD) seed mixes. For WD, WDr, and PD, n = 7, 1, and 2, respectively.

Sampling
Ticks were sampled with dry ice traps [11]. The traps were 8-liter coolers, measuring 33 cm × 24 cm × 22 cm (Igloo Coolers, Katy, TX, USA) with 13 mm diameter holes drilled into each side. The traps were loaded with 2 kg of dry ice. A 5 cm band of Shurtape Indoor/Outdoor tape (Hickory, NC, USA) was placed around the outside of the traps. Three habitats were sampled at each farm: the wildflower plot, a weedy field margin, and a nearby forest. One dry ice trap was placed in each sampling habitat at each location on each sampling date; the traps were placed at least 10 m apart. The traps were set between 9:00 a.m. and 12:00 p.m. and left in the field for 24 h. Each field was sampled once per month from April to July in 2018 and 2019 ( Figure 1). All ticks caught were placed in 95% ethanol and later identified by species and separated into nymphs and adults. Of the total 1731 A. americanum detected, 164 were taken from wildflower plots, 302 from weedy field margins, and 1265 from forest locations. No interaction between life stage and habitat was detected in either year of study. There was no difference in A. americanum abundance between wildflower plots and weedy field margins in 2018 and 2019 ( Figure 2). Amblyomma americanum were detected most often in the forest plots ( Figure 2), with significantly fewer A. americanum collected in wildflower plots than forest plots in both years (z = −5.23, p < 0.001; z = −3.89, p = 0.003). Forests consistently had the thickest duff layers, shortest vegetation, and most stable temperatures of the three habitat types sampled ( Table 2).

Environmental Variables
Temperature and relative humidity at the soil surface were recorded every half-hour for the full duration of trap deployment in each habitat at each field site during each round of sampling using a Hobo U23 Pro V2 data logger (Onset; Bourne, MA, USA). Vegetation height was measured at five locations during each sampling date. The height of herbaceous vegetation was measured at the trap location and 4 m away from the trap in each cardinal direction [32]. The duff depth was measured as the distance from the bare soil to the top of the organic matter on the ground and was measured after the last round of tick sampling each year at the same locations as the herbaceous vegetation height.

Statistical Analysis
To determine if wildflower plots increased A. americanum abundance relative to weedy field margins, a generalized linear mixed model fit to a negative binomial distribution was used to analyze these data. The interaction of the sampling habitat and tick life stage (nymph or adult) and their main effects were fixed factors. The field and the sampling date nested in field were set as random effects. Tukey's Honestly Significant Difference (HSD) was used to test for differences among means.
To investigate the effects of the measured environmental variables on tick abundance, a multimodel approach was used. A set of 10 a priori models was created with predictor environmental variables that are commonly associated with A. americanum. The variables tested were: habitat, duff depth, average vegetation height, average relative humidity, and temperature standard deviation. Each variable was tested alone, and then the habitat and the other environmental variables were tested together with main effect and interaction terms in the models. An additional intercept-only model was included as a null model. Models were constructed as generalized linear mixed effects models with a negative binomial distribution. Additionally, the field was included as a random effect, along with the year and sampling date nested within field. Models within four Akaike Information Criterion, adjusted for small sample size, points (AICc) of the top model were selected and averaged [36] using the package 'MuMIn' [37]. To reduce the likelihood of including uninformative parameters, the changes in AICc values were compared relative to changes in log likelihood values [38]. All analyses were done separately for nymphs and adults as they can have different responses to the environment [39].

Results
We collected 1165 nymphs and 566 adult A. americanum over the two years of sampling. Amblyomma americanum nymph abundance peaked in June of both years ( Figure 1). A peak in adult abundance was seen in May in 2018 and April in 2019 ( Figure 1). On average, more nymphs than adults were detected in 2018 (z = 2.91, p = 0.003). This effect was not statistically significant in 2019 (z = 1.85, p = 0.06) (Figure 1).
Of the total 1731 A. americanum detected, 164 were taken from wildflower plots, 302 from weedy field margins, and 1265 from forest locations. No interaction between life stage and habitat was detected in either year of study. There was no difference in A. americanum abundance between wildflower plots and weedy field margins in 2018 and 2019 ( Figure 2). Amblyomma americanum were detected most often in the forest plots ( Figure 2), with significantly fewer A. americanum collected in wildflower plots than forest plots in both years (z = −5.23, p < 0.001; z = −3.89, p = 0.003). Forests consistently had the thickest duff layers, shortest vegetation, and most stable temperatures of the three habitat types sampled (Table 2). The model with the interaction of habitat type and vegetation height was the top ranked model for predicting adult A. americanum abundance ( Table 3). The models containing habitat only and the interaction between habitat and duff depth were within 4 AICc points of the top model and were included in model averaging (Table 3). After model averaging, no effects were detected for duff depth (Table 4). Vegetation height had a significant different effect in weedy field margins compared to the other habitats. As the height of vegetation increased in weedy field margins, the abundance of A. americanum decreased (z = 2.4, p = 0.02) (Table 4, Figure 3).  The model with the interaction of habitat type and vegetation height was the top ranked model for predicting adult A. americanum abundance ( Table 3). The models containing habitat only and the interaction between habitat and duff depth were within 4 AICc points of the top model and were included in model averaging (Table 3). After model averaging, no effects were detected for duff depth (Table 4). Vegetation height had a significant different effect in weedy field margins compared to the other habitats. As the height of vegetation increased in weedy field margins, the abundance of A. americanum decreased (z = 2.4, p = 0.02) (Table 4, Figure 3).   Similar to adult A. americanum, the habitat by vegetation height model was the best predictor of nymph abundance. All other models were more than 4 AICc points higher ( Table 5). As vegetation height increased in the forest samples, so did A. americanum nymph abundance (z = 3.4, p = 0.001) ( Table 6). This contrasts to wildflower plots and weedy field margins, where nymph abundance decreased with increasing vegetation height (z = −2.08, p = 0.04) (Table 6, Figure 3).

Discussion
To our knowledge, this is the first study investigating the interaction of wildflower plots and tick abundance. In this study, wildflower plots planted for pollinator conservation did not

Discussion
To our knowledge, this is the first study investigating the interaction of wildflower plots and tick abundance. In this study, wildflower plots planted for pollinator conservation did not inadvertently constitute an ecosystem disservice by simultaneously increasing A. americanum abundance. While A. americanum were detected in wildflower plots, this habitat harbored fewer of them than weedy field margins. Therefore, wildflower plots do not pose a risk of augmenting on-farm A. americanum abundance.
Vegetation height is playing a role in the differences in adult A. americanum abundance between the sampling habitats. These changes could be related to the questing success of A. americanum adults on taller vegetation. Adult A. americanum could have higher success rates in finding a host in weedy field margins compared to the other habitats sampled. With taller vegetation, adult A. americanum would have more area to utilize for questing to attach to larger hosts like white-tailed deer. Many of the weedy field margins sampled were a transition zone from agricultural areas to forested ones. These transition areas are frequented by white-tailed deer as they move from areas of cover to open areas as part of their diurnal movement [40]. Adult A. americanum could be investing more in vertical movement in the habitat than horizontal since hosts are not likely to linger. This behavior was seen with I. scapularis in habitats that were deemed difficult for ticks to traverse due to the vertical habitat structure [41] With greater success in finding hosts, fewer A. americanum would be available to sample.
Amblyomma americanum nymph abundance decreased with taller vegetation in wildflower plots and weedy field margins but increased with taller vegetation in forested areas. Decreases in nymph abundance could be following a similar pattern as the adults; as vegetation height increased, so did questing success. The increase of A. americanum nymph abundance with increasing vegetation height in forested areas could be explained by the preference of white-tailed deer to use dense vegetation in forests for bedding sites [40]. With female white-tailed deer having a strong preference for their home range, they could be frequenting bedding areas and dropping engorged larvae. This could create nymphal hotspots in these areas. When the deer return to bed, A. americanum nymphs may have the time to successfully attach to the host. Ixodes scupalris more actively quest towards a host when the host is stationary [41]. Larger wildflower plots could potentially provide enough cover for deer to use as bedding sites, creating a similar situation. However, given the smaller size of the plots used in this study, this was unlikely to be occurring. A study with Peromyscus spp. mice found that 64% of the nests surveyed had I. scapularis present, and 87% of all larval ticks present had taken a blood meal [42]. Future studies could examine if specific sites where hosts remain immobile and which they frequently visit are attractive for immature stages of ticks.
In this study, duff depth was selected as a factor for adult A. americanum but was not significant with model averaging. A similar result was seen in Missouri, where duff layer depth was selected as a factor, but not a significant factor in determining adult A. americanum abundance [39]. However, duff depth is an important environmental factor for the survival of adult A. americanum as it can create a critical microclimate for preventing desiccation. Amblyomma americanum adults are sensitive to moisture loss and will seek out moist microclimates after losing only 10-15% of their body weight to counteract desiccation [43]. While the duff layer is important, its presence may be all that matters. In a previous study, A. americanum were collected from areas that had shallower duff layers than I. scapularis, but never from areas that had no duff layer [44]. Better quantification of the microclimate of the duff layer may also help detect its effects on A. americanum abundance, as the duff layer can be 2-3 • C cooler than the ambient air temperature [16].
The result that wildflower plots are not increasing on-farm A. americanum abundance is encouraging for both pollinator conservation and vector control. Further studies are needed to verify the results in other geographic areas and with different mixes of wildflowers. The mix of wildflower species in a pollinator habitat may have a large influence on the behaviors of tick hosts. A study in Florida observed that white-tailed deer browsed on all 11 wildflower species used in their pollinator mixes [45]. Of the species tested by Degroote et al. [45], two flower species were also present in this study: Coreopsis lanceolata L. and Rudbeckia hirta L. However, DeGroote et al. [45] found that these were the least and fifth least browsed wildflower species, respectively. If wildflower mixes have species that are attractive to white-tailed deer, tick abundance may likely increase. However, from this study, it is not known if the presence of A. americanum in the plots indicates that they can complete their lifecycle in this habitat, or if they are simply dropping off of their host. This could include sampling for hosts from within the different habitats to see what role they are playing in driving the difference in A. americanum abundance.
Different tick species may have varied levels of attraction to the same habitat. Within a 1-ha forested plot, A. americanum and Ixodes scapularis were distributed between two different sets of habitat conditions related to each species' tolerance to desiccation [44]. Amblyomma americanum was found in areas with a more open canopy and less shrubby understory compared to I. scapularis [44]. If dense stands of ground cover develop within wildflower plots, they could attract rodent hosts of I. scapularis [46], potentially increasing I. scapularis abundance within its range. This could be more problematic if conservation efforts are focused on the use of longer-lived woody plants, or if herbaceous wildflowers are not mowed regularly.

Conclusions
In summary, A. americanum were sampled from within wildflower plots, but they are not increasing the risk of exposure to A. americanum relative to weedy field margins. The role of hosts in moving A. americanum into the wildflower plots is an important factor in the success of A. americanum in colonizing these habitats which deserves further study. Understanding how the hosts of A. americanum utilize wildflower plots for cover and their preferences for different wildflower species as food could further inform the risks posed by wildflower plots.