Flagging versus dragging as sampling methods for nymphal Ixodes scapularis (Acari: Ixodidae)

Vol. 3, no. 1 Journal of Vector Ecology 13 Flagging versus dragging as sampling methods for nymphal Ixodes scapularis (Acari: Ixodidae) Eric L. Rulison 1*, Isis Kuczaj, Genevieve Pang, Graham J. Hickling 3, Jean I. Tsao, and Howard S. Ginsberg 1 Department of Plant Sciences and Entomology, University of Rhode Island, Kingston, RI 1, U.S.A. Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI, U.S.A. 3 Center for Wildlife Health, University of Tennessee, Knoxville, TN 3799, U.S.A. USGS Patuxent Wildlife Research Center, Coastal Field Station, University of Rhode Island, Kingston RI 1, U.S.A. * Current address: Caesar Kleberg Wildlife Research Institute, Texas A&M University-Kingsville, Kingsville TX 733, elrulison@gmail.com Received 7 November 1; Accepted January 13 ABSTRACT: The nymphal stage of the blacklegged tick, Ixodes scapularis (Acari: Ixodidae), is responsible for most transmission of Borrelia burgdorferi, the etiologic agent of Lyme disease, to humans in North America. From to fall of 1, we compared two commonly used techniques, flagging and dragging, as sampling methods for nymphal I. scapularis at three sites, each with multiple sampling arrays (grids), in the eastern and central United States. Flagging and dragging collected comparable numbers of nymphs, with no consistent differences between methods. Dragging collected more nymphs than flagging in some samples, but these differences were not consistent among sites or sampling years. The ratio of nymphs collected by flagging vs dragging was not significantly related to shrub density, so habitat type did not have a strong effect on the relative efficacy of these methods. Therefore, although dragging collected more ticks in a few cases, the numbers collected by each method were so variable that neither technique had a clear advantage for sampling nymphal I. scapularis. Journal of Vector Ecology 3 (1): 13-17. 13. Keyword Index: Dragging, flagging, Ixodes scapularis, nymphs. INTRODUCTION The blacklegged tick, Ixodes scapularis, is the primary vector of Lyme disease in the eastern and central United States, and its nymphal stage is responsible for most pathogen transmission to humans (Fish 1993). Nymphs live primarily in leaf litter of forested areas (Ginsberg and Ewing 199a), sometimes in close association with human dwellings (Maupin et al. 1991). Therefore, effective sampling of host-seeking or questing nymphal I. scapularis is critical to Lyme disease surveillance programs. Two of the most widely-used methods for sampling questing ticks are flagging and dragging (Sonenshine 1993). Flagging involves sweeping a cloth material (i.e., flannel, cotton) attached like a flag to a hand-held pole or dowel and swept through leaf litter or vegetation. Dragging involves pulling the equivalent material behind the investigator, typically by rope attached to a basal pole, with the pole horizontal and perpendicular to the direction of movement. Investigators have used flagging methods to sample I. scapularis (Ginsberg et al. ), dragging methods (Diuk-Wasser et al. ), and hybrid techniques that combine elements of both methods (Ginsberg and Ewing 199b, Bouseman et al. 199, Carroll and Schmidtmann 199, Goddard and Piesman ). Sonenshine (1993) suggested that flagging would be more effective at sampling ticks in leaf litter in areas with dense undergrowth (because the flag could be forced through the understory vegetation to contact the leaf litter, whereas the shrubs would interfere with dragging), while dragging would be more effective in more open areas (where a greater surface area of material would contact the tick environment). However, this recommendation has never been directly tested. In this study, we compare the efficacy of flagging and dragging for collection of nymphal I. scapularis at several locations in the eastern and central U.S. that differed in forest type, and particularly in density of underbrush. MATERIALS AND METHODS Ticks were sampled by flagging and dragging at three sites in the eastern and central U.S.: Cape Cod, Massachusetts, southern Rhode Island, and Fort McCoy Garrison in central Wisconsin. These locations included a variety of canopy cover, tree density, shrub density, understory, and ground cover types. The Cape Cod sites had pitch pine and mixed oak woodlands, with shrub layers dominated by bear oak. The Rhode Island sites had canopies dominated by red maple, white pine, and white oak, with rhododendrons and tree saplings in the shrub layer. The forest at Fort McCoy was dominated by oaks and maples, with a shrub layer of mostly tree saplings. Each sampling flag consisted of a 1 m piece of white cotton flannel attached to a 1.5 m wooden dowel. The attachment consisted of a sleeve sewn into the material through which the dowel was placed and secured with hose clamps (this attachment was not included in the 1 m ). Drags were similarly 1 m, but the dowel (1.1 m to 1.5 m) was attached to a rope pull at each end, and the trailing end of the drag was weighted with a. cm, 3.1 g mending plate at each corner. At each location, either two (MA and RI) or three (WI) sampling arrays were established, with the arrays separated by >1 km. The arrays at Fort McCoy were named Gauntlet, Stonehenge, and Valley; those at Cape Cod were named Lab and Eastham; and those in Rhode Island were named Kettle and Tuck. Each sampling

1 Journal of Vector Ecology June 13 array consisted of a 7 x 7 square grid of pin flags placed 15 m apart. Transects were oriented in one direction (e.g., north-south) and samples were taken on transects along each edge of the grid and between each row of flags, for a total of eight transects, each 9 m in length. Pilot studies were conducted at Fort McCoy from May to August,, in which the same transects were sampled weekly by flagging and dragging. In 11 and 1, sampling of each transect alternated between flagging and dragging on successive trips (even numbered transects were flagged and odd transects dragged on one trip; then even were dragged and odd flagged on the next trip). Field workers were deployed (switched between flagging and dragging samples) so as to avoid any bias from the sampling techniques of particular individuals. In 11, the Cape Cod (MA) and Fort McCoy (WI) sites were sampled approximately every three weeks from May through September, and in 1 all sites were sampled from May through August. All nymphal ticks were removed, stored in 95% ethanol, and their numbers recorded every 15 m. Ticks were later identified to species in the lab. Shrub density at each site was calculated according to Engeman et al. (199) using the distance from each pin flag to the second nearest shrub stalk. Tree density was calculated in similar fashion. density = (N 1) π/ (R i ) N = number of samples R i = distance to second plant from flag i Numbers of questing nymphs collected by flagging and dragging showed strong overdispersion, so the data were analyzed using general linear models with a log link (SAS 9.3, Cary NC, GENMOD procedure). Negative binomial distributions provided the best models (determined by AIC) and these results are reported here (significance assessed using Wald chi-square). The model parameters included arrays, sampling methods, sampling trips, and interactions among factors (no interactions were significant). We also tested consistency of collections by each method through the season at each sampling array using Wilcoxon matched-pairs signed-ranks tests (Sokal and Rohlf 195, Rohlf and Slice 1999), where the numbers collected by flagging vs dragging during each sampling trip was a matched pair. Simple linear regressions of the ratios of nymphal ticks collected by flagging vs dragging in relation to shrub and tree densities were performed using the Data Analysis subprogram in Excel (Microsoft Corporation, Redmond, WA). RESULTS NYMPHS PER TRANSECT (±SE) 5 15 5 15 5 3 GAUNTLET FORT MCCOY, WI - 11 STONEHENGE 15 5 3 VALLEY 15 5 3 Figure 1. Mean numbers of Ixodes scapularis nymphs (±SE) collected by flagging and dragging 9 m transects, Fort McCoy, WI, 11. Julian days on horizontal axes. The samples in WI (n = 1,353 nymphs) showed no overall difference in the numbers of nymphs collected by dragging and flagging (Wald chi-square =., df = 1, P =.59). Over the season, the numbers collected by dragging were consistently greater (Wilcoxon matched-pairs signed ranks tests) at the Gauntlet array (mean drag =.7, SE = 9.57, mean flag = 3.7, SE = 7.551, N = 9, t s = -.3, P =.), but not at the Stonehenge array (mean drag =.57, SE =.179, mean flag = 9.3, SE = 7.9, N = 7, t s = -.57, P =.3), nor at the Valley array (mean drag = 15., SE =.75, mean flag = 1., SE = 3.3, N = 9, t s = -.17, P =.3). The drag and flag collections at these arrays in 11 (n = nymphs) are shown in Figure 1. Trends in nymphal hostseeking activity over time generally were similar between the two methods at all three arrays, with peak nymphal host-seeking activity occurring in mid-june, but no overall differences between sampling methods (Wald chi-square =., df = 1, P =.91). There were no consistent differences over the season between dragging and flagging at the Gauntlet array (Wilcoxon test, N = 5, t s = -.9, P =.173) or the Valley array (N = 5, t s = -.135, P =.), but dragging consistently collected more ticks than flagging at Stonehenge (N = 5, t s = -1.753, P =.). The Cape Cod samples (n = 1 nymphs, Figure ) similarly showed no consistent difference between flagging and dragging (Wald chisquare =., P =.993), and there were no consistent differences over the season in the numbers of nymphs collected by dragging vs flagging at either the Eastham array (N = 5, t s = -.5, P =.33) or at the Lab array (N = 5, t s = -.5, P =.33). In the 1 samples, neither technique consistently collected greater numbers of nymphal ticks (Figure 3). The numbers of ticks collected by flagging did not differ significantly from dragging in Wisconsin (Wald chi-square =., df = 1, P =.15), Massachusetts (Wald chi-square =.19, df = 1, P =.7), or Rhode Island (Wald chi-square =.53, df = 1, P =.7). The effects of undergrowth were analyzed by comparing the ratio of the number of nymphs collected by flagging over the number collected by dragging as a function of shrub density at each site. The flag/drag ratio, though positive in slope (Figure ), was not significantly related to shrub density (r =.193, n = 15, P =.1). The flag/drag ratio also failed to show a significant relationship with tree density (r =.19, n = 13, P =.19). DISCUSSION The effectiveness of dragging did not differ consistently from flagging in our samples, although in the few cases when one M J J A S O

Vol. 3, no. 1 Journal of Vector Ecology 15 NYMPHS PER TRANSECT (±SE) CAPE COD, MA - 11 15 5 3 LAB LAB.5 3 1.5 EASTHAM 15 5 3 M J J A S O Figure. Mean numbers of Ixodes scapularis nymphs (±SE) collected by flagging and dragging 9 m transects, Cape Cod National Seashore, MA, 11. Julian days on horizontal axes. technique collected more nymphs in repeated samples over the season, it was dragging. Nevertheless, these differences were not consistent among sample arrays or years, and different techniques collected larger absolute numbers of nymphs in different samples. This variation in the number of nymphal ticks collected did not shift consistently from one sampling period to the next (Figures 1-3), suggesting that the actual number of ticks along each transect was not the major reason for the variability in the number captured. Rather, local factors such as weather conditions (Vail and Smith 199, ) and variations in litter depth and microtopography presumably affected the number questing at any one time, and this variability was reflected in the inconsistent differences between sampling methods. It is important to emphasize that this result applies specifically to sampling of I. scapularis nymphs. Indeed, Ginsberg and Ewing (199b) and Schulze et al. (1997) both found that various sampling techniques were differentially effective at collecting different species (A. americanum vs I. scapularis) and even different stages of I. scapularis. We concentrated on nymphal I. scapularis in this study because of its importance as a vector of Lyme disease. However, sampling programs for other ticks need specific assessments of sampling effectiveness of different techniques. For example, Fourie et al. (1995) found that flagging was more effective than dragging at collecting adult I. rubicundus, the Karoo WISCONSIN Gauntlet MASSACHUSETTS Lab 1 1 1 1 15 5 15 5 Stonehenge Eastham NYMPHS PER TRANSECT 1 1 15 5 Valley 1 1 NYMPHS PER TRANSECT 1 1 15 5 RHODE ISLAND Tuck 1 1 Figure 3. Mean numbers of Ixodes scapularis nymphs collected per transect by flagging and dragging 9 m transects at all sites, 1. Julian days on horizontal axes (e.g., June 1 = day 153, July 1 = day 13, August 1 = day 1). 15 5 15 5 Kettle 1 SAMPLES 1 SAMPLES 1 1

1 Journal of Vector Ecology June 13 F/D 1. 1....5 1 1.5 SHRUB DENSITY Figure. Ratio of Ixodes scapularis nymphs captured by flagging/ dragging (F/D) as a function of shrub density (shrubs m - ). Data points presented are from Wisconsin (-1), Cape Cod (11, 1) and Rhode Island (1) sample sites. paralysis tick, in South Africa. In our samples, the ratio of nymphs collected by flagging compared to dragging did not change significantly as the density of shrubs increased (Figure ). These data points are not strictly independent because some represented samples in different years from the same arrays. Nevertheless, the overall variability in this ratio was such that we could not confirm a clear trend in the relative effectiveness of these two techniques as shrub density increased. Dragging was difficult at our sites with the highest shrub densities because the drag frequently caught on the vegetation and often rode over the top of the shrubs, well above the leaf litter layer. Conversely, when unimpeded, drags may have collected more ticks than flags because a larger collecting surface area contacted the leaf litter, even if both methods used the same size material. Furthermore, we measured shrub density based on stem density at ground level but did not consider the spread of the various shrub species above the base, which could influence sampling efficacy. Finally, we did not characterize heterogeneity of the vegetation at the sampling arrays, which featured open and dense shrub areas arranged in complex patterns, which could have affected the efficacy of sampling. Nevertheless, the large degree of variability among samples outweighed any differences in sampling effectiveness between these two techniques. We emphasize that this conclusion applies primarily to leaf-litter dwelling ticks, such as I. scapularis nymphs, and not necessarily to ticks that quest higher in vegetation, such as Dermacentor variabilis, Amblyomma americanum, or I. scapularis adults. The flags and drags used in our study all consisted of square 1 m pieces of material for purposes of comparison. However, different dimensions and configurations of material can potentially alter performance. For example, Bouseman et al. (199) used a drag that was composed of weighted strips to foster contact with leaf litter when pulled through vegetation. Tack et al. (11) found that a whole blanket method collected more I. ricinus nymphs than a strip blanket technique. Carroll and Schmidtmann (199) developed a tick sweep, which was a flag attached to a rod angled at the end of a shaft that allowed a drag-type piece of material to be run through leaf litter. The configuration of any sampling device can be optimized based on the needs of the study. However, our results do not support a consistent difference between flagging and dragging methods in collecting leaf litter-dwelling ticks, and suggest that a choice between these sampling techniques for nymphal I. scapularis in any given study can be selected based on considerations other than sampling effectiveness in woodland habitat types. Acknowledgments We thank the staffs of Fort McCoy Garrison, Cape Cod National Seashore, the Kettle Pond Visitor Center (U.S. Fish and Wildlife Service), and Andrew Hamilton for permission to sample on their land and for logistical support. J. Bondesen, R. Burke, R. Gerhold, K. Kerr, L. Kramer, T. Lewis, F. Mackechnie, M. Mackenzie, L. Maestas, T. Moody, H. Edwards, W. Allen, J. Parmer, and A. Scholze assisted with planning, field sampling, and interpretation. Prof. Liliana Gonzalez provided statistical advice. Use of trade or product names in this paper does not imply endorsement by the U.S. Government. 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