Different Populations of Blacklegged Tick Nymphs Exhibit Differences in Questing Behavior That Have Implications for Human Lyme Disease Risk

May Different Populations of Blacklegged Tick Nymphs Exhibit Differences in Questing Behavior That Have Implications for Human Lyme Disease Risk Isis M. Arsnoe 0 1 Graham J. Hickling 0 1 Howard S. Ginsberg 0 1 Richard McElreath 0 1 Jean I. Tsao 0 1 0 1 Department of Fisheries and Wildlife, Michigan State University , East Lansing, Michigan , United States of America, 2 Center for Wildlife Health, University of Tennessee Institute of Agriculture , Knoxville , Tennessee, United States of America, 3 United States Geological Survey Patuxent Wildlife Research Center, Rhode Island Field Station, University of Rhode Island , Kingston , Rhode Island, United States of America, 4 Department of Anthropology and Center for Population Biology, University of California Davis , Davis, California , United States of America 1 Academic Editor: Ulrike Gertrud Munderloh, University of Minnesota, UNITED STATES Animal behavior can have profound effects on pathogen transmission and disease incidence. We studied the questing (= host-seeking) behavior of blacklegged tick (Ixodes scapularis) nymphs, which are the primary vectors of Lyme disease in the eastern United States. Lyme disease is common in northern but not in southern regions, and prior ecological studies have found that standard methods used to collect host-seeking nymphs in northern regions are unsuccessful in the south. This led us to hypothesize that there are behavior differences between northern and southern nymphs that alter how readily they are collected, and how likely they are to transmit the etiological agent of Lyme disease to humans. To examine this question, we compared the questing behavior of I. scapularis nymphs originating from one northern (Lyme disease endemic) and two southern (non-endemic) US regions at field sites in Wisconsin, Rhode Island, Tennessee, and Florida. Laboratory-raised uninfected nymphs were monitored in circular 0.2 m2 arenas containing wooden dowels (mimicking stems of understory vegetation) for 10 (2011) and 19 (2012) weeks. The probability of observing nymphs questing on these stems (2011), and on stems, on top of leaf litter, and on arena walls (2012) was much greater for northern than for southern origin ticks in both years and at all field sites (19.5 times greater in 2011; 3.6-11.6 times greater in 2012). Our findings suggest that southern origin I. scapularis nymphs rarely emerge from the leaf litter, and consequently are unlikely to contact passing humans. We propose that this difference in questing behavior accounts for observed geographic differences in the efficacy of the standard sampling techniques used to collect questing nymphs. These findings also support our hypothesis that very low Lyme disease incidence in southern states is, in part, a consequence of the type of hostseeking behavior exhibited by southern populations of the key Lyme disease vector. - Competing Interests: The authors have declared that no competing interests exist. The blacklegged or deer tick (Ixodes scapularis) vectors Borrelia burgdorferi, the etiological agent of Lyme disease (LD), which is the most common vector-borne disease in the United States (US) [1]. Despite the widespread presence of blacklegged ticks throughout the eastern US [2, 3], there is pronounced geographical variation in LD incidence in that region. Ninetyfive percent of human LD cases in the US are reported from Lyme endemic states in northeast and upper midwest regions [4]; LD incidence in southeast regions is over an order of magnitude lower [5]. We refer to this latitudinal variation in disease incidence in the eastern US as the Lyme Disease Gradient. Ixodes scapularis has three parasitic life stages (larva, nymph, adult), but only the bites of nymphs and adults transmit B. burgdorferi. There is neglible transovarial transmission of the pathogen therefore larvae are considered to be born uninfected [6]. The nymphal stage is regarded as the most epidemiologically important for LD transmission because of its small size and spring/summer seasonality that coincides with human outdoor activity [7, 8]. The density of infected nymphs (DIN) is considered a useful predictor of LD risk [915]. Factors that influence DIN (including tick survivorship [16], host composition [17, 18], and abiotic variables [16]) are thought to influence the magnitude of risk [11, 1921]. DIN is calculated by multiplying the density of nymphs collected in a given area by the B. burgdorferi infection prevalence of those nymphs [9, 14, 15, 22]. Ticks in such studies are typically collected by dragging or flagging a 1 m2 white cloth through the understory vegetation, as this is considered to be the most reliable and efficient method for sampling nymphal I. scapularis populations in the northeastern US [23, 24]. These methods intentionally target ticks that are questing (= host-seeking) on or above the leaf litter, as these are the ticks most likely to encounter humans. Flagging and dragging are less likely to collect ticks beneath the surface of the leaf litter; however, since such ticks are unlikely to encounter humans they presumably contribute little to LD risk. Quantification of DIN by flagging/dragging thus provides a useful index of human-nymphal encounter rates. Indeed, DIN is highly correlated with the Lyme Disease Gradient [14, 25], providing support for its use as an index of human LD risk. Several hypotheses for the Lyme Disease Gradient have been proposed. These include geographic variation in large-scale predictors such as climatic variables [2629], biodiversity [10, 11, 19, 20, 30], and tick genetics [31, 32], which are known and/or believed to influence the abundance of questing infected nymphs. These predictors help us understand the ultimate causes of risk variation, but for planning and executing effective intervention strategies, a mechanistic understanding of factors underlying the Lyme Disease Gradient is key. A key mechanistic factor that needs to be considered for the LD system is vector behavior. With vector-borne diseases, it is typically assumed that risk of contact with an infected vector, and therefore risk of disease, is proportional to the abundance of infected vectors [15, 33, 34]. Behavior of both hosts and vectors influences the likelihood of encounter and thus the risk of disease, and these behaviors might differ at different sites. A mechanistic hypothesis is that nymphal questing behavior varies regionally, leading to differences in tick/human contact rates that contribute to the Lyme Disease Gradient. At least three lines of evidence support this questing behavior hypothesis. First, drag/flag sampling efficacy differs between LD endemic and non-endemic regions. These standard methods readily collect all three tick life stages in northeastern and upper midwestern regions [3, 23, 35], whereas in southeastern regions, they collect very few nymphs even at sites where adult I. scapularis are readily flagged or dragged [3, 3638]. T (...truncated)