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  • All mouse and rabbit handling and tick feeding protocols were approved through Michigan State University’s Institutional Animal Care and Use Committee (AUF # 06/09-094-00). Permission for field site use and to conduct the research was obtained from Tall Timbers Research Station and Land Conservancy, the University of Tennessee’s Forest Resources AgResearch and Education Center (FRAEC) and Fort McCoy Military Installation. In 2011, we measured the climbing behavior of nymphal I. scapularis placed at a field site at Fort McCoy Garrison, Wisconsin (latitude 44.01°N), a LD endemic area. In 2012, we expanded our study to include two LD endemic and two non-endemic sites. The 2012 LD endemic sites comprised Fort McCoy plus a site near Kingston, Rhode Island (latitude 41.48°N); the non-endemic sites were near Oak Ridge, Tennessee (latitude 36.01°N) and at Tall Timbers Research Station, Florida (latitude 30.53°N). All sites were located in mixed deciduous forests. The forest at Fort McCoy was dominated by various oaks (Quercus spp. ), pines (Pinus spp.) and red maples (Acer rubrum), with a shrub layer of mostly tree saplings. The Rhode Island site was dominated by red maple (A. rubrum), white pine (Pinus strobus), and white oak (Q. alba), with tree saplings in the shrub layer. The Tennessee forest was dominated by upland oaks (Quercus spp. ), hickory (Carya spp.) and yellow poplar (Liriodendron tulipifera), with a mixed understory containing various saplings and several invasive understory species. The Florida forest was dominated by oak (Quercus spp. ), maple (Acer spp. ), interspersed with shortleaf pine and a shrub layer dominated by tree saplings. Average canopy cover estimates for the sites during the study months ranged from 86%-92%. Meteorological measurements (temperature and relative humidity) were recorded hourly at each site in both years, using paired iButton data loggers (Hygrochron, Dallas Semiconductor) placed just below the surface of the leaf litter level (0 cm) and above ground (10 cm). The laboratory-reared nymphs used in 2011 originated from 22 (WI = 8, SC = 14) engorged female I. scapularis collected in November 2010 from hunter harvested deer at check stations in Monroe County, Wisconsin (latitude 44.01°N) and Aiken County, South Carolina (latitude 33.56° N). Engorged females were allowed to oviposit in individual vials in humidity chambers at 21°C and 98% relative humidity at 16:8 (L:D) hour photoperiod conditions. At 2 to 7 weeks of age, the resulting larvae were fed on female laboratory mice (ICR (CD-1) strain, Mus musculus) and allowed to molt into nymphs; at 2 to 4 weeks of age, these nymphs were transferred to the field sites. Seven groups of nymphs (3 WI, 4 SC) from the 2011 colonies were used to propagate a second generation of nymphs that were used in the 2012 experiments. Two of these groups were siblings of the nymphs used in the 2011 experiments, while the remaining five groups were from other females collected at the same locations and time as the mothers of the 2011 nymphs (S4 Table). Nymphs were fed on female laboratory mice and engorged nymphs were housed individually in vials where they were allowed to molt into adults. Adults of a single origin (WI or SC) were then mated together on New Zealand White rabbits (Oryctolagus cuniculus) in November 2011. The resulting engorged females were maintained as described above through the ovipositional period. Resulting larvae were reared to nymphs using the 2011 protocol. In November 2011, 5 additional engorged females were collected from hunter killed deer at check stations in Hyde County, North Carolina (latitude 35.50°N) and Aiken County, South Carolina (latitude 33.56°N) to provide additional nymphs for the 2012 experiment. Experimental apparatus and questing observations: Ticks originating from Wisconsin (WI) and South Carolina (SC; 2011 and 2012) and North Carolina (NC; 2012) were placed at field sites in the eastern US (Wisconsin (WI) in 2011; WI, Rhode Island (RI), Tennessee (TN), and Florida (FL) in 2012). The design of the 2011 experiment consisted of 5 blocks, each containing 4 arenas. All naturally-occurring ticks were removed by heat-treating locally-obtained leaf litter before adding it to the arenas. Two arenas were not seeded with nymphs and served as experimental controls for the effectiveness of the arena barrier and leaf litter heat treatment. In 2011, there were 5 individual sightings of a single nymph (which was not removed when sighted) in the treated control arenas. No nymphs were recovered from the treated controls during the survival assessments. In 2012, to provide further assurance that all preexisting, local ticks were removed from the leaf litter after heat treatment and before the experimental nymphs were released, we conducted microdrags (pressing a 12 cm x 12 cm square of white flannel material against the leaf litter inside of the arenas) and carbon dioxide (CO2) assays (dry ice baits) in each arena at each site. In 2012, no nymphs were observed in control arenas at any of the four sites, and no nymphs were recovered from these arenas during end-of-study survival assessments. The arena design was modified from previously published apparatuses used for measuring Ixodes spp. questing behavior in natural field conditions [52, 74]. Each arena consisted of a 0.5 m diameter circle of 25 cm high aluminum flashing sunk ~7 cm into the ground. A 6 cm blockade of Tree Tanglefoot Insect Barrier (Contech) was applied to the top inner rim of the arenas to prevent ticks climbing out [74]. Inside the arenas we installed 24, 3.0 mm wide bamboo dowel rods (stems), of three heights, 5 cm, 10 cm, and 20 cm. The stems were spaced in a semi-regular pattern, with the height of stem at each position randomized. These stems served to mimic understory vegetation that ticks can climb in their natural environment. Arenas were grouped in blocks of four and surrounded by a 60 cm wire mesh (2.54 cm) barrier and covered with a wire mesh lid (S4 Fig). This excluded large and medium-sized terrestrial species and birds from the arenas, while the aluminum flashing walls provided a barrier to deflect smaller terrestrial vertebrates. In 2011, 16 arenas in Wisconsin each received 44–60 lab-reared nymphs of a single geographic origin (8 received WI nymphs; 8 received SC nymphs). Due to limited availability of lab-reared nymphs, some arenas contained nymphs from multiple mothers originating from the same geographic origin (S4 Table). Nymphs were deposited into the arenas on May 23, 2011. Observers, who were blind to the origin of nymphs in the arenas, recorded the number of nymphs visible on the stems during a two minute observation of the arena (Fig 3) at bi-hourly intervals during three 24-hour (June 15–16, July 7–8, July 29–30) and one 14-hour (July 5) sampling periods. Control arenas were checked in the same manner during each sampling visit. This sampling design was employed because it was not known whether I. scapularis nymphs from different origins might have divergent patterns of diel activity. The questing behavior of the SC versus WI nymphs was compared statistically based on the log-odds of their presence on stems. Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0127450.g003 Ixodes scapularis nymph questing on stem in experimental arena.1 cm of a 10 cm dowel is visible. Photo by G. Hickling. In 2012, the experimental design at each site consisted of four blocks of four arenas, each arena containing 37–62 nymphal ticks from a single geographic origin (5 from WI, 3 from NC, and 8 from SC) and two unseeded additional arenas which served as controls for the leaf litter and arena barriers. The WI site had two additional arenas containing SC derived ticks. Arenas were established using the same protocol as 2011, except that the number of stems used was reduced from 24 to 15 (5 of each of the same heights used in 2011) leaving a larger area of “stem-less” leaf litter adjacent to the arena walls. Spacing between stems remained the same in both years. This design was replicated at each of the four field site locations. Nymphs were deposited into the arenas during the 1st week of May, 2012. Nymph questing in arenas was recorded by observers blinded to nymph origin in the morning (approx. 0800 hours) and late afternoon (approx. 1600 hours) at weekly or biweekly intervals from May–September 2012. Additionally, a total of 18 midnight observations were carried out at 3 of the sites (FL, TN, and WI). We chose these times to conduct our observations based the periods of highest-activity of WI and SC nymphs observed in 2011 (S1 Fig). In 2012, we expanded our definition of nymph questing to include ticks on the leaf litter and arena wall, as well as on stems, after observing nymphs in these locations during the 2011 observations. We hypothesize that all such nymphs emerged from the leaf litter represent a potential risk to human hosts, not just those on stems. As ticks do not jump or fly, they must make direct contact with a host in order to attach and acquire a bloodmeal [75]. Ticks emerged from the leaf litter can instantly position their forelegs in the air to attach to a passing host. Ticks under the leaf litter (not emerged) would have difficulty making direct contact with hosts walking upon the leaf litter, as the leaf litter would create a barrier between the nymph’s forelegs and the host body. The questing behavior of the nymphs was again assessed by estimating the log-odds of observing emerged nymphs. In 2012, each site had its own set of observers. Once behavioral observations were complete (in July 30, 2011 and mid-September 2012, respectively), we assessed the relative survival of the nymphs in each arena by conducting searches of the arena litter. In 2011, we placed air-activated hand warmers (Grabber, Byron Center, Michigan), wrapped in white flannel into the arenas for 1.5–2 hours. Nymphs attracted to the heat were removed from the flannel and placed in 95% ethanol. A 12 cm x 12 cm square of white flannel material was then pressed against the leaf litter inside of the arenas (= microdrag) and rustling the leaf litter to stir it up and expose subsurface dwelling nymphs. After the initial microdrag, a second round of microdrags was performed, again stirring the leaf litter and with moving the cloth through the leaf litter to contact in the sub-surface layers. Individuals were placed in 95% ethanol to preserve their field collected condition. In 2012, the heat pack method was abandoned because it appeared to preferentially target WI ticks (captured 53.9% of all WI ticks recovered) over SC ticks (captured 6.0% of all SC ticks recovered) and survival was assessed using only the microdragging method. We used a Bayesian approach for predicting the log-odds of nymph questing behavior as a function of nymph geographic origin. No p-values are reported; but rather summaries of the posterior distributions generated from models using the data obtained. These posterior distributions describe the plausibility of possible parameter values generated from the model, given the data we observed [76]. Our goals were to quantify questing behavior and evaluate the strength of evidence for effect of geographic origin on the questing behavior of I. scapularis nymphs. We used a multilevel binomial regression model to predict the log-odds of observing questing nymphs in the arenas. Questing behavior was measured by counting the number of nymphs visible on stems (2011), or on stems, leaf litter and arena walls (2012) during a given two-minute observation of an arena. Similarly, survival was measured using a multilevel binomial regression model to predict the log-odds of recovering nymphs from arenas at the end of each study period. Survival was measured by tallying the number of nymphs recovered from arenas at the end of each experiment. We adopted a multilevel modeling approach (see Gelman and Hill [77] for an overview) for the reasons outlined by McElreath and Koster [76]; briefly, the approach simultaneously addresses our concerns regarding repeated measures and imbalanced sampling. The questing behavior models allowed for nymph questing to vary by individual arena, observation date, state of origin, and (in 2012) site of observation. The survival models allowed for nymph recovery to vary by individual arena, state of origin, and (in 2012) site of observation. Models were fitted using Stan 2.3.0 [78], a Hamiltonian Monte Carlo sampler, to draw samples from the joint posterior density of the parameters. We used weakly informative regularizing priors to analyze the data. The results we present are based on estimates derived from 3,000 samples of each parameter, after 1,000 samples for adaptation. Convergence was assessed by trace plots. To determine if the regression coefficients of the nymph origins were credibly different from one another, we estimated the posterior distribution of the difference between the coefficients. Coefficients were considered to be credibly different if the HDIs of the posterior distribution of their difference did not encompass zero [48]. Model code was generated using a convenience package for Rstan known as map2stan [79]. To visualize the results, predicted log-odds and the associated highest density interval (HDI) were back-transformed into probabilities. All statistical analyses were undertaken using R 3.1.0 (http://www.r-project.org).
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