Location, Location, Location: Geographic source of blacklegged tick (Ixodes scapularis) nymphs determines behavioral outcomes in laboratory studies

Location, Location, Location: Geographic source of blacklegged tick (Ixodes scapularis) nymphs determines behavioral outcomes in laboratory studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Location, Location, Location: Geographic source of blacklegged tick ( Ixodes scapularis ) nymphs determines behavioral outcomes in laboratory studies Karen C. Poh, Jessica E. Brown, Mia I. Esoldo, Erika T. Machtinger This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7312978/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Ixodes scapularis is considered a significant medical and veterinary arthropod pest, capable of transmitting several pathogens that cause disease in humans and animals. Previous work has identified two distinct populations of I. scapularis in the United States (northern and southern), characterized by differences in their genetics and behavior. This study aimed to characterize and compare the lateral movement and feeding behaviors of nymphal I. scapularis between the northern and southern populations in the United States. Using laboratory-reared ticks from BEI (northern), Oklahoma State University (southern), and field-collected ticks from central Pennsylvania (Mid-Atlantic), behavioral bioassays were conducted to quantify distances traveled and velocities in a one-hour time frame. Ticks from the northern lineage walked longer distances and at faster speeds compared to ticks from the southern lineage. Field-collected ticks from central Pennsylvania, located between what is considered the northern and southern populations, exhibited similar movement behaviors as ticks from the southern population, even though ticks from the Mid-Atlantic are geographically categorized as the northern population. To compare feeding behaviors, colony-reared white-footed mice ( Peromyscus leucopus ) were artificially infested with northern and southern ticks, and the percentages of infestation and feeding successes were compared. Northern ticks had higher success in infestations and feeding to repletion compared to southern ticks. These behavioral differences in movement and feeding patterns provide additional evidence for the hypothesis that geographically distinct populations of I. scapularis exist across the United States. Researchers should consider these population differences when selecting tick lineages for behavioral studies and other blacklegged tick research. Behavior parasite Lyme disease infestation genotypes ticks Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The blacklegged tick ( Ixodes scapularis Say, Acari: Ixodidae) is considered the most significant vector of tick-borne pathogens in North America, capable of transmitting Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic anaplasmosis), Babesia microti (human babesiosis), and Powassan virus (Eisen and Eisen 2018 ). Although blacklegged ticks are found throughout the northeastern, Mid-Atlantic, northcentral, and southern United States, these regions have varying levels of reported tick-borne diseases associated with the blacklegged tick (Rosenberg et al. 2018 ). Differences in disease incidence may be related to variations in the ecology of I. scapularis , resulting from differing demographic histories in their respective geographic regions (Norris et al. 1996 , Ginsberg et al. 2014 , Sakamoto et al. 2014 , Arsnoe et al. 2015 , 2019 ). The blacklegged tick comprises at least two distinct populations: northern and southern (Norris et al. 1996 , Van Zee et al. 2013 , Sakamoto et al. 2014 ). These populations exhibit notable differences in genetics, survival patterns, host attachment preferences, and questing behavior. Numerous studies have documented these regional variations in I. scapularis biology and ecology (Piesman and Spielman 1979 , Carey et al. 1980 , Mather et al. 1989 , Apperson et al. 1993 , Goddard 1993 , Norris et al. 1996 , Durden et al. 2002 , Piesman 2002, LoGiudice et al. 2003 , Goddard and Piesman 2006 , Van Zee et al. 2013 , Ginsberg et al. 2014 , Sakamoto et al. 2014 , Arsnoe et al. 2015 , 2019 , Curtis et al. 2020 , Tietjen et al. 2020 ). Understanding the differences between the two distinct lineages of blacklegged ticks could have important implications for prediction, prevention, and control. For example, information on questing and horizontal movement could potentially improve tick bite risk models by accounting for differences in movement per lineage (Hassett et al. 2022 , Marshall et al. 2025 ). Furthermore, understanding the feeding patterns of different tick populations on reservoir hosts could clarify predictions about tick-borne disease risk. Questing behavior (vertical movement) differs between I. scapularis populations in the United States, with northern populations questing more frequently and at greater heights than their southern counterparts (Arsnoe et al. 2015 , 2019 , Tietjen et al. 2020 ). While lateral movement has been evaluated separately for adult I. scapularis from Mississippi and New York (Goddard 1993 , Curtis et al. 2020 ), direct comparisons of horizontal movement between northern and southern lineages remain lacking, particularly for nymphal stages. Similarly, potential behavioral differences in host infestation and feeding success between these populations have not been systematically assessed. When evaluating tick behavior, it is important to consider the impact of the source population. Therefore, our study aims to identify differences in movement and feeding behavior of nymphal blacklegged ticks from these two distinct lineages. This study compared the horizontal movement (distance) and speed (velocity) of nymphal I. scapularis from northern and southern lineages, as well as an additional lineage that originates from an area that lies between the northern and southern populations (central Pennsylvania). Host infestation and feeding differences between northern and southern lineages were also evaluated. Materials and Methods Movement Assessment For this study, I. scapularis that are found in the northeast and central regions of the United States north of the Chesapeake Bay are considered the “northern” population, and ticks found south of this region are the “southern” population (Ginsberg et al. 2014 ). Nymphal I. scapularis ticks were acquired from three locations: (1) BEI Resources (ATCC, Manassas, VA), which was started with ticks collected in Rhode Island (northern ticks); (2) Oklahoma State University Tick Lab (Stillwater, OK), which was started using ticks collected in Oklahoma (southern ticks); and (3) several field locations in central Pennsylvania (Centre County, PA) (Mid-Atlantic ticks). After ticks were received or collected, they were stored in snap cap vials with modified lids and mesh to allow airflow (Levin and Schumacher 2016 ). Vials of ticks were then stored in a humidity chamber at 80–95% RH. The chamber was housed in an environmental chamber set at 22°C and a L:D setting of 16:8 (Troughton and Levin 2007 , Levin and Schumacher 2016 ). Once ready for observation in the experiment, individual ticks were removed from their vials using a pair of featherweight forceps or a paintbrush. To ensure ticks were alive before the start of the experiment, ticks that were actively moving in the vial were chosen. During each bioassay, ticks were observed between 0800 and 1300 h (Schulze et al. 2001 , Schulze and Jordan 2003 ). A similar temperature (20.3 ± 0.8°C) and relative humidity (85.5 ± 6.1%) were maintained for the bioassay using a portable heater and humidifier, respectively, within the humidity chamber. One nymphal tick lineage was randomly chosen for observation for each replicate. A tick was placed into a 100 x 15 mm glass petri dish (Pyrex, Corning, Inc., Glendale, AZ) and was given approximately five minutes to acclimate before recording began. Tick movement was recorded using the Basler acA1300-60gm GigE camera (Basler AG, Ahrensburg, Germany) at a frame rate of 30 frames per second and a resolution of 1280 x 1024 pixels. The camera feed was connected to a laptop running MediaRecorder 6 (Noldus, Wageningen, the Netherlands), which recorded and stored the video. The video recording was then imported into EthoVision XT 15 (Noldus, Wageningen, Netherlands) to quantify movement. After acclimation, the camera recording was started, and tick movement was tracked for one hour. Once completed, the tick was removed and not reused. While ticks that seemed active in the vials were chosen for the experiment, the tick was stimulated at the end of the experiment by breathing air to check that the ticks did not expire while being recorded. If ticks did not respond, the recording was not used. The petri dish was washed with unscented soap and water, dried, and then replaced under the camera. Multiple replicates were completed in one day, with all experiments ending by 1300 h. Preliminary trials of the experiment revealed that ticks reduced or completely stopped their movement after approximately 2.5-3 hours of the experiment starting, which would skew average velocity calculations. Before being imported into EthoVision, recordings were reviewed to ensure no artifact movements (i.e., movements other than the tick) were detected and that the camera maintained a clear view of the tick for the entire observation period. If either occurred, the recording was not used in the analysis. Once the videos passed inspection, they were imported into EthoVision XT 15 and analyzed by the software, which calculated the total distance the tick moved (in cm) and its average velocity (in cm/s). After removing videos with artifacts or obscured views, 60 replicates of each of the northern and Mid-Atlantic lineages and 58 replicates of the southern lineage were included in analyses. Total distances and average velocities of each tick population were compared using a one-way ANOVA, calculated using the aov package in the statistical program R (R Core Team 2024). Post-hoc pairwise comparisons were conducted using Tukey’s HSD. Significance was set at α = 0.05. Tick Feeding Behavior Assessment Mice were artificially infested with ticks to examine differences in tick infestation and feeding patterns between the northern and southern lineages of I. scapularis . White-footed mice ( Peromyscus leucopus ) between the ages of 3–24 months were used in the experiment. Animals used were from the F1 and F2 generations of an in-house, wild-caught colony of Peromyscus leucopus and housed according to approved mouse-rearing standards of The Pennsylvania State University. Housing and experimental procedures were conducted according to approved Institutional Animal Care and Use Committee (IACUC) protocols (PROTO202101808, PROTO202001598) and met the requirements of the Public Health Service Policy on Humane Care and Use of Laboratory Animals. A total of 66 animals (33 males, 33 females) were used in the experiments and included in the statistical analyses. In infestations involving northern ticks, 21 male and 23 female mice were used, while infestations with southern ticks used 12 male and 10 female mice. Because pathogens within field-collected ticks could not be verified before infestation, field-collected tick testing was not approved by the IACUC; thus, only northern and southern lineages of nymphal I. scapularis were used for the tick feeding behavior assessment. In each round of infestations, three mice were infested with ticks of either lineage as part of complementary projects evaluating rodent behavior to tick infestation. Infestation procedures followed Brown et al. (2025). To infest mice, nymphal ticks were added to white cotton crew socks, which were held open by small plastic cups. The number of ticks used varied based on lineage-specific attachment rates and experimental requirements. For the northern lineage, 15–30 ticks were used per sock, while for the southern lineage, 30–60 ticks were used per sock due to consistently lower attachment rates observed in preliminary trials. All socks in one round were infested with the same lineage of ticks. After mice were anesthetized and added to the sock, a square metal bulldog clip was used to close the sock. The cups holding the socks were then moved to secondary containment. The ticks were given three hours to infest the mice before the mice and attached ticks were moved to Noldus Phenotyper cages (Model PT3000; Noldus, Wageningen, Netherlands). Ticks remaining in the sock were counted and subtracted from the original number added (depending on the lineage) to obtain the approximate number of ticks that successfully infested the mice. The percentage of successful infestation was calculated as the number of ticks that infested mice divided by the original number of ticks added to the sock multiplied by 100. Mice were observed in the Phenotyper cages for four days, and the number of replete nymphs was counted daily. The percentage of successful feeding was calculated as the total number of replete ticks divided by the number of ticks that successfully infested mice, expressed as a percentage, and then multiplied by 100. Wilcoxon signed rank tests were performed in R to determine if the percentages of successful infestation and successful feeding differed between the northern and southern lineages of I. scapularis . Significance was set at α = 0.05. Results Overall, the distance traveled by each nymphal I. scapularis lineage was significantly different (p < 0.01, df = 2) (Fig. 1 ). In post-hoc analyses, ticks associated with the northern lineage traveled significantly farther in one hour (mean ± SE = 248.4 cm ± 20.8 cm) compared to ticks from the southern or Mid-Atlantic lineages (p < 0.01). Ticks with southern or Mid-Atlantic lineages traveled similar distances (p = 0.31), traveling approximately 155.5 ± 27.2 cm and 150.7 ± 14.4 cm, respectively, during the observation period. Like distances traveled, the average velocities of nymphal I. scapularis from various populations were significantly different (p = 0.05, df = 2) (Fig. 2 ). Nymphs from the northern lineage traveled at a higher velocity (0.078 ± 0.006 cm/s) compared to ticks with southern (p < 0.01, 0.050 ± 0.009 cm/s) and Mid-Atlantic lineages (p = 0.02, 0.064 ± 0.008 cm/s). Furthermore, nymphal ticks from the Mid-Atlantic had higher velocities compared to those from the south (p < 0.01). While the movement experiments were done in the absence of host signals and odors, which could influence movement patterns, the infestation and feeding trials illuminated significant differences between the northern and southern lineages of I. scapularis in the presence of a host. The northern population of I. scapularis were more likely to successfully infest white-footed mice during the artificial infestation process compared to ticks from the southern population, with 60.4% ± 18.7% and 22.3% ± 13.0% of ticks successfully infesting mice, respectively (p < 0.01, W = 921.5) (Fig. 3 ). Of the ticks that successfully infested mice, northern lineage ticks were more likely to feed to repletion compared to southern lineage ticks (35.2% ± 26.4% and 18.5% ± 27.1 of ticks that successfully infested mice feeding to repletion, respectively) (p < 0.01, W = 709.5) (Fig. 4 ). Discussion It is important to understand the behavior of the target animal when designing behavioral experiments. In this study, we demonstrated significant differences in movement among three lineages of blacklegged ticks and feeding behavior between the northern and southern lineages. Our data corroborate previous studies, where ticks from a northern lineage tended to move farther distances and at faster velocities than those from a southern lineage (Arsnoe et al. 2015 , 2019 , Tietjen et al. 2020 ). Interestingly, Mid-Atlantic ticks were more similar in movement behavior to the southern population of I. scapularis compared to those from the northern population, even though Mid-Atlantic ticks would geographically be classified as members of the northern population (east and north of the Chesapeake Bay) (Ginsberg et al. 2014 ). The Mid-Atlantic ticks were collected from central Pennsylvania, which borders the proposed division between northern and southern blacklegged tick populations, emphasizing the need for additional research to define these populations. It is possible that Mid-Atlantic ticks could be a hybrid of both northern and southern lineage ticks, where the Mid-Atlantic ticks exhibit shared characteristics or genes from both lineages. This is supported by evidence that I. dammini in the north (before being reclassified as I. scapularis ) and I. scapularis from the south readily hybridize in the laboratory (James and Oliver 1990 ). Genetically, Sakamoto et al. ( 2014 ) identified two distinct clades of ticks in the United States, one that includes ticks from the northern and southern collection region and another clade that only includes ticks collected from the south. While the northern and southern lineages may have distinct demographic histories, the authors’ data support a previous hypothesis that I. scapularis may have originated in the south and a small founder population migrated to the north during the Pleistocene era (Norris et al. 1996 , Van Zee et al. 2013 , Sakamoto et al. 2014 ). This series of events could possibly explain the behavioral differences of all three lineages, where the Mid-Atlantic ticks could be related to both the northern and southern lineages. Another possible explanation for differences in movement between the Mid-Atlantic and the northern lineages could be due to the effects of colonization, where behavioral differences can arise if they are raised in colony (northern lineage) versus the field (Mid-Atlantic). Differences in behavior and physiology have been reported in several insects and arthropods. For example, anthrophilic mosquitoes that are laboratory-reared may change host preferences if they are habitually fed blood from a non-human source, thereby adapting to a new host (Laarman 1958 , Spitzen and Takken 2005 ). Behavioral differences between laboratory-reared and field-collected jumping spiders have also been reported, where the authors argued that the developmental histories of animals can affect how they respond to treatments in the laboratory (Wiggins et al. 2018 ). Changes in tick physiology due to differences in developmental history have also been reported. Field-caught Dermacentor variabilis were found to be more tolerant of dehydration compared to laboratory-reared ticks since field-caught ticks are more likely to undergo selective pressure due to less than optimal rearing conditions (Yoder et al. 2012 ). To the authors’ knowledge, this is the first report on behavioral differences between colony-reared and field-caught I. scapularis . Due to significant differences between the lineages of I. scapularis , future behavioral and physiological research should include testing both colony-reared and field-caught ticks to validate results. Differences in infestation and feeding success by nymphal ticks could indicate host preference in response to host availability in the respective geographic regions, where immature northern ticks parasitize small mammals such as white-footed mice, voles, or chipmunks (Piesman and Spielman 1979 , Carey et al. 1980 , Mather et al. 1989 , LoGiudice et al. 2003 ), while the southern counterparts may parasitize small mammals and other animals including birds and lizards (Spielman et al. 1984 , Apperson et al. 1993 , Durden et al. 2002 , Piesman 2002, Goddard and Piesman 2006 ). Preferences for certain hosts may additionally account for the differences seen in successful infestations of white-footed mice. During animal infestations in this experiment, ticks from the southern lineage rarely attached to white-footed mice, even though they are considered permissive hosts for I. scapularis (Anderson and Magnarelli 1980 , Ostfeld et al. 1996 , Brown et al. 2023 , Caron-Lévesque and Careau 2023 ), with only 22.3% of the ticks infesting the mouse and only 18.5% of those ticks feeding successfully to repletion (Fig. 3 ). The results in this study deviate from results in a previous study establishing that nymphal I. scapularis from Great Island, Massachusetts and Statesboro, Georgia, exhibited similar feeding success rates and host preferences for both mice and lizards (James and Oliver 1990 ). Even when in close proximity to a host, southern I. scapularis rarely attached, which could be driven by preferences for certain host cues to identify their hosts, such as host odors or heat. Southern I. scapularis did not infest mice at the rate of the northern lineage. Still, there were some successful infestations, indicating that southern I. scapularis recognized that a host was available. Still, they may have shifted or adapted their host preferences to other hosts that are more abundant in their region (i.e., lizards) (Spielman et al. 1984 , Tietjen et al. 2020 ). Differences in heat production by hosts could additionally explain why I. scapularis from the southern lineage did not frequently attach to the white-footed mice, where endotherms (i.e., small mammals) may not elicit a response from ticks compared to ectotherms (i.e., lizards). One study reported that when southern I. scapularis were presented with a choice of a heating source or no heating source, the ticks did not show a preference for the heating element compared to the control (Otálora-Luna et al. 2022 ). While I. scapularis is a well-known vector of several pathogens in the United States, the behavioral response of I. scapularis to hosts is understudied, and comparing host preferences of two distinct tick lineages is even more so. Overall, tick behavior in response to host cues is complex and requires further research to understand the nuances in the ecological relationships between ticks and their hosts. Unknown pathogen infection status is one limitation of this study. Laboratory-reared ticks have been confirmed to be pathogen-free (Salazar 2015 ), however, field-collected ticks from the Mid-Atlantic region were not tested for pathogens. Pathogen infection in I. scapularis has been found to modify some behaviors (Benelli 2020 ). For example, nymphal I. scapularis infected with B. burgdorferi exhibited increased phototaxis and attraction to vertical surfaces in laboratory settings, but their questing height and ability to overcome physical obstacles were not significantly different from uninfected ticks (Lefcort and Durden 1996 ). When infected with Ehrlichia muris eauclairensis , nymphal I. scapularis moved faster during host-seeking compared to uninfected ticks (Aspinwall et al. 2025 ). In a different tick-borne disease system, Rickettsia -infected Dermacentor reticulatus females exhibited higher locomotor activity, resulting in longer trajectories compared to uninfected ticks (Pipová et al. 2023 ). The results from the present study showed that Mid-Atlantic ticks traveled faster than the southern lineage of ticks (Fig. 2 ), but both lineages traveled similar distances overall (Fig. 1 ). In contrast, the Mid-Atlantic ticks moved more slowly and traveled shorter distances compared to northern ticks. While the literature suggests that infected I. scapularis move faster, infection status would not entirely explain why the Mid-Atlantic ticks were still more similar to the southern lineage (e.g., slower and traveled less distance) compared to the northern lineage of pathogen-free ticks. In addition, 20–30% of nymphal blacklegged ticks in this region are infected with tick-borne pathogens (Brown et al. 2015 , Livengood et al. 2020 , Schwartz et al. 2022 ), but a bimodal response, suggesting infected and non-infected ticks behave differently, was not demonstrated in this study. However, discrepancies in tick behavior due to infection with pathogens in tick-borne disease systems highlight the need for future work on this topic. Another possible limitation of this study is that it is unknown whether the recorded movement is related to the effects of energy reserves on walking activity. For I. ricinus , ticks with higher fat content are expected to have more energy storage and thus, travel further than ticks with lower fat (Crooks and Randolph 2006 ). While colony-reared ticks may have more consistent ages and therefore energy storage, similar information was not available for field-collected Mid-Atlantic ticks, where differences in their life history (e.g., age, previous blood meals, distance traveled, time since last blood meal) could result in differences in energy budgets and behaviors (Alasmari and Wall 2020 ). While it would have been ideal to measure energy storage before the start of the trials, the current available protocols would result in specimen destruction. This work provides additional behavioral data that support the hypothesis of at least two distinct populations of blacklegged ticks in the United States. These differences have been previously supported by genetic, behavioral, and ecological studies comparing northern and southern populations of I. scapularis (Norris et al. 1996 , Van Zee et al. 2013 , Ginsberg et al. 2014 , Sakamoto et al. 2014 , Arsnoe et al. 2015 , 2019 , Tietjen et al. 2020 ). Collectively, this is the first report quantifying and comparing the horizontal movement of nymphal I. scapularis from two laboratory-reared populations representing different geographic regions and one field population from an area bordering the two regions. Furthermore, this research provides additional information on the differences in host infestation and feeding between two laboratory-reared colonies originating from different regions of the United States. These data provide empirical results on blacklegged tick movement and feeding behaviors that can be incorporated into tick bite risk or distribution models that account for ticks from different geographic regions. These results can also provide guidance for researchers conducting tick behavioral studies, where the choice of tick sources could affect interpretation of results and conclusions. Declarations Competing Interests The authors declare no competing interests. Ethical Approval Housing and experimental procedures involving Peromyscus spp. were conducted according to approved Institutional Animal Care and Use Committee (IACUC) protocols (PROTO202101808, PROTO202001598) and met the requirements of the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Funding Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI121495. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was additionally supported by the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04762 and Accession number 1025757. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer. Author Contribution Conceptualization: KCP, ETM; Methodology: KCP, JEB, MIE, ETM; Formal analysis and investigation: KCP, JEB, MIE, ETM; Writing - original draft preparation: KCP, JEB, MIE, ETM; Writing - review and editing: KCP, JEB, MIE, ETM; Funding acquisition: ETM; Resources: ETM; Supervision: KCP, ETM Acknowledgement The authors would like to thank Graham Hickling for sharing the protocol for effectively infesting mice with blacklegged ticks in the lab and Kylie Green for managing the wild-caught colony of white-footed mice. In addition, we would like to thank Arash Maleki and Jesse Evans for their assistance with running trials, collecting field ticks, and/or providing feedback and expertise on the experimental design. We would also like to thank Jake Perryman, Melissa Welker, Dr. Jeffery Dodds, Dr. Sima Bruggeman, and the rest of the Animal Resource Program staff for their vital roles in animal care. The following reagent was provided by the Centers for Disease Control and Prevention for distribution by BEI Resources, NIAID, NIH: Ixodes scapularis Nymph (Live), NR-44116. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Alasmari S, Wall R. 2020. Determining the total energy budget of the tick Ixodes ricinus . Experimental and Applied Acarology. Anderson JF, Magnarelli LA. 1980. Vertebrate host relationships and distribution of ixodid ticks (Acari: Ixodidae) in Connecticut, USA. 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Manual for maintenance of multi-host ixodid ticks in the laboratory. Exp Appl Acarol. 70(3):343–367. https://doi.org/10.1007/s10493-016-0084-8 Livengood J, Hutchinson ML, Thirumalapura N, et al. 2020. Detection of Babesia, Borrelia, Anaplasma, and Rickettsia spp. in Adult Black-Legged Ticks ( Ixodes scapularis ) from Pennsylvania, United States, with a Luminex Multiplex Bead Assay. Vector-Borne and Zoonotic Diseases. LoGiudice K, Ostfeld RS, Schmidt KA, et al. 2003. The ecology of infectious disease: Effects of host diversity and community composition on Lyme disease risk. Proceedings of the National Academy of Science. 100(2):567–571. https://doi.org/10.5586/aa.2009.011 Marshall DS, Poh KC, Reichard MV, et al. 2025. Spatial and temporal activity patterns of Amblyomma americanum . Parasites and Vectors. 18(12):1–10. Mather TN, Wilson ML, Moore SI, et al. 1989. Comparing the relative potential of rodents as reservoirs of the Lyme disease spirochete ( Borrelia burgdorferi ). American Journal of Epidemiology. 130(1):143–150. Norris DE, Klompen JSH, Keirans JE, et al. 1996. Population Genetics of Ixodes scapularis (Acari: Ixodidae) Based on Mitochondrial 16S and 12S Genes. Journal of Medical Entomology. 33(1):78–89. https://doi.org/10.1093/jmedent/33.1.78 Ostfeld RS, Miller MC, Hazler KR. 1996. Causes and Consequences of Tick ( Ixodes scapularis ) Burdens on White-Footed Mice ( Peromyscus leucopus ). Journal of Mammalogy. 77(1):266–273. https://doi.org/10.2307/1382727 Otálora-Luna F, Dickens JC, Brinkerhoff J, et al. 2022. Behavior of Nymphs and Adults of the Black-Legged Tick Ixodes scapularis and the Lone Star Tick Ambylomma americanum in Response to Thermal Stimuli. Insects. 13(2):130. https://doi.org/10.3390/insects13020130 Piesman J. 2002. Ecology of Borrelia burgdorferi sensu lato in North America. In: Gray J, Kahl O, Lane RS, et al., editors. Lyme Borreliosis: Biology, Epidemiology, and Control. 1st ed. Wallingford, United Kingdom: CABI Publishing. p. 223–249. [accessed 2025 Mar 12]. https://www.cabidigitallibrary.org/doi/abs/ 10.1079/9780851996325.0000 . Piesman J, Spielman A. 1979. Host-Associations and Seasonal Abundance of Immature Ixodes dammini in Southeastern Massachusetts. Annals of the Entomological Society of America. 72:829–832. https://doi.org/10.1093/aesa/72.6.829 Pipová N, Peňazziová K, Baňas M, et al. 2023. The Behavior of Rickettsia -Positive Dermacentor reticulatus Ticks under Laboratory Conditions. Life. 13(3):612. https://doi.org/10.3390/life13030612 Rosenberg R, Lindsey NP, Fischer M, et al. 2018. Vital Signs: trends in reported vectorborne disease cases — United States and territories, 2004–2016. Morbidity and Mortality Weekly Report. 67(17):496–501. https://doi.org/10.15585/mmwr.mm6717e1 Sakamoto JM, Goddard J, Rasgon JL. 2014. Population and Demographic Structure of Ixodes scapularis Say in the Eastern United States. Brissette CA, editor. PLoS ONE. 9(7):e101389. https://doi.org/10.1371/journal.pone.0101389 Salazar JL. 2015. Detection of tick-borne pathogens in lab reared tick colonies and wild populations [M.S.]. Oklahoma State University. [accessed 2025 Mar 14]. https://www.proquest.com/docview/1820918743/abstract/65C4608172314808PQ/1 . Schulze TL, Jordan RA. 2003. Meteorologically Mediated Diurnal Questing of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) Nymphs. Journal of Medical Entomology. 40(4):395–402. https://doi.org/10.1603/0022-2585-40.4.395 Schulze TL, Jordan RA, Hung RW. 2001. Effects of Selected Meteorological Factors on Diurnal Questing of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae). J Med Entomol. 38(2):318–324. https://doi.org/10.1603/0022-2585-38.2.318 Schwartz S, Calvente E, Rollinson E, et al. 2022. Tick-Borne Pathogens in Questing Blacklegged Ticks (Acari: Ixodidae) From Pike County, Pennsylvania. Rich S, editor. Journal of Medical Entomology. 59(5):1793–1804. https://doi.org/10.1093/jme/tjac107 [Software] R Core Team. 2024. R: A language and environment for statistical computing. http://www.r-project.org/ . Spielman A, Levine JF, Wilson ML. 1984. Vectorial Capacity of North American Ixodes Ticks. The Yale Journal of Biology and Medicine. 57:507–513. Spitzen J, Takken W. 2005. Malaria mosquito rearing – maintaining quality and quantity of laboratory-reared insects. Proceedings of the Netherlands Entomological Society Meeting. 16:95–100. Tietjen M, Esteve-Gasent MD, Li AY, et al. 2020. A comparative evaluation of northern and southern Ixodes scapularis questing height and hiding behavior in the United States. Parasitology.:1–34. https://doi.org/10.1017/s003118202000147x Troughton DR, Levin ML. 2007. Life Cycles of Seven Ixodid Tick Species (Acari: Ixodidae) Under Standardized Laboratory Conditions. Journal of Medical Entomology. 44(5):732–740. Van Zee J, Black WC, Levin M, et al. 2013. High SNP density in the blacklegged tick, Ixodes scapularis , the principal vector of Lyme disease spirochetes. Ticks and Tick-borne Diseases. 4(1–2):63–71. https://doi.org/10.1016/j.ttbdis.2012.07.005 Wiggins WD, Bounds S, Wilder SM. 2018. Laboratory-reared and field-collected predators respond differently to same experimental treatments. Behav Ecol Sociobiol. 72(2). https://doi.org/10.1007/s00265-017-2437-7 Yoder JA, Hedges BZ, Benoit JB. 2012. Water balance of the American dog tick, Dermacentor variabilis , throughout its development with comparative observations between field-collected and laboratory-reared ticks. International Journal of Acarology. 38(4):334–343. https://doi.org/10.1080/01647954.2011.647073 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Dec, 2025 Reviews received at journal 24 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers invited by journal 03 Sep, 2025 Editor assigned by journal 03 Sep, 2025 Submission checks completed at journal 19 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7312978","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511981105,"identity":"01368c1a-d457-4ce0-9fff-9297afae040f","order_by":0,"name":"Karen C. Poh","email":"","orcid":"","institution":"Pennsylvania State University","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"C.","lastName":"Poh","suffix":""},{"id":511981106,"identity":"771845bb-1d84-4043-9c88-9c46b301b89b","order_by":1,"name":"Jessica E. Brown","email":"","orcid":"","institution":"Pennsylvania State University","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"E.","lastName":"Brown","suffix":""},{"id":511981107,"identity":"cb68bed6-92b7-4c97-88a6-d9053c1d80f5","order_by":2,"name":"Mia I. Esoldo","email":"","orcid":"","institution":"Pennsylvania State University","correspondingAuthor":false,"prefix":"","firstName":"Mia","middleName":"I.","lastName":"Esoldo","suffix":""},{"id":511981108,"identity":"fa236ee3-3140-492c-b41d-6cbaee64ba25","order_by":3,"name":"Erika T. Machtinger","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYJCCAwkVEIYEcerZGBgPfDhDohbmgzPbSNHCL9+dcJh33h17gwPMB2/zEKNFso13w2Hebc8SNxxgS7YmSovBMbCWwwkGB3jMpInSYg/WMucw0GH834jTYsDGu+HgzIbDjBsO8LARp0XiWO6GAx+OPUuceZjN2HIOMVr4m89u/pBQc8ee73jzwxtviNECBQcYGJhJUA7VMgpGwSgYBaMAFwAAaT82qZKJbKIAAAAASUVORK5CYII=","orcid":"","institution":"Pennsylvania State University","correspondingAuthor":true,"prefix":"","firstName":"Erika","middleName":"T.","lastName":"Machtinger","suffix":""}],"badges":[],"createdAt":"2025-08-06 21:08:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7312978/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7312978/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90980140,"identity":"f97d9d46-ac10-48ff-954f-ecc4bff8ac71","added_by":"auto","created_at":"2025-09-10 09:13:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17056,"visible":true,"origin":"","legend":"\u003cp\u003eDistances moved by nymphal blacklegged ticks (\u003cem\u003eIxodes scapularis\u003c/em\u003e) from different lineages found in the United States. The “southern” lineage refers to ticks from the Oklahoma State University Tick Lab, with ticks collected from Oklahoma. The “Mid-Atlantic” lineage includes ticks collected from field locations in central Pennsylvania. The “northern” lineage refers to ticks from BEI Resources that were started from ticks collected in Rhode Island. Results from one-way ANOVA and post-hoc pairwise comparisons with Tukey’s HSD are provided. N = 58 ticks (southern), 60 (Mid-Atlantic), 60 (northern).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7312978/v1/35000963c422819a5278bccd.png"},{"id":90980135,"identity":"89e5598b-949e-4e8a-88b6-6a50eab92112","added_by":"auto","created_at":"2025-09-10 09:13:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16774,"visible":true,"origin":"","legend":"\u003cp\u003eAverage velocities of nymphal blacklegged ticks (\u003cem\u003eIxodes scapularis\u003c/em\u003e) from different lineages found in the United States. The “southern” lineage refers to ticks from the Oklahoma State University Tick Lab, with ticks collected from Oklahoma. The “Mid-Atlantic” lineage includes ticks collected from field locations in central Pennsylvania. The “northern” lineage refers to ticks from BEI Resources that were started from ticks collected in Rhode Island. Results from one-way ANOVA and post-hoc pairwise comparisons with Tukey’s HSD are provided. N = 58 ticks (southern), 60 (Mid-Atlantic), 60 (northern).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7312978/v1/c5f6e4bd5af4aa22bd59fe75.png"},{"id":90980137,"identity":"8c5c0fda-fec8-4074-8634-d42c1bf771fe","added_by":"auto","created_at":"2025-09-10 09:13:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14654,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of nymphal blacklegged ticks (\u003cem\u003eIxodes scapularis\u003c/em\u003e) from different lineages found in the United States that successfully infested white-footed mice after artificial infestation in a sock. The percentage was calculated as the number of ticks that were found on the mouse after the artificial infestation period divided by the number of ticks that were added to the sock with the mouse. The “northern” lineage refers to ticks from BEI Resources that were started from ticks collected in Rhode Island. The “southern” lineage refers to ticks from the Oklahoma State University Tick Lab, with ticks collected from Oklahoma. Results from the Wilcoxon signed rank test are provided. N = 44 mice (21 male, 23 female) (northern), 22 mice (12 male, 10 female) (southern).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7312978/v1/6b345dce5a75f8cc43eb085e.png"},{"id":90980590,"identity":"2e7b3791-51a5-48c9-95c5-bcee5276bbd2","added_by":"auto","created_at":"2025-09-10 09:21:43","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128781,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of nymphal blacklegged ticks (\u003cem\u003eIxodes scapularis\u003c/em\u003e) from different lineages found in the United States that successfully fed to repletion on white-footed mice after artificial infestation in a sock. The percentage was calculated as the number of ticks that fed to repletion on the mouse divided by the number of ticks that successfully infested the mouse after artificial infestation. The “northern” lineage refers to ticks from BEI Resources that were started from ticks collected in Rhode Island. The “southern” lineage refers to ticks from the Oklahoma State University Tick Lab, with ticks collected from Oklahoma. Results from the Wilcoxon signed rank test are provided. N = 44 mice (21 male, 23 female) (northern), 22 mice (12 male, 10 female) (southern).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7312978/v1/e58884c074d68029654b9a51.jpeg"},{"id":90982516,"identity":"32e9d8fc-bd26-44a2-9975-c35c112e553f","added_by":"auto","created_at":"2025-09-10 09:29:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":728692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7312978/v1/c69ca730-56f9-4b67-90e1-7b6360641ccf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLocation, Location, Location: Geographic source of blacklegged tick (\u003cem\u003eIxodes scapularis\u003c/em\u003e) nymphs determines behavioral outcomes in laboratory studies\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe blacklegged tick (\u003cem\u003eIxodes scapularis\u003c/em\u003e Say, Acari: Ixodidae) is considered the most significant vector of tick-borne pathogens in North America, capable of transmitting \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e (Lyme disease), \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e (human granulocytic anaplasmosis), \u003cem\u003eBabesia microti\u003c/em\u003e (human babesiosis), and Powassan virus (Eisen and Eisen \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Although blacklegged ticks are found throughout the northeastern, Mid-Atlantic, northcentral, and southern United States, these regions have varying levels of reported tick-borne diseases associated with the blacklegged tick (Rosenberg et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Differences in disease incidence may be related to variations in the ecology of \u003cem\u003eI. scapularis\u003c/em\u003e, resulting from differing demographic histories in their respective geographic regions (Norris et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Ginsberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Sakamoto et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Arsnoe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe blacklegged tick comprises at least two distinct populations: northern and southern (Norris et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Van Zee et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Sakamoto et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These populations exhibit notable differences in genetics, survival patterns, host attachment preferences, and questing behavior. Numerous studies have documented these regional variations in \u003cem\u003eI. scapularis\u003c/em\u003e biology and ecology (Piesman and Spielman \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Carey et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, Mather et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, Apperson et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Goddard \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Norris et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Durden et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Piesman 2002, LoGiudice et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Goddard and Piesman \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Van Zee et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ginsberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Sakamoto et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Arsnoe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Curtis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Tietjen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Understanding the differences between the two distinct lineages of blacklegged ticks could have important implications for prediction, prevention, and control. For example, information on questing and horizontal movement could potentially improve tick bite risk models by accounting for differences in movement per lineage (Hassett et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Marshall et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, understanding the feeding patterns of different tick populations on reservoir hosts could clarify predictions about tick-borne disease risk.\u003c/p\u003e\u003cp\u003eQuesting behavior (vertical movement) differs between \u003cem\u003eI. scapularis\u003c/em\u003e populations in the United States, with northern populations questing more frequently and at greater heights than their southern counterparts (Arsnoe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tietjen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While lateral movement has been evaluated separately for adult \u003cem\u003eI. scapularis\u003c/em\u003e from Mississippi and New York (Goddard \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Curtis et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), direct comparisons of horizontal movement between northern and southern lineages remain lacking, particularly for nymphal stages. Similarly, potential behavioral differences in host infestation and feeding success between these populations have not been systematically assessed.\u003c/p\u003e\u003cp\u003eWhen evaluating tick behavior, it is important to consider the impact of the source population. Therefore, our study aims to identify differences in movement and feeding behavior of nymphal blacklegged ticks from these two distinct lineages. This study compared the horizontal movement (distance) and speed (velocity) of nymphal \u003cem\u003eI. scapularis\u003c/em\u003e from northern and southern lineages, as well as an additional lineage that originates from an area that lies between the northern and southern populations (central Pennsylvania). Host infestation and feeding differences between northern and southern lineages were also evaluated.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMovement Assessment\u003c/h2\u003e\u003cp\u003eFor this study, \u003cem\u003eI. scapularis\u003c/em\u003e that are found in the northeast and central regions of the United States north of the Chesapeake Bay are considered the \u0026ldquo;northern\u0026rdquo; population, and ticks found south of this region are the \u0026ldquo;southern\u0026rdquo; population (Ginsberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Nymphal \u003cem\u003eI. scapularis\u003c/em\u003e ticks were acquired from three locations: (1) BEI Resources (ATCC, Manassas, VA), which was started with ticks collected in Rhode Island (northern ticks); (2) Oklahoma State University Tick Lab (Stillwater, OK), which was started using ticks collected in Oklahoma (southern ticks); and (3) several field locations in central Pennsylvania (Centre County, PA) (Mid-Atlantic ticks). After ticks were received or collected, they were stored in snap cap vials with modified lids and mesh to allow airflow (Levin and Schumacher \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Vials of ticks were then stored in a humidity chamber at 80\u0026ndash;95% RH. The chamber was housed in an environmental chamber set at 22\u0026deg;C and a L:D setting of 16:8 (Troughton and Levin \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Levin and Schumacher \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOnce ready for observation in the experiment, individual ticks were removed from their vials using a pair of featherweight forceps or a paintbrush. To ensure ticks were alive before the start of the experiment, ticks that were actively moving in the vial were chosen. During each bioassay, ticks were observed between 0800 and 1300 h (Schulze et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Schulze and Jordan \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A similar temperature (20.3\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.8\u0026deg;C) and relative humidity (85.5\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;6.1%) were maintained for the bioassay using a portable heater and humidifier, respectively, within the humidity chamber.\u003c/p\u003e\u003cp\u003eOne nymphal tick lineage was randomly chosen for observation for each replicate. A tick was placed into a 100 x 15 mm glass petri dish (Pyrex, Corning, Inc., Glendale, AZ) and was given approximately five minutes to acclimate before recording began. Tick movement was recorded using the Basler acA1300-60gm GigE camera (Basler AG, Ahrensburg, Germany) at a frame rate of 30 frames per second and a resolution of 1280 x 1024 pixels. The camera feed was connected to a laptop running MediaRecorder 6 (Noldus, Wageningen, the Netherlands), which recorded and stored the video. The video recording was then imported into EthoVision XT 15 (Noldus, Wageningen, Netherlands) to quantify movement. After acclimation, the camera recording was started, and tick movement was tracked for one hour. Once completed, the tick was removed and not reused. While ticks that seemed active in the vials were chosen for the experiment, the tick was stimulated at the end of the experiment by breathing air to check that the ticks did not expire while being recorded. If ticks did not respond, the recording was not used. The petri dish was washed with unscented soap and water, dried, and then replaced under the camera. Multiple replicates were completed in one day, with all experiments ending by 1300 h. Preliminary trials of the experiment revealed that ticks reduced or completely stopped their movement after approximately 2.5-3 hours of the experiment starting, which would skew average velocity calculations.\u003c/p\u003e\u003cp\u003eBefore being imported into EthoVision, recordings were reviewed to ensure no artifact movements (i.e., movements other than the tick) were detected and that the camera maintained a clear view of the tick for the entire observation period. If either occurred, the recording was not used in the analysis. Once the videos passed inspection, they were imported into EthoVision XT 15 and analyzed by the software, which calculated the total distance the tick moved (in cm) and its average velocity (in cm/s). After removing videos with artifacts or obscured views, 60 replicates of each of the northern and Mid-Atlantic lineages and 58 replicates of the southern lineage were included in analyses.\u003c/p\u003e\u003cp\u003eTotal distances and average velocities of each tick population were compared using a one-way ANOVA, calculated using the \u003cem\u003eaov\u003c/em\u003e package in the statistical program R (R Core Team 2024). Post-hoc pairwise comparisons were conducted using Tukey\u0026rsquo;s HSD. Significance was set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTick Feeding Behavior Assessment\u003c/h3\u003e\n\u003cp\u003eMice were artificially infested with ticks to examine differences in tick infestation and feeding patterns between the northern and southern lineages of \u003cem\u003eI. scapularis\u003c/em\u003e. White-footed mice (\u003cem\u003ePeromyscus leucopus\u003c/em\u003e) between the ages of 3\u0026ndash;24 months were used in the experiment. Animals used were from the F1 and F2 generations of an in-house, wild-caught colony of \u003cem\u003ePeromyscus leucopus\u003c/em\u003e and housed according to approved mouse-rearing standards of The Pennsylvania State University. Housing and experimental procedures were conducted according to approved Institutional Animal Care and Use Committee (IACUC) protocols (PROTO202101808, PROTO202001598) and met the requirements of the Public Health Service Policy on Humane Care and Use of Laboratory Animals. A total of 66 animals (33 males, 33 females) were used in the experiments and included in the statistical analyses. In infestations involving northern ticks, 21 male and 23 female mice were used, while infestations with southern ticks used 12 male and 10 female mice. Because pathogens within field-collected ticks could not be verified before infestation, field-collected tick testing was not approved by the IACUC; thus, only northern and southern lineages of nymphal \u003cem\u003eI. scapularis\u003c/em\u003e were used for the tick feeding behavior assessment.\u003c/p\u003e\u003cp\u003eIn each round of infestations, three mice were infested with ticks of either lineage as part of complementary projects evaluating rodent behavior to tick infestation. Infestation procedures followed Brown et al. (2025). To infest mice, nymphal ticks were added to white cotton crew socks, which were held open by small plastic cups. The number of ticks used varied based on lineage-specific attachment rates and experimental requirements. For the northern lineage, 15\u0026ndash;30 ticks were used per sock, while for the southern lineage, 30\u0026ndash;60 ticks were used per sock due to consistently lower attachment rates observed in preliminary trials. All socks in one round were infested with the same lineage of ticks.\u003c/p\u003e\u003cp\u003eAfter mice were anesthetized and added to the sock, a square metal bulldog clip was used to close the sock. The cups holding the socks were then moved to secondary containment. The ticks were given three hours to infest the mice before the mice and attached ticks were moved to Noldus Phenotyper cages (Model PT3000; Noldus, Wageningen, Netherlands). Ticks remaining in the sock were counted and subtracted from the original number added (depending on the lineage) to obtain the approximate number of ticks that successfully infested the mice. The percentage of successful infestation was calculated as the number of ticks that infested mice divided by the original number of ticks added to the sock multiplied by 100.\u003c/p\u003e\u003cp\u003eMice were observed in the Phenotyper cages for four days, and the number of replete nymphs was counted daily. The percentage of successful feeding was calculated as the total number of replete ticks divided by the number of ticks that successfully infested mice, expressed as a percentage, and then multiplied by 100. Wilcoxon signed rank tests were performed in R to determine if the percentages of successful infestation and successful feeding differed between the northern and southern lineages of \u003cem\u003eI. scapularis\u003c/em\u003e. Significance was set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOverall, the distance traveled by each nymphal \u003cem\u003eI. scapularis\u003c/em\u003e lineage was significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, df\u0026thinsp;=\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In post-hoc analyses, ticks associated with the northern lineage traveled significantly farther in one hour (mean\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SE\u0026thinsp;=\u0026thinsp;248.4 cm\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;20.8 cm) compared to ticks from the southern or Mid-Atlantic lineages (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Ticks with southern or Mid-Atlantic lineages traveled similar distances (p\u0026thinsp;=\u0026thinsp;0.31), traveling approximately 155.5\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;27.2 cm and 150.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;14.4 cm, respectively, during the observation period.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLike distances traveled, the average velocities of nymphal \u003cem\u003eI. scapularis\u003c/em\u003e from various populations were significantly different (p\u0026thinsp;=\u0026thinsp;0.05, df\u0026thinsp;=\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nymphs from the northern lineage traveled at a higher velocity (0.078\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.006 cm/s) compared to ticks with southern (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 0.050\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.009 cm/s) and Mid-Atlantic lineages (p\u0026thinsp;=\u0026thinsp;0.02, 0.064\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.008 cm/s). Furthermore, nymphal ticks from the Mid-Atlantic had higher velocities compared to those from the south (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhile the movement experiments were done in the absence of host signals and odors, which could influence movement patterns, the infestation and feeding trials illuminated significant differences between the northern and southern lineages of \u003cem\u003eI. scapularis\u003c/em\u003e in the presence of a host. The northern population of \u003cem\u003eI. scapularis\u003c/em\u003e were more likely to successfully infest white-footed mice during the artificial infestation process compared to ticks from the southern population, with 60.4% \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e 18.7% and 22.3% \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e 13.0% of ticks successfully infesting mice, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, W\u0026thinsp;=\u0026thinsp;921.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Of the ticks that successfully infested mice, northern lineage ticks were more likely to feed to repletion compared to southern lineage ticks (35.2% \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e 26.4% and 18.5% \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e 27.1 of ticks that successfully infested mice feeding to repletion, respectively) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, W\u0026thinsp;=\u0026thinsp;709.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is important to understand the behavior of the target animal when designing behavioral experiments. In this study, we demonstrated significant differences in movement among three lineages of blacklegged ticks and feeding behavior between the northern and southern lineages.\u003c/p\u003e\u003cp\u003eOur data corroborate previous studies, where ticks from a northern lineage tended to move farther distances and at faster velocities than those from a southern lineage (Arsnoe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tietjen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Interestingly, Mid-Atlantic ticks were more similar in movement behavior to the southern population of \u003cem\u003eI. scapularis\u003c/em\u003e compared to those from the northern population, even though Mid-Atlantic ticks would geographically be classified as members of the northern population (east and north of the Chesapeake Bay) (Ginsberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The Mid-Atlantic ticks were collected from central Pennsylvania, which borders the proposed division between northern and southern blacklegged tick populations, emphasizing the need for additional research to define these populations.\u003c/p\u003e\u003cp\u003eIt is possible that Mid-Atlantic ticks could be a hybrid of both northern and southern lineage ticks, where the Mid-Atlantic ticks exhibit shared characteristics or genes from both lineages. This is supported by evidence that \u003cem\u003eI. dammini\u003c/em\u003e in the north (before being reclassified as \u003cem\u003eI. scapularis\u003c/em\u003e) and \u003cem\u003eI. scapularis\u003c/em\u003e from the south readily hybridize in the laboratory (James and Oliver \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Genetically, Sakamoto et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) identified two distinct clades of ticks in the United States, one that includes ticks from the northern and southern collection region and another clade that only includes ticks collected from the south. While the northern and southern lineages may have distinct demographic histories, the authors\u0026rsquo; data support a previous hypothesis that \u003cem\u003eI. scapularis\u003c/em\u003e may have originated in the south and a small founder population migrated to the north during the Pleistocene era (Norris et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Van Zee et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Sakamoto et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This series of events could possibly explain the behavioral differences of all three lineages, where the Mid-Atlantic ticks could be related to both the northern and southern lineages.\u003c/p\u003e\u003cp\u003eAnother possible explanation for differences in movement between the Mid-Atlantic and the northern lineages could be due to the effects of colonization, where behavioral differences can arise if they are raised in colony (northern lineage) versus the field (Mid-Atlantic). Differences in behavior and physiology have been reported in several insects and arthropods. For example, anthrophilic mosquitoes that are laboratory-reared may change host preferences if they are habitually fed blood from a non-human source, thereby adapting to a new host (Laarman \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1958\u003c/span\u003e, Spitzen and Takken \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Behavioral differences between laboratory-reared and field-collected jumping spiders have also been reported, where the authors argued that the developmental histories of animals can affect how they respond to treatments in the laboratory (Wiggins et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Changes in tick physiology due to differences in developmental history have also been reported. Field-caught \u003cem\u003eDermacentor variabilis\u003c/em\u003e were found to be more tolerant of dehydration compared to laboratory-reared ticks since field-caught ticks are more likely to undergo selective pressure due to less than optimal rearing conditions (Yoder et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To the authors\u0026rsquo; knowledge, this is the first report on behavioral differences between colony-reared and field-caught \u003cem\u003eI. scapularis\u003c/em\u003e. Due to significant differences between the lineages of \u003cem\u003eI. scapularis\u003c/em\u003e, future behavioral and physiological research should include testing both colony-reared and field-caught ticks to validate results.\u003c/p\u003e\u003cp\u003eDifferences in infestation and feeding success by nymphal ticks could indicate host preference in response to host availability in the respective geographic regions, where immature northern ticks parasitize small mammals such as white-footed mice, voles, or chipmunks (Piesman and Spielman \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Carey et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, Mather et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, LoGiudice et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), while the southern counterparts may parasitize small mammals and other animals including birds and lizards (Spielman et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Apperson et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Durden et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Piesman 2002, Goddard and Piesman \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Preferences for certain hosts may additionally account for the differences seen in successful infestations of white-footed mice. During animal infestations in this experiment, ticks from the southern lineage rarely attached to white-footed mice, even though they are considered permissive hosts for \u003cem\u003eI. scapularis\u003c/em\u003e (Anderson and Magnarelli \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1980\u003c/span\u003e, Ostfeld et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Brown et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Caron-L\u0026eacute;vesque and Careau \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), with only 22.3% of the ticks infesting the mouse and only 18.5% of those ticks feeding successfully to repletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The results in this study deviate from results in a previous study establishing that nymphal \u003cem\u003eI. scapularis\u003c/em\u003e from Great Island, Massachusetts and Statesboro, Georgia, exhibited similar feeding success rates and host preferences for both mice and lizards (James and Oliver \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Even when in close proximity to a host, southern \u003cem\u003eI. scapularis\u003c/em\u003e rarely attached, which could be driven by preferences for certain host cues to identify their hosts, such as host odors or heat. Southern \u003cem\u003eI. scapularis\u003c/em\u003e did not infest mice at the rate of the northern lineage. Still, there were some successful infestations, indicating that southern \u003cem\u003eI. scapularis\u003c/em\u003e recognized that a host was available. Still, they may have shifted or adapted their host preferences to other hosts that are more abundant in their region (i.e., lizards) (Spielman et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Tietjen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDifferences in heat production by hosts could additionally explain why \u003cem\u003eI. scapularis\u003c/em\u003e from the southern lineage did not frequently attach to the white-footed mice, where endotherms (i.e., small mammals) may not elicit a response from ticks compared to ectotherms (i.e., lizards). One study reported that when southern \u003cem\u003eI. scapularis\u003c/em\u003e were presented with a choice of a heating source or no heating source, the ticks did not show a preference for the heating element compared to the control (Ot\u0026aacute;lora-Luna et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While \u003cem\u003eI. scapularis\u003c/em\u003e is a well-known vector of several pathogens in the United States, the behavioral response of \u003cem\u003eI. scapularis\u003c/em\u003e to hosts is understudied, and comparing host preferences of two distinct tick lineages is even more so. Overall, tick behavior in response to host cues is complex and requires further research to understand the nuances in the ecological relationships between ticks and their hosts.\u003c/p\u003e\u003cp\u003eUnknown pathogen infection status is one limitation of this study. Laboratory-reared ticks have been confirmed to be pathogen-free (Salazar \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), however, field-collected ticks from the Mid-Atlantic region were not tested for pathogens. Pathogen infection in \u003cem\u003eI. scapularis\u003c/em\u003e has been found to modify some behaviors (Benelli \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, nymphal \u003cem\u003eI. scapularis\u003c/em\u003e infected with \u003cem\u003eB. burgdorferi\u003c/em\u003e exhibited increased phototaxis and attraction to vertical surfaces in laboratory settings, but their questing height and ability to overcome physical obstacles were not significantly different from uninfected ticks (Lefcort and Durden \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). When infected with \u003cem\u003eEhrlichia muris eauclairensis\u003c/em\u003e, nymphal \u003cem\u003eI. scapularis\u003c/em\u003e moved faster during host-seeking compared to uninfected ticks (Aspinwall et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In a different tick-borne disease system, \u003cem\u003eRickettsia\u003c/em\u003e-infected \u003cem\u003eDermacentor reticulatus\u003c/em\u003e females exhibited higher locomotor activity, resulting in longer trajectories compared to uninfected ticks (Pipov\u0026aacute; et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The results from the present study showed that Mid-Atlantic ticks traveled faster than the southern lineage of ticks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), but both lineages traveled similar distances overall (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the Mid-Atlantic ticks moved more slowly and traveled shorter distances compared to northern ticks. While the literature suggests that infected \u003cem\u003eI. scapularis\u003c/em\u003e move faster, infection status would not entirely explain why the Mid-Atlantic ticks were still more similar to the southern lineage (e.g., slower and traveled less distance) compared to the northern lineage of pathogen-free ticks. In addition, 20\u0026ndash;30% of nymphal blacklegged ticks in this region are infected with tick-borne pathogens (Brown et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Livengood et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Schwartz et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but a bimodal response, suggesting infected and non-infected ticks behave differently, was not demonstrated in this study. However, discrepancies in tick behavior due to infection with pathogens in tick-borne disease systems highlight the need for future work on this topic.\u003c/p\u003e\u003cp\u003eAnother possible limitation of this study is that it is unknown whether the recorded movement is related to the effects of energy reserves on walking activity. For \u003cem\u003eI. ricinus\u003c/em\u003e, ticks with higher fat content are expected to have more energy storage and thus, travel further than ticks with lower fat (Crooks and Randolph \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). While colony-reared ticks may have more consistent ages and therefore energy storage, similar information was not available for field-collected Mid-Atlantic ticks, where differences in their life history (e.g., age, previous blood meals, distance traveled, time since last blood meal) could result in differences in energy budgets and behaviors (Alasmari and Wall \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While it would have been ideal to measure energy storage before the start of the trials, the current available protocols would result in specimen destruction.\u003c/p\u003e\u003cp\u003eThis work provides additional behavioral data that support the hypothesis of at least two distinct populations of blacklegged ticks in the United States. These differences have been previously supported by genetic, behavioral, and ecological studies comparing northern and southern populations of \u003cem\u003eI. scapularis\u003c/em\u003e (Norris et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Van Zee et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Ginsberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Sakamoto et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Arsnoe et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tietjen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Collectively, this is the first report quantifying and comparing the horizontal movement of nymphal \u003cem\u003eI. scapularis\u003c/em\u003e from two laboratory-reared populations representing different geographic regions and one field population from an area bordering the two regions. Furthermore, this research provides additional information on the differences in host infestation and feeding between two laboratory-reared colonies originating from different regions of the United States. These data provide empirical results on blacklegged tick movement and feeding behaviors that can be incorporated into tick bite risk or distribution models that account for ticks from different geographic regions. These results can also provide guidance for researchers conducting tick behavioral studies, where the choice of tick sources could affect interpretation of results and conclusions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003cp\u003eHousing and experimental procedures involving \u003cem\u003ePeromyscus\u003c/em\u003e spp. were conducted according to approved Institutional Animal Care and Use Committee (IACUC) protocols (PROTO202101808, PROTO202001598) and met the requirements of the Public Health Service Policy on Humane Care and Use of Laboratory Animals.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eResearch reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI121495. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was additionally supported by the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04762 and Accession number 1025757. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: KCP, ETM; Methodology: KCP, JEB, MIE, ETM; Formal analysis and investigation: KCP, JEB, MIE, ETM; Writing - original draft preparation: KCP, JEB, MIE, ETM; Writing - review and editing: KCP, JEB, MIE, ETM; Funding acquisition: ETM; Resources: ETM; Supervision: KCP, ETM\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Graham Hickling for sharing the protocol for effectively infesting mice with blacklegged ticks in the lab and Kylie Green for managing the wild-caught colony of white-footed mice. In addition, we would like to thank Arash Maleki and Jesse Evans for their assistance with running trials, collecting field ticks, and/or providing feedback and expertise on the experimental design. We would also like to thank Jake Perryman, Melissa Welker, Dr. Jeffery Dodds, Dr. Sima Bruggeman, and the rest of the Animal Resource Program staff for their vital roles in animal care. The following reagent was provided by the Centers for Disease Control and Prevention for distribution by BEI Resources, NIAID, NIH: Ixodes scapularis Nymph (Live), NR-44116.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlasmari S, Wall R. 2020. Determining the total energy budget of the tick \u003cem\u003eIxodes ricinus\u003c/em\u003e. Experimental and Applied Acarology.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnderson JF, Magnarelli LA. 1980. 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Water balance of the American dog tick, \u003cem\u003eDermacentor variabilis\u003c/em\u003e, throughout its development with comparative observations between field-collected and laboratory-reared ticks. International Journal of Acarology. 38(4):334\u0026ndash;343. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/01647954.2011.647073\u003c/span\u003e\u003cspan address=\"10.1080/01647954.2011.647073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-insect-behavior","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joir","sideBox":"Learn more about [Journal of Insect Behavior](http://link.springer.com/journal/10905)","snPcode":"10905","submissionUrl":"https://submission.nature.com/new-submission/10905/3","title":"Journal of Insect Behavior","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Behavior, parasite, Lyme disease, infestation, genotypes, ticks","lastPublishedDoi":"10.21203/rs.3.rs-7312978/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7312978/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eIxodes scapularis\u003c/em\u003e is considered a significant medical and veterinary arthropod pest, capable of transmitting several pathogens that cause disease in humans and animals. Previous work has identified two distinct populations of \u003cem\u003eI. scapularis\u003c/em\u003e in the United States (northern and southern), characterized by differences in their genetics and behavior. This study aimed to characterize and compare the lateral movement and feeding behaviors of nymphal \u003cem\u003eI. scapularis\u003c/em\u003e between the northern and southern populations in the United States. Using laboratory-reared ticks from BEI (northern), Oklahoma State University (southern), and field-collected ticks from central Pennsylvania (Mid-Atlantic), behavioral bioassays were conducted to quantify distances traveled and velocities in a one-hour time frame. Ticks from the northern lineage walked longer distances and at faster speeds compared to ticks from the southern lineage. Field-collected ticks from central Pennsylvania, located between what is considered the northern and southern populations, exhibited similar movement behaviors as ticks from the southern population, even though ticks from the Mid-Atlantic are geographically categorized as the northern population. To compare feeding behaviors, colony-reared white-footed mice (\u003cem\u003ePeromyscus leucopus\u003c/em\u003e) were artificially infested with northern and southern ticks, and the percentages of infestation and feeding successes were compared. Northern ticks had higher success in infestations and feeding to repletion compared to southern ticks. These behavioral differences in movement and feeding patterns provide additional evidence for the hypothesis that geographically distinct populations of \u003cem\u003eI. scapularis\u003c/em\u003e exist across the United States. Researchers should consider these population differences when selecting tick lineages for behavioral studies and other blacklegged tick research.\u003c/p\u003e","manuscriptTitle":"Location, Location, Location: Geographic source of blacklegged tick (Ixodes scapularis) nymphs determines behavioral outcomes in laboratory studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 09:13:39","doi":"10.21203/rs.3.rs-7312978/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-27T18:40:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T18:24:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88672590043906354558090569926080963673","date":"2025-09-08T14:11:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T19:52:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-03T19:51:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-19T04:38:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Insect Behavior","date":"2025-08-06T20:52:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-insect-behavior","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joir","sideBox":"Learn more about [Journal of Insect Behavior](http://link.springer.com/journal/10905)","snPcode":"10905","submissionUrl":"https://submission.nature.com/new-submission/10905/3","title":"Journal of Insect Behavior","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"13f07745-60ac-463f-82ec-92fbf0d7cb80","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T14:08:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 09:13:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7312978","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7312978","identity":"rs-7312978","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}