Northeastern Naturalist Vol. 24, No. 2 M.J. Buchholz and C.W. Dick 2017 97 2017 NORTHEASTERN NATURALIST 24(2):97–109 Ecology of Rodent–Ectoparasite Associations in South-Central Kentucky Matthew J. Buchholz1,2,* and Carl W. Dick1 Abstract - The goal of this study was to elucidate the host–ectoparasite associations of small-mammal communities in south-central Kentucky. Specifically we sought to determine whether host species, sex, and age as well as site or season affected the infestation of small mammals by parasitic arthropods. We captured small mammals from November 2014 to October 2015 using live traps in three 200 m x 50 m trapping grids within Western Kentucky University’s Green River Preserve. We identified captured small mammals to species and recorded standard measurements. Ectoparasites were removed and retained for identification. We collected 9 species of ectoparasites, including 3 ixodid ticks, 5 species of Siphonaptera, and 1 mesostigmatid mite, from 7 species of small mammals and calculated prevalence and mean intensity for each host–parasite association. Infestation rates of ectoparasites were generally low, but were affected by age and sex of the host, site, and season in different parasite taxa. We posit several natural- and life-history characteristics of hosts and parasites that likely contribute to the observed effects. The findings presented here provide an inventory of small-mammal and ectoparasite species in south-central Kentucky as well as insight into the dynamics of host–ectoparasite associations in the southeastern United States. Introduction Host–parasite associations are model systems for ecological and evolutionary studies. Each host is a well-defined unit of study with a sample community of ectoparasites, and individual hosts provide replicate samples (Presley 2011). Host body size, sex, population density, and other ecological and demographic characteristics can affect the quality and quantity of ectoparasite communities (Presley 2011). By examining the effects of each individual host’s characteristics on its ectoparasite assemblage, researchers can quantify these associations and understand how they may have developed over ecological and evolutionary time. Host–parasite associations often drive evolution and ecology of the parasite and host through a prolonged “arms race” (Roberts and Janovy 2009). In many systems, these relationships are complex and change with the diversity and distribution of host and parasite species present, as well as with the system’s abiotic factors. Rodents are important hosts for the larval and nymphal stages of hard ticks including Ixodes and Dermacentor spp., as well as numerous species of fleas (Kiffner et al. 2011, Krasnov et al. 2006). High global diversity of rodent species 1Department of Biology and Center for Biodiversity Studies, Western Kentucky University, Bowling Green, KY 42101. 2Current address - Department of Natural Resources Management, Texas Tech University, 2903 15th Street, Lubbock, TX 79409. *Corresponding author - matthew.buchholz@ttu.edu. Manuscript Editor: Howard Ginsburg Northeastern Naturalist 98 M.J. Buchholz and C.W. Dick 2017 Vol. 24, No. 2 and ectoparasites that infest them results in a wide range of associations affected by numerous characteristics of the host and parasite species, as well as other biotic and abiotic factors. Moreover, associations between rodents and ectoparasites are essential to understanding dynamics of infectious disease systems. Numerous pathogens of medical and veterinary concern are maintained within rodent reservoirs and transferred from host to host by ectoparasitic vectors. The etiologic agents for diseases such as Lyme, plague, Rocky Mountain spotted fever, anaplasmosis, flea-borne spotted fever, and many others are transmitted to humans and non-human animals through the feeding activities of ectoparasites (Bitam et al. 2010, Bratton and Corey 2005). Successful colonization of a host is associated with the likelihood of an ectoparasite encountering a host. Combes (1991) described this phenomenon in his conceptualization of an encounter filter. The encounter filter excludes potential hosts that a parasite would not encounter due to behavioral or ecological characteristics of the host and parasite, thus driving the evolution and ecology of the parasite to take advantage of hosts most likely to be encountered. Tactics such as occupying microhabitats of the host species allow ectoparasites to wait until a suitable host is present, subsequently allowing the parasite to colonize the host (Bitam et al. 2010, Oliver 1989, Parola and Raoult 2001). Social behavior of the host species can also facilitate transfer of ectoparasites from one host to another. Krasnov and Khokhlova (2001) found that fleas were easily transferred between rodent species when host individuals came into direct contact. The natural history of the host can also influence the likelihood of becoming infested with ectoparasites. Krasnov et al. (2011) speculated that the larger home range and wider dispersal of male vs. female rodents would cause increased occurrence of fleas on males. This project investigated host–parasite associations within small-mammal communities in south-central Kentucky. We sought to identify and quantify the small-mammal species present along with their ectoparasites by conducting a trapping survey and sampling captured rodents for ectoparasites. Due to the wide host breadth of the ectoparasites likely to be encountered in this study, we hypothesized that prevalence and mean intensity of infestation by ectoparasites would not vary among mammal species but would vary between sexes because males disperse further, thus increasing encounter rates of ectoparasites (Gaines and McClenaghan 1980). We hypothesized that ectoparasites would display aggregated distributions, with the majority of the ectoparasites found on relatively few individual small mammals. Due to the highly seasonal nature of tick life cycles, we hypothesized that the prevalence and mean intensity of ticks would vary by season, while indices of parasitism by fleas would not change by season. As fleas and ticks are not permanent parasites, we hypothesized that sub-adult and adult mammals would have equal prevalence and mean intensity of ectoparasitic infestation. Finally, we hypothesized that the composition of the small-mammal community would vary by season and site, potentially affecting the presence of parasites within sites. Northeastern Naturalist Vol. 24, No. 2 M.J. Buchholz and C.W. Dick 2017 99 Field-Site Description We sampled for this study on Western Kentucky University’s (WKU) Green River Preserve (GRP) located at 37°14'13"N, 85°59'32"W, just a few miles east of Mammoth Cave National Park. After being acquired in 2004, the GRP has been managed to restore and maintain natural habitats of south-central Kentucky. We conducted small-mammal trapping in 3 habitats within the GRP: young lowland forest (earlysuccessional trees less than 40 years old with a dense understory), early-successional old field (mix of grasses and forbs replanted after the field was removed from agricultural production), and mixed-age upland forest (late-successional trees with a scarce understory). Each habitat contained one trapping grid. Total precipitation during the 12-month study period was 141.88 cm (annual average = 132.72 cm). Seasonal low and high temperatures were -21° and 19°, -17° and 33°, 11° and 36°, and -9° and 34° C for winter, spring, summer, and fall respectively (NOAA 2016). Methods Small mammal trapping and sampling Each trapping grid was composed of 100 Sherman live traps (Model LNG; H.B. Sherman Traps, Tallahassee, FL) in a 200 m x 50 m grid with traps placed 10 m apart. We located trapping grids at least 400 m away from each other at their closest points. We baited traps with rolled oats and peanut butter. Traps were checked for captures and closed within 2 hours of sunrise and reopened ~2 hours before sunset for 3 consecutive days each month from November 2014 to October 2015, except for only 2 days in February 2015 due to heavy snowfall. We transported captured small mammals within the trap to an on-site laboratory for processing. We identified all captured small mammals as either an initial capture or recapture and recorded demographics and standard measurements for each individual. We distinguished between Peromyscus leucopus (Rafinesque) (White-footed Mouse) and P. maniculatus (Wagner) (Deer Mouse) by morphological characteristics, including degree of dorsoventral coloration, with all such determinations made by the first author for consistency. We then, carefully examined the captured small mammals by combing of the pelage and close examination of the ears, face, and anal region for any ectoparasites (attached and unattached), which we subsequently collected into vials of 70% ethanol. We housed and processed individual rodents separately as a precaution to prevent cross contamination by parasites. Following parasite sampling, mammals were anesthetized by isoflurane inhalation. We collected blood by irritation of the retro-orbital sinus with a Pasteur pipet and transferred the samples into a collection tube containing the anticoagulant K2-EDTA (Terumo Medical Products, Somerset, NJ) to be used in a separate study. Specimens were then ear tagged (Model 1005-1 Stamped Number; National Band & Tag Company, Newport, KY) with unique numbers and released at the site of capture. Small-mammal handling and sampling protocols were approved by the Western Kentucky University Institutional Animal Care and Use Committee in protocol #14-22. Northeastern Naturalist 100 M.J. Buchholz and C.W. Dick 2017 Vol. 24, No. 2 Ectoparasite identification All ectoparasites were stored in 70% ethyl alcohol for transport to WKU and sorted and enumerated under magnification. We identified ticks to genus and life stage using keys from the University of Rhode Island TickEncounter Resource Center Tick Identification Chart (URI 2016) and fleas to species by dichotomous key (Ewing and Fox 1943). Flea identifications were later confirmed by Dr. Ralph Eckerlin (Northern Virginia Community College, Springfield, VA). Data analyses All statistical analyses were conducted using the statistical program R (R Core Team 2015) with α = 0.05. We determined parasite presence/absence and intensity for each examination of an individual small mammal at the time of capture, and consider all sampling events to be independent. We recorded mammal species, sex, age (adult or sub-adult), site (trapping grid), and season (Dec–Feb = Winter, March–May = Spring, June–Aug = Summer, and Sep–Nov = Fall) for each sampling event. We calculated parasite prevalence (the proportion of examined hosts positive for parasites) and mean intensity (average number of parasites present on an infested host) for each parasite species individually as well as collectively for the tick and flea taxa. We examined the prevalence of ticks and fleas by identifying all individual rodents that harbored at least 1 tick or flea and creating 3-way contingency tables. Age and sex of the rodent were included along with parasite presence/absence for one analysis, and season and site were included as variables in another table for a separate analysis. We conducted analyses of the 3-way contingency tables by comparing generalized linear models involving different interactions with parasite presence/ absence. Interactions included the effect of each of the other 2 variables (age and sex or season and site) independent of the other and the effect of the interaction of the other 2 variables together. We then compared these models to a base model without any effects added by analysis of deviance using a likelihood ratio test. We made follow-up comparisons using the pairwise.G.test function in the R package “RVAideMemoire” (R Core Team 2015) with a Bonferroni correction. We also examined parasite prevalence with a G-test of independence comparing prevalence of each individual ectoparasite species among the different mammal species and made follow-up comparisons using the pairwise.G.test function in the R package “RVAideMemoire” (R Core Team 2015) with a Bonferroni correction. To examine whether mean intensity of tick or flea infestation was affected by host age, host sex, season, or site, we performed 2-way analyses of variance examining the interaction of age and sex and the interaction of season and site. Intensities were calculated by adding up all the ticks or fleas present on an individual small mammal regardless of species. We made post-hoc comparisons using a Tukey’s HSD test and compared intensities of each individual species of ectoparasite by a non-parametric ANOVA using resampling among the different mammal species. In addition, we examined the structure of the small-mammal community. Population size was estimated for each trapping grid during winter, spring, and summer Northeastern Naturalist Vol. 24, No. 2 M.J. Buchholz and C.W. Dick 2017 101 using the standard Schnabel population estimate. We calculated diversity of the small-mammal community of each trapping grid during each season using the Shannon-Wiener diversity index. We examined dissimilarity of the small-mammal communities between seasons and sites by calculating the Bray-Curtis dissimilarity index for each season–site pairing and using the advanced.procD.lm function within the R package “geomorph” (Adams and Otarola-Castillo 201 3). Results A total trapping effort of 10,500 trap nights resulted in 748 captures (7.12% trapping success). We captured 336 unique animals, comprising 7 species: Blarina brevicauda (Say) (Northern Short-Tailed Shrew), Microtus ochrogaster (Wagner) (Prairie Vole), Microtus pinetorum (Le Conte) (Woodland Vole), White- Footed Mouse, Deer Mouse, Reithrodontomys humulis (Audubon and Bachman) (Eastern Harvest Mouse), and Zapus hudsonius (Zimmermann) (Meadow Jumping Mouse) (Table 1). Of these, Northern Short-Tailed Shrew, Woodland Vole, and Meadow Jumping Mouse were infrequently captured and excluded from statistical analyses comparing prevalence and mean intensity of parasite infestation. Prevalence and mean intensities of occurrence of all parasite species observed during the study on each mammal species are presented in Table 2. Approximately 80% of the parasites collected during this study were taken from only 18% of the hosts that were examined. Prevalence of ticks did not vary by host age (deviance = 0.837; df = 4, 3; P = 0.360), sex (deviance = 1.427, df = 4 & 3, P = 0.232), or by age x sex interaction (deviance = 8.302, df = 4, P = 0.081) (Fig. 1A, B). However, prevalence of ticks varied by season (deviance = 64.946; df = 17, 14; P < 0.001), site (deviance = 13.667; df = 17, 15; P = 0.001), and by season x site interaction (deviance = 162.680, df = 17, P < 0.001) (Fig. 1C, D). Mean intensity of ticks varied by host age (F1 = 5.312, P = 0.025) but not by host sex (F1 = 2.463, P = 0.122) or by the interaction of age x sex (F1, 54 = 0.898, P = 0.348) (Fig. 1E, F). Mean intensity of ticks did not vary by season (F2 = 2.983, P = 0.059), site (F2 = 1.827, P = 0.171) , or by the interaction of Table 1. Total captures, unique individuals captured, individuals recaptured, and the average number of recaptures of recaptured individuals of the 7 small-mammal species captured during the study. Numbers in parentheses depict the percentage of the total for each column. Avg Total Individuals Individuals # of Mammal species captures captured recaptured recapt. Peromyscus maniculatus (White-footed Mouse) 327 (43.72) 126 (37.50) 71 (46.10) 2.82 Peromyscus leucopus (Deer Mouse) 318 (42.51) 131 (38.99) 68 (44.16) 2.75 Reithrodontomys humulis (Eastern Harvest Mouse) 65 (8.69) 45 (13.39) 11 (7.14) 1.82 Microtus ochrogaster (Prairie Vole) 32 (4.28) 29 (8.63) 3 (1.95) 1.33 Zapus hudsonius (Meadow Jumping Mouse) 4 (0.53) 3 (0.89) 1 (0.65) 1.00 Microtus pinetorum (Woodland Vole) 1 (0.13) 1 (0.30) - - Blarina brevicuada (Northern Short-tailed Shrew) 1 (0.13) 1 (0.30) - - Total 748 (100) 336 (100) 154 (100) 2.68 Northeastern Naturalist 102 M.J. Buchholz and C.W. Dick 2017 Vol. 24, No. 2 Table 2. Prevalence and mean intensity of the 9 species of ectoparasites collected during the study on P. leucopus (White-footed Mouse), P. maniculatus (Deer Mouse), M. ochrogaster (Prairie Vole), and R. humulis (Eastern Harvest Mouse), the 4 most common mammal species captured. I = Ixodes, A. a. = Amblyomma americanum (Lone Star Tick), D. v. = Dermacentor variabilis (American Dog Tick or Wood Tick), S. a. = Stenoponia americana (Baker), E. w. = Epitedia wenmanni (Rothschild), C. s. = Ctenophthalmus pseudagyrtes, O. l. = Orchopeas leucopus (Baker) (Rodent Flea), P. h = Peromyscopsylla hesperomys, and A. f. = Androlaelaps fahrenholzi. Ticks Fleas Mites I. spp. A. a. D. v. S. a. E. w. C. p. O. l. P. h. A. f. White-footed Mouse Prevalence 1.68% 0.55% 12.70% 4.97% 4.97% 2.21% 7.73% 17.13% 1.68% Mean intensity 1.00 2.00 3.26 1.22 1.00 1.00 1.64 1.68 1.33 Deer Mouse Prevalence 0.00% 0.52% 14.06% 8.83% 6.25% 4.17% 5.21% 21.88% 1.04% Mean intensity 0.00 5 2.81 1.44 1.17 1.13 1.3 1.67 1.00 Prairie Vole Prevalence 0.00% 0.00% 15.38% 0.00% 3.85% 15.38% 0.00% 0.00% 3.85% Mean intensity 0.00 0.00 3.25 0.00 1.00 1.25 0.00 0.00 1.00 Eastern Harvest Mouse Prevalence 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 1.92% 0.00% Mean intensity 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 Figure 1. Prevalence of ticks by (A) host sex, (B) host age, (C) season, and (D) site. Mean intensity of tick infestation by (E) host sex, (F) host age, (G) season, and (H) site. Lowercase letters depict statistically significant differences in mean intensity at α = 0.05. Error bars depict the standard error. Abbreviations of seasons are W (Winter), Sp (Spring), Su (Summer), and F (Fall). Abbreviations of sites are LF (lowland forest), OF (old field), and UF (upland forest). Northeastern Naturalist Vol. 24, No. 2 M.J. Buchholz and C.W. Dick 2017 103 season x site (F2, 52 = 0.563, P = 0.573) (Fig. 1G, H). Separate analyses conducted on Dermacentor variabilis (Say) (Amercian Dog Tick or Wood Tick) alone showed an effect of season (deviance = 68.536; df = 17, 14; P < 0.001), site (deviance = 12.419; df = 17, 14; P = 0.002) and season x site interaction (deviance = 165.820, df = 17, P < 0.001) on prevalence. However, prevalence of Wood Ticks did not vary by host age (deviance = 1.540; df = 4, 3; P = 0.215), sex (deviance = 2.264; df = 4, 3; P = 0.133), or by age x sex interaction (deviance = 8.984, df = 4, P = 0.061). Mean intensity of Wood Ticks varied by host age (F1 = 5.252, P = 0.026) and season (F2 = 3.369, P = 0.042), but not by host sex (F1 = 2.690, P = 0.107) interaction of age x sex (F1, 51 = 1.040, P = 0.313), site (F2 = 1.556, P = 0.221) or by the interaction of season x site (F2, 49 = 0.750, P = 0.478). While pooling of life stages may have confounded determination of seasonal variation, 90.2% of all ticks collected were larvae. Winter was excluded from the analyses of mean tick intensity by season because no individuals examined during that season harbored ticks. Prevalence of fleas did not vary by host age (deviance = 2.290; df = 4, 3; P = 0.130) or by age x sex interaction (deviance = 7.884, df = 4, P = 0.096), but did vary by host sex (deviance = 5.490; df = 4, 3; P = 0.019) (Fig. 2A, B). Prevalence of fleas did not vary by season (deviance = 4.143; df = 17, 14; P = 0.246), but did vary by site (deviance = 9.5989; df = 17, 15; P = 0.008) and by season x site interaction (deviance = 103.960, df = 17, P < 0.001) (Fig. 2C, D). Mean intensity of fleas did not vary by host sex (F1 = 2.538, P = 0.114), age (F1 = 1.499, P = 0.223) or by the interaction of Figure 2. Prevalence of fleas by (A) host sex, (B) host age, (C) season, and (D) site. Mean intensity of flea infestation by (E) host sex, (F) host age, (G) season, and (H) site. Lowercase letters depict statistically significant differences in mean intensity at α = 0.05. Error bars depict the standard error. Abbreviations of seasons are W (Winter), Sp (Spring), Su (Summer), and F (Fall). Abbreviations of sites are LF (lowland forest), OF (old field), and UF (upland forest). Northeastern Naturalist 104 M.J. Buchholz and C.W. Dick 2017 Vol. 24, No. 2 age x sex (F1, 124 = 3.480, P = 0.064) (Fig. 2E, F). Mean intensity of fleas did not vary by site (F2 = 0.554, P = 0.576) or season (F3 = 1.156, P = 0.330) but did vary by the interaction of season x site (F6, 121 = 2.756, P = 0.015) (Fig. 2G, H). Results of analyses comparing the prevalence and mean intensity of each parasite species on each mammal species are presented in Table 3. While prevalence of the 3 most common parasite species varied between host species, the mean intensity of infestation by any of the parasite species did not differ between hosts. Due to small sample size, ANOVAs were not conducted to compare mean intensity of Ixodes spp., Amblyomma americanum L. (Lone Star Tick), and Androlaelaps fahrenholzi (Berlese). Schnabel population estimates of the small-mammal population for each site ranged 48–83, 185–250, and 41–89 across seasons for the 3 trapping sites, respectively. Population size was not calculated for fall because the timing of the trapping period did not allow for inclusion of sufficient resampling events to calculate the Schnabel population estimate. Shannon-Wiener diversity index values ranged 0.5– 0.92, 0.99–1.41, and 0.5–0.8 across seasons for the 3 trapping sites, respectively. Dissimilarity of the composition of the small-mammal community as represented by trapping was not significant by season (F9, 6 = 0.631, P = 0.557), but was significant by site (F8, 6 = 2.212, P = 0.002) and by the interaction of season x site (Z = 1.789, P = 0.001). Post-hoc comparisons of the sites showed that the small-mammal composition of the early-successional old field site was significantly different than both the young lowland forest (P = 0.002) and mixed-age upland forest (P = 0.021) sites. In both comparisons, the field site was significantly more diverse in terms of species richness and evenness. Discussion Of the 9 species of parasites collected during this study, 6 showed no difference in prevalence among the 4 most abundant species of mammals. Interestingly, the 3 parasite species that did show variation in prevalence were all influenced by a general Table 3. Results of G-tests of independence and non-parametric ANOVAs comparing prevalence and mean intensity of parasite infestation among mammal species. G-tests of independence were all conducted with 3 degrees of freedom. Significant results are marked with an asterisk. Prevalence Mean intensity G P F df P Ticks Ixodes spp. 2.870 0.412 - - - Amblyomma americanum 0.255 0.968 - - - Dermacentor variabilis 10.930 0.012* 0.104 2, 51 0.925 Fleas Stenoponia americana 6.946 0.073 0.649 1, 23 0.332 Epitedia wenmanni 2.808 0.422 0.864 2, 19 0.532 Ctenophthalmus pseudagyrtes 9.675 0.022* 0.500 2, 13 0.850 Orchopeas leucopus 5.947 0.114 0.708 1, 22 0.483 Peromyscopsylla hesperomys 20.721 less than 0.001* 0.172 2, 71 0.898 Mites Androlaelaps fahrenholzi 1.977 0.577 - - - Northeastern Naturalist Vol. 24, No. 2 M.J. Buchholz and C.W. Dick 2017 105 lack of presence on Eastern Harvest Mouse. While many ticks and fleas are generalist in host choice and occur on multiple host species, some fleas and ticks are found more often on certain host species than others (Brunner and Ostfeld 2008, Durden and Kollars 1997, Krasnov et al. 2002). Specifically, Peromyscopsylla hesperomys (Baker) is more often associated with Peromyscus spp. and the relationship between Ctenophthalmus pseudagyrtes Baker and Prairie Vole observed here has previously been recorded in the south-central region of the US (Durden and Kollars 1997). As hypothesized, the parasites collected during this study had an aggregated pattern of distribution, with most of the parasites occurring on only a few host individuals. This finding provides support for the “80–20 rule” (Brunner and Ostfeld 2008, Hawlena et al. 2005). Female ixodid ticks deposit between 100 and 18,000 eggs in a single mass (Roberts and Janovy 2009). Small mammals that encounter an egg mass during larval hatching would likely be infested by many ticks, while those that do not happen upon a hatching egg mass may encounter few or no ticks. Additionally, host-seeking behaviors such as occupation of a particular small-mammal burrow or nest by ticks and fleas could expose certain small mammals to more ectoparasites than others (Bitam et al. 2010, Oliver 1989, Parola and Raoult 2001). Season affected the prevalence and mean intensity of parasitism of small mammals in this study. As hypothesized, tick prevalence was highly seasonal, with the vast majority of recorded tick infestations occurring in spring and summer. However, the overwhelming majority of ticks collected from small mammals during sampling were larval Wood Ticks. Thus, the observed effect of season on tick prevalence and the nearly significant effect on tick intensity are likely better explained by considering Wood Ticks alone instead of all 3 tick species together. Seasonal variation in Wood Ticks prevalence is likely explained by a life cycle resulting in high abundance of ticks questing for hosts during particular seasons when each life stage is present in the environment (Oliver 1989, Wilson and Spielman 1985). In Kentucky, larval Wood Tick activity is highest during March, April, and May (Kollars et al. 2000; URI 2016), which we defined as spring. Additionally, the abundance of Wood Tick nymphs and adults is highest during late spring and summer (Kollars et al. 2000, URI 2016). Additional seasonal patterns would likely have been observed if more Lone Star Ticks and Ixodes scapularis Say (Deer Tick) had been collected as the seasonal abundance of life stages of different species of ticks is not consistent (URI 2016). Site also affected the prevalence of tick infestation. Prevalence of tick infestation was higher in both forest sites than in the old field site. Deciduous forests provide the microclimatic conditions conducive to tick survival throughout their life cycle (Gray 1998). Additionally, while the old field site was previously planted to restore native prairie habitat, several exotic grass species have invaded the site including Dactylis spp. (Orchard Grasses), Setaria faberi Herrm. (Japanese Bristlegrass), and Fescue arundinacea Schreb. (Kentucky 31 Tall Fescue). Civitello et al. (2008) found that invasion by exotic grasses reduces survival of the Wood Tick, potentially decreasing its prevalence on small mammals in our old field site. Interestingly, the prevalence of ticks and fleas, as well as mean intensity of flea infestation varied Northeastern Naturalist 106 M.J. Buchholz and C.W. Dick 2017 Vol. 24, No. 2 with the interaction of season x site. This result suggests that the change in the site’s habitat characteristics throughout the year was directly affecting the parasite species, supporting previous findings (Krasnov et al. 2010; Leha ne 2005). Demographic characteristics of hosts have been known to influence the prevalence and intensity of parasitic infestation. In the present study, mean intensity of tick infestation was the only measure of parasitic infestation to vary with age of the host. The tick and flea species recorded in this study are all temporary or periodic feeders making them less likely to accumulate on the host over the host lifespan (Krasnov et al. 2010, Lehane 2005). However, variation in prevalence and intensity of temporary parasites by host age has been previously recorded. Krasnov et al. (2006) found variation in flea prevalence and species richness of fleas parasitizing several species of rodents by age, hypothesizing that the life-history characteristics of the host could cause older individuals to be more heavily parasitized. Increased age of the host may also be associated with increased body size, creating a larger “target” or resource for parasites to encounter and colonize (Krasnov et al. 2006). Additionally, dispersal of rodents that have matured to adults could influence the host encounter rate for temporary ectoparasites. For example, Peromyscus californicus (Gambel) (California Deermouse) has been observed to stay very close to its natal home range as juveniles and not disperse widely until adult age (Ribble 1992). Sex-biased parasitism has been recorded in numerous host–parasite systems involving arthropod, helminth, and unicellular parasites (Moore and Wilson 2002). Previous studies have shown that arthropods typically display male-biased parasitism (Moore and Wilson 2002). Because most arthropods rely on the host coming to the parasite instead of the parasite searching out a host, active hosts that cover larger spatial areas would likely encounter parasites more frequently (Bitam et al. 2010, Combes 1991, Parola and Raoult 2001). We hypothesized that males would have higher prevalence and mean intensity of flea and tick infestation due to male rodents having generally larger dispersal areas (Gaines and McClenaghan 1980). Although only 1 of the 4 host–parasite indices calculated was statistically significant, our results suggest that the rodent–ectoparasite system observed is at least skewed toward male hosts. While higher daily activity rates in female rodents has been recorded, theoretically providing them with more potential encounters with the ectoparasites found in their home range (Lightfoot 2008), the males’ larger dispersal area and home range should expose them to an overall greater population of ectoparasites. Another potential explanation for the observed male bias is that males are simply larger “targets” for ectoparasites. Many species of rodents display malebiased sexual size dimorphism (Schulte-Hostedde 2008). However, in the present study the variation in mass between sexes did not exceed 2 g (male Prairie Voles were 7.2% heavier than females on average). These small variations in mass may or may not have contributed to the male-biased parasitism observed in this study. Density of hosts in the environment can affect the distribution and composition of ectoparasite communities. Higher density of hosts has been correlated with increased species richness of ectoparasites (Krasnov et al. 2002). Increased density of hosts allows for species of parasites that may normally be out-competed by Northeastern Naturalist Vol. 24, No. 2 M.J. Buchholz and C.W. Dick 2017 107 other parasites or have minimal dispersal ability to find and colonize hosts. Additionally, high host density can result in a dilution effect (Kiffner et al. 2011). The overabundance of potential hosts with a limited population of ectoparasites could dilute the prevalence and intensity of parasitism as the parasites have many hosts to colonize. We calculated density of the small-mammal community at each trapping site. Small-mammal density in the early-successional old field site was calculated to be 185–250 individuals/ha across seasons. This high density of hosts and a dilution effect may explain the lower prevalence of tick and flea infestation in the old field than in either of the forest sites where small-mammal density did not exceed 89 individuals/ha. Results of dissimilarity analyses showed that composition of the representative small-mammal community was different in the early-successional old field site compared to either forest site. The difference in community composition is directly related to the abundance, species richness, and evenness of species at each site. Diversity of the small-mammal community was generally low at all 3 sites, but species diversity was highest in the field site. These findings are likely related to the composition of the vegetation at each site because different species of vegetation provide different habitat niches for small-mammal species. Species richness was also low during this study: R = 4, 5, and 3 for the lowland forest, old field, and upland forest sites, respectively. While low host species richness would normally result in low parasite richness, this correlation is likely offset by the wide host breadth of ectoparasites (Hechinger and Lafferty 2005). Our findings are similar to other surveys of small-mammal–ectoparasite relationships from nearby states in the southeastern US. Durden and Wilson (1991) and Clark and Durden (2002) found similar associations between the same smallmammal and ectoparasite species that were observed in this study in Tennessee and Mississippi, respectively. However, both studies found substantially higher ectoparasite species richness, with 16 and 15 species, respectively, compared to only 9 in our study, as well as substantially higher prevalence of Wood Ticks. A recent tick survey conducted in southern Indiana found substantially more Ixodes on small mammals (~40%) than what we found on small mammals examined for this study (Rynkiewicz and Clay 2014). However, while geographically close, these results do support the current known range of the Deer Tick (Eisen et al. 2016). Additional studies are needed to further elucidate the host–parasite associations of small mammals across Kentucky and the southeastern US. This study was limited to 3 trapping grids at 1 location and is thus limited in geographical coverage. Patterns uncovered here may or may not be extrapolated to other sites in the region. Furthermore, future studies that utilize multiple capture techniques may be able to obtain a more representative sample of the small-mammal community (Stephens and Anderson 2014), allowing a more thorough assessment of host–parasite associations. By monitoring host–parasite associations of small mammals and how they may change over time, we can elucidate the ecology and evolution of these systems and understand the risk of humans contracting pathogens that are maintained in and vectored by these systems. Northeastern Naturalist 108 M.J. 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