Southeastern Naturalist 597 A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 22001188 SOUTHEASTERN NATURALIST 1V7o(4l.) :1579,7 N–6o0. 44 Effect of Cuterebra fontinella (Mouse Bot Fly) on the Movement of Peromyscus leucopus (White-footed Mouse) Allison B. Johnson1, Tyler J. Barzee2,3, Kasey D. Holbert4, Samantha L. Poarch1,5, and Jonathan J. Storm1,* Abstract - Peromyscus leucopus (White-footed Mouse) is a common host for Cuterebra fontinella (Bot Fly), but few studies of this interaction in the southeastern US exist. We assessed the movement of White-footed Mice infested with Bot Flies at 9 riparian woodland sites in Spartanburg County, SC. Our objectives were to determine the prevalence of bot warbles, lumps under the skin containing Bot Fly larva, on White-footed Mice and if the warbles reduced mouse movement. We found that 17.4% of mice had bot warbles during the August trapping period, with a mean intensity of 1.21 ± 0.09 (SE) per mouse. Male and female mice did not differ in the prevalence of bot infestation. Bot-infested mice did not differ from uninfested mice in their mean squared distance from center of activity (MSD). During May, mice that later became infested with a bot warble in August, did not differ in MSD from mice that did not become infested, suggesting that greater movement does not heighten the risk of infestation. Our data show that bot warbles do not reduce the movement of White-footed Mice and our findings add to the growing consensus that Bot Flies do not have a strong negative effect on the ecology of White-footed Mice. Introduction Peromyscus leucopus Rafinesque (White-footed Mouse) is a common host for Cuterebra fontinella Clark (Mouse Bot Fly, hereafter Bot Fly). Female Bot Flies lay eggs on vegetation and debris near the ground. The sticky eggs adhere to a passing host and the larvae hatch in response to the host’s body heat. Larvae then enter the host’s body through an external opening (e.g., nostrils) and travel through the body cavity before embedding as a subcutaneous lump, termed a warble, typically in the inguinal region (Catts 1982, Slansky 2007). Once embedded, larvae grow to a mass of ~1.0 g, or roughly 5% of the body mass of the typical adult White-footed Mouse (Munger and Karasov 1994). Within White-footed Mouse populations, the prevalence and intensity of Bot Flies may differ by sex. For example, Brown and Fuller (2006) found that in environmentally stressful floodplain environments, bot warbles were more prevalent in males. Xia and Millar (1990) hypothesized that male mice should have a higher 1Division of Natural Sciences and Engineering, University of South Carolina Upstate, Spartanburg, SC, 29303. 2Department of Environmental Engineering and Earth Sciences, Clemson University, Clemson, SC 29631. 3Current address - Department of Biological and Agricultural Engineering, University of California Davis, Davis, CA 95616. 4Department of Biological Sciences, Clemson University, Clemson, SC 29631. 5Current address - University of Florida College of Veterinary Medicine, Gainesville, FL 32608. *Corresponding author - jstorm@uscupstate.edu. Manuscript Editor: Michael Cove Southeastern Naturalist A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 598 infestation rate because they have a larger home-range than females (Schug et al. 1991, Wolff 1989) and are more likely to passively encounter Bot Fly eggs. Most studies, however, have found no difference in infestation between sexes (Clark and Kaufman 1990, Cramer and Cameron 2006, Jaffe et al. 2005, Timm and Cook 1979, Wecker 1962). Despite the substantial size of bot warbles and their location within the inguinal region, they do not appear to hinder the distance moved by mice (Burns et al. 2005, Cramer and Cameron 2010, Goertz 1966, Hunter, 1972) nor do they alter food intake (Munger and Karasov 1994). Most studies, though, have focused on comparing the movement of infested mice to individuals that are not infested at the same time (but see Cramer and Cameron 2010). One problem with this approach is that it does not account for individual differences in movement. For example, individuals may move less because they are infested, or perhaps individuals that move less are less likely to become infested. In the laboratory, Peromyscus maniculatus Wagner (Deer Mice) infested with a single bot warble exhibited no difference from uninfested mice in their ability to locomote and evade predation from Mustela erminea L. (Ermine). Infested mice were less active than uninfested mice, though, and spent less time running (Smith 1978). Although there have been several field studies on the effect of Bot Flies on Whitefooted Mice (Cramer and Cameron 2006, Jaffe et al. 2005, Timm and Cook 1979, Wolf and Batzli 2001), relatively few have been done in the southeastern US (but see Jennison et al. 2006, Klein et al. 2010). In this study, we sought to determine: (1) the prevalence and intensity of Bot Flies in White-footed Mice, (2) whether bot warbles reduced the movement of infested mice, and (3) whether mice with larger movements before the bot season were more likely to become infested. Field-site Description Our 9 study sites were mature riparian woodlands in Spartanburg County, SC, in the northern Piedmont region of the state (Table 1). Study sites were separated by a mean ± SE of 14.9 km ± 1.1 km, and there were no movements of Whitefooted Mice between sites. Study sites were a mixture of public (n = 5) and private Table 1. Location of 9 study sites in Spartanburg County, SC during 2013–2015. Peromyscus leucopus (White-footed Mouse) infested with Cuterebra fontinella (Bot Fly) were encountered during at least 1 year at each site. Site Locality (latitude, longitude) Year(s) of Bot-infested mouse capture 1 34°47'58"N, 81°59'51"W 2014 2 34°48'18"N, 81°59'54"W 2014 3 34°55'40"N, 81°46'35"W 2013 4 34°56'2"N, 81°46'39"W 2013 5 34°59'45"N, 81°57'8"W 2013, 2014 6 34°59'9"N, 81°57'49"W 2013 7 35° 0'18"N, 81°57'47"W 2013 8 35° 0'19"N, 81°58'19"W 2014 9 35° 1'1"N, 81°59'1"W 2014, 2015 Southeastern Naturalist 599 A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 (n = 4) land. Each site consisted of mixed deciduous–coniferous forest dominated by Quercus alba L. (White Oak), Carya tomentosa (Lam. ex Poir.) Nutt. (Mockernut Hickory), Fagus grandifolia Ehrh. (American Beech), Liquidambar styraciflua L. (Sweetgum), and Pinus taeda L. (Loblolly Pine). The understory consisted of Lonicera japonica (Thunb.) (Japanese Honeysuckle), Smilax spp. (greenbriars), Microstegium vimineum (Trin.) A. Camus (Japanese Stiltgrass), and Vitis rotundifolia Michx. (Muscadine). Methods We live-captured White-footed Mice from 2013 to 2015. During each trapping session, we baited 125 Sherman live traps (7.6 cm x 8.9 cm x 22.9 cm; H.B. Sherman Traps, Inc., Tallahassee, FL) with a mixture of oatmeal, sunflower seeds, and bacon bits, and placed traps in a 5 m x 25 m grid (0.96 ha) with 10-m spacing between traps at each of the sites. We checked traps for 7 consecutive days during May and August. We trapped each site for 2–3 consecutive years. We ear-tagged mice (Monel #1005-1P, National Band and Tag Company, Newport, KY) and recorded standard measurements for each individual: sex, body mass, age class (juvenile or adult), hindfoot length, tail length, and the number of visible warbles. We used the combination of hindfoot and tail length, as well as mass and coloration, to aid in distinguishing White-footed Mice from Deer Mice and P. gossypinus Le Conte (Cotton Mouse) (Reed et al. 2004, Webster et al. 1985). We live-captured and handled animals following the guidelines of the American Society of Mammalogists (Sikes et al. 2016), and our procedures were approved by the University of South Carolina Animal Care and Use Committee (Protocol #2161-100806-042114). At each site, we calculated the prevalence of Bot Flies as the proportion of mice that were visibly infested and defined intensity as the number of visible bot warbles in each individual (Bush et al. 1997). We only calculated bot prevalence and intensity for sites that had at least 1 infested individual and only during the August period because we observed no instances of bot warbles in May. We used a chi-square goodness-of-fit test to test whether there was a difference in Bot Fly infestation between male and female mice. To determine the movement pattern of mice during a trapping session, we used the mean squared distance from center of activity (MSD; Slade and Swihart 1983). We used MSD because it is unbiased with regard to the number of times each individual is captured and it exhibits a positive relationship with home-range size (Slade and Russell 1998). Following the recommendation of Slade and Swihart (1983), we only included individuals in MSD calculations if they had been captured at least 3 times during a trapping session. MSD has been used in previous studies (Cramer and Cameron 2010, Klein and Cameron 2012) to estimate the effect of Bot Flies on the movement of White-footed Mice. Each individual’s MSD was calculated as: MSD = Σ([xi - x̅ ]2 + [yi - y̅ ]2) / n, Southeastern Naturalist A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 600 where n is the number of captures for a given individual, xi is the x coordinate of a given capture, yi is the y coordinate of a given capture, and an individual’s center of activity is represented as the mean of their x and y captures during the trapping session, x̅ and y̅ . We performed 2 separate analyses to determine whether Bot Flies reduced the MSD of infested mice. First, to control for seasonal variation in movement, we used a 2-way ANOVA to compare the MSD of infested and uninfested mice during August. Sex and bot status were the independent variables. Next, we controlled for individual variation by using a repeated-measures ANOVA to compare the MSD of mice captured in both May and August. In this latter analysis, bot status and trapping month were independent variables. Increased movement has the potential to increase the likelihood of bot infestation; thus, we used this repeated measures design to determine whether mice infested with a bot in August had a larger MSD during May than mice that did not become infested. MSD data can be biased for individuals whose center of activity is at the edge of the grid, as their home range and movements may be mostly outside the trapping area. This situation could lead to an artificially low MSD for these mice relative to individuals that have a center of activity in the middle of the grid (Cramer and Cameron 2010). To check for this bias within the May trapping period, we performed a 2-sample t-test comparing the MSD of individuals with a center of activity in the outer 10 m of the trapping grid to those with a center of activity in the middle of the grid. For the August data, we performed a 2-way ANOVA to determine if MSD was influenced by either the center of activity (edge or middle of grid) or bot status. We performed statistical analyses in Minitab 17 (Minitab Inc, State College, PA) and log-transformed data to achieve normality. Data are presented as mean ± SE. Results We captured infested mice in August during 2 years at 2 sites and during a single year at the other 7 sites (Table 1). Across all years, 17.4% of all mice captured (n = 218) in August were infested with 1–3 bot warbles. We found that 84.2% of infested mice (n = 38) had a single warble, with a mean intensity of 1.21 ± 0.09 SE warbles per mouse. We also found no strong difference in bot prevalence between male (n = 132, 21.2% infested) and female White-footed Mice (n = 86, 11.6% infested) (χ² = 2.75, df = 1, P = 0.097). When considering all White-footed Mice captured during August, we found no effect of sex (F1,140 = 0.78, P = 0.379; Fig. 1) or bot status (F1,140 = 0.05, P = 0.821) on the MSD. In addition, there was no interaction between sex and bot status (F1,140 = 2.27, P = 0.134) on the MSD. We used a repeated-measures ANOVA to compare the MSD of individuals captured in both May and August and found no effect of bot status (F1,90 = 0.17, P = 0.678; Fig. 2) or trapping month (F1,90 = 0.31, P = 0.581) on the MSD. Using a Tukey pairwise comparison, we found no difference in MSD during May for mice that did (141.4 m2 ± 70.0 m2) and did not (189.9 m2 ± 29.4 m2) have a bot warble in August (t = 0.93, P = 0.788). Southeastern Naturalist 601 A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 Figure 1. Mean ± SE squared distance from the center of activity (MSD) for Peromyscus leucopus (White-footed Mouse) at 9 field sites in Spartanburg County, SC, during August 2013–2015. Of the 94 males, 18 (19.2% of males) were infested by Cuterebra fontinella (Bot Fly), whereas 9 of the 50 females (18.0% of females) were infested. There was no effect of sex or bot warbles on the MSD. Figure 2. Mean ± SE squared distance from the center of activity (MSD) for Peromyscus leucopus (White-footed Mouse) captured during both May and August 2013–2015 in Spartanburg County, SC. Of the 48 individuals captured in May, 10 were subsequently infested with a Cuterebra fontinella (Bot Fly) in August, whereas 38 individuals were not infested in August. During May, there was no difference in MSD between mice that did and did not become infested in August. Southeastern Naturalist A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 602 During May, there was no difference between the MSD of mice with a center of activity at the edge versus those in the middle of the trapping grid (t = 1.36, df = 164, P = 0.177). During August, there was a significant effect of trapping-grid location on the MSD (F1,141 = 9.51, P = 0.002), with mice in the center of the grid having a 29.5% larger MSD than individuals from the edge (t = 3.08, P = 0.002). There was, however, no effect of bot status on the MSD (F1,141 = 0.230, P = 0.631) and no interaction between center of activity and bot status (F1,141 = 1.24, P = 0.268). Discussion Similar to previous studies, we observed bot warbles in White-footed Mice during August, but not May. Bot warbles are generally most abundant in mice during July–September (Catts 1982, Hunter 1972, Miller and Getz 1969). We observed variation across sites in bot presence, which likely resulted from the patchy dispersion of Bot Flies across the landscape (Catts 1982, Miller and Getz 1969). Prior studies have also found bot prevalence to be variable between sites (Miller and Getz 1969, Wolf and Batzli 2001). Across all years, the prevalence of bot infestation was 17.4%, which is similar to prior studies that found mean infestation rates of 3–25% (Clark and Kaufman 1990, Dunaway et al. 1967, Jaffe et al. 2005, Klein et al. 2010). We did not find a sex difference in bot prevalence, which agrees with the findings of several studies (Clark and Kaufman 1990, Cramer and Cameron 2006, Jaffe et al. 2005, Timm and Cook 1979). In addition, most individuals harbored a single bot, which agrees with previous reports (Cramer and Cameron 2006, 2010; Dunaway et al. 1967, Tim and Cook 1979; Wolf and Batzli, 2001). We found that infested mice did not have a reduced MSD relative to uninfested mice, a result found in previous studies (Burns et al. 2005, Cramer and Cameron 2010, Hunter 1972). Given that White-footed Mice are territorial (Barko et al. 2003), infested mice may have to move as much as uninfested mice in order to forage and defend their territory (Nupp and Swihart 1996). In addition, mice infested with a bot warble in August did not have a larger MSD in May than mice that did not become infested. This finding suggests that bot-infested mice are not individuals with a larger MSD that might increase their chance of acquiring a bot warble; rather it likely reflects the life history of Bot Flies, as the eggs are often deposited near a host burrow (Catts 1982) and mice do not have to move far to become infested. Similar to prior studies of White-footed Mice in the northern end of their range, our results suggest that Bot Flies do not reduce the movement of mice. Perhaps the short-term nature of bot infection, often lasting just 19–26 d (Catts 1982), is one reason for the lack of a change in movement. Future work should address whether Bot Flies reduce the speed of movement or alter the microhabitat use of foraging White-footed Mice. Changes in these behaviors may influence the survival and overall fitness of bot-infested mice. Acknowledgments We thank the following individuals for assistance with fieldwork: R. Dolewski, B. Doornbos, A. Faso, N. Hyatt, J. Johnson, T. Khleborod, C. Kross, D. Kunda, J. Kwasniewski, Southeastern Naturalist 603 A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 R. Lever, A. Modarres, J. Morrissey, A. O’Brien, D. Patel, E. Phifer, J. Price, A. Russell, M. Storm, M. Sudduth, S. Wilkes, and N. Varakin. We are grateful to J. Boyles, V. Connors, and J.O. Whitaker Jr for comments on the manuscript. We appreciate the University of South Carolina Office of Research for a Magellan Scholar fellowship provided to S.L. Poarch, the University of South Carolina Upstate Faculty Excellence Committee for funds provided to J.J. Storm, and the Office of Sponsored Awards and Research Support for funds provided to S.L. Poarch and A.B. Johnson. Literature Cited Barko, V.A., G.A. Feldhamer, M.C. Nicholson, and D.K. Davie. 2003. Urban habitat: A determinant of White-footed Mouse (Peromyscus leucopus) abundance in southern Illinois. Southeastern Naturalist 2:369–376. Brown, T.T., and C.A. Fuller. 2006. Stress and parasitism of White-footed Mice (Peromyscus leucopus) in dry and floodplain environments. Canadian Journal of Zoology 84:1833–1839. Burns, C.E., B.J. Goodwin, and R.S. Ostfeld. 2005. A prescription for longer life? Bot fly parasitism of the White-footed Mouse. Ecology 86:753–761. Bush, A.O., K.D. Lafferty, J.M. Lotz, and A.W. Shostak. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. Journal of Parasitology 83:575–583. Catts, E.P. 1982. Biology of New World bot flies: Cuterebridae. Annual Review of Entomology 27:313–338. Clark, B.K., and D.W. Kaufman. 1990. Prevalence of botfly (Cuterebra sp.) parasitism in populations of small mammals in eastern Kansas. American Midland Naturalist 124:22–30. Cramer, M.J., and G.N. Cameron. 2006. Effects of Bot Fly (Cuterebra fontinella) parasitism on a population of White-footed Mice (Peromyscus leucopus). Journal of Mammalogy 87:1103–1111. Cramer, M.J., and G.N. Cameron. 2010. Effects of bot fly parasitism on movements of Peromyscus leucopus. American Midland Naturalist 163:455–462. Dunaway, P.B., J.A. Payne, L.L. Lewis, and J.D. Story. 1967. Incidence and effects of Cuterebra in Peromyscus. Journal of Mammalogy 48:38–51. Goertz, J.W. 1966. Incidence of warbles in some Oklahoma rodents. American Midland Naturalist 75:242–245. Hunter, D.M., J.M. Webster, and R.M.F. Sadleir. 1972. Studies on ecology of Cuterebrid parasitism in deer mice. Canadian Journal of Zoology 50:25–29. Jaffe, G., D.A. Zegers, M.A. Steele, and J.F. Merritt. 2005. Long-term patterns of botfly parasitism in Peromyscus maniculatus, P. leucopus, and Tamias striatus. Journal of Mammalogy 86:39–45. Jennison, C.A., L.R. Rodas, and G.W. Barrett. 2006. Cuterebra fontinella parasitism on Peromyscus leucopus and Ochrotomys nuttalli. Southeastern Naturalist 5:157–164. Klein, G.P., C.C. Christopher, G.N. Cameron, and G.W. Barrett. 2010. Effect of bot fly parasitism on vertical habitat use by Peromyscus leucopus. Northeastern Naturalist 17:223–228. Klein, G.P., and G.N. Cameron. 2012. Effect of habitat gradients on space use by Whitefooted Mice (Peromyscus leucopus). Journal of Mammalogy 93:706–715. Miller, D.H., and L.L. Getz. 1969. Botfly infections in a population of Peromyscus leucopus. Journal of Mammalogy 50:277–283. Southeastern Naturalist A.B. Johnson, T. Barzee, K.D. Holbert, S.L. Poarch, and J.J. Storm 2018 Vol. 17, No. 4 604 Munger, J.C., and W.H. Karasov. 1994. Costs of bot fly infection in White-footed Mice: Energy and mass flow. Canadian Journal of Zoology 72:166–173. Nupp, T.E., and R.K. Swihart. 1996. Effect of forest-patch area on population attributes of White-footed Mice (Peromyscus leucopus) in fragmented landscapes. Canadian Journal of Zoology 74:467–472. Reed, A.W., P.K. Kennedy, M.L. Beck, and M.L. Kennedy. 2004. Using morphologic characters to identify Peromyscus in sympatry. American Midland Naturalist 152:190–195. Schug, M.D., S.H. Vessey, and A.I. Korytko. 1991. Longevity and survival in a population of White-footed Mice (Peromyscus leucopus). Journal of Mammalogy 72:360–366. Sikes, R.S., and the Animal Care and Use Committee of the American Society of Mammalogists. 2016. 2016 Guidelines of the American Society of Mammalogists for the use of wild mammals in research and education. Journal of Mammalogy 97:663–688. Slade, N.A., and L.A. Russell. 1998. Distances as indices to movements and home-range size from trapping records of small mammals. Journal of Mammalogy 79:346–351. Slade, N.A., and R.K. Swihart. 1983. Home-range indexes for the Hispid Cotton Rat (Sigmodon hispidus) in northeastern Kansas. Journal of Mammalogy 64:580–590. Slansky, F. 2007. Insect–mammal associations: Effects of cuterebrid bot fly parasites on their hosts. Annual Review of Entomology 52:17–36. Smith, D.H. 1978. Effects of bot fly (Cuterebra) parasitism on activity patterns of Peromyscus maniculatis in laboratory. Journal of Wildlife Diseases 14:40–51. Timm, R.M., and E.F. Cook. 1979. Effect of bot fly larvae on reproduction in White-footed Mice, Peromyscus leucopus. American Midland Naturalist 101:211–217. Webster, W.D., J.F. Parnell, and W.C. Biggs Jr. 1985. Mammals of the Carolinas, Virginia, and Maryland. The University of North Carolina Press, Chapel Hill, NC. 255 pp. Wecker, S.C. 1962. The effects of bot fly parasitism on a local population of the Whitefooted Mouse. Ecology 43:561–565. Wolf, M., and G.O. Batzli. 2001. Increased prevalence of Bot Flies (Cuterebra fontinella) on White-footed Mice (Peromyscus leucopus) near forest edges. Canadian Journal of Zoology 79:106–109. Wolff, J.O. 1989. Social behavior. Pp. 271–291, In G.L. Kirkland Jr. and J.N. Layne (Eds.) Advances in the Study of Peromyscus (Rodentia). Texas Tech University, Lubbock, TX. 366 pp. Xia, X., and J.S. Millar. 1990. Infestations of wild Peromyscus leucopus by bot fly larvae. Journal of Mammalogy 71:255–258.