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Immune dimorphism in wild mice of Monteverde, Costa Rica

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Immune dimorphism in wild mice of Monteverde, Costa Rica
Abbreviated Title:
Dimorfismo inmune en ratones silvestres de Monteverde, Costa Rica
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Thoene, Danielle M.
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Rodents ( lcsh )
Roedores ( lcsh )
Climate changes ( lcsh )
Cambios climáticos ( lcsh )
Immune system ( lcsh )
Sistema imunologico ( lcsh )
Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
EAP Fall 2016
EAP Otoño 2016
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Rodents are at risk as climate change and human interference promote the spread of infectious disease. As a result, understanding the relationship between disease and population characteristics is important in developing conservation and management strategies. I investigated the existence of immune dimorphism, which is the variation in immune response and function between sexes, in mice of the Monteverde region. I captured a total of 33 mice and 1 rat: Heteromys nubicolens (n=18), Peromyscus nudipes (n=13), Scotinomys teguina (n=1), and Oligoryzomys fulvescens (n=1). For each captured individual, I collected all visible ectoparasites and took a blood sample for further laboratory analysis. I calculated average ectoparasite abundance and leukocyte count and compared these values between males and females to test for the existence of immune dimorphism. No significant differences in average ectoparasite abundance or leukocyte count were found between sexes of the same species or among different species; therefore, my findings do not support the existence of immune dimorphism in wild mice of Monteverde. The lack of immune dimorphism has positive health and survival implications for the study species, such as greater resilience to outbreaks of disease. Since males are not disproportionately at risk to disease and parasitism, the ratio of males to females is more likely to be balanced which enhances the stability of a population. ( ,, )
Abstract:
Los roedores están en riesgo por el cambio climático y por la interferencia humana al promover la propagación de enfermedades infecciosas. Como resultado, entender la relación entre la enfermedad y las características de la población es importante en el desarrollo de estrategias de conservación y manejo. Investigué la existencia del dimorfismo inmune, que es la variación en la respuesta inmune entre machos y hembras, en ratones de la región de Monteverde. Capturé un total de 33 ratones y una rata: Heteromys nubicolens (n=18), Peromyscus nudipes (n=13), Scotinomys teguina (n=1), and Oligoryzomys fulvescens (n=1). Para cada individuo capturado, recogí todos los ectoparásitos visibles y recolecté una gota de sangre de cada individuo. Calculé la abundancia media de ectoparásitos y conté el número de leucocitos, luego comparé estos valores entre los machos y las hembras para estimar la existencia de dimorfismo inmune. No encontré diferencias significativas en la abundancia media de ectoparásitos ni tampoco en el recuento de leucocitos entre ambos sexos de la misma especie, o entre diferentes especies. Mis resultados no apoyan la existencia de dimorfismo inmune en ratones silvestres de Monteverde. La falta de dimorfismo inmune tiene implicaciones positivas para la salud y la supervivencia de las especies estudiadas, como una mayor resiliencia a brotes de enfermedades. Dado que los machos no están desproporcionalmente en riesgo de enfermedad y parasitismo, la proporción de machos a hembras es más probable que sea equilibrada, lo que mejora la estabilidad de una población.
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Student affiliation: University of California, Los Angeles
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Monteverde Institute
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Monteverde Institute
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Immune Dimorphism in Wild Mice of Monteverde, Costa Rica Danielle M. Thoene Institute of the Environment and Sustainability University of California, Los Angeles EAP Tropical Biology and Conservation, Fall 2016 16 December 2016 ABSTRACT R odents are at risk as climate ch ange and human interference promote the spread of infectious disease. As a result, understanding the relationship between disease and population characteristics is important in developing conservation and management strategies. I investig ated the existence of i mmune dimorphism, which is the variation in immune response and function between sexes, in mice of the Monteverde region. I captured a total of 33 mice and 1 rat : Heteromys nubicolens (n=18), Peromyscus nudipes (n=13), Scotinomys teg uina (n=1), and Oligoryzomys fulvescens (n=1). For each captured individual, I collected all visible e ctoparasites and took a blood sample f or further laboratory analysis I calculated average e ctoparasite abundance and leukocyte count and compared these v alues between males and females to test for the existence of immune dimorphism. No significant differences in average ectoparasite abundance or leukocyte count were found between sexes of the same spec ies or among different species ; therefore, m y findings do not support the existence of immune dimorphism in wild mice of Monteverde The lack of immune dimorphism has positive health and survival implications for the study species such as greater resilience to outbreaks of disease Since males are not disproportionately at risk to disease and parasitism the ratio of males to females is more likely to be balanced which enhances the stability of a population. RESUMEN Los roedores estn en riesgo por el cambio climtico y por la interferenc ia humana al promover la propagacin de enfermedades infecciosas. Como resultado, entender la relacin entre la enfermedad y las caractersticas de la poblacin es importante en el desarrollo de estrategias de conservacin y manejo. Investigu la existenci a del dimorfismo inmune, que es la variacin en la respuesta inmune entre machos y hembras, en ratones de la regin de Monteverde. Captur un total de 33 ratones y una rata: Heteromys nubicolens (n=18), Peromyscus nudipes (n=13), Scotinomys teguina (n=1), and Oligoryzomys fulvescens (n=1). Para cada individuo capturado, recog todos los ectoparsitos visibles y recolect una gota de sangre de cada individuo. Calcul la abundancia media de ectoparsitos y cont el nmero de leucocitos, luego compar estos va lores entre los machos y las hembras para estimar la existencia de dimorfismo inmune. No encontr diferencias significativas en la abundancia media de ectoparsitos ni tampoco en el recuento de leucocitos entre ambos sexos de la misma especie, o entre dife rentes especies. Mis resultados no apoyan la existencia de dimorfismo inmune en ratones silvestres de Monteverde. La falta de dimorfismo inmune tiene implicaciones positivas para la salud y la supervivencia de las especies estudiadas, como una mayor resili encia a brotes de enfermedades. Dado que los machos no estn desproporcionalmente en riesgo de enfermedad y parasitismo, la proporcin de machos a hembras es ms probable que sea equilibrada, lo que mejora la estabilidad de una poblacin.

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Immune Dimorphism in Wild Mice Thoene 2 Rod ents belong to the most diverse mammalian order and can be found in nearly every type of biome (Myers 2000). As a result, some species have become highly specialized and occupy specific ecological niches. For example, the newly described cloud dwelling spi ny pocket mouse ( Heteromys nubicolens ) is endemic to the highlands of the Tilaran range in Costa Rica and has become a target of conservation and research (Anderson and Timm 2006). Wild, native rodents such as H. nubicolens serve important ecological roles as they help to control pest populations while also serving as a food source for larger animals (Mihalca and Sandor 2013). Furthermore, rodents are also widely used in research and have aided human understanding of many co mplex physiological and ecological processes. Since mice and rats are important to both human and ecosystem health monitoring and maintaining healthy populations of native rodent species is in our best interest We must first understand the characteristi cs of local populations before we can develop effective management and conservation strategies For example, the ratio of males to females is particularly important since this may influence the resilience of a population to natural disasters or disease out breaks. However, understanding population dynamics is further complicated by the physiological, behavioral, and immunological differences between males and females (Khokhlova 2004). For example, estrogen in female mice may be partially responsible for high er concentrations of antibodies which aid in combating infections (Martin et al. 2007; Bhatia et al. 2014). In contrast males are typically more parasitized than females, possibly due to the immunosuppressive effects of testosterone (Bacelar 2011; Kowalsk i et al. 2015). In rodents, testosterone has been shown to reduce resistance to ticks by inhibiting development of T and B cells which are important in antibody production and defense against foreign cells (Hughes and Randolph 2001; Lai et al. 2012; Peckha m 2004). This marked difference in immune response between sexes is known as immune dimorphism (Bhatia et al 2014). Rodent immunolo gy studies often use leukocyte (white blood cell) c ount as an indicator of immunocompetence, or an efend against parasites or disease (Khokhlova et al. 2010; Doeing et al. 2003). Since literature on immune dimorphism in Central American rodents is very limited I am interested in addressing the following question: Does immune dimorphism exist in wild mi ce populations in Monteverde, Costa Rica? Leukocyte count and ectoparasite abundance are used as indicators of immune differences between sexes. If leukocyte abundance is similar between sexes, then I expect that each sex has similar white blood cell co mposition and immune response. If ectoparasite abundance is similar between sexes, then I expect that testosterone does not significantly impair immune system health and increase parasite susceptibility in males. However, a significant difference in leukoc yte count or ectoparasite abundance between males and females may indicate the existence of immune dimorphism in the study species. Materials and Methods Study Sites in Monteverde, Costa Rica I collected mice and rats at four different sites characterized by either secondary growth forest or forest near trail edges. The first study site was the Dwight and Rachel Crandell Memorial Reserve (DRCMR) in Monteverde. I placed eight Sherman live traps along the Bioluminosa tra il on 22 November and 19 traps along the same trail on 25 November 2016. The second study site was the Sendero de Los Nios in the Bosque Eterno de los Nios Reserve

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Immune Dimorphism in Wild Mice Thoene 3 located in Bajo del Tigre. I placed t wenty Sherman live traps along this trail for four consecutive nights from 26 November to 29 November 2016. The third study site was the Sendero Murcilagos in the Bosque Eterno de los Nios Reserve located in Bajo del Tigre. I placed t wenty three Sherman live traps along this trail on 30 November 2016 and 1 December 2016. The fourth study site was along the Sendero Principal near the Estacin Biolgica Monterverde (EBM). I placed e ight traps along this trail earlier in the month on 6 November 2016 In addi tion, I placed 40 Sherman live traps along this trail on 4 December 2016 in collaboration with visiting scientist Dr. David Ribble. Trapping Regime and Field Work I used a mixture of oatmeal and vanilla as bait and placed a small amount in each trap I placed t raps approximately 5 meters apart, with preference given to locations near logs and areas covered by foliage. Since rodents prefer to reduce their exposure to predators, I avoided placing traps in o pen areas I baited and opened traps between 16:0 0 and 17:00 each evening and checked them the following morning between 6:30 and 7:30. Each morning I recorded the status of the traps as: capture of rodent, trap closure, stolen bait, or no activity (Gonzalez 2009). Trap closure signifies a closed trap wi th no animal and the bait remaining. Bait stealing signifies either an open or closed trap with the bait missing. Lastly, no activity signifies an open trap with the bait still inside. The status of the traps over the course of the study period can be foun d in the Appendix. One trap night was defined as one trap set for one night, this metric was used to provide a baseline for comparison to other trapping studies. Federico Chincilla transferred each captured rodent from the Sh erman live trap to a cloth ba g, then I recorded the sex and species of each individual. Next, I took measurements of tail length, foot length, ear length, and weight. In addition, I took notes of unique physical characteristics such as scars or missing toes. Lastly, I cut a small sect ion of hair from the hind leg of each rodent in order to mark previously captured individuals. To collect a blood sample, I sterilized the end of the tail with rubbing alcohol and cut approximately two millimeters from the tail using sharp scissors. In ord er to create a thin blood film, I placed a single drop of blood on a microscope slide and smeared the drop using another microscope slide. The thin blood film was then stored in a slide box for later analysis in the laboratory. Lastly, all visible ectopara sites were collected with forceps and stored in a plastic vial with alcohol for later analysis. I returned captured rodents to their capture site in either a cloth bag or Sherman trap and released them back into the wild. Parasite Analysis I viewed the c ollected ectoparasites under a dissecting microscope and classified them into four morph otypes: ticks, fleas, mites, or eggs (Vickery 2011). I recorded t he abundance of each type of ectoparasite for each captured rodent Next, I prepared each thin blood f ilm by placing the slides in a methanol bath for 30 seconds. Next, I prepared the staining solution by mixing Giemsa stain and buffer in a 1:5 ratio. I covered s lides with the Giemsa buffer mixture and allowed them to sit for 30 minutes. Lastly, I rinsed slides with water and then left them to air dry. I viewed s tained slides using a compound microscope at 400x magnification. I chose five random fields of view within the monolayer region of the smear, counted all visible leukocytes,

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Immune Dimorphism in Wild Mice Thoene 4 and took the average of all five values to obtain average leukocyte abundance. I avoided analyzing a reas of the thin blood film that were too darkly stain ed or had many holes or streaks. Results This study consisted of 201 trap nights I captured 34 rodents, 1 3 were male and 21 were female (Table 1). Captured indivi duals belonged to four species: C loud dwelling spiny pocket mouse ( Heteromys nubicolens n=18), Mexican deer mouse ( Peromyscus nudipes n=13), N orthern pygmy rice rat ( Oligoryzomys fulvescens n=1), and ging mouse ( Scotinomys teguina n=1). I recaptured only one individual, a male H. nubicolens who I did not collect ectoparasites or take a blood sample from to prevent creating a duplicate data entry Ectoparasite abundance was collected for all 34 indivi duals, whereas blood samples were only collected from 29 individuals since I did not have permission to collect blood samples for the three individuals captured on 8 November 2016. In addition, I omitted the mouse captured on 25 November 2016 from blood an alysis due to issues properly staining the thin blood film. Table 1. Capture success over the course of the study period. The sites include Estacin Biolgica Monterverde (EBM), Dwight and Rachel Crandell Memorial Reserve (DRCMR), Sendero de Los Nios in Bajo del Tigre (BT N), and Sendero Murcilagos in Bajo del Tigre (BT M). Date Site Males Captured Females Captured Number of Traps 6 November EBM 1 2 8 22 November DRCMR 0 0 8 25 November DRCMR 1 0 19 26 November BT N 2 2 20 27 November BT N 1 0 20 28 November BT N 0 4 20 29 November BT N 3 1 20 30 November BT M 2 0 23 1 December BT M 1 3 23 4 December EBM 2 9 40 Total 13 21 201 Of the four ectoparasite morphotypes, ticks and ear m ites were found on all species. F leas were only found in P. nudipes whereas lice eggs were only found in H. nubicolens One H. nubicolens female and one P. nudipes female was found with a tick. Overall females of both H. nubicolens and P. nudipes were found carryi ng three different types of ectoparasites, whereas males of both species were found carrying only two different types of ectoparasites. Therefore, females displayed greater ectoparasite richness than their male counterparts Although females were found ca rrying a greater variety of ectoparasites, the average ectoparasite abundance per individua l was greater in males for H. nubicolens ( = 48.71 SD = 59.56 ) and P. nudipes ( = 25.25, SD = 24.02; Figure 1). I conducted an unpaired t test for each species to compare average ectoparasite abundance in males versus females. There were no statistically significant differences between sexes for H. nubicolens (t (16) = 0.22, p = 0.82674 )

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Immune Dimorphism in Wild Mice Thoene 5 or P. nudipes ( t (3 ) = 1.25, p = 0.29114 ) O. fulvescens and S. teguina wer e omitted from further statistical analysis since only one individual from each species was captured but their results are shown in Figure 1 Figure 1. Average ectoparasite abundance and morphotype composition is shown for males and females of each sp ecies. Although not visible in the figure, ticks are also present in H. nubicolens and P. nudipes females (Average ectoparasite abundance per individual is 0.09 and 0.11 respectively). For O. fulvescens leukocyte abundance was 5.6. For H. nubicolens average leukocyte abundance was higher in males ( = 11 .3, SD = 7.9 6) than females ( = 5.98, SD = 3.45), whereas P. nudipes females ( = 6.4, SD = 2.82 ) showed slightly higher average leukocyte abundance than males ( = 6.0, SD = 2.51 ; Figure 2). I co nducted an unpaired t test to compare average leukocyte count between males and females for each species. Leukocyte count did not differ significantly between males and females of H. nubicolens (t (6) = 1.56; p = 0.16955 ) or P. nudipes (t (9) = 0.21, p = 0 .83498).

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Immune Dimorphism in Wild Mice Thoene 6 Figure 2 Average Leukocyte Abundance. Values calculated from thin blood films viewed at 400X is given for males and females of each species. In summary, neither ectoparasite abundance nor leukocyte count significantly differs between males and females of the same species. In order to account for variation across species, I conducted an unpaired t test to compare ectoparasite abundance between males of H. nubicolens versus P. nudipes and found that they did not differ significantly ( t (9) = 0.74, p = 0.477961 ) I performed the same test comparing females and found no significant difference among the two species ( t (11 ) = 1.65, p = 0.12911) I also compared average leukocyte count between H. nubicolens and P. nudipes males ( t (7) = 1.09, p = 0.310463 ) and females ( t (17) = 0.28, p = 0.782476 ) and found no significant differences. All captured rodents regardless of sex or species, displayed no significant differences in either ectoparasite abundance or leukocyte count Discu ssion Ultimately, average ectoparasite abundance and leukocyte count were not significantly different between males and females within the same species or across different species. Similarities in parasite load and imm unocompetence between sexes do not s upport the existence of immune dimorphism in the principal study species, namely H. nubicolens and P. nudipes The absence of immune dimorphism in the study species has positive health and survival implications, such as greater resilience to outbreaks of d isease. Since males are not disproportionately at risk to disease and parasitism, the ratio of males to females is more likely to be balanced which enhances the stability of a population. The ratio of males to females in a population provides insight on i mportant factors such as birth rate, age structure and stability as previously mentioned ( Wedekind 2002 ) Therefore, the fact that approximately two thirds of the captured rodents were female has significant consequences. The sex ratio in rodents varies greatly throughout the year and fluctuates in

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Immune Dimorphism in Wild Mice Thoene 7 accordance with breeding season ( Hansson 1978 ); therefore, it is possible that the disproportionate number of females captured is the result of natural variation, however more research is needed to confirm this hypothesis. Ultimately, this result inspires many more questions, such as: do females have higher survival rates during specific times of the year or are females more inclined to visit novel food sources, such as the baited Sherman traps? Population structure and dynamics of local mice populations represents a promising field for further investigation. Studi es have shown that different arthropod parasites will produce variable responses in their hosts; for example, fur mites have been shown to promote anemia in mice, while saliva from hard bodied ticks inhibits inflammation and the release of anticoagulants ( Gillespie et al. 2000). Furthermore, ectoparasites may not provoke any substantial immune response in their host, which is the case between fleas and desert rodents (Khokhlova et al. 2010). Ultimately, the immune response of a host to a specific ectoparasi te is extremely variable; therefore, overall parasite abundance is not the most precise measure of immunocompetence Despite this, my findings on ectoparasite abundance still reveal signs of host specificity. In particular, fleas were only found on P. nudi pes and lice eggs were only found on H. nubicolens ; however, the origin behind these host parasite trends remains unknown. Future studies on this topic can involve exposing mice to a variety of ectoparasites in a controlled setting to improve our understan ding of this interaction. Although differences in leukocyte count between sexes was not significantly different, the relatively high average leukocyte count for H. nubicolens males should be explored further In the Monteverde region, o lder H. nubicolens males are captured more frequently than younger males ( D.R. personal obs .) Older individuals typically have higher leukocyte counts due to prolonged inflammation and arthritis; therefore, the data may have been skewed by this age bias trend ( Barnhart 2011 ). It is difficult to draw conclusions about the existence of immune dimorphism without the use of more advanced technology and procedures; however, future studies can make use of visual and physical observations to aid in understanding immune response. Li fe stage, mating system, and reproductive status of captured rodents provide valuable insight on expected hormone and leukocyte concentrations (Klein and Nelson 1998). Despite the limitations of my study, the results serve as the foundation for many n ew questions. For example, we have yet to determine which metrics are best suited for testing the existence of immune dimorphism. In addition, it remains unclear if immune dimorphism is present in other forms, such as blood or gastrointestinal parasitism. Further areas of research include investigating the relationship between arthropod parasites and the specif ic immune response they elicit. In conclusion, the adaptability of local mice and rat populations to changing environment al conditions remains uncert ain. Further studies on rodent populations and disease transmission are needed to inform best practices for conservation and management Acknowledgments First and foremost, I would like to thank Federico Chinchilla who served as my primary advisor as well as fieldwork partner, I would not have been able to handle mice or complete this project without his help. In addition, I would like to thank the Monteve rde Institute for providing for allowing me to work in the reserve, and to Marvin for allowing me to work in the area surrounding the Estacin Biolgica Montev erde. Lastly, I would like to thank visiting scientist

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Immune Dimorphism in Wild Mice Thoene 8 Dr. David Ribble for allowing me to collect data from rodents he was capturing for an independent research project. His expertise and support also made this project possible. Literature Cited Anderson, R. P., & Timm, R. M. 2006 A new montane species of spiny pocket mouse (Rodentia: Heteromyidae: Heteromys ) from northwestern Costa Rica. American Museum Novitates, 1 38. Bacelar, F. S., White, A., & Boots, M. 2011 Life history and mating system s select for male biased parasitism mediated through natural selection and ec ological feedbacks. Journal of T heoretical B iology, 269(1), 131 137. Barnhart, K. (2011, June 10). Comparative Hematology of Laboratory Animals [PDF]. M.D. Andersen Cancer Center Bhatia, A., Sekhon, H. K., & Kaur, G. 2014 Sex hormones and immune dimorphism. Th e Scientific World Journal Doeing, D. C., Boro wicz, J. L., & Crockett, E. T. 2003 Gender dimorphism in differential peripheral blood leukocyte counts in mice usin g cardiac, tail, foot, and saphenous vein puncture methods. BMC clinical pathology, 3(1), 1. Gillespie, R. D ., Mbow, M. L., & Titus, R. G. 2000 The immunomodulatory factors of bloodfeeding arthropod saliva. Parasite Immunology, 22(7), 319 331. doi:10.104 6/j.1365 3024.2000.00309.x Gonzalez, D. 2009 Rodent species richness in disturbed and natural habits [ EAP Course Book ]. Hansson, L. 1978 Sex ratio in small mammal populations as affected by the pattern of fluctuations. Acta Theriologica, 23, 203 212 doi:10.4098/at.arch.78 13 Hu ghes, V. L., & Randolph, S. E. 2001 Testosterone depresses innate and acquired resistance to ticks in natural rodent hosts: a force for aggregated distributions of parasites. Journal of Parasitology, 87(1), 49 54. Khokhlova I. S., Spinu, M., Krasnov, B. R., & Degen, A. A. 2004 Immune response to fleas in a wild desert rodent: effect of parasite species, parasite burden, sex of host and host parasitological experience. Journal of Experimental Biology, 207(16), 2725 2733. K hokhlova, I. S., Ghazaryan, L., Degen, A. A., & Krasnov, B. R. 2010 Infestation experience of a rodent host and offspring viability of fleas: variation among host parasite associations. Journal of Experimental Zoology Part A: Ecological Genetics and Physi ology, 313(10), 680 689.

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Immune Dimorphism in Wild Mice Thoene 9 Klein, S. L., & Nelson, R. J. 1998 Adaptive immune responses are linked to the mating system of arvicoline rodents. The American Naturalist, 151(1), 59 67. Kowalski, K., Bogdziewicz, M., Eichert, U., & Rychlik, L. 2015 Sex differences in flea infections among rodent hosts: is there a male bias? Parasitology research, 114(1), 337 341. Lai, J. J., Lai, K. P., Zeng, W., Chuang, K. H ., Altuwaijri, S., & Chang, C. 2012 Androgen receptor influences on body defense system via mo dulation of innate and adaptive immune systems: lessons from conditional AR knockout mice. The American journal of pathology, 181(5), 1504 1512. Martin, L. B. Weil, Z. M., & Nelson, R. J. 2007 Immune defense and reproductive pace of life in Peromyscus m ice. Ecology, 88(10), 2516 2528. Mi halca, A. D., & Sndor, A. D. 2013 The role of rodents in the ecology of Ixodes ricinus and associated pathogens in Central and Eastern Europe. Frontiers in cellular and infection microbiology, 3, 56. Myers, P. 2000 R odentia (rodents). Retrieved December 06, 2016, from http://animaldiversity.org/accounts/Rodentia/ Peckham, M. 2004 White Blood Cells. Retrieved from http://www.histology.leeds.ac.uk/blood/blood_wbc.php Ribble, D. (2016, December 5). Personal Observatio n. Costa Rica, Monteverde. Vickery, A. 2011 The dilution effect hypothesis of disease ecology in continuous and fragmented forests of Monteverde, Costa Rica [ EAP Course Book ]. Wedekind, C. 2002 Manipulating sex ratios for conservation: short term risks and long term benefits. Animal Conservation, 5(1), 13 20.

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Immune Dimorphism in Wild Mice Thoene 10 Appendix Table A Sherman trap status over the course of the study period. The sites include Estacin Biolgica Monterverde (EBM), Dwight and Rachel Crandell Memorial Reserve (DRCMR), Sende ro de Los Nios in Bajo del Tigre (BT N), and Sendero Murcilagos in Bajo del Tigre (BT M). Date Site Males Captured Females Captured Trap Closure Stolen Bait No Activity Number of Traps 6 Nov EBM 1 2 1 0 4 8 22 Nov DRCMR 0 0 0 1 7 8 25 Nov DRCMR 1 0 0 5 13 19 26 Nov BT N 2 2 3 7 6 20 27 Nov BT N 1 0 2 9 8 20 28 Nov BT N 0 4 0 5 11 20 29 Nov BT N 3 1 6 4 6 20 30 Nov BT M 2 0 12 3 6 23 1 Dec BT M 1 3 0 6 13 23 4 Dec EBM 2 9 11 0 18 40 Total 13 21 35 40 92 201