Sex Differences In Innate Immunity In Tree Swallows

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1 Grand Valley State University Student Summer Scholars Undergraduate Research and Creative Practice 2009 Sex Differences In Innate Immunity In Tree Swallows Bradley J. Houdek Grand Valley State University Michael P. Lombardo Grand Valley State University, Patrick A. Thorpe Grand Valley State University, Follow this and additional works at: Part of the Biology Commons Recommended Citation Houdek, Bradley J.; Lombardo, Michael P.; and Thorpe, Patrick A., "Sex Differences In Innate Immunity In Tree Swallows" (2009). Student Summer Scholars This Open Access is brought to you for free and open access by the Undergraduate Research and Creative Practice at It has been accepted for inclusion in Student Summer Scholars by an authorized administrator of For more information, please contact

2 REMINDER: THIS IS A DRAFT ONLY SEX DIFFERENCES IN INNATE IMMUNITY IN TREE SWALLOWS Bradley J. Houdek, Michael P. Lombardo, Patrick A. Thorpe Department of Biology, Grand Valley State University, Allendale, MI REMINDER: THIS IS A DRAFT ONLY

3 Abstract Evolutionary theory predicts that exposure to more diverse pathogens will lead to the evolution of more effective immune responses. The innate immune system defends the host from pathogens in a non-specific manner and is an important first-line of defense. We predicted that female Tree Swallows have more robust innate immunocompetence than males because females are exposed to more microbes during the breeding season than are males. This is because (a) females participate in extra-pair copulations with multiple males exposing them to sexually transmitted microbes (STMs) (e.g., bacteria, fungi, viruses), (b) the transmission of STMs during copulation is asymmetrical because ejaculates move from males to females, (c) Tree Swallow semen contains potentially pathogenic STMs, and (d) females spend more time in the nest than males. Additionally, elevated testosterone in males is a known correlate with suppressed immune function. We tested our prediction in the 2009 breeding season by conducting an assay of the innate immune system using whole-blood samples. A microbicidal assay using E. coli produced an index of the capacity of the blood to kill bacteria. Tree Swallow whole blood readily lyses E. coli, but there was no difference in mean E. coli lysis levels between males and females. However, females with higher lysis levels had less louse damage to feathers and older females with higher lysis levels had greater fledging success. These results suggest that while female Tree Swallows may not experience greater pathogen exposure, female innate immunocompetence may predict reproductive success. Introduction Ecological immunology examines the ecological and evolutionary relationships between parasites and their hosts. It has become increasingly clear to eco-immunologists that immunocompetence is of significant importance in shaping life history patterns (Viney et al. 2005), and is a key mechanism regulating host survival. Piersma (1997) postulated that life histories should tend to reflect the pathogenic environment as much as any other factor contributing to evolutionary change within a species. Additionally, Lochmiller and Deerenberg (2000) have gone so far as to suggest that immunocompetence; an organism s capacity to prevent or control infection (Owens and Wilson 1999), could be the most important fitness-influencing factor in many species. Within living organisms, pathogen defense is accomplished by a complex immune system, whose function is to defend against infection in a fitness-maximizing manner (Viney et al. 2005). Conventionally the vertebrate immune system is divided into innate and acquired

4 immunity. Acquired immunity develops throughout the lifetime of an individual as an adaptation to infection with a specific pathogen, and may even confer lifelong protection (Bonneaud et al. 2003). This lifelong protection is achieved through immunological memory, which allows the host to respond if the same antigen is introduced in the future (Zuk and Stoehr 2002). In general, the acquired branch is based on the activation of B-cells, T-cells, and the production of antibodies capable of highly-specific antigen binding (Schmid-Hempel 2003). Before activation of an acquired response, many pathogens are swiftly detected and destroyed by the innate immune system (Bonneaud et al. 2003). The innate immune system provides a rapidly inducible and non-specific first line of defense against pathogens, that works to neutralize them before an acquired response is triggered (Grieves et al. 2006; Lochmiller and Deerenberg 2000; Rickert 2005). Active components of the innate branch include natural killer cells, and a number of cells (e.g., macrophages, neutrophils, dendritic cells) capable of phagocytosing non-self materials (Viney et al. 2005). Unlike components of the acquired branch, innate components do not develop memory of specific antigens (Janeway et al. 1999), and are thus most important during a host s first encounter with a specific pathogen (Lee et al. 2006). Pathogen defense clearly confers benefits to host but may also incur numerous costs (e.g., developmental, energetic, nutritional), demanding it be optimized rather than maximized (Bonneaud et al. 2003; Stoehr 2007; Viney et al. 2005; Zuk and Stoehr 2002). Optimal resource allocation to pathogen defense, including not only maintaining a competent immune system but also being able to mount a successful immune response when challenged, will depend upon resource availability and tradeoffs with other competing life-history characteristics (Bonneaud et al. 2003; Sheldon and Verhulst 1996) like growth, thermoregulation, and reproduction. Schmid-

5 Hempel (2003) referred to this as the evolutionary cost of the immune system, since it too must evolve only at the expense of investment in other traits (a life-history tradeoff). A recently active subdivision of ecological immunology is one focused on sex differences in immunocompetence (Stoehr and Kokko 2006). In general, female vertebrates show greater immunocompetence than males (Grossman 1984; McGraw and Ardia 2005; Møller et al. 1998). This has historically been attributed to the immunosuppressive effects of elevated testosterone in males (i.e., the immunocompetence handicap hypothesis (ICHH): Folstad and Karter 1992), or to fundamental differences between the sexes in tradeoffs between pathogen defense and other costly life-history traits (Nunn et al. 2009; Rolff 2002). In these ways, sexual dimorphism in immunocompetence is ultimately the result of differences in how the sexes maximize fitness (Stoehr and Kokko 2006). If, as the susceptible male hypothesis (Rolff 2002) predicts, female fitness benefits more from longevity while male fitness benefits more from investment in current reproduction, females should evolve to increase survivorship through greater investment in immune defense relative to males. This study examined sex-biased variation in the innate immunocompetence of breeding adult Tree Swallows (Tachycineta bicolor). Since males and females of this species face different levels of pathogen exposure (reference), selection should shape immunocompetence of the sexes differently. We predicted that during the breeding season, female Tree Swallows would show greater innate immunocompetence than would males because of: 1)greater pathogen challenge due to unequal microbe transfer during copulation (Lombardo et al. 1999), 2) more exposure to nest microbes due to time spent in the nest during nest building, incubation, brooding, and nestling care (Robertson et al. 1992), and 3) less immuno-suppression by testosterone, as compared to males.

6 Methods Data were collected from adult Tree Swallows nesting on the campus of Grand Valley State University in Allendale, MI (42 57 N, W), from May July, Tree Swallows are aerial insectivores with a socially monogamous breeding system (Robertson et al. 1992). They are a good model species because they readily breed in nest boxes, and are tolerant of human activity and direct handling. The study site was located in an old agricultural field, and consisted of a 10 x 10, 100-box grid of standard wooden nest-boxes. Nest boxes were arranged in rows each spaced 20m apart from the next. Blood samples ranging from ~10-60 µl were collected from the brachial vein on the right wing of adult Tree Swallows. To ensure a sterile bleeding site, the area surrounding the brachial vein was cleared of interfering feathers, soaked liberally with 70% EtOH, swabbed with a fresh cotton ball, and allowed to air dry seconds. The brachial vein was punctured with a sterilized lancet, and blood was collected in heparinized capillary tubes and transported to the laboratory in sterile 50 ml Fisher tubes. All blood samples were collected within 3 minutes of handling, to avoid the effects of immunosuppression mediated by stress hormones such as corticosterone (reference). Following blood sample collection, mass, head-bill length, wing chord length, depth of tail forking, and the number of ectoparasite (louse) holes on wings and tails were recorded for each individual. Females were grouped into separate age classes based on plumage characteristics. SY denotes second-year females with no prior breeding experiences; ASY denotes after second-year females with at least one season of prior breeding experiences. For each nest, dates of clutch initiation (when first egg was laid) and clutch size were also recorded.

7 A microbicidal assays was performed using Tree Swallow whole blood, following Millet et al. (2007). Specifically, this assay measures the ability of innate components (e.g., natural antibodies, complement proteins, lysozyme) within plasma to clear a bacterial infection. All statistical analysis was completed using SPSS. Results Considering all complete mate-pairs sampled (n=34), we found no significant difference between sexes in mean proportion lysis (female: mean=0.83, SD=0.25; male: mean=0.80, SD=0.27; male vs. female: Z=-0.45, p=0.65), and no significant relationship between mate-pairs. This same pattern also held when all individuals sampled were considered (female: n=44, mean=0.84, SD=0.23; male: n=35, mean=0.80, SD=0.26; male vs. female: Mann-Whitney U=730.0, Wilcoxon W=1360.0, Z=-0.40, p=0.69). Within females, there was no significant difference between SY and ASY age classes in mean proportion E. coli lysis (SY: mean=0.84, SD=0.27; ASY: mean=0.85, p=0.21; SY vs. ASY: Mann-Whitney U=235.0, Wilcoxon W=560.0, Z=-0.06, p=0.95). For both sexes, there was no significant correlation between mean proportion lysis and clutch size, % hatch, % fledge, or dates of nest completion and clutch initiation. Individuals were grouped into separate kill levels according to their mean proportion lysis (high = proportion lysis >0.80, low = proportion lysis 0.80). Overall, high-kill females had significantly fewer total louse holes than did low-kill females (Mann-Whitney U=95.0, Wilcoxon W=623.0, Z=-2.59, p=0.01). For SY females, there was no significant correlation between kill level and clutch size, % hatch, % fledge, or dates of completion and clutch initiation (all p>0.05). For ASY females, there was no significant correlation between kill level and clutch size, %

8 hatch, or dates of nest completion and clutch initiation (all p>0.05); however, there was a significant relationship between kill level and % fledge, with high-kill females successfully fledging a greater proportion of nestlings. High-kill and low-kill males did not differ significantly from each other in number of total louse holes (Mann-Whitney U=127.0, Wilcoxon W=218.0, Z=-0.55, p=0.58). Within mate-pairs, there was no significant relationship between male mean proportion lysis and the age of his female partner (SY or ASY). There was no significant difference between SY and ASY females in proportion of eggs that successfully hatched (p=0.44) or proportion of nestlings that successfully fledged (p=0.95). However, SY and ASY females did differ significantly from each other in date of clutch initiation (p=0.01) and clutch size (p=0.01), with ASY females beginning clutch initiation earlier in the breeding season and laying larger clutches. For each individual we recorded mass, head-bill length, wing chord length, depth of tail forking, and the number of louse holes on wings and tail. For males, there was no correlation between date of sampling and any of these morphological features (all p>0.05). For females, there was no correlation between date of sampling and head-bill length, wing chord length, depth of tail forking, and number of louse holes (all p>0.05); however, some standardization was necessary because date of sampling had a significantly positive correlation with body mass (p=0.00). There were no significant differences between sexes in body mass (p=0.06), head-bill length (p=0.21), wing louse holes (p=0.06), tail louse holes (p=0.83), or total louse holes (p=0.59). However, the sexes were found to differ significantly in wing-chord length (p=0.00) and depth of tail-forking (p=0.00), with males possessing longer wings and deeper tail-forks. For both sexes, there was no significant correlation between mean proportion E. coli lysis and body mass, head-bill length, wing chord length, or depth of tail forking (all p>0.05). For males, there

9 was no significant correlation for between mean proportion E. coli lysis and any louse hole measurement. For females, while there was no significant correlation between E. coli lysis and wing louse holes, there was a significant negative correlation with both tail and total louse holes. Discussion Only recently have eco-immunologists developed interest in better understanding variation in the immune response, both within and across species boundaries. Using breeding adult Tree Swallows, we predicted that female Tree Swallows would show greater immunocompetence than would males because of lower levels of endogenous, immunesuppressing testosterone, and greater exposure to pathogens, following the idea that exposure to greater numbers of pathogens drives the evolution of increased immune defense. Contrary to our prediction, we found no difference between sexes in the ability of whole blood to lyse E. coli. Although means differed in the predicted direction, with females displaying greater immunocompetence than males, the difference was not significant (see Results). This lack of difference in proportion lysis, a measure of immunocompetence, between the sexes may be due to several factors. First, our sample population consisted exclusively of breeding individuals, enhancing the likelihood that we only sampled high quality individuals who were able to gain mates and maintain a breeding territory. Second, the high frequency of extra-pair mating in Tree Swallows (reference) may ameliorate any differences between the sexes in exposure to sexually transmitted pathogens.

10 References Bonneaud C, Mazuc J, Gonzalez G, Haussy C, Chastel O, Faivre B, Sorci G Assessing the cost of mounting an immune response. The American Naturalist 161(3): Folstad I, Karter A J Parasites, bright males, and the immunocompetence handicap. The American Naturalist 139: Frank M M, Miletic V D, Jiang H X Immunoglobulin in the control of complement action. Immunologic Research 22: Grieves T J, McGlothlin J W, Jawor J M, Demas G E, Ketterson E D Testosterone and innate immune function inversely covary in a wild population of breeding Dark-Eyed Juncos (Junco hyemalis). Functional Ecology 20: Grossman C J Regulation of the immune system by sex steroids. Endocr. Rev. 5: Hasselquist D, Marsh J A, Sherman P W, Wingfield J C Is avian humoral immunocompetence suppressed by testosterone? Behav. Ecol. Sociobiol 45: Janeway C A, Travers P, Walport M, Capra J C Immunobiology: the immune system in health and disease, 4 th edn. Current Biology Publications, London. Lee K A, Martin II L B, Hasselquist D, Ricklefs R E, Wikelski M Contrasting adaptive immune defenses and blood parasite prevalence in closely related Passer sparrows. Oecologia 150: Lochmiller R L, Deerenberg C Trade-offs in evolutionary immunology: just what is the cost of immunity? Oikos 88:87-98.

11 McGraw K J, Ardia D R Sex differences in carotenoid status and immune performance in zebra finches. Evolutionary Ecology Research 7: Millet S, Bennett J, Lee K A, Hau M, Klasing K C Quantifying and comparing constitutive immunity across avian species. Developmental and Comparative Immunology 31: Moller A P, Sorci G, Erritzoe J Sexual dimorphism in immune defense. American Naturalist 152: Moret Y Explaining variable costs of the immune response: selection for specific versus non-specific immunity and facultative life history change. Oikos 102: Nunn C L, Lindenfors P, Pursall E R, Rolff J On sexual dimorphism in immune function. Phil. Trans. R. Soc. B 364: Owens I, Wilson K Immunocompetence: a neglected life history trait or conspicuous red herring? Trends in Ecology and Evolution 14: Piersma T Do global patterns of habitat use and migration strategies coevolve with relative investments in immunocompetence due to spatial variation in parasite pressures (should this be a?). Oikos 80: Rickert R C Regulation of B lymphocyte activation by complement C3 and the B cell coreceptor complex. Current Opinions in Immunology 17: Rolff J Bateman s principle and immunity. Proc. R. Soc. Lond. B 269: Schmid-Hempel P Variation in immune defense as a question of evolutionary ecology. Proceedings of the Royal Society of London, B 270: Sheldon B C, Verhulst S Ecological immunology: costly parasite defenses and trade-offs in evolutionary ecology. Trends in Ecology and Evolution 11: Stoehr A M Inter- and intra-sexual variation in immune defence in the cabbage white butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Ecological Entomology 32: Stoehr A M, Kokko H Sexual dimorphism in immunocompetence: what does life-history theory predict? Behavioral Ecology 17: Viney M E, Riley E M, Buchanan K L Optimal immune responses: immunocompetence revisited. Trends in Ecology and Evolution 20(12): Zuk M Disease, endocrine-immune interactions, and sexual selection. Ecology 77:

12 Zuk M, Stoehr A M Immune defense and host life history. American Naturalist 160:S9- S Wilcoxon signed ranks, Z = -0.45, P = 0.65 Mean Proportion Lysis Male Sex Female Figure 1: There was no significant difference between mates in mean proportion lysis.

13 1.0 Proportion Male Lysis Spearman rho = 0.16, P = Proportion Female Lysis Figure 2: There was no significant correlation between mates in proportion lysis.

14 Mean Proportion Lysis by Category Proportion Lysis SY-females ASY-females All females Males Figure 3: There were no significant differences in proportion lysis between females of different age classes or between males and females (all P > 0.05).

15 Female Total Louse Holes x Proportion Lysis Male Total Louse Holes x Proportion Lysis Total Louse Holes 40 Spearman rho = -0.34, P = Proportion Lysis Total Louse Holes 70 Spearman rho = -0.02, P = Proportion Lysis Figure 4: There was a significant negative correlation between female total number of louse holes and proportion lysis. However, this relationship was absent in males. There were no significant differences between females and males in the number of louse holes in their wing and tail feathers.

16 ASY females 1.0 Mann-Whitney U = 29.5, P = 0.04 Proportion of Hatchling that Fledged Low ( 0.80) High (> 0.80) Lysis Level Figure 5: ASY females with high proportion lysis (> 0.80) fledged a significantly greater proportion of hatchlings than did those with low proportion lysis ( 0.80).

17 14 Mann-Whitney U = 95, P = Mean Total Louse Holes Low ( 0.80) High (> 0.80) Lysis Level Figure 6: Females with high proportion lysis (> 0.80) had significantly fewer louse holes than did females with low proportion lysis ( 0.80).

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