Impact of an introduced parasite on Darwin's finches

Invasive parasites are a growing problem as humans continue to traverse the globe. The impact invasive parasites have on naive host populations is the focus of my dissertation. To date, the Galapagos Islands remain one of the most well-preserved archipelagos, with no known extinctions of endemic bird species. However, the recent introduction of Philornis downsi, an obligate nest parasite, threatens birds across the islands, including the iconic Darwin's finches. Using an experimental manipulation of parasite abundance in nests, my work shows the detrimental effect P. downsi has on fledging success in medium ground finches (Geospiza fortis). I explore the mechanisms underlying these effects by investigating the impact of P. downsi on nestling growth and condition. I demonstrate that adult medium ground finches and seven other species of Darwin's finches produce P. downsi-specific antibodies. Nestling medium ground finches did not have detectable P. downsi-specific antibodies nor was there evidence of maternally transferred antibodies. Parental behavior also changed in response to P. downsi parasitism, though neither immunological nor behavioral responses were effective against P. downsi, and did not result in increased host reproductive success. Finally, using data from my three-year study, I present a model that predicts population viability of medium ground finches in light of the observed effects of P. downsi on host fitness. The model predicts that medium ground finches on the island of Santa Cruz are likely to go extinct within the next half century unless conservation efforts are able to significantly reduce P. downsi populations. My work highlights the dramatic impact an introduced parasite can have on naive host populations. Parasites with low host-specificity and high rates of dispersal, such as P. downsi, can maintain high levels of virulence. In combination with ineffective host defense mechanisms, introduced parasites can lead to severe host population declines, even extinctions.

IMPACT OF AN INTRODUCED PARASITE ON DARWIN'S FINCHES by Jennifer Amelia Hutchens Koop A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology The University of Utah August 2011 Copyright© Jennifer Amelia Hutchens Koop 2011 All Rights Reserved The Univers i t y o f Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Jennifer Amelia Hutchens Koop has been approved by the following supervisory committee members: Dale H. Clayton , Chair 5/25/2011 Date Approved M. Denise Dearing , Member 5/25/2011 Date Approved Wayne K. Potts , Member 5/25/2011 Date Approved Franz Goller , Member 5/25/2011 Date Approved Jeb P. Owen , Member 5/25/2011 Date Approved and by Neil J. Vickers , Chair of the Department of Biology and by Charles A. Wight, Dean of The Graduate School. ABSTRACT Invasive parasites are a growing problem as humans continue to traverse the globe. The impact invasive parasites have on naïve host populations is the focus of my dissertation. To date, the Galapagos Islands remain one of the most well-preserved archipelagos, with no known extinctions of endemic bird species. However, the recent introduction of Philornis downsi, an obligate nest parasite, threatens birds across the islands, including the iconic Darwin's finches. Using an experimental manipulation of parasite abundance in nests, my work shows the detrimental effect P. downsi has on fledging success in medium ground finches (Geospiza fortis). I explore the mechanisms underlying these effects by investigating the impact of P. downsi on nestling growth and condition. I demonstrate that adult medium ground finches and seven other species of Darwin's finches produce P. downsi-specific antibodies. Nestling medium ground finches did not have detectable P. downsi-specific antibodies nor was there evidence of maternally transferred antibodies. Parental behavior also changed in response to P. downsi parasitism, though neither immunological nor behavioral responses were effective against P. downsi, and did not result in increased host reproductive success. Finally, using data from my three-year study, I present a model that predicts population viability of medium ground finches in light of the observed effects of P. downsi on host fitness. The model predicts that medium ground iv finches on the island of Santa Cruz are likely to go extinct within the next half century unless conservation efforts are able to significantly reduce P. downsi populations. My work highlights the dramatic impact an introduced parasite can have on naïve host populations. Parasites with low host-specificity and high rates of dispersal, such as P. downsi, can maintain high levels of virulence. In combination with ineffective host defense mechanisms, introduced parasites can lead to severe host population declines, even extinctions. TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii LIST OF FIGURES .......................................................................................................... vii LIST OF TABLES...............................................................................................................x ACKNOWLEDGMENTS ................................................................................................. xi Chapter 1. INTRODUCTION ...................................................................................................1 Background..................................................................................................1 Chapter Summaries......................................................................................3 References....................................................................................................8 2. EXPERIMENTAL DEMONSTRATION OF THE FITNESS CONSEQUENCES OF AN INTRODUCED PARASITE OF DARWIN'S FINCHES............10 Abstract......................................................................................................11 Introduction................................................................................................11 Materials and Methods...............................................................................12 Results........................................................................................................14 Discussion..................................................................................................15 Acknowledgments......................................................................................16 References..................................................................................................16 3. ECOIMMUNITY IN DARWIN'S FINCHES: INVASIVE PARASITES TRIGGER ACQUIRED IMMUNITY IN THE MEDIUM GROUND FINCH (GEOSPIZA FORTIS)..................................................................18 Abstract......................................................................................................19 Introduction................................................................................................19 Results........................................................................................................20 Discussion..................................................................................................21 Methods......................................................................................................22 Acknowledgments......................................................................................24 References..................................................................................................24 vi 4. TEST FOR PARASITE-SPECIFIC IMMUNE RESPONSE IN MULTIPLE SPECIES OF DARWIN'S FINCHES .......................................................25 Abstract......................................................................................................25 Introduction................................................................................................26 Methods......................................................................................................29 Results........................................................................................................33 Discussion..................................................................................................38 Acknowledgments......................................................................................39 References..................................................................................................40 5. ARE DARWIN'S FINCHES SITTING DUCKS? INEFFECTIVE HOST DEFENSES AGASINT AN INTRODUCED PARASITE .......................43 Abstract......................................................................................................43 Introduction................................................................................................44 Methods......................................................................................................48 Results........................................................................................................57 Discussion..................................................................................................71 Acknowledgments......................................................................................77 References..................................................................................................77 6. THE DEMISE OF DARWIN'S FINCHES? A MODELING APPROACH TO ASSESS THE IMPACT OF AN INTRODUCED PARASITE ON HOST POPULATION VIABILITY.....................................................................82 Abstract......................................................................................................82 Introduction................................................................................................83 Methods......................................................................................................88 Results........................................................................................................95 Discussion..................................................................................................97 Acknowledgments....................................................................................102 Appendix..................................................................................................103 References................................................................................................106 7. CONCLUSION....................................................................................................110 References................................................................................................115 Appendices A: HOW BIRDS COMBAT ECTOPARASITES ...............................................118 B: DOES SUNLIGHT ENHANCE THE EFFECTIVENESS OF AVIAN PREENING FOR ECTOPARASITE CONTROL? ...............................150 LIST OF FIGURES Figure 2.1 Study organisms.....................................................................................................12 2.2 Comparison of the mean (± SE) number of P. downsi in lined and unlined nests........................................................................................................................14 2.3 Comparison of mean (± SE) growth parameters for nestlings in lined () and unlined () nests, including body mass (A), tarsus length (B), and outermost primary feather length (C) .....................................................................................14 2.4 Effect of liners on host fledging success................................................................15 3.1 Parasite-specific antibody response of Geospiza fortis .........................................20 3.2 Western blot of serum dilutions developed for houst sparrow IgY .......................23 3.3 Optimization of ELISAs for antigen and Darwin's finch serum ...........................23 4.1 Comparison of cross-reactivity of house sparrow antiserum with plasma from different Darwin's finch species ............................................................................35 4.2 Results of enzyme-linked immunosorbent assays (ELISA) to test whether multiple species of Darwin's finches mount P. downsi-specific antibody responses................................................................................................................36 5.1 Mean (± SE) anti-P. downsi antibody responses (optical density, OD) of females, males and nestlings from fumigated (gray bars) and control (hatched bars) nests........................................................................................................................58 5.2 Relationship between adult female anti-P. downsi antibody response and P. downsi abundance within control nests..................................................................59 5.3 Differences in female brooding (A, B) and nest sanitation (C, D) behaviors between fumigated and control nests.....................................................................61 viii 5.4 Results of linear effects mixed models used to predict the effect of nest treatment and nestling age on (A) mass, (B) tarsus length, and (C) outermost primary feather length .........................................................................................................65 5.5 Mean (± SE) hematocrit values for nestlings in fumigated and control nests .......68 5.6 Effect of nest treatment on host fledging success in 2010.....................................69 6.1 Diagram of model predicting annual reproductive fitness.....................................89 6.2 Distribution of extinction times over 1,000 model simulations for medium ground finches....................................................................................................................96 6.3 Mean extinction times as a function of parasite prevalence during wet years under both operational definitions of fledgling success...................................................98 6.4 Frequency plots of the number of nests to produce a number of fledglings under the following conditions of weather and P. downsi parasitism ...........................104 A.1 Crested Auklets (Aethia cristatella), such as the one shown here, emit a citrus-like odor that may deter ectoparasites.........................................................................122 A.2 (a) Preening Black Swan (Cygnus atratus) (b) Allopreening between Magellanic Penguins (Spheniscus magellanicus)...................................................................124 A.3 Natural and experimentally induced variation in the bill overhang.....................125 A.4 (a) Mean (± SE) number of lice on 26 adult pigeons in an experiment to test the impact of the bill overhang on preening efficiency. (b) SEM of an undamaged louse (Campanulotes compar), compared to lice that have had most of their legs removed (c), or been decapitated (d), or lacerated (e) by birds with normal overhangs.............................................................................................................126 A.5 Overhang lengths of Western Scrub-jays, in relation to ectoparasite abundance ............................................................................................................127 A.6 Variation in the structure of the pectinate claw, ranging from (a) the coarsely serrated claw of the American Dipper (Cinclus mexicanus) to (b) the finely serrated claw of the Magnificent Frigatebird (Fregata magnificens)..................127 A.7 Barn owls (Tyto alba) have a pectinate claw on their middle toe, which is used in scratching.............................................................................................................138 A.8 Southern ground-hornbill (Bucorvus leadbeateri) dusting itself.........................139 A.9 White-rumped shama (Copsychus malabaricus) sunning itself ..........................139 ix A.10 Jay(Garrulus glandarius) anting .........................................................................140 A.11 Mean (± SE) number of (a) feather mites and (b) lice on European starlings (Sturnus vulgaris) before and after experimental birds were allowed to engage in anting behavior.....................................................................................................141 A.12 Bearded vultures (Gypaetus barbatus) stain their plumage with soil rich in iron oxide; captive birds without access to such soil have white underparts..............142 B.1 Mean (± SE) percent time spent preening for each treatment group: sun exposed (Sun), shade (Shade), not bitted (NB), and bitted (B) .........................................157 B.2 Mean (± SE) number of (A) adult lice, and (B) adult and nymphal lice combined, at the end of the experiment: sun exposed (Sun), shade (Sun), not bitted (NB), and bitted (B)..............................................................................................................158 LIST OF TABLES Table 2.1. Tests of the impact of Philornis downsi on Darwin's Finches...............................13 4.1. Results of Dunnett's multiple comparison post-hoc test of slopes between Geospiza fortis and other species of Darwin's finches relative to their cross-reactivity with house sparrow antiserum ...............................................................34 5.1 Linear mixed-effects models to compare growth parameters of nestlings in fumigated and control nests ...................................................................................64 6.1 Summary of results from three-year study on the effects of P. downsi on medium ground finch fitness .................................................................................87 6.2 Parameter descriptions and estimates ....................................................................91 A.1 Occurrence of pectinate claws among 1421 study skins of birds representing 278 species in 250 genera (118 families, 23 orders)............................................129 A.2 Examples of birds known to dust.........................................................................138 ACKNOWLEDGMENTS My graduate school experience parallels a typical field season in the Galapagos. The extreme heat, cactus spines, fire ants and razor sharp lava rocks present challenges to the tasks at hand. However, upon completion, sitting back with caipirinha in hand, I realize how lucky I have been to experience it all, how grateful I am to those who have helped me, and how much fun I had along the way. Dale Clayton, my advisor and co-author, has always been there with bold ideas, loud gestures, and high expectations. His knowledge and enthusiasm have made me a better biologist and a stronger person. I am very grateful for his continued financial support, encouragement and fearlessness. I am also indebted to the post-docs and graduate students who have made up the Clayton lab over the past five years. I am especially thankful to Sarah Huber, who took me under her wing and showed me the challenges and rewards of doing field work in the Galapagos. It is largely due to Sarah that I am one of the lucky few who will fledge (pending committee approval, of course). I am thankful to Celine LeBohec, Sarah Knutie and Sarah Bush, who were able to join me in the field. I thank them for for their contributions to the projects as coauthors, and also for their perpetual optimism, even while picking spines out of some questionable body regions. I was also very lucky to be supported by wonderful assistants both in the Galapagos (Randy Cordova, Oliver Tisalema, Priscilla Espina, Roger and Mimi Clayton) and in Utah (especially Autumn Henry and Ale Alguilar). I thank my committee, Denise Dearing, Franz Goller, Jeb xii Owen, and Wayne Potts, for their helpful input and advice pertaining to this thesis. I would like to thank Peter Kim for his patience and help as a coauthor in creating the model presented in Chapter 6. I would also like to thank Fred Adler and Sean Laverty, for their patience and humor as statistical consultants and collaborators. The staff of the University of Utah biology department are amazing and have helped keep me on track through all the paperwork and logistical details. I would like to extend my sincerest thanks to my friends and family, who have dealt with me through the inevitable hiccups of graduate school life. Four GALS in particular, Lesley Chesson, Jael Malenke, Johanna Varner and Jessica Waite, provided unwavering support and friendship, not to mention tasty breakfast treats. I am grateful to my parents, who despite my not wanting to be an engineer or a nurse, have supported me as I ran around desert islands chasing birds and counting maggots. Finally, I thank my husband, David Koop, for his love, tolerance, patience, sympathy and encouragement, which was obvious regardless of the number of miles between us. I also want to thank the Galapagos National Park and the Charles Darwin Foundation for approving and supporting my research. Many thanks to Ivan Cabrera Sanchez for all the pre-dawn rides to our field site in the all-powerful Mobil Connan. This dissertation was financially supported in part by an NSF grant to DHC (DEB- 0816877), the Society for the Study of Evolution through DHC, a Frank A. Chapman grant from the American Museum of Natural History, Sigma Xi Grant-in-aid-of-research awards, an NSF research collaborative network grant, and an American Ornithologists' Union research award. Additional financial support came from the University of Utah Biology Department. CHAPTER 1 INTRODUCTION Background By definition, parasites are costly to their hosts. To minimize these costs, hosts have evolved defense mechanisms that include immunological, behavioral, physiological and morphological adaptations (Clayton et al., 2010, Hart, 1990). In turn, parasites evolve reciprocal adaptations of their own to escape host defenses (Bush & Clayton, 2006, Bush et al., 2010). Hosts and parasites coevolve through this type of arms race such that populations of both groups persist. However, when parasites encounter novel hosts, those hosts may not yet have effective defense mechanisms. In such circumstances, parasites have the upper hand and can have severe effects on host fitness (de Castro & Bolker, 2004). Observing the dynamics of novel host-parasite associations in wild populations is inherently difficult. For logistical and ethical reasons, experimental introductions of parasites to naïve host populations are usually restricted to laboratory settings. As human populations continue to grow and expand, however, introductions of parasites to novel host populations are becoming more frequent (Smith et al., 2006). Researchers can use these "natural experiments" to study the initial interactions of novel host-parasite associations in wild populations (Lafferty et al., 2005). The accidental nature of most introductions means that the effects of introduced parasites are often not noticed until 2 host populations begin to decline severely (McCallum & Dobson, 1995), at which point conservation priorities may preclude rigorous experimental study. Thus, it is extremely important that researchers take every opportunity to investigate novel host-parasite associations to better predict the impact of such encounters. The virulence of an introduced parasite, hereafter defined as the degree of the effect on host reproductive success, is often determined by attributes of both the parasite and the infected host (Toft, 1991). Island populations of hosts are particularly susceptible to the effects of introduced parasites (Reid & Miller, 1989). Restricted dispersal, low genetic diversity, and inbreeding depression can predispose island populations to extinction even in the absence of introduced parasites (Delannoy & Cruz, 1991, Frankham, 1998). These same characteristics limit variation in available host defense mechanisms, which can further relax selection on parasite virulence. Parasites with low host specificity and high dispersal can become quite virulent. The availability of alternative host populations or species means that introduced parasite populations can remain stable even if a given host population is driven to extinction (de Castro & Bolker, 2004). The recent introduction of a nest ectoparasite, Philornis downsi, to the Galapagos Islands, presents a rare opportunity to study the initial interactions of a novel host-parasite association. P. downsi was originally described from Trinidad and Brazil (Dodge & Aitken, 1968, Couri, 1985), and was introduced to the Galapagos as early as the 1960's. However, P. downsi was first observed in the nests of Darwin's finches in 1997. It is since been documented on 11 of 13 major islands in the Galapagos archipelago and in the nests of at least 14 species of birds, including 9 species of 3 Darwin's finches (Fessl & Tebbich, 2002, Wiedenfeld et al., 2007, Fessl et al., 2010, O'Connor et al., 2009). P. downsi have already been implicated in the severe decline of several Darwin's finch species (O'Connor et al., 2009, Grant et al., 2005). My dissertation examines the interactions between P. downsi and a relatively abundant species of Darwin's finch, the medium ground finch (Geospiza fortis). I use an experimental approach to investigate the effects of P. downsi on medium ground finch reproductive fitness (Chapter 2). Then, I investigate whether medium ground finches can mount parasite specific antibody-mediated immune responses to P. downsi and avian poxvirus, a pathogen that is also present in some populations of Darwin's finches (Chapter 3). I further validate the use of an immuno-assay using house sparrow antiserum to detect P. downsi-specific antibodies in seven species of Darwin's finches parasitized by P. downsi (Chapter 4). I then examine whether medium ground finch immunological and behavioral defense mechanisms are effective against P. downsi (Chapter 5). Finally, I use a population viability model to predict the persistence of medium ground finch populations in light of the observed effects of P. downsi parasitism on host survival (Chapter 6). Chapter Summaries Chapter 2: Experimental demonstration of the fitness consequences of an introduced parasite of Darwin's finches Chapter 2 investigates the effects of Philornis downsi on the fitness of medium ground finches. Several studies report that P. downsi, recently introduced to the Galápagos Islands, reduces fitness of its avian hosts. However, most of these studies are based on correlational or observational data. A single previous experimental study was 4 performed but with small sample sizes that required the authors to combine results across species (Fessl et al., 2006). While these studies were integral in bringing attention to the potential impact of this introduced parasite on native birds, a more rigorous experimental manipulation was needed to measure the direct effect of the parasite on host fitness. We performed a large-scale experimental study using nest liners to manipulate parasite abundance in the nests of medium ground finches. We quantified the impact of the parasite on nestling growth and fledging success. Nest liners significantly reduced, but did not completely eliminate P. downsi in nests. A reduction in parasite abundance resulted in a significant increase in the number of nests that successfully fledged young. Nestlings in parasite-reduced nests also tended to be larger prior to fledging. By using an experimental approach, our results confirm that P. downsi has significant negative effects on the fitness of medium ground finches. Furthermore, our results showed that a reduction in parasite load is sufficient to significantly increase fledging success, information that may be useful in the design of management plans for controlling P. downsi populations. Chapter 3: Ecoimmunity in Darwin's finches: invasive parasites trigger acquired immunity in the medium ground finch (Geospiza fortis) In Chapter 3, we investigate host immune responses against two classes of parasites, the ectoparasitic nest fly, Philornis downsi, and pox virus (Poxvirus avium). We developed an enzyme linked immunosorbent assay (ELISA) using house sparrow antiserum to test for the presence of parasite specific antibodies in the serum of medium ground finches. Finches from populations affected by pox had higher pox-specific antibody responses than finches from populations without visible symptoms of the virus. 5 Finches had higher Philornis-specific antibody responses during the breeding season, when exposure to the nest fly occurs, compared to finches prior to the breeding season. Female medium ground finches had higher Philornis-specific responses than males, consistent with increased exposure while females brood nestlings (males do not brood). This study was one of the first to show parasite-specific antibody responses to multiple classes (intracellular and ectoparasitic) of parasites in a wild population of avian hosts. Development of a parasite specific immuno-assay is the first step in determining whether Darwin's finches are able to defend themselves immunologically against introduced parasites. Chapter 4: Test for parasite-specific immune response in multiple species of Darwin's finches Chapter 4 validates the use of an immuno-assay to detect parasite specific antibodies in multiple species of Darwin's finches. We used house sparrow antiserum (Passer domesticus) to develop an enzyme-linked immunosorbent assay (ELISA) that detects parasite-specific antibodies in the serum of medium ground finches (Geospiza fortis) (Chapter 3). Here, we test whether this same technique can be used with serum from other species of Darwin's finches. We compared cross-reactivity of serum from seven species of Darwin's finches with antiserum from house sparrows using a total-IgY sandwich ELISA and tests of dilutional parallelism. Our results show that house sparrow antiserum cross-reacts well with serum from seven other species of Darwin's finches. We then tested whether these same seven host species produced parasite-specific antibodies against the introduced parasitic fly, Philornis downsi. All seven species are known hosts of this parasite and our results show that all seven species 6 produced P. downsi-specific antibodies. This is the first study to demonstrate a parasite-specific antibody response in a group of closely related wild host species. Validation of this technique and confirmation of the presence of P. downsi-specific antibodies in multiple species of Darwin's finches provides the necessary framework for comparative studies of immune defense against an introduced parasite. Chapter 5: Are Darwin's finches sitting ducks? Ineffective host defenses against an introduced parasite Chapter 5 investigates the presence and efficacy of immunological and behavioral defenses of the medium ground finch against the introduced parasite, Philornis downsi. Hosts can use a variety of defense mechanisms to mitigate the negative effects of parasitism. However, host populations that encounter introduced parasites may not yet have effective defense mechanisms. P. downsi is a hematophagous nest parasite recently introduced to the Galapagos Islands where it infests the nests of multiple species of land birds, including Darwin's finches. P. downsi negatively impacts nestling growth and fledging success, posing a serious threat to the reproductive fitness of its hosts. The goal of this study was to investigate whether medium ground finches possess defense mechanisms against P. downsi that are effective in mitigating at least some of the negative effects of this parasite. We used a fumigant to eliminate P. downsi from the nests of medium ground finches and monitored nestling growth and fledging success in fumigated and control nests. We used nest cameras to record parental and nestling behaviors during the day and nighttime and quantified P. downsi-specific antibody responses in parent and nestling finches. We found no evidence of effective behavioral defenses by parent or nestling finches, though observed 7 changes in behavior helped elucidate possible mechanisms by which P. downsi causes nestling mortality. Nestlings did not produce P. downsi-specific antibodies, nor were maternally transferred antibodies present when nestlings were five days old. Adult females in parasitized nests had a significantly stronger P. downsi-specific antibody response than females in unparasitized nests. Among females in parasitized nests, there was a weak correlation suggesting that greater adult female P. downsi-specific antibody responses decreased parasite abundance in nests. While all fumigated nests fledged at least one offspring, all control nests had complete nest failure (100% mortality). This results suggests that none of the observed behavioral or immunological responses to P. downsi were effective, at least during our study. Chapter 6: The demise of Darwin's finches? A modeling approach to assess the impact of an introduced parasite on host population viability In Chapter 6 we use a population viability model to predict the persistence of medium ground finches affected by the nest parasite, Philornis downsi. Introduced parasites and pathogens present one of the greatest threats to naïve host populations, especially those on islands. P. downsi has already been implicated in the severe population declines of several endangered Darwin's finch species. We develop a model largely based on data from our own three-year experimental study of the effects of P. downsi on medium ground finch reproductive fitness. The model predicts that extinction of medium ground finches on the island of Santa Cruz is likely within the next half-century, demonstrating the devastating impact P. downsi can have on even relatively large populations of finches. We use the predictions of our model to highlight the need for additional experimental research on the effects of P. downsi on other populations and 8 species of finches. By manipulating various parameters of the model we show the extent to which P. downsi prevalence needs to be reduced to increase the predicted time to host extinction beyond 100 years. The predictions of our model are meant to serve as a warning of the potential impact of this fly on Darwin's finches. We discuss conservation efforts currently underway to control P. downsi populations and hope that the predictions of our model reinforce the need for such intervention. References Bush, S. E. & Clayton, D. H. 2006. The role of body size in host specificity: reciprocal transfer experiments with feather lice. Evolution 60: 2158-2167. Bush, S. E., Kim, D., Reed, M. & Clayton, D. H. 2010. Evolution of cryptic coloration in ectoparasites. American Naturalist 176: 529-535. Clayton, D. H., Koop, J. A. H., Harbison, C. W., Moyer, B. R. & Bush, S. E. 2010. How birds combat ectoparasites. Open Ornithology Journal 3: 41-71. Couri, M. S. 1985. Considerações sobre as relações ecológicas das larvas de Philornis Meinert, 1890 (Diptera, Muscidae) com aves. Revista Brasileira de Entomologia 29: 17- 20. de Castro, F. & Bolker, B. 2004. Mechanisms of disease-induced extinction. Ecology Letters 8: 117-126. Delannoy, C. & Cruz, A. (1991) Philornis parasitism and nestling survival of the Puerto Rican sharp-shinned hawk. In: Bird-Parasite Interactions: Ecology, Evolution, and Behaviour (Loye, J. & Zuk, M., eds.). pp. 93-103. Oxford University Press, Oxford, UK Dodge, H. R. & Aitken, T. H. G. 1968. Philornis flies from Trinidad (Diptera: Muscidae). Journal of the Kansas Entomological Society 41: 134-154. Fessl, B., Kleindorfer, S. & Tebbich, S. 2006. An experimental study on the effects of an introduced parasite in Darwin's finches. Biological Conservation 127: 55-61. Frankham, R. 1998. Inbreeding and extinction: island populations. Conservation Biology 12: 665-675. Hart, B. L. 1990. Behavioral adaptations to pathogens and parasites: five strategies. Neuroscience and Biobehavioral Reviews 14: 273-294. 9 Lafferty, K. D., Smith, K. F., Torchin, M. E., Dobson, A. P. & Kuris, A. M. (2005) The role of infectious diseases in natural communities: what introduced species tell us. In: Species Invasions. Insights into Ecology, Evolution and Biogeography, (Sax, D. F., Stachowicz, J. J. & Gaines, S. D., eds.). pp. 111-134. Sinauer Associates, Sunderland, MA. McCallum, H. & Dobson, A. 1995. Detecting disease and parasite threats to endangered species and ecosystems. Trends in Ecology & Evolution 10: 190-194. Reid, W. V. & Miller, K. R. 1989. Keeping options alive: the scientific basis for conserving biodiversity. World Resources Institute, Washinton, DC. Smith, K. F., Sax, D. F. & Lafferty, K. D. 2006. Evidence for the role of infectious disease in species extinction and endangerment. Conservation Biology 20: 1349-1357. Toft, C. A. 1991. Current theory of host-parasite interactions. Oxford University Press, Oxford. CHAPTER 2 EXPERIMENTAL DEMONSTRATION OF THE FITNESS CONSEQUENCES OF AN INTRODUCED PARASITE OF DARWIN'S FINCHES Printed with permission from: Koop JAH, Huber SK, Laverty SM, Clayton DH (2011) Experimental demonstration of the fitness consequences of an introduced parasite of Darwin's finches. PLoS ONE 6(5):e19706. doi:10.371/journalpone.0019706. 11 -~- plPs one Experimental Demonstration of the Fitness Consequences of an Introduced Parasite of Darwin's Finches J e nnife r A. H. Koop'·, Sarah K. Huber'·, Sean M. Lave rty', Dal e H. Clllyton' .""*>gy.,...... ...... .-... ..1""" ...... Ci ........ .-_ .. d-. . .............. .,........-.'-"-' .. d""" .. ".-Ci .............. _ .. 01 ...... riLo &dtground: Introduoed para$lles a~ a pllrliculllr thol'at 10 vnall popul/ltions 01 ho~1$ IMng on i$lands beQus~ ~rtinctJon can oo;uo" lH:'fo<e 1>0$1$ """~ a ch .. r";~ 10 ~~ effe<:tMo defen~es. An e<peolmental approach in whid> para~ abundon« is .....,...;pulated in the foeld can ~ th~ rn<;ISt informotivo:' ~ns 01 aueuing a pII'a$II~s ""pact on the hoo~1. Th~ pII'asmc ty PI!iJorIlif doiinIi, ~entty Introdueed 10 the G31apllgQ$ 1!JIInd'!, ~ on neUling D3rv.in'~ fi~ MIl OIlier IMd bird!. ~,aI corrdotional ~ludies, and one ~i"""taI ~tud)l 01 mixed ~leso.o:"~<e'tI"al)'!''''f, ,eported that the files ,ed.a I>o$t fotneu. H,,~ ~ 'eporIlhe ~$UIIs 01 a IIlrge' ~ expeolmental ~tud)l 01 a ~1ngIe loP«les at a ~Ie $1«' 0.0:" a $Ingle ~ng~~wn. _hodology/Prlnct»I Rndlngt:We """'lpulated th~ ""und~ offlies in th~ I"IesI$ oI...-.ediumground fj~ (GMspiza brio) ""d qu""Ufoed the ""pilei oIth~ pII'as~ on ~tling growth MIl fledging ~uocess. W~ used nylon nest liners 10 ~ th~ numbe< 01 paras./les in 2-4- ne~f, Iea ..... g .... QIh" 2-4 ne~1$ as (Ontto/$. A llgnWocant ~Ion in me .... pII'as~ ""und~ led to a llgnjftrnnt in(J~~ in the numbe, 01 ~I$ tl"llll ~uoces~uII)I fledged young. Nestlings in pII'as~ reduced ~I$ also t",<Ied to be IIIrge' prior 10 fledging. CMldUJlotrtISIgnlllanc.: Our ~s confirm that P. doMlfi has llgnifocant rw:'9atM:' ~fIeCtS on the f~neu of ...-.edium ground ftnchef, and they may po~ a ~I'riousthol'at to QIh" ~pede$ of Darwin'~ ftnches. The~ data can help in the dellgn 01 ma""9<:'1"1"oo:'nt plans for contJOlling P. downfi in Darwin'~ f....cn breeding populations. CitoIMo. ~ JNl. _, sa. t-I. SM. a..- 00 UIl'1) u,..;""",~ 1lomaHt_ 01 ,ho fil .... c.......-.. 01.., l .. ox1Kod "" .... 01 o..wo.~ __ """ IN 6(51, • • 0_ .... ,10. m._.o_ Ed""" ..... "- _ 1Iri_ .. 01 u-.-. ..-d .. ........., ___ ,..10'111 -.,....I A", ".1.", ... _"" .... ... 101. CovPiIh" C '.11 "- " 01_ l!;, .. .., ................. ~ .. _ri ............ , ........ 01 .... c-... c......... .... ___ -.. .... m .. .... _--. ..... _-. ..... ~""' .,_-'""" ....-, .. ""11'''''...- .......... _.-Mod. F ...... Tho ..... __ ..... _S<BK • ...........,I ......... .,...., • • I19O ..... 0u.-00.6a17 .. OK:) ........ _""' .. _oI......-. Tho_ .... ... ' ... 'n_~ ..... «.,"'_ .... _._,o .................... ""'01' .. ~_ Com ...... ,_ Tho ...-, ..... _ . .. ,"" ... ,om ........ _"' __ '''-'~----''' • c.. ___ ""*'9r.,...... ...... ....-.-.-onCdtoy.o. _ ................. ,,_ d_ Introduction lnU"<><b:ed p=ooiteo and p.o.thog<", an: a n i....,..",ing pn>bIem a, """"""ic growth and Ir.ItIe """'""'" fW"\he< ".,.,.", ... ,;'i .. fOt" , ,.,..,ieo 10 ",-.de [1 1- SrnaD, encIemic populatioo>o of"'-... ,..,h '" Il>ooo "" islanck, an: particularly a\ risk i-om "U"Orl",,«I p.o."' .... and p.o.1hoge", bt:<a .... ~tion can ""COli" I><f<n """" hay< a clw>o:. 10 ~ .ir""......, de" ...... ('l ,31_ r ".. ""ample, \he in1n>dooai"" d a,ian malaria and .. mooquito "",tor 10 \he Hawaiian islands hay< 1>«" i~<;a1Od ., \he ra;>id ... tinction of oe>...-.I .nclemic /xmo:yo"e<"... lIf>t:<ieo [-4-.5 ,61- The Ga~ !.Ilands hay< fared 1><\\..-; ...,.... of \he birds .nclemk 10 \his a n;~lago hay< ouI1i:red • .o:~o>W,", d ... 10 p.o."'''''''' p.o.tl>og<", OYer re<:or<Ied his.,.,. Fl- However, reo:n\ _ ..... i-om in1n>dow:t:d paraoil<O and p.o.thog<", has \he J»l<ntial 10 ca .... .. ri"", f>OIlUIaW>. dedi ...... if r.o\ .nin<funo 18,g)- A p.o."'''' of particular """",.n, i, \he "".n~y "U"Orl",,«I fly, I'Id>n!itl""I<fi ~"': MllOcida<; Dodg<.t Ailhn) [10! -To our h>owIedg< , \he .. an: r.o O1udi .. d "'. fltne>O conoeq......., .. of p_ .......- '"' """" .,; Ihin \he na""'" rang< d \his fly_As 010 f rem \he Galapagoo, \he only other "",om. of P_ .......- an: frem T rinidad and Braz~ fl l1_ P_"""'-wao l>O\oboerv<d., \he neouofbi""., \he Galapagor until 1997 fl21_ P_ .......-i. nowh>own IOp.o."' •• a\ 1 .... 1-4- lIf>t:<ieo ofGaI~ IaOO I>ir<k, i»du.c!ing 9 lIf>t:<ieo d Darwin', r...,beo [12. 13 ,1-4-1_ It has \>o<ol bund "" ! 1 of 1bo 13 Gal:l.pagoo [.\lands pmpl«l [151 _ P_ I,."I<fi may I>< [W"Ily "'!'<"'IINe for "".n\ dedi .... of \he .nc\ang<r«I maIlgr<N< r...,h (Ct-*.,.. ............ ), 1bo .ndang<n:d n-"dium Ire< r...,h (Ct-*.,.. ... ,..-JWl. and \he warbler finch (CmI ...... f ... .oj [8,9,131_ P_ .......- i, an d>ligal< ".. p=ooil< d birds_ ~ \he adull ~ieo a t< fiOU1""""'U (\hey feed '"' d:<;aying mall.r), \he larva< an: sornHl.<malOfl>.ago .. p.o."' .... d .... Ilingo fl61 (Fig- lA~ P_ .......- larva. chew 1hrough \he skin d .... ~ingo and c""umo blood and other fui<ll fl61 (F"'I!- 18). ~ feed primarily al nighl; during \he day moot Ia".,.. burrow "10 \he "",I ma1<ria! [171_ Mull ~ieo lay \heir.ggo ., \he neoting mal<rial and nares (nootrio) d neoIlingo fl8,19)_ All<r 1bo .ggo ha1<h, \he larva< ccrnple., 1hr« j" .. aro, 1bo f ... \ d whl:h can! .... ., 1bo na"" d\he 12 FlfIUI'. I. StudyOl"9" .......... ... I_~ _11o __ "1!>on ... ol . mI Ie G<.l l.J.m...!" r O..U..nt Ido> gln _do /Gt_a<plZG_ trn~ ..t. . ptt/>I<t lt_o ......_...y 04 <...I.o Hnaog<<d<o-t;o: 1I>e __ lI><><_1 from P. d-..l ..... M. dcI :lo.1J71~0D19706qOO1 ..... or r.....,. in the .... ' .....rn..I.ilamatt .. the o>ata d..."u. ... <ad pers. iDIo oduIlhood 1201. s.oo..d and thi«I i~w,."3C~"" r, ..... y in the .... ~ wt..n. they-.u..ally P"P""'''''' IaI.er ...... ~ .. ><hili: 1Iieo. F ....... " .... .,c the iml*' .,c p . ..... on Ibo"win'. r""""," i,kntj"""" .... paraoit< ... p<>I...u..I ....... (Table I). s.....nl ...... report a owgo.li,'e """,1aD:>n 00_ P. ..... ~ uri ~ .1>0: ... f21,n,23,241 . Addieional _uda <q>an'~ dq«<o .,c .... Jai"", ~ ...... partial b<ood 1<>00) '-<d (WI. f~ P . ....... in ....... [12 ,13,14, 19]. \,"h~ ........ 1UdieI ha .... he<" i" .. gra1 "' bringing a, .. "lio" .. ,~ imf>'l<' of P. '-i on v,a ,;"", ind> opod .. , the 1><>1'''''' i. '" m • ........, ,ho: dncu1l"o:;t of the: paraoit<, ..... contrd)il'll br """" v.viabl .. Ilut ~ boo ""'""'linK '" ..... ' fai ...... (~ . ~al ~ OtICh .. rainfaD and food~, whi<h ,iii". .. flOm y_ "')/'I"a< 12~.26D· .n T.o. ............. the "",«_da panoilJt!.cfiotctd"«l on a boor, porim.eDtaI opprood> ;, --r 177.281. ~ be......, panoire ___ """ ..,.. ..... """ cad be dAcWI '" i,,"'l'"'" """"'- tbey do _ .......... tho ditUI efb:t (III 10, f...-. Fo< eampio, poorly ~ birdo ~aol have 11;«1' """""'" ol , panoi ... 1>0<_ ,hoy h.wo ...... ""'"IY '" i ........ in ... r ..... , wbie aIoo hr.,,,, low <q>o"O<b: ..... OtIC.,.. """""'" they haw ..... """"" to i..- id olli,,,.;,,, . ..,.. c_"""". ;, • ""'""'" C(ll"l"elatioo (to" II It .... an inflated one) be""""" pl. ...... abulXWlcO and ho:Nl fi(J1&. To dat<, joaot "n. pul.ili'hed _udy hao ."1"'....,,,, .. Dy ma"ip- WaIM P . ...... ..... ,,(\ .. "" .... ><\ I ..... ured i. "'pac' On Ibo"win'. il.m... F ... .1. ('29) .. ~...,;1UICd P ....... h>rn b .... ~ft#lis .-.. and dsh' G . .JJft- .-.. by f ..... ..." the _to with. 1'IIo~"""" ...... ""'. r~ ...,_~ the awhon monj . I(ftd ~ ~ ......... fWro-doyp<riod; they aIoo..-u..-l ....q ~obOn Ie..d .dd the ~.....:ao death _', con>pw<d 10 _""'ipIftI ...... ~ limUd um~ .. . iuo ""lui...d _10 p:IOida .. be ...... n """,,"""'" ""..,.,.. .... tbeir ....... ~ ,hat ..... dinp in ""';poed ..... _ .. haw; hiper ~ ~ • sipUfl<al1lly hiper S""",h ""., ..,.. .ipl"'.....,. pta .... ~ _ than .-II .... in ~ipt ....... u (T ..... I ~ H ....... <q>an ,ho: <auIu ol.Ia'S ... ","Ie .. p:ri .... ouI """" cI a ••• pecitt of O~'I r.",h aI. a single site ...... r a single: I:.o-.,.d"" .... ""'. w. , ..... ,;p .. lalM me abw>danc. cI ru.. in ,ho: .-. of medium 1',,,. ,,1 f."' .... rr;..~.f><riIJ ."d q" .. ",ifitd ,h~ "'pac' cI the po"' .... 00' 1 ..... 'svOWlh and a.dgil'll ''''''''' Ethics na!~n! AI prot ... "... ........, ~ by the UDM-niry ol Utah I .... u ........ Animal C ... ..,.. u.. Commi .... (pooto<oI fi(1I • ..".) 5I:udy sb and e!(peQnent, 1 dt-sign Our _..,.,. _ """"""""J....-y-Apd, 2IXIB at fJ G_I'"­...., "" SMca Cr\11 .. land in tho ("-1'"8"0 Arc .... , F..<:u.>d<w. G/n, ;, abun<lam .. ,.,. ... ['23), """' .. it buido ..... in ~i< '"'" <ac'; (q,_ d ........ ) and,j __ 1.5 to 4 m<ten """"" ... "",,,><\. (l.,,,,hsi .. "'ngeof...,." 2-~ 'lIP- ..,.. meOOation .,.riod i. """"""'a .. ly 12 dal", and l><Idinp,.,.oo 1(>-14 ~""~ .... prior ",fledging. BoIh .. _" GjinlWd ~. and d ..... ,''' .... , bu, only hWeo me ...... "Ill' and t.n;.,d bat<btd ... ..,.-ing. ~",poi .. olad"'" <;Itm ~<S'. but they do "'" ... tho ........... a~ fW]. W ... ard>td. U kmxU km ....,a b"""" G.p .... u ~ tho brucfinc ........ W. """'irond ...... J d43 ..... . .. oltbem-...:ud .. ,.,., cacti, by 3t diI"..,.." Ixwdins poi .. olfmcheo. ro.r-~) olthe ..... in our ..... pio we ........ .. bouu "'_' .. d..i,. tho .. """ period. AmIIt birdo we ..... at<! ...... the .... ..,.. r.1«i with • """,be""" 1.1" ... 1 metal band and """" pial';" ...,.",. bando r .... idemiliealion at • clio .. "",. At, .... ..... .............. ..-y """" day be!Ween the houn olO6OO and 1100, and tho """,ber of'll'.nd ~ we'" "".,..J,.d. N ... ....... iI>c ........ in ,ho: uperim .... if they we", dio:~ ber"", ,ho: egg> hatdled (n "+I '...u! or, in tho .,... cI fOW" .-.. _ ....... hatch"" ( ..... i ... ,1\5 daY' of ase, but 1heoe four ..... __ omi"«1 from all .. "alyo .. ol ~). W. c<»""ued I<)'~ , .... and pr« .. _,lin" (ot<: boolow) ,.,lil the d<leol ".od"" _ 10 day> of . ' to" .. ",a .all ol ... I~ died. Proc .. "" -tliJ'!lo 0l<I<l- than 10 days d • un 1I"i.,- p-tmaI"'" I1«IP'll [:10). ~ ....... thooldl:.: .-II",,,,a<I><d 10day>"'aF . ..... "'" .,...,.,..",....Jinco. G.p ..... haw;. oide~ """ maka iI. po;:ooabl. 10 ........ ddt<" ..."u.... r""" • ~ with binocoIoD. Ono< empty . ..... """" collo:ded 10 "'""'''' po ....... Nom -.. randoomIy _~ 10 the uperi"'"""" F""P{II ,, ~ ~ <r mnuol F""P (n ,,~ ..... } 10 ""'"' c-. rI ~ by 13 ....-'.F_ ...... ........ HIo~ --- -_. .~ .- ~, -' ""' ....... ....-. " , W, ""'_t..~ , , ......... "" ~ , ~, G __ • t.."m'" , , , ~, G __ IoJ9 .............. , c.m __. .. , (It) c.m __. . , (1l) ( " ) ~~ B-.r , (ll) "-(.-", , ~, {Y.'_d_.""_ ............ -. ...... _ ....... oot · ,_ ... 'Ilb._ ..... . _ ~ __ ... __ b _ 1"\1 ....... _ 'eo-........ ..... _.""IP" ... ___ ._ ..... ~ .. ''''''''- '-""""' ..... ,~ ·OOo __ _....... __dd bb.._ '_''- ....-. - ''- "Go ....... fuO'II"-- c.mc.-........, .......... c-. .... _ c..t_. __ "'-'- ftJ~ "- _ c.mc.-........., ~ .... c-. .... _ ua ............... c..t_. __ _ 'o_m'''''''"'''' ........ ''''''''''', a .ingl. palr of bird!, "'" U"<atmenl wall «""JOe<! be\Ween «prod"" ...... OO~,,- "The fk><n of ';q>erimenlal ".... we", r.1ed with a liner ~..aed &om a . maD "",tion of nyb> 'lOCking 'U"el<bed <;N<r a win: hoop (-9 cm in diam.lerj_ "The li""r """",nled .....,. of \he Ily larvae in \he 001."., of \he ..... , i-orn «aching \he ..... \lings_ "This a f'l'"O"Ch has been .11'''';"" in o\her experirn.nlal manipulafuno d ..... paraoi ... [311_ [ ;q>erimenlal ...... we", ti ued with line,.. within ,..., day d\he f ... egg hal<hing (a d~Kh of eggs ...,....,any hal<hes over \WO 10 fOW" dayo). "The lOW" ...... WI aln:ady c<»"';0>«1 ..... dings wben r ... , ma>aored we", aD aooigI>ed 10 "'" wilio>ed gn;mp bo:a .... \hey could have already be<n e>:J><>OOd ., para ..... P....,.,." larvae O<<:allionally crawled over \he line,... <:<>ming inlO c<»\at;1 with .....uingo. For \his «"""". line,.. we", c .... fuDy ."amO>ed and d ... 0>«1 or rq>laced .ach Un. \he ...... we", d>e<;W Any larvae bund and rem","", we", iO>dorled in final ",."',. of paraoi" aI:>w>c\an<; • • ,inc. these parasiteo may ha"" be<n able ., 10ed <» 0>eIIling. and may ha"" alreaed o>eoIling gn>W\h and """,,",,1 Nestling growth AI each ..... , cbed \he O>eIIling' we« ...,,;gbed with a digilal balanc. (O ha .... 0_1 g accw:a<"\'l_ [n aMiIi<». \he bllowing m ... ",men,. we« \.al::en wilh digi .. l c~ ... W.herl>raOO. 0 _01 mm ao:;W"at;yj: bill leng"'. b ~ l <lep"' . b ~ l widlh . ......... length, ancI1ength of \he o ... nnoot p.-imary ka\her &om whe", il .m.rged fr<;rn "'" din 10 .. diota! ~_ AI \he linl ,;,. aher ha"hl" g . ""'Uings we", aged t-odon body ...... ..,;ngdata i-orn B<;ooog [321. a, followo: '" 1.9 grams (1 day 01<1); 2-2_9 grams (2 dayo old); 3--3 _9 grams (3 dayo olcI). New ""'Uings we", m .... h:<\ iOOivWju.ally by <:OIoring a .,.naiI with a penn ...... nl m .... h:r_ AI Ihree 10 lOW" dayo of ago: \hey ...... giv<-n a oingle piau <:<>lor baOO_ When ""'\lings we« 0.1 least """" dayo of ago: \hey we", .f..1..e.d. with a nwrt>en:d Monel melal baOO and Ihree plastic <:<>lor Beea .... Darwin', tiO>d>eo ha"" aoy~ hal<hlng. \he facl WI we pro:eooed ".... <» ahemate dayo m.anl .".., birdo r 'oM day birds"1 we", p-<Xeued f,..\he r ... Un. 0.1 one day of ago: _ and on ocId day, ""'",afl:r - ""til \hey we", nine dayo old Other b irds , (" .... en day birQ "l ...... pro:eooed f,..\he f ... Un. 0.1 \'00 dayo d ago: - and on ..... n dayo \he ..... fler _ ""til \hey we", \en dayo old_ n,... \WO dala ... we", ..,.d 10 ~"'" growIh C""'" f,.. lined and wiliO>«l "",,"'.nU-Fledging Sl)(<<'SS Fledging wall conlinned by oJ:>sen.ing and O\enlj'ying birdo on \he ~, d \heir color baOOs aIler \hey lel\ \he neiL ParasitE' aburdalXE' Aft..- .ach...,.u,g boul we removed \he ..... and placed;' in a "aled plastic ~_ "The neol wall c .... fuDy diue<;l!:d within .ighl bo .... d collection and P_ '-'uilarvae . ~. 0.00 eck>oed pupal caoeo ...... COWlIed_ Fint i".,..,. la",,,,, . whl:h .... 100 ""all 10 dio:.m «liabIy in "'" "",I material . ...... nol iO>duded incOWl,. d paraoite al:>w>c\an<;e. TOIaI paraoite aI:>w>c\an<;. wall \he ,wn d ",<»<I and Ihlrd i".,..,. larvae . pupae. and .d"oed pupal caoeo_ Other \ypeI dlly larvae • • .g_ Sarcq>hagidae. we", O\en,.oed bul nol included in <0""" d lOla! parasile abw>c\an<;. bo:a .... Iheoe larvae .... nol paraoiliC; \hey 10ed on \he ....... d dead ""'\lings 1"1 Stati$ti(;ll analyses Stotisu:a1 analy ... we« <b>o in Pri"". v.3 _ct> (G<aphPad Software. [",, _I 0.00 R v_2_12 _2 (R Developm.nl Core T.am)_ Neotling growth wa, analyl!od ... ing n:greui"" and ~ I ........ For oome growIh param: ..... we also <:aI<>Ila1ed .11'"", ''''. Le. \he mean dill ........ c. in a gn>W\h param."r be\Ween \he lined and wilined "",,"'.n,. [331- W • ..,.d booutra;>ping (10 .000 rq>etiWno) ., ~"'" 95"" <:<>nfotle""" in\ervals around m.an .lr"", .. eo [33] - It wall r.ol ~hl. 10 analy:l. growth over Un • ..,;ng rq-..ealed m .... """ ANOVA ,.. GL\[M be<:a .... "",noM: morlality in one of \he groupo (>00"" pi,.. 10 fledging in wilined. beavily paraoitised ..... 10) made ... ~ .;,.. ""'Y ..,.,......., <;N<r tOn._ "The",bre, gr<lWIh data ...... IeIted f,.. an .11'"", of U"<almenl .. ~ by c~ \he ronal ,...:1 .... \ah:n f,.. HO>Cd ...... and wilined ....... wben ..... Uings we", nine,.. \en dayo old lll.i ..... n 14 ..... ~ings in oeven wilined ...... . ""ive<! lO al leaot nine dayo d >«. c~ 10 '1.6 ..... \lings ;n \We"" Iint:d ".. • . To a...,jd ~caW>. "" otS<d the m:an bnx>d val ... of nine aM I<n day old I>O$\lings in .ach"... 11>< data 10< ni .... aM I<n day old birds ""'" con-i>ined f,.. a nalyoi, omleu Ih<'" wall a n .11'«1 of age on Ih< growth param<t<, of inl<reol (do:\<rnlined via "'~011 a nalyoio). 11><", wa, an .11'«1 of age <»Iy in Ih< ca.., d oo«nnoOl primary f",th.o. Io~. """'h .~D """ "",t I><sw'- I<> .. yrnpt<>1< \y Dayo 9 aoo 10 (R'" 0.30. P" OJ)03). 11><.-.f"",. Ih< 1i:all><r daoa ""'" analy"led ""I""'>-1<1y f",. ...... C(Oltaining nine aoo I<n day dd ..... ~ings . Reluhl ParasitE' aburdar"«' P. '-'i wa, p«o<nl ;n H of 48 G-f .... .,.... (90"'). u,... pn:oumably did "",I """"nl ad""! ... fr<;rn laying eggs ;n ..... :.; I>owev<,. ;f line,.. red ... edlh< nwrto., of Of4'01'\wUUeo 10< larva. I<> f<ed. then Iint:d ...... oboWe! ha ..... had f""", paras ... than wiliO>Od ........ In OUPPOO1 d thlo p«diWon. "" 1OW>C! thalliO>Od ...... had . ignj"o;antly ""'" paras ... 1'<''''' than wiliO>Od ...... (m:an paras .. load :!: S[ "2 L 79 :!: 3-56 in Iint:d ........ c,""pawI 10 3750:!: • . 92 in wilined ..... to; W.kh·, I ...... 1"2.58, <If".l . p"O-Ol (Fig. 2)~ NE'Stling growth N .. llingo ;n liO>«! ...... ""'" "",I oignOo;antly ho:a,"" than ..... dings in wilined ...... (\ " 1.73. <If" 18, p"O. lO; Fig. 3A~ H""""",. an anaIy:oio of .110<1 oi"" "' ..... ale<! thaI 1>O$\li"le'l in liO>Od "... (m.an :!: SF~ 12 .7 :!:O .• gj ""'" 1.7 g "",-, ...... on "' ..... than .-ling, ;n wilined ....... (11.0:!:1.O gj. with a 95"- C I " -0.3 g 10 3.7 g. Th ... _\lings ;n liO>«! ...... <oWe! ""'1:' fr<;rn 3.7 g ho:a,"" than ..... ~ings ;n wiliO>Od ....... I<> 0.3 g Iight<r, I>owev<,. Iho:y ""'" ligh .... in <rly 5'" of Ih< booulraf> IWtlpleo.. Tarsus length did 1>01 .mer oignilicantly ho:twe<n .....u;ngs in lined (1&1.:!:0·:w....,1 ........... wilined ....... (17.23:!:0.H mOl) (I" Lfi.t.. <If" 18. p"OJ2; F't!. 38). I-k>w<-v<r. a naly:sio of.fli:<;1 , ;". oI>ow<d thal ..... \lings in liO>«! ....... had Iaroi 0.91 ....,~, than .....uing. ;n wiliO>«! ....... (95'" <:<>nfotlo:""" ;n ..... val" -0.09...., 10 1.97 mm). Tho: 95'" C I aroW>C! thlo .fli:<;1 , ;". O>d>:ated thaI ..... \lings in liO>Od ....... could ha ..... tan; "1' l<> 50 -~ 40 T ~ 30 ~ 1. 15 T ~ 20 -'- , z 10 0 Lned Unlined Treatment fIg... 2.C<>mpoorloonof "'" m_ n IjoSEI numb.of P. _"In 1In..:l and .... 1....:1 ....... dot 1 0.1 l11I)oofNlpon .. OOI9101igOO2 • A " " B 20 E " .5. " 14 ~ c 12 ~ ,• " ~ 8 6 4 c " E " 5 14 .. 12 c~ 10 ~ 8 • 6 "~• 4 2 0 I 2 3 4 5 6 7 8 9 10 Age (days) 1 2 345678 910 Age (days) 4 5 6 7 8 9 10 Age (days) flgu .. 3. Compoor"on of ...... n IjoSEI grow'" poor .......... lor ..... 1"'" In .. ..:I I()I and ...... ..:1 101 n .... , Indudlng bod)l ma .. LA I, ....... "9'" IBI, and o .... m .... primaI)' "'at!> • • "9'" Ic!. detlo.l l1VP<wn,"_DOI9106.\fJOl 15 un mm ~, on """"/1", ""'" .-Ii .. ., W>Imrd _to. A1rmatO.'tly, .-lins> ;n Iino:d ..... muId howe IM'O; "" ,., 0.09 nun oI>on« "'an _d .. ., Qnlinod _u. boot only .,4'110 0( Ih< boola"..., ~_ Ou .. ~ prio=ry rea""'" d "odd day" '><o'ling! ;n lined "... (12.6' :!:0.77 mm) ""' ... "',;I",,,dy 1c.'lI'" !han ""-< or "..d" "' unlined ' .... (9.02:!:0.82 ..... ) ('''3.13, df" 13, P "().()(NI; .'8. 3C~ o" .. ~ prima!)' "atb<n 0( """,,, day" ....... ;0 ID:d _IS (16.65:!: 1.18 ..... ) ..... ...., .pnnt)y I ..... than thooe 0(-,,,;0 W>Imrd ..... ( 11.67:!: 1.l0 mNj (,"2.21, <1"" 10, p"O.o5~ ... """pooil< _ .... of ~ .... , ....... principal «mpo;1llellU .~of~ le<>glh, bill ........ , and t.;1 dq><h ('l6J, ...... oI<d tha, PCI explained 685% d tho ...n..""" ~"2.a;~ H.,....,..., PC! did _ d~ ."""!Candy b«wreD _t .. in I_:and W>Imrd _ .. (I ::0.831 . df:: 18. p"o.42), _ ..... ~ . """" """' Fl@dging 5U«~S N .. 1lFogo ;0 lined ...... b.od .p&canll)' *",al<1' f1rdP"8'~ Ilw, _IliI>gI "' QnliI>«I ' ..... F-«b, of :H liI>«I ..... (33'110) w~.- .)'.OW >g. c~ 10';"" ""e d 24 (4'110) Qnlined ..... xact \col. p::O.02, .... .... ~ W. aIo<> c~ II>e "limbe, d ;oo;..;dual ,~ , .... ~ r ...... lined "' ..... ..wnt:d.-.:: 19d75 ........ (25'110) Worn ID:d ....... ""' .. iIIy IIodpl, """,puo1 ,., only du'ec 0(67 .......... (4'110) i-om Wlint:d ..... (p<o.OOI; F'S- 48). n..., tho ~l ..due""" in ~il< """,be< ..... a dear poo ..... im.-", on ........ umeoo. OisclSssion 0... .""rio .. ~e>petiDW:tllal ... d!he im ..... d 1'. __ on !he ilDeoo of Darwin'. f"" ..... Oar ... ~ .... m ...... io<d ~.rWi<>o, be<w«O 'I'K~ ....... >0:1 1"_, ~ ... ,., _nlify Ih<dn:a ....... d 1' ...... on par1\m<t<nd "'-tf_ W ..... riplblr:d ~.~ in a rtIa.¥OIyI-F m ... be<o( mediQm ~f""h ' .... "';''«'''' ~.-aIM<!han chemical r,.,.,;~ ... Ib .. <limina,;,'!! ""y poooible oicIo erwu or"",ti<;i1eo on '><oIIi'IjI gr<»<Ih t;>" olher f., .... ''''"' ro'''''. (34]. Inn n:<b:<rl ~il< abw>dan« by 42'11o. on """"'I'- ",. ft<b;:tion ",~il< .... led ,., a oigni&:ano inc .. _ ;n the "11m"'" 0( ..... thaI 1~1IIy ~)'OOI'>(- Our ....... ..., ........ .,ot".;,t, IlwM 0( FsoI " • . f'l9J, .. t.o ..... f" ..... b oipU"' .... incn_ in tho nwrt>e< <>f ..... ..... """",",illy I~ l"""'« .. -ben ~iI.. .... ... C>OrnpIot.<ty .~miowed """"'" Ihe .... <>f. fumipnt Oar _""r funbet ...... thai P. ....... IIao .......... elb:t OIl neoli .. ..,...-.h- \VbeD .... ...Ir:d tho i".....,. of ... ~ ""_ OIl ~ .......... ou .. """'. pnn..,. "alber ~ '" an;oo.. of,...,..m, ~ ...... d_ ~ .. "" ... Ne.li .. in W>Imrd neo. t..d ou ........ ' pnn.ty "athas thai ~ :10% ......... !han neolli ... ;n lmed ...... ~ ....... twdo 11<d(L"'tI f""" ....m...I neo. ~ howe ~1q:>«I " ......... F.alber ....... io a ................... o(~'" "' bi<do, beta .... r .......... "..,.. ...,.., r.Ipidly than overallbody ................... 1 .. 'I'~ r:n,3~. N.,IliI>gI "' wilined ..... abo .. n<le<l .. haw lo:)W<O' body rn-. and ohone, .... ~ than ,...w,. ;" 1.>«1 " ..... n.. .11' ... <>f P. Mutti (0' n.o:olli", ""'" and _ Je.ocoh ..., ........ e". whl>. <Xhe, "U<I", ~ lOr ....... d paraoilic n;.. "" .-Ii .. p-owth. In OW' "~, ~ ... ..... ined _ .. .....,;pod. _an <>f 1''110 ...., and .... d .... i ............. a .... "" d 5'110 ."""- than """'" in ~nt:d ...... In """'pon...., .... oIiot!: 1Il>II! .... ("'- _~ _ H """, __ (T ........ ~ pua,;,;>ed by bIo..flMs (~ ...... weishtd:Hi"" ... and ........... o-~."""- ....... unp-.IIIIiliz<d neo .... prior,., f1rdP"8 P7J8j. , A B i b ~ z f"" .. 4 . fI'ee, of I ........ on ho .. fledging lIO<CIOIL ~,w. .. "'" IOQI ",,_of (,1,) ".",00<1 !II nosd"ll' _. 00R0I1>¥t ... (,1,) fl. ",,_of n ... tn. flodgod one Of""". _ ..... (I)1ht IOQI n_ of fltdgIlng. f""" nos .. in N<h __ t oIoi:lo.ll711~_.oo19106.~ o..r .a ... """" that ~Iy ft<b;:"'tI po ...... abuo-daoo:< o leado IO • .-.du<tion ;n H<Olli .. body ..... , .......... f:'h, :and _, pm.a.y ......... ....... Only tho .-.du<tion in bhet ...... _ .... ticaIy "niI"o:aol; """--, tho rae. Ilw tho ....e c .. OIl body ..... :and ...... ~ ...... ~ in,~ and in tho """" ~iOII ao Ih< rlIK1 "" reatbet ..... ''«11'"'' ""', 1'. .... doto. in fan. mllft neo."'tI..,...-.h-o.. r data """-d "" ....... of.-JUlin "" tho biI ._ d neaJinss, ... .. ..... 1«1 by a principol """""",,"' anaJyoio. H~.!he bill ........ width and depth 0( ~ ind>ot are ~'IO inc .. _ """" Ii¢wly !han body maoo, .......... ,Id "';"1 chord [32\. Morpholoti<allr.lilJ oud> a, flicb. r ... lb .... "-''''''' qo.ic:lly ;" """'do. .... '3' to bo c>f>al>le oUyiI'!! ."., after ,hoty lea", ,he ,,.. •. Sirnbriy. n.o:olli .. wiIlt high body ...... ....., m<ll"e Ihly to """'"'" ai", ............. _tliIlgo wiIlt low bo<I,o rnM [39]_ ~ oduIu .... ll>ei< bib to cn.ck oeedI ft;>" food; '--' • ooed <no:1<iI1c abilily • _ '"' irnponaot in )'OI"lf; IIe<\PowI .......... oduIu <001 ..... feM .. tbem afier <bey .......... , (32] _ Body ............... known 10 prtdic. po1I~.....,..;...a1 in twdo pg,tq. ·fb •• d , •• , iI;, liIro<Iy thai ...... a .mal.me, d pat'aIi'''' "" .-Ii ... ", prior 10 ....... will pia« bio-do., • 16 ·ignifio;anl di<ad"an\.>g<. AlthOllgh we did r.ol m<»-w pool. fledging ourvival in oW" OIorly, il ;, _1>Ie \hal 6edgIings i-om oW" wili .... d.-. did 1>0\ ourviv< as weD as Iho larg<r fledglings i-om 1Ov:d ........ 11m., Iho ~" of P. ""-i <»- l>oot rqyodO><tiv • • ""' ... may ha,~ ..... ncbI b<yoOO "'" <l<m""lr.".d im1"'<1 <»- fledging _«OS- FW'\he< 0Iu.cty is o>eed:d 10 m"';\(>" pool.fledging .""' ... in omer 10 mOJO fWly W><Ienw>d long-l<ml .110<. of P. '-'i ",,"'oi\ison, in atIditi<»- 10 Iho more immedia t< im1"'<loflho """"ileo <»- growth and fledging_=-- \\b~. we did "",I tell Iho .11'"" of ...... Im.nl on grow"'­p. a.ram...... ...".al<dly over Iho de\-dq>m.nlal ""R><! of Iho ..... dings , Iho .me,...,,,," in growth we", 1>0\ """"",nl ""til ..... dings W<:r< olcIe< in a ny <:as< (F'I!. 3A---C ~ lbe Iat< a;>p<aran,;e of growIh dilr.", ...... belW<en ..... dings in HO>«! a O>d wilined.-. may ha..... been a \Yyprod"" of 0"- m.1hod d p.a.raoil< manipula\i<;o>. P . ......- eggs a O>d fnt in"arla".,.. "'" <hen fo_ in Iho ""reo (nootrio) of O>eIllings [19l . FOO" \his ... ,.,.,." "'" .... of nyl<»- line .. woWd r.ol """ ..... rily air"" Iho fnt inow" otago oflho """"it<. It is _1>Ie \hal yoomg O>eIllings in boIh liO>«! and wili .... d.-. ,,,,,,,,", .... «1 . imilar lovds d f..,1 ""W """"um a O>d, Ih.., oimilar .110< .. on grow"'- al an.arly age In <On.,.,.., ..... 1 lin ... inhibited oe<:<:>1><I and Ihird ""W larvae , whim . pend moot d Ihoir ~me in Iho .,.. ma\erial 11m., Iho i_I <»­..... dio>.g .... re~ in oW".u.cty may ha ..... been d ... primariy 10 0«X>l>CI and Ihird ""w larvae P . • omui ",,"'oi\ism may aff«1 ..... ~ings Ihrough oover.oI ...,.,.. mUlually .lIt;l ....... m«hani ...... BIood-ioeding p.a.raoil<O <;an lower bemoglobin c<»<.nu-a\i:o>o in"..uing .. cauoing ..... mia [.1 ,.2). DOOanie< tI,.r ('.! 1) fo_ a ""ga"'~ c"""IaWn belW«n P . ......­a bw><Un<;. a O>d bemoglobin <:<»><.nlrati<»- in ""an grow><! lind>et (Gfolii_, Table I). FeW tI,.r [29) bunt! \hal""'ings i-om """"Med '-'1<00ed 10 ha ..... lowe< bemoglobin <X>1>O:nU'aWno \han .>eotling. in unp=ooitized ........ Ahhough we did r.ol m ........ bemoglobin c<»<.nu-a~<»- in \hi •• 1U<Iy, 0"- more ....,...,1 wod: conti""" \hal O>eIllings in p.a.raoiti>ed ....... ha ..... lowe< hernalO<rh (b .... I"ed blood cdl.",:Jwn.) \han O>eIllings in unp=ooiti:l«l .-. (Kt:>q., ompot>Ii>l>ed cia ... ). P . • """,i may also air"" ..... Iling beha,;,.. a O>d impede c,.,ruo<»- . ignaling 10 p.a.ren ... Neotlings \hal "'" wealene<! by p.a.raoil<O may r.ol ha,~ ."",I>gh ..... rgy 10 beg f,.. food [.3) . N .. ding begging Refe r ence s 1_o<y KD,_ K>', .. T • • "';. M il, ""-M, "". .. ..., ,,. ~) n.. .. of -..... -........ ,-_. -............. -~ ........ . "'DF,~JJ,C""""","' . """"'_"""""" "'''''''' ............... """-",, . ................ '" sw..... _ . '" 111_1 \< ~ Wi""'; M , F ........... J, v ..... H, .... H ('>II(H) C ............ .,.,., _ .-..,1_ """-..... _ ......... _~ . "''''''II' _ "",~ .,." ........ "P"_.~~ .,. •• '...;I9f. . ,'a.Q. " ""..-.. 5, "'. G", ""- M, T __ • MK, """""_ In, .. ~ ('2OO'l) no. .... ~.--............. , __ ... . .. , """- 'l, ....... A., C_"" TIT, _ " H, ""- M, .... n.. "'....,. of Wi"'''' ""'_. """' .. , 0J000d u .... .-.y ....... '" lW-l.\O. < """- CT, 0. .. " "J, W_ KJ, ... WM 1'(l(1(li """-;,;,y~"'" -"'"' .. .."...... .. ...,-.. ................... J<-..... ~Wo1d"" 0.-. '" 1'/1-..... .\. _ ..... d:.,_ Rp..SG,C..r Ml, , ..... M (19106) n.. • ....-..,._ " ....... "',",-, ~ --. .. ......... ,-,.,.,.,. """"'"'~ - ........ -IO<W-,.. 6- w.."" "" P')O;'l n.. .... ~ __ dO-...... ..- of'" _~,...........--.. """"'" "-' , 101_1 .... ........ PC, ____ NIt, ...... "" ~) """"_ -..;.", ..... C ............ , ...... ' _ ..... _,,,........ __ ......,. ,,~_ ...... ,., ' ~'- '- C.-o,n.C .... ,TIR, ...... K, ""~,U' ('200~) ~""'" _""'''' ... ........ ... of _ of'" o..w;", ""'" .......... , ....... _ C ....................... """"_''''' __ 9- ""","", M, V.,... II, F ...... T""". SI'(I(H)Oo ... _~..-. -..y~ ... _"". c..~_ .. _.. _ .. ... C ............ ........ 00)0. '" 1-9. • inl<""l)' ;, <:OO'1'daled ~ Iho """,oml of bod """"' .. 1"'<"'ido in o""'r . p.de. ofbhn, [<44). Even if ..... Wngs "'" fed atIeq ... teIy , Ihooe in """"iti:led .-. may .dor ..... rg<tic coo. \hal ...... n1l1ally lead b det;reaoed ourvivaI. A """nl .u.cty bt 0 ' ConDOr tI,.r [17) rq>orted a""OIaJ>C. be/l.a.....". by ..... 1ling Inrwin'. f"""hes >:>ward P. lomui larva. in Iho ...... La ",,,,, W<:r< moot active al night; "..uing. kq>\ awake al nighl by ioeding larvae p<esUmably haY< Ie. ..... rgy lOr gn:>Wlh. P . ...."..- Ia".,.. may alto ane.:1 O>eIlling gro»U> 0>dire<Uy by aff«ting """"' .... beha,io<. Mull f.males irriIaled by ioeding w-.""" 00" by .... Ileoo ....-lingo, may <I"""" 10 0l0f> brooding young, deo'e_ ioeding visi. 10 Iho .,.., or abaOOon Iho ..... I.ntirely. FW'\her.u.cty ;, o>ee<Ied 10 inv .. tigat< Iho p"""imal med>aniorno W><Ie<lying c".. d P . ....,,..- p.a.raoitiorn on fledging _"",. 0 111' 1Iu.cty flll1her <l<m:>Il$\l'a\el Iho deva$ ... ting elrea \hal P. ......- has <»- 1>001 fledging _"",. Olly a oingl • .,.. fr<;rn Iho wilined ""a,,,,,,nl prod""ed fledglings \hal W<:r< oighled aIler lea'ing Iho ..... L A .2'" .. ""rim"" .... redO><tion in """' ... aI:>undaJ>C. wall ouJ)'w;;"nl 10 oignj"w;anlly i....,.._ "'" nwnber d .-. \hal fedged )'OOIDg. Th ... , c,.....",a\i<;ol .11',.,. aimed al c",U'<iIIing P. 1,.,,"'- may be eIf«tive ...... n if fly J>01ll1la \i<;o>. "'" oi~ red""ed bul 1>0\ """euarlly .limina>:d. FUlure moniIoring ;, o>ee<Ied b <I<.,nn;"" ""be""'r Iho i"""" of P. '-'i <»- ..... ling fiO>d>eo.:ales ~ 10 Iho level d pof>ulaWno and . p.de. [H,4GJ. lbere ;,.tiIl m""h IOleam aooul Iho «<>logy d P. '-'ibolh in .. native and inb'Odo>(>;(\ ~ rang ... Acknowledgment. \\'. "' .... tho Gallpop N ........ Pad, tho ChuI<> ~ F.,..,.., ..... Suah B.,.h, R_dyG>nl<wa, AIr- Faky, BUp F"',A...J~ H....oy, _dj</> 0-" fo.-__ bm> ol_...". Sp<QoI '~'o F..d ....... too- _"""""" "" " ... __ .,..,~ am to ....... ..,."....,... "*~ ..- """",,,* ('Holy n ,..."m ,ho mamoo<>'¥ Author Contribution. G>n<:<Md am d~ tho <>p< ......... JAlIK SKH DlIe PmOomod """""'-_JMIK SK/I DlIe ...... .,..,dthod_,JAlIK SKH SML DlIe. eo.._d ","_Jimo",'alJ/_"" _ DlIe. w_ ,ho po_ JAlIK SKH SML Dlle. 10 C .. _C .. _s ............ .I. .... __ 1, ........ «J, ..... ('lOOO) "' ~ ........ n... ... _ ~ ... , __ ~C .......... _ ....... ~ ... "-"""'" ""'~ .,.~ ............. '1\1 , ,.,_"" 11 F ..... ,c.;.~MS, T._. SI'OOI) __ """" .......... _., ... C...."....I._~.., M""'""'I-,.. .... ~.""7_= 1~ F ...... T ....... S('2\lO'l)_---.;· .""'..,. ....... """.... ....... c....".... _",..... . • _ .... ~. -..' .... 1«, "~~1 11 0"'-J,"'~ >]. ~' .1. ""~ S ('>\109) II ..... "--' "..-;, ... ".......,.'-~-.... -ry ..... - .. y ...... ....... o..w;", .. _ .... _(~~ _,.,;,y_"-""""" ''''~ " F ...... v .... IIG, v .... 1tJ', .................. _J, """""' ...... ... ('>\1101 ""'w., ....... __ o..w;", Im<. ",- ...-.............. .. ..... .. , ...... ,._ .... ....."..... T_ .. "'~ ... ...... S<ri<ry._ '0'''''_ I.\. l'o" ......... ""-J'*-. C ..... F ..... , _ "" 5, V_ JC ('>001) .--~ ..... _"'_""'IIyIl ..... ---.;~.., .......... ) .. ... C""- ........ _ "-""""".dorY 1~ 1<-19- 16- "--.' M MS . .n..". "- l) H"'"'''"''" ("D""P"o' .".".. M.._ ..._...) ., " _,,",_, "". "_" '"'" ....... ."...'.-.. ...... "-..... "",,-..... 17 0_"_'-_J, ..~ _ .. . _J,_"'.S('>\I101 V. ........, ...~ ___. . ~~. -... o,o.",-IlIe--w< ,'- """" IlR (1'/11) a.. ........ .....,. ~ .... ~ ...................... ... (llpo .... M __ ) ....... __ .... " , '*'>')9. 19- F ..... , .......... '.I. ""~ S ('lOOO) T .. , .. ..".... ~ ........ "--' (llpo"" M~ ".... ..... 1.,.."...' -.. _ .. .."..ti •• _ .... _ "-'"""'c!' 1", ",..,., 17 ... .,.....,...,~5<2OH1~ __ .. __ ..,. ... ,......-,.,...,I.W,.o.o..o.o.OVt .-....... p.;.p .... _~ .. Don.oO.._..-_ ~, -...J--I_ ... .- """;"y"' ~'~ 11 Oodooio< n, ~ 5, .. '"' • IlOOII _ .. of .. ""­,, __ IWooooOt --.,; ... ""'...potoo .... , ... -.... --..... .. n-w._"' ....... _ (.-_.~ _ .. ""~ ,,,-.-,. U 0._ " Y, p ..... , 1UBoooIori. 5 (2001) ..... _ .... -' _..,.,.... _ .... _..,.~ ............ .,.. III_ ......... ~._ . U. _..~..(.'..! "O"O"l'I.I-_'~. !lWft. .. _,... .... IWooooOt ...... .. ...-. ...- .... --"'J .. .. __ ..-_ (..,... ......... ..... "-"...- , .. , 111'- U. aa...-J.'- .. Y. ~.I(SI"" __ .... "_-_"_~, .,.'..-_T".."... .".. ."."..",.. 'N'o'_-. .- -'--­... .-ft, .-_(lfaJE ____ ... .. __ __ ""~m. H. .--.-P.Il.C''."',! .E-<oIo-o7 .... _oIno.-• .-__ -J' .,. .~......... H... ,_... "... _( 'ISlI .... .T...'.- - .. ,.......,. . ... E.__ -,.. .. II. _'.U'~.. _or• .-..,.I<D(tOOIIl. .._ "" ... ____ _ ,,_"'''''''''-___ ... 0 ...... .....,."",'_"~, .,. _ .. OU' ......... ... T_$IIOOIIAo .... __ -,. .... _ of ... ......,.",--... .. _._, ........... """'.-m'.j.H;'. !II. c_. PIl CO. ,) -.. ~..- .. n.,..;.' .... __ ....... ~ .. ....,... """ ~ .,.~ '_ . .. ., <O!-432, "- a...-........ """'" '.I'll, ,_ N N (200+) P .. _ ......... _ ........ _"" .... _ ........ _ t.-...IO, __ . Jt. .... I'T CO'll<) ""'""' .... ~"-'~"""'''''''''''''' n-w . .<..1" "_" ,"." """'''' .. ,. ,."..... ,,_ ~.)MnooI .7-., ..,.. , Moy 2011 I V ........ I ..... 5 I .'9106 CHAPTER 3 ECOIMMUNITY IN DARWIN'S FINCHES: INVASIVE PARASITES TRIGGER ACQUIRED IMMUNITY IN THE MEDIUM GROUND FINCH (GEOSPIZA FORTIS) Printed with permission from: Huber SK, Owen JP, Koop JAH, King MO, Grant PR, et al. (2010) Ecoimmunity in Darwin's finches: invasive parasites trigger acquired immunity in the medium ground finch (Geospiza fortis). PLoS ONE 5(1):e8605, doi:10.371/journalpone.0008605. 19 OPEN a ACCESS FrMly .".lla"" onilM ·~· PLos one Ecoimmunity in Darwin's Finches: Invasive Parasites Trigger Acquired Immunity in the Medium Ground Finch (Geospiza fortis) Sarah K. Huber' .... , Jeb P. Owen:!, Jennifer A. H. KOOp', Marisa O. King3 , Peter R. Grant4 , B. Rosemary Grant4, Dale H. Clayton 1 1 B..,1ogy Departmenl, Unr.er~tyofUtah, Sa~ la ~ City, Utah, Un~ed State5 of Amerka, 2 Dep;lnment of Entomology, Waos hington Stale Univerl ity, pojlma n, wa~ ingl<>n, United State of Amerka, 3 Sdlool of Biologkal Sdente5, Wa~ ington Stale UniYe<~y, pojlma n, Waos hington, Un ited State'! of Amerko1, 4 Dep;lnmenl of Et;oIogy a nd EYOlution;wy Biology, Printet<>n Univerl ~y, Prino;eton, New Jerley, United Stale'! of Amerka Abstract Background: Invasive parasites are a major threat to island populations of animals. Darwin's finches of the Galapagos Islands are under attack by introduced pox virus (Poxvirus avium) and nest flies (Philornis downssl We developed assays for parasite-specific antibody responses in Darwin's finches (GeospizQ fortis ), to test for relationships between adaptive immune responses to novel parasites and spatial-temporal variation in the occurrence of parasite pressure among G. fortis populations. MethodoJogy/Principal Findings: We developed enzyme-linked immunosorbent assays (EU$As) for the presence of antibodies in the serum of Darwin's finches specific to pox virus or Phiiornis proteins. We compared antibody levels between bird populations with and without evidence of pox infection (visible lesions), and among birds sampled before nesting (prior to nest-fly exposure) versus during nesting (with fly exposure). Birds from the Pox-positive population had higher levels d pox-binding antibodies. Philomis-binding antibody levels were higher in birds sampled during nesting. Female birds, which occupy the nest. had higher Philornis-binding antibody levels than males. The study was limited by an inability to confirm pox exposure independent of obvious lesions. However, the lasting effects of pox infection (e.g., scarring and lost digits) were expected to be reliable indicators of prior pox infection. Conclusions/Significance: This is the first demonstration, to our knowledge, of parasite-specific antibody responses to multiple classes of parasites in a wild population d birds. Darwin's finches initiated acquired immune responses to novel parasites. Our study has vital implications for invasion biology and ecological immunology. The adaptive immune response of Darwin's finches may help combat the negative effects of parasitism. Alternatively, the physiological cost of mounting such a response could outweigh any benefits, accelerating population decline. Tests of the fitness implications of parasite­specific immune responses in Darwin's finches are urgently needed. Citation: Huber SI(, Owen )P, Koop JAH, lOng MO, GIant PRo et al {201O} Eooinvnunity in Darwin's Findles: I""awe Parasite5 Trigger kquired Invnun~y in the Medi"'" Ground Find! {&ospoZQ "'"is}. PinS ONE 5{1 }: e860S. do-i:l0.l171/joumai.pone .OOO66OS Editor: lau""t I\o!-nia, BMSl-A"STAR, Singap..-e R..,.lved July 4, 2009; Acu pt.-d C«ember 4, 2009; Publi.Md Ja ..... ary 6, 2010 Copyright: C 2010 Huber et ... Thil iI an open-aoce'llS article dillJObuIed under the te rms of the Creative Common'S An rib""'n l ken ~, whic:h permil'l un~lJicted u ~, diltribut..,n, and reproduction in any med""', provided the original author and ~"<:e ;we credited. Funding: Wort. was ~pported by NSF {DEB-OI18794 and DEB-081687n and the Societyforthe Stu<!yof EvoI .. ..,n. The funderl had no role in srudydesign, dala oollettion and anai~~ ded~on to publilh, or ~p;vation of the ma ..... ~ript. Comptlting Im ..... u: The author"! have deda~ thai no oompeting inI~U exill. • E-mail: s;wahhuberCtnnc.edu ~ C .. ""t ad'he!< Biology Department, Randolpl>-Maoon College, A ~ land, Virginia, Un~ed State'! of Amenta Introduction Invasive parasites pose a serious threat to native animal populations, because hosts with no histol)' of exposure may lad. effective immune defenses. Invasive parasites are a particular threat to small, island populations [1 ,2]. For example, introduced malaria (Pfamwdium rt5ctum) has exacerbated the decline of Hawaiian honeycreeper species, many of which are now extinct [3,4]. Darwin's rllIches have recently been exjll.l6ed to two introduced parasites d: high conservation priority: avian pox vil\lS (P= iru.s <Ilium) and the nest fly lY! iIomis thu"'si (Figure lA, I B) [1 ,2]. Both of these parasites have been shown to have negative effects on host fitness of Galapagos birds [5,6,7,8,9, lO]. If birds are able :@: PlJ:lS ONE I www.plosone.org to mount an immune response to these novel pathogens, then they might ultimately be protected, to at least some degree, from the negative fitness consequences of parasi tism. Alternatively, the physiological costs of an induced immune response to these parasites may exceed the benefits of mitigating parasite damage and contribute to negative fitness consequences. Indeed, these contrasting p(l$$ibilities are a guiding fon:e behind resean;h within the fidd of ecological immundogy [II]. The prevalence of Avip«.: in the Galapagos Islands varies on a geographic $Cale. Over the past 35 years it has been absent or vel)' rare at Daphne Major and El Garrapatero, Santa CI\lZ Island. Daphne Major had episodic outbreaks of pox in 1983 and 2008 [12] , and during our study in 2008, we lOund 50% of birds to be January 2010 I Vol ume 5 I Issue 1 I e8605 20 Ecoimmunity Darwin's Rndle'S EI GSITSpatefO EI GalTspatefO Figure 1. Parasite-specifk antibody response of Gl'Ospizii fonis. (A) Medium ground filCh, Grospiza fortis, with pox lesion in front d eye. (B) G. fonis nestling with Philamis dow1I5i lesions in nostrils and ear. (0 Pox·binding antibody levels of adult birds on Daphne Major (n = 30) were higher than those of adult birds at EI Garrapatero (n = 113) (Mann Whitney U=61950, p< O.OOOI ). (D) Philomis-binding antibody levels of adult birds with a(tive nests at EI Garrapatero (n = 37) were higher than those of adult birds prior to nesting (n = 76) at the same site (U =800, p< O.OOOI). Antibody response is measured as the optical density (OD) at 4SOnm. Bars indicate mean::!: standard error. d oi:10.1371Ijoumal.pone.OO08605.g001 symptomatic for pox (I5 out of 30 birds had active lesions). The outbreak of pox on Daphne Major in 2008 was not seen at El Garrapaterro. In 2008 not a single bird at El Garrapaterro, out of 129 individuals captured, was symptomatic, and none of these birds showed evidence of prior pox infection (e.g., scars or missing digits). The differences in pox prevalence between these two localities, allowed us to examine h(M' infection inHuen.-;es pox­specifIC antibody levds in two populations with relatively similar histories of pox exposure. lYIiIomis d(}llmSi was fll"St detected in the GaIapagOl in 1964; however, presence d: the fly went relatively unnoticed until the late 1990's when large numbers oflruvae were discovered in the nests ofGaIapagos land birds, including Darwin's fllIdtes [13,14]. Adult flies are not parasitic, but larvae are obligate parasites that leed on the blood and ttuues d: nestling birds. Nestling Darwin's fllIdtes exposed to fly larvae have reduced survival and growth [8,9]. At El Garrapatero in 2008, 96% of 23 nests were infested with P. d(1UmJi. Ecological immunologists are exploring potential fitness trade­offi between immune delense against parasites and the physiolog­ical demands of other life-history traits (e.g. growth and reproduction). Although parasites are treated as a selective fon:e acting on the immune system, few studies within ecological immunology use parasi te-spe.-;ific assays of immune function [15]. Non-speciflC assays do not clarify interactions between the immune system and parasites [16,17]. As a result, non-spe.-;ific assays do not directly test fitness effects of immunological variation in the context of parasite pressure. Here we take the fll"St step in :@: PlJ:IS ONE I www.plosone.org 2 examining avian responses to introduced parasites directly, by demonstrating parasite-specific antibody responses to multiple .-;lasses of pa rasites in Darwin's firlChes. We developed assays for parasite-spec iflC antibody responses in the medium ground firw;h (GlWph.aJortU) (see Methods). Our goal was to test lOr relationships between adaptive immune responses to novel parasites and spatial­temporal variation in the ocwrrence of parasite pressure among G. .fortis populations. Our results demonstrate that Darwin's fllldtes produce antibodies against these invasive parasites, and that the immune responses are correlated with spatial-temporal variation in parasite pressure, both between fllldt populations, and between sexes. To our Knowledge, this is the fll"St time parasi te-spe.-;ific immune responses have been demonstrated rdative to multiple .-;lasses of parasites in a wild population of birds. Results Adult birds on Daphne Major had signifICantly higher levels of pox-binding antibodies than birds from El Garrapatero (mean:!:SE for Daphne Major=0.63:!:0.09 optical density (OD); mean:!:SE for El Garrapatero=0.20:!:0.02 00; Mann Whitney U=619.50; p<O.OOOI; Figure IC). When we compared lYIiIomis-speciflC antibody levels in adult birds sampled belOre nesting (prior to Phi{qr."u exposure) with a different set of individuals sampled during the nesting period, we found significantly greater levels of Philomis-spe.-;ific antibodies during the nesting period {mean:!:SE lOr nesting = 1.08:!:0.12 00; January 2010 I Volume 5 I Issue I I e8605 21 mean:!:SE lOr pre-nesting=0.64:!:0.07 00; Mann Whitney U=S(M).OO; p<O.OOOI; Figure lD). \Ve found no !leX difference in pox..,peciflC antibody levels (mean :!:SE for Daphne Major females=0.61:!:0.12 00; mean:!:SE for Daphne Major males=0.67:!:0.IS 00; Mann Whitney U =91.50, p=0.71), suggesting equal exposure of males and females to pox virus. In contrast, we found signifICantly higher Phi/Omu..,pecific antibody levels in females compared to males (mean :!:SE lOr El Garrapatero females = 0.99:!:0.11; mean:!:SE lOr El Garrapatero males = 0.58:!:0.06; Mann Whitney U= 1018.00, p=O.OOI). This result is consistent with adult females having increased exposure to P. doumsi when they brood offipring (males do not brood). Discussion Higher levels d pox-binding and R'lilorni.s-binding antibodies in Darwin's fillChes exposed to these parasites conflrlllS that these birds are capable of mounting parasite"'J>ecific adaptive immune responses to novel parasites. Importantly, these antibody responses are directed against parasites that represent distinct immunological demands (intracellular versus external), and which constitute a serious threat to Darwin's finches. From the J>ersJ>ective of vertebrate immunology, it is nO( unusual that G. fQrlu is able to develop antibodies against novel challenges. However, our data are unique in two respects. This study is the first demonstration, to our knowledge, d ectoparasite"'J>eciflC antibodies in a wild bird population. This study is also the first demonstration of parasite­sJ> eciflC antibodies directed against two distillCt classes of parasites (external and intracdlular) in a wild bird population. Within the foekl of ecological immunology, these observations are important because they establish a defmitive immunological link between actual parasites and an animal of ecological interest [Hi]. These data also raise intriguing questions about prevailing assumptions regarding the host-parasite interactions of P. d(1UmJi. We found no differellCes in the levels of pox-binding antibodies between male and female fmche$. This fmdi.ng agrees with the known ecology of avipox vil\ls, which is transmitted by mosquitoes, or through bird-bird contact [1,7], where no bias in transmission among the sexes would be expected. In contrast, we found significantly higher R'lilomis-SJ>eciflC antibody levels in females compared to males, which agrees with the exj>ected bias of higher female exposure to P. doumsi during female brooding on the nest. Thus, our data cast doubt on the assumption that adults are never bitten [IS]. The prevailing nO(ion that adults are nO( exj>Osed to \arval feeding is based primarily on twoobsetvations: {ij lesions from \arval feeding have not been observed on captured adult females; and (iij the scaly covering on the females legs is thought to prevent larvae from J>enetrating the female's skin. The absence of obvious lesions on females <:koes not rule out the p,')Slibility that adult females are bitten. Forexample, fewer than half of the nestlings in our study had visible lesionsalOOCiated with larvae feeding, even though nests were heavily parasitized and in many cases nestlings died (unpublished data). Second, while larvae likely could not J>enetrate the $Cales on female's legs, females might be vulnerable to larval feeding through their brood patch, which is completely devoid d a feather covering. Larvae may come into contact with the female's brood patch while she is sitting on nestlings, particularly when larvae are in the first or second instar and reside on the nestlings (e.g., in the nostrils or on the wing webbing) [IS]. Although the immunological data indicate feeding attempts on females do occur, we are not suggesting this is evidence that adult fmches are viable hosts for P. doumsi. Blood feeding attempts on :@: PlJ:IS ONE I www.plosone.org Ecoimmunity Darwin's Rnthes adult birds may consistently fail for a variety of physical and behavioral reasons. H(M'ever, if feeding attempts by latvae are occurring, it is reasonable to exjlect adult females are exJ>Osed to P. doumsi antigens that are stimulating an immune response. The ecological importance d this immune response deJ>ends on multiple unexjllored factors. For example, antibody devdopment by the female could confer a defensive advantage to offspring, if there is transfer d maternal antibodies to the chicks [19]. If females are exposed during the fIrSt clutch and produce antibodies, they might transfer these antibodies to the eggs of the ir second or third clutch. Alternatively, a stimulated antibody response in the female could produce a physiological demand that reduces energy available for foraging and subsequent breeding attempts in the season. A number of important immunological questions must be answered to address these p(l$$ible ecological outcomes. For example, how quickly are antibodies produced and how klllg do they J>ersist? Though anti-ectoparasite antibodies can be produced rapidly (I -week) and J>ersist up to two months without stimulation [20,21], the dynamics of anti-PI!ilomu antibodies remain to be determined. \Ve are currently attempting to detennine if maternal antibodies are transferred to G.fIJf'/U offipring, as well as the timing of primary and secondary immune responses to P. doumsi by female fmches through the breeding sea$On. A critical next step in understanding the relationship between parasite infection and antibody production is to examine how these factors affect fiuless. The only fiu'lelll data available for the effects of pox on Darwin's finches underscore the l'leed for a detailed study of sun;val in rdation toantiboo:tj response. Observations of G . .fortUon Daphne Major in 2009 found that II out of 14 birds with pox symptoms in 2008 survived to the next year, compared with 12 out of 19 birds without P(l< symptOlllS (Fisher's exact test: two-tailed p = 0.46). These data suggest P(l< might not have the same impact on Darwin's fUlChes as it <:koes on Galapagos Mocking birds [5,12,22,23]. However, long-tenn fitnelll eflects estimated in relation to short-te rm measures of prevalellCe are inadequate for several reasons. First, we do not know the severity d P(l< infection for individuals in our study. We only know that some birds on Daphne Majorwere exj>Osed, whereas birds at El Garrapatero were not exj>Osed over the course of our study. Variation in the intensity of exjlOSure is likely related to survival. Second, we do not know if birds that were unexj>Osed to pox at the time of sampling continued to be parasite-free. Finally, survival may be confounded by sex, age, condition, and breeding status, among other variables. For example, males and females might have different physiological responses to these diseases or the costs of breeding might be greater in Ol'le sex than the other. Forexample, some evidence suggests that males with prior P(l< exjlOSure might have decreased pairing success [7]. We emphasize the need lOr future studies that control for these factors and that exJ>erimentaUy test for the impact of parasite load and antibody production on fiu'less. For example, sun;val data for birds with controlled exjlOSure to pox can be compared between individuals with low versus high levds of anti-pox antibodies; these data would all(M' us to test the extent to which antibody production might be protective. Conversely, sun;val data for birds that are bl(M'n to be free of active pox infection can be compared between individuals with anti-pox antibodies and those without anti110x antibodies; these data would allow us to test whether antibody production might be costly. Studies such as these should be a major focus of future resean;h, lOr both JlOX and R'l ~ms. In summary, the assays presented here are valuable tools for exjlloring the ecological immunology of Darwin's fmches, and in helping to dete rmine the epidemiology d two critically itnilOrtant diseases threatenitlg avifauna itl the Galapagos archq>elago. Broadly, we exjlect this approach can be applied to other resean;h January 2010 I Volume 5 I Issue 1 I e8605 22 systems as well, which will strengthen studies that have typically relied on non-specific measures of immune function [16]. Methods Ethics Statement AU procedures were approved by the University d: Utah Institutional Animal Care and Use Committee (prO(oool # 07- 08IJIH). Sample Collection We studied birds at two sites in the Galapagos Islands: El Garrapaterro, Isla Santa Cruz, and Isla Daphne Major. Birds were sampled at El Garrapaterro from January-April 2008 and at Daphne Major on Man;h II , 2008. T hey were captured using mist nests, or Potter's traps, and each bird was individually marked with a combination of one aluminum ring and three daMe color bands. We noted whether birds had active pox lesions, or evidence of prior pox infection (e.g. , missing digits). We then collected a small volume d: blood by piercing the ulnar vein with a 27-gauge needle. Approximately 50 ~ of blood was collected with a capillary tube and expdled into centrifuge tubes. Centrifuge tubes were stored on ice in the fidd (approximately 6 hours), then transported to the laboratory where they were centrifuged. The senun was then pipetted off the top and stored at - 80°C. At El Garrapatero, we made focal observations ofindividual~ to determine pairing status and nest location. We checked nests every other day to dete rmine egg laying date, dutch size, and hatch date. \\'hen nests were no longer active (nestlings were predated, fledged, or died), the nests were dissected to obtain fresh lY! iIomu Mumsi larvae, which were placed in a centrifuge tube and stored at - 8O"C for future antigen extraction (see bdow). Adults sampled at El Garrapatero were assigned to one d: two groups: un-exposed or exposed. Un-exposed birds (n = 76) were individuals that I) had a nest but were sampled prior to the hatching d: their first brood, 2) females that did not have a brood patch (and thus were not breeding), or 3) umnated males that were sampled early in the breeding season. Exposed birds (n = 37) were those sampled while they had nestlings in the nest and Itad parasites present in the nest. No unexposed individuals were re­sampled during the nesting period, and no exposed individuals were sampled prior to the nesting period. For birds sampled at Daphne Major and El Garrapatero the $!X was detenni.lled based on plumage (black plumage lOr males and the presence of a brood patch for females) or by genotypi.llg. Blood samples of indivkluals lOr which we couk! nO( determine $!X (nonbreedi.llg females and young males have identical plumage) were sent to Avian Biotech Intemational (fallah.assee, Fl.) lOr genotyping via PCR. On Daphne Majorwe sampled 10 females and 20 males; at El Garrapatero we sampled 56fernales and 57 males. Comparisons of pox immune response were rnade between populations (Daphne Major versus El Garrapatero). We did not compare asymptomatic and symptornatic birds within populations because it was not possible to evaluate the timing of prior pox exposure from current symptoms alone. Asymptornatic individuals could have elevated antibody levels due to prior infection. Additionally, there is a lag between infection and the production of antibodies (10-12 days). Thus, symptornatic individuals could have low Pox-specific antibody levels due to sampling prior to antibody production. T hese factors confounded our abili ty to detect relevant differences in Pox-specific antibody levels within a population. In contrast, we were able to compare lY! iIomis-speciflC antibody levels between unexposed and exposed birds from El Garrapatero, :@: PlJ:IS ONE I www.plosone.o rg , Ecoimmunity Darwin's Rnthes because we could determine the timing of parasite exposure (nesting period), visually confirm the prese rlCe of the parasite, and obtain blood samples after the lag t~ required for up-regulation of any antibody response. Although pre-nesting birds could have been exposed to PhiI{)I'1fU in a previous breeding season, and thus have anti-lY! iIomu antibodies, we expected those antibody levels to be low (at or near background), owing to the breakdown of antibodies in the abserlCe of antigenic stimulation between breeding seasons [24]. Antigen Production First and second instar larvae of P. doumsi were used for antigen extraction. Larvae were placed into a centrifuge tube and rnacerated with 100 v.L of phosphate buffe red saline (PBS) and ImM EDTA. The tube was centrifuged at 14.8 thousand revolutions per minute, and the supernatant containing the extract was removed. The supematant was passed through a 0.2 micron f!lter and the protein concentration was estimated using a spectropltotometer. Th.e extract was diluted to a concentration of 0.613 mg mL - 1. For pox antigen we used a live vil\l$ vaccine for Fowl Pox Vil\l$ (FP-VAC; Intervet/Schering-PIough), following tests of binding by Darwin's fmch antibodies (see below) and based on the likely occurrerlCe of conserved antigens among Fowl Pox and Canary Pox [25]. Production of Secondary Antibody and Cross Reactivity with Darwin 's Finch Serum Anti-house-$parrow-immunoglobulin antisel\lm was produced by immuni1.ing rats with purifIed house sparrow (PQ.UIT M7N.$ticui) IgY (Yolk Immunoglobulin). House sparrow IgY was isolated using thiophilic inte raction chromatography (described in 26). The recovered fraction was analyzed via $O".">dium dodecyl sulfate polyacrylamide gel electro­phoresis (SDS-PAGE) on 12% slab-gels and stained with Coomassie Blue R-250 to confirm the presence of house sparrow IgY. Lyophilized house sparrow IgY was then re-dissolved in PBS at I v.g/~ and emulsifIed with an equal volume of complete Freund's adjuvant (CFA). Three rats received a subcutaneous prirnary irljection of house sparrow IgY with CFA (50 v.g of protein I 100 ~ emulsion was used per injection). Rats received booster shots containing house sparrow IgY with irlcomplete Freund's adjuvant (IFA) at 4-week intervals two times. Rats were exsanguinated 4 weeks after the final booster shot. Cross-reactivity between house sparrow IgY, Darwin's finch sel\lm and the rat antisel\lm was confrrmed using Western-Blot analysis. Briefly, purified IgY was separated usirlg SDS-PAGE and transferred on to a nitrocdlulose membrar>e for immunoblottirlg. Filters were blocked with casein blocking buffer lOr one hour at room temperature and then washed three tirnes in double deionized water (ddH20). T he blots were incubated for one hour at room temperature with rat-anti-house-$parr(M'-IgY (RotHOSP­IgY) and then washed three times again with ddH20. The blots were then irlCubated lOr another hour at room temperature with comme rdally prepared goat-anti-mouse antibody conjugated to horseradish peroxidase (GotM-hrp) {Bethyl Laboratories, Inc., Mongomery, TX) and then washed a final three times with ddH20 . The blots were analyzed using enharlCed chemilumine­se rlCe (Figure 2). Cross-reactivity between Darwin's fmch sel\lm arid RotHOSP­IgY was established via enzyme-linked immunosorbent assay (ELISA). Brie fly, 96-well ELISA plates were coated irl triplicate with 100 v.l of Darwin's firlCh sel\lm diluted at 1:100, 1:500, Jan uary 2010 I Volume 5 I Issue 1 I e8605 23 - lgY H - chain - lgY L - chain Figure 2. Western blot of serum dilutions developed for house sparrow IgY. Western blot d serum dikltions from Darwin's fil1(h (OF), house sparrow and chicken using antibody ma!i(ers developed for house sparrow IgY. Lane 1: OF serum 1 :10. Lane 2: OF serum 1:20. Lane 3: house sparrow serum 1:10. Lane 4: house sparrow serum 1:20. Lane 5 chk:ken serum 1 :10. Lane 6 chicken serum 1 :20. Image indk:ates cross rea:::tivity of house sparrow IgY deteaion antibody with Darwin's finch IgY. The lack of binding to chicken serum indi(ates no cross· reactivity with that speties. doi:l 0.1 371/joumal.pone.0008605.g002 l:lOOO, and 1:5000 in carbonate coating buffer {O.05 M, pH 9.6). The plates were incubated lOr one hour at 3rC on an orbital table before being washed three times with 200 ~ of wash solution per 0.6 0.6 A B 0 0 0 00.5 j 05 ~ 'E•0~ 0.4 ~~~ 0.4 ~ ~ ~ 0.3 0.3 1/100 1/500 111000 11100 Ecoimmunity Darwin's Rnches well. The plates were blocked with casein blod..ing buffer and agai.n incubated for one hour at 37°C on an orbital table. The RIXHOSP-IgY was diluted in sample buffer at 1:50, 1:100, 1:500 and I: 1000. After washing the plate three times, 100 v.l of the RIXHOSP-IgY was added to each Darwin's fllIch serum dilution, such that each serum dilution was tested against each RlXHOSP­IgY dilution. Plates were again incubated lOr one hour at 3rC on an orbital table and then washed three times. The secondary antibody, GaM-hrp, was diluted 1:1000 in sample buffer and 100 ~ of this solution was added to each well. The plates were incubated for one hour at 3rC on an orbital table and then washed a final three times. 100 ~ of peroxidase substrate (2,2' ­azino- bis-3-ethylbenzthiazoline-6-sulphonic acid, ABTS: Sigma cat. AI888) and peroxide was added to each well and the plates were covered with tinfoil and allowed to develop for one hour at room temperature before being read on a spectrophotometer using a 4Q5-nanometer fdter. Three blank wells were included on each plate, as well as three wells that measured non-<>peciflC binding, which quantifIed binding of RIXHOSPIgY and GaM-hrp to the respective antigen. These wells received all the reagents described aboye except for Darwin's fillCh serum. In this step, blocking buffer was used in place of serum. The mean absorbance of these wells was subtracted from the absorballCe measures determined aboye. Results from this ELISA indicated crossreactivity between Darwin's fillCh senHn and RIXHOSP-IgY. 1/500 lfl000 Pox Antigen Dilution Philomis Antigen Dilution 2.5 1.0 C D Cl 2.0 0 0 0 • ~ ~ 1.5 ." ~ ~ 0.5 ." 0 ~ 1.0 •• 0 ~ ~ ~ 0.5 0.0 0.0 11100 11500 1/1000 1/100 11500 lfl000 Darwin's Finch Serum Dilution Darwin's Finch Serum Dilution Figure 3. Optimization of EUSAs for antigen and Darwin's finch serum. Optk:aI density (00) values for optimization EUSAs of (Al Pox antigen dilutions and Darwin's fil1(h serum at 1/500, (8) Philomis antigen dilutions and Darwin's fil1(h serum at 1/500, (0 Darwin's fil1(h serum dilutions and PQx antigen at 1/1000, and (D) Darwin's finch serum dilutions and Philomis antigen at 1/1000. Decreasing amounts of antigen (A,B) and antibody ((,0) result in decreasing optical density values, indk:ating specific antibody-antigen binding. doi:l 0.1 371/joumal.pone.0008605.g003 ~: PlJ:IS ONE I www.plosone.org January 2010 I Volu~ 5 I Issue 1 I e8605 24 Cross Reactivity of Darwin's Finch Antibiodies and Parasite Antigen Cross-reactivity between Darwin's finch antibodies and Philomu doumsi protein, or Fowl Pox virus, was established via ELISA, using dilutions of Darwin's flllch sel\lm and antigen (Philomu protein or Fowl Pox vil\ls). Briefly, 96-well ELISA plates were coated in triplicate with 100 v.l of either Fowl Pox vil\lS in PBS, or Philomu extract, diluted at 1:100, 1:500, or 1:1000 in carbonate coating buffer (O.05 M, pH 9.6). Plates were inwbated for one hour at room temperature on an orbital table, and then washed five times in wash buffer. Wells were then coated with 200 ).II bovine serum albumin (BSA) blocking buffer, incubated lOr 30 minutes at room temperature 011 an orbital table, and then washed five times with wash buffe r. Each wdl was then loaded with 100 v.l of Darwin's flllch sel\lm (pooled sample) then diluted I: 100, I :500 or I: 1000 in sample buffer, such that each serum dilution was tested against each antigen dilution. Plates were irw;ubated for one hour at room temperature on an orbital table, and then washed (5 x) with wash buffe r. Next, lOO).II of Ra.HOSP-IgY {I: lOOOj was added to each well, followed by a OIle hour incubation at room temperature and wash (5 x). The second detection antibody (Ga.M-hrp, I: 1000) was then added, followed by a OIle hour irw;ubation at room temperature and washing (5x). Finally, 100 v.l of peroxidase substrate (tetramethylberl1.idine, TMB: Kirkegaard and Perry cat. 50-77-03) was added to each well. The plates were irw;ubated for exactly five mirlutes at room temperature and the reaction was stopped usirlg 100 v.l of 2 M H2S04 irl each wdl, before reading optical density on a spectrophotometer usirlg a 450-nanometer fdter. Based on optimizatiOlI results (Figure 3), a standard serum References Wi.kekki M, Foufq><>t>looJ, Varguo H, S",,]] H~) CalIJ"'gO$ Birdo and DiKaoa: I",·aon.·e Pathogeno ao ~all br bland Spocieo. £.col Soc 9(1 ): ~ (onlin~): hnp:II,,'WW.<Wog)'''nckociety .org/wl9lisoll arO Ca"""n CE, I'.:<:k SB, Sinclair BJ, Roq.,.,..Ahd> l, HO<Ig:Km CJ," at ('1006) Alien in""",: threa .. and OnpIicalio .. f", the con"""'alion of the CalIJ"'gO$ blando. Annal, f.ntomoI Soc America 99: 121-143 ,,,n '4>e' III C, ,,,n J4>er 5(;, CoIfML, Loird M (198&)The q>i2lXltioiogyand e<:oIogi<:al , ignifi",,,,,,, of malaria. in Hawaiian land hirdo. £.col Ma>ographo :;6' 327-344 4 ,,,n J4>er ID C, "an J4>er 5(;, Hansen WR ('1002) G> ~ogy and ell:<t of a.,un """ on Ha"",~an fo_ him.. Auk 119: 929--9+2 Varga> H (1987) .·""'l»enCf and elf""" of """fib: leoim, in CalaragO$ rnockingbirdo.. J Field Omith ;.1.\: 101-102 6 Pa.rf<er PC, Whi""""n NK., Miller RE ('1006) Consen-atim modi<i>e a> the Calilpogoo blan<k: Partnemg """"-''i>r.oI, J>OI"'Ialion and , . ....mary .:ientioll Auk 123: 62H3S Kkinclorfer S, Duclani"" RY (2()(l(ij In""" ... ing I""",.)ence of ",;an pa<m. in D"",;n', rlOCh .. and ill d'""" a> male paimg ' ''''''''''.J A,;an Bioi 'SI: 69-76 8 F .... B, Kleindorfer S, Te/Jbich S (2006) An ~ta.I ,rudy a> the ell:<:to of an introdO><:e<l J""Ute in Darwin', fOlcheo. Bioi Conserv 127: ~HI 9 HOOc SK (2008) fiT""" of the i1ltJ'<><h>c<:d I""""ire ~ J/imui on neotling gJ'O"th and rnona.lity in the m~ gnouncl fiDCh (GNpi:;ll.fortii). Bioi Conserv HI: 601--{;()9. 10 DOOanie<; RY, F .... B, KJeindorb S (2007) Int.:r.ann"" and in~o:; ,,,riatia> in in"""ity of the J""Utic fly, ~s J/imu~ in D"""in', fiDCheo. Bioi eonsen-"fun 139: 325--332 II Nom. K, E,,, .. MR (IW» f.ooIogi<:al mmunology: life hioto<y tradoxJf, and immune defense in him.. Behav £.col II: 19-26 12 Cr..", PR (198&) Ecology and r.."oh>tia> of Da,,,,in', rlOCh ... Pm""",n' Pmceton Unn."eroity Pre. 13 F .... B, Cow; ~IS, Te/Jbidl S (2001) ~ bmui Dod)," and Aitken, newto the Caliipa.goo IsIando (D;p.:r.., Muscrla~~ Srudia. Dipterologi<:a 8: 317-322 14 F .... B, Tebbich S ('1002) f'IliIMrtis btmui- a. n:<:entIy~"ered P':",""ire on the Caliipa.goo An:hipelag<>- a. threal for D"""in', rOlC"'" lhio 1# +l5--~1 :@: PlJ:IS ONE I www,plosone.org 6 Ecoimmunit)' Darwin's Rnthes dilution of I :500 was selected for the ELISAs of individual birds and a standard dilution of I: 1000 was selected for Po>< and Philomu antigens, which were tested separatdy. On each plate we irlduded three wdls for non-<>peciflC birldirlg, which quantified birldirlg of Ra.HOSP-IgY arid GaM-hrp to the respective antigen, These wells received all the reagents described aboye except for Darwin's firw;h serum. In this step, blocking buffer was used in place of serum. The mean absorbance of these wells was subtracted from the absorban(e measures determined aboye, Firlally, we calibrated absorbance values between plates using a positive controL In brief, each plate contai.ned the same refererw;e sample in triplicate, The refere,w;e sample absorbarw;e was compared across all plates, and we cakulated a correction factor for each plate to standardi1.e absorbance, These standard­ized values were used for subsequent analyses of immune response irl birds. Aclmowledgments We thank thor: GaUpagus National Park, thor: Charla Dan.;n Foondation, Ratady Cordova, Midade Rttd, and Sarah Bush, Author Contributions Conceived and des igned the ~erimenrs: SKHJPO DHC, Perfonned the ~erimenrs: SKH JAHK MOK PRG BRG DHC. Analj'ZW thor: data: SKH JID JAHK MOK DHC. Contribured reagenrs/marcriak/analysis rools: SKHJPOJAHK MOK PRG BRG DHC. Wrore the paper: SKH JID JAHK MOK PRG BRG DHC, I~ Ardia. DR, Sdlal K.A ('lOO8) EcommuDOlogy. In: Davison F, ~en B, Sehal ](A,~ .. A,;an ImmuDOlogy. New Vork: £!se,.;er. PI' HI---HI 16 0""" JP, Clay"" DH (2007) Wh""" an: the pa.r.uiteo in the PHA ""1',,,..,' Tmrlo £.col Ewl 22: 228-229. 17 Lind_ K.M, .·OIlbrwho J, Plim H, Wi.kekki M (~) ImmWldogical i"''''tment, re/Ie<;t J""Ute abundance in isJand J>OI"'Ialio .. ofD",,,,in', fiDCheo !'roc R Soc l.<:<ld B 271: 1~13-1~19 18 ...... B, Sinclair B, KJoindo<b S ('1006) The Iife-<ycleof fWtwoiJ"-ui(D;p.:r..' Muscrla~) I""""iti>.ing D"""in', fOlcheo and ill .""""" on "",ding ,,,,,;,::01 P"""'iroIogy 133: 7'»-747 19 BoWinier T, Suue"w V ('lOO8) Mar.:mal tra .. i:r of antibodies: raioing irnnmno·«:<>Iogy iJoueo. Tmrlo EcoI Ewl 23(~): 282-286 20 0."., .... 1 F, Lukeo S, Crubho8'er L (1989) Antibody-mediated """"'., of ",),"On, to A'l"" ~ .. Ian.:al f.,.,ding and chara<:terizatim oflan.:al an.n .'o/ia. Pa .... i' 36: 83-92 21 MW'ano T, Namiki K., Uchino T (1989) [)e,."eIOl"nen' of~ita.ting antihody in chic.keno experimenta.lly inb ted ",irh northern f""i mite, 0mitJa,0a)' .... 9''''"""'''' (A<;ari: Ma.cron)"lidat). Pwlr Sci 68: 842---t14~ 22 Cuny Rl, Cr.ant PR (1989) ~y of the """"""",..""Iy hn:<:ding Caliipagoo mo::kinghird (X_ ..... J-r-Ior) in a. cfimali<;ally ,:ariahle em-;I'OJ>­men'. J Anim £.coI;.I.\: +H·-463 23 Cran' BR, Cr.ant PR (1989) Ewluti>nary Dy~ of a. Natw:al PorWalia>' 'IDe ~)," Cacruo Finch of the CalIJ"'gO$. Chicagoll.: Uru.'enity ofcru"'go '= H Davis«> r, M;ogOl' KE, Ka.,pen B ('lOO8) Stl'U<;t\ln! and ."..,!utim of a.,un irnm""",!!"k>bi ... In: Davison .~ Ka.open B, Sehal ](A, <:do. Avian ImmuDOlogy. New Vork: E!se>;er. PI' 107-127 2~ SehnitlJeB> WM, Child).':al N, TriJ"'lhy ON (1988) Genomic and antigenic char.acteri>.ation of a.,~. Vrn, Reo 10: 65--76 26 Hans<m P, Scobie JA, Han.", B, Hoogenr.ud NJ (1998) l.o>Ialion and """'",,fun ofimmWlqrkibWi .. from chicken egg> u,;ng thiq>hilic int.:r.a<:ti>n ch~y. J ImmWlol Method, 21~: 1-7 January 2010 I Volume 5 I Issue 1 I e8605 CHAPTER 4 TEST FOR PARASITE-SPECIFIC IMMUNE RESPONSE IN MULTIPLE SPECIES OF DARWIN'S FINCHES Abstract Ecoimmunology aims to explain variation in immune responses within an ecological and evolutionary context. Traditionally, studies have used non-pathogenic agents to elicit nonspecific immune responses in hosts. Studies of immune responses to specific parasites are often limited to host species for which commercially produced detection antibodies are available. Recently, medium ground finches (Geospiza fortis) were shown to mount a parasite-specific antibody-mediated immune response to the introduced ectoparasite, Philornis downsi, using an indirect enzyme-linked immunosorbent assay (ELISA) with house sparrow (Passer domesticus) antiserum. Despite these two species of birds not being closely related, house sparrow antiserum cross-reacted well with medium ground finch serum. This study validates the use of house sparrow antiserum to quantify parasite-specific immune responses in other species of Darwin's finches. P. downsi was recently introduced to the Galapagos and is known to negatively affect nestling growth and fledging success in several species of finches. Validation of this immuno-assay with other species of Darwin's finches is the first step toward determining whether these species are also able to mount P. downsi-specific 26 antibody-mediated immune responses and in determining whether this immune response is a viable defense mechanism against P. downsi. Introduction The field of ecoimmunology explores variation in immune responses relative to tradeoffs with other life-history traits (Norris & Evans, 2000, Lochmiller & Deerenberg, 2000). Traditionally, studies have used derived substances (e.g., phytohaemagglutinin (PHA), keyhole limpet hemocyanin (KLH)) or attenuated pathogens (Newcastle disease virus vaccine (NDV)) to elicit various immune responses in host organisms (Hasselquist et al., 2001, Smits et al., 1999, Saino et al., 2002). This approach allows researchers to compare life-history traits between individuals while eliminating the confounding effects of a given parasite or pathogen on host fitness. In addition, the substances used to elicit these responses are commercially available and easy to use in a field setting (Martin et al., 2004). While this approach has provided useful insights about potential trade-offs between the immune system and other fitness components, it ignores the more complicated interactions that can occur between a host and parasite (Owen & Clayton, 2007, Kennedy & Nager, 2006, Norris & Evans, 2000, Owen et al., 2010). Immune responses to substrates, such as PHA or KLH, may not be comparable in longevity or intensity to immune responses elicited by actual parasites (Owen & Clayton, 2007). Thus, the field of ecoimmunology is evolving to assess the costs and benefits of host immune responses to relevant parasites and pathogens. Unfortunately, quantifying host immune responses to real parasites and pathogens is still largely limited by the techniques and reagents available for a given host species. Most work on avian immunology is still centered on poultry species due to their 27 agricultural and economic influence. The majority of commercially available antibody and immuno-assay products are designed for domestic chickens (Gallus gallus domesticus). Low cross-reactivity between chicken antiserum and many wild bird species limits the number of systems in which avian antibody responses can be studied using chicken antiserum. However, recent efforts have been made to design other antibody products for wild bird species (King et al., in press, Ilmonen et al., 2002, Hasselquist et al., 1999). King et al. (in press) created antiserum against purified house sparrow (Passer domesticus) immunoglobulin Y (IgY) and tested its cross-reactivity with a variety of other passerine and non-passerine species. Using tests of dilutional parallelism, the authors showed that house sparrow antiserum cross-reacted strongly with eight of 19 wild bird species tested. The development of house sparrow antiserum provided the opportunity to study antibody-mediated immune responses in rarer bird species, including Darwin's finches (Huber et al., 2010). For high-yield production of antiserum, destructive sampling of eggs is required to extract immunoglobulins from the yolk (De Meulenaer & Huyghebaert, 2001, King et al., in press). Since destructive sampling of large numbers of Darwin's finch eggs is undesirable, it is not feasible to develop antiserum specific to this group of birds. However, Huber et al. (2010) successfully quantified parasite-specific antibody-mediated immune responses (IgY) in populations of medium ground finches (Geospiza fortis) using house sparrow antiserum in enzyme-linked immunosorbent assays (ELISA). Their results showed that medium ground finches mount antibody-mediated immune responses specific to the recently introduced nest parasite, Philornis downsi. 28 Philornis downsi (Diptera: Muscidae) is a parasitic fly that was recently introduced to the Galapagos Islands, and which has the potential to affect many bird species, including all species of Darwin's finches (Fessl & Tebbich, 2002). Adult flies lay their eggs in the nests of birds. The eggs hatch and larvae blood-feed on nestling and adult birds as they progress through three instars. The larvae then pupate in the nest material and emerge as adult flies. Adult flies are nonparasitic and feed on organic matter (Dodge & Aitken, 1968, Couri, 1985). P. downsi has been documented on 11 of 13 major islands in the archipelago and in the nests of at least 14 species of birds, including 9 species of Darwin's finches (Wiedenfeld et al., 2007, Fessl & Tebbich, 2002). P. downsi significantly reduces nestling growth and fledging success in several finch species (Koop et al., in press, Fessl et al., 2006). In fact, P. downsi has been implicated in the recent severe declines of the critically endangered medium tree finch (O'Connor et al., 2009), mangrove finch (Fessl et al., 2010) and warbler finch (Grant et al., 2005). The goal of this study was to validate the use of house sparrow antiserum to detect antibody-mediated immune responses in several species of Darwin's finches. The high relatedness between species of Darwin's finches (Grant, 1986) suggests that house sparrow antiserum should cross-react similarly between species of Darwin's finches. Since P. downsi is known to parasitize multiple species of Darwin's finches, we would predict that these species will also mount P. downsi-specific antibody-mediated immune responses. 29 Methods Study site Our study was conducted January-April, 2009 on Santa Cruz Island in the Galapagos Archipelago, Ecuador. Santa Cruz Island has three main geographic areas, including an arid zone around the perimeter of the island, a humid, highland zone at the central peaks, and a transitional/agricultural zone between these. Samples were collected from 8 species of adult Darwin's finches where populations are most abundant. Samples from adult Geospiza fortis, G. magnirostris, G. scandens, and Platyspiza crassirostris were collected only in the arid zone. Certhidea olivacea and Camarhynchus parvulus were collected only in the highland zone. Cactospiza pallida and Geospiza fuliginosa were collected in both the highland and arid zones. We used mist nests to capture adult birds in each habitat. Upon capture, we collected a small blood sample (70 μl) via brachial veinipuncture. Blood was collected using a heparinized hematocrit tube and transferred to a 1.5 ml microcentrifuge tube for storage in a cooler of wet ice. Bleeding stopped within 1 minute of pressure being applied at the puncture sight. Birds were immediately released following processing. Within six hours of collection, each blood sample was spun by hand-crank centrifuge for 5 minutes. Plasma was extracted from each vial and transferred to a separate 1.5 ml microcentrifuge vial for storage. All vials were then placed in a -20°C freezer until the end of the field season. Upon return to the United States, blood samples were stored in a -80°C freezer until further processing. Huber (2010) found that female medium ground finches had greater P. downsi-specific immune responses than males. Therefore, we used plasma from females in our 30 study to maximize the likelihood of detecting P. downsi-specific antibodies. Furthermore, we used pooled samples of plasma from six individual females of each species of Darwin's finch for the assays. Cross-reactivity validation with αHOSP-IgY To validate that house sparrow antiserum (αHOSP-IgY) cross-reacted with plasma from various Darwin's finch species, we performed a sandwich ELISA for total IgY and a test of dilutional parallelism for each species (Plikaytis et al., 1994, Washburn et al., 2007). Pooled plasma from each species was used to make the following serial dilutions in sample buffer (Tris-buffered saline with 0.05% Tween 20): 1:1000, 1:2000, 1:5000, 1:10,000, 1:15,000, 1:20,000, 1:25,000, 1:50,000. All samples were run in triplicate. The following protocol is modified from King et al. (in press). Briefly, 96-well plates were coated with 100 μl/well of Rat-αHOSP-IgY plasma diluted 1:1000 in coating buffer (sodium bi-carbonate, 0.05M, pH 9.6) and incubated overnight at 4°C. Plates were loaded with 200μl/well of blocking buffer (Tris-buffered saline with bovine serum albumin, pH 8.0) and incubated at 37°C for 30 minutes on an orbital table. Between each of the following steps, plates were washed five times with wash buffer (Tris-buffered saline with Tween 20, pH 8.0), loaded as described below and incubated at 37°C on an orbital table for 1 hour. Plates were loaded with 100 μl/well with the eight finch species plasma dilutions (three wells per species per dilution). Plates were then loaded with 100 μl/we