Heightened Exposure to Parasites Favors the Evolution of Immunity in Brood Parasitic Cowbirds
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- Hahn, D.C. & Reisen, W.K. Evol Biol (2011) 38: 214. doi:10.1007/s11692-011-9112-0
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Immunologists and evolutionary biologists are interested in how the immune system evolves to fit an ecological niche. We studied the relationship between exposure to parasites and strength of immunity by investigating the response of two species of New World cowbirds (genus Molothrus, Icteridae), obligate brood parasites with contrasting life history strategies, to experimental arboviral infection. The South American shiny cowbird (M. bonariensis) is an extreme host-generalist that lays its eggs in the nests of >225 different avian species. The Central American bronzed cowbird (M. aeneus) is a relative host-specialist that lays its eggs preferentially in the nests of approximately 12 orioles in a single sister genus. West Nile virus provided a strong challenge and delineated immune differences between these species. The extreme host-generalist shiny cowbird, like the North American host-generalist, the brown-headed cowbird, showed significantly lower viremia to three arboviruses than related icterid species that were not brood parasites. The bronzed cowbird showed intermediate viremia. These findings support the interpretation that repeated exposure to a high diversity of parasites favors the evolution of enhanced immunity in brood parasitic cowbirds and makes them useful models for future studies of innate immunity.
KeywordsEcoimmunology Brood parasitism Cowbird West Nile virus Parasite-mediated selection Evolution of immunity
Pathogens which cause illness and mortality in animals influence the evolution of their host’s immune systems (Sheldon and Verhulst 1996; Moller 1997; Norris and Evans 2000). As a result, each species’ immune system reflects the pathogens historically encountered within its niche (Schmid-Hempel and Ebert 2003). Currently there is interest in determining the ecological factors that favor the evolution of increasingly effective immune defenses (Schulenburg et al. 2009). Examination of immunity in species with measurably higher or lower exposure to pathogens provides insight into the selective value of pathogen diversity (Buehler et al. 2008) and the plasticity of immunity as a life history trait (Zuk and Stoehr 2002).
Several investigators have examined immunity in birds whose niches are particularly parasite-rich and found evidence of enhanced immune function, including: (1) scavenging species that feed on carcasses have higher blood leukocyte concentrations than non-scavenging species (Blount et al. 2003); (2) ruddy turnstones (Arenaria interpres), which scavenge among rotting organic material, have higher levels of natural antibodies (IgY) than shorebird species in a different niche (Mendes et al. 2006); and (3) Galapagos finch populations on islands with greater parasite prevalence and/or infection intensity showed higher concentrations of natural antibodies and faster antibody responses than finches from islands with lower parasite exposure (Lindstrom et al. 2004). These studies suggest that species in parasite-riche niches may have enhanced immune systems and justify a direct test of immune defenses using experimental infection.
We propose that obligate brood parasitic birds are useful model organisms for studying the evolution of immunity, because they are exposed to a wide diversity of pathogens from their parenting bird species. “Parasites” is used here in its widest sense to include viruses, bacteria, protists, and eukaryotes (Schulenburg et al. 2009). Brood parasites have unusual exposure to other species’ parasites, because they do not build their own nests, but lay their eggs in the nests of other avian species and appropriate parental care from those species (Payne 1977; Johnsgard 1997; Davies 2000). For example, several investigators have shown that the young of brood parasites are frequently infested with the ectoparasites of the avian species in whose nests they hatch. Brown-headed cowbird fledglings frequently are infested with the louse species (Phthiraptera) of their foster parent species (Hahn et al. 2000; Hahn and Price 2001), as are young diederik cuckoos (Lindholm et al. 1998) and viduine finches (Balakrishnan and Sorenson 2007). Ectoparasites exert significant costs directly and as vectors of pathogens (Bartlett 1993; Aspoeck 2002), and they reduce avian survival and reproductive success (Lehmann 1993; Brown et al. 1995; Clayton and Tompkins 1995; Bize et al. 2004), so exposure to more ectoparasite species is likely an added cost for brood parasites. The more diverse the parenting avian species exploited by a brood parasite, the more diverse the pathogens and parasites to which its young are exposed (Hahn et al. 2000; Hahn and Price 2001), so host-generalists like some of the New World cowbirds are exposed to a particularly diverse array of parasites.
Young brood parasites also have elevated exposure to diverse oral microbes through their foster parent species during feeding and to diverse gut microbes via fecal contact in the nest, and these foreign oral and gut microbes of non-conspecifics may elicit an immune response. Salivary microbes were first suggested to be species-specific in chimney swifts (Chaetura pelagica), when adult saliva was found to be critical for nestling nutrition (Kyle and Kyle 1990), and both crop and gut microbiomes in avian (Godoy-Vitorino et al. 2008) and mammalian species (Ley et al. 2008) recently have been shown to be species-specific through sequencing.
The present study examines two additional species of cowbirds and extends our investigation of the relationship between enhanced immunity and obligate brood parasites. We experimentally challenged the South American shiny cowbird (M. bonariensis), an extreme host-generalist, and the Central American bronzed cowbird (M. aeneus), a relative host-specialist (Fig. 2). The shiny cowbird lays its eggs in the nests of >225 different avian host species and thus has been exposed to the diverse parasites associated with them (Lowther and Post 1999). In contrast, the bronzed cowbird (M. aeneus) has restricted its nest parasitism to approximately twelve oriole species in the sister genus Icterus over most of its evolutionary history (Friedmann 1929; Ellison and Lowther 2009), and thus has been exposed to a lower diversity of parasites and hence less selection pressure on its immune system. In the twentieth century the bronzed cowbird began to expand its host range and utilize more host species, but still prefers orioles (Ellison and Lowther 2009).
Our experiment followed the protocols of Reisen and Hahn (2007) to facilitate comparison of the current findings for the shiny and bronzed cowbirds with the previous results for brown-headed cowbird and the three non-parasitic blackbirds. We used the invading West Nile virus as the challenge pathogen, because it provided the strongest natural probe of host immune defenses. We also infected the shiny cowbird with the endemic Western equine encephalomyelitis (WEEV) and St. Louis encephalitis (SLEV) viruses.
We predicted that (1) shiny cowbirds would have robust immunity to these viruses similar to that of the brown-headed cowbird, reflecting their similar habits as extreme host-generalist brood parasites with a similar broad level of parasite exposure; (2) both shiny and bronzed cowbirds would exhibit stronger immunity than related, non-parasitic icterid species; and (3) the extreme host-generalist (shiny cowbird) would show a stronger immune response than the host-specialist brood parasite (bronzed cowbird) as a result of the former’s exposure to more diverse avian host species and associated parasites.
Materials and Methods
The shiny cowbird is a South American species with a range extending from Argentina north to Venezuela and the Caribbean (Fig. 2) (Jaramillo and Burke 1999). It is an extreme host-generalist brood parasite that is most closely related to the North American brown-headed cowbird (Friedmann 1929; Lowther 1993). Twenty shiny cowbirds were obtained in April 2007 from the Puerto Rican Department of Natural Resources, Mayaguez, P.R. which manages a cowbird control program to protect the nests of the threatened yellow-shouldered blackbird (Agelaius xanthomus).
The bronzed cowbird is a Central American species with a range extending into northwestern South America (Friedmann 1929) (Fig. 2). Bronzed cowbird began invading southern Texas and Arizona in the twentieth century, expanding its range and avian host species (Friedmann 1929; Kostecke et al. 2004). Five bronzed cowbirds were obtained in April 2007 from the Fort Hood cowbird control project which protects the nests of black-capped vireos (Vireo atricapillus).
Both cowbird species were transported by air to Los Angeles and taken to the University of California Arbovirus Field Station in Bakersfield, CA where they were prebled to establish that they had not been infected previously with the viruses to be tested. They were then banded and released into a mosquito-proof outdoor aviary to observe general health. They were fed a mixture of mixed wild bird seed and adjusted well to captivity.
We compared results from the current study to our previous data for after-hatching-year Brewer’s blackbirds, red-winged blackbirds and brown-headed cowbirds that were collected from mixed winter foraging flocks at several dairies near Bakersfield in Kern County, CA and experimentally infected during 2003–2005 (Reisen and Hahn 2007). Additional adult brown-headed cowbirds were obtained from grain-baited traps in the Coachella Valley, Riverside County, CA during 2004, and hatching year tricolored blackbirds were collected by mist netting at a nesting colony along the Kern River, Kern County, CA, during 2005 (Reisen and Hahn 2007).
All birds were trapped and transported under appropriate state and federal permits, and the infection of birds was done under Protocol 11184, approved by the Institutional Animal Care and Use Committee of the University of California, Davis. The use of arboviruses under BSL3 containment was approved under Biological Use Authorization #0554 by Environmental Health and Safety of the University of California, Davis, and USDA Permit #47901.
We used the following low passage virus strains that previously were used in our host competence studies (Reisen et al. 2003, 2005; Reisen and Hahn 2007). Multiple inocula were made so the same stock and passage viruses were used in all experiments.
WNV: NY99 strain isolated from a Flamingo that died in the Bronx Zoo (strain 35211 AAF 9/23/99) and passaged twice in Vero cells.
SLEV: Virus is grouped with WNV within the Japanese encephalitis virus serocomplex. We used the Kern217 strain isolated in Bakersfield, Kern County, California, during the outbreak of 1989 (Reisen et al. 1992).
WEEV: The Kern5547 strain of WEEV was isolated from mosquitoes collected in Bakersfield during 1983.
Three groups of shiny cowbirds (n = 6) each were infected with one of the above three viruses; bronzed cowbirds were infected only with WNV. Birds were inoculated subcutaneously in the cervical region with 100 μl of diluent containing 2.3–2.7 log10 plaque forming units (PFU) of virus. This dose is comparable to the amount of virus expectorated by infectious Culex tarsalis mosquitoes (Reisen et al. 2005) and was found to produce a viremia response in house finches similar to infection by mosquito bite (Reisen et al. 2000).
Infection was determined by the detection of a viremia or antibody response. Viremia was monitored on days 1–7 post infection (dpi) by jugular puncture using a 28 g syringe. The 100 μl blood sample was expelled immediately into 400 μl of virus diluent (phosphate buffered saline containing 20% fetal calf serum and antibiotics). Samples were clarified by centrifugation and then stored at −80°C until tested. Antibody levels were measured by collecting blood from birds weekly for 6 weeks post infection by jugular puncture (100 μl blood expelled immediately into 900 μl of saline). Surviving birds were necropsied at 6 weeks post infection and blood, lung, spleen and kidney tissues tested for virus.
Mortality was recorded daily. Viremia was measured by standard plaque assay using Vero cell culture (Kramer et al. 2002) with a minimal threshold of detection of 2 plaque forming units (PFU) per 100 μl or 1.7 log10 PFU/ml. Antibody in surviving birds was measured (1) weekly using an enzyme immunoassay (EIA) (Chiles and Reisen 1998) and reported as the ratio of the mean optical density of two positive wells over an antigen negative well (P/N ratio) for each bird, and (2) on weeks 4 and 6 post infection a plaque reduction neutralization test (PRNT), with positives neutralizing >80% of 75–100 PFU of virus at a dilution of ≥1:20. Necropsy was conducted on all birds that survived 6 weeks to assess and blood, lung, spleen and kidney tissues were tested for presence of viral RNA using RT-PCR.
Viremia expressed as log10 PFU per ml of blood was compared for each virus among avian species on 1–6 days post infection (dpi) using a repeated measures analysis of variance (ANOVA) (Hintze 1988). These analyses included viremia data from bird species used in our previous study (Reisen and Hahn 2007). Because there was significant interaction among mean WNV viremias for each species and time in dpi, peak means also were compared using a one-way ANOVA. In both analyses, avian species were grouped by Tukey-Kramer test to examine all pairs of means using the experimentwise error rate and the Studentized range distribution (Hintze 1988). Mortality was compared using Fisher’s Exact Probability test, and rate of recovery was assessed as time to ‘event’ data using the SAS procedure Proc Lifetest, which yields a Chi Square test statistic (SAS Institute 1999).
Shiny cowbirds experienced no mortality after infection with any of the viruses, and the control bird, which was bled and maintained concurrently, also survived. In contrast, 2 of 4 bronzed cowbirds died on days 5 and 6 post-infection after WNV. Shiny cowbird mortality was similar to that of brown-headed cowbird, which had experienced no mortality (Reisen and Hahn 2007). Together shiny and brown-headed cowbird showed lower mortality than bronzed cowbird (Fisher’s Exact Test, P = 0.035). Among the non-parasitic icterids one Brewer’s blackbird died (1/6), whereas red-winged blackbird and tricolored blackbird experienced no mortality (Reisen and Hahn 2007). Overall, cowbird mortality (2/19) was not different from that of the non-parasitic blackbirds (1/17) (Fisher’s Exact Test, P = 0.41).
Intensity of Infection and Time to Recovery
Comparison of immune responses of parasitic cowbirds and non-parasitic blackbirds (Icteridae) to West Nile, Western equine encephalomyelitis, and St. Louis encephalitis viruses
West Nile virus
Western equine encephalomyelitis virus
St. Louis encephalitis virus
Mean peak viremia ± SE (n) (2–4 dpi)1
Time to2 clearance (days)
Mean peak viremia ± SE (n) (1–2 dpi)1
Time to2 clearance (days)
Mean peak viremia ± SE(n) (2–4 dpi)1
Time to2 clearance (days)
3.9 ± 0.0.57b (6)
1.8 ± 0.42 (6)
1.7 ± 0.1 (6)
4.1 ± 0.47b (9)
5.2 ± 0.81ab (4)
6.5 ± 0.57a (6)
3.9 ± 0.42 (5)
4.5 ± 1.1 (5)
7.4 ± 0.57a (6)
5.6 ± 0.42 (5)
2.5 ± 0.8 (5)
7.0 ± 0.63a (5)
West Nile Virus
Shiny and bronzed cowbirds showed different viremia profiles after infection (Fig. 4). When tested by a repeated measures ANOVA, virus titers varied significantly among these 6 icterid species (F = 13.77, df = 5,30, P < 0.001) and days post infection (F = 34.6, df = 5,145, P < 0.001), and the species × days interaction term also was highly significant (F = 5.7, df = 25, 145, P < 0.001) indicating that the shape of the viremia profiles varied among the six species. Overall mean viremias for shiny and brown-headed cowbirds were significantly lower than those of the blackbird species that are not brood parasites; bronzed cowbirds were intermediate and not different from the other cowbirds or red-winged and tricolored blackbirds (P > 0.05, Tukey-Kramer test).
The day of peak viremia occurred during 1–4 dpi and varied markedly among species (Fig. 4). Shiny cowbirds’ peak viremia was on Day 3 post-infection and averaged 3.9 log10 PFU/ml, while the bronzed cowbirds’ peak viremia was on Day 4 post-infection and averaged 5.2 log10 PFU/ml, which is greater than an order of magnitude more than that of shiny cowbirds’ average. It is noteworthy that all bronzed cowbirds experienced peak viremias >4.9 log10 PFU/ml and that the two bronzed cowbirds that died did so after peak viremias of 5.3 and 6.8 log10 PFU/ml, respectively. Mean peak viremia for the six species (Fig. 4) differed significantly (F = 7.4, df = 5, 29, P < 0.001) when compared by one-way ANOVA. Grouping of means by Tukey-Kramer test agreed well with the overall comparisons described above (Table 1).
For shiny cowbirds, the mean time to recovery when viremia was eliminated, i.e. first day when plaque assay was negative (i.e., <1.7 log10 PFU/ml), was day 4.8, while the time to recovery in bronzed cowbirds was day 6 (Table 1). Shiny cowbirds and brown-headed cowbirds had similar rates of recovery (Chi square = 2.02, df = 1, P = 0.16) (Table 1), and both had faster recovery than the bronzed cowbird (Chi square = 5.79; df = 1; P = 0.02). The two host-generalist cowbirds also had faster rates of recovery than the Brewer’s blackbird, the non-parasitic species to which genus Molothrus is most closely related (Fig. 1b) (Chi square = 5.95; df = 1; P = 0.02). However, their rate of recovery was not different from that of red-winged cowbird or tricolored blackbird (Table 1).
Antibody titers measured the humoral immune response following infection.
WEEV: All shiny cowbirds failed to produce a detectable EIA response, but two had PRNT titers of 1:40 on week 4. The low antibody response is consistent with low viremia levels to WEEV.
SLEV: Shiny cowbirds produced a relatively strong, but ephemeral antibody response (Fig. 5), and by week 6 only 1 bird retained neutralizing antibody (titer = 1:40). The rapid antibody production contrasted with the low viremia levels to SLEV.
A final measure of the effectiveness of the immune system is total clearance of infectious virus (Janeway et al. 2001). The blood, lung, spleen and kidney tissues of all surviving shiny and bronzed birds were tested at 6 weeks post-infection for viral RNA using RT-PCR, and all were negative. As a positive control, we did detect viral RNA and isolated infectious virus from multiple tissues from a female bronzed cowbird that had died on day 4 post infection.
Viremia was the principal response that showed clear differences between the parasitic cowbirds and related non-parasitic blackbird species, and WNV provided the strongest challenge to the immune defenses. Our results support the hypothesis that exposure to an increased diversity of parasites favors the evolution of more effective immune defenses, because the shiny and brown-headed cowbirds showed significantly lower viremia responses than the non-parasitic blackbirds (Table 1). However, the intermediate viremia response of the bronzed cowbird suggests that there is a threshold exposure to parasites required to effect measurable changes in immunity. We suggest that the threshold for effective exposure can be explored further by examining the immune responses of an extreme host-specialist, the screaming cowbird (M. rufoaxillaris), the ancestral South American species that lays its eggs primarily in the nest of one host species, the bay-winged cowbird (Lanyon 1992; Davies 2000) (Fig. 1a). The limited exposure to parasites of this extreme host-specialist offers a greater contrast to the host-generalist cowbirds than does the intermediate exposure of the bronzed cowbird.
In the present study, the mortality data did not yield as clear a pattern of differences between cowbirds and non-parasitic blackbirds as did the viremia data. The host-generalist cowbirds showed significantly lower mortality than the relative host-specialist, bronzed cowbird, but mortality was not different between cowbirds and the non-parasitic blackbirds. However, viremia likely is a more meaningful immunity measure than mortality, because mortality is underestimated in laboratory studies like this one when individuals with high viremia recover more often than they would under natural conditions. Similarly, rate of recovery data did not show differences among species as great as did the viremia data. Shiny and brown-headed cowbirds had a similar rate of recovery, but faster than only Brewer’s blackbird. We suggest that cellular-level assays such as those described by Millet et al. (2007) may be required to distinguish differences in cowbird immunity, because these assays measure immune mechanisms directly, whereas viremia is an indirect indicator of the effectiveness of innate and cross-reacting acquired immune defenses. Our preliminary findings using such assays support the findings reported here and demonstrated significant differences between the brown-headed and bronzed cowbird and between both cowbirds and the non-parasitic red-winged blackbird (Hahn et al. 2009).
It is increasingly recognized that the immune system is complex (Adamo 2004) and each species has a ‘portfolio’ of immune defenses adapted to its niche (Schmid-Hempel 2003). Our results suggest that it is likely that the innate immune defenses were primarily responsible for the significantly lower viremia of the host-generalist cowbird species, because innate immunity determines the host’s response to pathogens at exposure and in the earliest stages of infection (Janeway et al. 2001), and the viruses tested here were eliminated in less than a week (Fig. 4). It appears likely that antibodies did not play a principal role in controlling these viremias, because the antibody response, part of the adaptive immune system, takes longer to activate and was low titered and variable (Fig. 6). Cowbirds are ‘fast pace-of-life’ species, which mature rapidly and reproduce prolifically, and such species typically favor ‘inexpensive’ and nonspecific immune defenses (Martin et al. 2007). Furthermore, innate immunity is also likely to be particularly important in cowbirds, because young cowbirds have elevated exposure to parasites at hatching due to their physical proximity to foster parents. Because the immune system in altricial nestlings is not yet mature and innate immune defenses predominate (Apanius 1998; Klasing and Leshchinsky 1998), innate immunity likely experiences significant selection in young cowbirds.
Comparison of diversity of louse species infestations on brown-headed cowbirds and on the songbird species that serve as their foster parents
Number of species
Number of birds
Louse species/avian species or population
1–2 louse species per population of each bird species
1–2 louse species per individual bird
1–2 louse species per individual bird
11 louse species per fledgling population
1–2 louse species individual bird
6 louse species per adult population
Wild species serving as model animal systems are of increasing interest in biomedical research, and the New World cowbirds offer an interesting system in which to explore how evolution shapes the design and function of immune responses. Particularly in this era of predicted global climate change, it is of practical value to understand the ecological conditions that favor the evolution of enhanced immune systems and to identify which species would be most resilient if disruptions in ecosystem relationships cause changes in disease prevalence and transmission (Harvell et al. 2002; Schwartz et al. 2006).
We thank Katsi Ramos Alvarez and Marilyn Colon, Division Recursos Terrestre, Puerto Rico for making shiny cowbirds available and arranging transport, and Scott Summers, The Nature Conservancy of Texas, for making bronzed cowbirds available and arranging transport. This research was funded, in part, by Research Grants RO1-39483, RO1-AI47855, and AI55607 from the National Institutes of Allergy and Infectious Diseases, NIH, the Coachella Valley and Kern Mosquito and Vector Control Districts, special funds for the Mosquito Research Program allocated annually through the Division of Agriculture and Natural Resources, University of California, and by base funds from USGS-Patuxent Wildlife Research Center. We thank staff of the Center for Vectorborne Diseases for excellent technical support: V.M. Martinez, H.D. Lothrop, S.S. Wheeler and B.D. Carroll assisted with bird collections; V.M. Martinez assisted with bird maintenance and bleeding; and Y. Fang, M. Shafii, S. Garcia, R.E. Chiles and S. Ashtari assisted with laboratory diagnostics. We thank R.B. Payne, S.M. Lanyon, D. Mock, and two anonymous reviewers for helpful comments on the manuscript. Use of trade, product, or firm names does not imply endorsement by the U.S. Government.