Ontogeny of innate and adaptive immune defense components in free-living tree swallows, Tachycineta bicolor

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Abstract

Little is known about the development of immune function in wild animals. We investigated the ontogeny of immune defense in a free-living bird, the tree swallow. We assessed total and differential leukocyte counts, natural antibodies, complement activity, in vivo skin swelling response, and in vitro lymphocyte proliferation and compared the levels of development between nestlings and young adults. We also assessed whether body condition explained variation in these immune components. We found some support for the prediction that innate defenses, which do not need to generate a broad repertoire of specific receptors, would reach adult levels earlier than adaptive defenses. In contrast, we found limited support for the prediction that adaptive defenses, which are thought to be more costly to develop, would be more related to body condition than innate defenses. We discuss our findings in the context of other studies on the ontogeny of immune function.

Introduction

Ecoimmunology is a rapidly growing field of evolutionary ecology that studies immune defense strategies and their relationship to the life history and ecology of organisms [1], [2], [3], [4]. A major aim of ecoimmunology is to understand the causes and elucidate the consequences underlying the broad variation in immune defense strategies observed within and among species. One key insight from such studies has been that immune function is costly in terms of energy and/or limiting nutrients (e.g., protein, carotenoids) and therefore traded-off with other costly functions such as growth and reproduction. An important aspect of ecoimmunology that has been relatively understudied is the age-specific variation in immune function [3].

Studies in humans, laboratory animals, and domestic animals indicate that the efficiency of immune defense mechanisms is compromised at both extremes of the lifespan due to development during early life [5] and deterioration or dysregulation in old individuals [6]. This leads to an increased vulnerability to pathogens among very young and very old individuals. One cause for the paucity of studies exploring the relationships between age and immune function in wild animals is the relative difficulty of correctly assigning ages to individuals. However, even for organisms important in the origin and advancement of ecoimmunology, and for which ages can be relatively easily assigned at least for part of the lifespan (e.g., nestling birds), information on age-related changes in immune function is still sparse.

Changes in circulating immunoglobulin (Ig) levels during the nestling period have been documented in a few wild bird species [7], [8], [9], showing the initial presence of maternal antibodies and the later increase of endogenous Ig levels during the first weeks of life. The skin swelling response to phytohemagglutinin (PHA) has been compared between nestlings and adults in several bird species, usually as part of studies for which the primary focus was not ontogeny. These studies show mixed results [10], with some reporting greater swellings in adults [10], [11] and some in nestlings [12], [13]. Therefore, although studies in free-living animals to date suggest that patterns of immune development may vary among species with different ecologies and life histories, they also highlight the paucity of knowledge on developmental immunology in the wild. In particular, most studies to date have measured immune function of immature animals only at a certain age (e.g., day 12 of the nestling period) instead of the ontogenetic development of this function, and have assessed only one immune component at a time, precluding the assessment of relative rates of development of different immune defense components.

Studies in humans and laboratory or domestic animals indicate that neonates and juveniles depend strongly on innate immune defenses and maternally derived immunoglobulins. Adaptive defenses, on the other hand, are poorly developed in neonates and juveniles and take more time to become fully functional [14], [15], [16]. Differential rates of development of these main arms of the immune system is likely explained by the different costs and processes involved in the ontogeny of each component [15], [16]. For example, the nutritional costs and time needed for the development of lymphocytes that mediate adaptive immunity are greater than those required by cells involved in innate immunity (e.g., granulocytes, monocytes), mainly because of the need to generate the immense diversity of lymphocytes with specific receptors that will recognize a broad range of potential threats (non-self) without reacting against self [15], [16].

In birds, most studies on developmental immunology have used chickens and other domesticated species with precocial development [14], [15]. Many of these species have been selected for increased production (e.g., broilers, layers), with consequent changes in immunological development [17], [18]. For example, broilers selected for fast body growth suffer from reduced immune defense and lifespan, while layers selected for a long productive life show increased immune defenses [18]. Therefore, studies on free-living birds in general, subjected to natural rather than artificial selection, and on species with altricial development in particular, are valuable additions to comparative avian immunology and ecoimmunology.

The aim of this study was to investigate the ontogeny of immune defense in a free-living, altricial bird, the tree swallow Tachycineta bicolor, by describing changes in several immune parameters during the nestling period and comparing the levels of immune development between nestlings and adults. Tree swallows are a common passerine bird species distributed throughout North America [20] that readily breed in nest-boxes. Females commonly lay clutches of 5–6 eggs, which hatch after approximately 15 days of incubation [20]. Most females start incubating before laying the ultimate or penultimate egg, resulting in asynchronous hatching [21]. As for all passerine species, hatchlings are altricial [22] and both parents feed the chicks for about 20 days (nestling period), after which young leave the nest (fledge) [20].

We measured components of both innate and adaptive immunity including some that are constitutive and some that are induced (Table 1). We followed the classification by Lee [4], except for natural antibodies, which we classified as innate defenses [19]. We predicted that innate defenses would reach adult levels earlier than adaptive defenses, given that innate defenses need not generate the vast repertoire of receptors required among lymphocytes. In addition, we assessed whether development of the various immune components was associated with body condition. We predicted that adaptive immune parameters, which are thought to be more costly to develop, would be more related to body condition than less costly innate parameters.

Section snippets

Field protocols and sample collection

We conducted this study in tree swallows breeding in nest-boxes near Ames, IA (42°1′N, 93°37′W). Nest-boxes were located near prairie and wetland areas, or near cropland and horticultural plantations. During the 2005 and 2006 breeding seasons we monitored nests closely to determine clutch completion date (i.e., date last egg is laid), exact hatch date (i.e., date first nestling emerges from egg = day 0), and therefore, exact nestling age. The small size of tree swallow nestlings precludes

Constitutive innate immunity

Total numbers of leukocytes per milliliter of blood were measured in broods 10–14 days old and one-year-old females. Neither the number of granulocytes (heterophils, eosinophils, and basophils) nor monocytes was correlated with body condition in nestlings or adults (all p > 0.16) and they did not differ significantly between these age groups (MANOVA including all leukocyte types, all p > 0.51, Table 2). Differential leukocyte counts were estimated for nestlings ranging from 5 to 19 days old and

Development of innate and adaptive immunity

We found mixed support for our prediction that innate immune components would develop earlier than adaptive components in nestling tree swallows. Total leukocyte counts and in vitro lymphocyte proliferation in response to PHA support the prediction, while the two measures of constitutive innate humoral immunity (natural antibodies and complement activity) and lymphocyte proliferation in response to ConA do not.

Of the different leukocyte types, only total lymphocyte counts were lower in

Acknowledgements

The authors thank the members of the Vleck lab, Frederic Angelier, and two anonymous reviewers for valuable comments on previous versions of this manuscript. Funding for this research came from grants to M.G.P. provided by the American Ornithologist’s Union, the Society for Integrative and Comparative Biology, Sigma Xi, and Sigma Delta Epsilon Graduate Women in Science.

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