Outdoor immunology: methodological considerations for ecologists

Abstract

Summary 1.Immune defence is an incredibly complicated system, but to understand how it functions in an ecological context is challenging. Our focus is to outline the diversity of ways to measure immune function for ecologists, and to provide some details on the limitations in interpretation of results. 2. There are two broad questions that ecological immunologists have to deal with. The first is what assays are appropriate for the research question of interest? Some researchers assume the biological relevance of an immune assay without investigation or a complete understanding of the immune response. Therefore, the second question is, what parasite challenge does one choose, and does a measurement of immune function reflect the response of the host towards that parasite? There are many assumptions, caveats, and pitfalls facing ecological immunologists, and investigating the relationships between immune assays and whole organismal defence will help us to understand variations in immune responses. 3. We provide an extensive listing of immune function measures, presenting examples from both the vertebrate and invertebrate literature, and wherever possible from non-model organisms. We also outline how these responses are part of an integrated immune defence and encourage thinking about immune function as a hierarchical defence model. We describe how immune responses interact with one another, identify concerns regarding when to measure an immune response, and describe general problems faced when trying to collect a measure of immune function in wild organisms. 4. Extrinsic factors influence immune measurements and the importance of parasites is often overlooked. We give several examples of how parasites interact with organism's immune systems, suggest greater inclusion of parasites into ecological immunology experiments, describe how micro-organisms may interact symbiotically with their host's immune system, and advocate the inclusion of tolerance and resistance in ecological immunological thinking.

Common measures of immune function vary with time of day and sampling protocol in five passerine species

“…More importantly, the observed pattern of differences in immune parameters and individual variation in this change profile are likely to reflect biologically meaningful differences between individuals and species; this suggests that studies of innate immune function may benefit from measuring immune time series, much as 'stress series protocols' have become common in the study of glucocorticoids because of the additional information gained from examining the change in an individual's glucocorticoid levels over time (Wingfield et al, 1995). For example, the magnitude of the change in certain immune parameters may be important to limit colonization by a pathogen, while the speed at which these parameters return to normal, or the relationship between these two measures, could be an important factor in determining the risk of self-harm due to the immune response (Boughton et al, 2011;Haine et al, 2008;Koenen et al, 2002;Korner and Schmid-Hempel, 2004). …”

“…the impact of variation in the immune response profile on an individual's ability to resist colonization or clear infection). In examining the impact of immune response profiles on the risks of self-harm stemming from activation of the immune response, it will be important to consider that self-harm can manifest in a variety of ways (Boughton et al, 2011), including but not limited to auto-reactive damage (Sadd and SivaJothy, 2006), auto-immunity (Day et al, 2007), increased use of important resources (i.e. energy, nutrients or antioxidants) (Klasing, 1998;Lee et al, 2006;Martin et al, 2003), opportunity costs (i.e.…”

“…I tested these hypotheses in five species of passerine, Galápagos fly catchers [Myiarchus magnirostris (GFC), Gould], medium ground finches [Geospiza fortis (MGF), Gould], small ground finches [Geospiza fuliginosa (SGF), Gould], small tree finches [Camarhynchus parvulus (STF), Gould] and yellow warblers [Setophaga petechial (YW), (Linnaeus)]. In these species, I focus on four measures commonly used in ecological immunology studies to evaluate the strength of individual immune defenses (Boughton et al, 2011): natural antibodies, complement, haptoglobin-like PIT54 acute phase protein and lysozyme acute phase protein.…”

“…To this end, scientists have developed a variety of methods to measure variation in immune function in a field setting (Boughton et al, 2011;Matson et al, 2006;Millet et al, 2007). While measuring immune function in an ecological context has yielded many insights, scientists continue to struggle with understanding what particular immune measurements indicate about the overall immune function of an individual (Boughton et al, 2011). One basic obstacle to interpreting field immune data is separating variation in immune function that is related to factors of biological interest from measurement artifacts, such as variation associated with handling stress (Buehler et al, 2008).…”

Captivity induces hyper-inflammation in the house sparrow (Passer domesticus)

“…To determine whether captivity impacted inflammation regulation in house sparrows, we compared responses to LPS in terms of: (1) leukocyte gene expression, (2) corticosterone (the predominant avian GC), and (3) circulating endotoxin among wild birds and birds held for 2 or 4weeks in captivity. By measuring the latter parameter, we hoped to obtain a functional readout of quantitative variation in gene expression (Boughton et al, 2011); quicker clearance of endotoxin L. B. Martin and others should be proportional to cytokine and/or TLR4 expression and presumably favorable because inflammation could subside more rapidly (Graham et al, 2005).…”