Integrative and Comparative , volume 51, number 4, pp. 563–576 doi:10.1093/icb/icr011

SYMPOSIUM

Pathogen Pressure Puts Immune Defense into Perspective Nicholas P. C. Horrocks,1 Kevin D. Matson and B. Irene Tieleman Animal Group, Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 11103, 9700 CC, Groningen, The Netherlands From the symposium ‘‘Bridging the Gap Between Ecoimmunology and Disease Ecology’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2011, at Salt Lake City, Utah. 1E-mail: [email protected] Downloaded from

Synopsis The extent to which organisms can protect themselves from disease depends on both the immune defenses they maintain and the pathogens they face. At the same time, immune systems are shaped by the they encounter, both over ecological and evolutionary time. Ecological immunologists often recognize these interactions, yet ecological currently lacks major efforts to characterize the environmental, host-independent, antigenic pressures to which all animals are exposed. Failure to quantify relevant diseases and pathogens in studies of ecological immunology http://icb.oxfordjournals.org/ leads to contradictory hypotheses. In contrast, including measures of environmental and host-derived commensals, pathogens, and other immune-relevant organisms will strengthen the field of ecological immunology. In this article, we examine how pathogens and other organisms shape immune defenses and highlight why such information is essential for a better understanding of the causes of variation in immune defenses. We introduce the concept of ‘‘operative protection’’ for understanding the role of immunologically relevant organisms in shaping immune defense profiles, and demonstrate how the evolutionary implications of immune function are best understood in the context of the pressures that diseases and pathogens bring to bear on their hosts. We illustrate common mistakes in characterizing by guest on December 9, 2011 these immune-selective pressures, and provide suggestions for the use of molecular and other methods for measuring immune-relevant organisms.

Integrating immunology and ecology Many factors influence, and can generate variation The bridges the divide between in- in, immune responses: these include sex, nutritional ternal and external environments, integrating an or- status, social dominance, exercise, and seasonality, as ganism’s and environment. In doing so, well as trade-offs in resource allocation between the the immune system acts as a barrier to infection and immune system and other physiological systems such disease, identifying threats and coordinating neces- as reproduction (Sadd and Schmid-Hempel 2009; sary responses. Despite its complexity, immunolo- Schulenburg et al. 2009). Over both ecological and gists have elucidated many of the cellular processes evolutionary timescales, however, the most enduring and specific mechanisms that allow the immune selective pressures on the immune system are the system to function. Yet our knowledge of how evo- myriad challenges posed by everything that lutionary pressures shape immune systems is still immune systems encounter. Particularly important incomplete. in terms of are interactions between the Ecological immunology promotes the use of im- immune system and organisms with the ability to munological measures to test ecological and evolu- live in, or on, a host and the potential to evolve in tionary hypotheses. The field arose from a desire to response to current immune defenses. We refer to explain the variation in immune function that is ob- the specific suite of components that generate these served within and among individuals, populations, evolutionary and ecological selective forces on the and species, across environments and over time. immune system as the ‘‘immunobiome’’ and their

Advanced Access publication June 20, 2011 ß The Author 2011. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: [email protected]. 564 N. P.C. Horrocks et al.

Fig. 1 A representation of the antigenic universe, which consists of all the possible antigens that any immune system could ever encounter. This universe includes antigenic, immunogenic, inflammatory, and toxic agents. Within the antigenic universe is the immu- nobiome. The immunobiome contains all the living organisms that can live in or on a host and with the potential to evolve in response to immune defenses. The immunobiome does not include other immuno-reactive particles such as dust that cannot multiply. Two major

components of the immunobiome, in terms of the evolution of the immune system, are commensals and pathogens. Since some Downloaded from commensals may be pathogenic under suitable conditions, these groups are not mutually exclusive. Immunobiome components that fall outside these two categories (dotted area) include environmental microbes such as ‘‘pseudo-commensals’’ (Rook 2009), which although regularly encountered by hosts, do not gain any benefit from their temporary association with a host, yet may still shape regulatory circuits of the immune system. Scaling of the different subsets is arbitrary. http://icb.oxfordjournals.org/ ability to shape immune defenses as ‘‘immunobiotic immunology. Rather than being relegated to anony- pressure’’ (Fig. 1). Understanding the interactions of mous, yet highly relevant sources of variation, the the immune system with immunobiomes is essential diverse constituents of the immunobiome and the for helping to explain patterns of immunological evolutionary pressures they exert must be seen as variation. central to ecoimmunological studies (Bordes and In light of immunological costs, animals should Morand 2009; Sadd and Schmid-Hempel 2009; match their immune defenses (any anatomical, Graham et al. 2011; Pedersen and Babayan 2011;). chemical, physiological, or behavioral barrier main- by guest on December 9, 2011 tained by an animal that inhibits or controls the es- Interactions with entire immunobiomes tablishment and reproduction of any element of the shape immune defenses, but pathogens immunobiome within or on the animal) to the threats that they face (Sheldon and Verhulst 1996; and commensals are particularly Tschirren and Richner 2006). However, the nature important of immunobiomes is poorly understood. For exam- Animals live in diverse and variable environments ple, does their basic composition differ among envi- and their immune systems must interact with and ronments? Which components most strongly shape respond to equally diverse and variable immuno- immune defenses in which hosts? Understanding biomes. However, across immunobiomes, two cate- these issues will be central to solving broader chal- gories of organisms that lie at all points along a lenges in immunology such as the consequences of continuum from benign, or even beneficial, to harm- emergent infectious diseases (Jones et al. 2008) or the ful, are expected to be particularly important sources consequences for health of altering commensal mi- of immunobiotic pressure (Fig. 1). At the detrimen- crobial communities (Blaser and Falkow 2009). We tal end of this continuum are pathogens. These propose that to advance ecological immunology, microparasites (viruses, bacteria, fungi, and protists) measures of the immunobiome that are independent and macroparasites (e.g., helminths, ticks, and lice) of immune indices should be developed and incor- can potentially harm host tissues through their in- porated into future studies. Much as data on avail- herent ability to breach immune defenses that nor- ability of food are required for an understanding of mally restrict other organisms. Pathogens seek to diet selection (e.g., Belovsky 1981) and environmen- circumvent host immune defenses and may disrupt tal temperature profiles are required for explaining normal immune processes (Tortorella et al. 2000; heat balance (e.g., Tieleman and Williams 2002), Finlay and McFadden 2006). This might include im- knowledge of immunobiomes and immune stimuli munosuppression (Babu et al. 2006; Jackson et al. is essential when testing hypotheses in ecological 2009) or shifting of the immune system toward a Pathogen pressure and immune defense 565 specific mix of defenses (Maizels and Yazdanbakhsh protection from infection and enhancement of fitness 2003). to an animal in the wild? We advocate that levels of Of equal importance to the evolution of the immune defense be considered relative to the immu- immune system, commensals normally sit at the nobiotic pressures that are encountered by an organ- benign end of the pathogenicity spectrum. ism, focusing on effectiveness of protection rather Benefitting from intimate associations with their than upon magnitude of response (see also e.g., host, commensals can also modulate immune re- Viney et al. 2005; Graham et al. 2011; Pedersen sponses and play an essential role in development and Babayan 2011). We refer to this of the immune system (Rakoff-Nahoum et al. 2004; immunobiome-specific assessment of immune de- Mazmanian et al. 2005; Rook 2009; Round and fense as ‘‘operative protection.’’ Operative protection Mazmanian 2009). Different commensal communi- encompasses the fitness-enhancing protection against ties might offer distinct advantages or disadvantages immunobiotic pressure (and immunopathology) af- in terms of immunomodulation (Jackson et al. 2009) forded by the immune system, relative to the immu- and colonization by pathogens (Stecher and Hardt nobiotic pressure under which an organism is placed 2008; Stecher et al. 2010). Under some circum- (Fig. 2). Put more simply, operative protection de- stances, organisms that normally behave commen- scribes the goodness of fit between the immunobiotic sally can become pathogenic (sensu ‘‘amphibiosis’’) pressure in a given environment and the immune Downloaded from (Rosebury 1962). Commensals that escape immune defenses of an animal in that environment. A mis- controls by inappropriately breaching defensive bar- match between immunobiotic pressure and immune riers (e.g., the intestinal epithelium) may also defenses could result in three outcomes. become de facto pathogens (Blaser and Falkow Inappropriately low operative protection (i.e., 2009). Overall, interactions with the entire immuno- immune defenses inadequate to match immunobiotic http://icb.oxfordjournals.org/ biome, encompassing organisms associated with the pressure) could lead to increased infection and dis- full range of the pathogenicity spectrum, shape both ease. Inappropriately high operative protection (i.e., immune defenses and immunobiotic components immune defenses exceeding the immunobiotic pres- themselves, and can have implications for health sure of the current environment) could lead to im- and survival (Round and Mazmanian 2009). munopathology and unnecessary expenditure of Hosts might encounter immunobiomes that vary energetic and nutritional resources on the immune temporally (e.g., at different times of the day, or system. This in turn might affect other physiological by guest on December 9, 2011 across seasons) or spatially (e.g., small scale differ- systems (e.g., reproduction) that must compete with ences in use of habitat, or large-scale differences the immune system for allocation of resources. In in biogeography). At ecological scales, immunobiotic both instances a reduction in fitness is expected. In pressures mould individual responses and physiolog- some specific instances, inappropriately high opera- ical condition. Immune function interacts with tive protection might indirectly correlate with in- and is influenced by each (pathogenic, com- creased fitness. For example, if an invasive species mensal, or otherwise) encountered over the life- leaves behind the immunobiome with which it time of an individual, from in utero or in ovo co-evolved (Torchin et al. 2003), then that species (Boulinier and Staszewski 2008; Grindstaff 2008) to might encounter a less-threatening immunobiome adulthood. Nonetheless, these relationships are ulti- that requires reduced investment in immune de- mately governed by an immune system that has fenses. The positive effects on fitness of this enemy been shaped through evolution. At evolutionary release might partially outweigh any negative fitness scales, differences in composition and function of consequences associated with superfluous immuno- immunobiomes and levels of exposure to them are logical investment. On balance, this excess invest- expected to lead to genetically-based changes in ment still has the potential to reduce fitness (i.e., the organization of the immune system, with immu- the gross fitness benefits of enemy release might be nobiotic pressures shaping immuno-defensive even greater than the net, realized fitness benefits). architecture. Therefore any mismatch in operative protection may be transient on an evolutionary timescale. In general Operative protection—balancing however, immune responses that are evolutionarily advantageous in one immunobiome may not be ad- immune defenses and immunobiotic vantageous in a different immunobiome. Operative pressure protection might change, for example, over an How can ecological immunologists best understand annual cycle or among environments, if immune what immune indices represent in terms of defense or immunobiotic pressures also vary. 566 N. P.C. Horrocks et al. Downloaded from http://icb.oxfordjournals.org/ Fig. 2 Operative protection requires the consideration of immune defenses in terms of immunobiotic pressures. Immune systems and immunobiomes are complex, and these multivariate systems interact to shape immunobiotic load, fitness and life-history. Immunobiotic load (or pathogen load in studies of single pathogens or parasites) relates to intensity of infection after the deployment of immune defenses. The broadly integrative and multidimensional nature of the ‘‘immunobiome operative protection approach’’ (left side of figure) offers several advantages over a ‘‘single pathogen approach’’ (right side of figure). Specific components of the immunobiome may not be universal, making operative protection particularly relevant in comparative studies. Furthermore, simultaneous measurement of multiple components of the immunobiome allows for the fact that the effects of immunobiotic load (e.g., co-infections) on fitness may be interactive rather than simply additive. The costs involved in shaping fitness or life-history trade-offs are also clearer when considering immunobiotic load than when considering the load of a single putative parasite or pathogen, which may impose negligible or even no by guest on December 9, 2011 fitness costs.

Identifying and understanding such differences in pathogen pressure (e.g., abundance, diversity) to eco- operative protection would be an important ad- logical or environmental variation (Table 1). These vancement over simply identifying variation in hypotheses have generally been tested using measures immune indices. The magnitude of an immune re- of either host-associated pathogens (i.e., pathogen sponse need not relate directly to fitness, while load) or immune defense (Table 1), but neither ap- knowledge of changes in operative protection identi- proach provides a complete and independent picture, fies when and where individuals, populations, or spe- or direct measures, of environmental pathogen pres- cies are most at risk from disease and infection. sure (Fig. 3).

Using pathogen load to test hypotheses about Current hypotheses about pathogen pathogen pressure pressure, a component of immunobiotic pressure, require additional data and In one approach, pathogen load is used as an indi- cator of exposure to pathogens and hence as a proxy more testing for pathogen pressure. Pathogen load is measured as Understanding operative protection requires insight the prevalence or intensity of infection by a single both into immune defenses and immunobiotic pres- pathogen, or as parameters of host-associated path- sure (Fig. 2). In terms of pressure, researchers have ogen guilds such as species richness (Table 1). A generally focused their thinking on only pathogens greater pathogen load is taken to indicate a stronger and parasites, and not on other components of an pathogen pressure. Yet pathogen load provides infor- immunobiome such as commensals. We refer to this mation about intensity of infection only after subset of immunobiotic pressure as ‘‘pathogen pres- immune defenses and other behavioral counterstra- sure.’’ Several hypotheses link differences in tegies have been deployed (Moyer et al. 2002). Pathogen pressure and immune defense 567

Table 1 Examples of studies testing predictions about pathogen pressure.

Variable examined References Studies that use pathogen load only to test hypotheses about pathogen pressure Cooperative breeding Poiani (1992) Sociality and group size Snaith et al. (2008) Migration Figuerola and Green (2000) Saline versus freshwater environments Figuerola (1999); Mendes et al. (2005); Piersma (1997) Latitude Rohde and Heap (1998); Guernier et al. (2004); Nunn et al. (2005); Salkeld et al. (2008) Studies that use immune measures only to test hypotheses about pathogen pressure Diet Blount et al. (2003) Sexual promiscuity Nunn (2002); Nunn et al. (2003) Cooperative breeding Spottiswoode (2008) Population size, group size or sociality Nunn (2002); Semple et al. (2002); Nunn et al. (2003); Wilson et al. (2003) ; Stow et al. (2007) Migration Møller and Erritzoe (1998)

Life-history strategy Nunn (2002) Downloaded from Substrate use Nunn (2002); Nunn et al. (2003) Risk of injury Semple et al. (2002) Tropical versus temperate environments Møller (1998) Continental versus insular environments Matson (2006) http://icb.oxfordjournals.org/ Saline versus freshwater environments Mendes et al. (2006)

Intrinsic to these studies are assumptions that reflect previous exposure to pathogens, current immune defense only involves eliminating or reduc- state of health or disease, or the degree of evolved

ing pathogen load (i.e., resistance); processes that protection (Matson 2006; Bradley and Jackson 2008). by guest on December 9, 2011 limit the damage caused by a given pathogen load Even immune assays that are seen as measuring fun- without necessarily reducing it (i.e., tolerance), are damental attributes of individuals (i.e., are signifi- neglected (Ra˚berg et al. 2009). Resistance, tolerance, cantly repeatable) (Buehler et al. 2008a) exhibit and pathogen pressure together dictate pathogen large amounts of unexplained variation. Further load. High pathogen loads could indicate high path- complications can arise when immune measures are ogen pressure, but also low (or compromised) not clearly linked to known aspects of the immuno- immune defenses, or tolerance to the measured en- biome. Even when identified, any such links are ex- tities (Fig. 3). Pathogen load need not always equate pected to be highly specific and might not extend to pathogen pressure. beyond the circumstances of a given study system. Since later studies often continue to cite initial re- Using measures of immune function to test ideas ports and poorly substantiated hypotheses as a basis about hypothesized pathogen pressure for further predictions and new conclusions, the In another approach, differences in among need for direct measures of immunobiotic pressure groups or locations are attributed to a priori assump- is real. Combined measures of pathogen load and tions regarding differences in pathogen pressure, but immune defenses may be more instructive about pathogen pressure is usually not directly measured broad patterns of potential pathogen pressure than (Table 1). This approach arises from theoretical pre- either measure alone. Nonetheless, conclusions re- dictions that relate the inherent costs of immune garding pathogen pressure drawn from just these defenses to the evolution of immune systems that combined data must still be regarded as incomplete optimally match pathogen pressure (Lochmiller and (Fig. 3). Deerenberg 2000; Bonneaud et al. 2003; Tschirren and Richner 2006). That is, when pathogen pressure An example: Immunobiotic and immune-defense is low, immune defenses are expected to be low, and perspectives lead to divergent predictions vice versa. It is unclear over short timescales, how- The study of immune defense as a life-history corre- ever, to what extent assays of immune function late is an active area of research in ecological 568 N. P.C. Horrocks et al. Downloaded from http://icb.oxfordjournals.org/

Fig. 3 Many studies investigating immunobiotic pressure actually focus just on pathogen pressure. However, pathogen pressure is rarely measured directly. Instead researchers rely on pathogen load or immune function (Table 1), without actually calibrating these indices with host-independent measures of pathogens. On their own, neither type of index consistently predicts pathogen pressure correctly. In the leftmost column, the birds (which could equally symbolize nonavian taxa) represent four host individuals, populations or species. In each case, pathogen loads (indicated by the number of pathogens in each bird) and immune defenses (indicated by the number of in each bird) have been measured independently of each other. More antibodies equate to stronger or more fitness-enhancing immune defenses; more pathogens equate to higher pathogen loads. Pathogen pressure is usually unknown in eco- by guest on December 9, 2011 logical immunology studies, but to illustrate our point we provide hypothetical values of pathogen pressure in the second column. More pathogens equate to greater environmental pathogen pressures. In the third and fourth columns, we list predictions about pathogen pressure based on typical assumptions: higher pathogen loads indicate a greater pathogen pressure, and stronger immune defenses also indicate a greater pathogen pressure. In the rightmost column, these predictions are evaluated in light of the hypothetical pathogen pressures.

immunology (Tella et al. 2002; Tieleman et al. 2005; confounded factors; manipulations aimed at directly Martin et al. 2006). For the purpose of these studies, altering immune defense could indirectly change related organisms from distinct environments and either composition of the immunobiome or levels with different life-history strategies are compared of exposure (Moyer et al. 2002). For example, exper- (Martin et al. 2004). However, life-history strategies imental which makes animals more tend to co-vary with environmental conditions (e.g., sedentary (Adelman et al. 2010) might increase ex- Tieleman et al. 2004) and immunobiotic pressure posure to vector-borne diseases through reduced might also co-vary with these conditions. As a anti-vector behaviors (e.g., less grooming). result, all three factors (life-history strategy, immu- Similarly, experimental removal of immunobiotic nobiotic pressure, and abiotic environment) are po- components (e.g., specific pathogens) can alter tentially correlated and are difficult to disentangle. immune phenotypes and might affect infections by Neglecting to explicitly measure immunobiotic pres- other pathogens or members of immunobiomes sure ignores the possibility that immunological var- (Ezenwa et al. 2010). iation among different environments may relate Using birds as an example, we illustrate one in- directly to differences in immunobiotic pressure stance of confounded factors in a comparative study. (Buehler et al. 2008b) and only indirectly to dispa- Specifically, we show how simplistic predictions rate life-history strategies. Experimental studies of about immune parameters differ depending on individual animals could involve similarly whether they are derived from the perspective of Pathogen pressure and immune defense 569 life-history strategy or immunobiotic pressure. studies of immunology (e.g., Sadd and Long-lived species generally have long development Schmid-Hempel 2009; Schulenburg et al. 2009; periods, which allow for diverse repertoires of Pedersen and Babayan 2011), but these ideas require -producing B- (Lee et al. further development. The assessment of operative 2008). Over a lifetime long-lived species potentially protection necessitates descriptions of immune de- encounter the same immunobiotic components re- fense profiles that are protective against all (or at peatedly, so antibody-mediated acquired immunity least a diverse range of) encountered pathogens and and immunological memory may be especially valu- other immunobiotic components. Immunobiomes able (Boots and Bowers 2004). Long-lived birds in- are central players, and an ‘‘immunobiome ap- clude those inhabiting the tropics (Wiersma et al. proach’’ must treat them as such. We envisage re- 2007), open oceans (Ricklefs 1990), and deserts searchers examining the diverse immunobiotic (Tieleman et al. 2004). However, these three environ- selective pressures that animals encounter, by explor- ments are predicted to differ in terms of immuno- ing the immunobiomes relevant to their study sys- biomes. Marine (Piersma 1997; Mendes et al. 2005) tems (Alcaide et al. 2010). Although a daunting task, and xeric (Moyer et al. 2002; Valera et al. 2003) smart use of relevant technologies will make it feasi- environments are hypothesized to be relatively ble. Such an integrative effort is comparable to the pathogen-free, while wet tropical environments Human Microbiome Project (HMP; http://nihroad- Downloaded from might harbor abundant and diverse pathogens map.nih.gov/hmp), which focuses on identifying all (Møller 1998; Guernier et al. 2004). From a human-associated microbes and analyzing the role of life-history perspective, these ‘‘slow-living’’ birds are these microbes in health and disease. Similar to the all predicted to invest similarly in immunity and self- HMP, we expect that the microbial component of maintenance (Lee 2006). Yet from the view point of the immunobiome will provide a particularly rich http://icb.oxfordjournals.org/ immunobiotic (and more specifically, pathogen) and diverse landscape to describe, since for wild an- pressure, tropical land birds are predicted to invest imals it remains unexplored. differently in immune defense than do oceanic or To start this daunting task, we suggest focusing desert birds. In fact, the extent to which environ- first on microbial pathogens and microbial pathogen ments truly differ in either pathogen pressure or pressure, before expanding to the rest of the micro- broader immunobiotic pressure is not at all clear. bial immunobiome (Appendix 1). A range of meth-

ods, including microbiological and metagenomic by guest on December 9, 2011 Measuring immunobiotic pressure: an techniques will be required for initial surveys and immunobiome-wide approach description. Subsequently, the most relevant patho- gens can be identified, and connections to specific Veterinarians, parasitologists, ecologists, and immu- components of immunity can be established, allow- nologists all think differently about the role of dis- ing the identification of protective immune pheno- ease. Often, single pathogens or diseases are types (Pedersen and Babayan 2011). Eventually, other identified or analyzed outside of the contexts of ecol- subsets of the immunobiome can also be evaluated ogy and evolution. In some field studies, one or (e.g., commensals, multicellular pathogens). Then, a more key pathogens may have been identified, and more complete picture can be drawn of how the the effects of these infections on fitness might be immunobiome shapes immune defenses, and conclu- clear. For the majority of wild hosts however, the sions regarding operative protection can be made fitness-reducing effects of most individual pathogens (Fig. 2). or parasites (or the fitness-enhancing effects of most commensals) are poorly understood. Studying the costs of infection of single pathogens is informative Description is innovation and should not be abandoned, but to understand From some perspectives (e.g., that of a parasitolo- immunobiotic pressure and operative protection we gist), attempting to measure all the organisms asso- advocate a broader strategy. Increasingly, more atten- ciated with a host might not appear particularly tion is being devoted to understanding multiple in- novel. From the viewpoint of ecological immunolo- fections, better reflecting the ecological context of gy, however, assembling detailed descriptions of individual animals, populations or species and their host- and habitat-associated immunobiomes is diseases (e.g., Jolles et al. 2008; Behnke et al. 2009; highly novel (Appendix 1). The first step in analyzing Ezenwa et al. 2010). Earlier authors have touched any community is to identify who is present and in upon the importance of incorporating ecological what numbers, and without this essential foundation measures (i.e., immunobiomes) into ecological progress in ecological immunology will be stunted. 570 N. P.C. Horrocks et al.

Furthermore, it is essential for understanding the se- multiple immune components must be measured lective pressures that immunobiomes exert on the (Norris and Evans 2000). Similarly, multiple axes immune system. While it may not be possible to (e.g., diversity and abundance of archaea, bacteria, measure all relevant factors, simultaneous measure- fungi and viruses) will be required to encapsulate ment of multiple components of the immunobiome the complexity of microbial immunobiotic pressure. will always be desirable because the effects of immu- Available molecular techniques allow this to be done nobiotic load (e.g., co-infections) on fitness may be (Appendix Table A1). Individual pathogenic strains interactive rather than simply additive (Jolles et al. can be identified and known markers or genes of 2008; Behnke et al. 2009; Ezenwa et al. 2010). As pathogens can be targeted. Of course, a sequencing such, description represents the first level of innova- approach alone does not guarantee that all pathogens tion in the immunobiome approach that we pro- and virulence-factors will be recognized. Moreover, mote, and will be a major advancement. By freeing sequence data has a limited capacity to predict the researchers from the constraints of microbial culture impact that a given immunobiotic element has on a techniques, molecular–genetic approaches dramati- particular host. Pathogenicity may vary depending cally increase the number of components of the on hosts’ characteristics at the individual, popula- immunobiome that can be surveyed. At the same tion, and species levels. Collaboration among parasi- time, the battery of indices available in comparative tologists, microbiologists, veterinarians, and other Downloaded from immunology continues to diversify. Integrating these wildlife-disease experts will be essential for overcom- indices of immunity with the newfound understand- ing these issues relating to methodology and ing of immunobiotic pressure will lead to a second interpretation. level of innovation. Linking broadly defined immu- A next step will then be to classify components of nobiotic pressures to evolution of the immune newly described immunobiomes according to their http://icb.oxfordjournals.org/ system and to operative protection remains an ulti- mode of action. This will more easily allow individ- mate goal. ual components of immunobiotic pressure to be re- lated to the particular classes of immune responses Collaboration is key that they elicit. At some levels this will be obvious (e.g., extracellular parasites should elicit extracellular A generalist approach to quantifying immunobiotic immune responses), but identifying which specific pressure is not without challenges, and requires care- components of the immunobiome elicit which spe- by guest on December 9, 2011 ful consideration of sampling schemes and laboratory cific responses may be less clear. On one hand, pair- techniques. For example, samples should be collected ing immune challenges and immune responses in with the interactions between host and immuno- this way is a nod to traditional immunology. On biome in mind. Outside surfaces of hosts and loca- the other hand, the comparative nature tions where external environment meets internal and ecosystem-wide scope of this ‘‘immunobiome physiology, such as mucous membranes, should be approach’’ firmly plants this research outside the targeted. Environmental substrates that hosts com- bounds of immunology and anchors it within monly contact, such as water sources and sleeping ecology. Joining these opposing factors will allow areas should be evaluated. The most probable for the development of more targeted immune routes of infection, such as ingestion with food, assays. Eventually, specific knowledge of immuno- must also be considered. Ideally, potential pathogens biotic pressures will direct choice of immune found in the wider environment, including micro- assay. A clearer understanding of which parameters parasites and macroparasites, will be linked to hosts of immunity are most important for hosts when through ecology and habitat. exposed to immunobiomes with different composi- In terms of methodology, adapting procedures al- tions (e.g., more or less diversity) will ultimately ready used for routine monitoring of microbial com- emerge. munities in other applications, such as in wastewater management, air-monitoring, and soil science, will be fruitful (Appendix 1). Likewise, reagents or meth- Future directions and broader impacts odologies already established in model and From the standpoint of ecological immunology, the commercially exploited animals may be suitable for necessity for measuring elements of the immuno- studies of wild and nonmodel animals (Abolins et al. biome is to gain a better handle on the selective 2011). Early pioneers of ecological immunology real- pressures that shape the immune systems and ized that if broad comparisons were to be made, then define the immune-strategies of animals living in the complexity of immune systems dictates that the wild. In a wider context, however, these novel Pathogen pressure and immune defense 571 evolutionary and ecological perspectives on immune Adelman JS, Bentley GE, Wingfield JC, Martin LB, Hau M. systems also have much to offer, both to ecologists 2010. Population differences in fever and sickness behaviors interested in the immune system and to immunolo- in a wild passerine: a role for . J Exp Biol 213:4099–109. gists who wish to place immunology in the context of a more ‘‘real’’ world. For example, studying the Alcaide M, Lemus JA, Blanco G, Tella JL, Serrano D, Negro JJ, Rodrı´guez A, Garcı´a-Montijano M. 2010. 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Table A1 Molecular approaches to measuring pathogens

Level/question being asked Possible methods for measuring pathogens Pathogens applicable to Comments Host–pathogen interaction:

 How does the presence of a single  Prevalence/abundance counts  Ecto- and endoparasites Does not reflect the complexity of pathogen assemblages. Ignores the type of pathogen correlate with possibility of interactions among co-infecting pathogens.  Microscopy (e.g., blood smears, coproscopy) immune function? Often unclear which immune parameter(s) is/are most appropriate to test.  51% microbial species  Culture-based techniques Any observed correlation may be between an unmeasured pathogen  qPCR with pathogen-specific primers (Whyte et al.  Any microbial species and immune function. 2002) Unlikely that single pathogen measures are representative of overall pathogen pressure. The link between host fitness and infection with individual pathogens may be very weak. Interaction of host with the pathogen assemblage (descriptive):  Prevalence/abundance counts Prevalence/abundance counts are laborious and sample size  How does the diversity of the path- affects the pathogen diversity recorded (Walther et al. 1995). Alongside  Microscopy ogen assemblage correlate with visual counts, diagnostic assays adapted from the livestock/pet industries immune function?  Culture-based techniques could be used to measure large pathogens (e.g., endoparasites)  All microbes, either one taxonomic (Traversa and Otranto 2009).  How does the abundance of compo-  Sequence-based metagenomics (community group at a time, or nents of the pathogen assemblage ‘‘fingerprinting’’): Fingerprinting methods are uninformative about the identity of members of simultaneously (i.e., microarray; con- correlate with immune function? the assemblages described; with some techniques (e.g., DGGE), – DGGE (Klomp et al. 2008) taining DNA sequences from candi- follow-up sequencing can achieve this. date community members of many – RISA (Ruiz-Rodrı´guez et al. 2009) Unrepresentative of true diversity; only the most abundant members are taxa e.g., bacteria, – TRFLP (Hackl et al. 2004) archaea, fungi, etc.) represented. The exception is bar-coded pyrosequencing, which allows deeper coverage of microbial diversity. – Gene sequence libraries (Corby-Harris et al. 2007) Provides some information about which features of the assemblage might – Bar-coded pyrosequencing (Cox-Foster et al. correlate with immune function. 2007) Unclear as to cause and effect; is diversity of the pathogen assemblage – Taxonomic Microarrays (Gentry et al. 2006) driven by immune defenses, or vice versa? (Bordes and Morand 2009). Interaction of the host with the pathogen assemblage (functional):  Function-based metagenomics:  All microbes, but more difficult with Markers of pathogenicity include pathogenicity islands, antibiotic resistance,  Which attributes or components of eukaryotic microorganisms due to virulence factors, genes associated with secretion systems, sequence the pathogen assemblage correlate – Large insert libraries (Treusch et al. 2004) amount of genetic information homology with other known pathogenic species (Finlay and Falkow with immune function? – Meta-transcriptomes—sequencing expressed ge- present. 1997). netic information e.g., mRNA, protein (Bailly et al.  Do microbial assemblages with higher Even though the identity of members of the pathogen assemblage may be Horrocks C. P. N. ‘‘pathogenic function’’ (e.g., greater 2007) initially unknown, it is still possible to examine functionality. number of pathogenic markers) cor- – Functional microarrays—containing DNA se- Provides more information about which features of the pathogen assem- relate with higher indices of host quences for genes of known function e.g., viru- blage might correlate with immune function e.g., diversity, abundance or immune defense? lence factors (Gentry et al. 2006) presence of certain members of the assemblage. Currently more expensive and technically advanced than other techniques.

qPCR, quantitative PCR; DGGE, denaturing gel gradient electrophoresis; RISA, ribosomal intergenic spacer analysis; TRFLP, terminal restriction fragment length polymorphism. tal. et

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