INTRODUCTION
Ecological physiology has received considerable attention in recent years due to a growing number of empirical and theoretical studies. The overall goal of this discipline is to evaluate the function of physiological mechanisms in the contexts of ecology and evolution. Multiple studies have attempted to determine how differences in physiological mechanisms influence the distribution and abundance of organisms in space and time (Bozinovic et al. 2011). By exploring the processes by which such mechanisms arise and are maintained, ecological physiology seeks to understand how physiological parameters are shaped by natural selection (McNab 2002; Bozinovic 2003; Withers et al. 2016).
Ecological physiology emerged in the early 1960s as an integrative, conceptually driven offshoot of comparative physiology. Studies conducted during the 1960s and 1970s established the importance of diversity in physiological mechanisms as a means of responding to different environmental conditions (see Schmidt-Nielsen 1997). The mid 1980s marked a crucial junction for this discipline, due to the incorporation of a phylogenetic, or evolutionary perspective into physiological analyses (Felsenstein, 1985). Today, research in ecological physiology draws upon these foundations to examine the adaptive and functional importance of physiological diversity; although reductionist analyses of the physical and chemical bases for physiological responses are important, they are only part of the larger ecological and evolutionary contexts in which animals respond to environmental conditions (McNab 2002).
Here, we evaluate the current status of research in ecological physiology, focusing on studies of wild mammals from Argentina that have been conducted over the last 25 years. Within this taxonomic scope, we consider multiple aspects of physiological response to changes in ecological conditions. Because ecological physiology is necessarily an integrative discipline, our review touches on an array of topics, with emphasis on energetics, water balance, endocrinology, and immunology. The research reviewed here includes studies of both free-living and captive animals, with emphasis on the complementarity of data collected in these two contexts. Because ecological physiology is a relatively new discipline in Argentina, this effort encompasses the vast majority of studies conducted in mammals over the last 25 years. Throughout this review, we seek to identify topics particularly in need of further investigation and we highlight taxa that appear to be well suited to such research. Our goal is to stimulate exciting new studies of mammalian ecophysiology that will carry this discipline through the next quarter century and beyond.
ENERGETICS
Ecological physiology provides an important bridge between mechanisms of response to environmental conditions and the ecological and evolutionary significance of those responses. Accordingly, this discipline has direct ties to how different ecological factors impose limits on the energy budgets of organisms. Both internal (e.g. digestive capacity) and external (e.g. ambient temperature, atmospheric pressure, water availability, social interactions) conditions can affect the flow of materials (Wiegert 1968) through impacts on the acquisition, absorption, allocation and expenditure of energy (Karasov 1986; Weiner 1992). In this context, understanding how animals work requires an understanding of their energy budget which, for autotrophic organisms, represents the integration of energy intake, storage and loss. Thus, a major goal of ecological physiology is to understand the factors that determine energy budgets (McNab 2002). In general, such factors can be divided into limits on the rate of resource acquisition (e.g. foraging rate, rates of digestion and absorption) and limits on energy expenditure (e.g. mechanical work, heat production, water loss, tissue growth; Weiner 1992). Energy acquisition and expenditure, however, do not function in the same way; while processes for energy intake occur in sequence, processes for energy expenditure are parallel and can be additive (Weiner 1992; Bacigalupe & Bozinovic 2002). As a result, we have divided the following review of energy budgets into consideration of factors affecting energy intake versus those affecting energy expenditure (Fig. 1).
Energy intake
We focus our review of energy intake on the digestive system, which is where energy is extracted from food and enters the physiological machinery of the focal organism. Clearly, digestive physiology is important and can influence key life history features such as growth, reproduction, and survival (Karasov 1986). Multiple studies have shown that the digestive system responds to changes in biotic as well as abiotic elements of the environment (Karasov & Douglas 2013), leading to the expectation that diges tive physiology is under strong selective pressure. Below, we consider several ways in which mammals may alter their digestive physiology to respond to changes in environmental conditions.
Dietary variation. One way that mammals may alter their digestive physiology and energy intake is through changes in food consumption. For example, when fed a low quality diet, captive Talas tuco-tucos (Ctenomys talarum) consumed more food and devoted more time to feeding than when provided with a high quality diet (Perissinotti et al. 2009). No morphological differences in the gastrointestinal tracts of these animals were observed (del Valle & López Mañanes 2008, 2011), suggesting that response to variable diet quality relies in large part on behavioral adjustments. This may include changes in the frequency of coprophagy (i.e. reingestion of fecal items), which is common in rodents, including C. talarum (Martino et al. 2007, Perissinotti et al. 2009). Because coprophagy in this species occurs across all diets, however, it is possible that reingestion of feces is driven more by acquisition of essential nutrients (e.g. vitamins and aminoacids) than energy intake (Martino et al. 2007; Perissinotti et al. 2009). Changes in diet composition in response to environmental conditions have also been reported for Phyllotis xanthopygus (Sassi et al. 2017). Members of this species captured at different elevations in the Andes varied between omnivorous and granivorous- frugivorous diets in relation to habitat conditions. Changes in digestive physiology and energy intake were also reported across the elevations sampled (Sassi & Novillo 2015). Although additional studies of these types are needed, we propose that changes in food resources and food consumption are common responses to variable environmental conditions that have important implications for energy intake.
Digestive efficiency. Digestive efficiency provides a critical link between extrinsic factors (e.g. food resources) and intrinsic processes such as extraction of energy from food resources. One of the primary determinants of digestive efficiency is the quality of food consumed (Karasov 1986; Bozinovic 1993). Within species, diet quality can affect both the morphology and physiology of the gastrointestinal tract. For example, gut length in the omnivorous sigmodontine rodent Akodon azarae increased when captive individuals were maintained on a low quality diet (del Valle et al. 2006); this change occurred in the anterior portion of the gut, where digestion and energy absorption typically take place. A low quality diet was also associated with digestive changes at the cellular level (Sabat et al. 1999); A. azarae fed a low quality diet displayed reduced sucrose-specific hydrolytic activity in the small intestine, indicating a down-regulation of the sucrase-isomaltase enzyme complex involved in digestion of carbohydrates (del Valle et al., 2006). Similar changes in digestive physiology were evident in A. azarae exposed to low ambient temperatures (del Valle et al. 2004), suggesting that changes in carbohydrate processing and thus digestive efficiency in these animals may be a general response to challenging environments. Diet-induced changes in digestive efficiency have also been reported for the herbivorous cavy Microcavia australis; Sassi et al. (2007) reported that among free-living populations of this species, differences in diet quality were associated with size variation in digestive organs, particularly the caecum. In captivity, differences in digestibility and retention time were observed in relation to differences in diet quality (Sassi et al. 2010), providing further evidence that digestive efficiency varies in response to the resources consumed.
Across species, fundamental differences in mam malian life history traits may also impact digestive efficiency. For example, the digestive system of bats is generally similar to that of non-volant mammals, with the exception of the large intestine, which is relatively short in chiropterans compared to other mammals (Caviedes-Vidal et al. 2007). This morphological difference may reflect a trade-off between a reduction in internal organ size that facilitates flight and an associated reduction in gut absorptive capacity (Price et al. 2015). As an apparent form of compensation for the reduced surface area available in the large intestine, bats absorb water-soluble nutrients (glucose, aminoacids) through a passive-transport system that occurs via paracellular junctions (Price et al. 2015). For example, studies of populations of the insectivorous bat Tadarida brasiliensis from Argentina revealed high levels of paracellular absorption (Fasulo et al., 2013, Caviedes-Vidal et al. 2008), a finding that was linked to an increased number of tight junctions in the intestine (Price et al. 2013). Indeed, T. brasiliensis has more villous area and a greater number of enterocytes per area than non-volant mammals (Zhang et al. 2015), which facilitate their reliance on paracellular absorption (Price et al. 2013). Thus, over evolutionary time scales, the challenges imposed by different mammalian life history patterns may also result in changes in the mechanisms underlying digestive efficiency.
Energy expenditure
Energy that is acquired by an organism can be spent in multiple ways. This includes expenditures required simply to maintain the most basic life processes such as respiration, as well as expenditures associated with more specialized activities such as thermogenesis, reproduction, or escape from predators. Below, we briefly review forms of energy expenditure that are common to mammals, with emphasis on how these expenditures may change in response to extrinsic environmental conditions.
Basal metabolic expenditures
In endotherms, one of the primary physiological variables used to assess minimum energy expenditure is basal metabolic rate (BMR). Estimates of BMR are generated under standardized conditions (e.g. thermoneutral, non-reproductive, inactive phase of circadian cycle) so as to provide a measure of the minimum energy necessary to maintain homeostasis (Hulbert & Else 2004). Studies of BMRs for Argentine mammals are largely restricted to rodents. One of the first such studies was conducted on A. azarae (Dalby & Heath 1976); this classic analysis estimated BMRs for captive individuals. Subsequently, Antinuchi and Busch (2000) estimated BMR for a larger number of members of this species that had been obtained directly from the field, resulting in values of BMR similar to those reported by Dalby & Heath (1976). In contrast to these surface-dwelling rodents, members of the genus Ctenomys are subterranean and, accordingly, display morpho-physiological features thought to reflect adaptations to life in underground burrows (Nevo 1999). Busch (1989) examined the metabolism of two species of ctenomyids, revealing that in both Ctenomys talarum and C. australis, BMR was lower than that for surface-dwelling rodents of similar body size.
Following these pioneering efforts, studies of Argentine mammals have increasingly focused on the ecological and evolutionary contexts underly ing diversity in physiological processes. Because environments are dynamic, variability in morpho- physiological responses (i.e. phenotypic plasticity; Spicer & Gaston, 1999) should improve an individual’s ability to cope with temporal changes in environmental conditions and multiple studies have now examined variability in the physiological responses of individuals. For example, Perissinotti et al. (2009) evaluated the effects of experimentally induced differences in diet quality and soil hardness on BMR in C. talarum while Luna et al. (2012) examined the effects of acclimation to different ambient temperatures on the same physiological parameter; while diet quality and soil hardness were associated with differences in BMR, ambient temperature had no apparent effect on this variable. Over somewhat longer time periods, seasonal variation in BMR was not detected for C. talarum (Meroi et al., 2014) although Tachinardi et al. (2017) reported that for C. aff. knighti, winter BMRs were lower if surface activity was restricted to the daytime, which is when above-ground activity by these animals is typically observed (Tomotani et al. 2012). Thus, variation in environmental conditions appears to elicit changes in individual metabolic physiology over multiple time scales.
In some species, particular life history traits (e.g. specialization for marine habitats) makes it difficult to quantify BMR; in these taxa, resting metabolic rate (RMR) is typically reported (Speakman et al. 2004; Withers et al., 2016). For example, Dassis et al. (2012) quantified RMR in Southern sea lions (Otaria ßavescens) while the animals were at rest on the ground, at rest floating in water, and swimming underwater. Estimates of RMR increased across these three conditions, as expected based on anticipated energetic costs of terrestrial versus marine conditions and resting versus active individuals. RMR has also been used to assess energetic expenditure by juvenile South American fur seals (Arctocephalus australis; Dassis et al. 2014). While BMR and RMR clearly differ in terms of the specific metabolic information provided, both measures have been used to assess the relative energetic costs of differences in activity and environmental conditions.
All of the above studies were based on analyses of intraspecific variation in BMR or RMR. Analyses of interspecific differences in metabolic rates are needed to identify large-scale patterns of energetic expenditure. Primary determinants of variation in BMR include body mass and phylogenetic background (Kleiber 1932; Hayssen & Lacy 1985; Mueller & Diamond 2001). Considerable variation in BMR, however, remains after correction for body mass and phylogeny (McNab 1988, 2009), suggesting that other factors also contribute to differences in BMR. These additional factors include ambient tempera ture (Naya et al. 2013), diet type (i.e. herbivores versus carnivores; McNab 1986), climatic factors (i.e. T a and precipitation; Rezende et al. 2004), latitude (Lovegrove 2003) and zoogeographical zones (Lovegrove 2000). To the best of our knowledge, the only studies of Argentine mammals to have examined interspecific variation in BMR have focused on the rodent genus Ctenomys rodents. Contrary to data from surface-dwelling rodents, BMRs in Ctenomys are not affected by differences in climate, soil conditions, habitat productivity or net primary productivity (Luna et al. 2009). This outcome is consistent with the long-standing expectation that subterranean environments tend to be relatively stable (Buffenstein 2000), leading to the prediction that among ctenomyids, environmental differences may affect energetic variables other than BMR (e.g. maximum metabolism: Luna et al. 2009, 2017). Clearly, further research is needed to understand how body size, phylogenetic history, and environmental conditions interact to shape the energetics of these and other Argentine mammals.
Thermoregulation
In mammals and other endotherms, body temperature (T b) reflects the balance between metabolic heat production and loss of that heat to the environment. Both of these parameters may vary over the course of the 24-hr cycle, leading to circadian variation in T b. For example, Casanave & Affanni (1994) reported that in the hairy armadillo Chaetophractus villosus, T b was lower in the morning than during the afternoon (35.1 °C and 36.1 °C, respectively). Factors thought to play key roles in regulating circadian patterns of T b include the light-dark cycle as well as daily fluctuations in ambient temperature and food availability (Refinetti 2010). For example, even among individual Ctenomys aff. knighti housed in complete darkness, Tachinardi et al. (2014) found daily fluctuations in T b that were synchronized with the 24-hr light-dark cycle. With regard to ambient temperature, Seitz and Puig (2018) reported that daily variation in external temperatures had a strong effect on T b in the hairy armadillo Zaedyus pichiy, although a comparable effect was not detected for two other species of hairy armadillos (Chaetophractus vellerosus, and Ch. villosus).
Heat production
In terms of metabolic heat production, total thermo genic capacity and the underlying components (i.e. shivering and non-shivering thermogenesis) have been quantified for only a single Argentine mammal, C. talarum. In this species, non-shivering thermogenesis (NST) did not vary with acclimation to low T as and did not appear to be the primary source of heat production (Luna et al. 2012). Similarly, maximum metabolic rate (MMR) did not differ with acclimation to cold or warm environments (Luna et al. 2012), suggesting that thermogenic capacity is already maximal in warm acclimation. Given that these subterranean rodents live in what are expected to be relatively stable thermal environments, the implications of these data for other, surface dwelling species are not clear. Comparative data from a greater range of taxa and from species occupying a greater diversity of habitats are required to evaluate fully the role of NST versus other forms of heat production in Argentine mammals.
Heat loss
Mammals use multiple mechanisms to reduce loss of metabolic heat, including huddling and changes in body fat or pelage. More specialized forms of reducing heat loss include torpor, which has been reported for some species of Argentine mammals. More specifically, Caviedes-Vidal et al. (1990) found that the mouse Calomys venustus became torpid when exposed to low T as, a response that may facilitate survival in environments with wide thermal fluctuations (Caviedes-Vidal et al. 1990). Torpor has also been reported for the Patagonian opossum (Lestodelphys halli), in which T b varied with cold exposure and periods of torpor persisted for up to 10 h (Geiser & Martin 2013). In other species, torpor appears to be triggered by variation in diet quality (i.e., energetic intake rather than heat loss). For example, when experimentally subjected to a low quality diet, armadillos (Z. pichyi) spent 6 h at sub-normal T b, while animals fed a standard diet allowed T b to drop for no more than 1 h (Superina & Jahn 2013). Z. pichyi is the only species of xenarthran that is known to hibernate, with hibernation bouts (interrupted by short periods of euthermia) lasting 75 h (Superina & Boily 2007). Thus, in addition to morphological adaptations, at least some Argentine mammals use modifications of metabolic rate to conserve heat.
Conversely, dissipation of metabolic heat may be a challenge for some species, particularly terrestrial taxa that are exposed to high ambient temperatures. Heat may be lost to the environment via direct physical routes (conduction, convection, radiation) or via evaporative water loss. Direct loss of heat is likely to be facilitated by properties of the pelage. For example, Jofré & Caviedes-Vidal (2003) reported seasonal variation in heat transfer via fur in Calomys musculinus, with loss of heat being greater during the summer. Although Cutrera & Antinuchi (2004) did not measure heat transfer directly, they reported seasonal changes in the density and length of fur in the subterranean rodent C. talarum, with both measures decreasing during summer. Heat dissipation may also be affected by changes in behavior that alter the surface area available for thermal conductance. Luna & Antenucci (2007a) found that, while digging, C. talarum did not display changes in T b in response to changes in ambient temperature; these authors proposed that the animals press their ventral surface against the substrate while digging, thereby increasing conductive heat loss. Increased contact with the substrate has also been proposed for the armadillo Chaetophractus villosus, which displayed decreased T b when experimentally covered with soil (Casanave & Affanni 1995).
Evaporative water loss may be particularly effective at high ambient temperatures because this mechanism does not rely on a thermal gradient (Withers et al. 2016) between an organism and its environment. Evaporative water loss (EWL; Stitt 1976) is a standardized physiological measurement used to estimate the loss of heat due to evaporation; mechanisms such as sweating, salivation or panting that increase EWL are expected to also increase evaporative heat loss. Among Argentine mammals, EWL in C. talarum remained constant below and within the animals’ thermoneutral zone but increased above thermoneutrality (Baldo et al. 2015). This outcome was not influenced by relative humidity (Baldo et al. 2016); because the air in underground burrows is typically very moist, it may impede evaporation, suggesting that other forms of heat loss such as conductance may be more important in subterranean species such as C. talarum (see Luna & Antenucci 2007b). More generally, because evaporative heat loss also results in loss of water, the relative importance of this cooling mechanism is expected to vary between arid and more mesic habitats.
Water balance and osmoregulation
Water is an essential component of the mammalian body and its abundance must be regulated to maintain homeostasis. In mammals, water is obtained both exogenously (e.g. direct intake or through food) and endogenously (e.g. production of metabolic water during catabolism). Loss of water occurs via excretion of urine and feces as well as through evap oration. Much of the research on water balance in mammals has focused on mechanisms for reducing water loss, such as producing urine that is significantly more concentrated than plasma (Schmidt- Nielsen 1997). The ability of mammalian kidneys to produce concentrated urine depends on the length of the loops of Henle and collecting ducts that traverse the renal medulla, with the lengths of both structures being directly related to the thickness of the renal medulla (see McNab 2002). Accordingly, medullary thickness has been used as an index of renal capacity to concentrate urine. Diaz & Ojeda (1999) compared renal structure across multiple species of Argentine rodents as a function of environmental water availability. These authors found that among rodents that occupy desert habitats, murid species (Salinomys delicatus, Andalgalomys olrogi, A. roigi, C. musculinus, Eligmodontia moreni, E. typus) had the greatest relative medullary thickness and most concentrated urine. Within octodontids, the salt flat specialist Tympanoctomys barrerae had a higher renal index and greater urine osmolarity than the closely related but somewhat more generalist Octomys mimax and Octodontomys gliroides (Diaz & Ojeda 1999, Diaz & Cortés 2003). Comparisons of several species of Akodon from habitats with different rainfall regimes revealed that the capacity to concentrate urine was higher in the more ariddwelling A. iniscatus and A. azarae than in the more mesic-living A. cursor (Antinuchi and Busch 1999). Baldo et al. (2019) observed that C. talarum is able to concentrate urine beyond the level required under conditions of low water availability. Diaz et al. (2001) found that food deprived Thylamys pusilla displayed the highest reported value of osmolarity for an insectivorous marsupial. Thus, studies of multiple mammal lineages provide morphological and physiological evidence that kidney function varies predictably with environmental aridity, presumably as a means of reducing water loss in drier habitats.
Other potential adaptations to reduce water loss include increased uptake of water vapor via aquapor ins in nasal turbinates (Gallardo et al. 2008; Castellar et al. 2015) and the absorption of water in the intestine (Gallardo et al. 2002; Bozinovic & Gallardo 2006). To date, no studies have been performed in Argentine mammals to evaluate the importance of either route in the conservation of body water. Finally, mammals may use a wide range of behavioral strategies (e.g. burrow construction, nocturnal activity) to reduce the probability of experiencing water imbalance (Bozinovic & Gallardo 2006); these potential adaptations also require additional study in Argentine mammals. More generally, future studies should explore the interplay between morphological, physiological and behavioral mechanisms for reduc ing water loss across a variety of environmental settings.
Non-basal energetics
Most animals spend the majority of their lives operating under non-basal metabolic conditions. As a result, measurement of energetic expenditure under a wide range of non-basal conditions is critical to understanding the actual energy budgets of most free-living mammals. To the best of our knowledge, only one species of Argentine mammal -C. talarum- has been studied with regard to how energetic costs change across different basic life history activities. As analyses of this species reveal, energetic demands can change markedly with activity, suggesting that studies of basal metabolic demands are not sufficient to understand the energy physiology of free-living animals.
Burrow excavation
C. talarum is subterranean, meaning that individuals spend the majority of their time in underground burrows that they excavate for themselves (Lacey et al. 2000). Digging new tunnels is thought to be very energetically costly (see Vleck 1979) and, in general, subterranean species are characterized by low BMRs compared to those for surface-dwelling rodents of similar body mass. The “cost-of-burrowing hypothesis” posits that reduced BMR may help to compensate for the metabolic demands imposed by digging to create new burrows or to modify existing tunnel systems (Vleck 1979, 1981). In contrast, the “thermal-stress hypothesis” argues that low BMR minimizes the risk of overheating in confined and poorly ventilated burrow environments in which convective and evaporative heat loss are restricted (McNab 1966, 1979). To evaluate these hypotheses, Luna & Antenucci (2006, 2007b) analyzed the effect of soil hardness on digging metabolic rates (DMR) in two species of ctenomyids. These authors reported that in both C. talarum and C. australis, DMR for animals living in soft soils increased as soil hardness increased; in contrast, metabolic rates for animals living in hard soils did not change. Further, in C. talarum, increasing the incline at which animals were digging to > 40° also resulted in an increase in DMR (Luna & Antenucci 2007c), suggesting that both soil conditions and burrow architecture affect the metabolic costs of burrow excavation. Energetic demands imposed by these factors may, in turn, interact with other extrinsic factors; Luna & Antenucci (2007a) demonstrated that metabolic rates while digging increased for C. talarum at low ambient temperatures and that the energy generated during digging was used in part to supply the energy required for thermoregulation. In contrast, at high temperatures, the increase of DMR did not affect T b, presumably because the animals made greater use of conductive heat loss when digging. Overall, these findings fail to provide clear support for either the cost-of- burrowing or thermal-stress hypotheses and, indeed, both ideas may contribute to the understanding of the metabolic costs of digging.
Reproduction
For female mammals, reproduction is one of the most energetically costly activities that an individ ual can undertake (Tomasi & Horton 1992), with reproduction accounting for as much as 50% of a fe- male’s annual energy budget (Kenagy 1987). During pregnancy, the energetic requirements of females increase due to the need to support fetal growth as well as the growth of maternal reproductive structures (Thompson 1992). Lactation is typically the most expensive portion of the reproductive cycle, with energy expenditures rising to greater than 2- 3 times those associated with gestation (Künkele 2000a;b). These estimates do not take into account the energetic costs of securing mates or providing other forms of maternal care, all of which may further enhance the costs of reproduction.
Considerable research has focused on the appar ent evolutionary trade-off between the duration of gestation versus lactation and the relative energetic requirements of each of these portions of the reproductive cycle, a dynamic that is expected to vary with general differences in life-history strategies (Thompson 1992). In C. talarum, females experience increased energetic demands during gestation and lactation, although the cost of these two stages of reproductive appear to be similar (Zenuto et al. 2002). The lack of a pronounced increase in energetic needs during lactation is thought to reflect the general reproductive biology of this species; like other hystricognath rodents, ctenomyids undergo a long period of gestation that results in the production of relatively precocial neonates (Weir 1974). The only other study to have examined the reproductive energetics of Argentine rodents revealed that in Akodon azarae, metabolic rates for pregnant and lactating females are higher than those for non-breeding females (Antinuchi and Busch 2001), al though the relative costs of gestation and lactation were not assessed for this species.
Growth
As indicated above, newborn ctenomyids tend to be more precocial than neonates in some other lineages of rodents (e.g. murids; Weir 1974). Although newborn C. talarum are largely furless and initially highly dependent on parental care, they quickly become mobile and begin eating solid food very early during their postnatal development. With regard to energetic demands, Zenuto et al. (2002a) reported that prior to postnatal day 10, the metabolic rates of pups increase although these individuals are not yet capable of maintaining a stable T b. Once young begin to eat solid food, their metabolic rates decrease; individuals achieve adult T b’s by 35-45 days after birth and display adult metabolic rates by∼90 days after birth (Zenuto et al. 2002; Cutrera et al. 2003). While neonates are clearly dependent on their mothers to maintain body temperature, short daily isolation of pups does not affect the acquisition of adult T b and metabolic rate (Baldo et al. 2014). Neonatal A. azarae are altricial and in this species, development of homeothermy is positively associated with litter size (Antinuchi and Luna 2002).
As should be evident, understanding energy budgets allows us to better compare the responses of organisms to the environments in which they live and, ultimately, to assess the evolution of physiological variation among species (McNab 2002). To facilitate such understanding, one of the primary goals of future research should be to examine physiological variation at different hierarchical levels-from individuals to species assemblages-so as to identify the sources and determinants of the diversity in physiological systems. Studies of the energetics of Argentine mammals provide a logical starting point for more comprehensive analyses of the physiology of individual species as well as more comparative analyses of physiological variation across larger taxonomic scales. Given the compelling evidence for rapid climatic changes at a global scale (IPCC 2013), these types of studies should become increasingly important to understanding current organismal reponses and, thus, predicting responses to future environmental change (Bradshaw & Holzapfel 2010; Somero 2010; Huey et al. 2012).
ECOLOGICAL ENDOCRINOLOGY
Hormones regulate numerous diverse activities including growth, development, reproduction, metabolic rate, water balance, blood homeostasis and adaptation to stress, and thus measuring hormone concentrations provides an important method for quantifying a wide spectrum of responses to environmental conditions (Wingfield 2018). Further, the quantification of hormones can serve as a proxy to evaluate many aspects of physical condition, survival, and reproductive success of individuals, for which it would otherwise be necessary to record ecological information (e.g. sex, age, reproductive condition) over extended time periods (Cooke et al 2013; Dantzer et al. 2014). In free-living mammals, obtaining such information is not easy due to the longevity of most species and the difficulties of trapping, marking and observing individuals in nature and thus hormonal estimates of physiology are particularly valuable to studies of these organisms.
Glucocorticoid hormones
In mammals, as in other vertebrates, stressful stimuli (e.g. drought, limited food, predator threats, social upheaval) trigger the activation of the hypothalamic-pituitary-adrenal (HPA) axis and, consequently, the production of glucocorticoid (GC) hormones, notably cortisol and/or corticosterone. In the short term, GC-mediated physiological and behavioral responses help animals to cope with environmental stressors, although prolonged exposure to stressors may negatively impact an individual’s condition, such that survival and reproduction are compromised (Sapolsky et al. 2000; Boonstra 2005). More specifically, depending on the severity of the challenges experienced and their effects on energetic needs and availability, individuals will face different degrees of allostasis, including potential allostatic overload (McEwen & Wingfield 2003). Given that GCs play a primary role in energy mobilization and mediate multiple regulatory functions –including response to stressors– recording changes in cortisol and/or costicosterone in relation to different ecological conditions and environmental challenges can provide critical information regarding the physiological condition of the animals under study (Vera et al. 2018; MacDougall-Shackleton et al. 2019).
Estimates of GC levels can be obtained from multiple sources. One of the most commonly assayed sources of GCs is blood plasma, especially to capture acute stress responses. While blood plasma provides a critical measure of circulating GC levels, using plasma requires that blood samples be obtained quickly (e.g. immediately after capture) to avoid including the acute pulse of GCs released as a consequence of capture and handling. Plasma levels of GCs have been assessed for two Argentine species of subterranean rodents: Ctenomys talarum (Vera et al. 2011a, 2012, 2013, 2018) and C. sociabilis (Woodruff et al. 2010). In addition, plasma measures of GCs have been obtained for guanacos (Lama guanicoe; Carmanchahi et al. 2011; Taraborelli et al. 2011, 2017) and vicuñas (Vicugna vicugna; Arzamendia & Vilá 2012; Marcoppido et al. 2017). In general, however, analyses of circulating GC levels remain scarce for mammals from Argentina and adjacent parts of South America.
Alternatively, levels of GC metabolites can be measured from urine, feces, saliva, hair, feathers, respiratory vapor, and baleen. Fecal samples in particular offer several advantages, namely avoidance of acute responses to capture, handling, restraint, anesthesia, and sample collection. Given that fecal samples capture information about GC production over a period of multiple hours, care must be taken to account for potential confounding factors such as differences in metabolism, sex, diet, reproductive status, and portion of the circadian cycle, each of which may influence the hormone concentrations of individuals (Goymann 2012; Romero & Reed 2005). Among Argentine mammals, measures of fecal GC metabolites have been completed for colonial tuco-tucos (C. sociabilis; Woodruff et al. 2010, 2013), domestic chinchillas (Chinchilla lanigera; Ponzio et al. 2004; Mastromonaco et al. 2015), pichis (Zaedyus pichiy; Superina et al. 2009), howler monkeys (Alouatta caraya; Cantarelli et al. 2017), and guanacos L. guanicoe (Ovejero Aguilar et al. 2016). Other materials that have been assayed include urine (C. lanigera: Ponzio et al. 2004; Mastromonaco et al. 2015) and baleen (Eubalaena australis; Fernández Ajó et al. 2018).
The relative abundance of cortisol and corticos terone may vary with the specific taxon under study. Among mammals, cortisol generally tends to be the most abundant GC; although corticosterone tends to dominate in Rodentia, this pattern varies among rodent species (see Woodruff et al. 2010; Vera et al. 2012, 2018). For example, while corticosterone was identified as the primary GC in plasma and fecal samples from female C. sociabilis (Woodruff et al. 2010), both GCs are found in plasma in C. talarum (Vera et al. 2011a). In the latter species, cortisol is responsive to acute and chronic stressors as well as to challenge with adrenocorticotropic hormone (ACTH) while corticosterone is not (Vera et al. 2011a, 2012, 2013, 2018). Further, inhibition by dexamethasone occurs for both GCs in free-living animals but only for cortisol in captive individuals (Vera et al. 2018). Finally, plasma levels for both GCs –in particular corticosterone– fluctuate seasonally and between years, resulting in a significant temporal variation in the ratio of circulating cortisol to corticosterone (Vera et al., 2011a, 2012, 2013).
Factors a ecting GC levels
Intrinsic and extrinsic factors, in conjunction with anthropogenic disturbances, can affect baseline GC levels as well as physiological responses to different stressors. Differences in GC levels may arise due to intrinsic attributes of individuals, including parameters such as sex, reproductive condition, ontogenetic history, and recent stressful experiences (Dantzer et al. 2014). Studies of free-living and captive mammals from Argentine have explored the effects of different intrinsic factors (Fig. 2). In at least two species (C. talarum; Vera et al. 2012; A. caraya; Cantarelli et al. 2017), cortisol levels were typically higher for females than for males, presumably due to the greater energetic demands on females associated with reproduction and or social interactions. Female guanacos also displayed higher levels of cortisol than males; potential explanations for this finding include sex-based differences in baseline GC levels as well as differential response to stressors (Carmanchahi et al. 2011). Among vicuñas, GC response to capture methods has been shown to vary between the sexes (Arzamendia et al. 2010). Changes in GC levels are expected to vary in relation to reproduction. Higher cortisol levels in male guanacos during the reproductive season were related to energetic demands of reproductive and associated social interactions (Ovejero Aguilar 2016). Both males and females of C. talarum showed higher cortisol levels during the reproductive season compared to other portions of the year (Vera et al. 2013). With regard to developmental experience, predatory prenatal stress did not influence the stress response to immobilization in juvenile C. talarum (Brachetta et al. 2018). Collectively, these findings suggest that information regarding basic phenotypic parameters is required to understand patterns of GC variation and physiological stress response in free-living populations of mammals.
GC levels may also be influenced by a variety of ecological and anthropogenic factors, operating independently or in conjunction with each other. Studies of free-living and captive mammals from Argentina have explored the effects of multiple such factors, including the following (Fig. 2):
a. Diet. Because GCs are involved in the regulation of energy balance, it is logical to predict that diet is an important determinant of GC levels in mammals (Reeder et al. 2004). Data from captive C. talarum appear to support this expectation; individuals fed with a low quality diet displayed increased cortisol levels (Vera et al. 2018). Diet likely impacts other physiologically important parameters such as glucose, triglycerides, and inflammatory response (Merlo et al. 2016a), suggesting that no single biomarker of physiological condition is sufficient for capturing the effects of this factor and thus future studies will benefit from recording multiple elements of response, as has been done for C. talarum (Vera et al. 2008, 2011a, 2013, 2018, Brachetta et al. 2018), guanacos (Carmanchahi et al. 2011; Taraborelli et al. 2017) and vicuñas (Arzamendia & Vilá 2012; Marcoppido et al. 2017).
b. Social relationships. The social environment in which an individual lives may affect the stressors that it experiences. In particular, while solitary animals may be more subject to stressors associated with food shortages or predator exposure, those living in groups face additional stressors arising due to regular social interactions with conspecifics. In this context, Woodruff et al. (2013) compared baseline GC levels in lone versus group living yearling female C. sociabilis, finding that in both captive and free-living populations, lone females had higher baseline GC levels. These authors suggest that while lone females must respond to all environmental challenges on their own, females in groups share some of these challenges, thus reducing the impact on each individual in a group, thereby underscoring the role that social environment may play in mediating response to challenge.
c. Predator threats. Predators affect prey directly through mortality as well as indirectly through impacts on behavior, foraging opportunities, and reproductive success; these more indirect effects may, in turn, be reflected in the stress physiology of individuals (Clinchy et al. 2013). To explore these impacts, analyses of GC levels in the baleen of southern right whales (E. australis: Fernández Ajó et al. 2018) have been completed for a population located near Península Valdez that has been subject to high calf mortality in recent years (Sironi et al. 2015). Loss of calves is thought to be related to attacks by Kelp Gulls (Larus dominicanus), which cause significant injuries while feeding on the skin and blubber of young animals (Marón et al. 2015). Sampling of baleen from stranded whales has revealed elevated levels of both cortisol and corticosterone, with a clear relationship between GC levels and the severity of wounds resulting from gull predation (Fernández Ajó et al. 2018, Dickens & Romero 2013). Thus, even sub-lethal predation may affect GC responses in free-living mammals. While changes in GC levels are associated with the mobilization of energy required for antipredatory behavioral responses, relationships between physiological and behavioral variables may be complex in vertebrates. Studies of captive
C. talarum have revealed that cortisol secretion increases when these animals are exposed to predator odor and immobilization (simulating capture by a predator). However, the moderate magnitude of the resulting endocrine response is consistent with predation pressure that is alleviated by the use of subterranean burrows by this species. This physio logical response was accompanied by changes in performance in an elevated plus maze, providing evidence that predator exposure impacts both GC production and behavioral response (Brachetta et al. 2019).
d. Anthropogenic activities. As indicated above, GC levels may be influenced by a variety of conditions, including whether individuals are free-living or housed in captivity. For example, wild female C. sociabilis displayed higher baseline corticosterone levels than captive conspecifics (Woodruff et al. 2010), potentially reflecting the greater challenges (e.g. securing food, avoiding predators) associated with natural environments (McEwen & Wingfield 2003). Consistent with this, once in captivity, cortisol and corticosterone levels decrease markedly in C. talarum, with this change being particularly pronounced for females. Clearly, captive housing affects GC physiology in both of these species, although further studies are needed to understand whether the observed decrease in GC levels in captive animals is part of a chronic stress response to captivity (Dickens & Romero 2013). Human activity, including the direct manipulation of wild species as well as the modification of nat ural environments, may have important effects on stress physiology (Madliger et al. 2016). For example, physical restraint of guanacos during shearing has been shown to be associated with increases in cortisol levels as well as increases in heart rate and body temperature (Carmanchahi et al. 2011, Taraborelli et al. 2011). Similarly, vicuñas captured for shearing displayed elevated levels of cortisol (Bonacic & Macdonald 2003). In contrast, analyses of fecal GC metabolites for black and gold howler monkeys (A. caraya) revealed no differences in cortisol levels between animals living in human-fragmented versus continuous forest habitats (Cantarelli et al. 2017). Overall, these types of studies are critical to the conservation of threatened species such as guanacos, vicuñas, and howler monkeys and their associated habitats. At the same time, these findings underscore the complexity and context dependency of interactions among human activity, environmental conditions, and stress physiology.
Reproductive hormones
In numerous populations of free-living mammals, hormone secretion varies temporally, often in accord with reproductive seasonality. In this context, changes in energetic demands are associated with reproductive activity, and as a consequence, both GCs and reproductive hormones are fundamental components of the endocrine response (Place & Kenagy; Boonstra 2005; Reeder & Kramer 2005). Changes in reproductive hormone levels as a function of reproductive status have been reported based on analyses of urine from A. caraya (Raño et al 2018), plasma and urine from C. talarum (Vera et al. 2011b, 2013; Fanjul & Zenuto 2012, 2017), plasma and feces from the armadillos Chaetophractus villosus and Ch. vellerosus (Luaces et al. 2011), urine and feces from chinchillas (Busso et al. 2005, 2007; Mastromonaco et al. 2015), and feces from pichis Z. pichiy (Superina & Jahn 2009; Superina et al. 2009) and guanacos (Ovejero Aguilar 2016). In male C. talarum, testosterone peaked during the reproductive season although levels were highly variable among individuals, ranging from barely detectable to extremely high compared to other mammals (Vera et al. 2011b, 2013). In contrast, progesterone levels in this species did not differ between the reproductive and non- reproductive seasons (Vera et al. 2013). In captivity, female reproductive behavior varied with progesterone and oestradiol levels, with both hormone levels and vaginal cytology affected by the presence of a male (Fanjul & Zenuto 2012). Although females displayed clear preferences for dominant males as mates, dominance was inversely related to cortisol levels and showed no relationship with testosterone levels (Fanjul & Zenuto 2017). Hormone levels may also vary as a function of lifetime reproductive trajectory. For example, longitudinal anal yses of urine samples from an elderly and a young female black and gold howler monkey revealed that the younger, reproductively active female had higher average values of progesterone metabolites and estrone conjugates, thereby providing the first evidence of potential reproductive senescence in free-living A. caraya (Raño et al. 2018).
Because successful reproduction is essential to the persistence of threatened species, understanding how environmental conditions affect production of reproductive and glucocorticoid hormones may improve our ability to protect such species. To this end, studies such as those by Busso et al. (2005, 2007), who used urine to examine gonadal endocrine activity in chinchillas, may provide valuable information for conservation breeding programs for this and other species that are endangered in the wild. Similarly, gonadal endocrine activity has been assessed in male and female pichis (Ch. villosus), which are also of conservation concern in Argentina (Superina & Jahn 2009; Superina et al. 2009). Because the morphology of these animals makes it difficult to monitor reproductive status based on external cues or (for females) vaginal smears, use of endocrine data to track hormonal (progesterone) changes in feces is an important tool for monitoring the reproductive physiology of armadillos (Luaces et al. 2011) and, potentially, other species of mammals.
Given that hormones regulate such diverse aspects of organismal function as development, reproduction, metabolic rate, water balance, and response to stressors, our understanding of different endocrine processes represents a powerful method for evaluating how individuals respond to changing environmental conditions. Despite the importance of such information, relatively little work has been completed on the eco-endocrinology of Argentina mammals. Indeed, the first studies on this topic–analyses of armadillos and chinchillas– were completed only fourteen years ago. Although studies of guanacos, vicuñas, tuco-tucos, howler monkeys and whales have followed, the number and diver- sity of mammalian taxa for which endocrine data are available remains limited. Conceptually, the primary topic that has been explored is response to stressors associated with diet, predation, social interactions, and anthropogenic activity (direct manipulation of animals as well as changes to their habitats). While a few of these studies have also assessed impacts on other physiological parameters such as reproduction, much remains to be learned about interactions among these systems and their implications for management of both threatened and pest species. We assert that ecological endocrinology represents an important but underutilized tool for monitoring free-living animals in nature as well as implementing better, more controlled laboratory experiments. The information reviewed here should serve as a foundation for future studies of the eco- endocrinology of mammals from Argentina.
ECOLOGICAL IMMUNOLOGY
All living organisms are exposed to a wide range of pathogens and thus have evolved complex physiological responses to resist or to tolerate these threats (Råberg et al. 2008); collectively, these different lines of defense against invading pathogens constitute the immune system (Demas & Nelson 2012). Although pathogens are expected to exert strong selective pressures on their hosts by affecting variables intimately linked to fitness, hosts exhibit great variability in the strength and efficiency of their immune strategies. The field of ecological immunology, or “ecoimmunology”, aims to understand this variability by examining the interactions between host physiology (i.e. immune function) and disease ecology (i.e. pathogen prevalence) in a wide range of environments and animal species. This field of study first emerged at the beginning of the 21st century, with early studies focusing primarily on birds (i.e. Martin et al. 2003, 2006a; Eraud et al. 2005); among mammals, emphasis to date has been on surface-dwelling rodents (i.e. Derting & Compton 2003; Martin et al. 2006b). Within Argentina, research in this discipline began approximately 10 years ago with studies that explored energetic aspects of immunity as well as trade-offs between immunity and other costly processes in a number of local vertebrate species, with emphasis on field and laboratory studies of non-model mammalian species such as tuco-tucos (Ctenomys talarum; Cutrera et al. 2010, 2011; Merlo et al. 2014a, 2016b, 2018) and capybaras (Hydrochoerus hydrochaeris; Eberhardt et al. 2013, 2015).
Variation in disease susceptibility may be driven by extrinsic factors such as temporal and geographic changes in pathogen prevalence but can also be the result of intrinsic changes in host immunity, as well as interactions among these elements. This variability in the magnitude and efficiency of host immune responsiveness may have a genetic basis (Kubinak & Potts 2012) and/or may be attributed to the balance between the obvious benefits of mounting an immune response and the costs of doing so, a trade-off that also mediates interactions between immunity and other costly activities such as reproduction (Demas et al. 2012, Fig. 3). In this context, the role of Major Histocompatibility Complex (MHC) genes in immune recognition as well as the forces driving the extremely high levels of polymorphism that characterize these loci (i.e. pathogen selection, Doherty & Zinkernagel 1975) have been studied in three Argentine species of subterranean rodent, the Patagonian tuco-tuco C. haigi, the colonial tuco-tuco C. sociabilis (Hambuch & Lacey 2002), and the talas tuco-tuco (C. talarum; Cutrera & Lacey 2006, Cutrera et al. 2011, 2012, 2014). Here, however, we focus on studies that have assessed the costs of immunity and the trade-offs involving immune function that are mediated by these costs, as well as the critical insights that these analyses have generated into immune system function in natural populations of mammals.
Costs of immunity
Immunity, just like other physiological processes, requires energy to be activated and to be maintained over time. Accordingly, immune defense is expected to confer both fitness benefits in the form of resistance against pathogen infections and substantial costs, which can be energetic and/or nutritional, among others (Lochmiller & Deerenberg 2000, Hasselquist & Nilsson 2012). As a result, variability in immune strategies is expected to arise from the trade-offs between allocating resources to immunity versus other costly processes (Sheldon & Verhulst 1996). Although this dynamic makes it essential to estimate the direct metabolic costs of the different components of the immune system, few studies have explored this topic in mammals of Argentina (Cutrera et al. 2010; Merlo et al. 2014a). One exception are analyses of C. talarum, in which the energetic consequences of triggering an immune response were assessed using a novel non-pathogenic antigen (sheep red blood cells, SRBC) to activate the adaptive immune response (Cutrera et al. 2010). These authors reported a significant increase in oxygen consumption among immune-challenged (injected with SRBC) compared to control animals; this increase in oxygen consumption corresponds to a 15% increase in the daily energetic expenditure (DEE) of a tuco-tuco, which is a substantial change that may have significant impacts on body condition in wild individuals. Among the components of immunity, inflammation, which is triggered primarily by the entry of microorganisms or tissue damage, is considered to be associated with a large energetic expenditure arising from the production of lymphocytes required for this type of cell-mediated adaptive response (Klasing & Leshchinsky 1999). To test this assumption, Merlo et al. (2014a) used a subcutaneous injection of phytohemagglutinin (PHA) to induce an inflammatory response in C. talarum. PHA is a plant lectin that is widely used in ecological studies because it permits assessment of the costs of inflammation without triggering additional costs associated with pathogen replication and tissue damage to the host (Martin et al. 2006c). Results from tuco-tucos (Merlo et al. 2014b) indicate that, contrary to what has been demonstrated for birds (Martin et al. 2006), PHA triggers both innate and adaptive immune responses (presence of lymphocytes, neutrophils, eosinophils, and macrophages at the site of injection). This local inflammatory response, however, is not associated with increased oxygen consumption, indicating that an immune challenge may not always result in a detectable energetic cost. (Merlo et al. 2014a).
Studies that manipulate resource availability have tended to confirm that triggering and maintaining the immune system is energetically costly. However, interpretation of these results is challenging due to the effect that food restriction has on general condition; the reported effects of food manipulation on immunity may instead reflect more general impacts on individual condition (the so-called “vicious circle”, see Beldomenico & Begon 2010). To explore this dynamic, Merlo et al. (2016a) assessed the effect of diet manipulations on nutritional state, the magnitude of the inflammatory response to PHA, and other multiple immune parameters (e.g. bacterial killing capacity, natural antibodies, and leukocyte profile) in C. talarum. Individuals under food restriction displayed an altered nutritional status as well as increased stress levels (higher neutrophil:lymphocyte ratios) and a marked reduction in the inflammatory response to PHA compared to individuals fed ad libitum. Supplementation with methionine, however, did not affect any of the parameters analyzed and endoparasite loads were not affected by any treatments. These findings support the assertion that food insufficiency can modulate immune responsiveness through lack of adequate essential nutrients, through metabolic fuel and energetic reserves, or through detrimental effects of the stress caused by nutrient limitation.
Although initial studies of response to PHA (Merlo et al. 2014a) reported no substantial associated energetic cost in C. talarum, the response to PHA did involve pronounced nutritional demands; this is in contrast to what has been found regarding the nutritional requirements of the adaptive anti-body response to SRBC in this species. Antibody response against SRBC (Cutrera et al. 2010) did not vary in magnitude between animals subjected to slight versus severe food restriction (Schleich et al. 2015), suggesting that energetically demanding processes are not always more compromised during periods of nutrient deficiency than are processes with lower energetic costs. Nonetheless, nutrient limitation may be one of the most important environmental factors affecting immunocompetence and hence disease resistance. For example, Agostini et al. (2017) used an experimental approach to evaluate the reciprocal effects of nutritional status and parasitism in two groups of black capuchin monkeys (Sapajus nigritus) from Iguazú National Park in Misiones, Argentina. Food availability was manipulated by provisioning monkeys with bananas, and parasite burdens were modified by applying antiparasitic drugs to selected individuals. Although no effect of antiparasitic drugs on physical condition was detected, parasite loads decreased in response to high levels of food availability, lending support to the prediction of the “vicious circle” hypothesis (Beldomenico & Begon 2010) that nutritional status should impact parasite dynamics, and hence, physical condition.
Immune function trade-offs
Growth. The energetic costs of mounting and maintaining an immune response can also be assessed indirectly by determining whether exposure to immune challenge triggers the re-allocation of energy away from other costly physiological processes such as growth and reproduction (Derting & Compton 2003). In turn, the magnitude and efficiency of an immune response may be negatively affected by these other processes. For example, in sexually dimorphic species, members of the larger sex may invest less in immune response, presumably because they have to allocate more energy towards growth (Fargallo et al. 2002; Chin et al. 2005). In addition to a negative relationship between these physiological demands (Fair et al. 1999), immune response of young animals is expected to be lower than that of adults due to immaturity of the immune system. However, when juvenile C. talarum were challenged with PHA, they mounted a greater inflammatory response than adults with no differences in magnitude between the sexes despite the marked sexual dimorphism in body size among adult of this species (Cutrera et al. 2014). Clearly, the energetics of immune response are more complex than originally envisioned and understanding the energetic consequences of such responses requires further investigation of multiple aspects of an organism’s physiology during different developmental stages and periods of varying energetic demands.
Reproduction. Life-history theory predicts that individuals will adjust their investment in current reproduction to the level at which the sum of current and future fitness is maximized (Stearns 1992). As a consequence, particularly energetically costly reproductive stages may depress immunocompetence. In C. talarum, the increase in oxygen consumption triggered by challenging animals with SRBC was lower for females during the breeding season compared to the non-breeding season. Interestingly, antibody titers for females did not decrease during the breeding season (Cutrera et al. 2010), suggesting that future work should examine the potential role of pregnancy in facilitating the production of Th-2 cells and humoral immunity in this species (Cutrera et al. 2010). When PHA-induced inflammatory response was assessed, however, no differences in energetic investment or in the magnitude of the swelling response were found between the breeding and non- breeding seasons or between the sexes. Thus, in C. talarum, the inflammatory response appears to be less costly than the adaptive immune response with the result that the former may not be subject to significant trade-offs with other energy-demanding processes such as reproduction (Merlo et al. 2014a).
Parasitic infections. Existing parasitic infections may enhance or suppress immune response to a new challenge (Page et al. 2006). The negative effect of parasitism on immune response may reflect either competition for host resources by different pathogens or a general impoverishment of host condition (Cox 2001). Alternatively, immune processes triggered by simultaneous challenges could interact directly via induced immunodepression by parasites or synergistic or antagonistic effects on different immune pathways (Cox 2001). Interactions among different pathogens remain difficult to under stand, which is probably why few parasitological or ecological studies have focused on more than one pathogen. Recently, however, Colombo et al. (2015a, b) found numerous interactions among loads of immature stages of the tick Amblyomma triste and other ectoparasites of Akodon azarae, suggesting that co-infections may be important for tick dynamics. Merlo et al. (2016b) examined the influence of multiple challenges on immune responsiveness in C. talarum by comparing the impacts of the natural parasite community plus those of experimental infection with a coccidian (Eimeria sp.) on PHA- induced inflammation and a variety of immune, condition, nutrition, and stress parameters. In this species, the inflammatory response triggered by PHA was impaired by infection with Eimeria, either alone or in conjunction with infection by a number of gastrointestinal nematodes. However, with the exceptions of erythrocyte sedimentation rate and the magnitude of local swelling (both proxies of inflammation), no other physiological parameters were affected by parasitism. This is in contrast to data from capybara, in which parasitism by Trypanosoma evansi was associated with detrimental effects on host body condition as well as altered biochemical and hematological parameters (Eberhardt et al. 2017). Collectively, these findings suggest that parasitism is a key factor modulating immune responsiveness in at least some species of mammals from Argentina.
Effects of life history
In a seminal publication on ecological immunology, Lee (2006) proposed that the relative effort spent on either the innate or the adaptive portions of the immune system may be influenced by a species’ life history. Specifically, Lee (2006) predicted that the relative magnitude of the activation of each of these portions of the immune response should be related to the pace of life of an organism. Further, a species’ relative metabolic rate may impose limits on the synthetic processes involved in the adaptive immune response (Brown et al. 2004; Auer et al. 2018). Therefore, “slow-living” species with long lifespans and high investment in offspring are expected to exhibit stronger adaptive immunity, particularly with regard to antibody production, which is slower but confers immunological “memory” against repeated infections, which are more likely to occur in long-lived species. Merlo et al. (2019) explored this prediction using studies of C. talarum which, among rodents, is a slow-living species (Antinuchi et al. 2007; Busch et al. 1989; Zenuto et al. 2001, 2002a,b) that is exposed to a high prevalence of infections by a relatively limited suite of parasites (Rossin & Malizia 2002). These traits led Merlo et al (2019) to predict that C. talarum should rely more strongly on the adaptive rather than the innate component of immunity. However, injecting animals with PHA (triggers both innate and adaptive responses in tuco-tucos; Merlo et al. 2014b) and SRBC (allows estimation of adaptive immune response), revealed that the magnitudes and energetic costs of each portion of the immune response were similar; this outcome was the same for animals treated with one or both challenges, suggesting the absence of an energetic or resource-based trade-off between the responses triggered by PHA and SRBC (Merlo et al. 2019).
Implications for animal health. Beldomenico and Begon (2016) defined the health of a wild animal as the “state of balance between the stressors (including parasites) and the physiological regulatory network of the host (including the stress response and the immune function)”. In this context, assessing generic indices of the health of wild animals in their natural habitats not only increases our knowledge of their natural history, but also provides information required to evaluate interactions between extrinsic (i.e. seasonal) and intrinsic (i.e. genetic, neural, endocrine) factors affecting immune response. Among ecoimmunologists, there has been a growing tendency to use multiple functional assays to estimate the combined strength of the different portions of the immune system (Adamo 2012). Other measures such as behavioral patterns or proxies of physiological condition may help to estimate disease resistance. One of the first studies of a mammal of Argentina to measure multiple biochemical and physiological pa rameters (blood cell counts, serum protein fractions, and spleen mass) and to relate these parameters to differences in sex, body condition, pregnancy status, and season was conducted in free-living capybara (Eberhardt et al. 2015). In captivity, food restricted capybara displayed poorer body condition, higher eosinophil counts, and lower helminth counts than control animals (Eberhardt et al. 2013). In contrast, among free-living animals, body condition was negatively associated with eosinophil counts. These comparisons underscore the importance of physiological data from wild populations as a reference for experimental manipulations conducted under more controlled conditions (i.e. captive settings).
Multivariate analyses of the factors associated with prevalence of immature stages of the tick A. triste on one of its main hosts in Argentina, the rodent A. azarae (Colombo et al. 2015a), revealed significant associations among larval tick burdens and several environmental factors, including the presence of cattle. This outcome underscores the importance of considering the ecoepidemiological implications of human agricultural practices on dynamics of parasites such as of A. triste, which is the vector for the human pathogen Rickettsia parkeri in the southern cone of South America. More recently, Merlo et al. (2018) explored the influence of condition of individual C. talarum on the magnitude of the inflammatory response induced by PHA. Their results provide further evidence that parasitism, reproductive condition and leukocyte counts affect response to this challenge, which is one of the most frequently-used tests to assess immunocompetence in wild species of mammals.
In sum, ecoimmunological studies conducted to date on Argentine mammals have generated multiple new insights into the factors that shape variation in immune responsiveness in wild species in their natu ral habitats. Further, these studies have contributed to improved conservation strategies for species subject to disease threats, such as brown howler monkeys (Alouatta guariba clamitans) and susceptibility to yellow fever (Agostini et al. 2014; Moreno et al. 2015). Application of ecoimmunological principles has also contributed to greater knowledge of host-parasite dynamics involving species of public health concern, such as relationships between A. triste, which carries Rikettsia, and its primary host, the rodent A. azarae (Colombo et al. 2015b). Finally, these studies have provided important information regarding the role of wild species as vectors of diseases that affect domestic animals (Marcoppido et al. 2010).
FUTURE DIRECTIONS
Conducting a review on the development and degree of advancement of a given scientific discipline is informative for many reasons. By assessing the extent of current understanding of a topic, questions and lines of inquiry in need of further development can be identified. Further, these efforts help to acknowledge the value of specific disciplines, such as the role of ecological physiology in understanding individual responses to changing environmental con ditions and associated impacts on population status, management and conservation. As a result, reviews such as this one allow us to carry out more robust research and contribute to increased knowledge of methods and tools needed to preserve biodiversity. In sum, periodically reviewing biological disciplines should facilitate the expansion of scientific knowledge, the conservation of our fauna, and the efficient management of natural systems.
To date, studies of the ecological physiology of Argentine mammals have focused on only a limited number of species. Not surprisingly, taxonomic eepresentation is particularly narrow for newer conceptual themes such as ecological immunology. Even more established topics such as energetics, however, have been explored primarily in rodents, most likely reflecting the challenges associated with monitoring many lineages of mammals in their natural habitats or establishing those species in the laboratory. Future studies will benefit from including a wider diversity of mammalian taxa as study subjects. This enhanced diversity will allow greater comparison across species, thereby facilitating our understanding of the roles of life history, habitat, and phylogeny in ecological physiology.
Field and laboratory studies are complementary and, together, allow a more comprehensive understanding of the factors affecting the ecological physiologyof animals. While not all mammal species are amenable to captive housing, more can be done to integrate data from free-living populations with information obtained under more controlled conditions. Such efforts are important because they generate valuable information on performance in both types of settings; contrasts between data sets are often particularly informative regarding the factors affecting physiological and other aspects of organismal function.
In all environments, use of multiple biomarkers is essential to characterizing the physiological profile of an organism. This includes assessment of body condition, health, and stress parameters, all of which may influence the specific physiological responses discussed here. Studies of Argentine mammals have begun to use multiple indicators of condition, recognizing that their interdependence reflects their role as regulatory components of a single, unified physiological system. We urge that this effort be expanded so as to improve our understanding of the diverse factors that drive observed variation in energy use, immune responsiveness, and ability to cope with stressful situations among members of free-living mammals in their natural habitats.