Robin W. Warne

Co-Infection by Chytrid Fungus and Ranaviruses in Wild and Harvested Frogs in the Tropical Andes

While global amphibian declines are associated with the spread of Batrachochytrium dendrobatidis (Bd), undetected concurrent co-infection by other pathogens may be little recognized threats to amphibians. Emerging viruses in the genus Ranavirus (Rv) also cause die-offs of amphibians and other ectotherms, but the extent of their distribution globally, or how co-infections with Bd impact amphibians are poorly understood. We provide the first report of Bd and Rv co-infection in South America, and the first report of Rv infections in the amphibian biodiversity hotspot of the Peruvian Andes, where Bd is associated with extinctions. Using these data, we tested the hypothesis that Bd or Rv parasites facilitate co-infection, as assessed by parasite abundance or infection intensity within individual adult frogs. Co-infection occurred in 30% of stream-dwelling frogs; 65% were infected by Bd and 40% by Rv. Among terrestrial, direct-developing Pristimantis frogs 40% were infected by Bd, 35% by Rv, and 20% co-infected. In Telmatobius frogs harvested for the live-trade 49% were co-infected, 92% were infected by Bd, and 53% by Rv. Median Bd and Rv loads were similar in both wild (Bd = 101.2 Ze, Rv = 102.3 viral copies) and harvested frogs (Bd = 103.1 Ze, Rv = 102.7 viral copies). While neither parasite abundance nor infection intensity were associated with co-infection patterns in adults, these data did not include the most susceptible larval and metamorphic life stages. These findings suggest Rv distribution is global and that co-infection among these parasites may be common. These results raise conservation concerns, but greater testing is necessary to determine if parasite interactions increase amphibian vulnerability to secondary infections across differing life stages, and constitute a previously undetected threat to declining populations. Greater surveillance of parasite interactions may increase our capacity to contain and mitigate the impacts of these and other wildlife diseases.

Capital Breeding and Allocation to Life-History Demands Are Highly Plastic in Lizards

The use of stored resources to fuel reproduction, growth, and self-maintenance in the face of uncertain nutrient availability is a tactic common to many organisms. The degree to which organisms rely on stored resources in response to varied nutrients, however, is not well quantified. In this study, we used stable isotope methods to quantify the use of stored versus incoming nutrients to fuel growth and egg and fat body development in lizards under differing nutrient regimes. We found that the degree of capital breeding is a function of an individual’s body condition. Furthermore, given sufficient income, lizards in poor condition can allocate simultaneously to storage, growth, and reproduction and “catch up” in body size and reproductive allocation to better-conditioned animals. Using natural variation in the δ13C of environmental nutrient pulses, we also found a high degree of variation in capital breeding in a lizard community. These findings demonstrate that capital breeding in lizards is not simply a one-way flow of endogenous stores to eggs but is a function of the condition state of individuals and seasonal nutrient availability. We use our findings to comment on capital breeding in lizards and the utility of the capital-income concept in general.

Exogenous stress hormones alter energetic and nutrient costs of development and metamorphosis

ABSTRACT Variation in environmental conditions during larval life stages can shape development during critical windows and have lasting effects on the adult organism. Changes in larval developmental rates in response to environmental conditions, for example, can trade off with growth to determine body size and condition at metamorphosis, which can affect adult survival and fecundity. However, it is unclear how use of energy and nutrients shape trade-offs across life-stage transitions because no studies have quantified these costs of larval development and metamorphosis. We used an experimental approach to manipulate physiological stress in larval amphibians, along with respirometry and 13C-breath testing to quantify the energetic and nutritional costs of development and metamorphosis. Central to larval developmental responses to environmental conditions is the hypothalamic–pituitary–adrenal/interrenal (HPA/I) axis, which regulates development, as well as energy homeostasis and stress responses across many taxa. Given these pleiotropic effects of HPA/I activity, manipulation of the HPA/I axis may provide insight into costs of metamorphosis. We measured the energetic and nutritional costs across the entire larval period and metamorphosis in a larval amphibian exposed to exogenous glucocorticoid (GC) hormones – the primary hormone secreted by the HPA/I axis. We measured metabolic rates and dry mass across larval ontogeny, and quantified lipid stores and nutrient oxidation via 13C-breath testing during metamorphosis, under control and GC-exposed conditions. Changes in dry mass match metamorphic states previously reported in the literature, but dynamics of metabolism were influenced by the transition from aquatic to terrestrial respiration. GC-treated larvae had lower dry mass, decreased fat stores and higher oxygen consumption during stages where controls were conserving energy. GC-treated larvae also oxidized greater amounts of 13C-labelled protein stores. These results provide evidence for a proximate cause of the physiological trade-off between larval growth and development, and provide insight into the energetic and nutrient costs that shape fitness trade-offs across life stages. Summary: The energetic and nutrient costs of glucocorticoid-accelerated development alter fitness trade-offs across life stages.

Immune function trade-offs in response to parasite threats

Immune function is often involved in physiological trade-offs because of the energetic costs of maintaining constitutive immunity and mounting responses to infection. However, immune function is a collection of discrete immunity factors and animals should allocate towards factors that combat the parasite threat with the highest fitness cost. For example, animals on dispersal fronts of expanding population may be released from density-dependent diseases. The costs of immunity, however, and life history trade-offs in general, are often context dependent. Trade-offs are often most apparent under conditions of unusually limited resources or when animals are particularly stressed, because the stress response can shift priorities. In this study we tested how humoral and cellular immune factors vary between phenotypes of a wing dimorphic cricket and how physiological stress influences these immune factors. We measured constitutive lysozyme activity, a humoral immune factor, and encapsulation response, a cellular immune factor. We also stressed the crickets with a sham predator in a full factorial design. We found that immune strategy could be explained by the selective pressures encountered by each morph and that stress decreased encapsulation, but not lysozyme activity. These results suggest a possible trade-off between humoral and cellular immunity. Given limited resources and the expense of immune factors, parasite pressures could play a key factor in maintaining insect polyphenism via disruptive selection.

Pouch brooding marsupial frogs transfer nutrients to developing embryos

Marsupial frogs have a unique reproductive mode in which females carry eggs enclosed in a sealed dorsal brood pouch. While most anurans are considered to be oviparous with lecithotrophic eggs, the extensively vascularized membrane of the brood pouch in marsupial frogs suggests potential opportunities for nutrient transfer. We tested for matrotrophy in the live-bearing Gastrotheca excubitor (Hemiphractidae), through feeding insects labelled with a 13C-fatty acid and a 15N-amino acid to brooding marsupial frogs. We observed significant increases of δ13C and δ15N in both maternal pouch tissues and embryos, suggesting nutrient transfer. Embryo dry mass also increased with developmental stage, providing further direct evidence for matrotrophy. These results suggest that in addition to gas exchange, the vascularized brood pouch membrane of G. excubitor also enables maternal nutrient transfer. This finding revealed a suspected but untested trait in the evolution of parental care in marsupial frogs, in contrast to previous work on Gastrotheca species that release tadpoles, and suggests greater complexity in reproductive and provisioning modes than previously thought.

Critical disease windows shaped by stress exposure alter allocation trade-offs between development and immunity

Ubiquitous environmental stressors are often thought to alter animal susceptibility to pathogens and contribute to disease emergence. However, duration of exposure to a stressor is likely critical, because while chronic stress is often immunosuppressive, acute stress can temporarily enhance immune function. Furthermore, host susceptibility to stress and disease often varies with ontogeny; increasing during critical developmental windows. How the duration and timing of exposure to stressors interact to shape critical windows and influence disease processes is not well tested. We used ranavirus and larval amphibians as a model system to investigate how physiological stress and pathogenic infection shape development and disease dynamics in vertebrates. Based on a resource allocation model, we designed experiments to test how exposure to stressors may induce resource trade-offs that shape critical windows and disease processes because the neuroendocrine stress axis coordinates developmental remodelling, immune function and energy allocation in larval amphibians. We used wood frog larvae (Lithobates sylvaticus) to investigate how chronic and acute exposure to corticosterone, the dominant amphibian glucocorticoid hormone, mediates development and immune function via splenocyte immunohistochemistry analysis in association with ranavirus infection. Corticosterone treatments affected immune function, as both chronic and acute exposure suppressed splenocyte proliferation, although viral replication rate increased only in the chronic corticosterone treatment. Time to metamorphosis and survival depended on both corticosterone treatment and infection status. In the control and chronic corticosterone treatments, ranavirus infection decreased survival and delayed metamorphosis, although chronic corticosterone exposure accelerated rate of metamorphosis in uninfected larvae. Acute corticosterone exposure accelerated metamorphosis increased survival in infected larvae. Interactions between stress exposure (via glucocorticoid actions) and infection impose resource trade-offs that shape optimal allocation between development and somatic function. As a result, critical disease windows are likely shaped by stress exposure because any conditions that induce changes in differentiation rates will alter the duration and susceptibility of organisms to stressors or disease.

Manipulation of Gut Microbiota Reveals Shifting Community Structure Shaped by Host Developmental Windows in Amphibian Larvae

Exploration of the importance of developmental windows for microbial colonization in diverse animal taxa, and tests of how these shape both animal microbiomes as well as host phenotypes promise to shed needed light on host-microbe interactions. The aims of this study were to explore how gut microbiota diversity of larval amphibians varies among species and across ontogeny, and to test if manipulation of gut colonization can reveal how microbiomes develop. We found that gut microbiomes differ among species and change across larval ontogeny, with distinctive differences between larvae, metamorphic animals, and juvenile frogs. Through applying a gnotobiotic protocol to eggs and cross-inoculating gut microbiomes between species, we demonstrated that microbiota can be transplanted among species and developmental stages. These results also demonstrated that microbial colonization at hatching is potentially formative for long term composition and function of amphibian gut microbiomes, suggesting that hatching may be a critical developmental window for colonization, similar to the effects of birth mode on human microbiomes. Specifically, our results suggest that either the egg jelly and/or capsules surrounding amphibian eggs are likely important sources for initial microbiome inoculation. Furthermore, we speculate these results suggest that vertical transmission may be important to amphibian microbiome establishment and development, as is common among many animal taxa. Taken together, our results suggest that explicit tests of how host developmental windows influence microbial colonization, and shape amphibian microbiomes across life stages promise to provide insight into the ecological and evolutionary dynamics of host-microbe interactions.

Behavioural phenotypes predict disease susceptibility and infectiousness

Behavioural phenotypes may provide a means for identifying individuals that disproportionally contribute to disease spread and epizootic outbreaks. For example, bolder phenotypes may experience greater exposure and susceptibility to pathogenic infection because of distinct interactions with conspecifics and their environment. We tested the value of behavioural phenotypes in larval amphibians for predicting ranavirus transmission in experimental trials. We found that behavioural phenotypes characterized by latency-to-food and swimming profiles were predictive of disease susceptibility and infectiousness defined as the capacity of an infected host to transmit an infection by contacts. While viral shedding rates were positively associated with transmission, we also found an inverse relationship between contacts and infections. Together these results suggest intrinsic traits that influence behaviour and the quantity of pathogens shed during conspecific interactions may be an important contributor to ranavirus transmission. These results suggest that behavioural phenotypes provide a means to identify individuals more likely to spread disease and thus give insights into disease outbreaks that threaten wildlife and humans.

A novel framework for predicting the use of facultative heterothermy by endotherms.

Expression of body temperature (Tb) is an important factor affecting fitness of all organisms, including endotherms (Angilletta et al., 2010; Kingsolver, 2009). At the extreme of the thermoregulatory spectrum in endotherms, the facultative heterothermic responses of torpor and hibernation have long received considerable attention and, without doubt, the vast majority of studies in this field have focused on the energetic benefits of abandoning homeothermy under certain situations (Geiser and Brigham, 2012). Recently, many authors have begun advocating for a move away from largely post hoc speculation based upon descriptive Tb patterns towards explicitly testing the costs and benefits of various thermoregulatory patterns seen in endotherms (e.g., Angilletta et al., 2010; Humphries et al., 2003), much like the work that has dominated the ectotherm literature for decades (Huey and Slatkin, 1976; Kingsolver, 2009). Here we give one example of a framework, drawing from several related subdisciplines, to predict which species should be most likely to use facultative heterothermy and to what degree it should be used. We begin with the idea that performance of endotherms is thermally dependent (Choi et al., 1998), as has been repeatedly shown in ectotherms (Angilletta, 2009). Body temperature likely affects organisms at every level of organization (Clarke, 2003) from protein synthesis and stability (Somero, 1995) to intercellular signaling (Socha et al., 2011) and performance of tissue groups (Bennett, 1985). Collectively, thermal dependence of molecular, cellular, and system level processes should thus lead to wholeorganism thermal dependence as well (Kassahn et al., 2009). With the adoption of new technology, evidence is mounting that considerable variation in Tb exists across a wide spectrum of mammals and birds (Boyles et al., 2013); given this, it makes sense that thermal sensitivity in performance should also vary among populations and species that show different thermoregulatory patterns (Angilletta et al., 2010). Specifically, performance of endotherms that show greater fluctuations in Tb during active periods should be less sensitive to those fluctuations than those that commonly maintain a very narrow range of Tbs. In other words, thermoregulation and thermal sensitivity should be coadapted (Fig. 1a and b). It is also logical to further predict that if decreased performance directly affects fitness, organisms should either (a) increase homeostatic responses as Tb decreases or (b) use facultative heterothermy to avoid the decreased fitness costs altogether. On the ecological timescale, organisms can maintain homeostasis though increased energetic expenditure and behavioral thermoregulation to return Tb to optimal or increased production of chaperone proteins to limit the effects of decreased Tb; chaperone protein production is also likely related to the process of seasonal thermal acclimation. On an evolutionary timescale, adaptations in homeostatic responses may be represented by altered reaction norms among populations and species in traits that include molecular (e.g., increased constitutive heat shock protein expression), physiological (e.g., increased metabolic rates), morphological (e.g., increased size) or ecological (e.g., range shifts). Expanding these two ideas (performance is thermally dependent and endotherms must respond in some way to decreased performance during the active phase) to make predictions about the conditions under which endotherms should use facultative heterothermy is relatively straightforward. Take two hypothetical species (or populations) with varying thermoregulatory characteristics: one that maintains relatively tight Tb during the active period (i.e, a thermal specialist) and one that allows its Tb to fluctuate several degrees (i.e, a thermal generalist) (Fig. 1a). The (admittedly over-simplistic) idea of a generalist-specialist trade-off predicts thermal specialists should experience a greater drop-off in performance as Tb decreases (Angilletta et al., 2006). Specialists should therefore either mount an earlier and greater homeostatic response (e.g., increased metabolic rate) or use other methods (e.g., torpor) to avoid the performance effects of fluctuations in Tb. Furthermore, specialists may reach the point where the attempted maintenance of homeostasis causes more harm than good (roughly analogous to the “homeostatic overload” defined by Romero et al. (2009)) faster than do generalists. Under this model, we counter-intuitively predict that species that are “good” thermoregulators should be more prone to facultative heterothermy to avoid the costs of fluctuating Tb during activity than those that are “poor” thermoregulators (assuming both species are capable of torpor). It may seem that we are arguing all species that maintain a very narrow range of Tbs should therefore also display torpor, but this is not the case. Our argument is broader in that we predict specialists should either use torpor or display earlier and greater homeostatic responses to fluctuations in Tb than generalists. In this regard, homeostatic responses such as increased molecular expression of stress proteins (e.g., heat shock proteins) and stress hormones (e.g., glucocorticoids) may play central roles in the expression of torpor. This is because generalists, for example, may express these molecules at relatively higher constitutive levels to buffer against greater fluctuations in Tb. While studies have shown increased constitutive levels of homeostatic molecules across populations exposed to increased levels of chronic thermal stress (Calabria et al., 2012; Schwartz and Bronikowski, 2013; Sørensen, 2010; Ulmasov et al., 1992), their role in regulating torpor expression have not been studied; this is an interesting avenue for future research. Of course framing these explorations within evolutionary history are essential. There are certainly many reasons why ‘homeothermic’ species do not use torpor: evolutionary history may preclude the use of torpor in some species, and tropical or