A host of issues: pseudoreplication in host-microbiota studies.

Pseudoreplication compromises the validity of research by treating non-independent samples as independent replicates. This review examines the prevalence of pseudoreplication in host-microbiota studies, highlighting the critical need for rigorous experimental design and appropriate statistical analysis. We systematically reviewed 115 manuscripts on host-microbiota interactions. Our analysis revealed that 22% of the papers contained pseudoreplication, primarily due to co-housed organisms, whereas 52% lacked sufficient methodological details. The remaining 26% adequately addressed pseudoreplication through proper experimental design or statistical analysis. The high incidence of pseudoreplication and insufficient information underscores the importance of methodological reporting and statistical rigor to ensure reproducibility of host-microbiota research.

The Costs of Living Together: Immune Responses to the Microbiota and Chronic Gut Inflammation.

While the vertebrate microbiota is critical to the normal function of many host traits, hosts may expend a large amount of energy to constrain and interface with their microbiota via their immune system to avoid the high fitness costs associated with gut dysbiosis, pathobionts, and opportunistic pathogens. All jawed vertebrates share mucosal immunity dedicated to isolating the microbiota, and a breakdown of this system can result in chronic gut inflammation. In humans, chronic gut inflammation negatively affects growth and development. There is little information available on the prevalence of chronic gut inflammation in wild animals, but given that animals with different life histories emphasize different immune responses, it follows that wild animals may vary in their susceptibility to chronic gut inflammation, and most animals will experience signaling that can lead to this state. These can be top-down signals originating from sources like the central nervous system or bottom-up signals originating from changes in the gut microbiota. The sources of these signals might include stress, developmental transitions, food restriction, and dietary shifts. Here, we briefly discuss host-microbiota interactions from the perspective of life history theory and ecoimmunology, focusing on the mucosal immune system and chronic gut inflammation. We also include future directions for research and the tools necessary to investigate them.

Manipulation of gut microbiota during critical developmental windows affects host physiological performance and disease susceptibility across ontogeny.

Colonization of gut microbiomes during early life can shape metabolism and immunity of adult animals. However, most data are derived from antibiotic-treated or germ-free laboratory mammals. Furthermore, few studies have explored how microbial colonization during critical windows influences a suite of other fitness-related traits in wild animals. This study tested whether hatching constitutes a critical developmental window for gut microbiome colonization in wild-caught amphibians and whether perturbations to gut microbiota at hatching shape fitness-related traits of larval growth, metabolism, metamorphosis and disease susceptibility. We sterilized wood frog eggs and then inoculated them with microbes from differing sources, including from another species (bullfrogs) that differ in disease resistance and life history. We measured development, growth and metabolic rates through metamorphosis among individuals from each microbial treatment. A separate group was exposed to an LD50 dose of ranavirus-an emerging disease-to test for microbiome effects on disease susceptibility. We also quantified rates of deformities to test for microbial treatment effects on overall health. Manipulation of microbiota on eggs altered the trajectory of gut microbiome communities across larval ontogeny, though disruption appeared to be transitory. While microbiome structure converged among the treatments by metamorphosis, the effects of disruption on host phenotypes persisted. Larvae inoculated with the bullfrog gut microbiota exhibited accelerated growth and development rates compared to controls. By contrast, sterilized larvae maintained in sterile water for several days after hatching exhibited greater disruption to their gut microbiota across ontogeny, as well as altered metabolism, more tail deformities, and were more likely to die when exposed to an LD50 dose of ranavirus compared to the other treatments. These results suggest perturbations to the microbiota during critical developmental windows can alter the trajectory of the gut microbiome, and have long-term effects on fitness-related traits in larval amphibians. These results suggest that explicit tests of how changes in the composition and abundance of the microbial community shape phenotypes across ontogeny in amphibians could shed light on host-microbe interactions in wildlife, as well as inform conservation efforts to mitigate emerging diseases.

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.

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

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.

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.

An improved method to assess the encapsulation response in arthropods.

Ecoimmunology explores how ecological factors and evolutionary processes influence immune responses across various taxa and how immune responses trade-off with other traits. Studying immune responses requires biologically meaningful immunoassays applicable to a broad range of taxa and are sensitive enough to detect changes in the immune response. Useful immunoassays should also correlate with immunocompetence and fitness. The encapsulation response, a complex immune mechanism in arthropods, serves as a robust method for ecoimmunological investigations. However, traditional methods to test the encapsulation response can require long training. This study introduces an innovative, cost-effective method for assessing the encapsulation immune response in arthropods, which simplifies the procedure by reducing the training time and skill required. Our modified device utilizes a pen and syringe assembly for inserting monofilaments into arthropod larvae. We compared our device against traditional methods. Despite the new method being 22% faster, it did not compromise the accuracy or effectiveness of the encapsulation response when compared with traditional techniques, demonstrating similar degrees of melanization and encapsulation. Our method allowed for more accessible participation by less experienced researchers, such as undergraduates, facilitating their involvement in ecoimmunological research.

Infection Causes Trade-Offs between Development and Growth in Larval Amphibians.

AbstractTrade-offs between life history traits are context dependent; they vary depending on environment and life stage. Negative associations between development and growth often characterize larval life stages. Both growth and development consume large parts of the energy budget of young animals. The metabolic rate of animals should reflect differences in growth and developmental rates. Growth and development can also have negative associations with immune function because of their costs. We investigated how intraspecific variation in growth and development affected the metabolism of larval amphibians and whether intraspecific variation in growth, development, and metabolic rate could predict mortality and viral load in larvae infected with ranavirus. We also compared the relationship between growth and development before and after infection with ranavirus. We hypothesized that growth and development would affect metabolism and predicted that each would have a positive correlation with metabolic rate. We further hypothesized that allocation toward growth and development would increase ranavirus susceptibility and therefore predicted that larvae with faster growth, faster development, and higher metabolic rates would be more likely to die from ranavirus and have higher viral loads. Finally, we predicted that growth rate and developmental rate would have a negative association. Intraspecific variation in growth rate and developmental rate did not affect metabolism. Growth rate, developmental rate, and metabolism did not predict mortality from ranavirus or viral load. Larvae infected with ranavirus exhibited a trade-off between developmental rate and growth rate that was absent in uninfected larvae. Our results indicate a cost of ranavirus infection that is potentially due to both the infection-induced anorexia and the cost of infection altering priority rules for resource allocation.

Early life disruption of the microbiota affects organ development and cytokine gene expression in threespine stickleback.

The microbiota that inhabits vertebrates exerts strong effects on host physiology and can be crucial to the development of a normal phenotype. This includes development of the immune system, somatic growth and maintenance, and morphogenesis. However, the genetic background of the host can also affect these life history traits. To this end, we investigated the effects of the microbiota on growth, development, and immune gene expression on two populations of threespine stickleback (Gasterosteus aculeatus), one anadromous and one freshwater. We tested the hypotheses that microbial colonization and the genetic background of the host would affect survival, cytokine gene expression, growth, and development. We raised in vitro crosses of stickleback larvae with and without conventional microbiota. We then exposed all these treatments to Vibrio anguillarum, a potential fish pathogen, in a full factorial design. We found stickleback raised without conventional microbiota had smaller swim bladders relative to those raised with conventional microbiota. Stickleback raised with conventional microbiota exhibited small increases in cytokine gene expression. We found no differences in growth or survival regardless of treatment. These results are consistent with other investigations that show microbiota disruption, in early life, can alter host organ and tissue development and immune responses.

Sex and life history shape the strength of cellular and humoral immune responses in a wing dimorphic cricket.

Immune function is a complex collection of responses that often trade-off with one another and with other life history traits, because of the high costs of mounting and maintaining immune responses. Animals, even those from the same populations, may emphasize different aspects of immune function depending on their habitat and phenotype. For example, host population density mediates the threat from density-dependent parasites. Animals at high densities may emphasize fast-acting humoral responses, while those at low densities may favor slower, but more specific, cellular responses. However, these predictions may be dependent on other life history traits, like sex, which is associated with variation in many immune responses. We used wing dimorphic Gryllus firmus crickets to test humoral responses, measured by lysozyme and phenoloxidase activities, and cellular immune responses, measured by encapsulation, between morphs and sex. We found that both morphs and sexes differed in aspects of immune function. Long wing morphs had stronger encapsulation responses than short winged morphs. Additionally, females exhibited higher PO activity than males, and by contrast, males had higher lysozyme activity than females. Our study suggests that G. firmus morphs prioritize different immune responses that may reflect a balancing between the costs of immunity and differing pathogen threats. Male and female crickets exhibit differences in humoral immune responses that may reflect their different life history demands.

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.

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.

Influence of Physiological Stress on Nutrient Stoichiometry in Larval Amphibians.

Exposure to environmental stressors alters animal phenotypes as well as nutrient metabolism, assimilation, and excretion. While stress-induced shifts in nutrient processes are known to alter organismal carbon (C) and nitrogen (N) stoichiometry, there has been little exploration of how environmental factors influence phosphorous (P). A better understanding of how P cycling varies with animal physiological state may provide insight into across-scale processes, because P is essential to animal function and ecological processes such as production and decomposition. We tested the effects of predator stress and exogenous glucocorticoids on C∶N∶P stoichiometry of larval amphibians. Glucocorticoids altered nutrient stoichiometry, apparently by modulating ossification and renal function. This reduced whole-body P and significantly increased N∶P. Additionally, elevated glucocorticoids caused a long-term reduction in P excretion. This reduction may reflect an initial unmeasured loss of P that glucocorticoids induce over acute timescales. In contrast, exposure to predator cues had no effect on larval C∶N∶P stoichiometry, which highlights that different stressors have varied effects on the endocrine stress response. Predation, in particular, is ubiquitous in the environment; thus, larvae responding to predators have conserved mechanisms that likely prevent or minimize physiological disruption. These results demonstrate the differing physiological roles of N and P, distinct nutrient demands associated with amphibian metamorphosis, and the contrasting effects that different environmental factors have on the physiological stress response. Our results also suggest that anthropogenic changes to the environment that induce chronic stress in amphibians could affect the biogeochemistry of nutrient-poor environments where they may act as keystone species.