Variability in environmental persistence but not per capita transmission rates of the amphibian chytrid fungus leads to differences in host infection prevalence.

Heterogeneities in infections among host populations may arise through differences in environmental conditions through two mechanisms. First, environmental conditions may alter host exposure to pathogens via effects on survival. Second, environmental conditions may alter host susceptibility, making infection more or less likely if contact between a host and pathogen occurs. Further, host susceptibility might be altered through acquired resistance, which hosts can develop, in some systems, through exposure to dead or decaying pathogens and their metabolites. Environmental conditions may alter the rates of pathogen decomposition, influencing the likelihood of hosts developing acquired resistance. The present study primarily tests how environmental context influences the relative contributions of pathogen survival and per capita transmission on host infection prevalence using the amphibian chytrid fungus (Batrachochytrium dendrobatidis; Bd) as a model system. Secondarily, we evaluate how environmental context influences the decomposition of Bd because previous studies have shown that dead Bd and its metabolites can illicit acquired resistance in hosts. We conducted Bd survival and infection experiments and then fit models to discern how Bd mortality, decomposition and per capita transmission rates vary among water sources [e.g. artificial spring water (ASW) or water from three ponds]. We found that infection prevalence differed among water sources, which was driven by differences in mortality rates of Bd, rather than differences in per capita transmission rates. Bd mortality rates varied among pond water treatments and were lower in ASW compared to pond water. These results suggest that variation in Bd infection dynamics could be a function of environmental factors in waterbodies that result in differences in exposure of hosts to live Bd. In contrast to the persistence of live Bd, we found that the rates of decomposition of dead Bd did not vary among water sources, which may suggest that exposure of hosts to dead Bd or its metabolites might not commonly vary among nearby sites. Ultimately, a mechanistic understanding of the environmental dependence of free-living pathogens could lead to a deeper understanding of the patterns of outbreak heterogeneity, which could inform surveillance and management strategies.

The biomechanics of tree frogs climbing curved surfaces: a gripping problem.

The adhesive mechanisms of climbing animals have become an important research topic because of their biomimetic implications. We examined the climbing abilities of hylid tree frogs on vertical cylinders of differing diameter and surface roughness to investigate the relative roles of adduction forces (gripping) and adhesion. Tree frogs adhere using their toe pads and subarticular tubercles, the adhesive joint being fluid-filled. Our hypothesis was that on an effectively flat surface (adduction forces on the largest 120 mm diameter cylinder were insufficient to allow climbing), adhesion would effectively be the only means by which tree frogs could climb, but on the 44 and 13 mm diameter cylinders, frogs could additionally utilise adduction forces by gripping the cylinder either with their limbs outstretched or by grasping around the cylinder with their digits, respectively. The frogs' performance would also depend on whether the surfaces were smooth (easy to adhere to) or rough (relatively non-adhesive). Our findings showed that climbing performance was highest on the narrowest smooth cylinder. Frogs climbed faster, frequently using a 'walking trot' gait rather than the 'lateral sequence walk' used on other cylinders. Using an optical technique to visualise substrate contact during climbing on smooth surfaces, we also observed an increasing engagement of the subarticular tubercles on the narrower cylinders. Finally, on the rough substrate, frogs were unable to climb the largest diameter cylinder, but were able to climb the narrowest one slowly. These results support our hypotheses and have relevance for the design of climbing robots.

Do host-associated gut microbiota mediate the effect of an herbicide on disease risk in frogs?

Environmental stressors, such as pollutants, can increase disease risk in wildlife. For example, the herbicide atrazine affects host defences (e.g. resistance and tolerance) of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), but the mechanisms for these associations are not entirely clear. Given that pollutants can alter the gut microbiota of hosts, which in turn can affect their health and immune systems, one potential mechanism by which pollutants could increase infection risk is by influencing host-associated microbiota. Here, we test whether early-life exposure to the estimated environmental concentration (EEC; 200 µg/L) of atrazine affects the gut bacterial composition of Cuban tree frog (Osteopilus septentrionalis) tadpoles and adults and whether any atrazine-induced change in community composition might affect host defences against Bd. We also determine whether early-life changes in the stress hormone corticosterone affect gut microbiota by experimentally inhibiting corticosterone synthesis with metyrapone. With the exception of changing the relative abundances of two bacterial genera in adulthood, atrazine did not affect gut bacterial diversity or community composition of tadpoles (in vivo or in vitro) or adults. Metyrapone did not significantly affect bacterial diversity of tadpoles, but significantly increased bacterial diversity of adults. Gut bacterial diversity during Bd exposure did not predict host tolerance or resistance to Bd intensity in tadpoles or adults. However, early-life bacterial diversity negatively predicted Bd intensity as adult frogs. Specifically, Bd intensity as adults was associated negatively with the relative abundance of phylum Fusobacteria in the guts of tadpoles. Our results suggest that the effect of atrazine on Bd infection risk is not mediated by host-associated microbiota because atrazine does not affect microbiota of tadpoles or adults. However, host-associated microbes seem important in host resistance to Bd because the early-life microbiota, during immune system development, predicted later-life infection risk with Bd. Overall, our study suggests that increasing gut bacterial diversity and relative abundances of Fusobacteria might have lasting positive effects on amphibian health.

Effects of temperature and force requirements on muscle work and power output.

Performance of muscle-powered movements depends on temperature through its effects on muscle contractile properties. In vitro stimulation of Cuban treefrog (Osteopilus septentrionalis) plantaris muscles reveals that interactions between force and temperature affect the mechanical work of muscle. At low temperatures (9-17°C), muscle work depends on temperature when shortening at any force, and temperature effects are greater at higher forces. At warmer temperatures (13-21°C), muscle work depends on temperature when shortening with intermediate and high forces (≥30% peak isometric tetanic force). Shortening velocity is most strongly affected by temperature at low temperatures and high forces. Power is also most strongly affected at low temperature intervals, but this effect is minimized at intermediate forces. Effects of temperature on muscle force explain these interactions; force production decreases at lower temperatures, increasing the challenge of moving a constant force relative to the muscle's capacity. These results suggest that animal performance that requires muscles to do work with low forces relative to a muscle's maximum force production will be robust to temperature changes, and this effect should be true whether muscle acts directly or through elastic-recoil mechanisms and whether force is prescribed (i.e. internal) or variable (i.e. external). Conversely, performance requiring muscles to shorten with relatively large forces is expected to be more sensitive to temperature changes.

Lack of vitamin B12 impairs innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles.

Vitamin B12 is a micronutrient required by a variety of organisms for healthy cellular functioning. Despite the systemic effects observed in cases of B12 deficiency, relatively little is known about how vitamin B12 affects immune health, especially in amphibians, which are declining at unprecedented rates. In this study, we tested how supplementing an algae diet with B12 affects the innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We found that innate immunity, as measured by a bacterial killing assay, was significantly more robust in B12-supplemented tadpoles than control tadpoles, but no significant differences were found in natural antibody production or hematocrit between groups. Adaptive immunity, as measured by Aeromonas hydrophila-specific IgY antibodies, was significantly greater in tadpoles challenged with A. hydrophila and supplemented with B12 than in control tadpoles, those only challenged with A. hydrophila, and those only given B12. Our results suggest that vitamin B12 is an important factor in maintaining a functional immune system in tadpoles, which may also be true for all vertebrates.

Batrachochytrium salamandrivorans Can Devour More than Salamanders.

Batrachochytrium salamandrivorans is an emerging fungus that is causing salamander declines in Europe. We evaluated whether an invasive frog species (Cuban treefrog, Osteopilus septentrionalis) that is found in international trade could be an asymptomatic carrier when exposed to zoospore doses known to infect salamanders. We discovered that Cuban treefrogs could be infected with B. salamandrivorans and, surprisingly, that chytridiomycosis developed in animals at the two highest zoospore doses. To fulfill Koch's postulates, we isolated B. salamandrivorans from infected frogs, exposed eastern newts (Notophthalmus viridescens) to the isolate, and verified infection and disease by histopathology. This experiment represents the first documentation of B. salamandrivorans chytridiomycosis in a frog species and substantially expands the conservation threat and possible mobilization of this pathogen in trade.

Scaling of work and power in a locomotor muscle of a frog.

Muscle work and power are important determinants of movement performance in animals. How these muscle properties scale determines, in part, the scaling of performance during movements, such as jump height or distance. Muscle-mass-specific work is predicted to remain constant across a range of scales, assuming geometric similarity, while muscle-mass-specific power is expected to decrease with increasing scale. We tested these predictions by examining muscle morphology and contractile properties of plantaris muscles from frogs ranging in mass from 1.28 to 20.60 g. Scaling of muscle work and power was examined using both linear regression on log10-transformed data (LR) and non-linear regressions on untransformed data (NLR). Results depended on the method of regression not because of large changes in scaling slopes, but because of changing levels of statistical significance using corrections for multiple tests, demonstrating the importance of careful consideration of statistical methods when analyzing patterns of scaling. In LR, muscle-mass-specific work decreased with increasing scale, but an accompanying positive allometry of muscle mass predicts constant movement performance at all scales. These relationships were non-significant in NLR, though scaling with geometric similarity also predicts constant jump performance across scales, because of proportional increases in available muscle energy and body mass. Both intrinsic shortening velocity and muscle-mass-specific power were positively allometric in both types of analysis. Nonetheless, scale accounts for little variation in contractile properties overall over the range examined, indicating that other sources of intraspecific variation may be more important in determining muscle performance and its effects on movement.