Amphibians exhibit extremely high hydric costs of respiration.

Terrestrial environments pose many challenges to organisms, but perhaps one of the greatest is the need to breathe while maintaining water balance. Breathing air requires thin, moist respiratory surfaces, and thus the conditions necessary for gas exchange are also responsible for high rates of water loss that lead to desiccation. Across the diversity of terrestrial life, water loss acts as a universal cost of gas exchange and thus imposes limits on respiration. Amphibians are known for being vulnerable to rapid desiccation, in part because they rely on thin, permeable skin for cutaneous respiration. Yet we have a limited understanding of the relationship between water loss and gas exchange within and among amphibian species. In this study, we evaluated the hydric costs of respiration in amphibians using the transpiration ratio, which is defined as the ratio of water loss (mol H2O d-1) to gas uptake (mol O2 d-1). A high ratio suggests greater hydric costs relative to the amount of gas uptake. We compared the transpiration ratio of amphibians with that of other terrestrial organisms to determine if amphibians had greater hydric costs of gas uptake relative to plants, insects, birds, and mammals. We also evaluated the effects of temperature, humidity, and body mass on the transpiration ratio both within and among amphibian species. We found that hydric costs of respiration in amphibians were two to four orders of magnitude higher than the hydric costs of plants, insects, birds, and mammals. We also discovered that larger amphibians had lower hydric costs than smaller amphibians, at both the species- and individual-level. Amphibians also reduced the hydric costs of respiration at warm temperatures, potentially reflecting adaptive strategies to avoid dehydration while also meeting the demands of higher metabolic rates. Our results suggest that cutaneous respiration is an inefficient mode of respiration that produces the highest hydric costs of respiration yet to be measured in terrestrial plants and animals. Yet, amphibians largely avoid these costs by selecting aquatic or moist environments, which may facilitate more independent evolution of water loss and gas exchange.

Parameterizing mechanistic niche models in biophysical ecology: a review of empirical approaches.

Mechanistic niche models are computational tools developed using biophysical principles to address grand challenges in ecology and evolution, such as the mechanisms that shape the fundamental niche and the adaptive significance of traits. Here, we review the empirical basis of mechanistic niche models in biophysical ecology, which are used to answer a broad array of questions in ecology, evolution and global change biology. We describe the experiments and observations that are frequently used to parameterize these models and how these empirical data are then incorporated into mechanistic niche models to predict performance, growth, survival and reproduction. We focus on the physiological, behavioral and morphological traits that are frequently measured and then integrated into these models. We also review the empirical approaches used to incorporate evolutionary processes, phenotypic plasticity and biotic interactions. We discuss the importance of validation experiments and observations in verifying underlying assumptions and complex processes. Despite the reliance of mechanistic niche models on biophysical theory, empirical data have and will continue to play an essential role in their development and implementation.

Development of an amphibian sperm biobanking protocol for genetic management and population sustainability.

Sperm cryopreservation is a vital tool in amphibian assisted reproductive technologies that aids in genetic and population management, specifically for at-risk species. Significant advancements have been made in the cryopreservation of amphibian sperm, yet there is little information on how the cryopreservation process influences fertilization and embryonic development. In this study, we tested several cryoprotective agents (CPAs) and freezing rates on sperm recovery, fertilization potential and embryo development using Fowler's toads (Anaxyrus fowleri) as a model amphibian species for application to at-risk anurans. Three cryoprotectant treatments were tested, which included 10% trehalose + 0.25% bovine serum albumin with (1) 5% N,N-dimethylformamide (DMFA); (2) 10% DMFA; or (3) 10% dimethyl sulfoxide (DMSO). Additionally, sperm in each cryoprotectant was frozen at two different rates, -32 to -45°C/min and -20 to -29°C/min. Post-thaw sperm analysis included motility, morphology, viability, fertilization success and embryo development. Results show that 10% DMFA produced significantly higher (P = 0.005) post-thaw sperm motility than 5% DMFA and was similar to 10% DMSO. Furthermore, sperm frozen at -32 to -45°C/min had significantly higher post-thaw motility (P < 0.001) compared to sperm frozen at -20 to -29°C/min. We also found that embryos fertilized with sperm frozen with 5% DMFA resulted in significantly higher (P = 0.02) cleavage than 10% DMSO, yet there was no other effect of CPA on fertilization or embryo development. Furthermore, embryos fertilized with sperm frozen at -32 to -45°C/min resulted in significantly higher cleavage (P = 0.001), neurulation (P = 0.001) and hatching (P = 0.002) numbers than sperm frozen at a rate of -20 to -29°C/min. Overall, eggs fertilized with frozen-thawed sperm produced 1327 tadpoles. These results provide insight towards a biobanking strategy that can be applied to imperilled species to preserve genetic lineages and bolster offspring genetic diversity for reintroduction.

Sperm Cryopreservation as a Tool for Amphibian Conservation: Production of F2 Generation Offspring from Cryo-Produced F1 Progeny.

Sperm cryopreservation and biobanking are emerging as tools for supporting genetic management of small and threatened populations in amphibian conservation programs. However, there is little to no evidence demonstrating reproductive maturity and viability of offspring generated with cryopreserved sperm, potentially limiting widespread integration of these technologies. The purpose of this report is to demonstrate that amphibian sperm can be cryopreserved and thawed to successfully produce individuals of an F1 generation that can reach adulthood and reproductive maturity, to generating viable gametes and an F2 generation. Species-specific exogenous hormones were administered to both F0 and F1 adults to stimulate spermiation and oviposition in the eastern tiger salamander (Ambystoma tigrinum), dusky gopher frog (Lithobates sevosa), and Puerto Rican crested toad (Peltophryne lemur). Sperm cells collected non-lethally from F0 adults were cryopreserved, thawed, and used for in vitro fertilization (IVF) to produce F1 offspring. Individuals of the F1 generation are shown to reach adulthood, express viable gametes, and produce offspring through facilitated breeding, or IVF. The production of amphibian F2 generations shown here demonstrates that amphibian sperm collected non-lethally can be banked and used to generate reproductively viable animals of subsequent generations, thus maintaining valuable genetic linages and diversity in threatened amphibian species. The incredible value that cryopreservation of sperm has for long-term genetic management aids in the sustainability of both in situ and ex situ conservation efforts for this taxon.

Near-Infrared Spectroscopy (NIRS) as a Method for Biological Sex Discrimination in the Endangered Houston Toad (Anaxyrus houstonensis).

Biological sex is one of the more critically important physiological parameters needed for managing threatened animal species because it is crucial for informing several of the management decisions surrounding conservation breeding programs. Near-infrared spectroscopy (NIRS) is a non-invasive technology that has been recently applied in the field of wildlife science to evaluate various aspects of animal physiology and may have potential as an in vivo technique for determining biological sex in live amphibian species. This study investigated whether NIRS could be used as a rapid and non-invasive method for discriminating biological sex in the endangered Houston toad (Anaxyrus houstonensis). NIR spectra (N = 396) were collected from live A. houstonensis individuals (N = 132), and distinct spectral patterns between males and females were identified using chemometrics. Linear discriminant analysis (PCA-LDA) classified the spectra from each biological sex with accuracy ≥ 98% in the calibration and internal validation datasets and 94% in the external validation process. Through the use of NIRS, we have determined that unique spectral signatures can be holistically captured in the skin of male and female anurans, bringing to light the possibility of further application of this technique for juveniles and sexually monomorphic species, whose sex designation is important for breeding-related decisions.