The wood frog (Rana sylvatica): An emerging comparative model for anuran immunity and host-ranavirus interactions.

The wood frog (Rana sylvatica) is widely distributed across North America and is the only amphibian found north of the Arctic Circle due to its remarkable ability to tolerate whole-body freezing. Recent mass mortalities attributable to Ranavirus spp. (family Iridoviridae) in wild juvenile wood frogs, coupled with the apparent high susceptibility of wood frogs to experimental infection with frog virus 3 (FV3), the type species of the Ranavirus genus, or FV3-like isolates underscore the serious threat ranaviruses poses to wood frog populations. Despite the ecological relevance and unique life history of wood frogs, our understanding of the wood frog immune system and antiviral response to ranaviral infections is in its infancy. Here we aim to (1) synthesize the limited knowledge of wood frog immune defences, (2) review recent progress in establishing the wood frog as a study system for ranavirus infection, and (3) highlight the future use of wood frogs as a model anuran to provide insight into the evolution of anuran immune systems and antiviral responses.

The amphibian invitrome: Past, present, and future contributions to our understanding of amphibian immunity.

Many amphibian populations are declining worldwide, and infectious diseases are a leading cause. Given the eminent threat infectious diseases pose to amphibian populations, there is a need to understand the host-pathogen-environment interactions that govern amphibian susceptibility to disease and mortality events. However, using animals in research raises an ethical dilemma, which is magnified by the alarming rates at which many amphibian populations are declining. Thus, in vitro study systems such as cell lines represent valuable tools for furthering our understanding of amphibian immune systems. In this review, we curate a list of the amphibian cell lines established to date (the amphibian invitrome), highlight how research using amphibian cell lines has advanced our understanding of the amphibian immune system, anti-ranaviral defence mechanisms, and Batrachochytrium dendrobatidis replication in host cells, and offer our perspective on how future use of amphibian cell lines can advance the field of amphibian immunology.

So, you want to create a frog cell line? A guide to establishing frog skin cell lines from tissue explants.

Skin is an important interface with the external environment and investigating amphibian skin cell biology will improve our understanding of how environmental factors such as pathogens and pollutants are contributing to global amphibian declines. There is a critical need for in vitro systems to facilitate conservation research in model and non-model amphibians and the creation of new amphibian cell lines will play a significant role in reducing or even replacing the use of live animals for in vivo studies by providing an in vitro alternative. Here, we detail an adapted protocol for the generation of spontaneously arising cell lines from frog skin tissues, without the need for immortalization steps. Expanding the amphibian invitrome will foster and expedite new research in amphibian gene function, cellular responses, host-pathogen interactions, and toxicology. The following customizations to traditional tissue explant generation procedures have facilitated the successful generation of adherent skin epithelial-like cell lines from Xenopus laevis and can be further adapted for use with different frog species, such as Rana sylvatica, and different tissues:•Osmotic adjustment of culture medium and solutions for different amphibian species.•Use of small tissue explants, instead of enzymatic digestion of tissues, and gentle spotting of these tissue explants onto the growth surface of tissue culture flasks to promote better tissue adherence.•Partial replacement of medium to allow accumulation of potential endogenous growth factors in cultures.

Composition of the North American Wood Frog (Rana sylvatica) Bacterial Skin Microbiome and Seasonal Variation in Community Structure.

While a number of amphibian skin microbiomes have been characterized, it is unclear how these communities might vary in response to seasonal changes in the environment and the corresponding behaviors that many amphibians exhibit. Given recent studies demonstrating the importance of the skin microbiome in frog innate immune defense against pathogens, investigating how changes in the environment impact the microbial species present will provide a better understanding of conditions that may alter host susceptibility to pathogens in their environment. We sampled the bacterial skin microbiome of North American wood frogs (Rana sylvatica) from two breeding ponds in the spring, along with the bacterial community present in their vernal breeding pools, and frogs from the nearby forest floor in the summer and fall to determine whether community composition differs by sex, vernal pond site, or temporally across season (spring, summer, fall). Taxon relative abundance data reveals a profile of bacterial phyla similar to those previously described on anuran skin, with Proteobacteria, Bacteroidetes, and Actinobacteria dominating the wood frog skin microbiome. Our results indicate that sex had no significant effect on skin microbiota diversity; however, this may be due to our limited female frog sample size. Vernal pool site had a small but significant effect on skin microbiota, but skin-associated communities were more similar to each other than to the communities observed in the frogs' respective pond water. Across seasons, diversity analyses suggest that there are significant differences between the bacterial skin microbiome of frogs from spring and summer/fall groups while the average α-diversity per frog remained consistent. These results illustrate seasonal variation in wood frog skin microbiome structure and highlight the importance of considering temporal trends in an amphibian microbiome, particularly for species whose life history requires recurrent shifts in habitat and behavior.