Transcriptional regulation of North American wood frog (Rana sylvatica) dorsal skin during development and in response to chronic exposure to a microplastics mixture

Abstract

Amphibian populations have been experiencing declines globally for decades due to factors such as habitat loss, invasive species, climate change, overexploitation, pollution, and disease. Skin is an important immune organ in amphibians, employing physical, chemical, microbiological, and immunological defences to prevent infection. Amphibian skin also undergoes dramatic morphological changes during metamorphosis. Therefore, it is important to gain a deeper understanding of the biological changes that amphibian skin undergoes during metamorphosis and whether these tightly regulated processes can be disrupted by environmental contaminants.

Microplastics are a persistent, ubiquitous environmental contaminant of increasing concern with a wide range of effects on exposed organisms. Previous studies have reported mortality, reduced growth, behavioural changes, and hepatotoxicity in amphibians exposed to microplastics. However, few studies have evaluated sub-organismal effects of microplastic exposure on amphibians, particularly in relation to the immune system. Furthermore, few studies have chronically exposed amphibians to an environmentally realistic microplastics mixture.

To address these gaps, an outdoor mesocosm experiment was performed where developing North American wood frogs (Rana sylvatica) were exposed from embryos throughout metamorphosis to a microplastics mixture consisting of an equal number of particles by count of polypropylene, polystyrene, and polyethylene terephthalate microplastics. Developing wood frogs were exposed to one of three concentrations: a 0× negative control of 0 g/L, a 1× treatment of 0.069 g/L, or a 10× treatment of 0.691 g/L of the microplastics mixture. This thesis reports on the transcriptomic analysis of dorsal skin of wood frogs from three different developmental timepoints: (1) after 50 days of exposure, average Gosner stage (GS) 34; (2) after 74 days of exposure, average GS 42; and (3) GS 45, 74 – 82 days of exposure.

The first analysis performed compared the dorsal skin transcriptome in the 0× unexposed wood frogs at different timepoints to evaluate changes in dorsal skin gene expression during development. Trends corresponding to known developmental phenomena such as keratinization of the skin and active ion transport nearing the end of metamorphosis were observed in the data. Downregulation of immune genes was also seen in the skin after the first timepoint, and is in line with the systemic immunosuppression metamorphosing amphibians undergo leading up to and during metamorphic climax. This result demonstrates that the systemic immunosuppression is paired with suppression of immune function in skin tissues.

The second analysis evaluated the effects of microplastic exposure on the dorsal skin transcriptome at each of the three selected developmental stages. Concentration- and timepoint-dependent effects were observed, with the greatest number of differentially expressed genes found in the 1× treatment at the first timepoint (GS ~34). Clusters of differentially expressed genes involved in mitochondrial function, protein synthesis, transmembrane transport, and immunity were found in at least one timepoint/treatment group. Developmental plasticity, morphological and behavioural changes during development, and a potential nonmonotonic dose response to microplastics in the wood frog are ideas proposed to account for the differences in response seen to different microplastic concentrations and at different developmental timepoints. Downregulation of immune genes was seen at the first developmental timepoint in response to the 1× microplastic treatment, suggesting that microplastics could cause developmental stage-specific immunosuppression in developing wood frogs. Because amphibian populations are already experiencing declines due to infectious diseases, it is important for future studies to continue to evaluate the potential for microplastics to act as a stressor and predispose amphibians to infection.