Effects of thermal acclimation on the proteome of the planarian Crenobia alpina from an alpine freshwater spring

Abstract

Species' acclimation capacity and their ability to maintain molecular homeostasis outside ideal temperature ranges will partly predict their success following climate change-induced thermal regime shifts. Theory predicts that ectothermic organisms from thermally stable environments have muted plasticity, and that these species may be particularly vulnerable to temperature increases. Whether such species retained or lost acclimation capacity remains largely unknown. We studied proteome changes in the planarian Crenobia alpina, a prominent member of cold-stable alpine habitats that is considered to be a cold-adapted stenotherm. We found that the species' critical thermal maximum (CTmax) is above its experienced habitat temperatures and that different populations exhibit differential CTmax acclimation capacity, whereby an alpine population showed reduced plasticity. In a separate experiment, we acclimated C. alpina individuals from the alpine population to 8, 11, 14 or 17°C over the course of 168 h and compared their comprehensively annotated proteomes. Network analyses of 3399 proteins and protein set enrichment showed that while the species' proteome is overall stable across these temperatures, protein sets functioning in oxidative stress response, mitochondria, protein synthesis and turnover are lower in abundance following warm acclimation. Proteins associated with an unfolded protein response, ciliogenesis, tissue damage repair, development and the innate immune system were higher in abundance following warm acclimation. Our findings suggest that this species has not suffered DNA decay (e.g. loss of heat-shock proteins) during evolution in a cold-stable environment and has retained plasticity in response to elevated temperatures, challenging the notion that stable environments necessarily result in muted plasticity.

Keywords: Crenobia alpina; Aquatic ectotherm; Molecular adaptation; Phenotypic plasticity; Proteomics; Thermal tolerance.

Fig. 1.

Critical thermal maximum (CT_max) and proteomics results. (A) Violin plots of CT_max values of three Crenobia alpina populations (n=31 per population; see Fig. S1 for information on sites) independent of acclimation temperature. P­-values are shown above the brackets (ANOVA). (B) Raincloud plot of CT_max values following thermal acclimation of the three C. alpina populations shown in A. (C) Non-metric multidimensional scaling (nMDS) ordination based on the relative abundance of 3399 proteins commonly identified across n=5 biological replicates per acclimation temperature in the alpine population (Alps) following 168 h acclimation to 8, 11, 14 or 17°C. (D) Heatmap of all heat shock proteins (HSPs) quantified in the alpine population's proteome following 168 h thermal acclimation to 8, 11, 14 or 17°C. Colours represent the log₂ label-free quantification (LFQ) values for each HSP measured in the 20 biological replicates of the alpine C. alpina population (columns; n=5 replicates per acclimation temperature).

Fig. 2.

Proteome changes of C. alpina following 168 h acclimation to sublethal temperatures. (A) GO enrichment results of proteins of higher abundance following acclimation (WGCNA modules: Tan, Purple and Magenta) for the Cellular Compartment (CC; top) and Biological Process (BP; bottom) GO category. (B) GO enrichment results of proteins of lower abundance following acclimation (WGCNA modules: Green and Blue) for the Molecular Function (MF; top), Cellular Compartment (CC, middle) and Biological Process (BP, bottom) GO category. Dashed lines indicate an adjusted P-value cut-off of 0.05. Colours represent the adjusted P-value and dot size indicates the number of significant proteins associated with the respective GO term. (C) Names (top) and statistics of WGCNA modules and their association with acclimation temperature, represented by Pearson's correlation coefficients (r) between module eigengene (ME) and acclimation temperature, the P-value of the correlation in parentheses and the number of proteins assigned to each module (n; with membership value >0.5). The plot in the middle shows the correlation between the ME expression of the Blue module and acclimation temperature.