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Current knowledge on the biological responses of freshwater fish under projected scenarios of climate change remains limited. Here, we examine differences in the protein configuration of two endemic Iberian freshwater fish species,  Squalius carolitertii  and the critically endangered  S .  torgalensis  that inhabit in the Atlantic-type northern and in the Mediterranean-type southwestern regions, respectively. We performed protein structure modeling of fourteen genes linked to protein folding, energy metabolism, circadian rhythms and immune responses. Structural differences in proteins between the two species were found for HSC70, FKBP52, HIF1α and GPB1. For  S .  torgalensis , besides structural differences, we found higher thermostability for two proteins (HSP90 and GBP1), which can be advantageous in a warmer environment. Additionally, we investigated how these species might respond to projected scenarios of 3° climate change warming, acidification (ΔpH = -0.4), and their combined effects. Significant changes in gene expression were observed in response to all treatments, particularly under the combined warming and acidification. While  S .  carolitertii  presented changes in gene expression for multiple proteins related to folding ( hsp90aa1 ,  hsc70 ,  fkbp4  and  stip1 ), only one such gene was altered in  S .  torgalensis  ( stip1 ). However,  S .  torgalensis  showed a greater capacity for energy production under both the acidification and combined scenarios by increasing  cs  gene expression and maintaining  ldha  gene expression in muscle. Overall, these findings suggest that  S .  torgalensis  is better prepared to cope with projected climate change. Worryingly, under the simulated scenarios, disturbances to circadian rhythm and immune system genes ( cry1aa ,  per1a  and  gbp1 ) raise concerns for the persistence of both species, highlighting the need to consider multi-stressor effects when evaluating climate change impacts upon fish. This work also highlights that assessments of the potential of endangered freshwater species to cope with environmental change are crucial to help decision-makers adopt future conservation strategies.

Protein analysis and gene expression indicate differential vulnerability of Iberian fish species under a climate change scenario