The effects of global warming are becoming increasingly evident in freshwater systems, and the impact of stress related to warming surface waters has been studied extensively on multiple levels of biological organization. However, the impact of warming on freshwater mussels (Bivalvia: Unionidae), a keystone species in freshwater systems, is poorly understood. A next generation sequencing approach (Ion Torrent platform) was undertaken to identify genes that were differentially expressed in response to temperature stress. Two species of freshwater mussel native to Alabama (USA), Villosa nebulosa and Villosa lienosa, were chosen due to differences in habitat range and conservation status. V. nebulosa is restricted to a relatively narrow range and is of moderate conservation risk, while V. lienosahas a large geographic range and is currently stable. Both species were subjected to mild heat shock (5°C above ambient 22°C) and tissue samples were collected at 3, 6, 24 hrs post heat stress and after 48 hr recovery. RNA samples from both treatments were pooled together for each species for transcriptomic sequencing and gene expression profiling via quantitative real-time PCR. qRT-PCR data on several stress-response genes were analyzed by comparing absolute molecule number for basal gene expression and experimental heat shock.
Results/Conclusions
An average of 330,000 sequence reads with a mean length of 100 base pairs was generated from both species. The sequence assembly yielded 17,637 and 21,657 contigs for V. nebulosa and V. lienosa, respectively. Contigs were blasted (blastx) against the NCBI nonredundant database and gene ontology annotation confirmed a significant number of genes related to heat shock, oxidative and cellular stress (HSP70i, HSP70c, HSP90, Ferritin, GST-pi, GST sigma). Preliminary results from gene expression profiling on HSP70i show comparatively higher basal expression in the more geographically abundant and thus more hypothetically robust V. lienosa, while V. nebulosa showed higher basal expression for HSP90, Ferritin, and GST-pi. Both species showed significant up-regulation of heat shock genes over the time series, but no significant regulation was seen in the oxidative stress genes. Further, the majority of genes showed down-regulation back to normal ambient levels after 48 hrs of recovery. These findings support the idea that minor increases in water temperature predicted in future warming scenarios can lead to a visible genetic response. Basal gene expression differed between the two species and appears to be under seasonal regulation. Finally, acute heat shock does not appear to lead to short-term oxidative stress.