4.2 Heat shock response between heat-tolerant and heat-sensitiveBrachionus species
Heat Shock Response, which involves the induction of heat shock protein (hsp ) genes, is a well known and evolutionary conserved mechanism present in both prokaryotes and eukaryotes (Feder & Hofmann, 1999). Induction of hsps has been connected to several stress conditions such as exposure to extreme temperatures, heavy metals, pathogens, and osmotic stress (De Jong et al., 2008). In the present study, expression of hsp related genes mirrored measured changes in the population growth rate across a temperature gradient. Population growth rate was low when hsp genes were up-regulated. This pattern was consistent in both species, showing that hsps genes are indeed part of a species’ stress response, when environmental conditions (here, temperature) are outside the ‘comfort zone’ for optimal growth.
Closely related species differentially adapted to cold vs. warm habitats have been found to express hsp genes differently also in other aquatic organisms such as the amphipods Eulimnogammarus. S pecies originated from a cold habitat when exposed to heat, up-regulated hsp genes already at lower temperatures compared to species from a warmer habitat (Bedulina et al. 2013). In the present study, closely related Brachionus species with differences in their ability to tolerate heat have been found to express hspgenes differently. More specifically, hsp genes were induced outside the temperature of optimal growth: in the heat-sensitive B. fernandoi, hsp genes were induced by heat, while in heat-tolerant B. calyciflorus s.s. the majority of hsp genes were induced at the lower end of temperature exposure (20 oC), indicating that 20 oC may already be cold stress for the heat-tolerant species.
Proteins of the hsp90 family serve to increase the available chaperons in the cells in order to recover from cellular stress and maintain structural integrity at high temperatures. In contrast to other hsp genes, their expression patterns supported a specific involvement in heat response, as genes encoding for hsp90 were up-regulated towards the higher temperature regime in both species. The expression ofhsp90 gene has been found to be temperature dependent also in other aquatic organisms, e.g. copepods, oysters (Schoville et al., 2012; Kim et al., 2017; Lim et al., 2016). This suggests that induction ofhsp90 gene along with heat might be a common mechanism in aquatic organisms. In B. calyciflorus s.s., genes encoding hsp20were also induced by high heat. Up-regulation of hsp20 genes has been reported previously from other Brachionus species and copepods as a response to elevated temperatures (Rhee et al., 2011; Seo, Lee, Park, & Lee, 2006). Transformed bacteria (Escherichia coli ) expressing the Brachionus hsp20 (Br-hsps20 ) gene had a 100 times increase in their survival compared to the non transformed ones under high heat-stress, indicating that Br-hsp20specifically contributes to increased thermal tolerance (Rhee et al., 2011). Up-regulation of hsp20 genes was found to also increase resistance to oxidative stress (Rhee et al. 2011). Possibly, an increase in expression of hsp20 reflects a cellular defense mechanism in response to different stressors that might be common amongBrachionus species.
A reversed expression pattern among our two species was found in genes encoding for hsp70 and hsp27 . In heat-tolerant B. calyciflorus s.s., genes encoding for hsp70 were significantly up-regulated towards the lower temperature, while in B. fernandoitowards the higher temperature. It seems that temperatures such as 20oC might constitute cold stress for warm adapted species and a stress response might be initiated under these conditions. In B. calyciflorus s.s., the same induction pattern withhsp70 genes followed genes encoding for hsp40 andhsp60 pointing towards a synergistic mechanism regulating the expression of these three genes as reported also in B. manjavacas(Smith et al., 2012). Such a synergistic relationship has been reported among hsp40 and hsp70 proteins, as hsp40 regulates the ATPase activity of hsp70 (Cintron & Toft, 2006). These genes were induced in B. manjavacas with increasing heat (Smith et al., 2012). This indicates that these genes are induced under any condition that constitutes temperature stress for a particular Brachionusspecies.