## 3 Results and Discussion
### Dynamics of Compatible Solutes Accumulation after Hypoosmotic or Hyperosmotic Shock  
In model fungi, the accumulation of compatible solutes has been analyzed during hypercosmic or hypoosmotic shock but there are few studies analyzing halophile fungi growing under optimal salinity conditions and changed to osmo-stressful conditions [105; 106]. Here we have termed these as stress-inducing shocks to highlight the difference from hyperosmic and hypoosmotic shocks between non-optimal growth conditions. We evaluated the accumulation of polyols and trehalose when _A. Sydowii_ was growing with 0.5 M NaCl and then exposed to a medium without salt or with 2.0 M NaCl. After a hyperosmotic shock, _S. cerevisiae_, _H. werneckii_, and _W. ichthyophaga_ prevalently accumulate glycerol. However, in _A. Sydowii_ cells under stable salinity conditions, we observed that the accumulation of glycerol occurred only at 2.0 M NaCl. This leads to questions as to whether glycerol or other osmolytes will be produced if mycelium is shifted from hypoosmotic conditions (without NaCl) to optimal salt conditions (0.5 M NaCl), which presumably do not represent a stressful condition for this fungus. Therefore, we determined the accumulation of compatible solutes of _A. Sydowii_ when exposed to hypoosmotic, hyperosmotic, and stress-inducing osmotic shocks. When cultures without NaCl were transferred to 0.5 M NaCl (Figure 4), the fungus responded after 2 h with a spike in the concentration of erythroid and arabitol, which diminished after 8 h to the levels encountered in mycelia cultured at 0.5 M NaCl. Trehalose and mannitol levels did not change, and glycerol was not produced, even though similar salinities induce its accumulation in other non-halophile Aspergillus such as _A. nidulans_ and _A. niger_[4; 107]. In contrast, the shifting of cultures without NaCl to 2.0 M NaCl induced a small glycerol accumulation after 8 h and a progressive decrease of mannitol concentration. This osmotic shock induced the expression of both _stps_ and _ccg-9_ genes (Figure 5), which did not correlate with trehalose accumulation. The gene _m2 dh_ did not change its expression, while the _mtld_ gene was downregulated after the shock, coinciding with the decrease of mannitol concentration. The _gpd_ transcripts were detected transiently between 2 and 8 h, which also coincides with the increase in glycerol after 8 h. These observations are different than in studies performed in _S. cerevisiae_ and _D. hansenii_, and others, where the intracellular concentration of glycerol increased after 30 min when the cells were exposed to a moderate osmotic shock (without salt to 0.5 M of NaCl) [54; 108; 109; 110]. After a hypoosmotic shock from 2.0 M NaCl to a medium without salt, trehalose was detected as early as 10 min, while glycerol rapidly disappeared, but there were not dramatic changes in total osmolyte concentration (Figure 4). Under this shock, the _ccg-9_ gene was upregulated soon after, while the _stps_ gene was downregulated after 8 h and the _gpd_ transcript levels decreased progressively (Figure 5). These changes alone do not explain the dynamics of trehalose and glycerol but are consistent with the physiological response of the mycelium in this scenario. When the hypoosmotic shock was from 2 M NaCl to a medium with 0.5 M NaCl, fluctuations were more evident, as erythroid concentrations spiked after 2 h and glycerol initially disappeared and later spiked also after 2 h from the shock. There was a spike of trehalose 48 h after this treatment for which we have no plausible explanation. Our results indicated that, when changed to a condition without salt, the concentrations of mannitol and arabitol spiked transiently at 2 h and returned to approximately the initial level after 24 h (Figure 4), which coincided with an initial upregulation of _m2 dh_ and to a lesser extent _tktB_ (Figure 5), but these might not be responsible for the observed osmolyte dynamics.