2.2. Desiccation Experimental Procedures

Groups of 50 individuals of each species were randomly assigned to either a water control group (C) or to a prior desiccation group (Figure 1). For this prior desiccation exposure, specimens were gently dried on blotting paper and then individually placed in open glass vials and exposed to 40 ± 5% RH and 20 °C for 6 h (E. jesusarribasi) or 3 h (N. baeticus) in the environmental chamber, at the constant temperature and light cycle conditions mentioned above (Figure 1). A relative humidity of 40% was selected because it represents the minimum air humidity in the natural habitats of these species. Temperature was set to 20 °C to avoid additional thermal stress in the experiment. Exposure times were set based on the differential survival times under desiccation shown by these species in a previous study [31], in which N. baeticus was more sensitive to desiccation than E. jesusarribasi. After prior desiccation, specimens were allowed to recover for 24 h by immersing them in water at their corresponding field optimum salinity. The control groups were simultaneously maintained in aquaria at the conditions described above. Then, specimens from both the control and prior desiccation groups were exposed simultaneously to subsequent desiccation of the same intensity and duration as the first desiccation treatment (40 ± 5% RH for 3 h in N. baeticus and 6h in E. jesusarribasi). These experimental groups are referred to as D1 (one desiccation phase) and D2 treatments (two desiccation phases), respectively (Figure 1). Survival was monitored after each experimental phase (i.e., prior desiccation, recovery underwater and subsequent desiccation).

Schematic representation of the experimental design showing the different stress exposure steps and timings for cuticular hydrocarbons (CHCs) sampling and total water loss rate (WLR) estimation.