*2.5. cDNA Synthesis and Real Time q-PCR*

For each sample, 1000 ng of total RNA was retrotranscribed with an iScript™ cDNA Synthesis kit (Bio-Rad, Milan, Italy), following the manufacturer's instructions. The variations in the expression of five genes involved in stress response (*hsp26*, *hsp60*, *hsp70*, *COXI,* and *COXIII* [49]; see Supplementary Figure S1) were evaluated for adults. For nauplii, the variations in the expression of four other genes involved in developmental and differentiation processes (*HAD-like*, *tcp*, *UCP2,* and *CDC48*, [49]) were also tested (Supplementary Figure S1).

Undiluted cDNA was used as a template in a reaction containing a final concentration of 0.3 mM for each primer and 1 × SensiFASTTM SYBR Green master mix (total volume of 10 μL) (Meridiana Bioline). PCR amplifications were performed in AriaMx Real-Time PCR instrument (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's instructions, using the following thermal profile: 95 ◦C for 10 min, one cycle for cDNA denaturation; 95 ◦C for 15 s and 60 ◦C for 1 min, 40 cycles for amplification; 95 ◦C for 15 s, one cycle for final elongation; and one cycle for melting curve analysis (from 60 ◦C to 95 ◦C) to verify the presence of a single product. Each assay included a no-template control for each primer pair. To capture intra-assay variability, all realtime qPCR reactions were carried out in triplicate. Fluorescence was measured using Agilent Aria 1.7 software (Agilent Technologies, Inc.). The relative expression ratios were calculated according to [50,51] using REST software (Version No., Relative Expression Software Tool, Weihenstephan, Germany; Equation (1)):

$$R = \frac{E\_{\text{target}}^{\Delta \text{C} \text{q target}} \left( {\text{Mean Control}} - {\text{Mean Sample}} \right)}{E\_{\text{refriger}}^{\Delta \text{C} \text{q reference} \ (\text{Mean Control} - \text{Mean Sample})}} \tag{1}$$

The expression of each gene was analyzed and internally normalized against *GAPDH* [49] using REST software (Relative Expression Software Tool, Weihenstephan, Germany) based on the Pfaffl method [50,51]. Relative expression ratios above 1.5 were considered as significant. The nonparametric Mann–Whitney test was applied to ΔCq (Cq gene of interest— Cq reference) values between treated and control samples (n = 3). *p*-Values < 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism Software (version 8.02 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com, accessed on 1 February 2021). Fold-change values were represented through a Heatmap generated by GraphPad Prism Software.
