*4.3. Expression of OvoA in Calanus finmarchicus and C. helgolandicus*

Expression of OvoA transcript was examined in the copepods *Calanus finmarchicus* and *Calanus helgolandicus* exposed to stress conditions and in *C. finmarchicus* across development using previously published data [28,29]. The datasets were searched for the OvoA sequences identified in this study for *C. finmarchicus* and *C. helgolandicus*. The expression data for OvoA obtained from the three datasets was normalized using the Reads Per Kilobase per Million mapped reads RPKM method. A 2-way ANOVA (*p* < 0.05) followed by post-hoc Tukey's test which was used to assess statistical significance in each study. A brief description of the datasets mined for the expression data is presented below.

The *C. finmarchicus* RNA-Seq dataset included expression data for females incubated for two and five days under three experimental diets: control (*Rhodomonas* sp.) and two doses (low and high) of the toxic dinoflagellate *Alexandrium fundyense* [24,50]. The toxic concentrations used for the dinoflagellate (LD = 50,000 cells L−1; HD = 200,000 cells L−1) were comparable with low and high bloom conditions reported in the Gulf of Maine [28]. Detailed methods for copepod collection, algal-incubation, RNA extraction and RNA-Seq processing are described in Roncalli et al. [28]. Briefly, females were exposed to the three diets for a total of 7 days with samples being harvested for RNA-Seq at day 2 and day 5 (three replicates/treatment). Expression was quantified by mapping each RNA-Seq library against the *C. finmarchicus* reference transcriptome (NCBI: PRJNA236528) using bowtie software (v.2.0.6). The second *C. finmarchicus* RNA-Seq dataset included expression data for six developmental stages: embryos, early nauplii (NII-NIII), early copepodites (CI), late copepodites (CIV), pre-adults (CV), and females (F) [50–52]. For each stage three samples were processed for RNA-Seq (exception CI and CIV with two replicates) and expression rate was measured by mapping each RNA-Seq library against the *C. finmarchicus* reference transcriptome (NCBI: PRJNA236528) using bowtie software (v.2.0.6).

Lastly, the *C. helgolandicus* dataset consisted of RNA-Seq data for laboratory-incubated females feeding for five days on the oxylipin-producing diatom *Skeletonema marinoi* and the control diet *Prorocentrum minimum*. Detailed methods for copepod collection, algalincubation experiments, transcriptome sequencing, de novo assembly and annotation, are described in [29]. In brief, *C. helgolandicus* females collected in the Gulf of Naples (Mediterranean Sea) were fed for five days with either *S. marinoi* or *P. minimum* at 1 mg C L−<sup>1</sup> (three replicates each). RNA-Seq libraries were pooled to generate a de novo assembly (NCBI: PRJNA640515) that was used to quantify expression levels by self-mapping using bowtie. Reads were normalized by length using the RPKM methods Reads Per Kilobase per Million mapped reads (RPKM).
