*4.1. In Silico Mining, Reciprocal Blast, and Protein Structural Analysis*

In silico search for putative OvoA encoding transcript was performed using a wellestablished vetting protocol that involves a mining, a reciprocal BLAST, and a structural motif analysis [43,44]. The transcriptome shotgun assembly (TSA) database on the National Center for Biotechnology Information (NCBI) was searched (October 2021) using the OvoA sequence from *Paracentrotus lividus* (AMM72581) as query (tblastn algorithm) limiting the results to Arthropoda (taxid: 6656). Among the hits, we focused on the marine organisms, mostly represented by crustacean including the Copepoda and Malacrostacea subphyla. For completeness, we also mined the Crustybase [45] to search additional available transcriptomes for other marine arthropods. Using *P. lividus* OvoA protein sequence we mined (tblastn algorithm) transcriptomes for the decapods *Eriocheir sinensis*, *Litopenaeus vannamei*, *Callinectes sapidus*, *Gecarcinus lateralis*, *Homarus americanus* whose references were not available on NCBI (Table S1). The transcripts encoding sequences OvoA for all the newly identified arthropods are provided in Supplementary File S3.

Reciprocal blast was used to confirm the identity of the putative OvoA transcripts by blasting each transcript against the non-redundant (nr) protein database. Briefly, each putative OvoA transcript was fully translated using ExPASy [46] and then the deduced protein was used to query the NCBI non-redundant (nr) protein database (blastp algorithm). Protein sequences were further inspected by searching the Pfam database for structural domains [47]. Specifically, we searched for the three expected conserved domains: the DinBlike domain, the FGE-sulfatase domain, and the methyltransferase 11 domain pertaining to the SAM-dependent methyl-transferase homologous superfamily. OvoA sequences from the calanoid *C. finmarchicus* and *C. helgolandicus* were aligned using ClustalW software (Galaxy version 2.1 [48]).
