**4. Conclusions**

The present work revealed for the first time a draft of the whole proteome of the sea anemone *B. verrucosa*. The shotgun proteomics analysis yielded most of the protein identified in a total of 412, whereas gel-based analyses provided less data but useful as complementary information. Altogether, both gel-based and gel-free approaches of proteomics analyses and functional bioinformatics analyses revealed three major groups of proteins belonging to "metabolic process", "binding" and "cell parts" GO categories. Unlike throughput analyses, only eight proteins were identified from two-dimensional electrophoresis combined with MALDI-TOF/TOF. These eight proteins comprised enzymes mainly involved in the glycolytic pathway, antioxidants activities and RNA degradation. Notably, according to the results of KEGG analysis a significant number of enzymes corresponded to the Biosynthesis of antibiotics pathway indicating the importance of the biological antimicrobial chemical defense mechanisms. Moreover, some potential toxins such as metalloproteinases, and neurotoxin such as SE-cephalotoxin were identified. The combination of proteomic evidences and the ecology of the species, shed light about its strategy to subdue preys like mussels. In this sense, the toxins seemingly act synergically. Metalloproteinase may produce a degradation of the tissues, aiding the diffusion of the neurotoxins to the target, producing muscle paralysis. Hence, this work constitutes a reference proteome for future studies in sea anemones, also given insight about its potential toxin production and its putative mechanism of action in feeding.

**Supplementary Materials:** The following are available online at www.mdpi.com/1660-3397/16/2/42/s1, Figure S1: Two-dimensional gel electrophoresis of insoluble fraction (IF) from *Bunodactis verrucosa*, Figure S2: Combined Graph obtained for GO Distribution by Level (2); Table S1: Proteins identified against custom cnidarians databases title; Table S2: Proteins identified as potential toxins; Table S3: Details of GO annotation and protein accession number obtained with the Balst2Go software; Table S4: Details of the KEGG analyses obtained with the Balst2Go software.

**Acknowledgments:** Dany Domínguez Pérez was supported by a Ph.D. gran<sup>t</sup> (SFRH/BD/80592/2011) from the Portuguese Foundation for Science and Technology (FCT—Fundação para a Ciência e a Tecnologia, Portugal). A. Campos work were supported respectively by Postdoc grants SFRH/BPD/92978/2013 and SFRH/BPD/103683/2014 from the FCT. Armando A Rodríguez was supported by an Alexander von Humboldt postdoctoral fellowship (3.2-KUB/1153731 STP and the Collaborative Research Centre 1279 funded by the German Research Foundation). We are grateful to Isabel Cunha and Daniela Almeida from CIIMAR, FCUP, University of Porto, for the help in the identification, sampling and transporting of *B. verrucosa* specimens. To Barbara Frazão, from Instituto Português do Mar e da Atmosfera, Lisbon, Portugal for suggestions and by providing information related the species studied. This study was funded in part by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT and the European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through the FCT under the project PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845), and by the Structured Programs of R&D&I INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources (NORTE-01-0145-FEDER-000035, Research Line NOVELMAR) and CORAL NORTE (NORTE-01-0145-FEDER-000036), and funded by the Northern Regional Operational Program (NORTE2020) through the ERDF.

**Author Contributions:** Conceived and Designed the experiments: D.D.-P., V.V., A.C., A.A. Supervised and Contributed Reagents/Materials: V.V., A.A. Two-dimensional gel electrophoresis and MALDI-TOF/TOF analysis: T.R.; A.C., H.O. Shotgun proteomic analysis: M.V.T., D.D.-P., A.C. Wrote the paper: D.D.-P., A.C., A.A.R. Performed the figures and tables: D.D.-P., A.C. Analyzed, discussed and revised the manuscript: A.A.R., M.T., H.O., T.R., V.V., A.A.

**Conflicts of Interest:** The authors declare no conflict of interest.
