*4.4. Recommendations for Future Research*

Culture success can be further improved by optimizing the period of culture. Optimal culture time can be determined by observing sponge growth rates over a period of two or more subsequent years. Page et al. [39], found reduced growth rates for *Mycale (Carmia) hentscheli* (Bergquist & Fromont) [47] over time. The growth rates in their study dropped from 2437% year−<sup>1</sup> to 1355% year−<sup>1</sup> from the first to the third culture period. Moreover, the growth rates of cloned sponges harvested from cultured explants should also be followed, as Page et al. [39], found reduced growth rates and even negative growth through repeated cloning (F0 to F2).

Other important aspects to include in future studies are seasonality (e.g., is the fast initial growth observed in this study season-influenced or a wound-healing response that is irrespective of season) and genotypic variability.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/1/29/s1, Figure S1: Overview of culture methods; (a) 1st trial; explants tie-wrapped to black PVC, experiment start (b) 1st trial; sponges tie-wrapped to white PVC, 1 year later explants splitting into two fragments and relocating position. (c) 2nd trial, polluted site; image of 50 × 50 cm plates, sponge explants secured with chicken wire (0–30◦ plates can be seen, 25 additional explants are on the other side of each plate). (d) 2nd trial, pristine site; infected explants shortly after seeding. (e) 3rd trial, polluted site; first upscaling of aquaculture of Chondrosia reniformis on vertical PVC plates. Explants are attached with gel-based polyacrylate superglue to PVC plates (10 plates per site). (f) 3rd trial, pristine site; explants after 4 months of culture. Some of the explants tend to travel towards the polypropylene plate and grow on it, but others prefer to stay on the PVC plates. Figure S2: Correlation between surface area (cm2) and wet weight (g) for Chondrosia reniformis (Pearson correlation, *r* = 0.92, *n* = 20, *p* = 0.000, two-tailed). Figure S3: Correlation between surface area (cm2) and volume (mL) for Chondrosia reniformis (Pearson correlation, *r* = 0.92, *n* = 20, *p* = 0.000, two-tailed). Table S1: Experimental design of aquaculture trials 2 and 3. In Trial 3, 10 explants were taken per parent sponge, five explants were attached to each plate.

**Author Contributions:** Conceptualization, M.G., A.S., J.M.d.G. and R.O.; Data curation, M.G.; Formal analysis, M.G., T.W. and R.O.; Funding acquisition, J.M.d.G. and R.O.; Investigation, M.G.; Methodology, M.G., A.S., J.M.d.G. and R.O.; Resources, R.O.; Supervision, R.O.; Validation, M.G., T.W. and R.O.; Visualization, M.G. and T.W.; Writing—original draft, M.G.; Writing—review & editing, T.W., A.S., J.M.d.G. and R.O.

**Funding:** This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. KBBE-2010-266033 (Project SPECIAL). Funding was also received from The Innovational Research Incentives Scheme of the Netherlands Organization for Scientific Research (NWO-VENI; 863.10.009; personal grant to Jasper M. de Goeij). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Acknowledgments:** Intergrup is acknowledged for providing the PVC plates. Special thanks to Laura Valderrama Ballesteros, Holger Kuehnhold, Tjitske Kooistra, Marretje Adriaanse and Mustafa Gökalp for dive support, Suha and Alev Gökalp, Yasemin and Hakan Akyuz for logistics, and Erdal Betin for continuous support.

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