*2.3. Mariculture Trials*

Within the period between April 2011 and December 2013, three subsequent mariculture trials were executed in order to develop and improve a culture method for *C. reniformis*.

1st mariculture trial, April 2011–June 2011: testing materials and attachment procedures—Sponge explants (*n* = 5 specimens per plate) were attached to four types of supports (autoclaved aerated concrete, white polyvinyl chloride (PVC), black PVC, and cemented PVC) using six different combinations of attachment methods and substrates (Table 1 (a)). The cementation of plates may improve attachment of the sponges to the support and enhance growth, since quartz and silica are found to promote collagen formation in sponges [34,40]. Accordingly, coarse sea-sand was used to make cement to cover the cemented-PVC supports. All of the supports were positioned in Meteoroloji Bay (Figure 1 Pr. 1) at 2–3 m water depth under overhangs or in crevices (i.e., not in direct sunlight) and fixed with diving weights.

2nd mariculture trial, June 2011–June 2012: testing culture plate orientation and site—Based on the results of the first trial, PVC plates were chosen for the second mariculture experiment. Explants (250 in total per site) were positioned on both sides of five 50 × 50 cm plates, with 25 explants on each side of every plate. In order to find the optimal positioning of the sponges, explants were cultured at nine different angles, under light (exposed side of plate) or dark (underside of plate) conditions, resulting in 10 different conditions (*n* = 1 plate per treatment) (0◦, 30◦, 45◦, 60◦, 90◦ light, 90◦, 120◦, 135◦, 150◦, and 180◦ dark; Supplementary Table S1, Figure 2a,b). In order to keep the sponges attached to the plates until natural attachment took place, the sponges were covered with chicken wire and left on the seabed for two days. After the attachment of the explants to the plates, the plates were mounted on a metal frame. Frames were installed in July 2011 at the two selected sites (Kargı Island (Figure 1 Pr. 2)—pristine and Guvercinlik Bay (Figure 1 Po. 1)—polluted) at a water depth of 10 m.

**Figure 2.** Schematic drawings of the culture platform designs. (**a**) 2nd trial—Top view; aluminum culture frame and attached 50 × 50 cm polyvinyl chloride (PVC) plates covered with chicken wire, each carrying 25 sponge explants on one side, totaling 50 sponges per plate. (**b**) 2nd trial—Side view; plates positioned at 9 different testing angles 0◦, 30◦, 45◦, 60◦, 90◦, 120◦, 135◦, 150◦, 180◦. 3rd trial, (**c**) 3rd trial—front view, and (**d**) 3rd trial—side view showing positioning of glued *C. reniformis* explants on 25 × 25 cm PVC plates. PVC plates were secured tightly to the aluminum frame from four corners with 6-mm thick fishing lines, blue PP plates were attached to the bottom sides of the PVC plates.

**Table 1.** Overview of the results of the three culture trials executed in between May 2011 and November 2013. (a) 1st trial, material test and attachment procedures, pristine site (b) 2nd trial, testing culture orientation and site (c) 3rd trial, assessment of productivity at the optimal culture orientation.


3rd mariculture trial, June 2012–July 2013: assessment of growth at the optimal culture orientation—Based upon the observations of the 2nd mariculture trial, it was decided to choose an angle of 90◦ for primary upscaling of the cultures. Two new frames were installed in early June 2012, one at the pristine site and one at the polluted site, each carrying 20 white PVC plates of 25 × 25 cm with a total of 100 explants of *C. reniformis* (5 per plate), attached using gel-based polyacrylate superglue. A gel-based polyacrylate superglue method was preferred over chicken wire to improve the handling time and reduce the weight and cost of the culture materials. In addition, horizontal blue polypropylene PP plates were placed underneath the 90◦ PVC plates to provide extra surface for the explants to attach and grow, should they fall (Figure 2c,d). Explants were grown for 13 months, photographed, and measured in June, July, August, September, October, and November 2012 (both sites), in March and May 2013 (polluted site only, due to weather restrictions), and in July 2013 (both sites).
