*2.4. Survival Rate Analysis and Sponge Explant Growth*

Survival rates in Trial 1 were assessed by visual observation. For Trials 2 and 3, survival was monitored by underwater photography. Explants were photographed using a Nikon D300s digital single lens reflex camera (Nikon Corporation, Tokyo, Japan) and a Sigma 10–20 mm wide-angle lens set (Sigma Corporation, Ronkonkoma, NY, USA), coupled with dedicated Ikelite housing and an DS160 substrobe (Ikelite, Indianapolis, IN, USA). Survival was calculated from the initial and final number (N) of explants residing on the PVC and/or PP plates, as follows:

$$\text{Survival} = (\text{N}\_{\text{final}} / \text{N}\_{\text{initial}}) \times 100 \tag{1}$$

For Trial 3, as described above, blue PP plates were used to collect detached sponges. To calculate survival data, sponges that had fallen onto the PP plates were pooled with the sponges that remained on the PVC plates. However, fallen sponges were excluded from the growth analysis, as PP may affect sponge growth differently from PVC.

For Trial 2, explant growth rates were calculated by measuring wet weights by using a scale (Sinbo SKS 4514) at the start and end of the experiment. To reduce measurement error, the sponges were briefly wiped with clean paper to remove seawater for a duration of approximately 10 s. Growth was calculated from initial and final explant wet weights (WW) as follows:

$$\text{Growth (\%)} = \text{((WW}\_{\text{final}} - \text{WW}\_{\text{initial}}) / \text{WW}\_{\text{initial}}) \times 100 \tag{2}$$

For Trial 3, growth was monitored by underwater photography using the same setup, as described above, for the monitoring of survival. Following each dive, recorded images were transferred to Photoshop CS5 software (Adobe Systems Incorporated, San Jose, CA, USA) and lens distortion was corrected using a Camera Raw 6.7.1 plug-in (Adobe Systems Incorporated, San Jose, CA, USA). The images were calibrated using known plate dimensions, peripheries of explants were marked, and surface area was calculated from pixel counts of the marked areas [39]. Growth was expressed as the increase in the number of pixels, calculated with the pixel counter function of the image editing software. At each time point, the growth in percentage increase in projected surface area was calculated from initial (at start of the time point) and final explant surface areas (A) as follows:

$$\text{Growth (\%)} = (\text{A}\_{\text{final}} - \text{A}\_{\text{initial}}) / \text{A}\_{\text{initial}} \text{)} \times 100\tag{3}$$

To assess the correlation between surface area growth to both biomass and volumetric growth, an additional 20 sponges of random size were collected from a neighboring site. For all 20 sponges, the wet weights were determined, as described above. In addition, volume was determined by measuring displaced seawater in a graduated cylinder. Finally, surface area was determined by using photography and a ruler as a reference, and photographs were processed, as described above.
