Centrifugation Speeds and Times from Freeman and Thacker (2011) Were Modified to Allow

Centrifugation speeds and times from Freeman and Thacker (2011) were modified to allow separation using an Eppendorf 5430 counter-top centrifuge with an Eppendorf F-

35-6-30 rotor holding 50 ml centrifuge tubes. For optimal purity of the sponge cell fraction, homogenized filtrate (containing dissociated sponge and microbial cells) from the Aplysina spp. and N. erecta, was centrifuged at 730 RCF (Relative Centrifugal

Force), for 2 minutes, while the filtrate from N. subtriangularis and C. caribensis was centrifuged for 2 minutes at 260 RCF and 1050 RCF, respectively. For all species, approximately 80% of the resulting supernatant (containing microbial cells) was carefully poured into a clean centrifuge tube and stored at 4 °C. The remaining supernatant was poured off and discarded to prevent contamination of the sponge cell fraction and the sponge pellet was resuspended in 10 ml of Calcium and Magnesium Free Artificial Sea

Water (CMFASW) and centrifuged at either 730 RCF (for C. caribensis), 470 RCF

(Aplysina spp. and N. erecta) or 260 RCF (N. subtriangularis) for 2 minutes, after which the supernatant was discarded. Following one more cycle of resuspension and centrifuging at these speeds (except for C. caribensis, which was centrifuged at 470 RCF for 2 minutes in this final cycle), the sponge pellet was resuspended in 1.5 ml of

CMFASW and added to a 1.5 ml microcentrifuge tube. These samples were centrifuged at 239 RCF for 3 minutes using an Eppendorf FA-45-30-11 rotor. The supernatant was carefully removed using a pipette and the sponge pellet was frozen for future analyses.

The supernatant remaining from the original centrifugation step (containing microbial cells) was centrifuged at 7200 RCF for 4 minutes to pellet the microbial cells. After removing the resulting supernatant, the pellet was resuspended in 1.5 ml of CMFASW, transferred to a 1.5 ml microcentrifuge tube, and then centrifuged at 6800 RCF for 5 minutes. The supernatant was removed and the microbial fraction was frozen for future isotope analyses.

New methods were developed for X. bocatorensis, which hosts filamentous cyanobacterial symbionts. Minced tissue was wrapped in 55 μm mesh and soaked in

CMFASW containing EDTA (CMFASW-E; Freeman and Thacker, 2011) for 30 min.

The sponge was homogenized using a mortar and pestle and the resulting slurry containing sponge and microbial cells was filtered through a Whatman No. 4 filter (20

-25 μm pore size) to remove undissociated cells. This filtrate was centrifuged for 2 min at

1050 RCF to obtain a pellet containing sponge cells and some cyanobacterial filaments.

Approximately 80 % of the supernatant containing microbial cells was poured into a clean 50 ml centrifuge tube and stored at 4 °C; the remainder of the supernatant was discarded. The mixed pellet was resuspended and centrifuged at 730 RCF for 2 min, followed by another cycle of resuspension and centrifugation at 450 RCF for 2 min to obtain a sponge cell pellet. The original supernatant containing microbial cells was centrifuged at 7200 RCF for 3 min to obtain a microbial pellet. Sponge and microbial pellets were transferred to microcentrifuge tubes and frozen until future analyses. The purity of sponge and microbial fractions of each species was assessed using light and epifluoresence microscopy (Freeman and Thacker, 2011). As outlined in Muscatine et al.

(1989), and Tanaka et al. (2006), these methods achieve nearly pure cell fractions. Using the methods above, we minimized contamination of the sponge or bacterial pellets by bacterial or sponge cells and measured fractions to contain ~ 95% of one cell type.