Symbiodiniaceae cell densities in feces of coral reef fish, sediments and seawater in Mo'orea, French Polynesia, July-August 2019

Individuals of nine fish species, sediments, and seawater were collected (n=6-14 per sample type or species) in July and August 2019 on two reef zones: the back reef (1-2 m depth) and fore reef (5-10 m depth), between LTER sites 1 and 2 of the Mo’orea Coral Reef (MCR) Long Term Ecological Research (LTER) site. We selected fish species that broadly differ in their level of corallivory (Ezzat et al., 2020; Harmelin-Vivien and Bouchon-Navaro, 1983; Rotjan and Lewis, 2008; Viviani et al., 2019) as obligate corallivores (butterflyfishes Chaetodon lunulatus, Chaetodon ornatissimus, Chaetodon reticulatus, and the filefish Amanses scopas), facultative corallivores (butterflyfishes Chaetodon citrinellus and Chaetodon pelewensis and the parrotfish Chlorurus spilurus) and grazer/detritivores (surgeonfishes Ctenochaetus flavicauda and Ctenochaetus striatus). Sample processing Following collection, all samples were sub-sampled and processed for Symbiodiniaceae density and viability. Feces were sampled from the hindgut of each fish (n=6-14 per species) so that Symbiodiniaceae condition (live versus dead) was assessed from cells that had passed through the entire fish digestive tract. Fecal samples for Symbiodiniaceae cell counts were preserved in 750 µl 10% formalin in 100 kDa-filtered seawater immediately after dissection out of each fish. Reef-associated seawater samples were collected from the fore reef and back reef (n=6 per reef zone) at a distance of 10-100 cm off the reef bottom and processed using a protocol modified from Littman et al (2008). Samples were filtered onto two separate 0.45-micron filters. One filter per sample was preserved for Symbiodiniaceae cell counts by resuspension in 3ml 5% formalin in 100 kDa-filtered seawater and vortexing at maximum speed for 10 minutes. Reef sediment samples were collected concomitantly with water column samples (n=6 per reef zone). Considering that Symbiodiniaceae populations in sediments are spatially heterogeneous (Littman et al., 2008), each individual sediment sample consisted of 500 ml of sediment pooled from ten sterile 50 ml conical tubes filled at five random locations in a 10m radius from where the associated seawater sample was collected. To best approximate the Symbiodiniaceae cells that would be available for uptake by nearby coral colonies, sediment collections were focused at the sediment-water interface (no deeper than 5 cm) and occurred less than 1m from live coral colonies. Sediments were immediately washed over a 120-micron nylon mesh using 500 ml 100 KDa filtered seawater and filtered through a 20-micron nylon mesh. The flow-through was then processed in the same manner as described above for the seawater samples. Symbiodiniaceae cell density and viability All cell count samples were homogenized using a Fisher Scientific homogenizer F150 for five seconds to break up cell clumps and subsequently filtered through 120 and 20 micron nylon filters to reduce the density of debris in samples. Symbiodiniaceae cells in fish feces, seawater and sediments were quantified using a Neubauer hemocytometer. All samples were stained with 0.16% trypan blue 5 minutes before processing to parse live from dead cells (Haslun et al., 2011; Strychar and Coates, 2004; Zhang et al., 2008). Individual cells were interpreted as having been ‘live’ at the time of fixation if, following staining, they retained a golden-brown color. Cells were interpreted as ‘dead’ at the time of fixation if staining turned them blue. During counts, cells were counted as Symbiodiniaceae if they were of the correct size (6-12 µm; LaJeunesse et al., 2018), had visible organelles, a coccoid shape, and a large pyrenoid accumulation body (Littman et al., 2008; Nitschke et al., 2016).