DOI: 10.5061/dryad.80gb5mkpd
title: Symbiodiniaceae cell densities in feces of coral reef fish, sediments and seawater in Mo'orea, French Polynesia, July-August 2019

description: Background: The microbiomes of foundation (habitat-forming)
 species such as corals and sponges underpin the biodiversity,
 productivity, and stability of ecosystems. Consumers shape communities of
 foundation species through trophic interactions, but the role of consumers
 in dispersing the microbiomes of such species is rarely examined. For
 example, stony corals rely on a nutritional symbiosis with single-celled
 endosymbiotic dinoflagellates (family Symbiodiniaceae) to construct reefs.
 Most corals acquire Symbiodiniaceae from the environment, but the
 processes that make Symbiodiniaceae available for uptake are not resolved.
 Here, we provide the first comprehensive, reef-scale demonstration that
 predation by diverse coral-eating (corallivorous) fish species promotes
 the dispersal of Symbiodiniaceae, based on symbiont cell densities and
 community compositions from the feces of four obligate corallivores, three
 facultative corallivores, two grazer/detritivores as well as samples of
 reef sediment and water. Results: Obligate corallivore feces are
 environmental hotspots of Symbiodiniaceae cells: live symbiont cell
 concentrations in such feces are 5–7 orders of magnitude higher than
 sediment and water environmental reservoirs. Symbiodiniaceae community
 compositions in the feces of obligate corallivores are similar to those in
 two locally abundant coral genera
 (Pocillopora and Porites), but differ from
 Symbiodiniaceae communities in the feces of facultative corallivores and
 grazer/detritivores as well as sediment and water. Combining our data on
 live Symbiodiniaceae cell densities in feces with in situ observations of
 fish, we estimate that some obligate corallivorous fish species release
 over 100 million Symbiodiniaceae cells per 100 m2 of reef per
 day. Released corallivore feces came in direct contact with coral colonies
 in the fore reef zone following 91% of observed egestion events, providing
 a potential mechanism for the transfer of live Symbiodiniaceae cells among
 coral colonies. Conclusions: Taken together, our findings show that fish
 predation on corals may support the maintenance of coral cover on reefs in
 an unexpected way: through the dispersal of beneficial coral symbionts in
 corallivore feces. Few studies examine the processes that make symbionts
 available to foundation species, or how environmental reservoirs of such
 symbionts are replenished. This work sets the stage for parallel studies
 of consumer-mediated microbiome dispersal and assembly in other sessile,
 habitat-forming species.
description: 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 <i>Chaetodon
 lunulatus</i>, <i>Chaetodon ornatissimus, Chaetodon
 reticulatus,</i> and the filefish <i>Amanses
 scopas</i>), facultative corallivores (butterflyfishes
 <i>Chaetodon citrinellus</i> and<i> Chaetodon
 pelewensis</i> and the parrotfish <i>Chlorurus
 spilurus</i>) and grazer/detritivores (surgeonfishes
 <i>Ctenochaetus flavicauda </i>and <i>Ctenochaetus
 striatus</i>). <b>Sample processing</b> 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. <b>Symbiodiniaceae cell density and viability</b> 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).
description: The file
 “Fishces_2019_cellcounts.csv“ contains densities of live (unstained by
 trypan blue; column ‘Live’) and dead (stained by trypan blue; column
 ‘Dead’) Symbiodiniaceae cells ml<sup>-1</sup> material (feces,
 sediment seawater), as determined through cell counts using a
 hemocytometer and compound microscope at 20x and 40x magnification.
 Reported cell densities are averages of 8 replicate counts. The code to
 replicate this analysis can be found on https://github.com/CorreaLab/Fishces_2020/blob/master/Symbiodiniaceae%20cell%20counts%20code.