A shift away from mutualism under food-deprived conditions in an anemone-dinoflagellate association

Introduction

The symbiosis between corals and photosynthetic dinoflagellates of the family Symbiodiniaceae allows for these cnidarians to construct reefs across Earth’s tropical seas (Wood, 1999; Stanley, 2003); prior to the emergence of these symbioses, although anthozoans were well diversified, they did not play a major role in reef construction (Gaetani & Gorza, 1989; Stanley & Swart, 1995; Russo et al., 1997; Russo et al., 2000; Keim & Schlager, 2001). Given the recent threats of climate change towards coral reef ecosystems, researchers have been making a concerted effort to better understand the fundamental biology of anthozoan-dinoflagellate symbioses (e.g., Mayfield & Gates, 2007; Mayfield et al., 2014), as well as why they bleach upon prolonged exposure to unfavorable environmental conditions (Mayfield et al., 2013; Mayfield, Fan & Chen, 2013). Unfortunately, investigations of the symbiotic relationship between hermatypic corals and Symbiodiniaceae dinoflagellates have been hampered by the slow growth of most corals, the difficulty of working with calcifying organisms, and the fact that, unlike some other animal-dinoflagellate symbioses, corals readily die upon becoming aposymbiotic (i.e., bleaching) and are consequently not amenable to inoculation and bleaching studies.

Anemones of the genus Exaiptasia also engage in symbiosis with Symbiodiniaceae dinoflagellates through mechanisms that are believed to be similar to those characteristic of the most basal coral-Symbiodiniaceae interactions (Lehnert, Burriesci & Pringle, 2012). They are consequently used often as model organisms for studies of coral-dinoflagellate symbioses due to the ease with which they can be maintained in culture in both symbiotic and aposymbiotic states (Weis, 2008; Weis et al., 2008; Sunagawa et al., 2009; Hambleton, Guse & Pringle, 2014); unlike corals, their symbiosis with dinoflagellates is considered facultative, and they are not generally believed to suffer adverse health effects in the aposymbiotic state (provided, presumably, that they are fed regularly).

Typically, the symbiosis between invertebrates and Symbiodiniaceae dinoflagellates is considered mutualistic (Muscatine, 1967; Lewin, 1982). The latter receive CO₂, phosphate, and ammonium (NH₄⁺) from animal catabolism (Cook, D’Elia & Muller-Parker, 1988; Lesser & Shick, 1989; Klumpp, Bayne & Hawkins, 1992; Baillie & Yellowlees, 1998; Yellowlees, Rees & Leggat, 2008; Fransolet, Roberty & Plumier, 2012); in turn, the animal hosts receive up to 95% of the dinoflagellate photosynthate (Trench, 1979; Muscatine, 1990), satisfying nearly all of their energetic needs (Fisher, Fitt & Trench, 1985; Falkowski et al., 1993). However, in some cases, these dinoflagellates have been shown to behave more like parasites (Leung & Poulin, 2008). In excavating sponges, a shift to parasitism was observed when irradiance was modified (Fang et al., 2017). In jellyfish, Symbiodiniaceae dinoflagellates become parasitic when acquired via human-induced horizontal transmission, whereas their association with the jellyfish appears mutualistic when the dinoflagellates are acquired naturally via vertical transmission (Sachs & Wilcox, 2006). As another example, in adults of the gastropod Strombus gigas, Symbiodiniaceae cells have been observed to be predominantly heterotrophic, bestowing a net cost to the host (Banaszak, Ramos & Goulet, 2013); similar behavior was observed in starved anemones kept in darkness, in which aposymbiotic individuals showed a lower mortality rate compared to symbiotic ones (Steen, 1986). Adverse effects have also been documented in anemones inoculated with heterologous symbionts (Matthews et al., 2017). Furthermore, Symbiodiniaceae dinoflagellates are mixotrophic when free-living (Jeong et al., 2012; Xiang et al., 2013; Xiang et al., 2015; Xiang et al., 2018), and such mixotrophic behavior has been observed in hospite (with the algae actually importing carbon from their hosts (Dunn et al., 2012).

Most members of the Apicomplexa, a sister group of dinoflagellates, are parasites (Coats, 1999; Lesser, Stat & Gates, 2013); however, the photosynthetic apicomplexan Chromera velia (Oborník & Lukeš, 2015) is allegedly capable of symbiotic interaction with corals (Venuleo, Práŝil & Giordano, 2017). Following the hypotheses put forth previously (e.g., Leung & Poulin, 2008; Wooldridge, 2010; Lesser, Stat & Gates, 2013), we hypothesized that we could directly demonstrate whether the interaction between the Symbiodiniaceae dinoflagellate Breviolum minutum and Exaiptasia pallida indeed shifts away from mutualism under certain environmental conditions. Specifically, we hypothesized that, by limiting food intake, we could change the behavior of Breviolum spp. in hospite and consequently alter the physiology of their anemone hosts over a multi-week timescale.

Materials and Methods

Anemone husbandry, dinoflagellate culture, and anemone inoculation

The sea anemones (E. pallida) used in this study were obtained from a single clonal line, PT-1, which was originally isolated from the husbandry center of the National Museum of Marine Biology and Aquarium (NMMBA; Peng et al., 2012). The aposymbiotic anemones were prepared via repeatedly cold shocking individuals until they were completely bleached (sensu Muscatine, Grossman & Doino, 1991), and the resulting aposymbiotic anemones were maintained in the dark and fed freshly hatched Artemia sp. nauplii weekly for several years. In order to confirm that there were no dinoflagellate cells within these anemones, juveniles (∼2–3 mm in height) were cultured under a 12-h light (40 µmol photons m⁻² s⁻¹): 12-h dark photoperiod at 25 ° C for two weeks. This period allowed residual dinoflagellate cells, if any, to replicate within the anemones, and anemones were examined for presence of dinoflagellates under a fluorescence stereomicroscope (AxioCam SteREO Discovery V8; Zeiss, Germany).

Upon verifying the absence of dinoflagellates, anemones were further cultured in seawater that had been piped in from offshore (N22 03 00.08, E120 41 42.88) and filtered; filtered seawater (FSW) was changed daily. These experimental organisms were then divided into four groups of 12 individuals: (1) aposymbiotic and starved anemones (ASA), (2) aposymbiotic and fed anemones (AFA), (3) inoculated and starved anemones (ISA), and (4) inoculated and fed anemones (IFA) (Fig. 1). The 12 anemones from each treatment were cultured separately in two 6-well cell culture plates (1 anemone per well). Anemones of the IFA and ISA groups were inoculated with the monoclonal homologous dinoflagellate B. minutum (ITS2 type B1; strain SBM2), which was originally isolated from an Exaiptasia anemone and had been cultured in the laboratory according to Peng et al. (2012) for several years in Guillard’s (f/2) media (without silica, cat. G0154, Sigma-Aldrich, USA) containing antibiotics (10 mg ml⁻¹ streptomycin and 10 units ml⁻¹ penicillin; cat. 15140-122, Gibco, USA) at 25 °C under the same photoperiod described above. For inoculation trials, we followed a previously established method to prepare symbiotic anemones and confirmed their successful uptake of (and later colonization by) dinoflagellates (Chen et al., 2016). Briefly, the cultured dinoflagellates in the early stationary phase were collected via centrifugation (800 xg for 5 min), re-suspended and diluted with 0.22-µm-FSW, and then 15 ml of the dinoflagellate cell solution (3 ×10⁵ cells ml⁻¹) were co-cultured with 50 aposymbiotic anemones in a sterile petri dish (90 × 15 mm).

After 1 hr of co-culture, the anemones were washed twice with FSW to remove any dinoflagellate cells that were not engulfed. The inoculated anemones were observed under the fluorescence stereomicroscope to ensure that dinoflagellates had been uptaken, and >90% of the anemones were successfully inoculated; only these were used in experiments. Consequently, all anemones (including the aposymbiotic controls of the ASA and AFA groups) were maintained in 6-well cell culture plates (1 anemone per well) for further observations. In order to prevent the buildup of bacteria, seawater was changed daily. After two weeks of inoculation, the AFA and IFA groups were fed with Artemia sp. nauplii (56 well⁻¹) once a week; as this was more than the anemones could consume, uneaten nauplii were removed after 30 min. Replicates of all four treatments were visually monitored, and growth and pedal disk measurements (described below) were made weekly.

Results

E. pallida anemones of the IFA and ISA groups were successfully inoculated with the monoclonal, cultured, and homologous Breviolum dinoflagellates (SBM2). One-hour post-inoculation, the Breviolum cells were observed in the gastric cavity of each anemone (Fig. 2A, 2G), and the dinoflagellate cells were initially concentrated in the column (with only a few present in the tentacles). After 1 to 2 weeks, the dinoflagellate cells proliferated (Fig. 2B–2C, 2H–2I; Fig. S1A–S1B), and the average dinoflagellate cell numbers were 126, 2,896, and 9,617 cells per anemone after 1 h, 1 week, and 2 weeks in the ISA groups, respectively, and 244, 3,113, and 10,687 cells per anemone, respectively, in the IFA group (Fig. S1A).

Two-weeks post-inoculation, the average dinoflagellate cell density within the anemones increased significantly over earlier observation times, nearly saturating the anemone tissues; the densities were similar in the ISA and IFA groups (17.6 ×10³ cells mm⁻² and 16.4 ×10³ cells mm⁻², respectively; Fig. S1B). After confirming that both groups of anemones were colonized to a similar degree, we began to feed those of the IFA group. The aposymbiotic control groups, AFA and ASA, remained symbiont-free for the entire duration of the study (see Fig. 2M–2R and 2S–2X for representative images.) From the 4th week onwards, the endosymbiont cell counts differences between IFA and ISA became more pronounced; this is evident from both images of representative anemones (Fig. 2D) and cell counts data per whole anemone (Fig. S1A). Curiously, when accounting for the reduced size of the ISA (normalizing to anemone surface area), the endosymbiont density, in fact, remained similar to that of IFA over time (Fig. S1B).

Overall, the two fed groups (IFA and AFA) grew to larger sizes, and feeding had a significant effect in the two-way, repeated measures ANOVA (Table 1); specifically, pedal disk analysis (Fig. 3) confirmed that the fed groups were appreciably larger from the 4th week post-inoculation onwards, and such differences were statistically significant by the 6th week (Fig. 3Q). By the 8th week, anemones of the IFA and AFA groups were ∼20- and ∼10-fold larger, respectively, than the ISA and ASA groups, respectively (Fig. 3Q); interestingly, anemones of the IFA and AFA were similarly sized. For the starved groups (ISA and ASA), the anemones did not grow appreciably over time (Fig. 3Q); in fact, pedal disk areas shrunk from 0.44 mm² at week 1 to 0.22 mm² at week 8 (ISA) and from 0.58 mm² to 0.27 mm², respectively (ASA), though these differences were not statistically significant.

The groups differed not only in their growth but also in their external morphology (Fig. 2). While the fed groups showed the typical anemone morphology (Figs. 2G–2L and 2S–2X), the starved ones demonstrated morphological changes, which were especially evident in the ISA group by the 4th week (Fig. 2A–2F); specifically, these individuals were characterized by smaller column sizes and reduced tentacle size and number (Fig. 2D; Fig. 4C). Four to five weeks after inoculation, anemones from the ISA group began to morph into spherical shapes. At this stage, it was still possible to distinguish the oral disk from the mesenteries. These anemones eventually formed tissue balls lacking in recognizable structure (Fig. 2E; Fig. 4D; Figs. 5A–5B); by the 8th week, 100% of the samples in this treatment group had formed tissue balls (Table 2). There was a statistically significant effect of treatment on likelihood of tissue ball formation (Table 2). Furthermore, 25% of the ISA had died by the 8th week. In contrast, anemones of no other treatments formed tissue balls, except for ASA after six months of observation (Figs. 4E–4H). In the tissue balls of the ISA, the host tissues eventually disintegrated (Fig. 5D); however, dinoflagellate cells appeared intact, and cell walls, chloroplasts, nuclei, and chromosomes were observed (Figs. 5E–5F). Even a dividing dinoflagellate cell was observed (Fig. 5F). Moreover, tissues ball did not bleach (Figs. 5A–5B), and the density of symbiotic dinoflagellates in the tissue ball actually increased (Fig. S1B).

Fed anemones (both inoculated and aposymbiotic) were further cultured for six months to allow them to (potentially) grow to full size. During this period, asexual reproduction (laceration) was documented, and the aposymbiotic (AFA) and inoculated (IFA) groups produced 9 and 30 lacerates, respectively.

Discussion

Regularly fed Exaiptasia-Breviolum holobionts increased in size and exhibited high asexual reproduction rates and low mortality rates (only 1 of 12 fed anemones died.). However, their ability to grow and reproduce cannot be solely attributed to their association with photosynthetic dinoflagellates; symbiotic anemones that were deprived of exogenously supplied food failed to grow (Figs. 1 and 2). Whether this was due to host malnourishment or, as we hypothesize herein, a shift in the behavior of the Breviolum populations in hospite, remains to be determined. Certainly nutrient labeling approaches (Cui et al., 2019) should be undertaken in the future to determine whether, for instance, Breviolum cease to translocate fixed carbon to the host anemones, or even uptake organic nutrients from them, under such starved conditions. Nevertheless, we now explore the potential shift away from mutualistic behavior by the homologous dinoflagellate B. minutum upon integration of our data with findings from the primary literature.

Steen (1986) revealed that Symbiodiniaceae dinoflagellates can survive extended periods of time in darkness due to their capacity to obtain organic nutrients from their anemone hosts. Moreover, the same study demonstrated that symbiotic anemones in complete darkness had a higher mortality rate than aposymbiotic ones. Baker et al. (2018) obtained similar results from working with corals (Orbicella faveolata); specifically, they found that seawater warming, even in non-limiting food conditions, induced the symbionts to (1) release less photosynthate and (2) increase the uptake of resources from the host. These studies suggest that Symbiodiniaceae heterotrophy in hospite can impose a fatal metabolic burden on their anthozoan hosts, which could explain why our symbiotic, albeit starved, anemones failed to grow and instead formed tissue balls (or even died) in many cases. The fact that aposymbiotic anemones that were fed, in contrast, continued to grow may signify that the dinoflagellates in the starved anemones were indeed compromising the health of their hosts. It is worth noting here that the starved-symbiotic anemones (ISA) did not bleach. Moreover, when the anemone tissues disintegrated, the symbiotic dinoflagellates still displayed normal cellular ultrastructure and were capable of dividing; the fact that they were survived and proliferated in disintegrating host tissue is further evidence for either commensal, or maybe even parasitic, behavior (Fig. 6).

There are several reports that have explored the potential for nitrogen limitation to be a means of inducing symbiont lipid biogenesis/accumulation (Peng et al., 2012) and density control (Cui et al., 2019) in Exaiptasia anemones. Under nitrogen-limited or deprived conditions, the dinoflagellates regularly accumulate lipid bodies (LBs), regardless of whether they are free-living or in hospite (Jiang, Pasaribu & Chen, 2014; Weng et al., 2014). In this study, there was no LB accumulation within dinoflagellate cells of disintegrating host anemones (Figs. 5D–5F). Instead, the morphology of these dinoflagellate cells was similar to the nutrient-sufficient control specimens of the afore-cited works. Given that dinoflagellate cells did not show signs of nitrogen starvation, but instead continued to grow under these conditions, it is possible that they were acquiring nitrogen from the dying host cells. Nutrient labeling approaches (sensu Cui et al., 2019) could surely aid in verifying this.

In Hydra, an asymbiotic cnidarian, starvation-induced autophagy is a critical survival mechanism under food-deprived conditions. Upon degrading their own nonessential cells/tissues to obtain amino acids, lipids, and other nutrients, their tentacles and overall body sizes shrink, and they cease undergoing asexual reproduction (Chera et al., 2009). In this study, the ASA and ISA groups behaved similarly to the starved Hydra, as their tentacle and body size decreased gradually. Since autophagy is a highly conserved process in all eukaryotes (Parzych & Klionsky, 2014), we suggest it may play a critical role in regulating the cellular stress responses and metabolic modulation of the starved anemone. Our findings might also suggest that the symbiotic dinoflagellate cells appear to accelerate the self-degrading/recycling mechanisms of their host anemones, or even benefit from this process, because the anemones of the ISA group only survived for 2 months, while the ASA group survived for 6 months.

In addition to the other instances of mutualism-to-parasitism shifts mentioned in the Introduction, it is worth noting that mutualism is typically thought to have evolved from parasitism, at least in the case of Symbiodiniaceae dinoflagellates (Stat, Morris & Gates, 2008). This (point #1), along with the aforementioned relationship between dinoflagellates and parasitic apicomplexans (point #2; Lesser, Stat & Gates, 2013) and the microalgal capacity for heterotrophy (point #3; Jeong et al., 2012; Xiang et al., 2015; Xiang et al., 2018) all lend credence to our hypothesis that the delicate metabolic and physiological equilibrium between invertebrates and intracellular dinoflagellates may transition away from being one of mutual benefit to both partners when certain environmental conditions deteriorate. Further work must be undertaken, though, in order to know whether the dinoflagellates within the starved anemones are also malnourished or if they are truly acting as parasites (i.e., absorbing nutrients from their dying hosts). Given that the marine regions in which coral reefs are found tend to be oligotrophic (and therefore of limited potential food supply), the answer to this question may have profound implications for the future survival of coral reef ecosystems.

Conclusions

Given that anemones that were inoculated with dinoflagellate endosymbionts and consequently starved failed to grow, and finally disintegrated into tissue balls, whereas those inoculated anemones that were fed instead continued to grow, we surmise that, under stressful conditions, the anemone-dinoflagellate endosymbiosis may shift away from one that benefits both partners. Whether this is due to truly parasitic behavior of the endosymbionts, rather than simply commensalism coupled with host starvation, remains to be determined and should be the focus of future studies. These findings and raised hypothesis, therefore, contribute to the field and how we view symbiosis across a spectrum of mutualistic and parasitic relationships.

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