Coral reefs are the most biodiverse marine ecosystems on our planet. Their immense productivity is driven by friendly relationships, or symbioses, between microbes called algae and the corals. Related organisms, such as anemones, also rely on these close associations. The algae use energy from sunlight to make sugars, cholesterol and other molecules that they supply to their host. In exchange, the host’s cells provide homes for the algae inside specialist, acidic structures called symbiosomes.

Corals and anemones particularly need cholesterol and other ‘sterol’ molecules from the algae, because they are unable to create these building blocks themselves. In mammals, a protein known as Niemann-Pick Type C2 (NPC2) transports cholesterol out of storage structures into the main body of the cell. Corals and anemones have many different, ‘atypical’ NPC2 proteins: some are produced more during symbiosis, and these are mainly found in symbiosomes. However, it was not known what role these NPC2 proteins play during symbioses.

Here, Hambleton et al. studied the symbioses that the anemone Aiptasia and the coral Acropora create with different strains of Symbiodiniaceae algae. The experiments found that the strain of algae dictated the mixture of sterols inside their hosts. The hosts could flexibly use different mixes of sterols and even replace cholesterol with other types of sterols produced by the algae. Atypical NPC2 proteins accumulated over time within the symbiosome and directly bound to cholesterol and various sterols the way other NPC2 proteins normally do. Further experiments suggest that, compared to other NPC2s, atypical NPC2 proteins may be better adapted to the acidic conditions in the symbiosome. Taken together, Hambleton et al. propose that atypical NPC2 proteins may play an important role in allowing corals to thrive in environments poor in nutrients.

The first coral reefs emerged over 200 million years ago, when the Earth still only had one continent. Having built-in algae that provide the organisms with nutrients is thought to be the main driver for the formation of coral reefs and the explosion of diversity in coral species. Yet these ancient relationships are now under threat all around the world: environmental stress is causing the algae to be expelled from the corals, leading to the reefs ‘bleaching’ and starving. The more is known about the details of the symbiosis, the more we can understand how corals have evolved, and how we could help them survive the crisis that they are currently facing.

Many plants and animals cultivate symbioses with microorganisms for nutrient exchange. Cnidarians, such as reef-building corals and anemones, establish an ecologically critical endosymbiosis with photosynthetic dinoflagellate algae (Douglas, 2010) (family Symbiodiniaceae) (LaJeunesse et al., 2018). Their symbionts reside within endo/lysosomal-like organelles, termed symbiosomes, and transfer photosynthetic products to their hosts (Muscatine, 1990; Yellowlees et al., 2008). In addition to sugars that mostly provide energy, recent studies hint at the importance of the transfer of various lipids including sterols (Crossland et al., 1980; Battey and Patton, 1984; Revel et al., 2016). Sterols are essential building blocks for the cell membrane and endomembrane systems, in the form of cholesterol and other sterol variants. Cnidarians are sterol auxotrophs (Baumgarten et al., 2015; Gold et al., 2016) that must acquire these essential compounds from diet and/or symbionts (Goad, 1981). In line with this, non-canonical variants of the conserved cholesterol transporter Niemann-Pick Type C2 (NPC2) are among the most up-regulated genes in symbiotic Exaiptasia pallida (commonly Aiptasia) and Anemonia viridis anemones (Dani et al., 2014; Lehnert et al., 2014; Kuo et al., 2010; Ganot et al., 2011; Wolfowicz et al., 2016). Dinoflagellates synthesize various sterols, many of which are found in symbiotic cnidarians (Bohlin et al., 1981; Withers et al., 1982; Ciereszko, 1989); however, the specific combinations of transferred sterols, as well as the mechanism of this transfer remain unknown. To what extent is the specific mix of transferred sterols controlled by the host, symbiont, or both – reflecting physiological relevance – and how is such selective transport achieved?

To answer these questions, we took advantage of the availability of distinct strains of Symbiodiniaceae symbionts with different and complex sterol compositions (Bohlin et al., 1981; Withers et al., 1982; Ciereszko, 1989), and of various hosts. Besides the coral Acropora digitifera, we investigated different host lines of the symbiotic anemone Aiptasia, an emerging model system for coral-algal symbiosis (Tolleter et al., 2013; Neubauer et al., 2017). We used gas chromatography/mass spectrometry (GC/MS) to semi-quantitatively profile relative sterol abundances in three compatible symbiont strains (Xiang et al., 2013; Hambleton et al., 2014), and related this to sterol abundances in the coral and in three Aiptasia laboratory lines (Grawunder et al., 2015), with or without symbionts (Figure 1, Figure 1—source data 1). First, to validate our assay and to show that algal sterols are indeed transferred to host tissue, we determined the host sterol composition without symbionts (aposymbiotic), in symbiosis with recent dietary input (two weeks since last feeding, ‘intermediate’), and in symbiosis with essentially no dietary input (five weeks since last feeding, ‘symbiotic’). For the Aiptasia F003 host line, this revealed a gradual transition from an initial aposymbiotic, food-derived cholesterol profile to a cholesterol-reduced, algal sterol-enriched symbiotic profile that was also found in the symbiont-free eggs (and is thus present in host tissue) (Figure 1A). We also compared the sterol composition of coral symbiotic polyps collected from the wild to that of their symbiont-free eggs, which again proved nearly identical sterol compositions (Figure 1A) and unambiguously revealed symbiont-to-host tissue transfer. Taken together, this suggests that symbiont-derived sterols can functionally replace dietary cholesterol without any further chemical conversion by the host. Moreover, the sterol content of the hosts is highly plastic, and sterols are used flexibly as they become available from food and/or symbionts.

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Figure 1—source data 1

Relative sterol compositions of samples in pie graphs.

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We next focused on the sterol compositions in different symbiont-host pairings, to determine how these would change upon switching of either symbiont or host line. To this end, we investigated the same Aiptasia line CC7 hosting distinct symbionts (SSA01 or SSB01, see Materials and methods) with different symbiont profiles; and the same symbiont (SSB01) in two distinct host lines (CC7 and H2), as well the symbiont CCMP2466 similar to that in Acropora. We found that Aiptasia CC7 hosting Symbiodiniaceae strain SSA01 contained a large proportion of stigmasterol-like sterol (dark blue, Figure 1B) when compared to campesterol (light blue, Figure 1B). In contrast, the same Aiptasia line hosting strain SSB01 contained minimal stigmasterol-like derivatives compared to campesterol, as well as the unique sterol gorgosterol (light blue and pink, respectively, Figure 1B), characterized by an unusual cyclopropyl group (Ciereszko, 1989). (Figure 1—figure supplement 1). A very similar sterol profile was observed when the same symbiont (SSB01) infected the H2 host line, indicating that the host sterol profile was largely symbiont-driven. Likewise, in Aiptasia line F003 hosting both SSA01 and SSB01, the sterol proportions reflect both symbionts: a dominance of stigmasterol-like sterol (reflecting SSA01) together with gorgosterol (reflecting SSB01) (Figure 1A). We also compared the sterol profile of Acropora colonies collected from the wild to that of a closely related symbiont CCMP2466 in laboratory culture and found a strong enrichment for gorgosterol and campesterol at the expense of stigmasterol-like sterols – highly reminiscent of the trend previously observed in the SSB01/CC7 and SSB01/H2 pairings (Figure 1A). We thus observed two major patterns of sterol transfer in our symbiont-host combinations – one enriching for stigmasterol-like sterols (combinations SSA01/CC7 and SSA01 +SSB01/F003), and another one enriching for gorgosterol and campesterol (combinations SSB01/CC7; SSB01/H2; and CCMP2466/Acropora). This suggests that selective sterol transfer and/or accumulation by the host may occur.

Moreover, symbionts may change their sterol synthesis profile as symbiotic vs. free-living cells. To address this, we separated anemone homogenates by centrifugation into symbiont-enriched (pellet, although substantial host tissue remained, Figure 1—figure supplement 2A and Figure 1—source data 1) and symbiont-depleted (supernatant) fractions, for which the sterol profiles could be directly compared to free-living symbionts cultured under similar conditions. This revealed that certain sterols were absent in symbiont-enriched pellets yet present in symbiont cultures (Figure 1C, Figure 1—figure supplement 2B). For example, stigmasterol/gorgosterol-like (dark purple) and ergost-tetrol-like sterol (light purple) are proportionally highly abundant in cultured symbionts, yet are basically absent in all pellet samples (Figure 1C, Figure 1—figure supplement 2B). This suggests that synthesis of at least some sterols changes in residence vs. in culture, providing further support that the symbiont has a major influence on which specific composition and proportion of the sterols are transferred during symbiosis. Further, cultured symbionts exhibited some degree of plasticity of sterol profiles under various culturing conditions (e.g. SSA01 in Figure 1A vs. Figure 1C).

To elucidate possible molecular mechanisms how symbiont-hosting cells may influence sterol transfer from the symbiont, we focused on non-canonical members of the highly conserved NPC2 protein family (Dani et al., 2014; Lehnert et al., 2014). The current hypothesis in the field is that non-canonical NPC2s may specifically facilitate transfer of symbiont-produced sterols in cnidarian-algal symbiosis (Revel et al., 2016; Baumgarten et al., 2015; Wolfowicz et al., 2016; Dani et al., 2017). However, NPC2s may serve other purposes, for example signaling (Baumgarten et al., 2015; Dani et al., 2017), and mechanistic analyses of NPC2 function are lacking. To characterize them further, we first compared the genomic complement of NPC2 homologues in symbiotic cnidarians to that of non-symbiotic metazoans, uncovering several previously unidentified homologues in the reef-building corals and other taxa (asterisks, Figure 2A, Supplementary file 1). A Bayesian tree reconstruction placed all canonical NPC2 family members (identified by three shared introns) on a large multifurcation, and all previously and newly identified non-canonical NPC2 (identified by the absence of introns due to retrotransposition [Dani et al., 2014]) to a basal position, most likely attracted by the Capsaspora outgroup NPC2s. This indicated higher sequence divergence of non-canonical NPC2s; and in line with this, they contain only around half as many residues under negative (purifying) selection (35 to 61) as canonical NPC2s and twice as many residues under positive (diversifying) selection (12 to 5) (Figure 2B). Our analysis also revealed that non-canonical NPC2 homologues are confined to cnidarians within the anthozoan class, as they did not appear in the earlier-branching sponge Amphimedon nor in the hydrozoans Hydra magnipapillata and Hydractinia echinata. Notably, the occurrence of non-canonical NPC2s appeared to correlate with symbiotic state: the symbiotic anthozoans (Aiptasia, Acropora, Montastrea) have several non-canonical NPC2 homologues (3, 3, and 2, respectively). In contrast, the non-symbiotic anemone Nematostella displays evolutionary traces of a single non-canonical NPC2, which either failed to expand or underwent higher loss (Figure 2A).

Figure 2

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We next investigated the expression of all Aiptasia NPC2s in vitro and in vivo (Figure 3). As determined by qPCR and Western blotting using custom-made antibodies (Figure 3—figure supplement 1), two of the three non-canonical NPC2 homologues displayed substantially higher expression at the transcript and protein levels in symbiotic but not aposymbiotic animals (closed blue symbols; Figure 3A+B). The third non-canonical NPC2 homologue was highly expressed in both symbiotic and aposymbiotic animals, yet more so in symbiotic animals. Conversely, canonical NPC2s were highly expressed in both symbiotic and aposymbiotic animals (closed red symbols). Likewise, the non-symbiotic anemone Nematostella exhibited ubiquitously high expression of canonical NPC2 genes (open red symbols), whereas the non-canonical NPC2 gene was highly expressed only upon feeding (open blue symbols). Aposymbiotic embryos of the symbiotic coral Acropora, as well as Nematostella embryos, contained maternally provided canonical NPC2 transcripts, suggesting that these are required for development (Figure 3—figure supplement 2). Notably, several canonical NPC2s in Aiptasia (XM_021046710, XM_021041174) and Nematostella (XM_001635452) may be ‘in transition’ to becoming non-canonical: they were expressed at intermediate abundances between the two groups, and they responded to symbiosis (Aiptasia) or feeding (Nematostella) (red square and triangles, Figure 3A + 3B). Some of their intron/exon structures reflected those of the non-canonical group (red triangles, Figure 2A). Immunofluorescence analysis revealed that the non-canonical NPC2s decorated intracellular symbionts in Aiptasia in vivo (Figure 3C + D), consistent with previous data in Anemonia viridis (Dani et al., 2014; Dani et al., 2017). The NPC2 signal appears to be restricted to the symbiosome and absent from the cytoplasm of the symbiont-containing cell (Figure 3—figure supplement 3). We noted that non-canonical NPC2s decorate some but not all symbionts (Dani et al., 2014; Dani et al., 2017), suggesting that at any given time, symbiosomes are a dynamic group of specialized organelles. To gain further insight into the NPC2-decorated symbiosome dynamics, we measured the spatio-temporal regulation of non-canonical NPC2s in Aiptasia larvae establishing symbiosis (‘infection’) with Symbiodiniaceae strain SSB01 (Hambleton et al., 2014; Bucher et al., 2016). Indeed, non-canonical NPC2 slowly decorated intracellular symbionts over time (Figure 3E, Figure 3—figure supplement 4). This localization ranged from weak ‘grainy’ patterns to stronger ‘halos’ around symbionts (arrows, Figure 3C). We quantified infection rates, symbiont load of individual larvae, and non-canonical NPC2 signal intensity (Figure 3F, Figure 3—figure supplement 5). We found that infection rates remained steady after removal of symbionts from the environment, whereas the proportion of larvae showing non-canonical NPC2 signal continued to increase to eventually include the majority of infected larvae (Figure 3F). Concordantly, the proportion of symbionts within each larva surrounded by NPC2 signal also increased over time, as did the signal strength (Figure 3F). Finally, infected larvae displaying any NPC2 signal generally contained a higher symbiont load than their infected, unlabelled counterparts (Figure 3—figure supplement 5). Thus, non-canonical NPC2 is increasingly expressed and recruited to symbionts over time, suggesting that non-canonical NPC2 function becomes important primarily once symbiosomes become ‘mature’.

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As a first step towards elucidating NPC2 function during symbiosis, we investigated the effect of global sterol transport inhibition by treating symbiotic and aposymbiotic adult Aiptasia with the drug U18666A, a competitive inhibitor of the NPC2 binding partner NPC1 that is required for efficient cholesterol egress from lysosomes (Liscum and Faust, 1989; Cenedella, 2009; Vance, 2010; Lu et al., 2015). Because of the profound effect of this drug on all cells and thus anemone physiology, severe effects are to be expected. Accordingly, we found that both symbiotic and aposymbiotic anemones appear to lose tissue and shorten their tentacles in a dose- and duration-dependent manner. However, symbiotic anemones showed such effects on host physiology faster than their aposymbiotic counterparts (Figure 3G, Figure 3—figure supplement 6). Moreover, symbiont density decreased in response to U18666A treatment (Figure 3H). We observed similar effects with A. digitifera juvenile primary polyps stably hosting Symbiodiniaceae strain SSB01 when exposed to increasing concentrations of U18666A (Figure 3—figure supplement 7). This suggests that inhibition of sterol transport affects symbiosis stability and may lead to loss of symbionts (‘bleaching’). Further, the disruption of global sterol transport compromises host tissues in all cases, emphasizing the importance of sterols in tissue homeostasis.

To test sterol-binding properties of Aiptasia NPC2, we compared the most conserved canonical NPC2 to the non-canonical NPC2 most up-regulated upon symbiosis (XM_021041171 to XM_021052404, respectively). We used lipidomics to quantify lipids bound by immunoprecipitated native or recombinant NPC2s (Figure 4, Figure 4—figure supplement 1) (after Li et al., 2010). Recombinant proteins were expressed in HEK 293T cells, after which cell lysates were mixed with Symbiodiniaceae SSB01 homogenates at either neutral conditions (pH 7) or acidic conditions reflecting the lysosome/symbiosome (pH 5). Under both conditions, canonical and non-canonical NPC2:mCherry fusion proteins bound symbiont-produced sterols significantly above the background levels of the control, mCherry alone (Figure 4A). The relative proportions of bound sterols generally exhibited equilibrium levels with the corresponding symbiont homogenate (Figure 4B). To validate sterol binding by non-canonical NPC2 in vivo, we also immunoprecipitated the native non-canonical NPC2 and bound sterols directly from homogenates of symbiotic Aiptasia. Again, we detected symbiont-produced sterols above background levels, validating our heterologous system and indicating that these proteins bind sterols in vivo during symbiosis (Figure 4C). These data indicate that, despite their evolutionary divergence, both types of Aiptasia NPC2s have the conserved function of binding sterols in lysosomal-like environments. Although we cannot rule out subtle differences in sterol binding dynamics between the two proteins, our results suggested no differential binding between canonical and non-canonical NPC2s, consistent with the observations that the sterol ligand and the residues lining the binding cavity tolerate considerable variations (Xu et al., 2007; Liou et al., 2006). Corroborating this, we were unable to detect any difference in the differential expression of canonical and non-canonical NPC2s between aposymbiotic and symbiotic state in three symbiont-Aiptasia pairings (Figure 4D).

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With data suggesting both NPC2 types can bind symbiont-produced sterols, we were therefore left with the question: what is the functional advantage of localizing non-canonical NPC2s specifically in the symbiosome? The mature symbiosome, where non-canonical NPC2 appears to function, remains poorly understood; however, extreme acidity appears to be a unique characteristic of these specialized cellular compartments. Whereas lumenal pH of classic lysosomes can range from 4.7 to 6 (Johnson et al., 2016), recent work indicates that mature symbiosomes in steady-state symbiosis are even more acidic (pH ~4) to promote efficient photosynthesis (Barott et al., 2015). We therefore sought to compare the stability/solubility of representative canonical and non-canonical NPC2s at different pH (Figure 4E + F, Figure 4—figure supplement 2). Interestingly, the patterns of extracted soluble proteins vary among NPC2s: canonical NPC2 appears in one predominant form at both pH’s (Figure 4E, red arrowhead), whereas one of the symbiosis-responsive non-canonical NPC2s (XM_021052412; Figure 3A + B) always appears in two forms in both conditions (Figure 4E, arrowheads and asterisks). Strikingly, the pattern for the other symbiosis-specific non-canonical NPC2 (XM_021052404; Figures 3A–G and 4A–C) is distinct between pH 7 and pH 5, with a consistently occurring additional band at higher pH (Figure 4E, blue arrow). Although we cannot rule out that the additional bands reflect degradation products, we favor the interpretation that they most likely represent distinct glycoforms, which also occur in vivo (Figure 3B). When quantifying the protein variant common to all samples (Figure 4E, arrowheads), we found that at pH 5, the non-canonical NPC2s were consistently more abundant in the soluble fraction than the canonical counterpart (Figure 4F). For all proteins tested, the ratio of the predominant soluble protein variant at pH 7 to that at pH 5 was always >1, indicating more solubility at pH 7. However, the ratio was higher for Aiptasia canonical NPC2 than for the non-canonical NPC2s, indicating that the former is relatively less soluble at pH 5 (Figure 4F). Taken together, symbiosis-responsive non-canonical NPC2 appears to be more soluble/stable than canonical NPC2 at a lower pH, likely characteristic of the symbiosome. In line with this, all Aiptasia non-canonical NPC2 proteins harbored glycosylation sites and a glycine followed by a histidine residue (Figure 2B), which may contribute to protein stability in acidic environments (Rudd et al., 1994; Hanson et al., 2009; Culyba et al., 2011). However, pH-dependent protein stability is difficult to predict and functional experiments are required to determine whether such motifs (or others) play a role for the adaptation to the symbiosome or not.

In summary, our data reveal that the transfer of complex mixtures of symbiont-derived sterols is a key feature of anthozoan photosymbiosis (Figure 4G), whereby the specific composition and proportion of transferred sterols appears to be under the control of both symbiont and host. While the non-canonical NPC2 sterol-binding proteins are part of the machinery transferring sterols from symbiont to host, they do not contribute to host sterol selection by differential expression or differential binding. Instead, our assays reveal the possibility of an increased tolerance to acidic conditions of non-canonical NPC2s and their late accumulation in the symbiosome, consistent with gradual enrichment upon increasing symbiosome acidification. We propose that whereas ubiquitously expressed canonical NPC2 homologues are ‘workhorses’ in sterol trafficking throughout the host, non-canonical NPC2s are spatiotemporally regulated to accumulate as the symbiosome matures, developing into a unique compartment optimized to promote the interaction and communication of the symbiotic partners (Figure 4G). This allows symbiotic cnidarians to flexibly use symbiont-produced sterols, with reef-building corals nearly fully substituting these for prey-derived cholesterol, supporting survival in nutrient-poor environments. More broadly, our findings indicate that carbon acquisition by lipid transfer, similar to other symbioses (Keymer et al., 2017), is a major driver of coral-algal symbiotic relationships as a means to adapt to various ecological niches by efficient exploitation of limited resources.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Elizabeth Ann Hambleton

   Centre for Organismal Studies (COS), Universität Heidelberg, Heidelberg, Germany

   Contribution

   Conceptualization, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing, Directed the research, Primarily conducted the experiments and analyses with contributions from IM with immunoprecipitations, pH experiments, cloning, symbiont genotyping, and Western blots, Conducted and analysed sterol-binding lipidomics with DK and TS

   For correspondence

   liz.hambleton@cos.uni-heidelberg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0204-5037

2. Victor Arnold Shivas Jones

   Centre for Organismal Studies (COS), Universität Heidelberg, Heidelberg, Germany

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing, Conducted the immunofluorescence, confocal imaging, and IF quantification

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8712-3454

3. Ira Maegele

   Centre for Organismal Studies (COS), Universität Heidelberg, Heidelberg, Germany

   Contribution

   Investigation, Visualization, Methodology, Writing—review and editing, Contributed towards conducting the experiments and analyses with immunoprecipitations, pH experiments, cloning, symbiont genotyping, and Western blots

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2324-3561

4. David Kvaskoff

   Heidelberg University Biochemistry Center (BZH), Universität Heidelberg, Heidelberg, Germany

   Contribution

   Data curation, Investigation, Methodology, Conducted and analysed sterol-binding lipidomics with EAH and TS

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5569-7226

5. Timo Sachsenheimer

   Heidelberg University Biochemistry Center (BZH), Universität Heidelberg, Heidelberg, Germany

   Contribution

   Investigation, Conducted and analysed sterol-binding lipidomics with DK and EAH

   Competing interests

   No competing interests declared

6. Annika Guse

   Centre for Organismal Studies (COS), Universität Heidelberg, Heidelberg, Germany

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing, Directed the research

   For correspondence

   annika.guse@cos.uni-heidelberg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8737-9206

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