Menthol-induced bleaching rapidly and effectively provides experimental aposymbiotic sea anemones (Aiptasia sp.) for symbiosis investigations

Experimental manipulation of the symbiosis between cnidarians and photosynthetic dinoflagellates (Symbiodinium spp.) is crucial to advancing the understanding of the cellular mechanisms involved in host-symbiont interactions, and overall coral reef ecology. The anemone Aiptasia sp. is a model for cnidarian-dinoflagellate symbiosis, and notably it can be rendered aposymbiotic (i.e. dinoflagellate-free) and re-infected with a range of Symbiodinium types. Various methods exist for generating aposymbiotic hosts; however, they can be hugely time consuming and not wholly effective. Here, we optimise a method using menthol for production of aposymbiotic Aiptasia. The menthol treatment produced aposymbiotic hosts within just 4 weeks (97-100% symbiont loss), and the condition was maintained long after treatment when anemones were held under a standard light: dark cycle. The ability of Aiptasia to form a stable symbiosis appeared to be unaffected by menthol exposure, as demonstrated by successful re-establishment of the symbiosis when anemones were experimentally re-infected. Furthermore, there was no significant impact on photosynthetic or respiratory performance of re-infected anemones.Keywords: Coral reefs, Symbiodinium, Cnidarian-dinoflagellate symbiosisKeywords: Coral reefs, Symbiodinium, Cnidarian-dinoflagellate symbiosi

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.journal articl

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Catastrophic Mortality on Inshore Coral Reefs of the Florida Keys Due to Severe Low-Temperature Stress

Coral reefs of the Florida Keys typically experience seasonal temperatures of 20–31 °C. Deviation outside this range causes physiological impairment of reef-building corals, potentially leading to coral colony death. In January and February 2010, two closely spaced cold fronts, possibly driven by an unusually extreme Arctic Oscillation, caused sudden and severe seawater temperature declines in the Florida Keys. Inshore coral reefs [e.g., Admiral Reef (ADM)] experienced lower sustained temperatures (i.e., \u3c 12 °C) than those further offshore [e.g., Little Grecian Reef (LG), minimum temperature = 17.2 °C]. During February and March 2010, we surveyed ADM and observed a mass die-off of reef-building corals, whereas 12 km away LG did not exhibit coral mortality. We subsequently measured the physiological effects of low-temperature stress on three common reef-building corals (i.e., Montastraea faveolata, Porites astreoides, and Siderastrea siderea) over a range of temperatures that replicated the inshore cold-water anomaly (i.e., from 20 to 16 to 12 °C and back to 20 °C). Throughout the temperature modulations, coral respiration as well as endosymbiont gross photosynthesis and maximum quantum efficiency of photosystem II were measured. In addition, Symbiodinium genotypic identity, cell densities, and chlorophyll a content were determined at the beginning and conclusion of the experiment. All corals were significantly affected at 12 °C, but species-specific physiological responses were found indicating different coral and/or Symbiodinium cold tolerances. Montastraea faveolata and P. astreoides appeared to be most negatively impacted because, upon return to 20 °C, significant reductions in gross photosynthesis and dark respiration persisted. Siderastrea siderea, however, readily recovered to pre-treatment rates of dark respiration and gross photosynthesis. Visual surveys of inshore reefs corroborated these results, with S. siderea being minimally affected by the cold-water anomaly, whereas M. faveolata and P. astreoides exhibited nearly 100% mortality. This study highlights the importance of understanding the physiological attributes of genotypically distinct coral-Symbiodinium symbioses that contribute to tolerance, recovery, and consequences to an environmental perturbation. These data also document effects of a rarely studied environmental stressor, possibly initiated by remote global climate events, on coral-Symbiodinium symbioses and coral reef communities

Sub-cellular imaging shows reduced photosynthetic carbon and increased nitrogen assimilation by the non-native endosymbiontDurusdinium trenchiiin the model cnidarian Aiptasia

Hosting different symbiont species can affect inter-partner nutritional fluxes within the cnidarian-dinoflagellate symbiosis. Using nanoscale secondary ion mass spectrometry (NanoSIMS), we measured the spatial incorporation of photosynthetically fixed(13)C and heterotrophically derived(15)N into host and symbiont cells of the model symbiotic cnidarian Aiptasia (Exaiptasia pallida) when colonized with its native symbiontBreviolum minutumor the non-nativeDurusdinium trenchii.Breviolum minutumexhibited high photosynthetic carbon assimilationpercell and translocation to host tissue throughout symbiosis establishment, whereasD. trenchiiassimilated significantly less carbon, but obtained more host nitrogen. These findings suggest thatD. trenchiihas less potential to provide photosynthetically fixed carbon to the host despite obtaining considerable amounts of heterotrophically derived nitrogen. These sub-cellular events help explain previous observations that demonstrate differential effects ofD. trenchiicompared toB. minutumon the host transcriptome, proteome, metabolome and host growth and asexual reproduction. Together, these differential effects suggest that the non-native host-symbiont pairing is sub-optimal with respect to the host's nutritional benefits under normal environmental conditions. This contributes to our understanding of the ways in which metabolic integration impacts the benefits of a symbiotic association, and the potential evolution of novel host-symbiont pairings

Phylogenetic analysis of cell-cycle regulatory proteins within the Symbiodiniaceae

In oligotrophic waters, cnidarian hosts rely on symbiosis with their photosynthetic dinoflagellate partners (family Symbiodiniaceae) to obtain the nutrients they need to grow, reproduce and survive. For this symbiosis to persist, the host must regulate the growth and proliferation of its symbionts. One of the proposed regulatory mechanisms is arrest of the symbiont cell cycle in the G1 phase, though the cellular mechanisms involved remain unknown. Cell-cycle progression in eukaryotes is controlled by the conserved family of cyclin-dependent kinases (CDKs) and their partner cyclins. We identified CDKs and cyclins in different Symbiodiniaceae species and examined their relationship to homologs in other eukaryotes. Cyclin proteins related to eumetazoan cell-cycle-related cyclins A, B, D, G/I and Y, and transcriptional cyclin L, were identified in the Symbiodiniaceae, alongside several alveolate-specific cyclin A/B proteins, and proteins related to protist P/U-type cyclins and apicomplexan cyclins. The largest expansion of Symbiodiniaceae cyclins was in the P/U-type cyclin groups. Proteins related to eumetazoan cell-cycle-related CDKs (CDK1) were identified as well as transcription-related CDKs. The largest expansion of CDK groups was, however, in alveolate-specific groups which comprised 11 distinct CDK groups (CDKA-J) with CDKB being the most widely distributed CDK protein. As a result of its phylogenetic position, conservation across Symbiodiniaceae species, and the presence of the canonical CDK motif, CDKB emerged as a likely candidate for a Saccharomyces cerevisiae Cdc28/Pho85-like homolog in Symbiodiniaceae. Similar to cyclins, two CDK-groups found in Symbiodiniaceae species were solely associated with apicomplexan taxa. A comparison of Breviolum minutum CDK and cyclin gene expression between free-living and symbiotic states showed that several alveolate-specific CDKs and two P/U-type cyclins exhibited altered expression in hospite, suggesting that symbiosis influences the cell cycle of symbionts on a molecular level. These results highlight the divergence of Symbiodiniaceae cell-cycle proteins across species. These results have important implications for host control of the symbiont cell cycle in novel cnidarian–dinoflagellate symbioses

The Influence of Symbiosis on the Proteome of the <i>Exaiptasia</i> Endosymbiont <i>Breviolum minutum</i>

The cellular mechanisms responsible for the regulation of nutrient exchange, immune response, and symbiont population growth in the cnidarian–dinoflagellate symbiosis are poorly resolved. Here, we employed liquid chromatography–mass spectrometry to elucidate proteomic changes associated with symbiosis in Breviolum minutum, a native symbiont of the sea anemone Exaiptasia diaphana (‘Aiptasia’). We manipulated nutrients available to the algae in culture and to the holobiont in hospite (i.e., in symbiosis) and then monitored the impacts of our treatments on host–endosymbiont interactions. Both the symbiotic and nutritional states had significant impacts on the B. minutum proteome. B. minutum in hospite showed an increased abundance of proteins involved in phosphoinositol metabolism (e.g., glycerophosphoinositol permease 1 and phosphatidylinositol phosphatase) relative to the free-living alga, potentially reflecting inter-partner signalling that promotes the stability of the symbiosis. Proteins potentially involved in concentrating and fixing inorganic carbon (e.g., carbonic anhydrase, V-type ATPase) and in the assimilation of nitrogen (e.g., glutamine synthase) were more abundant in free-living B. minutum than in hospite, possibly due to host-facilitated access to inorganic carbon and nitrogen limitation by the host when in hospite. Photosystem proteins increased in abundance at high nutrient levels irrespective of the symbiotic state, as did proteins involved in antioxidant defences (e.g., superoxide dismutase, glutathione s-transferase). Proteins involved in iron metabolism were also affected by the nutritional state, with an increased iron demand and uptake under low nutrient treatments. These results detail the changes in symbiont physiology in response to the host microenvironment and nutrient availability and indicate potential symbiont-driven mechanisms that regulate the cnidarian–dinoflagellate symbiosis