• Photosynthesis and other factors affecting the establishment and maintenance of cnidarian-dinoflagellate symbiosis. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Tran, C., Rosenfield, G. R., Cleves, P. A., Krediet, C. J., Paul, M. R., Clowez, S., Grossman, A. R., Pringle, J. R. 2024; 379 (1901): 20230079


    Coral growth depends on the partnership between the animal hosts and their intracellular, photosynthetic dinoflagellate symbionts. In this study, we used the sea anemone Aiptasia, a laboratory model for coral biology, to investigate the poorly understood mechanisms that mediate symbiosis establishment and maintenance. We found that initial colonization of both adult polyps and larvae by a compatible algal strain was more effective when the algae were able to photosynthesize and that the long-term maintenance of the symbiosis also depended on photosynthesis. In the dark, algal cells were taken up into host gastrodermal cells and not rapidly expelled, but they seemed unable to reproduce and thus were gradually lost. When we used confocal microscopy to examine the interaction of larvae with two algal strains that cannot establish stable symbioses with Aiptasia, it appeared that both pre- and post-phagocytosis mechanisms were involved. With one strain, algae entered the gastric cavity but appeared to be completely excluded from the gastrodermal cells. With the other strain, small numbers of algae entered the gastrodermal cells but appeared unable to proliferate there and were slowly lost upon further incubation. We also asked if the exclusion of either incompatible strain could result simply from their cells' being too large for the host cells to accommodate. However, the size distributions of the compatible and incompatible strains overlapped extensively. Moreover, examination of macerates confirmed earlier reports that individual gastrodermal cells could expand to accommodate multiple algal cells. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

    View details for DOI 10.1098/rstb.2023.0079

    View details for PubMedID 38497261

  • Role of the bicarbonate transporter SLC4γ in stony-coral skeleton formation and evolution. Proceedings of the National Academy of Sciences of the United States of America Tinoco, A. I., Mitchison-Field, L. M., Bradford, J., Renicke, C., Perrin, D., Bay, L. K., Pringle, J. R., Cleves, P. A. 2023; 120 (24): e2216144120


    Coral reefs are highly diverse ecosystems of immense ecological, economic, and aesthetic importance built on the calcium-carbonate-based skeletons of stony corals. The formation of these skeletons is threatened by increasing ocean temperatures and acidification, and a deeper understanding of the molecular mechanisms involved may assist efforts to mitigate the effects of such anthropogenic stressors. In this study, we focused on the role of the predicted bicarbonate transporter SLC4γ, which was suggested in previous studies to be a product of gene duplication and to have a role in coral-skeleton formation. Our comparative-genomics study using 30 coral species and 15 outgroups indicates that SLC4γ is present throughout the stony corals, but not in their non-skeleton-forming relatives, and apparently arose by gene duplication at the onset of stony-coral evolution. Our expression studies show that SLC4γ, but not the closely related and apparently ancestral SLC4β, is highly upregulated during coral development coincident with the onset of skeleton deposition. Moreover, we show that juvenile coral polyps carrying CRISPR/Cas9-induced mutations in SLC4γ are defective in skeleton formation, with the severity of the defect in individual animals correlated with their frequencies of SLC4γ mutations. Taken together, the results suggest that the evolution of the stony corals involved the neofunctionalization of the newly arisen SLC4γ for a unique role in the provision of concentrated bicarbonate for calcium-carbonate deposition. The results also demonstrate the feasibility of reverse-genetic studies of ecologically important traits in adult corals.

    View details for DOI 10.1073/pnas.2216144120

    View details for PubMedID 37276409

  • Molecular insights into the Darwin paradox of coral reefs from the sea anemone Aiptasia. Science advances Cui, G., Konciute, M. K., Ling, L., Esau, L., Raina, J. B., Han, B., Salazar, O. R., Presnell, J. S., Rädecker, N., Zhong, H., Menzies, J., Cleves, P. A., Liew, Y. J., Krediet, C. J., Sawiccy, V., Cziesielski, M. J., Guagliardo, P., Bougoure, J., Pernice, M., Hirt, H., Voolstra, C. R., Weis, V. M., Pringle, J. R., Aranda, M. 2023; 9 (11): eadf7108


    Symbiotic cnidarians such as corals and anemones form highly productive and biodiverse coral reef ecosystems in nutrient-poor ocean environments, a phenomenon known as Darwin's paradox. Resolving this paradox requires elucidating the molecular bases of efficient nutrient distribution and recycling in the cnidarian-dinoflagellate symbiosis. Using the sea anemone Aiptasia, we show that during symbiosis, the increased availability of glucose and the presence of the algae jointly induce the coordinated up-regulation and relocalization of glucose and ammonium transporters. These molecular responses are critical to support symbiont functioning and organism-wide nitrogen assimilation through glutamine synthetase/glutamate synthase-mediated amino acid biosynthesis. Our results reveal crucial aspects of the molecular mechanisms underlying nitrogen conservation and recycling in these organisms that allow them to thrive in the nitrogen-poor ocean environments.

    View details for DOI 10.1126/sciadv.adf7108

    View details for PubMedID 36921053

  • Conversion of oxybenzone sunscreen to phototoxic glucoside conjugates by sea anemones and corals. Science (New York, N.Y.) Vuckovic, D., Tinoco, A. I., Ling, L., Renicke, C., Pringle, J. R., Mitch, W. A. 2022; 376 (6593): 644-648


    The reported toxicity of oxybenzone-based sunscreens to corals has raised concerns about the impacts of ecotourist-shed sunscreens on corals already weakened by global stressors. However, oxybenzone's toxicity mechanism(s) are not understood, hampering development of safer sunscreens. We found that oxybenzone caused high mortality of a sea anemone under simulated sunlight including ultraviolet (UV) radiation (290 to 370 nanometers). Although oxybenzone itself protected against UV-induced photo-oxidation, both the anemone and a mushroom coral formed oxybenzone-glucoside conjugates that were strong photo-oxidants. Algal symbionts sequestered these conjugates, and mortality correlated with conjugate concentrations in animal cytoplasm. Higher mortality in anemones that lacked symbionts suggests an enhanced risk from oxybenzone to corals bleached by rising temperatures. Because many commercial sunscreens contain structurally related chemicals, understanding metabolite phototoxicity should facilitate the development of coral-safe products.

    View details for DOI 10.1126/science.abn2600

    View details for PubMedID 35511969

  • Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor. Proceedings of the National Academy of Sciences of the United States of America Cleves, P. A., Tinoco, A. I., Bradford, J., Perrin, D., Bay, L. K., Pringle, J. R. 2020


    Reef-building corals are keystone species that are threatened by anthropogenic stresses including climate change. To investigate corals' responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating many hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals or closely related cnidarians. CRISPR technology seems likely to alleviate this problem. Indeed, we show here that microinjection of single-guide RNA/Cas9 ribonucleoprotein complexes into fertilized eggs of the coral Acropora millepora can produce a sufficiently high frequency of mutations to detect a clear phenotype in the injected generation. Based in part on experiments in a sea-anemone model system, we targeted the gene encoding Heat Shock Transcription Factor 1 (HSF1) and obtained larvae in which >90% of the gene copies were mutant. The mutant larvae survived well at 27 °C but died rapidly at 34 °C, a temperature that did not produce detectable mortality over the duration of the experiment in wild-type (WT) larvae or larvae injected with Cas9 alone. We conclude that HSF1 function (presumably its induction of genes in response to heat stress) plays an important protective role in corals. More broadly, we conclude that CRISPR mutagenesis in corals should allow wide-ranging and rigorous tests of gene function in both larval and adult corals.

    View details for DOI 10.1073/pnas.1920779117

    View details for PubMedID 33168726

  • Insights into coral bleaching under heat stress from analysis of gene expression in a sea anemone model system. Proceedings of the National Academy of Sciences of the United States of America Cleves, P. A., Krediet, C. J., Lehnert, E. M., Onishi, M., Pringle, J. R. 2020


    Loss of endosymbiotic algae ("bleaching") under heat stress has become a major problem for reef-building corals worldwide. To identify genes that might be involved in triggering or executing bleaching, or in protecting corals from it, we used RNAseq to analyze gene-expression changes during heat stress in a coral relative, the sea anemone Aiptasia. We identified >500 genes that showed rapid and extensive up-regulation upon temperature increase. These genes fell into two clusters. In both clusters, most genes showed similar expression patterns in symbiotic and aposymbiotic anemones, suggesting that this early stress response is largely independent of the symbiosis. Cluster I was highly enriched for genes involved in innate immunity and apoptosis, and most transcript levels returned to baseline many hours before bleaching was first detected, raising doubts about their possible roles in this process. Cluster II was highly enriched for genes involved in protein folding, and most transcript levels returned more slowly to baseline, so that roles in either promoting or preventing bleaching seem plausible. Many of the genes in clusters I and II appear to be targets of the transcription factors NFkappaB and HSF1, respectively. We also examined the behavior of 337 genes whose much higher levels of expression in symbiotic than aposymbiotic anemones in the absence of stress suggest that they are important for the symbiosis. Unexpectedly, in many cases, these expression levels declined precipitously long before bleaching itself was evident, suggesting that loss of expression of symbiosis-supporting genes may be involved in triggering bleaching.

    View details for DOI 10.1073/pnas.2015737117

    View details for PubMedID 33168733

  • Impact of menthol on growth and photosynthetic function of Breviolum minutum (Dinoflagellata, Dinophyceae, Symbiodiniaceae) and interactions with its Aiptasia host. Journal of phycology Clowez, S., Renicke, C., Pringle, J. R., Grossman, A. R. 2020


    Environmental change, including global warming and chemical pollution, can compromise cnidarian (e.g., coral) -dinoflagellate symbioses and cause coral bleaching. Understanding the mechanisms that regulate these symbioses will inform strategies for sustaining healthy coral-reef communities. A model system for corals is the symbiosis between the sea anemone Exaiptasia pallida (common name Aiptasia) and its dinoflagellate partners (family Symbiodiniaceae). To complement existing studies of the interactions between these organisms, we examined the impact of menthol, a reagent often used to render cnidarians aposymbiotic, on the dinoflagellate Breviolum minutum, both in culture and in hospite. In both environments, the growth and photosynthesis of this alga were compromised at either 100 or 300M menthol. We observed reduction of PSII and PSI functions, the abundances of reaction-center proteins, and, at 300M menthol, of total cellular proteins. Interestingly, for free-living algae exposed to 100M menthol, an initial decline in growth, photosynthetic activities, pigmentation, and protein abundances reversed after 5-15 d, eventually approaching control levels. This behavior was observed in cells maintained in continuous light, but not in cells experiencing a light-dark regimen, suggesting that B. minutum can detoxify menthol or acclimate and repair damaged photosynthetic complexes in a light- and/or energy-dependent manner. Extended exposures of cultured algae to 300M menthol ultimately resulted in algal death. Most symbiotic anemones were also unable to survive this menthol concentration for 30 d. Additionally, cells impaired for photosynthesis by pre-treatment with 300M menthol exhibited reduced efficiency in re-populating the anemone host.

    View details for DOI 10.1111/jpy.13081

    View details for PubMedID 33025575

  • Cleavage-furrow formation without F-actin in Chlamydomonas. Proceedings of the National Academy of Sciences of the United States of America Onishi, M., Umen, J. G., Cross, F. R., Pringle, J. R. 2020


    It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.

    View details for DOI 10.1073/pnas.1920337117

    View details for PubMedID 32690698

  • Symbiont population control by host-symbiont metabolic interaction in Symbiodiniaceae-cnidarian associations. Nature communications Xiang, T. n., Lehnert, E. n., Jinkerson, R. E., Clowez, S. n., Kim, R. G., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. 2020; 11 (1): 108


    In cnidarian-Symbiodiniaceae symbioses, algal endosymbiont population control within the host is needed to sustain a symbiotic relationship. However, the molecular mechanisms that underlie such population control are unclear. Here we show that a cnidarian host uses nitrogen limitation as a primary mechanism to control endosymbiont populations. Nitrogen acquisition and assimilation transcripts become elevated in symbiotic Breviolum minutum algae as they reach high-densities within the sea anemone host Exaiptasia pallida. These same transcripts increase in free-living algae deprived of nitrogen. Symbiotic algae also have an elevated carbon-to-nitrogen ratio and shift metabolism towards scavenging nitrogen from purines relative to free-living algae. Exaiptasia glutamine synthetase and glutamate synthase transcripts concomitantly increase with the algal endosymbiont population, suggesting an increased ability of the host to assimilate ammonium. These results suggest algal growth and replication in hospite is controlled by access to nitrogen, which becomes limiting for the algae as their population within the host increases.

    View details for DOI 10.1038/s41467-019-13963-z

    View details for PubMedID 31913264

  • F-actin homeostasis through transcriptional regulation and proteasome-mediated proteolysis. Proceedings of the National Academy of Sciences of the United States of America Onishi, M., Pecani, K., Jones, T. 4., Pringle, J. R., Cross, F. R. 2018


    Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the "LRE motif." The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin-dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.

    View details for PubMedID 29941587