Characterization of Acantharea-Phaeocystis photosymbioses: distribution, abundance, specificity, maintenance and host-control

Microbial eukaryotes (protists) are important contributors to marine biogeochemistry and play essential roles as both producers and consumers in marine ecosystems. Among protists, mixotrophs—those that use both heterotrophy and autotrophy to meet their energy requirements—are especially important to primary production in low-nutrient regions. Acantharian protists (clades E & F) accomplish mixotrophy by hosting ​Phaeocystis spp.​ as algal endosymbionts and are extremely abundant in subtropical low-nutrient regions where they form productivity hotspots. Despite their ecological importance, acantharians remain understudied due to their structural fragility and inability to survive in culture. In order to overcome these challenges and illuminate key aspects of acantharian biology and ecology—including distribution, abundance, and specificity and specialization of symbioses—single-cell RNA sequencing methods were developed for acantharians and used alongside environmental metabarcode sequencing and high-throughput, in-situ imaging. Major findings from this thesis were that i) acantharian cell (> 250 µm) concentrations decrease with depth, which correlates to patterns in relative sequence abundances for acantharian clades with known morphologies but not for those lacking known morphology, and that ii) while individual acantharians simultaneously harbor multiple symbiont species, intra-host symbiont communities do not match environmental communities, providing evidence for multiple uptake events but against continuous symbiont turnover, and that iii) photosynthesis genes are upregulated in symbiotic Phaeocystis​, reflecting enhanced productivity in symbiosis, but DNA replication and cell-cycle genes are downregulated, demonstrating that hosts suppress symbiont cell division. Moreover, storage carbohydrate and lipid biosynthesis and metabolism genes are downregulated in symbiotic ​Phaeocystis​, suggesting fixed carbon is relinquished to acantharian hosts. Gene expression patterns indicate that symbiotic ​Phaeocystis​ is not nutrient limited and likely benefits from host-supplied ammonium and urea, thus providing evidence for nutrient transfer between hosts and symbionts. Interestingly, genes associated with protein kinase signaling pathways that promote cell proliferation are downregulated in symbiotic ​Phaeocystis​. Deactivation of these genes may prevent symbionts from overgrowing hosts and therefore represents a key component of maintaining the symbiosis. This research contributes new insights into the ecologically relevant photosymbioses between Acantharea and ​Phaeocystis​ and illustrates the benefits of combining single-cell sequencing and imaging technologies to illuminate important microbial relationships in marine ecosystems.Okinawa Institute of Science and Technology Graduate Universit

Differential Gene Expression Supports a Resource‐Intensive, Defensive Role for Colony Production in the Bloom‐Forming Haptophyte, Phaeocystis globosa

Phaeocystis globosa forms dense, monospecific blooms in temperate, northern waters. Blooms are usually dominated by the colonial morphotype—nonflagellated cells embedded in a secreted mucilaginous mass. Colonial Phaeocystis blooms significantly affect food‐web structure and function and negatively impact fisheries and aquaculture, but factors regulating colony formation remain enigmatic. Destructive P. globosa blooms have been reported in tropical and subtropical regions more recently and warm‐water blooms could become more common with continued climate change and coastal eutrophication. We therefore assessed genetic pathways associated with colony formation by investigating differential gene expression between colonial and solitary cells of a warm‐water P. globosa strain. Our results illustrate a transcriptional shift in colonial cells with most of the differentially expressed genes downregulated, supporting a reallocation of resources associated with forming and maintaining colonies. Dimethylsulfide and acrylate production and pathogen interaction pathways were upregulated in colonial cells, suggesting a defensive role for producing colonies. We identify several protein kinase signaling pathways that may influence the transition between morphotypes, providing targets for future research into factors affecting colony formation. This study provides novel insights into genetic mechanisms involved in Phaeocystis colony formation and provides new evidence supporting a defensive role for Phaeocystis colonies

Influence of Regional Oceanography and Hydrothermal Activity on Protist Diversity and Community Structure in the Okinawa Trough

Microbial eukaryotes (protists) contribute substantially to ecological functioning in marine ecosystems, but the relative importance of factors shaping protist diversity, such as environmental selection and dispersal, remains difficult to parse. Water masses of a back-arc basin with hydrothermal activity provide a unique opportunity for studying the effects of dispersal and environmental selection on protist communities. In this study, we used metabarcoding to characterize protist communities in the Okinawa Trough, a back-arc spreading basin containing at least twenty-five active hydrothermal vent fields. Water was sampled from four depths at fourteen stations spanning the length of the Okinawa Trough, including three sites influenced by nearby hydrothermal vent sites. While significant differences in community structure reflecting water depth were present, protist communities were mostly homogeneous horizontally. Protist communities in the bottom waters affected by hydrothermal activity were significantly different from communities in other bottom waters, suggesting that environmental factors can be especially important in shaping community composition under specific conditions. Amplicon sequence variants that were enriched in hydrothermally influenced bottom waters largely derived from cosmopolitan protists that were present, but rare, in other near-bottom samples, thus highlighting the importance of the rare biosphere

Effects of microbial evolution dominate those of experimental host-mediated indirect selection

Microbes ubiquitously inhabit animals and plants, often affecting their host’s phenotype. As a result, even in a constant genetic background, the host’s phenotype may evolve through indirect selection on the microbiome. ‘Microbiome engineering’ offers a promising novel approach for attaining desired host traits but has been attempted only a few times. Building on the known role of the microbiome on development in fruit flies, we attempted to evolve earlier-eclosing flies by selecting on microbes in the growth media. We carried out parallel evolution experiments in no- and high-sugar diets by transferring media associated with fast-developing fly lines over the course of four selection cycles. In each cycle, we used sterile eggs from the same inbred population, and assayed mean fly eclosion times. Ultimately, flies eclosed seven to twelve hours earlier, depending on the diet, but microbiome engineering had no effect relative to a random-selection control treatment. 16S rRNA gene sequencing showed that the microbiome did evolve, particularly in the no sugar diet, with an increase in Shannon diversity over time. Thus, while microbiome evolution did affect host eclosion times, these effects were incidental. Instead, any experimentally enforced selection effects were swamped by uncontrolled microbial evolution, likely resulting in its adaptation to the media. These results imply that selection on host phenotypes must be strong enough to overcome other selection pressures simultaneously operating on the microbiome. The independent evolutionary trajectories of the host and the microbiome may limit the extent to which indirect selection on the microbiome can ultimately affect host phenotype. Random-selection lines accounting for independent microbial evolution are essential for experimental microbiome engineering studies

Intra-host Symbiont Diversity and Extended Symbiont Maintenance in Photosymbiotic Acantharea (Clade F)

Photosymbiotic protists contribute to surface primary production in low-nutrient, open-ocean ecosystems and constitute model systems for studying plastid acquisition via endosymbiosis. Little is known, however, about host-symbiont dynamics in these important relationships, and whether these symbioses are mutualistic is debated. In this study, we applied single-cell sequencing methods and advanced fluorescent microscopy to investigate host-symbiont dynamics in clade F acantharians, a major group of photosymbiotic protists in oligotrophic subtropical gyres. We amplified the 18S rRNA gene from single acantharian hosts and environmental samples to assess intra-host symbiont diversity and to determine whether intra-host symbiont community composition directly reflects the available symbiont community in the surrounding environment. Our results demonstrate that clade F acantharians simultaneously host multiple species from the haptophyte genera Phaeocystis and Chrysochromulina. The intra-host symbiont community composition was distinct from the external free-living symbiont community, suggesting that these acantharians maintain symbionts for extended periods of time. After selectively staining digestive organelles, fluorescent confocal microscopy showed that symbionts were not being systematically digested, which is consistent with extended symbiont maintenance within hosts. Extended maintenance within hosts may benefit symbionts through protection from grazing or viral lysis, and therefore could enhance dispersal, provided that symbionts retain reproductive capacity. The evidence for extended symbiont maintenance therefore allows that Phaeocystis could glean some advantage from the symbiosis and leaves the possibility of mutualism

Paired high‐throughput, in situ imaging and high‐throughput sequencing illuminate acantharian abundance and vertical distribution

Acantharians (supergroup Rhizaria) can be important contributors to surface primary production and to carbon flux to the deep sea, but are often underestimated because their delicate structures are destroyed by plankton nets or dissolved by chemical fixatives. As they are also uncultured, relatively little is known about acantharian biology, especially regarding their life cycles. Here, we take a paired approach, bringing together high-throughput, in situ imaging and metabarcode sequencing, to investigate acantharian abundance, vertical distribution, and life history in the western North Pacific. Concentrations of imaged acantharian cells correlated well with relative abundances of 18S rRNA gene sequences from acantharians with known, recognizable morphologies, but not to sequences corresponding to acantharians with unknown morphology. These results suggest that morphologically undescribed clades may lack the characteristic star-shaped acantharian skeleton or are much smaller than described acantharians. The smaller size of acantharians imaged at depth supports current hypotheses regarding nonsymbiotic acantharian life cycles: cysts or vegetative cells release reproductive swarmer cells in deep water and juvenile cells grow as they ascend toward the surface. Moreover, sequencing data present the possibility that some photosymbiotic acantharians may also reproduce at depth, like their nonsymbiotic, encysting relatives, which is counter to previous hypotheses. Finally, in situ imaging captured a new acantharian behavior that may be a previously undescribed predation strateg

Extreme storms cause rapid but short‐lived shifts in nearshore subtropical bacterial communities

Climate change scenarios predict tropical cyclones will increase in both frequency and intensity, which will escalate the amount of terrestrial run‐off and mechanical disruption affecting coastal ecosystems. Bacteria are key contributors to ecosystem functioning, but relatively little is known about how they respond to extreme storm events, particularly in nearshore subtropical regions. In this study, we combine field observations and mesocosm experiments to assess bacterial community dynamics and changes in physicochemical properties during early‐ and late‐season tropical cyclones affecting Okinawa, Japan. Storms caused large and fast influxes of freshwater and terrestrial sediment – locally known as red soil pollution – and caused moderate increases of macronutrients, especially SiO2 and PO43−, with up to 25 and 0.5 μM respectively. We detected shifts in relative abundances of marine and terrestrially derived bacteria, including putative coral and human pathogens, during storm events. Soil input alone did not substantially affect marine bacterial communities in mesocosms, indicating that other components of run‐off or other storm effects likely exert a larger influence on bacterial communities. The storm effects were short‐lived and bacterial communities quickly recovered following both storm events. The early‐ and late‐season storms caused different physicochemical and bacterial community changes, demonstrating the context‐dependency of extreme storm responses in a subtropical coastal ecosystem

Preparing aquatic research for an extreme future: call for improved definitions and responsive, multidisciplinary approaches

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Aoki, L. R., Brisbin, M. M., Hounshell, A. G., Kincaid, D. W., Larson, E., Sansom, B. J., Shogren, A. J., Smith, R. S., & Sullivan-Stack, J. Preparing aquatic research for an extreme future: call for improved definitions and responsive, multidisciplinary approaches. Bioscience, 72(6), (2022): 508-520, https://doi.org/10.1093/biosci/biac020.Extreme events have increased in frequency globally, with a simultaneous surge in scientific interest about their ecological responses, particularly in sensitive freshwater, coastal, and marine ecosystems. We synthesized observational studies of extreme events in these aquatic ecosystems, finding that many studies do not use consistent definitions of extreme events. Furthermore, many studies do not capture ecological responses across the full spatial scale of the events. In contrast, sampling often extends across longer temporal scales than the event itself, highlighting the usefulness of long-term monitoring. Many ecological studies of extreme events measure biological responses but exclude chemical and physical responses, underscoring the need for integrative and multidisciplinary approaches. To advance extreme event research, we suggest prioritizing pre- and postevent data collection, including leveraging long-term monitoring; making intersite and cross-scale comparisons; adopting novel empirical and statistical approaches; and developing funding streams to support flexible and responsive data collection

Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition