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, 9, 1998
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Intra-host Symbiont Diversity and Extended Symbiont Maintenance in Photosymbiotic Acantharea (Clade F)

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

Margaret Mars Brisbin et al. Front Microbiol.

Abstract

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.

Keywords: Acantharea; Phaeocystis; Rhizaria; mutualism; photosymbiosis; plankton; protist; symbiosis.

Figures

FIGURE 1
FIGURE 1
Relative abundance of symbiotic Sequence Variants (SVs) in individual acantharian hosts (A) and environmental samples (B). (A) Each bar represents a single acantharian host and is labeled by collection location (st#, ECS cruise station number; Oki, Okinawa Island; Cat, Catalina Island) and sample ID. Individual acantharians contained 4–12 symbiotic SVs (mean = 8, SD = 2). (B) Each bar represents an environmental replicate from ECS cruise stations. Symbiotic SVs are colored by clade: green is Chrysochromulina, blue is Phaeocystis clade Phaeo2, pink is Phaeocystis cordata, gray/black is Phaeocystis jahnii, and purple is Phaeocystis, but not placed within known clades. Ten of the symbiotic SVs were not found in environmental samples and are marked by an asterisk in the legend. The figure was created with R package ggplot2.
FIGURE 2
FIGURE 2
Phylogenetic placement of symbiotic SVs identified from acantharian hosts. The phylogenetic tree was built from a MUSCLE v3.8.31 alignment of 21 symbiotic SVs and GenBank reference sequences representing the 5 haptophyte orders with MrBayes v3.2.7. The symbiotic SVs are highlighted to match the legend in Figure 1. Phaeocystis clades are color coded and include the Phaeo2 clade, which is an uncultured clade identified from symbiotic sequences from acantharians collected near Okinawa (Decelle et al., 2012b). Values associated with nodes are posterior probabilities as a percent after 100,000 generations (average SD of split frequencies = 0.019). The scale bar indicates 0.02 changes expected per site.
FIGURE 3
FIGURE 3
Phylogenetic placement of acantharian host SVs. The phylogenetic tree was built from a MUSCLE v3.8.31 alignment of the 5 dominant host SVs and GenBank reference sequences representing clades E and F Acantharea and the nasellarian radiolarian Triastrum aurivillii (outgroup). The tree was built with MrBayes v3.2.7. Values associated with nodes are posterior probabilities as a percent after 40,000 generations (average SD of split frequencies = 0.01). The scale bar indicates 0.03 changes expected per site.
FIGURE 4
FIGURE 4
Principal Coordinates Analysis of Bray-Curtis distances between symbiont communities within individual acantharian hosts. Point shape corresponds to host SV and color corresponds to the collection location of the host acantharians (n = 42, 1–14 per site). The symbiont communities associated with acantharians collected near Catalina Island and from ECS station 2 form clusters, while communities from the other locations do not cluster separately. A PERMANOVA by location (excluding st. 4 and st. 10 due to insufficient sample size) performed with the adonis function in the R package vegan on the Bray-Curtis distances between symbiont communities confirmed that collection location had a significant effect on symbiont community (p = 0.001, R2 = 0.47 after 999 permutations). Host SV also had a significant effect (p = 0.009, R2 = 0.19), but only SV 5 was significantly different when pairwise PERMANOVA comparisons were performed in Qiime2. Bray-Curtis distance and PCoA ordination were performed with R package phyloseq and the plot was rendered with R package ggplot2.
FIGURE 5
FIGURE 5
Principal Coordinates Analysis of Bray-Curtis distances between host-associated symbiont communities and free-living symbiont communities. PCoA plot from Bray-Curtis distances between ECS environmental free-living and the host-associated symbiotic community compositions, including all symbiotic SVs identified in this study. The two sample types cluster separately and the difference in community composition is statistically significant (PERMANOVA, p = 0.001, R2 = 0.326, 999 permutations). Analyses were performed with R packages phyloseq and vegan and plot was rendered with R package ggplot2.
FIGURE 6
FIGURE 6
Fluorescent confocal microscopy of acantharians and their symbionts. (A) Single optical slice displaying autofluorescence of symbiont chlorophyll (red) and halogen light imaging of an entire host, sample Oki.3A. (B) Maximum projection of a z-stack spanning the entire host sample Oki.4A, which contains 17 visible symbionts and 10 symbiotic sequence variants. Arrows indicate symbionts presenting the free-living phenotype: smaller cell diameter and two elongate, parietal chloroplasts, which is also visible in the inset image of a Phaeocystis globosa CCMP1528 cell in culture that was imaged following the same methods. Red fluorescence is chlorophyll autofluorescence. (C–E) Maximum projections of z-stacks spanning entire hosts collected near Okinawa in December 2017. Red fluorescence is symbiotic chlorophyll autofluorescence. Green fluorescent staining is LysoTracker Green, which selectively binds to low-pH digestive-organelles, including lysosomes and phagolysosomes. Symbionts are not held in phagolysosomes and lysosomes are not concentrated around symbionts, indicating that symbionts are not actively being digested. Scale bars are 10 μm in all panels.

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