Toxicity thresholds of nine herbicides to coral symbionts (Symbiodiniaceae)

Over 30 herbicides have been detected in catchments and waters of the Great Barrier Reef (GBR) and their toxicity to key tropical species, including the coral endosymbiotic algae Symbiodiniaceae, is not generally considered in current water quality guideline values (WQGVs). Mutualistic symbionts of the family Symbiodiniaceae are essential for the survival of scleractinian corals. We tested the effects of nine GBR-relevant herbicides on photosynthetic efficiency (ΔF/Fm′) and specific growth rate (SGR) over 14 days of cultured coral endosymbiont Cladocopium goreaui (formerly Symbiodinium clade C1). All seven Photosystem II (PSII) herbicides tested inhibited ΔF/Fm′ and SGR, with toxicity thresholds for SGR ranging between 2.75 and 320 µg L−1 (no effect concentration) and 2.54–257 µg L−1 (EC10). There was a strong correlation between EC50s for ΔF/Fm′ and SGR for all PSII herbicides indicating that inhibition of ΔF/Fm′ can be considered a biologically relevant toxicity endpoint for PSII herbicides to this species. The non-PSII herbicides haloxyfop and imazapic did not affect ΔF/Fm′ or SGR at the highest concentrations tested. The inclusion of this toxicity data for Symbiodiniaceae will contribute to improving WQGVs to adequately inform risk assessments and the management of herbicides in tropical marine ecosystems.

Catalysis of Chlorovirus Production by the Foraging of Bursaria truncatella on Paramecia bursaria Containing Endosymbiotic Algae

Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time; however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of chlorovirus titers over the controls and more foraging generated more chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of chlorovirus population sizes and dynamics.

Coral microbiome manipulation elicits metabolic and genetic restructuring to mitigate heat stress and evade mortality

Beneficial microorganisms for corals (BMCs) ameliorate environmental stress, but whether they can prevent mortality and the underlying host response mechanisms remains elusive. Here, we conducted omics analyses on the coral Mussismilia hispida exposed to bleaching conditions in a long-term mesocosm experiment and inoculated with a selected BMC consortium or a saline solution placebo. All corals were affected by heat stress, but the observed “post-heat stress disorder” was mitigated by BMCs, signified by patterns of dimethylsulfoniopropionate degradation, lipid maintenance, and coral host transcriptional reprogramming of cellular restructuration, repair, stress protection, and immune genes, concomitant with a 40% survival rate increase and stable photosynthetic performance by the endosymbiotic algae. This study provides insights into the responses that underlie probiotic host manipulation. We demonstrate that BMCs trigger a dynamic microbiome restructuring process that instigates genetic and metabolic alterations in the coral host that eventually mitigate coral bleaching and mortality.

The tropical coral Pocillopora acuta displays an unusual chromatin structure and shows histone H3 clipping plasticity upon bleaching

Background: Pocillopora acuta is a hermatypic coral with strong ecological importance. Anthropogenic disturbances and global warming are major threats that can induce coral bleaching, the disruption of the mutualistic symbiosis between the coral host and its endosymbiotic algae. Previous works have shown that somaclonal colonies display different levels of survival depending on the environmental conditions they previously faced. Epigenetic mechanisms are good candidates to explain this phenomenon. However, almost no work had been published on the P. acuta epigenome, especially on histone modifications. In this study, we aim at providing the first insight into chromatin structure of this species. Methods: We aligned the amino acid sequence of P. acuta core histones with histone sequences from various phyla. We developed a centri-filtration on sucrose gradient to separate chromatin from the host and the symbiont. The presence of histone H3 protein and specific histone modifications were then detected by western blot performed on histone extraction done from bleached and healthy corals. Finally, micrococcal nuclease (MNase) digestions were undertaken to study nucleosomal organization. Results: The centri-filtration enabled coral chromatin isolation with less than 2% of contamination by endosymbiont material. Histone sequences alignments with other species show that P. acuta displays on average ~90% of sequence similarities with mice and ~96% with other corals. H3 detection by western blot showed that H3 is clipped in healthy corals while it appeared to be intact in bleached corals. MNase treatment failed to provide the usual mononucleosomal digestion, a feature shared with some cnidarian, but not all; suggesting an unusual chromatin structure. Conclusions: These results provide a first insight into the chromatin, nucleosome and histone structure of P. acuta. The unusual patterns highlighted in this study and partly shared with other cnidarian will need to be further studied to better understand its role in corals.

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.

A novel nitrogen concentrating mechanism in the coral-algae symbiosome

Coral algal symbionts are hosted inside the symbiosome of gastrodermal cells, an intracellular compartment that isolates algae from the external environment and allows host cells to control the delivery of metabolites to their symbionts. However, the underlying molecular mechanisms are largely unknown. Here, we report the diel trafficking of NH3-transporting Rhesus (Rh) channels between the cytoplasm and the symbiosome membrane in the coral Acropora yongei, which matches established patterns of nitrogen delivery to endosymbionts. Heterologous expression in Xenopus oocytes established that A. yongei Rh (ayRhp1) is a channel that facilitates NH3 diffusion across membranes following its partial pressure gradient. Immunostaining revealed ayRhp1 is widely distributed throughout coral tissues and most abundantly present in oral ectodermal cells, desmocytes, and gastrodermal cells. In the latter, ayRhp1 was observed in the symbiosome membrane of alga-containing cells. Together with V-type H+-ATPases that make the symbiosome highly acidic (pH~4), ayRhp1 constitutes an NH4+-trapping mechanism analogous to that in mammalian renal tubule. Remarkably, ayRhp1 presence in the symbiosome membrane was higher during the day than the night. This indicates a regulatory mechanism that facilitates NH4+ delivery to alga during the day, likely to sustain high turnover rates of photosynthetic proteins, while restricting NH4+ delivery at night to maintain the endosymbiotic algae in a nitrogen-limited stage that stagnates their growth. The dynamic trafficking of proteins to and away from the symbiosome membrane is a previously unknown mechanism that contributes to metabolic regulation between symbiotic partners.Significance StatementThe endosymbiotic relationship between corals and algae relies on the coordinated exchange of metabolites. Disruption of these metabolic exchanges can result in interruption of the symbiosis; however, the underlying molecular mechanisms are poorly understood. Here we report that Acropora yongei coral host cells express ammonia-transporting channel proteins (ayRhp1), which traffic to and away from the symbiosome membrane surrounding the endosymbiotic algae. In conjunction with the acidic symbiosome microenvironment, this mechanism allows host cells to regulate nitrogen delivery to endosymbionts sustaining essential functions while restricting growth. This work provides novel mechanistic information about metabolic regulation of animal-algae symbioses, and advances our understanding of physiological mechanisms that might determine coral local adaptation, resilience, and vulnerability to environmental stress including climate change.

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 widespread and ecologically important. Investigating modern 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 3D subcellular imaging with photophysiology, carbon flux imaging and transcriptomics, we show that cell division of algal 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 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 upregulation 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. NanoSIMS analysis revealed major carbon allocation to plastids and transfer to the host cell. Invagination of the symbiosome into endosymbionts to optimize metabolic exchanges is strong evidence that the algal metamorphosis is irreversible. Hosts therefore trigger and unambiguously benefit from major bioenergetic remodeling of symbiotic microalgae with important consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step towards plastid acquisition.

Insights into coral bleaching under heat stress from analysis of gene expression in a sea anemone model system

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 NFκB 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.

Organic Carbon and Nitrogen Isoscapes of Reef Corals and Algal Symbionts: Relative Influences of Environmental Gradients and Heterotrophy

The elemental (C/N) and stable isotopic (δ13C, δ15N) compositions and compound-specific δ15N values of amino acids (δ15NAA) were evaluated for coral holobionts as diagnostic tools to detect spatiotemporal environmental heterogeneity and its effects on coral health. Hermatypic coral samples of eight species were collected at 12 reef sites with differing levels of pollution stress. The C/N ratios, δ13C values, and δ15N values of coral tissues and endosymbiotic algae were determined for 193 coral holobionts, and the amino acid composition and δ15NAA values of selected samples were analyzed. δ15N values were influenced most by pollution stress, while C/N ratios and δ13C values depended most strongly on species. The results imply that δ13C and δ15N values are useful indicators for distinguishing the ecological niches of sympatric coral species based on microhabitat preference and resource selectivity. Using δ15NAA values, the trophic level (TL) of the examined coral samples was estimated to be 0.71 to 1.53, i.e., purely autotrophic to partially heterotrophic. Significant portions of the variation in bulk δ15N and δ13C values could be explained by the influence of heterotrophy. The TL of symbionts covaried with that of their hosts, implying that amino acids acquired through host heterotrophy are translocated to symbionts. Dependence on heterotrophy was stronger at polluted sites, indicating that the ecological role of corals changes in response to eutrophication.

Molecular Investigation of Paramecium bursaria Endosymbiotic Algae: the First Records of Symbiotic Micractinium reisseri from Kamchatka

Paramecium bursaria is a symbiotic ciliate species which cells contain hundreds of algae enclosed in perialgal vacuoles. The aim of the present study was to identify endosymbiotic algal strains of P. bursaria and to define the geographical distribution of the identified species. We analyzed symbiotic strains of P. bursaria originating from distant geographical locations and housed at the Culture Collection of Ciliates and their Symbionts (CCCS) at St. Petersburg University. Based on the obtained results, we identified these strains as Micractinium reisseri , Chlorella vulgaris, and Chlorella variabilis. We did not confirm the occurrence of a division into American and European groups and we guess that this division is only contractual and corresponds to the amount of introns in the 18S rDNA, and that there is no strong correlation with the geographical location. We have demonstrated that the range of M. reisseri is greater than previously supposed. We identified algae strains originating from Southern Europe (Serbia), Western Asia, and from the Far East (Kamchatka) as M. reisseri. Moreover, we identified two strains originating from Europe as C. variabilis, which also contradicts the predetermes about a division into American and European groups.