Antibiotics reduce Pocillopora coral-associated bacteria diversity, decrease holobiont oxygen consumption and activate immune gene expression.

Corals are important models for understanding invertebrate host-microbe interactions; however, to fully discern mechanisms involved in these relationships, experimental approaches for manipulating coral-bacteria associations are needed. Coral-associated bacteria affect holobiont health via nutrient cycling, metabolic exchanges and pathogen exclusion, yet it is not fully understood how bacterial community shifts affect holobiont health and physiology. In this study, a combination of antibiotics (ampicillin, streptomycin and ciprofloxacin) was used to disrupt the bacterial communities of 14 colonies of the reef framework-building corals Pocillopora meandrina and P. verrucosa, originally collected from Panama and hosting diverse algal symbionts (family Symbiodiniaceae). Symbiodiniaceae photochemical efficiencies and holobiont oxygen consumption (as proxies for coral health) were measured throughout a 5-day exposure. Antibiotics altered bacterial community composition and reduced alpha and beta diversity, however, several bacteria persisted, leading to the hypothesis that these bacteria are either antibiotics resistant or occupy internal niches that are shielded from antibiotics. While antibiotics did not affect Symbiodiniaceae photochemical efficiency, antibiotics-treated corals had lower oxygen consumption rates. RNAseq revealed that antibiotics increased expression of Pocillopora immunity and stress response genes at the expense of cellular maintenance and metabolism functions. Together, these results reveal that antibiotic disruption of corals' native bacteria negatively impacts holobiont health by decreasing oxygen consumption and activating host immunity without directly impairing Symbiodiniaceae photosynthesis, underscoring the critical role of coral-associated bacteria in holobiont health. They also provide a baseline for future experiments that manipulate Pocillopora corals' symbioses by first reducing the diversity and complexity of coral-associated bacteria.

Functional Characterization of Hexacorallia Phagocytic Cells.

Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral Pocillopora damicornis and the sea anemone Nematostella vectensis. Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology.

Novel methods to establish whole-body primary cell cultures for the cnidarians Nematostella vectensis and Pocillopora damicornis.

Cnidarians are emerging model organisms for cell and molecular biology research. However, successful cell culture development has been challenging due to incomplete tissue dissociation and contamination. In this report, we developed and tested several different methodologies to culture primary cells from all tissues of two species of Cnidaria: Nematostella vectensis and Pocillopora damicornis. In over 170 replicated cell cultures, we demonstrate that physical dissociation was the most successful method for viable and diverse N. vectensis cells while antibiotic-assisted dissociation was most successful for viable and diverse P. damicornis cells. We also demonstrate that a rigorous antibiotic pretreatment results in less initial contamination in cell cultures. Primary cultures of both species averaged 12-13 days of viability, showed proliferation, and maintained high cell diversity including cnidocytes, nematosomes, putative gastrodermal, and epidermal cells. Overall, this work will contribute a needed tool for furthering functional cell biology experiments in Cnidaria.

The starlet sea anemone, Nematostella vectensis, possesses body region-specific bacterial associations with spirochetes dominating the capitulum.

Sampling of different body regions can reveal highly specialized bacterial associations within the holobiont and facilitate identification of core microbial symbionts that would otherwise be overlooked by bulk sampling methods. Here, we characterized compartment-specific associations present within the model cnidarian Nematostella vectensis by dividing its morphology into three distinct microhabitats. This sampling design allowed us to uncover a capitulum-specific dominance of spirochetes within N. vectensis. Bacteria from the family Spirochaetaceae made up 66% of the community in the capitulum, while only representing 1.2% and 0.1% of the communities in the mesenteries and physa, respectively. A phylogenetic analysis of the predominant spirochete sequence recovered from N. vectensis showed a close relation to spirochetes previously recovered from wild N. vectensis. These sequences clustered closer to the recently described genus Oceanispirochaeta, rather than Spirochaeta perfilievii, supporting them as members of this clade. This suggests a prevalent and yet uncharacterized association between N. vectensis and spirochetes from the order Spirochaetales.

The Complicated Evolutionary Diversification of the Mpeg-1/Perforin-2 Family in Cnidarians.

The invertebrate innate immune system is surprisingly complex, yet our knowledge is limited to a few select model systems. One understudied group is the phylum Cnidaria (corals, sea anemones, etc.). Cnidarians are the sister group to Bilateria and by studying their innate immunity repertoire, a better understanding of the ancestral state can be gained. Corals in particular have evolved a highly diverse innate immune system that can uncover evolutionarily basal functions of conserved genes and proteins. One rudimentary function of the innate immune system is defense against harmful bacteria using pore forming proteins. Macrophage expressed gene 1/Perforin-2 protein (Mpeg-1/P2) is a particularly important pore forming molecule as demonstrated by previous studies in humans and mice, and limited studies in non-bilaterians. However, in cnidarians, little is known about Mpeg-1/P2. In this perspective article, we will summarize the current state of knowledge of Mpeg-1/P2 in invertebrates, analyze identified Mpeg-1/P2 homologs in cnidarians, and demonstrate the evolutionary diversity of this gene family using phylogenetic analysis. We will also show that Mpeg-1 is upregulated in one species of stony coral in response to lipopolysaccharides and downregulated in another species of stony coral in response to white band disease. This data presents evidence that Mpeg-1/P2 is conserved in cnidarians and we hypothesize that it plays an important role in cnidarian innate immunity. We propose that future research focus on the function of Mpeg-1/P2 family in cnidarians to identify its primary role in innate immunity and beyond.

Lipopolysaccharide treatment stimulates Pocillopora coral genotype-specific immune responses but does not alter coral-associated bacteria communities.

Corals are comprised of a coral host and associated microbes whose interactions are mediated by the coral innate immune system. The diversity of immune factors identified in the Pocillopora damicornis genome suggests that immunity is linked to maintaining microbial symbioses while also being able to detect pathogens. However, it is unclear which immune factors respond to specific microbe-associated molecular patterns and how these immune reactions simultaneously affect coral-associated bacteria. To investigate this, fragments of P. damicornis and P. acuta colonies from Taiwan were subjected to lipopolysaccharide (LPS) treatment to stimulate immune responses and measure bacteria community shifts. RNA-seq revealed genotype-specific immune responses to LPS involving the upregulation of immune receptors, transcription factors, and pore-forming toxins. Bacteria 16S sequencing revealed significantly different bacteria communities between coral genotypes but no differences in bacteria communities were caused by LPS. Our findings confirm that Pocillopora corals activate conserved immune factors in response to LPS and identify transcription factors coordinating Pocillopora corals' immune responses. Additionally, the strong effect of coral genotype on gene expression and bacteria communities highlights the importance of coral genotype in the investigation of coral host-microbe interactions.