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Synopsis Single-cell RNA sequencing (scRNAseq) is a powerful tool to describe cell types in multicellular organisms across the animal kingdom. In standard scRNAseq analysis pipelines, clusters of cells with similar transcriptional signatures are given cell type labels based on marker genes that infer specialized known characteristics. Since these analyses are designed for model organisms, such as humans and mice, problems arise when attempting to label cell types of distantly related, non-model species that have unique or divergent cell types. Consequently, this leads to limited discovery of novel species-specific cell types and potential mis-annotation of cell types in non-model species while using scRNAseq. To address this problem, we discuss recently published approaches that help annotate scRNAseq clusters for any non-model organism. We first suggest that annotating with an evolutionary context of cell lineages will aid in the discovery of novel cell types and provide a marker-free approach to compare cell types across distantly related species. Secondly, machine learning has greatly improved bioinformatic analyses, so we highlight some open-source programs that use reference-free approaches to annotate cell clusters. Lastly, we propose the use of unannotated genes as potential cell markers for non-model organisms, as many do not have fully annotated genomes and these data are often disregarded. Improving single-cell annotations will aid the discovery of novel cell types and enhance our understanding of non-model organisms at a cellular level. By unifying approaches to annotate cell types in non-model organisms, we can increase the confidence of cell annotation label transfer and the flexibility to discover novel cell types. 

Abstract The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes. Initially characterized in vertebrate neutrophils, cells capable of ETosis have been discovered recently in diverse non-vertebrate taxa. To assess the conservation of ETosis between evolutionarily distant non-vertebrate phyla, we observed and quantified ETosis using the model ctenophoreMnemiopsis leidyiand the oysterCrassostrea gigas. Here we report that ctenophores – thought to have diverged very early from the metazoan stem lineage – possess immune-like cells capable of phagocytosis and ETosis. We demonstrate that bothMnemiopsisandCrassostreaimmune cells undergo ETosis after exposure to diverse microbes and chemical agents that stimulate ion flux. We thus propose that ETosis is an evolutionarily conserved metazoan defense against pathogens. 

Abstract The cluster of differentiation 36 (CD36) domain defines the characteristic ectodomain associated with class B scavenger receptor (SR-B) proteins. In bilaterians, SR-Bs play critical roles in diverse biological processes including innate immunity functions such as pathogen recognition and apoptotic cell clearance, as well as metabolic sensing associated with fatty acid uptake and cholesterol transport. Although previous studies suggest this protein family is ancient, SR-B diversity across Eukarya has not been robustly characterized. We analyzed SR-B homologs identified from the genomes and transcriptomes of 165 diverse eukaryotic species. The presence of highly conserved amino acid motifs across major eukaryotic supergroups supports the presence of a SR-B homolog in the last eukaryotic common ancestor. Our comparative analyses of SR-B protein structure identify the retention of a canonical asymmetric beta barrel tertiary structure within the CD36 ectodomain across Eukarya. We also identify multiple instances of independent lineage-specific sequence expansions in the apex region of the CD36 ectodomain—a region functionally associated with ligand-sensing. We hypothesize that a combination of both sequence expansion and structural variation in the CD36 apex region may reflect the evolution of SR-B ligand-sensing specificity between diverse eukaryotic clades. 

Cell suspension fluidics, such as flow cytometry (FCS) and fluorescence-activated cell sorting (FACS), facilitates the identification and precise separation of individual cells based on phenotype. Since its introduction, flow cytometry has been used to analyze cell types and cellular processes in diverse non-vertebrate taxa, including cnidarians, molluscs, and arthropods. Ctenophores, which diverged very early from the metazoan stem lineage, have emerged as an informative clade for the study of metazoan cell type evolution. We present standardized methodologies for flow cytometry-mediated identification and analyses of cells from the model ctenophoreMnemiopsis leidyithat can also be applied to isolate targeted cell populations. Here we focus on the identification and isolation of ctenophore phagocytes. Implementing flow cytometry methods in ctenophores allows for fine scale analyses of fundamental cellular processes conserved broadly across animals, as well as potentially revealing novel cellular phenotypes and behaviors restricted to the ctenophore lineage. 

Abstract 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. 

Stony coral tissue loss disease (SCTLD) is destructive and poses a significant threat to Caribbean coral reef ecosystems. Characterized by the acute loss of coral tissue, SCTLD has impacted over 22 stony coral species across the Caribbean region, leading to visible declines in reef health. Based on the duration, lethality, host range, and spread of this disease, SCTLD is considered the most devastating coral disease outbreak ever recorded. Researchers are actively investigating the cause and transmission of SCTLD, but the exact mechanisms, triggers, and etiological agent(s) remain elusive. If left unchecked, SCTLD could have profound implications for the health and resilience of coral reefs worldwide. To summarize what is known about this disease and identify potential knowledge gaps, this review provides a holistic overview of SCTLD research, including species susceptibility, disease transmission, ecological impacts, etiology, diagnostic tools, host defense mechanisms, and treatments. Additionally, future research avenues are highlighted, which are also relevant for other coral diseases. As SCTLD continues to spread, collaborative efforts are necessary to develop effective strategies for mitigating its impacts on critical coral reef ecosystems. These collaborative efforts need to include researchers from diverse backgrounds and underrepresented groups to provide additional perspectives for a disease that requires creative and urgent solutions. 

Reef-building corals contain a complex consortium of organisms, a holobiont, which responds dynamically to disease, making pathogen identification difficult. While coral transcriptomics and microbiome communities have previously been characterized, similarities and differences in their responses to different pathogenic sources has not yet been assessed. In this study, we inoculated four genets of the Caribbean branching coral Acropora palmata with a known coral pathogen ( Serratia marcescens ) and white band disease. We then characterized the coral’s transcriptomic and prokaryotic microbiomes’ (prokaryiome) responses to the disease inoculations, as well as how these responses were affected by a short-term heat stress prior to disease inoculation. We found strong commonality in both the transcriptomic and prokaryiomes responses, regardless of disease inoculation. Differences, however, were observed between inoculated corals that either remained healthy or developed active disease signs. Transcriptomic co-expression analysis identified that corals inoculated with disease increased gene expression of immune, wound healing, and fatty acid metabolic processes. Co-abundance analysis of the prokaryiome identified sets of both healthy-and-disease-state bacteria, while co-expression analysis of the prokaryiomes’ inferred metagenomic function revealed infected corals’ prokaryiomes shifted from free-living to biofilm states, as well as increasing metabolic processes. The short-term heat stress did not increase disease susceptibility for any of the four genets with any of the disease inoculations, and there was only a weak effect captured in the coral hosts’ transcriptomic and prokaryiomes response. Genet identity, however, was a major driver of the transcriptomic variance, primarily due to differences in baseline immune gene expression. Despite genotypic differences in baseline gene expression, we have identified a common response for components of the coral holobiont to different disease inoculations. This work has identified genes and prokaryiome members that can be focused on for future coral disease work, specifically, putative disease diagnostic tools. 

Abstract 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. 

Climate change induced heat stress has increased coral bleaching events worldwide. Differentially regulated immune genes are one of the primary responses to heat stress suggesting that immune activation is critical. However, the cellular immune mechanisms of coral bleaching is currently unknown, and it is still not known if the immune response documented during heat stress is a consequence of bleaching or is directly caused by the heat stress itself. To address this question, we have used two model system sea anemones (Order: Actiniaria): Exaiptasia diaphana and Nematostella vectensis . E. diaphana is an established sea anemone model for algal symbiont interaction, while N. vectensis is an established sea anemone model that lacks the algal symbiont. Here, we examined the effect of increased temperature on phagocytic activity, as an indication of immune function. Our data shows that immune cell activity increases during heat stress, while small molecule pinocytosis remains unaffected. We observed an increase in cellular production of reactive oxygen species with increasing temperatures. We also found that the cellular immune activity was not affected by the presence of the Symbiodiniaceae. Our results suggest that the immune activity observed in heat-stress induced bleaching in corals is a fundamental and basic response independent of the bleaching effect. These results establish a foundation for improving our understanding of hexacorallian immune cell biology, and its potential role in coral bleaching.