Ctenophores are an attractive phylogenetic lineage for studying animal evolution due to their early divergence from other metazoans. Among Ctenophora, Mnemiopsis leidyi is a model system for developmental, cellular, molecular genetic, and evolutionary studies. Until recently, many of these studies were conducted on wild-caught animals, limiting access to researchers on the coast. Here we present significant advancements towards culturing M. leidyi in laboratories without coastal access, enabling its wider use as an experimental and genetic model system. We detail updated feeding regimens that take advantage of co-culturing Brachionus rotifers with Apocyclops copepods, and quantify the reproductive output of our M. leidyi lab strain on this diet. Our updated feeding regimen maintains reproductive fitness comparable to wild-caught individuals. Importantly, we have eliminated the logistical complexities and costs of regularly feeding live larval fish to M. leidyi. Our updated protocols make it feasible to maintain continuous ctenophore cultures independent of access to both coastal populations of wild M. leidyi and larval fish culturing facilities.

The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes 1,2 . Initially characterized in vertebrate neutrophils, cells capable of ETosis have been discovered recently in diverse non-vertebrate taxa 3 4-6 . To assess the conservation of ETosis between evolutionarily distant non-vertebrate phyla, we observed and quantified ETosis using the model ctenophore Mnemiopsis leidyi and the oyster Crassostrea gigas . Here we report that ctenophores – thought to have diverged very early from the metazoan stem lineage 7-10 – possess immune cell types capable of phagocytosis and ETosis. We demonstrate that both Mnemiopsis and Crassostrea immune cells undergo ETosis after exposure to diverse microbes and chemical agents that stimulate ion flux. Our results support ETosis as an evolutionarily ancient metazoan defense against pathogens.

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 ctenophore Mnemiopsis leidyi that 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.

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.

A new species of Glossocephalus, G. aurantium sp. nov., is described based on two female specimens collected from the Monterey Canyon, California, eastern Pacific Ocean. It was found associated with an undescribed lobate ctenophore. Glossocephalus aurantium is readily distinguished from its congeners by the large crescent-shaped bulbous eye fields relative to the size of the head capsule. The pereopod morphology is most similar to G. rebecae, recently described from the same general region by Zeidler and Browne (2015), but that species is readily distinguished by it's relatively narrow crescent-shaped eye fields. Apart from it's prominent bulbous eye structures, G. aurantium is distinguished from G. milneedwardsi by the morphology of the gnathopods (not spoon-shaped). In addition, pereopods 5 and 6 are slender (not paddle-like) and the head capsule is relatively larger with a sharp rostrum (not rounded).

Despite long-standing experimental interest in ctenophores due to their unique biology, ecological influence and evolutionary status, previous work has largely been constrained by the periodic seasonal availability of wild-caught animals and difficulty in reliably closing the life cycle. To address this problem, we have developed straightforward protocols that can be easily implemented to establish long-term multigenerational cultures for biological experimentation in the laboratory. In this protocol, we describe the continuous culture of the Atlantic lobate ctenophore Mnemiopsis leidyi . A rapid 3-week egg-to-egg generation time makes Mnemiopsis suitable for a wide range of experimental genetic, cellular, embryological, physiological, developmental, ecological and evolutionary studies. We provide recommendations for general husbandry to close the life cycle of Mnemiopsis in the laboratory, including feeding requirements, light-induced spawning, collection of embryos and rearing of juveniles to adults. These protocols have been successfully applied to maintain long-term multigenerational cultures of several species of pelagic ctenophores, and can be utilized by laboratories lacking easy access to the ocean. We also provide protocols for targeted genome editing via microinjection with CRISPR–Cas9 that can be completed within ~2 weeks, including single-guide RNA synthesis, early embryo microinjection, phenotype assessment and sequence validation of genome edits. These protocols provide a foundation for using Mnemiopsis as a model organism for functional genomic analyses in ctenophores.

Abstract The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes 1,2. First characterized in vertebrate neutrophils, cells capable of ETosis have been recently discovered in several invertebrate taxa and the formation of invertebrate DNA traps has been most thoroughly examined in bivalves ​​3–5. Here we report that ctenophores – thought to have diverged very early from the metazoan stem lineage 6,7 – possess immune cell types capable of ETosis, suggesting that this cellular immune response behavior was likely present early in metazoan evolution. To assess conservation of ET activation between evolutionarily distant phyla, we deployed a comparative approach integrating data from the model ctenophore Mnemiopsis leidyi and the oyster Crassostrea gigas to develop a novel imaging analysis pipeline to quantify ETosis in large numbers of cells. We demonstrate that both Mnemiopsis and Crassostrea immune cells can undergo ETosis after exposure to diverse microbes and pharmacological stimuli. Our results suggest that the range of cellular immune behaviors and signaling cascades that produce extracellular DNA traps were likely present prior to the divergence of extant metazoan lineages and thus ETosis represents an evolutionarily ancient defense against pathogens existing at the dawn of animal multicellularity.

EDITORIAL article Front. Immunol., 31 January 2022Sec. Comparative Immunology Volume 13 - 2022 | https://doi.org/10.3389/fimmu.2022.816550

The ability to isolate, monitor, and examine specific cells of interest enables targeted experimental manipulations that would otherwise be difficult to perform and interpret in the context of the whole organism. In vitro primary cell cultures derived from ctenophores thus serve as an important tool for understanding complex cellular and molecular interactions that take place both within and between various ctenophore cell types. Here we describe methods for reliably generating and maintaining primary cell cultures derived from the lobate ctenophore Mnemiopsis leidyi that can be used for a wide variety of experimental applications.

Background The formation of the Isthmus of Panama and final closure of the Central American Seaway (CAS) provides an independent calibration point for examining the rate of DNA substitutions. This vicariant event has been widely used to estimate the substitution rate across mitochondrial genomes and to date evolutionary events in other taxonomic groups. Nuclear sequence data is increasingly being used to complement mitochondrial datasets for phylogenetic and evolutionary investigations; these studies would benefit from information regarding the rate and pattern of DNA substitutions derived from the nuclear genome. Results To estimate the genome-wide neutral mutation rate (µ), genotype-by-sequencing (GBS) datasets were generated for three transisthmian species pairs in Alpheus snapping shrimp. A range of bioinformatic filtering parameters were evaluated in order to minimize potential bias in mutation rate estimates that may result from SNP filtering. Using a Bayesian coalescent approach (G-PhoCS) applied to 44,960 GBS loci, we estimated µ to be 2.64E−9 substitutions/site/year, when calibrated with the closure of the CAS at 3 Ma. Post-divergence gene flow was detected in one species pair. Failure to account for this post-split migration inflates our substitution rate estimates, emphasizing the importance of demographic methods that can accommodate gene flow. Conclusions Results from our study, both parameter estimates and bioinformatic explorations, have broad-ranging implications for phylogeographic studies in other non-model taxa using reduced representation datasets. Our best estimate of µ that accounts for coalescent and demographic processes is remarkably similar to experimentally derived mutation rates in model arthropod systems. These results contradicted recent suggestions that the closure of the Isthmus was completed much earlier (around 10 Ma), as mutation rates based on an early calibration resulted in uncharacteristically low genomic mutation rates. Also, stricter filtering parameters resulted in biased datasets that generated lower mutation rate estimates and influenced demographic parameters, serving as a cautionary tale for the adherence to conservative bioinformatic strategies when generating reduced-representation datasets at the species level. To our knowledge this is the first use of transisthmian species pairs to calibrate the rate of molecular evolution from GBS data.

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.

The Krüppel-like factor (Klf) gene family encodes transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation and development in bilaterians. Although Klf genes have been shown to specify functionally various cell types in non-bilaterian animals, their role in early-diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here, we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis resulted in abnormal development of several organs, including the pharynx, tentacle bulbs and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation.

Invasion rates have increased in the past 100 y irrespective of international conventions. What characterizes a successful invasion event? And how does genetic diversity translate into invasion success? Employing a whole-genome perspective using one of the most successful marine invasive species world-wide as a model, we resolve temporal invasion dynamics during independent invasion events in Eurasia. We reveal complex regionally independent invasion histories including cases of recurrent translocations, time-limited translocations, and stepping-stone range expansions with severe bottlenecks within the same species. Irrespective of these different invasion dynamics, which lead to contrasting patterns of genetic diversity, all nonindigenous populations are similarly successful. This illustrates that genetic diversity, per se, is not necessarily the driving force behind invasion success. Other factors such as propagule pressure and repeated introductions are an important contribution to facilitate successful invasions. This calls into question the dominant paradigm of the genetic paradox of invasions, i.e., the successful establishment of nonindigenous populations with low levels of genetic diversity.

Coelenterazine is a key substrate involved in marine bioluminescence which is used for light-production by at least nine phyla. Some luminous animals, such as the hydromedusa Aequorea, lack the ability to produce coelenterazine endogenously and instead depend on dietary sources. Little is known about the source organisms or the metabolic process of coelenterazine biosynthesis. Here, we present evidence that ctenophores are both producers and suppliers of coelenterazine in marine ecosystems. Using biochemical assays and mass spectrometry analyses, we detected coelenterazine from cultured ctenophores fed with a non-luminous coelenterazine-free diet. We propose that ctenophores are an emerging model organism to study coelenterazine biosynthesis and the origins of bioluminescence.