A hybrid-capture approach to reconstruct the phylogeny of Scleractinia (Cnidaria: Hexacorallia).

A well-supported evolutionary tree representing most major lineages of scleractinian corals is in sight with the development and application of phylogenomic approaches. Specifically, hybrid-capture techniques are shedding light on the evolution and systematics of corals. Here, we reconstructed a broad phylogeny of Scleractinia to test previous phylogenetic hypotheses inferred from a few molecular markers, in particular, the relationships among major scleractinian families and genera, and to identify clades that require further research. We analysed 449 nuclear loci from 422 corals, comprising 266 species spanning 26 families, combining data across whole genomes, transcriptomes, hybrid capture and low-coverage sequencing to reconstruct the largest phylogenomic tree of scleractinians to date. Due to the large number of loci and data completeness (less than 38% missing data), node supports were high across shallow and deep nodes with incongruences observed in only a few shallow nodes. The "Robust" and "Complex" clades were recovered unequivocally, and our analyses confirmed that Micrabaciidae Vaughan, 1905 is sister to the "Robust" clade, transforming our understanding of the "Basal" clade. Several families remain polyphyletic in our phylogeny, including Deltocyathiidae Kitahara, Cairns, Stolarski & Miller, 2012, Caryophylliidae Dana, 1846, and Coscinaraeidae Benzoni, Arrigoni, Stefani & Stolarski, 2012, and we hereby formally proposed the family name Pachyseridae Benzoni & Hoeksema to accommodate Pachyseris Milne Edwards & Haime, 1849, which is phylogenetically distinct from Agariciidae Gray, 1847. Results also revealed species misidentifications and inconsistencies within morphologically complex clades, such as Acropora Oken, 1815 and Platygyra Ehrenberg, 1834, underscoring the need for reference skeletal material and topotypes, as well as the importance of detailed taxonomic work. The approach and findings here provide much promise for further stabilising the topology of the scleractinian tree of life and advancing our understanding of coral evolution.

Colour patterns influence symbiosis and competition in the anemonefish-host anemone symbiosis system.

Colour patterns in fish are often used as an important medium for communication. Anemonefish, characterized by specific patterns of white bars, inhabit host anemones and defend the area around an anemone as their territory. The host anemone is used not only by the anemonefish, but also by other fish species that use anemones as temporary shelters. Anemonefish may be able to identify potential competitors by their colour patterns. We first examined the colour patterns of fish using host anemones inhabited by Amphiprion ocellaris as shelter and compared them with the patterns of fish using surrounding scleractinian corals. There were no fish with bars sheltering in host anemones, although many fish with bars were found in surrounding corals. Next, two fish models, one with white bars and the other with white stripes on a black background, were presented to an A. ocellaris colony. The duration of aggressive behaviour towards the bar model was significantly longer than that towards the stripe model. We conclude that differences in aggressive behaviour by the anemonefish possibly select the colour patterns of cohabiting fish. This study indicates that colour patterns may influence not only intraspecific interactions but also interspecific interactions in coral reef ecosystems.

Never, Ever Make an Enemy Out of an Anemone: Transcriptomic Comparison of Clownfish Hosting Sea Anemone Venoms.

Sea anemones are predatory marine invertebrates and have diverse venom arsenals. Venom is integral to their biology, and is used in competition, defense, and feeding. Three lineages of sea anemones are known to have independently evolved symbiotic relationships with clownfish, however the evolutionary impact of this relationship on the venom composition of the host is still unknown. Here, we investigate the potential of this symbiotic relationship to shape the venom profiles of the sea anemones that host clownfish. We use transcriptomic data to identify differences and similarities in venom profiles of six sea anemone species, representing the three known clades of clownfish-hosting sea anemones. We recovered 1121 transcripts matching verified toxins across all species, and show that hemolytic and hemorrhagic toxins are consistently the most dominant and diverse toxins across all species examined. These results are consistent with the known biology of sea anemones, provide foundational data on venom diversity of these species, and allow for a review of existing hierarchical structures in venomic studies.

Comparison of sequence-capture and ddRAD approaches in resolving species and populations in hexacorallian anthozoans.

Genome-level sequencing is the next step in understanding species-level relationships within Anthozoa (soft corals, anemones, stony corals, and their kin) as morphological and PCR-directed (single-locus) sequencing methods often fall short of differentiating species. The sea anemone genus Metridium is a common northern temperate sea anemone whose species are difficult to differentiate using morphology alone. Here we use Metridium as a case study to confirm the low level of information available in six loci for species differentiation commonly sequenced for Actiniaria and explore and compare the efficacy of ddRAD and sequence-capture methods in species-level systematics and biogeographic studies. We produce phylogenetic trees from concatenated datasets and perform DAPC and STRUCTURE analyses using SNP data. The six conventional loci are not able to consistently differentiate species within Metridium. The sequence-capture dataset resulted in high support and resolution for both current species and relationships between geographic areas. The ddRAD datasets displayed ambiguity among species, and support between major geographic groupings was not as high as the sequence-capture datasets. The level of resolution and support resulting from the sequence-capture data, combined with the ability to add additional individuals and expand beyond the genus Metridium over time, emphasizes the utility of sequence-capture methods for both systematics and future biogeographic studies within anthozoans. We discuss the strengths and weaknesses of the genomic approaches in light of our findings and suggest potential implications for the biogeography of Metridium based on our sampling.

Characterising Functional Venom Profiles of Anthozoans and Medusozoans within Their Ecological Context.

This review examines the current state of knowledge regarding toxins from anthozoans (sea anemones, coral, zoanthids, corallimorphs, sea pens and tube anemones). We provide an overview of venom from phylum Cnidaria and review the diversity of venom composition between the two major clades (Medusozoa and Anthozoa). We highlight that the functional and ecological context of venom has implications for the temporal and spatial expression of protein and peptide toxins within class Anthozoa. Understanding the nuances in the regulation of venom arsenals has been made possible by recent advances in analytical technologies that allow characterisation of the spatial distributions of toxins. Furthermore, anthozoans are unique in that ecological roles can be assigned using tissue expression data, thereby circumventing some of the challenges related to pharmacological screening.

Transcriptomic Analysis of Four Cerianthid (Cnidaria, Ceriantharia) Venoms.

Tube anemones, or cerianthids, are a phylogenetically informative group of cnidarians with complex life histories, including a pelagic larval stage and tube-dwelling adult stage, both known to utilize venom in stinging-cell rich tentacles. Cnidarians are an entirely venomous group that utilize their proteinaceous-dominated toxins to capture prey and defend against predators, in addition to several other ecological functions, including intraspecific interactions. At present there are no studies describing the venom for any species within cerianthids. Given their unique development, ecology, and distinct phylogenetic-placement within Cnidaria, our objective is to evaluate the venom-like gene diversity of four species of cerianthids from newly collected transcriptomic data. We identified 525 venom-like genes between all four species. The venom-gene profile for each species was dominated by enzymatic protein and peptide families, which is consistent with previous findings in other cnidarian venoms. However, we found few toxins that are typical of sea anemones and corals, and furthermore, three of the four species express toxin-like genes closely related to potent pore-forming toxins in box jellyfish. Our study is the first to provide a survey of the putative venom composition of cerianthids and contributes to our general understanding of the diversity of cnidarian toxins.

A next generation approach to species delimitation reveals the role of hybridization in a cryptic species complex of corals.

BACKGROUND: Our ability to investigate processes shaping the evolutionary diversification of corals (Cnidaria: Anthozoa) is limited by a lack of understanding of species boundaries. Discerning species of corals has been challenging due to a multitude of factors, including homoplasious and plastic morphological characters and the use of molecular markers that are either not informative or have not completely sorted. Hybridization can also blur species boundaries by leading to incongruence between morphology and genetics. We used traditional DNA barcoding and restriction-site associated DNA sequencing combined with coalescence-based and allele-frequency methods to elucidate species boundaries and simultaneously examine the potential role of hybridization in a speciose genus of octocoral, Sinularia. RESULTS: Species delimitations using two widely used DNA barcode markers, mtMutS and 28S rDNA, were incongruent with one another and with the morphospecies identifications. When mtMutS and 28S were concatenated, a 0.3% genetic distance threshold delimited the majority of morphospecies. In contrast, 12 of the 15 examined morphospecies formed well-supported monophyletic clades in both concatenated RAxML phylogenies and SNAPP species trees of > 6000 RADSeq loci. DAPC and Structure analyses also supported morphospecies assignments, but indicated the potential for two additional cryptic species. Three morphologically distinct species pairs could not, however, be distinguished genetically. ABBA-BABA tests demonstrated significant admixture between some of those species, suggesting that hybridization may confound species delimitation in Sinularia. CONCLUSIONS: A genomic approach can help to guide species delimitation while simultaneously elucidating the processes generating coral diversity. Results support the hypothesis that hybridization is an important mechanism in the evolution of Anthozoa, including octocorals, and future research should examine the contribution of this mechanism in generating diversity across the coral tree of life.

Molecular phylogeny and diversity of sea pens (Cnidaria: Octocorallia: Pennatulacea) with a focus on shallow water species of the northwestern Pacific Ocean.

The order Pennatulacea, commonly known as sea pens, are colony-forming benthos belonging within subclass Octocorallia (Anthozoa, Cnidaria). Sea pens are found worldwide from shallow to deep waters, and they are important components in sandy and muddy environments. Thus far, there has been only one molecular study focusing on the phylogenetic relationships within the order Pennatulacea, which mainly treated deep-sea species, and thus information on shallow water species is still lacking. On a regional scale, the diversity of sea pens in the northwestern Pacific, including Japan and Palau, has not been well investigated. In this research, we aimed to: (1) more accurately resolve the phylogenetic relationships of sea pens with the inclusion of shallow water species, and (2) obtain a better understanding of the diversity of sea pens in Japan and Palau. Specimens were collected by SCUBA and dredging from the Ryukyu Islands in southern Japan, and from mainland Japan and Palau, and identified to at least the genus level by their morphological traits. Construction of phylogenetic trees with concatenated sequences including the mitochondrial mutS-like protein DNA mismatch repair gene mtMutS and the NADH dehydrogenase subunit 2 ND2 region were performed. The p-distances of mtMutS were calculated for estimation of species number following McFadden et al. (2011). Molecular data for 12 families and 20 genera of sea pens were used in this study. This most comprehensive study including shallow water taxa provided us with more knowledge of phylogenetic relationships. The resulting phylogenetic trees showed a topology distinguished by four large clades (clades 1-4). Families Veretillidae and Echinoptilidae are shown as not the earliest-diverging taxa. Virgulariidae and Scleroptilidae are shown as polyphyletic groups, and our results reconfirm that families Pennatulidae, Kophobelemnidae and Umbellulidae are not monophyletic groups. Overall, we collected and examined an estimated 18 species from the Ryukyu Islands, 16 species from mainland Japan, and five species from Palau. Some of these specimens represented new records from Ryukyu Islands and Palau. Previous records of these sea pens did not exist likely due to a lack of diversity research in sandy and muddy areas. These results demonstrate that many sea pens discoveries likely remain in shallow waters of the Pacific.

Cnidarian Interaction with Microbial Communities: From Aid to Animal's Health to Rejection Responses.

The phylum Cnidaria is an ancient branch in the tree of metazoans. Several species exert a remarkable longevity, suggesting the existence of a developed and consistent defense mechanism of the innate immunity capable to overcome the potential repeated exposure to microbial pathogenic agents. Increasing evidence indicates that the innate immune system in Cnidarians is not only involved in the disruption of harmful microorganisms, but also is crucial in structuring tissue-associated microbial communities that are essential components of the Cnidarian holobiont and useful to the animal's health for several functions, including metabolism, immune defense, development, and behavior. Sometimes, the shifts in the normal microbiota may be used as "early" bio-indicators of both environmental changes and/or animal disease. Here the Cnidarians relationships with microbial communities and the potential biotechnological applications are summarized and discussed.

Colorful Packages: Encapsulation of Fluorescent Proteins in Complex Coacervate Core Micelles.

Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs) were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs) than for Hydrozoa FPs derived from Aequorea victoria (avFPs). Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2) and Tag red fluorescent protein (TagRFP)) or to a pKa shift of the chromophore (FP variant mCherry).

A staging system for the regeneration of a polyp from the aboral physa of the anthozoan Cnidarian Nematostella vectensis.

BACKGROUND: As the sea anemone Nematostella vectensis emerges as a model for studying regeneration, new tools will be needed to assess its regenerative processes and describe perturbations resulting from experimental investigation. Chief among these is the need for a universal set of staging criteria to establish morphological landmarks that will provide a common format for discussion among investigators. RESULTS: We have established morphological criteria to describe stages for rapidly assessing regeneration of the aboral end (physa) of Nematostella. Using this staging system, we observed rates of regeneration that are temperature independent during wound healing and temperature dependent afterward. Treatment with 25 µM lipoic acid delays the progression through wound healing without significantly affecting the subsequent rate of regeneration. Also, while an 11-day starvation before amputation causes only a minimal delay in regeneration, this delay is exacerbated by lipoic acid treatment. CONCLUSIONS: A system for staging the progression of regeneration in amputated Nematostella physa based on easily discernible morphological features provides a standard for the field. This system has allowed us to identify both temperature-dependent and -independent phases of regeneration, as well as a nutritional requirement for normal regenerative progression that is exacerbated by lipoic acid.

Skimming genomes for systematics and DNA barcodes of corals.

Numerous genomic methods developed over the past two decades have enabled the discovery and extraction of orthologous loci to help resolve phylogenetic relationships across various taxa and scales. Genome skimming (or low-coverage genome sequencing) is a promising method to not only extract high-copy loci but also 100s to 1000s of phylogenetically informative nuclear loci (e.g., ultraconserved elements [UCEs] and exons) from contemporary and museum samples. The subphylum Anthozoa, including important ecosystem engineers (e.g., stony corals, black corals, anemones, and octocorals) in the marine environment, is in critical need of phylogenetic resolution and thus might benefit from a genome-skimming approach. We conducted genome skimming on 242 anthozoan corals collected from 1886 to 2022. Using existing target-capture baitsets, we bioinformatically obtained UCEs and exons from the genome-skimming data and incorporated them with data from previously published target-capture studies. The mean number of UCE and exon loci extracted from the genome skimming data was 1837 ± 662 SD for octocorals and 1379 ± 476 SD loci for hexacorals. Phylogenetic relationships were well resolved within each class. A mean of 1422 ± 720 loci was obtained from the historical specimens, with 1253 loci recovered from the oldest specimen collected in 1886. We also obtained partial to whole mitogenomes and nuclear rRNA genes from >95% of samples. Bioinformatically pulling UCEs, exons, mitochondrial genomes, and nuclear rRNA genes from genome skimming data is a viable and low-cost option for phylogenetic studies. This approach can be used to review and support taxonomic revisions and reconstruct evolutionary histories, including historical museum and type specimens.

Development of microsatellite markers and evaluation of the genetic diversity of the edible sea anemone Paracondylactissinensis (Cnidaria, Anthozoa) in China.

Paracondylactissinensis Carlgren, 1934 is a sea anemone with economic value in China. The wild population of P.sinensis has been shrinking due to overfishing and environmental pollution, which have caused price instability. In winter, the price of P.sinensis can reach 25 USD per kilogram. Up to now, there are no genetic markers developed for P.sinensis, preventing a further exploration of their population genetic diversity. In this study, the full-length transcriptome of P.sinensis was sequenced and microsatellite DNA markers (simple sequence repeats [SSRs]) were developed from those transcripts. A total of 52 primer pairs, which can amplify specific polymorphic bands in PCR experiments, were designed for the SSR markers. Genetic diversity and population genetics were analysed for P.sinensis populations collected from the coasts of Taizhou and Rizhao using six microsatellite DNA loci. While inbreeding was detected in both populations (Fis > 0), the overall number of alleles (Na = 11.3) and bottleneck analysis suggested that the genetic diversity of P.sinensis has not been greatly impacted. Clustering analyses using STRUCTURE, principal coordinate analysis and unweighted pair group method with arithmetic mean tree revealed that the Taizhou population diverged from the Rizhao population; however, the genetic differentiation between the populations was moderate. Human-mediated commercial activities may be the principal reasons for the gene flow between the populations. Our study provides the first evaluation of the genetic resources of wild P.sinensis populations in China, which can serve as a useful reference for future comparative studies on population genetics and may guide policy-makers in initiating strategies for germplasm conservation and artificial breeding.

Larval swimming in the sea anemone Nematostella vectensis is sensitive to a broad light spectrum and exhibits a wavelength-dependent behavioral switch.

In nearly all animals, light-sensing mediated by opsin visual pigments is important for survival and reproduction. Eyeless light-sensing systems, though vital for many animals, have received relatively less attention than forms with charismatic or complex eyes. Despite no single light-sensing organ, the sea anemone Nematostella vectensis has 29 opsin genes and multiple light-mediated behaviors throughout development and reproduction, suggesting a deceptively complex light-sensing system. To characterize one aspect of this light-sensing system, we analyzed larval swimming behavior at high wavelength resolution across the ultraviolet and visual spectrum. N. vectensis larvae respond to light at least from 315 to 650 nm, which is a broad sensitivity range even compared to many animals with complex eyes. Planktonic swimming is induced by ultraviolet (UV) and violet wavelengths until 420 nm. Between 420 and 430 nm a behavioral switch occurs where at wavelengths longer than 430 nm, larvae respond to light by swimming down. Swimming down toward the substrate is distinct from light avoidance, as animals do not exhibit positive or negative phototaxis at any wavelength tested. At wavelengths longer than 575 nm, animals in the water column take increasingly longer to respond and this behavior is more variable until 650 nm where larval response is no different from the dark, suggesting these longer wavelengths lie outside of their sensitivity range. Larval swimming is the only motile stage in the life history of N. vectensis, and increased planktonic swimming could lead to greater dispersal range in potentially damaging shallow environments with short-wavelength light exposure. Longer wavelength environments may indicate more suitable substrates for metamorphosis into the polyp stage, where the individual will remain for the rest of its life. Future work will test whether this robust behavior is mediated by multiple opsins.

A new gorgonian Pseudopterogorgiananjiensis sp. nov. (Cnidaria, Octocorallia, Gorgoniidae) from the Nanji Islands, China.

Members of the genus Pseudopterogorgia Kükenthal, 1919 are widely distributed in shallow water of the Indo-West Pacific. During an investigation of benthic biodiversity in the subtidal zone surrounding the Nanji Islands in the East China Sea, two specimens of Pseudopterogorgia were collected and described as a new species based on an integrated morphological-molecular approach. Pseudopterogorgiananjiensis sp. nov. is most similar to P.fredericki Williams & Vennam, 2001 in the irregular branching form and indistinct scaphoids, but differs by the coenenchymal sclerite content of distinct capstans and a few warty spindles and radiates (vs. mostly warty spindles and a few capstans), and a purplish colony (vs. white, pink to deep rose). Molecular phylogenetic analyses, based on the mtMutS-COI gene sequences, delineated a monophyletic clade encompassing all assessed Pseudopterogorgia species. Within this clade, P.nanjiensis sp. nov. showed a close phylogenetic affinity with both P.fredericki and P.australiensis Ridley, 1884.

Taxonomy and Molecular Phylogeny of the Sea Anemone Macrodactyla (Haddon, 1898) (Cnidaria, Actiniaria), with a Description of a New Species from Singapore.

Sea anemones (Cnidaria, Actiniaria) are a successful group of marine invertebrates found in a diverse range of environments globally. In spite of their ubiquity, identities for many sea anemones remain unverified, especially those from the Indo-West Pacific region. Here, we clarify the taxonomy of the poorly known Macrodactyla aspera, a shallow-water species first described from the Torres Straits in northern Australia. We re-describe M. aspera based on new morphological and molecular data gathered from the type specimen, other museum vouchers, and from fresh material collected from Singapore. We tested the monophyly of Macrodactyla using three mitochondrial (12S, 16S and cox3) and one nuclear (28S) marker based on three congeners, recovering this genus to be polyphyletic. As a consequence, we transferred M. doreensis to the genus Heteractis, and describe a new species, Macrodactyla fautinae sp. nov. While both M. aspera and M. fautinae sp. nov. share the same arrangement and number of complete mesenteries, a similar distribution of cnidae, and are not symbiotically associated with any other biota, M. fautinae sp. nov. has perforated, lobe-like verrucae on its column, and lacks nematocyst batteries on its tentacles, unlike M. aspera. These two species also occur in similar habitats in Singapore. Finally, because M. aspera strongly resembles Dofleinia armata, the latter species flagged as a danger to public health due to its ability to inflict painful stings, we tested the relationship between these species and found them not to be closely related. However, tentacles of M. aspera, like D. armata, are densely covered with nematocyst batteries and harbour large nematocysts; we infer that M. aspera may also be capable of delivering stings that endanger public health. This study builds upon a growing number of studies that aim to ascertain identities and systematics of sea anemones historically reported from the Indo-West Pacific. Our findings will facilitate accurate species identification, which is crucial for advancing research, formulating conservation measures, and protecting public health.

Nematostella vectensis exemplifies the exceptional expansion and diversity of opsins in the eyeless Hexacorallia.

BACKGROUND: Opsins are the primary proteins responsible for light detection in animals. Cnidarians (jellyfish, sea anemones, corals) have diverse visual systems that have evolved in parallel with bilaterians (squid, flies, fish) for hundreds of millions of years. Medusozoans (e.g., jellyfish, hydroids) have evolved eyes multiple times, each time independently incorporating distinct opsin orthologs. Anthozoans (e.g., corals, sea anemones,) have diverse light-mediated behaviors and, despite being eyeless, exhibit more extensive opsin duplications than medusozoans. To better understand the evolution of photosensitivity in animals without eyes, we increased anthozoan representation in the phylogeny of animal opsins and investigated the large but poorly characterized opsin family in the sea anemone Nematostella vectensis. RESULTS: We analyzed genomic and transcriptomic data from 16 species of cnidarians to generate a large opsin phylogeny (708 sequences) with the largest sampling of anthozoan sequences to date. We identified 29 opsins from N. vectensis (NvOpsins) with high confidence, using transcriptomic and genomic datasets. We found that lineage-specific opsin duplications are common across Cnidaria, with anthozoan lineages exhibiting among the highest numbers of opsins in animals. To establish putative photosensory function of NvOpsins, we identified canonically conserved protein domains and amino acid sequences essential for opsin function in other animal species. We show high sequence diversity among NvOpsins at sites important for photoreception and transduction, suggesting potentially diverse functions. We further examined the spatiotemporal expression of NvOpsins and found both dynamic expression of opsins during embryonic development and sexually dimorphic opsin expression in adults. CONCLUSIONS: These data show that lineage-specific duplication and divergence has led to expansive diversity of opsins in eyeless cnidarians, suggesting opsins from these animals may exhibit novel biochemical functions. The variable expression patterns of opsins in N. vectensis suggest opsin gene duplications allowed for a radiation of unique sensory cell types with tissue- and stage-specific functions. This diffuse network of distinct sensory cell types could be an adaptive solution for varied sensory tasks experienced in distinct life history stages in Anthozoans.

Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis.

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here, we investigate factors that influence asexual reproduction in the burrowing sea anemone Nematostella vectensis, which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral-aboral axis of the polyp. Homeobox transcription factors and components of the TGFß, Notch, and FGF signalling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed, and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction in Nematostella, with implications for understanding the reproductive and regenerative biology of other cnidarian species.

Tentacle patterning during Exaiptasia diaphana pedal lacerate development differs between symbiotic and aposymbiotic animals.

Exaiptasia diaphana, a tropical sea anemone known as Aiptasia, is a tractable model system for studying the cellular, physiological, and ecological characteristics of cnidarian-dinoflagellate symbiosis. Aiptasia is widely used as a proxy for coral-algal symbiosis, since both Aiptasia and corals form a symbiosis with members of the family Symbiodiniaceae. Laboratory strains of Aiptasia can be maintained in both the symbiotic (Sym) and aposymbiotic (Apo, without algae) states. Apo Aiptasia allow for the study of the influence of symbiosis on different biological processes and how different environmental conditions impact symbiosis. A key feature of Aiptasia is the ease of propagating both Sym and Apo individuals in the laboratory through a process called pedal laceration. In this form of asexual reproduction, small pieces of tissue rip away from the pedal disc of a polyp, then these lacerates eventually develop tentacles and grow into new polyps. While pedal laceration has been described in the past, details of how tentacles are formed or how symbiotic and nutritional state influence this process are lacking. Here we describe the stages of development in both Sym and Apo pedal lacerates. Our results show that Apo lacerates develop tentacles earlier than Sym lacerates, while over the course of 20 days, Sym lacerates end up with a greater number of tentacles. We describe both tentacle and mesentery patterning during lacerate development and show that they form through a single pattern in early stages regardless of symbiotic state. In later stages of development, Apo lacerate tentacles and mesenteries progress through a single pattern, while variable patterns were observed in Sym lacerates. We discuss how Aiptasia lacerate mesentery and tentacle patterning differs from oral disc regeneration and how these patterning events compare to postembryonic development in Nematostella vectensis, another widely-used sea anemone model. In addition, we demonstrate that Apo lacerates supplemented with a putative nutrient source developed an intermediate number of tentacles between un-fed Apo and Sym lacerates. Based on these observations, we hypothesize that pedal lacerates progress through two different, putatively nutrient-dependent phases of development. In the early phase, the lacerate, regardless of symbiotic state, preferentially uses or relies on nutrients carried over from the adult polyp. These resources are sufficient for lacerates to develop into a functional polyp. In the late phase of development, continued growth and tentacle formation is supported by nutrients obtained from either symbionts and/or the environment through heterotrophic feeding. Finally, we advocate for the implementation of pedal lacerates as an additional resource in the Aiptasia model system toolkit for studies of cnidarian-dinoflagellate symbiosis.

Contribution of Maternal and Paternal Transmission to Bacterial Colonization in Nematostella vectensis.

Microbial communities confer multiple beneficial effects to their multicellular hosts. To evaluate the evolutionary and ecological implications of the animal-microbe interactions, it is essential to understand how bacterial colonization is secured and maintained during the transition from one generation to the next. However, the mechanisms of symbiont transmission are poorly studied for many species, especially in marine environments, where the surrounding water constitutes an additional source of microbes. Nematostella vectensis, an estuarine cnidarian, has recently emerged as model organism for studies on host-microbes interactions. Here, we use this model organism to study the transmission of bacterial colonizers, evaluating the contribution of parental and environmental transmission to the establishment of bacterial communities of the offspring. We induced spawning in adult male and female polyps of N. vectensis and used their gametes for five individual fertilization experiments. While embryos developed into primary polyps, we sampled each developmental stage and its corresponding medium samples. By analyzing the microbial community compositions of all samples through 16S rRNA gene amplicon sequencing, we showed that all host tissues harbor microbiota significantly different from the surrounding medium. Interestingly, oocytes and sperms are associated with distinct bacterial communities, indicating the specific vertical transmission of bacterial colonizers by the gametes. These differences were consistent among all the five families analyzed. By overlapping the identified bacterial ASVs associated with gametes, offspring and parents, we identified specific bacterial ASVs that are well supported candidates for vertical transmission via mothers and fathers. This is the first study investigating bacteria transmission in N. vectensis, and among few on marine spawners that do not brood larvae. Our results shed light on the consistent yet distinct maternal and paternal transfer of bacterial symbionts along the different life stages and generations of an aquatic invertebrate.