The genome of the colonial hydroid Hydractinia reveals that their stem cells use a toolkit of evolutionarily shared genes with all animals.

Hydractinia is a colonial marine hydroid that shows remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, Hydractinia symbiolongicarpus and Hydractinia echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell-type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from nonself.

The genome of the colonial hydroid Hydractinia reveals their stem cells utilize a toolkit of evolutionarily shared genes with all animals.

Hydractinia is a colonial marine hydroid that exhibits remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, H. symbiolongicarpus and H. echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from non-self.

Cnidofest 2022: hot topics in cnidarian research.

The second annual Cnidarian Model Systems Meeting, aka "Cnidofest", took place in Davis, California from 7 to 10th of September, 2022. The meeting brought together scientists using cnidarians to study molecular and cellular biology, development and regeneration, evo-devo, neurobiology, symbiosis, physiology, and comparative genomics. The diversity of topics and species represented in presentations highlighted the importance and versatility of cnidarians in addressing a wide variety of biological questions. In keeping with the spirit of the first meeting (and its predecessor, Hydroidfest), almost 75% of oral presentations were given by early career researchers (i.e., graduate students and postdocs). In this review, we present research highlights from the meeting.

Coevolution of the Tlx homeobox gene with medusa development (Cnidaria: Medusozoa).

Cnidarians display a wide diversity of life cycles. Among the main cnidarian clades, only Medusozoa possesses a swimming life cycle stage called the medusa, alternating with a benthic polyp stage. The medusa stage was repeatedly lost during medusozoan evolution, notably in the most diverse medusozoan class, Hydrozoa. Here, we show that the presence of the homeobox gene Tlx in Cnidaria is correlated with the presence of the medusa stage, the gene having been lost in clades that ancestrally lack a medusa (anthozoans, endocnidozoans) and in medusozoans that secondarily lost the medusa stage. Our characterization of Tlx expression indicate an upregulation of Tlx during medusa development in three distantly related medusozoans, and spatially restricted expression patterns in developing medusae in two distantly related species, the hydrozoan Podocoryna carnea and the scyphozoan Pelagia noctiluca. These results suggest that Tlx plays a key role in medusa development and that the loss of this gene is likely linked to the repeated loss of the medusa life cycle stage in the evolution of Hydrozoa.

Chromosome-level genome assembly of Hydractinia symbiolongicarpus.

Hydractinia symbiolongicarpus is a pioneering model organism for stem cell biology, being one of only a few animals with adult pluripotent stem cells (known as i-cells). However, the unavailability of a chromosome-level genome assembly has hindered a comprehensive understanding of global gene regulatory mechanisms underlying the function and evolution of i-cells. Here, we report the first chromosome-level genome assembly of H. symbiolongicarpus (HSymV2.0) using PacBio HiFi long-read sequencing and Hi-C scaffolding. The final assembly is 483 Mb in total length with 15 chromosomes representing 99.8% of the assembly. Repetitive sequences were found to account for 296 Mb (61%) of the total genome; we provide evidence for at least two periods of repeat expansion in the past. A total of 25,825 protein-coding genes were predicted in this assembly, which include 93.1% of the metazoan Benchmarking Universal Single-Copy Orthologs (BUSCO) gene set. 92.8% (23,971 genes) of the predicted proteins were functionally annotated. The H. symbiolongicarpus genome showed a high degree of macrosynteny conservation with the Hydra vulgaris genome. This chromosome-level genome assembly of H. symbiolongicarpus will be an invaluable resource for the research community that enhances broad biological studies on this unique model organism.

Localization of Multiple Jellyfish Toxins Shows Specificity for Functionally Distinct Polyps and Nematocyst Types in a Colonial Hydrozoan.

Hydractinia symbiolongicarpus is a colonial hydrozoan that displays a division of labor through morphologically distinct and functionally specialized polyp types. As with all cnidarians, their venoms are housed in nematocysts, which are scattered across an individual. Here, we investigate the spatial distribution of a specific protein family, jellyfish toxins, in which multiple paralogs are differentially expressed across the functionally specialized polyps. Jellyfish toxins (JFTs) are known pore-forming toxins in the venoms of medically relevant species such as box jellyfish (class Cubozoa), but their role in other medusozoan venoms is less clear. Utilizing a publicly available single-cell dataset, we confirmed that four distinct H. symbiolongicarpus JFT paralogs are expressed in nematocyst-associated clusters, supporting these as true venom components in H. symbiolongicarpus. In situ hybridization and immunohistochemistry were used to localize the expression of these JFTs across the colony. These expression patterns, in conjunction with known nematocyst type distributions, suggest that two of these JFTs, HsymJFT1c-I and HsymJFT1c-II, are localized to specific types of nematocysts. We further interpret JFT expression patterns in the context of known regions of nematogenesis and differential rates of nematocyst turnover. Overall, we show that JFT expression patterns in H. symbiolongicarpus are consistent with the subfunctionalization of JFT paralogs across a partitioned venom system within the colony, such that each JFT is expressed within a specific set of functionally distinct polyp types and, in some cases, specific nematocyst types.

The Phylogenetic Position of the Enigmatic, Polypodium hydriforme (Cnidaria, Polypodiozoa): Insights from Mitochondrial Genomes.

Polypodium hydriforme is an enigmatic parasite that belongs to the phylum Cnidaria. Its taxonomic position has been debated: whereas it was previously suggested to be part of Medusozoa, recent phylogenomic analyses based on nuclear genes support the view that P. hydriforme and Myxozoa form a clade called Endocnidozoa. Medusozoans have linear mitochondrial (mt) chromosomes, whereas myxozoans, as most metazoan species, have circular chromosomes. In this work, we determined the structure of the mt genome of P. hydriforme, using Illumina and Oxford Nanopore Technologies reads, and showed that it is circular. This suggests that P. hydriforme is not nested within Medusozoa, as this would entail linearization followed by recirculation. Instead, our results support the view that P. hydriforme is a sister clade to Myxozoa, and mt linearization in the lineage leading to medusozoans occurred after the divergence of Myxozoa + P. hydriforme. Detailed analyses of the assembled P. hydriforme mt genome show that: (1) it is encoded on a single circular chromosome with an estimated size of ∼93,000 base pairs, making it one of the largest metazoan mt genomes; (2) around 78% of the genome encompasses a noncoding region composed of several repeat types; (3) similar to Myxozoa, no mt tRNAs were identified; (4) the codon TGA is a stop codon and does not encode for tryptophan as in other cnidarians; (5) similar to myxozoan mt genomes, it is extremely fast evolving.

Venom system variation and the division of labor in the colonial hydrozoan Hydractinia symbiolongicarpus.

Cnidarians (jellyfish, hydroids, sea anemones, and corals) possess a unique method for venom production, maintenance, and deployment through a decentralized system composed of different types of venom-filled stinging structures called nematocysts. In many species, nematocyst types are distributed heterogeneously across functionally distinct tissues. This has led to a prediction that different nematocyst types contain specific venom components. The colonial hydrozoan, Hydractinia symbiolongicarpus, is an ideal system to study the functional distribution of nematocyst types and their venoms, given that they display a division of labor through functionally distinct polyps within the colony. Here, we characterized the composition and distribution of nematocysts (cnidome) in the different polyp types and show that the feeding polyp (gastrozooid) has a distinct cnidome compared to the reproductive (gonozooid) and predatory polyp (dactylozooid). We generated a nematocyst-specific reporter line to track nematocyst development (nematogenesis) in H. symbiolongicarpus, and were able to confirm that nematogenesis primarily occurs in the mid-region of the gastrozooid and throughout stolons (tubes of epithelia that connect the polyps in the colony). This reporter line enabled us to isolate a nematocyst-specific lineage of cells for de novo transcriptome assembly, annotate venom-like genes (VLGs) and determine differential expression (DE) across polyp types. We show that a majority of VLGs are upregulated in gastrozooids, consistent with it being the primary site of active nematogenesis. However, despite gastrozooids producing more nematocysts, we found a number of VLGs significantly upregulated in dactylozooids, suggesting that these VLGs may be important for prey-capture. Our transgenic Hydractinia reporter line provides an opportunity to explore the complex interplay between venom composition, nematocyst diversity, and ecological partitioning in a colonial hydrozoan that displays a division of labor.

Phylogenetic and Selection Analysis of an Expanded Family of Putatively Pore-Forming Jellyfish Toxins (Cnidaria: Medusozoa).

Many jellyfish species are known to cause a painful sting, but box jellyfish (class Cubozoa) are a well-known danger to humans due to exceptionally potent venoms. Cubozoan toxicity has been attributed to the presence and abundance of cnidarian-specific pore-forming toxins called jellyfish toxins (JFTs), which are highly hemolytic and cardiotoxic. However, JFTs have also been found in other cnidarians outside of Cubozoa, and no comprehensive analysis of their phylogenetic distribution has been conducted to date. Here, we present a thorough annotation of JFTs from 147 cnidarian transcriptomes and document 111 novel putative JFTs from over 20 species within Medusozoa. Phylogenetic analyses show that JFTs form two distinct clades, which we call JFT-1 and JFT-2. JFT-1 includes all known potent cubozoan toxins, as well as hydrozoan and scyphozoan representatives, some of which were derived from medically relevant species. JFT-2 contains primarily uncharacterized JFTs. Although our analyses detected broad purifying selection across JFTs, we found that a subset of cubozoan JFT-1 sequences are influenced by gene-wide episodic positive selection compared with homologous toxins from other taxonomic groups. This suggests that duplication followed by neofunctionalization or subfunctionalization as a potential mechanism for the highly potent venom in cubozoans. Additionally, published RNA-seq data from several medusozoan species indicate that JFTs are differentially expressed, spatially and temporally, between functionally distinct tissues. Overall, our findings suggest a complex evolutionary history of JFTs involving duplication and selection that may have led to functional diversification, including variability in toxin potency and specificity.

The evolution and development of coloniality in hydrozoans.

Hydrozoan colonies display a variety of shapes and sizes including encrusting, upright, and pelagic forms. Phylogenetic patterns reveal a complex evolutionary history of these distinct colony forms, as well as colony loss. Within a species, phenotypic variation in colonies as a response to changing environmental cues and resources has been documented. The patterns of branching of colony specific tissue, called stolons in encrusting colonies and stalks in upright colonies, are likely under the control of signaling mechanisms whose changing expression in evolution and development are responsible for the diversity of hydrozoan colony forms. Although mechanisms of polyp development have been well studied, little research has focused on colony development and patterning. In the few studies that investigated mechanisms governing colony patterning, the Wnt signaling pathway has been implicated. The diversity of colony form, evolutionary patterns, and mechanisms of colony variation in Hydrozoa are reviewed here.

Frizzled3 expression and colony development in hydractiniid hydrozoans.

Hydractiniid hydrozoan colonies are comprised of individual polyps connected by tube-like stolons or a sheet-like mat. Mat and stolons function to integrate the colony through continuous epithelia and shared gastrovascular cavity. Although mechanisms of hydrozoan polyp development have been well studied, little is known about the signaling processes governing the patterning of colonies. Here we investigate the Wnt receptor family Frizzled. Phylogenetic analysis reveals that hydrozoans possess four Frizzled orthologs. We find that one of these genes, Frizzled3, shows a spatially restricted expression pattern in colony-specific tissue in two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, in a manner that corresponds to their distinct colony forms (stolonal mat in Hydractinia and free stolons in Podocoryna). Interestingly, Frizzled3 was lost in the genome of Hydra, which is a solitary polyp and thus lacks colony-specific tissue. Current evidence suggests that the Wnt signaling pathway plays a key role in the evolution of colony diversity and colony loss in Hydrozoa.

A cnidarian parasite of salmon (Myxozoa: Henneguya) lacks a mitochondrial genome.

Although aerobic respiration is a hallmark of eukaryotes, a few unicellular lineages, growing in hypoxic environments, have secondarily lost this ability. In the absence of oxygen, the mitochondria of these organisms have lost all or parts of their genomes and evolved into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Cnidaria, the myxozoan Henneguya salminicola, has no mitochondrial genome, and thus has lost the ability to perform aerobic cellular respiration. This indicates that these core eukaryotic features are not ubiquitous among animals. Our analyses suggest that H. salminicola lost not only its mitochondrial genome but also nearly all nuclear genes involved in transcription and replication of the mitochondrial genome. In contrast, we identified many genes that encode proteins involved in other mitochondrial pathways and determined that genes involved in aerobic respiration or mitochondrial DNA replication were either absent or present only as pseudogenes. As a control, we used the same sequencing and annotation methods to show that a closely related myxozoan, Myxobolus squamalis, has a mitochondrial genome. The molecular results are supported by fluorescence micrographs, which show the presence of mitochondrial DNA in M. squamalis, but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic environment is not unique to single-celled eukaryotes, but has also evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transition from an aerobic to an exclusive anaerobic metabolism.

Cassiosomes are stinging-cell structures in the mucus of the upside-down jellyfish Cassiopea xamachana.

Snorkelers in mangrove forest waters inhabited by the upside-down jellyfish Cassiopea xamachana report discomfort due to a sensation known as stinging water, the cause of which is unknown. Using a combination of histology, microscopy, microfluidics, videography, molecular biology, and mass spectrometry-based proteomics, we describe C. xamachana stinging-cell structures that we term cassiosomes. These structures are released within C. xamachana mucus and are capable of killing prey. Cassiosomes consist of an outer epithelial layer mainly composed of nematocytes surrounding a core filled by endosymbiotic dinoflagellates hosted within amoebocytes and presumptive mesoglea. Furthermore, we report cassiosome structures in four additional jellyfish species in the same taxonomic group as C. xamachana (Class Scyphozoa; Order Rhizostomeae), categorized as either motile (ciliated) or nonmotile types. This inaugural study provides a qualitative assessment of the stinging contents of C. xamachana mucus and implicates mucus containing cassiosomes and free intact nematocytes as the cause of stinging water.

Expansion of a single transposable element family is associated with genome-size increase and radiation in the genus Hydra.

Transposable elements are one of the major contributors to genome-size differences in metazoans. Despite this, relatively little is known about the evolutionary patterns of element expansions and the element families involved. Here we report a broad genomic sampling within the genus Hydra, a freshwater cnidarian at the focal point of diverse research in regeneration, symbiosis, biogeography, and aging. We find that the genome of Hydra is the result of an expansion event involving long interspersed nuclear elements and in particular a single family of the chicken repeat 1 (CR1) class. This expansion is unique to a subgroup of the genus Hydra, the brown hydras, and is absent in the green hydra, which has a repeat landscape similar to that of other cnidarians. These features of the genome make Hydra attractive for studies of transposon-driven genome expansions and speciation.

A new mitochondrial gene order in the banded cusk-eel Raneya brasiliensis (Actinopterygii, Ophidiiformes).

The complete mitochondrial genome of the banded cusk-eel, Raneya brasilensis (Kaup, 1856), was obtained using next-generation sequencing approaches. The genome sequence was 16,881 bp and exhibited a novel gene order for a vertebrate. Specifically, the WANCY and the nd6 - D-loop regions were re-ordered, supporting the hypothesis that these two regions are hotspots for gene rearrangements in Actinopterygii. Phylogenetic reconstructions confirmed that R. brasiliensis is nested within Ophidiiformes. Mitochondrial genomes are required from additional ophidiins to determine whether the gene rearrangements that we observed are specific to the genus Raneya or to the subfamily Ophidiinae.

Nonclonal coloniality: Genetically chimeric colonies through fusion of sexually produced polyps in the hydrozoan Ectopleura larynx.

Hydrozoans typically develop colonies through asexual budding of polyps. Although colonies of Ectopleura are similar to other hydrozoans in that they consist of multiple polyps physically connected through continuous epithelia and shared gastrovascular cavity, Ectopleura larynx does not asexually bud polyps indeterminately. Instead, after an initial phase of limited budding in a young colony, E. larynx achieves its large colony size through the aggregation and fusion of sexually (nonclonally) produced polyps. The apparent chimerism within a physiologically integrated colony presents a potential source of conflict between distinct genetic lineages, which may vary in their ability to access the germline. To determine the extent to which the potential for genetic conflict exists, we characterized the types of genetic relationships between polyps within colonies, using a RAD-Seq approach. Our results indicate that E. larynx colonies are indeed comprised of polyps that are clones and sexually reproduced siblings and offspring, consistent with their life history. In addition, we found that colonies also contain polyps that are genetically unrelated, and that estimates of genome-wide relatedness suggests a potential for conflict within a colony. Taken together, our data suggest that there are distinct categories of relationships in colonies of E. larynx, likely achieved through a range of processes including budding, regeneration, and fusion of progeny and unrelated polyps, with the possibility for a genetic conflict resolution mechanism. Together these processes contribute to the reevolution of the ecologically important trait of coloniality in E. larynx.

A genome wide survey reveals multiple nematocyst-specific genes in Myxozoa.

BACKGROUND: Myxozoa represents a diverse group of microscopic endoparasites whose life cycle involves two hosts: a vertebrate (usually a fish) and an invertebrate (usually an annelid worm). Despite lacking nearly all distinguishing animal characteristics, given that each life cycle stage consists of no more than a few cells, molecular phylogenetic studies have revealed that myxozoans belong to the phylum Cnidaria, which includes corals, sea anemones, and jellyfish. Myxozoa, however, do possess a polar capsule; an organelle that is homologous to the stinging structure unique to Cnidaria: the nematocyst. Previous studies have identified in Myxozoa a number of protein-coding genes that are specific to nematocytes (the cells producing nematocysts) and thus restricted to Cnidaria. Determining which other genes are also homologous with the myxozoan polar capsule genes could provide insight into both the conservation and changes that occurred during nematocyst evolution in the transition to endoparasitism. RESULTS: Previous studies have examined the phylogeny of two cnidarian-restricted gene families: minicollagens and nematogalectins. Here we identify and characterize seven additional cnidarian-restricted genes in myxozoan genomes using a phylogenetic approach. Four of the seven had never previously been identified as cnidarian-specific and none have been studied in a phylogenetic context. A majority of the proteins appear to be involved in the structure of the nematocyst capsule and tubule. No venom proteins were identified among the cnidarian-restricted genes shared by myxozoans. CONCLUSIONS: Given the highly divergent forms that comprise Cnidaria, obtaining insight into the processes underlying their ancient diversification remains challenging. In their evolutionary transition to microscopic endoparasites, myxozoans lost nearly all traces of their cnidarian ancestry, with the one prominent exception being their nematocysts (or polar capsules). Thus nematocysts, and the genes that code for their structure, serve as rich sources of information to support the cnidarian origin of Myxozoa.

A new transcriptome and transcriptome profiling of adult and larval tissue in the box jellyfish Alatina alata: an emerging model for studying venom, vision and sex.

BACKGROUND: Cubozoans (box jellyfish) are cnidarians that have evolved a number of distinguishing features. Many cubozoans have a particularly potent sting, effected by stinging structures called nematocysts; cubozoans have well-developed light sensation, possessing both image-forming lens eyes and light-sensitive eye spots; and some cubozoans have complex mating behaviors, including aggregations, copulation and internal fertilization. The cubozoan Alatina alata is emerging as a cnidarian model because it forms predictable monthly nearshore breeding aggregations in tropical to subtropical waters worldwide, making both adult and larval material reliably accessible. To develop resources for A. alata, this study generated a functionally annotated transcriptome of adult and larval tissue, applying preliminary differential expression analyses to identify candidate genes involved in nematogenesis and venom production, vision and extraocular sensory perception, and sexual reproduction, which for brevity we refer to as "venom", "vision" and "sex". RESULTS: We assembled a transcriptome de novo from RNA-Seq data pooled from multiple body parts (gastric cirri, ovaries, tentacle (with pedalium base) and rhopalium) of an adult female A. alata medusa and larval planulae. Our transcriptome comprises ~32 K transcripts, after filtering, and provides a basis for analyzing patterns of gene expression in adult and larval box jellyfish tissues. Furthermore, we annotated a large set of candidate genes putatively involved in venom, vision and sex, providing an initial molecular characterization of these complex features in cubozoans. Expression profiles and gene tree reconstruction provided a number of preliminary insights into the putative sites of nematogenesis and venom production, regions of phototransduction activity and fertilization dynamics in A. alata. CONCLUSIONS: Our Alatina alata transcriptome significantly adds to the genomic resources for this emerging cubozoan model. This study provides the first annotated transcriptome from multiple tissues of a cubozoan focusing on both the adult and larvae. Our approach of using multiple body parts and life stages to generate this transcriptome effectively identified a broad range of candidate genes for the further study of coordinated processes associated with venom, vision and sex. This new genomic resource and the candidate gene dataset are valuable for further investigating the evolution of distinctive features of cubozoans, and of cnidarians more broadly.