Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing.

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 genomics uncover contrasting scenarios of genetic diversity in a widespread marine invader.

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.

Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis.

Hox genes play crucial roles in establishing regional identity along the anterior-posterior axis in bilaterian animals, and have been implicated in generating morphological diversity throughout evolution. Here we report the identification, expression, and initial genomic characterization of the complete set of Hox genes from the amphipod crustacean Parhyale hawaiensis. Parhyale is an emerging model system that is amenable to experimental manipulations and evolutionary comparisons among the arthropods. Our analyses indicate that the Parhyale genome contains a single copy of each canonical Hox gene with the exception of fushi tarazu, and preliminary mapping suggests that at least some of these genes are clustered together in the genome. With few exceptions, Parhyale Hox genes exhibit both temporal and spatial colinearity, and expression boundaries correlate with morphological differences between segments and their associated appendages. This work represents the most comprehensive analysis of Hox gene expression in a crustacean to date, and provides a foundation for functional studies aimed at elucidating the role of Hox genes in arthropod development and evolution.

The maternal-zygotic transition and zygotic activation of the Mnemiopsis leidyi genome occurs within the first three cleavage cycles.

The maternal-zygotic transition (MZT) describes the developmental reprogramming of gene expression marked by the degradation of maternally supplied gene products and activation of the zygotic genome. While the timing and duration of the MZT vary among taxa, little is known about early-stage transcriptional dynamics in the non-bilaterian phylum Ctenophora. We sought to better understand the extent of maternal mRNA loading and subsequent differential transcript abundance during the earliest stages of development by performing comprehensive RNA-sequencing-based analyses of mRNA abundance in single- and eight-cell stage embryos in the lobate ctenophore Mnemiopsis leidyi. We found 1,908 contigs with significant differential abundance between single- and eight-cell stages, of which 1,208 contigs were more abundant at the single-cell stage and 700 contigs were more abundant at the eight-cell stage. Of the differentially abundant contigs, 267 were exclusively present in the eight-cell samples, providing strong evidence that both the MZT and zygotic genome activation (ZGA) have commenced by the eight-cell stage. Many highly abundant transcripts encode genes involved in molecular mechanisms critical to the MZT, such as maternal transcript degradation, serine/threonine kinase activity, and chromatin remodeling. Our results suggest that chromosomal restructuring, which is critical to ZGA and the initiation of transcriptional regulation necessary for normal development, begins by the third cleavage within 1.5 hr post-fertilization in M. leidyi.

Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi.

We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective.

Broad phylogenomic sampling improves resolution of the animal tree of life.

Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.

The Global Invertebrate Genomics Alliance (GIGA): developing community resources to study diverse invertebrate genomes.

Over 95% of all metazoan (animal) species comprise the "invertebrates," but very few genomes from these organisms have been sequenced. We have, therefore, formed a "Global Invertebrate Genomics Alliance" (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture.

Extracellular DNA traps in a ctenophore demonstrate immune cell behaviors in a non-bilaterian.

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 ctenophore Mnemiopsis leidyi and the oyster Crassostrea 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 both Mnemiopsis and Crassostrea immune 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.

Molecular phylogenetic evidence for the reorganization of the Hyperiid amphipods, a diverse group of pelagic crustaceans.

Within the crustaceans, the Amphipoda rank as one of the most speciose extant orders. Amphipods have successfully invaded and become major constituents of a variety of ecosystems. The hyperiid amphipods are classically defined as an exclusively pelagic group broadly inhabiting oceanic midwater environments and often having close associations with gelatinous zooplankton. As with other amphipod groups they have largely been classified based on appendage structures, however evidence suggests that at least some of these characters are the product of convergent evolution. Here we present the first multi-locus molecular phylogenetic assessment of relationships among the hyperiid amphipods. We sampled 51 species belonging to 16 of the 23 recognized hyperiidian families for three nuclear loci (18S, 28S, and H3) and mitochondrial COI. We performed both Bayesian Inference and Maximum Likelihood analyses of concatenated sequences. In addition, we also explored the utility of species-tree methods for reconstructing deep evolutionary histories using the Minimize Deep Coalescence (MDC) approach. Our results are compared with previous molecular analyses and traditional systematic groupings. We discuss these results within the context of adaptations correlated with the pelagic life history of hyperiid amphipods. Within the infraorder Physocephalata (Bowman and Gruner, 1973) we inferred support for three reciprocally monophyletic clades; the Platysceloidea, Vibilioidea, and Phronimoidea. Our results also place the enigmatic Cystisomatidae and Paraphronimidae at the base of the infraorder Physosomata (Bowman and Gruner, 1973) suggesting that Physosomata as traditionally recognized is paraphyletic. Based on our multilocus phylogeny, major rearrangements to existing taxonomic groupings of hyperiid amphipods are warranted.

Another new species of Glossocephalus (Crustacea: Amphipoda: Hyperiidea: Oxycephalidae) from the Monterey Bay region, California, USA.

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).

Phylogenetic and Protein Structure Analyses Provide Insight into the Evolution and Diversification of the CD36 Domain "Apex" among Scavenger Receptor Class B Proteins across Eukarya.

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.

MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi.

BACKGROUND: MicroRNAs play a vital role in the regulation of gene expression and have been identified in every animal with a sequenced genome examined thus far, except for the placozoan Trichoplax. The genomic repertoires of metazoan microRNAs have become increasingly endorsed as phylogenetic characters and drivers of biological complexity. RESULTS: In this study, we report the first investigation of microRNAs in a species from the phylum Ctenophora. We use short RNA sequencing and the assembled genome of the lobate ctenophore Mnemiopsis leidyi to show that this species appears to lack any recognizable microRNAs, as well as the nuclear proteins Drosha and Pasha, which are critical to canonical microRNA biogenesis. This finding represents the first reported case of a metazoan lacking a Drosha protein. CONCLUSIONS: Recent phylogenomic analyses suggest that Mnemiopsis may be the earliest branching metazoan lineage. If this is true, then the origins of canonical microRNA biogenesis and microRNA-mediated gene regulation may postdate the last common metazoan ancestor. Alternatively, canonical microRNA functionality may have been lost independently in the lineages leading to both Mnemiopsis and the placozoan Trichoplax, suggesting that microRNA functionality was not critical until much later in metazoan evolution.

Isolation and Maintenance of In Vitro Cell Cultures from the Ctenophore Mnemiopsis leidyi.

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.

De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis.

BACKGROUND: Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod Daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod Parhyale hawaiensis. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models. RESULTS: To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of P. hawaiensis. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid Acyrthosiphon pisum. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against nr (E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to D. pulex sequences but not to sequences of any other animal. Annotation of several hundred genes revealed P. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways. CONCLUSIONS: The amphipod P. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as D. pulex and P. hawaiensis, as well as the need for development of further substantial crustacean genomic resources.

Neuroanatomy of sea spiders implies an appendicular origin of the protocerebral segment.

Independent specialization of arthropod body segments has led to more than a century of debate on the homology of morphologically diverse segments, each defined by a lateral appendage and a ganglion of the central nervous system. The plesiomorphic composition of the arthropod head remains enigmatic because variation in segments and corresponding appendages is extreme. Within extant arthropod classes (Chelicerata, Myriapoda, Crustacea and Hexapoda--including the insects), correspondences between the appendage-bearing second (deutocerebral) and third (tritocerebral) cephalic neuromeres have been recently resolved on the basis of immunohistochemistry and Hox gene expression patterns. However, no appendage targets the first ganglion, the protocerebrum, and the corresponding segmental identity of this anterior region remains unclear. Reconstructions of stem-group arthropods indicate that the anteriormost region originally might have borne an ocular apparatus and a frontal appendage innervated by the protocerebrum. However, no study of the central nervous system in extant arthropods has been able to corroborate this idea directly, although recent analyses of cephalic gene expression patterns in insects suggest a segmental status for the protocerebral region. Here we investigate the developmental neuroanatomy of a putative basal arthropod, the pycnogonid sea spider, with immunohistochemical techniques. We show that the first pair of appendages, the chelifores, are innervated at an anterior position on the protocerebrum. This is the first true appendage shown to be innervated by the protocerebrum, and thus pycnogonid chelifores are not positionally homologous to appendages of extant arthropods but might, in fact, be homologous to the 'great appendages' of certain Cambrian stem-group arthropods.

Base-substitution mutation rate across the nuclear genome of Alpheus snapping shrimp and the timing of isolation by the Isthmus of Panama.

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.

Functional Characterization of Hexacorallia Phagocytic Cells.

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

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Evidence for de novo Biosynthesis of the Luminous Substrate Coelenterazine in Ctenophores.

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.

Conservation of arthropod midline netrin accumulation revealed with a cross-reactive antibody provides evidence for midline cell homology.

Although many similarities in arthropod CNS development exist, differences in axonogenesis and the formation of midline cells, which regulate axon growth, have been observed. For example, axon growth patterns in the ventral nerve cord of Artemia franciscana differ from that of Drosophila melanogaster. Despite such differences, conserved molecular marker expression at the midline of several arthropod species indicates that midline cells may be homologous in distantly related arthropods. However, data from additional species are needed to test this hypothesis. In this investigation, nerve cord formation and the putative homology of midline cells were examined in distantly related arthropods, including: long- and short-germ insects (D. melanogaster, Aedes aeygypti, and Tribolium castaneum), branchiopod crustaceans (A. franciscana and Triops longicauditus), and malacostracan crustaceans (Porcellio laevis and Parhyale hawaiensis). These comparative analyses were aided by a cross-reactive antibody generated against the Netrin (Net) protein, a midline cell marker and regulator of axonogenesis. The mechanism of nerve cord formation observed in Artemia is found in Triops, another branchiopod, but is not found in the other arthropods examined. Despite divergent mechanisms of midline cell formation and nerve cord development, Net accumulation is detected in a well-conserved subset of midline cells in branchiopod crustaceans, malacostracan crustaceans, and insects. Notably, the Net accumulation pattern is also conserved at the midline of the amphipod P. hawaiensis, which undergoes split germ-band development. Conserved Net accumulation patterns indicate that arthropod midline cells are homologous, and that Nets function to regulate commissure formation during CNS development of Tetraconata.