Finding food: how generalist predators use contact-chemosensory information to guide prey preferences.

Understanding the processes that guide carnivores in finding and selecting prey is a fundamental, unresolved challenge in sensory biology. To our knowledge, no published work has yet revealed the complete structural identities of compounds that cue preferences by generalist predators for different prey species. With this research imperative in mind, we determined the chemistry driving consumer preferences for live, intact, prey. The present study used two generalist predatory species (sea stars, Pisaster ochraceus; whelks, Acanthinucella spirata), along with two foundation prey species (mussels, Mytilus californianus; barnacles, Balanus glandula), inhabiting rocky, wave-swept shores. Each prey species is known to secrete either a 29.6 kDa (named "KEYSTONEin") or a 199.6 kDa (named "MULTIFUNCin") glycoprotein as a contact-chemical cue. Here, experimental manipulations utilized faux prey consisting of cleaned barnacle or mussel shells infused with KEYSTONEin, MULTIFUNCin, or seawater (control) gels. Whelks exhibited a strong penchant for MULTIFUNCin over KEYSTONEin, irrespective of shell type. In contrast, sea stars generally preferred KEYSTONEin over MULTIFUNCin, but this preference shifted depending on the experimental context in which they encountered physical (shell) and chemical (glycoprotein) stimuli. This study ultimately demonstrates clear and contrasting chemical preferences between sea stars and whelks. It highlights the importance of experimental setting in determining chemical preferences. Finally, it shows that prey preferences by these predators hinge only on one or two contact-protein cues, without the need for quality coding via fluid-borne compounds, low-molecular-weight substances, or mixture blends.

Sulfated Polyhydroxysteroid Glycosides from the Sea of Okhotsk Starfish Henricia leviuscula spiculifera and Potential Mechanisms for Their Observed Anti-Cancer Activity against Several Types of Human Cancer Cells.

Three new monosulfated polyhydroxysteroid glycosides, spiculiferosides A (1), B (2), and C (3), along with new related unsulfated monoglycoside, spiculiferoside D (4), were isolated from an ethanolic extract of the starfish Henricia leviuscula spiculifera collected in the Sea of Okhotsk. Compounds 1-3 contain two carbohydrate moieties, one of which is attached to C-3 of the steroid tetracyclic core, whereas another is located at C-24 of the side chain of aglycon. Two glycosides (2, 3) are biosides, and one glycoside (1), unlike them, includes three monosaccharide residues. Such type triosides are a rare group of polar steroids of sea stars. In addition, the 5-substituted 3-OSO3-α-L-Araf unit was found in steroid glycosides from starfish for the first time. Cell viability analysis showed that 1-3 (at concentrations up to 100 µM) had negligible cytotoxicity against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cells. These compounds significantly inhibited proliferation and colony formation in HCT 116 cells at non-toxic concentrations, with compound 3 having the greatest effect. Compound 3 exerted anti-proliferative effects on HCT 116 cells through the induction of dose-dependent cell cycle arrest at the G2/M phase, regulation of expression of cell cycle proteins CDK2, CDK4, cyclin D1, p21, and inhibition of phosphorylation of protein kinases c-Raf, MEK1/2, ERK1/2 of the MAPK/ERK1/2 pathway.

Complete Mitochondrial Genomes and Phylogenetic Analysis of Genus Henricia (Asteroidea: Spinulosida: Echinasteridae).

The genus Henricia is known to have intraspecific morphological variations, making species identification difficult. Therefore, molecular phylogeny analysis based on genetic characteristics is valuable for species identification. We present complete mitochondrial genomic sequences of Henricia longispina aleutica, H. reniossa, and H. sanguinolenta for the first time in this study. This study will make a significant contribution to our understanding of Henricia species and its relationships within the class Asteroidea. Lengths of mitochondrial genomes of the three species are 16,217, 16,223, and 16,194 bp, respectively, with a circular form. These genomes contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a D-loop. The gene order and direction aligned with other asteroid species. Phylogenetic relationship analysis showed that our Henricia species were in a monophyletic clade with other Henricia species and in a large clade with species (Echinaster brasiliensis) from the same family. These findings provide valuable insight into understanding the phylogenetic relationships of species in the genus Henricia.

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes, adopting a shape named 'scutoid' that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here, we use live imaging of the sea star embryo coupled with deep learning-based segmentation to dissect the relative contributions of cell density, tissue compaction and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing immediately after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

The complete mitochondrial genome of Poraniopsis inflata (Asteroidea: Valvatida: Poraniidae) from Dokdo Island, Korea.

This study presents the complete mitochondrial genome sequence of Poraniopsis inflata, providing valuable information on its genetic and taxonomic studies. Through next-generation sequencing, we successfully obtained the complete mitogenome of P. inflata, spanning a length of 16,322 bp. This genome structure encompasses 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The phylogenetic analysis, based on a dataset of 13 PCG sequences, illuminated the phylogenetic relationships of P. inflata with other species of class Asteroidea and a species of echinoderm classes. The maximum likelihood phylogenetic tree showed that P. inflata closely clustered with Linckia laevigata. By revealing its mitochondrial genome and positioning it within the Asteroidea lineage, this study provides insights into the phylogenetic context of P. inflata.

A reference genome for ecological restoration of the sunflower sea star, Pycnopodia helianthoides.

Wildlife diseases, such as the sea star wasting (SSW) epizootic that outbroke in the mid-2010s, appear to be associated with acute and/or chronic abiotic environmental change; dissociating the effects of different drivers can be difficult. The sunflower sea star, Pycnopodia helianthoides, was the species most severely impacted during the SSW outbreak, which overlapped with periods of anomalous atmospheric and oceanographic conditions, and there is not yet a consensus on the cause(s). Genomic data may reveal underlying molecular signatures that implicate a subset of factors and, thus, clarify past events while also setting the scene for effective restoration efforts. To advance this goal, we used Pacific Biosciences HiFi long sequencing reads and Dovetail Omni-C proximity reads to generate a highly contiguous genome assembly that was then annotated using RNA-seq-informed gene prediction. The genome assembly is 484 Mb long, with contig N50 of 1.9 Mb, scaffold N50 of 21.8 Mb, BUSCO completeness score of 96.1%, and 22 major scaffolds consistent with prior evidence that sea star genomes comprise 22 autosomes. These statistics generally fall between those of other recently assembled chromosome-scale assemblies for two species in the distantly related asteroid genus Pisaster. These novel genomic resources for P. helianthoides will underwrite population genomic, comparative genomic, and phylogenomic analyses-as well as their integration across scales-of SSW and environmental stressors.

Tracing the history of cell types.

A study of sea urchin and sea star larvae paves the way for understanding how cell types evolve and give rise to novel morphologies.

Sea stars resist wasting through active immune and collagen systems.

Epidemics are becoming more common and severe, however, pinpointing the causes can be challenging, particularly in marine environments. The cause of sea star wasting (SSW) disease, the ongoing, largest known panzootic of marine wildlife, is unresolved. Here, we measured gene expression longitudinally of 24 adult Pisaster ochraceus sea stars, collected from a recovered site, as they remained asymptomatic (8 individuals) or naturally progressed through SSW (16 individuals) in individual aquaria. Immune, tissue integrity and pro-collagen genes were more highly expressed in asymptomatic relative to wasting individuals, while hypoxia-inducible factor 1-α and RNA processing genes were more highly expressed in wasting relative to asymptomatic individuals. Integrating microbiome data from the same tissue samples, we identified genes and microbes whose abundance/growth was associated with disease status. Importantly, sea stars that remained visibly healthy showed that laboratory conditions had little effect on microbiome composition. Lastly, considering genotypes at 98 145 single-nucleotide polymorphism, we found no variants associated with final health status. These findings suggest that animals exposed to the cause(s) of SSW remain asymptomatic with an active immune response and sustained control of their collagen system while animals that succumb to wasting show evidence of responding to hypoxia and dysregulation of RNA processing systems.

Gecko adhesion based sea star crawler robot.

Over the years, efforts in bioinspired soft robotics have led to mobile systems that emulate features of natural animal locomotion. This includes combining mechanisms from multiple organisms to further improve movement. In this work, we seek to improve locomotion in soft, amphibious robots by combining two independent mechanisms: sea star locomotion gait and gecko adhesion. Specifically, we present a sea star-inspired robot with a gecko-inspired adhesive surface that is able to crawl on a variety of surfaces. It is composed of soft and stretchable elastomer and has five limbs that are powered with pneumatic actuation. The gecko-inspired adhesion provides additional grip on wet and dry surfaces, thus enabling the robot to climb on 25° slopes and hold on statically to 51° slopes.

Coelomic fluid of asteroid echinoderms: Current knowledge and future perspectives on its utility for disease and mortality investigations.

Coelomic fluid surrounds the internal organs of asteroid echinoderms (asteroids, otherwise known as sea stars or starfish) and plays an essential role in the immune system, as well as in the transport of respiratory gases, nutrients, waste products, and reproductive mediators. Due to its importance in physiology and accessibility for nonlethal diagnostic sampling, coelomic fluid of asteroids provides an excellent sample matrix for health evaluations and can be particularly useful in disease and mortality investigations. This is especially important in light of recent increases in the number of affected individuals and species, larger geographic scope, and increased observed frequency of sea star wasting events compared with historic accounts of wasting. This review summarizes the current knowledge about coelomocytes, the effector cell of the asteroid immune system; coelomic fluid electrolytes, osmolality, acid-base status and respiratory gases, and microbiota; and genomic, transcriptomic, and proteomic investigations of coelomic fluid. The utility of coelomic fluid analysis for assessing stressor responses, diseases, and mortality investigations is considered with knowledge gaps and future directions identified. This complex body fluid provides an exciting opportunity to increase our understanding of this unique and ecologically important group of animals.

Intra- and Interspecific Foraging and Feeding Interactions in Three Sea Stars and a Gastropod from the Deep Sea.

Competitive interactions come in a variety of forms and may be modulated by the size and number of individuals involved, and/or the resources available. Here, intra- and interspecific competitive behaviours for food (i.e., foraging/food search and feeding/food ingestion) were experimentally characterized and quantified in four co-existing deep-sea benthic species. Three sea stars (Ceramaster granularis, Hippasteria phrygiana, and Henricia lisa) and one gastropod (Buccinum scalariforme) from the bathyal Northwest Atlantic were investigated using video trials in darkened laboratory conditions. A range of competitive or cooperative behaviours occurred, depending on species (conspecific or heterospecific), comparative body size, and the number of individuals involved. Contrary to expectations, small individuals (or smaller species) were not always outcompeted by larger individuals (or larger species) when foraging and feeding. Moreover, faster species did not always outcompete slower ones while scavenging. Overall, this study sheds new light on scavenging strategies of co-existing deep-sea benthic species in food-limited bathyal environments, based on complex behavioural inter- and intraspecific relationships.

Top predator sea stars are the benthic equivalent to polar bears of the pelagic realm.

The marine pelagic compartment spans numerous trophic levels and consists of numerous reticulate connections between species from primary producers to iconic apex predators, while the benthic compartment is perceived to be simpler in structure and comprised of only low trophic level species. Here, we challenge this paradigm by illustrating that the benthic compartment is home to a subweb of similar structure and complexity to that of the pelagic realm, including the benthic equivalent to iconic polar bears: megafaunal-predatory sea stars.

Sex-specific expression of pheromones and other signals in gravid starfish.

BACKGROUND: Many echinoderms form seasonal aggregations prior to spawning. In some fecund species, a spawning event can lead to population outbreaks with detrimental ecosystem impacts. For instance, outbreaks of crown-of-thorns starfish (COTS), a corallivore, can destroy coral reefs. Here, we examine the gene expression in gravid male and female COTS prior to spawning in the wild, to identify genome-encoded factors that may regulate aggregation and spawning. This study is informed by a previously identified exoproteome that attracts conspecifics. To capture the natural gene expression profiles, we isolated RNAs from gravid female and male COTS immediately after they were removed from the Great Barrier Reef.  RESULTS: Sexually dimorphic gene expression is present in all seven somatic tissues and organs that we surveyed and in the gonads. Approximately 40% of the exoproteome transcripts are differentially expressed between sexes. Males uniquely upregulate an additional 68 secreted factors in their testes. A suite of neuropeptides in sensory organs, coelomocytes and gonads is differentially expressed between sexes, including the relaxin-like gonad-stimulating peptide and gonadotropin-releasing hormones. Female sensory tentacles-chemosensory organs at the distal tips of the starfish arms-uniquely upregulate diverse receptors and signalling molecules, including chemosensory G-protein-coupled receptors and several neuropeptides, including kisspeptin, SALMFamide and orexin. CONCLUSIONS: Analysis of 103 tissue/organ transcriptomes from 13 wild COTS has revealed genes that are consistently differentially expressed between gravid females and males and that all tissues surveyed are sexually dimorphic at the molecular level. This finding is consistent with female and male COTS using sex-specific pheromones to regulate reproductive aggregations and synchronised spawning events. These pheromones appear to be received primarily by the sensory tentacles, which express a range of receptors and signalling molecules in a sex-specific manner. Furthermore, coelomocytes and gonads differentially express signalling and regulatory factors that control gametogenesis and spawning in other echinoderms.

Feeding Ecology of Odontaster validus under Different Environmental Conditions in the West Antarctic Peninsula.

To study how Odontaster validus can influence the spatial structure of Antarctic benthic communities and how they respond to disturbance, it is necessary to assess potential dietary shifts in different habitats. We investigated the diets of O. validus from Maxwell Bay and South Bay in the West Antarctic Peninsula. A multifaceted approach was applied including in situ observations of cardiac stomach everted contents, isotopic niche, and trophic diversity metrics. Results confirm the flexible foraging strategy of this species under markedly different environmental conditions, suggesting plasticity in resource use. The data also showed evidence of isotopic niche expansion, high δ15N values, and Nacella concinna as a common food item for individuals inhabiting a site with low seasonal sea ice (Ardley Cove), which could have significant ecological implications such as new trophic linkages within the Antarctic benthic community. These results highlight the importance of considering trophic changes of key species to their environment as multiple ecological factors can vary as a function of climatic conditions.

Single-cell RNA-sequencing analysis of early sea star development.

Echinoderms represent a broad phylum with many tractable features to test evolutionary changes and constraints. Here, we present a single-cell RNA-sequencing analysis of early development in the sea star Patiria miniata, to complement the recent analysis of two sea urchin species. We identified 20 cell states across six developmental stages from 8 hpf to mid-gastrula stage, using the analysis of 25,703 cells. The clusters were assigned cell states based on known marker gene expression and by in situ RNA hybridization. We found that early (morula, 8-14 hpf) and late (blastula-to-mid-gastrula) cell states are transcriptionally distinct. Cells surrounding the blastopore undergo rapid cell state changes that include endomesoderm diversification. Of particular import to understanding germ cell specification is that we never see Nodal pathway members within Nanos/Vasa-positive cells in the region known to give rise to the primordial germ cells (PGCs). The results from this work contrast the results of PGC specification in the sea urchin, and the dataset presented here enables deeper comparative studies in tractable developmental models for testing a variety of developmental mechanisms.

Live imaging of echinoderm embryos to illuminate evo-devo.

Echinoderm embryos have been model systems for cell and developmental biology for over 150 years, in good part because of their optical clarity. Discoveries that shaped our understanding of fertilization, cell division and cell differentiation were only possible because of the transparency of sea urchin eggs and embryos, which allowed direct observations of intracellular structures. More recently, live imaging of sea urchin embryos, coupled with fluorescence microscopy, has proven pivotal to uncovering mechanisms of epithelial to mesenchymal transition, cell migration and gastrulation. However, live imaging has mainly been performed on sea urchin embryos, while echinoderms include numerous experimentally tractable species that present interesting variation in key aspects of morphogenesis, including differences in embryo compaction and mechanisms of blastula formation. The study of such variation would allow us not only to understand how tissues are formed in echinoderms, but also to identify which changes in cell shape, cell-matrix and cell-cell contact formation are more likely to result in evolution of new embryonic shapes. Here we argue that adapting live imaging techniques to more echinoderm species will be fundamental to exploit such an evolutionary approach to the study of morphogenesis, as it will allow measuring differences in dynamic cellular behaviors - such as changes in cell shape and cell adhesion - between species. We briefly review existing methods for live imaging of echinoderm embryos and describe in detail how we adapted those methods to allow long-term live imaging of several species, namely the sea urchin Lytechinus pictus and the sea stars Patiria miniata and Patiriella regularis. We outline procedures to successfully label, mount and image early embryos for 10-16 h, from cleavage stages to early blastula. We show that data obtained with these methods allows 3D segmentation and tracking of individual cells over time, the first step to analyze how cell shape and cell contact differ among species. The methods presented here can be easily adopted by most cell and developmental biology laboratories and adapted to successfully image early embryos of additional species, therefore broadening our understanding of the evolution of morphogenesis.

Optimizing CRISPR/Cas9-based gene manipulation in echinoderms.

The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.

In the footsteps of sea stars: deciphering the catalogue of proteins involved in underwater temporary adhesion.

Sea stars adhere strongly but temporarily to underwater substrata via the secretion of a blend of proteins, forming an adhesive footprint that they leave on the surface after detachment. Their tube feet enclose a duo-gland adhesive system comprising two types of adhesive cells, contributing different layers of the footprint and de-adhesive cells. In this study, we characterized the catalogue of sea star footprint proteins (Sfps) in the species Asterias rubens to gain insights in their potential function. We identified 16 Sfps and mapped their expression to type 1 and/or type 2 adhesive cells or to de-adhesive cells by double fluorescent in situ hybridization. Based on their cellular expression pattern and their conserved functional domains, we propose that the identified Sfps serve different functions during attachment, with two Sfps coupling to the surface, six providing cohesive strength and the rest forming a binding matrix. Immunolabelling of footprints with antibodies directed against one protein of each category confirmed these roles. A de-adhesive gland cell-specific astacin-like proteinase presumably weakens the bond between the adhesive material and the tube foot surface during detachment. Overall, we provide a model for temporary adhesion in sea stars, including a comprehensive list of the proteins involved.

The first records of sea star wasting disease in Crossaster papposus in Europe.

Sea star wasting disease (SSWD) refers to a suite of gross pathological signs observed in Asteroidea species. It presents to varying degrees as abnormal posture, epidermal ulceration, arm autotomy and eversion of viscera. We report observations of SSWD in the sunstar Crossaster papposus, the first observations of its kind in Europe. While the exact cause of SSWD remains unknown, studies have proposed pathogenic and environmental-stress pathways for disease outbreaks. Although the present observations do not support a precise aetiology, the presence of SSWD in a keystone predator may have wide reaching ecological and management implications.