SAIBR: a simple, platform-independent method for spectral autofluorescence correction.

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.

Somatostatin-type and allatostatin-C-type neuropeptides are paralogous and have opposing myoregulatory roles in an echinoderm.

Somatostatin (SS) and allatostatin-C (ASTC) are inhibitory neuropeptides in chordates and protostomes, respectively, which hitherto were identified as orthologs. However, echinoderms have two SS/ASTC-type neuropeptides (SS1 and SS2), and here, our analysis of sequence data indicates that SS1 is an ortholog of ASTC and SS2 is an ortholog of SS. The occurrence of both SS-type and ASTC-type neuropeptides in echinoderms provides a unique context to compare their physiological roles. Investigation of the expression and actions of the ASTC-type neuropeptide ArSS1 in the starfish Asterias rubens revealed that it causes muscle contraction (myoexcitation), contrasting with myoinhibitory effects of the SS-type neuropeptide ArSS2. Our findings suggest that SS-type and ASTC-type neuropeptides are paralogous and originated by gene duplication in a common ancestor of the Bilateria, with only one type being retained in chordates (SS) and protostomes (ASTC) but with both types being retained in echinoderms. Loss of ASTC-type and SS-type neuropeptides in chordates and protostomes, respectively, may have been due to their functional redundancy as inhibitory regulators of physiological processes. Conversely, the retention of both neuropeptide types in echinoderms may be a consequence of the evolution of a myoexcitatory role for ASTC-type neuropeptides mediated by as yet unknown signaling mechanisms.

A single-cell RNA-seq analysis of early larval cell-types of the starfish, Patiria pectinifera: Insights into evolution of the chordate body plan.

Ambulacrarians (echinoderms and hemichordates) are a sister group to chordates; thus, their larval cell-types may provide clues about evolution of chordate body plans. Although most genic information accumulated to date pertains to sea urchin embryogenesis, starfish embryogenesis represents a more ancestral mode than that of sea urchins. We performed single-cell RNA-seq analysis of cell-types from gastrulae and bipinnarial larvae of the starfish, Patiria pectinifera, and categorized them into 22 clusters, each of which is composed of cells with specific, shared profiles of development-relevant gene expression. Oral and aboral ectoderm, apical plate, hindgut or archenteron, midgut or intestine, pharynx, endomesoderm, stomodeum, and mesenchyme of the gastrulae, and neurons, ciliary bands, enterocoel and muscle of larvae were characterized by expression profiles of at least two relevant transcription factor genes and signaling molecular genes. Expression of Hox2, Hox7, Hox9/10, and Hox11/13b was detected in cells of clusters that form the larval enterocoel. By comparing homologous gene expression profiles in chordate embryos, we discuss and propose how the chordate body plan evolved from a deuterostome ancestor, from which the echinoderm body plan also evolved.

A description of the bat star nervous system throughout larval ontogeny.

Larvae represent a distinct life history stage in which animal morphology and behavior contrast strongly to adult organisms. This life history stage is a ubiquitous aspect of animal life cycles, particularly in the marine environment. In many species, the structure and function of the nervous system differ significantly between metamorphosed juveniles and larvae. However, the distribution and diversity of neural cell types in larval nervous systems remains incompletely known. Here, the expression of neurotransmitter and neuropeptide synthesis and transport genes in the bat star Patiria miniata is examined throughout larval development. This characterization of nervous system structure reveals three main neural regions with distinct but overlapping territories. These regions include a densely innervated anterior region, an enteric neural plexus, and neurons associated with the ciliary band. In the ciliary band, cholinergic cells are pervasive while dopaminergic, noradrenergic, and GABAergic cells show regional differences in their localization patterns. Furthermore, the distribution of some neural subtypes changes throughout larval development, suggesting that changes in nervous system structure align with shifting ecological priorities during different larval stages, before the development of the adult nervous system. While past work has described aspects of P. miniata larval nervous system structure, largely focusing on early developmental timepoints, this work provides a comprehensive description of neural cell type localization throughout the extensive larval period.

Rare Ophiuroid-Type Steroid 3ß,21-, 3ß,22-, and 3α,22-Disulfates from the Slime Sea Star Pteraster marsippus and Their Colony-Inhibiting Effects against Human Breast Cancer Cells.

Two new steroid 3ß,21-disulfates (1, 2) and two new steroid 3ß,22- and 3α,22-disulfates (3, 4), along with the previously known monoamine alkaloid tryptamine (5) were found in the ethanolic extract of the Far Eastern slime sea star Pteraster marsippus. Their structures were determined on the basis of detailed analysis of one-dimensional and two-dimensional NMR, HRESIMS, and HRESIMS/MS data. Compounds 1 and 2 have a Δ22-21-sulfoxy-24-norcholestane side chain. Compounds 3 and 4 contain a Δ24(28)-22-sulfoxy-24-methylcholestane side chain, which was first discovered in the polar steroids of starfish and brittle stars. The influence of substances 1-4 on cell viability, colony formation, and growth of human breast cancer T-47D, MCF-7, and MDA-MB-231 cells was investigated. It was shown that compounds 1 and 2 possess significant colony-inhibiting activity against T-47D cells, while compounds 3 and 4 were more effective against MDA-MB-231 cells.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

A New Brominated Isocoumarin from the Marine Starfish-Associated Fungus Aspergillus sp. WXF1904.

Based on the one strain many compounds strategy, a new brominated isocoumarin, 5-bromo-6,8-dihydroxy-3,7-dimethylisocoumarin (1), along with four new natural products, methyl 3-bromo-2,4-dihydroxy-6-methylbenzoate (2), methyl 2-bromo-4,6-dihydroxybenzoate (3), (E)-3-(3-bromo-4-hydroxyphenyl) acrylic acid (4) and 4-hydroxy-3-methyl-6-phenyl-2H-pyran-2-one (5), and four known compounds, methyl orsellinate (6), 4-hydroxy-3-methyl-6-(1-methyl-1-propenyl)-2H-pyran-2-one (7), pilobolusate (8) and cis-ferulic acid (9), were isolated from the ethyl acetate extract of the fungus Aspergillus sp. WXF1904 under the condition of adding bromine salt to the production medium. The structures of the new compounds were established by analysis of NMR and MS data. Compounds (1-9) were evaluated for inhibitory activity of acetylcholinesterase and pancreatic lipase, the new compound 1, known compounds 6 and 7 displayed weak inhibitory activity against acetylcholinesterase, compounds 2, 5, 7 and 8 showed weak inhibitory activity against pancreatic lipase.

Detection of TALEN-mediated genome cleavage during the early embryonic stage of the starfish Patiria pectinifera.

BACKGROUND: Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish. RESULTS: We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization. CONCLUSION: The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results.

Marine Envenomation in Okinawa: Overview and Treatment Concept.

Okinawa prefecture is a popular tourist destination due to its beaches and reefs. The reefs host a large variety of animals, including a number of venomous species. Because of the popularity of the reefs and marine activities, people are frequently in close contact with dangerous venomous species and, thus, are exposed to potential envenomation. Commonly encountered venomous animals throughout Okinawa include the invertebrate cone snail, sea urchin, crown-of-thorns starfish, blue-ringed octopus, box jellyfish, and fire coral. The vertebrates include the stonefish, lionfish, sea snake, and moray eel. Treatment for marine envenomation can involve first aid, hot water immersion, antivenom, supportive care, regional anesthesia, and pharmaceutical administration. Information on venomous animals, their toxins, and treatment should be well understood by prehospital care providers and physicians practicing in the prefecture.

Mechanism underlying retinoic acid-dependent metamorphosis in the starfish.

The evolution of the biphasic life cycle in marine invertebrates has attracted considerable interest in zoology. We recently provided evidence that retinoic acid (RA) is involved in the regulation of metamorphosis in starfish. It also functions in life cycle transitions of jellyfish (cnidaria). Thus, documenting the evolutionarily conserved role of RA in such transitions will help to trace the life cycle evolution of bilaterians and cnidarians. In this study, we examined the molecular mechanisms by which RA signaling is involved in the commencement of metamorphosis in starfish. First, we measured RA levels during the larval and metamorphosis stages by liquid chromatography-tandem mass spectrometry. We found that all-trans RA levels in the larval body are high before larvae acquire competence for metamorphosis, suggesting that the commencement of metamorphosis is not controlled by increased RA synthesis. Furthermore, the suppression of rar gene expression by TALEN-mediated gene knockout revealed that RA receptor (RAR) is essential for metamorphosis. These observations suggest that the initiation of metamorphosis is regulated at the level of synthesized RA to activate RAR. We discuss the divergence of ligand molecules and receptors during the evolution of life cycle regulation.

pmar1/phb homeobox genes and the evolution of the double-negative gate for endomesoderm specification in echinoderms.

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.

Marine heatwaves and upwelling shape stress responses in a keystone predator.

Climate change increases the frequency and intensifies the magnitude and duration of extreme events in the sea, particularly so in coastal habitats. However, the interplay of multiple extremes and the consequences for species and ecosystems remain unknown. We experimentally tested the impacts of summer heatwaves of differing intensities and durations, and a subsequent upwelling event on a temperate keystone predator, the starfish Asterias rubens. We recorded mussel consumption throughout the experiment and assessed activity and growth at strategically chosen time points. The upwelling event overall impaired starfish feeding and activity, likely driven by the acidification and low oxygen concentrations in the upwelled seawater. Prior exposure to a present-day heatwave (+5°C above climatology) alleviated upwelling-induced stress, indicating cross-stress tolerance. Heatwaves of present-day intensity decreased starfish feeding and growth. While the imposed heatwaves of limited duration (9 days) caused slight impacts but allowed for recovery, the prolonged (13 days) heatwave impaired overall growth. Projected future heatwaves (+8°C above climatology) caused 100% mortality of starfish. Our findings indicate a positive ecological memory imposed by successive stress events. Yet, starfish populations may still suffer extensive mortality during intensified end-of-century heatwave conditions.

Captivity induces a sweeping and sustained genomic response in a starfish.

Marine animals in the wild are often difficult to access, so they are studied in captivity. However, the implicit assumption that physiological processes of animals in artificial environments are not different from those in the wild has rarely been tested. Here, we investigate the extent to which an animal is impacted by captivity by comparing global gene expression in wild and captive crown-of-thorns starfish (COTS). In a preliminary analysis, we compared transcriptomes of three external tissues obtained from multiple wild COTS with a single captive COTS maintained in aquaria for at least 1 week. On average, an astonishingly large 24% of the coding sequences in the genome were differentially expressed. This led us to conduct a replicated experiment to test more comprehensively the impact of captivity on gene expression. Specifically, a comparison of 13 wild with 8 captive COTS coelomocyte transcriptomes revealed significant differences in the expression of 20% of coding sequences. Coelomocyte transcriptomes in captive COTS remain different from those in wild COTS for more than 30 days and show no indication of reverting back to a wild state (i.e. no evidence of acclimation). Genes upregulated in captivity include those involved in oxidative stress and energy metabolism, whereas genes downregulated are involved in cell signalling. These changes in gene expression indicate that being translocated and maintained in captivity has a marked impact on the physiology and health of these echinoderms. This study suggests that caution should be exercised when extrapolating results from captive aquatic invertebrates to their wild counterparts.

Molecular Identification and Cellular Localization of a Corticotropin-Releasing Hormone-Type Neuropeptide in an Echinoderm.

BACKGROUND: Corticotropin-releasing hormone (CRH) mediates physiological responses to stressors in mammals by triggering pituitary secretion of adrenocorticotropic hormone, which stimulates adrenal release of cortisol. CRH belongs to a family of related neuropeptides that include sauvagine, urotensin-I, and urocortins in vertebrates and the diuretic hormone DH44 in insects, indicating that the evolutionary origin of this neuropeptide family can be traced to the common ancestor of the Bilateria. However, little is known about CRH-type neuropeptides in deuterostome invertebrates. METHODS: Here, we used mass spectrometry, mRNA in situ hybridization, and immunohistochemistry to investigate the structure and expression of a CRH-type neuropeptide (ArCRH) in the starfish Asterias rubens (phylum Echinodermata). RESULTS: ArCRH is a 40-residue peptide with N-terminal pyroglutamylation and C-terminal amidation, and it has a widespread pattern of expression in A. rubens. In the central nervous system comprising the circumoral nerve ring and 5 radial nerve cords, ArCRH-expressing cells and fibres were revealed in both the ectoneural region and the hyponeural region, which contains the cell bodies of motoneurons. Accordingly, ArCRH immunoreactivity was detected in innervation of the ampulla and podium of locomotory organs (tube feet), and ArCRH is the first neuropeptide to be identified as a marker for nerve fibres located in the muscle layer of these organs. ArCRH immunoreactivity was also revealed in protractile organs that mediate gas exchange (papulae), the apical muscle, and the digestive system. CONCLUSIONS: Our findings provide the first insights into CRH-type neuropeptide expression and function in the unique context of the pentaradially symmetrical body plan of an echinoderm.

Improving coral cover using an integrated pest management framework.

Integrated pest management (IPM) leverages our understanding of ecological interactions to mitigate the impact of pest species on economically and/or ecologically important assets. It has primarily been applied in terrestrial settings (e.g., agriculture), but has rarely been attempted for marine ecosystems. The crown-of-thorns starfish (CoTS), Acanthaster spp., is a voracious coral predator throughout the Indo-Pacific where it undergoes large population increases (irruptions), termed outbreaks. During outbreaks CoTS act as a pest species and can result in substantial coral loss. Contemporary management of CoTS on the Great Barrier Reef (GBR) adopts facets of the IPM paradigm to manage these outbreaks through strategic use of direct manual control (culling) of individuals in response to ecologically based target thresholds. There has, however, been limited quantitative analysis of how to optimize the implementation of such thresholds. Here we use a multispecies modeling approach to assess the performance of alternative CoTS management scenarios for improving coral cover trajectories. The scenarios examined varied in terms of their ecological threshold target, the sensitivity of the threshold, and the level of management resourcing. Our approach illustrates how to quantify multidimensional trade-offs in resourcing constraints, concurrent CoTS and coral population dynamics, the stringency of target thresholds, and the geographical scale of management outcomes (number of sites). We found strategies with low target density thresholds for CoTS (≤0.03 CoTS min-1 ) could act as "Effort Sinks" and limit the number of sites that could be effectively controlled, particularly under CoTS population outbreaks. This was because a handful of sites took longer to control, which meant other sites were not controlled. Higher density thresholds (e.g., 0.04-0.08 CoTS min-1 ), tuned to levels of coral cover, diluted resources among sites but were more robust to resourcing constraints and pest population dynamics. Our study highlights trade-off decisions when using an IPM framework and informs the implementation of threshold-based strategies on the GBR.

Phytoplankton community structure and environmental factors during the outbreak of Crown-of-Thorns Starfish in Xisha Islands, South China Sea.

The "larval starvation hypothesis" proposed that the growing frequency of Crown-of-Thorns Starfish (CoTS) outbreaks could be attributed to increased availability of phytoplankton. However, comprehensive field investigation on the living environment of CoTS larvae and the availability of phytoplankton are still lacking. A cruise was conducted in June 2022 in Xisha Islands, South China Sea, to study the interaction between environmental conditions and phytoplankton communities during CoTS outbreak period. The average concentrations of dissolved inorganic phosphorus (0.05 ± 0.01 µmol L-1), dissolved inorganic nitrogen (0.66 ± 0.8 µmol L-1) and chlorophyll a (0.05 ± 0.05 µg L-1) suggested that phytoplankton may be limited for CoTS larvae in Xisha Islands. Microscopic observation and high-throughput sequencing were used to study the composition and structure of the phytoplankton communities. Bacillariophyta predominated in phytoplankton communities with the highest abundance and species richness. 29 dominant species, including 4 species with size-range preferred by CoTS larvae, were identified in Xisha Islands. The diversity index of all stations indicated a species-rich and structure-stable phytoplankton community in Xisha Islands during the period of CoTS outbreak, which may contribute to CoTS outbreak. These findings revealed the structure of phytoplankton community and environmental factors in the study area during CoTS outbreak, providing the groundwork for future research into the causes and processes of CoTS outbreak.

A Relaxin-Like Gonad-Stimulating Peptide Appears in the Early Development of the Starfish Patiria pectinifera.

Relaxin-like gonad-stimulating peptide (RGP) is a hormone with gonadotropin-like activity in starfish. This study revealed that spawning inducing activity was detected in an extract of brachiolaria larvae of Patiria pectinifera. Spawning inducing activity in the extract was due to P. pectinifera RGP (PpeRGP), not 1-methyladenine. The expression of PpeRGP mRNA was also found in brachiolaria. Immunohistochemical observation with specific antibodies for PpeRGP showed that PpeRGP was distributed in the peripheral adhesive papilla of the brachiolaria arms. In contrast, PpeRGP was not detected in the adult rudiment or ciliary band regions, which are present in the neural system. These findings strongly suggest that RGP exists in the larvae before metamorphosis. Because gonads are not developed in starfish larvae, it seems likely that RGP plays another role other than gonadotropic action in the early development of starfish.

Gonadotropic activity of a second relaxin-type peptide in starfish.

In starfish, a relaxin-like gonad-stimulating peptide (RGP) acts as a gonadotropin that triggers gamete maturation and spawning. In common with other relaxin/insulin superfamily peptides, RGP consists of an A- and a B-chain, with cross-linkages mediated by one intra- and two inter-chain disulfide bonds. In this study, a second relaxin-like peptide (RLP2) was identified in starfish species belonging to the orders Valvatida, Paxillosida, and Forcipulatida. Like RGP, RLP2 precursors comprise a signal peptide and a C-peptide in addition to the A- and B-chains. However, a unique cysteine motif [CC-(3X)-C-(10X)-C] is present in the A-chain of RLP2, which contrasts with the cysteine motif in other members of the relaxin/insulin superfamily [CC-(3X)-C-(8X)-C]. Importantly, in vitro pharmacological tests revealed that Patiria pectinifera RLP2 (Ppe-RLP2) and Asterias rubens RLP2 (Aru-RLP2) trigger shedding of mature eggs from ovaries of P. pectinifera and A. rubens, respectively. Furthermore, the potencies of Ppe-RLP2 and Aru-RLP2 as gonadotropic peptides were similar to those of Ppe-RGP and Aru-RGP, respectively, and the effect of RLP2 exhibited partial species-specificity. These findings indicate that two relaxin-type peptides regulate spawning in starfish and therefore we propose that RGP and RLP2 are renamed RGP1 and RGP2, respectively.

The C-terminally amidated relaxin-like gonad-stimulating peptide in the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin, consisting of A- and B-chain. Recently, an RGP ortholog (Asc-RGP) from Astropecten scoparius in the order Paxillosida was found to harbor an amidation signal (Gly-Arg) at the C-terminus of the B-chain (Mita et al., 2020a). Two cleavage sites were also predicted within the signal peptide of the Asc-RGP precursor. Thus, four kinds of analogs (Asc-RGP-NH2(S), Asc-RGP-GR(S), Asc-RGP- NH2(L), Asc-RGP-GR(L) were hypothesized as natural Asc-RGPs. To identify the natural Asc-RGP, an extract of radial nerve cords from A. scoparius was analyzed using reverse-phase high-performance liquid chromatography and MALDI-TOF-mass spectrometry. The molecular weight of Asc-RGP was 4585.3, and those of A- and B-chains were 2511.8 and 2079.8, respectively. This strongly suggests that natural RGP in A. scoparius is Asc-RGP-NH2(S). Asc-RGP-NH2(S) stimulated 1-methyladenine and cyclic AMP production in isolated ovarian follicle cells of A. scoparius. On the other hand, the concentrations of four synthetic Asc-RGP analogs required for the induction of spawning in 50% of ovarian fragments were almost the same. The size and C-terminal amidation of the B-chain might not be important for spawning-inducing activity. C-terminally amidated RGPs in the B-chain were also observed in other species of starfish belonging to the order Paxillosida, particularly the family Astropectinidae, but not the family Luidiidae.

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.