A Putative ‘Pre-Nervous’ Endocannabinoid System in Early Echinoderm Development

Phenotypic plasticity, the ability of a genotype to produce different phenotypes in response to environmental conditions, plays a crucial role in adaptation and evolution and can occur during development or adulthood. Sea urchin larvae exhibit developmental plasticity by adjusting their arm length in response to food availability. In this study, we investigated the phenotypic responses of three sea urchin species: Paracentrotus lividus and Arbacia lixula from the Mediterranean Sea, and Strongylocentrotus purpuratus from the Pacific Ocean. In all species, we observed that larvae reared under a 12h light:12h dark cycle exhibited phenotypic responses to food availability. However, the response was suppressed in larvae reared under constant darkness, suggesting that light has a role in mediating this phenotypic plasticity. Moreover, larvae grown in constant darkness were generally smaller than those exposed to light, with the magnitude of this effect varying among species, indicating that light exposure influences not only plasticity but also baseline growth rates. These findings underscore the utility of sea urchins as a model for studying ecological and evolutionary processes shaping phenotypic responses and suggest that light has an important impact on development and growth in sea urchins.

Many species of marine invertebrate larvae settle and metamorphose in response to chemicals produced by organisms associated with the adult habitat, and histamine is a cue for larvae of the sea urchin Holopneustes purpurascens. This study investigated the effect of histamine on larval metamorphosis of six sea urchin species. Histamine induced metamorphosis in larvae of three lecithotrophic species (H. purpurascens, Holopneustes inflatus and Heliocidaris erythrogramma) and in one planktotrophic species (Centrostephanus rodgersii). Direct comparisons of metamorphic rates of lecithotrophic and planktotrophic larvae in assays cannot be made due to different proportions of larvae being competent. Histamine (10 μM) induced metamorphosis in 95% of larvae of H. purpurascens and H. inflatus after 1 h, while the coralline alga Amphiroa anceps induced metamorphosis in 40–50% of these larvae. Histamine (10 μM) and A. anceps induced 40 and 80% metamorphosis, respectively, in the larvae of H. erythrogramma after 24 h. Histamine (10 μM) and the coralline alga Corallina sp. induced 30 and 70% metamorphosis, respectively, in the larvae of C. rodgersii after 24 h. No metamorphosis of any larval species occurred in seawater controls. Larvae of two planktotrophic species (Tripneustes gratilla and Heliocidaris tuberculata) did not metamorphose in response to histamine. Seagrasses, the host plants of H. inflatus, induced rapid metamorphosis in larvae of the two Holopneustes species, and several algae induced metamorphosis in C. rodgersii larvae. Histamine leaching from algae and seagrasses may act as a habitat marker and metamorphic cue for larvae of several ecologically important sea urchin species.

To screen for immune indicators closely related to disease resistance, two species of sea urchin susceptible to black mouth disease (Strongylocentrotus intermedius, S. intermedius ♀ × Heliocidaris crassispina ♂) and three species of sea urchin resistant to black mouth disease (H. crassispina, H. crassispina ♀ × S. intermedius ♂ and Mesocentrotus nudus) were artificially infected with the black mouth pathogen Vibrio echinoideorum. The phagocytosis-related immune indices of the five sea urchin species were compared at different time points post-infection. The results demonstrated that the parameters such as apoptotic rate of phagocytes, mean contribution value (MCV) of single effective phagocyte on Acid Phosphatase (ACP), Reactive Oxygen Species (ROS), and Total Antioxidant Capacity (T-AOC) of the five sea urchin species first increased and then decreased after infection. The key time points were 3 h to 6 h and 48 h post-infection when the black mouth disease-resistant and susceptible sea urchins demonstrated differences. At 3 h or 6 h post-infection, the up-regulation folds in MCV of ACP, ROS and T-AOC of black mouth disease-resistant sea urchins were considerably higher than that of the susceptible sea urchins. At 6 h post-infection, the apoptosis rate and the phagocytic index (PI) of the black mouth disease-resistant sea urchins were significantly higher than those of the susceptible sea urchins (p < 0.05). At 48 h post-infection, the necrosis rate of phagocytes, MCV of ACP and MCV of ROS of the black mouth disease-resistant sea urchins were significantly lower than those of the susceptible sea urchins (p < 0.05). The apoptosis and necrosis rate of phagocytes, PI, and MCV on ACP, ROS may be used as indicators of disease resistance in sea urchins. Disease resistance standards in immune indices can be summarized as phagocytosis increases greatly in the early infection stage and decreases timely to a normal level after killing the pathogen in a short period.

Alkaline digestive systems are well described for some insect species and their larval stages. More recently, larvae of the members of ambulacraria superphylum consisting of echinoderms and hemichordates were also discovered to have highly alkaline midguts (pH 9.5-10.5) with the underlying acid-base regulatory mechanisms largely unknown. Using pharmacological inhibition of acid-base transporters in conjunction with ion-selective microelectrode measurements and pH-sensitive dyes, we investigated intracellular and extracellular pH regulatory mechanisms of midgut epithelial cells of a sea urchin (Strongylocentrotus purpuratus) larva. Our findings suggest that vacuolar-type H+-ATPase (inhibited by bafilomycin a1), carbonic anhydrase (inhibited by acetazolamide), anion-exchangers (inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid or DIDS), and soluble adenylyl cyclase (inhibited by KH7) play crucial roles in cellular acid-base regulation as well as midgut alkalization. Ammonia excretion rates were decreased in the presence of bafilomycin and colchicine, pointing toward vesicular [Formula: see text] trapping and exocytosis mechanism in eliminating nitrogenous proton equivalents from midgut cells. Finally, midgut perfusion studies revealed ouabain-sensitive luminal [Formula: see text] uptake, suggesting a role for Na+/K+-ATPase-mediated ammonia transport in midgut alkalization. This comprehensive pharmacological analysis provides a new working model relying on the CO2/[Formula: see text] and NH3/[Formula: see text] buffer systems for midgut alkalization in the sea urchin larva. These findings are discussed in the context of other alkalizing systems with strong implications for the conserved role of [Formula: see text] and NH3-driven mechanism of midgut alkalization across the animal kingdom.NEW & NOTEWORTHY Sea urchin larvae evolved highly alkaline conditions in their digestive tracts, and the underlying acid-base regulatory mechanisms are little understood. Here we present evidence that the process of luminal alkalization is cAMP-dependent. Furthermore, our data point toward the involvement of bicarbonate and ammonia in regulating midgut fluid pH. These results identified a novel mechanism for luminal alkalization in the digestive tract of a marine animal with strong implications for other alkalizing systems in animals.

The plasticity of the nervous system plays a crucial role in shaping adaptive neural circuits and corresponding animal behaviors. Understanding the mechanisms underlying neural plasticity during development and its implications for animal adaptation constitutes an intriguing area of research. Sea urchin larvae offer a fascinating subject for investigation due to their remarkable evolutionary and ecological diversity, as well as their diverse developmental forms and behavioral patterns. We conducted immunochemical and histochemical analyses of serotonin-containing (5-HT-neurons) and dopamine-containing (DA-positive) neurons to study their developmental dynamics in two sea urchin species: Mesocentrotus nudus and Paracentrotus lividus. Our approach involved detailed visualization of 5-HT- and DA-positive neurons at gastrula-pluteus stages, coupled with behavioral assays to assess larval upward and downward swimming in the water column, with a focus on correlating cell numbers with larval swimming ability. The study reveals a heterochronic polymorphism in the appearance of post-oral DA-positive neuroendocrine cells and confirms the stable differentiation pattern of apical 5-HT neurons in larvae of both species. Notably, larvae of the same age exhibit a two- to four-fold difference in DA neurons. An increased number of DA neurons and application of dopamine positively correlate with larval downward swimming, whereas 5-HT-neurons and serotonin application induce upward swimming. The ratio of 5-HT/DA neurons determines the stage-dependent vertical distribution of larvae within the water column. Consequently, larvae from the same generation with a higher number of DA-positive neurons tend to remain at the bottom compared to those with fewer DA-positive neurons. The proportion of 5-HT and DA neurons within larvae of the same age underlies the different potentials of individuals for upward and downward swimming. A proposed model illustrates how coordination in humoral regulation, based on heterochrony in DA-positive neuroendocrine cell differentiation, influences larval behavior, mitigates competition between siblings, and ensures optimal population expansion. The study explores the evolutionary and ecological implications of these neuroendocrine adaptations in marine species.

Morphogenesis, the process by which the tissues and organs of the embryo are properly shaped, is a fundamental feature of development. In the sea urchin, the formation of the calcified enodoskeleton is a major morphogenetic event. Differentiation of the skeletogenic primary mesenchyme cells (PMCs) has been considered to occur in two phases: the autonomous specification of PMCs followed by signal-dependent patterning of PMCs and the embryonic skeleton they produce. Autonomous specification creates a homogenous population of PMCs, but the later differentiation of these cells is influenced by extrinsic signals that provide essential positional information. Recent studies showed that ectodermal growth factors are critically involved in the guidance of PMC migration and skeletal differentiation. However, a better understanding of the various signaling pathways that regulate skeletogenesis and their role in PMC gene expression remains to be established. This study examines the regulation of morphogenesis by signaling pathways, using skeletogenesis in the sea urchin embryo as a model. The aim of this study was to identify and study the roles of extrinsic signals in regulating PMC gene expression, focusing on the later, signal-dependent phase of PMC differentiation. By analyzing and classifying spatial expression patterns of 39 genes preferentially expressed in PMCs, I find that: 1) these genes are expressed non-uniformly within the PMC syncytium, reflecting a widespread influence of locally activated signals; 2) regions with elevated gene expression correlate with sites of rapid biomineral deposition at each stage; 3) non-uniform expression of genes within the PMC syncytium is controlled by multiple signal in a precise temporal sequence. I also provide evidence that ectoderm-derived VEGF signaling regulates gene expression in PMCs via the MAPK pathway on the ventral side of the embryo. Additionally, my work has identified an essential role for TGF-β signaling in skeletogenesis. Previous studies indicate that a complete repertoire of TGF-β signaling components is present in the sea urchin genome and TgfbrII mRNA is preferentially expressed in PMCs at the early gastrula stage. In this study, I show that TgfbrII mRNA is specifically expressed in the PMC lineage from the hatched blastula to the mid-gastrula stage. Perturbation experiments indicate that TgfbrII is activated by the single, sensu stricto TGF-β ligand in sea urchins and is required for skeletogenesis in the sea urchin embryo. I also show that the late activity of Alk4/5/7, the putative Type I receptor, regulates skeletogenesis in a dose-dependent manner. Isolation and in vitro culture of PMCs demonstrates that both Alk4/5/7 and TgfbrII function cell autonomously in these cells. I provide evidence that TGF-β-TgfbrII signaling is not involved in dorsal-ventral axis patterning or PMC specification; instead, this pathway plays a selective role in later skeletal patterning. Taken as a whole, my research demonstrates that skeletogenesis is regulated by a much more diverse suite of signaling pathways than was previously appreciated. These findings significantly expand our understanding of the complex regulation of skeletal morphogenesis by extrinsic signals during embryonic development.

In paper are presented drawings and pictures of sea urchins from original works of authors who determined and documented new species from Istria. TARAMELLI (1874), BITTNER (1880), OPPENHEIM (1901), TONIOLO (1909) and DEGLI INNOCENTI (1924 a, b) established in Paleogene beds of Istria 18 new species of Eocene sea urchins. Determined were four species of regular sea urchins of genera Stereocidaris, Phyllacanthus, "Eucidaris" and Arachniopleurus, and fourteen species of irregular sea urchins of nine genera - Conoclypus, Echinolampas, Rhyncholampas, Gitolampas, Ditremaster, Pericosmus, Prenaster, Brissopsis and Cyclaster. The highest species diversity was recorded within genus Echinolampas. New sea urchins species originate prevailingly in the Middle Eocene - Lutetian beds of localities in the narrower and broader surroundings of Koper, Buzet, Roč, Grdoselo, Pazin, Pićan, Čepić and Labin.

This chapter discusses primarily methods for tests with four species of North American echinoids including three species of sea urchins and the closely related sand dollar of the Pacific Coast. These species include: Purple urchin: Arbacia punctulata, Purple urchin: Strongylocentrotus purpuratus, Green urchin: Strongylocentrotus droebachiensis and Sand dollar: Dendraster excentricus. Sea urchin sperm cell tests are usually surrogate tests in both of these respects: they are intended to provide toxicity information for the protection of other marine organisms and the typical 60-min duration of the sperm test exposure probably exceeds real-world exposure conditions for sea urchin sperm. The chief advantages of the sea urchin sperm cell test are its rapidity, small test volume, and great potential for using many test treatments and multiple replications of each treatment. In practice, the standardization of the sea urchin sperm cell test has seen a number of variations on the basic design.

Many animals display nephridial structures for the ultrafiltration of metabolic waste. However, a nephridial equivalent and an excretory system are not generally recognized for echinoderms. Podocytes are nephridial cells that function in ultrafiltration of body fluids. Limited ultrastructural analyses of echinoderms identify cells with podocyte morphology in the axial organ and in the left coelom of larval sea urchins. Echinoid internal anatomy suggests that the excretory system functions by ultrafiltration in the axial organ, as well as filtrate flow via the water vascular system for excretion through the madreporite; however, these reports are based on morphology. To verify podocytes in the axial organ, orthologues of podocyte-specific genes were evaluated in the sea urchin genome and RNAseq data sets. To verify excretion from the madreporite, fluorescein was used as a tracer for nephridial clearance, and was injected into the main body cavity of sea urchins. Results showed that genes encoding proteins that function in podocytes of vertebrates are expressed specifically in the axial organ of sea urchins, in agreement with orthologue expression in the nurse shark kidney. However, fluorescein clearance from the body cavity shows elimination from the anus rather than the madreporite. This leads to the hypothesis that fluorescein and metabolic waste clearance occur through ultrafiltration by podocytes in the axial organ, but that the filtrate flows into the haemal system and the haemal capillaries in the intestinal walls, from which fluid is transferred to the intestinal lumen for elimination through the anus. Future testing is proposed to evaluate fluorescein filtration from the blastocoel of larvae into the left coelom, and for excretion by small or juvenile echinoids that have undergone tissue clearance to visualize the route of fluorescein flow within the internal anatomy of cleared, intact sea urchins.

Monosaccharides and trace elements, as essential components of enzymes, hormones, and vitamins, play indispensable roles in biological systems. Sea urchins, being ecologically and nutritionally significant marine invertebrates, offer a valuable model for studying the distribution and functions of these compounds. However, systematic investigations into interspecies variations in sea urchins remain limited. To address this, we conducted a comparative analysis of four commercially important sea urchin species from China— Strongylocentrotus intermedius, Mesocentrotus nudus, Glyptocidaris crenularis , and Anthocidaris crassispina . Using high-performance liquid chromatography (HPLC), we profiled eight monosaccharide components in sea urchin gonads, identifying glucose and mannose as the predominant components. Our analysis revealed significant interspecies differences in composition. Furthermore, we determined complete profiles of 20 trace elements in both gonads and intestines, distinguishing biologically essential elements from potentially toxic ones. Importantly, species-specific patterns were identified, including elevated Ca in the gonads of M. nudus , high Fe in the intestines of G. crenularis , and relatively high concentrations of Cd and As across intestinal tissues. In summary, this study systematically characterized the compositional differences of monosaccharides and trace elements in four commercial sea urchin species, providing baseline data for understanding physiological adaptation mechanisms and serving as a reference for assessing the nutritional quality of sea urchin products.

Sea urchin Mesocentrotus nudus, Glyptocidaris crenularis, and Strongylocentrotus intermedius gonad protein isolates (mnGPIs, gcGPIs, and siGPIs) were extracted by isoelectric solubilization/precipitation (ISP) from the defatted gonads, and their functional properties were compared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed the similar protein pattern between each protein isolate and defatted gonad, indicating the high efficiency of ISP processing for protein recovery. Amino acid profileconfirmed that the mnGPIs and siGPIs could be potential sources of essential amino acid in nature. As regard to functional properties, mnGPIs showed higher water- and oil- holding capacities followed bysiGPIs and gcGPIs and all protein isolates presented great foaming property. As for emulsifying activity index (EAI), mnGPIs, gcGPIs, and siGPIs showed the minimum solubility and EAI at pH 5, 3, and 4, respectively, and behaved a pH-dependent manner. The gcGPIs revealed the highest EAI from pH 6 to 8 among the samples. In addition, circular dichroism showed increased content of β-sheet at the expense of α-helix and β-turn, suggesting the structure denaturation of the protein isolates. Indeed, no statistical difference was observed between secondary structure of mnGPIs and siGPIs. Moreover, ISP processing increased free sulfhydryl content of sea urchin protein isolates, but no difference was observed among the samples. Furthermore, siGPIs revealed the highest amount of total sulfhydryl and disulfide bonds, whereas both defatted gonads and protein isolates from G. crenularis presented the maximum surface hydrophobicity. These results suggest that gonad protein isolates from three species of sea urchin possess various functionalities and therefore can be potentially applied in food system. PRACTICAL APPLICATION: Sea urchin M. nudus, G. crenularis, and S. intermedius gonads are edible, whereas the functional properties of protein isolates from sea urchin gonad remain unknown. In this case, the extraction and comparison of three species of sea urchin gonad protein isolates will not only confirm functional properties but also screen food ingredients with suitable functions. In this study, functionalities of protein isolates derived from M. nudus, G. crenularis, and S. intermedius gonads would provide potential application in bakery food and meat products or as emulsifier candidates in food system.

The effects of 3 different microalgae species on the growth, metamorphosis and MYP gene expression of two sea urchins, Strongylocentrotus intermedius and S. nudus

IL17 cytokines are central mediators of mammalian immunity. In vertebrates, these factors derive from diverse cellular sources. Sea urchins share a molecular heritage with chordates that includes the IL17 system. Here, we characterize the role of epithelial expression of IL17 in the larval gut-associated immune response. The purple sea urchin genome encodes 10 IL17 subfamilies (35 genes) and 2 IL17 receptors. Most of these subfamilies are conserved throughout echinoderms. Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance. IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype, indicating a potential feedback. A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families. The larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism.

The Wnt genes comprise a family of secreted glycoproteins involved in cell-cell signaling and pattern formation during the development of a variety of organisms. We have begun to examine Wnt gene expression in sea urchins that exhibit alternative modes of larval development but produce similar adults. Here we describe the isolation of five Wnt sequences from indirect- and direct-developing sea urchin species using a PCR-based strategy and library screening. Phylogenetic and distance analyses indicate that the five sequences represent sea urchin Wnt-1, -4, and -5 orthologs. Wnt-5 sequences were isolated from three sea urchin species and show a significantly faster rate of evolution than do their counterparts in jawed vertebrates. The genomic structure of the Wnt-5 locus was also examined, and its organization is similar to that of Wnt genes from insects and vertebrates. The temporal expression of all three sea urchin Wnt orthologs during sea urchin development was examined by RNA gel blots or RNase protection assays. Transcripts from all three sea urchin Wnts are detected at various developmental stages of both indirect- and direct-developing species. These data support the view that sea urchin Wnt genes exhibit many conserved aspects and at least three orthologs are developmentally regulated in both indirect- and direct-developing sea urchin embryos.

BackgroundLight is essential for various biological activities. In particular, visual information through eyes or eyespots is very important for most of animals, and thus, the functions and developmental mechanisms of visual systems have been well studied to date. In addition, light-dependent non-visual systems expressing photoreceptor Opsins have been used to study the effects of light on diverse animal behaviors. However, it remains unclear how light-dependent systems were acquired and diversified during deuterostome evolution due to an almost complete lack of knowledge on the light-response signaling pathway in Ambulacraria, one of the major groups of deuterostomes and a sister group of chordates.ResultsHere, we show that sea urchin larvae utilize light for digestive tract activity. We found that photoirradiation of larvae induces pyloric opening even without addition of food stimuli. Micro-surgical and knockdown experiments revealed that this stimulating light is received and mediated by Go(/RGR)-Opsin (Opsin3.2 in sea urchin genomes) cells around the anterior neuroectoderm. Furthermore, we found that the anterior neuroectodermal serotoninergic neurons near Go-Opsin-expressing cells are essential for mediating light stimuli-induced nitric oxide (NO) release at the pylorus. Our results demonstrate that the light>Go-Opsin>serotonin>NO pathway functions in pyloric opening during larval stages.ConclusionsThe results shown here will lead us to understand how light-dependent systems of pyloric opening functioning via neurotransmitters were acquired and established during animal evolution. Based on the similarity of nervous system patterns and the gut proportions among Ambulacraria, we suggest the light>pyloric opening pathway may be conserved in the clade, although the light signaling pathway has so far not been reported in other members of the group. In light of brain-gut interactions previously found in vertebrates, we speculate that one primitive function of anterior neuroectodermal neurons (brain neurons) may have been to regulate the function of the digestive tract in the common ancestor of deuterostomes. Given that food consumption and nutrient absorption are essential for animals, the acquirement and development of brain-based sophisticated gut regulatory system might have been important for deuterostome evolution.