The Effect of Synthetic Polyamine BPA-C8 on the Fertilization Process of Intact and Denuded Sea Urchin Eggs.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

Effects of seismic water guns on the peristomial membrane of sea urchins (Arbacia lixula, Linnaeus 1758).

The seismic water gun is widely used and plays an important role in seabed imaging acquisition; however, acoustic impacts on marine organisms are currently poorly understood. The aim of this study was to analyse the biochemical responses on the peristomial membrane (PM) of the sea urchin, Arbacia lixula, when exposed to water gun shots in open water. The PM (located around the mouth) is involved in vital functions, such as nutrition and protection. Individuals of sea urchins (n = 7 for each time slot) were sampled before, at the end, and at intervals of 3 h and 24 h after acoustic emission (duration of 20 min). Significant increases in superoxide dismutase, peroxidase, esterase and alkaline were observed immediately after water gun shots, highlighting an increase in the oxidative and inflammatory state of the tissue. Our results showed that acoustic impacts could interfere with PM vital functions, compromising the health, survival and ultimately the conservation of the species. Understanding these effects is crucial to predicting consequences on sea urchin populations and marine ecosystems.

Population genomic structure of the sea urchin Diadema africanum, a relevant species in the rocky reef systems across the Macaronesian archipelagos.

The sea urchin Diadema africanum is a macro-herbivore found in the rocky reef systems of the West African region and Macaronesian archipelagos. Over several decades, high densities of this species have generated marine barrens in certain areas at the Canary Islands. In contrast, more recently, during the last few years, the species has suffered mass mortality events that continue to the present day. In this study, we used 9,109 Single Nucleotide Polymorphisms (SNPs) and a fragment of a mitochondrial gene to evaluate the species' population structure, effects of mass mortalities on its diversity, and potential local adaptation across the Canary Islands and Cabo Verde. Our research provides compelling evidence of low genomic diversity and homogeneity across the studied area for neutral markers, along with recent demographic fluctuations. The high connectivity among distant areas likely allows a rapid recovering of the populations from local mortality events. Interestingly, we also observed genomic sub-structure from 405 SNPs identified as candidate loci under selection for seawater temperature. The lack of divergence among distant sites and the low diversity found can be attributed to the species' divergence from a small ancestral genomic pool, followed by a contemporary demographic expansion, and ongoinggene flow.

miR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

The people behind the papers - Carolyn Remsburg and Jia Song.

miR-31, an evolutionarily conserved microRNA, has been studied in different contexts, such as myogenesis, skeletogenesis and cancer; however, its role in post-transcriptional regulation during development is still unclear. In a new study, Jia Song and colleagues find that miR-31 regulates local translation of cytoskeletal remodelling transcripts at the mitotic spindle to ensure proper cell division during sea urchin embryogenesis. To find out more about the story behind the paper, we caught up with first author Carolyn Remsburg and Jia Song, Associate Professor at the University of Delaware, USA.

A natural multifunction and multiscale hierarchical matrix as a drug-eluting scaffold for biomedical applications.

Sea urchin spines are biogenic single crystals of magnesium calcite that are stiff, strong, damage tolerant and light and have a bicontinuous porous structure. Here, we showed that the removal of their intraskeletal organic matrix materials did not affect the compressive mechanical properties and generated an open porosity. This matrix was able to adsorb and release oxytetracycline, a broad-spectrum antibiotic. The drug-loaded sea urchin matrix induced bacterial cell death after 4 and 8 hours of incubation of both Gram-negative E. coli and Gram-positive S. aureus strains and this process induces an inhibition of bacterial cell adhesion. In conclusion, this study shows that thermally treated sea urchin spines are a compressive resistant and lightweight matrix able to load drugs and with potential use in spine fusion, a challenging application that requires withstanding high compressive loading.

Combinatorial regulatory states define cell fate diversity during embryogenesis.

Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15-40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.

Toxic Effects of Biogenic and Synthesized Silver Nanoparticles on Sea Urchin Echinometra lucunter Embryos.

Due to their broad-spectrum antimicrobial action and ease of synthesis, silver nanoparticles (AgNP) are one of the most widely used nanomaterials in different industrial and ecological areas. AgNP are released into marine ecosystems, nevertheless, their ecotoxicological effects have been overlooked. In this study, we evaluated the toxic effects of biogenic and synthesized AgNP (AgNPIBCLP11 and AgNPSINT) on sea urchin Echinometra lucunter embryos and compared them with the metal precursor silver nitrate (AgNO3). Fertilized eggs were exposed to five concentrations of the test compounds and a negative control for 48 h under controlled conditions. The IC50-48 h of AgNPIBCLP11, AgNPSINT and AgNO3 were 0.31, 4.095, and 0.01 µg L-1, evidencing that both AgNP are less toxic than AgNO3, and that AgNPSINT is less toxic than the AgNPIBCLP11. Toxicity to E. lucunter embryos could be explained by the fact that Ag affects DNA replication and induces the formation of pores in the cellular wall, leading to apoptosis.

Visualizing metabolic regulation using metabolic biosensors during sea urchin embryogenesis.

Growing evidence suggests that metabolic regulation directly influences cellular function and development and thus may be more dynamic than previously expected. In vivo and in real-time analysis of metabolite activities during development is crucial to test this idea directly. In this study, we employ two metabolic biosensors to track the dynamics of pyruvate and oxidative phosphorylation (Oxphos) during the early embryogenesis of the sea urchin. A pyruvate sensor, PyronicSF, shows the signal enrichment on the mitotic apparatus, which is consistent with the localization patterns of the corresponding enzyme, pyruvate kinase (PKM). The addition of pyruvate increases the PyronicSF signal, while PKM knockdown decreases its signal, responding to the pyruvate level in the cell. Similarly, a ratio-metric sensor, Grx-roGFP, that reads the redox potential of the cell responds to DTT and H2O2, the known reducer and inducer of Oxphos. These observations suggest that these metabolic biosensors faithfully reflect the metabolic status in the cell during embryogenesis. The time-lapse imaging of these biosensors suggests that pyruvate and Oxphos levels change both spatially and temporarily during embryonic development. Pyruvate level is increased first in micromeres compared to other blastomeres at the 16-cell stage and remains high in ectoderm while decreasing in endomesoderm during gastrulation. In contrast, the Oxphos signal first decreases in micromeres at the 16-cell stage, while it increases in the endomesoderm during gastrulation, showing the opposite trend of the pyruvate signal. These results suggest that metabolic regulation is indeed both temporally and spatially dynamic during embryogenesis, and these biosensors are a valuable tool to monitor metabolic activities in real-time in developing embryos.

An RNA interference approach for functional studies in the sea urchin and its use in analysis of nodal signaling gradients.

Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variegatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods are described for the design of specific DsiRNAs that lead to destruction of targeted mRNA. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.

Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

The first high-quality genome assembly and annotation of Anthocidaris crassispina.

Anthocidaris crassispina is a very popular edible sea urchin distributed along the coast of the South China Sea. In this study, we performed whole-genome sequencing and generated a chromosome-level assembly of this species. The total length of the genomic contig sequence was 891.02 Mb, and contig N50 was 808.15 kb when Hifiasm was used for assembly. The Hi-C library was constructed and sequenced, yielding approximately 68.61 Gb of data. After Hi-C assembly, approximately 886.72 Mb of sequence was able to be mapped onto 21 chromosomes, accounting for 99.52% of the total genome length. Among the sequences located on the chromosomes, those for which the order and direction could be determined accounted for approximately 826.82 Mb, or 93.24% of the total length. These results provide valuable resources for further study of A. crassispina at the genetic level.

Morphological and Molecular Evidence of an Intergeneric Host-Range in Clavisodalis sentifer (Crustacea: Copepoda: Taeniacanthidae) Associated with Diadematid Sea Urchins from the Western Pacific.

Sea urchins have a wide variety of symbionts on their body surfaces and inside their bodies. Copepods of the genus Clavisodalis (Taeniacanthidae) collected from the esophagus of sea urchins of the genera Diadema and Echinothrix in southern Japan were identified based on their morphological characteristics, and molecular analysis was conducted to determine whether genetic variation occurs in copepods from different localities and hosts. Morphological observations identified individuals from southern Japan as Clavisodalis sentifer Dojiri and Humes, 1982, making this the first record of this species in the northern hemisphere and the first record of its genus in Japan. Morphological and molecular analysis suggested that the copepod specimens collected from multiple hosts across two genera would be the same species. Considering the typically observed high level of host specificity among taeniacanthid copepods, the utilization of hosts from two genera by C. sentifer is noteworthy.

Investigating intraspecific variability in the biological responses of sea urchins (Paracentrotus lividus) to seawater acidification.

Alterations in seawater chemistry posed by acidification may lead to immunological and antioxidant defence impairment in sea urchins, with differences among local populations. Here, we analyzed the effects of reduced pH on Paracentrotus lividus, with a multibiomarker approach, and the possible intraspecific variations in sea urchin responses. Two groups of animals with different ecological histories (i.e., the pattern of environmental characteristics and pressures experienced throughout the organism's lifetime) were maintained at ambient pH and pH reduced of 0.4 units for 8 months. Changes in gonadosomatic index (GSI), immunological, and oxidative stress biomarkers were assessed in coelomic fluid, gonads, and digestive tract. Animals maintained at reduced pH showed limited impact of seawater acidification compared to the ambient pH condition. However, sea urchins from the two sites were differently influenced by the seawater pH (as shown by multivariate analyses). GSI and immunological and antioxidant status were differentially modulated between the two sexes, with generally higher values in females, but differences between sexes in relation to the pH of exposure were limited. Overall, our findings highlight that the impact of environmental stressors may differ in sea urchins from different locations. This has implications for the maintenance of P. lividus wild populations under future global change scenarios.

Integrative multi-omics increase resolution of the sea urchin posterior gut gene regulatory network at single-cell level.

Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing to obtain the necessary information for drafting the Strongylocentrotus purpuratus (Sp) posterior gut GRN. Here, we present an update to the GRN using: (1) a single-cell RNA-sequencing-derived cell atlas highlighting the 2 day-post-fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at the single-cell level; (2) a set of putative cis-regulatory modules and transcription factor-binding sites obtained from chromatin accessibility ATAC-seq data; and (3) interactions directionality obtained from differential bulk RNA sequencing following knockdown of the transcription factor Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1-positive cells in the 2 dpf gastrula embryo. Overall, our data suggest the complex connectivity of the posterior gut GRN and increase the resolution of gene regulatory cascades operating within it.

Effect of harvesting season and location on the microbial quality and community composition of the edible sea urchin (Echinus esculentus) gonads.

Despite the crucial role of microbial community composition in the quality and stability of seafood, little emphasis has been given to the microbiota profile of sea urchin gonads. This study investigates the microbial quality and community composition of sea urchin gonads (Echinus esculentus) as a function of harvesting season (autumn, winter, spring, and summer) and location (one site proximal to urban activity areas while the other is located in open water close to the coastline). Significant season-dependent variations were found in psychrotrophic and aerobic plate counts, with higher counts in summer, followed by autumn, spring, and winter. H2S-producing bacteria and Pseudomonas spp. counts were unaffected by harvesting season or location. Sea urchin gonad microbial composition proved resilient and dynamic, primarily shaped by seasonal variations, and minimally influenced by location. Winter and spring samples exhibited higher diversity than autumn and summer. Key genera like Pseudomonas, Psychromonas, Vibrio, Chryseobacterium, Shewanella, and Photobacterium varied seasonally. Pseudomonas, Vibrio, and Photobacterium are crucial in assessing microbial quality and safety due to their roles as specific spoilage organisms (SSOs) and, in some cases, human pathogens. Though relative abundances differed slightly between locations, harvesting location did not notably impact microbial community shaping in gonads. However, the results suggest that harvesting locations near areas with urban activity may lead to contamination with specific bacterial species, possibly due to water quality variations. These findings emphasize the importance of considering seasonality when evaluating sea urchin gonad microbial quality. Identifying key genera enhances insights into potential SSOs and human pathogens, enhancing food safety considerations in the consumption of raw or lightly processed sea urchin gonads and guiding the development of preservation methods to extend shelf life.

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

Screening and purification of antimicrobial materials from coelomic fluid of sea urchin, Heliocidaris crassispina.

Marine organisms, such as sea urchins like Heliocidaris crassispina, produce bioactive substances with antimicrobial activity to protect themselves from the high density of microorganisms in their habitats. One such substance, Echinochrome A (Ech A), has been isolated from various sea urchins' shells and spines using strong acidic solutions and organic solvents. Ech A, however, has not been reported from the coelomic fluid of H. crassispina. In this study, we report the antimicrobial activity of H. crassispina coelomic fluid extract against various microbes, evaluating its potential for purifying potent antimicrobial materials. Upon confirming the extract as a promising source of antimicrobial materials, we isolated antimicrobial compounds from the extract. A series of HPLC steps were taken to purify antimicrobial materials from the H. crassispina coelomic fluid extract, resulting in the isolation of two single absorbance peaks showing antimicrobial activity against Staphylococcus aureus. One peak consisted of a single antimicrobial compound with a molecular weight (MW) corresponding to Ech A, while the other peak comprised five MWs inferred to be those of Ech A and its oxidative products. The elution of Ech A in two separate peaks may be attributable to the presence of Ech A's isomer, as reported in several previous studies. The use of the environmentally friendly extraction method in procurement of Ech A from the coelomic fluid would contribute to the implementation of risk-reducing extraction method for researchers studying Ech A from sea urchins.

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.

Trace metals pollution and trophic position of three sea urchin species in the Gulf of California.

Sea urchin species are ecologically important in the Gulf of California and are becoming popular as a local fishery due to their commercial value. The most abundant species are Echinometra vanbrunti, Eucidaris thouarsii, and Tripneustes depressus. The objective of this study was to evaluate cadmium, lead, copper, zinc, and iron concentrations, as well as stable isotope values in these sea urchin species in the Santa Rosalia mining area (STR), in three sites: Punta Gorda, Punta el Aterrizaje, and Punta Salina. The highest Fe concentration (100.2 mg kg-1) was found in E. vanbrunti, while the highest concentrations of Pb (15.1 mg kg-1), Cu (14.5 mg kg-1), and Zn (347.7 mg kg-1) were recorded in E. thouarsii, and the highest Cd concentration (10.8 mg kg-1) was found in T. depressus. The main health risk of trace metal pollution in STR may be caused by Cd and Pb. δ15N and δ13C values were higher in E. thouarsii and T. depressus, respectively; E. thouarsii has the highest trophic position. Specimen size was not related to metal concentrations, but a positive relationship was observed between specimen size and isotopic values in T. depressus. The three species showed different bioaccumulation patterns for the metals analyzed. Additionally, collection sites and seasons play an important role in the variability of metal concentration.