Leachates of weathering plastics from an urban sandy beach: Toxicity to sea urchin fertilization and early development.

Plastic leachates have chemical and biological implications for marine environments. This study experimentally evaluated acute effects of weathering plastic leachates (0, 25, 50, 75 and 100 %) on fertilization and early development of the sea urchin Lytechinus variegatus. Fertilization, embryonic and larval development were drastically inhibited (~75 %) when gametes were exposed to intermediate and high leachate concentrations or delayed when exposed to the lowest concentration. Fertilization and first cleavage stages were highly affected by exposure to intermediate and high leachate concentrations. None of the cells incubated at concentrations from 50 % reached blastula stage, suggesting that embryonic development was the most sensitive stage. Abnormalities in embryos and larvae were observed in all leachate treatments. Chemical analysis detected high concentration of bisphenol A, which may induce these observed effects. Our results highlight the potential threats of plastic pollution to sea urchin populations, which may severely affect the structure and functioning of coastal ecosystems.

Voltage-gated sodium channel activity mediates sea urchin larval skeletal patterning through spatial regulation of Wnt5 expression.

Defining pattern formation mechanisms during embryonic development is important for understanding the etiology of birth defects and to inform tissue engineering approaches. In this study, we used tricaine, a voltage-gated sodium channel (VGSC) inhibitor, to show that VGSC activity is required for normal skeletal patterning in Lytechinus variegatus sea urchin larvae. We demonstrate that tricaine-mediated patterning defects are rescued by an anesthetic-insensitive version of the VGSC LvScn5a. Expression of this channel is enriched in the ventrolateral ectoderm, where it spatially overlaps with posterolaterally expressed Wnt5. We show that VGSC activity is required to spatially restrict Wnt5 expression to this ectodermal region that is adjacent and instructive to clusters of primary mesenchymal cells that initiate secretion of the larval skeleton as triradiates. Tricaine-mediated Wnt5 spatial expansion correlates with the formation of ectopic PMC clusters and triradiates. These defects are rescued by Wnt5 knockdown, indicating that the spatial expansion of Wnt5 is responsible for the patterning defects induced by VGSC inhibition. These results demonstrate a previously unreported connection between bioelectrical status and the spatial control of patterning cue expression during embryonic pattern formation.

Increasing Temperature Results in Higher Allocation of Energy to Protein Synthesis in Sea Urchin Larvae (Lytechinus pictus).

AbstractIt is well established that metabolic processes change with temperature and size. Yet the underlying physiological mechanisms are less well understood regarding how such processes covary within a species and particularly so for developmental stages. Physiological analysis of larvae of the sea urchin Lytechinus pictus revealed that protein was the major biochemical substrate supporting metabolism. The complex dynamics of protein synthesis, turnover, and accretion changed during growth, showing a sevenfold decrease in the ratio of protein accretion to protein synthesis (protein depositional efficiency). To test hypotheses of physiological variation with rising temperature, larvae were reared over a temperature range experienced by this species in its ambient habitat. The thermal sensitivity of protein synthesis was greater than respiration (thermal sensitivity values of 3.7 and 2.4, respectively). Bioenergetic calculations revealed a disproportionate increase in energy allocation toward protein synthesis with rising temperature. These differential temperature sensitivities result in metabolic trade-offs of energy acquisition and expenditure, thereby altering physiological homeostasis. Such insights are of value for improving predictions about limits of biological resilience in a warming ocean.

Alterations induced by titanium dioxide nanoparticles (nano-TiO2) in fertilization and embryonic and larval development of the tropical sea urchin Lytechinus variegatus.

The release of nanomaterials into the environment is the cause of an emerging concern. Titanium dioxide nanoparticles (nano-TiO2) among the most produced nanomaterials, has been documented in marine coastal areas posing a threat on marine biota. Sea urchin embryos are recognized as suitable bioindicators in ecological risk assessment and recently for nanomaterials. This study investigated the impact of nano-TiO2 on fertilization, embryonic and larval development of the tropical sea urchin Lytechinus variegatus in a range of concentrations (0.005-5 µg/mL) which includes environmentally relevant ones. The behavior of nano-TiO2 in tropical natural seawater was determined by dynamic light scattering (DLS) and toxicity was evaluated through fertilization and embryotoxicity tests, and morphological/morphometric analyses of sea urchin's larvae. Limited toxicity was recorded for nano-TiO2 in tropical sea urchin embryos and larvae, except for effects at the gastrula stage at 0.005 µg/mL. Large agglomerates of nano-TiO2 (5 µg/mL) were observed adhering onto sea urchin larvae thus probably preventing nanoparticles uptake at the highest concentrations (>0.005 µg/mL). Environmental levels of nano-TiO2 are able to cause toxicity on tropical sea urchin L. variegatus embryos with potential consequences on populations and their ecological role in tropical coastal areas.

Functional annotation of a hugely expanded nanos repertoire in Lytechinus variegatus, the green sea urchin.

Nanos genes encode essential RNA-binding proteins involved in germline determination and germline stem cell maintenance. When examining diverse classes of echinoderms, typically three, sometimes four, nanos genes are present. In this analysis, we identify and annotate nine nanos orthologs in the green sea urchin, Lytechinus variegatus (Lv). All nine genes are transcribed and grouped into three distinct classes. Class one includes the germline Nanos, with one member: Nanos2. Class two includes Nanos3-like genes, with significant sequence similarity to Nanos3 in the purple sea urchin, Strongylocentrotus purpuratus (Sp), but with wildly variable expression patterns. The third class includes several previously undescribed nanos zinc-finger genes that may be the result of duplications of Nanos2. All nine nanos transcripts occupy unique genomic loci and are expressed with unique temporal profiles during development. Importantly, here we describe and characterize the unique genomic location, conservation, and phylogeny of the Lv ortholog of the well-studied Sp Nanos2. However, in addition to the conserved germline functioning Nanos2, the green sea urchin appears to be an outlier in the echinoderm phyla with eight additional nanos genes. We hypothesize that this expansion of nanos gene members may be the result of a previously uncharacterized L1-class transposon encoded on the opposite strand of a nanos2 pseudogene present on chromosome 12 in this species. The expansion of nanos genes described here represents intriguing insights into germline specification and nanos evolution in this species of sea urchin.

Assessment of sediment porewater toxicity in Biscayne National Park with sea urchin (Lytechinus variegatus) embryos.

The sea urchin embryo development toxicity test was used to investigate toxicity of the benthic substrate in Biscayne National Park (BISC). Twenty-five sites were selected based upon a high potential for anthropogenic stressor input (e. g., hydrocarbons, personal care products, nutrients, etc.) or proximity to coral reef habitats. We found that sediment interstitial water (porewater) was toxic to urchin embryos at 22 of 25 sites. Healthy sites included two coral reefs (Anniversary Reef and Marker 14 Reef) and Turkey Point Channel. Discrete areas of BISC have highly toxic sediments and the presence of sediment contaminants could negatively impact reproduction, growth and population density of benthic invertebrates, such as corals. Results of the sea urchin embryo development toxicity test can be used as a baseline assessment for monitoring improvements or degradation in ecosystem health and could be a valuable tool to investigate the suitability of degraded habitats for future reef restoration. Since the last comprehensive environmental assessment of BISC was performed in 1999, further investigation into the sources of toxicity at BISC is needed.

Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus.

Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

Microbial Composition and Genes for Key Metabolic Attributes in the Gut Digesta of Sea Urchins Lytechinus variegatus and Strongylocentrotus purpuratus Using Shotgun Metagenomics.

This paper describes the microbial community composition and genes for key metabolic genes, particularly the nitrogen fixation of the mucous-enveloped gut digesta of green (Lytechinus variegatus) and purple (Strongylocentrotus purpuratus) sea urchins by using the shotgun metagenomics approach. Both green and purple urchins showed high relative abundances of Gammaproteobacteria at 30% and 60%, respectively. However, Alphaproteobacteria in the green urchins had higher relative abundances (20%) than the purple urchins (2%). At the genus level, Vibrio was dominant in both green (~9%) and purple (~10%) urchins, whereas Psychromonas was prevalent only in purple urchins (~24%). An enrichment of Roseobacter and Ruegeria was found in the green urchins, whereas purple urchins revealed a higher abundance of Shewanella, Photobacterium, and Bacteroides (q-value < 0.01). Analysis of key metabolic genes at the KEGG-Level-2 categories revealed genes for amino acids (~20%), nucleotides (~5%), cofactors and vitamins (~6%), energy (~5%), carbohydrates (~13%) metabolisms, and an abundance of genes for assimilatory nitrogen reduction pathway in both urchins. Overall, the results from this study revealed the differences in the microbial community and genes designated for the metabolic processes in the nutrient-rich sea urchin gut digesta, suggesting their likely importance to the host and their environment.

Developmental single-cell transcriptomics in the Lytechinus variegatus sea urchin embryo.

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.

Colorless spherule cells and lysozyme contribute to innate immunological responses in the sea urchin Lytechinus variegatus, exposed to bacterial challenge.

The sea urchin Lytechinus variegatus is considered a good candidate for aquaculture, but bacterial diseases are a major challenge in culture conditions. The innate immunological defenses of L. variegatus to bacterial challenges were assessed through hematology parameters, in vitro phagocytosis, lysozyme activity and total plasma protein concentrations in cell-free coelomic fluid. Adult sea urchins were inoculated with Microccocus lysodeikticus, Escherichia coli and Vibrio parahaemolyticus in the cavity coelomic. Filtrated and sterile seawater (FSW) injected and non-injected sea urchins were used as control groups. Righting time, external aspects and behavior of sea urchins were evaluated. Twenty-four hours post-inoculation, we found an increase in the population of colorless spherule cells (CLS), phagocytosis, and humoral responses in sea urchins challenged by bacterial inoculations. Righting time was not affected by the treatments and apparent external signs of disease were not observed at least during 96h post-inoculation. The immunological system of L. variegatus quickly eliminated pathogenic microorganisms. CLS and lysozyme activity cooperate in the immune defenses of L. variegatus, showing an extraordinary efficiency for adjusting the immune defenses under stress caused by microbes. We recommend that the cellular and humoral markers serve as routine tests to monitor health status in sea urchins.

Glyconectin Cell Adhesion Epitope, ß-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos.

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 ß-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

New techniques for creating parthenogenetic larvae of the sea urchin Lytechinus pictus for gene expression studies.

BACKGROUND: Sea urchins are model organisms for studying the spatial-temporal control of gene activity during development. The Southern California species, Lytechinus pictus, has a sequenced genome and can be raised in the laboratory from egg to egg in 4 to 5 months. RESULTS: Here, we present new techniques for generating parthenogenetic larvae of this species and include a gallery of photomicrographs of morphologically abnormal larvae that could be used for transcriptomic analysis. CONCLUSIONS: Comparison of gene expression in parthenogenotes to larvae produced by fertilization could provide novel insights into gene expression controls contributed by sperm in this important model organism. Knowledge gained from transcriptomics of sea urchin parthenogenotes could contribute to parthenogenetic studies of mammalian embryos.

Impacts of fishing on the Caribbean white sea urchin, Lytechinus variegatus, in Margarita Island, Venezuela.

The Caribbean white sea urchin, L. variegatus, is locally harvested in Margarita Island and other locations of southeastern Venezuela. The recent reduction of densities raises concerns about potential impacts for overfishing. Densities of L. variegatus were estimated at Impact and Control locations between late-2012 and mid-2015 to 1) test temporal changes, comparing with 1997-1998 estimations, 2) the effect of seasonal closure on resource recovery and 3) the effect of local expansion. The results suggest that, after a period of fourteen years, an important decrease in L. variegatus densities occurred, as well as a lack of population recovery in fishing areas during seasonal closures. Furthermore, during the monitoring program, it was observed fishing activities in one Control location that subsequently showed clear patterns of population depletion, like other Impact locations. Fishing impact on L. variegatus populations is severe, persistent and expands over time without evidence of recovery, therefore it is expected that local populations of L. variegatus would collapse under current exploitation levels. However, due to the L. variegatus life history and its distribution range, recovery of impacted populations could be possible via recruitment of planktonic larvae if effective management actions are imposed. It is recommended to exercise more regulations on fishing activities and to execute management measures that allow recovering the stocks to maintain local populations of sea urchin.

Chromosomal-Level Genome Assembly of the Painted Sea Urchin Lytechinus pictus: A Genetically Enabled Model System for Cell Biology and Embryonic Development.

The painted urchin Lytechinus pictus is a sea urchin in the family Toxopneustidae and one of several sea urchin species that are routinely used as an experimental research organism. Recently, L. pictus has emerged as a tractable model system for establishing transgenic sea urchin lines due to its amenability to long term laboratory culture. We present the first published genome of L. pictus. This chromosomal-level assembly was generated using Illumina sequencing in conjunction with Oxford Nanopore Technologies long read sequencing and HiC chromatin conformation capture sequencing. The 998.9-Mb assembly exhibits high contiguity and has a scaffold length N50 of 46.0 Mb with 97% of the sequence assembled into 19 chromosomal-length scaffolds. These 19 scaffolds exhibit a high degree of synteny compared with the 19 chromosomes of a related species Lytechinus variegatus. Ab initio and transcript evidence gene modeling, combined with sequence homology, identified 28,631 gene models that capture 92% of BUSCO orthologs. This annotation strategy was validated by manual curation of gene models for the ABC transporter superfamily, which confirmed the completeness and accuracy of the annotations. Thus, this genome assembly, in conjunction with recent high contiguity assemblies of related species, positions L. pictus as an exceptional model system for comparative functional genomics and it will be a key resource for the developmental, toxicological, and ecological biology scientific communities.

An early global role for Axin is required for correct patterning of the anterior-posterior axis in the sea urchin embryo.

Activation of Wnt/ß-catenin (cWnt) signaling at the future posterior end of early bilaterian embryos is a highly conserved mechanism for establishing the anterior-posterior (AP) axis. Moreover, inhibition of cWnt at the anterior end is required for development of anterior structures in many deuterostome taxa. This phenomenon, which occurs around the time of gastrulation, has been fairly well characterized, but the significance of intracellular inhibition of cWnt signaling in cleavage-stage deuterostome embryos for normal AP patterning is less well understood. To investigate this process in an invertebrate deuterostome, we defined Axin function in early sea urchin embryos. Axin is ubiquitously expressed at relatively high levels in early embryos and functional analysis revealed that Axin suppresses posterior cell fates in anterior blastomeres by blocking ectopic cWnt activation in these cells. Structure-function analysis of sea urchin Axin demonstrated that only its GSK-3ß-binding domain is required for cWnt inhibition. These observations and results in other deuterostomes suggest that Axin plays a crucial conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates.

Toposome: Source of antimicrobial molecules in the gonads of the sea urchin Lytechinus variegatus (Lamarck, 1816).

Sea urchins live in a challenging environment that requires rapid and efficient responses against pathogens and invaders. This response may be also important in reproductive processes once males and females release their gametes into water. In addition, the gonads are organs with dual function: reproductive organ and nutrient reserve, therefore it needs efficient protective mechanisms to preserve the nutrients as well as the reproductive cells. The aim of this study was to evaluate the presence and characterize antimicrobial molecules in the male and female gonads of the sea urchin Lytechinus variegatus. Through HPLC purification, antimicrobial activity test and mass spectrometry several antimicrobial molecules were found in the gonads of both gender. Computational in silico analyses showed that they are fragments of a glycoprotein called toposome, also known as major yolk protein (MYP) which is one of the major proteins found in the gonads. Although different functions have been reported for this protein, this is the first description of a direct antimicrobial activity in Lytechinus variegatus. The results indicate that when undergoing proteolysis the toposome generates different fragments with antimicrobial activity which may indicate the importance of a rapid defense response strategy against invading microorganisms in the gonads used by both males and females sea urchins.

Comparative study of coelomocytes from Arbacia lixula and Lythechinus variegatus: Cell characterization and in vivo evidence of the physiological function of vibratile cells.

The knowledge on echinoderm coelomocytes has increased in recent years, but researchers still face a complex problem: how to obtain purified cells. Even flow cytometry being useful to address coelomocytes in suspension, the need for a method able to provide isolated cells is still noteworthy. Here, we use Imaging Flow Cytometry (IFC) to characterize the coelomocytes of two sea urchin species - Arbacia lixula and Lytechinus variegatus - and obtain gates to isolate cell populations. Then, we used these gates to study the physiological response of A. lixula coelomocytes during an induced immune challenge with Escherichia coli. An analysis of area and aspect ratio parameters of the flow cytometer allowed the identification of two main cell populations in the coelomic fluid: circular and elongated cells. A combination of this method with nucleus labeling using propidium iodide allowed the determination of gates containing isolated subpopulations of vibratile cells, red spherulocytes, and two phagocytes subpopulations in both species. We observed that during an induced bacterial immune challenge, A. lixula was able to modulate coelomocyte frequencies, increasing the phagocytes and decreasing red spherulocytes and vibratile cells. These results indicate that vibratile cells and red spherulocytes act by immobilizing and stoping bacterial growth, respectively, cooperating with phagocytes in the immune response. The use of IFC was fundamental not only to identify specific gates for the main coelomic subpopulations but also allowed the investigation on how echinoids modulate their physiological responses during immune challenges. Furthermore, we provide the first experimental evidence about the role of vibratile cells, corroborating its involvement with the immune system.

Embryo, larval, and juvenile staging of Lytechinus pictus from fertilization through sexual maturation.

BACKGROUND: Sea urchin embryos have been used for more than a century in the study of fertilization and early development. However, several of the species used, such as Strongylocentrotus purpuratus, have long generation times making them suboptimal for transgenerational studies. RESULTS: Here, we present an overview of the development of a rapidly developing echinoderm species, Lytechinus pictus, from fertilization through sexual maturation. When grown at room temperature (20°C) embryos complete the first cell cycle in 90 minutes, followed by subsequent cleavages every 45 minutes, leading to hatching at 9 hours postfertilization (hpf). The swimming embryos gastrulate from 12 to 36 hpf and produce the cells which subsequently give rise to the larval skeleton and immunocytes. Larvae begin to feed at 2 days and metamorphose by 3 weeks. Juveniles reach sexual maturity at 4 to 6 months of age, depending on individual growth rate. CONCLUSIONS: This staging scheme lays a foundation for future studies in L. pictus, which share many of the attractive features of other urchins but have the key advantage of rapid development to sexual maturation. This is significant for multigenerational and genetic studies newly enabled by CRISPR-CAS mediated gene editing.

Genomic insights of body plan transitions from bilateral to pentameral symmetry in Echinoderms.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.

Choosing trash instead of nature: Sea urchin covering behavior.

Notwithstanding impacts of marine debris on fauna by ingestion and suffocation, little is known about debris-related behavior. Lytechinus variegatus is a common sea urchin known for its covering behavior. We hypothesized that L. variegatus would select more marine debris (i.e. litter) than natural material as cover and we also expected that the selected natural and artificial material would be different in weight, sizes and transparency. We haphazardly collected marine debris and natural material on 20 individuals of L. variegatus and on the bottom, around each individual. All sampled material was weighed, measured and classified regarding opacity, nature (natural or artificial). Our results showed that i) sea urchins picked more litter than natural objects, ii) proportional weight of litter carried by urchins was significantly larger than expected by chance, iii) when considering all objects (on urchins and on the bottom) litter was heavier, wider and less opaque than natural material and iv) litter carried by the urchins were wider and less opaque than natural material. We suggest that litter can influence urchin's protection against sunlight, camouflage and ballast and that sea urchins with covering behavior might be used as indicators of marine debris in coastal and deep waters.