Strongylocentrotus Purpuratus Research Articles

Energetic context determines the effects of multiple upwelling-associated stressors on sea urchin performance

Globally, kelp forests are threatened by multiple stressors, including increasing grazing by sea urchins. With coastal upwelling predicted to increase in intensity and duration in the future, understanding whether kelp forest and urchin barren urchins are differentially affected by upwelling-related stressors will give insight into how future conditions may affect the transition between kelp forests and barrens. We assessed how current and future-predicted changes in the duration and magnitude of upwelling-associated stressors (low pH, dissolved oxygen, and temperature) affected the performance of purple sea urchins (Strongylocentrotus purpuratus) sourced from rapidly-declining bull kelp (Nereocystis leutkeana) forests and nearby barrens and maintained on habitat-specific diets. Kelp forest urchins were of superior condition to barrens urchins, with ~ 6–9 times more gonad per body mass. Grazing and condition in kelp forest urchins were more negatively affected by distant-future and extreme upwelling conditions, whereas grazing and survival in urchins from barrens were sensitive to both current-day and all future-predicted upwelling, and to increases in acidity, hypoxia, and temperature regardless of upwelling. We conclude that urchin barren urchins are more susceptible to increases in the magnitude and duration of upwelling-related stressors than kelp forest urchins. These findings have important implications for urchin population dynamics and their interaction with kelp.

Identification of miRNAs in sea urchin Strongylocentrotus purpuratus larvae response to pH stress

MicroRNAs (miRNAs) are a class of small, endogenous, non-coding RNAs that regulate gene expression through transcriptional repression of messenger RNA. They play significant roles in many physiological and biochemical processes in eukaryotes. Ocean acidification can impact the development, survival, growth and physiology of many marine organisms. Here, we performed miRNA transcriptome analysis of the sea urchin Strongylocentrotus purpuratus larvae exposed to CO2-driven seawater acidification. We generated 10.6 and 10.8 million clean reads from the malformed S. purpuratus larva after CO2 treatment and the larvae with the normal bone development respectively. A total of 682 conserved and 17 novel miRNAs were identified. Target genes of the differential expression miRNAs were also predicted, which contained growth-related genes (collagenase, collagen and HSP70-binding protein 1), spicule formation-related gene (carbonic anhydrase transcript variant X1) and skeletogenesis-related genes (breast carcinoma amplified sequence 2). Target genes of the differentially expressed miRNAs were used to perform KEGG pathway analysis and were found to be involved in the proteasome and oxidative phosphorylation. These results provide a relatively large number of miRNAs transcriptome resource and provide a foundation for further analyses on the functional and molecular mechanisms of S. purpuratus larvae impacted by ocean acidification.

Variation in purple sea urchin (Strongylocentrotus purpuratus) morphological traits in relation to resource availability.

Flexible resource investment is a risk sensitive reproductive strategy where individuals trade resources spent on reproduction for basic metabolic maintenance and survival. This study examined morphological variation in herbivorous sea urchin grazers across a mosaic landscape of macroalgae dominated habitats interspersed with patches of sea urchin barrens to determine whether sea urchins shift energy allocation in response to food limitation. Extensive underwater surveys of habitat attributes (e.g., sea urchin density, algae cover) were paired with detailed laboratory assays (e.g., sea urchin dissections) to determine how resource abundance affects energy allocation between reproductive capacity and body structure in the purple sea urchin, Strongylocentrotus purpuratus. We found that: (1) sea urchins had a more elongate jaw structure relative to body size in habitats void of macroalgae (i.e., barrens), (2) sea urchin reproductive capacity (i.e., gonad index) was lower in barrens and the barrens habitat was primarily comprised of encrusting algae, and (3) sea urchin jaw morphology (i.e., lantern index) and reproductive capacity (i.e., gonad index) were inversely related. These results suggest that sea urchins respond to macroalgae limited environments by shifting energy allocation between reproductive capacity and modifications of the foraging apparatus, which may explain the ability of sea urchins to acquire food in resource-limited environments.

Complexation reduces nickel toxicity to purple sea urchin embryos (Strongylocentrotus purpuratus), a test of biotic ligand principles in seawater

The potential for Ni toxicity in seawater is of concern because of mining and processing activities in coastal regions. Determining Ni speciation is vital to understanding and predicting Ni toxicity and for bioavailability-based nickel risk assessment. The goal of this study was to characterize the complexation of Ni in relation to toxicity using embryological development of purple sea urchin (S. purpuratus). It was predicted that free ion [Ni2+] would be a better predictor of toxicity than total dissolved Ni concentrations (NiD). Synthetic ligands with known logKf values (Ethylenediaminetetraacetic acid (EDTA), Nitrilotriacetic acid (NTA), tryptophan (TRP), glutamic acid (GA), histidine (HD), and citric acid (CA)) were used to test the assumptions of the biotic ligand model (BLM) for Ni in seawater. [NiD] was measured by graphite furnace atomic absorption spectroscopy (GFAAS) and Ni2+ was first quantified using the ion-exchange technique (IET) and then concentrations were measured by GFAAS; [Ni2+] was also estimated using aquatic geochemistry modelling software (Visual Minteq). The mean EC50 values for [NiD] in unmodified artificial seawater control was 3.6 µM (95% CI 3.0–4.5) [211 µg/L 95% CI 176–264] and the addition of ligands provided protection, up to 6.5-fold higher [NiD] EC50 for EDTA. Compared to the control, measured EC50 values based on total dissolved nickel were higher in the presence of ligands. As predicted by BLM theory, [Ni2+] was a better predictor of Ni toxicity with 17% variability in EDTA and CA media while there was 72% variability in the prediction of Ni toxicity with total dissolved Ni. The results of this research provide support for the application of BLM- based prediction models for estimating Ni impacts in seawater.

Sea urchin microbiomes vary with habitat and resource availability

AbstractSea urchins are key grazers in coastal seas, where they can survive a variety of conditions and diets, enhancing their ecological impact on kelp forests and other ecosystems. Using 16S rRNA gene sequencing, we characterized bacterial communities associated with guts of the two dominant sea urchin species in southern California, the red urchin Mesocentrotus franciscanus, and the purple urchin Strongylocentrotus purpuratus. Our results show that the two urchin species have distinct gut microbiomes that vary with habitat. The taxonomic composition of their microbiomes suggests that they may facilitate digestion of food and be a source of nutrition themselves. These results highlight the role of microbiomes within macroorganisms as an extended ecological trait, and suggest that microbes may be crucial to resource use and partitioning in co‐occurring species.

Na+/H+ exchangers differentially contribute to midgut fluid sodium and proton concentration in the sea urchin larva.

Regulation of ionic composition and pH is a requisite of all digestive systems in the animal kingdom. Larval stages of the marine superphylum Ambulacraria, including echinoderms and hemichordates, were demonstrated to have highly alkaline conditions in their midgut with the underlying epithelial transport mechanisms being largely unknown. Using ion-selective microelectrodes, the present study demonstrated that pluteus larvae of the purple sea urchin have highly alkaline pH (pH∼9) and low [Na+] (∼120 mmol l-1) in their midgut fluids, compared with the ionic composition of the surrounding seawater. We pharmacologically investigated the role of Na+/H+ exchangers (NHE) in intracellular pH regulation and midgut proton and sodium maintenance using the NHE inhibitor 5-(n-ethyl-n-isopropyl)amiloride (EIPA). Basolateral EIPA application decreased midgut pH while luminal application via micro-injections increased midgut [Na+], without affecting pH. Immunohistochemical analysis demonstrated a luminal localization of NHE-2 (SpSlc9a2) in the midgut epithelium. Specific knockdown of spslc9a2 using Vivo-Morpholinos led to an increase in midgut [Na+] without affecting pH. Acute acidification experiments in combination with quantitative PCR analysis and measurements of midgut pH and [Na+] identified two other NHE isoforms, Spslc9a7 and SpSlc9a8, which potentially contribute to the regulation of [Na+] and pH in midgut fluids. This work provides new insights into ion regulatory mechanisms in the midgut epithelium of sea urchin larvae. The involvement of NHEs in regulating pH and Na+ balance in midgut fluids shows conserved features of insect and vertebrate digestive systems and may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.

CRISPR/Cas9 mutagenesis reveals a role for ABCB1 in gut immune responses to Vibrio diazotrophicus in sea urchin larvae.

The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host-microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment.

Chronic Toxicity of Cobalt to Marine Organisms: Application of a Species Sensitivity Distribution Approach to Develop International Water Quality Standards.

Water quality standards for cobalt (Co) have yet to be developed for the European Union or the United States. The primary objective of the present study was to produce a data set comprising marine Co toxicity data that could be used by both the European Union and the United States to determine a predicted-no-effect concentration and ambient water quality criteria, respectively. Ten marine species, ranging from algae to fish, were subjected to chronic Co toxicity tests that were designed to meet international water quality testing standards. Chronic 10% effect concentration values ranged from a low of 1.23 µg dissolved Co/L for red algae (Champia parvula) to a high of 31 800 µg dissolved Co/L for sheepshead minnow (Cyprinodon variegatus). The species sensitivity ranking for chronic marine Co exposure was as follows (from most to least sensitive): C. parvula > Neanthes arenaceodentata (polychaete) > Americamysis bahia (mysid shrimp) > Skeletonema costatum (marine diatom) > Dendraster excentricus (sand dollar) > Mytilus galloprovincialis (mussel) > Strongylocentrotus purpuratus (purple sea urchin) > Crassostrea gigas (oyster) > Dunaliella tertiolecta (marine flagellate) > C. variegatus. Chronic test results indicated that invertebrate and plant species were substantially more sensitive to Co exposure than fish. The chronic toxicity data were used to calculate a species sensitivity distribution, from which a hazardous concentration 5th percentile of 7.09 µg dissolved Co/L (95% CI 0.025-47.3 µg Co/L) was derived. Environ Toxicol Chem 2021;40:1405-1418. © 2021 SETAC.

Pulsed exposure toxicity testing: Baseline evaluations and considerations using copper and zinc with two marine species

Methods to assess environmental impacts from episodic discharges on receiving water bodies need a more environmentally relevant and scientifically defensible toxicity test design. Many permittees are regularly required to conduct 96-h toxicity tests on discharges associated with events that are generally less than 24 h in duration. Current standardized methods do not adequately reflect these episodic discharge conditions at either the point of compliance nor as it mixes with the receiving environment. In order to evaluate more representative biological effects, an alternative toxicity approach is described incorporating pulsed exposures of effluents and subsequent transfer of test organisms to clean water for the remainder of the test. This pulsed exposure protocol incorporates a slight modification to USEPA Whole Effluent Toxicity (WET) chronic and acute methods for two marine species, purple sea urchin embryos, Strongylocentrotus purpuratus, and juvenile mysid shrimp Americamysis bahia. Tests were performed with toxicants using standard static (96 h) and pulsed (6, 12, and 26 h) exposures. Following pulsed exposures, organisms were transferred to uncontaminated seawater for the remainder of the 96-h test period. Results for these species and endpoints indicated that the sensitivity of these species to copper and zinc were up to two orders of magnitude greater using standard continuous exposures compared to shorter pulsed exposures. Additional considerations assessed included timing of the onset of a pulse and latent effects following an exposure. This modified approach requires minimal modification to current standard methods and increases the realism to more accurately assess toxic effects resulting from episodic discharges.

Experimental assessment of the impacts of ocean acidification and urchin grazing on benthic kelp forest assemblages

Ocean acidification (OA) is likely to differentially affect the biology and physiology of calcifying and non-calcifying taxa, thereby potentially altering key ecological interactions (e.g., facilitation, competition, predation) in ways that are difficult to predict from single-species experiments. We used a two-factor experimental design to investigate how multispecies benthic assemblages in southern California kelp forests respond to OA and grazing by the purple sea urchin, Strongylocentrotus purpuratus. Settlement tiles accrued natural mixed assemblages of algae and invertebrates in a kelp forest off San Diego, CA for one year before being exposed to OA and grazing in a laboratory experiment for two months. Space occupying organisms were identified and pooled into six functional groups: calcified invertebrates, non-calcified invertebrates, calcified algae, fleshy algae, sediment, and bare space for subsequent analyses of community structure. Interestingly, communities that developed on separate tile racks were unique, despite being deployed close in space, and further changes in community structure in response to OA and grazing depended on this initial community state. On Rack 1, we found significant effects of both pCO2 and grazing with elevated pCO2 increasing cover of fleshy algae, but sea urchin grazers decreasing cover of fleshy algae. On Rack 2, we found a ~ 35% higher percent cover of sediment on tiles reared in ambient pCO2 but observed ~27% higher cover of bare space in the high pCO2 conditions. On Rack 3, we found an average of 45% lower percent cover of calcified sessile invertebrates at ambient pCO2 than in high pCO2 treatments on Rack 3. Net community calcification was 137% lower in elevated pCO2 treatments. Kelp sporophyte densities on tiles without urchins were 74% higher than on tiles with urchins and kelp densities were highest in the elevated pCO2 treatment. Urchin growth and grazing rates were 49% and 126% higher under ambient than high pCO2 conditions. This study highlights consistent negative impacts of OA on community processes such as calcification and grazing rates, even though impacts on community structure were highly context-dependent.

Behavioral responses across a mosaic of ecosystem states restructure a sea otter–urchin trophic cascade

Consumer and predator foraging behavior can impart profound trait-mediated constraints on community regulation that scale up to influence the structure and stability of ecosystems. Here, we demonstrate how the behavioral response of an apex predator to changes in prey behavior and condition can dramatically alter the role and relative contribution of top-down forcing, depending on the spatial organization of ecosystem states. In 2014, a rapid and dramatic decline in the abundance of a mesopredator (Pycnopodia helianthoides) and primary producer (Macrocystis pyrifera) coincided with a fundamental change in purple sea urchin (Strongylocentrotus purpuratus) foraging behavior and condition, resulting in a spatial mosaic of kelp forests interspersed with patches of sea urchin barrens. We show that this mosaic of adjacent alternative ecosystem states led to an increase in the number of sea otters (Enhydra lutris nereis) specializing on urchin prey, a population-level increase in urchin consumption, and an increase in sea otter survivorship. We further show that the spatial distribution of sea otter foraging efforts for urchin prey was not directly linked to high prey density but rather was predicted by the distribution of energetically profitable prey. Therefore, we infer that spatially explicit sea otter foraging enhances the resistance of remnant forests to overgrazing but does not directly contribute to the resilience (recovery) of forests. These results highlight the role of consumer and predator trait-mediated responses to resource mosaics that are common throughout natural ecosystems and enhance understanding of reciprocal feedbacks between top-down and bottom-up forcing on the regional stability of ecosystems.

After 15 years, no evidence for trophic cascades in marine protected areas.

In marine ecosystems, fishing often targets predators, which can drive direct and indirect effects on entire food webs. Marine reserves can induce trophic cascades by increasing predator density and body size, thereby increasing predation pressure on populations of herbivores, such as sea urchins. In California's northern Channel Islands, two species of sea urchins are abundant: the red urchin Mesocentrotus franciscanus, which is targeted by an economically valuable fishery, and the virtually unfished purple urchin Strongylocentrotus purpuratus. We hypothesized that urchin populations inside marine reserves would be depressed by higher predation, but that red urchins would be less affected due to fishing outside reserves. Instead, our analyses revealed that purple urchin populations were unaffected by reserves, and red urchin biomass significantly increased in response to protection. Therefore, urchin biomass overall has increased inside reserves, and we found no evidence that giant kelp is positively affected by reserves. Our results reveal the overwhelming direct effect of protecting fished species in marine reserves over indirect effects that are often predicted but seldom clearly documented. Indirect effects due to marine reserves may eventually occur in some cases, but very effective predators, large reserves or extended time periods may be needed to induce them.

Aquatic toxicity evaluations of PFOS and PFOA for five standard marine endpoints

Per- and poly-fluoroalkyl substances (PFAS) are emerging contaminants that are coming under increasing scrutiny. Currently, there is a paucity of effects data for marine aquatic life, limiting the assessment of ecological risks and compliance with water quality policies. In the present study, the toxicity of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) to four standard marine laboratory toxicity testing species, encompassing five endpoints, were evaluated: 1) 96-h embryo-larval normal development for the purple sea urchin (Strongylocentrotus purpuratus); 2) 48-h embryo-larval normal development and normal survival for the Mediterranean mussel (Mytilus galloprovincialis); 3) 96-h survival of opossum shrimp (Americamysis bahia); and 4) 24-h light output for the bioluminescent dinoflagellate Pyrocystis lunula. All species were tested using standard United States Environmental Protection Agency (USEPA) and/or American Society for Testing and Materials (ASTM) International protocols. For PFOS and PFOA, the order of species sensitivity, starting with the most sensitive, was M. galloprovincialis, S. purpuratus, P. lunula, and A. bahia. The range of median lethal or median effect concentrations for PFOS (1.1–5.1 mg L−1) and PFOA (10–24 mg L−1) are comparable to the relatively few toxicity effect values available for marine species. In addition to providing effects data for PFOA and PFOS, this study indicates these species and endpoints are sensitive to PFAS such that their use will be appropriate for deriving toxicity data with other PFAS in marine ecosystems.

Sea Urchin as a Universal Model for Studies of Gene Networks.

The purple sea urchin Strongylocentrotus purpuratus has been used for over 150 years as a model organism in developmental biology. Using this model species, scientists have been able to describe, in detail, the mechanisms of cell cycle control and cell adhesion, fertilization, calcium signaling, cell differentiation, and death. Massive parallel sequencing of the sea urchin genome enabled the deciphering of the main components of gene regulatory networks during the activation of embryonic signaling pathways. This knowledge helped to extrapolate aberrations in somatic cells that may lead to diseases, including cancer in humans. Furthermore, since many, if not all, developmental signaling pathways were shown to be controlled by non-coding RNAs (ncRNAs), the sea urchin organism represents an attractive experimental model. In this review, we discuss the main discoveries in the genetics, genomics, and transcriptomics of sea urchins during embryogenesis with the main focus on the role of ncRNAs. This information may be useful for comparative studies between different organisms, and may help identify new regulatory networks controlled by ncRNAs.

CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin

We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-β signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.

Modularity and hierarchy in biological systems: Using gene regulatory networks to understand evolutionary change.

Modularity and hierarchy are important theoretical concepts in biology, and both are useful frameworks to understand the evolution of complex systems. Gene regulatory networks (GRNs) provide a powerful mechanistic model for modularity in animal development, as they are made up of modular (or self-contained) circuits, which are deployed in a hierarchical manner over time. Over the years, studies in the sea urchin, Strongylocentrotus purpuratus, have provided an illustrative example of how these regulatory circuits are responsible for processes such as cell differentiation and cell state specificity. However, GRNs are themselves made up of a nested series of interactions, as each gene can be regulated by multiple cis-regulatory elements, which can be further broken down into distinct transcription factor binding sites (TFBS). As a result, modularity can be applied to each "level" of this complex hierarchy. Throughout the literature, there is considerable discussion about the roles modular circuits, modular enhancers, and modular TFBS play in evolution, yet there is little discussion about how these nested interactions operate as a whole. In this chapter, we discuss how modular changes at different levels of the GRN hierarchy affect animal development and aim to provide a unified framework to understand the role of modularity in evolution.

Seasonal Growth of the Purple Sea Urchin Heliocidaris crassispina Revealed by Sequential Fluorochrome Tagging.

Many studies have applied fluorochrome tagging to examine the growth of animals with calcified skeletons, but most of them have used only a single tag to determine the annual growth rate. We used sequential fluorochrome tagging to study the seasonal growth of the purple sea urchin Heliocidaris crassispina in Hong Kong waters from February 2012 to February 2013. Sea urchins ranging from 18.9 to 42.7 mm in test diameter had a yearly growth from 0.6 to 13.0 mm. During that year, the sea urchins grew from 0.6 to 5.0 mm in test diameter during the first six months, and from 0.4 to 10.2 mm in test diameter in the second six months. The seasonal differences in growth were confirmed using the von Bertalanffy model. The growth was clear for young sea urchins, especially for individuals less than 5 years old, but was not evident for sea urchins older than 7 years. The seasonal differences in growth were probably related to the reproductive cycle and the seasonal differences in environmental conditions. Our empirical results provide the first evidence of seasonal changes in growth for H. crassispina, demonstrating the usefulness of sequential fluorochrome tagging in studying the growth of sea urchins in the field. We also identify the problem of low recovery of tagged individuals and provide recommendations to improve the tagging procedure.

Effects of dead conspecifics, hunger states, and seasons on the foraging behavior of the purple urchin Heliocidaris crassispina

Trophic cascades are a powerful result of predator-prey relationships in an ecosystem. In aquatic environments, the signals associated with predators and predation are used by prey as a cue to avoid encountering predators when foraging for food. These behavioral cues can be powerful enough to control prey populations and indirectly protect primary producers. Here, we evaluated the effects of cues associated with predation on the purple urchin Heliocidaris crassispina and examined effects of hunger state and season, using time-lapse photography. A series of laboratory and in situ manipulative experiments were conducted to determine patterns of foraging behavior and behavioral modifications. We showed that starved urchins were less sensitive to predation cues compared to normally fed urchins. Field experiments indicated that 70% of fed urchins fled when exposed to a predation cue (presence of a dead urchin) whereas starved urchins remained regardless of the cue, supporting results from the laboratory using dead urchin and algal cues. Sea urchin activity and feeding rates were lower in winter-spring than in summer-autumn. Results suggest that hunger state has a large influence over the behavioral response of sea urchins, while also being affected by season due to metabolic control. In general, starvation appears to override predator avoidance behaviors, which exposes prey species to higher risks of predation.

Influence of environmentally relevant concentrations of Zn, Cd and Ni and their binary mixtures on metal uptake, bioaccumulation and development in larvae of the purple sea urchin Strongylocentrotus purpuratus

Metal accumulation, disturbance of Ca2+ homeostasis, and occurrence of abnormalities are well-established consequences of single metal exposure during early development stages of sea urchins. However, the effects caused by low concentrations of metals and metal mixtures need to be better understood in marine invertebrates. Therefore, the present study investigated the effects of environmentally relevant concentrations of Zn (9 μg/L), Cd (30 μg/L) and Ni (5 μg/L) in single and binary exposures (Zn + Cd, Cd + Ni and Ni + Zn) to the early life stages of the purple sea urchin Strongylocentrotus purpuratus. Endpoints checked in all treatments after 48-h exposure were unidirectional metal influx rates, bioaccumulation, and Ca2+ influx rates. Additionally, the presence of abnormal larvae and developmental delay was evaluated at 24 h, 48 h and 72 h of exposure. Unidirectional influx rates of all three metals were significantly higher than control background rates in all single exposures and binary mixtures, and were generally not different between them. Net accumulation (body burden) of both Zn and Cd increased significantly as a result of their respective single exposures, while Ni accumulation decreased considerably. When Zn or Cd were presented in binary exposures with other metals, the net accumulations of Zn or Cd were reduced relative to single exposures to these metals, whereas this did not occur for Ni accumulation. Thus, bioaccumulation proved to be a better metric than influx rate measurements to analyze metal competition at a whole organism level at these low metal concentrations. Short-term Ca2+ influx also did not appear to be a sensitive metric, as the measured rates did not vary among all single and binary exposures, with the exception of a lower rate in Ni + Zn binary exposure. A critical aspect observed was the relationship between bioaccumulation versus influx measurements, which proved positive for Cd, but negative for Zn and Ni, demonstrating possible capacities for both Zn and Ni regulation by sea urchin larvae. Increases in larval abnormalities relative to controls occurred only after binary mixtures, starting at 48 h exposure and maintained until 72 h. However, delay of the sea urchin development by the presence of gastrula stage at 72 h exposure occurred in Zn and Ni single exposures and all metal mixtures, with very high abnormal development when Ni was present.

Comparative Neurobiology of Biogenic Amines in Animal Models in Deuterostomes

In this study, we review the current knowledge on the presence of biogenic amines and their potential role as neurotransmitters in the nervous system of three groups of invertebrate deuterostomes: tunicates, cephalochordates and echinoderms. In addition to a general overview of biogenic amines in each subphylum we point most of our attention on a few species, as the sea squirts Ciona intestinalis, Ciona robusta, Phallusia mammillata and Ciona savignyi (tunicates), the lancelet amphioxus Branchiostoma lanceolatum and Branchiostoma floridae (cephalochordates), and the sea urchin Strongylocentrotus purpuratus (echinoderms). The choice of these species lies in the fact that they are currently the most studied invertebrate deuterostomes in the research field of Evolutionary Developmental biology (EvoDevo). Providing a comparative picture of the expression and role of neurotransmitters in deuterostomes will contribute to understand the evolution of these neural signalling systems. Such an approach represents a new frontier of comparative neuroanatomy and neurobiology, and a prerequisite to uncover the homology of neuronal structures and circuits in deuterostomes with such a different body plan organization and complexity.