Evolution of Maternal Provisioning and Development in the Ophiuroidea: Egg Size, Larval Form and Parental Care.

The Ophiuroidea is the most speciose class of echinoderms and has the greatest diversity of larval forms, but we know less about the evolution of development (evo-devo) in this group than for the other echinoderm classes. As is typical of echinoderms, evo-devo in the Ophiuroidea resulted in the switch from production of small eggs and feeding (planktotrophic) larvae to large eggs and non-feeding (lecithotrophic) larvae. Parental care (ovoviviparity or viviparity/matrotrophy) is the most derived life history. Analysis of egg data for 140 species (excluding viviparity and facultative planktotrophy) indicated a bimodal distribution in egg volume corresponding to planktotrophy and lecithotrophy + ovoviviparity, with three significant egg size groups due to the very large eggs of the ovoviviparous species. The marked reduction in fecundity in species with extremely large eggs is exemplified by the ovoviviparous species. Egg size in the two species with facultative planktotrophy were intermediate with respect to the two modes. Identifying the ancestral larval life history pattern and the pathways in the switch from feeding to non-feeding larvae is complicated by the two patterns of metamorphosis seen in species with planktotrophic development: Type I (ophiopluteus only) and Type II (ophiopluteus + vitellaria larva). The variability in arm resorption at metamorphosis across ophiuroid families indicates that the Type I and II patterns may be two ends of a morphological continuum. This variability indicates ancestral morphological plasticity at metamorphosis followed by canalization in some taxa to the vitellaria as the metamorphic larva. Vestigial ophiopluteal traits in lecithotrophic ophioplutei and vitellaria indicate evolution from the ancestral (feeding larva) state. Parental care has evolved many times from an ancestor that had a planktonic ophiopluteus or vitellaria and is often associated with hermaphroditism and paedomorphosis. A secondary reduction in egg size occurred in the viviparous species.

Investigating seed bank potential of crustose coralline algae using DNA metabarcoding.

To examine the potential for the autogenic ecosystem engineers, crustose coralline algae (CCA), to serve as seed banks or refugia for life stages of other species, it is critical to develop sampling protocols that reflect the diversity of life present. In this pilot study on two shallow water species of CCA collected from Raoul Island (Kermadec Islands; Rangitahua) New Zealand, we investigated two preservation methods (ethanol vs. silica gel), sampled inner and outer regions of the crusts, and used DNA metabarcoding and seven genes/gene regions (16S rRNA, 18S rRNA, 23S rRNA, cox1, rbcL, and tufA genes and the ITS rRNA region) to develop a protocol for taxa identification. The results revealed immense diversity, with typically more taxa identified within the inner layers than the outer layers. As highlighted in other metabarcoding studies and in earlier work on rhodoliths (nodose coralline algae), reference databases are incomplete, and to some extent, the use of multiple markers mitigates this issue. Specifically, the 23S rRNA and rbcL genes are currently more suitable for identifying algae, while the cox1 gene fares better at capturing the diversity present inclusive of algae. Further investigation of these autogenic ecosystem engineers that likely act as marine seed banks is needed.

Ocean acidification impacts sperm swimming performance and pHi in the New Zealand sea urchin Evechinus chloroticus.

In sea urchins, spermatozoa are stored in the gonads in hypercapnic conditions (pH<7.0). During spawning, sperm are diluted in seawater of pH>8.0, and there is an alkalinization of the sperm's internal pH (pHi) through the release of CO2 and H+. Previous research has shown that when pHi is above 7.2-7.3, the dynein ATPase flagellar motors are activated, and the sperm become motile. It has been hypothesized that ocean acidification (OA), which decreases the pH of seawater, may have a narcotic effect on sea urchin sperm by impairing the ability to regulate pHi, resulting in decreased motility and swimming speed. Here, we used data collected from the same individuals to test the relationship between pHi and sperm motility/performance in the New Zealand sea urchin Evechinus chloroticus under near-future (2100) and far-future (2150) atmospheric PCO2 conditions (RCP 8.5: pH 7.77, 7.51). Decreasing seawater pH significantly negatively impacted the proportion of motile sperm, and four of the six computer-assisted sperm analysis (CASA) sperm performance measures. In control conditions, sperm had an activated pHi of 7.52. Evechinus chloroticus sperm could not defend pHi in future OA conditions; there was a stepped decrease in the pHi at pH 7.77, with no significant difference in mean pHi between pH 7.77 and 7.51. Paired measurements in the same males showed a positive relationship between pHi and sperm motility, but with a significant difference in the response between males. Differences in motility and sperm performance in OA conditions may impact fertilization success in a future ocean.

Detecting Phytoplankton Cell Viability Using NIR Raman Spectroscopy and PCA.

Raman spectroscopy has long been suggested as a potentially fast and sensitive method to monitor phytoplankton abundance and composition in marine environments. However, the pitfalls of visible detection methods in pigment-rich biological material and the complexity of their spectra have hindered their application as reliable in situ detection methods. In this study we combine 1064 nm confocal Raman spectroscopy with multivariate statistical analysis techniques (principle component analysis and partial leas-squares discriminant analysis) to reliably measure differences in the cell viability of a diatom species (Chaetoceros muelleri) and two haptophyte species (Diacronema lutheri and Tisochrysis lutea) of phytoplankton. The low fluorescence background due to this combined approach of NIR Raman spectroscopy and multivariate data analysis allowed small changes in the overall spectral profiles to be reliably monitored, enabling the identification of the specific spectral features that could classify cells as viable or nonviable regardless of their species. The most significant differences upon cell death were shown by characteristic shifts in the carotenoid bands at 1527 and 1158 cm-1. The contributions from other biomolecules were less pronounced but revealed changes that could be identified using this combination of techniques.

Does a complex life cycle affect adaptation to environmental change? Genome-informed insights for characterizing selection across complex life cycle.

Complex life cycles, in which discrete life stages of the same organism differ in form or function and often occupy different ecological niches, are common in nature. Because stages share the same genome, selective effects on one stage may have cascading consequences through the entire life cycle. Theoretical and empirical studies have not yet generated clear predictions about how life cycle complexity will influence patterns of adaptation in response to rapidly changing environments or tested theoretical predictions for fitness trade-offs (or lack thereof) across life stages. We discuss complex life cycle evolution and outline three hypotheses-ontogenetic decoupling, antagonistic ontogenetic pleiotropy and synergistic ontogenetic pleiotropy-for how selection may operate on organisms with complex life cycles. We suggest a within-generation experimental design that promises significant insight into composite selection across life cycle stages. As part of this design, we conducted simulations to determine the power needed to detect selection across a life cycle using a population genetic framework. This analysis demonstrated that recently published studies reporting within-generation selection were underpowered to detect small allele frequency changes (approx. 0.1). The power analysis indicates challenging but attainable sampling requirements for many systems, though plants and marine invertebrates with high fecundity are excellent systems for exploring how organisms with complex life cycles may adapt to climate change.

Surveillance of densoviruses and mesomycetozoans inhabiting grossly normal tissues of three Aotearoa New Zealand asteroid species.

Asteroid wasting events and mass mortality have occurred for over a century. We currently lack a fundamental understanding of the microbial ecology of asteroid disease, with disease investigations hindered by sparse information about the microorganisms associated with grossly normal specimens. We surveilled viruses and protists associated with grossly normal specimens of three asteroid species (Patiriella regularis, Stichaster australis, Coscinasterias muricata) on the North Island / Te Ika-a-Maui, Aotearoa New Zealand, using metagenomes prepared from virus and ribosome-sized material. We discovered several densovirus-like genome fragments in our RNA and DNA metagenomic libraries. Subsequent survey of their prevalence within populations by quantitative PCR (qPCR) demonstrated their occurrence in only a few (13%) specimens (n = 36). Survey of large and small subunit rRNAs in metagenomes revealed the presence of a mesomycete (most closely matching Ichthyosporea sp.). Survey of large subunit prevalence and load by qPCR revealed that it is widely detectable (80%) and present predominately in body wall tissues across all 3 species of asteroid. Our results raise interesting questions about the roles of these microbiome constituents in host ecology and pathogenesis under changing ocean conditions.

Effect of acclimation on thermal limits and hsp70 gene expression of the New Zealand sea urchin Evechinus chloroticus.

Seawater temperature is projected to increase globally due to climate change, affecting physiological responses, fitness and survival of marine organisms. Thermal tolerance studies are critical to determine the ability of animals to adapt to future environmental conditions. In this study, we aimed to determine if the thermal limits of the New Zealand Evechinus chloroticus would shift with animal's thermal history. We tested the effect of six thermal regimes on the righting ability, temperature of loss of righting (TLOR), median lethal temperature (LT50), lethal temperature (LT) and the gene expression of the heat shock protein 70 (hsp70) of the New Zealand sea urchin E. chloroticus when exposed to a thermal shock of 1 °C day-1 (duration of 7-16 days depending on the treatment). Treatments consisted of laboratory acclimation for one and four weeks to 18 °C and 24 °C (mean winter (15 °C) and summer temperature (21 °C) + 3 °C of warming, respectively), compared to non-acclimated sea urchins collected during winter (14.6 °C) and summer seasons (20.4 °C). Thermal history did not have a significant effect on the righting ability of E. chloroticus (TLOR ranged between 28 and 29 °C for all treatments) and LT50 (ranged between 29 and 30 °C for all treatments). However, LT of E. chloroticus collected during winter season was significantly lower than animals acclimated for one week at 18 °C. Maximum expression of hsp70 mRNA (Tmax) was observed at around 27-28 °C regardless of treatment; however, relative hsp70 mRNA levels were significantly higher in animals acclimated for four weeks at 24 °C. Despite proving to be a thermotolerant species with LTs around 30 °C, E. chloroticus was unable to increase thermal tolerance and Tmax when acclimated to high temperatures, suggesting that E. chloroticus may have a limited adaptive capacity to modify its phenotype; however, evolutionary adaptations may allow E. chloroticus to adapt to future ocean temperatures.

The first mitochondrial genomes of endosymbiotic rhabdocoels illustrate evolutionary relaxation of atp8 and genome plasticity in flatworms.

The first three mitochondrial (mt) genomes of endosymbiotic turbellarian flatworms are characterised for the rhabdocoels Graffilla buccinicola, Syndesmis echinorum and S. kurakaikina. Interspecific comparison of the three newly obtained sequences and the only previously characterised rhabdocoel, the free-living species Bothromesostoma personatum, reveals high mt genomic variability, including numerous rearrangements. The first intrageneric comparison within rhabdocoels shows that gene order is not fully conserved even between congeneric species. Atp8, until recently assumed absent in flatworms, was putatively annotated in two sequences. Selection pressure was tested in a phylogenetic framework and is shown to be significantly relaxed in this and another protein-coding gene: cox1. If present, atp8 appears highly derived in platyhelminths and its functionality needs to be addressed in future research. Our findings for the first time allude to a large degree of undiscovered (mt) genomic plasticity in rhabdocoels. It merits further attention whether this variation is correlated with a symbiotic lifestyle. Our results illustrate that this phenomenon is widespread in flatworms as a whole and not exclusive to the better-studied neodermatans.

Description and ecophysiology of a new species of Syndesmis Silliman, 1881 (Rhabdocoela: Umagillidae) from the sea urchin Evechinus chloroticus (Valenciennes, 1846) Mortensen, 1943 in New Zealand.

A new rhabdocoel of the genus Syndesmis Silliman, 1881 (Umagillidae) is described from the intestine of the New Zealand sea urchin Evechinus chloroticus (Valenciennes, 1846) Mortensen, 1943a. This new species, Syndesmis kurakaikina n. sp., is morphologically distinct and can easily be recognised by its very long (±1 mm) stylet and its bright-red colour. In addition to providing a formal description, we present some observations on reproduction and life history of this new species. Fecundity is comparable to that of other umagillids and the rate of egg production and development increases with temperature. Hatching in this species is induced by intestinal fluids of its host. Relevant to global warming, we assessed the effect of temperature on survival, fecundity, and development. The tests indicate that Syndesmis kurakaikina n. sp. is tolerant of a wide range of temperatures (11-25 °C) and that its temperature optimum lies between 18.0 and 21.5 °C. Egg viability is, however, significantly compromised at the higher end of this temperature range, with expelled egg capsules often being deformed and showing increasingly lower rates of hatching. Given this, a rise in global temperature might increase the risk of Syndesmis kurakaikina n. sp. infecting new hosts and would possibly facilitate the spread of these endosymbionts.

Dwarf brooder versus giant broadcaster: combining genetic and reproductive data to unravel cryptic diversity in an Antarctic brittle star.

Poecilogony, or multiple developmental modes in a single species, is exceedingly rare. Several species described as poecilogenous were later demonstrated to be multiple (cryptic) species with a different developmental mode. The Southern Ocean is known to harbor a high proportion of brooders (Thorson's Rule) but with an increasing number of counter examples over recent years. Here we evaluated poecilogony vs. crypticism in the brittle star Astrotoma agassizii across the Southern Ocean. This species was initially described from South America as a brooder before some pelagic stages were identified in Antarctica. Reproductive and mitochondrial data were combined to unravel geographic and genetic variation of developmental modes. Our results indicate that A. agassizii is composed of seven well-supported and deeply divergent clades (I: Antarctica and South Georgia; II: South Georgia and Sub-Antarctic locations including Kerguelen, Patagonian shelf, and New Zealand; III-VI-VII: Patagonian shelf, IV-V: South Georgia). Two of these clades demonstrated strong size dimorphism when in sympatry and can be linked to differing developmental modes (Clade V: dwarf brooder vs. Clade I: giant broadcaster). Based on their restricted geographic distributions and on previous studies, it is likely that Clades III-VI-VII are brooders. Clade II is composed of different morphological species, A. agassizii and A. drachi, the latter originally used as the outgroup. By integrating morphology, reproductive, and molecular data we conclude that the variation identified in A. agassizii is best described as crypticism rather than poecilogony.

The role of the hyaline spheres in sea cucumber metamorphosis: lipid storage via transport cells in the blastocoel.

BACKGROUND: For echinoderms with feeding larvae, metamorphic and post-settlement success may be highly dependent on larval nutrition and the accumulation of energetic lipids from the diet. In contrast to the sea urchins, starfish and brittle stars within the Phylum Echinodermata, sea cucumber metamorphosis does not involve formation of a juvenile rudiment, but instead there is a rearrangement of the entire larval body. Successful metamorphosis in sea cucumbers is often associated with the presence in the late auricularia stage of an evolutionary novelty, the hyaline spheres (HS), which form in the base of the larval arms. Known since the 1850s the function of these HS has remained enigmatic-suggestions include assistance with flotation, as an organizer for ciliary band formation during metamorphosis and as a nutrient store for metamorphosis. RESULTS: Here using multiple methodologies (lipid mapping, resin-section light microscopy, lipid and fatty acid analyses) we show definitively that the HS are used to store neutral lipids that fuel the process of metamorphosis in Australostichopus mollis. Neutral lipids derived from the phytoplankton diet are transported by secondary mesenchyme cells ("lipid transporting cells", LTC), likely as free fatty acids or lipoproteins, from the walls of the stomach and intestine through the blastocoel to the HS; here, they are converted to triacylglycerol with a higher saturated fatty acid content. During metamorphosis the HS decreased in size as the triacylglycerol was consumed and LTC again transported neutral lipids within the blastocoel. CONCLUSION: The HS in A. mollis functions as a nutrient storage structure that separates lipid stores from the major morphogenic events that occur during the metamorphic transition from auricularia-doliolaria-pentactula (settled juvenile). The discovery of LTC within the blastocoel of sea cucumbers has implications for other invertebrate larvae with a gel-filled blastocoel and for our understanding of lipid use during metamorphosis in marine invertebrates.

Repeated measurement of Mo2 in small aquatic organisms: a manual intermittent flow respirometer using off-the-shelf components.

Measurement of rates of oxygen consumption ( Mo2) in small aquatic embryos or larvae (<1 mm) in response to altered environmental conditions has traditionally been challenging. Here, using modifications of a commercially available fluorescent optode flow-through cell (FTC; PreSens FTC-PSt3) and routine laboratory supplies (syringes, stopcocks, tubing), we have constructed a manual intermittent flow respirometer (MIFR) that allows measurement of Mo2 in small numbers of individuals when sequentially exposed to different environmental conditions (e.g., changes in seawater pH) through a gravity-driven media replacement perfusion system. We first show that the FTC can be used in "static" mode while incubating small numbers of embryos/larvae contained within the planar oxygen sensor (POS) chamber with Nitex filters. We then demonstrate the use of the MIFR by exposing larval echinoderms ( Fellaster zelandiae, Evechinus chloroticus, and Centrostephanus rodgersii) to seawater equilibrated with elevated CO2 and measured Mo2 during acute and chronic exposure to hypercapnia. This MIFR method will allow investigators to address questions regarding the respiratory physiology of small aquatic animals, such as the thresholds for metabolic depression in embryonic and larval forms. NEW & NOTEWORTHY A manual intermittent flow respirometer (MIFR), allowing media exchange in a flow-through cell containing small aquatic organisms, permits repeated measurement of Mo2 of individuals not only in a single medium (e.g., technical replication), but also in different media (here, high CO2-equilibrated seawater), enabling measurement of acute physiological responses to changed conditions. This versatile technique has wide-ranging implications for the study of the Mo2 response of aquatic organisms in the face of climate change.

Evaluation of the impact of a residency in an independent pharmacy in rural Mississippi.

OBJECTIVES: To evaluate the impact of a postgraduate year 1 (PGY1) community pharmacy residency program on clinical pharmacy service implementation and enhancement in a rural Mississippi community. SETTING: An independent rural community pharmacy in Canton, MS. PRACTICE DESCRIPTION AND INNOVATION: Delivery of clinical pharmacy services provided by PGY1 community residents 1 day a week to an underserved population during an 18-month period. EVALUATION: Economic impact of a community pharmacy residency on the pharmacy's revenue stream determined by calculating an estimated dollar amount generated by clinical pharmacy services. RESULTS: By providing services 1 day a week, the residents were able to directly contribute an estimated $8000 of revenue from vaccinations and medication therapy management services. In addition, residents provided point-of-care testing, facilitated group education, developed a medication synchronization program, and assisted with physician outreach. CONCLUSION: Overall, community pharmacy residencies can contribute to the generation of revenue in rural independent settings and may also offer an opportunity to generate revenue in different areas that were not present before residency implementation, thereby improving access to care for patients.

Comparison of Pharmacist and Physician Managed Annual Medicare Wellness Services.

BACKGROUND: Medicare Annual Wellness Visits (AWV) are a benefit provided for Medicare beneficiaries to increase focus on wellness and preventive measures. Pharmacists can conduct AWVs, which offers a potential avenue for outpatient revenue generation. PROGRAM DESCRIPTION: To compare a composite of interventions and screenings and revenue generated by a pharmacist with those made by a physician during a subsequent AWV. A report generated through the electronic health record was used to determine AWVs conducted by a pharmacist or 3 participating physicians from December 2013 to March 2016, including revenue generated. Through electronic chart review, documentation was accessed to quantify and categorize the number and types of referrals, health advice, laboratory tests, procedures, vaccinations, and screenings that were recommended during each patient's AWV. OBSERVATIONS: The pharmacist performed 19 subsequent visits, and the 3 physicians performed 89 subsequent visits. Overall, the composite of interventions and screenings was significantly higher in the pharmacist group than the physician group (P = 0.03). More interventions were made in the areas of health advice (P = 0.020), vaccine recommendations (P = 0.009), and screenings in the pharmacist group (P < 0.001). The physicians ordered significantly more laboratory tests per visit (P < 0.001). The pharmacist was reimbursed on average $105 per visit versus $99 per visit for the physicians. IMPLICATIONS: Pharmacist-provided AWVs are at least comparable to those provided by physicians and offer an additional access point for valuable services for Medicare beneficiaries. DISCLOSURES: There was no financial contribution to this study. Riche reports participation in the Speaker's Bureau for Merck and the Speaker's Bureau and Advisory Board for Novo Nordisk. The authors have no other conflicts of interest to report pertinent to this research. This data has not been previously published in any other location. Richie, Sewell, Malinowski, Jackson, and Fleming were involved in study design and manuscript preparation/approval. Jackson was involved in data collection, and Richie and Sewell were involved in data collection and data analysis. Sewell and Richie had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Can we predict ectotherm responses to climate change using thermal performance curves and body temperatures?

Thermal performance curves (TPCs), which quantify how an ectotherm's body temperature (Tb ) affects its performance or fitness, are often used in an attempt to predict organismal responses to climate change. Here, we examine the key - but often biologically unreasonable - assumptions underlying this approach; for example, that physiology and thermal regimes are invariant over ontogeny, space and time, and also that TPCs are independent of previously experienced Tb. We show how a critical consideration of these assumptions can lead to biologically useful hypotheses and experimental designs. For example, rather than assuming that TPCs are fixed during ontogeny, one can measure TPCs for each major life stage and incorporate these into stage-specific ecological models to reveal the life stage most likely to be vulnerable to climate change. Our overall goal is to explicitly examine the assumptions underlying the integration of TPCs with Tb , to develop a framework within which empiricists can place their work within these limitations, and to facilitate the application of thermal physiology to understanding the biological implications of climate change.

Maternal Lipid Provisioning Mirrors Evolution of Reproductive Strategies in Direct-Developing Whelks.

The energetic input that offspring receive from their mothers is a well-studied maternal effect that can influence the evolution of life histories. Using the offspring of three sympatric whelks: Cominella virgata (one embryo per capsule); Cominella maculosa (multiple embryos per capsule); and Haustrum scobina (multiple embryos per capsule and nurse-embryo consumption), we examined how contrasting reproductive strategies mediate inter- and intraspecific differences in hatchling provisioning. Total lipid content (as measured in µg hatchling(-1) ± SE) was unrelated to size among the 3 species; the hatchlings of H. scobina were the smallest but had the highest lipid content (33.8 ± 8.1 µg hatchling(-1)). In offspring of C. maculosa, lipid content was 6.6 ± 0.4 µg hatchling(-1), and in offspring of C. virgata, it was 21.7 ± 3.2 µg hatchling(-1) The multi-encapsulated hatchlings of C. maculosa and H. scobina were the only species that contained the energetic lipids, wax ester (WE) and methyl ester (ME). However, the overall composition of energetic lipid between hatchlings of the two Cominella species reflected strong affinities of taxonomy, suggesting a phylogenetic evolution of the non-adelphophagic development strategy. Inter- and intracapsular variability in sibling provisioning was highest in H. scobina, a finding that implies less control of allocation to individual hatchlings in this adelphophagic developer. We suggest that interspecific variability of lipids offers a useful approach to understanding the evolution of maternal provisioning in direct-developing species.

Low coverage sequencing of three echinoderm genomes: the brittle star Ophionereis fasciata, the sea star Patiriella regularis, and the sea cucumber Australostichopus mollis.

BACKGROUND: There are five major extant groups of Echinodermata: Crinoidea (feather stars and sea lillies), Ophiuroidea (brittle stars and basket stars), Asteroidea (sea stars), Echinoidea (sea urchins, sea biscuits, and sand dollars), and Holothuroidea (sea cucumbers). These animals are known for their pentaradial symmetry as adults, unique water vascular system, mutable collagenous tissues, and endoskeletons of high magnesium calcite. To our knowledge, the only echinoderm species with a genome sequence available to date is Strongylocentrotus pupuratus (Echinoidea). The availability of additional echinoderm genome sequences is crucial for understanding the biology of these animals. FINDINGS: Here we present assembled draft genomes of the brittle star Ophionereis fasciata, the sea star Patiriella regularis, and the sea cucumber Australostichopus mollis from Illumina sequence data with coverages of 12.5x, 22.5x, and 21.4x, respectively. CONCLUSIONS: These data provide a resource for mining gene superfamilies, identifying non-coding RNAs, confirming gene losses, and designing experimental constructs. They will be important comparative resources for future genomic studies in echinoderms.

Effects of warm acclimation on physiology and gonad development in the sea urchin Evechinus chloroticus.

The physiology of the New Zealand sea urchin Evechinus chloroticus was evaluated through feeding, respiration, growth and gonad growth in adult animals acclimated for 90days at 18°C (annual mean temperature) and 24°C (ambient summer temperature (21°C) +3°C). Measured parameters with representative rates of assimilation efficiency were used to calculate scope for growth (SfG) for each treatment. All physiological parameters were negatively affected at 24°C, showing a decrease in feeding rate which coincided with negative growth and gonad development at the end of the acclimation period, and a decrease in respiration rate suggesting metabolic depression. Histology of gonad samples after the acclimation period also showed no gametic material in animals acclimated at 24°C. All animals acclimated at 24°C had negative growth, differing from the calculated SfG which indicated that the animals had sufficient energy for production. The results suggest that calculated SfG in echinoderms should be used together with actual measurements of growth in individuals as, by itself, SfG may underestimate the actual effect of ocean warming when animals are exposed to stressful conditions. Overall, considering the total loss of reproductive output observed in E. chloroticus at higher temperatures, an increase in seawater temperature could dramatically influence the persistence of northern populations of this species, leading to flow-on effects in the subtidal ecosystem.

Real-time 2D visualization of metabolic activities in zebrafish embryos using a microfluidic technology.

Non-invasive and real-time visualization of metabolic activities in living small model organisms such as embryos and larvae of zebrafish has not yet been attempted largely due to profound analytical limitations of existing technologies. Historically, our capacity to examine oxygen gradients surrounding eggs and embryos has been severely limited, so much so that to date, most of the articles characterizing in situ oxygen gradients have described predominantly mathematical simulations. These drawbacks can, however, be experimentally addressed by an emerging field of microfluidic Lab-on-a-Chip (LOC) technologies combined with sophisticated optoelectronic sensors. In this work, we outline a proof-of-concept approach utilizing microfluidic living embryo array system to enable in situ Fluorescence Ratiometric Imaging (FRIM) on developing zebrafish embryos. The FRIM is an innovative method for kinetic quantification of the temporal patterns of aqueous oxygen gradients at a very fine scale based on signals coming from an optical sensor referred to as a sensor foil. We envisage that future integration of microfluidic chip-based technologies with FRIM represents a noteworthy direction to miniaturize and revolutionize research on metabolism and physiology in vivo.

Ocean acidification and fertilization in the antarctic sea urchin Sterechinus neumayeri: the importance of polyspermy.

Ocean acidification (OA), the reduction of the seawater pH as a result of increasing levels of atmospheric CO2, is an important climate change stressor in the Southern Ocean and Antarctic. We examined the impact of OA on fertilization success in the Antarctic sea urchin Sterechinus neumayeri using pH treatment conditions reflective of the current and near-future "pH seascape" for this species: current (control: pH 8.052, 384.1 µatm of pCO2), a high CO2 treatment approximating the 0.2-0.3 unit decrease in pH predicted for 2100 (high CO2: pH 7.830, 666.0 µatm of pCO2), and an intermediate medium CO2 (pH 7.967, 473.4 µatm of pCO2). Using a fertilization kinetics approach and mixed-effect models, we observed significant variation in the OA response between individual male/female pairs (N = 7) and a significant population-level increase (70-100%) in tb (time for a complete block to polyspermy) at medium and high CO2, a mechanism that potentially explains the higher levels of abnormal development seen in OA conditions. However, two pairs showed higher fertilization success with CO2 treatment and a nonsignificant effect. Future studies should focus on the mechanisms and levels of interindividual variability in OA response, so that we can consider the potential for selection and adaptation of organisms to a future ocean.