CO2 induced seawater acidification impacts sea urchin larval development I: elevated metabolic rates decrease scope for growth and induce developmental delay

Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129 Pa, 1271 μatm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.     

Response of larval barnacle proteome to CO(2)-driven seawater acidification

The majority of benthic marine invertebrates have a complex life cycle, during which the pelagic larvae select a suitable substrate, attach to it, and then metamorphose into benthic adults. Anthropogenic ocean acidification (OA) is postulated to affect larval metamorphic success through an altered protein expression pattern (proteome structure) and post-translational modifications. To test this hypothesis, larvae of an economically and ecologically important barnacle species Balanus amphitrite, were cultured from nauplius to the cyprid stage in the present (control) and in the projected elevated concentrations of CO₂ for the year 2100 (the OA treatment). Cyprid response to OA was analyzed at the total proteome level as well as two protein post-translational modification (phosphorylation and glycosylation) levels using a 2-DE based proteomic approach. The cyprid proteome showed OA-driven changes. Proteins that were differentially up or down regulated by OA come from three major groups, namely those related to energy-metabolism, respiration, and molecular chaperones, illustrating a potential strategy that the barnacle larvae may employ to tolerate OA stress. The differentially expressed proteins were tentatively identified as OA-responsive, effectively creating unique protein expression signatures for OA scenario of 2100. This study showed the promise of using a sentinel and non-model species to examine the impact of OA at the proteome level.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.     

Exogastrulation induced by heavy water in sea urchin larvae.

Replacement of H2O with D2O in seawater causes exogastrulation in larvae of the sea urchins, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius and S. nudus. When larvae at any stages before mesenchymal blastula stage are transferred to 40% D2O-seawater all of them develop gradually to exogastrulae and finally up to plutei with evaginated archenterons. Effects of D2O are partly reversible at limited steps of the way to exogastrulation. Fertilisation and cleavage are not affected appreciably by D2O (50% or less) except for the delay of cleavage.     

Physiological and induced apoptosis in sea urchin larvae undergoing metamorphosis

Paracentrotus lividus embryos at the early pluteus stage undergo spontaneous apoptosis. Using a TUNEL (TdT-mediated dUTP Nick-End Labelling) assay on whole mount embryos, we showed that there was a different distribution of the apoptotic cells in different optical sections. Not more than 20% of cells in plutei were spontaneously apoptotic, as confirmed by the counts of dissociated ectoderm and intestine cells. Observation of larva stages closer to metamorphosis confirmed that apoptosis is a physiological event for the development of the adult. In particular, larvae at different developmental stages showed apoptotic cells in the oral and aboral arms, intestine, ciliary band and both apical and oral ganglia. Moreover, we found that the number of apoptotic cells decreased in later larva stages, possibly because in the organism approaching metamorphosis, a smaller number of cells needs to be eliminated. Furthermore, combined phorbol ester (TPA) and heat shock treatment enhanced apoptosis by increasing the number of cells involved in the phenomenon.     

Acetylcholinesterase in the sea urchin Lytechinus variegatus: characterization and developmental expression in larvae

Acetylcholinesterase (AChE) in the echinoid Lytechinus variegatus has been characterized. Kinetic parameters V(max), K(m), K(ss), and b were 2594+/-1048 nmol ATCh hydrolyzed/min/mg tissue wet weight, 185+/-11 microM, 308+/-100 mM, and 0.2, respectively for the substrate ATCh and 17.8+/-6.87 nmol BTCh hydrolyzed/min/mg tissue wet weight, 654+/-424 microM, 36+/-31 mM, and 0.6, respectively for BTCh. Pharmacologic analyses were performed with four inhibitors of cholinesterases, physostigmine, BW284c51, ethopropazine, and iso-OMPA, and yielded IC(50) values of 106+/-4 nM, 718+/-118 nM, 2.57+/-0.6 mM, and >0.0300 M, respectively. Both kinetic and pharmacologic results confirmed the existence of AChE in larval L. variegatus. Dimeric and tetrameric globular forms (determined by velocity sedimentation on sucrose gradients) were present in L. variegatus larvae. Activity of AChE increased significantly as larvae progressed in development from embryos to eight-arm larvae. Acetylcholinesterase activity of F1 larvae derived from sea urchins collected from wild populations and of F1 larvae derived from sea urchins cultured in the laboratory and fed two different diets suggest that the nutritional and/or environmental history of the adult sea urchin affect the developmental progression of AChE activity in the F1 offspring.     

Actin gene expression in developing sea urchin embryos.

We show that the synthesis of actin is regulated developmentally during early sea urchin embryogenesis and that the level of synthesis of this protein parallels the steady-state amounts of the actin messenger ribonucleic acids (RNA). An in vitro translation and RNA blotting analysis of embryo RNA from several stages of early development indicated that during the first 8 h after fertilization there was a low and relatively constant level of actin messenger RNA in the embryo. Between 8 and 13 h of development, the amount of actin messenger RNA began to increase both in the cytoplasm and on polysomes, and by 18 h the amounts of actin message per embryo had risen between approximately 10- and 25-fold in the cytoplasm and between 15- and 40-fold on polysomes. Two size classes of actin messenger RNA (2.2 and 1.8 kilobases) were identified in unfertilized eggs and in all of the developmental stages examined. The amount of each actin message class increased over a similar time interval during early development. However, the amounts of these size classes in the cytoplasm relative to each other shifted between the earliest stages examined (2 to 5 h) and the hatching blastula stage (18 h), with the ratio of the 1.8-kilobase actin messenger RNA to the 2.2-kilobase actin messenger RNA increasing almost threefold during this period.     

Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

Archenteron formation induced by ascorbate and alpha-ketoglutarate in sea urchin embryos kept in SO2- 4 -free artificial seawater

In permanent blastulae of the sea urchin, which were obtained by culture in SO^2?₄-free artificial seawater from the time of fertilization, ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, induced the formation of archenteron. By adding either ascorbate or α-ketoglutarate to the SO^2?₄-free culture at 12 hr of fertilization, spherical embryos with archenteron were obtained by successive 12 hr cultures at 20°C. The embryos thus obtained did not develop to plutei. Archenteron formation induced by these compounds in SO^2?₄-free-cultured embryos, as well as in the normal embryos, was inhibited by α,α′-dipyridyl, an inhibitor of protocollagen proline hydroxylase. Glutamate, malate, citrate, and fumarate did not stimulate archenteron formation in SO^2?₄-free cultured embryos. In the SO^2?₄-free-cultured embryos exposed to [¹⁴C]proline, considerable radioactivity was found in hot trichloroacetic acid-extractable proteins but the radioactivity of [¹⁴C]hydroxyproline residue, produced by hydroxylation of proline residue of protocollagen, was markedly lower than that in normal embryos. In the presence of ascorbate and α-ketoglutarate, the radioactivity of [¹⁴C]hydroxyproline residue became high and was lowered by α,α′-dipyridyl. Archenteron formation induced by ascorbate and α-ketoglutarate in the embryos kept in SO^2?₄-free artificial seawater probably results from the stimulated protocollagen hydroxylation.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

The effect of salinity on larval survival and development in the sea urchin Echinometra lucunter

Summary

Reductions in salinity can have adverse effects on larval development and larval survival in some invertebrate taxa but not others. Salinity tolerance of larvae may be particularly important in echinoderms because they are both poor ion regulators and stenohaline. I examined the effect of six levels of salinity (15, 18, 21, 24, 27 and 33 PSU) on survival and rate of development of larvae in the subtropical sea urchin Echinometra lucunter. In the short-term, mortality rate was significantly lower in 33 PSU than in all other salinities except 27 PSU, and it was significantly greater in 15 and 18 PSU than in all higher salinities. In the long-term, daily and cumulative mortality were significantly greater in 15 PSU than in most other salinities over 11 days of development (except for cumulative mortality in 18 PSU). They were significantly greater in 18 PSU than in 21 PSU or 33 PSU over a period of 13 days. Furthermore, daily mortality was significantly greater in 18 PSU than in 24 PSU or 27 PSU at 13 d after fertilization. Daily and cumulative mortality were significantly lower in 33 PSU than in 21, 24 or 27 PSU over a period of 17 days. Although in the control (33 PSU) 75% of larvae completed development to the 8-arm stage at 35 d, no larvae developed further than the 4-arm stage in 18, 21, 24 or 27 PSU; in 15 PSU, ~60% of larvae did not develop further than swimming blastulae. Since prolonged exposure to salinities as high as 27 PSU (frequently recorded in the adult habitat) can result in great larval losses, adaptive behaviours that prevent larvae from entering water layers of low salinity will enhance their chance for survival.     

The stunting effect of a high CO2 ocean on calcification and development in sea urchin larvae,a synthesis from the tropics to the poles

The stunting effect of ocean acidification on development of calcifying invertebrate larvae has emerged as a significant effect of global change. We assessed the arm growth response of sea urchin echinoplutei, here used as a proxy of larval calcification, to increased seawater acidity/pCO₂ and decreased carbonate mineral saturation in a global synthesis of data from 15 species. Phylogenetic relatedness did not influence the observed patterns. Regardless of habitat or latitude, ocean acidification impedes larval growth with a negative relationship between arm length and increased acidity/pCO₂ and decreased carbonate mineral saturation. In multiple linear regression models incorporating these highly correlated parameters, pCO₂ exerted the greatest influence on decreased arm growth in the global dataset and also in the data subsets for polar and subtidal species. Thus, reduced growth appears largely driven by organism hypercapnia. For tropical species, decreased carbonate mineral saturation was most important. No single parameter played a dominant role in arm size reduction in the temperate species. For intertidal species, the models were equivocal. Levels of acidification causing a significant (approx. 10–20+%) reduction in arm growth varied between species. In 13 species, reduction in length of arms and supporting skeletal rods was evident in larvae reared in near-future (pCO₂ 800+ µatm) conditions, whereas greater acidification (pCO₂ 1000+ µatm) reduced growth in all species. Although multi-stressor studies are few, when temperature is added to the stressor mix, near-future warming can reduce the negative effect of acidification on larval growth. Broadly speaking, responses of larvae from across world regions showed similar trends despite disparate phylogeny, environments and ecology. Larval success may be the bottleneck for species success with flow-on effects for sea urchin populations and marine ecosystems.