Gamete cryobanks for laboratory research: Developing a rapid and easy-to-perform protocol for the cryopreservation of the sea urchin Paracentrotus lividus (Lmk, 1816) spermatozoa

Gamete cryopreservation is a biotechnology that can guarantee a continuous supply of gametes, regardless of the seasonal reproductive cycle. In this study we developed a protocol for the cryopreservation of the sea urchin Paracentrotuslividus spermatozoa, with a view to the creation of cryobanks of semen to be used as a model system in laboratory research and ecotoxicological tests. All the key phases of the procedure were separately considered and the effect on sperm motility was evaluated by means of computer assisted analysis. The best results were obtained using 7% dimethylsulfoxide in 1% NaCl plus 0.04 M trehalose as the extender, at a freezing rate of −20 °C/min. On thawing, in semen samples cryopreserved in accordance with this protocol the velocity parameters of the sub-population of rapid sperm (best performing spermatozoa) did not significantly differ from semen on collection; in addition also the fertilization ability was restored, and about 50% of normal developed plutei larvae were obtained by thawed semen. The developed protocol is rapid and easy-to-perform; moreover, the use of gametes from reared urchins makes it unnecessary to continuously collect specimens from natural populations, making this procedure a promising starting point for the creation of alternative and more sustainable methodologies in laboratory research on sea urchin gametes and embryos.     

鲈鱼胚胎的玻璃化冷冻保存

本文对鲈鱼(Lateolabrax japonicus)胚胎进行了玻璃化冷冻保存研究,筛选出了浓度较低、玻璃化程度较稳定的5种玻璃化液,冷冻时形成玻璃化的概率在48．1％～100％,在35～43℃的水浴中解冻时保持玻璃化的概率在44．4％～63．0％;玻璃化液VSD2在解冻时保持玻璃化的概率最高。对鲈鱼神经胚、20对肌节胚、尾芽胚、心跳胚、出膜前胚在玻璃化液VSD2中的适应能力及适合于玻璃化冷冻的胚胎时期进行了比较,结果显示：不同时期胚胎对玻璃化液的耐受能力不同,鲈鱼神经胚耐受能力最低,心跳胚耐受能力最强,出膜前期胚次之,心跳胚和出膜前胚适合于进行玻璃化冷冻。对0．5mol/L蔗糖的洗脱时间进行了选择,结果显示,洗脱10～20min效果较好。利用玻璃化程度较好的VSD2对鲈鱼不同时期胚胎进行超低温(-196℃)冷冻,获得了2．1％～27．9％的透明胚。将鲈鱼心跳胚冷冻解冻后获2粒复活胚,培养至出膜期,成活42～50h;出膜前期胚在冷冻解冻后有1粒胚复活,并且孵化出鱼苗[动物学报49(6)：843～850,2003]。     

Advances in the cryopreservation of sea-urchin embryos: Potential application in marine water quality assessment

Among the most widely used biological techniques in marine pollution assessment, the sea-urchin embryo–larval bioassay is in an advanced developmental stage. Cryopreservation might help to overcome the problem of the spawning seasonality and therefore strengthen the use of those embryo–larval bioassays. This work investigates different factors influencing cryopreservation of sea-urchin embryos, including the cooling rates and holding temperatures, the seeding, or the impact of plunging into liquid nitrogen. The blastula stage yielded better results than the fertilised egg, and the most effective cryoprotectants combination was dimethyl sulfoxide 1.5 M plus trehalose 0.04 M. The optimised protocol developed begins with an initial hold at 4 °C for 2 min, followed by cooling at −1 °C min⁻¹ to −12 °C. At this point a seeding step was incorporated with a 2 min hold, followed by a second cooling at −1 °C min⁻¹ to −80 °C. After a final hold of 2 min the cryovials are transferred into liquid nitrogen for storage. Although the cryopreservation processes might cause a delay in the development of sea-urchin embryos, high larval growth (71–98% of controls) was obtained when cryopreserved blastulae were further incubated for 72–96 h in artificial seawater. We conclude that embryo–larval bioassays with cryopreserved sea-urchin blastulae are suitable for use in marine pollution monitoring programs and may be considered as an acceptable solution to the reproductive seasonality of sea-urchin species.     

Phylogeography of the sea urchin Paracentrotus lividus (Lamarck) (Echinodermata:Echinoidea): first insights from the South Tyrrhenian Sea

The sea urchin Paracentrotus lividus (Lamarck) (Echinodermata: Echinoidea) is an Atlanto-Mediterranean species abundant in the littoral zone, where it occurs in the sublittoral down to 20 m. The aim of our work is to investigate the genetic patterns of P. lividus along the South Tyrrhenian coasts. Five specimens were collected in six localities, from the Gulf of Naples and the Cilento coast. The nuclear rDNA ITS2 spacer and the two mitochondrial genes 16S and COI were used for the analysis. The three markers utilised did not show any structure among populations from the Gulf of Naples. All populations appear to be polyphyletic, with Cilento samples more differentiated from the others. This suggests the existence of phylogeographic structure at larger geographic scale. Absence of genetic structure has been interpreted taking into consideration theoretical dispersal of the planktotrophic larvae, which can survive for 4–8 weeks before settlement, marine current patterns and persistence of the species in the area.     

Carotenoids in the sea urchin Paracentrotus lividus: occurrence of 9'-cis-echinenone as the dominant carotenoid in gonad colour determination

Regular sampling of wild Paracentrotus lividus was carried out over a 12-month period to examine seasonal effects on the pigment profile and content of the gonads, especially in comparison to gonad colour. The major pigments detected in the gut wall were breakdown products of fucoxanthin, namely fucoxanthinol and amarouciaxanthin A. Lower levels of other dietary carotenoids (lutein and β-carotene) together with some carotenoids not found in the diet, namely isozeaxanthin and echinenone ( 20% total carotenoid) were also detected in the gut wall. The presence of echinenone in the gut wall demonstrates that this organ acts as a major site of carotenoid metabolism. Echinenone is the dominant carotenoid in the gonads, accounting for approx. 50–60% of the total pigment. Both all-trans and 9′-cis forms of echinenone were detected in both the gut wall and in the gonad, with levels of the 9′-cis form typically 10-fold greater than the all-trans form in the gonad. The detection of large levels of 9′-cis-echinenone in wild sea urchins is unexpected due to the absence of 9- or 9′-cis forms of carotenoids in the natural, algal, diet. Whilst echinenone clearly contributes towards gonad pigmentation, levels of this carotenoid, cannot be directly linked to a qualitative assessment of gonad colour in terms of market acceptability. Indeed, unacceptable gonad colouration can be seen with both very low and high levels of echinenone and total carotenoid. The presence of 9′-cis-echinenone as the major carotenoid contributing to the pigmentation/colour of the gonad is an important observation in terms of developing artificial diets for urchin cultivation.     

dbEST-derived SSR markers in sea urchin (Hemicentrotus pulcherrimus)

Sea urchin is an important model organism for evolutionary biology, embryology, and gene regulation study. We developed and evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Strongylocentrotus purpuratus and Hemicentrotus pulcherrimus. Characteristics of nine EST-SSR loci were investigated using 41 Hemicentrotus pulcherrimus individuals. The number of alleles per locus ranged from two to five. The observed heterozygosity ranged from 0.122 to 0.7805, while the expected heterozygosity ranged from 0.4472 to 0.7696. These loci and markers will be useful for population genetics and systemic evolution among species of sea urchin.     

Isolation of elatol from Laurencia microcladia and its palatability to the sea urchin Echinometra lucunter

Elatol was isolated as the major compound from the red alga Laurencia microcladia Kütz. collected in Southern Brazil. This is the first report of elatol in this species. We also investigated the herbivore behaviour of the black sea urchin Echinometra lucunter (Linnaeus, 1758) towards L. microcladia, Ulva fasciata Delile and Gracilaria domingensis (Kütz.) Sond. ex Dickie through live algal multiple-choice feeding assay, as well as artificial feeding assay. The sea urchins ate the crude algae L. microcladia and pellets containing the powdered algae, extract and all tested concentrations of elatol, suggesting that this seaweed and its main compound are palatable for E. lucunter.     

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata

Summary Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK₂ cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were either one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK₂ cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.     

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages.     

Cryopreservation of marine thraustochytrids (Labyrinthulomycetes)

In this research, the viability of three marine thraustochytrid isolates (fungoid protists) (WSG05, W15 and WH3) were investigated after freezing in liquid nitrogen. Five cryopreservative combinations containing horse serum, glycerol and dimethylsulfide (Me₂SO) were used. The thraustochytrids were assessed directly after removal from liquid nitrogen and cell concentration measured for 10 days post-thawing. Results indicated that a combination of horse serum and Me₂SO were the most effective cryoprotectants for each of the strains tested. Glycerol was only successful in producing growth in one of the strains once thawed.The protocols developed and tested in this study may have further application for cryopreserving other isolates in this class.     

Salinity sensitivity of early embryos of the Antarctic sea urchin, <Emphasis Type="Italic">Sterechinus neumayeri</Emphasis>

Embryos and larvae of the Antarctic sea urchin, Sterechinus neumayeri, have received considerable experimental attention assessing impacts of low temperature on development; however, salinity effects are not well documented because heretofore, the Antarctic coastal marine environment has been remarkably stenohaline. In this study, subtle decreases of 2 and 4 parts per 1,000 in standard salinity were tested to see if the developmental rate of S. neumayeri embryos would be impacted by a potential hyposmotic stress. At 30 psu, significantly fewer embryos (2 individuals out of 198 tested) reached morula stage by 36 h post-fertilization in comparison embryos in control treatments at 34 psu. Antarctic sea urchins are an important component of marine environments due to their grazing activities. Reductions in larval recruitment success due to the influx of freshwater from melting ice shelves resulting from global climate change could have far-reaching impacts on benthic ecosystem structure in Antarctica.     

Characterization of the bacterial communities associated with the bald sea urchin disease of the echinoid Paracentrotus lividus

The microbial communities involved in the bald sea urchin disease of the echinoid Paracentrotus lividus are investigated using culture-independent techniques. Lesions of diseased specimens from two locations in France, La Ciotat (Mediterranean Sea) and Morgat (Atlantic Ocean), are examined by Scanning Electron Microscopy (SEM) and the diversity of their microbiota is analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene clones libraries construction. Microscopic observations demonstrated that only the central area of the lesions is invaded by bacteria but not the peripheral zone and the surrounding healthy tissues. Molecular analysis identified at least 24 bacterial genomospecies in bald sea urchin lesions: 5 are Alphaproteobacteria, 10 are Gammaproteobacteria, 8 are CFB bacteria and 1 is a Fusobacteria. Out of them, 4 are observed in both locations while 10 occur only in the Atlantic Ocean and 10 only in the Mediterranean Sea. Gammaproteobacteria are the most represented in clones libraries from both locations, with respectively 65% and 43% of the total clones. CFB and Alphaproteobacteria accounted for the majority of the remaining clones and were detected by DGGE in virtually all samples from both stations. Our results demonstrate that bacterial communities observed on diseased individuals of the same echinoid species but originating from distinct locations are not similar and thus support the hypothesis that bacteria involved in the worldwide echinoid disease commonly called the bald sea urchin disease are opportunistic and not specific.     

Kinetics of RNA-synthesis in the 16-cell stage of the sea urchinParacentrotus lividus

Summary The concentration of RNA synthesised in the prolonged interphase of micromeres at the 16 cell stage is about 9 times higher per unit volume than in the other blastomeres. Since there are cytoplasmic connections between micromeres and macromeres, a transfer of the micromere RNA by simple diffusion is to be expected.     

Membrane permeability characteristics and osmotic tolerance limits of sea urchin (Evechinus chloroticus) eggs

Development of effective cryopreservation protocols relies on knowledge of the fundamental cryobiological characteristics for a particular cell type. These characteristics include osmotic behaviour, membrane permeability characteristics, and osmotic tolerance limits. Here, we report on measures of these characteristics for unfertilized and fertilised eggs of the sea urchin (Evechinus chloroticus). In NaCl solutions of varying osmolalities, sea urchin eggs behaved as ideal linear osmometers. The osmotically inactive volume (vb) was similar for unfertilized and fertilised eggs, 0.367+/-0.008 (mean+/-SE) and 0.303+/-0.007, respectively. Estimates of water solubility (Lp) and solute permeability (Ps) and their respective activation energies (Ea) for unfertilized and fertilised eggs were determined following exposure to cryoprotectant (CPA) solutions at different temperatures. Irrespective of treatment, fertilised eggs had higher values of Lp and Ps. The presence of a CPA decreased Lp. Among CPAs, solute permeability was highest for propylene glycol followed by dimethyl sulphoxide and then ethylene glycol. Measures of osmotic tolerance limits of the eggs revealed unfertilized eggs were able to tolerate volumetric changes of -20% and +30% of their equilibrium volume; fertilised eggs were able to tolerate changes +/-30%. Using membrane permeability data and osmotic tolerance limits, we established effective methods for loading and unloading CPAs from the eggs. The results of this study establish cryobiological characteristics for E. chloroticus eggs of use for developing an effective cryopreservation protocol. The approach we outline can be readily adapted for determining cryobiological characteristics of other species and cell types, as an aid to successful cryopreservation.     

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral–aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral–aboral gene regulatory network. A model for oral–aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Lefty's function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral–aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.     

The isolation of isohistones by preparative gel electrophoresis from embryos of the sea urchin Parechinus angulosus

Ten isohistones from the embryo of the sea urchin Parechinus angulosus have been isolated by preparative gel electrophoresis and characterised by electrophoretic mobility in two detergent systems, amino acid composition and partial sequences. This brings the total number of different histones identified which are synthesized at one or the other time during the life cycle of the sea urchin to a minimum of 24 structurally characterised polypeptides.     

Bacterial phylotypes associated with the digestive tract of the sea urchin Paracentrotus lividus and the ascidian <Emphasis Type="Italic">Microcosmus</Emphasis> sp.

We used sequencing and phylogenetic analysis of PCR-amplified 16S rRNA genes from bacteria that are associated with the esophagus/pharynx, stomach and intestine of two marine sympatric invertebrates but with different feeding mechanisms, namely the sea urchin Paracentrotus lividus (grazer) and the ascidian Microcomus sp. (suspension feeder). Amplifiable DNA was retrieved from all sections except the pharynx of the ascidian. Based on the inferred phylogeny of the retrieved sequences, the sea urchin’s esophagus is mainly characterized mostly by bacteria belonging to α-, γ-Proteobacteria and Bacteriodetes, most probably originating from the surrounding environment. The stomach revealed phylotypes that belonged to γ- and δ-Proteobacteria, Verrucomicrobia and Fusobacteria. Since the majority of their closest relatives are anaerobic species and they could be putative symbionts of the P. lividus stomach, in which anaerobic conditions also prevail. Seven out of eight phylotypes found in the sea urchin’s intestine belonged to sulfate reducing δ-Proteobacteria, and one to γ-Proteobacteria, with possible nutritional activities, i.e. degradation of complex organic compounds which is beneficial for the animal. The bacterial phylotypes of the ascidian digestive tract belonged only to the phyla of Actinobacteria and Proteobacteria. The stomach phylotypes of the ascidian were related to pathogenic bacteria possibly originating from the water column, while the intestine seemed to harbour putative symbiotic bacteria that are involved in the degradation of nitrogenous and other organic compounds, thus assisting ascidian nutrition. The text was submitted by the authors in English.     

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.