A transcriptomic examination of encased rotifer embryos reveals the developmental trajectory leading to long-term dormancy; are they "animal seeds"?

BACKGROUND: Organisms from many distinct evolutionary lineages acquired the capacity to enter a dormant state in response to environmental conditions incompatible with maintaining normal life activities. Most studied organisms exhibit seasonal or annual episodes of dormancy, but numerous less studied organisms enter long-term dormancy, lasting decades or even centuries. Intriguingly, many planktonic animals produce encased embryos known as resting eggs or cysts that, like plant seeds, may remain dormant for decades. Herein, we studied a rotifer Brachionus plicatilis as a model planktonic species that forms encased dormant embryos via sexual reproduction and non-dormant embryos via asexual reproduction and raised the following questions: Which genes are expressed at which time points during embryogenesis? How do temporal transcript abundance profiles differ between the two types of embryos? When does the cell cycle arrest? How do dormant embryos manage energy? RESULTS: As the molecular developmental kinetics of encased embryos remain unknown, we employed single embryo RNA sequencing (CEL-seq) of samples collected during dormant and non-dormant embryogenesis. We identified comprehensive and temporal transcript abundance patterns of genes and their associated enriched functional pathways. Striking differences were uncovered between dormant and non-dormant embryos. In early development, the cell cycle-associated pathways were enriched in both embryo types but terminated with fewer nuclei in dormant embryos. As development progressed, the gene transcript abundance profiles became increasingly divergent between dormant and non-dormant embryos. Organogenesis was suspended in dormant embryos, concomitant with low transcript abundance of homeobox genes, and was replaced with an ATP-poor preparatory phase characterized by very high transcript abundance of genes encoding for hallmark dormancy proteins (e.g., LEA proteins, sHSP, and anti-ROS proteins, also found in plant seeds) and proteins involved in dormancy exit. Surprisingly, this period appeared analogous to the late maturation phase of plant seeds. CONCLUSIONS: The study highlights novel divergent temporal transcript abundance patterns between dormant and non-dormant embryos. Remarkably, several convergent functional solutions appear during the development of resting eggs and plant seeds, suggesting a similar preparatory phase for long-term dormancy. This study accentuated the broad novel molecular features of long-term dormancy in encased animal embryos that behave like "animal seeds".

Dormancy in Embryos: Insight from Hydrated Encysted Embryos of an Aquatic Invertebrate.

Numerous aquatic invertebrates remain dormant for decades in a hydrated state as encysted embryos. In search for functional pathways associated with this form of dormancy, we used label-free quantitative proteomics to compare the proteomes of hydrated encysted dormant embryos (resting eggs; RE) with nondormant embryos (amictic eggs; AM) of the rotifer Brachionus plicatilisA total of 2631 proteins were identified in rotifer eggs. About 62% proteins showed higher abundance in AM relative to RE (Fold Change>3; p = 0.05). Proteins belonging to numerous putative functional pathways showed dramatic changes during dormancy. Most striking were changes in the mitochondria indicating an impeded metabolism. A comparison between the abundance of proteins and their corresponding transcript levels, revealed higher concordance for RE than for AM. Surprisingly, numerous highly abundant dormancy related proteins show corresponding high mRNA levels in metabolically inactive RE. As these mRNAs and proteins degrade at the time of exit from dormancy they may serve as a source of nucleotides and amino acids during the exit from dormancy. Because proteome analyses point to a similarity in functional pathways of hydrated RE and desiccated life forms, REs were dried. Similar hatching and reproductive rates were found for wet and dried REs, suggesting analogous pathways for long-term survival in wet or dry forms. Analysis by KEGG pathways revealed a few general strategies for dormancy, proposing an explanation for the low transcriptional similarity among dormancies across species, despite the resemblance in physiological phenotypes.

The making of an embryo in a basal metazoan: Proteomic analysis in the sea anemone Nematostella vectensis.

Cnidarians are widely distributed basal metazoans that play an important role in the marine ecosystem. Their genetic diversity and dispersal depends on successful oogenesis, fertilization and embryogenesis. To understand the processes that lead to successful embryogenesis in these basal organisms, we conducted comparative proteomics on the model sea anemone Nematostella vectensis. We examined four developmental stages from oocyte maturation through early embryogenesis, as well as the oocyte jelly sac in which fertilization and embryogenesis take place. Our analysis revealed 37 stage-specifically expressed proteins, including cell cycle, cellular energy related and DNA replication proteins and transcription regulators. Using in situ hybridization, we show that within the mesenteria, two cell types support successful oocyte development and embryogenesis. Large somatic supporting cells synthesize vitellogenin, the most abundant egg yolk protein within the oocyte, whereas mesenteria gland cells synthesize mucin 5B, which was found to be the main component of the jelly sac. These findings shed light on the sexual reproduction program in cnidarians and suggest a high conservation with proteins governing oogenesis in Bilateria.

Biochemical diversification through foreign gene expression in bdelloid rotifers.

Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%-9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex.

Insight into molecular pathways of retinal metabolism, associated with vitellogenesis in zebrafish.

Retinal is the main retinoid stored in oviparous eggs of fish, amphibians, and reptiles, reaching the oocytes in association with vitellogenins, the yolk precursor proteins. During early presegmentation stages of zebrafish embryos, retinal is metabolized to retinoic acid (RA), which regulates genes involved in cell proliferation, differentiation, and tissue function and is therefore essential for normal embryonic development. While synthesis of vitellogenin and its regulation by 17ß-estradiol (E(2)) were extensively investigated, pathways for retinal synthesis remain obscure. We determined the expression pattern of 46 candidate genes, aiming at identifying enzymes associated with retinal synthesis, ascertaining whether they were regulated by E(2), and finding pathways that could fulfill the demand for retinoids during vitellogenesis. Genes associated with retinal synthesis were upregulated in liver (rdh10, rdh13, sdr) and surprisingly also in intestine (rdh13) and ovary (rdh1, sdr), concomitantly with higher gene expression and synthesis of vitellogenins in liver but also in extrahepatic tissues, shown here for the first time. Vitellogenin synthesis in the ovary was regulated by E(2). Gene expression studies suggest that elevated retinal synthesis in liver, intestine, and ovary also depends on cleavage of carotenoids (by Bcdo2 or Bmco1), but in the ovary it may also be contingent on higher uptake of retinol from the circulatory system (via Stra6) and retinol synthesis from retinyl esters (by Lpl). Decrease in oxidation (by Raldh2 or Raldh3) of retinal to RA and/or degradation of RA (by Cyp26a1) may also facilitate higher hepatic retinal levels. Together, these processes enable meeting the putative demands of retinal for binding to vitellogenins. Bioinformatic tools reveal multiple hormone response elements in the studied genes, suggesting complex and intricate regulation of these processes.

Design and characterization of genetically engineered zebrafish aquaporin-3 mutants highly permeable to the cryoprotectant ethylene glycol.

BACKGROUND: Increasing cell membrane permeability to water and cryoprotectants is critical for the successful cryopreservation of cells with large volumes. Artificial expression of water-selective aquaporins or aquaglyceroporins (GLPs), such as mammalian aquaporin-3 (AQP3), enhances cell permeability to water and cryoprotectants, but it is known that AQP3-mediated water and solute permeation is limited and pH dependent. To exploit further the possibilities of using aquaporins in cryobiology, we investigated the functional properties of zebrafish (Danio rerio) GLPs. RESULTS: Water, glycerol, propylene glycol and ethylene glycol permeability of zebrafish Aqp3a, -3b, -7, -9a, -9b, -10a and -10b, and human AQP3, was examined. Expression in Xenopus laevis oocytes indicated that the permeability of DrAqp3a and -3b to ethylene glycol was higher than for glycerol or propylene glycol under isotonic conditions, unlike other zebrafish GLPs and human AQP3, which were more permeable to glycerol. In addition, dose-response experiments and radiolabeled ethylene glycol uptake assays suggested that oocytes expressing DrAqp3b were permeated by this cryoprotectant more efficiently than those expressing AQP3. Water and ethylene glycol transport through DrAqp3a and -3b were, however, highest at pH 8.5 and completely abolished at pH 6.0. Point mutations in the DrAqp3b amino acid sequence rendered two constructs, DrAqp3b-T85A showing higher water and ethylene glycol permeability at neutral and alkaline pH, and DrAqp3b-H53A/G54H/T85A, no longer inhibited at acidic pH but less permeable than the wild type. Finally, calculation of permeability coefficients for ethylene glycol under concentration gradients confirmed that the two DrAqp3b mutants were more permeable than wild-type DrAqp3b and/or AQP3 at neutral pH, resulting in a 2.6- to 4-fold increase in the oocyte intracellular concentration of ethylene glycol. CONCLUSION: By single or triple point mutations in the DrAqp3b amino acid sequence, we constructed one mutant with enhanced ethylene glycol permeability and another with reduced pH sensitivity. The DrAqp3b and the two mutant constructs may be useful for application in cryobiology.

Late embryogenesis abundant (LEA) proteins in nondesiccated, encysted, and diapausing embryos of rotifers.

Two genes encoding for late embryogenesis abundant proteins (LEAs) are expressed in encysted diapausing embryos (or resting eggs) of rotifers (Brachionus plicatilis O.F. Müller) and females forming them. The two genes (bpa-leaa and bpa-leab) share approximately 50% of their nucleotides sequence, and bpa-leaa is more than twofold longer than bpa-leab. The deduced amino acid sequences show high abundance of alanine, glycine, lysine, and glutamic acid; a hydropathy index of lower than one; and a relatively high (81-82%) predicted probability of forming alpha-helices in their secondary structure, all of which are characteristic features of LEAs. The predicted molecular masses of bpa-LEAA ( approximately 67 kDa) and bpa-LEAB ( approximately 27 kDa) are similar to the molecular mass determined by Western-blot analyses, suggesting a low probability of posttranslational modifications. In silico analysis reveals that the two LEAs resemble group 3 LEAs based on the repeats for 11mer motifs, although they also display several putative amino acids typical of the 20mer motif of group 1 LEAs. The rotifer LEAs do not contain a predicted target sequence and are more likely localized in the cytosol. LEAs were expressed in resting eggs and females producing them, but not in other female forms or males. LEA transcripts and proteins are degraded during hatching, suggesting that LEAs are developmentally programmed during resting egg formation and hatching. LEAs probably equip the resting eggs to withstand desiccation if that occurs during dormancy. The present study expands our knowledge about the biological pathways associated with formation of rotifer resting eggs and also demonstrates the occurrence of LEAs in dormant, nondesiccated, encysted animal embryos.

Oogenesis in teleosts: how eggs are formed.

One of the major objectives of the aquaculture industry is the production of a large number of viable eggs with high survival. Major achievements have been made in recent years in improving protocols for higher efficiency of egg production and viability of progeny. Main gaps remain, however, in understanding the dynamic processes associated with oogenesis, the formation of an egg, from the time that germ cells turn into oogonia, until the release of ova during spawning in teleosts. Recent studies on primordial germ-cells, yolk protein precursors and their processing within the developing oocyte, the deposition of vitamins in eggs, structure and function of egg envelopes and oocyte maturation processes, further reveal the complexity of oogenesis. Moreover, numerous circulating endocrine and locally-acting paracrine and autocrine factors regulate the various stages of oocyte development and maturation. Though it is clear that the major regulators during vitellogenesis and oocyte maturation are the pituitary gonadotropins (LH and FSH) and sex steroids, the picture emerging from recent studies is of complex hormonal cross-talk at all stages between the developing oocyte and its surrounding follicle layers to ensure coordination of the various processes that are involved in the production of a fertilizable egg. In this review we aim at highlighting recent advances on teleost fish oocyte differentiation, maturation and ovulation, including those involved in the degeneration and reabsorption of ovarian follicles (atresia). The role of blood-borne and local ovarian factors in the regulation of the key steps of development reveal new aspects associated with egg formation.

Revealing genes associated with vitellogenesis in the liver of the zebrafish (Danio rerio) by transcriptome profiling.

BACKGROUND: In oviparous vertebrates, including fish, vitellogenesis consists of highly regulated pathways involving 17beta-estradiol (E2). Previous studies focused on a relatively small number of hepatic expressed genes during vitellogenesis. This study aims to identify hepatic genes involved in vitellogenesis and regulated by E2, by using zebrafish microarray gene expression profiling, and to provide information on functional distinctive genes expressed in the liver of a vitellogenic female, using zebrafish as a model fish. RESULTS: Genes associated with vitellogenesis were revealed by the following paired t-tests (SAM) comparisons: a) two-month old vitellogenic (Vit2) females were compared with non-vitellogenic (NV) females, showing 825 differentially expressed transcripts during early stages of vitellogenesis, b) four-month old vitellogenic (Vit4) females were compared with NV females, showing 1,046 differentially expressed transcripts during vitellogenesis and c) E2-treated males were compared with control males, showing 1,828 differentially expressed transcripts regulated by E2. A Venn diagram revealed 822 common transcripts in the three groups, indicating that these transcripts were involved in vitellogenesis and putatively regulated by E2. In addition, 431 transcripts were differentially expressed in Vit2 and Vit4 females but not in E2-treated males, indicating that they were putatively not up-regulated by E2. Correspondence analysis showed high similarity in expression profiles of Vit2 with Vit4 and of NV females with control males. The E2-treated males differed from the other groups. The repertoire of genes putatively regulated by E2 in vitellogenic females included genes associated with protein synthesis and reproduction. Genes associated with the immune system processes and biological adhesion, were among the genes that were putatively not regulated by E2. E2-treated males expressed a large array of transcripts that were not associated with vitellogenesis.The study revealed several genes that were not reported before as being regulated by E2. Also, the hepatic expression of several genes was reported here for the first time. CONCLUSION: Gene expression profiling of liver samples revealed 1,046 differentially expressed transcripts during vitellogenesis of which at least ~64% were regulated by E2. The results raise the question on the regulation pattern and temporal pleiotropic expression of hepatic genes in vitellogenic females.

Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis.

BACKGROUND: Microscopic monogonont rotifers, including the euryhaline species Brachionus plicatilis, are typically found in water bodies where environmental factors restrict population growth to short periods lasting days or months. The survival of the population is ensured via the production of resting eggs that show a remarkable tolerance to unfavorable conditions and remain viable for decades. The aim of this study was to generate Expressed Sequence Tags (ESTs) for molecular characterisation of processes associated with the formation of resting eggs, their survival during dormancy and hatching. RESULTS: Four normalized and four subtractive libraries were constructed to provide a resource for rotifer transcriptomics associated with resting-egg formation, storage and hatching. A total of 47,926 sequences were assembled into 18,000 putative transcripts and analyzed using both Blast and GO annotation. About 28-55% (depending on the library) of the clones produced significant matches against the Swissprot and Trembl databases. Genes known to be associated with desiccation tolerance during dormancy in other organisms were identified in the EST libraries. These included genes associated with antioxidant activity, low molecular weight heat shock proteins and Late Embryonic Abundant (LEA) proteins. Real-time PCR confirmed that LEA transcripts, small heat-shock proteins and some antioxidant genes were upregulated in resting eggs, therefore suggesting that desiccation tolerance is a characteristic feature of resting eggs even though they do not necessarily fully desiccate during dormancy. The role of trehalose in resting-egg formation and survival remains unclear since there was no significant difference between resting-egg producing females and amictic females in the expression of the tps-1 gene. In view of the absence of vitellogenin transcripts, matches to lipoprotein lipase proteins suggest that, similar to the situation in dipterans, these proteins may serve as the yolk proteins in rotifers. CONCLUSION: The 47,926 ESTs expand significantly the current sequence resource of B. plicatilis. It describes, for the first time, genes putatively associated with resting eggs and will serve as a database for future global expression experiments, particularly for the further identification of dormancy related genes.

Facing Adversity: Dormant Embryos in Rotifers.

An in-depth look at the basic aspects of dormancy in cyclic parthenogenetic organisms is now possible thanks to research efforts conducted over the past two decades with rotifer dormant embryos. In this review, we assemble and compose the current knowledge on four central themes: (1) distribution of dormancy in animals, with an overview on the phylogenetic distribution of embryo dormancy in metazoans, and (2) physiological and cellular processes involved in dormancy, with a strong emphasis on the dormant embryos of cyclically parthenogenetic monogonont rotifers; and discussions of (3) the selective pressures and (4) the evolutionary and population implications of dormancy in these animals. Dormancy in metazoans is a widespread phenomenon with taxon-specific features, and rotifers are among the animals in which dormancy is an intrinsic feature of their life cycle. Our review shows that embryo dormancy in rotifers shares common functional pathways with other taxa at the molecular and cellular level, despite the independent evolution of dormancy across phyla. These pathways include the arrest of similar metabolic routes and the usage of common metabolites for the stabilization of cellular structures and to confer stress resistance. We conclude that specific features of recurrent harsh environmental conditions are a powerful selective pressure for the fine-tuning of dormancy patterns in rotifers. We hypothesize that similar mechanisms at the organism level will lead to similar adaptive consequences at the population level across taxa, among which the formation of egg banks, the coexistence of species, and the possibility of differentiation among populations and local adaptation stand out. Our review shows how studies of rotifers have contributed to improved knowledge of all of these aspects.

Metabolomics reveals novel insight on dormancy of aquatic invertebrate encysted embryos.

Numerous aquatic invertebrates survive harsh environments by displaying dormancy as encysted embryos. This study aimed at determining whether metabolomics could provide molecular insight to explain the "dormancy syndrome" by highlighting functional pathways and metabolites, hence offering a novel comprehensive molecular view of dormancy. We compared the metabolome of morphologically distinct dormant encysted embryos (resting eggs) and non-dormant embryos (amictic eggs) of a rotifer (Brachionus plicatilis). Metabolome profiling revealed ~5,000 features, 1,079 of which were annotated. Most of the features were represented at significantly higher levels in non-dormant than dormant embryos. A large number of features was assigned to putative functional pathways indicating novel differences between dormant and non-dormant states. These include features associated with glycolysis, the TCA and urea cycles, amino acid, purine and pyrimidine metabolism. Interestingly, ATP, nucleobases, cyclic nucleotides, thymidine and uracil, were not detected in dormant resting eggs, suggesting an impairment of response to environmental and internal cues, cessation of DNA synthesis, transcription and plausibly translation in the dormant embryos. The levels of trehalose or its analogues, with a role in survival under desiccation conditions, were higher in resting eggs. In conclusion, the current study highlights metabolomics as a major analytical tool to functionally compare dormancy across species.

Alterations in micro-ribonucleic acid expression profiles reveal a novel pathway for estrogen regulation.

Estrogens are steroid hormones that have been implicated in a variety of cellular and physiological processes in the development of diseases such as cancer and are also known to be associated with the effects of endocrine disrupting chemicals. Here we show that 17beta-estradiol (E(2)) alters microRNA (miRNA) expression profiles in the adult zebrafish (Danio rerio). An association between E(2) and the expression of 25 miRNAs was found 12 h after treatment. Among the most up-regulated miRNAs were miR-196b and let-7h, and the most down-regulated miRNAs included miR-130c and miR-101a. Tissue-specific changes in the transcripts levels of estrogen receptors (Esr1, Esr2a, and Esr2b) and miRNAs were found after hormone treatment. The most up-regulated miR-196b and its precursors are highly expressed in the skin and showed similar tissue-specific expression patterns after treatment, indicating a common pattern of regulation by E(2). MiR-196b was shown to fine-tune the expression of its target gene Hoxb8a after treatment in whole-body homogenates. Taken together, our results suggest a novel pathway for the multifunctional and pleiotropic effects of estrogens and open new directions for future investigations of their association with miRNAs involved in estrogen-regulated physiological processes and diseases.

Apolipocrustacein, formerly vitellogenin, is the major egg yolk precursor protein in decapod crustaceans and is homologous to insect apolipophorin II/I and vertebrate apolipoprotein B.

BACKGROUND: In animals, the biogenesis of some lipoprotein classes requires members of the ancient large lipid transfer protein (LLTP) superfamily, including the cytosolic large subunit of microsomal triglyceride transfer protein (MTP), vertebrate apolipoprotein B (apoB), vitellogenin (Vtg), and insect apolipophorin II/I precursor (apoLp-II/I). In most oviparous species, Vtg, a large glycolipoprotein, is the main egg yolk precursor protein. RESULTS: This report clarifies the phylogenetic relationships of LLTP superfamily members and classifies them into three families and their related subfamilies. This means that the generic term Vtg is no longer a functional term, but is rather based on phylogenetic/structural criteria. In addition, we determined that the main egg yolk precursor protein of decapod crustaceans show an overall greater sequence similarity with apoLp-II/I than other LLTP, including Vtgs. This close association is supported by the phylogenetic analysis, i.e. neighbor-joining, maximum likelihood and Bayesian inference methods, of conserved sequence motifs and the presence of three common conserved domains: an N-terminal large lipid transfer module marker for LLTP, a DUF1081 domain of unknown function in their central region exclusively shared with apoLp-II/I and apoB, and a von Willebrand-factor type D domain at their C-terminal end. Additionally, they share a conserved functional subtilisin-like endoprotease cleavage site with apoLp-II/I, in a similar location. CONCLUSION: The structural and phylogenetic data presented indicate that the major egg yolk precursor protein of decapod crustaceans is surprisingly closely related to insect apoLp-II/I and vertebrate apoB and should be known as apolipocrustacein (apoCr) rather than Vtg. These LLTP may arise from an ancient duplication event leading to paralogs of Vtg sequences. The presence of LLTP homologs in one genome may facilitate redundancy, e.g. involvement in lipid metabolism and as egg yolk precursor protein, and neofunctionalization and subfunctionalization, e.g. involvement in clotting cascade and immune response, of extracellular LLTP members. These protein-coding nuclear genes may be used to resolve phylogenetic relationships among the major arthropod groups, especially the Pancrustacea-major splits.

Isolation and functional characterization of a new shrimp ovarian peritrophin with antimicrobial activity from Fenneropenaeus merguiensis.

Shrimp ovarian peritrophin (SOP), a major protein in jelly layer (JL) and cortical rods (CRs), is proposed to play a role in the protection of spawned eggs. The full sequence of SOP cDNA from Fenneropenaeus merguiensis (Fm-SOP) shares approximately 50% identity with other SOP sequences and contains several putative chitin-binding or peritrophin-A domains. Interestingly, Fm-SOP contains a putative 61-amino acid propeptide located at the N-terminal end, downstream of a 19-amino acid signal peptide, which is unique among penaeid SOP sequences described so far. This 61-amino-acid sequence constitutes a putative chitin-binding domain with six conserved cysteines, and is cleaved at a dibasic recognition site for a furin (subtilisin-like endoprotease). Expression analyses indicated that Fm-SOP mRNA is abundant in early vitellogenic ovaries and scarce in late-vitellogenic ovaries. Conversely, Fm-SOP protein is the most abundant at the end of vitellogenesis. To investigate its biological function, a recombinant Fm-SOP was expressed to generate a glycosylated protein in Spodoptera frugiperda Sf9 cells (rSOP-Sf9) and a nonglycosylated protein (rSOP-Ec) in Escherichia coli. rSOP-Sf9 and rSOP-Ec were found to bind to chitin, similarly to the native protein extracted from F. merguiensis ovaries. Most interestingly, rSOP-Ec displayed a chitinase activity and efficiently inhibited the growth of Vibrio harveyi and Staphylococcus aureus, with minimum inhibitory concentrations of 2.4 and 15.7 microM, respectively. This first report shows that a major component of CR and JL is biologically active against known pathogens and predicts a significant role of JL in the protection of the spawned eggs against pathogens.

Long-term survival of hydrated resting eggs from Brachionus plicatilis.

BACKGROUND: Several organisms display dormancy and developmental arrest at embryonic stages. Long-term survival in the dormant form is usually associated with desiccation, orthodox plant seeds and Artemia cysts being well documented examples. Several aquatic invertebrates display dormancy during embryonic development and survive for tens or even hundreds of years in a hydrated form, raising the question of whether survival in the non-desiccated form of embryonic development depends on pathways similar to those occurring in desiccation tolerant forms. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, Illumina short read sequencing was used to generate transcription profiles from the resting and amictic eggs of an aquatic invertebrate, the rotifer, Brachionus plicatilis. These two types of egg have very different life histories, with the dormant or diapausing resting eggs, the result of the sexual cycle and amictic eggs, the non-dormant products of the asexual cycle. Significant transcriptional differences were found between the two types of egg, with amictic eggs rich in genes involved in the morphological development into a juvenile rotifer. In contrast, representatives of classical "stress" proteins: a small heat shock protein, ferritin and Late Embryogenesis Abundant (LEA) proteins were identified in resting eggs. More importantly however, was the identification of transcripts for messenger ribonucleoprotein particles which stabilise RNA. These inhibit translation and provide a valuable source of useful RNAs which can be rapidly activated on the exit from dormancy. Apoptotic genes were also present. Although apoptosis is inconsistent with maintenance of prolonged dormancy, an altered apoptotic pathway has been proposed for Artemia, and this may be the case with the rotifer. CONCLUSIONS: These data represent the first transcriptional profiling of molecular processes associated with dormancy in a non-desiccated form and indicate important similarities in the molecular pathways activated in resting eggs compared with desiccated dormant forms, specifically plant seeds and Artemia.

Fibrin microbeads loaded with mesenchymal cells support their long-term survival while sealed at room temperature.

Efficient transfer of progenitor cells without affecting their survival is a key factor in any practical cell therapy. Fibrin microbeads (FMB) were developed as hard biodegradable cell carriers. The FMB could efficiently isolate mesenchymal stem cells (MSCs) from different sources and support the expansion of matrix-dependent cell types in a three-dimensional culture in slow rotation. The cells on FMB could also undergo induced differentiation for their eventual implantation to enhance tissue regeneration. FMB loaded with isolated human MSC (hMSC) were sealed in tubes topped up with medium. Almost full cell survival was recorded when the sealed cells were maintained in room temperature for up to 10 days, followed by a recovery period of 24 hrs at optimal conditions. Assay of cells recovery after such long room temperature storage showed ∼80%-100% survival of the cells on FMB, with only a marginal survival of cells that were kept in suspension without FMB in the same conditions. The hMSC that survived storage at room temperature preserved their profile of mesenchymal cell surface markers, their rate of proliferation, and their differentiation potential. The cell protective effect was not dependent on the presence of serum in the storage medium. It was clearly shown that over-expression of hypoxia induced factor-1α in hMSC with time, which may have protected the sealed cells on FMB at room temperature storage, was not necessarily related to extreme hypoxic stress. Foreskin normal fibroblasts on FMB sealed at room temperature were similarly protected, but with no elevation of their hypoxia-induced factor-1α expression. The results also show that FMB, unlike other commercially available cell carriers, could be used for delivery and shipping of progenitor cells at room temperature for extended time intervals. This could be highly useful for cell transfer for therapeutic application and for simplified cell transfer between different research centers.

Gilthead sea bream (Sparus auratus) and European sea bass (Dicentrarchus labrax) expressed sequence tags: Characterization, tissue-specific expression and gene markers.

The gilthead sea bream, Sparus auratus, and the European sea bass, Dicentrarchus labrax, are two of the most important marine species cultivated in Southern Europe. This study aimed at increasing genomic resources for the two species and produced and annotated two sets of 30,000 expressed sequence tags (EST) each from 14 normalized tissue-specific cDNA libraries from sea bream and sea bass. Clustering and assembly of the ESTs formed 5268 contigs and 12,928 singletons for sea bream and 4573 contigs and 13,143 singletons for sea bass, representing 18,196 and 17,716 putative unigenes, respectively. Assuming a similar number of genes in sea bass, sea bream and in the model fish Gasterosteus aculeatus genomes, it was estimated that approximately two thirds of the sea bream and the sea bass transcriptomes were covered by the unigene collections. BLAST sequence similarity searches (using a cut off of e-value <10(-5)) against fully the curated SwissProt (and TrEMBL) databases produced matches of 28%(37%) and 43%(53%) of the sea bream and sea bass unigene datasets respectively, allowing some putative designation of function. A comparative approach is described using human Ensembl peptide ID homolog's for functional annotation, which increased the number of unigenes with GO terms assigned and resulted in more GO terms assigned per unigene. This allowed the identification of tissue-specific genes using enrichment analysis for GO pathways and protein domains. The comparative annotation approach represents a good strategy for transferring more relevant biological information from highly studied species to genomic resource poorer species. It was possible to confirm by interspecies mRNA-to-genomic alignments 25 and 21 alternative splice events in sea bream and sea bass genes, respectively. Even using normalized cDNA from relatively few pooled individuals it was possible to identify 1145 SNPs and 1748 microsatellites loci for genetic marker development. The EST data are being applied to a range of projects, including the development microarrays, genetic and radiation hybrid maps and QTL genome scans. This highlights the important role of ESTs for generating genetic and genomic resources of aquaculture species.

Expression of genes associated with retinoid metabolism in the trout ovarian follicle.

In vertebrates producing oviparous eggs, retinoids and their precursor molecules need to be deposited in oocytes during vitellogenesis. While most studies focus on the transport of retinoids and carotenoids formed outside the fish ovary and their deposition within the developing oocyte, recent investigations in mammalian species suggest the ovary is an important site for retinoid and carotenoid metabolism. Therefore, we investigated the expression of six genes (bcmo1, bcdo2, rbp1, lrat, rbp4, and stra6) associated with retinoids and carotenoids in juvenile and adult trout ovaries. Except for bcdo2, these genes were expressed in the ovary. Expression of stra6 was detected in the ovary but not in the liver. Gene expression levels of bcmo1 and stra6 were significantly higher in juvenile ovaries, in contrast to those of rbp1, rbp4, and lrat, which were similar in all tested ovarian stages. The mean values of the relative mRNA levels of the tested genes differed between the ovary and the liver. Gene transcripts of rbp4 and bcmo1 were identified by in situ hybridization in the theca layer, and all five genes were expressed in the granulosa, stromal cells, and only the early vitellogenic oocyte. The occurrence of retinol-binding protein in the theca and granulosa cells and within oocytes at all developmental stages was revealed by immunocytochemistry. These results indicate that ovarian cells express genes putatively associated with cleavage of beta-carotene, storage and mobilization of retinyl-esters, and of retinol-binding protein synthesis, suggesting a novel pathway for providing retinoids and carotenoids to developing fish ovarian follicles.