Isotopic enrichment in Rhizoprionodon porosus (Poey, 1861) and Carcharhinus porosus (Ranzani, 1840) embryos.

Isotopic values of two Caribbean sharpnose shark Rhizoprionodon porosus litters (Poey, 1861) with two and three embryos and one litter of 11 smalltail shark Carcharhinus porosus embryos showed enriched 15 N and 13 C compared to their mothers. In R. porosus, embryonic isotope values were 3.06 ± 0.07‰ and 0.69 ± 0.15‰ greater than their mothers' for δ15 N and δ13 C, respectively, whereas in C. porosus, δ15 N and δ13 C were 1.79 ± 0.09‰ and 1.31 ± 0.17‰ greater in embryos than their mothers.

Protein Purification and Western Blot Detection from Single Zebrafish Embryo.

Characterization of a protein of interest during development is essential for functional studies. A general strategy for understanding the function of a particular protein involves the generation of null mutations, or treatment with drugs, that interfere with its activity. To demonstrate that the synthesis, stability, or activity of a protein has been affected, accurate and efficient detection of low amounts of protein is essential. This can be achieved by immunohistochemistry or by western blot. Here we describe a method for the detection of proteins from single de-yolked zebrafish embryos. This procedure includes a fixation step and the concomitant elimination of lipids from the yolk cell. We show that this approach allows the rapid analysis of proteins in embryos without having to manually remove the yolk. This method provides a convenient alternative for genotyping of mutant embryos as early as the 128 cell stage. In addition, in drug- or morpholino-treated embryos, the correlation between the penetrance of a phenotype and the concentration of a protein present may be established.

Studies on Toxicity of Suspensions of CdTe Quantum Dots to Biomphalaria glabrata Mollusks.

Quantum dots have generated great interest because of their optical properties, both to life sciences and electronics applications. However, possible risks to the environment associated with these nanoparticles are still under investigation. The present study aimed to evaluate the toxicity of suspensions of cadmium telluride (CdTe) quantum dots to Biomphalaria glabrata mollusks, a very sensitive aquatic environmental bioindicator for physical and chemical agents. Toxicity was examined by using embryos and adult mollusks as well as hemocytes. The distribution of cadmium in the organs of adults was also assessed. Effects of the stabilizing agent of the quantum dots were also evaluated. Animals were exposed to suspensions of quantum dots for 24 h, at concentrations varying from 1.2 to 20 nM for embryos and from 50 to 400 nM for adult mollusks. Results showed that suspensions of quantum dots induced malformations and mortality in embryos and mortality in adults, depending on the concentration applied. In the cytotoxicity study, hemocyte apoptosis was observed in adults exposed to the highest concentration of quantum dots applied as well as to the stabilizing agent. Cell binucleation and micronucleus frequencies were not significative. Bioaccumulation evaluation revealed that quantum dots targeted the digestive gland (hepatopancreas). Taken together, outcomes suggested that specific nano-effects related directly not only to composition but also to the aggregation of quantum dots may be mediating the observed toxicity. Thus B. glabrata was determined to be a very sensitive species for interpreting possible nano-effects in aquatic environments. Environ Toxicol Chem 2019;38:2128-2136. © 2019 SETAC.

Dose inseminante e resfriamento de embriões de peixes de água doce / Insemination dose and conservation of freshwater fish embryos

A aquicultura tem sido utilizada como importante ferramenta para suprir o constante aumento doconsumo de pescado. Um dos principais aspectos para a intensificação da produção piscícola, acompanhada dasustentabilidade tanto econômica quanto ambiental, é a utilização de técnicas de propagação artificial. Dessaforma, para otimizar a reprodução artificial têm-se investido em estudos afim de definir a proporção ideal deespermatozoides por ovócito e os melhores métodos de conservação para embriões de espécies de peixes de águadoce. Objetivou-se fazer uma breve revisão sobre as doses inseminantes e a biotecnologia de resfriamento deembriões de peixes de água doce que já foram estabelecidas.

Aquaculture has been used as an important tool to meet the steady increase in fish consumption. Amajor aspect for the intensification of fish production, accompanied by both economic and environmentalsustainability, is the use of techniques of artificial propagation. Thus, to optimize artificial reproduction havebeen invested in research in order to define the optimal ratio the sperm to oocyte and the best conservationmethods for embryos species of freshwater fish. The objective was to make a brief review of the inseminationdoses cooling biotechnology freshwater fish embryos that have already been established.

Dose inseminante e resfriamento de embriões de peixes de água doce / Insemination dose and conservation of freshwater fish embryos

A aquicultura tem sido utilizada como importante ferramenta para suprir o constante aumento doconsumo de pescado. Um dos principais aspectos para a intensificação da produção piscícola, acompanhada dasustentabilidade tanto econômica quanto ambiental, é a utilização de técnicas de propagação artificial. Dessaforma, para otimizar a reprodução artificial têm-se investido em estudos afim de definir a proporção ideal deespermatozoides por ovócito e os melhores métodos de conservação para embriões de espécies de peixes de águadoce. Objetivou-se fazer uma breve revisão sobre as doses inseminantes e a biotecnologia de resfriamento deembriões de peixes de água doce que já foram estabelecidas.(AU)

Aquaculture has been used as an important tool to meet the steady increase in fish consumption. Amajor aspect for the intensification of fish production, accompanied by both economic and environmentalsustainability, is the use of techniques of artificial propagation. Thus, to optimize artificial reproduction havebeen invested in research in order to define the optimal ratio the sperm to oocyte and the best conservationmethods for embryos species of freshwater fish. The objective was to make a brief review of the inseminationdoses cooling biotechnology freshwater fish embryos that have already been established.(AU)

A descriptive study of the occurrence and significance of lipids in Taenia hydatigena eggs.

The aim of this work was to investigate the lipid content of Taenia hydatigena eggs and to evaluate the role of lipids in the maintenance of embryo viability. The total lipid content of the egg was 4.5% (w/w). Five classes of neutral lipids were identified: esterified cholesterol, free cholesterol, triacylglycerols, diacylglycerols and free fatty acids. Our results suggest that triacylglycerols play a key role in the maintenance of embryo viability. In addition, we found that T. hydatigena eggs remain metabolically active by mobilisation of stored triacylglycerols. This study contributes to the understanding the survival strategies of a member of the Taeniidae family in the environment outside the host.

Fluorescent visualization of macromolecules in Drosophila whole mounts.

The ability to determine the expression dynamics of individual genes "in situ" by visualizing the precise spatial and temporal distribution of their products in whole mounts by histochemical and immunocytochemical reactions has revolutionized our understanding of cellular processes. Drosophila developmental genetics was one of the fields that benefited most from these technologies, and a variety of fluorescent methods were specifically designed for investigating the localization of developmentally important proteins and cell markers during embryonic and post embryonic stages of this model organism. In this chapter we present detailed protocols for fluorescence immunocytochemistry of whole mount embryos, imaginal discs, pupal retinas, and salivary glands of Drosophila melanogaster, as well as methods for fluorescent visualization of specific subcellular structures in these tissues.

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.

Carbohydrate accumulation and utilization by oocytes of Rhodnius prolixus.

The processes of accumulation and mobilization of carbohydrate stores in eggs of Rhodnius prolixus were analyzed. During oogenesis, the total amounts of glycogen, glucose, and trehalose increased with an accumulation of proteins, especially when oocytes grew from 1.0 to 1.5 mm in length. At 2.0 mm length, when oocytes were ready for oviposition, nutrient reserves did not increase appreciably and trehalose content decreased. Mating did not affect the final content of carbohydrates or proteins in oocytes of mated and virgin females. A trehalase activity was detected in follicles containing vitellogenic oocytes, 1.0 and 1.5 mm length, in both mated and virgin females. This activity was extremely low in chorionated, 2.0-mm oocytes. After oviposition, glycogen content decreased in fertilized eggs, but not in unfertilized ones, and some was present in newly hatched nymphs. Glucose content remained constant in unfertilized eggs, but increased in fertilized ones, while total protein amount was constant in both groups after egg laying.

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.

Molecular preservation in Late Cretaceous sauropod dinosaur eggshells.

Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells.

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

BACKGROUND: The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. RESULTS: Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. CONCLUSIONS: Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online.

Immunohistochemical localisation of a galectin from Bufo arenarum ovary.

Galectins are a group of soluble animal lectins that exhibit specificity for beta-galactosides and conserve sequence homology in the carbohydrate-recognition domain. The galectin from Bufo arenarum ovary showed a strong cross-reaction with the lectin of 14.5 kDa purified from embryos at early blastula stage. In this paper, we studied the immunohistochemical localisation of the galectin of 14.5 kDa from ovary of the toad B. arenarum in adult ovary sections. We also analysed the immunohistochemical localisation of the embryonic lectin during early development using the antiserum anti-ovary galectin. In the ovary, oocytes in the previtellogenic stage showed strong reactivity in the nucleus and the cortex but not in the cytoplasm. Oocytes in the stage of primary vitellogenesis exhibited a similar pattern in the nuclear and cortical areas but showed immunostaining in the cytoplasm. Intense nuclear staining was detected in oocytes in the stage of late vitellogenesis and in mature oocytes, which also presented strong reactions in the yolk platelets that completely covered the cytoplasm. In blastula embryos the staining was found in the blastomeres, the yolk platelets and the blastocoele. Each lectin localisation is discussed in relation to potential biological roles in the corresponding tissues.

The expression of Brachyury (T) during gastrulation in the marsupial frog Gastrotheca riobambae.

Gastrulation in the marsupial frog Gastrotheca riobambae has been analyzed by the distribution of the Brachyury (T) protein. Comparison with other amphibians provides mechanistic insights, since G. riobambae develops slowly and has the most divergent mode of amphibian gastrulation, producing an embryonic disk. The T pattern indicates that the prospective mesoderm is superficial, as in many amphibians. The dorsal blastopore lip could not be identified by the expression of T, or by morphological criteria, thus it is unknown whether Gastrotheca embryos have a dorsal organizer before or after blastopore closure. The circumblastoporal and notochordal expression of T, which are temporally contiguous in Xenopus, are separated in Gastrotheca, implying that distinct regulatory mechanisms may control the expression of T in its two domains. The separation of the T pattern also indicates that involution at the blastopore is separate from notochord formation. In addition, extension of the archenteron and notochord occurs after blastopore closure, suggesting that dorsal convergence and extension have been delayed until after blastopore closure. Therefore, dorsal convergence and extension need not be the cause of blastopore closure in Gastrotheca. The separation of gastrulation events in embryos that have not been experimentally manipulated, such as those of Gastrotheca, helps in understanding the distinct nature of gastrulation processes.

Differential effects of retinoic acid and a retinoid antagonist on the spatial distribution of the homeoprotein Hoxb-7 in vertebrate embryos.

An antibody raised against the recombinant Xenopus laevis Hoxb-7 protein (López and Carrasco [1992] Mech. Dev. 36:153-164) recognizes the 30 kDa translation product of the Hoxb-7 gene in X. laevis and the cognate nuclear protein in chicken embryos. The X. laevis Hoxb-7 protein was expressed maternally and zygotically. Treatment of X. laevis and chicken embryos with either all-trans retinoic acid (RA) or the retinoid antagonist Ro 41-5253 (Ro; Apfel et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:7129-7133) during early development induced malformations of the neural tube and complementary changes in the expression domain of the homeoprotein Hoxb-7. Treatment of X. laevis embryos with retinoic acid during gastrulation induced an anterior shift of the Hoxb-7 expression domain and was correlated with an enlargement of rhombomere r7. In addition to a reduction in rhombomere numbers and of forebrain size, various malformations involving all three germ layers were observed. Treatment of X. laevis embryos with the antagonist Ro before or during gastrulation caused a progressive reduction of the Hoxb-7 domain and also dose-dependent malformations of all three germ layers. RA or Ro treatment of chicken embryos from the beginning of gastrulation caused changes of the Hoxb-7 expression domain very similar to those observed in X. laevis. In particular, either a dose-dependent loss of the Hoxb-7 protein in the neural tube or an ectopic expression in the forebrain region was observed. The results of this study indicate that endogenous retinoids regulate the spatial expression of homeobox-containing genes in vertebrates.