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The early embryonic development of the green crucian carp (Carassius auratus indigentiaus subsp. nov.) was observed to study its timing and characteristics. The fertilized eggs are round, slightly yellow, and viscous demersal. The egg diameter after water swelling was 1.47 ± 0.04 mm. Embryonic development can be divided into eight stages according to its major characteristics: blastoderm formation, mitotic, blastula, gastrula, neurula, blastopore closure, organogenetic and hatching stages. Under a water temperature of 24 ± 1 °C, salinity of 35 ± 1, and pH of 7.4 ± 0.5, the blastoderm began to form 35 min after fertilization. It entered the mitotic stage at 55 min, blastula stage at 220 min, gastrula stage at 460 min, neurula stage at 675 min, blastopore closure stage at 700 min, organogenetic stage at 900 min and hatching stage at 3390 min. The total length of newly hatched larvae was 4.07 ± 0.35 mm. Regression models of growth characteristics were obtained. The full-length growth rate was fastest from 15 to 26 days, with an average of 0.396 mm/day. Compared with other cyprinid fishes, green crucian carp exhibited some distinct characteristics in certain stages of embryonic development. The eye primordium developed before the sarcomere, and the heart rate was relatively high before the member stage. Yolk fluctuation was observed during the multi-cell phase of embryonic development. The sarcomere formed after the eye primordium. The heart rate in the hatching phase was 136 beats/min. This study provides a reference for embryonic development in green crucian carp, which will assist its large-scale cultivation. [ABSTRACT FROM AUTHOR]

Mitosis-promoting factor (MPF) is a complex formed by cyclin B (cyclin B) and cyclin-dependent kinase (cdc2). To investigate the role of MPF in the reproduction of Exopalaemon carinicauda, we cloned its full-length cDNA of the Ec-cyclin B and Ec-cdc2 genes. We analyzed their molecular characteristics and expression profiles during ovarian and embryonic development. The results showed that the Ec-cyclin B gene was 1194 bp long and encoded a 397 amino acid (aa) long protein. However, Ec-cdc2 was 900 bp long, which encoded 299 aa with a conserved cyclin binding motif PSTAIRE. The phylogenetic tree analysis showed that Ec-cyclin B had the highest homology with the cyclin B of Macrobrachium rosenbergii (81.06%). In comparison, Ec-cdc2 had the highest homology with the cdc2 of E. modestus (96.80%). Ec-cyclin B showed the highest expression in the ovary, whereas Ec-cdc2 was the highest in the hepatopancreas, followed by the ovary. In the five stages of ovarian development, Ec-cyclin B and Ec-cdc2 expression levels reach the highest at stage ?(p < 0.05). Overall, the expression of these two genes first increased and then decreased at different embryonic developmental stages. Therefore, these findings suggested that cyclin B and cdc2 played an essential role in the ovarian and embryonic development of E. carinicauda. [ABSTRACT FROM AUTHOR]

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts. [ABSTRACT FROM AUTHOR]

Transcription factors play important roles in the development of the intestinal epithelium and its ability to respond to endocrine, nutritional, and microbial signals. Hepatocyte nuclear factor 4 family nuclear receptors are liganded transcription factors that are critical for the development and function of multiple digestive organs in vertebrates, including the intestinal epithelium. Zebrafish have 3 hepatocyte nuclear factor 4 homologs, of which, hnf4a was previously shown to mediate intestinal responses to microbiota in zebrafish larvae. To discern the functions of other hepatocyte nuclear factor 4 family members in zebrafish development and intestinal function, we created and characterized mutations in hnf4g and hnf4b. We addressed the possibility of genetic redundancy amongst these factors by creating double and triple mutants which showed different rates of survival, including apparent early lethality in hnf4a; hnf4b double mutants and triple mutants. RNA sequencing performed on digestive tracts from single and double mutant larvae revealed extensive changes in intestinal gene expression in hnf4a mutants that were amplified in hnf4a; hnf4g mutants, but limited in hnf4g mutants. Changes in hnf4a and hnf4a; hnf4g mutants were reminiscent of those seen in mice including decreased expression of genes involved in intestinal function and increased expression of cell proliferation genes, and were validated using transgenic reporters and EdU labeling in the intestinal epithelium. Gnotobiotics combined with RNA sequencing also showed hnf4g has subtler roles than hnf4a in host responses to microbiota. Overall, phenotypic changes in hnf4a single mutants were strongly enhanced in hnf4a; hnf4g double mutants, suggesting a conserved partial genetic redundancy between hnf4a and hnf4g in the vertebrate intestine. [ABSTRACT FROM AUTHOR]

Temperatures experienced during early ontogeny significantly influence fish phenotypes, with clear consequences for the wild and reared stocks. We examined the effect of temperature (17, 20, or 23 °C) during the short embryonic and yolk-sac larval period, on the swimming performance and skeleton of metamorphosing Gilthead seabream larvae. In the following ontogenetic period, all fish were subjected to common temperature (20 °C). The critical swimming speed of metamorphosing larvae was significantly decreased from 9.7 ± 0.6 TL/s (total length per second) at 17 °C developmental temperature (DT) to 8.7 ± 0.6 and 8.8 ± 0.7 TL/s at 20 and 23 °C DT respectively (p < 0.05). Swimming performance was significantly correlated with fish body shape (p < 0.05). Compared with the rest groups, fish of 17 °C DT presented a slender body shape, longer caudal peduncle, terminal mouth and ventrally transposed pectoral fins. Moreover, DT significantly affected the relative depth of heart ventricle (VD/TL,p < 0.05), which was comparatively increased at 17 °C DT. Finally, the incidence of caudal-fin abnormalities significantly decreased (p < 0.05) with the increase of DT. To our knowledge, this is the first evidence for the significant effect of DT during the short embryonic and yolk-sac larval period on the swimming performance of the later stages. [ABSTRACT FROM AUTHOR]

Copper has demonstrated utility in multiple industrial applications for its high conductivity and antibacterial/antiviral properties. However, numerous findings have suggested potential hazards regarding pathogenesis. This study was conducted to demonstrate the application of zebrafish (Danio rerio) as a cost-effective biological assay to detect environmental pollution, i.e., heavy metal of coins. We demonstrated that zebrafish larvae exposed to copper-plated coins or copper (II) ion solution elicited a consistent phenotype of early mortality without signs of morphological defects in surviving individuals. Copper ion solution served as a standard to (1) corroborate copper exposure from coins and (2) demonstrate proportional increase in early mortality phenotype according to concentration. We found that 5 μM CuSO4·5H2O was the minimal concentration to elicit the observed phenotypes from copper toxicity. This study aimed to demonstrate how a simple protocol involving wild-type zebrafish larvae could provide an economical solution to water monitoring in areas of rapid technological advancement and increasing environmental concerns, especially in communities without access to expensive analytical methods. [ABSTRACT FROM AUTHOR]

Despite the known teratogenic effects of alcohol (ethanol) on the developing human fetus, the prevalence of fetal alcohol spectrum disorder (FASD) is not decreasing. Appropriate treatment for this life-long disease has not been developed, and even diagnostic biomarkers are unavailable. FASD remains a large unmet medical need. Numerous animal models have been developed to mimic FASD and study potential underlying biological mechanisms. However, most of these models focused on neuronal phenotypes. Given that glial cells represent the majority of cells in the vertebrate brain, and given the increasingly appreciated roles they play in a myriad of neuronal functions as well as CNS disorders, we decided to investigate potential embryonic alcohol exposure induced changes in them. Building upon a previously introduced zebrafish model of milder and most prevalent forms of FASD, we investigated the effect of a 2-hour-long exposure to alcohol (1% vol/vol bath concentration) employed at the 24th hour postfertilization stage of development of zebrafish on a number of glial cell-related phenotypes. We studied oligodendrocyte, astrocyte as well as microglia-related phenotypes using immunohistochemistry, lipid, and enzyme activity analyses. We report significant changes in wide-spread glial cell phenotypes induced by embryonic alcohol exposure in the zebrafish brain and conclude that the zebrafish will advance our understanding of the mechanisms of this devastating disorder. [ABSTRACT FROM AUTHOR]

During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13–16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

Vitamin A (VA) is an essential dietary nutrient for fish and plays an important role in a range of physiological processes including vision, reproduction, embryogenesis, growth and differentiation and maintenance of epithelial cells. Knowledge of effects of VA on aquatic organisms is limited, but new information continues to be published. The aim of the present review is to present an overview of the dietary requirements and functions of VA in fish of commercial importance in aquaculture production. The effects of VA on reproduction and early embryonic development are described, and the estimated dietary requirements for VA of several species of fish are discussed. New research findings on how VA requirements vary with the life stages of fish are explored that will expand understanding of the complex role of VA. Sources of VA in feeds are described, and the potential need to consider how changes in fish feed formulations towards higher use levels of sustainable ingredients may affect dietary VA supplementation recommendations is discussed. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of heavy metals, such as copper, zinc, iron, cobalt, chromium, aluminium, manganese and molybdenum, on zebrafish egg hatching rate and the subsequent survival rate of the larvae. Each metal was used in the various concentrations of0.05 mg/L, 50 mg/L and 500 mg/L to detect the threshold limit of tolerance. The study's results showed significant differences in the hatching and survival rate of larvae between the control group and most of the experimental groups when adding heavy metals in various concentrations. The highest concentration (500 mg/L) of the induced heavy metals tested in this study resulted in total egg or larval mortality within 96 hr and significantly lower egg or larval life expectancy. Among the tested metals, zinc and molybdenum had the most significant impact on hatching time, in contrast to iron that did not differentiate the results from the control group. Copper, cobalt, chromium, aluminium and manganese did affect zebrafish eggs depending on their exposure concentrations, which differentiated the results from the control group. Also, the study's findings demonstrate a significant dependence of the hatched egg heart rate on the metal concentration, and in particular, higher concentrations of metals correspond to higher heart rate. Zinc, Molybdenum and copper was proved to be the most toxic heavy metals causing increased hazard rate to mortality up to 96 hr and shorter life expectancy. Finally, skeletal deformities that occasionally observed did not significantly affect the total life expectancy of the hatched eggs. [ABSTRACT FROM AUTHOR]

Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates. [ABSTRACT FROM AUTHOR]

The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. In this study, we used the embryonic zebrafish model to assess the impacts of a 3.5 GHz RFR on biology- a frequency typically used by 5G-enabled cell phones and lies within the 4G and 5G bandwidth. We established a plate-based exposure setup for RFRs, exposed developing zebrafish to 3.5 GHz RFR, specific absorption rate (SAR) ≈ 8.27 W/Kg from 6 h post fertilization (hpf) to 48 hpf, and measured a battery of morphological and behavioral endpoints at 120 hpf. Our results revealed no significant impacts on mortality, morphology or photomotor response and a modest inhibition of startle response suggesting some levels of sensorimotor disruptions. This suggests that the cell phone radiations at low GHz-level frequencies are likely benign, with subtle sensorimotor effects. Through this assessment, we have established a robust setup for zebrafish RFR exposures readily amenable to testing various powers and frequencies. Future developmental exposure studies in zebrafish will evaluate a wider portion of the radio frequency spectrum to discover the bioactive regions, the potential molecular targets of RFR and the potential long-term effects on adult behavior. [ABSTRACT FROM AUTHOR]

Elasmobranchs are among the species most threatened by overfishing and a large body of evidence reports their decline around the world. As they are large predators occupying the highest levels of marine food webs, their removal can alter the trophic web dynamic through predatory release effects and trophic cascade. Suitable management of threatened shark species requires a good understanding of their behaviour and feeding ecology. In this study we provide one of the first assessments of the trophic ecology of the "vulnerable" smooth-hounds Mustelus mustelus and M. punctulatus in the Central Mediterranean Sea, based on stomach contents and stable isotope analyses. Ontogenetic diet changes were addressed by comparing the feeding habits of three groups of individuals: juveniles, maturing and adults. Our results highlighted that the two species share a similar diet based mostly on the consumption of benthic crustaceans (e.g. hermit crabs). Their trophic level increases during ontogeny, with adults increasing their consumption of large-sized crustaceans (e.g. Calappa granulata, Palinurus elephas), cephalopods (e.g. Octopus vulgaris) and fish (e.g. Trachurus trachurus). Our results provide also evidence of ontogenetic shifts in diet for both species showing a progressive reduction of interspecific trophic overlap during growth. The results of this study contribute to improve the current knowledge on the trophic ecology of these two threatened sharks in the Strait of Sicily, thus providing a better understanding of their role in the food web. [ABSTRACT FROM AUTHOR]

Hybridization is widespread in fish and constitutes an important mechanism in fish speciation. There is, however, little knowledge about hybridization in mormyrids. F1-interspecies hybrids between Campylomormyrus tamandua ♂ × C. compressirostris ♀ were investigated concerning: (1) fertility; (2) survival of F2-fish and (3) new gene combinations in the F2-generation concerning the structure of the electric organ and features of the electric organ discharge. These F1-hybrids achieved sexual maturity at about 12–13.5 cm total length. A breeding group comprising six males and 13 females spawned 28 times naturally proving these F1-fish to be fertile. On average 228 eggs were spawned, the average fertilization rate was 47.8%. Eggs started to hatch 70–72 h after fertilization, average hatching rate was 95.6%. Average mortality rate during embryonic development amounted to 2.3%. Average malformation rate during the free embryonic stage was 27.7%. Exogenous feeding started on day 11. In total, we raised 353 normally developed larvae all of which died consecutively, the oldest specimen reaching an age of 5 months. During survival, the activities of the larval and adult electric organs were recorded and the structure of the adult electric organ was investigated histologically. [ABSTRACT FROM AUTHOR]

This study describes the fetal mummification process in two embryos of a 310 cm total length scalloped hammerhead shark Sphyrna lewini caught in southeastern Brazil, in December 2017. Fourteen embryos were observed in total, in which two males in the left uterus presented different stages of mummification. Both mummified embryos were covered by an exudate (i.e., a mucous substance), indicating a hematic mummification process. All embryos were at the placentotrophic stage of development, indicating that they were close to parturition. An intrinsic characteristic is suggested as possible etiology for this condition, such as umbilical torsion, because both embryos were at different sizes and, therefore, at different development stages. In addition, the sample size did not allow the authors to presume any pollution effect once only one female was observed. Finally, fetal mummification and other embryonic development disorders might have populational impacts due to reduction in embryo survival and, consequently, recruitment. For this reason and considering that S. lewini is categorized as a "critically endangered species," this study's results have conservational relevance. [ABSTRACT FROM AUTHOR]

A laboratory flume was constructed to examine substrate effects on aquatic development. The flume was designed as a once‐through system with a submerged cobble‐filled corebox. Lake whitefish (Coregonus clupeaformis) embryos and temperature probes were deployed at multiple sites within the cobble and in the open water channel. Embryos were incubated in the flume for two different experimental periods: one to examine substrate impacts during natural lake cooling (37 days: 5 December 2016 to 10 January 2017) and the second to investigate substrate effects while administering a twice weekly 1 h heat shock (51 days: 11 January to 2 March 2017). During incubation, no significant difference was found in the average temperature between locations; however, temperatures were more stable within the cobble. Following both incubation periods, embryos retrieved from the cobble were significantly smaller in both dry mass and body length by up to 20%. These results demonstrate differences between embryos submerged in a cobble substrate and in the open water column, highlighting the need to consider the physical influences from the incubation environment when assessing development effects as part of any scientific study or environmental assessment. [ABSTRACT FROM AUTHOR]

All extant holocephalans (Chimaeroidei) have lost the ability to make individual teeth, as tooth germs are not part of the embryonic development of the dental plates or of their continuous growth. Instead, a hypermineralized dentine with a unique mineral, whitlockin, is specifically distributed within a dentine framework into structures that give the dental plates their distinctive, species‐specific morphology. Control of the regulation of this distribution must be cellular, with a dental epithelium initiating the first outer dentine, and via contact with ectomesenchymal tissue as the only embryonic cell type that can make dentine. Chimaeroids have three pairs of dental plates within their mouth, two in the upper jaw and one in the lower. In the genera Chimaera, Hydrolagus and Harriotta, the morphology and distribution of this whitlockin within each dental plate differs both between different plates in the same species and between species. Whitlockin structures include ovoids, rods and tritoral pads, with substantial developmental changes between these. For example, rods appear before the ovoids and result from a change in the surrounding trabecular dentine. In Harriotta, ovoids form separately from the tritoral pads, but also contribute to tritor development, while in Chimaera and Hydrolagus, tritoral pads develop from rods that later are perforated to accommodate the vasculature. Nevertheless, the position of these structures, secreted by the specialized odontoblasts (whitloblasts), appears highly regulated in all three species. These distinct morphologies are established at the aboral margin of the dental plate, with proposed involvement of the outer dentine. We observe that this outer layer forms into serially added lingual ridges, occurring on the anterior plate only. We propose that positional, structural specificity must be contained within the ectomesenchymal populations, as stem cells below the dental epithelium, and a coincidental occurrence of each lingual, serial ridge with the whitlockin structures that contribute to the wear‐resistant oral surface. [ABSTRACT FROM AUTHOR]

Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrugactivating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the lightinducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control. [ABSTRACT FROM AUTHOR]

Plasma concentrations of progesterone (P4) and 17β‐oestradiol (E2) in juvenile, pre‐ovulatory, early, mid‐ or late pregnancy stages of female blue sharks Prionace glauca were analysed. Concentrations of P4 were significantly higher in pregnant than in non‐pregnant individuals, whereas E2 concentrations increased with embryonic and follicular development. A highly accurate (86.1%) random forest classification model was developed to predict shark pregnancy. It is proposed that hormone concentrations could be used for the subsequent non‐lethal determination of female P. glauca reproductive state. [ABSTRACT FROM AUTHOR]

Homology-directed (HD) genome modification offers an opportunity to precisely modify the genome. Despite reported successful cases, for many loci, precise genome editing remains challenging and inefficient in vivo. Here we report an effort to precisely knock-in a GFP reporter into gad locus mediated by CRISPR/Cas9 system in the zebrafish Danio rerio. PCR artifact was detected in testing for homologous recombination (HR), but was mitigated by optimizing PCR condition and decreasing the injected targeting plasmid concentration. Under this optimized condition, time course analysis revealed a decline of the HR-positive embryos at embryogenesis progressed. GFP signals also diminished at later developmental stages. The GFP signals were consistent with PCR detection, both of which suggested the loss of targeted insertion events at later stages. Such loss of insertion might be one underlying reason for the inability to obtain germ-line transgenic lines with GFP knocked into the gad locus. Our results suggest that the low HR efficiency associated with CRISPR-mediated knock-in is in part due to loss of insertion after targeted integration into the gad locus. [ABSTRACT FROM AUTHOR]

The regional identity of organs and organ systems along the anterior-posterior axis during embryonic development is patterned, in part, by Hox genes, which encode transcription factor proteins that activate or repress the expression of downstream target genes. Divergent nested Hox gene expression patterns may have had a role in facilitating morphological divergence of structures, such as the pharyngeal jaw apparatus, among evolutionarily divergent teleost fishes. Recent studies from several evolutionarily divergent teleosts, such as the Japanese Medaka (Oryzias latipes) and the Nile Tilapia (Oreochromis niloticus), have shown the presence of divergent expression patterns of several Hox genes within paralog groups 2-5 between these species. Specifically, these expression patterns were documented in the pharyngeal arches, which give rise to the pharyngeal jaw apparatus. While the expression patterns of several Zebrafish (Danio rerio) Hox genes that are orthologous to those of Medaka and Tilapia have been documented within the developing hindbrain and pharyngeal arches, many still have yet to be documented, especially within the pharyngeal arches during the postmigratory cranial neural crest cell stages. Here, we present the expression patterns of six Zebrafish Hox genes, hoxc3a, d3a, a4a, d4a, b5a, and c5a, within the pharyngeal arches during a postmigratory cranial neural crest cell stage and compare them to their orthologous genes of Medaka and Tilapia at similar stages. We show that while hoxc3a, d3a, and c5a of Zebrafish are absent from the pharyngeal arches, hoxa4a, d4a, and b5a show divergent expression patterns from their orthologs in Medaka and Tilapia. These observed divergences may be, in part, responsible for the divergent pharyngeal jaw apparatus structures exhibited by these fishes. [ABSTRACT FROM AUTHOR]

Pericellular and extracellular proteoglycans play an important role in modulating morphogen gradients and signal transductions. Chondroitin sulfate proteoglycan 4 (Cspg4) is a membrane spanning proteoglycan expressed in immature progenitor cells and cancer cells. Cspg4 participates in cellular events such as proliferation, migration and signal transduction, and these events are generally important for embryo development. In this study, we characterized Cspg4 for its roles in zebrafish embryonic development. Our results demonstrated that cspg4 was maternally expressed from 0 to 3 hours post fertilization (hpf) and expressed in the anterior and posterior embryo end after 9 hpf. Knocking-down cspg4 resulted in a shorter anterior-posterior axis than control embryo, which could be rescued by co-injecting wnt11 mRNA suggesting that Cspg4 regulates body axis organization through modulating the Wnt/planar cell polarity signaling pathway. In addition, overexpressing cspg4 caused cyclopia. The Cspg4 transmembrane domain mutant embryo phenocopied the global over-expression of cspg4 mRNA and led to cyclopia with a very low penetrance. Our results demonstrated that the quantitatively and spatially accurate distribution of Cspg4 is critical for body axis and midline development during gastrulation. [ABSTRACT FROM AUTHOR]

The objective of our study was to investigate the effects of heavy metals on the fertilizing capacity of exposed zebrafish sperm, on embryonic survival, and on occurrence of embryonic deformities following fertilization with exposed sperm. It is important to test heavy metals because they are well-known pollutants. Sperm of externally fertilizing species can get in contact with pollutants found in aquatic environment. Zebrafish sperm, despite its advantages, has seldom been used in in vitro toxicological studies and no reports are available regarding the fertilizing capacity of exposed sperm. Zebrafish sperm was stripped and exposed to concentrations of the tested heavy metals (Zn2+, Cd2+, Cr3+, Cu2+, Ni2+, Hg2+, As3+) for 30 or 120 minutes. Calculated half-maximal effective concentration (EC50) values do not differ significantly from those calculated for motility for any of the tested heavy metals, which means fertilization rate can indicate the toxicity of the given substance following exposure of sperm. Thus, its application as in vitro toxicological end point is reasonable. The survival of embryos and embryonic development have not been affected by the exposure of spermatozoa, which means all alterations in spermatozoa caused by heavy metals have been expressed before 24 hours post fertilization. [ABSTRACT FROM AUTHOR]

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia. • The first report on the possible soluble CD4 in cobia. • Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. • The results confirmed a maternal transfer of CD4-2 homologues to cobia embryos and larvae. [ABSTRACT FROM AUTHOR]

Egg quality and early embryonic development are important aspects for successful artificial propagation of fish species of ecological and economical values, and therefore have been intensively researched. It has been demonstrated that several maternal factors including TGF-β, cyclin B, follicle cell transcripts and gonadal peptides like inhibins influence fish egg quality. Inhibins are considered gonadal hormones that act on the anterior pituitary cells to regulate gonadotropin hormone secretion and as a paracrine substance that affects steroidogenesis and folliculogenesis. There are several intra-ovarian growth factors identified and characterized in fish that are important components of the brain-pituitary-gonadal (BPG) axis communication network. The role of inhibins on the reproductive cycle and its regulation by the hormonal negative feedback mechanism from gonads and how this inhibits activin signaling and suppresses the pituitary FSH production has been well established in higher vertebrates but is poorly understood in fish. Many advances have been made to elucidate the endocrine regulations involved during oogenesis in higher vertebrates. Neutralization of endogenous inhibins helps in many reproductive processes, including programed production of high-quality oocytes in large quantity, improved embryonic development and increased survival of offsprings. In fish, little is known about inhibin structure and endocrine regulation despite its immense importance in reproduction of female mammals. This review focuses on recent advances in the field of inhibins in fish with special emphasis on its structure, receptor interaction, mechanisms of action and its physiological role of in the oogenesis and reproductive cycle. [ABSTRACT FROM AUTHOR]

E2f5 is a member of the E2f family of transcription factors that play essential roles during many cellular processes. E2f5 was initially characterized as a transcriptional repressor in cell proliferation studies through its interaction with the Retinoblastoma (Rb) protein for inhibition of target gene transcription. However, the precise roles of E2f5 during embryonic and post-embryonic development remain incompletely investigated. Here, we report that zebrafish E2f5 plays critical roles during spermatogenesis and multiciliated cell (MCC) differentiation. Zebrafish e2f5 mutants develop exclusively as infertile males. In the mutants, spermatogenesis is arrested at the zygotene stage due to homologous recombination (HR) defects, which finally leads to germ cell apoptosis. Inhibition of cell apoptosis in tp53; e2f5 double mutants rescued ovarian development, although oocytes generated from the double mutants were still abnormal, characterized by aberrant distribution of nucleoli. Using transcriptome analysis, we identified dmc1, which encodes an essential meiotic recombination protein, as the major target gene of E2f5 during spermatogenesis. E2f5 can bind to the promoter of dmc1 to promote HR, and overexpression of dmc1 significantly increased the fertilization rate of e2f5 mutant males. Besides gametogenesis defects, e2f5 mutants failed to develop MCCs in the nose and pronephric ducts during early embryonic stages, but these cells recovered later due to redundancy with E2f4. Moreover, we demonstrate that ion transporting principal cells in the pronephric ducts, which remain intercalated with the MCCs, do not contain motile cilia in wild-type embryos, while they generate single motile cilia in the absence of E2f5 activity. In line with this, we further show that E2f5 activates the Notch pathway gene jagged2b (jag2b) to inhibit the acquisition of MCC fate as well as motile cilia differentiation by the neighboring principal cells. Taken together, our data suggest that E2f5 can function as a versatile transcriptional activator and identify novel roles of the protein in spermatogenesis as well as MCC differentiation during zebrafish development. Author summary: E2f family of transcription factors play essential roles during many cellular processes by activating or inhibiting target gene expression. E2F5 is recognized as a transcriptional repressor during cell-cycle progression. However, the role of E2f5 during embryonic development has not been fully investigated. Here, we show that E2f5 is required both for spermatogenesis and multiciliated cell differentiation in zebrafish. Specifically, E2f5 regulates homologous recombination, a central event in meiosis, by transactivating the expression of dmc1. Loss of E2f5 leads to spermatogenesis defects due to meiosis arrest. In addition, E2f5 plays an essential role during the differentiation of multiciliated cells and ion transporting principal cells in the pronephric ducts (embryonic zebrafish kidney). We showed that principal cells were devoid of cilia in the proximal straight tubule of pronephric ducts, while ectopic cilia developed in these cells due to lateral inhibition defects in the absence of E2f5 protein. Our data suggest that E2f5 functions as a versatile transcriptional activator during vertebrate development. [ABSTRACT FROM AUTHOR]

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF–Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF–Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF–Y or TALE defines genes in specific developmental processes, such that NF–Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF–Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF–Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA. • NF–Y and TALE factors occupy genomic elements during zygotic genome activation. • Genomic elements defined by NF–Y and TALE occupancy act as enhancers in vivo. • NF–Y and TALE have both shared and independent transcriptional targets. • Combinatorial use of TALE and NF–Y factors controls distinct genetic programs. [ABSTRACT FROM AUTHOR]

The circadian clock is a cell-autonomous time-keeping mechanism established gradually during embryonic development. Here, we generated a transgenic zebrafish line carrying a destabilized fluorescent protein driven by the promoter of a core clock gene, nr1d1, to report in vivo circadian rhythm at the single-cell level. By time-lapse imaging of this fish line and 3D reconstruction, we observed the sequential initiation of the reporter expression starting at photoreceptors in the pineal gland, then spreading to the cells in other brain regions at the single-cell level. Even within the pineal gland, we found heterogeneous onset of nr1d1 expression, in which each cell undergoes circadian oscillation superimposed over a cell type–specific developmental trajectory. Furthermore, we found that single-cell expression of nr1d1 showed synchronous circadian oscillation under a light–dark (LD) cycle. Remarkably, single-cell oscillations were dramatically dampened rather than desynchronized in animals raised under constant darkness, while the developmental trend still persists. It suggests that light exposure in early zebrafish embryos has significant effect on cellular circadian oscillations. A transgenic zebrafish line, nr1d1-VNP, enables the monitoring of single-cell circadian rhythms in live zebrafish; using this fish line, the authors find that light exposure in early development initializes rather than synchronizes single-cell oscillators. [ABSTRACT FROM AUTHOR]

The red claw crayfish (Cherax quadricarinatus) is an emerging and important commercial species in several countries, and is also a potential biological model in crustacean biology. However, its molecular embryonic development mechanism remains largely unknown because of a lack of genomic resources and systematic research. A comprehensive and integrated transcriptomic analysis is necessary to reveal the cell biological function, gene expression profiles, and embryo patterning that occur during embryogenesis. In the present study, transcriptomic profiles of C. quadricarinatus embryos during three developmental stages were investigated by high-throughput Illumina sequencing technology, and the genes related to development were further analyzed. In total, 49,436 unigenes were assembled and clustered, in which 13,727 were annotated in the Nonredundant database, 5087 were classified based on Gene Ontology annotations, and 2735 were associated with 189 Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, gene expression differences among the embryos stages were analyzed, and 6658 differentially expressed genes (DEGs) were identified. In total, 3300, 5211, and 1262 DEGs were identified between the eye pigments forming stage (EP) and prepare-hatching stage (PH), EP and larvae (L), as well as PH and L; meanwhile, 1595, 2540 and 680 DEGs were annotated, respectively. The fundamental developmental genes related to apoptosis, neurogenesis, and segmentation, as well as signaling pathways related to Hedgehog, MAPK, Wnt, TGF-β and Notch, showed higher expression during the EP stage than in other two stages, indicating that the EP stage has more active biological processes than the latter stages. This transcriptome studies gene expression at different stages of embryonic development and the datasets provide a basis for understanding crustacean developmental biology and guiding seedling production. [ABSTRACT FROM AUTHOR]

The differences in hatching rate, incubation period, viability of 24 h posthatch larvae and total mortality rate were tested through one-way ANOVA, followed by Tukey's I post hoc i test ( I p i < .05). The relationship between temperature and hatching rate, temperature and viability of 24hpost-hatch larvae, and temperature and total mortality rate was all modelled by a quadratic equation ( I r i SP 2 sp > .90; Figure). According to the total hatching rate observed, viability of 24 h posthatch larvae and total mortality rate, we can concluded that the optimal conditions of temperature for incubation eggs ranged from 24 to 28°C. [Extracted from the article]

Recently, essential oils from moringa seeds have been evaluated for their different biological potentials. However, their effect in embryonic development, either in human or animals, is not largely known. The current work was designed to investigate the developmental toxicity and in vivo anti-angiogenic effects of essential oils from Moringa oleifera and M. peregrina seeds on zebrafish (Danio rerio) embryos. Fatty acid composition of both oils revealed higher levels of unsaturated ω-9 oleic acid (67.1 and 73.57% for M. oleifera and M. peregrina , respectively). On the other hand, major saturated fatty acids (palmitic, stearic and arachidic) recorded comparable combined levels (15.42 and 14.81% for M. oleifera and M. peregrina , respectively). Seed oils from M. oleifera and M. peregrina were toxic to zebrafish embryos at LD 50 values of 21.24 ± 0.44 and 25.11 ± 0.547 μg/mL, respectively. However, sub-lethal doses of oils (10–20 μg/mL) showed stacking of blood cells around yolk area of treated embryos. Further investigation to this phenotype in zebrafish transgenic line Tg(fli1:EGFP) has confirmed malformed blood vessels in 100% treated embryos. In conclusion, essentials oils from M. oleifera and M. peregrina seeds represent an anti-angiogenic potential for combating cancer development. • Chemical composition and fatty acid composition of essential oils from M. oleifera and M. peregrina seeds are reported. • Both oils showed toxicity against zebrafish embryos. • Neurotoxicity during the development of zebrafish embryos is reported. • Seed oils showed promising anti-angiogenic effects against both wild- and transgenic-type of zebrafish embryos. [ABSTRACT FROM AUTHOR]

In this study, the embryonic development of Syngnathus schlegeli is first described based on external morphological characteristics and then the genetic relationship between embryos is investigated using a microsatellite-based approach. Embryonic development proceeds from fertilized egg to prerelease of larva and can be categorized into four periods: early embryogenesis, eye development, snout formation and larva, which can be further divided into 15 stages. Embryos in the brood pouch are all in the same developmental stage. However, multi-maternity is observed in all sampled broods. Microsatellite-based parentage analysis suggests that pregnant males had, per male, mated with and received eggs from 3 to 7 females, with average being 4.3. The embryos of full sibs within a brood pouch were clustered, and seven patterns of parentage were observed. Combining previous studies with the evidence obtained from genotyped eggs inside the brood pouch in this study, we can confirm that the mating pattern of S. schlegeli is polygamous. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 are critical signalling mediators and play pivotal roles in the innate immune and inflammatory responses mediated by TLR/IL-1R. In the present study, two IRAK family members, On IRAK1 and On IRAK4, were identified in the Nile tilapia Oreochromis niloticus with a conserved N-terminal death domain and a protein kinase domain, similar to those of other fishes and mammals. The gene structures of On IRAK1 and On IRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. On IRAK1 and On IRAK4 were broadly expressed in all of the tissues tested, with the highest expression levels being observed in the blood and the lowest expression levels being observed in the liver. Both genes could be detected from 2 d post-fertilization (dpf) to 8 dpf during embryonic development. Moreover, the expression levels of On IRAK1 and On IRAK4 were clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, Poly I: C, S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. The overexpression of On IRAK1 and On IRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, On IRAK1 significantly upregulated On Myd88-induced NF-κB activation, while On IRAK4 had no effect on On Myd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that On Myd88 did not interact with either On IRAK1 or On IRAK4 and that On IRAK1 did not interact with On IRAK4. Taken together, these findings suggest that On IRAK1 and On IRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion. • On IRAK1 and On IRAK4 gene were identified in Nile tilapia and were upregulated after S. agalactiae challenge. • On IRAK1 and On IRAK4 induced by LPS, Poly I: C, wild and mutant type S. agalactiae in in vitro. • On Myd88 did not interact with either On IRAK1 or On IRAK4, and On IRAK1 did not interact with On IRAK4. • Overexpression of On IRAK1 and On IRAK4 significantly increased NF-κB activation. • On IRAK1 significantly upregulated the NF-κB activity induced by On Myd88. [ABSTRACT FROM AUTHOR]

Hydroethanolic preparations of Acmella oleracea is used in the north of Brazil as a female aphrodisiac. Thus, the objective of this study was to evaluate the action of the hydroethanolic extract of Acmella oleracea (EHFAo) flowers (21.873 and 44.457 mg/kg) and spilanthol (3 mg/kg) administered orally on reproductive performance and effects on the embryonic development of zebrafish F1 generation. It was observed that in the groups in which males and females received EHFAo and spilanthol, the spawning was interrupted, whereas in the groups in which only the females were treated, spawning occurred during the 21 days. Thus, in the histopathological evaluation of the gonads, it was possible to observe that the percentage of mature cells in the spermatozoa and females was significantly reduced. Only the embryo groups in which parental generation was treated with EHFAo showed lethal and teratogenic effects. On the other hand, the parental groups treated with the spilanthol presented only the lethality. Spilanthol and some metabolites showed good oral availability and important toxicological properties. Thus, it is suggested that the treatment of parental generation of zebrafish with EHFAo and spilanthol caused severe changes in the gonads and on fertility. However, on the embryo, the most striking effects in the development were recorded in the groups in which the parental generation was treated with the EHFAo, while the spilanthol influenced the lethality of the embryos. [ABSTRACT FROM AUTHOR]

Post-transcriptional gene regulation through the recognition of specific elements in mRNAs is an important determinant of gene expression. The cis elements are recognised by RNA binding proteins (RBPs) and/or small non-coding RNAs, which then orchestrate a range of processes such as mRNA localization, translational control, and degradation. RNA regulation is critical for development and disruptions in regulatory mechanisms can cause disease. While mutations in numerous RBPs have been linked to diseases in humans, the contribution of mutations in RNA elements to disease manifestation is largely unknown. Danio rerio (zebrafish), a fish model is a widely used vertebrate system to study development and disease. Here, we describe how state-of-the-art genomics tools combined with in vivo functional studies in zebrafish have facilitated the discovery of RNA elements, many of which are functionally conserved. We also highlight the potential of zebrafish to model human diseases and for drug discovery. • RNA elements and RNA-binding proteins in Zebrafish. • RNA localization, translation and stability in zebrafish embryogenesis. • Zebrafish models of diseases linked to 3'UTR elements, RBP and RNP granules. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish. [ABSTRACT FROM AUTHOR]

Since the size of newly hatched larval fish is directly related to egg size, small differences in initial egg size can be critical to survival and further development of offspring. Underlying processes causing size variation in fish offspring are still not entirely understood. In this study we investigated whether the spatial position of an individual egg within a clutch affects size variation in two benthic spawning coral reef fishes, the clownfishes Amphiprion ocellaris and A. frenatus. To evaluate the effects of within-clutch position on embryonic development, egg growth metrics and protein content were analysed on day 2, 5 and 8 after deposition (adp). Additionally the activities of the key metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) were investigated to evaluate the physiological status of the embryos. Central eggs of A. frenatus were significantly longer and heavier than peripheral eggs only on day 2 and 5 adp (2.07 mg, 2.59 mm vs. 1.84 mg, 2.49 mm). No significant differences were observed in A. ocellaris between eggs originating from a central or peripheral (5 mm from edge) position (1.33 mg, 2.26 mm vs. 1.15 mg, 2,18 mm). Diameter of the eyes did not differ between the two fish species nor between different positions, for any age group. The protein content of eggs (7.5% of wet weight) was independent of age, position and species. Enzymatic activity increased from 2 adp until peak activity was observed for both enzymes on day 8 adp, independent from position. The range of CS- and LDH-activity was 0.3–13.0 and 0.2–71.7 U g-1 wet weight, respectively. Significant differences in enzymatic activity were observed between age groups in both species, which in connection with significantly larger eggs of A. frenatus at day 2 and 5 adp could hint at a better O2 supply of central eggs. Potential implications for captive breeding are given. [ABSTRACT FROM AUTHOR]

Slc25a17 is known as a peroxisomal solute carrier, but the in vivo role of the protein has not been demonstrated. We found that the zebrafish genome contains two slc25a17 genes that function redundantly, but additively. Notably, peroxisome function in slc25a17 knockdown embryos is severely compromised, resulting in an altered lipid composition. Along the defects found in peroxisome‐associated phenotypic presentations, we highlighted that development of the swim bladder is also highly dependent on Slc25a17 function. As Slc25a17 showed substrate specificity towards coenzyme A (CoA), injecting CoA, but not NAD+, rescued the defective swim bladder induced by slc25a17 knockdown. These results indicated that Slc25a17 acts as a CoA transporter, involved in the maintenance of functional peroxisomes that are essential for the development of multiple organs during zebrafish embryogenesis. Given high homology in protein sequences, the role of zebrafish Slc25a17 may also be applicable to the mammalian system. [ABSTRACT FROM AUTHOR]

The aim of this study was to make ultrasound observations of 2 captive reef manta rays (Mobula alfredi), thereby collecting the first known data regarding the embryonic development of reef manta rays throughout almost the entire gestation period. The gestation period and water temperature at parturition were ~1 year and ~27°C, both of which are consistent with observations of wild individuals. On the basis of embryonic features observed through ultrasound, 3 developmental stages were recognized: early (0-80 d after copulation), middle (80-150 d after copulation), and late (150-360 d after copulation). The middle stage was distinguished from the early stage by the presence of large, wing- shaped pectoral fins, cephalic lobes, and buccal pumping, whereas the late stage was distinguished from the middle stage by the overlapping of the right and left pectoral fins and cessation of motion of the entire body in utero. These criteria will be useful for determining the gestation stages of wild reef manta rays. [ABSTRACT FROM AUTHOR]

The present study provides the first comprehensive embryonic development of the freshwater Syngnathid fish species, Microphis deocata (Hamilton), a Near Threatened pipefish endemic to the Brahmaputra River drainage in Northeast India and Bangladesh. Microphis deocata is a Gastrophori species as the males develop an abdominal brood pouch. Mature individuals were collected and maintained in well‐aerated aquaria under controlled conditions to induce natural spawning. The number of eggs within the males' brood pouch ranged from 17 to 22 (for n = 10), measuring 0.7–1.0 mm in diameter. A total of 10 developmental stages could be recognized under four developmental periods namely, early embryogenesis, eye development, snout formation and juvenile. However, sensitivity, and therefore mortality, while handling of this species restricted the study from reporting the exact time intervals for stages following the blastodisc formation ~48 hr post fertilization. A newborn larvae measures ~14 mm and is free‐swimming with distinct dorsal fin (with 31–32 rays) and a sector‐shaped caudal fin (with 8–9 rays). The study aims to provide baseline information on the embryology of M. deocata in culture condition which will be helpful for future studies on conservation biology, population status and management of this species. [ABSTRACT FROM AUTHOR]

Populations of anadromous European smelt Osmerus eperlanus (L.) are declining across its geographical range in northern Europe, but no practical culture techniques exist to develop stock enhancement programmes for this species. In this study, a culture protocol is described to rear fish from fertilised eggs to mature adults in 2 years involving the use of 'green water', live feed and artificial diets. The sequence of embryonic development for eggs incubated at 10°C/0 ppt was described and photographed. To determine the optimum conditions for larval culture, fertilised eggs were reared at a range of salinities (0–20 ppt) and temperatures (5–18°C) until first feeding. Best hatching success (ca. 97%), size at hatch (ca. 0.8 mm) and survival to first feeding (ca. 96%) of larvae were achieved under combined conditions of low salinity (0–0 ppt) and temperature (5–10°C). No larvae survived a salinity of 20 ppt. The time taken from fertilisation to hatch (FtH) and hatching duration (HD) were temperature‐dependent ranging from 42 days FtH and 10 days HD at 5ºC, to 10 days FtH and 2 days HD at 18°C irrespective of salinity. The results indicate that conservation programmes could utilise existing salmonid hatchery facilities (i.e. freshwater, ≤10°C water temperature) for stock enhancement. Since on‐growing of smelt involves the logistical and technical problems of live feed production, it is recommended that smelt enhancement programme utilise freshwater hatchery facilities to rear fish until hatching, and then stock out onto known spawning grounds in rivers allowing hatched larvae to drift into estuaries to complete the larval and juvenile phases. This approach would minimise the time spent in the hatchery post‐hatching, eliminate the need for live food production, prevent the development of predator‐naïve fish, and hence would mimic the natural life cycle of the species as closely as possible. [ABSTRACT FROM AUTHOR]

2,2′,3,5′,6‐Pentachlorobiphenyl (PCB‐95) is an environmental neurotoxicant. There is accumulated evidence that some neurotoxic effects of PCB‐95 are caused by increased spontaneous Ca2+ oscillations in neurons resulting from modifying ryanodine receptors (RyR) in calcium‐releasing channels. However, there are large gaps in explaining brain and other developmental malformations on embryonic PCB‐95 exposure. In the present study, we address those deficiencies by studying the toxic effects of PCB‐95 using zebrafish as an ontogenetic model. To characterize these effects, zebrafish embryos with intact chorions were exposed to 4 different concentrations of PCB‐95 (0.25, 0.5, 0.75, and 1 ppm) for 3 consecutive days. The controls were maintained in 0.5 × E2 medium or egg water and in 0.1% (v/v) dimethyl sulfoxide (DMSO)/0.5 × E2 medium or egg water. PCB‐95‐treated groups showed dose‐dependent decreases in survival and hatching rates, with increased rates of developmental malformations when compared to controls. These include morphological malformations, brain cell necrosis, and smaller eye sizes at 5 d post fertilization. These data suggest potential mechanisms underlying the abnormal behavior observed in a visual stimulus assay. The present study provides insight into PCB‐95‐induced developmental toxicity and supports the use of the zebrafish model in understanding the effects of PCB‐95 exposure. Environ Toxicol Chem 2019;39:162–170. © 2019 SETAC [ABSTRACT FROM AUTHOR]

In this study, we cloned the complementary (c)DNA sequences of tumour necrosis factor receptor (TNFR)‐associated factor 3 (traf3) in Nile tilapia, Oreochromis niloticus. The expression patterns of the traf3 gene were investigated and preliminary functional analyses were performed. In healthy fish, traf3 transcript was broadly expressed in all examined tissues, with the highest expression level in the blood and the lowest in the liver. The traf3 gene reached its highest expression at 8 days post‐fertilisation (dpf) during embryonic development. Moreover, we found that expression of traf3 was clearly altered following stimulation with Streptococcus agalactiae in vivo and that traf3 could be induced by lipopolysaccharides (LPS), Poly I: C and S. agalactiae WC1535 in Nile tilapia macrophages. Overexpression in 293T cells showed that Traf3 protein was mainly distributed in the cytoplasm and could significantly increase nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) activation. Taken together, these results implied that traf3 could play important roles in the immune response to pathogen invasion. [ABSTRACT FROM AUTHOR]

Background: Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking. Results: Here, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf. Conclusions: Our results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos. [ABSTRACT FROM AUTHOR]

Embryos of the annual killifish Austrofundulus limnaeus are the most anoxia-tolerant vertebrate. Annual killifish inhabit ephemeral ponds, producing drought and anoxia-tolerant embryos, which allows the species to persist generation after generation. Anoxia tolerance and physiology vary by developmental stage, creating a unique opportunity for comparative study within the species. A recent study of small ncRNA expression in A. limnaeus embryos in response to anoxia and aerobic recovery revealed small ncRNAs with expression patterns that suggest a role in supporting anoxia tolerance. MitosRNAs, small ncRNAs derived from the mitochondrial genome, emerged as an interesting group of these sequences. MitosRNAs derived from mitochondrial tRNAs were differentially expressed in developing embryos and isolated cells exhibiting extreme anoxia tolerance. In this study we focus on expression of mitosRNAs derived from tRNA-cysteine, and their subcellular and organismal localization in order to consider possible function. These tRNA-cys mitosRNAs appear enriched in the mitochondria, particularly near the nucleus, and also appear to be present in the cytoplasm. We provide evidence that mitosRNAs are generated in the mitochondria in response to anoxia, though the precise mechanism of biosynthesis remains unclear. MitosRNAs derived from tRNA-cys localize to numerous tissues, and increase in the anterior brain during anoxia. We hypothesize that these RNAs may play a role in regulating gene expression that supports extreme anoxia tolerance. [ABSTRACT FROM AUTHOR]

Lipids exert substantial influences on vertebrate embryogenesis, but their metabolic dynamics at detailed structural levels remains elusive, primarily owing to the lack of a tool capable of resolving their huge structural diversity. Herein, we present the first large-scale and spatiotemporal monitoring of unsaturated lipids with C=C specificity in single developing zebrafish embryos enabled by photochemical derivatization and tandem mass spectrometry (MS). The lipid isomer composition was found extremely stable in yolk throughout embryogenesis, while notable differences in ratios of C=C location (e.g., PC 16:0_16:1 (7) vs. 16:0_16:1 (9)) and fatty acyl composition isomers (e.g., PC 16:1_18:1 vs. 16:0_18:2) were unveiled between blastomeres and yolk from zygote to 4 h post fertilization (hpf). From 24 hpf onwards, lipid isomer compositions in embryo head and tail evolved distinctively with development, suggesting a meticulously regulated lipid remodeling essential for cell division and differentiation. This work has laid the foundation for functional studies of structurally defined lipids in vertebrate embryology. [ABSTRACT FROM AUTHOR]

Banded killifish (Fundulus diaphanus) are a small, freshwater fish that have a wide distribution in eastern North America and are considered a species-at-risk on the island of Newfoundland. We posit that because Newfoundland's summer climate is much cooler than other locations at similar latitudes, there may be different constraints on banded killifish reproduction. We measured embryonic development under four temperatures (10, 16, 22, and 28 °C) and two conductivities (0.6 and 1.2 mS/cm). Warmer temperatures led to more developed embryos prior to death in embryos that ultimately did not hatch, higher hatch success, faster hatch time, and fewer thermal units to hatch. Conductivity and temperature interacted to affect hatch size. Therefore, banded killifish are likely challenged by the low temperature and conductivity conditions in Newfoundland which may result in reproductive constraints, and perhaps complete cohort failures in relatively cool summers. [ABSTRACT FROM AUTHOR]

Here, we explored cold‐shock‐induced haploid androgenesis in Japanese pufferfish (Takifugu rubripes) and analysed chromosomes by fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) in sex determination locus and microsatellite genotypes to verify all‐male inheritance. We also observed early embryonic development to have an insight into the cytological mechanism of cold‐shock‐induced gynogenesis. The chromosome number of control group was diploid (2n = 44), while in cold‐shock group haploid (1n = 22) and triploid (3n = 66) embryos were detected. Cellular DNA content flow cytometry showed that the rate of haploid induction was 90%. One signal of nucleolar organizing region (NOR) was detected by silver nitrate staining and FISH using rDNA probe in androgenetic haploids from cold‐shock group. SNP analysis revealed that about half of haploid embryos examined had the G genotype indicating male‐specific Y chromosome. Microsatellite genotyping showed that 26 out of 29 haploid embryos from cold‐shock group exclusively inherited paternally derived genotypes. Cytological observation revealed that both second polar body and egg nucleus were located on the blastodisc surface at 60 min post fertilization in cold‐shock group, suggesting simultaneous extrusion of both nuclei. [ABSTRACT FROM AUTHOR]

Context: Oridonin, isolated from the leaves of Isodon rubescens (Hemsl.) H.Hara (Lamiaceae), has good antitumor activity. However, its safety in vivo is still unclear. Objective: To investigate the preliminary safety of oridonin in zebrafish. Materials and methods: Embryo, larvae and adult zebrafish (n = 40) were used. Low, medium and high oridonin concentrations (100, 200 and 400 mg/L for embryo; 150, 300 and 600 mg/L for larvae; 200, 400 and 800 mg/L for adult zebrafish) and blank samples were administered. At specific stages of zebrafish development, spontaneous movement, heartbeat, hatching rate, etc., were recorded to assess the developmental effects of oridonin. VEGFA, VEGFR2 and VEGFR3 gene expression were also examined. Results: Low-dose oridonin increased spontaneous movement and hatching rate with median effective doses (ED50) of 115.17 mg/L at 24 h post-fertilization (hpf) and 188.59 mg/L at 54 hpf, but these values decreased at high doses with half maximal inhibitory concentrations (IC50) of 209.11 and 607.84 mg/L. Oridonin decreased heartbeat with IC50 of 285.76 mg/L at 48 hpf, and induced malformation at 120 hpf with half maximal effective concentration (EC50) of 411.94 mg/L. Oridonin also decreased body length with IC50 of 324.78 mg/L at 144 hpf, and increased swimming speed with ED50 of 190.98 mg/L at 120 hpf. The effects of oridonin on zebrafish embryo development may be attributed to the downregulation of VEGFR3 gene expression. Discussions and conclusions: Oridonin showed adverse effects at early stages of zebrafish development. We will perform additional studies on mechanism of oridonin based on VEGFR3. [ABSTRACT FROM AUTHOR]

As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90β, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90β were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach. • The expression patterns of HSP70, HSC70, HSP90α and HSP90β in different embryonic development stages of loach were analyzed. • The HSP proteins exhibited deferentially expressed in response to the different pathogenic invasion of loach. • Our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach. [ABSTRACT FROM AUTHOR]