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Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted. Image 1 • Mortality occur when zebrafish embryo and eleuthero-embryos are exposed to warfarin. • Warfarin exposed embryos showed mammalian warfarin embryopathy features. • 766 genes were found differentially expressed (564 up- and 202 down-regulated). • Warfarin exposure activated oxidative stress and apoptosis pathways. • Rat eradication actions should be programmed outside the fish spawning season. [ABSTRACT FROM AUTHOR]

The European eel is a critically endangered species that cannot be reproduced in captivity yet. Artificial maturation of female European eels can be achieved via a laborious and expensive procedure, including weekly injections with pituitary extracts for up to 6 months. The success rate is highly variable and a minimally invasive method for early selection of responsive eels would prevent the unnecessary and lengthy treatment of non-responding individuals. Since sexual maturation of European eels is accompanied by morphological changes of the pectoral fin, we examined whether fin could be used to monitor the response to the hormone treatment. Farmed eels were subjected to weekly injections with pituitary extracts and representative groups were sampled at 0 and 14–18 weeks of hormone treatment. Responders and non-responders were identified based on the gonado-somatic index. Transcriptomes of pectoral fin samples obtained at the start and end of the trial were mapped using Illumina RNAseq. Responders showed 384 and non-responders only 54 differentially expressed genes. Highly stringent selection based on minimum expression levels and fold-changes and a manual re-annotation round yielded 23 up-regulated and 21 down-regulated maturation marker genes. The up-regulated markers belong to five categories: proteases, skin/mucus structural proteins, steroid hormone signaling, tyrosine/dopamine metabolism and lipid metabolism. The down-regulated markers are either blood markers or lectin-related genes. In conclusion, pectoral fin transcriptomes are a rich source of indicator markers for monitoring hormone induced sexual maturation of female European eels. In addition, these markers provide important new insight into several fundamental processes in eel biology. [ABSTRACT FROM AUTHOR]

High infection levels due to third-stage larvae of the anisakid nematode Contracaecum osculatum have been documented in cod from the eastern part of the Baltic sea during the latest decades. The nematode larvae mainly infect the liver of Baltic cod and prevalence of infection has reached 100% with a mean intensity up to 80 parasites per host in certain areas and size classes. Low condition factors of the cod have been observed concomitant with the rise in parasite abundance suggesting a parasitic effect on growth parameters. To investigate any association between parasite infection and physiological status of the host we performed a comparative transcriptomic analysis of liver obtained from C. osculatum infected and non-infected cod. A total of 47,025 predicted gene models showed expression in cod liver and sequences corresponding to 2084 (4.43%) unigenes were differentially expressed in infected liver when compared to non-infected liver. Of the differentially expressed unigenes (DEGs) 1240 unigenes were up-regulated while 844 unigenes were down-regulated. The Gene Ontology (GO) enrichment analysis showed that 1304 DEGs were represented in cellular process and single-organism process, cell and cell part, binding and catalytic activity. As determined by the Kyoto Encyclopedia of Gene and Genomes (KEGG) Pathways analysis, 454 DEGs were involved in 138 pathways. Ninety-seven genes were related to metabolic pathways including carbohydrate, lipid, and amino acid metabolism. Thirteen regulated genes were playing a role in immune response such as Toll-like receptor signaling, NOD-like receptor signaling, RIG-I-like receptor signalling and thirty-six genes were associated with growth processes. This indicates that the nematode infection in Baltic cod may affect on molecular mechanisms involving metabolism, immune function and growth. • Contracaecum osculatum infection is associated with regulation of cod genes. • A total of 2084 unigenes were differentially expressed in the infected liver compared to non-infected. • A total of 1240 unigenes were up-regulated while 844 were down-regulated. • Of these were 97, 13, and 36 genes related to metabolism, immune responses, and growth, respectively. [ABSTRACT FROM AUTHOR]

Abstract Despite efforts to restore Atlantic sturgeon in European rivers, aquaculture techniques result in animals with high post-release mortality due to, among other reasons, their low tolerance to increasing water temperature. Marker genes to monitor heat stress are needed in order to identify heat-resistant fish. Therefore, an Atlantic sturgeon cell line was exposed to different heat shock protocols (30 °C and 35 °C) and differences in gene expression were investigated. In total 3020 contigs (∼1.5%) were differentially expressed. As the core of the upregulated contigs corresponded to heat shock proteins (HSP), the heat shock factor (HSF) and the HSP gene families were annotated in Atlantic sturgeon and mapped via Illumina RNA sequencing to identify heat-inducible family members. Up to 6 hsf and 76 hsp genes were identified in the Atlantic sturgeon transcriptome resources, 16 of which were significantly responsive to the applied heat shock. The previously studied hspa1 (hsp70) gene was only significantly upregulated at the highest heat shock (35 °C), while a set of 5 genes (hspc1, hsph3a , hspb1b , hspb11a, and hspb11b) was upregulated at all conditions. Although the hspc1 (hsp90a) gene was previously used as heat shock-marker in sturgeons, we found that hspb11a is the most heat-inducible gene, with up to 3296-fold higher expression in the treated cells, constituting the candidate gene markers for in vivo trials. Highlights • The heat shock response of an Atlantic sturgeon cell line was determined by RNAseq. • The core of the upregulated contigs corresponded to heat shock proteins (HSP). • 16 out of 76 HSP genes were significantly responsive to the applied heat shock. • hspb11a was the most heat-inducible gene. [ABSTRACT FROM AUTHOR]

Abstract The parasite Ichthyophthirius multifiliis infecting skin, fins and gills of a wide range of freshwater fish species, including rainbow trout, is known to induce a protective immune response in the host. Although a number of studies have reported activation of several immune genes in infected fish host, the immune response picture is still considered incomplete. In order to address this issue, a comparative transcriptomic analysis was performed on infected versus uninfected rainbow trout gills and it showed that a total of 3352 (7.2%) out of 46,585 identified gene sequences were significantly regulated after parasite infection. Of differentially expressed gene sequences, 1796 genes were up-regulated and 1556 genes were down-regulated. These were classified into 61 Gene Ontology (GO) terms and mapped to 282 reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Infection of I. multifiliis induced a clear differential expression of immune genes, related to both innate and adaptive immunity. A total of 268 (6.86%) regulated gene sequences were known to take part in 16 immune-related pathways. These involved pathways related to the innate immunity such as the Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration. Elevated transcription of genes encoding the TLR 8 gene and chemokines (CCL4, CCL19, CCL28, CXCL8, CXCL11, CXCL13, CXCL14) was recorded indicating their roles in recognition of I. multifiliis and subsequent induction of the inflammatory response, respectively. A number of upregulated genes in infected gills were associated with antigen processing/presentation and T and B cell receptor signaling (including B cell marker CD22 involved in B cell development). Overall the analysis supports the notion that I. multifiliis induces a massive and varied innate response upon which a range of adaptive immune responses are established which may contribute to the long lasting protection of immunized rainbow trout. Highlights • Immune mechanisms in rainbow trout towards Ichthyophthirius multifiliis were elucidated by transcriptomic analysis. • A total of 3352 genes were regulated following Ich exposure. • Of differentially expressed genes, 1796 genes were up-regulated and 1556 genes were down-regulated. • Totally 268 regulated genes were known to take part in 16 immune-related pathways. [ABSTRACT FROM AUTHOR]

Complete sexual maturation of European eels ( Anguilla anguilla ) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4–6 months and the response ranges from “unresponsive” to final maturation and ovulation. Reproductive success could be significantly increased via early selection of responders based on predictive markers and minimally invasive sampling methods. To get a better understanding of the genetic background of ovarian maturation of the European eel we performed a pilot deep-sequencing transcriptome analysis of ovarian tissue derived from a yellow eel, a prepubertal silver eel and a post-spawning matured eel. Two key players in steroidogenesis were strongly correlated with advanced sexual maturation, namely P450c17 and liver receptor homolog-1, suggesting that blood plasma steroids might qualify as minimally invasive markers for early detection of responders. Since the predictive value of plasma sex steroid levels for final maturation of the European eel had not yet been carefully examined, we performed an extensive artificial maturation trial. Farmed silver eels were treated with pituitary extracts and sampled at multiple time intervals. Expression of steroidogenesis-related genes in ovarian tissue of responding and non-responding eels after four weekly injections with pituitary extract was compared using a custom-built microarray and RNAseq. Increased expression of 17β-hsd1 was strongly linked to sexual maturation. Blood plasma levels of sex steroids were measured using ELISAs. We show that a 2.5-fold increase in blood-plasma estradiol level after 4 weekly pituitary extract injections is a strong predictor of final sexual maturation of female European eel. [ABSTRACT FROM AUTHOR]

Abstract: The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines. [Copyright &y& Elsevier]

• The gonadotropin receptors (LHR and FSHR) were cloned from Russian sturgeon. • Both recombinant sturgeon gonadotropins activated both receptors. • Sturgeon fshr and lhr have similar expression profiles throughout oogenesis. Sturgeons are being used in aquaculture because wild populations are now endangered due to overfishing for caviar. A challenge in working with sturgeon as an aquacultured species is its long and slow reproductive development. Reproduction is a hormonally regulated process that involves hierarchical signaling between the brain, pituitary gland, and gonads. In an effort to better understand the hormonal regulation of sturgeon reproduction, we have cloned the Russian sturgeon (st), Acipenser gueldenstaedtii, luteinizing hormone receptor (stLHR) and follicle stimulating hormone receptor (stFSHR) and measured their expression from previtellogenic to mature ovarian follicles. Sturgeon LHR and FSHR expression was elevated in early-vitellogenic and mature follicles compared with pre-vitellogenic and mid-vitellogenic follicles, and only LHR expression increased during late-vitellogenesis. Recombinant sturgeon FSH and LH both activated sturgeon LHR and FSHR in a cAMP reporter assay. Further molecular characterization of these receptors was accomplished by in silico modeling and cAMP reporter assays using heterologous recombinant gonadotropins from human and piscine species. There was no apparent trend in heterologous LH and/or FSH activation of the sturgeon LHR or FSHR. These data suggest that permissive activation of LHR and FSHR are a consequence of some yet undetermined biological characteristic(s) of different piscine species. [ABSTRACT FROM AUTHOR]