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Plant-specialized metabolism represents an inexhaustible source of active molecules, some of which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range of valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case for the pachysiphine derivatives which show interesting antitumor and anti-Alzheimer activities but accumulate at very low levels in several Tabernaemontana species. Unfortunately, genome data in Tabernaemontanaceae are lacking and knowledge on the biogenesis of pachysiphine-related MIAs in planta remains scarce, limiting the prospects for the biotechnological supply of many pachysiphine-derived biopharmaceuticals. Here, we report a raw version of the toad tree (Tabernaemontana elegans) genome sequence. These new genomic resources led to the identification and characterization of a couple of genes encoding cytochrome P450 with pachysiphine synthase activity. Our phylogenomic and docking analyses highlight the different evolutionary processes that have been recruited to epoxidize the pachysiphine precursor tabersonine at a specific position and in a dedicated orientation, thus enriching our understanding of the diversification and speciation of the MIA metabolism in plants. These gene discoveries also allowed us to engineer the synthesis of MIAs in yeast through the combinatorial association of metabolic enzymes resulting in the tailor-made synthesis of non-natural MIAs. Overall, this work represents a step forward for the future supply of pachysiphine-derived drugs by microbial cell factories., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The taxa Ixodes ricinus and Ixodes inopinatus are sympatric in Tunisia. The genetics underlying their morphological differences are unresolved. In this study, ticks collected in Jouza-Amdoun, Tunisia, were morphologically identified and sequenced using Oxford Nanopore Technologies. Three complete genome assemblies of I. inopinatus and three of I. ricinus with BUSCO scores of ∼98% were generated, including the reconstruction of mitochondrial genomes and separation of both alleles of the TRPA1, TROSPA and calreticulin genes. Deep sequencing allowed the first descriptions of complete bacterial genomes for " Candidatus Midichloria mitochondrii", Rickettsia helvetica and R. monacensis from North Africa, and the discovery of extensive integration of parts of the Spiroplasma ixodetis and " Ca . M. mitochondrii" into the nuclear genome of these ticks. Phylogenetic analyses of the mitochondrial genome, the nuclear genes, and symbionts showed differentiation between Tunisian and Dutch ticks, but high genetic similarities between Tunisian I. ricinus and I. inopinatus . Subtraction of the genome assemblies identified the presence of some unique sequences, which could not be confirmed when screening a larger batch of I. ricinus and I. inopinatus ticks using PCR. Our findings yield compelling evidence that I. inopinatus is genetically highly similar, if not identical, to sympatric I. ricinus. Defined morphological differences might be caused by extrinsic factors such as micro-climatic conditions or bloodmeal composition. Our findings support the existence of different lineages of I. ricinus as well of its symbionts/pathogens from geographically dispersed locations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa ("kratom"), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ∼290 μg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential., Competing Interests: Declaration of competing interest L.G.H., J.Z., J.D.K. and M.K.J. have financial interests in Biomia. J.D.K. also has financial interests in Amyris, Lygos, Demetrix, Napigen, Apertor Pharmaceuticals, Maple Bio, Ansa Biotechnologies, Berkeley Yeast and Zero Acre Farms respectively. R.P.D. and W.J.V. have financial interest in Future Genomics Technologies. All other authors have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study, we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Bacterial aggregates carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post-infection. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the inflammatory response of zebrafish larvae infected with the surrogate species M. marinum . We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis . The generated extensive transcriptome data sets will be of great use to add translational value to zebrafish as a model for infection of tuberculosis using the M. marinum infection system. In addition, we identify new marker genes such as dusp8 and CD180 that are induced by M. tuberculosis infection in zebrafish and in human macrophages at later stages of infection that can be further investigated.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae , one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae , we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non- Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Vincent Courdavault reports financial support was provided by Horizon Europe. Michael Krogh Jensen reports a relationship with BioMIA that includes: board membership and employment. Ron Dirks reports a relationship with Future Genomics Technologies that includes: board membership and employment. Hans Jensen reports a relationship with Future Genomics Technologies that includes: board membership and employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine metabolomics, proteomics, transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product synthesis. We identify a medium chain dehydrogenase/reductase (MDR) that produces a mixture of four diastereomers of yohimbanes including the well-known yohimbine and rauwolscine. In addition to this multifunctional yohimbane synthase (YOS), an MDR synthesizing mainly heteroyohimbanes and the short chain dehydrogenase vitrosamine synthase also display a yohimbane synthase side activity. Lastly, we establish that the combination of geissoschizine synthase with at least three other MDRs also produces a yohimbane mixture thus shedding light on the complex mechanisms evolved for the synthesis of these plant bioactives., (© 2023. The Author(s).)

Allopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ovulation in European eel is induced by injection of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) as the maturation-inducing hormone (MIH). Female eels need to ovulate within 18 h after injection to release good quality eggs. Progesterone (P), as an upstream precursor of DHP, may promote endogenous DHP production and improve egg quality. The purpose of this study was therefore to compare treatment of P with DHP on batch level, in vitro , to determine dose-response effects, and in vivo , at a single dose. For the in vitro experiment, ovarian tissue was extracted and placed in culture plates containing hormone-free medium and media supplemented with the treatment: DHP at 1, 10 and 100 ng mL -1 , or P at 10, 100 and 1,000 ng mL -1 . At the start of incubation, the folliculated oocytes were sampled for histology, microscopy and qPCR. After incubation for 12 and 18 h, the oocytes were sampled for microscopy and qPCR analysis. For the in vivo experiment, females were either injected with DHP or P at a dose of 2 mg kg -1 to assess their effects on ovulation and reproductive success. At the moment of release, eggs were sampled for RNA sequencing to compare effects of DHP and P on the expression of genes involved in egg quality aspects. Remaining eggs were fertilized and larval viability was recorded. Both DHP and P were able to induce GVBD (DHP at 10 and 100 ng mL -1 , P at 100 and 1,000 ng mL -1 ) in vitro . Expression of genes involved in oocyte maturation and ovulation was similar in vitro for both DHP and P treatments. Regarding the in vivo results, RNAseq results reflected similar DHP and P effects on the expression of genes involved in egg quality aspects. Females injected with either DHP or P ovulated, released eggs, and were equally able to produce larvae without any differences in reproductive success. Our results support the conclusion that DHP and P work equally well in vitro and in vivo . P is more attractive to apply as the price is 3,000 times lower than the price of DHP., Competing Interests: Author IG was employed by the company Rara Avis. Author RD was employed by the company Future Genomics Technologies B.V. Author WS was employed by the company Palingkwekerij Koolen B.V. Author LH was employed by the company Wageningen Eel Reproduction Experts B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jéhannet, Palstra, Meijerhof, Schipper, Giménez, Dirks, Swinkels, Heinsbroek and Komen.)

The Madagascar periwinkle, Catharanthus roseus , belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution., Competing Interests: Competing interests: Ron P. Dirks and Hans J. Jansen are CEO and CTO of Future Genomics Technologies, respectively., (Copyright: © 2022 Cuello C et al.)

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V. minor mainly accumulates vincamine, which is commercially used as a nootropic. Using a combination of Oxford Nanopore Technologies long read- and Illumina short-read sequencing, a 679,098 Mb V. minor genome was assembled into 296 scaffolds with an N50 scaffold length of 6 Mb, and encoding 29,624 genes. These genes were functionally annotated and used in a comparative genomic analysis to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. Furthermore, homology-based monoterpene indole alkaloid gene predictions together with a metabolic analysis across 4 different V. minor tissue types guided the identification of candidate monoterpene indole alkaloid genes. These candidates were finally used to identify monoterpene indole alkaloid gene clusters, which combined with synteny analysis allowed for the discovery of a functionally validated vincadifformine-16-hydroxylase, reinforcing the potential of this dataset for monoterpene indole alkaloids gene discovery. It is expected that access to these resources will facilitate the elucidation of unknown monoterpene indole alkaloid biosynthetic routes with the potential of transferring these pathways to heterologous expression systems for large-scale monoterpene indole alkaloid production., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

The Apocynaceae tree Voacanga thouarsii, native to southern Africa and Madagascar, produces monoterpene indole alkaloids (MIA), which are specialized metabolites with a wide range of bioactive properties. Voacanga species mainly accumulates tabersonine in seeds making these species valuable medicinal plants currently used for industrial MIA production. Despite their importance, the MIA biosynthesis in Voacanga species remains poorly studied. Here, we report the first genome assembly and annotation of a Voacanga species. The combined assembly of Oxford Nanopore Technologies long-reads and Illumina short-reads resulted in 3,406 scaffolds with a total length of 1,354.26 Mb and an N50 of 3.04 Mb. A total of 33,300 protein-coding genes were predicted and functionally annotated. These genes were then used to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. A transposable element (TE) analysis showed the highest proportion of TE in Voacanga thouarsii compared with all other MIA-producing plants. In a nutshell, this first reference genome of V. thouarsii will thus contribute to strengthen future comparative and evolutionary studies in MIA-producing plants leading to a better understanding of MIA pathway evolution. This will also allow the potential identification of new MIA biosynthetic genes for metabolic engineering purposes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Ca. Neoehrlichia mikurensis is widely prevalent in I. ricinus across Europe and has been associated with human disease. However, diagnostic modalities are limited, and much is still unknown about its biology. Here, we present the first complete Ca. Neoehrlichia mikurensis genomes directly derived from wildlife reservoir host tissues, using both long- and short-read sequencing technologies. This pragmatic approach provides an alternative to obtaining sufficient material from clinical cases, a difficult task for emerging infectious diseases, and to expensive and challenging bacterial isolation and culture methods. Both genomes exhibit a larger chromosome than the currently available Ca. Neoehrlichia mikurensis genomes and expand the ability to find new targets for the development of supportive laboratory diagnostics in the future. Moreover, this method could be utilized for other tick-borne pathogens that are difficult to culture.

The genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura, and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed on > 130 host plants, including several economically important crops. The 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long-read data and Illumina short read), a high-quality genome assembly was achieved (N50 = 1.1 Mb). An official gene set (18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq datasets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression combined with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed four potential genes of interest (three of them Spodoptera-specific) upregulated during first- and third-instar larval stages for targeted pest-outbreak management. The beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provide critical insights into the biology of this devastating polyphagous insect pest species worldwide. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

In eels, large variations in larval mortality exist, which would impede the viable production of juvenile glass eels in captivity. The transcriptome of European eel larvae was investigated to identify physiological pathways and genes that show differential regulation between non-viable vs. viable larvae. Expression of genes involved in inflammation and host protection was higher, suggesting that non-viable larvae suffered from microbial infection. Expression of genes involved in osmoregulation was also higher, implying that non-viable larvae tried to maintain homeostasis by strong osmoregulatory adaptation. Expression of genes involved in myogenesis, neural, and sensory development was reduced in the non-viable larvae. Expression of the major histocompatibility complex class-I ( mhc1 ) gene, M-protein ( myom2 ), the dopamine 2B receptor ( d2br ), the melatonin receptor ( mtr1 ), and heat-shock protein beta-1 ( hspb1 ) showed strong differential regulation and was therefore studied in 1, 8, and 15 days post-hatch (dph) larvae by RT-PCR to comprehend the roles of these genes during ontogeny. Expression patterning of these genes indicated the start of active swimming (8 dph) and feed searching behavior (15 dph) and confirmed immunocompetence immediately after hatching. This study revealed useful insights for improving larval survival by microbial control and salinity reduction.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 10 6 /tank; 70 dinospores/mL) under controlled conditions (26-28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis., (Copyright © 2021. Published by Elsevier B.V.)

Ceratothoa oestroides (Cymothoidea, Isopoda) is a generalist crustacean parasite that negatively affects the economic sustainability of European sea bass ( Dicentrarchus labrax ) aquaculture in the North-East Mediterranean. While mortalities are observed in fry and fingerlings, infection in juvenile and adult fish result in approximately 20% growth delay. A transcriptomic analysis (PCR array, RNA-Seq) was performed on organs (tongue, spleen, head kidney, and liver) from infected vs. Ceratothoa -free sea bass fingerlings. Activation of local and systemic immune responses was detected, particularly in the spleen, characterized by the upregulation of cytokines (also in the tongue), a general reshaping of the immunoglobulin (Ig) response and suppression of T-cell mediated responses. Interestingly, starvation and iron transport and metabolism genes were strongly downregulated, suggesting that the parasite feeding strategy is not likely hematophagous. The regulation of genes related to growth impairment and starvation supported the growth delay observed in infected animals. Most differentially expressed (DE) transcripts were exclusive of a specific organ; however, only in the tongue, the difference between infected and uninfected fish was significant. At the attachment/feeding site, the pathways involved in muscle contraction and intercellular junction were the most upregulated, whereas the pathways involved in fibrosis (extracellular matrix organization, collagen formation, and biosynthesis) were downregulated. These results suggest that parasite-inflicted damage is successfully mitigated by the host and characterized by regenerative processes that prevail over the reparative ones., Competing Interests: RD and ES are employed by the company Future Genomics Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Piazzon, Mladineo, Dirks, Santidrián Yebra-Pimentel, Hrabar and Sitjà-Bobadilla.)

A longer on-land rearing period of Gilthead seabream Sparus aurata before transfer to sea-cages would allow the farmer to benefit from exercise-enhanced growth, resilience, and robustness as induced by increasing water flow in the tanks. In this study, the physiological effects of flow-conditioning were investigated by subjecting large groups of experimental fish to minimal flow or to flow regimes inducing swimming exercise at 1 or 2 body length (BL) s -1 for a period of 8 months (February-October) in 1,500 L tanks. Fish representing the three treatment groups were then used for: (1) a stress challenge netting test and plasma cortisol measurement (baseline, peaking, and recovery levels), (2) blood plasma measurements of glucose, triglycerides, lactate, cholesterol, growth hormone (GH), and insulin-like growth factor 1 (IGF1), and (3) heart and muscle gene expression of the GH and IGF1 receptors and the muscle transcriptome by deep RNA sequencing (RNAseq). Fish size after 8 months of flow conditioning was 92 ± 27 g body weight (BW) for fish under minimal flow, 106 ± 24 g BW (+15%) at 1 BL s -1 , and 125 ± 27 g BW (+36%) at 2 BL s -1 . Flow conditioning at 1 BL s -1 provided optimal conditions for growth and uniformity, but also stress (lowest baseline plasma cortisol), robustness (higher condition factor and larger hearts), and energy mobilization (increased plasma glucose). Although flow enhanced growth linearly with swimming speed, also the percentage of lordotic fish increased with exercise, particularly high for swimming at 2 BL s -1 . The absence of important differences in plasma GH and IGF1, and expression levels of their receptors in heart and white skeletal muscle, indicated that other factors may be involved in growth enhancement. RNAseq of the white skeletal muscle showed upregulated expression of genes involved in muscle contraction, muscle development and its molecular regulation, and immune genes that may play a role in the muscle repair mechanism. An exercise regime of swimming at 1 BL s -1 can be considered as optimal for farming robust seabream although the increase of skeletal deformities should be avoided., Competing Interests: Author RD was employed by the company Future Genomics Technologies B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Palstra, Roque, Kruijt, Jéhannet, Pérez-Sánchez and Dirks.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Monogenean flatworms are the main fish ectoparasites inflicting serious economic losses in aquaculture. The polyopisthocotylean Sparicotyle chrysophrii parasitizes the gills of gilthead sea bream (GSB, Sparus aurata) causing anaemia, lamellae fusion and sloughing of epithelial cells, with the consequent hypoxia, emaciation, lethargy and mortality. Currently no preventive or curative measures against this disease exist and therefore information on the host-parasite interaction is crucial to find mitigation solutions for sparicotylosis. The knowledge about gene regulation in monogenean-host models mostly comes from freshwater monopysthocotyleans and almost nothing is known about polyopisthocotyleans. The current study aims to decipher the host response at local (gills) and systemic (spleen, liver) levels in farmed GSB with a mild natural S. chrysophrii infection by transcriptomic analysis., Results: Using Illumina RNA sequencing and transcriptomic analysis, a total of 2581 differentially expressed transcripts were identified in infected fish when compared to uninfected controls. Gill tissues in contact with the parasite (P gills) displayed regulation of fewer genes (700) than gill portions not in contact with the parasite (NP gills) (1235), most likely due to a local silencing effect of the parasite. The systemic reaction in the spleen was much higher than that at the parasite attachment site (local) (1240), and higher than in liver (334). NP gills displayed a strong enrichment of genes mainly related to immune response and apoptosis. Processes such as apoptosis, inflammation and cell proliferation dominated gills, whereas inhibition of apoptosis, autophagy, platelet activation, signalling and aggregation, and inflammasome were observed in spleen. Proteasome markers were increased in all tissues, whereas hypoxia-related genes were down-regulated in gills and spleen., Conclusions: Contrasting forces seem to be acting at local and systemic levels. The splenic down-regulation could be part of a hypometabolic response, to counteract the hypoxia induced by the parasite damage to the gills and to concentrate the energy on defence and repair responses. Alternatively, it can be also interpreted as the often observed action of helminths to modify host immunity in its own interest. These results provide the first toolkit for future studies towards understanding and management of this parasitosis.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Forced sustained swimming exercise at optimal speed enhances growth in many fish species, particularly through hypertrophy of the white skeletal muscle. The exact mechanism of this effect has not been resolved yet. To explore the role of cortisol, we first subjected wild-type zebrafish to an exercise protocol validated for exercise-enhanced growth, and showed that exercised zebrafish, which indeed showed enhanced growth, had higher cortisol levels than the non-exercised controls. A central role was therefore hypothesized for the steroid hormone cortisol acting through the Glucocorticoid receptor (Gr). Second, we subjected wild-type zebrafish and zebrafish with a mutant Gr to exercise at optimal, suboptimal, and super-optimal speeds and compared them with non-exercised controls. Exercised zebrafish showed growth enhancement at all speeds, with highest growth at optimal speeds. In the Gr mutant fish, exercise resulted in growth enhancement similar to wild-type zebrafish, indicating that cortisol signaling through Gr cannot be considered as a main determinant of exercise-enhanced growth. Finally, the transcriptome of white skeletal muscle tissue was analyzed by RNA sequencing. The results of this analysis showed that in the muscle tissue of Gr mutant fish a lower number of genes is regulated by exercise than in wild-type fish (183 vs. 351). A cluster of 36 genes was regulated by exercise in both wild-type and mutant fish, and in this cluster genes involved in transcriptional regulation and protein ubiquitination were overrepresented. Because these two processes appear to be regulated in both wild type and mutant fish, which both display exercise-enhanced growth, we suggest that they play an important role in the growth of muscles upon exercise.

Seaweeds may represent immuno-stimulants that could be used as health-promoting fish feed components. This study was performed to gain insights into the immunomodulatory effects of dietary seaweeds in Atlantic salmon. Specifically tested were 10% inclusion levels of Laminaria digitata (SW1) and a commercial blend of seaweeds (Oceanfeed ® ) (SW2) against a fishmeal based control diet (FMC). Differences between groups were assessed in growth, feed conversion ratio and blood parameters hematocrit and hemoglobin. After a LPS challenge of fish representing each of the three groups, RNAseq was performed on the head kidney as major immune organ to determine transcriptomic differences in response to the immune activation. Atlantic salmon fed with dietary seaweeds did not show major differences in performance in comparison with fishmeal fed fish. RNAseq resulted in ∼154 million reads which were mapped against a NCBI Salmo salar reference and against a de novo assembled S. salar reference for analyses of expression of immune genes and ontology of immune processes among the 87,600 cDNA contigs. The dietary seaweeds provoked a more efficient immune response which involved more efficient identification of the infection site, and processing and presentation of antigens. More specifically, chemotaxis and the chemokine-mediated signaling were improved and therewith the defense response to Gram-positive bacterium reduced. Specific Laminaria digitata effects included reduction of the interferon-gamma-mediated signaling. Highly upregulated and specific for this diet was the expression of major histocompatibility complex class I-related gene protein . The commercial blend of seaweeds caused more differential expression than Laminaria digitata and improved immune processes such as receptor-mediated endocytosis and cell adhesion , and increased the expression of genes involved in response to lipopolysaccharide and inflammatory response . Particularly, expression of many important immune receptors was up-regulated illustrating increased responsiveness. NF-kappa-B inhibitor alpha is an important gene that marked the difference between both seaweed diets as Laminaria digitata inhibits the expression for this cytokine while the blend of seaweeds stimulates it. It can be concluded that the inclusion of seaweeds such as Laminaria digitata can have important modulatory effects on the immune capacity of Atlantic salmon resulting in a more efficient immune response.

To enable selection of novel chemicals for new processes, there is a recognized need for alternative toxicity screening assays to assess potential risks to man and the environment. For human health hazard assessment these screening assays need to be translational to humans, have high throughput capability, and from an animal welfare perspective be harmonized with the principles of the 3Rs (Reduction, Refinement, Replacement). In the area of toxicology a number of cell culture systems are available but while these have some predictive value, they are not ideally suited for the prediction of developmental and reproductive toxicology (DART). This is because they often lack biotransformation capacity, multicellular or multi- organ complexity, for example, the hypothalamus pituitary gonad (HPG) axis and the complete life cycle of whole organisms. To try to overcome some of these limitations in this study, we have used Caenorhabditis elegans (nematode) and Danio rerio embryos (zebrafish) as alternative assays for DART hazard assessment of some candidate chemicals being considered for a new commercial application. Nematodes exposed to Piperazine and one of the analogs tested showed a slight delay in development compared to untreated animals but only at high concentrations and with Piperazine as the most sensitive compound. Total brood size of the nematodes was also reduced primarily by Piperazine and one of the analogs. In zebrafish Piperazine and analogs showed developmental delays. Malformations and mortality in individual fish were also scored. Significant malformations were most sensitively identified with Piperazine, significant mortality was only observed in Piperazine and only at the higest dose. Thus, Piperazine seemed the most toxic compound for both nematodes and zebrafish. The results of the nematode and zebrafish studies were in alignment with data obtained from conventional mammalian toxicity studies indicating that these have potential as developmental toxicity screening systems. The results of these studies also provided reassurance that none of the Piperazines tested are likely to have any significant developmental and/or reproductive toxicity issues to humans when used in their commercial applications., (Copyright © 2017. Published by Elsevier Ltd.)

Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was 'immune response', including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories 'glucose metabolism' and 'ROS metabolism' barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category 'extracellular matrix', the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced responsiveness to other challenges, like a nematode infection.

We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable nanopore sequencing and light weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.

One of the major challenges in evolutionary biology is to understand the mechanisms underlying morphological dimorphism and plasticity, including the genomic basis of traits and links to ecology. At the yellow eel stage of the European eel (Anguilla anguilla), two morphotypes are found: broad- and narrow-heads. This dimorphism has been linked to dietary differences, with broad-heads feeding on harder, larger prey than narrow-heads. However, recent research showed that both morphotypes could be distinguished at the glass eel stage, the nonfeeding predecessor of the yellow eel stage, implying that nondietary factors play a role in the development of this head shape dimorphism. Here, we used transcriptome profiling (RNAseq) to identify differentially expressed genes between broad- and narrow-headed glass eels. We found 260 significantly differentially expressed genes between the morphotypes, of which most were related to defence and immune responses. Interestingly, two genes involved in growth (soma and igf2) were significantly upregulated in narrow-heads, while nine genes involved in chemotaxis showed significant differential expression. Thus, we found support for the observation that head shape is associated with somatic growth, with fast-growing eels developing a narrower head. Additionally, observations in the wild have shown that slow-growers prefer freshwater, while fast-growers prefer brackish water. The differential expression of genes involved in chemotaxis seems to indicate that glass eel growth rate and habitat choice are linked. We hypothesize that two levels of segregation could take place in the European eel: first according to habitat choice and second according to feeding preference., (© 2017 John Wiley & Sons Ltd.)

Background: The introduction of the MinION TM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae ( Torulopsis vartiovaarae ) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes., Competing Interests: Competing interests: HJJ and CVH are members of the Nanopore Community, and have previously received flow cells free of charge, as well as travel expense reimbursements from Oxford Nanopore Technologies.

Background : The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods : We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results : In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae ( Torulopsis vartiovaarae ) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions : The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes., Competing Interests: Competing interests: HJJ and CVH are members of the Nanopore Community, and have previously received flow cells free of charge, as well as travel expense reimbursements from Oxford Nanopore Technologies.

Background: The common carp (Cyprinus carpio) is the oldest, most domesticated and one of the most cultured fish species for food consumption. Besides its economic importance, the common carp is also highly suitable for comparative physiological and disease studies in combination with the animal model zebrafish (Danio rerio). They are genetically closely related but offer complementary benefits for fundamental research, with the large body mass of common carp presenting possibilities for obtaining sufficient cell material for advanced transcriptome and proteome studies., Results: Here we have used 19 different tissues from an F1 hybrid strain of the common carp to perform transcriptome analyses using RNA-Seq. For a subset of the tissues we also have performed deep proteomic studies. As a reference, we updated the European common carp genome assembly using low coverage Pacific Biosciences sequencing to permit high-quality gene annotation. These annotated gene lists were linked to zebrafish homologs, enabling direct comparisons with published datasets. Using clustering, we have identified sets of genes that are potential selective markers for various types of tissues. In addition, we provide a script for a schematic anatomical viewer for visualizing organ-specific expression data., Conclusions: The identified transcriptome and proteome data for carp tissues represent a useful resource for further translational studies of tissue-specific markers for this economically important fish species that can lead to new markers for organ development. The similarity to zebrafish expression patterns confirms the value of common carp as a resource for studying tissue-specific expression in cyprinid fish. The availability of the annotated gene set of common carp will enable further research with both applied and fundamental purposes.

This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10°C (first 4weeks), 15°C (next 3weeks) and 20°C (last 6weeks); T15: 15°C (first 4weeks) and 20°C (last 9weeks); and T20: constant 20°C for the duration of the experiment. At 10°C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was ≥15°C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11βHSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2). Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was ≥15°C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories "response to DNA damage stimulus" and "cellular response to stress" illustrated the damaging effect of the nematode infection. This study has identified a range of cellular processes in the swimbladder of silver eels that appear to be altered by nematode infection. These altered cellular processes could contribute to detrimental changes in swimbladder function that, in turn, may lead to impairment of spawning migration.

Complete sexual maturation of European eels (Anguilla anguilla) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4-6months 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20β-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone β subunit (lhβ) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17β-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.

Reproduction of many temperate fishes is seasonal and maturation and spawning of gametes are under photothermal control. Reproductive success of first generation (G1) common sole Solea solea in captivity has been low. In this study, the sexual maturation status has been assessed during the prespawning months in G1 sole that were housed (a) outdoor under the natural photoperiod and temperature, or (b) indoor under artificial photothermal induction. Maturation was assessed in male and female G1 broodstock in November as controls, after which the remaining population was divided over two outdoor flow-through tanks placed in a pond and two indoor recirculating aquaculture system (RAS) tanks. Subsequently, maturation status (gonadosomatic index GSI and plasma levels of testosterone T and 17β-estradiol E2) was assessed in one tank for each condition in January, February and during spawning in early April, while fish in the other tank were not disturbed in achieving reproductive success. Quantitative real-time PCR was performed to determine species-specific gonadotropin mRNA expression in females. Successful G1 spawning and egg fertilisation occurred in all experimental tanks. Gonadal development was similar under both conditions. Higher E2 and T levels were found in indoor housed females. Gonadotropin expression revealed similar profiles between outdoor and indoor housed females. G1 sole could be reproduced in the outdoor tanks under the natural photoperiod and in the indoor tanks under artificial simulation of this regime that includes a potentially crucial chilling period of 2-3 months at 5-7 °C., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this review, we present an overview of the recent advances of genomic technologies applied to studies of fish species belonging to the superclass of Osteichthyes (bony fish) with a major emphasis on the infraclass of Teleostei, also called teleosts. This superclass that represents more than 50% of all known vertebrate species has gained considerable attention from genome researchers in the last decade. We discuss many examples that demonstrate that this highly deserved attention is currently leading to new opportunities for answering important biological questions on gene function and evolutionary processes. In addition to giving an overview of the technologies that have been applied for studying various fish species we put the recent advances in genome research on the model species zebrafish and medaka in the context of its impact for studies of all fish of the superclass of Osteichthyes. We thereby want to illustrate how the combined value of research on model species together with a broad angle perspective on all bony fish species will have a huge impact on research in all fields of fundamental science and will speed up applications in many societally important areas such as the development of new medicines, toxicology test systems, environmental sensing systems and sustainable aquaculture strategies.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

European eels (Anguilla anguilla) migrate ~6000km towards their spawning area in the Sargasso Sea. Based on the recent discovery that males swim even more efficiently than females, it was predicted that males also would be able to swim ~6000km within six months. Additionally, eels do not mature naturally in captivity due to strong neural inhibition. Earlier, it was hypothesized that swimming exercise is a natural trigger to induce sexual maturation and may even result in full maturation. In the present study two groups of farmed male silver eels were subjected to either endurance swimming or resting for up to 6months. It was found that male eels were able to swim continuously for a total distance of 6670km within 6months. The body weight decrease in swimming and resting males after 6months was similar (<30g) underlining the extreme low energy cost of swimming. In contrast to our expectation long-term swimming did not induce sexual maturation in farmed silver eels, suggesting that swimming alone is not sufficient as a trigger for sexual maturation. In conclusion, male eels are efficient long distance swimmers and likely able to cover the distance to the Sargasso Sea within the expected time span of 6months., (© 2013.)

Background: Sensitivity and throughput of transcriptomic and proteomic technologies have advanced tremendously in recent years. With the use of deep sequencing of RNA samples (RNA-seq) and mass spectrometry technology for protein identification and quantitation, it is now feasible to compare gene and protein expression on a massive scale and for any organism for which genomic data is available. Although these technologies are currently applied to many research questions in various model systems ranging from cell cultures to the entire organism level, there are few comparative studies of these technologies in the same system, let alone on the same samples. Here we present a comparison between gene and protein expression in embryos of zebrafish, which is an upcoming model in disease studies., Results: We compared Agilent custom made expression microarrays with Illumina deep sequencing for RNA analysis, showing as expected a high degree of correlation of expression of a common set of 18,230 genes. Gene expression was also found to correlate with the abundance of 963 distinct proteins, with several categories of genes as exceptions. These exceptions include ribosomal proteins, histones and vitellogenins, for which biological and technical explanations are discussed., Conclusions: By comparing state of the art transcriptomic and proteomic technologies on samples derived from the same group of organisms we have for the first time benchmarked the differences in these technologies with regard to sensitivity and bias towards detection of particular gene categories in zebrafish. Our datasets submitted to public repositories are a good starting point for researchers interested in disease progression in zebrafish at a stage of development highly suited for high throughput screening technologies.

Hormones secreted from the pituitary gland regulate important processes such as development, growth and metabolism, reproduction, water balance, and body pigmentation. Synthesis and secretion of pituitary hormones are regulated by different factors from the hypothalamus, but also through feedback mechanisms from peripheral organs, and from the pituitary itself. In the European eel extensive attention has been directed towards understanding the different components of the brain-pituitary-gonad axis, but little is known about the regulation of upstream processes in the pituitary gland. In order to gain a broader mechanistic understanding of the eel pituitary gland, we have performed RNA-seq transcriptome profiling of the pituitary of prepubertal female silver eels. RNA-seq reads generated on the Illumina platform were mapped to the recently assembled European eel genome. The most abundant transcript in the eel pituitary codes for pro-opiomelanocortin, the precursor for hormones of the melanocortin system. Several genes putatively involved in downstream processing of pro-opiomelanocortin were manually annotated, and were found to be highly expressed, both by RNA-seq and by qPCR. The melanocortin system, which affects skin color, energy homeostasis and in other teleosts interacts with the reproductive system, has so far received limited attention in eels. However, since up to one third of the silver eel pituitary's mRNA pool encodes pro-opiomelanocortin, our results indicate that control of the melanocortin system is a major function of the eel pituitary.

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 © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The Japanese eel is a much appreciated research object and very important for Asian aquaculture; however, its genomic resources are still limited. We have used a streamlined bioinformatics pipeline for the de novo assembly of the genome sequence of the Japanese eel from raw Illumina sequence reads. The total assembled genome has a size of 1.15 Gbp, which is divided over 323,776 scaffolds with an N50 of 52,849 bp, a minimum scaffold size of 200 bp and a maximum scaffold size of 1.14 Mbp. Direct comparison of a representative set of scaffolds revealed that all the Hox genes and their intergenic distances are almost perfectly conserved between the European and the Japanese eel. The first draft genome sequence of an organism strongly catalyzes research progress in multiple fields. Therefore, the Japanese eel genome sequence will provide a rich resource of data for all scientists working on this important fish species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Freshwater eels (genus Anguilla), especially the species inhabiting the temperate areas such as the European, American and Japanese eels, are important aquaculture species. Although artificial reproduction has been attempted since the 1930s and large numbers of studies have been conducted, it has not yet fully succeeded. Problems in eel artificial breeding are highly diverse, for instance, lack of basic information about reproduction in nature, no appropriate food for larvae, high mortality, and high individual variation in adults in response to maturation induction. Over the last decade, genomic data have been obtained for a variety of aquatic organisms. Recent technological advances in sequencing and computation now enable the accumulation of genomic information even for non-model species. The draft genome of the European eel Anguilla anguilla has been recently determined using Illumina technology and transcriptomic data based on next generation sequencing have been emerging. Extensive genomic information will facilitate many aspects of the artificial reproduction of eels. Here, we review the progress in genome-wide studies of eels, including additional analysis of the European eel genome data, and discuss future directions and implications of genomic data for aquaculture.

Significant declines in eel stocks have been noted in many parts of the world. Because eel aquaculture is dependent on wild-caught juveniles, there is a need to achieve artificial reproduction. Adult eel maturation is currently induced by repeated injections of purified gonadotropin (human chorionic gonadotropin [hCG]) or pituitary extract. Thus the determination of the biological efficacy and quantification of internal levels of gonadotropic hormones is important for optimizing artificial reproduction protocols. To quantify the plasma levels of biologically functional gonadotropic hormones, we developed a bioassay for luteinizing hormone (LH) and follicle-stimulating hormone (FSH) based on the stable expression of receptors in HEK293 cells of the Japanese eel Anguilla japonica LH (ajLHR) and the European eel Anguilla anguilla FSH (aaFSHR), respectively. Such cells also contain a firefly luciferase reporter gene driven by a cAMP-responsive element (CRE-Luc). We found that the obtained stable cells, with ajLHR, responded linearly to a more than 100,000-fold concentration range of hCG diluted in saline. The cells with aaFSHR showed a linear response to a 1000-fold concentration range of salmon pituitary extract mixed with saline. The biological functionality of the LH and FSH bioassays was validated using hCG, human FSH, and pituitary extracts from salmon, carp and eel. Since the toxins in eel plasma damaged the HEK293 cells, the protocol was adapted to selectively inactivate the toxins by heating at 37°C for 24h. This process successfully enabled the monitoring of hormone levels in blood plasma sampled from hCG-injected eels. In this paper, we describe the development of gonadotropin bioassays that will be useful for improving reproduction protocols in eel aquaculture., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Research on common carp, Cyprinus carpio, is beneficial for zebrafish research because of resources available owing to its large body size, such as the availability of sufficient organ material for transcriptomics, proteomics, and metabolomics. Here we describe the shot gun sequencing of a clonal double-haploid common carp line. The assembly consists of 511891 scaffolds with an N50 of 17 kb, predicting a total genome size of 1.4-1.5 Gb. A detailed analysis of the ten largest scaffolds indicates that the carp genome has a considerably lower repeat coverage than zebrafish, whilst the average intron size is significantly smaller, making it comparable to the fugu genome. The quality of the scaffolding was confirmed by comparisons with RNA deep sequencing data sets and a manual analysis for synteny with the zebrafish, especially the Hox gene clusters. In the ten largest scaffolds analyzed, the synteny of genes is almost complete. Comparisons of predicted exons of common carp with those of the zebrafish revealed only few genes specific for either zebrafish or carp, most of these being of unknown function. This supports the hypothesis of an additional genome duplication event in the carp evolutionary history, which--due to a higher degree of compactness--did not result in a genome larger than that of zebrafish.

The enigmatic life cycle and elongated body of the European eel (Anguilla anguilla L., 1758) have long motivated scientific enquiry. Recently, eel research has gained in urgency, as the population has dwindled to the point of critical endangerment. We have assembled a draft genome in order to facilitate advances in all provinces of eel biology. Here, we use the genome to investigate the eel's complement of the Hox developmental transcription factors. We show that unlike any other teleost fish, the eel retains fully populated, duplicate Hox clusters, which originated at the teleost-specific genome duplication. Using mRNA-sequencing and in situ hybridizations, we demonstrate that all copies are expressed in early embryos. Theories of vertebrate evolution predict that the retention of functional, duplicate Hox genes can give rise to additional developmental complexity, which is not immediately apparent in the adult. However, the key morphological innovation elsewhere in the eel's life history coincides with the evolutionary origin of its Hox repertoire.