Temperature training improves transcriptional homeostasis after heat shock in juvenile Atlantic sturgeon (Acipenser oxyrinchus).

Exposure to high temperatures can lead to thermotolerance in fish, which is hypothesized to potentially improve post-release survival in species under restocking programs, like Atlantic sturgeon. The aim of this study was to determine whether Atlantic sturgeon juveniles exposed to a 4-week temperature treatment respond differently to a subsequent heat shock than juveniles exposed to heat shock for the first time (naive fish). Response to heat shock was assessed by mapping the liver transcriptome. In total, 838 unique contigs were differentially expressed between the trained and the control group (592 downregulated, 261 upregulated, and 15 down- or upregulated, depending on the condition), corresponding to genes involved in the response to heat, tissue damage, proteolysis, and metabolism. Temperature-trained fish showed 2-4-fold fewer dysregulated contigs than naive fish, indicating their ability to maintain and recover homeostasis faster. During heat shock, hspc1 was upregulated in both experimental groups, while hspa1 and dnaja4 were exclusively upregulated in the control. Overall, compensatory mechanisms were observed in addition to the heat shock response. Only two genes, fgg and apnl, were upregulated at nearly all timepoints in both groups. Peptidases were more strongly downregulated in control fish, which also showed a reduction in lipid metabolism during recovery. Keratins, pck1, gadd45ga, and gadd45gb were differentially expressed between trained and control fish, and due to their roles in tissue protection and ER stress reduction, they might be responsible for the maintenance of the transcriptional homeostasis observed in trained fish.

Acting locally - affecting globally: RNA sequencing of gilthead sea bream with a mild Sparicotyle chrysophrii infection reveals effects on apoptosis, immune and hypoxia related genes.

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.

Transcriptomic analysis of Baltic cod (Gadus morhua) liver infected with Contracaecum osculatum third stage larvae indicates parasitic effects on growth and immune response.

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.

Deep transcriptome analysis of the heat shock response in an Atlantic sturgeon (Acipenser oxyrinchus) cell line.

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.

Transcriptomic analysis of immunity in rainbow trout (Oncorhynchus mykiss) gills infected by Ichthyophthirius multifiliis.

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.

Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

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.

A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation.

Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. A large network of molecular chaperones ensures protein quality control. Here, we show that within the Hsp70, Hsp110, and Hsp40 (DNAJ) chaperone families, members of a subclass of the DNAJB family (particularly DNAJB6b and DNAJB8) are superior suppressors of aggregation and toxicity of disease-associated polyglutamine proteins. The antiaggregation activity is largely independent of the N-terminal Hsp70-interacting J-domain. Rather, a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential. The SSF-SST region is involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with histone deacetylases (HDAC4, HDAC6, SIRT2). Inhibiting HDAC4 reduced DNAJB8 function. DNAJB8 is (de)acetylated at two conserved C-terminal lysines that are not involved in substrate binding, but do play a role in suppressing protein aggregation. Combined, our data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation.

Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel (Anguilla anguilla); Biomarkers for broodstock selection.

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 17ß-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.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

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.

Temperature modulates testis steroidogenesis in European eel.

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 aa11ßHSD) 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.

Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

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.

Robotic injection of zebrafish embryos for high-throughput screening in disease models.

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.

Identification of molecular markers in pectoral fin to predict artificial maturation of female European eels (Anguilla anguilla).

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.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism.

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.

Male European eels are highly efficient long distance swimmers: effects of endurance swimming on maturation.

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.

The induction of oocyte maturation and ovulation in the European eel (Anguilla anguilla): in vitro and in vivo comparison of progesterone with 17α,20ß-dihydroxy-4-pregnen-3-one.

Ovulation in European eel is induced by injection of 17α,20ß-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.

The Rauvolfia tetraphylla genome suggests multiple distinct biosynthetic routes for yohimbane monoterpene indole alkaloids.

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.

Assembly and Comparison of Ca. Neoehrlichia mikurensis Genomes.

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.

Plant- and Bacteria-Derived Compounds with Anti-Philasterides dicentrarchi Activity.

Philasterides dicentrarchi is a scuticociliate that causes high mortalities in farmed fish. Although vaccination is an effective method to prevent scuticociliatosis caused by the homologous serotype, a universal vaccine has not been developed yet. Many compounds have been shown to be toxic to this ciliate species; moreover, most of them are toxic to aquatic life and cannot be used to prevent the disease. We have evaluated the toxicity to P. dicentrarchi of several compounds of natural origin to be used to reduce parasite levels in the seawater. Ciliates were exposed to several compound concentrations, and the mortality was determined at several incubation times. Tomatine, plumbagin and 2',4'-dihydroxychalcone displayed the highest anticiliate activity, with a dose-dependent response. The effects of these compounds on the EPC cell line were also evaluated, finding that 2',4'-dihydroxychalcone displayed the lowest toxicity to fish cells. At 7.54 µM, 2',4'-dihydroxychalcone inhibited 50% parasite growth but only killed about 10% of EPC cells after 24 h incubation. Finally, we evaluated the toxicity of Pseudomonas H6 surfactant (PS) to P. dicentrarchi, finding that PS was toxic to the ciliate but showed lower toxicity to EPC cells. At a concentration of 7.8 µg/mL (LC50 for the ciliate after 3 h incubation), PS killed 14.9% of EPC cells. We conclude that 2',4'-dihydroxychalcone, and PS could be used to reduce parasite levels in seawater, thus decreasing the risk of scuticociliatosis infection in cultured fish.

Genome Assembly of the Medicinal Plant Voacanga thouarsii.

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.