A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis.

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.

Estimates of recent and historical effective population size in turbot, seabream, seabass and carp selective breeding programmes.

BACKGROUND: The high fecundity of fish species allows intense selection to be practised and therefore leads to fast genetic gains. Based on this, numerous selective breeding programmes have been started in Europe in the last decades, but in general, little is known about how the base populations of breeders have been built. Such knowledge is important because base populations can be created from very few individuals, which can lead to small effective population sizes and associated reductions in genetic variability. In this study, we used genomic information that was recently made available for turbot (Scophthalmus maximus), gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax) and common carp (Cyprinus carpio) to obtain accurate estimates of the effective size for commercial populations. METHODS: Restriction-site associated DNA sequencing data were used to estimate current and historical effective population sizes. We used a novel method that considers the linkage disequilibrium spectrum for the whole range of genetic distances between all pairs of single nucleotide polymorphisms (SNPs), and thus accounts for potential fluctuations in population size over time. RESULTS: Our results show that the current effective population size for these populations is small (equal to or less than 50 fish), potentially putting the sustainability of the breeding programmes at risk. We have also detected important drops in effective population size about five to nine generations ago, most likely as a result of domestication and the start of selective breeding programmes for these species in Europe. CONCLUSIONS: Our findings highlight the need to broaden the genetic composition of the base populations from which selection programmes start, and suggest that measures designed to increase effective population size within all farmed populations analysed here should be implemented in order to manage genetic variability and ensure the sustainability of the breeding programmes.

Assessment of the Simultaneous Use of Biomaterials in Transalveolar Sinus Floor Elevation: Prospective Randomized Clinical Trial in Humans.

Implants inserted in the posterior maxilla frequently need additional surgery for successful bone augmentation. One of the most common procedures for this is transalveolar sinus floor elevation. There are different protocols for this procedure, and there is controversy over the simultaneous application of grafting material upon elevating. In this prospective randomized clinical study in humans, a total of 49 transalveolar sinus floor elevations were performed in 49 different patients, divided into a control group (without graft, 25 patients) and a test group (with graft, 24 patients). The analyzed variables were obtained through digital orthopantomography on day 0 (day of surgery) and 18 months after surgery. These measurements showed a tendency towards greater vertical bone gain in the test group, but this was not statistically significant. Therefore, considering that sinus elevation and implant placement without the application of grafts is a successful treatment with fewer complications, a critical assessment of the need for these biomaterials is necessary.

Disentangling Genetic Variation for Resistance and Endurance to Scuticociliatosis in Turbot Using Pedigree and Genomic Information.

Selective breeding for improving host responses to infectious pathogens is a promising option for disease control. In fact, disease resilience, the ability of a host to survive or cope with infectious challenge, has become a highly desirable breeding goal. However, resilience is a complex trait composed of two different host defence mechanisms, namely resistance (the ability of a host to avoid becoming infected or diseased) and endurance (the ability of an infected host to survive the infection). While both could be targeted for genetic improvement, it is currently unknown how they contribute to survival, as reliable estimates of genetic parameters for both traits obtained simultaneously are scarce. A difficulty lies in obtaining endurance phenotypes for genetic analyses. In this study, we present the results from an innovative challenge test carried out in turbot whose design allowed disentangling the genetic basis of resistance and endurance to Philasterides dicentrarchi, a parasite causing scuticociliatosis that leads to substantial economic losses in the aquaculture industry. A noticeable characteristic of the parasite is that it causes visual signs that can be used for disentangling resistance and endurance. Our results showed the existence of genetic variation for both traits (heritability = 0.26 and 0.12 for resistance and endurance, respectively) and for the composite trait resilience (heritability = 0.15). The genetic correlation between resistance and resilience was very high (0.90) indicating that both are at a large extent the same trait, but no significant genetic correlation was found between resistance and endurance. A total of 18,125 SNPs obtained from 2b-RAD sequencing enabled genome-wide association analyses for detecting QTLs controlling the three traits. A candidate QTL region on linkage group 19 that explains 33% of the additive genetic variance was identified for resilience. The region contains relevant genes related to immune response and defence mechanisms. Although no significant associations were found for resistance, the pattern of association was the same as for resilience. For endurance, one significant association was found on linkage group 2. The accuracy of genomic breeding values was also explored for resilience, showing that it increased by 12% when compared with the accuracy of pedigree-based breeding values. To our knowledge, this is the first study in turbot disentangling the genetic basis of resistance and endurance to scuticociliatosis.

"A Zebra in the Water": Inspiring Science in Spain.

The zebrafish model can play a role in education because of its suitability for manipulation and attractiveness to students compared to traditional lecture-based instruction. Furthermore, zebrafish offer advantages over other model species. Seeing as fewer and fewer students are entering science degree programs, this project has been developed to encourage scientific vocations among secondary school students. To do so, an aquarium was given to 114 schools so that they could look after adult zebrafish, mate them, and visualize embryo development. For training on more sophisticated techniques, a virtual tool was developed to simulate a real genetics laboratory on a personal computer. Results based on teachers' feedback indicate that the students were fully dedicated to the project and achieved better understanding of genetic concepts and techniques. These results demonstrate the potential of alternative teaching methods for engaging students in science learning.

Microarray analysis of the inflammatory and immune responses in head kidney turbot leucocytes treated with resveratrol.

A DNA oligo-microarray enriched in genes and involved in inflammatory and immune responses was used to evaluate the effects of resveratrol on gene expression in turbot head kidney leucocytes. Leucocytes were cultured for 3, 6 and 24 h, in the presence or absence of resveratrol, or were stimulated with the membrane fraction of the parasite Philasterides dicentrarchi or with the membrane plus resveratrol. Gene expression changed considerably in control cells, and several of the regulated genes were related to inflammatory and immune responses and to the cytoskeleton. Similar changes in gene expression occurred in control cells and in cells stimulated with P. dicentrarchi membrane fraction. Treatment with resveratrol induced changes in the expression (mostly down-regulation) of several genes involved in immune responses and inflammation. Thus, the down-regulation of the transcription factor PU.1, pentraxin-multidomain protein, heme oxygenase 1, S100 calcium-binding protein A-16 (S100A16) and the signal transducer and activator of transcription 4 was observed after all three incubation times. The down-regulation of the suppressor of cytokine signalling 3a, LPS-induced tumour necrosis alpha, hepcidin, metallothionein, TLR8 and the calcium dependent lectin A was observed after 3 and 6 h. Resveratrol also decreased the expression of CCL20, IL-8, apolipoprotein E and glutathione S-transferase after incubation for 6 and 24 h, and of TNF-α after incubation for 3 and 24 h. Resveratrol also induced strong regulation of several cytoskeleton-related genes. The use of the turbot oligo-microarray enabled us to discover genes whose expression was not previously suspected of being modulated by this polyphenol.

Gene expression profiles of spleen, liver, and head kidney in turbot (Scophthalmus maximus) along the infection process with Philasterides dicentrarchi using an immune-enriched oligo-microarray.

We evaluated the expression profiles of turbot in spleen, liver, and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver, and 90 in head kidney, in at least 1 of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and downregulated gene groups. Upregulated genes were mostly associated to immune functions, while downregulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry.

Gene expression profiles of the spleen, liver, and head kidney in turbot (Scophthalmus maximus) along the infection process with Aeromonas salmonicida using an immune-enriched oligo-microarray.

We evaluated the expression profiles of turbot in the spleen, liver, and head kidney across five temporal points of the Aeromonas salmonicida infection process using an 8 × 15 K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 471 differentially expressed (DE) genes (17.3% of the whole microarray), 223 in the spleen, 246 in the liver, and 125 in the head kidney, in at least one of the five temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using Gene Ontology terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions in accordance to previous studies with this pathogen in other fish species. A high proportion of DE genes were organ specific (77.1%), but their associated GO functions were rather similar in the three organs. The most striking difference in functional distribution was observed between the up- and down-regulated gene groups. Up-regulated genes were mostly associated to key immune functions while down-regulated ones mainly involved metabolism- and transport-related genes. Genetic response appeared clustered in groups of genes with similar expression profiles along the temporal series. The spleen showed the most clustering while the liver and head kidney displayed a higher diversification. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to A. salmonicida to achieve more resistant broodstocks for turbot industry.

Design and performance of a turbot (Scophthalmus maximus) oligo-microarray based on ESTs from immune tissues.

An expressed sequence tag database from immune tissues was used to design the first high-density turbot (Scophthalmus maximus) oligo-microarray with the aim of identifying candidate genes for tolerance to pathogens. Specific oligonucleotides (60 mers) were successfully designed for 2,716 out of 3,482 unique sequences of the database. An Agilent custom oligo-microarray 8 x 15 k (five replicates/gene; eight microarrays/slide) was constructed. The performance of the microarray and the sources of variation along microarray analysis were examined on spleen pools of controls and Aeromonas salmonicida-challenged fish at 3 days postinfection. Only 48 out of 2,716 probes did not show signal of hybridization on the 32 microarrays employed, thus demonstrating the consistency of the bioinformatic applications of our database. An asymmetric hierarchical design was employed to ascertain the noise associated with biological and technical (RNA extraction, labeling, hybridization, slide, and dye bias) factors using 1C and 2C labeling approaches. The high correlation coefficient between replicates at most factors tested demonstrated the high reproducibility of the signal. An analysis of random-effects variance revealed that technical variation was mostly negligible, and biological variation represented the main factor, even using pooled samples. One-color approach performed at least as well as 2C, suggesting their usefulness due to its higher design flexibility and lower cost. A relevant proportion of genes turn out to be differentially labeled depending on fluorophore, which alerts for the likely need of swapping replication in 2C experiments. A set of differentially expressed genes and enriched functions related to immune/defense response were detected at 3 days postchallenging.

Identification of the major sex-determining region of turbot (Scophthalmus maximus).

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.