Study of genetic factors and temperature influence on sex determination and differentiation in turbot

Sex, as intuitive and simple as it may seem to us, poses some of the most interesting and complex questions when studying life. Sex is an intrinsic characteristic of most eukaryote species which eventually has led to the appearance of two differentiated adult phenotypes or sexes, males and females. This distinction rules a huge part of our lives and is the origin of important evolutionary processes based on intra-sex competition or inter-sex conflict due to sexual antagonism. Furthermore, sex is an important character for a plethora of species involved in human activities, for example in aquaculture many fish species present sex size dimorphisms where one sex grows faster than the other, and so knowing how sex is determined in each species is of the outmost interest. Traditionally, sex determination has been considered a cascade process with a master gene at the top, but recent findings have suggested that, instead, it might be a network process where different genetic and environmental factors can alter gonad fate, which in turn would be connected with a huge number of different sex determination mechanisms in vertebrates, especially in poikiloterms. In this new view of sex, the different players involved in sex differentiation gain relevance and their study may help us understanding how the fate of the gonad is determined. In this work, we have studied sex differentiation in turbot, a flatfish with a marked sex dimorphism where females grow faster than males. This species presents genetic sex determination, but also temperature effects on sex ratios have been reported, which seem to be family-dependant. Our aim was to study sex differentiation in turbot to gain knowledge about how sex is determined in this species and also in a broader sense in fish. This work consists of expression studies in turbot gonads using two different techniques: real-time PCR and microarrays. First of all, the real time PCR technique was setup for gonad development studies in turbot. The different methods available for reference gene stability calculation and efficiency determination were assessed. Then, using this information we performed an extensive expression study on turbot sex differentiation ranging from undifferentiated to differentiated gonads at three different temperatures. We found that the first molecular signs of sex differentiation are observed at 90 days post fertilization and that three genes, cyp19a1a, amh and vasa, can be used to sex turbot at this stage. Furthermore, the expression of genes involved in germ cell development pointed towards their involvement in early sex differentiation and possibly sex determination. Temperature effects on sex differentiation were also assessed in this study. A higher proportion of females was obtained at cold temperatures and several genes showed temperature dependant expression changes. Finally, to complete our study, we also performed a microarray analysis in turbot gonad samples from undifferentiated individuals to male and female juveniles. Female gonads were found to be more different from undifferentiated gonads than those of males, requiring the regulation of a large number of genes and the involvement of different processes including epigenetic mechanisms. Furthermore, the involvement of known sex differentiation genes and previously unrelated genes in sex differentiation was observed. This study has widened our knowledge on sex differentiation in turbot in particular and in fish in general, helping to understand the role of many genes involved in sex differentiation across the whole vertebrate taxa and pointing towards other genes which have been connected with sex for the first time. Our data suggest that a network model might be more accurate to explain sex determination in turbot, where the environment can interact with genetic factors and modify gonad fate

Sex separation unveils the functional plasticity of the vomeronasal organ in rabbits

Chemosensory cues are vital for social and sexual behaviours and are primarily detected and processed by the vomeronasal system (VNS), whose plastic capacity has been investigated in mice. However, studying chemosensory plasticity outside of laboratory conditions may give a more realistic picture of how the VNS adapts to a changing environment. Rabbits are a well-described model of chemocommunication since the discovery of the rabbit mammary pheromone and their vomeronasal organ (VNO) transcriptome was recently characterised, a first step to further study plasticity-mediated transcriptional changes. In this study, we assessed the plastic capacity of the rabbit male and female VNO under sex-separation vs. sex-combined scenarios, including adults and juveniles, to determine whether the rabbit VNO is plastic and, if so, whether such plasticity is already established at early stages of life. First, we characterised the number of differentially expressed genes (DEGs) between the VNO of rabbit male and female under sex-separation and compared it to sex-combined individuals, both in adults and juveniles, finding that differences between male and female were larger in a sex-separated scenario. Secondly, we analysed the number of DEGs between sex-separated and sex-combined scenarios, both in males and females. In adults, both sexes showed a high number of DEGs while in juveniles only females showed differences. Additionally, the vomeronasal receptor genes were strikingly downregulated in sex-separated adult females, whereas in juveniles upregulation was shown for the same condition, suggesting a role of VRs in puberty onset. Finally, we described the environment-modulated plastic capacity of genes involved in reproduction, immunity and VNO functional activity, including G-protein coupled receptors. Our results show that sex-separation induces sex- and stage-specific gene expression differences in the VNO of male and female rabbit, both in adults and juveniles. These results bring out for the first time the plastic capacity of the rabbit VNO, supporting its functional adaptation to specifically respond to a continuous changing environment. Finally, species-specific differences and individual variability should always be considered in VNO studies and overall chemocommunication research

Comparative expression analysis in mature gonads, liver and brain of turbot (Scophthalmus maximus) by cDNA-AFLPS

Turbot is one of the most important farmed fish in Europe. This species exhibits a considerable sexual dimorphism in growth and sexual maturity that makes the all-female production recommended for turbot farming. Our knowledge about the genetic basis of sex determination and the molecular regulation of gonad differentiation in this species is still limited. Our goal was to identify and compare gene expression and functions between testes and ovaries in adults in order to ascertain the relationship between the genes that could be involved in the gonad differentiation or related to the sex determination system. The identification of differentially expressed sex related genes is an initial step towards understanding the molecular mechanisms of gonad differentiation. For this, we carried out a transcriptome analysis based on cDNA-AFLP technique which allowed us to obtain an initial frame on sex-specific gene expression that will facilitate further analysis especially along the critical gonad differentiating period. With the aim of widening the study on sex-biased gene expression we reproduced the same experiments in two somatic tissues: liver and brain. We have selected the liver because it is the most analyzed one regarding sexual dimorphic gene expression and due to its importance in steroid hormones metabolism and the brain because the functional relationship between brain and gonad is documented. We found slight but important differences between sexes which deserve further investigationThis research work was financially supported by the Xunta of Galicia (07MMA004200PR) to A. Viñas. X. Taboada was supported by a fellowship from the European Social Fund and Consellería de Educación e Ordenación Universitaria- Xunta de Galicia (Spain)S

Inhibition of gamma-secretase promotes axon regeneration after a complete spinal cord injury

In a recent study, we showed that GABA and baclofen (a GABAB receptor agonist) inhibit caspase activation and promote axon regeneration in descending neurons of the sea lamprey brainstem after a complete spinal cord injury (Romaus-Sanjurjo et al., 2018a). Now, we repeated these treatments and performed 2 independent Illumina RNA-Sequencing studies in the brainstems of control and GABA or baclofen treated animals. GABA treated larval sea lampreys with their controls were analyzed 29 days after a complete spinal cord injury and baclofen treated larvae with their controls 9 days after the injury. One of the most significantly downregulated genes after both treatments was a HES gene (HESB). HES proteins are transcription factors that are key mediators of the Notch signaling pathway and gamma-secretase activity is crucial for the activation of this pathway. So, based on the RNA-Seq results we subsequently treated spinal cord injured larval sea lampreys with a novel gamma-secretase inhibitor (PF-3804014). This treatment also reduced the expression of HESB in the brainstem and significantly enhanced the regeneration of individually identifiable descending neurons after a complete spinal cord injury. Our results show that gamma-secretase could be a novel target to promote axon regeneration after nervous system injuriesGrant sponsors: FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación (Grant number: BFU-2017-87079-P) and the Xunta de Galicia (Grant number: ED431C 2018/28). DR was supported by the BBSRC Institute Strategic Programme Grants to the Roslin Institute (BB/P013732/1, BB/P013740/1, and BB/P013759/1)S

Analysis of qPCR reference genes stability determination methods and a practical approach for efficiency calculation on a turbot (Scphthalmus maximus) gonad dataset

Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industryVersión del edito

RNA-seq analysis of early enteromyxosis in turbot (Scophthalmus maximus): new insights into parasite invasion and immune evasion strategies

Enteromyxum scophthalmi, an intestinal myxozoan parasite, is the causative agent of a threatening disease for turbot (Scophthalmus maximus, L.) aquaculture. The colonisation of the digestive tract by this parasite leads to a cachectic syndrome associated with high morbidity and mortality rates. This myxosporidiosis has a long pre-patent period and the first detectable clinical and histopathological changes are subtle. The pathogenic mechanisms acting in the early stages of infection are still far from being fully understood. Further information on the host–parasite interaction is needed to assist in finding efficient preventive and therapeutic measures. Here, a RNA-seq-based transcriptome analysis of head kidney, spleen and pyloric caeca from experimentally-infected and control turbot was performed. Only infected fish with early signs of infection, determined by histopathology and immunohistochemical detection of E. scophthalmi, were selected. The RNA-seq analysis revealed, as expected, less intense transcriptomic changes than those previously found during later stages of the disease. Several genes involved in IFN-related pathways were up-regulated in the three organs, suggesting that the IFN-mediated immune response plays a main role in this phase of the disease. Interestingly, an opposite expression pattern had been found in a previous study on severely infected turbot. In addition, possible strategies for immune system evasion were suggested by the down-regulation of different genes encoding complement components and acute phase proteins. At the site of infection (pyloric caeca), modulation of genes related to different structural proteins was detected and the expression profile indicated the inhibition of cell proliferation and differentiation. These transcriptomic changes provide indications regarding the mechanisms of parasite attachment to and invasion of the host. The current results contribute to a better knowledge of the events that characterise the early stages of turbot enteromyxosis and provide valuable information to identify molecular markers for early detection and control of this important parasitosisThis study was funded by the Spanish Ministry of Economy and Competitiveness (AGL 2009-13282-C02-01 and -02; AGL2015-67039-C3-1-R and AGL2015-67039-C3-3-R), the European Regional Development Fund (ERDF, European Union) and Xunta de Galicia (Spain) local government (GRC2014/010 and GPC2015/34). Diego Robledo was supported by a FPU fellowship funded by the Spanish Ministry of Education, Culture and Sport. Paolo Ronza was supported by a grant from the scientific network “INMUNOGENOM”, funded by Xunta de Galicia (REDES GI-1251)S

Integrative Transcriptome, Genome and Quantitative Trait Loci Resources Identify Single Nucleotide Polymorphisms in Candidate Genes for Growth Traits in Turbot

Growth traits represent a main goal in aquaculture breeding programs and may be related to adaptive variation in wild fisheries. Integrating quantitative trait loci (QTL) mapping and next generation sequencing can greatly help to identify variation in candidate genes, which can result in marker-assisted selection and better genetic structure information. Turbot is a commercially important flatfish in Europe and China, with available genomic information on QTLs and genome mapping. Muscle and liver RNA-seq from 18 individuals was carried out to obtain gene sequences and markers functionally related to growth, resulting in a total of 20,447 genes and 85,344 single nucleotide polymorphisms (SNPs). Many growth-related genes and SNPs were identified and placed in the turbot genome and genetic map to explore their co-localization with growth-QTL markers. Forty-five SNPs on growth-related genes were selected based on QTL co-localization and relevant function for growth traits. Forty-three SNPs were technically feasible and validated in a wild Atlantic population, where 91% were polymorphic. The integration of functional and structural genomic resources in turbot provides a practical approach for QTL mining in this species. Validated SNPs represent a useful set of growth-related gene markers for future association, functional and population studies in this flatfish speciesThis work was funded by Spanish Ministry of Economy and Competitiveness and European Regional Development Funds (AGL2012-35904), and Ministry of Science and Innovation (Consolider Ingenio, Aquagenomics, CSD200700002). DR was supported by a FPU fellowship funded by Spanish Ministry of Education, Culture and Sport. Thanks to Lucía Ínsua for technical assistance. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics for the generation of the sequencing data, and the Spanish National Genotyping Center (CEGEN-ISCIII)-USC node for SNP genotyping support. We acknowledge the support of the Centro de Supercomputación de Galicia (CESGA) in the completion of this workS

Spinal cord RNA-seq data after a baclofen treatment in mice with a spinal cord injury

Spinal cord injury (SCI) leads to severe functional deficits. Currently, there are no available pharmacological treatments to promote neurological recovery in SCI patients. Recent work from our group has shown that a baclofen treatment can promote functional recovery after a compression SCI in mice [1]. Here, we provide transcriptomic (RNA-seq) data from adult mouse spinal cords collected 7 days after a compression SCI and baclofen (vs vehicle) administration. The Illumina NovaSeq 6000 platform was used to generate the raw transcriptomic data. In addition, we also present bioinformatic analyses including differential gene expression analysis, enrichment analyses for various functional annotations (gene ontology, KEGG and BioCarta pathways or InterPro domains) and transcription factor targets. The raw RNA-seq data has been uploaded to the NCBI Sequence Read Archive (SRA) database (Bioproject ID PRJNA886048). The data generated from the bioinformatic analyses is contained within the articleThis work was supported by the Wings for Life Spinal Cord Research Foundation (grant reference WFL-ES-03/19), the Portuguese Foundation for Science and Technology (FCT) EXPL/MED-FAR/1529/2021 to N. de Sousa and through the Scientific Employment Stimulus to N. Silva and S. Monteiro (CEECIND/04794/2017 and CEECIND/01902/2017) and Grant PID2020-115121GB-I00 funded by MCIN/AEI/10.13039/501100011033 to A. Barreiro-IglesiasS

Resultados preliminares de la influencia de la temperatura de cultivo sobre la proporción de sexos en el rodaballo (Scophthalmus maximus L.)

Las larvas de tres familias de rodaballos se cultivaron a temperaturas de 15'C, 18'C y 22'C desde el día dos hasta día 90 de vida y a temperatura ambiente hasta el dia 210. En las familias 1 y 2 las proporciones sexuales, determinadas por el fenotipo, oscilaron en los tres grupos entre el 40%- 60%,y no se observaron diferencias siqníñcaffvas entre los diferentes grupos de temperatura (p>0.05). En la familia 3, el porcentaje de hembras fue mayor que el de machos en los tres grupos de temperatura, y además se observó diferencia significativa (p<0.05) entre el grupo de peces cultivados a temperatura fría con respecto a los cultivados en agua ambiente. En tas familias 1 y 2 el sexo genético coincidió en gran medida con~ sexo fenolípico, siendo la discrepancia menor del 10%. Sin embargo, en la familia 3 se observó que el 36,5% de los machos genéticos eran hembras a 15'C, el 29% a 18ºC y el 18% a 23'C. Los resultados sugieren la interacción temperatura-familia en la determinación sexual del rodaballo, que debe ser confirmada en un mayor número de familias.Larvae from three families of turbot were cultured at 15'C, 18'C and 22'C from 2 to 90 days old, and then at ambient temperature until210 daysold. Regarding families 1 and 2, \he sexual proportions determined by \he phenotype varied between 40%·60%, regardless of \he cunure temperature (p>O.05). Gontrary, in family 3, \he percentage of females was higher than for males in all three cunure temperatures. Furthermore, \here were differences (¡¡<O.05) between families cultured at 15'C and 18'C. For families 1 and 2, sex determined by genotype was similarto \hat detennined by the phenotype, wi\h differences <1 0%. Gontrary, for family 3,\he \he percentage of males determined by \he genotype \hat were phenotipycatt¡ females was 36, 29 and 18% for animals cunured at 15'C, 18'C and 22'C, respectively. Results suggest an interaction temperature-family in \he turbot sex determination which should be checked on a higher numberoffamilies

Formas de Hispanidad

Este texto presenta estudios sobre las múltiples formas de hispanidad, desarrollados en los últimos años por destacados investigadores del mundo hispánico que, poco a poco, han estado construyendo un nuevo espacio de investigación para una creciente y activa comunidad científica. En este libro el lector encontrará estudios con enfoques desde la ciencia política, la teoría política, la historia, la filosofía, la sociología, la economía, los estudios literarios y culturales, entre otras perspectivas académicas. Los aportes de cada aproximación teórica y disciplinar están orientados al logro de una meta común: la de reconstruir y reinterpretar la tradición histórica hispánica, desmantelando prejuicios ideológicamente provocados, con el fin de comprender los fenómenos políticos que la caracterizan. Por las mismas razones este libro se sitúa en el debate sobre las formas de escritura de la historia, que no es sólo un debate de teoría de la historia sino también de filosofía de lo histórico