Effects of Enteromyxum spp. (Myxozoa) infection in the regulation of intestinal E-cadherin: Turbot against gilthead sea bream.

Enteromyxoses are relevant diseases for turbot and gilthead sea bream aquaculture. The myxozoan parasites invade the intestinal mucosa, causing a cachectic syndrome associated with intestinal barrier alteration; nonetheless, their pathological impact is different. Turbot infected by Enteromyxum scophthalmi develop more severe intestinal lesions, reaching mortality rates of 100%, whereas in E. leei-infected gilthead sea bream, the disease progresses slowly, and mortality rates are lower. The mechanisms underlying the different pathogenesis are still unclear. We studied the distribution and expression changes of E-cadherin, a highly conserved protein of the adherens junctions, in the intestine of both species by immunohistochemistry and quantitative PCR, using the same immunohistochemical protocol and common primers. The regular immunostaining pattern observed in control fish turned into markedly irregular in parasitized turbot, showing an intense immunoreaction at the host-parasite interface. Nevertheless, E-cadherin gene expression was not significantly modulated in this species. On the contrary, no evident changes in the protein distribution were noticed in gilthead sea bream, whereas a significant gene downregulation occurred in advanced infection. The results contribute to the understanding of the different host-parasite interactions in enteromyxoses. Host and parasite cells appear to establish diverse relationships in these species, which could underlie the different pathological picture.

Gene expression analysis of Ruditapes philippinarum haemocytes after experimental Perkinsus olseni zoospore challenge and infection in the wild.

The production of Manila clam (Ruditapes philippinarum) is seriously threatened by the protistan parasite Perkinsus olseni. We characterized and compared gene expression of Manila clam haemocytes in response to P. olseni in a time-course (10 h, 24 h, 8 d) controlled laboratory challenge (LC), representing the first step of infection, and in a more complex infection in the wild (WI), using a validated oligo-microarray containing 11,232 transcripts, mostly annotated. Several immune-genes involved in NIK/NF-kappaB signalling, Toll-like receptor signalling and apoptosis were activated at LC-10 h. However, down-regulation of genes encoding lysozyme, histones, cathepsins and heat shock proteins indicated signals of immunodepression, which persisted at LC-24 h, when only down-regulated genes were detected. A rebound of haemocyte activity occurred at LC-8 d as shown by up-regulation of genes involved in cytoskeleton organization and cell survival. The WI study showed a more complex picture, and several immune-relevant processes including cytoskeleton organization, cell survival, apoptosis, encapsulation, cell redox- and lipid-homeostasis were activated, illustrating the main mechanism of host response. Our results provide useful information, including potential biomarkers, to develop strategies for controlling Manila clam perkinsosis.

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.

De novo transcriptome assembly of Perkinsus olseni trophozoite stimulated in vitro with Manila clam (Ruditapes philippinarum) plasma.

The protistan parasite Perkinsus olseni is a deadly causative agent of perkinsosis, a molluscan disease affecting Manila clam (Ruditapes philippinarum), having a significant impact on world mollusc production. Deciphering the underlying molecular mechanisms in R. philippinarum-P. olseni interaction is crucial for controlling this parasitosis. The present study investigated the transcriptional expression in the parasite trophozoite using RNA-seq. Control and treatment (in vitro challenged with Manila clam-plasma) P. olseni trophozoite RNA were extracted and sequenced on the Illumina HiSeq 2000 instrument using a 100-bp paired-end sequencing strategy. Paired reads (64.7 million) were de novo assembled using Trinity, and the resultant transcripts were further clustered using CAP3. The re-constructed P. olseni transcriptome contains 47,590 unique transcripts of which 23,505 were annotated to 9764 unique proteins. A large number of genes were associated with Gene Ontology terms such as stress and immune-response, cell homeostasis, antioxidation, cell communication, signal transduction, signalling and proteolysis. Among annotated transcripts, a preliminary gene expression analysis detected 679 up-regulated and 478 down-regulated genes, linked to virulence factors, anti-oxidants, adhesion and immune-response molecules. Genes of several metabolic pathways such as DOXP/MEP, FAS II or folate biosynthesis, which are potential therapeutic targets, were identified. This study is the first description of the P. olseni transcriptome, and provides a substantial genomic resource for studying the molecular mechanisms of the host-parasite interaction in perkinsosis. In this sense, it is also the first evaluation of the parasite gene expression after challenge with clam extracellular products.

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones.

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.

Immunohistochemical detection and gene expression of TNFα in turbot (Scophthalmus maximus) enteromyxosis.

Enteromyxum scophthalmi (Myxozoa) constitutes one of the most devastating pathogens for turbot (Scophthalmus maximus, L.) aquaculture. This parasite causes a severe intestinal parasitosis that leads to a cachectic syndrome with high morbidity and mortality rates for which no therapeutic options are available. Presence of inflammatory infiltrates, increased apoptotic rates and epithelial detaching have been described at intestinal level, as well as leukocyte depletion in lymphohaematopoietic organs. Previous investigations on enteromyxosis in turbot showed the high susceptibility of this species to the parasite and reported the existence of a dysregulated immune response against the parasite. The pleiotropic cytokine tumour necrosis factor alpha (TNFα) plays a major role in immune response and is involved in a wide range of biological activities. In teleost, the gene expression of this cytokine has been found regulated under several pathological conditions. Teleost TNFα shows some analogous functions with its mammalian counterparts, but the extent of its activities is still poorly understood. Cytokines are generally considered as a double-edge sword and TNFα has been implicated in the pathogenesis of different inflammatory diseases as well as in wasting syndromes described in mammals. The aim of this work was to analyse the expression of TNFα during enteromyxosis with molecular (Q-PCR) and morphological (immunohistochemistry) tools. Kidney, spleen and pyloric caeca from turbot with moderate and severe infections were analysed and compared to healthy naïve fish. TNFα expression was increased in both spleen and kidney in the earlier stages of the disease, whereas in severely infected fish, the expression decreased, especially in kidney. At the intestinal level, an increase in the number of TNFα-positive cells was noticed, which was proportional to the infiltration of inflammatory cells. The results demonstrate the involvement of TNFα in the immune response to E. scophthalmi in turbot, which could be related to the development of the clinic signs and lesions.

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

BACKGROUND: 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 industry. RESULTS: We analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency. CONCLUSION: Our results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.

RNA-seq analysis reveals significant transcriptome changes in turbot (Scophthalmus maximus) suffering severe enteromyxosis.

BACKGROUND: Enteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis. RESULTS: A huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism. CONCLUSIONS: This transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen.

A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus).

BACKGROUND: Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry. RESULTS: Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immune-related tissues after several parasitic challenges. The resulting database ("Turbot 2 database") was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences ("Turbot 3 database"), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50-90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified. CONCLUSIONS: The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.

An Expressed Sequence Tag (EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework for comparative genomics across model and farmed teleosts.

BACKGROUND: The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies. RESULTS: A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups. CONCLUSIONS: The new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species.

The first high-density genetic map of common cockle (Cerastoderma edule) reveals a major QTL controlling shell color variation.

Shell color shows broad variation within mollusc species and despite information on the genetic pathways involved in shell construction and color has recently increased, more studies are needed to understand its genetic architecture. The common cockle (Cerastoderma edule) is a valuable species from ecological and commercial perspectives which shows important variation in shell color across Northeast Atlantic. In this study, we constructed a high-density genetic map, as a tool for screening common cockle genome, which was applied to ascertain the genetic basis of color variation in the species. The consensus genetic map comprised 19 linkage groups (LGs) in accordance with the cockle karyotype (2n = 38) and spanned 1073 cM, including 730 markers per LG and an inter-marker distance of 0.13 cM. Five full-sib families showing segregation for several color-associated traits were used for a genome-wide association study and a major QTL on chromosome 13 associated to different color-traits was detected. Mining on this genomic region revealed several candidate genes related to shell construction and color. A genomic region previously reported associated with divergent selection in cockle distribution overlapped with this QTL suggesting its putative role on adaptation.

QTL detection for Aeromonas salmonicida resistance related traits in turbot (Scophthalmus maximus).

BACKGROUND: Interactions between fish and pathogens, that may be harmless under natural conditions, often result in serious diseases in aquaculture systems. This is especially important due to the fact that the strains used in aquaculture are derived from wild strains that may not have had enough time to adapt to new disease pressures. The turbot is one of the most promising European aquaculture species. Furunculosis, caused by the bacterium Aeromonas salmonicida, produces important losses to turbot industry. An appealing solution is to achieve more robust broodstock, which can prevent or diminish the devastating effects of epizooties. Genomics strategies have been developed in turbot to look for candidate genes for resistance to furunculosis and a genetic map with appropriate density to screen for genomic associations has been also constructed. In the present study, a genome scan for QTL affecting resistance and survival to A. salmonicida in four turbot families was carried out. The objectives were to identify consistent QTL using different statistical approaches (linear regression and maximum likelihood) and to locate the tightest associated markers for their application in genetic breeding strategies. RESULTS: Significant QTL for resistance were identified by the linear regression method in three linkage groups (LGs 4, 6 and 9) and for survival in two LGs (6 and 9). The maximum likelihood methodology identified QTL in three LGs (5, 6 and 9) for both traits. Significant association between disease traits and genotypes was detected for several markers, some of them explaining up to 17% of the phenotypic variance. We also identified candidate genes located in the detected QTL using data from previously mapped markers. CONCLUSIONS: Several regions controlling resistance to A. salmonicida in turbot have been detected. The observed concordance between different statistical methods at particular linkage groups gives consistency to our results. The detected associated markers could be useful for genetic breeding strategies. A finer mapping will be necessary at the detected QTL intervals to narrow associations and around the closely associated markers to look for candidate genes through comparative genomics or positional cloning strategies. The identification of associated variants at specific genes will be essential, together with the QTL associations detected in this study, for future marker assisted selection programs.

Detection of growth-related QTL in turbot (Scophthalmus maximus).

BACKGROUND: The turbot (Scophthalmus maximus) is a highly appreciated European aquaculture species. Growth related traits constitute the main goal of the ongoing genetic breeding programs of this species. The recent construction of a consensus linkage map in this species has allowed the selection of a panel of 100 homogeneously distributed markers covering the 26 linkage groups (LG) suitable for QTL search. In this study we addressed the detection of QTL with effect on body weight, length and Fulton's condition factor. RESULTS: Eight families from two genetic breeding programs comprising 814 individuals were used to search for growth related QTL using the panel of microsatellites available for QTL screening. Two different approaches, maximum likelihood and regression interval mapping, were used in order to search for QTL. Up to eleven significant QTL were detected with both methods in at least one family: four for weight on LGs 5, 14, 15 and 16; five for length on LGs 5, 6, 12, 14 and 15; and two for Fulton's condition factor on LGs 3 and 16. In these LGs an association analysis was performed to ascertain the microsatellite marker with the highest apparent effect on the trait, in order to test the possibility of using them for marker assisted selection. CONCLUSIONS: The use of regression interval mapping and maximum likelihood methods for QTL detection provided consistent results in many cases, although the high variation observed for traits mean among families made it difficult to evaluate QTL effects. Finer mapping of detected QTL, looking for tightly linked markers to the causative mutation, and comparative genomics are suggested to deepen in the analysis of QTL in turbot so they can be applied in marker assisted selection programs.

Microsatellite marker development in the protozoan parasite Perkinsus olseni.

The analysis of an enriched partial genomic library and of public expressed sequence tag (EST) resources allowed the characterization of the first microsatellite loci in the protozoan parasite Perkinsus olseni. Clonal cultures from laboratory isolates derived from infected clams Ruditapes decussatus (from Spain), R. philippinarum (from Spain and Japan), and Austrovenus stutchburyi (from New Zealand) were used for the characterization of 12 microsatellites. Low variation was detected at most loci, with the number of alleles at polymorphic loci ranging from 2 to 7 (average 3.20 +/- 0.51) and gene diversity from 0.11 to 0.79 (average 0.40 +/- 0.07). Preliminary results show that (1) isolates of P. olseni are diploid cells, and (2) multiple infections can occur within a single host. Eight of the loci analyzed successfully cross-amplified in the congeneric species P. mediterraneus. These microsatellite markers will be useful to analyze in detail the population genetic structure of P. olseni, crucial for the efficient management of this parasitic disease.

Blood Transcriptomics of Turbot Scophthalmus maximus: A Tool for Health Monitoring and Disease Studies.

Blood transcriptomics is emerging as a relevant tool to monitor the status of the immune system and assist in diagnosis, prognosis, treatment and pathogenesis studies of diseases. In fish pathology, the potential of transcriptome profiling of blood is still poorly explored. Here, RNA sequencing was applied to analyze the blood transcriptional profile of turbot (Scophthalmus maximus), the most important farmed flatfish. The study was conducted in healthy specimens and specimens parasitized by the myxozoan Enteromyxum scophthalmi, which causes one of the most devastating diseases in turbot aquaculture. The blood of healthy turbot showed a transcriptomic profile mainly related to erythrocyte gas transportation function, but also to antigen processing and presentation. In moderately infected turbot, the blood reflected a broad inhibition of the immune response. Particularly, down-regulation of the B cell receptor signaling pathway was shared with heavily parasitized fish, which showed larger transcriptomic changes, including the activation of the inflammatory response. Turbot response to enteromyxosis proved to be delayed, dysregulated and ineffective in stopping the infection. The study evinces that blood transcriptomics can contribute to a better understanding of the teleost immune system and serve as a reliable tool to investigate the physiopathological status of fish.

A multidisciplinary approach to identify priority areas for the monitoring of a vulnerable family of fishes in Spanish Marine National Parks.

BACKGROUND: Syngnathid fishes (Actinopterygii, Syngnathidae) are flagship species strongly associated with seaweed and seagrass habitats. Seahorses and pipefishes are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently Data Deficient according to the IUCN (2019), requiring more biological and ecological research. This study provides the first insights into syngnathid populations in the two marine Spanish National Parks (PNIA-Atlantic- and PNAC-Mediterranean). Fishes were collected periodically, marked, morphologically identified, analysed for size, weight, sex and sexual maturity, and sampled for stable isotope and genetic identification. Due the scarcity of previous information, habitat characteristics were also assessed in PNIA. RESULTS: Syngnathid diversity and abundance were low, with two species identified in PNIA (Hippocampus guttulatus and Syngnathus acus) and four in PNAC (S. abaster, S. acus, S. typhle and Nerophis maculatus). Syngnathids from both National Parks (NP) differed isotopically, with much lower δ15N in PNAC than in PNIA. The dominant species were S. abaster in PNAC and S. acus in PNIA. Syngnathids preferred less exposed sites in macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. The occurrence of very large specimens, the absence of small-medium sizes and the isotopic comparison with a nearby population suggest that the population of Syngnathus acus (the dominant syngnathid in PNIA) mainly comprised breeders that migrate seasonally. Mitochondrial cytochrome b sequence variants were detected for H. guttulatus, S. acus, and S. abaster, and a novel 16S rDNA haplotype was obtained in N. maculatus. Our data suggest the presence of a cryptic divergent mitochondrial lineage of Syngnathus abaster species in PNAC. CONCLUSIONS: This is the first multidisciplinary approach to the study of syngnathids in Spanish marine NPs. Habitat preferences and population characteristics in both NPs differed. Further studies are needed to assess the occurrence of a species complex for S. abaster, discarding potential misidentifications of genus Syngnathus in PNAC, and evaluate migratory events in PNIA. We propose several preferential sites in both NPs for future monitoring of syngnathid populations and some recommendations for their conservation.

A rapid and simple method for constructing stable mutants of Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. Study of mutant phenotypes is fundamental for understanding gene function. The methodologies developed to inactivate A. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double (or more) gene knockout mutant strains of A. baumannii. RESULTS: We describe here a rapid and simple method of obtaining A. baumannii mutants by gene replacement via double crossover recombination, by use of a PCR product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. To demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes (omp33, oxyR, and soxR) by this method. In addition, we disrupted one of these genes (omp33) by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining A. baumannii mutants. Comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after 10 passages in broth without selective pressure. Moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in A. baumannii. CONCLUSIONS: This study provides a rapid and simple method of obtaining stable mutants of A. baumannii free of foreign plasmidic DNA, which does not require cloning steps, and enables construction of multiple gene knockout mutants.

Population Genomics in Rhamdia quelen (Heptapteridae, Siluriformes) Reveals Deep Divergence and Adaptation in the Neotropical Region.

Rhamdia quelen, a Neotropical fish with hybridization between highly divergent mitochondrial DNA (mtDNA) lineages, represents an interesting evolutionary model. Previous studies suggested that there might be demographic differences between coastal lagoons and riverine environments, as well as divergent populations that could be reproductively isolated. Here, we investigated the genetic diversity pattern of this taxon in the Southern Neotropical Basin system that includes the La Plata Basin, Patos-Merin lagoon basin and the coastal lagoons draining to the SW Atlantic Ocean, through a population genomics approach using 2b-RAD-sequencing-derived single nucleotide polymorphisms (SNPs). The genomic scan identified selection footprints associated with divergence and suggested local adaptation environmental drivers. Two major genomic clusters latitudinally distributed in the Northern and Southern basins were identified, along with consistent signatures of divergent selection between them. Population structure based on the whole set of loci and on the presumptive neutral vs. adaptive loci showed deep genomic divergence between the two major clusters. Annotation of the most consistent SNPs under divergent selection revealed some interesting candidate genes for further functional studies. Moreover, signals of adaptation to a coastal lagoon environment mediated by purifying selection were found. These new insights provide a better understanding of the complex evolutionary history of R. quelen in the southernmost basin of the Neotropical region.

The Teleost Thymus in Health and Disease: New Insights from Transcriptomic and Histopathological Analyses of Turbot, Scophthalmus maximus.

The thymus is a primary lymphoid organ that plays a pivotal role in the adaptive immune system. The immunobiology of the thymus in fish is considered to be similar to that of mammals, but it is actually poorly characterized in several cultured teleost species. In particular, while investigations in human and veterinary medicine have highlighted that the thymus can be affected by different pathological conditions, little is known about its response during disease in fish. To better understand the role of the thymus under physiological and pathological conditions, we conducted a study in turbot (Scophthalmus maximus), a commercially valuable flatfish species, combining transcriptomic and histopathological analyses. The myxozoan parasite Enteromyxum scophthalmi, which represents a major challenge to turbot production, was used as a model of infection. The thymus tissues of healthy fish showed overrepresented functions related to its immunological role in T-cell development and maturation. Large differences were observed between the transcriptomes of control and severely infected fish. Evidence of inflammatory response, apoptosis modulation, and declined thymic function associated with loss of cellularity was revealed by both genomic and morphopathological analyses. This study presents the first description of the turbot thymus transcriptome and provides novel insights into the role of this organ in teleosts' immune responses.

New insights into the Manila clam - Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges.

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum-P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosis.