Profiling of infection specific mRNA transcripts of the European seabass Dicentrarchus labrax

<p>Abstract</p> <p>Background</p> <p>The European seabass (<it>Dicentrarchus labrax</it>), one of the most extensively cultured species in European aquaculture productions, is, along with the gilthead sea bream (<it>Sparus aurata</it>), a prospective model species for the Perciformes which includes several other commercially important species. Massive mortalities may be caused by bacterial or viral infections in intensive aquaculture production. Revealing transcripts involved in immune response and studying their relative expression enhances the understanding of the immune response mechanism and consequently also the creation of vaccines. The analysis of expressed sequence tags (EST) is an efficient and easy approach for gene discovery, comparative genomics and for examining gene expression in specific tissues in a qualitative and quantitative way.</p> <p>Results</p> <p>Here we describe the construction, analysis and comparison of a total of ten cDNA libraries, six from different tissues infected with <it>V. anguillarum </it>(liver, spleen, head kidney, gill, peritoneal exudates and intestine) and four cDNA libraries from different tissues infected with Nodavirus (liver, spleen, head kidney and brain). In total 9605 sequences representing 3075 (32%) unique sequences (set of sequences obtained after clustering) were obtained and analysed. Among the sequences several immune-related proteins were identified for the first time in the order of Perciformes as well as in Teleostei.</p> <p>Conclusion</p> <p>The present study provides new information to the Gene Index of seabass. It gives a unigene set that will make a significant contribution to functional genomic studies and to studies of differential gene expression in relation to the immune system. In addition some of the potentially interesting genes identified by <it>in silico </it>analysis and confirmed by real-time PCR are putative biomarkers for bacterial and viral infections in fish.</p

Genomic Organization, Molecular Diversification, and Evolution of Antimicrobial Peptide Myticin-C Genes in the Mussel (Mytilus galloprovincialis)

Myticin-C is a highly variable antimicrobial peptide associated to immune response in Mediterranean mussel (Mytilus galloprovincialis). In this study, we tried to ascertain the genetic organization and the mechanisms underlying myticin-C variation and evolution of this gene family. We took advantage of the large intron size variation to find out the number of myticin-C genes. Using fragment analysis a maximum of four alleles was detected per individual at both introns in a large mussel sample suggesting a minimum of two myticin-C genes. The transmission pattern of size variants in two full-sib families was also used to ascertain the number of myticin-C genes underlying the variability observed. Results in both families were in accordance with two myticin-C genes organized in tandem. A more detailed analysis of myticin-C variation was carried out by sequencing a large sample of complementary (cDNA) and genomic DNA (gDNA) in 10 individuals. Two basic sequences were detected at most individuals and several sequences were constituted by combination of two different basic sequences, strongly suggesting somatic recombination or gene conversion. Slight within-basic sequence variation detected in all individuals was attributed to somatic mutation. Such mutations were more frequently at the C-terminal domain and mostly determined non-synonymous substitutions. The mature peptide domain showed the highest variation both in the whole cDNA and in the basic-sequence samples, which is in accordance with the pathogen recognition function associated to this domain. Although most tests suggested neutrality for myticin-C variation, evidence indicated positive selection in the mature peptide and C-terminal region. Three main highly supported clusters were observed when reconstructing phylogeny on basic sequences, meiotic recombination playing a relevant role on myticin-C evolution. This study demonstrates that mechanisms to generate molecular variation similar to that observed in vertebrates are also operating in molluscs

Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus

10 páginas, 7 figuras, 3 tablasDespite of the impact that viruses have on aquatic organisms, relatively little is known on how fish fight against these infections. In this work, the brain gene expression pattern of sea bream (Sparus aurata) in response to nodavirus infection was investigated. We used the supression subtractive hybridization (SSH) method to generate a subtracted cDNA library enriched with gene transcripts differentially expressed after 1 day post-infection. Some of the ESTs from the infected tissues fell in gene categories related to stress and immune responses. For the reverse library (ESTs expressed in controls compared with infected tissues) the most abundant transcripts were of ribosomal and mitochondrial nature. Several ESTs potentially induced by virus exposure were selected for in vivo expression studies. We observed a clear difference in expression between infected and control samples for two candidate genes, ubiquitin conjugating enzyme 7 interacting protein, which seems to play an important role in apoptosis and the interferon induced protein with helicase C domain 1 (mda-5) that contributes to apoptosis and regulates the type I IFN production, a key molecule of the antiviral innate response in most organismsThis research was partially supported by EU Projects (QLK2-CT-2002-01691, QLK5-CT-2002-51499) and Xunta de Galicia (PGIDIT02PXIC40205PM). LP-B is supported by a FPU fellowship from Ministerio de Educación y Ciencia, Spain.Peer reviewe

Nodavirus encephalopathy in turbot (Scophthalmus maximus): inflammation, nitric oxide production and effect of anti-inflammatory compounds [Poster]

8th International Symposium on Viruses of Lower Vertebrates, Santiago de Compostela (España), 26-29 abril 2010N

Nodavirus increases the expression of Mx and inflammatory cytokiness in fish brain

8 páginas, 1 tabla, 4 figurasNodavirus has become a serious pathogen for a wide range of cultured marine fish species. In the present work, the expression of genes related to immune and inflammatory responses of sea bream (Sparus aurata L.), considered as non susceptible species, was studied both in vitro and in vivo. No replication of the virus was observed in head kidney macrophages and blood leukocytes. Moreover, the enhancement of expression of several immune genes (tumor necrosis factor alpha (TNFα), interleukin-1-beta (IL-1β), interferon-induced Mx protein) was not detected in both head kidney macrophages and blood leucocytes in response to an in vitro infection with nodavirus. However, in vivo, nodavirus was detected 1 day post-infection (p.i.) by a reverse transcription-polymerase chain reaction (RT-PCR) in blood, liver, head kidney and brain of experimentally infected sea bream, while its presence clearly decreased in blood after 3 days p.i. Also, a transitory increment of the expression of TNFα and IL-1β was detected in the brain of intramuscular (i.m.) infected sea bream 3 days p.i. In head kidney, the over expression of TNFα was only observed 1 day p.i. The expression of Mx, an interferon induced gene, was increased in brain and head kidney of infected sea bream, reaching values of 1300-fold compared to controls in brain three days post-infection. For comparative purposes, we analyzed the expression of the same genes on a susceptible species, such as sea bass (Dicentrarchus labrax) and, although the same pattern of expression was observed both in brain and kidney, the magnitude was different mainly in the case of brain, the key organ of the infection, where higher expression of TNFα and lower expression of Mx compared with control was observedThis research was partially supported by the Commission of the European Communities QLRT-2002- 01691 and CT-2003-501984Peer reviewe

Effect of hexachlorocyclohexane isomers on some soil microbiological properties

[ES] Se estudió el efecto de la contaminación con altas cantidades de un residuo compuesto de isómeros de 1,2,3,4,5,6-hexaclorociclohexano (HCHs) (10, 50 y 100 g HCHs kg-1 suelo) sobre la densidad en el suelo de microorganismos heterótrofos, algas y cianobacterias fijadoras de N2 , sobre algunos microorganismos implicados en los ciclos de C y N (amonificantes, amilolíticos, y celulolíticos) así como sobre la capacidad de las comunidades microbianas edáficas para degradar varios sustratos carbonados (perfiles fisiológicos de las comunidades microbianas, CCLP). Estas propiedades microbianas se analizaron a las 2 semanas y los 2 y 4 meses de la aplicación del contaminante. También se estudió el efecto de la contaminación con HCHs aplicado a distintas concentraciones y en forma de partículas de distinto tamaño, sobre la respiración del suelo. A corto plazo tras la adición del contaminante al suelo, la densidad de los microorganismos heterótrofos aumentó, la abundancia de algas descendió y la población de cianobacterias no vario significativamente. A los 4 meses de la contaminación el efecto de las concentraciones de contaminante más altas (50 y 100 g HCHs kg-1 suelo) sobre los grupos microbianos mencionados fue negativo. El residuo de HCHs tuvo un efecto sobre la población amonificante similar a la observada en los microorganismos heterótrofos. Las densidades de amilolíticos y celulolíticos no se vieron afectadas o aumentaron, a corto plazo después de la adición de contaminante, aunque, a largo plazo (4 meses tras la contaminación) experimentaron una reducción con las concentraciones más altas de HCHs. A los dos meses de la contaminación, la capacidad de las comunidades microbianas del suelo para degradar sustratos carbonados (CCLP) se vio negativamente afectada por el residuo de HCHs. Cabe resaltar el descenso en la degradación de aminoácidos, que fue especialmente importante en los suelos con 50 y 100 g HCHs kg-1 suelo. El residuo de HCHs también afectó a la respiración del suelo, aunque el efecto dependió de la cantidad y tamaño de partícula del contaminante y de la presencia o ausencia de microorganismos procedentes de un emplazamiento contaminado con HCHs por largo tiempo (adaptados a la contaminación con HCHs). Cuando estuvieron presentes microorganismos adaptados a HCHs y el contaminante se aplicó en forma de partículas 4-10 mm, la respiración del suelo se vio estimulada con concentraciones entre 20 y 100 g HCHs kg-1 suelo e inhibida con concentraciones mayores de 200 g HCHs kg-1 suelo. Al aplicar el residuo de HCHs en forma de partículas < 1mm, el contaminante no estimuló la respiración y la redujo a concentraciones de 100 g HCHs kg-1 suelo o superiores. La adición de HCHs en forma de partículas de 4-10 mm en concentraciones de 100 g HCHs kg-1 suelo o superiores a un suelo con microorganismos no adaptados al contaminante redujo la respiración del suelo y con concentraciones menores no se modificó la cantidad de CO2 desprendida.[EN] The effect of heavy contamination with a residue composed of 1,2,3,4,5,6-hexachlorocyclohexane isomers (HCHs) (10, 50 and 100 g HCHs kg-1 soil) on the densities of cultivable soil heterotrophs, algae and N2 -fixing cyanobacteria, on soil microorganisms involved in the N and C cycles (ammonifiers, amylolytics and cellulolytics) as well as on the ability of soil microbial communities to degrade several C substrates (community level physiological profiles, CCLP) was studied. These microbial properties were analysed 2 weeks, and 2 and 4 months, after the soil was artificially contaminated. The effect of HCHs residue applied at different concentrations, and in the form of particles of different sizes on soil respiration was studied. The influence of the presence or absence of microorganisms from a long term polluted site on the effect of HCHs on soil respiration was also analysed. In the short term after the addition of the contaminant to the soil, the density of heterotrophs increased, the abundance of algae decreased, while the population of cyanobacteria did not change significantly. Four months after contamination the effect of the highest concentrations of HCHs (50 and 100 g HCHs kg-1 soil) on these microbial groups was negative. The HCH residue had a similar effect on the ammonifying population to that on heterotrophs. The densities of amylolytics and cellulolytics were either not modified, or were stimulated by the contaminant in the short term but, in the longer term (4 months after the contamination), were inhibited by HCHs applied at the highest concentrations. Two months after contamination, the ability of soil microbial communities to degrade C substrates (CCLP) was negatively affected by HCHs. It was noteworthy the decrease in the ability for degrading aminoacids, which was particularly important in the soils with 50 and 100 g HCHs kg-1 soil. HCHs also affected soil respiration but the effect depended on the HCH concentration, particle size of the contaminant, and on the presence or absence of microorganisms from a long term HCH-contaminated site (adapted to HCH contamination). When microorganisms adapted to HCH contamination were present and the contaminant was applied in form of particles of 4-10 mm, the soil respiration was stimulated by HCH at concentrations between 20 and 100 g HCH kg-1 and inhibited with concentrations higher than 200 g HCH kg-1 soil. If the HCHs were applied as particles < 1 mm, soil respiration was not stimulated by the contaminant and decreased with concentrations of 100 g HCH kg-1 or higher. The addition of HCHs in form of particles of 4-10 mm at concentration of 100 g HCH kg-1 or higher to a soil with microorganisms non-adapted to the contaminant reduced soil respiration, while lower concentrations of contaminant did not affect the amount of CO2 evolvedThe research was funded by the Consellería de Medio Ambiente and the Conselleria de Innovacion e Industria of the Xunta de Galicia (PGIDIT03PXIC40001P and INCITE08PXIB200136PR) and by the Ministerio de Ciencia y Tecnología and Ciencia e Innovación (REN2002- 04507-C02-02/ TECNO and CTM2009- 14576-C02-01)Peer reviewe

Suppression subtractive hybridization in head kidney of sea bass (Dicentrarhus labrax) infected with nodavirus allows the characterization of new lectin receptors

Poster.-- 7th International Symposium on Viruses of Lower Vertebrates, Oslo 22-25 April 2007Sea bass nervous necrosis virus is the causative agent of the viral nervous necrosis disease which causes high losses in the sea bass cultures in the world. In this work, gene expression of the head kidney of sea bass (Dicentrarchus labrax) in response to nodavirus infection was investigated. We used the Suppression Subtractive Hybridization (SSH) method to generate a subtracted cDNA library enriched with gene transcripts differentially expressed after 4 hours post infection. 206 ESTs were sequenced and classified in different categories. Among the genes related with the immune system, we focused on the lectin genes, in particular, two c-type lectins (one included in the group II and named SbCLA and other included in the group VII and named SbCLB) and one galectin (Sbgalectin-1) which were characterized for the first time in sea bass. Lectins are proteins that bind to specific carbohydrates structures and can be involved in numerous cellular processes including playing key roles in innate immunity. Expression studies in viva allowed us to confirm the SSH results, since these genes showed a significant increase after 4 hours postinfection. This increment was observed at 72 hours post-infection only for the SbCLB and Sbgalectin-1 genes. Our results suggest that the SSH strategy is a powerful approach to identify genes putatively involved in the nodavirus pathogenesis in sea bassN