MgC1q, a novel C1q-domain-containing protein involved in the immune response of Mytilus galloprovincialis

9 páginas, 6 figuras, 1 tablaIn this study, we present the characterization of a newly identified gene, MgC1q, revealed in suppression subtractive hybridization and cDNA libraries from immunostimulated mussels. Based on sequence homology, molecular architecture and domain similarity, this new C1q-domain-containing gene may be classified as a member of the C1q family and, therefore, part of the C1q–TNF superfamily. The expression of MgC1q was detected all along the mussel ontogeny, being detectable within 2 h post-fertilization, with a notable increase after 1 month and continuing to increase until 3 months. Measurable transcript levels were also evident in all analyzed tissues of naïve adult mussels, and the hemocytes showed the highest expression levels. Experimental infection of adult mussels with Gram positive or Gram negative bacteria significantly modulated the MgC1q expression, and confirmed it as an immune-related gene. Intra- and inter-individual sequence analyses revealed extraordinary diversity of MgC1q at both the DNA and cDNA levels. While further research is needed to define its function, our data indicate that MgC1q is a pattern recognition molecule able to recognize pathogens during innate immune responses in Myitilus galloprovincialis. The high sequence variability suggests that somatic diversification of these nonself recognition molecules could have occurred.This work has been funded by the EU Integrated Project FOOD-CT-2005-007103 and AGL2008-05111/ACU from the Spanish Ministerio de Ciencia e Innovación. Camino Gestal wishes to acknowledge additional funding from the Spanish Ministerio de Educación y Ciencia through the “Ramón y Cajal” Contract.Peer reviewe

Characterization of a birnavirus isolated from diseased turbot cultured in Spain

7 pages, 5 figures, 2 tables.During 1989, light but persistent mortalities were detected in a turbot Scophthalmus maximus L. farm in Galicia (northwestern Spain) and a virus with the characteristics of a birnavirus was isolated. The purpose of this study was to characterize the viral agent and determine the susceptibility of turbot to this virus. Electron microscopic examination revealed that the particles were isometric, hexagonal and unenveloped with an average diameter of 58 to 60 nm. The molecular weights of the RNA segments were 1.9 and 2.0 x 10(up to 6) daltons. The cells most susceptible to the turbot isolate were the CHSE-214, FHM and RTG-2 lines and the optimal temperature range for its replication was 15 to 2OºC. The RNA and polypeptide electropherotypes show that this virus resembles the Ab serotype of infectious pancreatic necrosis virus (IPNV); however, it differs in that it replicates in the FHM cell line and is not neutralized by antisera to the classical serotypes of IPNV. Infectivity trials conducted in turbot of dlfferent sizes indicated that the virus produced mortality only in small fish (2 g), although the larger fish (30 g) harbored the virus for at least 35 d. Fish inoculated with this isolate showed no pancreatic necrosis although necrosis of the hematopoietic elements of the kidney and spleen was detected.This work was supported by Grants MAR 89-0270 from the Comision Interministenal de Ciencia y Tecnologia (CICYT), and by XUGA 70708888 from Xunta de Galicia, Spain. Beatriz Novoa acknowledges the Ministerio de Educacion y Ciencia (Spain) for a research fellowship.Peer reviewe

Comparative analysis of the acute response of the trout, O. mykiss, head kidney to in vivo challenge with virulent and attenuated infectious hematopoietic necrosis virus and LPS-induced inflammation

Background: The response of the trout, O. mykiss, head kidney to bacterial lipopolysaccharide (LPS) or active and attenuated infectious hematopoietic necrosis virus (IHNV and attINHV respectively) intraperitoneal challenge, 24 and 72 hours post-injection, was investigated using a salmonid-specific cDNA microarray. Results: The head kidney response to i.p. LPS-induced inflammation in the first instance displays an initial stress reaction involving suppression of major cellular processes, including immune function, followed by a proliferative hematopoietic-type/biogenesis response 3 days after administration. The viral response at the early stage of infection highlights a suppression of hematopoietic and protein biosynthetic function and a stimulation of immune response. In fish infected with IHNV a loss of cellular function including signal transduction, cell cycle and transcriptional activity 72 hours after infection reflects the tissue-specific pathology of IHNV infection. attIHNV treatment on the other hand shows a similar pattern to native IHNV infection at 24 hours however at 72 hours a divergence from the viral response is seen and replace with a recovery response more similar to that observed for LPS is observed. Conclusion: In conclusion we have been able to identify and characterise by transcriptomic analysis two different types of responses to two distinct immune agents, a virus, IHNV and a bacterial cell wall component, LPS and a 'mixed' response to an attenuated IHNV. This type of analysis will lead to a greater understanding of the physiological response and the development of effective immune responses in salmonid fish to different pathogenic and pro-inflammatory agents

Study of diseases and the immune system of bivalves using molecular biology and genomics

24 páginas, 3 tablasEnvironmental chemico-physical factors, pathogens, and biological interactions constantly affect organism physiology and behavior. Invertebrates, including bivalve mollusks do not possess acquired immunity. Their defense mechanisms rely on an innate, non-adaptive immune system employing circulating cells and a large variety of molecular effectors. The mechanisms underlying host defense depend on the presence of functional proteins in appropriate quantities, within a crucial time window. These proteins are encoded by genes whose transcription is tightly coordinated by complex programs of gene expression. Currently available advanced techniques allow the evaluation of this gene expression, expanding our understanding of the behavior and function of cells and tissues under varying conditions. In particular, DNA microarray technology enables measurement of a large predetermined set of known genes or sequences. Expressed sequence tag sequencing from redundant, normalized, subtractive hybridization libraries is a robust method for sampling the protein encoding genes that are expressed within a tissue. The elimination of microorganisms by defense cells is a dynamic process that involves integrating synthesis of granule proteins during differentiation, migration onto sites of infection, phagocytosis and killing of microorganisms, modulation of their effector cells, and finally apoptosis. Understanding how this complex biological process is regulated can best be addressed using a systems biology approach to the study of organisms and populations in order to more effectively decipher the continuous challenge between two genomes, i.e., evolving host-pathogen interactionsPeer reviewe

The evolution and appearance of c3 duplications in fish originate an exclusive teleost c3 gene form with anti- inflammatory activity

12 páginas, 6 figuras, 3 tablas.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThe complement system acts as a first line of defense and promotes organism homeostasis by modulating the fates of diverse physiological processes. Multiple copies of component genes have been previously identified in fish, suggesting a key role for this system in aquatic organisms. Herein, we confirm the presence of three different previously reported complement c3 genes (c3.1, c3.2, c3.3) and identify five additional c3 genes (c3.4, c3.5, c3.6, c3.7, c3.8) in the zebrafish genome. Additionally, we evaluate the mRNA expression levels of the different c3 genes during ontogeny and in different tissues under steady-state and inflammatory conditions. Furthermore, while reconciling the phylogenetic tree with the fish species tree, we uncovered an event of c3 duplication common to all teleost fishes that gave rise to an exclusive c3 paralog (c3.7 and c3.8). These paralogs showed a distinct ability to regulate neutrophil migration in response to injury compared with the other c3 genes and may play a role in maintaining the balance between inflammatory and homeostatic processes in zebrafishThis work has been funded by the project CSD2007-00002 “Aquagenomics” from the Spanish Ministerio de Ciencia e Innovación, the ITN 289209 “FISHFORPHARMA” (EU) and project 201230E057 from the Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC).Peer reviewe

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

Mytilus galloprovincialis Myticin C: A Chemotactic Molecule with Antiviral Activity and Immunoregulatory Properties

Previous research has shown that an antimicrobial peptide (AMP) of the myticin class C (Myt C) is the most abundantly expressed gene in cDNA and suppressive subtractive hybridization (SSH) libraries after immune stimulation of mussel Mytilus galloprovincialis. However, to date, the expression pattern, the antimicrobial activities and the immunomodulatory properties of the Myt C peptide have not been determined. In contrast, it is known that Myt C mRNA presents an unusual and high level of polymorphism of unidentified biological significance. Therefore, to provide a better understanding of the features of this interesting molecule, we have investigated its function using four different cloned and expressed variants of Myt C cDNA and polyclonal anti-Myt C sera. The in vivo results suggest that this AMP, mainly present in hemocytes, could be acting as an immune system modulator molecule because its overexpression was able to alter the expression of mussel immune-related genes (as the antimicrobial peptides Myticin B and Mytilin B, the C1q domain-containing protein MgC1q, and lysozyme). Moreover, the in vitro results indicate that Myt C peptides have antimicrobial and chemotactic properties. Their recombinant expression in a fish cell line conferred protection against two different fish viruses (enveloped and non-enveloped). Cell extracts from Myt C expressing fish cells were also able to attract hemocytes. All together, these results suggest that Myt C should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases

New Insights into the Apoptotic Process in Mollusks: Characterization of Caspase Genes in Mytilus galloprovincialis

Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes’ tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature