Publications by authors named "Camino Gestal"

18 Publications

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First detection of OsHV-1 in the cephalopod Octopus vulgaris. Is the octopus a dead-end for OsHV-1?

J Invertebr Pathol 2021 Feb 14:107553. Epub 2021 Feb 14.

Marine Molecular Pathobiology Group, Marine Research Institute, Spanish National Research Council, Eduardo Cabello 6, 36208 Vigo, Spain. Electronic address:

The ostreid herpes virus (OsHV-1), associated with massive mortalities in the bivalve Crassostrea gigas, was detected for the first time in the cephalopod Octopus vulgaris. Wild adult animals from a natural breeding area in Spain showed an overall prevalence of detection of 87.5% between 2010 and 2015 suggesting an environmental source of viral material uptake. Overall positive PCR detections were significantly higher in adult animals (p = 0.031) compared to newly hatched paralarvae (62%). Prevalence in embryos reached 65%. Sequencing of positive amplicons revealed a match with the variant OsHV-1 µVar showing the genomic features that distinguish this variant in the ORF4. Gill tissues from adult animals were also processed for in situ hybridization and revealed positive labelling. Experimental exposure trials in octopus paralarvae were carried out by cohabitation with virus injected oysters and by immersion in viral suspension observing a significant decrease in paralarval survival in both experiments. An increase in the number of OsHV-1 positive animals was detected in dead paralarvae after cohabitation with virus injected oysters. No signs of viral replication were observed based on lack of viral gene expression or visualization of viral structures by transmission electron microscopy. The octopus response against OsHV-1 was evaluated by gene expression of previously reported transcripts involved in immune response in C. gigas suggesting that immune defences in octopus are also activated after exposure to OsHV-1.
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http://dx.doi.org/10.1016/j.jip.2021.107553DOI Listing
February 2021

Phylogenomics Identifies a New Major Subgroup of Apicomplexans, Marosporida class nov., with Extreme Apicoplast Genome Reduction.

Genome Biol Evol 2021 Feb;13(2)

Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada.

The phylum Apicomplexa consists largely of obligate animal parasites that include the causative agents of human diseases such as malaria. Apicomplexans have also emerged as models to study the evolution of nonphotosynthetic plastids, as they contain a relict chloroplast known as the apicoplast. The apicoplast offers important clues into how apicomplexan parasites evolved from free-living ancestors and can provide insights into reductive organelle evolution. Here, we sequenced the transcriptomes and apicoplast genomes of three deep-branching apicomplexans, Margolisiella islandica, Aggregata octopiana, and Merocystis kathae. Phylogenomic analyses show that these taxa, together with Rhytidocystis, form a new lineage of apicomplexans that is sister to the Coccidia and Hematozoa (the lineages including most medically significant taxa). Members of this clade retain plastid genomes and the canonical apicomplexan plastid metabolism. However, the apicoplast genomes of Margolisiella and Rhytidocystis are the most reduced of any apicoplast, are extremely GC-poor, and have even lost genes for the canonical plastidial RNA polymerase. This new lineage of apicomplexans, for which we propose the class Marosporida class nov., occupies a key intermediate position in the apicomplexan phylogeny, and adds a new complexity to the models of stepwise reductive evolution of genome structure and organelle function in these parasites.
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http://dx.doi.org/10.1093/gbe/evaa244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875001PMC
February 2021

Phylogenetic analysis of apicomplexan parasites infecting commercially valuable species from the North-East Atlantic reveals high levels of diversity and insights into the evolution of the group.

Parasit Vectors 2018 01 25;11(1):63. Epub 2018 Jan 25.

School of Biosciences, Cardiff University, Cardiff, CF10 3AX, UK.

Background: The Apicomplexa from aquatic environments are understudied relative to their terrestrial counterparts, and the seminal work assessing the phylogenetic relations of fish-infecting lineages is mostly based on freshwater hosts. The taxonomic uncertainty of some apicomplexan groups, such as the coccidia, is high and many genera were recently shown to be paraphyletic, questioning the value of strict morphological and ecological traits for parasite classification. Here, we surveyed the genetic diversity of the Apicomplexa in several commercially valuable vertebrates from the North-East Atlantic, including farmed fish.

Results: Most of the sequences retrieved were closely related to common fish coccidia of Eimeria, Goussia and Calyptospora. However, some lineages from the shark Scyliorhinus canicula were placed as sister taxa to the Isospora, Caryospora and Schellakia group. Additionally, others from Pagrus caeruleostictus and Solea senegalensis belonged to an unknown apicomplexan group previously found in the Caribbean Sea, where it was sequenced from the water column, corals, and fish. Four distinct parasite lineages were found infecting farmed Dicentrarchus labrax or Sparus aurata. One of the lineages from farmed D. labrax was also found infecting wild counterparts, and another was also recovered from farmed S. aurata and farm-associated Diplodus sargus.

Conclusions: Our results show that marine fish apicomplexans are diverse, and we highlight the need for a more extensive assessment of parasite diversity in this phylum. Additionally, parasites recovered from S. canicula were recovered as basal to their piscine counterparts reflecting hosts phylogeny.
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http://dx.doi.org/10.1186/s13071-018-2645-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785827PMC
January 2018

Morphological and Molecular Characterization of Aggregata spp. Frenzel 1885 (Apicomplexa: Aggregatidae) in Octopus vulgaris Cuvier 1797 (Mollusca: Cephalopoda) from Central Mediterranean.

Protist 2017 11 11;168(5):636-648. Epub 2017 Sep 11.

Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Napoli, Italy.

Coccidian parasites of the genus Aggregata are known to parasitize cephalopods as definitive hosts, however one of the genus members, A. octopiana, has shown an unresolved phylogeny within the same definitive host, the common octopus (Octopus vulgaris). Our study represents a large-scale investigation aimed at characterizing morphological traits and phylogeny of A. octopiana isolated from O. vulgaris inhabiting three distinct geographic areas of the central Mediterranean: The Adriatic, Ionian and Tyrrhenian Seas. The morphology of sporogonic stages of the parasite in octopus tissues was assessed by light and electron microscopy; molecular characterization has been carried out using the 18S rRNA locus. Our results support the hypothesis that two morphologically and genetically different A. octopiana infect O. vulgaris in the investigated areas of the Mediterranean Sea. Additional nuclear and mitochondrial markers for Aggregata should provide further information and better resolution of its phylogeny.
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http://dx.doi.org/10.1016/j.protis.2017.08.002DOI Listing
November 2017

Epigenetic DNA Methylation Mediating Early Development: Effect of Essential Fatty Acids Enriched Diet.

Front Physiol 2017 16;8:292. Epub 2017 May 16.

Aquatic Molecular Pathobiology Group, Instituto de Investigaciones Marinas (Consejo Superior de Investigaciones Científicas)Vigo, Spain.

The common octopus, , is a good candidate for aquaculture but a sustainable production is still unviable due to an almost total mortality during the paralarvae stage. DNA methylation regulates gene expression in the eukaryotic genome, and has been shown to exhibit plasticity throughout life cycle, changing profiles from paralarvae to adult stages. This pattern of methylation could be sensitive to small alterations in nutritional and environmental conditions during the species early development, thus impacting on its health, growth and survival. In this sense, a full understanding of the epigenetic mechanisms operating during development would contribute to optimizing the culture conditions for this species. Paralarvae of were cultured over 28 days post-hatching (dph) using two different sp. based diets: control and a long chain polyunsaturated fatty acids (LC-PUFA) enriched diet. The effect of the diets on the paralarvae DNA global methylation was analyzed by Methyl-Sensitive Amplification Polymorphism (MSAP) and global 5-methylcytosine enzyme-linked immunosorbent assay (ELISA) approaches. The analysis of different methylation states over the time revealed a global demethylation phenomena occurring along early development being directly driven by the age of the paralarvae. A gradual decline in methylated loci (hemimethylated, internal cytosine methylated, and hypermethylated) parallel to a progressive gain in non-methylated (NMT) loci toward the later sampling points was verified regardless of the diet provided and demonstrate a pre-established and well-defined demethylation program during its early development, involving a 20% of the MSAP loci. In addition, a differential behavior between diets was also observed at 20 dph, with a LC-PUFA supplementation effect over the methylation profiles. The present results show significant differences on the paralarvae methylation profiles during its development and a diet effect on these changes. It is characterized by a process of demethylation of the genome at the paralarvae stage and the influence of diet to favor this methylation loss.
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http://dx.doi.org/10.3389/fphys.2017.00292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432645PMC
May 2017

Guidelines for the Care and Welfare of Cephalopods in Research -A consensus based on an initiative by CephRes, FELASA and the Boyd Group.

Lab Anim 2015 Jul;49(2 Suppl):1-90

Division of Biomedical Sciences, St George's University of London, London, UK Association for Cephalopod Research 'CephRes', Italy.

This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of ∼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.
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http://dx.doi.org/10.1177/0023677215580006DOI Listing
July 2015

De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

PLoS One 2014 16;9(10):e107873. Epub 2014 Oct 16.

Departamento de Biotecnología y Acuicultura. Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Vigo, Spain.

Background: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis.

Results: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills.

Conclusion: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107873PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199593PMC
June 2015

The role of DNA methylation on Octopus vulgaris development and their perspectives.

Front Physiol 2014 24;5:62. Epub 2014 Feb 24.

Departamento de Bioquímica, Xenética e Inmunoloxía, Facultade de Bioloxía, Universidade de Vigo Vigo, Spain.

DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP), firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern changes with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since no significant effect was revealed in the analyses of molecular variance (AMOVA) performed. Our observations highlight the importance of epigenetic mechanisms in the first developmental steps of the common octopus and opens new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for aquaculture.
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http://dx.doi.org/10.3389/fphys.2014.00062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3932432PMC
March 2014

Cephalopods in neuroscience: regulations, research and the 3Rs.

Invert Neurosci 2014 Mar 3;14(1):13-36. Epub 2014 Jan 3.

Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy,

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.
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http://dx.doi.org/10.1007/s10158-013-0165-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938841PMC
March 2014

Proteomic characterization of the hemolymph of Octopus vulgaris infected by the protozoan parasite Aggregata octopiana.

J Proteomics 2014 Jun 23;105:151-63. Epub 2013 Dec 23.

Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Eduardo Cabello, 6, 36208 Vigo, Spain. Electronic address:

Unlabelled: The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The common octopus Octopus vulgaris has a high economic relevance and potential for aquaculture. However, disease outbreaks provoke serious reductions in production with potentially severe economic losses. In this study, a proteomic approach is used to analyze the immune response of O. vulgaris against the coccidia Aggregata octopiana, a gastrointestinal parasite which impairs the cephalopod nutritional status. The hemocytes and plasma proteomes were compared by 2-DE between sick and healthy octopus. The identities of 12 differentially expressed spots and other 27 spots without significant alteration from hemocytes, and 5 spots from plasma, were determined by mass spectrometry analysis aided by a six reading-frame translation of an octopus hemocyte RNA-seq database and also public databases. Principal component analysis pointed to 7 proteins from hemocytes as the major contributors to the overall difference between levels of infection and so could be considered as potential biomarkers. Particularly, filamin, fascin and peroxiredoxin are highlighted because of their implication in octopus immune defense activity. From the octopus plasma, hemocyanin was identified. This work represents a first step forward in order to characterize the protein profile of O. vulgaris hemolymph, providing important information for subsequent studies of the octopus immune system at molecular level and also to the understanding of the basis of octopus tolerance-resistance to A. octopiana.

Biological Significance: The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The study herein presented is focused to the comprehension of the octopus immune defense against a parasite infection. Particularly, it is centered in the host-parasite relationship developed between the octopus and the protozoan A. octopiana, which induces severe gastrointestinal injuries in octopus that produce a malabsorption syndrome. The common octopus is a commercially important species with a high potential for aquaculture in semi-open systems, and this pathology reduces the condition of the octopus populations on-growing in open-water systems resulting in important economical loses. This is the first proteomic approach developed on this host-parasite relationship, and therefore, the contribution of this work goes from i) ecological, since this particular relationship is tending to be established as a model of host-parasite interaction in natural populations; ii) evolutionary, due to the characterization of immune molecules that could contribute to understand the functioning of the immune defense in these highly evolved mollusks; and iii) to economical view. The results of this study provide an overview of the octopus hemolymph proteome. Furthermore, proteins influenced by the level of infection and implicated in the octopus cellular response are also showed. Consequently, a set of biomarkers for disease resistance is suggested for further research that could be valuable for the improvement of the octopus culture, taken into account their high economical value, the declining of landings and the need for the diversification of reared species in order to ensure the growth of the aquaculture activity. Although cephalopods are model species for biomedical studies and possess potential in aquaculture, their genomes have not been sequenced yet, which limits the application of genomic data to research important biological processes. Similarly, the octopus proteome, like other non-model organisms, is poorly represented in public databases. Most of the proteins were identified from an octopus' hemocyte RNA-seq database that we have performed, which will be the object of another manuscript in preparation. Therefore, the need to increase molecular data from non-model organisms is herein highlighted. Particularly, here is encouraged to expand the knowledge of the genomic of cephalopods in order to increase successful protein identifications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.
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http://dx.doi.org/10.1016/j.jprot.2013.12.008DOI Listing
June 2014

Molecular phylogenetic analysis of the coccidian cephalopod parasites Aggregata octopiana and Aggregata eberthi (Apicomplexa: Aggregatidae) from the NE Atlantic coast using 18S rRNA sequences.

Eur J Protistol 2013 Aug 13;49(3):373-80. Epub 2013 Mar 13.

Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Eduardo Cabello 6, 36208 Vigo, Spain.

The coccidia genus Aggregata is responsible for intestinal coccidiosis in wild and cultivated cephalopods. Two coccidia species, Aggregata octopiana, (infecting the common octopus Octopus vulgaris), and A. eberthi, (infecting the cuttlefish Sepia officinalis), are identified in European waters. Extensive investigation of their morphology resulted in a redescription of A. octopiana in octopuses from the NE Atlantic Coast (NW Spain) thus clarifying confusing descriptions recorded in the past. The present study sequenced the 18S rRNA gene in A. octopiana and A. eberthi from the NE Atlantic coast in order to assess their taxonomic and phylogenetic status. Phylogenetic analyses revealed conspecific genetic differences (2.5%) in 18S rRNA sequences between A. eberthi from the Ria of Vigo (NW Spain) and the Adriatic Sea. Larger congeneric differences (15.9%) were observed between A. octopiana samples from the same two areas, which suggest the existence of two species. Based on previous morphological evidence, host specificity data, and new molecular phylogenetic analyses, we suggest that A. octopiana from the Ria of Vigo is the valid type species.
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http://dx.doi.org/10.1016/j.ejop.2012.11.005DOI Listing
August 2013

A new dicyemid from Octopus hubbsorum (Mollusca:Cephalopoda:Octopoda).

J Parasitol 2011 Apr 21;97(2):265-9. Epub 2010 Oct 21.

Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain.

A new species of dicyemid mesozoan is described from Octopus hubbsorum Berry, 1953, collected in the south of Bahia de La Paz, Baja California Sur, México. Dicyema guaycurense n. sp. is a medium-size species that reaches about 1,600 µm in length. It occurs in folds of the renal appendages. The vermiform stages are characterized as having 22 peripheral cells, a conical calotte, and an axial cell that extends to the base of the propolar cells. Infusoriform embryos consist of 39 cells; 1 nucleus is present in each urn cell and the refringent bodies are solid. This is the first of a dicyemid species from a host collected in the Gulf of California.
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http://dx.doi.org/10.1645/GE-2577.1DOI Listing
April 2011

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

Dev Comp Immunol 2010 Sep 6;34(9):926-34. Epub 2010 Mar 6.

Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain.

In 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 2h 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.
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http://dx.doi.org/10.1016/j.dci.2010.02.012DOI Listing
September 2010

Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection.

Fish Shellfish Immunol 2010 Apr 1;28(4):640-8. Epub 2010 Jan 1.

Ecosystèmes Lagunaires, UMR 5119, Université de Montpellier 2-CNRS, cc093, place E. Bataillon, F-34095 Montpellier cedex 05, France.

Mussels live in diverse coastal environments experience various physical, chemical and biological conditions, which they counteract with functional adjustments and heritable adaptive changes. In order to investigate possible differences in immune system capabilities, we analyzed by qPCR the expression levels of 4 immune genes (defensin, mytilin B, myticin B, lysozyme) and HSP70 in the Mediterranean mussel, Mytilus galloprovincialis collected in 3 European farming areas {Atlantic Ocean-Ría de Vigo-Spain (RV), French Mediterranean Gulf of Lion-Palavas-Prévost lagoon (PP) and Northern Adriatic Sea-Venice-Italy (VI)} in response to one injection of one of the 3 bacterial species (Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus), and to heat shock or cold stress. We confirmed that the 5 genes are constitutively expressed in hemocytes, defensin being the less expressed, myticin B the highest. As suspected, the same gene resulted differently expressed according to mussel group, with the biggest difference being for HSP70 and lysozyme and lowest expression of all the 5 genes in mussels from RV. In addition, gene expression levels varied according to the challenge. Most frequent effect of bacterial injections was down-regulation, especially for mytilin B and myticin B. Heat shock enhanced transcript levels, particularly in mussels from RV, whereas cold stress had no effect. In situ hybridization of labelled probes on mussel hemocytes indicated that bacterial injections did not change the mRNA patterns of defensin and myticin B whereas mytilin B mRNA almost disappeared. In conclusion, these results demonstrated that constitutive level, nature and intensity of immune gene expression regulations strongly depended from mussel group, and support the concept of gene-environment interactions.
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http://dx.doi.org/10.1016/j.fsi.2009.12.022DOI Listing
April 2010

Analysis of differentially expressed genes in response to bacterial stimulation in hemocytes of the carpet-shell clam Ruditapes decussatus: identification of new antimicrobial peptides.

Gene 2007 Dec 15;406(1-2):134-43. Epub 2007 Aug 15.

Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Eduardo Cabello 6, Vigo, Spain.

Suppression subtractive hybridization was used to identify differentially expressed genes in hemocytes from carpet-shell clam Ruditapes decussatus stimulated with a mixture of dead bacterial strains. Putative function could be assigned to 100 of the 253 sequenced cDNAs. Based on sequence homologies, 3.16% of the total identified genes were possibly related to immune functions. Clam myticin isoforms 1, 2 and 3, and clam mytilin, with similarity with myticins and mytilins previously reported on Mytilus galloprovincialis were identified and characterized for the first time in clams. The analysis of their expression levels by quantitative PCR showed that they were induced by bacterial challenge. The results obtained in this work could be the first step leading to the understanding of molecular mechanisms by which these economically important marine bivalves respond to pathogens.
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http://dx.doi.org/10.1016/j.gene.2007.07.030DOI Listing
December 2007

High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host-pathogen interactions.

Dev Comp Immunol 2008 26;32(3):213-26. Epub 2007 Jun 26.

Department of Biology, University of Trieste, Trieste, Italy.

Small cationic antimicrobial peptides (AMPs) are host defense molecules detected in virtually all groups of organisms. To investigate the immune response mechanisms of Mytilus galloprovincialis, primary and suppression subtractive hybridization libraries were prepared from hemolymph of mussels injected with heat-inactivated bacteria or poly I:C, the latter mimicking viral infection. After DNA sequencing, sequence processing and similarity searching, a remarkable abundance of AMP mRNAs were identified. In detail, 25.9% and 32.4% AMP sequences from mussels infected with bacteria and 43.4% and 40.6% from mussels stimulated with poly I:C were detected by selective amplification of 180 differentially expressed genes and random sequencing of 967 cDNA clones, respectively. The 232 ESTs matching with myticin A and B (Mytilus spp.) displayed considerable sequence variability and revealed a third cluster proposed here as myticin C. Phenetic analysis of the translated myticin ESTs yielded 74 and 25 variants of the precursor and active peptide, respectively, and confirmed the high polymorphism of the new form. Myticin C shows typical features of the CSalphabeta AMP family (eight-cysteine array and secretory signal peptide) as well as amino acid variation, mainly in the anionic C-terminal region. The sequencing of one intronic region from genomic DNA, allowed us to detect 13 variants in 9 individual mussels referring them to one gene only. In addition to hemolymph, myticin C transcripts were detected in various mussel tissues, oocytes and early larval stages. The striking sequence variability and expression levels of myticins in mussels confirm the fundamental role of these natural antibiotics in the ancient host-pathogen interplay of mutual inhibition, evasion and adaptation strategies.
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http://dx.doi.org/10.1016/j.dci.2007.05.008DOI Listing
April 2008

Perkinsoide chabelardi n. gen., a protozoan parasite with an intermediate evolutionary position: possible cause of the decrease of sardine fisheries?

Environ Microbiol 2006 Jun;8(6):1105-14

Department of Cell Biology, Institute of Biomedical Sciences, ICBAS, University of Porto, Largo Prof. Abel Salazar no 2, 4099-003 Porto, Portugal.

Phenotypic scrutiny on the life cycle of Icthyodinium chabelardi (Perkinsoide chabelardi n. gen.) based on ultrastructural techniques, and molecular phylogenetic analysis of RNA gene sequences, were carried out in order to elucidate the taxonomic position of this parasite. The absence of plastid, presence of trichocysts, and chromosomes or chromatin condensed and low in number, suggested that this protozoan could be considered a dinoflagellate syndinial parasite. However, the life cycle, schizogonic divisions and structure of schizonts inside the host, the nuclei without the typical dinoflagellate appearance, presence of rhoptrias-like structures, a possible pseudo-conoid, and the biflagellated spore, resembled those of the genus Perkinsus. Phylogenetic analysis of genes transcribing for the RNA forming the small subunit and the large subunit suggests that this parasite has an ambiguous evolutionary position within the group formed by dinoflagellates, perkinsids and syndinials. Because of differences with dinoflagellates and similarities with perkinsids, we propose to change the generic name to P. chabelardi n. gen. High stationary infection prevalence on Sardina pilchardus eggs was observed. This protozoan parasite caused the death of all the infected sardine eggs, and therefore a high impact in the recruitment of this fishery in the Atlantic coast is expected.
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http://dx.doi.org/10.1111/j.1462-2920.2006.01008.xDOI Listing
June 2006

Ultrastructural and molecular characterization of Haplosporidium montforti n. sp., parasite of the European abalone Haliotis tuberculata.

J Invertebr Pathol 2006 May 24;92(1):23-32. Epub 2006 Mar 24.

Department of Cell Biology, Institute of Biomedical Sciences (ICBAS/UP) and Laboratory of Protoparasitology, CIIMAR/UP, University of Porto, Portugal.

A new member of the parasitic phylum Haplosporidia, which was found infecting the connective tissue, gill, digestive gland, and foot muscle of Haliotis tuberculata imported from Ireland and experimentally grown in Galicia (NW Spain), is described. Scanning electron microscopy, transmission electron microscopy, and molecular characterization of the small subunit ribosomal RNA (SSU rRNA) gene were carried out to confirm the description of this species. The ultrastructural morphology of the spores and their surrounding ornaments attached to the spore wall was described from light, scanning, and transmission electron microscopy observations. Systemic infection with uninucleated and multinucleated plasmodia containing spherical nuclei was observed among several sporocysts containing the different spore maturation stages. The spores were spherical to slightly ellipsoidal (2.42 +/- 0.5 x 2.31 +/- 0.6 microm). The apical zone of the spore wall was modified into a complex opercular system covering a circular orifice that measured about 0.5 microm in diameter. The operculum was connected to the spore wall by a hinge. The spore wall was about 110 nm thick, with 4 filaments (20-28 microm long). The filaments were composed of the same material that formed the wall. The cross-sections through the base of these filaments showed T-like and X-like sections. Internally, the uninucleated endosporoplasm contained typical haplosporidian structures, such as, haplosporosomes, a spherulosome, and mitochondria with vesicular cristae. The SSU rRNA gene sequence was different from previously reported haplosporidian SSU rRNA gene sequences, corroborating morphological data that this was an undescribed species. Based on differences from previously described haplosporidians in ultrastructural characteristics of the spore and SSU rRNA gene sequence, we describe the abalone haplosporidian as Haplosporidium montforti n. sp.
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http://dx.doi.org/10.1016/j.jip.2006.02.002DOI Listing
May 2006