Camino Gestal

Anisakis infestation in marine fish and cephalopods from Galician waters: an updated perspective

Abstract. A total of 2,673 fresh specimens of cephalopod and fish representing 35 species were obtained from commercial local fisheries in Galician waters (NW Spain). They were examined for anisakid nematodes by digestion of the muscle and elution of the viscera and whole body cavity. All larval nematodes recovered were identified by light microscopy and multilocus electrophoresis as belonging to the species Anisakis simplex sensu stricto and A. pegreffii. Encysted larvae mostly occurred in the viscera but were also found in the flesh of squid and fish. Demographic values for larval nematodes are discussed in relation to host preferences and the ecological niche of both anisakid species at the sampling area. Primary recommendations are also expressed concerning the effects of current fishing and aquaculture practices on the Anisakis problem.

Characterization of a C3 and a factor B-like in the carpet-shell clam, Ruditapes decussatus.

The alternative pathway is considered to be the most ancient route for activation of the complement system. Herein, we report the characterization of C3 and factor B-like proteins in the clam Ruditapes decussatus, termed Rd-C3 and Rd-Bf-like. The Rd-C3 is a three-chain protein, similar to other protoC3 proteins, and the Rd-Bf-like is composed of two complement control protein modules (CCP domains) that differ from other described Bf proteins. The inoculation of clams with live bacteria did not result in induction of these functions, but inhibited the expression of Rd-C3 and Rd-Bf-like.

Study of Diseases and the Immune System of Bivalves Using Molecular Biology and Genomics

Environmental 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 interactions.

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

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.

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

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.

Differentially expressed genes of the carpet shell clam Ruditapes decussatus against Perkinsus olseni

Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a reverse subtraction were carried out to identify up- and down-regulated genes in both haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for further investigation into the functional basis of resistance to pathogens, have been detected for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of ESTs with potential similarity with known genes was 97, 42 among them were related with immunity and stress related functions. The pattern of expression of the immune selected genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams and compared with samples from an in vitro infection of clam haemocytes with Perkinsus zoospores. The maximum expression was found 1h post infection. The complete open reading frames of selected sequences (Rd adiponectin-C1q and Rd DAD-1) were determined. Our results provide new insights into the molecular basis of host-pathogen interactions in R. decussatus.

Parasites in commercially-exploited cephalopods (Mollusca, Cephalopoda) in Spain: an updated perspective

Abstract This paper represents an updated review of the published and unpublished records of parasites collected from cephalopod molluscs (teuthoid squids, cuttlefish and octopods) in waters off Spain, with special mention for those inhabiting coastal and shelf waters off Galicia (NW Spain). The examination of about 1600 cephalopods in the southeastern North Atlantic Ocean revealed a new parasite species Stellicola hochbergi , 37 new host records for Atlanto-Iberian waters, and 18 new host records for the world Ocean (three for coccidian Aggregatidae; three and one for cestodes Phyllobothriidae and Tentaculariidae; five and one for nematodes Anisakidae and Cystidicolidae; one and four for copepods Lichomolgidae and Pennellidae). Results suggests the important role of cephalopods as intermediate or final hosts in the life cycles of ten systematic groups of parasites.

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

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.

De Novo Transcriptome Sequencing of the Octopus vulgaris Hemocytes Using Illumina RNA-Seq Technology: Response to the Infection by the Gastrointestinal Parasite Aggregata octopiana

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.

What makes a cephalopod a suitable host for parasite? The case of Galician waters

Abstract We analyse the cephalopod–parasite systems taking into account the abiotic and biotic factors that may affect the host risk of infection (contact) with parasites. On the basis of 2000 individuals comprising 10 cephalopod species collected at a microgeographic area (Galician waters, NW Spain), an attempt is made to find associations between parasite relative species diversity (RSD) and cephalopod life cycle characteristics. Additionally, an index of similarity and the ratios of adult/immature (A/I) and specialist/generalist (S/G) regarding the parasite condition were used to define cephalopod ecological groupings at those species with similar risk of becoming infected with a given parasite fauna. Results show the existence of three ecological groupings (coastal, intermediate, and nerito-oceanic), which suggest that the ecological niche of a cephalopod species is more important in determining its risk of parasitic infection than is phylogeny.