Publications

  • Sheila Castellanos-Martínez, David Arteta, Susana Catarino, Camino Gestal
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    ABSTRACT: 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.
    PLoS ONE 10/2014; 9(10). DOI:10.1371/journal.pone.0107873 · 3.53 Impact Factor
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    Sheila Castellanos-Martínez, Angel P Diz, Paula Alvarez-Chaver, Camino Gestal
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    ABSTRACT: 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-frames translation of an octopus hemocytes 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. 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 molluscs; 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 needed of the diversification of reared species in order to ensure the growth of the aquaculture activity. Although cephalopods are model species for biomedical studies and posses 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' hemocytes 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.(1.)
    Journal of proteomics 06/2014; 105:151-163. DOI:10.1016/j.jprot.2013.12.008 · 3.93 Impact Factor
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    Eva Díaz-Freije, Camino Gestal, Sheila Castellanos-Martínez, Paloma Morán
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    ABSTRACT: 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.
    Frontiers in Physiology 02/2014; 5:62. DOI:10.3389/fphys.2014.00062
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    S Castellanos-Martínez, M Prado-Alvarez, A Lobo-da-Cunha, C Azevedo, C Gestal
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    ABSTRACT: The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm ± 1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm ± 0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.
    Developmental and comparative immunology 11/2013; DOI:10.1016/j.dci.2013.11.013 · 3.71 Impact Factor
  • Sheila Castellanos-Martínez, Camino Gestal
    Journal of Experimental Marine Biology and Ecology 09/2013; 447:14-22. DOI:10.1016/j.jembe.2013.02.007 · 2.48 Impact Factor
  • S. Castellanos-Martínez, C. Gestal
    Fish &amp Shellfish Immunology 06/2013; 34(6):1699. DOI:10.1016/j.fsi.2013.03.189 · 3.03 Impact Factor
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    S. Castellanos-Martínez, A.P. Diz, P. Álvarez-Chaver, C. Gestal
    Fish &amp Shellfish Immunology 06/2013; 34(6):1643. DOI:10.1016/j.fsi.2013.03.027 · 3.03 Impact Factor
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    Sheila Castellanos-Martínez, Marcos Pérez-Losada, Camino Gestal
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    ABSTRACT: 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. Published by Elsevier GmbH.
    European Journal of Protistology 03/2013; 49(3). DOI:10.1016/j.ejop.2012.11.005 · 2.34 Impact Factor
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    ABSTRACT: Indirect methods such as length–frequency analysis have not been recommended for growth estimation in octopus (length is not a good measure of size for soft-bodied organisms; size is not a good indicator of age because of the high interindividual variability in growth rates and an asymptotic growth is often assumed). However, these methods are still applied in various places where octopus fisheries exist because they are low cost and easy to apply in most fisheries where there are no financial resources or scientific capacity to use direct methods. The purpose of this study was to investigate whether length–frequency analysis is an appropriatemethod for determining the growth pattern of Octopus bimaculatus fromthe Gulf of California. We tested the widely used methods ELEFAN I and NSLCA, and a modal progression analysis with a multimodel approach. The results showed that the growth pattern of O. bimaculatus was reasonably described using these analyses. First, the suitability of using mantle length as a measure of size was confirmed through a significant length–weight relationship. A length–age key was also generated in terms of probability to take into account the variability in growth rates, and with the multimodel approach it was not necessary to assume asymptotic growth. Population size structure is the most readily obtained and probably the most commonly used or only available information in a large number of fisheries. Therefore, the usefulness of length–frequency analyses should not be underestimated when direct methods are available.
    Journal of Shellfish Research 12/2012; 31(4):1173-1181. DOI:10.2983/035.031.0428 · 1.10 Impact Factor
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    Sheila Castellanos-Martinez, M Carmen Gómez, F G Hochberg, Camino Gestal, Hidetaka Furuya
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    ABSTRACT: 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.
    Journal of Parasitology 04/2011; 97(2):265-9. DOI:10.1645/GE-2577.1 · 1.26 Impact Factor

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