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

Estudio de genes involucrados en procesos de muerte celular apoptótica y desarrollo larvario en mejillón (Mytilus galloprovincialis)

La acuicultura es una actividad de gran importancia socieconómica para las áreas costeras de muchos países. Ofrece oportunidades que promueven el empleo y actúan como motor de desarrollo contribuyendo al alivio de la pobreza, reduciendo la sobreexplotación de los recursos naturales costeros y mejorando la seguridad alimentaria. Sin embargo, la gran variedad de enfermedades infecciosas existentes en la actualidad que afectan a instalaciones de cultivos de todo el planeta, ponen en riesgo el crecimiento de la producción acuícola. En este sentido, la adopción de una estrategia basada en la prevención de enfermedades, más que un tratamiento terapéutico, es crucial para la expansión de una actividad acuícola sostenible y compatible con el medio ambiente. Asimismo, una estrategia de esas características es fundamental para lograr la obtención de productos acuícolas saludables al mismo tiempo que garantiza el bienestar de los animales. En el caso de la acuicultura de moluscos marinos, la actividad acuícola se desarrolla, normalmente, de manera extensiva en el propio medio natural en el que los organismos habitan; por lo que el empleo de antibióticos u otros tratamientos químicos se presenta como inviable, poniendo de manifiesto la absoluta necesidad de desarrollar programas eficientes de prevención de enfermedades para moluscos bivalvos. Estos animales son seres filtradores que viven en una gran variedad de ambientes diversos donde abundan otros organismos de naturaleza comensal, oportunística y patógena compuesta por endoparásitos como Mytilicola y Urastoma; protozoos como Bonamia, Haplosporidium, Marteilia o Perkinsus spp; virus como los herpes-virus o iridovirus y bacterias del género Nocardia o Vibrio, entre otras. Estos microorganismos pueden mermar las defensas inmunes de los bivalvos durante periodos críticos y producir episodios de mortalidades masivas que llevan asociados grandes pérdidas económicas. Algunos grupos de investigación vienen centrando sus esfuerzos, desde hace varios años, en la búsqueda de estrategias que ayuden a mejorar el estado inmunológico de los moluscos bivalvos frente a las enfermedades, estresantes ambientales o contaminantes de origen antropogénico pero desafortunadamente, el conocimiento actual sobre el funcionamiento del sistema inmune de estos organismos es limitado. El mejillón mediterráneo (Mytilus galloprovincialis) (Lamarck, 1819) a diferencia de otras 15especies de bivalvos de interés acuícola como la ostra o la almeja, no se ha visto implicado en eventos de mortalidades a gran escala. Se trata de una especie altamente resistente, capaz de adaptarse a diferentes ambientes y soportar factores externos de diversa índole siendo empleado, frecuentemente, como biomarcador de contaminación ambiental. La descripción de genes y proteínas que participan de manera activa en las respuestas inmunes, puede aportar información de gran interés acerca de los mecanismos implicados en la protección de los moluscos bivalvos, como el mejillón, frente a factores externos. Varios son los genes relacionados con la defensa inmune identificados en este molusco que presentan una gran similitud respecto a sus homólogos descritos en vertebrados pero, en general, el número de genes caracterizados en bivalvos, en comparación con otros grupos taxonómicos, es muy reducido. Uno de los procesos que ha sido escasamente investigado en moluscos bivalvos es la apoptosis. La muerte celular programada o apoptosis, consiste en la eliminación de células dañadas o infectadas a través de un proceso considerado vital para el desarrollo embrionario, sistema inmune y mantenimiento de la homeostasis de los tejidos. Se ha visto que fallos en el mecanismo apoptótico en humanos, conducen al desencadenamiento de enfermedades como el cáncer, Parkinson o Alzheimer. La red apoptótica en moluscos es compleja y presenta características que la convierten en un proceso único respecto a otros organismos. Éstas podrían estar relacionadas con el hecho de que se trate de organismos sedentarios que están continuamente expuestos a cambios ambientales y otros factores de diversa naturaleza. En este sentido, el mejillón mediterráneo es un invertebrado con potencial para ser empleado como modelo animal en el estudio de los mecanismos implicados en las estrategias de defensa, ya sea durante los estadíos más tempranos de desarrollo o en organismos en edad adulta. En el presente trabajo se recogen los resultados obtenidos durante el estudio de identificación y caracterización, tanto desde un punto de vista estructural como funcional, de quince nuevos genes que se expresan activamente a lo largo del desarrollo del mejillón, frente a patógenos, compuestos genotóxicos y estresantes ambientales como contaminantes o radiación ultravioleta

Estudio de genes involucrados en procesos de muerte celular apoptótica y desarrollo larvario en mejillón (Mytilus galloprovincialis)

243 páginasPeer reviewe

Genes of the mitochondrial apoptotic pathway in Mytilus galloprovincialis

13 páginas, 6 figuras, 2 tablasBivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress.This work was partially funded through the EU Projects BIVALIFE (266157) and REPROSEED (245119).Peer reviewe

Involvement of pore-forming molecules in immune defense and development of the Mediterranean mussel (Mytilus galloprovincialis)

15 páginas, 9 figuras, 3 tablasThe membrane attack complex and perforin (MACPF) superfamily is one of the largest families of poreforming molecules. Although MACPF proteins are able to destruct invading microbes, several MACPF proteins play roles in embryonic development, neural migration or tumor suppression. We describe two apextrin-like proteins (ApelB and ApelP) and one MACPF-domain-containing protein (Macp) in Mytilus galloprovincialis. The two apextrin-like proteins did not present any conserved domain. The Macp protein contained the membrane/attack complex domain and its signature motif. Gene expression during larval development was analyzed by RT-PCR. There was a stage-specific up-regulation of the three proteins, suggesting that they play a role in development. Apextrin-like proteins were highly expressed at blastula and trochophore stage, whereas Macp was expressed at veliger stage. RT-PCR revealed up-regulation of the three genes in tissues and hemocytes from adults treated with bacteria and pathogen-associated molecular patterns, suggesting that they may be involved in the immune response.This work has been funded by the EU Projects IMAQUANIM (CT- 2005-007103), REPROSEED (245119) and by the National project AGL2008-05111/ACU from the Spanish Ministerio de Ciencia e Innovación.Peer reviewe

A: Structural characterization of the Bcl-2 family proteins.

<p>The Bcl domain is boxed. The four BH regions and the transmembrane domain are indicated in gray. <b>B: </b><b>Structural characterization of the BI-1 protein.</b> The BI-1 domain is boxed, and the family signature is highlighted. The six transmembrane domains are indicated. Double ER membrane retention signals are highlighted in black.</p

A: Structural characterization of the p53 protein.

<p>The three domains of p53 are highlighted. The black triangles show the conserved phosphorylation sites. The four DNA-binding domains are boxed. The TETRAMER domain contains the NES. NLS I, NLS II and NLS III refer to the three nuclear localization signals. <b>B: Structural characterization of the PDRP protein.</b> Only a Prefoldin 2 domain is present. These domains are indicated in bold letters, and the location of this domain in <i>M. galloprovincialis</i> is highlighted.</p

Modulation of apoptosis induced through UV light in mussel hemocytes.

<p><b>A:</b> Quantification of the ROS production through the measurement of the mean fluorescence in granulocytes (R1 region) and hyalinocytes (R2 region) immediately after irradiation. The results are shown as the mean ± SD of eight biological samples <b>B</b>: Effects of antioxidant treatments (NAC and PDTC) before the induction of apoptosis. <b>C</b>: Analysis of apoptosis levels after treatments with CsA and ZVAD-FMK. The results in B and C are shown as the mean ± SD of twelve biological samples. (*) Significant differences compared with non-irradiated control cells. (a) Significant differences compared with UV-treated control cells.</p

A: Schema representing the major apoptotic pathways in mollusks.

<p>The blue symbols indicate the genes described in bivalves <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Campos1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Romero1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Tait1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Widlak1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Akaike1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Simon1" target="_blank">[65]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-ProiettiDeSantis1" target="_blank">[69]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Jernelov1" target="_blank">[84]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Fleischer1" target="_blank">[89]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061502#pone.0061502-Zhu1" target="_blank">[102]</a>. The white symbols indicate the available ESTs, and the black symbols indicate genes that have not been described in mollusks. The red lines indicate inhibition. <b>B</b>: M<b>orphological changes in mussel hemocytes after UV-induced apoptosis</b>. The nuclear morphology and chromatin condensation was evaluated using confocal microscopy at 3 and 24 h after irradiation. Scale bar 7.5 µm.</p