Search for new biomarkers of tolerance to Perkinsus olseni parasite infection in Ruditapes decussatus clams.

The grooved carpet shell (Ruditapes decussatus) is a clam species with high economic and social importance in several European and Mediterranean countries. Production of this species suffered a decline caused by biotic (parasite infection) and abiotic factors (environmental factors, stress, poor management methods and intensive culture of the introduced species Ruditapes philippinarum). The protozoan parasite Perkinsus olseni is also responsible for the decline of production, being nowadays one of the major issues for clam culture. Molecular biomarkers that might represent tolerance of R. decussatus to P. olseni have already been uncovered, shedding light in a possible production improvement by selecting those clams with a strongest immune response. In the present study, new tolerance biomarkers to P. olseni infection in R. decussatus were identified. The haemolymph proteomic profiles of naturally non/low-infected (tolerant) and highly-infected (susceptible) clams by the parasite across several heavy affected areas of Europe were characterized through a shotgun proteomics approach. Also, the mechanisms that might be involved in the responses against the disease in chronic infections were explored. Proteins related to energy restoration and balance, metabolic regulation, energy accumulation, ROS production, lysosomal activity, amino acid synthesis, proteolytic activity, iron regulation, iron withholding, and immune response modulation were significantly regulated in susceptible clams. In the tolerant group, proteins related to phagocytosis regulation, control of cell growth and proliferation, gonadal maturation, regulation of apoptosis, growth modulation, response to oxidative stress, iron regulation, shell development and metabolic regulation were significantly expressed. In summary, the protein expression profile of tolerant individuals suggests that an efficient pathogen elimination mechanism coupled to a better metabolic regulation leads to a tolerance to the parasite infection by limiting the spread through the tissues.

Evaluation of the Ruditapes decussatus immune response after differential injected doses of Perkinsus olseni.

Ruditapes decussatus is a highly appreciated clam species, with a high demand, especially in European countries. However, natural populations of this species have been affected by over-exploitation, abiotic factors and also by diseases mainly caused by bacteria, virus and protozoans. Within the latter group of pathogens, Perkinsus spp. has been attracting attention for causing relevant mortality rates on bivalves around the world. From all species of this genus, Perkinsus olseni is one of the most impactful for R. decussatus, as it may cause chronic infection with fatal consequences under adverse conditions. Therefore, in this study, naïve clams were injected with two doses of P. olseni in order to better understand the response of R. decussatus against this parasite. So, one of the main goals of this experiment was to evaluate the differential response of the clams at gene expression level after injection and also to monitor the possible mortalities related to parasite. Results indicated that the methods applied for injection and detection of parasite load are effective. Furthermore, altogether, R. decussatus gene expression studies indicate differences on expression of some of the addressed genes among distinct levels of infection over time, which require further studies to confirm if these genes could be used as molecular markers of P. olseni infection. According to the results obtained, Adiponectin-C1q seems to be a promising gene to be used as molecular marker of chronic infection in clams haemolymph.

Extracting protein from microalgae (Tetraselmis chuii) for proteome analysis.

Microalgae have high potential as a resource for sustainable and green protein for food or bioactive molecules. Nonetheless, despite the high protein content of microalgae (40 - 70% dry weight) progress in the characterization of their protein composition remains challenging. This is due to the highly variable chemical composition of microalgae strains and factors such as their rigid thick cell wall, polysaccharide content, protein stability, pH. The method described herein was developed to optimize protein extraction for proteome analysis of microalgae (Tetraselmis chuii) biomass. The effects on protein solubility of solvent type (organic, denaturing, and non-denaturing) combined with three customized microalgae disruption methods were investigated. The proteome targeted high quality protein extracts were for hydro-soluble proteins recovered by cell disruption using bead milling coupled to centrifugation (protein yield ≈ 13%). The developed method is inexpensive, efficient (yielding high-quality protein extracts with a low content of interfering compounds) and from an industrial perspective easily scalable and compatible with other applications. To add value to the end product we additionally propose the use of stabilizing agents to maintain protein solubility during refrigerated storage and a method targeting the fractionation of low molecular weight proteins. • An inexpensive easy-to-do 5 step protocol for microalgae protein extracts. • A protein extraction method free from dangerous or highly polluting chemicals. • Production of high yield aqueous protein extracts suitable for proteomics.

An index-based method for evaluating seismic retrofitting techniques. Application to a reinforced concrete primary school in Huelva.

A project named PERSISTAH (Projetos de Escolas Resilientes aos SISmos no Território do Algarve e de Huelva, in Portuguese) is being developed. It aims to cooperatively assess and improve the seismic vulnerability of primary schools in the Algarve (Portugal) and Huelva (Spain). A large number of schools have to be analysed. In order to determine which seismic retrofitting technique is optimal, an index-based method is presented in this paper. It considers three parameters: first, the efficiency of the seismic retrofitting technique in relation to the structural improvement obtained; second, the cost of the implementation of the retrofitting technique; and third, the architectural impact. It should be mentioned that a specific measurement for each solution according to its geometry has been performed. Also, coefficients to consider the singularities of each analysis and the importance of the parameters (number of buildings, typology, available funds, etc.) in the study are considered. The most representative primary school of Huelva has been chosen to test the index-based method. The most suitable retrofitting techniques for this type of buildings have been tested. The retrofitting technique which most increased the seismic performance has been the addition of X and V bracings within the building's bays. Furthermore, the analyses have revealed that adding the retrofitting elements in the most vulnerable direction of the building provides a high efficiency. The results have also shown that implementing techniques of lower architectural impact gives acceptable results. The analysis of the mean damage level index has shown that the building would experiment a severe damage. All the retrofitting techniques applied have reduced it, at least, up to moderate damage. Finally, it should be noted that the position of the retrofitting elements is also paramount for providing an optimal retrofitting.