The activities included in the project are:

A0: Communication Plan.
A0.1. Definition of the Communication Plan and development of the project’s dissemination actions. A communication strategy will be developed in the early stages of the project, which will include methods, protocols and tools for the communication of the results to the aquaculture sector, Administration and general audience.
A0.2. Organisation of specific workshops with the authorities to provide useful information on the management of resources.
A0.3. Publication of articles in the newsletter of the Galician Mussel Regulatory Council and distribution among producers.
A0.4. Informative meetings with the sector.
A0.5. Organisation of final meetings to disseminate the results and transmit knowledge of the project with the participation of entities from the sector and the general public.
A0.6. Publication of press releases and an informative video subtitled of the project showing its objectives, as well as the results obtained.

Activity A1. Identification of the repertoire of dispensable genes.

The CSIC publication on the mussel genome (Gerdol et al., 2020) constituted a new paradigm in the genomics of metazoans. Presence-absence variation (PAV) implies that mussels contain genes that are structural and basic to their biology ( core genes), which are present in all individuals, and “dispensable” genes, which may or may not be present. This characteristic seems to be associated with local adaptation and the great invasive capacity of mussels, as they are enriched in immune and stress response functions (Saco et al., 2023). However, the small number of sequenced individuals is a limitation to understanding the biological significance of this phenomenon.

The high variability in the genome of this species, of 38% of different genes between individuals, affects the genetic structure of populations, but the advantages of having or not having certain genes for their survival are still unknown. This activity will address PAV in mussel populations in five geographical locations. The results will give a picture of genetic variability and plasticity in the face of future threats.

A1.1. Sampling in the most important cultivation areas in Galicia (Ría de Arousa and Ría de Pontevedra) and in the other locations where different environmental conditions and anthropogenic pressures are recorded (Basque Country, Valencian Community and Andalusia).
A1.2. Mussel sequencing. Illumina DNA sequencing will be used to analyse the dispensable genes of mussels collected in Galicia, the Basque Country, the Valencian Community and Andalusia.
A1.3. Comparison of dispensable genes between different populations. This analysis has been established in the Immunology and Genomics research group and consists of the use of a reference genome and the mapping of sequences of the resequenced individuals to evaluate coverage values. This makes it possible to determine whether a gene is present or absent in the genome of each of the individuals analyzed.

Activity A2. Determination of the epigenetic signature.

Together with the genomic plasticity provided by the presence-absence of genes, epigenetic modifications offer a parallel pathway for adaptation to the environment. Environmental stress produces epigenetic variations that can change gene expression patterns and cause favorable or detrimental phenotypic variations. Epigenetic changes could provide a means of rapid adaptation.

A2.1. Evaluation of the epigenetic signatures of mussels from different populations. Nanopore RNA/DNA sequencing will be used to identify the methylated regions.
A2.2. Comparison of epigenetic marks between the different populations. Based on the different environmental conditions, the research team will study the differentially methylated regions (DMRs) between the populations.

Activity A3. Study of the baseline transcriptome.

RNA sequencing (RNA-seq) is one of the most useful technologies for understanding an organism’s response. The baseline transcriptomic profile of individuals from the different geographical locations studied in this project is unknown. This basal transcriptome constitutes the response of mussels to their usual conditions.

A3.1. Study of the basal transcriptome of mussels from different populations. RNA sequencing will be performed using the Illumina platform.
A3.2. Comparison of transcriptomes of all individuals. The reference genome will be used to carry out the RNA-seq and differential gene expression analyses. The specific routes modulated in each population will be analyzed by means of enrichment analysis.
A3.3. Metatranscriptomic analysis to identify microorganisms associated with each of the mussel populations. In this activity, the work protocols developed in the group on microbiome studies through metatranscriptomics analysis will be used (Rey-Campos et al., 2022).

Activity A4. Identification of the most resilient populations.

Once the baseline genetic, epigenetic and transcriptomic information is known, they will correlate it with resilience using biotic and abiotic stimuli. The objective is to establish whether some populations are more resilient or better adapted than others.

A4.1. Collection of mussel specimens in Galicia and in the community in which the most divergent genetic and transcriptomic information has been detected in activities 1, 2 and 3. The animals will acclimatize to laboratory conditions.
A4.2. Treatment of populations with an increase in temperature and combination with infection with the pathogen Vibrio splendidus.
A4.3. Evaluation of the mortality patterns of the two populations in the face of the different challenges.
A4.4. Transcriptome to evaluate the response after the double stimulus.

Activity A5. Project coordination.

A5.1. Coordination and monitoring meetings: The members of the project grouping will meet periodically (every six months) to analyse and discuss the appropriate development of the project.
A5.2. Technical and economic reports: Development of all the control and monitoring documents required by the financing entity.