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A. Preparatory actions, elaboration of management plans and/or of action plans


ACTION A.1: Planning correction measures

Report with suggested corrective measures that could be implemented in the Alfacs and Fangar Bays in the Ebro Delta, on the Comunidad Valenciana coast, Mar Menor, French Mediterranean coastline, Italian coastline, and Greek coasts.

This will include the current situation of the species in the area and recommendations about specific actions that can improve the conservation of individuals in the long term. The regional administration and the responsible managers demand tools to help find solutions for the different pressures to which the population is subject. The transfer of knowledge and the development of actions for the conservation of the population are urgent


ACTION A.2: Location of optimum sites

The identification of optimal sites for conservation actions is crucial to achieve successful reintroductions/reinforcements in Pinna nobilis populations. Two scenarios for P. nobilis will be considered in this action: sanctuary areas (free of pathogens) and areas in the open Mediterranean sea (ideal for resistant individuals).

In the first case, based on updated knowledge, optimal sites will be explored considering environmental conditions that prevent the spread of the pathogen (e.g., salinity) but are compatible with the survival of P. nobilis (dissolved oxygen, temperature, additional impacts, etc.).

On the other hand, additional optimal sites will be prospected in the Mediterranean open sea, aiming to find suitable places to host P. nobilis-resistant individuals (leveraging previous knowledge of high densities, MPAs, etc.).

An additional synergy of this action, with broad implications at the local and European ecological levels, is that once the environmental extremes have been characterized, the identified optimal areas could be used for conservation projects involving other endangered species. The information will be made public, and there is a strong commitment within the project partners to apply the solutions developed not only during but also after the LIFE Pinnarca project. This way, the ecological impact of the project extends beyond the target species.

C. Conservation actions


ACTION C.1: Installation of larvae collectors

The objective of this action is to place larval collectors designed to collect fan mussel larvae that settle directly from the pelagic larval stage onto the mesh in the collectors. This process will indicate larval availability and potential recruitment in the benthos. When recruits are obtained, it will be possible to work with them in captivity maintenance (Action C7) and repopulation of study areas, which are free of the parasite and considered adequate for reintroduction (Action C4, D1).


ACTION C.2: Exhaustive shallow census

In areas where the die-off has already decimated populations, it is of utmost interest to search for possible survivors, who could be resistant to the disease. In the following actions (C4), these survivors will be aggregated in optimum sites with increased survival probabilities.

The partners will conduct a census of shallow (<30 m depth) areas to evaluate the prevalence of mortality episodes. Propulsion techniques, such as underwater scooters or aquaplanes, will be used along with real-time geo-referencing from a support boat.

ACTION C.3: Deep area census

Deep fan mussel populations live between 30 and 60 m depth in protected areas from anchoring or trawl fishing. These populations have seldom been monitored because limited resources have usually been directed to study shallow populations, yielding better outcomes for the invested effort. Furthermore, deep dives are shorter and multiply the necessity of safety procedures. 

The few deep populations studied are distributed in smaller areas and usually have lower densities than those located in shallower waters (densities of individuals usually peak between 10 and 15 m depth), altogether discouraging the development of systematic monitoring between 30 and 60 m depth. However, the higher the depth, the longer water temperatures are maintained low, and the lower the maximum temperatures reached in summer. It has been observed that temperatures above 14ºC increase the capacity of infection by H. pinnae. As a consequence, experts have indicated that the probability of finding alive individuals is higher in deep waters, which should be urgently explored in the search for fan mussels.

Specific methodologies adapted to the conditions of each area will be applied to census these deep populations by the partners of the consortium in the present proposal.


ACTION C.4: Translocation of individuals

Genetic diversity is fundamental for the maintenance of healthy populations, but the only viable fan mussel populations occur in reservoirs without connectivity among them (coastal lagoons and estuaries). These reservoirs are threatened by anthropic action and climate change. Before the MME, the species could follow metapopulation cycles of extinction and repopulation, but the lack of connectivity among them makes this impossible by natural means at present.

The slow decay of the populations without the possibility of natural repopulation in the following years and decades would extinguish all repositories in the medium term. We propose an alternative connectivity mechanism among populations through the translocation of individuals until new options (e.g., artificial reproduction of resistant individuals) are put into play. Additionally, some individuals could be reimplanted in areas optimal for the survival of fan mussels but that had not been colonized by the species before the occurrence of the MME, increasing the number of reservoirs.

In areas affected by H. pinnae, the few survivors are scattered hundreds of kilometers apart, preventing their reproduction. Finally, thousands of individuals live at risk in the reservoirs, and their longevity could be more than doubled just by translocating them to nearby safer areas.

ACTION C.5: Comparative genomincs

Samples of fan mussel tissues will be provided by most partners of the consortium and treated for subsequent DNA analyses by IOPR. This will involve tissues obtained with our safe method of sampling from extant P. nobilis individuals from different Mediterranean locations, the closely related and resistant Pinna rudis, and a few resistant hybrids of nobilis-rudis that have been detected since the epizootic event (Vasquez-luis et al., 2021).

Genotyping of P. nobilis individuals will provide insights into the genetic structure of populations for further conservation actions, such as ensuring the genetic diversity of the conserved animals. This action also aims to identify loci putatively involved in disease resistance by:

(1) comparing a group of healthy/resistant individuals with a group of a posteriori ill/dead individuals 

(2) comparing the genome of P. nobilis (Bunet et al., 2021) with the phylogenetically related pinnae P. rudis, known to be resistant to the parasite

Noteworthy, it will be necessary to (3) identify the presence of the pathogen prior to RAD sequencing by dissecting and analyzing dead tissues or detecting the presence of potential pathogens in healthy and morbid individuals following the protocols of Catanese et al., 2018 for Haplosporidium pinnae and Carella et al., 2018 for Mycobacterium sp.


ACTION C.6: Actions for environmental improvement in P. nobilis sanctuary areas

The objective of this action is to increase the survival and reduce the threats to fan mussel populations in the remaining sanctuaries where P. nobilis still survives. These sanctuaries occupy areas heavily impacted by anthropogenic factors such as pollution, fishing, recreational activities, etc. Different actions will be implemented in each country in the main sanctuaries observed. Once the effectiveness of one of these actions is proven for the recovery of fan mussel populations or the associated benthic habitat (based on associated monitoring actions), the information will be transferred to institutional managers and scientists across the species distribution range facing similar problems. This will allow for its implementation and replication on a large scale.

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ACTION C.7: Tank maintennce of healthy individuals

Two replicate actions with 20 individuals each will be carried out at the marine station of IMEDMAR-UCV and the installations of Murcia Aquarium. The individuals will be maintained throughout the entire project in closed circuits. Both institutions have experience in the maintenance of fan mussels in captivity. The objective of the action is to develop culture protocols to maximize the possibilities of captive reproduction while ensuring the long-term survival of stabilized individuals. Once reproduction in captivity is achieved, the same methodologies learned can be applied to the few resistant individuals to produce seed with a higher potential of being resistant to the disease. The seed could then be released into open waters to recover fan mussel populations. A common protocol will be used for the evaluation of growth rates and the maturation degree of individuals to establish the best possible diet for the captive maintenance of the species.

ACTION C.8: Treatment assays and analysis

The individuals will be analyzed for their tolerance to environmental variables and their response to the infection and treatment of the disease. This action will help save individuals from affected populations, increasing the pool of genetic variability. It has been observed that cold temperatures (<14ºC) slow down the infection and dispersion capacity of H. pinnae, and that extreme salinities (such as those observed in coastal lagoons and deltas) could prevent infection by the protozoan. Furthermore, some haplosporidians do not withstand extremely low temperatures, which eventually kill them. Treatments involving low temperatures and extreme salinities will be assayed in infected individuals to try to cure them in indoor facilities.

D. Monitoring of the impact of the project actions


ACTION D.1: Follow up of juvenile individuals

Translocated juveniles will be periodically monitored to control their survival. These juveniles could come from the artificial collectors (Action C1) or from areas within reservoirs that were inappropriate for long-term survival, such as the juveniles translocated in the Alfacs Bay (Action C4). The juveniles from collectors installed in reservoir areas will be implanted and followed in the same areas. Those from open waters, whose numbers are expected to be low a priori (<20/country and season), will be taken to indoor installations (Action C7) to control their survival, quarantine them, and use them for future indoor experimentation (Action C8).


ACTION D.2: Follow up of resistant individuals

Monitoring of adult individuals will be conducted monthly. Following mortality episodes, living individuals will be consistently monitored and studied. Resistance traits play a crucial role in minimizing the harm caused by disease, either by preventing infection or limiting subsequent pathogen growth and development within the host through avoidance or clearance of infection. In this task, each partner will use these animals for biopsies to assess the presence of pathogen load, with some specimens (defined in collaboration with the partners) sent to France for immunological studies. Italy, during this phase, will define the genetic variability of P. nobilis in the area covering the western Mediterranean and Adriatic Sea regions. Several resistant survival individuals have been identified at various sites along the coast of the Balearic Islands and will be continuously monitored. Potentially resistant individuals from the infected area of the Alfacs Bay (Ebro Delta) will be transplanted within a fenced area provided by the Coastal Administration.

ACTION D.3: Follow up of Mar Menor sanctuary population

The monitoring of permanent sampling plots will allow us to evaluate the population dynamics as well as the health level of the animals. Three fixed stations will be established in locations with high densities of Pinna nobilis. At each station, all individuals will be marked and measured. Non-invasive methods shall be used to measure the physiological state of the specimens, ensuring no harm to them. It is crucial to consider that, given the current high bacterial load of the lagoon, any action poses a high risk, and manipulation of animals causing stress or wounds could be fatal.

Twice a year, demographic measures (recruitment and mortality rates and population growth rates), health variables, description and quantification of the epiphytic community, position and orientation with respect to the substrate, and search for recruits will be carried out. Additionally, physicochemical variables will be collected, and photographs will be taken of each individual for later image analysis.

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ACTION D.4: Follow up of the effectiveness of sediment fences

Supervision of fence deployment at the Ebro Delta will be conducted by IRTA to ensure that fences are set in the desired place and have an adequate length. The surrounding area of the St. Joan Tower is regarded as an optimal place for this pilot study since the site is located between two discharge channels from rice field irrigation and naturally hosts a population of several hundred pen shells. Therefore, the improvement of habitat and water quality in this area will directly favor the condition status of these individuals and might enhance local recruitment.

Monthly monitoring will be conducted to determine the effectiveness of sediment fences and to address any possible damages. Assessment parameters for increased habitat quality will include an increase in the percent cover of vegetation, sediment accretion, physicochemical variables (salinity and temperature continuously monitored using a Hobo data logger, and oxygen levels, pH, and alkalinity at each sampling date using a multiparametric probe), and water nutrient concentrations compared to areas without sediment fences. The area will also be surveyed for potential recruitment and improvement of the vegetation cover and shoot density of the seagrass Cymodocea nodosa, the main local habitat for pen shell recruitment.

ACTION D.5: Monitoring and evaluation of the impact of the project actions

The conservation actions and the project as a whole aim to implement mitigation measures that prevent the total extinction of Pinna nobilis in the short to medium term. On the other hand, the monitoring and evaluation of the communication strategy (Action E) will allow documentation of the strategy's efficiency, enabling the implementation of necessary measures to achieve the proposed objectives in this aspect of the project. In this context, the evaluation of the impact of the communication actions has the final objective of measuring the effectiveness of the undertaken communication measures. Accordingly, Action D5 will periodically analyze the results achieved and produce periodic reports on them.

ACTION D.6: Development of a socioeconomic impact study

Development of a socioeconomic impact study to evaluate the achievement of the project's objectives and results in terms of the social perception of the project. This study aims to evaluate the cost-efficient replicability or transferability of the actions and results, along with the measures taken to ensure the actual replication or transfer of successful pilot/demonstration actions. This will make it possible to objectively analyze the impact of the project concerning the initial situation and the achievements in these aspects during the project's development.

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