An emergency situation for pen shells in the Mediterranean: The Adriatic Sea, one of the last Pinna nobilis shelters, is now affected by a mass mortality event.

We identified areas with high individual densities of the pen shell, Pinna nobilis, in two areas along the Croatian Adriatic coast. The surveys carried out in 2018 and 2019 showed population densities of approximately 9 to 13 individuals/100 m2. However, in 2019 a mass mortality event (MME) causing 36% to 100% mortality of this bivalve species was observed in the surveyed Croatian bays. The parasite Haplosporidium pinnae was identified by histological and molecular methods in all affected sampled individuals, while Mycobacterium sp. and Gram negative bacilli were detected in some affected and live bivalves. This finding constitutes the first record of these pathogens affecting P. nobilis in the middle Adriatic, confirming the continuous spread of the disease. Previously, the Adriatic water body was considered to be a natural shelter against the MME caused by pathogens in pen shell populations because of its distinct ecological features. The Adriatic Sea is a semi-closed water body with the largest continental shelf in the Mediterranean Sea, and due to its geomorphology and bathymetry, it is a sea with distinct characteristics. Monitoring plans and further studies in the Adriatic bays are now a priority for mitigating the high risk of extinction and working toward the conservation of this protected species.

Viral encephalopathy and retinopathy (VER) disease in Epinephelus marginatus from the Balearic Islands marine protected areas.

This is the first description of a betanodavirus infection in the dusky grouper Epinephelus marginatus within the marine protected areas (MPAs) of the Balearic Islands. Histopathology techniques were employed to describe neurological lesions in infected fish. Abnormal swimming, mortality, and neurological lesions were detected in all analysed grouper individuals. Virus particles were observed by means of transmission electron microscopy. Reverse transcription of RNA1 and RNA2 followed by cDNA amplification and sequencing allowed viral classification. Phylogenetic analysis showed the isolates from wild E. marginatus of the Balearic Islands MPAs to be closely related to Dicentrarchus labrax and Mullus barbatus strains from Cyprus and Italy. Although vertical transmission from infected spawners has been described as the major route for nodavirus infection, we point out in this work that horizontal transmission among sub-clinical fishes after migration or commercial import for aquaculture production could play a major role in the spreading of the disease in MPAs.

Haplosporidium pinnae sp. nov., a haplosporidan parasite associated with mass mortalities of the fan mussel, Pinna nobilis, in the Western Mediterranean Sea.

This study provides morphological and molecular characterization of a new species, Haplosporidium pinnae), very likely responsible for mass mortality of fan mussels, Pinna nobilis, in the Western Mediterranean Sea. The parasite was found in dead or moribund P. nobilis but did not occur in healthy fan mussels from locations that were not affected by abnormal mortality. Histological examination of infected fan mussels showed uninucleate cells of a haplosporidan parasite throughout the connective tissue and hemolymph sinuses of the visceral mass and binucleate cells and, rarely, multinucleate plasmodia were also detected in the connective tissue. Additionally, stages of sporulation occurred in the epithelium of the host digestive gland tubules. Spores were slightly ellipsoidal with a hinged operculum in one pole. Typical haplosporosomes were not found with TEM but vesicles with two concentric membranes resembling haplosporosomes were abundant in the cytoplasm of the multinucleate plasmodia occurring in host digestive gland tubules. SEM analysis showed multiple structures on the spore surface; some spores had two or four long tape-like filaments attached to the spore wall. Phylogenetic analysis based on the SSU rDNA sequence placed this parasite within a large clade including species of the order Haplosporida, not in the Bonamia/Minchinia subclade or the subclade containing most Haplosporidium species, but within a subclade of Haplosporidium sp. from Penaeus vannamei. Our results suggested that H. pinnae and the parasite of P. vannamei may represent a distinct new genus within the order Haplosporida.

Update of information on perkinsosis in NW Mediterranean coast: Identification of Perkinsus spp. (Protista) in new locations and hosts.

This study addressed perkinsosis in commercially important mollusc species in the western Mediterranean area. Perkinsus olseni was found in Santa Gilla Lagoon (Sardinia) infecting Ruditapes decussatus, Cerastoderma glaucum and Venerupis aurea, in Balearic Islands infecting Venus verrucosa and in Delta de l'Ebre (NE Spain) parasitising Ruditapes philippinarum and R. decussatus. Perkinsus mediterraneus was detected infecting Ostrea edulis from the Gulf of Manfredonia (SE Italy) and Alacant (E Spain), V. verrucosa and Arca noae from Balearic Islands and Chlamys varia from Balearic Islands, Alacant and Delta de l'Ebre.

Consumer beliefs regarding farmed versus wild fish.

Aquaculture is a food-producing activity, alternative to traditional extractive fishing, which still acts as a reference for most consumers. The main objective of the present paper was to study which consumer beliefs, regarding farmed versus wild fish, hinder the potential development of the aquaculture sector. To achieve this purpose the study was organized into two complementary steps: a qualitative approach (focus groups) aimed at assessing consumer perception about wild and farmed fish and to identify the salient beliefs that differentiate them; and a quantitative approach (survey by means of a questionnaire) to validate the results obtained in the focus group discussions over a representative sample of participants (n = 919). Results showed that participants perceive clear differences between farmed and wild fish. Although no significant differences between both kinds of fish were detected on safety, in general farmed fish was perceived to be less affected by marine pollution, heavy metals and parasites. In the contrary, wild fish was considered to have healthier feeding, to contain fewer antibiotics and to be fresher, healthier, less handled and more natural. Beliefs related to quality were in favour of wild fish, while those related to availability and price were in favour of farmed fish. Significant differences were observed in the perception of both kinds of fish depending on the consumers' objective knowledge about fish, on the level of education, age and gender and on the three segments of consumers identified: "Traditional/Conservative", "Connoisseur", "Open to aquaculture". The results provided could play an important role when planning and designing efficient marketing strategies for promoting farmed fish by adapting the information provided to the perception of each segment of consumers identified by the present study.

Internal transcribed spacer as effective molecular marker for the detection of natural hybridization between the bivalves Pinna nobilis and Pinna rudis.

The Pinna nobilis, a Mediterranean mollusc, has suffered population declines due to a massive mortality event associated with various factors including the parasite Haplosporidium pinnae. Some populations show resilience, possibly due to local environmental conditions. In this study, a molecular multiplex PCR method was developed using species-specific primers targeting Internal Transcribed Spacer (ITS) regions of P. nobilis and P. rudis, allowing accurate species identification and hybrid detection. Samples from Mediterranean areas were analysed, including putative hybrids and individuals from five other bivalve species. DNA was isolated, ITS regions were amplified and sequenced, and phylogenetic analyses confirmed species differentiation and primer specificity. The multiplex-PCR successfully identified P. nobilis, P. rudis, and their hybrids based on distinct amplicon patterns. This study highlights the value of molecular tools in species conservation, especially for monitoring and managing hybridization, supporting effective biodiversity conservation strategies.

Measuring activity-rest rhythms under different acclimation periods in a marine fish using automatic deep learning-based video tracking.

Most organisms synchronize to an approximately 24-hour (circadian) rhythm. This study introduces a novel deep learning-powered video tracking method to assess the stability, fragmentation, robustness and synchronization of activity rhythms in Xyrichtys novacula. Experimental X. novacula were distributed into three groups and monitored for synchronization to a 14/10 hours of light/dark to assess acclimation to laboratory conditions. Group GP7 acclimated for 1 week and was tested from days 7 to 14, GP14 acclimated for 14 days and was tested from days 14 to 21 and GP21 acclimated for 21 days and was tested from days 21 to 28. Telemetry data from individuals in the wild depicted their natural behavior. Wild fish displayed a robust and minimally fragmented rhythm, entrained to the natural photoperiod. Under laboratory conditions, differences in activity levels were observed between light and dark phases. However, no differences were observed in activity rhythm metrics among laboratory groups related to acclimation period. Notably, longer acclimation (GP14 and GP21) led to a larger proportion of individuals displaying rhythm synchronization with the imposed photoperiod. Our work introduces a novel approach for monitoring biological rhythms in laboratory conditions, employing a specifically engineered video tracking system based on deep learning, adaptable for other species.

Reproductive characteristics and gametogenic cycle of the scleractinian coral Dendrophyllia ramea.

The present study marks a pioneering investigation into the reproductive cycle of the scleractinian coral Dendrophyllia ramea. This is one of the first reproduction studies conducted in the Mediterranean Sea for a colonial azooxanthellate coral. Coral samples were collected in 2017 (May and October) and 2018 (February and July) in the Alborán Sea (SW Mediterranean). This location was selected due to its rarity as one of the few sites where this species thrives at depths shallower than 40 m. These samples were used to study the sexual patterns, fertilization mechanisms and gametogenic cycles by means of histological techniques. To broaden the scope, Sea Surface Temperature (SST) and Chlorophyll-a (Chl-a) data from open access databases have been considered to explore the potential influence of these environmental factors as triggers for gamete development and spawning time. The findings cast D. ramea as a gonochoric species, since no hermaphroditic specimens were observed among the analysed samples. Additionally, the lack of larvae and embryos in any of the analysed polyps, suggest that this species is fertilised externally. Two oocyte cohorts have been detected simultaneously, hinting at a yearly reproductive cycle, characterised by a prolonged oocyte maturation and seasonal spawning period taking place between August and October. Nevertheless, D. ramea display a low fecundity compared to other scleractinians inhabiting deep waters. Lastly, the early stages of gametogenesis seem to be coupled with the highest Chl-a values (i.e., March and December), whereas spawning takes place throughout the warmest period of the year (August to October).

Chronotypes-personality behavioural syndromes in wild marine fish.

Chronotypes, the individual differences in daily activity timing, have profound associations with numerous physiological processes. Despite this, the covariance between chronotypes and other aspects of an individual's behaviour has been infrequently explored in non-human animals. This study delves into individual's variation across four axes of personality in a controlled environment, utilising the pearly razorfish, a model species for fish chronotype studies. We identified behavioural types across the aggressiveness continuum and established behavioural syndromes amongst exploration, activity, and boldness, irrespective of body size and condition. Subsequent to this, the experimental subjects were reintroduced to their natural habitat and individually tracked using high-resolution technology to ascertain their chronotypes. Our results revealed that whilst the exploration-activity-boldness syndrome bore no correlation with chronotypes, a significant association was observed between aggressiveness and chronotype. Hence, individuals with later awakening times and rest onsets were more aggressive than their counterparts with earlier awakening times and rest onsets. This study provides pioneering evidence linking fish chronotypes with other behavioural traits, such as aggressiveness, suggesting that behavioural variation could be potentially linked to the individuals' variation in internal clocks and the environmental variables influencing their expression.

Disparate behavioral types in wild and reared juveniles of gilthead seabream.

Fish differ consistently in behavior within the same species and population, reflecting distinct behavioral types (BTs). Comparing the behavior of wild and reared individuals provides an excellent opportunity to delve into the ecological and evolutionary consequences of BTs. In this work, we evaluated the behavioral variation of wild and reared juvenile gilthead seabreams, Sparus aurata, a highly relevant species for aquaculture and fisheries. We quantified behavioral variation along the five major axes of fish behavioral traits (exploration-avoidance, aggressiveness, sociability, shyness-boldness, and activity) using standardized behavioral tests and a deep learning tracking algorithm for behavioral annotation. Results revealed significant repeatability in all five behavior traits, suggesting high consistency of individual behavioral variation across the different axes in this species. We found reared fish to be more aggressive, social and active compared to their wild conspecifics. Reared individuals also presented less variance in their aggressiveness, lacking very aggressive and very tame individuals. Phenotypic correlation decomposition between behavioral types revealed two different behavioral syndromes: exploration-sociability and exploration-activity. Our work establishes the first baseline of repeatability scores in wild and reared gilthead seabreams, providing novel insight into the behavior of this important commercial species with implications for fisheries and aquaculture.

Food determines ephemerous and non-stable gut microbiome communities in juvenile wild and farmed Mediterranean fish.

Novel insights were provided by contrasting the composition of wild and farmed fish gut microbiomes because the latter had essentially different environmental conditions from those in the wild. This was reflected in the gut microbiome of the wild Sparus aurata and Xyrichtys novacula studied here, which showed highly diverse microbial community structures, dominated by Proteobacteria, mostly related to an aerobic or microaerophilic metabolism, but with some common shared major species, such as Ralstonia sp. On the other hand, farmed non-fasted S. aurata individuals had a microbial structure that mirrored the microbial composition of their food source, which was most likely anaerobic, since several members of the genus Lactobacillus, probably revived from the feed and enriched in the gut, dominated the communities. The most striking observation was that after a short fasting period (86 h), farmed gilthead seabream almost lost their whole gut microbiome, and the resident community associated with the mucosa had a very much reduced diversity that was highly dominated by a single potentially aerobic species Micrococcus sp., closely related to M. flavus. The results pointed to the fact that, at least for the juvenile S. aurata studied, most of the microbes in the gut were transient and highly dependent on the feed source, and that only after fasting for at least 2 days could the resident microbiome in the intestinal mucosa be determined. Since an important role of this transient microbiome in relation to fish metabolism could not be discarded, the methodological approach needs to be well designed in order not to bias the results. The results have important implications for fish gut studies that could explain the diversity and occasional contradictory results published in relation to the stability of marine fish gut microbiomes, and might provide important information for feed formulation in the aquaculture industry.

Pinna nobilis in suboptimal environments are more tolerant to disease but more vulnerable to severe weather phenomena.

We examined a disease outbreak of the fan mussel, Pinna nobilis (L.), in the Alfacs Bay (South Ebro Delta, Spain) during a period of two years in three zones exposed to a summer salinity gradient resulting from agricultural freshwater discharges and distance to the open sea. Long-term monitoring was also conducted in Fangar Bay (North Ebro Delta), featuring lower salinities and no evidence of disease. Results showed that the salinity gradient of Alfacs Bay (37.4-35.7) was associated to cumulative mortality (100% near the mouth, 43% in middle regions, and 13% in inner regions), thus hindering the spread of pathogens. Young specimens showed to be more tolerant to disease than large adults but become vulnerable over time. In Fangar Bay, lower salinities (30.5-33.5) prevented the disease but individuals were highly vulnerable to Storm Gloria which caused 60% mortality in 3 weeks, and ~100% in 6 weeks.

Reduced Antioxidant Response of the Fan Mussel Pinna nobilis Related to the Presence of Haplosporidium pinnae.

The endemic fan mussel (Pinna nobilis) in the Mediterranean Sea is at high risk of disappearance due to massive mortality events. The aim of the study was to evaluate the antioxidant response of P. nobilis collected in the Balearic Islands (Western Mediterranean) before and after the mass mortality event. Individuals collected before (between 2011 and 2012) and after (between 2016 and 2017) the event were analyzed by histological, molecular, and biochemical methods to compare pathogenic loads and biochemical responses. All the individuals collected during 2016-2017 presented symptoms of the disease and were positive for Haplosporidium pinnae, while acid-fast bacteria or/and Gram-negative bacteria were detected in some individuals of both sampling periods. The activities of the antioxidant enzymes catalase and superoxide dismutase in the gills were significantly lower in P. nobilis affected with the parasite compared to those in the asymptomatic ones, while levels of malondialdehyde, as an indicator of lipid peroxidation, were higher in infected individuals. When analyzing the differential effects of H. pinnae and Mycobacterium sp. on P. nobilis, it was observed that significant effects on biomarkers were only observed in the presence of H. pinnae. Co-infection of P. nobilis by H. pinnae with other pathogens such as Mycobacterium sp. constitutes a serious problem due to its high mortality rate in the Balearic Island waters. This concerning situation for P. nobilis is favored by a reduction in antioxidant defenses related to H. pinnae infection that induces oxidative stress and cell damage.

Reversible morphological changes in a juvenile marine fish after exposure to predatory alarm cues.

Chemical cues from predators induce a range of predator-induced morphological defences (PIMDs) observed across fish taxa. However, the mechanisms, consistency, direction and adaptive value of PIMDs are still poorly studied. Here, we have tested if predatory cues can induce changes in the body shape of the juvenile marine fish Sparus aurata reared under controlled conditions without the presence of predators by exposing individuals to the olfactory stimulus of a fish predator. We tested our hypothesis using a nested replicated before-after-control-impact experiment, including recovery (potential reversibility) after the cessation of the predator stimulus. Differences in the size-independent body shape were explored using landmark-based geometric morphometrics and revealed that, on average, individuals exposed to a predatory cue presented deeper bodies and longer caudal regions, according to our adaptive theoretical predictions. These average plastic responses were reversible after withdrawal of the stimulus and individuals returned to average body shapes. We, therefore, provide evidence supporting innate reversible PIMDs in marine naive fish reared under controlled conditions. The effects at the individual level, including fitness and the associated applied implications, deserve further research.

Correction: Real-Time PCR based test for the early diagnosis of Haplosporidium pinnae affecting fan mussel Pinna nobilis.

[This corrects the article DOI: 10.1371/journal.pone.0212028.].

Real-Time PCR based test for the early diagnosis of Haplosporidium pinnae affecting fan mussel Pinna nobilis.

Noble pen shell or fan mussel, Pinna nobilis Linnaeus (1758), protected since 1992, was incorporated into the Spanish Catalogue of Threatened Species (Category: Vulnerable, Royal Decree 139/2011). The status is presently in the process of being catalogued as critically endangered, pending approval by Spanish Government (https://www.mapama.gob.es/es/biodiversidad/participacion-publica/Borrador_OM_situacion_critica.aspx). The International Union for the Conservation of Nature (IUCN) alerted the countries of the Mediterranean basin to the "emergent situation" due to serious mortality events suffered by the fan mussel, putting it in serious risk of extinction. Thus, emergency actions have been implemented by Spanish authorities in which several research institutes from all over the country are involved. The parasite, Haplosporidium pinnae, was recently characterized by histology, TEM, SEM and molecular biology techniques and it was considered responsible for the mass mortality of P. nobilis in the Mediterranean Sea. In this context, the aim of this study has been to develop species-specific quantitative PCR (qPCR) protocol carrying out a fast, specific and effective molecular diagnose of H. pinnae. In this sense, the detection limit for qPCR was equal to 30 copies of SSU rDNA / ng of DNA using plasmid alone and when 100ng DNA of non-infected oyster were added. The qPCR assay revealed that 94% of the 32 analysed mantle tissues of fan mussel were infected by H. pinnae, showing a high sensitivity and specificity for its detection (100% if we don't consider negative and too much degraded samples). This technique will allow us to make quicker follow-ups of the disease, allowing us to get a better understanding of its evolution in order to help in the rescue of P. nobilis populations.

Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain).

In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.

Comparing the catch composition, profitability and discard survival from different trammel net designs targeting common spiny lobster (Palinurus elephas) in a Mediterranean fishery.

In the Balearic Islands, different trammel net designs have been adopted to promote fisheries sustainability and reduce discards. Here, we compare the catch performance of three trammel net designs targeting the spiny lobster Palinurus elephas in terms of biomass, species composition and revenue from commercial catches and discards. Designs differ in the netting fiber type (standard polyfilament, PMF, or a new polyethylene multi-monofilament, MMF) and the use of a guarding net or greca, a mesh piece intended to reduce discards. Catches were surveyed by an on-board observer from 1,550 netting walls corresponding to 70 nets. The number of marketable species captured indicated that the lobster trammel net fishery has multiple targets, which contribute significantly to the total revenue. The discarded species ranged from habitat-forming species to elasmobranches, but the magnitude of gear-habitat interactions on the long term dynamics of benthos remains unclear. No relevant differences in revenue and weight of discards were detected after Bayesian analyses. However, the species composition of discards was different when using greca. Interestingly, high immediate survival was found for discarded undersized lobsters, while a seven day survival assessment, using captive observation, gave an asymptotic estimate of survival probability as 0.64 (95% CI [0.54-0.76]). Therefore, it is recommended that it would be beneficial for this stock if an exemption from the EU landing obligation regulation was sought for undersized lobsters in the Balearic trammel net fishery.

Anthropogenic chemical cues can alter the swimming behaviour of juvenile stages of a temperate fish.

Human pressure on coastal areas is affecting essential ecosystems including fish nursery habitats. Among these anthropogenic uses, the seasonal increment in the pressure due to leisure activities such as coastal tourism and yachting is an important environmental stressor in many coastal zones. These pressures may elicit understudied impacts due to, for example, sunscreens or other seasonal pollutants. The island of Majorca, northwest Mediterranean Sea, experiences one of the highest number of tourist visits per capita in the world, thus the surrounding coastal habitat is subject to high anthropogenic seasonal stress. Studies on early stages of fishes have observed responses to coastal chemical cues for the selection or avoidance of habitats. However, the potential interferences of human impacts on these signals are largely unknown. A choice chamber was used to determine water type preference and behaviour in naïve settled juvenile gilt-head sea bream (Sparus aurata), a temperate species of commercial interest. Fish were tested individually for behavioural changes with respect to water types from potential beneficial habitats, such as seawater with extract of the endemic seagrass Posidonia oceanica, anthropogenically influenced habitats such as water extracted from a commercial and recreational harbour and seawater mixed with sunscreen at concentrations observed in coastal waters. Using a Bayesian approach, we investigated a) water type preference; b) mean speed; and c) variance in the movement (as an indicator of burst swimming activity, or "sprint" behaviour) as behavioural descriptors with respect to water type. Fish spent similar percentage of time in treatment and control water types. However, movement descriptors showed that fish in sunscreen water moved slower (98.43% probability of being slower) and performed fewer sprints (90.1% probability of having less burst in speed) compared to control water. Less evident increases in sprints were observed in harbour water (73.56% more sprints), and seagrass (79.03% more) in comparison to control water. When seagrass water was tested against harbour water, the latter elicited a higher number of sprints (91.66% increase). We show that juvenile gilt-head seabream are able to react to a selection of naturally occurring chemically different odourscapes, including the increasingly important presence of sunscreen products, and provide a plausible interpretation of the observed behavioural patterns.