Sanctuaries are not inviolable: Haplosporidium pinnae as responsible for the collapse of the Pinna nobilis population in Lake Faro (central Mediterranean).

The rapid spread of the protozoan Haplosporidium pinnae is having a strong negative effect on Pinna nobilis populations. A case study on a residual population in Lake Faro (Sicily, Central Mediterranean), whose long-term monitoring has revealed a dramatic decline following the 2018-2020 mass mortality event, is presented. In the framework of such monitoring, we performed tissue sampling on nine living P. nobilis, detecting the pathogen in seven of them. In contrast, other pathogens associated with P. nobilis disease in other areas, i.e., Mycobacterium spp. and Vibrio mediterranei, were not recorded. The surviving individuals (approximately twenty) showed that brackish areas only weakly mitigate the effects of H. pinnae disease and might not be resolutive. Nevertheless, the results show that Lake Faro may constitute one of the last Mediterranean P. nobilis sanctuaries.

Haplosporidium pinnae Parasite Detection in Seawater Samples.

In this study, we investigated the presence of the parasite Haplosporidium pinnae, which is a pathogen for the bivalve Pinna nobilis, in water samples from different environments. Fifteen mantle samples of P. nobilis infected by H. pinnae were used to characterize the ribosomal unit of this parasite. The obtained sequences were employed to develop a method for eDNA detection of H. pinnae. We collected 56 water samples (from aquaria, open sea and sanctuaries) for testing the methodology. In this work, we developed three different PCRs generating amplicons of different lengths to determine the level of degradation of the DNA, since the status of H. pinnae in water and, therefore, its infectious capacity are unknown. The results showed the ability of the method to detect H. pinnae in sea waters from different areas persistent in the environment but with different degrees of DNA fragmentation. This developed method offers a new tool for preventive analysis for monitoring areas and to better understand the life cycle and the spread of this parasite.

DNA metabarcoding for identification of species used in fish burgers.

The absence of morphological identification characters, together with the complexity of the fish supply chain make processed seafood vulnerable to cases of species substitution. Therefore, the authentication and the traceability of such products play a strategic role in ensuring quality and safety. The aim of the present study was to detect species used in the production of multi-species fish burgers and to evaluate mislabelling rates, using a DNA metabarcoding approach by sequencing a fragment of the 16S rRNA mitochondrial gene. The study highlighted the presence of 16 marine and 2 mammalian taxa with an overall mislabelling rate of 80%, including cases of species substitution, the undeclared presence of molluscs and of taxa whose use is not permitted by current Italian legislation. The presence of swine DNA as well as the inclusion of undeclared taxa potentially causing allergies raise concerns regarding consumer safety and protection regarding ethical or religious issues. Overall, the study shows that the application of DNA metabarcoding is a promising approach for successfully enforcing traceability systems targeting multi-species processed food and for supporting control activities, as a guarantee of an innovative food safety management system.

The Nursehound Scyliorhinus stellaris Mitochondrial Genome-Phylogeny, Relationships among Scyliorhinidae and Variability in Waters of the Balearic Islands.

The complete mitochondrial DNA sequence of the Nursehound Scyliorhinus stellaris has been determined for the first time and compared with congeneric species. The mitogenome sequence was 16,684 bp in length. The mitogenome is composed of 13 PCGs, 2 rRNAs, 22 transfer RNA genes and non-coding regions. The gene order of the newly sequenced mitogenome is analogous to the organization described in other vertebrate genomes. The typical conservative blocks in the control region were indicated. The phylogenetic analysis revealed a monophyletic origin of the Scyliorhininae subfamily, and within it, two subclades were identified. A significant divergence of Scyliorhinus spp. together with Poroderna patherinum in relation to the group of Cephaloscyllium spp. was observed, except for Scyliorhinus torazame, more related to this last cited clade. A hypothesis of a divergent evolution consequent to a selective pressure in different geographic areas, which lead to a global latitudinal diversity gradient, has been suggested to explain this phylogenetic reconstruction. However, convergent evolution on mitochondrial genes could also involve different species in some areas of the world.

An incubation water eDNA method for a non-destructive rapid molecular identification of Pinna nobilis and Pinna rudis bivalve juveniles.

The pen shell Pinna nobilis is critically endangered due to a disease that has affected all open water populations since late 2016. Collection of early spats is considered a fundamental step for pen shell conservation. However, the identification between P. nobilis and P. rudis juveniles by morphology is a very difficult task. Furthermore, due to the small size of juveniles and high sensitivity to handling, the sampling for this purpose must not damage individuals. As a consequence, the application of molecular techniques for conservation strategies to identify threatened and endangered bivalve species is every day more and more necessary. In this study, we present the development of a multiplex-PCR procedure for the rapid identification of two Pinna species from eDNA water samples. Using species-specific primers, designed in the rRNA16S and rRNA12S mitochondrial genes, identification of species was obtained by cellular or extracellular DNA dissolved in water and differentiated based on the size of the amplified DNA fragments. • Development of a molecular multiplex-PCR procedure for the rapid identification of two Pinna species from eDNA water samples • Using specie-specific primers, the different species can be differentiated basing on the size of the amplified DNA fragments • This technique removes many of the limitations commonly associated with sampling of threatened and endangered juvenile bivalves for conservation strategies (sampling does not damage individuals).

Mitogenome sequence comparison in the endangered congeneric Pinna nobilis and Pinna rudis bivalves.

BACKGROUND: The pen shells Pinna nobilis and Pinna rudis are large wedge-shaped bivalve molluscs. Both species are threatened by different anthropogenic pressures. In the last few years, P. nobilis populations have significantly reduced due to massive mortality events. The complete mitochondrial DNA sequences of these congeneric species have been determined and compared for the first time. RESULTS: The mitogenome sequences of P. nobilis and P. rudis were 18,919 bp and 18,264 bp in length, respectively. Each mitogenome is composed of 12 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA (tRNAs) genes and non-coding regions. A putative Adenosine Triphosphate synthase subunit 8 gene could only be proposed for P. nobilis. Both newly sequenced mitogenomes present a conserved gene order between them, comparable to the closely related Atrina pectinata, but global arrangement greatly differs from other available bivalve mitochondrial sequences. Multiple copies of tRNA-Cys were identified, located in different positions probably due to mechanisms of mitochondrial genome rearrangements, and detected 2 and 3 times in P. rudis and in P. nobilis, respectively. CONCLUSION: A close relationship was shown between Pinna species and Atrina pectinata and a consistent clustering showing a monophyletic origin of Pinnidae family sequences was evidenced. The mitochondrial genomes will provide a valuable genetic resource for further studies on population genetics and species identification.

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.

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.

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.].

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.

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.

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.

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.

Morphological and Molecular Characterization of Aggregata spp. Frenzel 1885 (Apicomplexa: Aggregatidae) in Octopus vulgaris Cuvier 1797 (Mollusca: Cephalopoda) from Central Mediterranean.

Coccidian parasites of the genus Aggregata are known to parasitize cephalopods as definitive hosts, however one of the genus members, A. octopiana, has shown an unresolved phylogeny within the same definitive host, the common octopus (Octopus vulgaris). Our study represents a large-scale investigation aimed at characterizing morphological traits and phylogeny of A. octopiana isolated from O. vulgaris inhabiting three distinct geographic areas of the central Mediterranean: The Adriatic, Ionian and Tyrrhenian Seas. The morphology of sporogonic stages of the parasite in octopus tissues was assessed by light and electron microscopy; molecular characterization has been carried out using the 18S rRNA locus. Our results support the hypothesis that two morphologically and genetically different A. octopiana infect O. vulgaris in the investigated areas of the Mediterranean Sea. Additional nuclear and mitochondrial markers for Aggregata should provide further information and better resolution of its phylogeny.

Insights on the drivers of genetic divergence in the European anchovy.

Anchovies represent the largest world's marine fish catches and the current threats on their populations impose a sustainable exploitment based on sound scientific information. In the European anchovy (Engraulis encrasicolus), the existence of several populations has been proposed but a global view is missing. Using a multidisciplinary approach, here we assessed the divergence among different ecotypes and its possible causes. SNPs have revealed two functionally distinct ecotypes overlapping in the Central Mediterranean, with one ecotype confined near the river estuaries. The same SNPs outliers also segregated two distinct populations in the near Atlantic, despite their large spatial distance. In addition, while most studies suggested that adaptation to low salinity is key to divergence, here we show that the offshore ecotype has higher environmental tolerance and an opportunistic feeding behaviour, as assessed by the study of environmental conditions, anchovy diet and trophic levels, and passive egg dispersal. These results provide insights into the anchovy evolutionary history, stressing the importance of behaviour in shaping ecotypes.

Biocomplexity in Populations of European Anchovy in the Adriatic Sea.

The sustained exploitation of marine populations requires an understanding of a species' adaptive seascape so that populations can track environmental changes from short- and long-term climate cycles and from human development. The analysis of the distributions of genetic markers among populations, together with correlates of life-history and environmental variability, can provide insights into the extent of adaptive variation. Here, we examined genetic variability among populations of mature European anchovies (n = 531) in the Adriatic (13 samples) and Tyrrhenian seas (2 samples) with neutral and putative non-neutral microsatellite loci. These genetic markers failed to confirm the occurrence of two anchovy species in the Adriatic Sea, as previously postulated. However, we found fine-scale population structure in the Adriatic, especially in northern areas, that was associated with four of the 13 environmental variables tested. Geographic gradients in sea temperature, salinity and dissolved oxygen appear to drive adaptive differences in spawning time and early larval development among populations. Resolving adaptive seascapes in Adriatic anchovies provides a means to understand mechanisms underpinning local adaptation and a basis for optimizing exploitation strategies for sustainable harvests.

Octopus vulgaris (Cuvier, 1797) in the Mediterranean Sea: Genetic Diversity and Population Structure.

The common octopus, Octopus vulgaris Cuvier 1797, is a largely exploited cephalopod species in the Mediterranean Sea and the Atlantic Ocean, as well as along the coasts of Africa, Brazil and Japan, where its taxonomic identity is still debated. The assessment of its genetic structure is a pressing need to correctly manage the resource and to avoid overfishing and collapsing of local stocks. Here we analysed genetic variation and population structure of O. vulgaris using thirteen microsatellite loci in seven sampling localities from the Mediterranean Sea and one from the Atlantic Ocean. We also used a DNA barcoding approach by COI gene fragment to understand the phylogenetic relationships among the specimens here investigated and the ones whose sequences are available in literature. Our results reveal high levels of allelic richness and moderate heterozygosity in all samples investigated, and a pronounced differentiation of the Atlantic and Sicilian specimens. This latter aspect seems to support the isolation of the biota within the Strait of Messina. A certain degree of differentiation was detected among the other geographic samples within the Mediterranean Sea, which is more compatible with an island model than isolation by distance. The occurrence of null alleles affected more genetic diversity indices than population structure estimations. This study provides new insights about the genetic diversity and structure of O. vulgaris in the area of interest, which can be used as guidelines for a fisheries management perspective.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 April 2010 - 31 May 2010.

This article documents the addition of 396 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anthocidaris crassispina, Aphis glycines, Argyrosomus regius, Astrocaryum sciophilum, Dasypus novemcinctus, Delomys sublineatus, Dermatemys mawii, Fundulus heteroclitus, Homalaspis plana, Jumellea rossii, Khaya senegalensis, Mugil cephalus, Neoceratitis cyanescens, Phalacrocorax aristotelis, Phytophthora infestans, Piper cordulatum, Pterocarpus indicus, Rana dalmatina, Rosa pulverulenta, Saxifraga oppositifolia, Scomber colias, Semecarpus kathalekanensis, Stichopus monotuberculatus, Striga hermonthica, Tarentola boettgeri and Thermophis baileyi. These loci were cross-tested on the following species: Aphis gossypii, Sooretamys angouya, Euryoryzomys russatus, Fundulus notatus, Fundulus olivaceus, Fundulus catenatus, Fundulus majalis, Jumellea fragrans, Jumellea triquetra Jumellea recta, Jumellea stenophylla, Liza richardsonii, Piper marginatum, Piper aequale, Piper darienensis, Piper dilatatum, Rana temporaria, Rana iberica, Rana pyrenaica, Semecarpus anacardium, Semecarpus auriculata, Semecarpus travancorica, Spondias acuminata, Holigarna grahamii, Holigarna beddomii, Mangifera indica, Anacardium occidentale, Tarentola delalandii, Tarentola caboverdianus and Thermophis zhaoermii.

Evolutionary relatedness of mackerels of the genus Scomber based on complete mitochondrial genomes: strong support to the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as distinct species.

Mackerels of the genus Scomber are commercially important species, but their taxonomic status is still controversial. Although previous phylogenetic data support the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as separate species, it is only based on the analysis of partial mitochondrial and nuclear DNA sequences. In an attempt to shed light on this relevant issue, we have determined the complete mitochondrial DNA sequence of S. colias, S. japonicus, and Scomber australasicus. The total length of the mitogenomes was 16,568 bp for S. colias and 16,570 bp for both S. japonicus and S. australasicus. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs, and 22 tRNAs) and organization similar to that observed in Scomber scombrus and most other vertebrates. The major noncoding region (control region) ranged between 865 and 866 bp in length and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Scomber species with regard to other scombrid fish. The major finding of this study is that S. colias and S. japonicus were significantly grouped in distinct lineages within Scomber cluster, which phylogenetically constitutes evidence that they may be considered as separate species. Additionally, molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.