Microplastic Presence in the Digestive Tract of Pearly Razorfish Xyrichtys novacula Causes Oxidative Stress in Liver Tissue.

Plastic pollution in the oceans is a growing problem, with negative effects on exposed species and ecosystems. Xyrichtys novacula L. is a very important fish species both culturally and economically in the Balearic Islands. The aim of the present study was to detect and categorise the presence of microplastics (MPs) in the digestive tract of X. novacula, as well as the existence of oxidative stress in the liver. For this purpose, the fish were categorised into two groups based on the number of MPs observed in the digestive tracts: a group with no or low presence of MPs (0-3 items) and a group with a higher presence of MPs (4-28 items). MPs were found in 89% of the specimens analysed, with a dominance of fibre type and blue colour. Regarding the type of polymer, polycarbonate was the most abundant, followed by polypropylene and polyethylene. For the group with a greater presence of MPs, the activities of the antioxidant enzymes glutathione peroxidase and glutathione reductase, as well as the phase II detoxification enzyme glutathione s-transferase, were higher than the activities observed in fish with little to no presence of MPs. The activities of catalase and superoxide dismutase and the levels of malondialdehyde did not show significant differences between both groups. In conclusion, these results demonstrate the presence of MPs in the digestive tract of X. novacula and the existence of an antioxidant and detoxification response, mainly based on the glutathione-based enzymes.

First detection of microplastics in Xyrichtys novacula (Linnaeus 1758) digestive tract from Eivissa Island (Western Mediterranean).

Plastic waste and its ubiquity in the oceans represent a growing problem for marine life worldwide. Microplastics (MPs) are ubiquitous in the sea and easily enter food webs. Xyrichtys novacula L. is one of the main target species of recreational fishing in the Balearic Islands, Spain. In the present study, the quantity of MPs in gastrointestinal tracts of X. novacula from two different areas (a marine protected area (MPA) and a non-protected area) of Eivissa Island (in the Balearic archipelago) has been assessed, as well as MPs evaluation within the sediment of both areas. The results showed that over 80% of sampled individuals had MPs in their gut with an average of 3.9 ± 4.3 plastic items/individual. Eighty percent of these plastics were fibres, while the rest were fragments. Although the sediment of the non-protected area had a significant higher presence of MPs, no significant differences in the number of MPs were observed in X. novacula from both areas. The µ-FT-IR analysis showed that the main polymers in the sediments were polycarbonate (PC) and polypropylene (PP), whereas in the digestive tract of fish PC, PP, polyethylene, polystyrene and polyester. In conclusion, practically all X. novacula specimens presented MPs in their digestive tract regardless if the capture zone was in a MPAs or not. These results highlight the ubiquity of MPs in coastal marine areas, and further studies might be necessary to evaluate further implications of MP presence in this species.

Mitochondrial genome sequencing of marine leukaemias reveals cancer contagion between clam species in the Seas of Southern Europe.

Clonally transmissible cancers are tumour lineages that are transmitted between individuals via the transfer of living cancer cells. In marine bivalves, leukaemia-like transmissible cancers, called hemic neoplasia (HN), have demonstrated the ability to infect individuals from different species. We performed whole-genome sequencing in eight warty venus clams that were diagnosed with HN, from two sampling points located more than 1000 nautical miles away in the Atlantic Ocean and the Mediterranean Sea Coasts of Spain. Mitochondrial genome sequencing analysis from neoplastic animals revealed the coexistence of haplotypes from two different clam species. Phylogenies estimated from mitochondrial and nuclear markers confirmed this leukaemia originated in striped venus clams and later transmitted to clams of the species warty venus, in which it survives as a contagious cancer. The analysis of mitochondrial and nuclear gene sequences supports all studied tumours belong to a single neoplastic lineage that spreads in the Seas of Southern Europe.

In humans and other animals, cancer cells divide excessively, forming tumours or flooding the blood, but they rarely spread to other individuals. However, some animals, including dogs, Tasmanian devils and bivalve molluscs like clams, cockles and mussels, can develop cancers that are transmitted from one individual to another. Despite these cancers being contagious, each one originates in a single animal, meaning that even when the cancer has spread to many individuals, its origins can be traced through its DNA. Cancer contagion is rare, but transmissible cancers seem to be particularly common in the oceans. In fact, 7 types of contagious cancer have been described in bivalve species so far. These cancers are known as 'hemic neoplasias', and are characterized by the uncontrolled division of blood-like cells, which can be released by the host they developed in, and survive in ocean water. When these cells encounter individuals from the same species, they can infect them, causing them to develop hemic neoplasia too There are still many unanswered questions about contagious cancers in bivalves. For example, how many species do the cancers affect, and which species do the cancers originate in? To address these questions, Garcia-Souto, Bruzos, Díaz et al. gathered over 400 specimens of a species of clam called the warty venus clam from the coastlines of Europe and examined them for signs of cancer. Clams collected in two regions of Spain showed signs of hemic neoplasia: one of the populations was from the Balearic Islands in the Mediterranean Sea, while the other came from the Atlantic coast of northwestern Spain. Analyzing the genomes of the tumours from each population showed that the cancer cells from both regions had likely originated in the same animal, indicating that the cancer is contagious and had spread through different populations. The analysis also revealed that the cancer did not originally develop in warty venus clams: the cancer cells contained DNA from both warty venus clams and another species called striped venus clams. These two species live close together in the Mediterranean Sea, suggesting that the cancer started in a striped venus clam and then spread to a warty venus clam. To determine whether the cancer still affected both species, Garcia-Souto, Bruzos, Díaz et al. screened 200 striped venus clams from the same areas, but no signs of cancer were found in these clams. This suggests that currently the cancer only affects the warty venus clam. These findings confirm that contagious cancers can jump between clam species, which could be threat to the marine environment. The fact that the cancer was so similar in clams from the Atlantic coast and from the Mediterranean Sea, however, suggests that it may have emerged very recently, or that human activity helped it to spread from one place to another. If the latter is the case, it may be possible to prevent further spread of these sea-borne cancers through human intervention.

Assessment of the effect of long-term exposure to microplastics and depuration period in Sparus aurata Linnaeus, 1758: Liver and blood biomarkers.

The constant increase in plastic pollution has attracted great attention in recent years due to its potential detrimental effects on organisms and ecosystems. While the consequences of ingestion of large plastic litter are mostly understood, the impacts resulting from a long-term exposure and a recovery period of microplastics (MPs) are still limited. The aims were to monitor oxidative stress, detoxification and inflammatory biomarkers in liver, plasma and erythrocytes of Sparus aurata exposed during 90 days to low-density polyethylene (LDPE)-MPs enriched diet (10% by weight) followed by 30 days of depuration. Exposure to LDPE-MPs progressively activates the antioxidant and detoxification system and induces an inflammatory response in liver and plasma, whereas no significant changes were observed in erythrocytes. The plasma activities of catalase, myeloperoxidase (MPO), lysozyme and the levels of malondialdehyde (MDA) as maker of lipid peroxidation significantly increased after exposure to LDPE-MPs for 90 days compared to the control group. The activities of all antioxidant enzymes - catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase-, the detoxification enzyme glutathione s-transferase, MPO, the production of reactive oxygen species and the levels of MDA were also significantly increased in liver after MPs exposure. Additionally, all these biomarkers tended to recover during the depuration period, most of them reaching similar levels to those of the control group. In conclusion, the ingestion of a diet containing LDPE-MPs for 90 days induced a progressive increase in oxidative stress and inflammation biomarkers in liver and plasma of S. aurata but not in erythrocytes, which tended to regain control values when not exposed to MPs for 30 days. The present study contributes to a better understanding of the toxic effects of MPs in S. aurata and highlights the usefulness of plasma that can be obtained in a minimally invasive way to monitor these effects.

Microplastic ingestion in reared aquaculture fish: Biological responses to low-density polyethylene controlled diets in Sparus aurata.

During the last years, ingestion of microplastics (MPs) has been quantified in marine species both with an ecological and commercial interest at sea and under experimental conditions, highlighting the importance to assess MP ingestion in commercially and aquaculture important species such as gilthead seabream (Sparus aurata) fish. In order to study the ingestion of MPs in a commercially valuable species, gilthead seabreams were exposed to an enriched diet with virgin and weathered low-density polyethylene (LDPE) pellets for three months followed by a detoxification period of one month of no exposure to MP enriched diets. Our results indicate that MP ingestion in these fishes increased with exposure time, and differences were found between treatments, showing the highest ingestion values after three months of exposure to MP enriched diets and in the weathered treatment. However, after one month of detoxification, no MPs were found in the gastrointestinal tracts of fish, reflecting no long-term retention of MPs in Sparus aurata digestive system. According to results from this study, exposure of fish to MP enriched diets does not affect fish size neither the Fulton's condition index as both parameters increased with time in all treatments (control, virgin and weathered). Both carbon and nitrogen isotopic signatures decreased with fish size in all treatments which could be related to an increase of nitrogen deposition efficiency in fish muscle with a high protein assimilation during the first months of Sparus aurata.

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.

Long-term exposure to microplastics induces oxidative stress and a pro-inflammatory response in the gut of Sparus aurata Linnaeus, 1758.

Environmental pollution from plastic debris is a major global concern, being a potential threat to marine organisms and ecosystems. The accumulation of microplastics (MPs) in the oceans has notable ecological implications due to their long persistence, their potential ecotoxicity, and their ability to adsorb other pollutants and act as vectors of pathogens. Nevertheless, whereas the number of investigations documenting the presence of MPs in wild fish has increased, less studies have addressed the toxicological effects associated with the ingestion of MPs in long-term laboratory conditions. The aim of the present study was to assess the physiological response of gilthead seabream (Sparus aurata) exposed to low-density polyethylene (LDPE) MPs during a 90-day exposure followed by an extra 30 days of depuration through the application of oxidative stress biomarkers in the gut. No changes were observed in the Fulton condition factor of fish associated with MP intake. The activities of antioxidant enzymes and glutathione s-transferase and the levels of reduced glutathione progressively increased throughout the study in the MPs-fed group compared to the control group, reaching the highest values at 90 days. Similarly, the activity of the pro-inflammatory enzyme, myeloperoxidase, and the levels of oxidative damage markers -malondialdehyde and protein carbonyls-also increased after 90 days of exposure to an enriched diet with MPs. During the 30-day depuration period, all the biomarkers analysed tended to normalize, with the majority recovering values similar to those of the control group. In conclusion, MPs exposure during 90 days to S. aurata induced oxidative stress and a pro-inflammatory response in gut, and were able to recover after the exposure to MPs was removed.

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.].

Perkinsus mediterraneus infection induces oxidative stress in the mollusc Mimachlamys varia.

Perkinsus mediterraneus is a protozoan parasite that can cause marine mollusc diseases known as perkinsosis being a serious threat for clam cultures worldwide. The aim of the present study was first to determine the Perkinsus species infecting the variegated scallop Mimachlamys varia and then to evaluate the existence of oxidative stress in gills of M. varia according to different degrees of infection. DNA sequencing confirmed that P. mediterraneus was the species infecting M. varia. ROS production was progressively increasing with the degree of infection although the differences were only significant in the high-infected group. Low degree of infection significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and nitrite levels with respect to the control group. In the high-infected group, a significant increase was evidenced in all analysed enzymes, catalase, SOD, glutathione reductase and GST. Non-significant differences in MDA levels were observed between the control and low-infected groups; however, a significant increase in MDA levels was observed in the high-infected group. In conclusion, the infection by Perkinsus mediterraneus in M. varia induces oxidative stress and an antioxidant response directly related to the infection degree that can contribute to the pathogenicity of the infection.

Levels of radionuclide concentrations in benthic invertebrate species from the Balearic Islands, Western Mediterranean, during 2012-2018.

Baseline levels of radionuclides in the marine environment of the Balearic Islands in Western Mediterranean have not been reported in literature. Because of their ecological role and acknowledged sensitivity to pollutants, herein, the activity concentrations of 210Po, 40K, 210Pb, 90Sr, and 234Th were measured in two types of benthic invertebrate species (mussels and sea urchins) sampled during 2012-2018. The activity concentrations of 210Po, 40K, 210Pb, and 234Th ranged from 38 ±â€¯1 to 325 ±â€¯11 Bq kg-1 dry weight (d.w.), 220 ±â€¯10 to 996 ±â€¯46 Bq kg-1 d.w., ND (lower than the limit of detection) to 55 ±â€¯8 Bq kg-1 d.w., and ND to 70 ±â€¯15 Bq kg-1 d.w., respectively. In all cases, no artificial 90Sr activity was detected in the collected samples. The committed effective dose to humans was calculated to be in the range of 48-640 µSv year-1.

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.