Molecular characterization of Lernathropus kroyeri from intensive aquaculture and pathophysiology of infested sea bass.

The copepod Lernathropus kroyeri constitutes one of the major parasites for the Mediterranean aquaculture, infesting the sea bass Dicentrarchus labrax causing thus disruptions of growth performance and occasionally mortalities. Despite the large spread and the high frequency of this parasite in mariculture farms of Eastern Mediterranean, L. kroyeri genetic profile from aquaculture as well as the pathophysiological response of D. labrax have not been studied so far. Keeping this in mind, in the present study we investigated the L. kroyeri infestation on D. labrax from two farms in Greece, examining both healthy and heavy parasitized individuals. Assays included histopathology, phylogenetic reconstruction of the parasite and physiological response of the fish by the means of antioxidant, inflammatory metabolic and stress related gene expression analysis at both mRNA and protein levels. Genetic analysis indicated that L. kroyeri composes a monophyletic group, highly phylogenetically distant from other congeneric groups. Heavy infested D. labrax witnessed a significantly increased immune response that further led to oxidative stress and metabolic alterations. Overall, our results demonstrate the, seasonally independent, high infestation of this parasitic copepods, which continue to affect Mediterranean intensive aquaculture systems.

Influence of temperature on embryonic development of Pontastacus leptodactylus freshwater crayfish, and characterization of growth and osmoregulation related genes.

Narrow clawed crayfish, Pontastacus (Astacus) leptodactylus, represents an ecologically and economically valuable freshwater species. Despite the high importance of artificial breeding for conservation purpose and aquaculture potential, hatching protocols have not been developed so far in this species. Further, limited knowledge exists regarding the artificial egg incubation, the temperature effect on embryonic development, hatching synchronization and hatching rate. In the present study we investigated the temperature increase (from 17 oC to 22oC) effects in two different embryonic developmental stages of P. leptodactylus. Furthermore, two primer pairs for the Fibroblast Growth Factor Receptor 4 (FGFR4) gene cDNA amplification were successfully designed, characterising for the first time the FGFR4 gene in P. leptodactylus in relation to different developmental stages and temperatures. Apart from the FGFR4 gene, the Na+/K+-ATPase α-subunit expression was also explored. Both the FGFR4 and Na+/K+-ATPase α-subunit expression levels were higher in embryos closer to hatching. Egg incubation at 22oC for seven days led to significant increase of FGFR4 expression in embryos from earlier developmental stages. Nevertheless, temperature increase did not affect FGFR4 expression in eggs from latter developmental stages and Na+/K+-ATPase α-subunit expression in all developmental stages. Temperature increase represents therefore probably a promising strategy for accelerating hatching in freshwater crayfish particularly in early developmental stages. Specifically, our results indicate that FGFR4 expression increased in embryonic stages closer to hatching and that temperature influences significantly its expression in embryos from earlier developmental stages. Overall, these findings can provide a better understanding of artificial egg incubation of P. leptodactylus, and therefore can be employed for the effective management of this species, both for economic and biodiversity retention reasons.

Histopathology and Phylogeny of the Dinoflagellate Hematodinium perezi and the Epibiotic Peritrich Ciliate Epistylis sp. Infecting the Blue Crab Callinectes sapidus in the Eastern Mediterranean.

Bioinvasions constitute both a direct and an indirect threat to ecosystems. Direct threats include pressures on local trophic chains, while indirect threats might take the form of an invasion of a microorganism alongside its host. The marine dinoflagellate Hematodinium perezi, parasitizing blue crabs (Callinectes sapidus), has a worldwide distribution alongside its host. In Greece, fluctuations in the blue crab population are attributed to overexploitation and the effects of climate change. The hypothesis of the present study was that blue crab population reductions cannot only be due to these factors, and that particular pathogens may also be responsible for the fluctuations. To investigate this hypothesis, both lethargic and healthy blue crab specimens were collected from three different fishing sites in order to assess the health status of this important species. Together with the lethargic responses, the hemolymph of the infested crabs presented a milky hue, indicating the first signs of parasitic infestation with H. perezi. The histopathological results and molecular identification demonstrated the effect of the presence of H. perezi in the internal organs and their important role in the mortality of blue crabs. Specifically, H. perezi, in three different tissues examined (heart, gills, hepatopancreas), affected the hemocytes of the species, resulting in alterations in tissue structure. Apart from this dinoflagellate parasite, the epibiotic peritrich ciliate Epistylis sp. was also identified, infecting the gills. This study represents the first detection of H. perezi in the eastern Mediterranean, demonstrating that this is the main causative agent of blue crab mortality on Greek coastlines.

Increased seawater temperature triggers thermal, oxidative and metabolic response of Ostrea edulis, leading to anaerobiosis.

Bivalves are among the marine organisms most influenced by climate change. Despite the flat oyster's Ostrea edulis high economic value, its culture is developed on a very small scale, since this species possesses a strong susceptibility to abiotic stressors. Due to climate change, temperature is one of the most critical environmental parameters for the welfare of the Mediterranean basin's marine inhabitants. The present study's purpose was to investigate the physiological performance of the Mediterranean's native O. edulis as it faces exposure to different temperatures. Since juveniles are more susceptible to abiotic stressors, this experimental procedure was focused on young individuals. The seawater temperatures studied included a standard control temperature of 21 °C (often observed in several marine areas throughout the Mediterranean), as well as increased seawater temperatures of 25 °C and 28 °C, occasionally occurring in shallow Mediterranean waters inhabited by bivalve spat. These were selected since the tissues of O. edulis becomes partly anaerobic in temperatures exceeding 26 °C, while cardiac dysfunction (arrhythmia) emerges at 28 °C. The results demonstrate that temperatures above 25 °C trigger both the transcriptional upregulation of hsp70 and hsp90, and the antioxidant genes Cu/Zn sod and catalase. Enhancement of thermal tolerance and increased defense against increased ROS production during thermal stress, were observed. As the intensity and duration of thermal stress increases, apoptotic damage may also occur. The increased oxidative and thermal stress incurred at the highest temperature of 28 °C, seemed to trigger the switch from aerobic to anaerobic metabolism, reflected by higher pepck mRNA expressions and lower ETS activity.

The characterization of toll-like receptor repertoire in Pinna nobilis after mass mortality events suggests adaptive introgression.

The fan mussel Pinna nobilis is currently on the brink of extinction due to a multifactorial disease mainly caused to the highly pathogenic parasite Haplosporidium pinnae, meaning that the selection pressure outweighs the adaptive potential of the species. Hopefully, rare individuals have been observed somehow resistant to the parasite, stretching the need to identify the traits underlying this better fitness. Among the candidate to explore at first intention are fast-evolving immune genes, of which toll-like receptor (TLR). In this study, we examined the genetic diversity at 14 TLR loci across P. nobilis, Pinna rudis and P. nobilis × P. rudis hybrid genomes, collected at four physically distant regions, that were found to be either resistant or sensitive to the parasite H. pinnae. We report a high genetic diversity, mainly observed at cell surface TLRs compared with that of endosomal TLRs. However, the endosomal TLR-7 exhibited unexpected level of diversity and haplotype phylogeny. The lack of population structure, associated with a high genetic diversity and elevated dN/dS ratio, was interpreted as balancing selection, though both directional and purifying selection were detected. Interestingly, roughly 40% of the P. nobilis identified as resistant to H. pinnae were introgressed with P. rudis TLR. Specifically, they all carried a TLR-7 of P. rudis origin, whereas sensitive P. nobilis were not introgressed, at least at TLR loci. Small contributions of TLR-6 and TLR-4 single-nucleotide polymorphisms to the clustering of resistant and susceptible individuals could be detected, but their specific role in resistance remains highly speculative. This study provides new information on the diversity of TLR genes within the P. nobilis species after MME and additional insights into adaptation to H. pinnae that should contribute to the conservation of this Mediterranean endemic species.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Seasonality in Synergism with Multi-Pathogen Presence Leads to Mass Mortalities of the Highly Endangered Pinna nobilis in Greek Coastlines: A Pathophysiological Approach.

Mortalities of Pinna nobilis populations set at risk the survival of the species from many Mediterranean coastline habitats. In many cases, both Haplosporidium pinnae and Mycobacterium spp. are implicated in mass mortalities of P. nobilis populations, leading the species into extinction. In the context of the importance of these pathogens' role in P. nobilis mortalities, the present study investigated two Greek populations of the species hosting different microbial loads (one only H. pinnae and the second both pathogens) by the means of pathophysiological markers. More specifically, the populations from Kalloni Gulf (Lesvos Island) and from Maliakos Gulf (Fthiotis), seasonally sampled, were chosen based on the host pathogens in order to investigate physiological and immunological biomarkers to assess those pathogens' roles. In order to determine if the haplosporidian parasite possesses a major role in the mortalities or if both pathogens are involved in these phenomena, a variety of biomarkers, including apoptosis, autophagy, inflammation and heat shock response were applied. The results indicated a decreased physiological performance of individuals hosting both pathogens in comparison with those hosting only H. pinnae. Our findings provide evidence for the synergistic role of those pathogens in the mortality events, which is also enhanced by the influence of seasonality.

The impact of ascidian biofouling on the farmed Mediterranean mussel Mytilus galloprovincialis physiology and welfare, revealed by stress biomarkers.

In biofouling communities, ascidians are among the most damaging species, presenting severe threats, such as depressed growth rates and decreased chances of lower survival, to shellfish aquaculture. However, little is known concerning the fouled shellfish physiology. In an effort to obtain information for the magnitude of stress caused by ascidians to farmed Mytilus galloprovincialis, five seasonal samplings took place in a mussel aquaculture farm suffering from ascidian biofoulants, in Vistonicos Bay, Greece. The dominant ascidian species were recorded and several stress biomarkers, including Hsp gene expression at both mRNA and protein levels, as well as MAPKs levels, and enzymatic activities of intermediate metabolism were examined. Almost all investigated biomarkers revealed elevated stress levels in fouled mussels compared to non-fouled. This enhanced physiological stress seems to be season-independent and can be attributed to the oxidative stress and/or feed deprivation caused by ascidian biofouling, thus illuminating the biological impact of this phenomenon.

Effects of dietary substitution of fishmeal by black soldier fly (Hermetia illucens) meal on growth performance, whole-body chemical composition, and fatty acid profile of Pontastacus leptodactylus juveniles.

Freshwater crayfish are considered as aquatic products of high quality and high nutritional value. The increasing demand has led to populations reduction in several locations throughout their range. Thus, the development of appropriate rearing conditions is considered necessary, among which, optimization of their diet is a basic part. Towards this direction, in the present study, a 98-day feeding trial was carried out to evaluate the impact of dietary fishmeal substitution by Hermetia illucens meal on Pontastacus leptodactylus juveniles kept under laboratory conditions. Insect meals represent an environmentally friendly alternative solution, considered as a high-value feed source, rich in nutrients such as protein and fat. Three dietary regimens were utilized with a fishmeal-based without Hermetia meal (HM) defined as the control diet (HM0), and two diets, the first with 50% (HM50) and the second with 100% (HM100) of fishmeal substitution by HM, respectively. Growth performance, whole-body composition, and fatty acid profiles of individuals were studied in the different treatments. At the end of the feeding trial, statistically significant differences were observed in the mean survival rate (SR), specific growth rate (SGR), feed conversion ratio (FCR) and weight gain (WG) values. More specifically, animals fed with HM-based diets had higher mean SR, while the control group performed better regarding FCR and SGR. The HM inclusion in the diet significantly altered the whole-body chemical composition of the crayfish signifying a different metabolic utilization compared to fishmeal (FM). The fatty acid analysis revealed that 16:0 (palmitic acid) was the predominant saturated fatty acid (SFA), 18:1ω9 (oleic acid) was found to be the main monounsaturated fatty acid (MUFA), while 18:2ω6 (linoleic acid) represented the major polyunsaturated fatty acid (PUFA) followed by C20:3 cis ω3 (cis-11-14-17-eicosatrienoate) and C22:6 cis ω3 (cis-4,7,10,13,16,19-Docosahexaenoic) fatty acids. The inclusion of dietary HM significantly reduced the contents of ∑SFAs, ∑PUFAs and ∑ω6 fatty acids, as well as those of C22:6 cis ω3 and increased the ω6/ω3 and hypocholesterolemic to hypercholesterolemic ratios in the body. In parallel with improvements in balanced diets and in culture conditions that need to be optimised for rearing of freshwater crayfish, our study provides new data that enlighten the suitability of insect meals in the nutrition of P. leptodactylus.

Investigation of the highly endangered Pinna nobilis' mass mortalities: Seasonal and temperature patterns of health status, antioxidant and heat stress responses.

Recently, P. nobilis populations have suffered a tremendous reduction, with pathogens potentially playing a crucial role. Considering its highly endangered status, mechanisms leading to mass mortalities were examined in one or multiple pathogens infected populations. Thus, seasonal antioxidant enzymatic activities, hsp70 and catalase mRNA levels, were investigated in two different Greek populations, during mass mortality events in summer of 2020. Samples were collected from Fthiotis and Lesvos during February (ToC 14 ± 1.2 and 15 ± 1 respectively), April (ToC 18 ± 1.2 and 17 ± 1.3 respectively), and June (ToC 24.5 ± 1.5 and 21.5 ± 1.5 respectively) 2020. In July of the same year (ToC 26.5 ± 1.7 in Fthiotis and 24.5 ± 1.7 in Lesvos), no live specimens were found. All biochemical parameters and phylogenetic analysis suggest that pathogen infection increases P. nobilis sensitivity to water temperature, subsequently leading to mass mortality. The latter was obvious in Fthiotis individuals, in which Haplosporidium pinnae was also observed with Mycobacterium spp., compared to Lesvos individuals.

The Coding Mitogenome of the Freshwater Crayfish Pontastacus leptodactylus (Decapoda:Astacidea:Astacidae) from Lake Vegoritida, Greece and Its Taxonomic Classification.

Pontastacus leptodactylus (Eschscholtz, 1823) (Decapoda:Astacidea:Astacidae) constitutes an ecologically and economically highly important species. In the present study, the mitochondrial genome of the freshwater crayfish P. leptodactylus from Greece is analyzed for the first time, using 15 newly designed primer pairs based on available sequences of closely related species. The analyzed coding part of the mitochondrial genome of P. leptodactylus consists of 15,050 base pairs including 13 protein-coding genes (PCGs), 2 ribosomal RNA gene (rRNAs), and 22 transfer RNA genes (tRNAs). These newly designed primers may be particularly useful in future studies for analyzing different mitochondrial DNA segments. Based on the entire mitochondrial genome sequence, compared to other haplotypes from related species belonging in the same family (Astacidae) available in the GenBank database, a phylogenetic tree was constructed depicting the phylogenetic relationships of P. leptodactylus. Based on the results, the genetic distance between Astacus astacus and P. leptodactylus is smaller than the genetic distance between Austropotamobius pallipes and Austropotamobius torrentium, despite the fact that the latter two are classified within the same genus, questioning the phylogenetic position of A. astacus as a different genus than P. leptodactylus. In addition, the sample from Greece seems genetically distant compared with a conspecific haplotype available in the GenBank database, possibly implying a genetic distinction of P. leptodactylus from Greece.

Virulence Genes and In Vitro Antibiotic Profile of Photobacterium damselae Strains, Isolated from Fish Reared in Greek Aquaculture Facilities.

Bacteria belonging to the species Photobacterium damselae are pathogens of cultured marine fish, causing diseases of high importance, such as Pasteurellosis. Thus, they are considered a major threat to the aquaculture sector. Despite the great importance of fish mariculture for the Greek economy, the distribution and abundance of these bacteria are not well documented in aquaculture units in Greece. Keeping this in mind, the scope of the present study was to investigate the presence, antibiotic profile, and virulence of Photobacterium bacteria originating from a representative sample of mariculture units throughout Greece. Samples were collected from diseased fish belonging to three different cultured fish species, namely Sparus aurata, Dicentrarchus labrax, and Pagrus pagrus, from both the Aegean and the Ionian Sea. Tissue samples were cultured in agar media, and bacteria were molecularly identified using both bacterial universal and species-specific primer pairs for Photobacterium spp. Additionally, the identified strains were characterized for the presence of virulence genes as well as antibiotic profiles. According to the results, the aforementioned bacteria are distributed in the Greek aquaculture units and are characterized by high pathogenicity based on the abundance of virulence genes. Furthermore, the majority of the detected strains exhibit some level of antibiotic resistance. In summary, our results indicate the need for systematic surveillance and study of their antibiotic profiles in Greek aquaculture since these bacteria constitute a major threat to the sector.

Are Marine Heatwaves Responsible for Mortalities of Farmed Mytilus galloprovincialis? A Pathophysiological Analysis of Marteilia Infected Mussels from Thermaikos Gulf, Greece.

Marine heatwaves (excessive seawater temperature increases) pose high risk to bivalves' health and farming. The seawater temperature increase is responsible for various pathogen population expansions causing intense stress to marine organisms. Since the majority of knowledge so far derives from laboratory experiments, it is crucial to investigate stress responses in field conditions in order to understand the mechanisms leading to bivalves' mortality events after exposure to temperature extremes. Thus, we evaluated the pathophysiological response of the Mediterranean mussel Mytilus galloprovincialis originating from mortality events enhanced by intense heatwaves in Thermaikos Gulf, north Greece, along with Marteilia refrigens infection. Mussels that have been exposed to high environmental stressors such as high temperature were examined for various molecular and biochemical markers, such as hsp70, bax, bcl-2, irak4 and traf6 gene expression, as well as the enzymatic activity of the hsp70, hsp90, bax, bcl-2, cleaved caspases, TNFa and ll-6 proteins. Furthermore, histopathology and molecular positivity to Marteilia sp. were addressed and correlated with the gene expression results. Our findings elucidate the molecular and biochemical pathways leading to mortality in farmed mussels in the context of Marteilia infection, which according to the results is multiplied by heatwaves causing a significant increase in pathophysiological markers.

Aquaponics as a Promising Strategy to Mitigate Impacts of Climate Change on Rainbow Trout Culture.

The impact of climate change on both terrestrial and aquatic ecosystems tends to become more progressively pronounced and devastating over the years. The sector of aquaculture is severely affected by natural abiotic factors, on account of climate change, that lead to various undesirable phenomena, including aquatic species mortalities and decreased productivity owing to oxidative and thermal stress of the reared organisms. Novel innovative technologies, such as aquaponics that are based on the co-cultivation of freshwater fish with plants in a sustainable manner under the context of controlled abiotic factors, represent a promising tool for mitigating the effect of climate change on reared fish. The rainbow trout (Oncorhynchus mykiss) constitutes one of the major freshwater-reared fish species, contributing to the national economies of numerous countries, and more specifically, to regional development, supporting mountainous areas of low productivity. However, it is highly vulnerable to climate change effects, mainly due to the concrete raceways, in which it is reared, that are constructed on the flow-through of rivers and are, therefore, dependent on water's physical properties. The current review study evaluates the suitability, progress, and challenges of developing innovative and sustainable aquaponic systems to rear rainbow trout in combination with the cultivation of plants. Although not commercially developed to a great extent yet, research has shown that the rainbow trout is a valuable experimental model for aquaponics that may be also commercially exploited in the future. In particular, abiotic factors required in rainbow trout farming along, with the high protein proportion required in the ratios due to the strict carnivorous feeding behavior, result in high nitrate production that can be utilized by plants as a source of nitrogen in an aquaponic system. Intensive farming of rainbow trout in aquaponic systems can be controlled using digital monitoring of the system parameters, mitigating the obstacles originating from extreme temperature fluctuations.

Pollution Indicators and HAB-Associated Halophilic Bacteria Alongside Harmful Cyanobacteria in the Largest Mussel Cultivation Area in Greece.

Taking into consideration the essential contribution of Mytilus galloprovincialis farming, it is of rising importance to add knowledge regarding bacterial species occurrence in water samples from aquaculture zones from the point of view of both the organism and public health. In the present study, we investigated the bacterial community existing in water samples from six Mytilus galloprovincialis aquaculture areas in the Thermaikos gulf, northern Greece, that may provoke toxicity in aquatic organisms and humans and may indicate environmental pollution in mussel production as well as algal blooms. Bacterial species were identified molecularly by sequencing of a partial 16s rRNA segment and were analyzed phylogenetically for the confirmation of the bacterial taxonomy. The results obtained revealed the presence of four bacterial genera (Halomonas sp., Planococcus sp., Sulfitobacter sp., and Synechocystis sp.). Members of the Halomonas and Sulfitobacter genera have been isolated from highly polluted sites, Planococcus bacteria have been identified in samples derived directly from plastic debris, and Synechocystis bacteria are in line with microcystin detection. In this context, the monitoring of the bacteria community in mussel aquaculture water samples from the Thermaikos gulf, the largest mussel cultivation area in Greece, represents an indicator of water pollution, microplastics presence, algal blooms, and toxin presence.

The current situation and potential effects of climate change on the microbial load of marine bivalves of the Greek coastlines: an integrative review.

Global warming affects the aquatic ecosystems, accelerating pathogenic microorganisms' and toxic microalgae's growth and spread in marine habitats, and in bivalve molluscs. New parasite invasions are directly linked to oceanic warming. Consumption of pathogen-infected molluscs impacts human health at different rates, depending, inter alia, on the bacteria taxa. It is therefore necessary to monitor microbiological and chemical contamination of food. Many global cases of poisoning from bivalve consumption can be traced back to Mediterranean regions. This article aims to examine the marine bivalve's infestation rate within the scope of climate change, as well as to evaluate the risk posed by climate change to bivalve welfare and public health. Biological and climatic data literature review was performed from international scientific sources, Greek authorities and State organizations. Focusing on Greek aquaculture and bivalve fisheries, high-risk index pathogenic parasites and microalgae were observed during summer months, particularly in Thermaikos Gulf. Considering the climate models that predict further temperature increases, it seems that marine organisms will be subjected in the long term to higher temperatures. Due to the positive linkage between temperature and microbial load, the marine areas most affected by this phenomenon are characterized as 'high risk' for consumer health.

How Safe to Eat Are Raw Bivalves? Host Pathogenic and Public Health Concern Microbes within Mussels, Oysters, and Clams in Greek Markets.

Raw-bivalves consumption is a wide trend in Mediterranean countries. Despite the unambiguous nutritional value of seafood, raw consumption of bivalves may involve risks that could pose a significant threat to consumers' health. Their filter-feeding behavior is responsible for the potential hosting of a wide variety of microorganisms, either pathogenic for the bivalves or public health threats. Under this prism, the current study was conducted in an effort to evaluate the risk of eating raw bivalves originating from the two biggest seafood markets in Thessaloniki, the largest production area of bivalves in Greece. Both microbiological and molecular methodologies were applied in order to assess the presence of various harmful microbes, including noroviruses, Bonamia, Marteilia, Esherichia coli, Salmonella, and Vibrio. Results indicated the presence of several Vibrio strains in the analyzed samples, of which the halophilic Vibrio harveyi was verified by 16S rRNA sequencing; other than this, no enteropathogenic Vibrio spp. was detected. Furthermore, although Esherichia coli was detected in several samples, it was mostly below the European Union (EU) legislation thresholds. Interestingly, the non-target Photobacterium damselae was also detected, which is associated with both wound infections in human and aquatic animals. Regarding host pathogenic microorganisms, apart from Vibrio harveyi, the protozoan parasite Marteilia refrigens was identified in oysters, highlighting the continuous infection of this bivalve in Greece. In conclusion, bivalves can be generally characterized as a safe-to-eat raw food, hosting more bivalve pathogenic microbes than those of public health concern.

Pathophysiological Responses of Pinna nobilis Individuals Enlightens the Etiology of Mass Mortality Situation in the Mediterranean Populations.

Due to the rapid decrease of Pinna nobilis populations during the previous decades, this bivalve species, endemic in the Mediterranean Sea, is characterized as 'critically endangered'. In addition to human pressures, various pathogen infections have resulted in extended reduction, even population extinction. While Haplosporidium pinnae is characterized as one of the major causative agents, mass mortalities have also been attributed to Mycobacterium sp. and Vibrio spp. Due to limited knowledge concerning the physiological response of infected P. nobilis specimens against various pathogens, this study's aim was to investigate to pathophysiological response of P. nobilis individuals, originating from mortality events in the Thermaikos Gulf and Lesvos and Limnos islands (Greece), and their correlation to different potential pathogens detected in the diseased animals. In isolated tissues, several cellular stress indicators of the heat shock and immune response, apoptosis and autophagy, were examined. Despite the complexity and limitations in the study of P. nobilis mortality events, the present investigation demonstrates the cumulative negative effect of co-infection additionally with H. pinnae in comparison to the non-presence of haplosporidian parasite. In addition, impacts of global climate change affecting physiological performance and immune responses result in more vulnerable populations in infectious diseases, a phenomenon which may intensify in the future.

The Implication of Vibrio Bacteria in the Winter Mortalities of the Critically Endangered Pinna nobilis.

Pinna nobilis populations, constituting the largest bivalve mollusk endemic to the Mediterranean, is characterized as critically endangered, threatened by extinction. Among the various factors proposed as etiological agents are the Haplosporidium pinnae and Mycobacterium sp. parasites. Nevertheless, devastation of the fan mussel populations is still far from clear. The current work is undertaken under a broader study aiming to evaluate the health status of Pinna nobilis population in Aegean Sea, after the mass mortalities that occurred in 2019. A significant objective was also (a) the investigation of the etiological agents of small-scale winter mortalities in the remaining populations after the devastating results of Haplosporidium pinnae and Mycobacterium sp. infections, as well as (b) the examination of the susceptibility of the identified bacterial strains in antibiotics for future laboratory experiments. Microbiological assays were used in order to detect the presence of potential bacterial pathogens in moribund animals in combination with molecular tools for their identification. Our results provide evidence that Vibrio bacterial species are directly implicated in the winter mortalities, particularly in cases where the haplosporidian parasite was absent. Additionally, this is the first report of Vibrio mediterranei and V. splendidus hosted by any bivalve on the Greek coastline.

Gut Symbiotic Microbial Communities in the IUCN Critically Endangered Pinna nobilis Suffering from Mass Mortalities, Revealed by 16S rRNA Amplicon NGS.

Mass mortality events due to disease outbreaks have recently affected almost every healthy population of fan mussel, Pinna nobilis in Mediterranean Sea. The devastating mortality of the species has turned the interest of the research towards the causes of these events. After the haplosporidan infestation and the infection by Mycobacterium sp., new emerging pathogens have arisen based on the latest research. In the present study, a metagenomic approach of 16S rRNA next generation sequencing (NGS) was applied in order to assess the bacterial diversity within the digestive gland of diseased individuals as well as to carry out geographical correlations among the biodiversity of microbiome in the endangered species Pinna nobilis. The specimens originated from the mortalities occurred in 2019 in the region of Greece. Together with other bacterial genera, the results confirmed the presence of Vibrio spp., assuming synergistic effects in the mortality events of the species. Alongside with the presence of Vibrio spp., numerous bacterial genera were detected as well, including Aliivibrio spp., Photobacterium spp., Pseudoalteromonas spp., Psychrilyobacter spp. and Mycoplasma spp. Bacteria of the genus Mycoplasma were in high abundance particularly in the sample originated from Limnos island representing the first time recorded in Pinna nobilis. In conclusion, apart from exclusively the Haplosporidan and the Mycobacterium parasites, the presence of potentially pathogenic bacterial taxa detected, such as Vibrio spp., Photobactrium spp. and Alivibrio spp. lead us to assume that mortality events in the endangered Fan mussel, Pinna nobilis, may be attributed to synergistic effects of more pathogens.