Pinna nobilis in the Greek seas (NE Mediterranean): on the brink of extinction?

The Mediterranean endemic fan mussel Pinna nobilis is suffering an ongoing basin-scale mass mortality event (MME) since 2016. As most Mediterranean populations have collapsed, the species has been declared as Critically Endangered in the IUCN Red List of threatened species. In an effort to track the progress of the MME and provide updated information on the status of the species in the Greek seas, data collected through dedicated surveys and opportunistic assessments during 2019 and 2020 have been compiled. During surveys conducted at 258 sites, a total of 14,589 fan mussels were recorded, of which 81.1% were dead. Of the remaining 2,762 live individuals, 256 were juveniles. Two marine areas that still sustain living populations were identified, namely Kalloni Gulf (Lesvos Island), and Laganas Bay (Zakynthos Island). The inner part of Kalloni Gulf appears to maintain the largest surviving population of the species in the eastern Mediterranean, with an abundance estimate of 684,000 individuals (95% confidence interval: 322,000-1,453,000). Solitary, potentially resistant, scattered individuals were recorded at several sites. Other previously abundant populations that had been assessed in the past, specifically those of Lake Vouliagmeni (Korinthiakos Gulf), Souda Bay (Crete) and Gera Gulf (Lesvos Island) with a total of ~350,000 individuals, have now been wiped out. Our results document the collapse of most P. nobilis populations throughout the Greek seas. The MME has progressed substantially between early 2019 and mid-2020, as indicated by the increase in mortality at sites consecutively monitored multiple times. This work highlights the urgent need for continuous monitoring of surviving populations and calls for immediate implementation of an effective protection and management strategy that will ensure the persistence of surviving individuals and the production of resistant offspring

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

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.info:eu-repo/semantics/acceptedVersio

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.This work was supported by the University of Toulon and Toulon Provence Méditerranée (TPM) related to the PINORES project, the University Institute of Technology of the University of Toulon under the grant ‘CARTT’ and by the European Union's LIFE programme through the project LIFE PINNARCA (NAT/ES/001265). Fabio Scarpa, Marco Casu and Daria Sanna acknowledge the support of NBFC to the University of Sassari, funded by the Italian Ministry of University and Research, PNRR, Missione 4, Componente 2, ‘Dalla ricerca all'impresa’, Investimento 1.4 Project CN00000033.Peer reviewe

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

Tropical Shrimp Biofloc Aquaculture within Greenhouses in the Mediterranean: Preconditions, Perspectives, and a Prototype Description

Biofloc technology (BFT) offers an innovative eco-friendly approach that is particularly applicable in shrimp farming. Penaeus vannamei is the most important seafood species in terms of global economic value. Nevertheless, its increasing global demand highlights the necessity for sustainable production of P. vannamei shrimps outside their native range, assuring the avoidance of genetic pollution risk. Towards this direction, the present study focuses on the feasibility of tropical shrimp species aquaculture in indoor systems evaluating BFT application in temperate zones. The achievability of P. vannamei cultivation inside greenhouses in temperate latitudes is thoroughly examined and a representative experimental biofloc setup for P. vannamei within a greenhouse in Northern Greece is demonstrated. Nevertheless, there are two major limitations, related to economy and ecology, namely the energy demand for high seawater temperature and the fact that most reared shrimps are non-indigenous species setting risk for genetic pollution, respectively. To overcome the former, energy-saving measures such as tank and greenhouse insulation in combination with a microclimate chamber construction were implemented to optimize water temperature at minimal cost. Concerning the latter, there is clear evidence that P. vannamei populations cannot be established in the Mediterranean, setting aside any environmental risk. Overall, based on the developed and tested pilot prototype, employment of optimal management practices, innovative manufacturing and clean energy alternatives, and the utilization of ecosystem services could reduce the environmental impact and maximize the profitability of biofloc operations. These actions could probably permit sustainable and economically viable farming of P. vannamei employing BFT within greenhouses in the Mediterranean

The Coding Mitogenome of the Freshwater Crayfish <i>Pontastacus leptodactylus</i> (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

Partial Substitution of Fresh Microalgae with Baker’s Yeast (<i>Saccharomyces cerevisiae</i>) Enhances the Growth of Juvenile <i>Ostrea edulis</i> and <i>Ruditapes decussatus</i>

The hatchery culture of bivalve mollusks depends on feeding with fresh microalgae which represent up to 50% of the production costs. We investigated the growth performance of juvenile Ostrea edulis and Ruditapes decussatus under 15% and 30% replacement of microalgae with Saccharomyces cerevisiae. Metabolic indices were measured along with weight-specific growth rate and condition index for 28 days. 15% substitution led to great results, whereas 30% yeast-fed treatments displayed poor growth and a depressed metabolism

The Influence of Water Nitrate Concentration Combined with Elevated Temperature on Rainbow Trout <i>Oncorhynchus mykiss</i> in an Experimental Aquaponic Setup

Intensive recirculating systems are a fast-developing sector of aquaculture. While several warm-water fish have been reared in aquaponics, almost no data are available for cold-water species. The determination of nitrate toxicity thresholds in recirculating aquaculture is crucial. Different pollutants are typically more toxic at elevated temperatures. We investigated the performance of Oncorhynchus mykiss under two different nitrate levels and two temperatures. We applied a 2 × 2 factorial design, where fish (9.78 ± 0.51 g) were exposed to nitrate concentrations of 40 or 110 mg/L NO3− and to temperatures of 17 °C or 21 °C for 20 days. This study focused on understanding the physiological responses of rainbow trout to relatively low nitrate levels under heat stress in order to investigate the feasibility of integrating this species into commercial aquaponics. The growth, condition, and expression of genes involved in metabolism, heat shock, antioxidant, and immune response were assessed in the liver, together with the activities of enzymes related to glucose and fatty acid metabolism. High nitrate levels at 17 °C affected the condition but did not alter growth, leading to increased glycolytic potential and, occasionally, a greater reliance on lipid oxidation. Antioxidant defense was mainly induced due to high nitrates and the similar expression patterns of antioxidant genes observed under high nitrate at both 17 °C and 21 °C. Warm exposure decreased condition and growth, leading to greatly reduced glucokinase transcription, irrespective of the nitrate levels. Exposure to 21 °C and high nitrate led to equivalent growth and condition as well as to a milder inflammatory response combined with metabolic readjustments (enhancement of glycolytic and lipid oxidation pathways) compared to the low nitrates at 21 °C. Based on the results, rearing at a temperature close to 21 °C should be avoided for fingerling growth, while NO3− concentration until 110 mg/L may not have severe impacts on fingerling health and growth at 17 °C. In addition, rainbow trout fingerlings can tolerate a 20-day exposure at 21 °C and NO3− up to 110 mg/L. Additional factors should always be considered, such as specific water quality parameters, for a comprehensive approach to assessing the feasibility of rainbow trout aquaculture in aquaponics

Are Marine Heatwaves Responsible for Mortalities of Farmed <i>Mytilus galloprovincialis</i>? A Pathophysiological Analysis of <i>Marteilia</i> 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