Heat hardening enhances mitochondrial potential for respiration and oxidative defence capacity in the mantle of thermally stressed Mytilus galloprovincialis

Ectotherms are exposed to a range of environmental temperatures and may face extremes beyond their upper thermal limits. Such temperature extremes can stimulate aerobic metabolism toward its maximum, a decline in aerobic substrate oxidation, and a parallel increase of anaerobic metabolism, combined with ROS generation and oxidative stress. Under these stressful conditions, marine organisms recruit several defensive strategies for their maintenance and survival. However, thermal tolerance of ectothermic organisms may be increased after a brief exposure to sub- lethal temperatures, a process known as "hardening". In our study, we examined the ability of M. galloprovincialis to increase its thermal tolerance under the effect of elevated temperatures (24, 26 and 28 °C) through the "hardening" process. Our results demonstrate that this process can increase the heat tolerance and antioxidant defense of heat hardened mussels through more efficient ETS activity when exposed to temperatures beyond 24 °C, compared to non-hardened individuals. Enhanced cell protection is reflected in better adaptive strategies of heat hardened mussels, and thus decreased mortality. Although hardening seems a promising process for the maintenance of aquacultured populations under increased seasonal temperatures, further investigation of the molecular and cellular mechanisms regulating mussels’ heat resistance is required

Heat hardening enhances metabolite-driven thermoprotection in the Mediterranean mussel Mytilus galloprovincialis

Introduction: Temperature affects organisms’ metabolism and ecological performance. Owing to climate change, sea warming constituting a severe source of environmental stress for marine organisms, since it increases at alarming rates. Rapid warming can exceed resilience of marine organisms leading to fitness loss and mortality. However, organisms can improve their thermal tolerance when briefly exposed to sublethal thermal stress (heat hardening), thus generating heat tolerant phenotypes.Methods: We investigated the “stress memory” effect caused by heat hardening on M. galloprovincialis metabolite profile of in order to identify the underlying biochemical mechanisms, which enhance mussels’ thermal tolerance.Results: The heat hardening led to accumulation of amino acids (e.g., leucine, isoleucine and valine), including osmolytes and cytoprotective agents with antioxidant and anti-inflammatory properties that can contribute to thermal protection of the mussels. Moreover, proteolysis was inhibited and protein turnover regulated by the heat hardening. Heat stress alters the metabolic profile of heat stressed mussels, benefiting the heat-hardened individuals in increasing their heat tolerance compared to the non-heat-hardened ones.Discussion: These findings provide new insights in the metabolic mechanisms that may reinforce mussels’ tolerance against thermal stress providing both natural protection and potential manipulative tools (e.g., in aquaculture) against the devastating climate change effects on marine organisms

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

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

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

Assessment of Stocking Activities on the Native Brown Trout Populations from Nestos River (Southern Balkans) Inferred by mtDNA RFLP and Sequencing Analyses

Alien fish introductions, conducted towards the ichthyofauna enhancement in local drainages, have been occasionally proved harmful for the indigenous freshwater fish populations. The present study was designed to assess the impact of stocking activities, carried out in the past decades with fingerlings originating from Acheloos river hatcheries, on the native trout (Salmo sp.) populations of Nestos River, Greece. Trout specimens collected from several tributaries of Nestos River and were analyzed by means of PCR-RFLP and sequencing targeting the mitochondrial ND5-ND6 genes and the entire control region, respectively. It should be mentioned that trouts from Acheloos mainly belong to the marmoratus mitochondrial DNA (mtDNA) lineage, while the autochthonous trouts from Nestos belong to the Adriatic lineage. Both methodologies demonstrated that most samples from the three tributaries located at the lower part of Nestos constitute offspring of the fingerlings transferred from Acheloos hatcheries. Therefore, these tributaries have been strongly affected by stocking activities with a potential complete loss of their autochthonous trout. On the other hand, it seems that trout populations from higher altitude tributaries have not been affected by stockings. Hence, efforts should be undertaken in order to prevent the prevalence of the non-indigenous translocated Salmo in higher altitude tributaries, in conjunction with a management plan designed for the total trout populations from the area, speaking of which it has been recently included to the National Park of Rodopi Mountains

Genetic structure and molecular identification of Mediterranean mussel populations (Mytilus galloprovincialis), from the Eastern Mediterranean Sea

Genetic variation of mussels (Mytilus galloprovincialis) in central–eastern Mediterranean Sea was investigated in this thesis, by means of nuclear and mitochondrial DNA (mtDNA) molecular markers analyses. Mussels, collected from Greece, Italy, Croatia and Turkey, were genotyped at 10 microsatellite loci and their genetic structure was also investigated by mtDNA sequence analysis. In addition, the potential presence of other Mytilus species in eastern Mediterranean Sea was examined. Finally a fast and low-cost method was developed for the reliable identification of the Mediteranean mussel from Mytilus chilensis, that is imported in Greece as a deep frozen unshelled product.Στην παρούσα διατριβή διερευνήθηκε η γενετική σύσταση πληθυσμών του μυδιού (Mytilus galloprovincialis) από την κεντρική και ανατολική Μεσόγειο, μέσω της ανάλυσης του πυρηνικού και του μιτοχονδριακού DNA, με χρήση σύγχρονων μοριακών τεχνικών. Στο πυρηνικό DNA εξετάστηκαν 10 μικροδορυφορικοί τόποι ενώ για την ανάλυση του mtDNA εφαρμόστηκε η μέθοδος της εύρεσης της πρωτοδιάταξης των νουκλεοτιδίων (αλληλούχιση) σε πληθυσμούς από την Ελλάδα, την Ιταλία, την Κροατία και την Τουρκία. Επιπλέον εκτιμήθηκε η επίδραση ανθρωπογενών παραγόντων στη γενετική σύσταση των πληθυσμών που συλλέχθηκαν. Τέλος εξετάστηκε η ενδεχόμενη παρουσία άλλου είδους του γένους Mytilus στην κεντρική και ανατολική Μεσόγειο, ενώ αναπτύχθηκε και μια ταχεία - χαμηλού κόστους μέθοδος για την αξιόπιστη διάκριση του Μεσογειακού μυδιού από το εισαγόμενο μύδι της Χιλής (Mytilus chilensis)

First insights towards the population genetic structure and the phylogeographic status of the horse mussel (Modiolus barbatus) from the eastern Mediterranean

The horse mussel Modiolus barbatus is a marine benthic bivalve, distributed mainly in the Mediterranean basin, that constitutes a fishery product of high economic importance and a promising candidate for aquaculture. The current study provides the first insights regarding the genetic profile of M. barbatus populations from the eastern Mediterranean, by analysis of a partial segment of the mitochondrial COI gene in individuals collected from five sampling localities within the Aegean Sea. To the best of our knowledge, the derived haplotypes represent the first DNA barcodes of M. barbatus from the entire Mediterranean region. Maximum likelihood phylogenetic analysis revealed that M. barbatus from the North Sea and M. barbatus from the eastern Mediterranean may not belong to the same species and as a consequence, there might be three species of the genus Modiolus in Europe. On the other hand, eastern Mediterranean M. barbatus haplotypes were found to be more closely related to the Asian-Pacific Modiolus species. All geographic populations analysed displayed high levels of genetic diversity, in terms of haplotype and nucleotide diversity and a considerable number of unique alleles. Divergence among populations was found at generally low levels, corresponding with the majority of pairwise Fst values not being significant. These findings suggest no population structure and high levels of gene flow, a common feature observed in marine bivalves with long pelagic larval phases. © Copyright Marine Biological Association of the United Kingdom 2018