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

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

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

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

Conservation physiology of marine fishes: state of the art and prospects for policy

The state of the art of research on the environmental physiology of marine fishes is reviewed from the perspective of how it can contribute to conservation of biodiversity and fishery resources. A major constraint to application of physiological knowledge for conservation of marine fishes is the limited knowledge base; international collaboration is needed to study the environmental physiology of a wider range of species. Multifactorial field and laboratory studies on biomarkers hold promise to relate ecophysiology directly to habitat quality and population status. The 'Fry paradigm' could have broad applications for conservation physiology research if it provides a universal mechanism to link physiological function with ecological performance and population dynamics of fishes, through effects of abiotic conditions on aerobic metabolic scope. The available data indicate, however, that the paradigm is not universal, so further research is required on a wide diversity of species. Fish physiologists should interact closely with researchers developing ecological models, in order to investigate how integrating physiological information improves confidence in projecting effects of global change; for example, with mechanistic models that define habitat suitability based upon potential for aerobic scope or outputs of a dynamic energy budget. One major challenge to upscaling from physiology of individuals to the level of species and communities is incorporating intraspecific variation, which could be a crucial component of species' resilience to global change. Understanding what fishes do in the wild is also a challenge, but techniques of biotelemetry and biologging are providing novel information towards effective conservation. Overall, fish physiologists must strive to render research outputs more applicable to management and decision-making. There are various potential avenues for information flow, in the shorter term directly through biomarker studies and in the longer term by collaborating with modellers and fishery biologists.EU COST Action FA1004 Conservation Physiology of Marine Fishesinfo:eu-repo/semantics/publishedVersio

Interactions of temperature and pH on the regulatory properties of pyruvate kinase from organs of a marine mollusc

The effects of low temperature assay (5 °C) on the properties of the aerobic (low phosphate) vs. anoxic (high phosphate) forms of pyruvate kinase (PK) from foot muscle and gill of the whelk Busycon canaliculatum (L.) were assessed at two pH values, pH 7.00 and 7.25, and compared to control conditions of 20 °C and pH 7.00 (all assayed in imidazole buffer). When pH was held constant at 7.00, the decrease in assay temperature to 5 °C had large effects on the measured kinetic parameters of all PK forms, as compared to 20 °C and pH 7.00. However, when assay pH was allowed to rise, from 7.00 to 7.25, with the temperature decrease to 5 °C there were fewer alterations of kinetic parameters and quantitatively smaller changes to enzyme properties. It appears, then, that when pH rises with decreasing temperature following alphastat predictions, kinetic properties of PK are largely conserved. Low temperature, at either pH value, had several significant effects on PK properties. For example, low temperature raised the S0.5 for phosphoenolpyruvate of PK-anoxic from gill by 3-6 fold and decreased the I50 Mg · ATP for PK-anoxic from foot by the same amount. Arrhenius plots of PK activity for the gill PK forms showed a distinct break at 10 °C; > 10 °C Q10 was 2.5 whereas < 10 °C Q10 was 8.4. Temperature-dependent changes in all cases affected enzyme properties in a manner that would restrict enzyme function at low temperature

Seawater carbonate chemistry and processes during experiments with marine mussel, Mytilus galloprovincialis, 2005

In the context of future scenarios of progressive accumulation of anthropogenic CO2 in marine surface waters, the present study addresses the effects of long-term hypercapnia on a Mediterranean bivalve, Mytilus galloprovincialis. Sea-water pH was lowered to a value of 7.3 by equilibration with elevated CO2 levels. This is close to the maximum pH drop expected in marine surface waters during atmosextracellular pHric CO2 accumulation. Intra- and extracellular acid-base parameters as well as changes in metabolic rate and growth were studied under both normocapnia and hypercapnia. Long-term hypercapnia caused a permanent reduction in haemolymph pH. To limit the degree of acidosis, mussels increased haemolymph bicarbonate levels, which are derived mainly from the dissolution of shell CaCO3. Intracellular pH in various tissues was at least partly compensated; no deviation from control values occurred during long-term measurements in whole soft-body tissues. The rate of oxygen consumption fell significantly, indicating a lower metabolic rate. In line with previous reports, a close correlation became evident between the reduction in extracellular pH and the reduction in metabolic rate of mussels during hypercapnia. Analysis of frequency histograms of growth rate revealed that hypercapnia caused a slowing of growth, possibly related to the reduction in metabolic rate and the dissolution of shell CaCO3 as a result of extracellular acidosis. In addition, increased nitrogen excretion by hypercapnic mussels indicates the net degradation of protein, thereby contributing to growth reduction. The results obtained in the present study strongly indicate that a reduction in sea-water pH to 7.3 may be fatal for the mussels. They also confirm previous observations that a reduction in sea-water pH below 7.5 is harmful for shelled molluscs

Synergistic effects of acute warming and low pH on cellular stress responses of the gilthead seabream Sparus aurata

The present study assesses the resilience of the Mediterranean gilthead seabream (Sparus aurata) to acute warming and water acidification, using cellular indicators of systemic to molecular responses to various temperatures and CO2 concentrations. Tissue metabolic capacity derived from enzyme measurements, citrate synthase, 3-hydroxyacyl CoA dehydrogenase (HOAD), as well as lactate dehydrogenase. Cellular stress and signaling responses were identified from expression patterns of Hsp70 and Hsp90, the phosphorylation of p38 MAPK, JNKs and ERKs, from protein ubiquitylation and finally from the levels of transcription factor Hif-1α as an indicator of systemic hypoxemia. Exposure to elevated CO2 levels at temperatures higher than 24 °C generally caused an increase in fish mortality above the rate caused by warming alone, indicating effects of the two factors and a failure of acclimation and thus the limits of phenotypic plasticity to be reached. As a potential reason, tissue-dependent induction and stabilization of Hif-1α indicate hypoxemic conditions. Their exacerbation by enhanced CO2 levels is linked to the persistent expression of Hsp70 and Hsp90, oxidative stress and activation of MAPK and ubiquitin pathways. Antioxidant defence is enhanced by expression of catalase and glutathione reductase, however, leaving superoxide dismutase suppressed by elevated CO2 levels. On longer timescales in specimens surviving warming and CO2 exposures, various metabolic adjustments initiate a preference to oxidize lipid via HOAD for energy supply. These processes indicate significant acclimation up to a limit and a time-limited capacity to survive extreme conditions passively by exploiting mechanisms of cellular resilience