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.

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.

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.

Ultrafast Photophysics of Regioisomeric Triphenylamine Dyes Having Dipolar D-π-A-A and Octupolar D-(π-A-A)3 Character with a Benzothiazole Unit in Matched or Mismatched Orientation.

Benzothiazole is among prominent electron-withdrawing heteroarene moieties used in a variety of π-conjugated molecules. Its relative orientation with respect to the principal dipole vector(s) of chromophores derived thereof is crucial, affecting photophysical and nonlinear optical properties. Here we compare the photophysics and ultrafast dynamics of dipolar and octupolar molecules comprising a triphenylamine electron-donating core, ethynylene π-conjugated linker(s) and benzothiazole acceptor(s) having the matched or mismatched orientation (with respect to the direction of intramolecular charge transfer), while a carbaldehyde group is attached as an auxiliary acceptor. Among chromophores without the auxiliary acceptor, stronger fluorescence solvatochromism and faster excited state dynamics are exhibited for the derivatives with the mismatched geometry. On the contrary, introduction of the auxiliary acceptor to the benzothiazole unit enhances the intramolecular charge transfer ICT (featuring ultrafast dynamics of the excited state) for the matched geometry. The data confirm the crucial role of the relative orientation of asymmetric heteroaromatic unit (regioisomeric effect) in dipolar as well as in multipolar molecules in tuning linear and nonlinear optical properties as well as excited state dynamics.

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.

Red porgy's (Pagrus pagrus) cellular physiology and antioxidant defense in response to seasonality.

Physiological stress patterns of marine organisms in their natural habitats are considerably complex in space and time. These patterns can eventually contribute in the shaping of fish' thermal limits under natural conditions. In the view of the knowledge gap regarding red porgy's thermal physiology, in combination with the characterization of the Mediterranean Sea as a climate change ''hotspot'', the aim of the present study was to investigate this species biochemical responses to constantly changing field conditions. To achieve this goal, Heat Shock Response (HSR), MAPKs pathway, autophagy, apoptosis, lipid peroxidation and antioxidant defense were estimated and exhibited a seasonal pattern. In general, all the examined biochemical indicators expressed high levels parallel to the increasing seawater temperature in spring, although several bio-indicators have shown increased levels when fish were cold-acclimatized. Similar to other sparids, the observed patterns of physiological responses in red porgy may support the concept of eurythermy.

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.

Metabolic remodeling caused by heat hardening in the Mediterranean mussel Mytilus galloprovincialis.

Organisms can modify and increase their thermal tolerance faster and more efficiently after a brief exposure to sublethal thermal stress. This response is called 'heat hardening' as it leads to the generation of phenotypes with increased heat tolerance. The aim of this study was to investigate the impact of heat hardening on the metabolomic profile of Mytilus galloprovincialis in order to identify the associated adjustments of biochemical pathways that might benefit the mussels' thermal tolerance. Thus, mussels were exposed sequentially to two different phases (heat hardening and acclimation phases). To gain further insight into the possible mechanisms underlying the metabolic response of the heat-hardened M. galloprovincialis, metabolomics analysis was complemented by the estimation of mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK) and alternative oxidase (AOX) implicated in the metabolic pathways of gluconeogenesis, glycolysis and redox homeostasis, respectively. Heat-hardened mussels showed evidence of higher activity of the tricarboxylic acid (TCA) cycle and diversification of upregulated metabolic pathways, possibly as a mechanism to increase ATP production and extend survival under heat stress. Moreover, formate and taurine accumulation provide an antioxidant and cytoprotective role in mussels during hypoxia and thermal stress. Overall, the metabolic responses in non-heat-hardened and heat-hardened mussels underline the upper thermal limits of M. galloprovincialis, set at 26°C, and are in accordance with the OCLTT concept. The ability of heat-hardened mussels to undergo a rapid gain and slow loss of heat tolerance may be an advantageous strategy for coping with intermittent and often extreme temperatures.

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.

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.

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.

Treatment with ascorbic acid normalizes the aerobic capacity, antioxidant defence, and cell death pathways in thermally stressed Mytilus galloprovincialis.

Considering temperature's upcoming increase due to climate change, combined with the fact that Mediterranean mussels Mytilus galloprovincialis (Lamarck, 1819) live at their upper limits [critical temperatures (Tc) beyond 25 °C], we cannot be sure of this species' sustainable future in the Mediterranean Sea. Deviation from optimum temperatures leads to cellular damage due to oxidative stress. Although ascorbic acid (AA) is a major scavenger of reactive oxygen species (ROS), its capacity to minimize oxidative stress effects is scarcely studied in aquatic organisms. Thus, treatment with 5 mM and 10 mM AA of thermally stressed molluscs had been employed in order to examine its antioxidant capacity. While 5 mM had no effect, 10 mM normalized COX1 and ND2 relative mRNA levels, and superoxide dismutase (SOD), catalase, and glutathione reductase (GR) enzymatic activity levels in both examined tissues: posterior adductor muscle (PAM) and mantle. ATP levels, probably providing the adequate energy for antioxidant defence in thermally stressed mussels, is also normalized under 10 mM AA treatment. Moreover, autophagic indicators such as LC3 II/I and SQSTM1/p62 levels are normalized, indicating autophagy amelioration. Apoptosis also seems to be inhibited since both Bax/Bcl-2 and cleaved caspase substrate levels decrease with 10 mM AA treatment. Therefore, treatment of mussels with AA seems to produce threshold effects, although the precise underlying mechanisms must be elucidated in future studies. These findings show that treatment of mussels with effective antioxidants can be useful as a strategic approach for the reduction of the deleterious effects on mussels' summer mortality in aquaculture zones.

Energetic, antioxidant, inflammatory and cell death responses in the red muscle of thermally stressed Sparus aurata.

For a comprehensive understanding of fish responses to increasing thermal stress in marine environments, we investigated tissue energetics, antioxidant levels, inflammatory and cell death responses in Sparus aurata (gilthead seabream) red muscle during exposure to elevated temperatures (24 °C, 26 °C, 30 °C) compared to the control temperature of 18 °C. Energetic aspects were assessed by determining lactate, glucose and lipids levels in blood plasma, ATP, ADP and AMP levels, and AMPK phosphorylation as an indicator of regulatory changes in energy metabolism, in tissue extracts. Oxidative defence was assessed by determining superoxide dismutase, catalase and glutathione reductase maximum activities. Moreover, xanthine levels were determined as an indicator of purine conversion to xanthine and associated ROS production. In the context of inflammatory response and cell death due to oxidative stress, pro-inflammatory cytokines (IkBα phosphorylation, IL-6 and TNFα) levels, and LC3 II/I ratio and SQSTM1/p62 as indicators of autophagic-lysosomal pathway were also determined. A recovery in the efficacy of ATP production after a marked decrease during the 1st day of exposure to 24 °C is observed. This biphasic pattern is paralleled by antioxidant enzymes' activities and inflammatory and autophagy responses, indicating a close correlation between ATP turnover and stress responses, which may benefit tissue function and survival. However, exposure beyond 24 °C caused tissue's antioxidant capacity loss, triggering the inflammatory and cell death response, leading to increased fish mortality. The results of the present study set the thermal limits of the gilthead seabream at 22-24 °C and establish the used cellular and metabolic indicators as tools for the definition of the extreme thermal limits in marine organisms.

The anticoagulant activity of enoxaparin sodium during on-line hemodiafiltration and conventional hemodialysis.

To study and compare the anticoagulant activity of enoxaparin sodium during on-line hemodiafiltration (OL-HDF) and conventional hemodialysis (C-HD). Enoxaparin was administered as an anticoagulant to 21 hemodialysis patients at the beginning of a single 4-hour OL-HDF session as an intravenous bolus dose of 80 IU/kg [DOSAGE ERROR CORRECTED] On-line hemodiafiltration was performed using a high-flux polyester polymer alloy dialyzer and a total of 18 L replacement fluid (session A). One week later, the study was repeated in the same patients during a single 4-hour session of C-HD using a low-flux polysulfone dialyzer (session B). Blood samples for the measurement of Hb, blood urea and nitrogen (BUN), activated partial thromboplastin time (APTT), and anti-Xa levels were taken before each study session and 5-minute postdialysis. In 13 more patients, the same study was performed during OL-HDF using a high-flux polysulfone dialyzer (session C). No differences were found between sessions A, B, and C when predialysis values for Hb, BUN, APTT, and anti-Xa were compared. The mean postdialysis APTT and anti-Xa values were 32.5+/-3.8 seconds and 0.19+/-0.11 IU/mL, respectively, in session A, 39.0+/-5.0 seconds and 0.71+/-0.17 IU/mL in session B, and 33.8+/-3.1 seconds and 0.35+/-17 IU/mL in session C (A vs. B, P<0.0001, for both parameters, A vs. C, P<0.003 for anti-XA, and B vs. C, P<0.005, for both parameters). The anticoagulant activity of enoxaparin sodium is decreased significantly during a 4-hour OL-HDF session compared with to a similar session of C-HD. The degree of the reduction seems to depend on the dialyzer's membrane.

Veno-occlusive disease prophylaxis with fresh frozen plasma and heparin in bone marrow transplantation.

Fresh frozen plasma (FFP) contains natural anticoagulants, such as antithrombin (AT) and Protein C (Prot-C). We hypothesized that FFP given in addition to heparin, could potentially replace the consumption of endogenous anticoagulants occurring during conditioning and moreover, corrected AT levels could augment heparin's anticoagulant function. This could therefore result in an effective anti-VOD prophylaxis. In this study, we retrospectively analyzed the incidence of hepatic VOD in 403 consecutive bone marrow transplants (BMTs) comparing 2 prophylactic regimens and no prophylaxis. Patients received no prophylaxis (70/403), heparin-only (27/403) or heparin+2FFP daily during conditioning (306/403). VOD was significantly lower in the heparin+FFP group (5.9%) compared to heparin (20%) and no prophylaxis group (15.7%) [p<0.01]. Day 8 AT and Prot-C levels, were lower in the VOD- compared to the non-VOD group (AT: 69+/-26% vs. 89+/-19%, Prot-C:68+/-26% vs. 91+/-29%, respectively, p=0.001). In a multivariate logistic regression, risk factors for developing VOD were: the administration of >2 hepato-nephrotoxic drugs, previous history of hepatitis B or C and number of BMT. Multivariate analysis in a subset of 198 patients (all having recorded AT, Prot-C), demonstrated as VOD-related factors, the low day 8 Prot-C, number of BMT>1 and prior abdominal radiotherapy. Our study implies that FFP during conditioning, in addition to heparin, potentially has an anti-VOD prophylactic effect, presumably by minimizing the drop of natural anticoagulants around day 8. In order to evaluate if there truely is a beneficial effect of heparin+FFP in VOD prophylaxis, we have initiated a prospective randomized trial.

Heparin solution locked in acute hemodialysis catheters: impact on activated partial thromboplastin time.

Fifteen patients on hemodialysis, wearing acute dual lumen hemodialysis catheters (DLHCs) locked with a 4 ml heparin solution (HS) containing 7,500 IU of conventional heparin (3,750 IU/lumen), were studied. After the preexisting HS was aspirated and discarded, 10 ml of blood was withdrawn from each lumen in a syringe. Then, two 3 ml blood samples (A and B) were withdrawn in a row from the venous lumen; a third blood sample (C) was withdrawn from a peripheral vein, and the 20 ml of blood in the syringe was returned to the patient. Patient to control ratios of activated partial thromboplastin time (aPTT) in samples A, B, and C were 2.87 +/- 1.04, 2.02 +/- 0.85, and 1.06 +/- 0.14, respectively (p < 0.002 comparing A to B and B to C). In these patients, we also studied the effect of the same HS on the aPTT, 10 minutes after the filling of the DLHCs postdialysis. A blood sample (H1) was withdrawn at the end of the session; 10 minutes after injecting each lumen of the DLHC with a 2 ml HS containing 3,750 IU of heparin, a second blood sample (H2) was drawn from a peripheral vein. The aPTT ratios in samples H1 and H2 were 1.15 +/- 0.13 and 3.58 +/- 0.61 respectively (p < 0.0001). We concluded that even after the aspiration of 15 ml from the venous lumen of a DLHC filled with a 2 ml HS containing 3,750 IU of heparin, the next blood sample remains contaminated by heparin. Filling each lumen of a DLHC with the same HS results in a significant increment of the aPTT 10 minutes later.