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.

Evidence for Beneficial Physiological Responses of the Land Snail Cornu aspersum to Probiotics' (Lactobacillus plantarum) Dietary Intervention.

A large variety of microorganisms ingested with food constitute animals' intestinal microflora, enhancing and maintaining the homeostasis of the host. Rations enriched with probiotics are a method recommended to prevent undesirable conditions. To date, research has been limited to farmed animals and reared fish, creating a knowledge gap concerning the effect of probiotics on the growth rate, physiological responses, and energy metabolism of invertebrates such as the land snail Cornu aspersum. Herein, juvenile snails (26.23 ± 0.5 mm shell diameter and 8.23 ± 1.02 g body weight) were fed L. plantarum probiotic-enriched rations in two different proportions (1.25 mg and 2.5 mg), and their growth rate was monitored for three months. Additionally, the RNA/DNA and Bax/Bcl-2 ratios, HSP gene expression and protein levels, and ND2 expression, were measured in the hepatopancreas, digestive tract, and mantle. Although the snails' growth rate was not affected, the RNA/DNA ratio presented an increase in various tissues, indicating an intense physiological response. Also, probiotic administration demonstrated low levels of the Bax/Bcl-2 ratio. HSP levels were higher in the presence of probiotics, probably signaling an attempt by the animal to face potentially stressful situations. Finally, ND2 expression levels in the hepatopancreas indicate intense metabolic and antioxidant activity.

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.

Association of MTNR1A and GDF9 gene allelles with the reproductive performance, response to oestrus induction treatments and prolificacy, in improved and non-improved local indigenous sheep breeds.

Sheep farming plays a crucial role for Mediterranean countries, wherefrom a plethora of dairy products is produced. Yet, numerous indigenous sheep breeds in temperate latitudes such as the Mediterranean present a serious drawback that milk production is impaired by the seasonality of reproduction. Towards the efforts for reduction of this phenomenon, the purpose of this study was to evaluate and compare different oestrus synchronization treatments, as well as to associate two genes alleles', namely the MTNR1A and the GDF9, with the response to those treatments. Three indigenous breeds were investigated (Florina, Chios and Karagouniko sheep) and inside each breed three different oestrus synchronization treatments were applied (A: intravaginal sponges, B: GNRH use and C: male effect). In group A, Florina ewes expressed oestrus at 90% in July and fecundity was 85%. Karagouniko and Chios ewes exhibited an oestrus expression of 100% with fecundity rates at 95% and 99%, respectively. In group B, Florina ewes expressed oestrus at 60% with fecundity at 57%, Karagouniko ewes expressed oestrus at 65% with fecundity at 54%, whereas Chios breed animals expressed oestrus at 87% with fecundity rate at 85%. In group C, 68% of the Florina breed expressed oestrus 20-25 days post ram induction, whereas this proportion was 84% and 94%, for Karagouniko and Chios breed, respectively. For the molecular analysis, partial segments of the two genes were sequenced and analysed, whereas alleles were scored based on the detected SNPs. All frequencies of the four detected SNPs in MTNR1A gene were statistically and significantly different in ewes that expressed oestrus in comparison with ewes that did not express oestrus in Florina and Karagouniko breeds concerning all treatments. Two SNP's were detected in GDF9 gene, G1 and FecG , from which, only the FecG mutation exhibited statistically significant difference in twins and triplets than in singles in Florina and Karagouniko breeds.

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.

Probiotics, Prebiotics, and Synbiotics Utilization in Crayfish Aquaculture and Factors Affecting Gut Microbiota.

Aquaculture is affected by numerous factors that may cause various health threats that have to be controlled by the most environmentally friendly approaches. In this context, prebiotics, probiotics, and synbiotics are frequently incorporated into organisms' feeding rations to ameliorate the health status of the host's intestine, enhancing its functionality and physiological performance, and to confront increasing antimicrobial resistance. The first step in this direction is the understanding of the complex microbiome system of the organism in order to administer the optimal supplement, in the best concentration, and in the correct way. In the present review, pre-, pro-, and synbiotics as aquaculture additives, together with the factors affecting gut microbiome in crayfish, are discussed, combined with their future prospective outcomes. Probiotics constitute non-pathogenic bacteria, mainly focused on organisms' energy production and efficient immune response; prebiotics constitute fiber indigestible by the host organism, which promote the preferred gastrointestinal tract microorganisms' growth and activity towards the optimum balance between the gastrointestinal and immune system's microbiota; whereas synbiotics constitute their combination as a blend. Among pro-, pre-, and synbiotics' multiple benefits are boosted immunity, increased resistance towards pathogens, and overall welfare promotion. Furthermore, we reviewed the intestinal microbiota abundance and composition, which are found to be influenced by a plethora of factors, including the organism's developmental stage, infection by pathogens, diet, environmental conditions, culture methods, and exposure to toxins. Intestinal microbial communities in crayfish exhibit high plasticity, with infections leading to reduced diversity and abundance. The addition of synbiotic supplementation seems to provide better results than probiotics and prebiotics separately; however, there are still conflicting results regarding the optimal concentration.

High-Resolution Melting (HRM) Analysis for Rapid Molecular Identification of Sparidae Species in the Greek Fish Market.

The red porgy (Pagrus pagrus) and the common dentex (Dentex dentex) are Sparidae species of high commercial value, traded in the Greek market. In some cases, fish species identification from Greek fisheries is difficult for the consumer due to the strong morphological similarities with their imported counterparts or closely related species such as Pagrus major, Pagrus caeroleustictus, Dentex gibbosus and Pagellus erythrinus, especially when specimens are frozen, filleted or cooked. Techniques based on DNA sequencing, such as COI barcoding, accurately identify species substitution incidents; however, they are time consuming and expensive. In this study, regions of mtDNA were analyzed with RFLPs, multiplex PCR and HRM in order to develop a rapid method for species identification within the Sparidae family. HRM analysis of a 113 bp region of cytb and/or a 156 bp region of 16s could discriminate raw or cooked samples of P. pagrus and D. dentex from the aforementioned closely related species and P. pagrus specimens sampled in the Mediterranean Sea when compared to those fished in the eastern Atlantic. HRM analysis exhibited high accuracy and repeatability, revealing incidents of mislabeling. Multiple samples can be analyzed within three hours, rendering this method a useful tool in fish fraud monitoring.

Development of Multiplex PCR and Melt-Curve Analysis for the Molecular Identification of Four Species of the Mullidae Family, Available in the Market.

The authentication of food products and the verification of their identity are of major importance for consumers. Food fraud through mislabeling is an illegal practice consisting of the substitution of an expensive food product by a relatively cheaper one, misleading false labelling of their origin and adulteration in processed or frozen products. This issue is particularly of high importance concerning fish and seafood, which are easily adulterated primarily due to difficult morphological identification. Fish species of the Mullidae family are considered among the most high-valued seafood products traded in Greece and Eastern Mediterranean in general, in terms of the price and demand. Specifically, the red mullet (Mullus barbatus) and the striped red mullet (Mullus surmuletus) are both indigenous in the Aegean (FAO Division 37.3.1) and the Ionian (FAO Division 37.2.2) Seas, with high levels of consumers' preferences. However, they could be easily adulterated or misidentified by the invasive Aegean Sea Lessepsian migrator goldband goatfish (Upeneus moluccensis) as well as by the imported West African goatfish (Pseudupeneus prayensis). Keeping this in mind, we designed two novel, time-saving and easy-to-apply multiplex PCR assays and one multiple Melt-Curve analysis real-time PCR for the identification of these four species. These methodologies are based on species-specific primers targeting single nucleotide polymorphisms (SNPs) detected via sequencing analysis of the mitochondrial cytochrome C oxidase subunit I (CO1) and of the cytochrome b (CYTB) genes in newly collected individuals, with additional comparison with congeneric and conspecific haplotypes obtained from the GenBank database. Both methodologies, targeting CO1 or CYTB, utilize one common and four diagnostic primers, producing amplicons of different length that are easily and reliably separated on agarose gel electrophoresis, yielding a single clear band of diagnostic size for each species or a certain Melt-Curve profile. The applicability of this cost-effective and fast methodology was tested in 328 collected specimens, including 10 cooked samples obtained from restaurants. In the vast majority (327 out of the 328) of the specimens tested, one single band was produced, in agreement with the expected products with a single exception a M. barbatus sample that was identified as M. surmuletus, the identity of which was confirmed using sequencing, indicating erroneous morphological identification. The developed methodologies are expected to contribute to the detection of commercial fraud in fish authentication.

Applicability evaluation of mtDNA based molecular identification in mosquito species/subspecies/biotypes collected from Thessaloniki, Greece.

The genus Culex, containing many described species, plays a role as a vector for diseases of medical and veterinary importance worldwide. Among these species, Culex pipiens is one of the most widespread mosquitoes and is classified into two biological forms (biotypes), named as Culex pipiens pipiens and Culex pipiens molestus. Due to similar morphological structure between these biotypes, morphological identification is inadequate. Thus, molecular methods have been developed and are considered more reliable, some of which are based on analyses of mitochondrial DNA. The aim of the present study was to evaluate the applicability and reliability of mtDNA based molecular identification methodologies. Initially, mosquito specimens (n = 100) collected from Thessaloniki, Greece were morphologically analyzed. Then, mitochondrial cox1 sequencing and PCR-RFLP methods were used to confirm the morphological identification results as well as to discriminate species and subspecies/biotype of Culex pipiens complex. According to morphological identification results, Culex pipiens complex (n = 92), Culex modestus (n = 6) and Culex theileri (n = 2) were detected. Using mtDNA sequencing, all Culex modestus and Culex theileri samples were confirmed whereas 86 of Culex pipiens complex were detected as Culex pipiens but surprisingly the remaining six of them were detected as Culex quinquefasciatus. Among Culex pipiens specimens, PCR-RFLP detected that frequency of Culex pipiens pipiens (85%; 85/100) was very high compared to Culex pipiens molestus (1%, 1/100). In conclusion, this study shows the necessity of use of molecular methods beside morphological methods for especially specimens detected as Culex pipiens. Also, it was shown that mtDNA PCR-RFLP methodology represents a well-established alternative for Culex biotypes identification.

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.

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.

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.

Isolation of Listeria monocytogenes from poultry red mite (Dermanyssus gallinae) infesting a backyard chicken farm in Greece.

The poultry red mite (PRM), Dermanyssus gallinae, is arguably the most harmful, ubiquitous haematophagous ectoparasite infesting egg-laying hens. PRM is a vector of various microorganisms, with some being important for food microbiology and public health. The present study aimed to investigate the presence of specific pathogens, including Escherichia coli, Salmonella spp. and Listeria spp., carried by PRM infesting a chicken farm in Greece. Mites were caught using cardboard traps (Avivet), and 100 unwashed PRM were homogenized and used for microbiological cultures. Microbiological cultures were carried out on general and selective substrates to detect the above-mentioned bacteria. Specifically for Listeria spp., DNA was extracted from bacteria grown in Tryptone Soya Yeast Extract Agar using a commercial kit. The hly gene encoding the Listeriolysin O protein was amplified by PCR. Mites were identified as D. gallinae using morphological keys as well as by COI DNA barcoding. Microbiological cultures and PCR assays were positive for Listeria monocytogenes. No other bacteria were detected. The current study constitutes the first molecular isolation of L. monocytogenes from D. gallinae, confirming that PRM can carry this food-borne pathogen. PRM control measures and hygiene practices should be applied to minimize any possible contamination risk of poultry products with L. monocytogenes and safeguard public health.