Genetic prospective of a local invader: the strange pattern of Pontastacus leptodactylus population structure in Greece and Turkey based on microsatellite DNA.

BACKGROUND: The combination of the increasing demand of freshwater crayfish exports, the reduced population sizes due to overfishing, the crayfish plague epidemics and the habitat degradation, have led to unrecorded translocations of Pontastacus leptodactylus in Greek lakes. METHODS AND RESULTS: In the present study, the genetics of five narrow clawed crayfish (P. leptodactylus) populations were studied, namely three translocated populations inhabiting in Northern Greece, one native Greek population from Evros river and one potential progeny source population from Turkey. Nine microsatellite loci previously designed for the specific species were investigated, in order to assess the levels of genetic diversity and further to confirm the origin of these translocated populations some decades after the translocation events. Our results confirmed that the source population for the translocated Greek population is the Turkish lake Egirdir. Further, despite the low values of the number of alleles, heterozygosity, and FST the populations were generally diverse, providing evidence for local adaptation. CONCLUSIONS: The low values of FIS for the translocated populations in combination with the high values of gene flow, possibly indicate the existence of re-introducing events. Apart from the translocated populations, high levels of genetic diversity and heterozygosity were observed in Evros population, suggesting it as a possible unit for future conservation purposes both as a donor population for reintroduction purposes as well as a unique gene pool protection source. To the best of our knowledge this is the first study dealing with the genetic composition of Greek P. leptodactylus populations from Nothern Greece, operating as a first step towards the development of proper management practices for restocking events and monitoring of translocated populations.

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.

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.

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.

Antioxidant and DNA-Protective Activity of an Extract Originated from Kalamon Olives Debittering.

Table olives are a major component of the Mediterranean diet and are associated with many beneficial biological activities, which are mainly related to their phenolic compounds. Olive fruit debittering process defines the quantitative and qualitative composition of table olives in biophenols. The aim of the present study was to evaluate the in vitro antioxidant capacity and DNA-protective activity of an extract originated from brine samples, according to the Greek style debbitering process of Kalamon olive fruits. The main phenolic components determined in the brine extract were hydroxytyrosol (HT), verbascoside (VERB) and tyrosol (T). The in vitro cell-free assays showed strong radical scavenging capacity from the extract, therefore antioxidant potential. At cellular level, human endothelial cells (EA.hy296) and murine myoblasts (C2C12) were treated with non-cytotoxic concentrations of the brine extract and the redox status was assessed by measuring glutathione (GSH), reactive oxygen species (ROS) and lipid peroxidation levels (TBARS). Our results show cell type specific response, exerting a hormetic reflection at endothelial cells. Finally, in both cell lines, pre-treatment with brine extract protected from H2O2-induced DNA damage. In conclusion, this is the first holistic approach highlighted table olive wastewaters from Kalamon- Greek style debittering process, as valuable source of bioactive compounds, which could have interesting implications for the development of new products in food or other industries.

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.

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.

Determination of Redox Status in Different Tissues of Lambs and Kids and Their in-between Relationship.

The objective of this study was to assess the resting values of the physiological oxidative stress exhibited by lambs and kids reared in Greece, and the potential correlations between redox biomarker levels in blood and other tissues (liver, diaphragm, quadriceps, psoas major muscle). For this purpose, lambs and kids at different developmental stages (d.s.) were used. The latter corresponded to four live weight categories (LWC), each representing 25%, 35%, 70% and 100% of mature body weight. In each of the above tissues, the levels of five common redox biomarkers were determined: glutathione (GSH), catalase (CAT), total antioxidant capacity (TAC), thiobarbituric reactive substances (TBARS), and protein carbonyls (CARBS). The results revealed that lambs and kids belonging to the 35% LWC had weaker endogenous antioxidant pools, while animals in the 70% and 100% LWC had elevated intrinsic antioxidant defense systems. Blood redox biomarkers were associated with the respective ones measured in the diaphragm, liver, quadriceps, and psoas major of both species. Importantly, TBARS levels in blood of animals in the 25% and 100% LWC are correlated with the TBARS levels in all other tissues tested. Blood antioxidant parameters might be used as potential biomarkers to predict the antioxidant status of tissues that affect meat quality. The latter would facilitate quality assessment prior to slaughter, allowing for timely nutritional interventions that can improve meat products.

Pollution Indicators and HAB-Associated Halophilic Bacteria Alongside Harmful Cyanobacteria in the Largest Mussel Cultivation Area in Greece.

Taking into consideration the essential contribution of Mytilus galloprovincialis farming, it is of rising importance to add knowledge regarding bacterial species occurrence in water samples from aquaculture zones from the point of view of both the organism and public health. In the present study, we investigated the bacterial community existing in water samples from six Mytilus galloprovincialis aquaculture areas in the Thermaikos gulf, northern Greece, that may provoke toxicity in aquatic organisms and humans and may indicate environmental pollution in mussel production as well as algal blooms. Bacterial species were identified molecularly by sequencing of a partial 16s rRNA segment and were analyzed phylogenetically for the confirmation of the bacterial taxonomy. The results obtained revealed the presence of four bacterial genera (Halomonas sp., Planococcus sp., Sulfitobacter sp., and Synechocystis sp.). Members of the Halomonas and Sulfitobacter genera have been isolated from highly polluted sites, Planococcus bacteria have been identified in samples derived directly from plastic debris, and Synechocystis bacteria are in line with microcystin detection. In this context, the monitoring of the bacteria community in mussel aquaculture water samples from the Thermaikos gulf, the largest mussel cultivation area in Greece, represents an indicator of water pollution, microplastics presence, algal blooms, and toxin presence.