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.

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.

Seasonal energy investment and metabolic patterns in a farmed fish.

The present research focuses on the seasonal changes in the energy content and metabolic patterns of red porgy (Pagrus pagrus) sampled in a fish farm in North Evoikos Gulf (Greece). The study was designed in an effort to evaluate the influence of seasonality in several physiological feauteres of high commercial importance that may affect feed intake and growth. We determined glycogen, lipids and proteins levels, and cellular energy allocation (CEA) as a valuable marker of exposure to stress, which integrates available energy (Ea) and energy consumption (Ec). Metabolic patterns and aerobic oxidation potential were based on the determination of glucose transporter (GLU), carnitine transporter (CTP), L-lactate dehydrogenase (L-LDH), citrate synthase (CS), cytochrome C oxidase subunit IV isoform 1 (COX1) and 3-hydroxyacyl CoA dehydrogenase (HOAD) relative gene expression. To integrate metabolic patterns and gene expression, L-LDH, CS, COX and HOAD activities were also determined. For further estimation of biological stores oxidized during seasonal acclimatization, we determined the blood levels of glucose, lipids and lactate. The results indicated seasonal changes in energy content, different patterns in gene expression and reorganization of metabolic patterns during cool acclimatization with increased lipid oxidation. During warm acclimatization, however, energy consumption was mostly based on carbohydrates oxidation. The decrease of Ec and COX1 activity in the warm exposed heart seem to be consistent with the OCLTT hypothesis, suggesting that the heart may be one of the first organs to be limited during seasonal warming. Overall, this study has profiled changes in energetics and metabolic patterns occurring at annual temperatures at which P. pagrus is currently farmed, suggesting that this species is living at the upper edge of their thermal window, at least during summer.

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.

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.

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.

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.

The current situation and potential effects of climate change on the microbial load of marine bivalves of the Greek coastlines: an integrative review.

Global warming affects the aquatic ecosystems, accelerating pathogenic microorganisms' and toxic microalgae's growth and spread in marine habitats, and in bivalve molluscs. New parasite invasions are directly linked to oceanic warming. Consumption of pathogen-infected molluscs impacts human health at different rates, depending, inter alia, on the bacteria taxa. It is therefore necessary to monitor microbiological and chemical contamination of food. Many global cases of poisoning from bivalve consumption can be traced back to Mediterranean regions. This article aims to examine the marine bivalve's infestation rate within the scope of climate change, as well as to evaluate the risk posed by climate change to bivalve welfare and public health. Biological and climatic data literature review was performed from international scientific sources, Greek authorities and State organizations. Focusing on Greek aquaculture and bivalve fisheries, high-risk index pathogenic parasites and microalgae were observed during summer months, particularly in Thermaikos Gulf. Considering the climate models that predict further temperature increases, it seems that marine organisms will be subjected in the long term to higher temperatures. Due to the positive linkage between temperature and microbial load, the marine areas most affected by this phenomenon are characterized as 'high risk' for consumer health.

First report of canine Astrovirus and Sapovirus in Greece, hosting both asymptomatic and gastroenteritis symptomatic dogs.

BACKGROUND: Astrovirus, Norovirus and Sapovirus are widely distributed viruses in humans and animals worldwide. They have frequently been associated with disease, mainly of gastroenteric nature. In dogs, these viruses have been detected both in symptomatic and asymptomatic animals, mainly of young age. METHODS: In the present epidemiologic study, we investigated the presence of canine Astrovirus (CAstV), canine Norovirus (canine NoV) and canine Sapovirus (Canine SaV) in saliva and stools of 201 domestic dogs originating from throughout Greece, based on two different molecular methods, i.e. conventional and SYBR-Green Real-time RT-PCR. The samples derived from young and adult asymptomatic and symptomatic animals. CAstV was detected in 15/201 (7.5%) and 29/201 (15%) of the examined dogs using conventional RT-PCR and SYBR-Green Real time RT-PCR, respectively. RESULTS: The prevalence of the virus was higher at healthy dogs, with a slight discrepancy of the two methods on the aspect of age (67% young dogs with the method of conventional RT-PCR, versus 52% adult positive dogs with the method of SYBR-Green Real-time RT-PCR). Canine SaV was detected in 52/201 (23%) of the dogs (mainly young and asymptomatic), with the method of SYBR-Green Real-time RT-PCR only, while canine NoV was not detected in any sample with either of the two methods applied. Sequencing of the CAstV positive samples resulted in the acquisition of one CAstV sequence. Phylogenetic analysis confirmed the results, clustering the CAstV sequence with homologous canine hosting sequences from other countries. CONCLUSIONS: CAstV and Canine SaV were proved to circulate in Greek dogs. SYBR-Green Real time RT-PCR showed greater sensitivity in the detection of these viruses. Additionally, we were able to specify the CAstV strain that circulates in Greece, through phylogenetic analysis. To our knowledge, this is the first epidemiological study of CAstV and canine SaV in dogs in Greece, as well as the first time detected in dogs from Greece.

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.

Brucella spp. distribution, hosting ruminants from Greece, applying various molecular identification techniques.

BACKGROUND: Brucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers. RESULTS: Only one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples. CONCLUSIONS: The present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.

Origin, demographics, inbreeding, phylogenetics, and phenogenetics of Karamaniko breed, a major common ancestor of the autochthonous Greek sheep.

Greece has a long history in autochthonous sheep, the genetic ancestry of which has been associated with four subtypes known to inhabit Greece at the end of the nineteenth century. Among them, the Karamaniko breed is still surviving, however endangered. This study was designed in order to (a) determine the phylogenetic status, (b) to evaluate the levels of inbreeding, and (c) to assess the genetic basis of coat color of Karamaniko breed. For these purposes, the mitochondrial cyt b gene was sequenced, the AFLP methodology was applied, and the MC1R was genotyped, respectively, in 72 female sheep from the Karamaniko breed. Four different novel cyt b haplotypes were defined and three MC1R genotypes were scored, whereas inbreeding levels estimated using AFLPs by the means of relatedness coefficient (r) were 0.287, with gene diversity at the levels of 0.105. Phylogenetic analysis indicated an eastern Asian tropical and subtropical origin of the Karamaniko breed, close with breeds originating from central Turkey, or a clustering within western European or Mediterranean sheep, mirroring a recent genetic divergence, with a non-random spread towards the formation of lowland breeds. The MC1R genotypes were all associated with the white coat color, in which selective breeding has probably been based on traditional morphological characters. Finally, levels of inbreeding do not constitute an indication for a particular mating plan to prevent unpleasant phenomena such as inbreeding depression, probably because of the special attention paid by the farmers towards the avoidance of relative recurrent mating.

Molecular analysis of Greek isolates of cucumber mosaic virus from vegetables shows a low prevalence of satellite RNAs and suggests the presence of host-associated virus strains.

Cucumber mosaic virus (CMV) is a generalist pathogen that infects many economically important crops in Greece. The present study was designed to evaluate the genetic variability of Greek CMV isolates in combination with their satellite RNAs (satRNAs). To achieve this goal, 77 CMV isolates were collected from symptomatic Greek vegetables, mainly tomatoes and cucurbits, alongside their neighboring crops, during a four-year period from 2015 to 2018. Phylogenetic analysis of a partial coat protein (CP) gene segment revealed that all of the isolates belong to CMV subgroups IA and IB and that they are closely related to previously reported Greek isolates. It should be noted, however, that the latter mainly included tomato isolates. Network analysis of the evolutionary relationships among the CP sequences of the Greek isolates in comparison to the corresponding sequences obtained from the GenBank database indicated two predominant common ancestors and at least three differentiated peripherals, and possibly host-associated (tomatoes, legumes, cucurbits) haplogroups (strain groups). More specifically, host-adaptive evolution can be postulated regarding the tomato isolates in subgroup IB. Necrogenic or non-necrogenic satRNAs were detected in four samples from tomato and melon, and this is the first report of non-necrogenic satRNAs in CMV in Greece.

Advances in understanding the impacts of global warming on marine fishes farmed offshore: Sparus aurata as a case study.

Monitoring variations in proteins involved in metabolic processes, oxidative stress responses, cell signalling and protein homeostasis is a powerful tool for developing hypotheses of how environmental variations affect marine organisms' physiology and biology. According to the oxygen- and capacity-limited thermal tolerance hypothesis, thermal acclimation mechanisms such as adjusting the activities of enzymes of intermediary metabolism and of antioxidant defence mechanisms, inducing heat shock proteins (Hsps) or activating mitogen-activated protein kinases may all shift tolerance windows. Few studies have, however, investigated the molecular, biochemical and organismal responses by fishes to seasonal temperature variations in the field to link these to laboratory findings. Investigation of the impacts of global warming on fishes farmed offsore, in the open sea, can provide a stepping stone towards understanding effects on wild populations because they experience similar environmental fluctuations. Over the last 30 years, farming of the gilthead sea bream Sparus aurata (Linnaeus 1758) has become widespread along the Mediterranean coastline, rendering this species a useful case study. Based on available information, the prevailing seasonal temperature variations expose the species to the upper and lower limits of its thermal range. Evidence for this includes oxygen restriction, reduced feeding, reduced responsiveness to environmental stimuli, plus a range of molecular and biochemical indicators that change across the thermal range. Additionally, close relationships between biochemical pathways and seasonal patterns of metabolism indicate a connection between energy demand and metabolic processes on the one hand, and cellular stress responses such as oxidative stress, inflammation and autophagy on the other. Understanding physiological responses to temperature fluctuations in fishes farmed offshore can provide crucial background information for the conservation and successful management of aquaculture resources in the face of global change.

Correlation between intermediary metabolism, Hsp gene expression, and oxidative stress-related proteins in long-term thermal-stressed Mytilus galloprovincialis.

Long-term exposure of Mytilus galloprovincialis to temperatures beyond 26°C triggers mussel mortality. The present study aimed to integratively illustrate the correlation between intermediary metabolism, hsp gene expression, and oxidative stress-related proteins in long-term thermally stressed Mytilus galloprovincialis and whether they are affected by thermal stress magnitude and duration. We accordingly evaluated the gene expression profiles, in the posterior adductor muscle (PAM) and the mantle, concerning heat shock protein 70 and 90 (hsp70 and hsp90), and the antioxidant defense indicators Mn-SOD, Cu/Zn-SOD, catalase, glutathione S-transferase, and the metallothioneins mt-10 and mt-20. Moreover, we determined antioxidant enzyme activities, oxidative stress through lipid peroxidation, and activities of intermediary metabolism enzymes. The pattern of changes in relative mRNA expression levels indicate that mussels are able to sense thermal stress even when exposed to 22°C and before mussel mortality is initiated. Data indicate a close correlation between the magnitude and duration of thermal stress with lipid peroxidation levels and changes in the activity of antioxidant enzymes and the enzymes of intermediary metabolism. The gene expression and increase in the activities of antioxidant enzymes support a scenario, according to which exposure to 24°C might trigger reactive oxygen species (ROS) production, which is closely correlated with anaerobic metabolism under hypometabolic conditions. Increase and maintenance of oxidative stress in conjunction with energy balance disturbance seem to trigger mussel mortality after long-term exposure at temperatures beyond 26°C. Eventually, in the context of preparation for oxidative stress, certain hypotheses and models are suggested, integrating the several steps of cellular stress response.

Identification of Leishmania Species in Naturally Infected Sand Flies from Refugee Camps, Greece.

High infection rates of Leishmania donovani and L. tropica were detected in Phlebotomus spp. sand flies collected from refugee camps in Greece, indicating increased risk of infection among local populations. Detection and treatment of leishmaniasis, community education, and vector control are essential measures to prevent pathogen transmission and protect public health.

Mitochondrial control region variability in Mytilus galloprovincialis populations from the central-Eastern Mediterranean Sea.

The variable domain 1 (VD1) domain of the control region and a small segment of the rrnaL gene of the F mtDNA type were sequenced and analyzed in 174 specimens of Mytilus galloprovincialis. Samples were collected from eight locations in four Central-Eastern (CE) Mediterranean countries (Italy, Croatia, Greece and Turkey). A new primer, specific for the F mtDNA type, was designed for the sequencing procedure. In total 40 different haplotypes were recorded, 24 of which were unique. Aside from the two populations situated in Thermaikos gulf (Northern Aegean, Greece), relatively high levels of haplotype and nucleotide diversity were estimated for both Central and Eastern Mediterranean populations. Eight out of the 40 haplotypes were shared by at least three populations while two of them were found in all populations. ΦST and cluster analysis revealed lack of structuring among CE Mediterranean populations with the exception of those located at the Sea of Marmara and Croatian coast which were highly differentiated. Apart from the species' inherit dispersal ability, anthropogenic activities, such as the repeated translocations of mussel spat, seem to have played an important role in shaping the current genetic population structure of CE M. galloprovincialis mussels.

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.

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.