Utility of dairy microbiome as a tool for authentication and traceability.

Milk microbiome contributes substantially to the formation of specific organoleptic and physicochemical characteristics of dairy products. The assessment of the composition and abundance of milk microbiota is a challenging task strongly influenced by many environmental factors. Specific dairy products may be designated by the Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) labeling, which however, occasionally fail to differentiate them according to specific quality characteristics, which are defined by different microbiota-driven reactions. Combining the above limitations, the scope of the present study, was to summarize the existing information toward three main issues. First, to assess the influence level of the diet type and grazing to rumen-GI tract, mammary gland, and udder microbiome formation in ruminants. Second, to discuss the factors affecting milk microbiota, as well as the effect of the endo-mammary route on milk microbial taxa. Lastly, to evaluate "milk microbiome" as a tool for product differentiation, according to origin, which will contribute to a more robust PDO and PGI labeling. Although the limitations are still a matter of fact (especially considering the sample collection, process, evaluation, and avoidance of its contamination), significant progress has been made, regarding the identification of the factors affecting dairy products' microbiota and its core composition. In conclusion, although so far not totally efficient in dairy products molecular identification, with the progress in soil, water, plant, and animal host's microbiota assembly's characterization, microbiomics could provide a powerful tool for authentication and traceability of dairy products.

A Sister Species for the Blue Crab, Callinectes sapidus? A Tale Revealed by Mitochondrial DNA.

The Atlantic blue crab, Callinectes sapidus, is acknowledged as one of the worst invasive alien species in the Mediterranean, impacting biodiversity and human activities. Native to the western Atlantic, it has expanded to European coastal waters since the early 1900s. Despite its ecological and commercial importance, genetic research on this species is limited. Here we show a comprehensive investigation of the genetic variation and evolutionary history in Callinectes sapidus using 667 mitochondrial COI gene sequences. Our dataset encompasses 36 newly generated sequences from previously understudied Mediterranean sites and 631 from worldwide locations obtained from the GenBank public database. Our findings reveal two distinct, but closely related, genetic groups within the species' distribution range, suggesting the occurrence of a potential species complex. Furthermore, in the Mediterranean, low levels of genetic variability were observed except for substantial haplotypic differentiation in Turkish samples. This study depicts the global genetic diversity and evolutionary patterns of Callinectes sapidus, offering new insights into the taxonomic status of the species.

Heat Hardening Ameliorates Apoptotic and Inflammatory Effects Through Increased Autophagy in Mussels.

The severity, frequency, and duration of extreme events, in the context of global warming, have placed many marine ecosystems at high risk. Therefore, the application of methods that can mediate the impacts of global warming on marine organisms seems to be an emerging necessity in the near term. In this context, enhancing the thermal resilience of marine organisms may be crucial for their sustainability. It has been shown that the repeated time-limited exposure of an organism to an environmental stimulus modifies its response mode, thus enhancing resilience and allowing adaptation of the physiological and developmental phenotype to environmental stress. In the present study, we investigated the "stress memory" effect caused by heat hardening on Mytilus galloprovincialis cellular pathways to identify the underlying biochemical mechanisms that enhance mussel thermal tolerance. Heat hardening resulted in increased ETS activity and ATP production and increased autophagic performance at all elevated temperatures (24 °C, 26 °C, and 28 °C). Furthermore, at these increased temperatures, apoptosis and inflammation remain at significantly lower levels in pregnant individuals than in nonhardened individuals. Autophagy, as a negative regulator of apoptosis, may lead to decreased damage to surrounding cells, which in turn alleviates inflammatory effects. In conclusion, the exposure of mussels to heat hardening seems to provide a physiological response that enhances heat tolerance and increases cell survival through increased energy production and reduced cell death and inflammatory responses. The latter can be utilized for the management and conservation of aquatic species of economic value or endangered status.

Prevalence of Neospora caninum and associated risk factors in semi-intensive female water buffaloes reared in Greece, using an ELISA kit on milk samples.

Neospora caninum is an apicomplexan (family: Sarcocystidae) protozoan parasite with a global distribution. In the N. caninum life cycle, dogs and other related canids are the definitive hosts, while other animals such as water buffaloes (Bubalus bubalis) constitute the intermediate host for this parasite. In many countries, the water buffalo is of high economic importance, providing valuable high-quality products for human needs. Although knowledge concerning the prevalence of this parasite in intermediate animal host populations is crucial, data from water buffalo are scarce. Keeping this in mind, the aim of this study was to examine the presence and assess the prevalence rates of N. caninum infection in water buffaloes in Northern Greece, where this animal husbandry sector started to raise, as well as to determine associated risk factors, with the application of a commercially available Neospora ISCOM ELISA test kit, developed for the detection of antibodies against N. caninum in milk samples The study was conducted during January-June 2023 in a total of 172 individual raw milk samples, collected from dairy buffaloes, reared under a semi extensive system, in three different farms. Information on the status of N. caninum infection in buffaloes from Greece was so far unknown with a lack of epidemiological or risk factors associated. For the detection of N. caninum, the commercially available Neospora ISCOM ELISA test kit (SANOVIR ®, Svanova Biotech AB, Uppsala, Sweden) was utilized. Results demonstrated the presence of N. caninum in water buffaloes from Greece for the first time. All positive N. caninum animal were asymptomatic, with absence of any disease signs. The overall prevalence of infection was 22.10%, whereas the main risk factors include the presence of dogs as well as the low biosecurity measures.

First detection of Cupriavidus gilardii in a bovine neonatal diarrhea outbreak.

BACKGROUND: Cupriavidus gilardii is an aerobic, gram-negative, motile, glucose-nonfermenting bacillus, first described in 1999. Typically, it exhibits low pathogenicity in humans, causing opportunistic infections primarily in individuals with compromised immune systems. This bacterium has been also found in various environmental sources such as plants and contaminated soils. Notably, there have been no documented cases of C. gilardii infections in animals. CASE PRESENTATION: This case report outlines a bovine neonatal diarrhea outbreak that occurred in Northern Greece, during which C. gilardii was isolated. Faecal samples from 5-day-old calves were collected and transported to the laboratory for further examination. Bacterial culture and next generation sequencing techniques were employed to confirm the presence of this bacterium in the samples. Following the isolation and identification of C. gilardii from the samples, an autogenous vaccine was produced and administered to the cows within the farm. Subsequent to vaccination, a progressive reduction in calf diarrhea and deaths was observed, leading to their eventual complete resolution. To the best of our knowledge, this represents the first documentation of C. gilardii isolation from cases of bovine neonatal diarrhea. CONCLUSION: This case report presents the first isolation case of C. gilardii from animal samples and more specifically from calf faecal samples. It represents an important observation, providing evidence that this opportunistic human pathogen could contribute to clinical symptoms in animals.

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.

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.

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.

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.

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.

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.

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.

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.

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.