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Fish microbiome science is progressing fast, but it is biased toward farmed or laboratory fish species against natural fish populations, which remain considerably underinvestigated. We analyzed the midgut bacterial microbiota of 45 specimens of 12 fish species collected from the Gyaros Island marine protected area (Aegean Sea, Greece). The species belong to seven taxonomic families and are either herbivores or omnivores. Mucosa midgut bacterial diversity was assessed by amplicon metabarcoding of the 16S rRNA V3–V4 gene region. A total of 854 operational taxonomic units (OTUs) were identified. In each fish species, between 2 and 18 OTUs dominated with cumulative relative abundance ≥ 70%. Most of the dominating bacterial taxa have been reported to occur both in wild and farmed fish populations. The midgut bacterial communities were different among the 12 fish species, except for Pagrus pagrus and Pagellus erythrinus, which belong to the Sparidae family. No differentiation of the midgut bacterial microbiota was found based on feeding habits, i.e., omnivorous vs. carnivorous. Comparing wild and farmed P. pagrus midgut bacterial microbiota revealed considerable variation between them. Our results expand the gut microbiota of wild fish and support the host species effect as the more likely factor shaping intestinal bacterial microbiota.

Geothermal springs are essentially un-affected by environmental conditions aboveground as they are continuously supplied with subsurface water with little variability in chemistry. Therefore, changes in their microbial community composition and function, especially over a long period, are expected to be limited but this assumption has not yet been rigorously tested. Toward closing this knowledge gap, we applied whole metagenome sequencing to 17 water samples collected between 2010 and 2016 from the Thermopyles sulfur-rich geothermal springs in central Greece. As revealed by 16S rRNA gene fragments recovered in the metagenomes, Epsilonproteobacteria-related operational taxonomic units (OTUs) dominated most samples and grouping of samples based on OTU abundances exhibited no apparent seasonal pattern. Similarities between samples regarding functional gene content were high, with all samples sharing >70% similarity in functional pathways. These community-wide patterns were further confirmed by analysis of metagenome-assembled genomes (MAGs), which showed that novel species and genera of the chemoautotrophic Campylobacterales order dominated the springs. These MAGs carried different pathways for thiosulfate or sulfide oxidation coupled to carbon fixation pathways. Overall, our study showed that even in the long term, functions of microbial communities in a moderately hot terrestrial spring remain stable, presumably driving the corresponding stability in community structure. This article is protected by copyright. All rights reserved.

Η παρούσα Διδακτορική διατριβή, επικεντρώθηκε στη μελέτη των αυτόχθονων βακτηριακών κοινοτήτων του γαστρεντερικού συστήματος έξι (6) ειδών εκτρεφόμενων ιχθύων (Sparus aurata, Dicentrarchus labrax, Diplodus puntazzo, Pagrus pagrus, Argyrosomous regius και Salmo salar). Σκοπός αποτέλεσε η ανίχνευση παραγόντων που συνθέτουν τις γαστρεντερικές βακτηριακές τους κοινότητες (γεωγραφική θέση – απόσταση, συνθήκες εκτροφής κλπ.), την εύρεση κοινών βακτηριακών ειδών σε αλλοπάτριους και συμπάτριους πληθυσμούς ιχθύων και ο προσδιορισμός του λειτουργικού τους ρόλου στο γαστρεντερικό τους σύστημα. Η βακτηριακή ανίχνευση στο DNA των δειγμάτων που χρησιμοποιήθηκαν στην παρούσα διδακτορική διατριβή πραγματοποιήθηκε με μεθόδους αλληλούχισης νέας γενιάς (Roche 454 FLX titanium και Illumina MiSeq 2x300), στοχεύοντας την περιοχή V3-V4 του γονιδίου 16S rRNA.Η μελέτη των γαστρεντερικών βακτηριακών κοινοτήτων σε εκτρεφόμενα άτομα (4-6) αλλοπάτριων πληθυσμών ιχθύων (S. aurata και D. labrax), από πέντε διαφορετικές και απομακρυσμένες μεταξύ τους μονάδες εκτροφής στην Ελλάδα (Ηγουμενίτσα, Αταλάντη, Γιάλτρα, Χίο, Χανιά), έδειξε ότι η γεωγραφική θέση – απόσταση, δεν επηρέασε σημαντικά τη δομή και σύνθεση των γαστρεντερικών βακτηριακών κοινοτήτων σε διαφορετικούς πληθυσμούς του ίδιου είδους, αλλά ούτε μεταξύ των διαφορετικών ειδών ιχθύων. Μάλιστα ανεξαρτήτου γεωγραφικής θέσης οι ιχθυοπληθυσμοί του κάθε είδους παρουσίασαν μια μικρή ομάδα κοινών OTUs (8 OTUs στο S. aurata και 10 στο D. labrax), ενώ 5 OTUs βρέθηκαν κοινά και στα δύο είδη, υποδεικνύοντας την παρουσία κοινών βακτηριακών εκπροσώπων ακόμα και μεταξύ διαφορετικών ειδών ιχθύων. Ο λόγος του μικρού αριθμού κοινών OTUs πιθανόν να οφείλεται στις ατομικές μεταβολές που παρατηρήθηκαν στις γαστρεντερικές βακτηριακές κοινότητες, ακόμα και σε άτομα μεταξύ των ίδιων πληθυσμών και πιθανόν να σχετίζονται με γενετικούς παράγοντες. Τα βακτηριακά είδη στο γαστρεντερικό σύστημα του κάθε είδους ιχθύος φαίνεται να παρουσιάζουν συνεργατικές αλληλεπιδράσεις και διαδραματίζουν διαφορετικούς βιολογικούς ρόλους στο κάθε είδος που μελετήθηκε (S. aurata και D. labrax).Για περαιτέρω μελέτη της επίδρασης της γεωγραφικής θέσης στην σύνθεση και δομή των γαστρεντερικών βακτηριακών κοινοτήτων των ιχθύων, μελετήθηκαν δείγματα μεσέντερου από άτομα (3-5) συμπάτριων εκτρεφόμενων πληθυσμών των ειδών ιχθύων D. puntazzo, P. pagrus, A. regius, S. aurata και D. labrax. Τα αποτελέσματα έδειξαν, ότι παρόλο που οι γαστρεντερικές βακτηριακές τους κοινότητες δεν παρουσίασαν στατιστικώς σημαντικές διαφορές, μόνο ένα μικρό ποσοστό των OTUs (3,9%) βρέθηκε να είναι κοινό μεταξύ των προαναφερθέντων ειδών ιχθύων. Τα αποτελέσματα αυτά, υποδεικνύουν επιλεκτικές πιέσεις, ευνοώντας την εποίκιση και ανάπτυξη συγκεκριμένων βακτηριακών ειδών στο γαστρεντερικό σύστημα των πέντε ειδών ιχθύων.Τα αποτελέσματα που προέκυψαν από τη μελέτη της επίδρασης των συνθηκών εκτροφής στην πρώτη αποίκιση και διαδοχή των βακτηριακών κοινοτήτων σε ιχθύδια των ειδών S. aurata και S. salar, υπέδειξαν ομοιότητες μεταξύ των εκτρεφόμενων ειδών και των βακτηριακών κοινοτήτων του νερού εκτροφής και των παρεχόμενων τροφών. Ωστόσο πλήρη επικάλυψη μεταξύ αυτών δεν παρατηρήθηκε. Και στις δύο περιπτώσεις, σημαντική παρατήρηση αποτέλεσε η ανίχνευση OTUs στα γονιμοποιημένα αυγά των δύο εκτρεφόμενων ειδών, που δεν ανιχνεύθηκαν στο νερό εκτροφής, υποδεικνύοντας την πιθανή προέλευση τους από το μικροβίωμα των γεννητόρων. Γενικά η τροφή δεν επηρέασε σημαντικά την σύνθεση και δομή των βακτηριακών κοινοτήτων κατά τα χρονικά σημεία που μελετήθηκαν, ούτε στην περίπτωση της υποκατάστασης τουιχθυελαίου με έλαιο φυτικής προέλευσης, σε διατροφική μεταχείριση του είδους S. salar. Αντιθέτως, στο είδος αυτό ο παράγοντας της ηλικίας φάνηκε να επιδρά στη βακτηριακή ποικιλομορφία του γαστρεντερικού του συστήματος.Θα πρέπει να αναφερθεί ότι μέσα από τη φυλογενετική ανάλυση που πραγματοποιήθηκε στις βακτηριακές κοινότητες όλων των ειδών που μελετήθηκαν, καταγράφηκε επικράτηση των βακτηριακών φύλων Proteobacteria, Firmicutes, Actinobacteria και Bacteroidetes. Τα OTUs που ανιχνεύθηκαν σχετίζονται με βακτηριακά είδη που απομονώθηκαν από παρόμοια περιβάλλοντα, ενώ μερικά από αυτά (Weissela cibaria, Pseudomonas veronii, Ruegeria mobile κλπ.), όπως έχει καταγραφεί μέσα από άλλες εργασίες, φαίνεται να παρέχουν προβιοτικές ιδιότητες στα είδη ιχθύων που μελετήθηκαν εδώ.Συνολικά η παρούσα Διδακτορική διατριβή, συμβάλει στην διεύρυνση των γνώσεων μας για την σύνθεση και δομή των γαστρεντερικών βακτηριακών κοινοτήτων των ιχθύων, στις πιθανές βιολογικές τους σχέσεις και προσδιόρισε τους παράγοντες που τη μεταβάλλουν.

The aim of this study is to investigate the effect of three daily fish feeding frequencies, two, four and eight times per day (FF2, FF4, and FF8, respectively) on growth performance of sea bass (Dicentrarchus labrax)and lettuce plants (Lactuca sativa) reared in aquaponics. 171 juvenile sea bass with an average body weight of 6.80 ± 0.095 g were used, together with 24 lettuce plants with an average initial height of 11.78 ± 0.074 cm over a 45-day trial period. FF2 fish group showed a significantly lower final weight, weight gain and specific growth rate than the FF4 and FF8 groups. Voluntary feed intake was similar for all the three feeding frequencies treatmens (p > 0.05). No plant mortality was observed during the 45-day study period. All three aquaponic systems resulted in a similar leaf fresh weight and fresh and dry aerial biomass. The results of the present study showed that the FF4 or FF8 feeding frequency contributes to the more efficient utilization of nutrients for better growth of sea bass adapted to fresh water while successfully supporting plant growth to a marketable biomass.

The aquaculture industry is currently looking for alternative, sustainable diets that provide similar or better growth for the reared species. We investigated whether replacing fishmeal with yellow mealworm (Tenebrio molitor) meal in the supplied diets of the gilthead sea bream (Sparus aurata), European sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss) causes similar shifts in the bacterial gut communities of these farmed fish species. The diversity of the gut bacterial 16S rRNA gene revealed the presence of 598 operational taxonomic units (OTUs) belonging to all of the major phyla known to exist in the gut of these three fish species, such as Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. Although no statistically significant differences of diversity indices in all three species was found, there was a differential shift in the dominant bacteria in the gut of each species before and after the dietary meal replacement. In S. aurata and D. labrax diversity indices remained practically unchanged before and after the replacement, while for O. mykiss the five-fold increase of the Simpson dominance D index and the almost two-fold decrease of the Shannon H index, suggested that a more specialized gut bacterial community was favoured for this species after the replacement. Also, this replacement resulted in the occurrence of higher number of OTUs which were absent before the replacement in the gut of S. aurata (62.2% of all its OTUs) and D. labrax (60.0% of all its OTUs) compared to O. mykiss (33.0% of all its OTUs) suggesting that insect meal replacement resulted in novel nutritional niches in the gut of S. aurata and D. labrax compared to O. mykiss. Our results indicate that the most desirable fish diet substitution differentially affects the gut microbiota in different hosts, implying that a species-specific tailor-made approach in diet manipulations should be considered in the future.

Backgound: The present study investigated the effect of different lipid source in the feed on the colonization and the bacterial succession in early life stages (fertilized eggs until 93 days post first feeding) of S. salar. The two diets used in this study, FD (fish oil based diet) and VD (vegetable oil based diet), were formulated to cover the fish nutritional requirements and except the lipid source the components were identical between them.Hindgut samples collected at 0, 35, 65 and 93 days post first feeding (dpff). Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also sampled in order to assess a possible contribution of their microbiota to the colonization of the gut. To analyze the composition of the bacterial communities, the Illumina MiSeq platform was used. Results: S. salar growth variables (mean wet weight and total length) did not differ significantly during the experiment (p> 0.05) across replicate tanks and between dietary treatments. The analysis of the 16S rDNA sequencing data revealed a total of 4548 unique OTUs, affiliated in 21 bacterial phyla. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla. 13 OTUs were shared among all S. salar samples independent of life stage and diet treatment. Similarity percentages analysis (SIMPER) based on Bray–Curtis distance, showed that the average dissimilarity among the groups of the same life stages was 76.0%, whereas the average dissimilarity within groups of the same dietary treatment was 78.5% (FD) and 83.6% (VD). Conclusion: Feeding on either fish oil or vegetable oil-based diets, did not result in significant differences in the intestinal microbiota. The composition of gut microbiota did not differ significantly between the two dietary treatments, but changed with age, and each stage was characterized by different dominant bacteria. These OTUs are related to species that provide different functions and have been isolated from a variety of environments. Finally, this study revealed the occurrence of a core microbiota independent of the studied life stages and diet during the early life stages of Atlantic salmon.

Geothermal springs are barely affected by environmental conditions aboveground as they are continuously supplied with subsurface water with little variability in chemistry. Therefore, changes in their microbial community composition and function, especially over a long period, are expected to be limited but this assumption has not yet been rigorously tested. Toward closing this knowledge gap, we applied whole metagenome sequencing to 17 water samples collected between 2010 and 2016 (two to four samples per year) from the Thermopyles sulfur geothermal springs in central Greece. As revealed by 16S rRNA gene fragments recovered in the metagenomes, Epsilonproteobacteria-related operational taxonomic units (OTUs) dominated most samples, while grouping of samples based on OTU abundances exhibited no apparent seasonal pattern. Similarities between samples regarding functional gene content were high, especially in comparison to other surface water systems in Greece, with all samples sharing >70% similarity in functional pathways. These community-wide patterns were further confirmed by analysis of metagenome-assembled genomes (MAGs), which showed - in addition- that novel species and genera of the chemoautotrophic Campylobacterales order dominated the springs. These MAGs carried different pathways for thiosulfate and/or sulfide oxidation coupled to carbon fixation pathways. Overall, our study showed that even in the long term, functions of microbial communities in a moderately hot terrestrial spring remain stable, driving presumably the corresponding stability in community structure.

Despite the technical progress in high-throughput sequencing technologies, defining the sample size which is capable of yielding representative inferences in metabarcoding analysis still remains debatable. The present study addresses the influence of individual variability in assessing dietary effects on fish gut microbiota parameters and estimates the biological sample size that is sufficient to imprint a statistically secure outcome. European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) were fed three alternative animal protein diets and a fishmeal control diet. Gut microbiota data from 12 individuals per diet, derived from Illumina sequencing of the V3-V4 region of the 16S rRNA gene, were randomized in all possible combinations of n-1 individuals. Results in this study showcased that increasing the sample size can limit the prevalence of individuals with high microbial load on the outcome and can ensure the statistical confidence required for an accurate validation of dietary-induced microbe shifts. Inter-individual variability was evident in the four dietary treatments where consequently misleading inferences arose from insufficient biological replication. These findings have critical implications for the design of future metabarcoding studies and highlight the urgency in selecting an adequate sample size able to safely elucidate the dietary effects on fish gut microbial communities.

Chitinolytic bacteria are widespread in marine and terrestrial environment, and this is rather a reflection of their principle growth substrate’s ubiquity, chitin, in our planet. In this paper, we investigated the development of naturally occurring bacterial biofilms on the exoskeleton of the shrimp Melicertus kerathurus during its degradation in sea water. During a 12-day experiment with exoskeleton fragments in batch cultures containing only sea water as the growth medium at 18 °C in darkness, we analysed the formation and succession of biofilms by scanning electron microscopy and 16S rRNA gene diversity by next generation sequencing. Bacteria belonging to the γ- and α-Proteobacteria and Bacteroidetes showed marked (less or more than 10%) changes in their relative abundance from the beginning of the experiment. These bacterial taxa related to known chitinolytic bacteria were the Pseudolateromonas porphyrae, Halomonas aquamarina, Reinekea aestuarii, Colwellia asteriadis and Vibrio crassostreae. These bacteria could be considered as appropriate candidates for the degradation of chitinous crustacean waste from the seafood industry as they dominated in the biofilms developed on the shrimp’s exoskeleton in natural sea water with no added substrates and the degradation of the shrimp exoskeleton was also evidenced.

The reciprocal interaction between host organisms' physiology and their gut microorganism community is of great interest in aquatic animal biology and aquaculture but for crustaceans, it remains understudied. This study enhances our understanding of this community of microorganisms as it changes during the molt cycle. Because crustaceans shed a major component of their gut, and the associated microbiome, with each molt this adds a level of complexity heretofore unexamined. We have identified the bacterial communities that are affected by the changing gut environment and that may in turn, exert some control over aspects of the molt cycle. We investigated the structural changes of the resident gut bacterial communities, using the diversity of the 16S rRNA gene by 454 pyrosequencing, in the freshwater prawn Macrobrachium rosenbergii during its four-stage molt cycle. The number of bacterial operational taxonomic units (OTUs) increased from stages A to C. Stage C the intermolt and longest lasting stage was different in the gut bacterial community structure having the (a) highest number of total OTUs, (b) highest number of unique and newly introduced OTUs, (c) highest percentage of estimated specialists OTUs, i.e. that are more ecologically restricted. Moreover, stage C was characterized by greater contribution of Actinobacteria-related and unaffiliated OTUs. The most dominant OTUs found in stage C of the gut of M. rosenbergii were related to microorganisms involved in fermentation and food material processing originating from similar, i.e. gut, or habitats of terrestrial and freshwater animals. Thus, the distinct gut bacterial communities found in molting stage C corroborate with the physiological significance of this molting stage. The abiotic factors and the exact role of the corresponding specific bacterial communities in the animal's physiology and growth are areas that remain to be elucidated. Statement of relevance A thorough understanding of M. rosenbergii digestive physiology is essential to achieve better growth performance when cultured. This paper provides information that could be useful for developing effective strategies to manipulate gut microbial communities to promote prawn's growth and health and improve aquaculture productivity.

This study investigates Greek consumers' behavior towards organic aquaculture products, identifying possible distinct consumer groups and examining possible linkages between consumer characteristics and marketing aspects of organic aquaculture. Using data from an in-person field survey, a two-step cluster analysis demonstrated different attitudes among consumers with different profiles and also identified two distinct consumer groups differentiated primarily by income: the low-potential consumers and the high-potential consumers, representing 34% and 66% of the total sample, respectively. This study provides evidence that consumers' socioeconomic and demographic characteristics are key factors determining the consumers' purchasing behavior for organic aquaculture products. Thus, a market segmentation strategy is needed to allow organic aquaculture production to take advantage of the significant potential for growth in the near future.

One of the most widely reared fish in the Mediterranean Sea is Sparus aurata. The succession of S. aurata whole-body microbiota in fertilized eggs, five, 15, 21 and 71 days post hatch (dph) larvae and the contribution of the rearing water and the provided feed (rotifers, Artemia sp. and commercial diet) to the host&rsquo, s microbiota was investigated by 454 pyrosequencing of the 16S rRNA gene diversity. In total, 1917 bacterial operational taxonomic units (OTUs) were found in all samples. On average, between 93 &plusmn, 2.1 and 366 &plusmn, 9.2 bacterial OTUs per sample were found, with most of them belonging to Proteobacteria and Bacteroidetes. Ten OTUs were shared between all S. aurata stages and were also detected in the rearing water or diet. The highest OTU richness occurred at the egg stage and the lowest at the yolk sac stage (5 dph). The rearing water and diet microbial communities contributed in S. aurata microbiota without overlaps in their microbial composition and structure. The commercial diet showed higher contribution to the S. aurata microbiota than the rearing water. After stage D71 the observed microbiota showed similarities with that of adult S. aurata as indicated by the increased number of OTUs associated with &gamma, Proteobacteria and Firmicutes.

The reconstructed Lake Karla, Greece, has been undergoing its water-filling period since November 2009. In this paper, we aimed at investigating whether the unicellular eukaryotes, including the toxic/parasitic ones, that have been found during mass fish kills in the lake (March–April 2010), persist during the first warm period of the lake (May, August, November 2010). Given that microscopic characterization of some of these eukaryotes is not adequate for their identification, we analysed the 18S rRNA gene diversity of plankton samples. All the found phylotypes belonged to the phyla of Mesomycetazoa, Chlorophyta, Fungi, Alveolata, Cercozoa, Cryptophyta and Stramenopiles. Some members of these groups seem to persist in Lake Karla as they have been found in early spring as well. These microscopic eukaryotes are either ichthyotoxic/parasitic (e.g. Pfiesteria sp./Pseudopfiesteria shumwayae, some Fungi, Mesomycetazoa, Lagenidium sp., Cercozoa) or indicative of hyper-eutrophic conditions (e.g. Oocystis sp., Scenedesmus spp.) and were rather abundant during the first spring–autumn period of the lake’s refilling process. These complex microscopic communities are expected to shape highly dynamic and variable food webs with the risk of repeated fish kills.