Core vs. diet -associated and postprandial bacterial communities of the rainbow trout (Oncorhynchus mykiss) midgut and feaces

ACKNOWLEDGMENTS: Eleni Mente was awarded a visiting fellowship by Marine Alliance for Science and Technology Scotland (MASTS). Part of Eleni’s Nikouli’s work in this paper was carried out under the program “Scholarships of IKY in the Marine and Inland Management of Water Resources” and was co-funded by EEA grants– Financial Mechanism 2009-2014 (85%) and the General Secretariat for Investments and Development (15%). The authors declare no competing interests.Peer reviewedPublisher PD

Bacterial biofilm development during experimental degradation of <em>Melicertus kerathurus</em> exoskeleton in seawater

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

Gut Bacterial Communities in Geographically Distant Populations of Farmed Sea Bream (Sparus aurata) and Sea Bass (Dicentrarchus labrax)

This study investigated the profile of the autochthonous gut bacterial communities in adult individuals of Sparus aurata and Dicentrarchus labrax reared in sea cages in five distantly located aquaculture farms in Greece and determine the impact of geographic location on them in order to detect the core gut microbiota of these commercially important fish species. Data analyses resulted in no significant geographic impact in the gut microbial communities within the two host species, while strong similarities between them were also present. Our survey revealed the existence of a core gut microbiota within and between the two host species independent of diet and geographic location consisting of the Delftia, Pseudomonas, Pelomonas, Propionibacterium, and Atopostipes genera

Composition, structure and ecophysiological role of bacterial communities at gastrointestinal tract of aquacultured fish species

Studies have shown that host, diet and habitat can affect fish gut bacterial diversity, but even under the same environmental conditions, significant differences may occur. Up-to-date studies on fish bacterial symbionts are related to the effect of diet on the structure of these bacterial communities. This thesis studied for the first time the autochthonous gut bacterial diversity of Sparus aurata and Dicentrarchus labrax individuals originating from five distantly located aquaculture installations in Greece, in order to determine the impact of geographic location and to reveal their core gut microbiota.Moreover, we assessed whether five fish (Sparus aurata, Dicentrarchus labrax, Diplodus puntazzo, Pagrus pagrus, Argyrosomous regius) species cultured on the same site and fed with isoenergetic and isoproteic diets differ in their gut bacterial communities. Finally, we also investigated the host-associated bacterial succession during the early embryonic stages and first feeding in farmed Sparus aurata and Salmo salar populations.We analyzed the 16S rRNA gene diversity of Bacteria, targeting the V3-V4 region by using next generation sequencing techniques (454 pyrosequencing, illumina). Data analyses resulted in no significant geographic impact in the gut bacterial communities within the two allopatric host species (S. aurata, D. labrax), while strong similarities between them were also present. Our survey revealed the existence of a core gut microbiota within and between them independent of diet and geographic location consisting of the Delftia, Pseudomonas, Pelomonas, Propionibacterium, and Atopostipes genera.Results also revealed that the OTUs richness between the five sympatric host species (S. aurata, D. labrax, D. puntazzo, P. pagrus, A. regius) did not vary significant, but only 4% of the found OTUs belong to the core gut microbiota, indicating selective pressure within the gut, favoring the establishment and development of specific bacterial species in the gastrointestinal system in each of the five fish species. Results regarding the host associated bacterial succession in S. aurata and S. salar larviculture showed significantly higher species richness in rearing water samples. OTUs were shared between larvae and environmental samples (rearing water and diet), no overlap was detected between their bacterial communities.Overall this thesis contributes to broadening our knowledge on the composition and structure of the gut bacterial communities in fish species, their possible biological roles and also in understanding the factors that influence interactions between hosts and gut microbiota.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

Gut Bacterial Communities in Geographically Distant Populations of Farmed Sea Bream (Sparus aurata) and Sea Bass (Dicentrarchus labrax)

This study investigated the profile of the autochthonous gut bacterial communities in adult individuals of Sparus aurata and Dicentrarchus labrax reared in sea cages in five distantly located aquaculture farms in Greece and determine the impact of geographic location on them in order to detect the core gut microbiota of these commercially important fish species. Data analyses resulted in no significant geographic impact in the gut microbial communities within the two host species, while strong similarities between them were also present. Our survey revealed the existence of a core gut microbiota within and between the two host species independent of diet and geographic location consisting of the Delftia, Pseudomonas, Pelomonas, Propionibacterium, and Atopostipes genera

Host-Associated Bacterial Succession during the Early Embryonic Stages and First Feeding in Farmed Gilthead Sea Bream (Sparus aurata)

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

Freshwater-adapted sea bass Dicentrarchus labrax feeding frequency impact in a lettuce Lactuca sativa aquaponics system

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

Reshaping gut bacterial communities after dietary Tenebrio molitor larvae meal supplementation in three fish species

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

Dietary Lipid Effects on Gut Microbiota of First Feeding Atlantic Salmon (Salmo salar L.)

Decline in fish oil and fish meal availability has forced the aquaculture sector to investigate alternative and sustainable aquafeed ingredients. Despite that several studies have evaluated the effect of fish oil replacement in aquaculture fish species, there is a knowledge gap on the effects of alternative dietary lipid sources on the gut microbiota in early life stages of Salmo salar. The present study evaluated the influence of dietary administration of two different lipid sources (fish oil and vegetable oil) on the intestinal microbiota of first feeding Atlantic salmon (S. salar) up to 93 days post first feeding (dpff). The two diets used in this study, FD (fish oil diet) and VD (blend of rapeseed, linseed and palm oils diet), were formulated to cover the fish nutritional requirements. Apart from the lipid source, the rest of the feed components were identical in the two diets. Hindgut samples were collected at 0, 35, 65, and 93 dpff. Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also collected in order to assess a possible contribution of their microbiota to the colonization and bacterial succession of the fish intestines. To analyze the bacterial communities, amplicon sequencing was used targeting the V3–V4 region of the 16S rRNA gene. The findings indicate that feeding on either fish oil or vegetable oil-based diet, fish growth variables (mean wet weight and total length) did not differ significantly during the experiment (p > 0.05). No significant differences were also found between the two dietary groups, regarding their gut bacteria composition, after the analysis of the 16S rRNA sequencing data. Instead, gut microbiota changed with age, and each stage was characterized by different dominant bacteria. These operational taxonomic units (OTUs) were related to species that provide different functions and have been isolated from a variety of environments. The results also show little OTUs overlap between the host and rearing environment microbiota. Overall, this study revealed the occurrence of a core microbiota in early life of Atlantic salmon independent of the feed-contained oil origin