The gills and skin microbiota of five pelagic fish species from the Atlantic Ocean.

The gills and skin microbiota and microbiome of wild fish remain far more under-investigated compared to that of farmed fish species, despite that these animal-microbe interactions hold the same ecophysiological roles in both cases. In this study, the gills and skin bacterial microbiota profiles and their presumptive bacterial metabolisms were investigated in five open-sea fishes: bullet tuna (Auxis sp.), common dolphinfish (Coryphaena hippurus), Atlantic little tunny (Euthynnus alletteratus), Atlantic bonito (Sarda sarda) and Atlantic white marlin (Kajikia albida). Gills and skin tissues were collected from two to three individuals per species, from specimens caught by recreational trolling during summer of 2019, and their bacterial 16S rRNA gene diversity was analysed by high-throughput sequencing. The gills bacterial communities among the five species were clearly different but not the skin bacterial microbiota. The dominant operational taxonomic units belonged to the Moraxellaceae, Pseudomonadaceae, Rhodobacteraceae, Staphylococcaceae and Vibrionaceae families. Despite the differences in taxonomic composition, the presumptive bacterial metabolisms between the gills and skin of the five fishes investigated here were ≥ 94% similar and were dominated by basic metabolism, most likely reflecting the continuous exposure of these tissues in the surrounding seawater.

Midgut Bacterial Microbiota of 12 Fish Species from a Marine Protected Area in the Aegean Sea (Greece).

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.

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.

Gut Microbiota of Five Sympatrically Farmed Marine Fish Species in the Aegean Sea.

In this study, we hypothesized that sympatrically grown farmed fish, i.e. fish which experience similar environmental conditions and nutritionally similar diets, would have more convergent gut microbiota. Using a "common garden" approach, we identified the core microbiota and bacterial community structure differences between five fish species farmed in the same aquaculture site on the west coast of the Aegean Sea, Greece. The investigated individuals were at similar developmental stages and reared in adjacent (< 50 m) aquaculture cages; each cage had 15 kg fish m-3. The diets were nutritionally similar to support optimal growth for each fish species. DNA from the midgut of 3-6 individuals per fish species was extracted and sequenced for the V3-V4 region of the bacterial 16S rRNA. Only 3.9% of the total 181 operational taxonomic units (OTUs) were shared among all fish. Between 5 and 74 OTUs were unique to each fish species. Each of the investigated fish species had a distinct profile of dominant OTUs, i.e. cumulative relative abundance of ≥ 80%. Co-occurrence network analysis for each fish species showed that all networks were strongly dominated by positive correlations between the abundances of their OTUs. However, each fish species had different network characteristics suggesting the differential significance of the OTUs in each of the five fish species midgut. The results of the present study may provide evidence that adult fish farmed in the Mediterranean Sea have a rather divergent and species-specific gut microbiota profile, which are shaped independently of the similar environmental conditions under which they grow.

Imprinting statistically sound conclusions for gut microbiota in comparative animal studies: A case study with diet and teleost fishes.

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.

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

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'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 ± 2.1 and 366 ± 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 γ-Proteobacteria and Firmicutes.

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.

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

This study investigated the impact of different dietary ingredients, with different protein/lipid sources, on midgut and faeces bacteria community structures just before feeding and 3 h after feeding a single meal to individual rainbow trout (Oncorhynchus mykiss). Fish were kept in experimental rearing facilities and fed ad libitum twice daily for 5 weeks. Fish were fed three different commercial diets, which contained variations of high or low marine fishmeal/fish oil content. DNA was extracted from midgut and faeces samples for analysis of their bacterial 16S rRNA gene diversity by targeting the V3-V4 region with 454 pyrosequencing. A total of 332 unique bacterial operational taxonomic units (OTUs) were revealed in all samples. However, each sample was dominated (>80% relative abundance) by 2-14 OTUs, with the single most dominant OTU having >30% dominance, indicating that only a few bacteria were fundamental in terms of relative abundance in each treatment. Fifteen OTUs occurred in all samples (core microbiota). The majority of these OTUs belonged to the Proteobacteria, Firmicutes or Tenericutes, and were associated with other animal gut environments. The faecal material and the midgut samples had few overlaps in their shared OTUs. A postprandial response in the gut bacterial community structure 3 h after feeding highlights how dietary stimulation induces structural changes in the microbiota profiles in the established gut bacteria. This study showed that feeding O. mykiss different diets and even single meals lead to perturbations in the established gut bacteria of O. mykiss.

Bacterial biofilm development during experimental degradation of Melicertus kerathurus 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.