Research Article
2 July 2020

Changes in the Microbiome of Mariculture Feed Organisms after Treatment with a Potentially Probiotic Strain of Phaeobacter inhibens

ABSTRACT

The Phaeobacter genus has been explored as probiotics in mariculture as a sustainable strategy for the prevention of bacterial infections. Its antagonistic effect against common fish pathogens is predominantly due to the production of the antibacterial compound tropodithietic acid (TDA), and TDA-producing strains have repeatedly been isolated from mariculture environments. Despite many in vitro trials targeting pathogens, little is known about its impact on host-associated microbiomes in mariculture. Hence, the purpose of this study was to investigate how the addition of a TDA-producing Phaeobacter inhibens strain affects the microbiomes of live feed organisms and fish larvae. We used 16S rRNA gene sequencing to characterize the bacterial diversity associated with live feed microalgae (Tetraselmis suecica), live feed copepod nauplii (Acartia tonsa), and turbot (Scophthalmus maximus) eggs/larvae. The microbial communities were unique to the three organisms investigated, and the addition of the probiotic bacterium had various effects on the diversity and richness of the microbiomes. The structure of the live feed microbiomes was significantly changed, while no effect was seen on the community structure associated with turbot larvae. The changes were seen primarily in particular taxa. The Rhodobacterales order was indigenous to all three microbiomes and decreased in relative abundance when P. inhibens was introduced in the copepod and turbot microbiomes, while it was unaffected in the microalgal microbiome. Altogether, the study demonstrates that the addition of P. inhibens in higher concentrations, as part of a probiotic regime, does not appear to cause major imbalances in the microbiome, but the effects were specific to closely related taxa.
IMPORTANCE This work is an essential part of the risk assessment of the application of roseobacters as probiotics in mariculture. It provides insights into the impact of TDA-producing Phaeobacter inhibens on the commensal bacteria related to mariculture live feed and fish larvae. Also, the study provides a sequencing-based characterization of the microbiomes related to mariculture-relevant microalga, copepods, and turbot larvae.

INTRODUCTION

The rearing of aquatic animals and plants for human consumption, i.e., aquaculture, is a rapidly growing industry due to the increasing demand for high-quality protein to feed the growing world population (1, 2). In finfish rearing, animals undergo multiple developmental stages from eggs through larvae to juveniles and, finally, adult fish. During this process, fish are supplied with different food sources, which may include live feed such as algae, rotifers, Artemia, and copepods. The live feed itself feeds on microalgae, and hence, multiple trophic layers are involved. In many cases, fish farming has become a high-throughput process, with individual companies producing thousands of tons of fish annually, and by 2030, fish production from aquaculture is expected to reach an annual output of 109 million tons (2). Along with the sustainable development goals of the United Nations, there is an increasing focus on the sustainable production of food, ending hunger while protecting wild fish populations (3).
One of the major bottlenecks in fish production is disease outbreaks, and approximately 55% of infections are caused by pathogenic bacteria (4) that are typically introduced with supply water (5), broodstock, humans, and plankton feed (6). Particularly, vibrios such as Vibrio splendidus, Vibrio harveyi, Vibrio vulnificus, and Vibrio anguillarum are of major concern to mariculture facilities, as they cause severe fish diseases and mortalities (7, 8). This is predominantly an issue related to marine fish larvae where several species are reared in nutrient-rich green water tanks, feeding on live feed (6, 9, 10). Pathogenic Vibrio spp. are naturally associated with zooplankton (1114), and they can also easily proliferate in cultures of phytoplankton used as feed for the live feed (9). Thus, live feed organisms can act as vectors of opportunistic pathogenic vibrios.
Major crashes of fish larval populations are most likely due to detrimental interactions (dysbiosis) in the microbial communities associated with the fish larvae (15). Microbial communities respond quickly to changes in environmental factors such as oxygen concentrations, nutrient levels, pH, salinity, or accumulated toxic compounds (1619). In the event of an imbalance in the system, e.g., rapid increase in nutrient levels and temperature, opportunistic pathogens proliferate. The pathogens have traditionally been controlled by disinfection of the rearing tanks (20), sterilization of the rearing water (21), deployment of antibiotics (22), and, in recent years, by vaccination of the fish (23). However, the latter is not effective at the larval stage due to their underdeveloped immune systems (23). Thus, sustainable alternatives to antibiotics are sought (2224). One proposed alternative is the use of probiotics, which are live microorganisms that provide a health benefit to the host when administered in adequate amounts (25). The potential application of probiotics in aquaculture as a prophylactic and acute treatment of disease outbreaks has been studied, primarily focusing on the gut microbiome of the farmed animal (26). Currently, the majority of commercially available probiotics for aquaculture are based on mono- or mixed cultures of Firmicutes (3), which have been successful in humans and livestock, although not adapted to the diverse aquatic environments. Proteobacteria such as Shewanella spp. and tropodithietic acid (TDA)-producing members of the Roseobacter group have been studied extensively for their bioactivity and probiotic potential (3, 2729) as alternative candidates of marine origin.
Pure TDA is a bactericidal antibiotic at high concentrations (30), but it can also act as a signaling molecule (quorum sensing) affecting behavior such as motility, biofilm formation, and secondary metabolite production in TDA producers (31). Its antibacterial effect is due to its ability to act as an antiporter (32), and resistance does not develop easily (30, 33). Roseobacter organisms producing TDA are indigenous to microbiomes of marine eukaryotes, including micro- and macroalgae (34, 35), zooplankton (36), sponges (37), and mollusks (38, 39), where they are believed to control the abundance of pathogenic community members. However, increasing their abundance has only had subtle effects on the microbiomes associated with microalgae and oysters (40). While some community members were unaffected, Vibrio spp. and Pseudoalteromonas spp. were shown to diminish in the presence of Phaeobacter inhibens (40), and the presence of TDA or TDA producers has also caused a decrease in the relative abundances of closely related members of the Rhodobacteraceae family in microalgal microbiomes (41, 42).
TDA-producing Phaeobacter spp. of the Roseobacter group have been isolated from multiple mariculture facilities rearing mollusks and various marine finfish (38, 43, 44), and they have been able to antagonize fish pathogens in live feed cultures (9, 44, 45) without affecting growth or survival of the feed organisms (9, 10). Most importantly, addition of 106 to 107 CFU ml−1 can decrease mortality of turbot and cod larvae when challenged with pathogenic vibrios (9, 46, 47). The selective impact on host-associated microbiomes (40), along with the lack of resistance development despite their global occurrence in microbiomes (30, 33), including mariculture microbiomes, highlights the applicability of Phaeobacter spp. as probiotics. However, perturbations with probiotic levels of Phaeobacter spp. could potentially cause imbalance and thereby give rise to proliferation of other pathogens than vibrios. Furthermore, little is currently known about mariculture microbiomes, and hence, the purpose of this study was to investigate how the addition of TDA-producing P. inhibens affects microbiomes related to host organisms present in the food webs found in mariculture systems.

RESULTS

The impact of the TDA producer P. inhibens strain DSM 17395 on the microbiomes of mariculture-relevant marine microalgae, copepods, and fish eggs/larvae was determined by sequencing 16S rRNA gene V4 region amplicons and analyzing their taxonomic composition and diversity over 4 days; each coculture, as well as control cultures without addition of the probiotic, was sampled four times, at 0 h (T0), 24 h (T24), 48 h (T48), and 96 h (T96). The coculture experiment with turbot was initiated with eggs, which all hatched within 48 h of incubation; up to 2% of the eggs were hatched at time point 24 h.

P. inhibens in nontreated and treated microbial communities.

Amplicon sequence variants (ASVs) containing the added P. inhibens were detectable in all three microbiomes at all time points. Relative abundances varied from 0.05 to 1.7%, with the highest relative abundance in copepods (Table 1). Addition of P. inhibens, as expected, changed the relative abundances observed. In Tetraselmis suecica cultures, this increased from approximately 1% to 10% and was stable over the 96 h. In copepods, P. inhibens constituted between 30 and 50% in relative abundances, and in turbot larvae, this was approximately 20%. ASVs containing the added P. inhibens were removed from the data sets before the subsequent analyses to assess the potential changes in the indigenous background microbiome.
TABLE 1
TABLE 1 Relative abundance of P. inhibens in microbiomes associated with T. suecica, A. tonsa, and S. maximus eggs/larvae over timea
Eukaryotic hostRelative abundance (%) of Phaeobacter inhibens for:
Nontreated control at time:Probiotic-treated culture
T0T24T48T96T0T24T48T96
T. suecica (microalga)0.09 ± 0.050.06 ± 0.010.11 ± 0.070.06 ± 0.018.66 ± 0.849.56 ± 0.619.14 ± 0.6511.87 ± 2.43
A. tonsa (copepod)1.66 ± 2.490.22 ± 0.020.45 ± 0.020.15 ± 0.0155.57 ± 1.6242.97 ± 1.3634.95 ± 2.5029.83 ± 1.98
S. maximus (turbot)0.15 ± 0.060.06 ± 0.020.06 ± 0.020.05 ± 0.001NTb20.82 ± 2.7618.28 ± 0.7915.62 ± 1.50
a
The relative abundances were pooled ASVs identified as P. inhibens in each culture prior to calculation of average and standard deviation for two or three replicate cultures.
b
NT, not tested.

Effects of P. inhibens treatment on microbial community composition.

The taxonomy and relative abundance of the ASVs were used to assess the community composition at different taxonomic levels, i.e., phylum and genus levels. The community of the microalga T. suecica was dominated by bacteria from the phyla Proteobacteria, particularly members of the Rhodobacterales order, and Bacteroidetes, particularly members of the Flavobacteriales order (Fig. 1A). Other observed orders above 2% relative abundance included Alteromonadales, Burkholderiales, Caulobacterales, Rhizobiales, Sphingomonadales, Phycisphaerales, and Cytophagales. Burkholderiales (>2%) were only present in the initial microbiomes (T0), while Rhizobiales (>2%) appeared after 96 h of incubation. No obvious changes occurred at the order level due to the addition of P. inhibens, and hence, incubation time was the main driver of the observed changes in community composition at this taxonomic level. Similarly, no obvious changes occurred at the genus level (Fig. S1).
FIG 1
FIG 1 The composition of bacterial communities associated with T. suecica (A), A. tonsa (B), and Scophthalmus maximus (C) eggs/larvae in response to the addition of probiotic P. inhibens DSM 17395. All eggs were hatched after 48 h of incubation, while 98 to 100% were at the egg stage prior to the 48-h sampling. Each bar represents a culture which was sampled upon 0, 24, 48, and 96 h incubation; the T. suecica and A. tonsa cultures were in triplicates, and the S. maximus were in duplicates. The compositions of individual microbiomes are illustrated as relative abundances of all the bacterial orders observed in the cultures of microalga with or without P. inhibens. Only orders with abundance above 2% were included (the remaining low abundance orders are represented by the distance up to 1.00). Amplicon sequence variants (ASVs) containing the added P. inhibens were removed from the data set prior to plotting. Controls, untreated controls; treatment, probiotic P. inhibens.
The Acartia tonsa bacterial community composition was dominated by Proteobacteria, particularly members of the orders Alteromonadales and Oceanospirillales (Fig. 1B). Rhodobacterales, Rhodospirillales, and Flavobacteriales members were also present in all samples, though in lower relative abundances. Desulfobacterales (>2%) only occurred in the initial microbiome (T0), while Caulobacterales (>2%) increased in relative abundance in the microbiome after 96 h of incubation. The addition of P. inhibens decreased the relative abundance of Rhodobacterales and Rhodospirillales. Within the Rhodobacterales order, relative abundances of sequences assigned to the genera Ruegeria and Celeribacter were reduced from 2.88% to 7.27% to 0.60% to 2.27% and from 1.64% to 3.53% to 0.24% to 0.54%, respectively (Fig. S1). Furthermore, Alteromonadales increased initially (T24) in the probiotic group, though their dominance decreased over time. Hence, in this biological system, both time and the probiotic treatment affected the composition of the bacterial community at the order level.
The turbot egg and larval microbiomes were dominated by Proteobacteria, particularly Gammaproteobacteria of the orders Alteromonadales and Vibrionales (Fig. 1C). Vibrionales were most prominent in the initial egg microbiome (T0; relative abundance, 46.2% to 46.9%), though their relative abundance decreased to 14.3% to 19.2% thereafter and remained at the same level throughout the experiment. Concurrently, the relative abundance of Alteromonadales increased in abundance after 24 h of incubation. Rhodobacterales (>2%) appeared in the control samples at 24 h. Both Rhodobacterales (only in the controls) and Oceanospirillales increased in relative abundance, while Alteromonadales decreased over time. Pseudomonadales (>2%) occurred in the microbiome following 48 h of incubation and remained throughout the experiment. The bacterial community associated with turbot eggs/larvae receiving probiotic treatment did not contain members of the Rhodobacterales order (>2%) after the removal of the P. inhibens ASV. Altogether, the most prominent change occurred within the first 24 h of the experiment (establishment phase), and the bacterial community was stable from this point onward. The presence of P. inhibens decreased the relative abundance of other Rhodobacterales bacteria (mainly the genus Pseudophaeobacter; Fig. S1 in the supplemental material), but otherwise, the community was mainly affected by incubation time.

Impact of P. inhibens treatment on bacterial microbiome richness and diversity.

The total number of observed ASVs was similar across systems, with 857 in the microalgal system, 1,014 in the copepod system, and 801 in the fish larval system after removal of chloroplasts and P. inhibens-related ASVs. The richness and diversity of bacterial communities associated with the eukaryotic organisms were at the same general level with minor variations over time regardless of host organisms (Table 2). Estimated ASV richness (Chao1) of the microalgal microbiome ranged from 126 to 166 in the controls and 132 to 173 in the cultures exposed to the probiotic (Table 2). The richness of the untreated copepod microbiome was initially 179 to 225 ASVs, though it dropped to 154 to 157 after 24 h and remained at this level throughout the monitoring period (Chao1; 133 to 182; Table 2). The probiotic-treated group followed the same trend; the richness of the initial microbiome (T0) was 132 to 153 ASVs, followed by a decrease to 110 to 126 (T24) and an increase to 132 to 157 ASVs over the remaining 72 h (T96). A slight effect of probiotic treatment was observed in this microbiome, as the estimated richness was lower in treated copepods compared to the controls at all time points, though the difference was only significant at time points 0 h (t test; P = 0.014) and 24 h (t test; P = 0.017). The turbot egg microbiome richness was initially 119 to 162 ASVs (T0; Table 2). From time point 24 h to 96 h, both treatment groups increased richness from 122 to 154 ASVs to 172 to 199 ASVs, respectively. Altogether, these data indicate that the richness of the mariculture microbiomes is relatively low, regardless of the host and treatment.
TABLE 2
TABLE 2 Alpha diversity measures for microbiomes of T. suecica, A. tonsa nauplii, and turbot eggs and larvae treated with P. inhibens DSM 17395 and those without treatment (controls)a
Eukaryotic hostTime point (h)Chao1 richness for:Shannon diversity index for:
ControlTreatmentP valueControlTreatmentP value
AvgSDAvgSDAvgSDAvgSD
T. suecica (microalga)0148.0815.80148.144.440.99553.470.043.390.030.0453
 24147.6816.69145.085.470.81053.470.033.410.020.0209
 48140.4213.91158.4714.300.19213.450.053.370.020.0725
 96156.182.52141.017.810.03283.470.033.420.020.0366
A. tonsa (copepod)0202.0722.93141.2810.640.01413.420.013.010.031.83E−05
 24155.971.71119.308.140.01672.960.032.700.080.0053
 48149.8913.95134.045.850.14373.160.013.030.060.0727
 96169.1113.42147.2213.290.11523.410.043.440.030.4108
S. maximus (turbot; eggs and larvae)0140.7830.01NTNTNT2.390.08NTNTNT
24128.177.36141.2018.30NT3.090.013.090.03NT
48184.0110.09141.7325.55NT3.420.063.130.02NT
96186.4219.07190.9612.65NT3.510.043.390.02NT
a
NT, not tested.
Similar patterns were observed with respect to diversity (Shannon diversity index). The microalgal microbiome diversity remained stable for the untreated controls (Shannon index, 3.39 to 3.51) and cultures treated with P. inhibens (Shannon index, 3.35 to 3.43) throughout the experiment (Table 2). The difference in diversity was significant between the controls and treatment at time points 0 h (t test; P = 0.045), 24 h (t test; P = 0.021), and 96 h (t test; P = 0.037), although the P value was close to the alpha level (0.05). In the copepod microbiome, the diversity was initially at the same level as the microalgal microbiome (Shannon index, 3.42 to 3.43), though it dropped slightly to a Shannon index between 2.92 and 2.99 within 24 h (Table 2). The diversity increased to the initial level after 96 h of incubation. A similar pattern was observed for the copepod cultures supplemented with P. inhibens (Table 2); the initial Shannon diversity index was 2.98 to 3.04, dropping to a range of 2.61 to 2.76 and subsequently exhibiting an increase equivalent to the final level of the untreated controls (3.41 to 3.48). As for the changes observed in OTU richness estimates, the difference in diversity was significant between the controls and treatment at time points 0 h (t test; P = 0.000018) and 24 h (t test; P = 0.0053). The initial turbot egg microbiome diversity was lower than the microalgal and copepod microbiome diversity (Shannon index, 2.33 to 2.45 at T0), though it increased steadily during the incubation period of 96 h (in both controls and treated samples). Altogether, these observations demonstrate that P. inhibens has subtle yet significant effects on the richness and diversity of the microbiomes associated with the live feed, i.e., microalgae and copepods. Due to low replication levels, further studies have to be conducted to support the observed trends in the fish larval microbiomes.

Impact of P. inhibens on community structure.

Unconstrained ordinations, i.e., principal-coordinate analysis (PCoA), on Bray-Curtis distances were used to assess the impact of P. inhibens on community structure of the microbiomes associated with the three mariculture-related eukaryotes (Fig. 2). The community structure shifted during incubation time for all three microbial communities, regardless of treatment (permutational multivariate analysis of variance [PERMANOVA]; R2 values between 0.24112 and 0.44059; P = 0.001). The microalgal microbial community structures treated with probiotics were significantly different from the untreated controls (PERMANOVA; R2 = 0.31982; P = 0.001; Fig. 2A). This was also observed in the copepod-associated microbiome (PERMANOVA; R2 = 0.21008; P = 0.001; Fig. 2B), though time was a bigger driver than treatment (R2 [time] = 0.44059 > R2 [treatment] = 0.21008). However, the turbot larval microbial community structure was not significantly affected by the presence of P. inhibens (PERMANOVA; R2 = 0.1021; P = 0.06; Fig. 2C). Hence, incubation time was a major driver of the microbial community structure across all samples, and the impact of the probiotic treatment depended on the trophic layer at hand; the biggest impact occurred in the live feed, while the fish larval community structure was unaffected.
FIG 2
FIG 2 Community structure of microbial communities associated with three mariculture-relevant host organisms. Principal coordinate analysis (PCoA) on Bray-Curtis distances between samples from microbiomes associated with T. suecica (A), A. tonsa nauplii (B), and turbot eggs and larvae (C). The shape of the data point indicates treatment; microbial communities exposed to probiotic P. inhibens DSM 17395 (triangles) or sterile media (untreated control, circles). Each community was sampled at time point 0 h (red), 24 h (yellow), 48 h (green), and 96 h (blue).

Impact of P. inhibens on specific taxa.

At the order level, P. inhibens DSM 17395 decreased the relative abundance of Rhodobacterales in two of the microbiomes (Fig. 1B and C), while the effects on the algal microbiome were minor. Therefore, differences at the ASV level (100% sequence similarity, no clustering) were investigated to elucidate which of the most abundant members were affected. No major impact on the relative abundance of the most abundant ASVs was observed in the microbiomes of T. suecica due to treatment (Fig. S2), and thus, alterations in community structure were mainly confined to low-abundance ASVs. In the A. tonsa microbiome, the most abundant Halomonas spp. were slightly lower in abundance in the P. inhibens-treated samples, but still dominating (Fig. 3). Members of the Rhodobacteraceae family, such as Ruegeria sp. and Celeribacter sp., decreased in relative abundance in the presence of P. inhibens, which is in line with the observations in the community composition analysis (Fig. 1B and Fig. S1). Members of the Saccharospirillaceae family and Hyphomonas spp. were initially lower in relative abundance in both treatment groups but increased over time. In contrast to the microalgal and copepod microbiomes, the samples from the more dynamic fish eggs/larvae system clustered according to time rather than treatment (Fig. S3). No major changes were observed due to treatment, but changes over time were observed, confirming the PCoA (Fig. 2C). Some Colwellia sp. ASVs disappeared as a function of incubation time, while others increased in relative abundance. Other Alteromonadales bacteria, such as Psychrobium sp. and Alteromonas sp., increased. Vibrio spp. had high relative abundances in the initial microbiome (T0), though they decreased as a function of incubation time. No effect of treatment was observed on the vibrios. Altogether, the occurring changes due to the presence of P. inhibens were unique to the eukaryotic host, and the largest changes were observed in the copepod microbiome (Fig. 3).
FIG 3
FIG 3 Heat map indicating the log10(x + 1)-transformed relative abundances of the 30 most abundant ASVs in the A. tonsa (AT) microbiome in response to the addition of probiotic P. inhibens DSM 17395 (P). Untreated controls are included (C). Each microbiome was sampled at time point 0 h, 24 h, 48 h, and 96 h. The VSEARCH classified SILVA annotations are listed next to the individual ASV, and the Bray-Curtis distances are represented as dendrograms.

DISCUSSION

TDA-producing roseobacters can function as probiotics in mariculture due to their efficient killing of common pathogens (9, 44, 45) and protection of vibrio-challenged fish larvae (9, 46, 47). Since the natural microbiomes in these systems are imperative for the health of, e.g., algae (48), addition of probiotics should selectively remove pathogens while leaving the commensal majority of the population unaffected. Our results demonstrate that the impact of P. inhibens on the microbiome is highly dependent on the commensal microbiome composition and the inoculation level, with a greater impact on the bacterial community structure at the lower levels of the food web.
Three eukaryotes, green microalgae, copepods, and turbot larvae, were selected to represent different important levels in the food web, including feed for live feed, live feed, and reared fish, found in mariculture. Several studies have been conducted on microalgal microbiomes and how roseobacters interact with these unicellular eukaryotes (4951). Despite T. suecica being widely used and produced in hatcheries, the microbial community associated with this microalga is not well studied. Biondi et al. (52) observed that the T. suecica microbiome was dominated by Proteobacteria, particularly members of the Roseobacter group, but also Rhizobiales and Bacteroidetes (Flavobacteriales). This is similar to our findings and also to findings for another mariculture-relevant microalgal genus, Nannochloropsis (53). The A. tonsa microbiome was also dominated by Proteobacteria, particularly Gammaproteobacteria, in this study. This has previously been observed in copepods from the North Atlantic Ocean (54). Cultivation-based methods have found that Vibrio spp. was dominating (14, 55); however, the order Vibrionales was below the 2% relative abundance cutoff in our community composition analysis, indicating that the relative abundance of these bacteria is likely overestimated in cultivation-dependent studies. Moisander et al. (54) also observed that Rhodobacteraceae dominated the transient food microbiome and proposed that they contribute to copepod nutrition. Members of the Rhodobacterales order were also found at high relative abundances in our copepod system, though Alteromonadales and Oceanospirillales bacteria exhibited the highest relative abundances. These differences in the composition of the copepod-associated community are likely due to differences in the composition of the bacterial community in the immediate environment (natural versus laboratory cultivation) and to different methodologies applied (cultivation dependent versus cultivation independent).
The culturable bacteria of turbot eggs and larvae have been studied for decades, and the isolates have been dominated by members of the Vibrionales and Aeromonadales orders (56, 57). While we observed Vibrionales in the egg microbial community, the Aeromonadales were not abundant (below the 2% relative abundance cutoff) in any of the samples. In contrast, we observed high relative abundances of Alteromonadales. However, most studies on aquaculture microbiomes are cultivation based, and poor correlations between culture-dependent and -independent microbiome investigations were also observed by Fjellheim et al. in cod larval microbiomes (58).
In concordance with a previous study of the microalgal Emiliania huxleyi microbiome (40), we found that the effects of probiotic treatment did not cause major changes to the community across orders. Interestingly, the addition of P. inhibens reduced the relative abundance of closely related taxa from the Rhodobacterales order in the copepod and fish larvae microbiomes, while they were unaffected in the microbiome of T. suecica. Effects on closely related Rhodobacterales members have been observed in the haptophyte E. huxleyi microbiome exposed to the same P. inhibens strain (40). Several genera of the Rhodobacteraceae family, namely, Sulfitobacter, Phaeobacter, Pelagicola, and Loktanella, were reduced or absent in the presence of another TDA-producing strain of P. inhibens (2.10 variant) in the diatom Thalassiosira rotula microbiome (42). In our study, among the 30 most abundant taxa in the copepod and turbot microbiomes, unclassified genera of the Rhodobacteraceae family, Ruegeria spp., Celeribacter spp., and Pseudophaeobacter spp. decreased in relative abundance. Addition of pure TDA to cultures of Nannochloropsis salina has also been shown to decrease the relative abundance of Rhodobacteraceae at relatively low concentrations (31.25 to 500 nM) (41), which could indicate that TDA is causing the observed decrease of Rhodobacteraceae members in the copepod and turbot microbiomes. Potentially, production and/or sensing of TDA is involved in the interspecies competition within the Roseobacter group in which specific species or strains are more susceptible than others. Vibrio spp. and Pseudoalteromonas spp. were reduced by the presence of P. inhibens DSM 17395 in the microalgal E. huxleyi microbiome (40), though in the present study, the orders Vibrionales and Alteromonadales were unaffected compared to the controls. Majzoub et al. (42) found that the T. rotula microbiome exposed to a P. inhibens 2.10 variant (NCV12a1), with reduced antagonistic effect, developed in the same way as microbiomes exposed to the original bioactive strain, and factors other than TDA could be responsible for our observations. Altogether, these results indicate that closely related roseobacters compete for the same niches and that the impact is dependent on the eukaryotic host as well as the abundance of the roseobacters present in the commensal microbiome. However, as the sampling of the coculture systems was different between the tested systems, planktonic bacteria could be washed away from the copepods and fish eggs/larvae during sampling and therefore not captured as highly abundant in the sequences, while more planktonic bacteria would remain in the pelleting of the microalgal culture. Further studies should reveal how specific interactions determine which species prevail.
All microbiomes had similar richness and diversity indices. Bakke et al. (59) reported that richness and diversity varied throughout the life stages of cod larvae. While the turbot larvae in this study were younger, the alpha diversity measure was similar to the observations by Bakke et al. (59). The richness and diversity of the rearing water (i.e., green water prepared with algae, Nannochloropsis oculata, and paste) and live feed (copepod, A. tonsa, and rotifers, Brachionus ‘Nevada’) were much higher than observed in the larval microbiome (59) and the live feed assessed in this study. However, this is most likely due to experimental differences; this study was conducted in laboratory, small-scale cultures, while the study by Bakke et al. (59) was conducted in large-scale, aquaculture flowthrough systems. The addition of the probiotic treatment in this study had a slight yet significant effect on the alpha diversity of the live feed. Dittmann et al. (40) observed that treatment with, and the inoculation density of, P. inhibens DSM 17395 did not impact the richness and diversity of the low-complexity microalgal E. huxleyi microbiome. In contrast, the more complex oyster microbiome increased in richness when P. inhibens had been added to the system, though the diversity was unaffected. How the observed changes in alpha diversity of host-associated microbiomes translate to the overall health state of the host organism needs further scrutiny.
The microbial communities associated with the three microbiomes were generally dynamic and changed over time, which is in concordance with previous studies (42, 59, 60). The addition of P. inhibens had a significant impact on the microbiome structure of T. suecica and A. tonsa. In contrast, the microbiome associated with the turbot larvae was more affected by incubation time compared to probiotic treatment. All eggs hatched within the first 48 h of the experiment, and thereby, a sudden increase in nutrients has likely occurred. In contrast, no nutrients were added to the microalgal and copepod systems, and thus, nutrients from the medium and the eukaryotic hosts were slowly consumed, and competition likely increased. The minor impact of P. inhibens addition to the turbot egg and larval microbiome would indicate that the addition of probiotics would not cause dysbiosis in a healthy larval microbiome and a subsequent population crash. To determine this fully, longer trials would have to be conducted, monitoring the overall health of the larvae. The minor impact on the larval microbiome might also mean that the probiotic is less efficient at this level. Vibrio spp. are commonly reported as detrimental pathogens to fish larvae (7, 8), while they are also part of the commensal microbiome (56, 57). In this study, the high relative abundance of Vibrionales in the turbot microbiome was due to relatively few ASVs belonging to the Vibrio genus, and the abundance of these ASVs did not change regardless of treatment (from day 1 through day 4). We added P. inhibens at concentrations at which a probiotic effect had been observed in previous challenge trials (9, 47, 61). In those trials, vibrios were reduced in numbers if not kept at inoculation level (9, 47, 61), depending on the initial concentration of Vibrio spp. (9). Combined, these results would suggest that the addition of P. inhibens, or the presence of inherent, closely related taxa, can keep vibrios in the fish microbiome at a stable level; however, this does not necessarily eliminate potential pathogens from the system. The effect is likely dose dependent, which was observed in a previous study of the microalgal E. huxleyi microbiome (40). Altogether, these data emphasize the need for investigating the optimal addition of probiotic P. inhibens in relation to dose and which part of the food web to add the probiotic treatment to in order to obtain the most efficient protection against opportunistic pathogens while keeping the effects on the commensal microbiome to a minimum. The addition of the probiotic is likely more efficient at the live feed stages such as microalgae or zooplankton where P. inhibens establishes itself and changes the structure. However, results based on nested PCRs on low-biomass samples, such as those presented in this study, are prone to biases and vulnerable to contaminations. Hence, it is not possible to conclude yet whether any changes would be beneficial or detrimental to the microbiome or its function. Broader omics studies should elucidate this in the future.
In conclusion, the addition of TDA-producing P. inhibens caused significant changes to the microbiome structure of the live feed but had little effect on the order-level composition and various effects on diversity and richness of the microbial communities associated with microalgae and copepods. No effect was seen on the community structure associated with turbot larvae. Particularly, the relative abundances of closely related taxa from the Roseobacter group were reduced as a function of probiotic treatment, but only in the copepod and turbot larval microbiomes. Vibrio spp. were highly abundant in the turbot microbiome, and these were kept at a stable level, though not eliminated, which indicates that the probiotic effect toward vibrios is likely dose dependent. Hence, the effect of adding a probiotic bacterium such as P. inhibens to the microbiome of mariculture-related eukaryotes is not likely to cause major perturbations to the existing microbial communities.

MATERIALS AND METHODS

Bacterial cultivation.

P. inhibens DSM 17395 (38, 62, 63) was grown in half-strength yeast extract, tryptone, and sea salts broth (one-half YTSS medium [2 g/liter Bacto yeast extract, 1.25 g/liter Bacto tryptone, 20 g/liter Sigma sea salts]) (64). Liquid cultures were incubated under agitation (250 rpm) at 25°C or room temperature. When grown on solid substrates, marine agar (MA; Difco marine agar 2216) or one-half YTSS agar (one-half YTSS with 15 g/liter agar) was used.

Algae-Phaeobacter coculturing.

A nonaxenic strain of the green microalgae T. suecica was obtained from the aquaculture facility Selonda Aquaculture SA, Athens, Greece. It was grown in sterilized f/2 medium (65) without Na2SiO3 but with 5 mM NH4Cl in 1 liter of 3% Instant Ocean sea salt (Aquarium Systems Inc., Sarrebourg, France). This modified f/2 will from this point be referred to as f/2.
The cell density of T. suecica in the stock culture was determined using an improved Neubauer counting chamber. The cells were reinoculated in f/2 medium at a final concentration of approximately 5 × 105 algae ml−1 before splitting into six cultures of 600 ml in 1-liter Erlenmeyer flasks. Three overnight cultures of P. inhibens DSM 17395 in one-half YTSS were adjusted to an optical density at 600 nm (OD600nm) of 1.0 and washed once in f/2 medium (7,000 rpm, 3 min). In triplicates, cocultures of T. suecica were inoculated with P. inhibens DSM 17395 at a final concentration of 4.06 × 106 ± 1.05 × 106 CFU ml−1 (equivalent to 8 P. inhibens cells per algal cell), verified by plate spreading dilutions on MA. The remaining three cultures of T. suecica were treated with sterile 2% Instant Ocean and served as controls. The cultures were incubated stagnant at 18°C with white fluorescent light (1,623 μmol m−2 s−1 photosynthetically active radiation [PAR]). The cultures were sampled at 0 h, 24 h, 48 h, and 96 h for algal abundance determinations and for biomass to be used in DNA extractions. For abundance measures, 1 ml coculture was fixed in 1% 0.2 μm filtered glutaraldehyde (final concentration), and the cell numbers were determined using an improved Neubauer counting chamber. For DNA extraction, 100 ml of each culture was pelleted (8,000 × g for 5 min at 25°C) and resuspended in 1 ml lysis buffer (400 mM sodium chloride, 750 mM sucrose, 20 mM EDTA, 50 mM Tris-HCl, 1 mg ml−1 lysozyme, pH 8.5) (66) and stored at −80°C until extraction.

Copepod-Phaeobacter coculturing.

A. tonsa eggs were kindly provided by B. W. Hansen, Roskilde University, and stored at 5°C until use. Three days before the experiment, eggs were inoculated in 3% Instant Ocean and incubated at 18°C with white fluorescent light (1,623 μmol m−2 s−1 PAR). The density of A. tonsa nauplii in the culture was determined using a Sedgewick rafter counting cell, and the culture was adjusted to 2 nauplii per ml using 3% Instant Ocean. Seven cultures of 30 ml adjusted nauplii culture were set up in 50-ml Falcon tubes. In triplicates, overnight cultures of P. inhibens DSM 17395 in one-half YTSS were inoculated into the A. tonsa nauplii culture to a level of 0.5% (equivalent to 5 × 106 CFU ml−1, verified by plate spreading on MA). Three A. tonsa cultures were treated with sterile one-half YTSS and served as controls. The last culture was used untreated for quantification of live A. tonsa. All cocultures were incubated horizontally with shaking (60 rpm) at 18°C with white fluorescent light (1,623 μmol m−2 s−1 PAR) and sampled at days 0, 1, 2, and 4. Before sampling, each tube was mixed by inversion, and 5 ml culture (equivalent to 10 A. tonsa nauplii) was taken out for filtration onto a MontaMil polycarbonate membrane filter (pore size, 0.2 μm; diameter, 47 mm). The filters were transferred to cryo tubes, flooded in sucrose lysis buffer, and stored at −80°C until extraction.

Phaeobacter coculturing of turbot eggs and larvae.

Nonaxenic turbot eggs were received from France Turbot hatchery L’Epine (Noirmoutier Island, France) with 24 h of transport before conducting the experiment. One hundred eggs were transferred to four petri dishes (20 cm diameter, glass) containing sterile-filtered (0.22-μm filter) seawater adjusted to a salinity of 34 ‰ with Sigma sea salts (catalog no. S9883; Sigma) and a temperature of 15°C. The final volume was 200 ml. An overnight culture of P. inhibens DSM 17395 in one-half YTSS was washed one time in 2% sterile Instant Ocean (7,000 rpm for 3 min). In duplicates, cocultures of turbot eggs were inoculated with P. inhibens DSM 17395 at a final concentration of 1 × 107 CFU ml−1 in the seawater (equivalent to 2 × 107 P. inhibens cells per egg), verified by plate spreading dilutions on one-half YTSS agar. The remaining two cultures of eggs were treated with an equivalent volume of sterile 2% Instant Ocean and served as controls. The experiment was initiated with 0% of the eggs being hatched. After 24 h of incubation, 0% to 2% of the eggs were hatched, while all the eggs were hatched after 48 h of incubation. Biomass samples for DNA extraction were taken at days 0, 1, 2, and 4 by transferring 15 eggs from each culture to a cryo tube. Transferred seawater was removed, and the eggs were resuspended in sucrose lysis buffer and stored at −80°C until extraction. At each sampling time point, the number of eggs that had hatched was noted.

DNA extraction and PCR amplification.

Extractions were performed using the phenol-chloroform-based protocol described by Dittmann et al. (40). The gDNA was eluted in Tris-EDTA (TE) buffer and incubated at 4°C overnight. Quality and quantity were assessed by absorption (DeNovix DS-11+; DeNovix Inc., Wilmington, DE, USA) and fluorescence (Qubit dsDNA BR assay; Invitrogen by Thermo Fisher Scientific Inc., Eugene, OR, USA) spectroscopy. The DNA was diluted to the same concentration (15 ng/μL) for all samples—except samples with lower DNA yield, which were used undiluted—prior to application in a nested PCR of the 16S rRNA V4 region. The universal primers 27F and 1492R (67) were applied for the initial amplification of the 16S rRNA gene using the TEMPase Hot Start 2× master mix blue II (Ampliqon; catalog no. 290806); 75 ng gDNA was used as the template for each reaction except for samples with lower yield, where the added amount was down to 10 ng. The PCR program was 1 cycle of denaturation for 15 min at 95°C, 35 cycles of denaturation (95°C for 30 s), annealing (51°C for 30 s), elongation (72°C for 2 min), and, finally, 1 cycle of extended elongation (72°C for 5 min). The PCR products were used as templates in the subsequent PCR amplification of the V4 region using the primers 515F-Y (GTGYCAGCMGCCGCGGTAA) (68) and 806R (GGACTACNVGGGTWTCTAAT) (69). The V4 PCRs were run in duplicates using the Kapa HiFi HotStart readymix (Roche; catalog no. 07958935001) and the PCR program of 1 cycle of denaturation for 3 min at 95°C, 25 cycles of denaturation (98°C for 20 s), annealing (53°C for 15 s), elongation (72°C for 15 s), and, finally, 1 cycle of extended elongation (72°C for 1 min). The PCR products of the duplicates were pooled prior to purification (AmPure XP PCR purification; Agencourt Bioscience Corporation, Beverly, MA, USA) and subsequent quality and quantity assessment (as described above).

Amplicon sequencing and bioinformatics data analysis.

Amplicons were indexed and prepared for 250PE Illumina MiSeq sequencing at the sequencing core at the Novo Nordisk Foundation Center for Biosustainability, Kongens Lyngby, Denmark. The raw demultiplexed reads were checked for quality and trimmed using AfterQC (70) default settings, i.e., trim front and tail based on auto-detected quality; per-base quality trimming using Phred score ≥ 20; minimum sequence length, 35 bp; maximum number of n = 5; and filtering of sequences with a Phred score below 20. The trimmed reads were processed through the QIIME2 pipeline (version 2019.1) (71) run in a Docker virtual machine (Docker, Inc., Palo Alto, CA). In brief, the reads were imported along with metadata. The DADA2 (72) plugin for QIIME2 was used for removing PhiX, denoising, merging of paired reads, merging duplicate sequences, removal of chimeric sequences, and construction of the amplicon sequence variant (ASV) table. Taxonomy of the ASVs was assigned by global alignment against the SILVA database (v132 SSU release; V4 fraction extracted reference sequences using the primers applied in this study) using the VSEARCH consensus taxonomy classifier (73). The ASV table and taxonomy were extracted from the QIIME2 format using the QIIME tools export and convert, followed by import into R (v3.5.2) along with the metadata. ASVs classified as chloroplasts were filtered using the dplyr and tidyr packages for R. ASVs containing the added P. inhibens DSM 17395 were classified as Rhodobacteraceae by VSEARCH; these were identified based on their relative abundances in the probiotic-treated samples compared to the controls as well as 100% similarity of the representative sequence to P. inhibens strain DSM 17395 (publicly available at GenBank under accession no. CP002976.1). Two ASVs with relative abundances of 0.02% to 0.1% in controls and 3.2% to 7.6% in samples treated with probiotics were determined to contain the added P. inhibens bacteria in the T. suecica microbiome. Four (ASVs) with relative abundances of 0% to 2.3% in controls and 0.2% to 32.4% in samples treated with probiotics were determined to contain the added P. inhibens bacteria in the A. tonsa microbiome. Five ASVs with relative abundances of 0% to 0.1% in controls and 0.08% to 11.9% in samples treated with probiotics were determined to contain the added P. inhibens bacteria in the turbot microbiome. To reduce any biasing effects of the increased abundance of the added probiont, these ASVs were excluded from all samples in analyses of composition and alpha and beta diversity measures, thus focusing the analyses on the background microbiome. For relative abundances of P. inhibens in all samples, see Table 1.
The community composition of each microbiome was analyzed and visualized using the functions of the phyloseq and qqplot2 packages. These packages were also used to calculate measures of alpha diversity (Chao1 estimated richness and Shannon diversity index) and beta diversity (Bray-Curtis distances) of data rarefied to even sampling depths of 68,163 for the T. suecica data set, 62,049 for the copepod data set, and 85,621 for the turbot egg/larval data set. The richness and diversity estimates were calculated based on the unfiltered data set using 100 iterations, and statistically significant difference was determined using F and t tests. Multivariate analysis was conducted on unfiltered data using unconstrained ordination, i.e., PCoA, on Bray-Curtis distances (community dissimilarities), and PERMANOVA using the adonis function from the vegan R package on the Bray-Curtis distances was applied to test the significance of treatment and time (control versus probiotics, time in days, 999 permutations).

Data availability.

The demultiplexed sequencing reads were deposited in the Sequence Read Archive (SRA) at NCBI under the accession number PRJNA558217.

ACKNOWLEDGMENTS

We thank Nancy Dourala, Selonda Aquaculture, Greece, for providing cultures of T. suecica, Benni W. Hansen, Department of Science and Environment, Roskilde University, Denmark, for providing copepod eggs, and France Turbot hatchery L’Epine for turbot eggs.
The present study was funded by the Technical University of Denmark (PhD grant for K.K.D.) and the Danish Council for Strategic Research, Program Commission on Health, Food and Welfare (12-132390; ProAqua). The study was supported by a grant from the Danish National Research Foundation (DNRF137).
We declare no conflict of interests.

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Information & Contributors

Information

Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 86Number 142 July 2020
eLocator: e00499-20
Editor: Shuang-Jiang Liu, Chinese Academy of Sciences
PubMed: 32385083

History

Received: 27 February 2020
Accepted: 3 May 2020
Published online: 2 July 2020

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Keywords

  1. 16S rRNA amplicon sequencing
  2. Phaeobacter
  3. Roseobacter
  4. aquaculture
  5. microbial community composition
  6. microbiome
  7. tropodithietic acid

Contributors

Authors

Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Bastian Barker Rasmussen https://orcid.org/0000-0002-2523-9304
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Jette Melchiorsen
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Lone Gram
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

Editor

Shuang-Jiang Liu
Editor
Chinese Academy of Sciences

Notes

Address correspondence to Mikkel Bentzon-Tilia, [email protected].

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