Supplementation of diets with seaweeds and commercially available aquafeed supplements had some subtle effects on the diversity and composition of hindgut microbial communities in the rabbitfish Siganus fuscescens, particularly at the genus level, although half of the genera were unknown. The relative abundance of Fusobacterium sp. was enhanced in the hindgut of fish fed diets supplemented with seaweed or other functional ingredients, whereas dietary supplementation reduced the abundance of the genus Arcobacter sp. (which was on average five times more abundant in the control fish). Overall, however, the hindgut microbiomes remained remarkably consistent between treatments including the control fish, suggesting either (i) that the dietary supplementations (3%) were insufficient to elicit a strong change in the fish GI microbiomes, or (ii) the existence of a stable, core microbiome in S. fuscescens. In this study, all fish sampled shared at least 63 out of a total of 113 ASVs identified in our bioinformatics pipeline. When the sequence data from the current study to those reported in two recently published papers on the hindgut microbiomes in the same fish species, despite some clear differences, 55 out of a total of 174 ASVs were shared and 13 of those represented between 66% and 85% of the total relative abundance. These observations from geographically and temporally distinct populations provide strong evidence for a core microbiome in this omnivorous subtropical fish, which appeared surprisingly stable, despite experimental manipulations of diet in the current empirical work at levels that are known to be able to fundamentally change the outcomes of production and other fish traits [35, 54]. Furthermore, given the number of different seaweed species screened (eleven) alongside other aquafeed products (four) and the very broad differences in both the proximate compositions and natural products chemistry between the various dietary supplements provided, the maintenance of the majority of the GI microbiome in these fish provides unusually comprehensive evidence for a core microbiome in this candidate aquaculture species.
Effects of diet on the hindgut microbiome of S. fuscescens
Some previous studies have observed dramatic effects of experimental diets on the gut microbiomes of farmed fish [25, 27, 29], whereas others have found that microbiomes of fish are relatively stable and do not show much overall change to dietary manipulation [38, 42, 55]. The studies that detected strong changes typically supplemented fish diets with probiotics or other functional feeds for 4–8 weeks experiments, longer than our experiments. However, Wong, et al. [38] fed Rainbow trout experimental diets including grains for a period of 10 months and observed only subtle changes in the fish microbial communities. Lyons, et al. [55] supplemented the diet of rainbow trout but in this case with microalgal meal at a level of 5% and found that, whilst addition increased diversity, the overall structure of microbiomes in the distal guts of the fish were not significantly altered. In another study similar to ours, Zhang, et al. [42] found that supplementing Siganus canaliculatus [a color morph of S. fuscescens; 56] with 10% Ulva pertusa and other additives for a period of 8 weeks did not significantly alter the microbial diversity in the gut content of the fish. They concluded that a strong core microbiome constituted of 86 operational taxonomical units (OTUs) which was shared across all fish regardless of the dietary treatment [42].
Although, all the seaweeds tested in this screening trial seemed to affect the hindgut microbiome composition of the mottled rabbitfish, the lack of impact at a high level was surprising given the breadth of biochemical compounds produced by these taxonomically diverse organisms and their known impacts on microorganisms in nature [57–59] and experiments [60–62]. The microbiome of seaweed varies greatly from its surrounding environment and between seaweed species. One mechanism involved in shaping the microbiome of seaweed is the biochemical composition of the surfaces of seaweed and the metabolites they produce. For example the red seaweed Delisea pulchra has been reported to be mainly colonised by Gram negative bacteria and this has been linked to the seaweed’s production of N – acyl – homoserine lactones which inhibit the signal pathways in Gram negative bacteria [63].
Seaweed have also been used as prebiotics in animal feeds, including for fish, to induce microbial shifts in the gut of its host towards a more beneficial bacterial assemblage [64–66]. Although, these studies focused on the prebiotic effects of the seaweed complex polysaccharides (e.g. sodium alginate, agar and carrageenan) there is one clear example from land animals that seaweed secondary metabolites can also have strong effects on the gut microbiome of it hots. Feeding ruminants the red seaweed Asparagopsis taxiformis quickly (e.g. 3 days) changed the rumen microbial composition leading to drastic reduction of methanogenic Archaea and as a result led to reductions in enteric methane emissions [67, 68]. This was the result of supplementing the diets of the ruminants (ovine and bovine) with up to 5% A. taxiformis which produces and accumulates halogenated compounds including the anti-methanogenic bromoform [69]. For these reasons, the bioactive and prebiotic potential of seaweeds are receiving increasing levels of attention [70].
Although seaweed are known to be a diverse group (~ 10,000 species) producing a wide range of secondary metabolites with bioactive properties, there is a gap in the literature regarding their potential as dietary supplement to shape intestinal microbiome of animals including fish [71, 72]. Even if the effects of dietary seaweed supplementation on the fish microbiome in our feeding trial were not as pronounced as expected, this study was the first to screen multiple seaweed species including some known for their secondary metabolites (e.g. Asparagopsis taxiformis, Caulerpa taxifolia and Laurencia obtusa). The potential influence of seaweed chemistry was well illustrated by one seaweed species in particular: fish fed diets supplemented with the green alga Caulerpa taxifolia more closely resembled those fed the control diet than the other seaweed treatments. In particular the genera Cetobacterium sp., Treponema sp. and Fusobacterium sp. were all enhanced in hindguts of fish that received C. taxifolia and this difference was significant for Fusobacterium sp.. This seaweed produces many interesting bioactive compounds [73] and its presence on reefs can completely alter sediment microbiomes through chemical modifications of the substrate [see 74 and references therein]. This seaweed is typically avoided by the native herbivorous fish (Girella tricuspidata) and invertebrate grazers in Australia [75] and can be toxic to invertebrates forced to consume it in feeding trials [75, 76]. Another seaweed avoided by marine herbivores due to its production and storage of bioactives is the seaweed A. taxiformis [77], which also stood out from the other treatments in our trial as it resulted in the highest abundance of Romboutsia sp. (2.5 times higher than the other dietary supplementations or control) in the hindgut microbiome of the fish that were fed it.
The Fusobacterium genus includes some important human pathogens that have been implicated in diseases such as colorectal cancer [78] and have been identified in GI microbiomes of other commercially important, warm water fish previously [20]. Additionally, although the role of Fusobacterium sp. in fish remains largely unknown [20], this genus has demonstrated enzymes for the breakdown of carbohydrates in three fish species [79]. Our observation of them in our fish supports the hypothesis put forward by Larsen et al, (2014) that these may be normal members of microbiomes in the guts of many, diverse fish species. The microbial taxonomic composition of the fish hindgut revealed other bacterial taxa associations typically far removed from fish, seaweed or marine settings. The genus Arcobacter sp. is usually associated with water from sewage [80] or farm effluents such as piggeries [81] but can be common in marine invertebrates such as crabs and oysters [82] and includes some intestinal pathogens of both humans and fish [83, 84]. In our study, it was particularly abundant in fish fed the ‘control’ diets without any supplementation (five times more abundant). This observation suggests that the seaweeds may perform a prebiotic-like role, which enhances the growth of more favourable bacteria, thereby preventing the growth of potential pathogens such as Arcobacter sp.. Although our fish were caught near the coast and could have been subjected to farm effluents through river flow during high rainfall events, this ASV was also found - albeit in lower abundances - in the hindgut of the fish on the remote One Tree Island site from the study from Nielsen, et al. [15] and on the two sites from the west coast of Australia from study by Jones, et al. [17], suggesting that it may be a commensal or symbiotic member of the core microbiome, or a widespread, opportunistic pathogen that dietary inclusion of seaweed keeps under control.
The vast majority of host associated intestinal bacteria within any host organism remain unidentified (in the current study and the other two used as comparisons, 53% of the ASVs > 1% abundance could not identified to the genus level) and their functions are largely unknown. For, example despite efforts to increase genera identification, the unknown bacterial genera in the human gut as part of the Human Microbiome Project and the Human Gastrointestinal Bacteria Genome Collection still represent 40% of the recovered genera as of August 2018 [85]. Nonetheless, various intestinal bacteria are known to benefit their host by aiding in digestion, metabolic processes, growth and development, immune responses and resilience to stress and other factors via the production of diverse compounds including short chain fatty acids and vitamins (79). For example, the vitamin B-12 producing Cetobacterium spp. was detected in most of the fish in the current study. Although this bacterium is generally associated with freshwater fish such as the grass carp, Ctenopharyngodon idellus, its detection could be related to the presence of functional genes associated with protein digestion within the genome of Cetobacterium spp. [86], which support host responses to dietary change [87]. Since our fish were wild-caught and, prior to capture, presumably browsed mainly on seaweed, Cetobacterium sp. might have been over-represented because these bacteria were supporting their hosts’ transition to new diets.
The appearance of Treponema sp. in all our fish was another unexpected observation. Treponema sp. has been described in the hindgut of S. fuscescens previously [15], and was also found in the hindgut of the fish from WA [17], yet is predominantly reported from the GI tracts of pre-industrial, traditional and agrarian human populations including pygmies and Amazonians [88] and other primates, terrestrial mammals and termites [89]. Although of significant interest in human microbiome research because this genus seems to have been eradicated from human GI tracts by unknown processes linked to industrialization, in the context of rabbitfish, this taxon might indicate the presence of a bacterial community using compounds such as xylan, xylose, carboxymethyldcellulose or hemicellulose which are likely present in aquafeed. This bacterium was overrepresented in our samples compared to the wild fish where it was absent (GBR) or present only at minute levels (0.1% and 0.2% for the Shark Bay and Kimberley fish respectively), particularly in those fish fed C. taxifolia supplements, to potentially assist the host in obtaining nutrients from these novel types of food, as β-1,3-xylan is a major component in the cell wall of this seaweed.
In the current study, the experimental design (which was optimised to include as many different seaweeds and aquafeed supplements as possible), lacked statistical power in some cases due to the unexpectedly stable microbiome of these fish overall, the lack of genus assignment for half of the recovered ASVs and, the high level of between-individual variation within treatments, thereby limiting the ability to resolve some of the statistically observed differences. Furthermore given the extraordinary ecological breadth of ‘fish’ (marine, estuarine, freshwater, diadromous lifestyles, carnivorous, omnivorous and herbivorous trophic levels, benthic pelagic, cryptic lifestyles, etc.) and the multiple, complex ways in which their environments (wild [polar, temperate, tropical, shallow, deep, etc.] or aquaculture), and biology (e.g. genetics, life history stage) can affect their gut microbiomes, more research is needed to better understand the general influences of diet on the composition, diversity and function of microbiomes within the GI tracts of fish to enable generalisations about the influence of diet.
Effects of temporal and spatial variation on the hindgut microbiome of S. fuscescens
When comparing the three populations, the hindgut microbiomes from rabbitfish on the GBR seemed more distinct than other populations. Overall, the hindgut microbiome composition of the fish from our study (Sunshine Coast) more closely resembled those from Shark Bay in Western Australia, which is at a similar latitude. Surprisingly our fish were more similar to fish from the tropical Kimberley site than the tropical GBR fish, which were collected from a site much closer than the former.
Potential explanations for these groupings are that all of the seaweed genera fed to our fish have tropical, subtropical or temperate distributions and are common on the eastern coast of Australia, with many also occurring on the west [90]. It is therefore possible that some of the similarities between the wild populations and our seaweed treated fish, may be the result of the fish at those locations feeding on similar diets. Another explanation for the similarities observed between our fish and those from Shark Bay could be similar abiotic and biotic factors, given that the Kimberley and GBR sites are both tropical and the Sunshine Coast and Shark Bay sites are both sub-tropical locations. Furthermore, the fish from our screening trial were collected near shore (< 1 km away), as were the fish from Shark Bay, whereas the fish from the Kimberley site were approximately 25 km from the coast and finally the most distinct hindgut microbiome was found in the fish from One Tree Island on the GBR which is about 70 km from the coast. The impact of rivers, agriculture and other human or land associated impacts may be clearer in nearshore areas, which could explain some of the differences observed here.
Similarly low spatial and temporal variation was observed in the gut microbiomes of larvae from another rabbitfish species, Siganus guttatus, across 3 sites separated by up to 390 km across a three year sampling program [91]. The gut microbiota of zebrafish Danio reiro, from genetically distinct wild and domesticated populations were strikingly similar despite very different environmental and dietary conditions and the authors speculated that shared intestinal features from the two groups of fish led to the selection of specific bacteria taxa resulting in strongly similar gut microbiome regardless of the origin or domestication status [92].
Because of vastly different biotic and abiotic conditions, along with different sampling times and teams, distance from the coast and a myriad of other possible differences across the three studies, many factors not considered here would likely influence GI microbiomes in the sampled fish so consequentially, the authors from this study will refrain from drawing any broad conclusions regarding specific supplements used in this study, or even broad geographical patterns from these comparisons. However, the overlap between hindgut microbiomes from fish in the three studies against the backdrop of such biological and physical variation, provides further compelling evidence for the existence of a core microbiome in S. fuscescens.
Does Siganus fuscescens have a core microbiome?
Despite experimental manipulations of diet with a broad and comprehensive list of taxonomically and chemically diverse seaweeds and samples originating from populations in locations separated by up to 4000 km around the Australian coast (the Sunshine Coast in southeast Queensland; present study, Shark Bay and the Kimberley site in Western Australia; Jones, et al. [17], and the Great Barrier Reef; Nielsen, et al. [15]), the mottled rabbitfish in Australia maintained nearly one third of their hind gut microbiota in common, of which 35 ASVs were identified as being part of a core microbiome in this species. Identifying a core microbiome is one of the first steps required to link microbial community structure and diversity to its function and importantly, the role it plays for its host [93]. The existence of a conserved group of bacterial taxa indicates that the functions they provide, persistently throughout multiple populations (and in our case, despite diet manipulation and starvation), could be essential to the development or homeostasis of the host. However, 80% of the identified core ASVs were not identified at the genus level, making it difficult at this stage to speculate on the function they may play. Core microbiomes have been identified in other marine organisms, including corals [94, 95], seaweeds [96] and other cultivated fish [38]. Understanding how these microbial taxa benefit fish could help expedite the development of a new aquaculture industry for Siganids, of which many species including S. fuscescens, are being considered for culture in different countries around the world given their flexible dietary nature and tolerance to a broad range of environmental factors [45–48].
Evidence for core microbiomes has been observed in studies of other rabbitfish species [42, 91]. Consistent with our observations, these authors reported both overlap and variation between locations and/or populations and speculated that if the core microbiome related to functionality, rather than taxonomy per se then the functional redundancy within the core microbiome could be expected. Functional redundancy has been identified in core microbiomes from other marine organisms, including a seaweed [97], so investigating the functional profiles of these ‘core’ taxa could be a sensible way to progress towards a better understanding of the roles microbiomes play for their hosts.
Microbiomes and the sustainable development of aquaculture
The existence of a core microbiome would suggest that the members of that microbiome were essential to the normal development or homeostasis of the host (79). Understanding the functions core microbiota play for their hosts will provide valuable insights for the development of sustainable aquaculture practises. For example, understanding the roles of gut microbiomes in herbivorous fish may help to facilitate the transition of carnivorous fish onto more sustainable plant-based diets. It is possible that microbiome manipulations could be applied via microbiome transfer or targeted probiotic strains delivered in the feed. Although, interspecific transfer of microbial communities is a novel area of microbiome research, early results are promising and suggest the possibility of establishing a stable microbiome from one donor species in the GI tract of a recipient host belonging to a completely different species. This has been trialled between humans and pigs and between fish and rats [98]. In one example, microbial community transfer between two rats: an herbivore (Neotoma albigula) and an omnivorous laboratory rat, (Rattus norvegicus) led to the development in the latter a newly acquired ability to degrade oxalate, a nephrotoxin found in plant materials, which persisted for 9 months after the transplant [99]. Microbiome manipulations such as these have the potential to greatly enhance the sustainability and resilience of the aquaculture industry.
‘Designer microbiomes’ have been proposed as a potential solution to the global problem of coral bleaching due to thermal stress. van Oppen and Blackall [100] and others have proposed that probiotics (either naturally-occurring or genetically engineered microbiomes) could be delivered to corals to enhance their tolerance to thermal stress. Warming ocean waters are also problematic for aquaculture because of associated reductions in the thermal niches of high latitude fish (e.g. Atlantic salmon Salmo salar), thus restricting the availability of places in which they can be farmed, while more temperate and subtropical species tend to be more tolerant to temperature fluctuations [101, 102]. It is possible that designer microbiomes of the future could also address this issue by extending the thermal tolerance of commercially valuable fish species.
A potential barrier to the use of microbiome manipulations for sustainable aquaculture is that a significant proportion of host-associated bacteria have never been identified, in our study and others [15, 17, 103–105]. For example, one ASV from our study could only be taxonomically ascribed with confidence at the Kingdom level, yet it accounted for > 30% of the total abundance of the microbes in some fish populations. Furthermore, this represents a key knowledge gap as functional traits of many of these microbes is almost entirely unknown thus highlighting a priority area for future research into holobionts in ecological or any applied context, including aquaculture.
No clear or consistent effects of fish size on hindgut microbiomes of S. fuscescens
Many researchers have reported on the importance of life history stage in the diversity and structure of gut microbiomes (Egerton et al. 2018). Due to the haphazard nature of our sampling, the fish collected for the feeding trial ranged between 15–21 cm in length and 70 g – 189 g in mass. Fish size can be used as a proxy for fish age, however when fish mass or fish lengths were included as covariates (comparing fish fed different diets and fish from different populations/studies), it did not explain any of the variation in microbial diversity or composition. This could suggest that by the time they are 15 cm + in length, the microbiome of S. fuscescens is fairly stable.