Core Intestinal Microbiome Richness of Coral Reef Damselshes (Actinopterygii: Pomacentridae) Reects Trophic Guild

Background: Fish harbour diverse microbiomes within their gastro-intestinal system that affect the host’s digestion, nutrition and immunity and facilitate resource partitioning in coral reef ecosystems. Despite the great taxonomic diversity of sh, little is understood about sh microbiome diversity and the factors that determine its structure and composition. Damselsh are important coral reef sh species that play a strong role in determining algae and coral structure of reefs. Broadly, damselsh belong to either of two trophic guilds based on whether they are planktivorous or algae-farming. In this study, we use 16s rRNA sequencing to interrogate the intestinal microbiome of 10 damselsh species (Pomacentridae) from the Great Barrier Reef to compare the composition of their intestinal bacterial assemblages across the planktivorous and algae-farming trophic guilds. Results: We identify core intestinal bacterial taxa for each host sh species. Gammaproteobacteria, belonging to the genus Actinobacillus, were detected in 80 % of sampled individuals and suggests a possible core member of pomacentrid microbiomes. Core microbiomes of algae-farming species were more diverse than planktivorous species with farming species sharing 35 ± 22 ASVs and planktivorous sharing 7 ± 3 ASVs. We also provide evidence for signicant shifts in bacterial community composition along the intestines. We show that Bacteroidia, Clostridia and Mollicutes bacteria are more abundant in the anterior intestinal regions while Gammaproteobacteria are generally highest in the stomach. Finally, we highlight differences in microbiomes associated with both trophic guilds. Algae-farming and planktivorous damselsh host species signicantly differed in their composition of bacteria belonging to Vibrionaceae, Lachnospiraceae and Pasteurellaceae. Conclusions: Our results demonstrate that the richness of the core intestinal bacterial communities of damselsh reects host species diet and trophic guild, whereby algae-farming hosts have larger and more diverse core microbiomes than planktivorous hosts. We suggest that algae-farming damselsh within the same species share bacterial taxa that reect their specialised diets.


Background
Fishes represent the greatest taxonomic diversity of vertebrates, and despite our understanding of the importance of intestinal microbiota of terrestrial vertebrates, we still lack an understanding of sh microbiome diversity and functioning [3]. Largely, sh microbiome studies have centred around species with commercial value, including trout, salmon and carp [4]. For example, gastrointestinal sh microbiomes are known to be important in intestinal cell proliferation [5,6], nutrition [3,7] and immunity [8][9][10]. These studies show that the intestines of shes harbour a large abundance and diversity of bacteria [11] and regulation of this diversity is important in the maintenance of host health through a complex set of microbe -microbe interactions and microbe -host interactions [1,2].
There are many factors that affect the structure of sh gastrointestinal microbiomes [3,4]. These include host-related factors such as genetic attributes, size, age, sex [12][13][14], host phylogeny [15][16][17],environmental factors (such as water quality) [18][19][20] and host diet [15,17,18]. Studies that investigate intestinal microbiome changes have mostly been concentrated on the impact of sh foods on species of aquaculture importance [21,22], although a few studies have investigated wild sh populations [15,23]. Bacterial symbiont diversi cation in wild herbivorous surgeon sh intestines is thought to be an important driver of host niche-partitioning [24,25], suggesting that intestinal microbiomes can in uence the trophic ecology of coral reefs and facilitate resource partitioning in these hyper-diverse ecosystems.
There is increasing evidence that herbivorous shes have distinct microbiomes as compared to omnivorous and carnivorous shes [26]. Herbivorous and carnivorous sh diets are known to cause shifts in intestinal microbiomes; shes with plant-based diets have intestinal microbiomes dominated by Firmicutes, such as Clostridium, while shes with fat-based diets have microbiomes dominated by protease producing Proteobacteria [27][28][29][30]. In addition, the diversity of herbivorous sh intestinal microbiomes is higher than omnivorous and carnivorous host species under similar environmental conditions [31], suggesting that host feeding behaviour has a signi cant effect on sh intestinal microbiomes.
Damsel shes (Pomacentridae) are a diverse and abundant group of coral reef shes [32,33], and they are among the most widely studied family of reef shes [34,35]. Broadly, damsel shes are grouped into either planktivorous or herbivorous trophic guilds, although some herbivorous species may also feed on zooplankton [36]. Many herbivorous damsel shes that inhabit reef crest environments are territorial, and they cultivate palatable algae within their territories, which they aggressively defend from other species.
In this study, we investigate the intestinal microbial diversity of ten species of planktivorous and algaefarming damsel shes, two guilds of damsel shes that signi cantly impact coral reef trophic dynamics.
Planktivorous damsel shes play a key role transferring energy from the plankton to higher tiers of food chain, while algae-farming damsel shes in uence sediment and algae dynamics on coral reefs and may increase the presence of coral disease associated pathogens within their territories [35,40,49,[52][53][54]. The aim of this study is to describe the taxonomic composition of planktivorous and algae-farming damsel sh intestinal microbiomes. Thus, we hypothesise that differences in intestinal microbial communities will re ect the differences between these two feeding guilds. Speci cally, across the different host species and feeding guilds, we examined (1) the phylogenetic differences in microbial communities, (2) the core microbial members, and (3) the changes in microbial community structure along the length of the intestinal tract.

Species collections and dissections
Fishes were collected from the Heron Island lagoon in the southern Great Barrier Reef, Australia (23°26'53"S, 151°56'52"E) in January and February of 2015. Collections occurred at a depth of 1-8 m adjacent to the Heron Island Research Station. Three individuals of ten damsel sh species of similar lengths were randomly collected across the two trophic guilds (Table 1). Each trophic guild was represented by ve species and 15 individuals. Collections were conducted on SCUBA, and the planktivorous species were collected using a barrier net, while the algae-farming species were collected by speargun. Following collections, the shes were immediately placed on ice and transported to Heron Island Research Station. In the laboratory under sterile conditions, shes were weighed, measured and photographed, then the gastrointestinal tract was removed, and the gut length was recorded and photographed. The entire gut was xed in 4% DNA/RNA free paraformaldehyde and sterile phosphatebuffered saline for 12 hours, then it was stored in DNA/RNA free water. Ampli cation of the 16S V1-V3 rRNA gene region was done using the primers 27F (5'-AGRGTTTGATCMTGGCTCAG-3') [55] and 519R (5'-GTNTTACNGCGGCKGCTG-3') [56] with barcodes on the forward primer. These genes were ampli ed in a 30 cycle PCR (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 °C for 3 minutes, followed by 28 cycles of 94 °C for 30 seconds, 53 °C for 40 seconds and 72 °C for 1 minute, after which a nal elongation step at 72 °C for 5 minutes was performed. After ampli cation, PCR products were checked in 2% agarose gel to determine the success of ampli cation and the relative intensity of bands. Multiple samples were pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were puri ed using calibrated Ampure XP beads. Then the pooled and puri ed PCR products were used to prepare a DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at the Molecular Research LP (MR DNA; Texas, USA) on a MiSeq™ System following the manufacturer's guidelines.
Amplicon sequence data were sorted by the sample and demultiplexed using demux for QIIME 2 (version 2018.11; Bolyen et al. 2018). Sequences were screened for quality, trimmed at 450 bp after removal of primer sequences and assigned as amplicon sequence variants (ASVs) [58] using DADA2 [59]. Taxonomy of the ASVs was determined using a pre-trained, naїve Bayes classi er [60] and the q2-feature-classi er plugin [61]. The classi er was trained on the target 480 bp region of sequences in the Greengenes 13_8 99% database. ASV clusters were arranged in a phylogenetic tree using FastTree [62,63] and visualised using Interactive Tree of Life [64]. The feature table, metadata and taxonomic classi cations were exported from QIIME 2 in .biom format [65], and the rooted phylogenetic tree was exported in .nwk format.
The closest known sequences and the origin of selected ASVs were identi ed through a BLASTN-based search against the GenBank nr/nt database.

Statistical analysis
The exported feature table and phylogenetic tree were imported into R version 3.5.2 (R Core Team 2019) and stored as a phyloseq object [66] for downstream analyses. All ASVs not assigned to phylum were ltered from the data, and those designated as chloroplasts or cyanobacteria were removed and stored as a separate object for further analysis. Samples were rare ed to minimum sampling depth for diversity analyses; however, non-rare ed data were used for multivariate modelling [67,68]. Multivariate generalised linear models were used to test for signi cant differences in bacterial communities among host sh species, trophic guild and location along intestines using mvabund in R [69,70]. Bacterial taxa were grouped by class when examining microbiome changes along the length of the intestinal tract. Bacterial community data were tted to negative binomial distributions and tested using log-likelihood ratios (LRT) via 999 simulations using Monte Carlo resampling. A nested analysis of variance (ANOVA) used to test the role of gut location when accounting for species variation. Traditional distance-based ordination methods to visualise variation across communities, such as non-metric multidimensional scaling (NMDS) and principal coordinate analysis (PCoA) may confound trends [71]. To avoid these issues, the R package boral [72] was utilised to explain the bacterial community composition of each sample through a set of latent variables. Bacterial community data were tted to a negative binomial distribution, and the model was run with two latent variables to account for residual variation for each of the major phyla detected in the samples. Venn diagrams were produced using the VennDiagram package [73].

Results
A total of 1,254,909 sequences were detected in 119 samples after denoising and trimming of all chloroplast, mitochondria sequences and host DNA. Among these sequences, 3,776 ASVs were detected; 39.4% of which belonged to the Phyla Proteobacteria, 26.2% to Bacteroidetes, 13.4% to Firmicutes and 12.6% to Planctomycetes. The 20 most abundant ASVs accounted for 41% of the total number of detected sequences. The most common ASV belonged to the genus Actinobacillus and accounted for 9.9% of the total detected sequences ( Table 2). A further two unknown members of Mollicutes and Pasteurellacea accounted for 9.9 and 3.8% of sequences, respectively. An ordination analysis revealed that most host sh species have distinct Proteobacteria, Bacteroidetes and Firmicutes communities (Fig. 2). Abudefduf sexfasciatus and Abudefduf whitleyi displayed high variation in Proteobacteria communities while the two trophic guilds have similar community composition. Bacteroidetes are distinct for A. sexfasciatus and Stegastes apicalis, with no discernible patterns between the two trophic guilds. Communities of Firmicutes are the most distinct between host species, although some host species, such as S. apicalis, Chromis atripectoralis and A. whitleyi, are variable in composition. However, there is reasonable separation of the two trophic guilds in terms of Firmicutes community composition .

Core Microbiomes
Most ASVs occur in less than 30% of sampled individuals across all host species (Fig. 3a). 13 bacterial ASVs are found in more than 30% of sampled individuals; therefore, they may represent core members of pomacentrid microbiomes ( Table 3). The most common ASV in this study belongs to the genus Actinobacillus, which occurs in more than 80% of sampled individuals, albeit at a low abundance in many individuals, with the highest abundances in the planktivorous damsel shes Acanthochromis polyacanthus and P. moluccensis. Table 3 Taxonomic composition of core ASVs occurring in more than 80% of sampled individuals. Accession numbers for closest GenBank sequences (similarity given in brackets) are supplied. Occurrence and relative abundances were generated from rare ed data. Signi cant variation in the richness of core bacterial assemblages for each sh species (de ned as ASVs that are shared between all sampled individuals for each species) were also detected (Fig. 3b) Fig. 1). There is high richness of core Pasteurellaceae and Vibrionales ASVs, with 10 and 21 core members, respectively. High diversity of an unknown clade of Gammproteobacteria is also reported for P. moluccensis and Pomacentrus wardi hosts .
There are 61 core ASVs belonging to the Bacteroidetes, 28 of which occur in S. apicalis and 38 in P. perspicillatus ( Supplementary Fig. 2). An unknown clade of Flavobacteriales and a diverse consortium of Rikenellaceae are core members of S. apicalis, while P. perpicillatus has a diverse core assemblage of ASVs belonging to the family Flavobacteriaceae. One ASV belonging to Spirochaetes, Brevinema andersonii, is a core member of S. nigricans and C. atripectoralis, while a Tenericutes ASV belonging to Mollicutes is a core member of all host species except the planktivorous damsel shes A. polyacanthus and A. sexfasciatus ( Supplementary Fig. 3). There is a rich consortium of core Firmicutes ASVs for S. apicales and S. nigricans, which includ members of the Erysipelotrichaceae, Ruminococcaceae and Lachnospiraceae families.  (Fig. 5).

Effect of trophic guild on microbiomes
There is a signi cant difference between trophic guilds and microbiome composition (LRT= -0.021, P = 0.001). Most bacterial ASVs are unique to either of the trophic guilds of the host sh, with only 124 ASVs common to both guilds (Fig. 5). 78 bacterial ASVs, belonging to 20 families, are important drivers of this relationship. There are marked differences in abundances of ASVs belonging to Vibrionaceae, Lachnospiraceae and Pasteurellaceae. Two Vibrio sp. (Vibrionaceae) are more common in planktivorous host species, and ve members of Actinobacillus are more abundant in algae-farming host species. None of the ASVs occurred exclusively in the planktivorous or algae-farming damsel shes, suggesting that this relationship is driven by host species rather than trophic guild.

Discussion
This study reveals that algae-farming damsel sh species have taxonomically richer core microbiomes than planktivorous species. This result is likely attributable to the specialised feeding behaviour of these species where they largely consume a narrow range of turf algae species [37,39,49], unlike planktivorous species which are adapted to a more opportunistic feeding strategy. We also provide evidence that algaefarming damsel sh tend to have more diverse intestinal microbiomes than planktivorous species. These results show that microbiome structure of host sh species that have specialised feeding behaviour have acquired specialised intestinal bacteria and further research is needed to investigate how microbiome specialisation effects host digestion and metabolism.
Like many other species of marine sh, the intestinal microbiomes, the damsel sh microbiomes presented here were dominated by members of Proteobacteria, Bacteroidetes, Firmicutes and Planctomycetes. For example, surgeon sh, parrot sh and rabbit sh intestinal microbiomes from the Red Sea also consist of diverse assemblages of Firmicutes and Proteobacteria [15]. Another dominant ASV in the damsel sh microbiome belonging to Mollicutes (Tenericutes) resembled bacteria detected in rabbit sh intestines [23]. The number of highly similar bacterial ASVs shared among pomacentrids, acanthurids and siganids may re ect the similar feeding behaviours of these coral reef shes. For instance, algae-farming damsel shes may also ingest prey items other than algae, such as zooplankton [36] or other invertebrates [75]. The functional roles of these seemingly important microbial taxa warrant further attention in order to understand the potential consequences on host metabolism and health.
Damsel sh microbiomes were largely dominated by Gammaproteobacteria of the Pasteurellaceae, with one ASV occurring in more than 80% of sampled shes and representing almost 10% of total detected sequences. Although this ASV currently represents an unknown species of the Actinobacillus genus, a 98% similar sequence has been collected from the intestines of surgeon shes in Saudi Arabia [25], suggesting these taxa are important components of reef sh microbiomes. Members of Pasteurellaceae have also been recorded in high abundances in adult damsel shes and cardinal shes collected around Lizard Island, Australia [76], and they are deemed important components of tropical planktivorous sh gut microbiomes [77]. Gammaproteobacteria are also very abundant on the skin of many coral reef shes [78]. The prevalence of Pasteurellaceae amongst the damsel shes in this study, as well as other reef shes, provides additional evidence that Pasteurellaceae are important members of coral reef associated sh microbiomes.
Algae-farming damsel shes had larger core microbiomes than the planktivorous damsel shes, and these core microbiomes were speci c to each host species. For example, P. wardi and P. moluccensis had diverse, but different strains of Gammaproteobacteria, while D. perspicillatus and S. apicalis had large Bacteroidetes core communities but were dominated by Flavobacteriaceae and Rikenellaceae, respectively. Different species of territorial damsel shes farm and consume different species of algae [37,49], and the large differences in their specialized microbiomes may re ect these narrow dietary preferences. Conversely, the small core microbiomes of the planktivorous damsel shes may re ect the high variation in consumed plankton of each species, suggesting these shes have opportunistic feeding behaviours. These results, however, do not support the notion that sh with greater diet variability have more diverse microbiomes [26]. In fact, the damsel sh with narrow, algae-farming feeding behaviours tended to have the greatest diversity of intestinal bacterial, suggesting that the host may select microbial populations that include specialised bacteria that enhance the digestion and absorption of nutrients from speci c algal diets.
Recent evidence suggests a high degree of resource partitioning in sh communities which is a key mechanism that facilitates the high diversity of coral reefs [79,80]. The largely distinct microbiomes of each host species presented in this study may re ect the high degree of resource partitioning found in coral reef communities, whereby different species of damsel sh may be consuming different size classes of zooplankton [79], farm different algal species [37,49] or occupy different trophic niches [80]. The similarity between closely related host species and microbiomes, such as P. wardi and P. moluccensis, also demonstrates that phylogeny may in uence the intestinal microbiomes of damsel shes [15][16][17]78].
Interestingly, Photobacterium damselae, Vibrio harveyi, Vibrio ponticus and other Vibrio sp. were prevalent amongst the damsel shes sampled in this study. These bacteria represent potential pathogenic members of Vibrionacaea and have been detected in many shes of aquaculture importance, including Chromis punctipinnis [81], Lutjanus argentimaculatus [82], Seriola dumerili [83], Scophthalmus maximus [84,85], Sparus aurata [86], Solea quinqueradiata [87] and Solea senegalensis [88]. Although identi ed as Vibrio harveyi in the GreenGenes database, GenBank revealed there was high similarity of these sequences to other members of the Harveyi clade, such as Vibrio owensii [83]. There are thought to be up to 11 species of Vibrio belonging to this clade [89], most of which are pathogens of sh, shrimp and coral [90][91][92]. Given the apparent healthy state of the sampled shes and the high abundances of potentially pathogenic Vibrionacaea in the sh guts, we provide support to the idea that these organisms are natural components of healthy sh microbiomes and are opportunistic pathogens in shes only under speci c conditions [82,93].
The facultative anaerobic bacterial classes Bacteroidia, Clostridia and Mollicutes were generally in higher abundance in the mid and posterior intestinal regions than the stomach. Differences in microbiomes along the intestinal tract have been recorded in the rabbit sh Siganus fuscescens [94], with midgut communities more representative of the environmental sources and hindguts hosting a microbiome more specialised to anaerobic conditions and fermentation [95]. The increase in Bacteroidia, Clostridia and Mollicutes along the intestines may be due some members of the class being mutualistic components of the sh gastrointestinal ora. Some members of Bacteroidetes are known to breakdown polysaccharides and metabolise the derived sugars [96], while members of Clostridium are known to metabolise cellulose [30]. Our results con rm the increased prevalence of anaerobic bacteria in the hindgut of damsel shes, which probably consists of taxa responsible for the fermentation and metabolism of complex molecules before being absorbed by the host [3].

Conclusions
In this study, we demonstrate that damsel shes have diverse intestinal microbial communities whereby the core members of a species re ect diet and trophic guild. We show that algae-farming damsel shes have more rich core microbiomes, which may re ect the more specialised diets of these species. We also provide evidence that damsel sh mid and posterior intestines have higher abundances of facultative anaerobic bacteria that are known to play important roles in fermentation and cellulose breakdown.
These ndings add to a growing body of literature that suggests that host sh feeding behaviour has a strong in uence on the composition of intestinal microbiomes.

Declarations
Ethics approval and consent to participate The authors declare that they have no competing interests.

Funding
Not applicable Authors' contributions CRJK analysed and interpreted the amplicon sequence data and was the major contributor in writing the manuscript and preparing gures and tables. JMC undertook the eldwork and collected all specimens, performed gut dissections, tissue biopsies and provided feedback on the manuscript. JHC was involved with the initial synthesis and design of this study and provided feedback on the manuscript. WL and TDA were involved with the initial synthesis and design of this study, provided the facilities to undertake laboratory work and provided feedback on the manuscript. All authors read and approved the nal manuscript. Figure 1 Mean observed richness, Shannon diversity and evenness for each sh species. Planktivorous host species are shaded red and algae-farming species shaded green. n=3.   Venn diagrams depicting the number of shared ASVs for each trophic guild (left) and for each region of the intestine (right).

Supplementary Files
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