Intestinal Microbiome Richness of Coral Reef Damselfishes (Actinopterygii: Pomacentridae).

Fish gastro-intestinal system harbors diverse microbiomes that affect the host's digestion, nutrition, and immunity. Despite the great taxonomic diversity of fish, little is understood about fish microbiome and the factors that determine its structure and composition. Damselfish are important coral reef species that play pivotal roles in determining algae and coral population structures of reefs. Broadly, damselfish belong to either of two trophic guilds based on whether they are planktivorous or algae-farming. In this study, we used 16S rRNA gene sequencing to investigate the intestinal microbiome of 5 planktivorous and 5 algae-farming damselfish species (Pomacentridae) from the Great Barrier Reef. We detected Gammaproteobacteria ASVs belonging to the genus Actinobacillus in 80% of sampled individuals across the 2 trophic guilds, thus, bacteria in this genus can be considered possible core members of pomacentrid microbiomes. Algae-farming damselfish had greater bacterial alpha-diversity, a more diverse core microbiome and shared 35 ± 22 ASVs, whereas planktivorous species shared 7 ± 3 ASVs. Our data also highlight differences in microbiomes associated with both trophic guilds. For instance, algae-farming damselfish were enriched in Pasteurellaceae, whilst planktivorous damselfish in Vibrionaceae. Finally, we show shifts in bacterial community composition along the intestines. ASVs associated with the classes Bacteroidia, Clostridia, and Mollicutes bacteria were predominant in the anterior intestinal regions while Gammaproteobacteria abundance was higher in the stomach. Our results suggest that the richness of the intestinal bacterial communities of damselfish reflects host species diet and trophic guild.

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 fish microbiome diversity and functioning (Clements et al. 2014). Largely, fish microbiome studies have centered around species with commercial value, including trout, salmon, and carp (Wang et al. 2018). For example, gastrointestinal fish microbiomes are known to be important in intestinal cell proliferation (Rawls et al. 2004; Cheesman et al. 2011), nutrition ( Ray et al. 2012; Clements et al. 2014), and immunity (Bates et al. 2006; Bates et al. 2007; Galindo-Villegas et al. 2012). These studies show that the intestines of fishes harbor a large abundance and diversity of bacteria (Nayak 2010) and the regulation of this diversity is important in the maintenance of host health through a complex set of microbe-microbe and microbe-host interactions ( Neish 2009; Foster et al. 2017).

There are many factors that affect the structure of fish gastrointestinal microbiomes (Clements et al. 2014; Wang et al. 2018). These include host-related factors such as genetic attributes, size, age, sex (Bolnick et al. 2014; Li et al. 2016; Stephens et al. 2016), host phylogeny (Sullam et al. 2012; Li et al. 2014; Miyake et al. 2015), environmental factors (such as water quality) (Hagi et al. 2004; Sullam et al. 2012; Neuman et al. 2016), and host diet (Miyake et al. 2015; Neuman et al. 2016). Studies that investigated intestinal microbiome changes have mostly focused on the impact of fish foods on species of aquaculture importance (Ringø et al. 2006; Martin-Antonio et al. 2007), although a few studies have investigated wild fish populations (Miyake et al. 2015; Zhang et al. 2018). For instance, bacterial symbionts diversification in wild herbivorous surgeonfish intestines is thought to be an important driver of host niche-partitioning (Miyake et al. 2016; Ngugi et al. 2017), suggesting that intestinal microbiomes can influence the trophic ecology of coral reefs and facilitate resource partitioning in these hyper-diverse ecosystems. However, the involvement of intestinal bacteria in wild fish physiology remains largely unknown.

There is increasing evidence that herbivorous fishes have distinct microbiomes as compared to omnivorous and carnivorous fishes (Givens et al. 2015). Herbivorous and carnivorous diets are known to cause shifts in intestinal fish microbiomes; fishes with plant-based diets have intestinal microbiomes dominated by Firmicutes, such as Clostridium, while fishes with fat-based diets have microbiomes dominated by protease-producing Proteobacteria (Desai et al. 2012; Ingerslev et al. 2014; Liu et al. 2016). In addition, the diversity of herbivorous fish intestinal microbiomes is higher than omnivorous and carnivorous host species under similar environmental conditions (He et al. 2013), suggesting that host feeding behavior has a significant effect on fish intestinal microbiomes.

Damselfishes (Pomacentridae) are a diverse and abundant group of coral reef fishes (Cooper et al. 2009; Campbell et al. 2018), and they are among the most widely studied families (Choat 1991; Emslie et al. 2019). Broadly, damselfishes are grouped into either planktivorous or algae-farming trophic guilds, although some herbivorous species may also feed on zooplankton (Eurich et al. 2019). Planktivorous damselfishes play a key role in transferring energy from the plankton to higher tiers of the food chain, while algae-farming damselfishes influence sediment and algae dynamics on coral reefs and may increase the presence of coral disease-associated pathogens within their territories (Casey et al. 2015; Casey et al. 2015; Emslie et al. 2019; Randazzo Eisemann et al. 2019, Tebbett et al. 2020; Blanchette et al. 2019). Algae-farming species can be differentiated based on the algal composition within their territories, and they are divided into several behavioral guilds, including indeterminate grazers, extensive grazers, and intensive grazers (Hata and Kato 2004; Emslie et al. 2012; Casey et al. 2015; Emslie et al. 2019). Indeterminate and extensive grazers feed both on macroalgae and turf, while intensive grazers maintain distinct areas of turf algae through selective grazing and weeding of unpalatable algae (Gibson et al. 2001; Emslie et al. 2012). Intensive grazing damselfish are also referred to as algae farmers. Research on intensive grazers has focused on competition (Eurich et al. 2018), patterns of co-existence (Eurich et al. 2018; Eurich et al. 2018; Eurich et al. 2019), behavioral interactions (Kasumyan 2009; Weimann et al. 2018), and their role in structuring algae and coral communities (Klumpp et al. 1987; Ceccarelli et al. 2005; Ceccarelli 2007; Gochfeld 2010; Casey et al. 2014; Casey et al. 2015).

In this study, we investigated and described the intestinal microbial diversity of ten species of planktivorous and algae-farming damselfishes, two guilds that significantly impact coral reef trophic dynamics. We hypothesized that differences in intestinal microbial communities will reflect the differences between these two trophic guilds. Specifically, across the different host species and trophic guilds, we examined (1) differences in bacterial communities across fish species and trophic guilds, (2) core microbial members, and (3) changes in microbial community structure along the length of the intestinal tract.

Methods

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 2015. Collections occurred at a depth of 1–8 m adjacent to the Heron Island Research Station. Three individuals of ten sympatric damselfish species (Abudefduf sexfasciatus, A.whitleyi, Acanthochromis polyacanthus, A. polyacanthus, Chromis atripectoralis, Dischistodus pseudochrysopoecilus, D. perspicillatus, Pomacentrus moluccensis, P. wardi, Stegastes apicalis, and S. nigricans) of similar lengths were randomly collected across the two trophic guilds planktivorous and algae-farming. Each trophic guild was represented by 5 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 using a speargun. Following collections, the fishes were immediately placed on ice and transported to Heron Island Research Station. In the laboratory under sterile conditions, fishes were weighed, measured and photographed, then the gastrointestinal tract was removed, and the gut length was recorded and photographed. The entire gut was fixed in 4% DNA/RNA free paraformaldehyde and sterile phosphate-buffered saline for 12 h, then it was stored in DNA/RNA free water.

DNA extraction, amplification, and sequencing

Samples were transported to James Cook University for subsampling along each intestinal tract and DNA extraction. Under sterile conditions, standardized biopsy cores (3 × 3 mm) were taken from four locations along the intestinal tract: the stomach, the anterior intestine, the mid-intestine, and the posterior intestine. DNA was extracted from tissue biopsies using a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) following the manufacture's guidelines. A nanodrop was used to record the quality (260/280 ratio) and quantity (ng/μL) of DNA from each extraction.

Amplification of the 16S V1-V3 rRNA gene region was done using the primers 27F (5′-AGRGTTTGATCMTGGCTCAG-3′) (Ludwig 2007) and 519R (5′-GTNTTACNGCGGCKGCTG-3′) (Lane et al. 1985) with barcodes on the forward primer. These 16S rRNA genes were amplified using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification 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 purified using calibrated Ampure XP beads. Then the pooled and purified 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 V2 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) using DADA2 (Callahan et al. 2016). Taxonomy of the ASVs was determined using a pre-trained, naїve Bayes classifier (Pedregosa F) and the q2-feature-classifier plugin (Bokulich et al. 2018). The classifier 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 (Price et al. 2010) and visualized using Interactive Tree of Life 3.6.1 (Letunic and Bork 2016). The feature table, metadata, and taxonomic classifications were exported from QIIME 2 in .biom format and the rooted phylogenetic tree was exported in .nwk format. The closest known sequences and the origin of selected ASVs were identified through a BLASTN-based search against the GenBank nr/nt database.

Statistical analysis

The feature table and phylogenetic tree were imported into R version 3.5.2 and stored as a phyloseq object (McMurdie and Holmes 2013) for downstream analyses. All ASVs not assigned to phylum were filtered from the data, and those designated as chloroplasts or cyanobacteria were removed and stored as a separate object for further analysis. Samples were rarefied to minimum sampling depth for alpha-diversity analyses, which was estimated using the R package vegan (Oksanen et al. 2017). Non-rarefied data were used for generalized linear model (GLM) analysis (McMurdie and Holmes 2014; McMurdie 2018). Data used for principal component analysis (PCA), betadisper-test and PERMANOVA were computed using centered log-transformed Euclidean distance matrices of the non-rarefied ASV table. Differences in alpha-diversity between trophic guilds were tested via t-test. Multivariate GLM was used to test for significant differences in bacterial communities among host fish species, trophic guild, and location along intestines using mvabund in R (Wang et al. 2012). PCA, betadisper-test and PERMANOVA were used to test differences in the communities of Proteobacteria, Bacteroidetes, and Firmicutes among fish species and between the two trophic guilds. Bacterial taxa were grouped by class when examining microbiome changes along the length of the intestinal tract. Bacterial community data were fitted 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 trophic guild and gut location when accounting for species variation. Venn diagrams were produced using the VennDiagram package (Chen and Boutros 2011).

Results

A total of 1,254,909 sequences were detected in 119 samples after denoising and removing all chloroplast, mitochondria, and uncharacterized sequences. 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 1). Two unknown species of Mollicutes and Pasteurellacea accounted for 6.9 and 3.8% of sequences, respectively.

Table 1

Sequence abundance and taxonomy for each ASVs representing more than 1% of total sequences. Accession numbers for closest GenBank sequences (similarity given in brackets) are supplied.

ASV	Phylum	Lowest taxonomic division	Number of sequences	Proportion of total (%)	GenBank accession number
b727	Proteobacteria	Actinobacillus sp.	124,499	9.9	KT952745 (97.5%)
5647	Tenericutes	Mollicutes	87,057	6.9	HG971018 (96.3%)
94ba	Proteobacteria	Pasteurellacea	47,527	3.8	KT952745 (93.5%)
3023	Firmicutes	Ruminococcaceae	26,355	2.1	MG488771 (98.8%)
6350	Tenericutes	Mycoplasmataceae	24,219	1.9	LN612674 (91.5%)
9b2f	Proteobacteria	Pasteurellacea	24,219	1.9	KT952745 (91.9%)
d532	Proteobacteria	Alteromonadales	23,877	1.9	KT952746 (100.0%)
5a8a	Proteobacteria	Vibrio ponticus	22,112	1.8	MG524941 (100%)
7936	Proteobacteria	Alteromonadales	15,147	1.2	KT952746 (99.8%)
596f	Proteobacteria	Gammaproteobacteria	14,436	1.2	LC121875 (88.4%)
73d1	Proteobacteria	Vibrio sp.	13,977	1.1	KT952854 (98.7%)
6013	Proteobacteria	Pasteurellacea	13,435	1.1	KT952745 (92.3%)
af86	Firmicutes	Clostridium colinum	13,177	1.1	KC993540 (94.2%)

Different ASV richness was detected for each fish species with observed ASVs (t = −3.15, P = <0.01) and Shannon index (t = −3.68, P = <0.01) differing significantly between the two trophic guilds. The damselfish D. perspicillatus had the greatest mean richness of ASVs, with a total of 322 ± 17 ASVs per individual (Fig. 1). The species with the lowest ASV richness were C. atripectoralis and A. sexfasciatus with 47 ± 21 and 30 ± 8 ASVs per individual, respectively (Fig. 1). Shannon diversity was greatest for two algae-farming species D. perspicillatus and S. apicalis and lowest for the planktivorous species A. polyacanthus and P. moluccensis. PCA biplots, betadisper-test, and PERMANOVA revealed that the beta-diversity of Proteobacteria, Bacteroidetes, and Firmicutes communities differed among fish species and trophic guilds (Fig. 2; Table 2).

Fig. 1

Observed richness and Shannon diversity for each fish species. Planktivorous host species are shaded red and algae-farming species are shaded green.

Fig. 2

PCA biplots showing individual fish intestinal microbiomes for Proteobacteria, Bacteroidetes, and Firmicutes. Ordinations are divided by fish species (left) and trophic guild (right).

Table 2

Results of betadisper-test and PERMANOVA testing the beta-diversity of Proteobacteria, Bacteroidetes, and Firmicutes communitites across fish species and between trophic guilds.

	Fish species		Trophic guild
	betadisper	PERMANOVA	betadisper	PERMANOVA
Proteobacteria	P = 0.084	F = 1.86; p = 0.001***	p = 0.039*	F = 3.52; p = 0.001***
Bacteroidetes	P = 0.269	F = 1.78; p = 0.001***	p = 0.233	F = 2.41; p = 0.001***
Firmicutes	P = 0.001***	F = 2.17; p = 0.001***	p = 0.355	F = 3.92; p = 0.001***

Core microbiomes

In line with previous studies that investigated the core microbiome of other organisms (Ainsworth et al. 2015; Ricci et al. 2022), we choose a minimum threshold of 30% for this metric. Most ASVs occurred in less than 30% of sampled individuals across all fish species (Fig. 3a). A total of 13 bacterial ASVs were found in more than 30% of sampled individuals; therefore, they may represent the 30% core microbiome of pomacentrid investigated in this study (Table 3). The most common ASV in this study belonged to the genus Actinobacillus, which occurred in more than 80% of sampled individuals (Table 3), albeit at a low abundance in many individuals, with the highest abundances in the planktivorous damselfishes A. polyacanthus and P. moluccensis.

Fig. 3

(A) Core members of the microbiome (blue) at different threshold levels. The variable community represents ASVs occurring in less than 30% of sampled individuals. (B) Venn diagrams depicting the number of ASVs shared between whole microbiomes of the three sampled individuals for each fish species. The top row represents planktivorous species and bottom row represent algae-farming species.

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 rarefied data.

ASV	Phylum	Lowest taxonomic division	Occurrence (%)	Relative abundance	GenBank accession number
b727	Proteobacteria	Actinobacillus sp.	83.3	0.083	KT952745 (97.5%)
94ba	Proteobacteria	Actinobacillus sp.	53.3	0.017	KT952745 (93.5%)
9bd9	Proteobacteria	Photobacterium damselae	43.3	0.013	CP035457 (100%)
5647	Tenericutes	Mollicutes	40.0	0.022	HG971018 (96.3%)
a832	Proteobacteria	Photobacterium damselae	40.0	0.008	CP018297 (100%)
73d1	Proteobacteria	Vibrio sp.	40.0	0.010	KT952854 (98.7%)
9b2f	Proteobacteria	Actinobacillus porcinus	40.0	0.018	KT952745 (91.9%)
6c33	Proteobacteria	Spirobacillales	37.7	0.002	KU578602 (100%)
dc1c	Proteobacteria	Vibrio sp.	37.7	0.004	CP033144 (100%)
5a8a	Proteobacteria	Vibrio ponticus	37.7	0.019	MG524941 (100%)
762a	Bacteroidetes	Lutimonas sp.	30.0	0.001	MG488523 (99.6%)
ca47	Proteobacteria	Vibrio harveyi	30.0	0.009	CP033144 (100%)
6013	Proteobacteria	Pasteurellaceae	30.0	0.007	KT952745 (92.3%)

The core bacterial assemblages of each fish species (defined as ASVs that were shared between all sampled individuals for each species) were composed of a variable number of ASVs (Fig. 3b). For example, there were 70 bacterial ASVs shared between the three sampled individuals of D. perspicillatus and only two ASVs shared between the three A. sexfasciatus individuals. Core microbiomes within fish species were richer in algae-farming species than planktivorous species (Fig. 3b), with algae-farming species sharing 35 ± 22 ASVs and planktivorous species sharing only 7 ± 3 ASVs (Wilcox test W = 25, p < 0.01).

Core ASVs that occurred in all three individuals of a fish species belonged to the phyla Bacteroidetes, Firmicutes, Tenericutes, Spirochaetes, Planctomycetes, Proteobacteria, and Verrucomicrobia. Core ASVs belonging to Coraliomargarita sp. and Verruco-5 (Verrucomicrobia), Pirellulaceae (Planctomycetes), and Desulfovibrionaceae (Deltaproteobacteria) occurred in all three sampled D. perspicillatus individuals (Supplementary Figure S1). We also detected high diversity of an unknown clade of Gammaproteobacteria in P. moluccensis and P. wardi damselfish. There were 61 core ASVs belonging to the Bacteroidetes, 28 of which occur in S. apicalis and 38 in D. perspicillatus (Supplementary Figure S2). An unknown clade of Flavobacteriales and a diverse consortium of Rikenellaceae were core members of S. apicalis, while D. perpicillatus had a diverse core assemblage of ASVs belonging to the family Flavobacteriaceae. One ASV belonging to Spirochaetes, Brevinema andersonii, was a core member of S. nigricans and C. atripectoralis, while a Tenericutes ASV belonging to Mollicutes was a core member of all fish species except the planktivorous damselfishes A. polyacanthus and A. sexfasciatus (Supplementary Figure S3). There was a rich consortium of core Firmicutes ASVs for S. apicales and S. nigricans, which included members of the Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae families.

Bacterial shifts along the intestinal tract

The interaction between the trophic guild and intestinal region had a significant influence on the gut bacterial community composition (LRT = 152, P = 0.001; Supplementary Table 1). The abundance of nine classes of bacteria changed significantly across the different fish species and locations along the intestinal tract (LRT = −0.0229, P < 0.001; Fig. 4; Supplementary Table 2). Members of Gammaproteobacteria were especially common throughout the planktivorous intestinal tracts, but we also found them along all the intestines regions of the algae-farming species D. perspicillatus, D. pseudochrysopoecilus, and P. wardi (Fig. 4). In intestinal regions where Gammaproteobacteria were uncommon, members of Bacteroidia and Clostridia were generally found at higher abundances—especially for algae-farming species (Fig. 4). Members of the Mollicutes and Planctomycetia were more common throughout the intestinal tracts of algae-farming hosts than planktivorous species although their abundances were generally lowest within the stomach region (Fig. 4). The stomach had 286 unique bacterial ASVs, the anterior intestine 753, while 1,139 and 656 ASVs were only found in the mid and posterior intestines, respectively (Fig. 5). Only 19 ASVs were common in the stomach and posterior intestine while 152 ASVs were found throughout the intestine (Fig. 5).

Fig. 4

Changes in abundance of selected bacterial Classes along the four locations along the intestine of each species of damselfish as determined by nested multivariate generalized linear models. Intestinal locations include stomach (S), anterior intestine (AI), mid-intestine (MI), and the posterior intestine (PI). The top row represents planktivorous species and bottom row represent algae-farming species.

Fig. 5

Venn diagrams depicting the number of shared ASVs for each trophic guild (left) and for each region of the intestine (right).

Effect of the trophic guild on microbiomes

There was a significant difference in the microbiome composition between trophic guilds (LRT = −0.021, P < 0.001; Supplementary Table 2). Most bacterial ASVs were unique to either of the trophic guilds, with only 124 ASVs common to both guilds (Fig. 5). A total of 78 bacterial ASVs, belonging to 20 families, were important drivers of this relationship. There were marked differences in abundances of ASVs belonging to Vibrionaceae, Lachnospiraceae, and Pasteurellaceae. Two Vibrio sp. (Vibrionaceae) were more common in planktivorous species, and five ASVs of Actinobacillus (Pasteurellaceae) were more abundant in algae-farming species.

Discussion

Our data show that algae-farming damselfish species have richer microbiomes than planktivorous species (Fig. 1) and this result is also reflected in their core bacterial community (Fig. 3). This result is likely attributable to the specialized feeding behavior of algae-farming species, which largely consume a narrow range of turf algae species (Hata and Kato 2004; Casey et al. 2014), unlike planktivorous species that are adapted to a more opportunistic feeding strategy. These results suggest that the microbiome structure of fish species with specialized feeding behavior has acquired specific intestinal bacteria and further research is needed to investigate how microbiome specialization affects host digestion and metabolism. We also note that other processes that were not tested in our study such as host phylogeny and functional traits could influence the composition of damselfish intestinal bacteria and ultimately influence fish physiology.

We found that similar to what was recorded in many other species of marine fish, the damselfish intestinal microbiome was dominated by members of Proteobacteria, Bacteroidetes, Firmicutes, and Planctomycetes (Table 1). For example, surgeonfish, parrotfish, and rabbitfish intestinal microbiomes from the Red Sea also consist of diverse assemblages of Firmicutes and Proteobacteria (Miyake et al. 2015). Another dominant ASV in the damselfish microbiome belonging to Mollicutes (Tenericutes) resembled bacteria detected in rabbitfish intestines (Zhang et al. 2018). The number of highly similar bacterial ASVs shared among pomacentrids, acanthurids, and siganids may reflect the similar feeding behaviors of these coral reef fishes. For instance, algae-farming damselfishes may also ingest prey items other than algae, such as zooplankton (Eurich et al. 2019) or other invertebrates (Letourneur et al. 1997). The functional roles of these seemingly important microbial taxa warrant further attention in order to understand the potential consequences on host metabolism and health.

Damselfish microbiomes were largely dominated by the family Pasteurellaceae in the phylum Gammaproteobacteria, with one ASV (b727) occurring in more than 80% of sampled fishes and representing almost 10% of the total detected sequences (Tables 1 and 3). Although this ASV currently represents an unknown species in the Actinobacillus genus, a 98% similar sequence has been retrieved from the intestines of surgeonfishes in Saudi Arabia (Miyake et al. 2016), suggesting that Actinobacillus are common members of reef fish microbiomes. Bacteria in the genus Pasteurellaceae have also been recorded in high abundances in adult damselfishes and cardinalfishes collected around Lizard Island, Australia (Parris et al. 2016), and they are deemed as common components of tropical planktivorous fish gut microbiomes (Egerton et al. 2018). The prevalence of Pasteurellaceae amongst the damselfishes in this study, as well as in other reef fishes, provides additional evidence that Pasteurellaceae are likely important members of coral reef-associated fish microbiomes.

Algae-farming damselfishes had more observed ASVs and larger core microbiomes than planktivorous species (Figs. 1 and 3), and these core microbiomes were specific to each host species (Fig. 3). For example, P. wardi and P. moluccensis microbiomes were dominated by different taxa of Gammaproteobacteria, while D. perspicillatus and S. apicalis had large Bacteroidia core communities but were dominated by Flavobacteriaceae and Rikenellaceae, respectively. Different species of algae-farming damselfishes consume different species of algae (Casey et al. 2014), and the large differences in their specialized microbiomes may reflect these narrow dietary preferences. Conversely, the small core microbiomes of the planktivorous damselfishes may reflect the high variation in consumed plankton of each species, suggesting these fishes have opportunistic feeding behaviors. These results, however, do not support the notion that fish with greater diet variability have more diverse microbiomes (Givens et al. 2015). In fact, the damselfish with narrow, algae-farming feeding behaviors tended to have the greatest diversity of intestinal bacteria, suggesting that the host-microbiome interactions may select for specialized bacteria that enhance the digestion and absorption of nutrients from specific algal diets. The richer microbiome of algae-farming fishes could also reflect the necessity of this trophic guild to be associated with a pool of symbionts that facilitate the breakdown of algal cellulose. We also acknowledge that some of the bacteria we retrieved from the damselfish intestine could have been associated with the food recently ingested by the fish and, therefore, not being part of the damselfish microbiome.

Evidence suggests a high degree of resource partitioning in fish communities, which is a key mechanism that facilitates the high diversity of coral reefs (Casey et al. 2019; Leray et al. 2019). The largely distinct microbiomes of each host species presented in this study may reflect the high degree of resource partitioning found in coral reef communities, whereby different species of damselfish may be consuming different size classes of zooplankton (Leray et al. 2019), farm different algal species (Casey et al. 2014), or occupy different trophic niches (Casey et al. 2019). The similarity between closely related host species and microbiomes, such as P. wardi and P. moluccensis, also demonstrates that phylogeny may influence the intestinal microbiomes of damselfishes (Sullam et al. 2012; Miyake et al. 2015; Neuman et al. 2016; Chiarello et al. 2018).

Interestingly, Photobacterium damselae, Vibrio harveyi, Vibrio ponticus, and other Vibrio sp. were prevalent amongst the damselfishes sampled in this study (Table 3). These bacteria represent potential pathogenic members of Vibrionacaea and have been detected in many fishes of aquaculture importance, including Chromis punctipinnis (Love et al. 1981), Lutjanus argentimaculatus (Reshma et al. 2018), Seriola dumerili (Nishiki et al. 2018), Scophthalmus maximus (Montes et al. 2003), Sparus aurata (Vera 1991), and Solea senegalensis (Terceti et al. 2016). Although identified as Vibrio harveyi in the GreenGenes database, GenBank revealed there was a high similarity of these sequences to other members of the Harveyi clade, such as Vibrio owensii (Nishiki et al. 2018). It is thought that there are up to 11 species of Vibrio belonging to this clade (Urbanczyk et al. 2013), most of which are pathogens of fish, shrimp, and coral (Thompson et al. 2004; Austin and Zhang 2006; Ushijima et al. 2012). Given the apparently healthy state of the sampled fishes and the high abundances of potentially pathogenic Vibrionacaea in the fish guts, we provide support to the idea that these organisms are natural components of healthy fish microbiomes and are opportunistic pathogens in fishes only under specific conditions (Rivas et al. 2013; Reshma et al. 2018). Future studies should also investigate the involvement of algae-farming damselfish in the spreading of pathogens across reef organisms. For instance, it has recently been reported that the seagrass pathogen Labyrinthula was present in the skeleton of a common coral species (Ricci et al. 2021) and probably infected the abundant endolithic algae living in the coral skeleton (Ricci et al. 2019; Iha et al. 2020; Tandon et al. 2022; Ricci et al. 2022). Thus, it is possible that damselfishes grazing near alive corals were the medium that allowed the pathogen Labyrinthula to infect the corals’ endolithic algae.

The facultative anaerobic bacterial classes Bacteroidia, Clostridia, and Mollicutes were generally in higher abundance in the mid and posterior intestinal regions than in the stomach (Fig. 4). Differences in microbiomes along the intestinal tract have been recorded in the rabbitfish Siganus fuscescens (Nielsen et al. 2017), with midgut communities more representative of the environmental sources and hindguts hosting a microbiome more specialized to anaerobic conditions and fermentation (Jones et al. 2018). The increase in Bacteroidia, Clostridia, and Mollicutes along the intestines may be due to some members of these bacterial classes being mutualistic components of the fish gastrointestinal microbiome. Some members of Bacteroidia are known to breakdown polysaccharides and metabolize the derived sugars (Xu et al. 2003), while members of Clostridium are known to metabolize cellulose (Liu et al. 2016). Our results confirm the increased prevalence of anaerobic bacteria in the hindgut of damselfishes, which probably consists of taxa responsible for the fermentation and metabolism of complex molecules before being absorbed by the host (Clements et al. 2014). We also note that Actinobacillus sp. that could breakdown cellulose via fermentation (Almqvist et al. 2016) were more abundant in the gut of algae-farming damselfish, suggesting that these bacteria could aid the digestion of fish in this trophic guild.

Conclusions

In this study, we show that damselfishes have diverse intestinal microbial communities whereby the bacterial richness of a species reflects diet and trophic guild. We show that algae-farming damselfishes have richer bacterial alpha-diversity and core microbiomes, which may reflect the more specialized diets of this trophic guild. We also provide evidence that damselfish mid and posterior intestines have higher abundances of facultative anaerobic bacteria that are known to play important roles in fermentation and cellulose breakdown. These findings add to a growing body of literature that suggests that host fish feeding behavior has a strong influence on the composition of intestinal microbiomes.

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