Marine Ecology Progress Series 518:209
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Vol. 518: 209–223, 2015 MARINE ECOLOGY PROGRESS SERIES Published January 7 doi: 10.3354/meps11034 Mar Ecol Prog Ser Comparison of the gut microbiomes of 12 bony fish and 3 shark species Carrie E. Givens1,2, Briana Ransom1,3, Nasreen Bano1,4, James T. Hollibaugh1,* 1Department of Marine Sciences, University of Georgia, Athens, GA 30602, USA 2Present address: US Geological Survey Michigan Water Science Center, 6520 Mercantile Way, Lansing, MI 48911, USA 3Present address: Oglethorpe County High School, 749 Athens Rd, Lexington, GA 30648, USA 4Present address: USDA ARS, 950 College Station Rd, Athens, GA 30605, USA ABSTRACT: We used massively parallel sequencing (pyrosequencing) of 16S rRNA genes to com- pare the composition of microbial communities in the guts of 12 bony fish and 3 shark species. The species analyzed encompass herbivores and carnivores with varied digestive physiologies, are classified as pelagic and demersal species, and reside in estuarine to marine environments. We also compared the gut microbial assemblages of wild and cultured Fundulus heteroclitus and of juvenile and adult Lagodon rhomboides. A total of 1 214 355 sequences were filtered, denoised, trimmed, and then sorted into operational taxonomic units (OTUs) based on 97% sequence simi- larity. Bacteria representing 17 phyla were found among the sampled fish, with most fish hosting between 7 and 15 phyla. Proteobacteria OTUs were present in all fish and often dominated the libraries (3.0 to 98%; average: 61%). Firmicutes were also prevalent, but at a lower relative abun- dance, ranging between 1.3 and 45% (average: 17%). In most cases, the gut microflora of individ- ual fish of a given species contained many of the same OTUs; however, some species (e.g. great barracuda) shared few OTUs among the individuals sampled. Although no single OTU was shared among all fish species, many of the OTUs present in one species’ core group were also found in the core groups of other species. Several OTUs were consistently found in the guts of multiple species, suggesting that these OTUs may be important contributors to fish gut functions such as digestion, nutrient absorption, and immune response. KEY WORDS: Fish gut · Gut microbiome · 16S rRNA · Gut microflora · 454-pyrosequencing · Shark gut · Core gut microbiome Resale or republication not permitted without written consent of the publisher INTRODUCTION have documented obligate anaerobes as part of the gut microbial assemblage (Trust et al. 1979, Ringø et Skin, gills, eggs, and intestinal tracts of fish all har- al. 1995). Izvekova et al. (2007) reviewed studies of bor abundant populations of bacteria (MacFarlane et fish gut microflora published between 1929 and 2006 al. 1986, Cahill 1990) that impact the overall health and found that of the 73 bacterial taxa documented and physiology of the host. Fish intestines in particu- 53% were Gram-negative aerobes, 34% were Gram- lar harbor large and diverse populations of bacteria positive aerobes, 8.2% were Gram-negative anaer- (Austin & Austin 1987, Cahill 1990, Ringø et al. 1995). obes, and 4.1% were Gram-positive anaerobes. Most studies have shown that this gut microflora Direct comparisons between past studies are ham- varies among fish species, and that the dominant pered by inconsistencies in the methods used. Stud- bacteria are typically either aerobes or facultative ies conducted prior to ~2005 relied on culture-based anaerobes (Ringø et al. 1995). However, some studies techniques to enumerate and identify bacteria *Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 210 Mar Ecol Prog Ser 518: 209–223, 2015 (Newman et al. 1972, MacFarlane et al. 1986, gut (Sugita et al. 1988, Cahill 1990). Within a species’ Spanggaard et al. 2000, Aschfalk & Müller 2002, natural habitat, stable environmental conditions may Verner-Jeffreys et al. 2003, Al-Harbi & Naim Uddin lead to the establishment of a stable gut microflora 2004, Martin-Antonio et al. 2007, Skrodenyte˙- that is representative of the ‘natural flora’ of the spe- Arbacˆiauskiene˙ 2007). These studies have provided cies (Lynch & Hobbie 1988, Oxley et al. 2002). How- valuable insights into the composition of microbial ever in culture systems, conditions of diet, water communities and have yielded isolates for detailed quality, and population density may be very different physiological investigation; however, they are likely from those of the natural habitat. This may result in to have provided biased assessments of the micro- differences between the gut microflora of wild and bial community composition, as typically <1% of the cultured populations of the same species, and cells known to be present by direct microscopic indeed, MacFarlane et al. (1986) observed that farm- enumeration produce colonies on solid media (Fer- raised fish had a simpler gut flora than their wild guson et al. 1984, Head et al. 1998), which were for- counterparts. merly a crucial step in identifying bacteria. With that Several studies have shown that many herbivorous caveat, Table S1 (in the Supplement at www. int- fish such as the pinfish Lagodon rhomboides under - res.com/ articles/suppl/ m518 p209 _supp .pdf) lists the go an ontogenetic diet shift, transitioning from car- dominant gut micro flora reported in published stud- nivorous juveniles to either omnivorous or herbivo- ies of a variety of fresh- and saltwater fish species rous adults (Benavides et al. 1994, Muñoz & Ojeda from wild and cultured populations. Most of these 2000, Gallagher et al. 2001). Luczkovich & Stellwag studies only examined a single fish species and (1993) indicated that this ontogenetic shift in diet used a variety of culture-dependent and culture- resulted in both qualitative and quantitative variabil- independent me tho dologies to assess micro flora ity in the composition of the L. rhomboides gut micro - community composition. flora. Considering the likely significance of gut Based on this review of the literature (Table S1), microflora in digestion and nutrient acquisition, fish the gut microbiomes of most fish are dominated by adapted to a carnivorous lifestyle likely have gut γ-Proteobacteria such as Aeromonas sp., Escherichia microbial assemblages that are different from those coli, Photobacterium sp., Pseudomonas sp., and Vib- that feed on plant material. rio sp. However, some fish such as the Atlantic We used massively parallel sequencing (pyrose- salmon Salmo salar (Holben et al. 2002) and the long- quencing) of Bacteria 16S rRNA genes to test hypo - jawed mudsucker Gillichythys mirabilis (Bano et al. theses about the relationship between gut microflora 2007) have intestinal microflora dominated by Tener- composition and lifestyle in 12 bony fish and 3 shark icutes (Mycoplasma sp.). Lactic acid bacteria (mainly species, selected to encompass a wide range of Lactobacillus sp.) have also been found to be minor lifestyles. The fish species sampled include both her- components of the gut microflora of both freshwater bivores and carnivores, represent varied digestive and marine fish (Izvekova et al. 2007). Unlike bony physiologies, are classified as pelagic or demersal fish, there has been little research on the gut micro- species, and reside in estuarine to marine environ- biomes of sharks. One culture-dependent study ments. We also included 3 species of sharks that, found that Photobacterium damselae was a normal unlike bony fish, have a short intestine incorporating member of their gut microflora (Grimes et al. 1985), a spiraled valve (Budker & Whitehead 1971) that but there have been no culture-independent analy- increases the intestinal surface area and allows for ses of the shark gut microbiome. increased absorption (Castro & Huber 2003). The gut microbial community can respond to a variety of factors affecting the host, including chang- ing environmental conditions such as temperature MATERIALS AND METHODS and salinity (Yoshimizu & Kimura 1976, MacFarlane et al. 1986), developmental stage (Verner-Jeffreys et Fish collection al. 2003, Romero & Navarrete 2006), digestive physi- ology (Cahill 1990), and feeding strategy (Uchii et al. Table 1 lists the species used in this study, along 2006). Some of the gut microflora appear to be tran- with their phylogenetic classification, feeding strate- sient, while other bacteria seem to be permanent res- gies, common habitats, and digestive physiologies. In idents (Kim et al. 2007). Resident gut microflora are addition to the 15 fish species used for interspecific those bacteria from the diet or environment that are comparison, we also compared wild and cultured able to colonize, persist, and proliferate within the Fundulus heteroclitus (mummichogs) and juvenile Table 1. Fish species sampled in this study, including their habitat (preferred environment and salinity range), overall feeding strategy (C: carnivore, H: herbivore, O: omnivore; based on Froese & Pauly 2011) with specific categories in brackets, and digestive physiology Common Name ID Species Order Family Habitat Feeding Digestive strategy physiology Mummichog MC Fundulus heteroclitus Cyprinodonti- Fundulidae Benthopelagic; O Simple tube formes freshwater-marine Pinfish PF Lagodon rhomboides Perciformes Sparidae Demersal; C/Ha (herbivore, Differentiated, brackish-marine invertivore) elongated intestine Givens etal.:Comparisonoffishgutmicroflora Silver perch SP Bairdiella chrysoura Perciformes Sciaenidae Demersal; brackish- C (invertivore) Differentiated marine Black sea bass BSB Centropristis striata Perciformes Serranidae Reef-associated;