The gut virome of the protochordate model organism, Ciona intestinalis subtype A
Introduction
In recent years, numerous studies have defined the importance of host-associated microorganisms, including both bacteria and viruses. The bacterial component of this ‘microbiome’ often outnumbers host cells by an order of magnitude (Turnbaugh et al., 2007) and can influence host nutrient acquisition and metabolism (Nicholson et al., 2012, Tremaroli and Backhed, 2012). The increased recognition of the contributions of bacterial communities to the biology and physiology of animals has motivated our perception of animals as complex metaorganisms (Rohwer et al., 2002, Rosenberg and Zilber-Rosenberg, 2011, Theis et al., 2016). This concept was first introduced into biology to describe the coral animal and all of its associated microbes (Rohwer et al., 2002). Since then, many animal models, including humans, have been documented to contain a stable, core community of bacteria on or within their bodies (Dishaw et al., 2014, Roeselers et al., 2011, Sabree et al., 2012, Schmitt et al., 2012, Turnbaugh and Gordon, 2009), the disruption of which often results in disease (Cho and Blaser, 2012, Sobhani et al., 2011, Tamboli et al., 2004). While bacteria have historically predominated microbiome studies, recent efforts are recognizing a vital role for viruses as well (Abeles and Pride, 2014, Grasis et al., 2014, Minot et al., 2011, Reyes et al., 2010, Thurber et al., 2017). The viruses associated with animal hosts are present as both free viral particles and/or stably integrated proviruses (Feschotte and Gilbert, 2012) and can shape the health of the metaorganism through pathogenesis (Davies et al., 2016) or through influencing the metabolic potential of both the animal host and its associated microbes (Roossinck, 2011). In addition to host immunity (Cullender et al., 2013, Thaiss et al., 2016), nutrient availability (Cohen et al., 2015), and bacterial competition (Flint et al., 2007), viruses, the majority of which are bacteriophages (i.e. phages), likely affect the structure of animal-associated bacterial communities through lytic infection and prophage integration and/or induction. In this report, we describe the gut virome of a marine filter-feeding invertebrate protochordate, Ciona intestinalis subtype A, cataloging for the first time the viral communities associated with these early extant chordates and establishing a tractable model system in which to pursue future studies examining the complex dynamics between metazoan hosts and their associated bacterial and viral communities.
Ciona is a well-known developmental model system that has been adapted recently for studies of host-microbe interactions within the gut ecosystem (Dishaw et al., 2014, Dishaw et al., 2011, Dishaw et al., 2016, Liberti et al., 2014). This animal is a marine filter-feeding protochordate that derives nutrients from particulates in the water column, including phytoplankton (Coleman, 1991). Ciona is amenable to germ-free mariculture (Leigh et al., 2016) and has been shown previously to maintain a core bacterial community in its gut (Dishaw et al., 2014). Here we report the presence of temporally stable viruses within this metabolically active environment. We also describe evidence for compartmentalization of both viruses and bacteria, with distinct members occupying the stomach, midgut, and hindgut. The observation that Ciona maintains a virome with many predicted hosts matching members of the core microbiome and a prevalence of prophages indicates a role for phages as major players in shaping host-associated bacterial communities within the dynamic gut ecosystem of these animals.
Section snippets
Tissue collections
Adult Ciona were wild-harvested in San Diego, California, USA, and shipped overnight to the laboratory in Florida. Upon arrival, ten animals were randomly selected, five of which were immediately harvested for gut tissue while gut contents were cleared from the remaining five in 100 kD filtered virus-free seawater for 24 h, with water changes every 3 h for the first 12 h. In 2014, entire guts were dissected and snap-frozen in liquid nitrogen, while in 2015, guts were trisected (stomach, midgut and
Ciona gut virome
The seven following Ciona gut virome samples were sequenced: SDC 14 (cleared guts from 2014); cleared stomach (SC), midgut (MC), and hindgut (HC) tissues from 2015 (referred to as SDC 15 when pooled); and uncleared (“full”) stomach (SF), midgut (MF), and hindgut (HF) tissues from 2015 (referred to as SDF 15 when pooled). The virome sequencing and single sample assembly statistics are shown in Table 1. In 2015, additional viromes were sequenced from the surrounding seawater from the collection
Discussion
Utilizing metagenomics, we report here on the gut virome of the filter-feeding marine protochordate, Ciona intestinalis subtype A, a close invertebrate relative of vertebrates. Similar to other studied marine organisms, Ciona contains bacterial and viral communities that are distinct from the surrounding environment. In the extensively studied marine environment, phage outnumber bacteria approximately 10:1 (Suttle, 2005). Within the gut mucus environment, phage numbers are estimated to be
Data availability
Viral data were deposited in both MetaVir (IDs 7811 (SF), 8143 (SC), 7815 (MF), 7814 (MC), 7812 (HF), 7910 (HC), 7816 (CB), 7819 (MB), and 8255 (SDC14)) and MG-RAST (IDs 4707275.3 (SF), 4707280.3 (SC), 4707282.3 (MF), 4734670.3 (MC), 4707277.3 (HF), 4707278.3 (HC), 4707281.3 (CB), 4707279.3 (MB), 4734673.3 (SDC14)). Bacterial data were deposited in MG-RAST (IDs 4751381.3 (SF), 4751379.3 (SC), 4751382.3 (MF), 4751386.3 (MC), 4751377.3 (HF), and 4751377.3 (HC)).
Funding
These studies were supported by a grant from the National Science Foundation (IOS-1456301) to L.J.D. and M.B. and by a National Science Foundation Graduate Research Fellowship (Award No. 1144244) to B.A.L.
Acknowledgements
The authors would like to thank Matthew Sullivan and his group for training on the iVirus pipeline as well as Ben Bolduc in particular for his constant help in navigating iVirus. The authors would also like to thank Ryan Schenck for his digital illustration of the Ciona anatomy.
References (92)
- et al.
Molecular bases and role of viruses in the human microbiome
J. Mol. Biol.
(2014) - et al.
Innate and adaptive immunity interact to quench microbiome flagellar motility in the gut
Cell Host Microbe
(2013) - et al.
Ocean viruses: rigorously evaluating the metagenomic sample-to-sequence pipeline
Virology
(2012) The ecology of Cytophaga-Flavobacteria in aquatic environments
FEMS Microbiol. Ecol.
(2002)- et al.
Disease-specific alterations in the enteric virome in inflammatory bowel disease
Cell
(2015) - et al.
Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae)
Virology
(2001) - et al.
Persicivirga ulvanivorans sp. nov., a marine member of the family Flavobacteriaceae that degrades ulvan from green algae
Int. J. Syst. Evol. Microbiol.
(2011) - et al.
Bacteriophage adhering to mucus provide a non-host-derived immunity
Proc. Natl. Acad. Sci. U. S. A.
(2013) - et al.
iVirus: facilitating new insights in viral ecology with software and community data sets imbedded in a cyberinfrastructure
ISME J.
(2017) - et al.
Genome and proteome characterization of the psychrophilic Flavobacterium bacteriophage 11b
Extremophiles
(2007)
Metagenomic analyses of an uncultured viral community from human feces
J. Bacteriol.
DADA2: High-resolution sample inference from Illumina amplicon data
Nat. Methods
Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae
PLoS One
VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R
BMC Bioinform.
The human microbiome: at the interface of health and disease
Nat. Rev. Genet.
A molecular analysis of fecal and mucosal bacterial communities in irritable bowel syndrome
Dig. Dis. Sci.
Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist
Proc. Natl. Acad. Sci. U. S. A.
Encyclopedia of Marine Animals
progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement
PLoS One
Temperate phages both mediate and drive adaptive evolution in pathogen biofilms
Proc. Natl. Acad. Sci. U. S. A.
Identifying bacterial genes and endosymbiont DNA with Glimmer
Bioinformatics
A role for variable region-containing chitin-binding proteins (VCBPs) in host gut-bacteria interactions
Proc. Natl. Acad. Sci. U. S. A.
The gut of geographically disparate Ciona intestinalis harbors a core microbiota
PLoS One
Gut immunity in a protochordate involves a secreted immunoglobulin-type mediator binding host chitin and bacteria
Nat. Commun.
A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes
Nat. Commun.
Phage therapy of coral white plague disease: properties of phage BA3
Curr. Microbiol.
Endogenous viruses: insights into viral evolution and impact on host biology
Nat. Rev. Genet.
A virophage at the origin of large DNA transposons
Science
Interactions and competition within the microbial community of the human colon: links between diet and health
Environ. Microbiol.
Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis
Nat. Rev. Microbiol.
Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (Diptera: Culicidae) (L.)
Parasit. Vectors
Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1
ISME J.
Species-specific viromes in the ancestral holobiont Hydra
PLoS One
QUAST: quality assessment tool for genome assemblies
Bioinformatics
The Human Virome
Metagenomics of the Human Body
Twelve previously unknown phage genera are ubiquitous in global oceans
Proc. Natl. Acad. Sci. U. S. A.
Complete genome sequences of two Persicivirga bacteriophages, P12024S and P12024L
J. Virol.
Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data
Bioinformatics
MG-RAST: a metagenomics service for analysis of microbial community structure and function
Methods Mol. Biol.
Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses
Appl. Environ. Microbiol.
Comparative genomic analysis of 18 Pseudomonas aeruginosa bacteriophages
J. Bacteriol.
Localization of bacteria in the gastrointestinal tract: a possible explanation of intestinal spirochaetosis
Infect. Immun.
Generation of germ-free Ciona intestinalis for studies of gut-microbe interactions
Front. Microbiol.
Isolation and characterization of a Shewanella phage-host system from the gut of the tunicate, Ciona intestinalis
Viruses
Expression of Ciona intestinalis variable region-containing chitin-binding proteins during development of the gastrointestinal tract and their role in host-microbe interactions
PLoS One
Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations
ISME J.
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