Metavirome Sequencing of the Termite Gut Reveals the Presence of an Unexplored Bacteriophage Community

Abstract

The Formosan subterranean termite; Coptotermes formosanus is nutritionally dependent on the complex and diverse community of bacteria and protozoa in their gut. Although, there have been many studies to decipher the taxonomic and functional diversity of bacterial communities in the guts of termites, their bacteriophages remain unstudied. We sequenced the metavirome of the guts of Formosan subterranean termite workers to study the diversity of bacteriophages and other associated viruses. Results showed that the termites harbor a virome in their gut comprised of varied and previously unknown bacteriophages. Between 87-90% of the predicted dsDNA virus genes by Metavir showed similarity to the tailed bacteriophages (Caudovirales). Many predicted genes from the virome matched to bacterial prophage regions. These data are suggestive of a virome dominated by temperate bacteriophages. We predicted the genomes of seven novel Caudovirales bacteriophages from the termite gut. Three of these predicted bacteriophage genomes were found in high proportions in all the three termite colonies tested. Two bacteriophages are predicted to infect endosymbiotic bacteria of the gut protozoa. The presence of these putative bacteriophages infecting endosymbionts of the gut protozoa, suggests a quadripartite relationship between the termites their symbiotic protozoa, endosymbiotic bacteria of the protozoa and their bacteriophages. Other than Caudovirales, ss-DNA virus related genes were also present in the termite gut. We predicted the genomes of 12 novel Microviridae phages from the termite gut and seven of those possibly represent a new proposed subfamily. Circovirus like genomes were also assembled from the termite gut at lower relative abundance. We predicted 10 novel circovirus genomes in this study. Whether these circoviruses infect the termites remains elusive at the moment. The functional and taxonomical annotations suggest that the termites may harbor a core virome comprised of the bacteriophages infecting endosymbionts of the gut protozoa.

Keywords: bacteriophages; endosymbionts; metavirome; symbiosis; termite.

Figure 1

Classification and abundance (%) of various virus types observed in the guts of the termite workers from three different colonies. (A) Percent of taxonomic assignment of all viral genes (B) Percent of taxonomic assignment of dsDNA virus related genes. (C) Percent taxonomic assignment of ssDNA virus related genes. (D) Percent taxonomic assignment of Caudovirales related genes. The data were generated using Metavir-2 server by comparing the predicted proteins to the NCBI virus protein database. Only top BLAST hits with an e-value of 10⁻⁵ or less were used (normalized for each category).

Figure 2

Taxonomic distribution (%) of the predominant dsDNA bacteriophage genes from the guts of termite workers from three different colonies (normalized). The data were generated using Metavir-2 server by comparing the predicted proteins to the NCBI virus protein database. Only top BLAST hits with an e-value of 10⁻⁵ or less were used.

Figure 3

A maximum likelihood phylogenetic tree of large terminase subunit of type terminase_6. The nodes with a bootstrap value of 70% or more are indicated by a circular symbol. Sequences from the termite gut are colored purple. Bacteriophages: red. Firmicutes: orange. Spirochetes: dark blue. Gammaproteobacteria: bright green. Bacteroidetes: dark green. Actinomycetes: pink. Alphaproteobacteria: light blue. Termite Group I bacterium: sky blue. Others: black.

Figure 4

(A) Comparative genomic analysis of LSPY01000004 and LSPY01000006 with Azobacteroides phage ProJPt-1Bp1. All three circular genomes have been rearranged so that the start codon of a conserved hypothetical protein is the first base in the sequence. (B) Comparative genomic analysis of LSPZ01000002 with Bacillus phage AR9 and Yersinia phage phi R137. All three circular genomes have been rearranged so that the start codon of the large terminase subunit is the first base in the sequence. The figures were generated using Easyfig software with tblastx. The structural genes are indicated in red, DNA metabolism related genes are indicated in blue and RNA polymerase genes are indicated in orange. All the other genes are indicated in sky blue color.

Figure 5

A maximum likelihood phylogenetic tree of Microviridae VP1 from the guts of workers from three termite colonies along with the comparative genomic analysis of closely related Microviruses. Nodes with a bootstrap score of more than 70% are indicated by gray circles. For this figure, only VP1 genes from putative full Microviridae genomes were used. VP1 is colored red, VP2: green, VP4: blue. An extra ORF found after the VP1gene of Sukshmaviriane is colored pink.

Figure 6

Unrooted maximum likelihood phylogenetic tree of Circoviridae replication initiation protein. The hosts of the Circoviruses are displayed in a picture next to the sequence. Environmental Circoviruses are shown in green. Insect related Circoviruses are shown in red.