Metagenomics of the midgut microbiome of Rhipicephalus microplus from China

Abstract

Background: Ticks, which are ectoparasites of animals, may carry multiple pathogens. The cattle tick Rhipicephalus microplus is an important bovine parasite in China. However, the midgut microbiome of R. microplus from China has not been characterized via metagenomic methods.

Methods: Rhipicephalus microplus were collected from cattle in the city of Changsha in Hunan province, China. The DNA of the midgut contents was extracted from fully engorged adult female R. microplus. A DNA library was constructed and sequenced using an Illumina HiSeq sequencing platform. SOAPdenovo software was used to assemble and analyze the clean data. The latent class analysis algorithm applied to system classification by MEGAN software was used to annotate the information on the species' sequences. DIAMOND software was used to compare unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and functional annotation was carried out based on the results of the comparison.

Results: The dominant phyla in the five samples were Firmicutes, Proteobacteria, and Actinobacteria. Streptococcus, Mycobacterium, Anaplasma, Enterococcus, Shigella, Lactobacillus, Brachyspira, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were the dominant genera in the five samples. The endosymbiotic bacterium Wolbachia was also detected in all of the samples. Mycobacterium malmesburyense, Streptococcus pneumoniae, Anaplasma phagocytophilum, Enterococcus faecium, Shigella sonnei, Enterococcus faecalis, Lactobacillus casei, Brachyspira hampsonii, Pseudomonas syringae, Enterobacter cloacae, and Lactococcus garvieae were the dominant species in the five samples. In addition to these bacterial species, we also detected some eukaryotes, such as Rhizophagus irregularis, Enterospora canceri, Smittium culicis, Zancudomyces culisetae, Trachipleistophora hominis, and viruses such as orf virus, human endogenous retrovirus type W, enzootic nasal tumor virus of goats, bovine retrovirus CH15, and galidia endogenous retrovirus in all of the samples at the species level. The results of the annotated KEGG pathway predictions for the gene functions of the midgut microflora of R. microplus indicated genes involved in lipid and amino acid metabolism, infectious diseases (e.g., Streptococcus pneumonia infection, human granulocytic anaplasmosis, Shigella sonnei infection, Salmonella enterica infection, and pathogenic Escherichia coli infection), and cancer.

Conclusions: Our study revealed that the midgut microbiome of R. microplus is not only composed of a large number of bacteria, but that a portion also comprises eukaryotes and viruses. The data presented here enhance our understanding of this tick's midgut microbiome and provide fundamental information for the control of ticks and tick-borne diseases.

Keywords: Gene function; Metagenomics; Microbiome; Midgut; Rhipicephalus microplus.

Fig. 1

Venn diagram analysis of the number of genes of the samples of Rhipicephalus microplus. Each circle represents a sample; the numbers in the overlapping areas indicate the number of genes shared between the samples. The numbers in the non-overlapping areas indicate the number of unique genes in the sample

Fig. 2

Microbial population characteristics of the 10 most abundant phyla in the five samples of Rhipicephalus microplus

Fig. 3

Microbial population characteristics of the 10 most abundant genera in the five samples of Rhipicephalus microplus

Fig. 4

Number of genes and abundance clustering heat map at the genus level. a Heat map of the annotated unigene statistics. Sample names are given on the horizontal axis, and species’ information on the vertical axis; different colors represent the number of unigenes. b Relative abundance clustering heat map at the genus level. Sample information is given on the horizontal axis; the vertical axis gives the species’ information. The cluster tree on the left of the figure is the species cluster tree; the value corresponding to the middle heat map is the Z-value obtained after the relative abundance of each species row is standardized

Fig. 5

Annotations of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the number of genes of the midgut microbiota in the five samples of Rhipicephalus microplus at level 1. The black font on the ordinate indicates KEGG level 1, the colored fonts indicate the specific pathway at this level, and the abscissa indicates the number of genes in the pathway

Fig. 6

Relative abundance of midgut microbial genes involved in metabolism in the five samples of Rhipicephalus microplus at level 2 in KEGG pathway annotation

Fig. 7

Relative abundance of midgut microbial genes involved in human diseases in the five samples of Rhipicephalus microplus at level 2 in KEGG pathway annotation