Tick-Borne Pathogens Shape the Native Microbiome Within Tick Vectors

Abstract

Ticks are blood-feeding arthropods and transmit a variety of medically important viral, bacterial, protozoan pathogens to animals and humans. Ticks also harbor a diverse community of microbes linked to their biological processes, such as hematophagy, and hence affect vector competence. The interactions between bacterial and/or protozoan pathogens and the tick microbiome is a black-box, and therefore we tested the hypothesis that the presence of a protozoan or bacterial pathogen will alter the microbial composition within a tick. Hence, this study was designed to define the microbial composition of two tick species, Hyalomma (H.) anatolicum and Rhipicephalus (R.) microplus. We used a combination of PCR based pathogen (Anaplasma marginale and Theileria species) and symbiont (Wolbachia species) identification followed by metagenomic sequencing and comparison of the microbial communities in PCR positive and negative ticks. A total of 1786 operational taxonomic units was identified representing 25 phyla, 50 classes, and 342 genera. The phylum Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota were the most represented bacteria group. Alpha and beta diversity were not significantly affected in the presence or absence of Theileria sp. and A. marginale as see with H. anatolicum ticks. Interestingly, bacterial communities were significantly reduced in Theileria sp. infected R. microplus ticks, while also exhibiting a significant reduction in microbial richness and evenness. Putting these observations together, we referred to the effect the presence of Theileria sp. has on R. microplus a "pathogen-induced dysbiosis". We also identify the presence of Plasmodium falciparum, the causative agent of human malaria from the microbiome of both H. anatolicum and R. microplus ticks. These findings support the presence of a "pathogen-induced dysbiosis" within the tick and further validation experiments are required to investigate how they are important in the vector competence of ticks. Understanding the mechanism of "pathogen-induced dysbiosis" on tick microbial composition may aid the discovery of intervention strategies for the control of emerging tick-borne infections.

Keywords: Anaplasma marginale; Francisella; Hyalomma anatolicum; Pakistan; Rhipicephalus microplus; Theileria sp.; Wolbachia; microbiome; ticks.

Figure 1

16S bacteria abundance profiles of H. anatolicum and R. microplus. (A,B) Bacteria abundance at phylum. (C,D). Bacteria abundance at the family level. (E,F) Bacteria abundance at the genus level. Hyalomma anatolicum (left panel), R. microplus (right panel).

Figure 2

18S eukaryote abundance profiles of H. anatolicum and R. microplus. (A) The relative abundance of identified eukaryote species from Theileria sp. positive and uninfected H. anatolicum ticks. (B) The relative abundance of identified eukaryote species from Theileria sp. positive, A. marginale positive, and uninfected R. microplus ticks.

Figure 3

Alpha diversity analysis in R. microplus ticks. (A) Estimation of species richness using the observed operational taxonomic (OTUs) metrics (Kruskal–Wallis H-test, df = 1, p-value = 0.003). (B) Estimation of species evenness using the Shannon diversity index (Kruskal–Wallis H-test, df = 1, p-value = 0.005). Ticks found to be positive for Theileria sp. showed the least diversity using both measures.

Figure 4

Estimation of differences in the microbial communities as a measure of Beta diversity analysis in R. microplus ticks. (A) Principal coordinate analysis (PCoA) of the Bray–Curtis distance matrix (PERMANOVA, F-value: 4.2171; R: 0.33161; p-value < 0.005). (B) Principal coordinate analysis (PCoA) Jaccard distance matrix (PERMANOVA, F-value: 3.2588; R: 0.27714; p-value < 0.005).

Figure 5

Pattern correlation analysis of top 7 bacteria genera in R. microplus ticks. Ticks positive with Theileria sp. showed a positive correlation with the presence of Bacillus.

Figure 6

Heat map and classical univariate compositional analysis in R. microplus ticks. (A) Heat map correlation analysis between A. marginale positive, Theileria sp. positive and uninfected ticks. Log-transformed count of (B) Acinetobacter (FDR = 0.19277, df = 1, p-value = 0.007), (C) Staphylococcus (FDR = 0.19277, df = 1, p-value = 0.007), and (D) Bacillus (FDR = 0.0002, df = 1, p-value = 3.94e-223) in A. marginale positive, Theileria sp. positive and uninfected R. microplus ticks.