Characterization and manipulation of the bacterial community in the midgut of Ixodes ricinus

Abstract

Background: Ticks are obligate hematophagous arthropods transmitting a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, primarily in the ovaries and the midgut. In the previous study on Ixodes ricinus, we used a culture-independent approach and showed a diverse but quantitatively poor midgut bacterial microbiome. Our analysis also revealed the absence of a core microbiome, suggesting an environmental origin of the tick midgut microbiota.

Methods: A bacterial analysis of the midgut of adult females collected by flagging from two localities in the Czech Republic was performed. Using the culture-independent approach, we tested the hypothesis that the midgut microbiome is of the environmental origin. We also cultured indigenous bacteria from the tick midgut and used these to feed ticks artificially in an attempt to manipulate the midgut microbiome.

Results: The midgut showed a very low prevalence and abundance of culturable bacteria, with only 37% of ticks positive for bacteria. The culture-independent approach revealed the presence of Borrelia sp., Spiroplasma sp., Rickettsia sp., Midichloria sp. and various mainly environmental Gram-positive bacterial taxa. The comparison of ticks from two regions revealed that the habitat influenced the midgut bacterial diversity. In addition, the midgut of ticks capillary fed with the indigenous Micrococcus luteus (Gram-positive) and Pantoea sp. (Gram-negative) could not be colonized due to rapid and effective clearance of both bacterial taxa.

Conclusions: The midgut microbiome of I. ricinus is diverse but low in abundance, with the exception of tick-borne pathogens and symbionts. The environment impacts the diversity of the tick midgut microbiome. Ingested extracellular environmental bacteria are rapidly eliminated and are not able to colonize the gut. We hypothesize that bacterial elimination triggered in the midgut of unfed adult females is critical to maintain low microbial levels during blood-feeding.

Keywords: Capillary feeding; Culturing; High-throughput sequencing; Ixodes ricinus; Microbiome; Microbiome manipulation; Midgut.

Fig. 1

Bacteria identified by culturing and culture-independent methods in the midgut of individual Ixodes ricinus adult females collected from Brno and Ceske Budejovice. Bacterial CFUs were log transformed. Bacterial genera from the culture-independent method are represented by the relative abundance (%). Black arrows show an example of the bacterial taxon detected in individual ticks by both methods. Abbreviations: CFU, Colony-forming units

Fig. 2

Rarefaction curves of 16S rDNA sequences in the midgut of Ixodes ricinus

Fig. 3

Effect of location on bacterial species richness (a), Shannon diversity index (b) and Faith’s PD (c) of the midgut bacterial community of Ixodes ricinus. Abbreviations: se, Standard error

Fig. 4

Bacterial community composition in individual tick midguts. PCoA plots generated using the Bray-Curtis index. Abbreviations: PCoA, Principal coordinate analysis

Fig. 5

Mean bacterial relative abundance (%, at phylum level) associated with the geographic location (a) and in individual tick midgut samples (b). Sequences that were classified as “Bacteria_unclassified” and phyla with low abundance were grouped into Other_Bacteria

Fig. 6

Mean relative abundance and prevalence of 50 most abundant taxa in the midgut of Ixodes ricinus adult females detected by the culture-independent method

Fig. 7

Colony-forming counts of bacteria ingested (in red) and isolated (in blue) from the midgut of the same individual of Ixodes ricinus adult females after 2 h of capillary feeding. a Pantoea sp., b Micrococcus luteus. Chryseobacterium indologenes was used as a positive control. The results represent the median for 10 individual ticks. Asterisks indicate statistically significant difference at *** P < 0.001 and ****P < 0.0001

Fig. 8

Fluorescent microscopy of capillary-fed GFP-labeled bacteria in the midgut of Ixodes ricinus adult females. a, b Staphylococcus aureus, c, d Escherichia coli. Abbreviations: GFP, Green fluorescent protein