Identification of Tick Ixodes ricinus Midgut Genes Differentially Expressed During the Transmission of Borrelia afzelii Spirochetes Using a Transcriptomic Approach

Abstract

Lyme borreliosis is an emerging tick-borne disease caused by spirochetes Borrelia burgdorferi sensu lato. In Europe, Lyme borreliosis is predominantly caused by Borrelia afzelii and transmitted by Ixodes ricinus. Although Borrelia behavior throughout tick development is quite well documented, specific molecular interactions between Borrelia and the tick have not been satisfactorily examined. Here, we present the first transcriptomic study focused on the expression of tick midgut genes regulated by Borrelia. By using massive analysis of cDNA ends (MACE), we searched for tick transcripts expressed differentially in the midgut of unfed, 24h-fed, and fully fed I. ricinus nymphs infected with B. afzelii. In total, we identified 553 upregulated and 530 downregulated tick genes and demonstrated that B. afzelii interacts intensively with the tick. Technical and biological validations confirmed the accuracy of the transcriptome. The expression of five validated tick genes was silenced by RNA interference. Silencing of the uncharacterized protein (GXP_Contig_30818) delayed the infection progress and decreased infection prevalence in the target mice tissues. Silencing of other genes did not significantly affect tick feeding nor the transmission of B. afzelii, suggesting a possible role of these genes rather in Borrelia acquisition or persistence in ticks. Identification of genes and proteins exploited by Borrelia during transmission and establishment in a tick could help the development of novel preventive strategies for Lyme borreliosis.

Keywords: Borrelia afzelii; Ixodes ricinus; RNAi; massive analysis of cDNA ends (MACE); midgut; tick; transcriptome.

Figure 1

Scheme of sample preparation for massive analysis of cDNA ends (MACE) analysis. The uninfected larvae, originating from three individual females fed on a single guinea pig, were mixed and fed on B. afzelii-infected or uninfected mice. The nymphs then were fed on uninfected mice and dissected for midguts (150–220 nymphs for each group) at the three indicated time points. The MACE analyses were performed on six different RNA pools (MACE 1-6).

Figure 2

Expression of tick midgut genes is altered in the presence of Borrelia afzelii. Volcano plots showing differentially expressed tick transcripts analyzed by MACE at individual time points. (A) Unfed nymphs (B) Nymphs fed for 24 h (C) Fully fed nymphs. n = number of differentially expressed transcripts. Total differentially expressed transcripts (black), upregulated transcripts (red; p-value ≤ 0.05 and log2 fold change ≥ 2.3), and downregulated transcripts (blue; p-value ≤ 0.05 and log2 fold change ≤ −2.3). up = total upregulated transcripts, down = total downregulated transcripts, MG, midgut.

Figure 3

Expression of selected transcripts can be verified by technical and biological qRT-PCR validations. (Upper) (A) Venn diagram of the top-score differentially expressed B. afzelii-infected nymph midgut transcripts (fold change ≥ 5 fold and expression ≥ 5 transcripts per million). The upregulated transcripts are marked by a red arrow, downregulated by a blue arrow. (B–H) qRT-PCR profiles (relative expression) of seven biologically validated transcripts were significantly upregulated by B. afzelii infection (Mann-Whitney test). The biological validations were carried out on 10 individual tick populations. Each dot represents expression in a single nymph population. In each graph, cDNA with the highest expression was set as 100. The tick elongation factor was used as a housekeeping gene. (Lower) Summary table of the validated transcripts. In total, 22 transcripts from different time points of feeding (see Results for the selection criteria) were analyzed by the technical and biological validations. UF, unfed; 24hrs, fed for 24hrs; FF, fully fed. P < 0.05 (*), P < 0.01 (**).

Figure 4

Effect of gene silencing by RNA interference on nymph feeding and Borrelia afzelii transmission. (Upper) Silencing of five tick genes in uninfected nymphs. (A) Evaluation of the silencing level by qRT-PCR (each group represents a mix of five fully fed nymphs). (B) Weights of individual fully fed nymphs. Each dot represents a single tick. (C) Duration of nymph feeding. (D) Molting success of fully fed nymphs into adults (percentage of molted nymphs fed on each mouse; biological triplicates). (Lower) Summary table of two transmission experiments with the gene-silenced B. afzelii-infected nymphs. Numbers indicate total qRT-PCR positive/total mouse tissues during the infection (ear week 2) and after mice scarification (week 3). dsGFP was used as a negative control. A decrease of positivity by >25% is highlighted in red.