Fig 1.
The tick I. ricinus possesses a complete Toll pathway.
(A) Simplified schematic of the I. ricinus NF-κB signaling pathways (Toll and IMD). Proteins characterized in this work are highlighted in blue. Dashed lines indicate missing key components. P = phosphorylation. (B) Domain structure of two NF-κB and two I-κB proteins identified in I. ricinus transcripts. RHD = Rel homology domain, IPT = Immunoglobulin-like fold, Plexins, Transcription factors, NLS = nuclear localization signal, PEST = P/E/S/T-rich sequence, a signal peptide for protein degradation. (C) Phylogenetic tree of selected arthropod NF-κBs. Unrooted tree of NF-κB amino acid sequences reconstructed by the Neighbor-Joining method (NJ) based on alignment across the RHD domain with ClustalX. Asterisks indicate Relish sequences lacking ankyrin domains. Alignment and sequence descriptions can be found in the S2 Data and S1 Text. Numbers at branches represent bootstrap support using NJ with 1,000 repeats each (bars = 0.1 substitutions per site). (D) Relative expression (qRT-PCR) of tick nf-κb, i-κb, and defensin (defIR; tick antimicrobial protein; further identified as a Toll pathway read-out gene) in tissues of half-fed (fed for five days) adult ticks (top) and different developmental stages (bottom) normalized to tick elongation factor 1 (ef). Results represent the mean of three independent biological replicates. The highest individual value for a given gene in each panel was set to 100% and all other values were expressed relative to this value.
Fig 2.
The tick Toll pathway components regulate expression of immune genes.
(A) Efficacy of gene silencing in the fully-fed nymphs (whole bodies) measured by qRT-PCR. Each dot represents a pool of five nymphs. (B-C) Identification of the Toll pathway read-out effector gene. Relative expression (qRT-PCR) of defIR (B) and other immune genes (C) in the dsRNA-injected, fully-fed nymphs. Gene expression in the dsGFP control was set as 1. dl = dorsal, KD = knockdown. *P ≤ 0.05; **P ≤ 0.01.
Fig 3.
The tick Toll pathway senses E. coli bacteria, but not Borrelia afzelii spirochetes.
(A) Relative expression (qRT-PCR) of defIR in the unfed nymphs 24 hours after injection of microbes. Each dot represents a pool of ten nymphs. (B) Efficacy of myd88 silencing by RNAi in the unfed nymphs 14 days after injection of dsRNA and 24 hours after injection of microbes. Results represent the mean of five biological replicates. (C) Relative expression of defIR in the unfed nymphs 14 days after injection of dsRNA and 24 hours after injection of microbes. (D) Relative expression of defIR in the unfed nymphs 24 hours after injection of B. afzelii. (E) A scheme of B. afzelii transmission experiment. (F) PCR detection of B. afzelii in murine tissues four weeks after infestation with ten infected nymphs pre-injected with dsRNA. KD = knockdown. *P ≤ 0.05; **P ≤ 0.01; n.s. = not significant P ≥ 0.05.
Fig 4.
The tick Toll pathway regulates acquisition of Babesia microti by tick nymphs.
(A) Detection of B. microti (B.m.) and mouse DNA by PCR in the nymph midgut and salivary glands at various days post detachment (DPD). Each sample represents a single tick. (B) Confocal microscopy of salivary glands 0 DPD after feeding on mice infected with B. microti. (C) Transmission electron microscopy of infected salivary glands 0 DPD. Red arrows indicate infected acinar cells. (D) Relative expression of defIR in infected nymphs (nymphs fed on B. microti-infected mice) 0, 3, and 6 DPD. (E) A scheme of the B. microti acquisition experiment. (F) Relative quantity (qRT-PCR) of B. microti in the salivary glands (SG) of nymphs pre-injected with dsRNA analyzed 6 DPD. The results were normalized to I. ricinus ferritin 2 (fer2). rel = relish, dl = dorsal. *P ≤ 0.05; ****P ≤ 0.0001; n.s. = not significant P ≥ 0.05.