Fig 1.
Haller’s organ is the key sensor for cinnamaldehyde repellency in ticks.
A. Cinnamaldehyde repellency test within two hours in different stages of H. longicornis. The repellency percentages of cinnamaldehyde against larvae, nymphs, and female adults were 99.17%, 100%, and 80.67%, respectively. Samples were obtained from unfed developmental stages of ticks at 7–15 d after molting (****p<0.0001). B. Scanning electron microscope images of different tick stages. a. larvae, b. nymph, c. female adult. The scale bars = 50 μm. The orange arrow points to the sensory hair of the anterior pit. C. Response of ticks to cinnamaldehyde repellency after removal of the first pair of legs. These data show the repellent response throughout 120 min, where the repellency percentage declined from 97.33% to 0 at the nymph stage and from 93.75% to 49.67% at the female adult stage. Data are expressed as percentages (*p<0.05, ***p<0.001). D. EAG response of ticks to different concentrations of cinnamaldehyde. From the initial cinnamaldehyde solution to a 10-6 dilution as −0.035, −0.025, −0.019, −0.018, −0.016, −0.015, −0.013 mV. In contrast, there was no response to paraffin oil (Control group, as solvent) (−0.0073 mV) (p<0.05).
Fig 2.
A. Relationships among HL-IR homologs from other species by phylogenetic analysis using maximum likelihood. HL-IR is marked with black circles. B. Transcription analysis of HL-IR during different developmental stages and in different tissues of female adult ticks. SN: ganglion, FB: fat body, SG: salivary glands, OV: ovary, MG: midgut. Data are presented after ANOVA and multiple comparisons (p < 0.05). C. Subcellular localization of HL-IR. Green Fluorescence: the protein containing the EGFP fluorescent tag. Red Fluorescence: Na+/K+-ATPase (Cell membrane). Blue Fluorescence: Cell nucleus. The scale bars = 10 μm.
Fig 3.
HL-IR is crucial for ticks to recognize cinnamaldehyde.
A. Determination of HL-IR transcript levels after cinnamaldehyde stimulation (***p < 0.001). B. Expression of HL-IR in Haller’s organ by in situ hybridization. A synthetic Luciferase probe was used as a control. Red Fluorescence shows HL-IR; Blue Fluorescence shows the cell nucleus. The scale bars = 50 μm. C. Confirmation of RNAi. Transcription level (left) and Expression levels (right) of HL-IR after RNAi. D. Calculation of cinnamaldehyde repellency after RNAi. The repellency rate decreased from 100%, 100%, 100% to 53.33%, 50%, 50% (****p < 0.0001). E. EAG detection of cinnamaldehyde after RNAi. The EAG response decreased from −0.0332 mv to −0.0156 mv (****p < 0.0001).
Fig 4.
Cinnamaldehyde is a specific ligand for HL-IR.
A. Binding curve and Scatchard equation for HL-IR. The ratio of bound/free 1-NPN gradually decreased as the concentration of 1-NPN increased, accompanied by a corresponding rise in fluorescence intensity. B. competitive combination curves of four ligands with HL-IR. The figure highlights how the fluorescence intensity decreases with increasing ligand cinnamaldehyde concentration. Fluorescence competitive binding is demonstrated by HL-IR and cinnamaldehyde. C. Molecular docking map (3D) and site prediction of cinnamaldehyde with HL-IR. ASN218 and PHE219 amino acid positions were expected to be the sites of intermolecular interactions with cinnamaldehyde. D. Competitive combination curves of cinnamaldehyde with mutation of HL-IR. The figure illustrates that only the fluorescent competition binding experiment failed following the mutation at 218.