Synergistic attraction of Western black-legged ticks, Ixodes pacificus, to CO2 and odorant emissions from deer-associated microbes

Abstract

Foraging ticks reportedly exploit diverse cues to locate their hosts. Here, we tested the hypothesis that host-seeking Western black-legged ticks, Ixodes pacificus, and black-legged ticks, I. scapularis, respond to microbes dwelling in sebaceous gland secretions of white-tailed deer, Odocoileus virginianus, the ticks' preferred host. Using sterile wet cotton swabs, microbes were collected from the pelage of a sedated deer near forehead, preorbital, tarsal, metatarsal and interdigital glands. Swabs were plated on agar, and isolated microbes were identified by 16S rRNA amplicon sequencing. Of 31 microbial isolates tested in still-air olfactometers, 10 microbes induced positive arrestment responses by ticks, whereas 10 others were deterrent. Of the 10 microbes prompting arrestment by ticks, four microbes-including Bacillus aryabhattai (isolates A4)-also attracted ticks in moving-air Y-tube olfactometers. All four of these microbes emitted carbon dioxide and ammonia as well as volatile blends with overlapping blend constituents. The headspace volatile extract (HVE) of B. aryabhattai (HVE-A4) synergistically enhanced the attraction of I. pacificus to CO₂. A synthetic blend of HVE-A4 headspace volatiles in combination with CO₂ synergistically attracted more ticks than CO₂ alone. Future research should aim to develop a least complex host volatile blend that is attractive to diverse tick taxa.

Keywords: Ixodes pacificus; Ixodes scapularis; Odocoileus virginianus; microbes; sebaceous gland secretions; semiochemicals.

Figure 1.

Graphical illustration depicting the locations of forehead, preorbital, tarsal, metatarsal and interdigital excretory glands of white-tailed deer from which swabs were taken for microbe identifications.

Figure 2.

Graphical illustrations of (a) still-air 3-chamber olfactometers (1–3) housed in a plexiglass box (4; 33.5 × 49.5 × 9.1 cm high) and (b) a moving-air Y-tube olfactometer mounted within a cloth-covered scaffold (57 × 36 × 123 cm; not shown) to standardize visual cues. The lateral chambers (1a, 1b; 2a, 2b; 3a, 3b; each 9.0 × 3.1 cm) of each still-air olfactometer received a 1 cm disc of agar inoculated with a test microbe, or not (control), and a moist sterile cotton ball (5) to elevate the relative humidity. For each bioassay replicate, two ticks were placed in the central chamber of an olfactometer, and after 24 h in darkness their position in the olfactometer was scored (see methods for detail). The Pyrex glass Y-tube olfactometer (6; diameter: 2.0 cm; length of main stem and side arms: 19.5 cm and 11.5 cm, respectively) was held by a clamp (7) and fitted with Y-shaped bamboo skewers (8) as a walk-on substrate for the ticks, with a metal wire (9)—serving as a weight—to prevent physical contact of skewers with the olfactometer. For each replicate, a disc of agar inoculated with a bacterium, or not (control), was placed on sterile filter paper (10) at the orifice of side arms. To initiate a bioassay, a tick was placed on the bottom of the bamboo skewers, and a rubber stopper (11)—connected to a PVC pipe (12) and a vacuum pump—was inserted into the stem of the olfactometer, resulting in a flow (0.2 l·min⁻¹) of humidified air (40–70%) through the olfactometer. Each tick that within 12 min walked > 8.0 cm into a treatment or control side arm as its first choice was considered a responder.

Figure 3.

Arrestment of the ticks Ixodes pacificus and I. scapularis in still-air, three-chamber olfactometer experiments 1–20 (n = 15 or 30) (figure 2a) in response to microbes collected near excretory glands of white-tailed deer (figure 1). Treatment and control stimuli consisted of microbe-inoculated and sterile agar, respectively. In each replicate, two ticks were tested and the proportion of ticks choosing the lateral chamber with the treatment or with the control stimulus was calculated. An asterisk (*) denotes a significant proportion of ticks arrested in the treatment or the control chamber (χ² tests on proportional responses; * = p < 0.10, ** = p < 0.05, *** = p < 0.01).

Figure 4.

Attraction of the ticks Ixodes pacificus and I. scapularis in Y-tube binary choice olfactometer bioassays (figure 2b) in response to microbes collected near excretory glands of white-tailed deer (figure 1). Treatment and control stimuli consisted of microbe-inoculated and sterile agar, respectively (χ² tests on absolute numbers; * = p < 0.10, *** = p < 0.01).

Figure 5.

Responses of Ixodes pacificus and I. scapularis in Y-tube olfactometers (figure 2b) to (i) microbe-derived gases (Exps. 40–42), (ii) Tenax/Carbosieve headspace volatile extract of Bacillus aryabhattai (A4) (HVE-A4) (Exp. 43), (iii) HVE-A4 plus CO₂ (4% in medical air) versus CO₂ (I. pacificus: Exps. 44–46; I. scapularis: 47–48), (iv) HVE-A4 plus CO₂ versus HVE-A4 (Exp. 49) and (v) synthetic HVE-A4 plus CO₂ versus CO₂ (Exps. 50–51) (χ² tests on absolute numbers; * = p < 0.05, ** = p < 0.01). Note: (1) synthetic blend composition reported in table 1; (2) all volatile blends and corresponding solvent controls were applied in 10 µl aliquots; (3) 4.8 PHE = amount of volatiles released during 1 h from 4.8 plates of agar inoculated with B. aryabhattai.