Unfed 2 to 3-month old female adult A. americanum, D. variabilis, R. sanguineus, and I. scapularis were obtained from the Oklahoma State University tick rearing facility. They were kept at room temperature and >95% RH until used for experiments. Ticks were kept at 28 °C and >90% RH prior to experiments.
Induction of dermal secretion by heat probe
Ticks were immobilized on a flat surface with double-sided sticky tape (3M Scotch permanent mounting tape, MN, USA). The dorsal surface of the tick was exposed to heat by a direct contact with a heat probe made of 28-gauge nichrome 80 wire for 1–5 sec., depending on the experiments specified. The temperature of the wire was controlled by a current controller (Stoelting Co, IL, USA) and the temperature was monitored by a thermal camera (SeekTermal, CompactXR).
In order to measure the threshold temperature for secretion response in A. americanum, we treated the ticks with different temperatures for 1 sec. with the temperature increasing from 30 to 70 oC by 5 oC interval. Eight ticks were tested at each temperature to avoid acclimation or sensitization of ticks to prior exposure to the heat probe. Another modified method was used for measuring the heat sensitivity in the test including other Metastriata ticks (A. americanum, R. sanguineus and D. variabilis). In this test, individual ticks were exposed to the probe repeatedly with sequential increments of the probe temperature. In each trial the probe temperature was sequentially increased by ~3 to 4 °C (n=10 for R. sanguineus, n=12 for A. americanum, and n=12 for D. variabilis). If the dermal secretion was not observed before 5 seconds of contact at the given temperature, the probe was detached from the tick and the next higher temperature was set for another contact that was followed by ~ 1 min. interval. A control group was treated with the probe at room temperature (RT) to ensure the response was not due to mechanical simulation by contact from the probe. During this operation, the thermal image was recorded and analyzed by SeekThermal application software (V184.108.40.206) and the image processing software (V220.127.116.11).
To assess the impact of dermal secretion on the body cooling rate, a set of 5 ticks per group were used; a group with no sweat and with sweat. Both were treated with the probe at the temperature between 40-42 °C. Ticks reported as; no sweat means they did not exhibit the secretion after 5 seconds of the heat probe contact. Thermal images were analyzed as 20 frames per second using ImageJ 1.53a 40. The cooling rate was assessed by the average pixel values of 3 different regions of interest (ROI) surrounding the probe contact point on the tick surface. The average pixel value of the ROI was used for accurate estimation of temperature. The rate of lowering the temperature from 40 °C in each tick were fitted to an ExpDec1 curve (OriginPro 2020b, Fig 3b) and values obtained for each rate of decay, A1/t1, were used in a Student’s T-test for the analysis (Fig 3b). ExpDec1 regression provided the fits with R values in the range of 0.97 to 0.99. The regression and the data analyses were conducted in OriginPro 2020b (18.104.22.168).
To assess the amount of weight loss after exhaustive dermal secretion, the ticks kept in high RH (>95%) for one day were weighted before and after the contact with heat probe at 55–58°C for 5 sec. The ticks after the treatments were placed on the high RH for recovery and monitored for survivorship.
Pharmacology of dermal secretion
To identify the neural or hormonal components involved in dermal excretion, we injected a series of biogenic amines, neuropeptides, and secondary signaling messengers. The chemicals used for biogenic amines were: Octopamine ((±)-Octopamine hydrochloride, Sigma, Cas#00250), Norepinephrine ((±)-Norepinephrine (+)-bitartrate salt, Sigma, Cas#3414-63-9), Dopamine (Dopamine hydrochloride, Sigma, Cas#H8502), Serotonin (5-hydroxytriptamine hydrochloride, Sigma Cas#H9523), for neuropeptides were: SIFamide (AYRKPPFNGSIFamide, 41,42), MIP-1 (Mioinhibitory peptide-1, ASDWNRLSGMWamide, 41,42), Proctolin (RYLPT, ELV-1 (Elevenin, LDCRKYPFYYRCRGISA, 43), for secondary signaling messengers were: Dibutyryl cAMP (Dibutyryl adenosine 3',5'-cyclic monophosphate sodium salt, Santa Cruz Biotechnology, Cas# 16980-89-5), Forskolin (Sigma, Cas # F3917), Dibutyryl cGMP (Santa Cruz Biotechnology, Cas# 51116-00-8) SNAP (N-(acetyloxy)-3-nitrosothiovaline, Cayman chemicals Cas#67776-06-1).
The compounds dissolved in water were injected through the base of the second coxal segment from lateral side using a Nanoject III nano-injector (Drummond Scientific Company, PA, USA) (Table 1). All injections were made for 10 nL unless it is specified. The strong secretion inducer, serotonin, was further tested for obtaining the full dose-responses.
We tested the role of Na+/K+-ATPase in the dermal secretion by a pretreatment of the tick with Oubaian, a Na+/K+-ATPase inhibitor. Ticks were injected with 10 nl of 100 µM Oubaian 30 min before injection of serotonin (10 nl of 1mM). In rare cases (3 out of 20), the ticks that showed dermal secretion in Ouabain injection were excluded in data analysis because they were considered as the response to mechanical stimulation by insertion of the needle, which also occurred in water control.
Localization of dermal secretion and the anatomy
Naïve A.mericanum females were used to visualize the structure of the dermal gland on the dorsal and ventral dermal integuments. Immobilized ticks were placed on a double-sided sticky tape and treated by a heat contact to the legs. Dorsal dermal secretion was observed under fluorescent light with CFP filter set (excitation BP436/7, dichromatic mirror 455, and emission filter 470LP). The majority of dermal secretions displayed fluorescence drops with the CFP filter set. For internal view of the dermal glands, naïve ticks were injected with a local anesthetic, 20 µl of 740 mM of Procaine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA), to prevent the dermal secretion triggered in the processes of dissection, which empties the gland contents. Ten minutes after procaine injection, the dorsal integument of ticks was removed with a surgical scalpel to visualize the internal glands. External and internal images were captured using a camera (DFC400) attached to a stereo microscope (M205FA; Leica, Heerbrugg, Switzerland) with the CFP filter set.
For confocal imaging of the glands, cellular structures, and molecular components, dorsally opened tissues were washed with PBST fixed in 4% paraformaldehyde for 3 hours. The tissues were then incubated with 5% normal goat serum (Jackson ImmunoResearch), containing the target antibodies overnight at room temperature. Immunohistochemistry (IHC) was performed using beta-tubulin mouse antibody (GenScript, Piscataway, NJ, USA) at a final concentration of 0.5 mg/ml, mouse monoclonal antibody (a5) raised against chicken Na/K-ATPase (Developmental Study Hybridoma Bank, University of Iowa) at 4.4 µg/ml. To localize Na/K-ATPase, we used a procedure already established by our laboratory 44. Following primary antibody incubation, tissues were washed with PBST and subsequently incubated overnight at room temperature with the secondary antibody, goat-anti-mouse IgG antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA). In addition, goat polyclonal antibody against horseradish peroxidase (HRP) conjugated with Cyanine Cy™3 (8 µg/ml, Jackson Immunoresearch, West Grove, PA. USA), containing 5% NGS, was used. The HRP-antibody has been used for characterization of the tissues having neural properties in insect 45, due to its immunoreactivity against an N-linked oligosaccharide epitope expressed on neuronal glycoproteins in insects 46. After the secondary antibody incubations, tissues were washed with PBST, incubated in 300nM 4′,6′-diamino-2-phenylindole (DAPI, Sigma) or 2.5 µg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA, US) and 40 fold dilution of Phalloidin conjugated with Alexa Fluor™ 555 (used for actin staining), (Molecular Probes, Eugene, OR, USA) or 5 µg/ml CellMaskTM (Invitrogen, Carlsbad, CA, US) for 10 minutes, washed for 30 minutes and then mounted in glycerol. Images were captured with a confocal microscope (Zeiss LSM 700).
In microtome sections to visualize the cuticle integument and epidermal layers, ticks were cut into 2–3 pieces directly alive or after snap-freezing using liquid Nitrogen. Ticks were fixed for 3 hours at room temperature in non-alcoholic Bouin’s fixative and washed with PBS containing 0.5% Triton X-100 (PBST). Samples were dehydrated with series of increasing ethanol solution (50 to 95%) and an additional cuticle plasticization step was conducted by placing samples in n-Butanol (3 hours incubation at room temperature with rotation). Following dehydration, samples were placed in 100% chloroform for overnight at 60 °C and transferred to paraffin for up to 96 hrs. Tissue sections were made by using a Leica microtome at 8 to 10 µm thickness and placed on a slide with 0.5% gelatin. Samples were dried in an incubator at 40 °C overnight. Deparaffinization was conducted using xylene, tissues were rehydrated with decreasing series of ethanol solutions (95 to 50%) and washed with PBST.
For visualization of dermal layers under bright field, staining with methylene blue (10 seconds) was conducted, using solution II from the Hema 3TM staining kit (ProtocolTM, Fisher Scientific, Waltham, MA, USA). Slides were visualized using a Nikon Eclipse E800 compound microscope.
Data will be deposited in the public repository if the manuscript is accepted for publication.