Ixodes ricinus females were used as experimental model organisms. In total, 620 adult female I. ricinus ticks were used. Ticks were collected in spring 2018 from vegetation by the flagging method with a white cotton blanket (1 m2). The ticks were collected in a forest park in Kosice city in eastern part of Slovakia (48,745 °N; 21,277 °E). In the laboratory, all collected ticks were determined to the species level according to Siuda and separated to individual tubes according to developmental stage and gender 21.
All collected ticks were kept in polypropylene tubes under stable conditions in an environmental chamber at 16°C and 90% relative air humidity under 16:8-h light-dark regime. All ticks were maintained free from the influence of external stimuli, such as odor, that could affect their behavior.
Radiation-shielded tube test
A total 280 female I. ricinus ticks were placed into the Radiation Shielded Tube test (RST), where females had a choice between a shielded and unshielded part. Altogether 140 female ticks were exposed to 900 MHz RF EMF in the RST arenas for 24 hours. A control group of I. ricinus ticks (140 individuals) were placed in RST tubes under the same conditions without RF - EMF exposure. For the detailed description of RST arenas see Vargová et al., 201818.
Source of radiation for RST
We focused on the artificially emitted electromagnetic field with 900 MHz frequencies. These frequencies represent the range frequency limits that are used in common telecommunications services. The 900 MHz frequency band is used in the Global System for Mobile Communications (GSM).
A signal generator (N5183A, Agilent, MY) was used as a source of RF-EMF connected to the Double-Ridged Waveguide Horn Antenna HF907 (Rohde and Schwarz, Munich, Germany). The output power of the generator was calculated and placed at a distance 2 m from the antenna with a power flux density of 1 mW/m2 which is 0.6 V/m on the electric field intensity. The temperature in the anechoic chamber was 22°C and the humidity was 60%, measured by using a LabQuest 2 unit (Vernier Software & Technology, Oregon, USA).
Experimental procedure of RST
In a single RST test, 10 female ticks I. ricinus were placed directly in the middle of the test tube. 14 tubes were used to conduct an irradiation or control sample tests. The total number of females used in the irradiation experiment was 140. They were exposed to 900 MHz RF-EMF. The control group consisted of 140 ticks and they were not exposed to RF-EMF. The experiments lasted for 24 hours. Finally, the total number of ticks in both individual arms of RST was collected. The experiments were performed in the darkness, to exclude the influence of light to individual’s behavior.
Radiation-shielded circular open field arena
In the specifically modified behavioral arena, 2 zones with different experimental conditions: 1. with EMF and 2. EMF free were established (Fig. 1). The arena is of a circular base with a diameter of 100 cm and a height of 50 cm made of white opaque polypropylene (PP), in a position 1 meter above the floor. Copper foil 35 Micron Double-sided on 1.6 mm epoxy glass laminate desk (DSCED in short) served as EMF shielding. The DSCED semicircle attached to the bottom of the arena half-circular and perpendicular to the base the rectangle shape divided the space of the interest into shielded/unshielded environments. There was no barrier in the arena between the two halves so that the ticks could move freely. The cell phone was used as an EMF source. It was placed on the unshielded side, 0.2 m from the center of circular projection to the floor (in Fig. 1 depicted as a dashed line).
Source of radiation for circular arena
As a source of RF-EMF common cell phone registered in the cellular telecommunications network was used. 900 MHz frequency RF - EMF emission was provided by an ongoing call on the cell phone (model Samsung J5 2016, The South Korea). The 900 MHz mode was performed by changing the phone settings by selecting the network mode 2G only. The phone operation at maximum power was achieved by limiting the intensity of the GSM base station external signal to reach the lowest received signal strength. The availability of the external GSM signal was controlled inside the chamber simply by the position of the electromagnetic shielding on the entrance door. The lowest achieved operating intensity of the signal was 0.037 V/m as measured by a signal analyzer (Spectran HF 60105, Aaronia, DE). Generated by the cell phone during a call, the maximum intensity of EMF in the unshielded part of the arena was 0.61 V/m, while in the shielded one it was 0.09 V/m, both measured in situ by the signal analyzer.
Experimental procedure of circular arena
Ten individual female I. ricinus ticks were placed on the centerline of the circular arena, where ticks had a choice between a shielded and unshielded part. The experiment lasted 120 min, after this time the emission on EMF was stopped and ticks were counted in the shielded and unshielded part and collected into polypropylene tubes with 70% ethanol. Altogether 20 experiments were performed.
The movement of the ticks in the arena was recorded by a CCD camera (model Panasonic HC-X920 Japan). The camera was equipped with a wide-angle 29.8 mm lens and the aperture F1.5 was used. The recording was made in Full HD resolution. The recording rate of 1 frame per second was established experimentally. It provided sufficiently detailed sequences for further tick movement analysis. Up to 12x optical zoom and 5-axis image stabilization were used. The individual experimental observations were recorded and stored in a separate record.
The experimental arena was placed in the anechoic chamber (model 1710 − 100, Comtest, The Netherlands) which ensured no external electromagnetic fields to be used to violate the experiment. All experiments were performed in an anechoic chamber. Temperature in the anechoic chamber was 22°C and air humidity was 60%, measured by using a LabQuest 2 unit (Vernier Software & Technology, Oregon, USA). The interior was illuminated by the pair of low power EMF free lights placed in two opposite corners of the ceiling of the chamber.
Feature extraction and statistical analysis
We used a camera-based method for quantifying track length and the time spent in irradiated or shielded part of the arena. To analyze recorded tick’s movement sequences the software tool has been used, discussed elsewhere. The recorded area was 1920x1080 pixels, out of which a circular arena was extracted.
Both horizontal and vertical neighbor pixels’ displacement represents approx. 1 mm distance in real measure in these directions.
Ticks in the arena were tracked and digitized information saved in the data file, i.e. tick’s identity and x,y-coordinates, too. The coordinates represented the center of the tick’s body (center of the gravity). The tracking performance is depicted in Fig. 2. Shadowed area means EMF shielded part of the arena.
The spatio-temporal evidence of ticks inside the arena during the experiment was analyzed. The arena digital representation was divided into the shielded and unshielded parts. The analysis consists of the registration of the tick's digitized coordinate in that specific part where it belongs. It was realized in a cycle over all digitized coordinates.
The trial time was divided to 10-minute periods. Digitized path data of 160 individuals were used to calculate several parameters in these time parts: (1) the total track length (TTL) as the sum of track length contributions walked by ticks included in the experiment; (2) the total track length in the irradiated zone (TTL_I); (3) the total track length in the shielded zone (TTL_S) zone. Similarly, during the temporal observation individuals’ evidence in the shielded and unshielded parts was performed. Additional parameters were calculated: (4) percentage of the time spent in the arena (PIZ); (5) percentage of the time spent in the irradiated zone (PIZ_I); (6) in the shielded zone (PIZ_S). The descriptive statistics was calculated and compared.