Effects of Electromagnetic Radiation on NeuropeptideTranscript Levels in the Synganglion of Ixodes ricinus

Ticks were divided into 24 experimental groups, 5 ticks per group. To ensure the proper hydration of ticks, each group was placed in a plastic 2 mL tube with moistened filter paper before irradiation. The irradiation was conducted in an anechoic chamber (model 1710–100, Comtest Engineering, Leyde, Netherlands), ensuring no external electromagnetic fields affected the experiment. A signal generator (N5183A, Agilent Technologies, Kuala Lumpur, Malaysia) was used as a source of radiofrequency electromagnetic field (RF EMF) connected to a Double-Ridged Waveguide Horn Antenna HF907 (Rohde and Schwarz, Munich, Germany). The antenna was placed approximately 2 m from the experimental apparatus containing ticks.During the experiment, ticks were irradiated (Table 1) with a constant, polarized, unmodified electromagnetic field at a 900 MHz frequency and two different intensities—2 V/m and 40 V/m. The duration of the exposure was 10 min, 1 h, 3 h and 24 h. Together, 4 control groups of ticks were placed into the anechoic chamber for the designated times but without irradiation.A pool of 5 synganglia was used for each experimental condition. Total RNA was isolated using an RNeasy® Micro Kit (Qiagen, Venlo, Netherlands). The concentration and purity of obtained RNA were measured using a NanoDrop ONE (Thermo Scientific, Waltham, MA, USA). The obtained RNA was reverse-transcribed to cDNA using a RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Scientific), using oligo (dT) primers. To determine the change in the mRNA levels, qRT-PCR was performed using a LightCycler® 480 II (Roche, Meylan, France) thermocycler. LightCycler® 480 SYBR® Green I Master mastermix (Roche) was used each 20 μL reaction was prepared from 10 μL of mastermix, 7 μL of PCR-grade H2O, 1 μL of 10 μM forward and 1 μL of 10 μM reverse primer, and 1 μL of template cDNA. Primers for qRT-PCR are listed in Table 2. The nomenclature of studied neuropeptides and their genes is according to Coast and Schooley 2011. As a reference gene, the ribosomal protein S4 (rps4) was used.