Capacitance-voltage (C-V) measurements were carried out using a Fluke PM6306 programmable RCL meter (Fluke Calibration, Everett, WA, USA) with 1 MHz.
Current was measured using a Fluke 189 True RMS Multimeter in series with a 1 Megohm resistor.
Samples were collected from the caudal peduncle vein by using a 20- or 21-gauge, 1.5-inch Vacutainer needle (Becton Dickinson Vacutainer Systems, Rutherford, NJ, USA) or from a fluke vein by using a 21-gauge, 1-inch butterfly needle.
A serum sample was tested at Leiden University Medical Center by using an in-house immunofluorescence assay and ELISA with Fasciola spp. antigens, which are likely to show cross-reactivity with other liver flukes (9 ).
All temperatures were measured using a calibrated Fluke 51 electronic thermometer.
The current (I) flowing through the tissue was measured using a multimeter (Fluke 189 True RMS Multimeter, Everett, Washington, USA; Fig. 1).
The dead flukes were assessed by disturbing the flukes using a needle.
The reference end-systolic volume (ESV) was calculated by determination of the stroke volume (SV) using a gas flow analyser (VT Plus HF, Fluke, Everett, WA) and using the relation SV = EDV – ESV.
Surface temperature was verified using an infrared thermometer (accuracy 0.01°C, Fluke 63; Fluke UK Ltd., Norwich, UK) before and after each replicate of these experiments.
Total RNA from whole body of each fluke was extracted by using RNeasy Protect Mini Kit (Qiagen) with an on-column DNAse step (Macherey Nagel) as previously described [ 75].
