Effects of low frequency rectangular electric pulses on Trichoplax (Placozoa)

Abstract

The effect of extremely low frequency electric and magnetic fields (ELF-EMF) on plants and animals including humans is quite a contentious issue. Little is known about ELF-EMF effect on hydrobionts, too. We studied the effect of square voltage waves of various amplitude, duration, and duty cycle, passed through seawater, on Trichoplax organisms as a possible test laboratory model. Three Placozoa strains, such as Trichoplax adhaerens (H1), Trichoplax sp. (H2), and Hoilungia hongkongensis (H13), were used in experiments. They were picked at the stationary growth phase. Arduino Uno electronics platform was used to generate a sequence of rectangular pulses of given duration and duty cycle with a frequency up to 2 kHz. Average voltage up to 500 mV was regulated by voltage divider circuit. Amlodipine, an inhibitor of calcium channel activity, was used to check the specificity of electrical pulse effect on voltage-gated calcium channels in Trichoplax. Experimental animals were investigated under a stereo microscope and stimulated by current-carrying electrodes placed close to a Trichoplax body. Variations in behavior and morphological characteristics of Trichoplax plate were studied. Stimulating and suppressing effects were identified. Experimental observations were recorded using photo and video techniques. Motion trajectories of individual animals were tracked. Increasing voltage pulses with fixed frequency of 20 Hz caused H2 haplotype individuals to leave “electrode zone” within several minutes at a voltage of 25 mV. They lost mobility in proportion to voltage rise and were paralyzed at a voltage of 500 mV. Therefore, a voltage of 50 mV was used in further experiments. An animal had more chance to move in various directions in experiments with two electrodes located on one side instead of both sides of Trichoplax. Direction of motion was used as a characteristic feature. Trichoplax were observed to migrate to areas with low density of electric field lines, which are far from electrodes or behind them. Animals from old culture were less sensitive to electrical stimulus. H2 strain was more reactive than H1 strain and especially than H13 strain; it demonstrated stronger physiological responses at frequencies of 2 Hz and 2 kHz with a voltage of 50 mV. Motion patterns and animal morphology depended on the duration of rectangular stimulation pulses, their number, amplitude, and frequency. Effects observed varied over a wide range: from direct or stochastic migration of animals to the anode or the cathode or away from it to their immobility, an increase of optical density around and in the middle of Trichoplax plate, and finally to Trichoplax folding and detach from the substrate. Additional experiments on Trichoplax sp. H2 with pulse duration of 35 ms and pulse delay of 1 ms to 10 s showed that the fraction of paralyzed animals increased up to 80 % with minimum delay. Nevertheless, in the presence of amlodipine with a concentration of 25 nM, almost all Trichoplax remained fast-moving for several minutes despite exposure to voltage waves. Experimental animals showed a total discoordination of motion and could not leave an “electrode trap”, when amlodipine with a concentration of 250 nM was used. Further, Trichoplax plate became rigid, which appeared in animal shape invariability during motion. Finally, amlodipine with a concentration of 50 μM caused a rapid folding of animal plate-like body into a pan in the ventral-dorsal direction and subsequent dissociation of Trichoplax plate into individual cells. In general, the electrical exposure applied demonstrated a cumulative but a reversible physiological effect, which, as expected, is associated with activity of voltage-gated calcium channels. Amlodipine at high concentration (50 μM) caused Trichoplax disintegration; at moderate concentration (250 nM), it disrupted the propagation of activation waves that led to discoordination of animal motion; at low concentration (25 nM), it prevented an electric shock.