In the first set of experiments, the activity of nerves in NI-Ctr group was evaluated. The activity of the left cercal nerve (LCN) under the left cercus (LC) stimulation from the left side was 38.95% higher than LCN activity when LC was stimulated from the right side (Fig. 1a) (p = 0.0065). Right cercus (RC) responded better to stimulations from the right side, and activity of right cercal nerve (RCN) when RC was stimulated from right side was similar to the activity of LCN, when LC was stimulated from the left (Fig. 1a,b). These results showed that LC responds the most to left side stimulation, whereas the highest response of RC occurs under stimulation from the right side of the body.
When LC was stimulated from the left, the activity of the connective nerve (CON) was 53.37% lower than the activity of LCN after stimulation from the same direction (p = 0.01; Fig. 1a). A similar reduction in CON activity was observed when LC was stimulated from the right: the activity of CON was 52.90% lower than LCN activity after receiving the stimulation from the same side (p = 0.0032). Original representative recordings of LCN and CON activity when LC was stimulated from the left side are presented in Fig. 1c and 1d, respectively.
RC ablation resulted in the loss of the ability to perceive stimulation (Fig. 1b,e). Shortly after RC ablation, LCN, and CON activities, regardless of the side of LC stimulation, were not significantly different from the pre-injury values. That indicates that immediately after RC ablation, the perception of the right-sided stimulation was limited, and the enhancement of the response was not observed.
In the I-Ctr group, LCN activity after LC stimulation from the left side was constant over a period of 3 weeks (Fig. 2a). The slight increase in nerve activity, observed in the 3rd week of the experiment, was not significant. However, the activity of LCN after stimulation from the right side increased over time (Fig. 2b). After 3 weeks, LCN activity was 94.93% higher than the activity before the injury (p = 0.043). In the I-Ctr group, changes in the activity of CON were also observed. When LC was stimulated from the left, CON activity increased (Fig. 2c); particularly, 2 weeks after the injury, the activity was 92.95% higher than pre-injury (p = 0.036). When LC was stimulated from the right, CON activity remained at the pre-injury level up to the end of the observations (Fig. 2d).
LCN and CON activities were evaluated in cockroaches exposed to electromagnetic field (I-EMF group). In this group, LCN activity in response to stimulation of LC from the left side was higher by 55.96%, 59.12%, and 34.89% after 1, 2, and 3 weeks, respectively, than in the I-Ctr animals, (p = 0.005 for 1 week after injury, p = 0.017 after 2 weeks; Fig. 3a). These values were also higher by 63.85%, 69.26%, and 56.57% than the pre-injury values (p = 0.018 after 1 week, p = 0.014 after 2 weeks and 0.042 after 3 weeks).
The increase in LCN activity was also observed when LC was stimulated from the right side (Fig. 3b). LCN activity was higher by 110.05%, 65.62%, and 75.33%, respectively, than the activity in the I-Ctr group at corresponding times (p = 0.012 after 1 week, p = 0.030 after 2 weeks, p = 0.017 after 3 weeks) and was higher by 162.07%, 125.46% and 241.79%, respectively, than the pre-injury LCN activity (p = 0.003 for 1 week, p = 0.034 for 2 weeks and p < 0.001 for 3 weeks).
The EMF induced an increase of CON activity (Fig. 3c,d). When LC was stimulated from the left, the CON activity was only slightly higher than the activity of the nerve found in the I-Ctr group. However, the CON activity for the I-EMF group were higher than the pre-injury nerve activity by 109.85%, and 161.65%, respectively, after 2, and 3 weeks (p = 0.01 for 1 week, p = 0.004 for 2 weeks, p = 0.001 for 3 weeks) (Fig. 3c).
When LC was stimulated from the right side, an increase in CON activity was also observed (Fig. 3d). The values were 71.38%, 89.51% and 152.21% higher than the values in I-Ctr group, after 1, 2 and 3 weeks, respectively (p = 0.023 after 3 weeks), moreover the values were 146.36%, 128.58% and 196.18% higher than CON activity measured before the injury (p = 0.005 for 1 week, p = 0.024 for 2 weeks and p = 0.004 for 3 weeks).
To evaluate whether the perception of the right-sided stimulation was overtaken by the left side of the escape system, the ratio of LCN activity induced by the right sided stimulation in respect to the stimulation from the left side was calculated – R/L ratio (Fig. 3e). For the I-Ctr group, the ratio increased from 0.36 ± 0.07, before injury, to 0.51 ± 0.08 in one week after injury, and stayed constant afterwards: in two weeks − 0.50 ± 0.14 and in three weeks − 0.52 ± 0.11. In I-EMF group, this ratio increased to 0.61 ± 0.07 in one week after injury, to 0.62 ± 0.09 after two weeks and to 0.87 ± 0.09 three weeks after injury (p = 0.014). Higher values of the ratio indicate better perception of right-sided stimulation by the left side of the escape system. It should be emphasized that the increase over time of the R/L ratio was only observed for the I-EMF group, and not for the I-Ctr group.
To verify if the effect of EMF exposure was not accidental or non-specific, TGF-β was used as a positive control (Fig. 3f). The magnitude of the bioelectrical activity of CON was evaluated when LC was stimulated from right side since the EMF exposure affected the most CON activity.
When TGF-β was administrated, CON activity was found higher by 86.74%, 65.93%, and 95.32% in respect to the I-Ctr group, for 1, 2, and 3 weeks, respectively (p = 0.017 for 3 weeks), and the values were found increased by 68.43%, 100.13%, and 246.8% when compared to pre-injury values (p = 0.008 for 3 weeks). CON activity after TGF-β treatment was not significantly different from the activity registered when insects were exposed to EMF (see Fig. 3d).
TGF-β impact on the R/L ratio was also evaluated (Fig. 3g). The level of R/L ratio was equal to 0.62 ± 0.05 one week after injury, 0.60 ± 0.15 after two weeks, and 0.90 ± 0.14 after three weeks. In the third week of the experiment, the R/L ratio was significantly higher in insects treated with TGF-β in respect to pre-injury (p = 0.009) and did not differ from the ratio evaluated in insects exposed to EMF (see Fig. 3e).
I-Ctr cockroaches deprived of the RC, three weeks after the injury, traveled a distance similar to that of NI-Ctr insects. Moreover, they remained close to the arena borders similarly to the NI-Ctr animals (Fig. 4a,b,f). The distance traveled by cockroaches deprived of RC and exposed to EMF for three weeks was slightly higher than the distance reached by insects before the injury and significantly higher than that in I-Ctr insects (p = 0.03). However, the I-EMF insects remained at the arena border for a time similar to the NI-Ctr and I-Ctr groups (Fig. 4.a,b,g)
The insects deprived of RC were significantly more active 24 hours after the injury than the NI-Ctr animals (p = 0.002) (Fig. 4c,d,e). Three weeks after the injury in the I-Ctr group, the percentage of motionless returned to the pre-injury value (Fig. 4c,f). Surprisingly, in the I-EMF group, the immobility was significantly lower than in the I-Ctr group and NI-Ctr group (p = 0.027 and p = 0.038 respectively; Fig. 4c,g).