The authors have declared that no competing interests exist.
Some natural alkaloids, e.g. capsaicin and camphor, are known to induce a desensitization state, causing insensitivity to pain or noxious temperatures in mammals by acting on TRP receptors. Our research, for the first time, demonstrated that a phenomenon of pharmacological blockade of heat sensitivity may operate in American cockroach,
Capsaicin from chili pepper is known to alter animals’ thermoregulatory processes. In 1970, Jancsó-Gábor et al. [
Later it was demonstrated that thermoregulatory changes induced by capsaicin were mediated by the TRPV1 receptor, long before this channel received its current name [
Exposure of TRPV1 channels to capsaicin not only causes their activation, but also induces the release of proinflammatory peptides, such as substance P or calcitonin gene-related peptide, and the generation of impulses transmitted via central fibers to the spinal cord where they are perceived as pain. A state of high-dose capsaicin-induced neuronal insensitivity to stimuli, which would normally activate TRPV-expressing neurons, is termed desensitization [
Nociceptive responses to heat were studied in
In the present study, the thermal response of
Experiments were performed on adult American cockroaches,
The test compounds (Sigma Aldrich) were dissolved in ethyl alcohol and diluted to obtain the desired concentrations: capsaicin 0.0001mM and 0.1mM, capsazepine 0.0001mM, menthol 2mM, thymol 1mM, camphor 15mM, and AITC 3mM. Optimal drug concentrations for use in the following experiments were determined based on preliminary results and literature data. The control group was treated with water. To exclude the effect of solvent, one group of cockroaches was exposed to ethyl alcohol in the same concentration as used to the test drug solution (1%; vehicle group). The tested substances (10μl) were applied under the wings, on the mesothorax.
To evaluate insect escape times from noxious heat, we constructed a ‘heat box’ (5×7×5 cm) (
Heat box used to measure the latency to escape from noxious temperature (50°C) in cockroaches.
Two different experimental designs were used (
Five-trial heat box test. Cockroaches were examined for five days (7 different drug groups; n = 13–60 for each treatment group). Each day they were applied with test drugs and tested in the heat-box (1 drug per subject/1 heat-box trial per day).
Single-trial heat box test. The second part of experiment was performed to exclude effects of heat stress and learning on cockroach responses to agonists and antagonists of thermo-TRP. The test drugs were administered to the cockroaches once daily for five days, but the testing in the heat box was performed only after administration of the fifth dose on day five (one time in the heat box) (n = 16–39 for each treatment group; 1 drug per subject/ single trial on day five).
The scheme of the experimental design.
We have found that some TRP agonists resulted in increased time spent at high ambient temperatures (heat-box test), thus we decided to determine whether ‘sensitized’ insects alter their thermal preferences in the normal temperature range (10–40°C). A novel set of cockroaches was used in the behavioral thermoregulation experiment.
Experiments were performed in a thermal gradient using an identical method as described previously [
The test compounds affected cockroach responses to noxious high temperature. In order to assess whether the response might be a result of secondary effects of drug action on oxidative stress, lipid peroxidation by TBARS assay and catalase activity was measured.
The TBARS assay quantifies oxidative stress by measuring the level of lipid peroxidation. To assess the lipid peroxidation level, spectrophotometric quantitation of the thiobarbituric reaction product, malondialdehyde (MDA), was used [
Catalase activity (CAT) was measured as described by Orta-Zavalza et al. [
The concentration of protein in the cockroach homogenates was determined by the Bradford method [
Kolmogorov-Smirnov test showed that the obtained data are not normally distributed, therefore the results were analyzed using non-parametric tests. The Mann-Whitney test showed that the there were no significant differences in escape time between males and females, so the subsequent analyses were performed on data obtained for both sexes. All analyses were performed with IBM SPSS Statistics 24 software. Data are presented as mean±SEM.
The effect of test compounds on latency to escape from heat plate was assessed using the Kruskal–Wallis test. If Kruskal–Wallis testing revealed a significant difference, a pairwise Mann-Whitney U test was carried out. Values were considered significant after adjustment of multiple testing with Holm’s sequential correction for multiple comparisons.
The differences between effects of separate doses of each tested drug in five-trial heat-box test (escape time of each dose was compared against each previous dose) were determined with Wilcoxon signed-rank test followed by Holm’s sequential correction for multiple comparisons.
The comparison between the effect of treatments on thermal preferences (means from 24 hour observation) was made using Kruskal–Wallis test followed by pairwise Mann-Whitney U test with Holm’s sequential correction for multiple comparisons.
The effect of the test drugs on MDA level and catalase activity was assessed with pairwise Mann-Whitney U tests after Kruskal–Wallis tests. Multiple testing adjustment after Mann-Whitney U tests were made with Holm’s sequential correction for multiple comparisons.
Cockroaches remained at 50°C for a significantly longer period of time after five doses of the test compound (Kruskal-Wallis test: χ2 = 40.8; df = 8; P<0.001) (
Latency to escape from noxious heat—50°C (s; mean ± SEM) after administration of water (Con), vehicle (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ) in American cockroaches. Insects were exposed to the test compound and placed at 50°C once a day for five days–each dose administration and exposure to heat was repeated every 24 hours—five trial heat box test. Letters indicate results statistically significant vs. control (A) or solvent (B) group in each tested day (Mann-Whitney U test with Holm adjustment).
n | Con 1x | V 1x | C 0.1 1x | C100 1x | M 1x | T 1x | CMF 1x | AITC 1x | CPZ 1x | |
Con 1x | 60 | - | 0.38 | 0.22 | 0.14 | 0.13 | 0.02 | 0.53 | 0.40 | |
V 1x | 40 | 0.38 | - | 0.75 | 0.58 | 0.46 | 0.09 | 0.11 | 0.07 | 0.08 |
C0.1 1x | 50 | 0.22 | 0.75 | - | 0.80 | 0.67 | 0.13 | 0.03 | 0.01 | 0.07 |
C100 1x | 50 | 0.14 | 0.58 | 0.80 | - | 0.75 | 0.18 | 0.04 | 0.01 | 0.21 |
M 1x | 19 | 0.13 | 0.46 | 0.67 | 0.75 | - | 0.50 | 0.04 | 0.02 | 0.63 |
T 1x | 18 | 0.02 | 0.09 | 0.13 | 0.18 | 0.50 | - | 0.01 | 0.01 | 0.55 |
CMF 1x | 60 | 0.53 | 0.11 | 0.01 | 0.04 | 0.04 | 0.01 | - | 0.69 | |
AITC 1x | 62 | 0.40 | 0.07 | 0.03 | 0.01 | 0.02 | 0.01 | 0.69 | - | |
CPZ 1x | 50 | 0.08 | 0.07 | 0.21 | 0.63 | 0.55 | - | |||
n | Con 3x | V 3x | C 0.1 3x | C100 3x | M 3x | T 3x | CMF 3x | AITC 3x | CPZ 3x | |
Con 3x | 55 | - | 0.68 | 0.36 | 0.07 | 0.62 | 0.18 | 0.19 | 0.83 | |
V 3x | 35 | 0.68 | - | 0.15 | 0.05 | 0.38 | 0.26 | 0.17 | 0.56 | |
C0.1 3x | 46 | 0.36 | 0.15 | - | 0.45 | 0.69 | 0.76 | 0.02 | 0.91 | 0.40 |
C100 3x | 46 | 0.07 | 0.05 | 0.45 | - | 0.39 | 0.72 | 0.19 | 0.31 | 0.11 |
M 3x | 16 | 0.62 | 0.38 | 0.69 | 0.39 | - | 0.75 | 0.03 | 0.74 | 0.80 |
T 3x | 16 | 0.18 | 0.26 | 0.76 | 0.72 | 0.75 | - | 0.11 | 0.94 | 0.39 |
CMF 3x | 50 | 0.02 | 0.19 | 0.03 | 0.11 | - | 0.09 | |||
AITC 3x | 52 | 0.19 | 0.17 | 0.91 | 0.31 | 0.74 | 0.94 | 0.09 | - | 0.26 |
CPZ 3x | 49 | 0.83 | 0.56 | 0.40 | 0.11 | 0.80 | 0.39 | 0.26 | - | |
n | Con 5x | V 5x | C 0.1 5x | C100 5x | M 5x | T 5x | CMF 5x | AITC 5x | CPZ 5x | |
Con 5x | 46 | - | 0.46 | 0.02 | 0.35 | 0.86 | 0.04 | |||
V 5x | 28 | 0.46 | - | 0.02 | 0.21 | 0.19 | 0.40 | 0.40 | ||
C0.1 5x | 33 | 0.02 | - | 0.28 | 0.01 | 0.04 | 0.49 | 0.10 | 0.56 | |
C100 5x | 40 | 0.02 | 0.21 | 0.28 | - | 0.03 | 0.12 | 0.05 | 0.53 | 0.06 |
M 5x | 13 | 0.35 | 0.19 | 0.01 | 0.03 | - | 0.50 | 0.08 | ||
T 5x | 14 | 0.86 | 0.40 | 0.04 | 0.12 | 0.50 | - | 0.19 | 0.007 | |
CMF 5x | 34 | 0.49 | 0.05 | - | 0.01 | 0.8 | ||||
AITC 5x | 45 | 0.04 | 0.40 | 0.10 | 0.53 | 0.08 | 0.19 | 0.01 | - | 0.01 |
CPZ 5x | 42 | 0.56 | 0.06 | 0.007 | 0.75 | 0.01 | - |
P-values for Mann-Whitney U test of data representing the latency to escape from 50°C in American cockroaches exposed to the first, third and fifth dose* of water (Con), vehicle (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ). Values in bold show significant results after Holm adjustment (total number of tests to see whether drug-treated groups are comparable with respect e.g. vehicle group was N = 8).
1Kruskal-Wallis test results: the first dose: χ2 = 26.9, df = 8, p = 0.001; the second dose: χ2 = 8.52, df = 8, p = 0.39; the third dose: χ2 = 19.2, df = 8, p = 0.01; the fourth dose: χ2 = 15.0, df = 8, p = 0.06; the fifth dose: χ2 = 40.8, df = 8, p<0.0001.
* Mann-Whitney U test was not carried out for the second and fourth dose, as Kruskal-Wallis test revealed no significant differences between groups.
First-second | First-third | First-fourth | First-fifth | Second-third | Second-fourth | Second-fifth | Third-fourth | Third-fifth | Fourth-fifth | |
---|---|---|---|---|---|---|---|---|---|---|
Con | 0.65 | 0.97 | 0.84 | 0.98 | 0.77 | 0.76 | 0.32 | 0.89 | 0.39 | 0.09 |
V | 0.29 | 0.30 | 0.06 | 0.35 | 0.37 | 0.71 | 0.75 | 0.53 | 0.33 | 0.44 |
C 0.1 | 0.29 | 0.02 | 0.008 | 0.37 | 0.08 | 0.01 | 0.35 | 0.27 | ||
C100 | 0.32 | 0.03 | 0.04 | 0.04 | 0.18 | 0.37 | 0.69 | 0.73 | ||
M | 0.04 | 0.07 | 0.3 | 0.86 | 0.96 | 0.68 | 0.15 | 0.16 | 0.29 | 0.20 |
T | 0.04 | 0.06 | 0.16 | 0.33 | 0.84 | 0.73 | 0.3 | 0.68 | 0.20 | 0.43 |
CMF | 0.12 | 0.04 | 0.52 | 0.20 | 0.24 | |||||
AITC | 0.34 | 0.15 | 0.16 | 0.59 | 0.07 | 0.01 | 0.14 | 0.70 | 0.71 | 0.92 |
CPZ | 0.14 | 0.01 | 0.04 | 0.11 | 0.70 | 0.03 |
P-values for statistical comparisons between the values obtained on different days (from first to fifth) within the same treatment, for each treatment with Wilcoxon signed-rank test. Values in bold show significant results after Holm adjustment (total number of tests to see whether groups treated on the one day treatment are comparable with groups treated on other tested days was N = 10). Cockroaches were treated with: water (Con), vehicle (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ).
Differences in escape time between cockroaches treated with the fifth dose of capsaicin, camphor, and capsazepine were not significant, however camphor-induced latency to escape was significantly longer than in insects exposed to menthol and thymol. Capsazepine induced significant changes to menthol, but not thymol (
Capsaicin, camphor, and capsazepine induced changes in cockroach responses to high ambient temperatures, significantly prolonging time spent at 50°C (Kruskal-Wallis test: χ2 = 37.79; df = 8; P<0.001) (
Substance tested |
Con | V | C 0.1 | C 100 | M | T | CMF | AITC | CPZ |
---|---|---|---|---|---|---|---|---|---|
Latency to escape from heat box (s; mean±SEM) | 3.54±0.49 |
4.36±0.54 |
17.86±4.28 |
14.51±2.99 |
5.23±1.17 |
7.17±1.89 |
18.34±3.98 |
7.62±1.83 |
36.21±8.64 |
Difference to control (water) group |
- | 0.82 | 14.32* | 10.97* | 1.69 | 3.63 | 14.79* | 4.08 | 32.67 |
Difference to alcohol (solvent) group |
0.82 | - | 0.87 | 2.81 | 3.25 | ||||
35 | 37 | 31 | 30 | 16 | 18 | 39 | 39 | 19 | |
Number of individuals that did not leave heat box | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 |
Number of dead individuals | 1 | 1 | 3 | 1 | 3 | 0 | 1 | 0 | 0 |
Escape from heat box after administration with five doses (each dose applied every 24 hours) of water (Con), alcohol (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ) in American cockroach. Insects were placed at 50°C only after the fifth dose of the test drug–single trial heat box test. Values are mean±SEM.
*indicates differences statistically significant to control and vehicle (bold) groups after Holm adjustment (total number of tests to see whether drug-treated groups are comparable with respect e.g. vehicle group was N = 8).
There is no significant difference in latency to escape between two respective groups–cockroaches exposed to single or five trial heat box test after the administration of the last fifth doses of the substances, except for vehicle-treated groups (Mann-Whitney U test: U = 318, Z = -2.65, P = 0.001).
Cockroaches exposed to five dosings of a test compound (each drug was applied once every 24 hours) were able to freely choose ambient temperature in a thermal gradient. Vehicle-treated cockroaches spent most of time at a mean temperature of 26.77±0.15°C (
Ambient temperature preferred (°C; mean±SEM) by American cockroaches exposed to vehicle, capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), capsazepine 0.0001mM (CPZ), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF) and allyl isothiocyanate 3mM (AITC). Cockroaches were exposed to the test compounds for five days–each dose was repeated every 24 hours and then the insect was placed in the thermal gradient for 24 hours (n = 12 for each substance). * indicates values statistically significant from vehicle group (Mann-Whitney U test with Holm adjustment, * p<0.05; **p<0.01; *** p<0.001).
As shown in
Con | V | C 0.1 | C 100 | M | T | CMF | AITC | CPZ | |
---|---|---|---|---|---|---|---|---|---|
MDA (μmol/mg protein) | |||||||||
4.04 | 2.62 | 9.19 | 7.86 | 4.26 | |||||
±0.89 | ±0.63 | ±2.58 | ±0.84 | ±3.52 | ±1.66 | ±3.03 | ±1.78 | ±0.43 | |
N = 17 | N = 16 | N = 17 | N = 19 | N = 17 | N = 17 | N = 19 | N = 20 | N = 12 | |
13.94 | 10.91 | 10.82 | 7.91 | 7.45 | 9.63 | 6.87 | |||
±3.16 | ±0.85 | ±1.43 | ±1.01 | ±1.52 | ±0.70 | ±1.46 | ±1.60 | ±1.60 | |
N = 21 | N = 12 | N = 12 | N = 12 | N = 8 | N = 10 | N = 11 | N = 19 | N = 12 | |
Catalase activity (U/mg protein) | |||||||||
20.00 | 45.69 | 48.73 | 52.44 | 52.74 | 57.30 | 59.96 | 22.61 | 52.71 | |
±4.50 | ±7.75 | ±3.89 | ±2.51 | ±5.15 | ±2.68 | ±3.43 | ±3.03 | ±2.12 | |
N = 18 | N = 17 | N = 17 | N = 19 | N = 17 | N = 17 | N = 18 | N = 20 | N = 12 | |
88.49 | 105.9 | 65.60 | 63.38 | 62.69 | 87.46 | 81.73 | |||
±2.02 | ±1.94 | ±1.17 | ±1.25 | ±1.20 | ±1.05 | ±1.46 | ±0.79 | ±1.55 | |
N = 15 | N = 12 | N = 12 | N = 12 | N = 12 | N = 10 | N = 11 | N = 19 | N = 12 |
Malondialdehyde concentration (MDA; μmol/mg protein) and catalase activity (U/mg protein) in cockroaches exposed to single-trial and five-trial heat box test treated with water (Con), alcohol (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ). Values are mean±SEM.
In the five-trial test cockroaches showed increased level of MDA comparing to single-trial test, but only in the control (U = 70, Z = -3.2, P = 0.001) and vehicle-alone (U = 6, Z = -4.2, P<0.001) groups, while thymol treatment resulted in MDA level decline after five doses (U = 13, Z = -3.6, P<0.001). Capsaicin, capsazepine, menthol and camphor did not affect MDA levels compared to the vehicle-treated group, however there was a significant decline in the lipid peroxidation marker level in thymol (U = 2, Z = -3.8, P<0.001) and AITC-treated groups (U = 29, Z = -3.4, P = 0.001).
Cockroaches in the single-trial heat box test showed a significant increase in catalase activity versus the water-treated group (Kruskal-Wallis test: χ2 = 61.98; df = 8; P<0.001). However, there were no differences between vehicle (ethyl alcohol) and treated groups (
Catalase activity in cockroaches exposed to five-trial test was significantly higher than that observed in insects exposed to single-trial heat box test in three drug groups (control: U = 39, Z = -3.1, P = 0.01; capsaicin 0.0001mM: U = 2, Z = -4.4, P<0.001; capsazepine: U = 6, Z = -3.78, P<0.001).
Ambient temperature perception plays an essential role in the life of ectotherms, because it affects their ability to survive, reproduce, and develop. Perception of ambient temperatures allows insects to regulate their body temperature behaviorally, and therefore enables them to defend against variable temperatures. Our experiments showed that some TRP agonists and antagonists: capsaicin, capsazepine, and camphor may induce changes in insect perception and response to high ambient temperatures.
Pharmacological blockade of heat sensitivity, observed as a weaker response to noxious heat, occurred in cockroaches exposed to five doses of capsazepine. Although capsazepine is one of the most extensively studied TRPV1 antagonists, it is characterized by moderate potency and limited selectivity [
Our results demonstrate that cockroaches exposed to repeated doses of camphor tend to stay much longer at high ambient temperature (50°C) than insects that were not exposed to the alkaloid. This suggests that cockroaches treated with camphor became, to some extent, insensitive to noxious ambient temperatures. Contrary, it can be considered that the sensitized state to the action of this compound occurred in the examined insects, as the absence of the effect on escape latency after first administration was observed, and the development of the substantial effect on escape latency after the fifth administration. Camphor is a product of the fragrant camphor tree and is known for its antimicrobial and anti-nociceptive effects [
Capsaicin also changed cockroaches’ response to high ambient temperature, however the significant effect was observed only in single-trial heat box test. Capsaicin is known to activate the mammalian heat receptor TRPV1, and in high doses it causes TRPV1 desensitization and loss of the ability to perceive high ambient temperatures [
To ascertain that the diminished heat sensitivity in capsaicin, capsazepine and camphor-treated cockroaches is not an effect of other possible causes, such as general toxicity, central nervous suppression or partial motor paralysis, we conducted additional behavioral test–response of exposed insects to noxious cold. Data are presented inS
AITC is known to activate two warmth receptors in insects–dTRPA1 and Painless, in vitro [
It is worth emphasizing that cockroaches, after exposure to five doses of capsazepine and camphor, showed a significant increase in time spent at 50°C, irrespectively of type of experimental schedule–single or five-trial heat box test. This implies that these drugs may facilitate development of a strong suppression of the heat sensitivity. The effect of capsaicin on escape latency was less pronounced. This may suggest development of the sensitized state to the action of these compounds or on the other hand, a desensitized state to noxious heat. Babcock et al. [
Cockroaches exposed to repeated dosing of the drugs showed a preference for higher ambient temperatures versus the vehicle-treated group and control (water-treated) group. The interesting observation was that although submicromolar capsaicin induced changes in cockroach thermoregulation against noxious heat in single-trial heat box test, it did not significantly affect thermal preferences in normal temperature range. Cockroaches treated with five doses of submicromolar capsaicin prefer similar temperatures to non-treated insects (
Therefore, the question arises, why do cockroaches exposed to multiple doses of 0.1 mM capsaicin, capsazepine, and camphor tend to stay in warmer regions of a thermal gradient, since they also demonstrate signs of suppression of the heat sensitivity to noxious heat? It may be assumed that capsazepine and camphor may act on structures other than TRPV. Capsazepine was shown to activate TRPA1 [
It seems that the effect of capsazepine action is also highly species-specific. It was demonstrated that human and guinea pig TRPV1 response to capsaicin, noxious heat, and protons was inhibited by capsazepine, while for rat TRPV1 capsazepine blocked the response only to capsaicin, but not to low pH (the blockade of response to heat was weaker) [
TRPV1 activation by capsaicin was demonstrated to increase subsequent oxidative stress, which may contribute to elevated pain sensation [
In order to determine whether the cockroach response to noxious heat could be affected by a secondary effect of the drugs, we examined the level of lipid peroxidation and catalase activity as markers of oxidative state. In the single-trial heat box test cockroaches treated with capsaicin showed an increase in MDA levels, suggesting that this drug exert some toxic effect. However, higher MDA levels were also observed in cockroaches exposed to menthol or thymol, neither of which affected cockroach response to heat. Moreover, in cockroaches exposed to heat for five trials, some drugs (thymol and AITC) significantly lowered the MDA level, irrespectively of action on thermal perception. Therefore, we can exclude that the increased oxidative stress induced significant changes in cockroach heat response. We could also mention that thymol and AITC were shown to exhibit some antioxidant activity [
Our results demonstrate that an increase in time spent at 50°C (single versus five heat box trials) induces oxidative stress, but only in control and vehicle- treated groups. The levels of MDA in capsaicin, capsazepine, menthol, camphor and AITC-treated cockroaches did not differ between one or multiple exposures to heat. Moreover, the tested drugs did not affect catalase activity compared to vehicle, irrespectively of time spent at noxious heat (a single trial or five trials). Only AITC induced a significant reduction in catalase activity in cockroaches exposed to 50°C during five trial test. AITC was shown to reduce oxidative stress and induce increase in glutathione-S-transferase activity in
The toxicity of the tested compounds was low. The observed mortality levels of cockroaches exposed to five doses of the test drugs was appreciable, but appears to have resulted not from the drugs but from vehicle and the experimental paradigm itself, as the highest 15% mortality occurred after vehicle-alone and camphor treatments only. Capsazepine, which induced the strongest sensitization effect, induced only 8% mortality. Other studies confirm low fumigant and contact toxicity of camphor in German cockroach [
Our results show, for the first time, that repeated exposure to capsazepine, camphor and capsaicin induces a state of pharmacological blockade of heat sensitivity. Cockroaches became sensitized to the action of these compounds what is observed as the development of the substantial effect on escape latency after the fifth administration. Moreover, behavioral thermoregulation of such ‘sensitized’ insect in normal temperature range is altered.
Latency to escape from noxious heat—50°C (s; raw data) after administration of water (Con), vehicle (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ) in American cockroaches. Single and five-trial heat box test.
(XLSX)
Temperatures preferred by cockroaches exposed to test drugs after repeated application (5 doses).
(XLSX)
Malondialdehyde concentration (MDA; μmol/mg protein) in cockroaches exposed to single-trial and five-trial heat box test treated with water, alcohol, capsaicin 0.0001mM and 0.1mM, menthol 2mM, thymol 1mM, camphor 15mM, allyl isothiocyanate 3mM and capsazepine 0.0001mM.
(XLSX)
Catalase activity (U/mg protein) in cockroaches exposed to single-trial and five-trial heat box test treated with water, alcohol, capsaicin 0.0001mM and 0.1mM, menthol 2mM, thymol 1mM, camphor 15mM, allyl isothiocyanate 3mM and capsazepine 0.0001mM.
(XLSX)
Latency to escape from noxious cold—5°C (s; raw data) after administration of water (Con), vehicle (V), capsaicin 0.0001mM (C 0.1) and 0.1mM (C100), menthol 2mM (M), thymol 1mM (T), camphor 15mM (CMF), allyl isothiocyanate 3mM (AITC) and capsazepine 0.0001mM (CPZ) in American cockroaches. Single-trial cold plate test.
(XLSX)
This study was funded by the grant from the National Science Centre, Poland (grant no. 2016/23/D/NZ4/01394). The authors would like to thank the Reviewers for valuable comments that greatly improved this manuscript.