The examination performed before the chemical treatment revealed the presence of ixodid ticks at all prospected localities. At localites of Kaćka in 2020, two tick species were identified: I. ricinus (nymph and adult stages) and Dermacentor marginatus (Sulzer, 1776) in adult stage. At localities of Fruška Gora National Park in 2021, three tick species were identified: nymph and adult stages of I. ricinus and adult stage of D. marginatus and Haemaphysalis punctata (Canestrini and Fanzago, 1878). Ixodes ricinus was the dominant species at all investigated localities. It is to be emphasized that before the treatment, all prospected localities included in the study were characterized by high density of tick population expressed as number of ticks sampled by flag/hour (Tables 1 and 2).
Table 1
Number of mobile stages of ticks collected by flag/hour method in pre and post-treatment days in localities of Kaćka Forest treated with Perme Plus® (permethrin + tetramethrin)
Test days | Locality Kaćka Forest 1 | Locality Kaćka Forest 2 | Locality Kaćka Forest 3 | Locality Kaćka Forest 4 | Control | Analysis of variance |
A | N | A + N | A | N | A + N | A | N | A + N | A | N | A + N | A + N | F(4,25) | p |
-2 | 28.5 | 7.5 | 36.00 ± 1.79 a - | 20 | 9 | 29.00 ± 1.41 a ! | 26 | 6 | 32.00 ± 2.83 a * | 20 | 5 | 25.00 ± 2.10 a # | 49.00 ± 1.90 a + | 119.86 | < 0.01 |
1 | 6 | 3 | 9.00 ± 0.89 d - | 5 | 3 | 8.00 ± 1.10 b -* | 4 | 2 | 6.00 ± 1.41 b ! | 7 | 0 | 7.00 ± 1.26 d *! | 48.00 ± 2.61 a + | 796.69 | < 0.01 |
7 | 1.5 | 3 | 4.50 ± 1.05 e - | 2 | 3 | 5.00 ± 0.89 c - | 2 | 2 | 4.00 ± 1.55 b - | 4 | 1 | 5.00 ± 1.41 d - | 42.00 ± 1.41 c + | 1010.42 | < 0.01 |
14 | 0 | 12 | 12.00 ± 2.10 c - | 0 | 3 | 3.00 ± 1.41 d ! | 0 | 1 | 1.00 ± 0.89 c # | 0 | 10 | 10.00 ± 1.67 c * | 45.00 ± 1.26 b + | 821.64 | < 0.01 |
21 | 0 | 18 | 18.00 ± 3.35 b - | 0 | 4 | 4.00 ± 1.26 cd ! | 0 | 6 | 6.00 ± 1.79 b ! | 0 | 14 | 14.00 ± 2.00 b * | 33.00 ± 2.00 d + | 167.50 | < 0.01 |
Analysis of variance | F(4,25) | 217.83 | | | 468.55 | | | 289.39 | | | 127.09 | 68.833 | | |
p | < 0.01 | | | < 0.01 | | | < 0.01 | | | < 0.01 | < 0.01 | | |
A = mean number of adults, N = mean number of nymphs, A + N = mean number of ticks ± Sd Mean values within the same column labeled with different letters are significantly different (Duncan Multiple Range Test for α = 0.05) Mean values within the same row labeled with different symbols are significantly different (Duncan Multiple Range Test for α = 0.05) Meteorological data during the treatment: Average air temperature 12°C; Average air humidity 65%; Wind speed 1–2 m/s |
Table 2
Number of mobile stages of ticks collected by flag/hour method in pre and post-treatment days in localities of Fruška Gora treated with Icon® 10CS (lambda cyhalothrin)
Test days | Locality Letenka | Locality Andrevlje | Locality Testera | Locality Stražilovo | Control | Analysis of variance |
A | N | A + N | A | N | A + N | A | N | A + N | A | N | A + N | A + N | F(4,25) | p |
-2 | 18 | 4 | 22.00 ± 1.41 a *! | 19 | 8 | 27.00 ± 1.41 a - | 18 | 3 | 21.00 ± 1.26 a ! | 15 | 8 | 23.00 ± 1.41 a * | 31.00 ± 0.89 a + | 61.43 | < 0.01 |
1 | 4 | 1 | 5.00 ± 1.41 b - | 6 | 0 | 6.00 ± 0.63 b - | 4 | 0 | 4.33 ± 1.03 b - | 5 | 0 | 5.00 ± 1.41 b - | 27.00 ± 2.37 b+ | 261.39 | < 0.01 |
7 | 3 | 0 | 3.00 ± 0.89 c -* | 1 | 1.5 | 2.50 ± 0.84 c -* | 2 | 0 | 2.00 ± 0.63 c * | 4 | 0 | 4.00 ± 1.41 bc - | 25.00 ± 2.00 b+ | 373.86 | < 0.01 |
14 | 0 | 3 | 3.00 ± 0.89 c - | 1 | 2 | 3.00 ± 0.63 c - | 0 | 1 | 1.00 ± 0.63 c * | 0 | 3 | 3.00 ± 0.89 c - | 22.50 ± 1.87 c+ | 410.59 | < 0.01 |
21 | 0 | 3 | 3.00 ± 0.63 c - | 0 | 2 | 2.00 ± 1.26 c - | 0 | 2 | 2.00 ± 0.63 c - | 1 | 2 | 3.00 ± 0.63 c - | 21.00 ± 2.28 c+ | 257.62 | < 0.01 |
Analysis of variance | F(4,25) | 346.00 | | | 670.88 | | | 552.33 | | | 311.67 | 24.27 | | |
p | < 0.01 | | | < 0.01 | | | < 0.01 | | | < 0.01 | < 0.01 | | |
A = mean number of adults, N = mean number of nymphs, A + N = mean number of ticks ± Sd Mean values within the same column labeled with different letters are significantly different (Duncan Multiple Range Test for α = 0.05) Mean values within the same row labeled with different symbols are significantly different (Duncan Multiple Range Test for α = 0.05) Meteorological data during the treatment (April 26th 2020): Average air temperature 15°C; Average air humidity 63%; Wind speed 0–1 m/s |
The presence of only one Borrelia species, B. afzelii was identified in I. ricinus ticks collected at the localities of Letenka, Testera and Andrevlje, whereas two Borrelia species, B. garinii and B. lusitaniae were identified in ticks collected from the locality Kaćka. In I. ricinus collected at the locality of Stražilovo, the presence of Borrelia DNA was detected, yet without successful identification at species level.
Differences between the mean numbers of ticks recorded in localities of Kaćka Forest (Table 1) at the pre-treatmant day (day − 2) were highly significant (p < 0.01). The highest mean number of ticks was recorded in the control site and the lowest and the lowest in the locality Kaćka 4 (49.00 ± 1.90 and 25.00 ± 2.10 ticks per flag hour, respectively). In all localities intendend for the treatment with Perme Plus®, both adults and nymphs were recorded and the adults represented the dominant mobile stage.
One day after the treatment with Perme Plus® the mean number of sampled ticks was significantly reduced at all treated localites (from 6.00 ± 1.41 to 9.00 ± 0.89) while in the untreated locality (control) it didn’t significantly change (48.00 ± 2.61). Comparison of the obtained results on the first post-treatment day revealed highly significant differences between the localities (F (4, 25) = 796.69; p < 0.01). Duncan's multiple range test demonstrated that the number of ticks in the control was significantly higher than in all treated localities. Significant differences were also deteced between locality Kaćka Forest 1 compared to both Kaćka Forest 3 and 4, as well as between Kaćka Forest 2 and 3. In relation to the stage composition, both adult and nymphs were present at all treated localities exept Kaćka Forest 4, where only adults were recorded.
Seven days after the treatment with Perme Plus®, mean numbers of ticks in the treated localities were not significantly different (4.00 ± 1.55 to 5.00 ± 1.41) while at the control locality the results were still significantly higher (42.00 ± 1.41). Furthermore, on the 7th post-treatment day significant reduction of mean number of ticks comparing to the previous evaluation term was recorded in the control and at the localities Kaćka Forest 1 and 2. At localities Kaćka Forest 3 and 4 the mean number of ticks also decreased, but not significantly. Regarding the stage composition of samples, adults and nymphs were recorded at all treted localities.
Two weeks after the treatment (day 14) the differences in the mean number of ticks among all localities, including the control were highly significant. Moreover, all sampled ticks on the 14th post-treatment day were in nymphal stage. Within the treated localities, the lowest number of ticks was recorded at Kaćka Forest 3 (1.00 ± 0.89), the highest at Kaćka Forest (12.00 ± 2.10). The significant increasing trend of population density was demonstrated in the control but also at Kaćka Forest 1 and 4. At the same time, the number of ticks in the other two treated localities remained in the trend of significant declining.
Regardless the increasing trend of tick number recorded at all treated localities on the 21st post-treatment day, the obtained mean values (4.00 ± 1.26 to 18.00 ± 3.35) were still significantly lower than in the untreated locality (33.00 ± 2.00). The increase of tick numbers was significant at all treated localities except Kaćka Forest 2. Qualitative and quantitative stage composition of collected ticks on the 21st day after the treatment with Perme Plus® again revealed that the population consisted of nymphal stage, exclusivelly.
Regarding the second group of localities of Fruska Gora intended for testing the efficacy of Icon® 10CS, the analysis of variance demonstrated that the differences in the mean number of ticks per flag hour at the pre-treatmant day (day − 2) were also highly significant (F = 61.43; p < 0.01 ), as in Kaćka Forest in the same evaluation term (Table 2). Mean number of ticks ranged between 21.00 ± 1.26 (locality Testera) and 31.00 ± 0.89 (control). On the other hand, Duncan's multiple range test demonstrated that the mean number of ticks at Letenka was not significantly different from the results for Testera and Stražilovo. Both adult and nymphal stages were recorded at all localities at the pre-treatment sampling term.
One day after the treatment with Icon® 10CS the mean number of sampled ticks at all treated localites was significantly reduced (4.33 ± 1.03 to 6.00 ± 0.63 at Testera and Andrevlje, respectivelly) and Duncan's multiple range test revealed that there were no statistical differences between the treated localities. Although the mean number of ticks at the control (untreated) locality (27.00 ± 2.37) was lower in comparison with the pre-treatment day, it was still significantly higher than in all localities treated with Icon® 10CS. Regarding the stage composition, adult stage was present at all of the treated localities, while nymphal stage was recorded only at the locality Letenka.
Seven days after the treatment with Icon® CS, the continual decreasing trend of mean number of ticks was remarked at all treated localities, with statistically significant differences, detected by Duncun's test in comparison with the results obtained on the 1st post-treatment day at all treated localities except at Stražilovo. The recorded mean number of ticks at the treated localities was between 2.00 ± 0.63 (Testera) and 4.00 ± 1.41 (Stražilovo), and in all cases it was significantly lower then in the control (25.00 ± 2.00). Among the treated localities, significant differences detected by Duncun's test were only observed between Stražilovo and Testera. Adult stage was present in samples from all of the treated localities, while the nymphal stage was present only in the samples from Andrevlje.
On the 14th evaluation day the mean number of ticks at all treated localities with Icon® CS10 was stil low (from 1.00 ± 0.63 to 3.00 ± 0.89) and significantly lower than in the control locality. Moreover, at the locality Testera the mean number of sampled ticks was the lowest and significantly different from the values recorded at other treated localities. Compared to the 7th post-treatment day the population density at all treated localities did not significantly change. The nympahal stage was present in all of the treated localities, while both adults and nymphs were recorded only at Andrevlje.
On the 21st post-treatment day, the mean number of ticks in all localites didn’t significantly change, compared to the previous evaluation term. Additionally, all of the treated localities had significantly lower number of ticks then the control. Also, there were no significant differences among the treated localities. The nympahal stage was present in all of the treated localities, while both adults and nymphs were recorded only at Stražilovo.
Treatment effects of applied biocide Perme Plus®, expressed by the reduction of the number of ticks during the post-treatment evaluation period revealed highly significant differences at each treated locality of Kaćka Forest (Table 3).
Table 3
Reduction of population density of ticks (%) evaluated in different post-treatment days in localities of Kaćka Forest treated with Perme Plus® (treatment conducted on April 26th, 2020)
Post treatment days | Localities | Analysis of variance |
Kaćka forest 1 | Kaćka forest 2 | Kaćka forest 3 | Kaćka forest 4 | F(3,20) | p |
1 | 74.43 ± 0.13 b − | 71.83 ± 0.21 b − | 80.87 ± 0.45 bc + | 71.53 ± 0.23 a − | 6.183 | < 0.01 |
7 | 85.53 ± 0.26 a + | 79.92 ± 0.28 b +− | 85.79 ± 0.67 b + | 76.64 ± 0.94 a − | 3.750 | 0.028 |
14 | 63.78 ± 0.46 c * | 89.15 ± 0.90 a − | 97.80 ± 1.43 a + | 56.17 ± 0.81 b * | 48.011 | < 0.01 |
21 | 25.85 ± 1.04 d − | 79.45 ± 1.12 b + | 72.19 ± 1.09 c + | 13.56 ± 4.85 c − | 37.005 | < 0.01 |
Analysis of variance | F (3,20) | 98.131 | 8.123 | 19.581 | 33.931 | | |
p | < 0.01 | < 0.01 | < 0.01 | < 0.01 | | |
Values are presented by means ± standard deviation Mean values within the same column labeled with different letters are significantly different (Duncan Multiple Range Test for α = 0.05) Mean values within the same row labeled with different symbols are significantly different (Duncan Multiple Range Test for α = 0.05) In the process of statistical analysis, the % values of reduction rates were transformed in arcsin √% and afterwards, for easier interpretation converted in %, as presented here |
Post-treatment results obtained 24 hours after the treatment indicated that the tick population reduction at all four treated localities of Kaćka Forest were within the interval of satisfying effects (70% − 90%). The analysis of variance for the efficacy of Perme Plus® at the 1st post-treatment revealed high statistical significance with respect to locality as independent variable (F(3,20) = 6.183; p < 0.01). The highest reduction of tick population was recorded at the locality Kaćka forest 3 (80.87%), and according Duncun’s test, here the reduction was significantly different from the values obtained at all other treated localities of Kaćka Forest. Reduction rates of localities Kaćka Forest 1, 2 and 3 were not significantly different.
On the 7th post-treatment day, the reduction of tick population was still satisfying at all localities (from 76.64–85.79%). The highest reduction of tick population at assessment on the 7th post-treatment day was recorded again at the locality Kaćka Forest 3. Anyhow, the reduction rates obtained at localities Kaćka Forest 1, 2 and 3 were not significantly different according Duncun’s test, while in the case of Kaćka Forest 4 the reduction was significantly lower than in Kaćka Forest 1 and 3. Comparing to the first post-treatment day, the reduction rate increased in all localities, but the increase was significant only at Kaćka Forest 1.
On the 14th post-treatment day higly significant differences in the reduction rates were detected among the treated localities (F (3, 20) = 48.011; p < 0.01). In localities Kaćka forest 2 and 3 the reduction rate significantly increased, while at Kaćka Forest 1 and 4 it the reduction shown significant decreasing tendency. The highest reduction rate, classified as good (> 90%) was recorded at Kaćka Forest 3 (97.80%), followed by Kaćka Forest 2 with satisfying reduction (89.15%), while at Kaćka Forest 1 and 4 the reduction was weak (< 70%).
On Day 21 post-treatment, highly statistically significant differences of the reduction rate (F(3,20) = 37.005; p < 0.01) were observed between localities of Kaćka Forest and in all of them the reduction significantly decreased. However, satisfying effects were still recorded at localities Kaćka Forest 2 and 3 (79.45% and 72.19%, respectively), while at localities Kaćka Forest 1 and 4 the efficacy substantially decreased (25.85% and 13.56%, respectively). In this evaluation term, Duncun`s test demonstrated that the reduction at localities Kaćka Forest 2 and 3 were not significantly different. Also the reduction rates obtained at Kaćka forest 1 and 4 were not significantly different.
Based on the presented results in Table 3, it can be observed that at two treated localities (Kaćka Forest 2 and 3) the treatment efficacy expressed as reduction rate had the increasing trand until the 14th post-treatment day and afterwards the efficacy significantly decreased, although the reduction at the 21st day was satisfying. In the other two localities, the satisfying treatment effects of Perme Plus® were observed until the 7th post-treatment day, and afterwards the reduction significantly decreased to values classified as weak.
Post-treatment results for the applied biocide Icon® 10CS based on lambda-cyhalothrin (Table 4) demonstrated that at first assessment (24 hours after the treatment), the efficacy ranged between 74.26% and 76.38%, and thus the effects at all localities were classified as satisfying, and not significantly different (F(3,20) = 0.239; p = 0.868).
Table 4
Reduction of population density of ticks (%) evaluated in different post-treatment days in localities of Fruška Gora treated with Icon® 10CS (treatment conducted on April 29th, 2021)
Post treatment days | Localities | Analysis of variance |
Letenka | Andrevlje | Testera | Stražilovo | F(3,20) | p |
1 | 74.26 ± 0.51 b + | 74.41 ± 0.12 b + | 76.38 ± 0.42 c + | 75.55 ± 0.31 a + | 0.239 | 0.868 |
7 | 83.20 ± 0.55 a + − | 88.62 ± 0.39 a + | 88.16 ± 0.65 b + | 78.17 ± 1.68 a − | 3.273 | 0.042 |
14 | 81.52 ± 0.42 a − | 84.67 ± 0.30 a − | 94.68 ± 1.67 a + | 82.36 ± 0.35 a − | 8.534 | < 0.01 |
21 | 79.75 ± 0.41 ab − | 91.32 ± 2.39 a + | 86.20 ± 0.37 b + − | 80.64 ± 0.38 a − | 4.060 | 0.021 |
Analysis of variance | F (3,20) | 2.874 | 7.518 | 9.801 | 1.171 | | |
p | 0.062 | < 0.01 | < 0.01 | 0.345 | | |
Values are presented by means ± standard deviation Mean values within the same column labeled with different letters are significantly different (Duncan Multiple Range Test for α = 0.05) Mean values within the same row labeled with different symbols are significantly different (Duncan Multiple Range Test for α = 0.05) In the process of statistical analysis, the % values of reduction rates were transformed in arcsin √% and afterwards, for easier interpretation converted in %, as presented here |
On the 7th post-treatment day, the increasing trend of reduction of tick population was recorded at all localities. Despite significant differences between the localities (F(3,20) = 3.273; p = 0.042), the reduction rate at all localities was classified as satisfying (from 78.17–88.62%). Compared to the 1st post-treatment evaluation day, the treatment efficacy significantly increased at all localities, except Stražilovo. Moreover, according to Duncun`s test, the reduction rate at Stražilovo was significantly lower compared to Testera and Andrevlje.
Fourteen days after the treatment, the highest efficacy, classified as good was recorded at the locality Testera (94.68%), while in other localities the treatment effects were lower (from 81.52–84.67%) but still satisfying. Only at the locality Testera the efficacy significantly increased compared to the previous evaluation term. The increased efficacy was also recorded at Stražilovo but according to the Duncun`s test results the increase was not significant. In other two localities, the reduction of tick population decreased but not significantly when compared to the results obtained on the 7th post-treatment evaluation day.
On the final, 21st post-treatment evaluation day, the highest reduction rate, classified as good was recorded at Andrevlje (91.32%), while the reduction rate at other treated localities was lower but still satisfying. Compared to the results obtained at the 14th day, the increase of the population reduction was only recorded at Andrevlje, but anyhow Duncun`s test revealed that the increase was not significant. In other localities, the efficacy decreased but only at the locality Testera the reduction rate was significantly lower than at the previous post-treatment evaluation term. The lowest efficacy was recorded at Letenka, where the reduction rate of 79.75% was significantly lower compared to Andrevlje, but not to other localities. In contrast to the efficacy results for Perme Plus®, Icon® 10CS provided satisfying efficacy rate during the entire evaluation period of 21 days.
Comparison of the efficacy trends of Perme Plus® and Icon® 10CS (Fig. 1) demonstrated similar initial effects until the 7th post-treatment day, when the reduction rate of treated tick populations for both applied products achieved the value which, transformed to percentages was about 84%. In the following post-treatment period, the reduction of population density treated with Icon® 10CS continued to increase until the day 14.5 when the maximal reduction of 87.12% was achieved. Later on, the population reduction rate gradually decreased. According the resulting trend formula for Icon® 10CS, the expected efficacy one month after the treatment (on the 30th day) was still satisfying (70.71%). The trend line for Perme Plus® demonstrated that the maximal reduction rate of the population density was achieved on the day 8.5 and it corresponded to the value of 84.36%. In the following period, the decreasing of the treatment efficacy demonstrated by the trend line for Perme Plus® was intensive and more expressed than in the case of Icon® 10CS. According the resulting trend formula for Perme Plus®, the population reduction rate on the 17th post-treatment day declined to the corresponding value of 68.59%, thus the treatment effects starting from this post-treatment day were weak. Finally, on the 30th day any post-treatment effects of Perme Plus® could be expected (reduction equal to 0%).