Chemical composition of the oils
Chemical components analysis of the essential oils revealed that the predominant chemical compound found on N. crispa oil was 1,8-Cineole (57.68%). Predominant essential oil compounds found on S. hortensis were carvacrol (34.75%), gamma-terpinene (34.28%) and para-cymene (16.96%). L-phellandrene (34.18%), Carvone (23.68%) and limonene (21.46%) were the main compounds identified in A. graveolens oil (Table 1).
Table 1 Chemical composition of the essential oils of N. crispa, S. hortensis and A. graveolens
Plant
|
Component
|
aRRI
|
Composition %
|
Plant
|
Component
|
a RRI
|
Composition %
|
N. crispa
|
|
|
|
|
Para-cymene
|
1028
|
16.98
|
|
Alpha-pinene
|
939
|
2.50
|
|
Limonene
|
1034
|
0.74
|
|
Sabinene
|
974
|
2.08
|
|
1,8-cineole
|
1038
|
0.28
|
|
Beta-pinene
|
982
|
6.54
|
|
Gamma-terpinene
|
1062
|
34.28
|
|
Beta-myrcene
|
993
|
0.42
|
|
Menthol
|
1176
|
0.50
|
|
Para-cymene
|
1028
|
1.43
|
|
Thymol
|
1298
|
0.27
|
|
Limonene
|
1034
|
1.37
|
|
Carvacrol
|
1308
|
34.75
|
|
1,8-cineole
|
1038
|
57.68
|
|
Trans-caryophyllene
|
1427
|
0.29
|
|
Gamma-terpinene
|
1062
|
1.19
|
|
Caryophyllene oxide
|
1592
|
0.23
|
|
Linalool
|
1098
|
1.26
|
A. graveolens
|
|
|
|
|
Terpinene-4-ol
|
1183
|
1.74
|
|
Beta-myrcene
|
993
|
0.74
|
|
Alpha terpineol
|
1195
|
4.45
|
|
L-phellandrene
|
1006
|
34.18
|
S. hortensis
|
|
|
|
|
Para-cymene
|
1028
|
5.53
|
|
Alpha-pinene
|
939
|
1.96
|
|
Limonene
|
1034
|
21.46
|
|
Beta-pinene
|
982
|
0.87
|
|
Gamma-terpinene
|
1062
|
0.77
|
|
Beta-myrcene
|
993
|
1.88
|
|
Alpha-terpineol
|
1190
|
5.57
|
|
Alpha-phellandrene
|
1007
|
0.33
|
|
Carvone
|
1251
|
23.68
|
|
Alpha-terpinene
|
1019
|
2.77
|
|
Carvacrol
|
1308
|
0.79
|
a RRI: Relative retention index
Oil effects on T. urticae
Mortality rates of T. urticae adults were significantly different among oil concentrations of N. crispa (Wald χ2 = 115.9; df = 4; P <0.001), S. hortensis (Wald χ2 = 102.5; df = 4; P <0.001) and A. graveolens (Wald χ2 = 105.2; df = 4; P <0.001). The highest concentrations resulted in an average mortality of about 90% after 24 h exposure (Fig. 1). No mortality was recorded in control group.
Probit analysis data of acute toxicity of T. urticae adults in response to the three oils revealed the median lethal concentration (LC50) values, which were the highest for S. hortensis and the lowest for N. crispa (Table 2).
Table 2 Median lethal concentration (LC50) estimated using probit analysis for adult female T. urticae exposed to N. crispa, S. hortensis and A. graveolens for 24 h
Pesticide
|
LC (50) (µl liter-1)
|
95% Confidence limits
|
Slope ± SE
|
χ2
|
df
|
N. crispa
|
32.04
|
29.01‒35.47
|
2.86±0.24
|
0.93
|
3
|
S. hortensis
|
136.00
|
126.07‒146.24
|
3.85±0.35
|
1.24
|
3
|
A. graveolens
|
73.07
|
64.66‒82.48
|
2.32±0.20
|
3.05
|
3
|
Side-effects on biochemical parameters of A. swirskii
Calculation of total available energy as the sum of the energy contents, shows that treatments with the oils of N. crispa, S. hortensis and A. graveolens significantly reduced the total carbohydrates content (F3,8 = 6.16; P < 0.018), lipid content (F3,8 = 6.30; P < 0.017) and protein content (F3,8 = 9.75; P < 0.005) in the females of A. swirskii compared to the control treatment. However treatments of S. hortensis and A. graveolens did not reduce lipid content in comparison to the control treatment (Fig. 2).
Exposure of A. swirskii adults to the three essential oils had a significant effect on the detoxification enzyme activity of GST enzyme (F3,8 = 5.15; P < 0.028) and the α-esterase enzyme (F3,8 = 47.56; P < 0.0001), but not on the β-esterase enzyme (F3,8 = 1.16; P < 0.38) (Fig. 3). All oil treatments resulted in a higher α-esterase enzyme activity compared to the control, with the highest levels for S. hortensia, whereas the GST enzyme activity was only higher for the A. graveolis oil treatment compared to the control treatment (Fig. 3).
Side-effects on demographic parameters of A. swirskii
The application of N. crispa, S. hortensis and A. graveolens oils considerably affected developmental time, longevity and total life span of the progeny of A. swirskii females (Table 3). Duration of egg and larva stages of female and male from females exposed to the three treatments were not significantly different from the untreated control, but with an exception of N. crispa effects on the larva stage of females. There was no significant difference between treatments and control for protonymph and deutonymph duration of female and male (Table 3). However, developmental time of females was significantly prolonged in all treatments compared to the control treatment, but this was not the case for males. Female longevity compared to control was most significantly reduced by oil application of N. crispa and A. graveolens followed by S. hortensis. A similar effect was observed for males. Each of three tested treatments significantly reduced the total life span of both genders compared to the control treatment, with most impact from N. crispa and A. graveolens (Table 3).
Table 3 Mean (±SE) developmental time, longevity and total life span (days) of offspring from females of A. swirskii from control or treatments of N. crispa, S. hortensis and A. graveolens
Treatments
|
Sex/stage
|
N. crispa
|
S. hortensis
|
A. graveolens
|
Control
|
F value*
|
Female
|
|
|
|
|
Egg duration
|
1.76 ± 0.12a
|
1.73 ± 0.10a
|
1.70 ± 0.11a
|
1.48 ± 0.11a
|
33.36
|
Larva duration
|
1.33 ± 0.11a
|
1.27 ± 0.10ab
|
1.25 ± 0.10ab
|
1.09 ± 0.06b
|
30.54
|
Protonymph
|
1.81 ± 0.11a
|
1.82 ± 0.13a
|
1.71 ± 0.13a
|
1.74 ± 0.16a
|
3.99
|
Deutonymph
|
2.00 ± 0.17a
|
1.91 ± 0.17a
|
1.85 ± 0.17a
|
1.87 ± 0.17a
|
3.32
|
Developmental time
|
6.90 ± 0.23a
|
6.73 ± 0.22b
|
6.51 ± 0.179a
|
6.17 ± 0.29c
|
3122.13
|
Longevity
|
18.10 ± 0.22c
|
19.32 ± 0.19b
|
18.20 ± 0.17c
|
23.09 ± 0.22a
|
3122.13
|
Total life span
|
25.00 ± 0.35c
|
26.05 ± 0.28b
|
24.71 ± 0.26c
|
29.26 ± 0.32a
|
1026.86
|
Male
|
Egg duration
|
1.82 ± 0.15a
|
1.67 ± 0.14a
|
1.61 ± 0.14a
|
1.53 ± 0.12a
|
13.55
|
Larva duration
|
1.41 ± 0.16a
|
1.39 ± 0.14a
|
1.28 ± 0.11a
|
1.24 ± 0.11a
|
7.82
|
Protonymph
|
1.88 ± 0.15a
|
1.83 ± 0.12a
|
1.72 ± 0.14a
|
1.76 ± 0.11a
|
5.53
|
Deutonymph
|
1.65 ± 0.12a
|
1.61 ± 0.12a
|
1.56 ± 0.12a
|
1.53 ± 0.15a
|
3.04
|
Developmental time
|
6.76 ± 0.34a
|
6.50 ± 0.34a
|
6.17± 0.34a
|
6.06 ± 0.20a
|
19.21
|
Longevity
|
17.04 ± 0.23c
|
18.33 ± 0.24b
|
17.50 ± 0.32c
|
22.35 ± 0.44a
|
1048.89
|
Total life span
|
23.71 ± 0.43c
|
24.83 ± 0.26b
|
23.67 ± 0.33c
|
28.41 ± 0.49a
|
598.96
|
Means followed by different letters in the same row are significantly different by using paired bootstrap test based on CI of difference (P < 0.05)
*F(df,n): female (3,82), male (3,66)
Reproduction and population growth parameters of A. swirskii
N. crispa and A. graveolens treatments resulted in a significantly prolonged pre-oviposition time compared to the control treatment (Table 4). Oviposition time was significantly reduced compared to the control in treatments with an exception of S. hortensis. Total fecundity of A. swirskii was negatively affected by all oil treatments. A similar effect was observed for the post-oviposition time (Table 4).
Table 4 Mean (±SE) reproductive period and total fecundity of offspring from females of A. swirskii from control or treatments of N. crispa, S. hortensis and A. graveolens
Treatments
|
|
N. crispa
|
S. hortensis
|
A. graveolens
|
Control
|
F value*
|
Pre-oviposition (day)
|
3.48 ± 0.11ab
|
3.27 ± 0.13bc
|
3.65 ± 0.11a
|
3.17 ± 0.08c
|
80.87
|
Oviposition (day)
|
11.38 ± 0.26b
|
12.41 ± 0.21a
|
10.90 ± 0.26b
|
12.91 ± 0.18a
|
357.17
|
Post-oviposition (day)
|
4.92 ± 0.10b
|
5.15 ± 0.13b
|
5.35 ± 0.13b
|
6.20 ± 0.11a
|
278.00
|
Total fecundity (offspring)
|
11.57 ± 0.24c
|
12.59 ± 0.22b
|
11.40 ± 0.27c
|
13.57 ± 0.26a
|
359.66
|
Means followed by different letters in the same row are significantly different by using paired bootstrap test based on CI of difference (P < 0.05)
*F(df,n): (3,82)
Age-specific survival rate (lx) and age-specific fecundity of the total population (mx) exposed to essential oils were compared with populations in the control treatment (Fig. 4). Regardless the developmental stage, lx represents the probability that an egg will survive to age x, and the curve of the age-specific survival rate is a simplified form of the curves of age-stage survival rate. Total life span averaged 29.26 days for the untreated females and 25.00 days, 26.05 days and 24.71 days for the females treated with highest concentrations of N. crispa, S. hortensis and A. graveolens, respectively. Comparison of the survival (lx) of untreated mites and those treated with essential oils of N. crispa and A. graveolens revealed 4.9 and 5.30 % mortality in the immature stages, with 95.1 and 94.7 % chance of reaching adulthood, respectively. However the mites treated with S. hortensis showed no mortality in immature stages, with 100 % chances of reaching adulthood.
Maximum mx of 0.78 eggs/female/day was observed on day 11 for untreated mites. For mites treated with N. crispa, S. hortensis and A. graveolens, mx was approximately 0.61, 0.60 and 0.63 eggs/female/day, respectively, which occurred on days 15, 13 and 16 of life span, respectively. Based on the age-stage specific survival rate of both untreated and treated individuals of A. swirskii, the probability that an egg will survive to age x and develop to stage j was illustrated. Compared to the control, N. crispa and A. graveolens increased the pre-oviposition period. Male adults emerged simultaneously with females.
The age-stage survival rate (sxj) represents the probability that an egg of A. swirskii will survive to age x and stage j. There is an overlap in the curves at different developmental periods among the individuals in all treatments. The highest female survival rate was observed in control compared with other treatments and 57.50% of eggs normally developed to the adult stage (Fig. 5).
There was no significant effect of the highest concentration of essential oils on the population parameters including intrinsic rate of increase (r), the finite rate of increase (λ), the net reproductive rate (R0), the gross reproductive rates (GRR) and the mean generation time (T) (Table 5).
Table 5 Mean (±SE) population parameters of the females of Amblyseius swirskii from control or treatments of N. crispa, S. hortensis and A. graveolens
|
Treatments
|
|
N. crispa
|
S. hortensis
|
A. graveolens
|
Control
|
F value*
|
Intrinsic rate of increase, r (day-1)
|
0.11 ± 0.01a
|
0.12 ± 0.01a
|
0.11 ± 0.01a
|
0.13 ± 0.01a
|
42.11
|
Finite rate of increase, λ (day-1)
|
1.12 ± 0.01a
|
1.13 ± 0.01a
|
1.12 ± 0.01a
|
1.14 ± 0.01a
|
42.56
|
Net reproductive rate, R0 (offspring)
|
6.07 ± 0.92a
|
6.92 ± 0.99a
|
5.70 ± 0.90a
|
7.80 ± 1.09a
|
36.55
|
Gross reproductive rate, GRR (offspring)
|
6.47 ± 094a
|
6.95 ± 0.99a
|
6.04 ± 0.93a
|
7.80 ± 0.09a
|
23.10
|
Mean generation time, T (day)
|
15.93 ± 0.23a
|
15.85 ± 0.23a
|
15.58 ± 0.25a
|
15.23 ± 0.29a
|
60.70
|
Means followed by different letters in the same row are significantly different by using paired bootstrap test based on CI of difference (P < 0.05)
*F(df,n): (3,156)
The age-stage-specific life expectancy (exj: the period that an individual of age x and stage j is expected to survive) of A. swirskii individuals was affected by treatments. According to the exj curve of newborns (e01), predatory mite was expected to live 20.00, 21.04, 19.66 and 25.26 days in N. crispa, S. hortensis and A. graveolens and control, respectively (Fig. 6).