Sublethal Effects of Atropa belladonna Herbal Extract on Biological Performance of the Two-Spotted Spider Mite Tetranychus urticae Koch (Acari: Tetranychidae)

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is one of the most important pests of agricultural products. Using chemical pesticides is one of the main methods for its management. Due to undesirable effects of synthetic pesticides, this research was conducted to achieve alternative safe compounds. In this study sublethal effect of Atropa belladonna herbal extract including LC10, LC20, LC30 and distilled water were evaluated on biological parameters (such as r, λ, GRR, R0 and T) of T. urticae. The experiments were done under laboratory condition at 25±2 °C, 60±5% RH, and a photoperiod of 16:8 (L: D) hours. The crude data were analyzed based on age-stage, two-sex life table analysis. The results indicated significant reduction in female’s duration of maturation, oviposition period and total fecundity by increasing examined dose. The highest and lowest values of the net reproductive rate (R0) were obtained 38.41 and 18.08 offspring/individual in control and LC30, respectively. The maximum values of intrinsic rate of increase (r) was 0.2201 day that occurred on control treatment while the minimum values was obtained 0.1937 day in LC30 concentration. Finite rate of increase (λ) had significantly descended with concentration enhancing from LC20 to LC30, compared with the control. The results demonstrated that Atropa belladonna herbal extract could be incorporated in integrated pest management (IPM) programs of T. urticae.


Introduction
The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is an serious pest of many greenhouse plants and field crops (Cote et al., 2002;Sedaratian et al., 2009;Khanamani et al., 2013;Maleknia et al., 2016).The phytophagous mite T. urticae damage may include webbing, fine stippling, leaf yellowing, leaf crop, and even plant death (Helle & Sabelis, 1985).This pest with a short life span, high reproductive ability, multiple births and plant nutrition can stop plant growth and reduce the quality and quantity of the crop (Saeedi & Arbabi, 2007).In severe infection a large amount of thin webbing is produced that may completely cover infested areas of leaves (Zhang, 2003).Control of this mite species is obviously difficult and mostly relies on the use of pesticides (Zhao, 2000).
The wide use of pesticides leads to affect non-target organisms (Croft, 1990), human safety (Garcia-Marı & Gonzalez-Zamora, 1999), the emergence of secondary pests (Elzen, 2001) and development of resistance (Brattsten et al., 1986).Also because of the adverse effect of pesticide use, alternative control methods are being researched for T. urticae (Yorulmaz et al., 2014).Plant compounds such as extracts were used as insecticides (Ofuya & Okuku, 1994;Kim et al., 2003), acaricidals (Rim & Jee, 2006;Fernandes & Freitas, 2007).The Atropa belladonna L. (Solanaceae) is a perennial herb that is cultivated throughout the world.This plant belongs to the Caspian region and grows on the edge of forests and sub-trees.The geographical distribution of this species is in Europe, Turkey and northern Iran (Rita & Animesh, 2011).The scientific name A. belladonna is one of the most important medicinal plants of the Solanaceae family (Berdai et al., 2012).It has natural habitats in the northern regions of Iran.The berries are sweet and are consumed by animals that disperse the seeds in their droppings, even though they contain toxic alkaloids (Kay, 1992).A number of studies have been conducted for evaluating the lethal and sublethal effects of various herbal extracts, on two-spotted spider mite (Choi et al., 2004;Wang et al., 2007;Kumral et al., 2010;Pavela, 2015), but no reports have been made about the effects of this plant extract on two-spotted spider mite.The effect of pesticides on insects usually is estimated in experimental conditions based on LC 50 .Sublethal effect studies have been used to assess the selectivity of pesticides to beneficial arthropods (Teodoro et al., 2005;Poletti et al., 2007).
However, the common method for estimating the effect of pesticides on insects only addresses the effects of lethality and mortality data, so demographic toxicological studies or toxicological evaluation tests on insect life table that provide more accurate information can be recommended.In these studies, the effects of pesticides on a (homogeneous) same age group of survivors from the impact of the pesticide are investigated and the survival, fecundity and mortality of this group are studied until the end of lifespan.It also provides a measure of population growth rates.The use of the inherent rate of population increase as a biometric parameter has been recommended in toxicological studies (Boykin & Campbell, 1982).Therefore, in predicting the effect of pesticide on the insect, life-history tests are more reliable than their lethal dose estimation.Only essential oils derived from some plants can be good alternative methods to controlling this pest.Essences, secondary metabolites of plants with aromatic compounds, have low molecular weight compounds and volatile compounds with proven pesticide properties (Bakkali et al., 2008).A number of essential oils act selectively and do not affect non-target organisms.So far the properties of several herbal essential oils are studied for controlling two-spotted spider mite (Cavalccanti et al., 2010;Han et al., 2010).Also, many studies have been done on the sublethal effects of herbal essential oils, such as repulsion effects, nutritional inhibition, breeding prevention and breeding (Choi et al., 2004).Many of these plant compounds are able to affect the growth and reproduction parameters of pests and their various growth stages (Tsolakis & Ragusa, 2008).According to the researches done so far the effect of A. belladonna herbal extract has not been reported on two-spotted spider mite.
Consequently, taking the activity of this herbal extract, in this experiment, the effects of sublethal concentrations of Atropa belladonna herbal extract on T. urticae were investigated using demographic toxicological analysis.Being a part of systematic evaluation of the acaricidal potential of the plants, the objective of this study was to evaluate and validate the effects of sublethal concentrations of A. belladonna herbal extract on demographic parameters of T. urticae, to effective and low-risk control of two-spotted spider mite that is eco-friendly.

Host Plant Rearing
Beans plants (phaseolus vulgaris L. var.Khomein) were planted as host plant for T. urticae rearing.The plants were reared in pots with 15 cm diameter under laboratory conditions at 25±2 °C, 60±5% R.H. and a photoperiod of 16:8 (L:D) hours.

Mite Colonies
Primary population of T. urticae was collected from infested greenhouses of rose with scientific name Rosa hybrida from greenhouse around Pakdasht (South eastern of Tehran, Iran) and then grown on beans planted in the greenhouse.The colony of T. uricae was placed in the growth chamber with a temperature of 25±2 °C, a relative humidity of 60±5% and a photoperiod period of 16:8 h (light:dark).

Preparation of Herbal Extract
The Atropa belladonna plants were collected from the forests of Golestan province.The leaves were first crushed and then 40 grams of leaves were extracted with ethanol (300 milliliters 70 percent) for 3 hours at each turn using Soxhlet device.The solvent was isolated using a rotary machine at 40 degrees Celsius at 120 rpm.

Concentration-Response Bioassay
To determine the effective dose of A. belladonna, were used five concentrations .The experiments were assessed according to leaf dip bioassay method (Helle & Overmeer, 1985).The mortality covering the range of 10-90%.The leaf discs were dipped for 15 seconds into solution of herbal extract.The control leaf discs were dipped in distilled water only.Then, leaf discs dried in room condition for about 3 hours.Twenty same age (24-h-old) adults mite (from both sexes) were transferred to leaf discs with a diameter of 3 cm and placed in Petri dishes with diameter of 6 cm.Mortality of adult was evaluated after 24 h.Mites were considered as dead if after touching with a small fine brush, they could not crawl and were non-functional.All experimentas were conducted in the laboratory condition at 25±2 °C, 60±5% RH and a photoperiod period of 16:8 h (L:D).In this study four replicates were used for each concentration.There were five concentrations and four replicates per concentration.

Effect of Sublethal Concentrations on Biological Parameters of Offspring From Treated Females
In order to evaluate the sublethal effects of A. belladonna herbal extract on T. uricae, bean leaf discs were treated with sublethal concentrations including LC 10 , LC 20 , LC 30 and allowed to dry for 2 h.One hundred same-aged (24-h-old) gravid females were used for each treatment.After 24 hours, the surviving females were separately transferred to leaf disc with 3 cm diameter on wet filter paper in Petri dishes.After 24 hours, one egg as the basis was placed in each Petri dish and other eggs laid by female and mite were removed from Petri dish.
The newly-emerged females were coupled with males for mating after the adult emergence.Finally, the fecundity of females was daily recorded and population parameters were calculated in both male and females until the death of the last sample.In order to avoid creating tension in mites (due to aging) leaves of beans were replaced by fresh leaves every 48 h.The Petri dishes were checked every 24 hours and changes such as developmental time and survival of them were recorded until the death of the last female.

Data Analysis
The value of LC 50 and sublethal concentrations, was calculated using the software SPSS ver.19.0.To compare the significance of the LC 50 95% of the confidence interval were used.The raw data obtained from the two-spotted spider mite life cycle for analyzing and determining the parameters of the life table according to the Chi (1988), and Chi and Liu (1988) by using the computer program, TWO-SEX_MsChart (Chi, 2016).The means of the latter parameters were compared by using the paired bootstrap test at the 5% significance level (Riahi et al., 2017;Khanamani et al., 2017).Differences between means were compared with the Turkey's test was carried out using SAS (SAS Institute 2002).The age-specific fecundity (m x ), age-specific survival rate (l x ), age-stage specific survival rate (S xj ) (where, x = age in day and j = stage) and the population growth parameters were calculated accordingly: where, k is the number of stages.
The intrinsic rate of increase is estimated by using the iterative bisection method from the following equation: ∑ e -r(x+1) with age indexed from 0 to ω (Goodman, 1982).

Concentration-Response Bioassay
The sublethal concentrations of Atropa belladonna herbal extract (LC 10 , LC 20 and LC 30 ) for T. urticae were estimated to be (1650, 2300 and 3000 ppm), respectively.No mortality was observed in control.The value of p is greater than 0.05, indicating the biometric alignment of the lines, and the Chi-squared value also confirms it. jas.ccsenet.

Develo
The impac in Note.Means within a row followed by the same letter are not significantly different (Turkey-kramer p < 0.05).

Reproduction
According to Table 2 the duration of oviposition period in treated females with different concentrations significantly reduced in compared to control.However, no significant difference was observed for pre-oviposition period between untreated and treated cohort.In this study, an increase in the dosage of leaf extract was followed by a significant decrease in oviposition period.The shortest period of oviposition (7.06 [F = 542.3,df = 3, P < 0.0001] days) was recorded for mites that treated with LC 30 concentration and the longest oviposition time (10.74 [F = 542.3,df = 3, P < 0.0001] days) was recorded for distilled water.Our findings showed that fecundity of female mites significantly decreased under the influence of sublethal concentrations of A. belladonna herbal extract.
The lowest value of eggs laid per female was found in the LC 30 treatment.Therefore, the treated mites with this concentration will produce the least population in their second generation.However according to Binaghi et al (2014) the total fecundity of two-spotted spider mite in control group were 64.44 per female and 32.46 and 31.08 eggs for cumin and clove essential oil treatments respectively.Also the results of present study is consistent that the findings reported by Castagnoli et al. (2005), Numa et al. (2011) and Sivira et al. (2011), who observed that increasing the concentration of the extract applied to T. urticae adults, cause to reducing the fercundity of the phytophagous females.In another study, Refaat et al. (2002) investigated two essential oils, Ocimum basilicum L.
(Lamiaceae) and Lavandula of Ficinalis Chaix (Lam-iaceae) on T. urticae.Their results showed that both essential oils have significant reduction in the total number of eggs laid by this mite.Note.TPOP: total pre-oviposition period.Means within a row followed by the letter are not significantly different.The ±SE were estimated by using 100,000 bootstraps and compared by using paired bootstraps test at 5% significance level.

Population Parameters
The demographic parameters of T. urticae affected by sublethal treatments of A. belladonna herbal extract are depicted in Table 3. GRR decreased significantly with increasing sublethal concentrations.Net reproductive rate (R 0 ) in the LC 30 dose was significantly inferior to other treatments.Given that the amount of R 0 depends on the number of eggs produced and the survival rate, the data indicated a low survival and oviposition rate in the treated population.The highest and lowest values of R 0 were 18.08 and 38.41 offspring/individual, respectively, for control and LC 30 treatment.Assessed intrinsic rate of increase for mites influenced by sublethal doses was ranged of 0.2201 to 0.1937 day -1 .Also, in mites treated with different doses of Atropa belladonna herbal extract, the values of r and λ were significantly less than control.
A. belladonna herbal extract caused a reduction in longevity, survival and fecundity of T. urticae and thereby causes a reduction in r m .The higher r value, population increase is faster and growth period is shorter.The results that obtained from this study is consistent with reported by Gholamzadeh Chitgar et al. ( 2013) that noted population parameters of T. urticae that treated by Thymus vulgaris essential oil had a significant difference at r, R 0 , T and λ parameters.Therefore, it can be concluded that the doses used from the extract reduce the growth potential of TSSM in comparison with the control.The results are similar to those reported by Binaghi et al. ( 2014) that cumin and clove oil reduces the growth potential of two spotted spider mite population.The average of a mean generation time needs 16.55 and 14.93 days in control and LC 30 concentration.
The lowest average generation length was observed for the LC 30 dose of the treated mites (P < 0.0001, df = 3).Kumral et al. (2013) reported that leaf extract of D. stramonium had toxic and repellent effect both on P. ulmi and its predator Stethorus gilvifrons (Muls.)(Coleoptera: Coccinellidae).Note.Means within a row followed by the same letter are not significantly different.The ±SE were estimated by using 100,000 bootstraps and compared by using paired bootstraps test at 5% significance level.

Survival and Fecundity
Figure 2, compares the l x , of T. urticae at different concentration of Atropa belladonna herbal extract.The total life time for untreated mites was scored 26 days, while it was 25, 24 and 22 days for the mites treated at LC 10 , LC 20 and LC 30 of experimental concentrations.
The age-specific fecundity (m x ) curves for both treated and untreated mites are depicted in Figure 3.The maximum values of m x was 4.74 eggs/individual/day for control which was occurred on 21 th day of lifespan.This value were estimated to be 3.95, 3.69, 3.39 eggs/individual/day that were happened on 17, 15, 15 th day which were recorded for the mites treated at LC 10 , LC 20 and LC 30 , respectively.
The age-stage survival rate (S xj ) indicated chance that a T. uricae egg will survive to age x and stage j (Figure 4).This curve indicated overlaps due to the varation in different life stages of T. urticae.The highest female and male survival rate obtained 69% and 23% in control (female) and LC 10 , LC 20 and LC 30 (male) treatment respectively., 2014).This study provides the population parameters and demographic data related to offspring in treated T. urticae with sublethal concentrations of Atropa belladonna herbal extract.Our results clearly showed that Atropa belladonna herbal extract can be consid-ered as an acaricide against the two spotted spider mite, causing reduction in fecundity and longevity in the laboratory at concentrations.This herbal extract is a promising natural alternative for the control of T. urticae.Pesticides based on plant or their constituents have demonstrated efficacy against a range of stored product pests (Gholamzadeh Chitgar et al., 2013).
The results of this study showed that the application of the Atropa belladonna herbal extract has effects on the Tetranychus urticae population.The LC 30 concentration caused a higher mortality in mites population than LC 20 and LC 10 .It also reduced the parameters such as the immature period.Also the life span both of the sex.This herbal extract has an impact on population growth and reproductive rates of TSSM and can cause a decrease in the population treated in the next generation.Consequently, it is recommended that apply sublethal concentrations of Atropa belladonna herbal extract at lower rates could lead to efficient control of T. urticae.

Figure 2 .Figure 3 .FecundityFigure 4 .
Figure 2. Age-specific survivorship (lx) of offspring of the treated and untreated mites of Tetranychus urticae by of Atropa belladonna extract

Table 1 .
Effects of sublethal concentrations of Atropa belladonna herbal extract on developmental times, adult longevity, and total life span (day±SE) of Tetranychus urticae

Table 2 .
Effects of sublethal concentration of Atropa belladonna herbal extract on reproductive parameters of Tetranychus urticae