Introduction

One of the major concerns to IPM practitioners is pesticides adaptability with biological agents. Hence, the knowledge on mode of action of pesticides on non-target insects, human health and the environment are necessary (Stark et al. 2004; Barzman et al. 2015). Consequently, understanding the response of natural enemies to pesticides has great importance for the development of IPM programs. Indeed, many studies have been conducted on various pesticides to beneficial organisms (Kavousi and Talebi 2003; Lucas et al. 2004; Medina et al. 2003; Rezaei et al. 2007). In field crops, sub-lethal concentrations usually occur after the initial insecticide application. These concentrations depend on how often insecticides are applied, and how they are degraded by biotic and abiotic factors, such as rainfall, temperature and sunlight. Sublethal effects may impair many physiological and behavioral traits of the exposed arthropods (Desneux et al. 2007). Indeed, sublethal impacts can be observed on life span, weight loss, behavior, mature and immature times, developmental rates, sex ratio, and mutation of offspring and fertility rates (Rezaei et al. 2007; Stark et al. 2004; Desneux et al. 2007). The impairment of key behavioral and physiological traits may strongly affect population dynamics (Stark and Banks 2003; Tan et al. 2012). Therefore, accurate assessment of sublethal effects is important for a complete analysis of impact of pesticides in agro-ecosystems, as well as on their selectivity towards non-target organisms (Stark et al. 1998).

The green lacewing, Chrysoperla carnea, Stephens (Neoroptera, Chrysopidae) is one of the most important general predators of insect pests that attack aphids, thrips, coccids, whiteflies and eggs of lepidopterans and coleopterans in different agro-ecosystems (Golmohammadi et al. 2009). Currently, it is one of the most important biological agents that is commercially available (Medina et al. 2003; Tauber et al. 2000). Geographical distribution, broad host range, potency of suitable compatibility in agro-ecosystems, high searching ability, easy mass rearing in laboratory, high reproductive potential and broad resistance to numerous insecticides especially in larval and pupal stages cause the green lacewing to be considered as an important biocontrol agent (Azema and Mirabzadeh 2004).

The objectives of the current research were evaluating sublethal effects of three insecticides on first instar larvae of green lacewing, C. carnea and their biological characteristics using life table parameters. Results were used to identify the most selective compounds in order to propose them for use in IPM programmes in combination with 57augmentative releases of C. carnea. We also measured the enzyme activities of the C. carnea as biomarkers of sublethal exposure to insecticides (Rumpf et al. 1997; Booth et al. 2007).

To do this, we used three insecticides with different modes of action. Flupyradifurone is a rather new systemic insecticide which is classified as a butenolide subgroup in IRAC MoA Group 4D acting as Nicotinic acetylcholine receptor (nAChR) competitive modulators (Jeschke et al. 2015). Clothianidin is another neonicotinoid systemic insecticide with same mode of action which is categorized in IRAC MoA Group 4A. Tebufenozide is non-steroidal ecdysteroid agonist insecticide (Dhadialla et al. 1998). According to the IRAC MoA classification, it belongs to the group 18, as ecdysone receptor agonist.

Materials and methods

Rearing of insects

The Mediterranean flour moth, Ephestia kuehniella Zell (Lep., Pyralidae) adults (for the rearing of C. carnea) were provided by the Iranian Research Institute of Plant Protection (IRIPP). For rearing of E. kuehniella, a big plastic container with a net cloth (70 cm in diameter × 25 cm high) was used. In each dish, a layer of mixed wheat flour and wheat bran plus wheat yeast (2.5: 0.5: 40; kg: kg: g) were added, then one gram of E. kuehniella eggs was spread uniformly on it. The plastic containers were kept in controllable conditions consisting of 25 ± 1 °C, 60 ± 5% and a photoperiod 16: 8 h (D: L). After oviposition of adults, the eggs were collected and refrigerated (specify the temperature) for as long as they were required to feed the green lacewing.

The initial colony of green lacewing, C. carnea, adults were provided from Khorasan Razavi Agriculture and Natural Resources Research Center. For rearing of the C. carnea, plastic cylinder containers, covered with a net cloth, were used. The adults were fed on artificial diet containing yeast, honey and distilled water (4:7:5 g/g/ml) and their larvae were fed with eggs of E. kuehniella, daily. In order to prevent cannibalism in the colony, the larvae were placed in containers with a soft cloth layer and larvae were positioned between these layers. Each layer had 15 larvae of C. carnea and enough eggs of E. kuehniella. After emergence of pupae, they were collected and were transferred to another container. Rearing containers were maintained at conditions of 25 ± 1 C°, 60 ± 5% and a photoperiod 16: 8 h (L: D).

Determination of the activity of esterase enzymes

Materials included: Tris-base, glycin, alpha-naphthal and beta-naphthol from the Merck company (Germany) and alpha-naphthyl acetate, beta-naphthyl acetate and fast blue RR from Sigma chemical company (Germany). Activity of the esterase enzymes was measured with the method of Van Asperen (1962) with some modification. First of all, in order to prepare the extract of the enzymes, the insects (N = 20 insects) were homogenized in each treatment with200 μl 0.1 M sodium phosphate buffer with pH 7. The solution was then centrifuged at 10,000 rpm for 15 min at4°C, and the supernatant was transferred to fresh vials. The 20 μl of the enzyme extract was kept at 200 μl of a substrate of alpha-naphthyl acetate or beta-naphthyl acetate at a concentration of 0.3 mM to the micro plate wells at 25 °C for 20 min. Then, after adding 50 μl fast blue RR solution with SDS 5% to the solution for 20 min, the samples were absorbed in 405 nm for alpha-esterase and at 492 nm for beta-esterase (with double beam spectrophotometer, model Perkin Elmer). Measurement of esterase activity was calculated using the standard curve of alpha-naphthol and beta-naphthol, which are reaction products, in micromole per minute per milligram protein.

Insecticides

The insecticides used in this study were Flupyradifurone (Sivanto®, SC 48%, Bayer Crop Science), Tebufenozide (Mimic®, SC 20%, Sumitomo Japan) and Clothianidin (Poncho®, WG 20%, BayerCrop Science). All tested compounds were prepared by the Iranian Research Institute of Plant Protection (IRIPP).

Bioassays

After preliminary experiments, the range of concentrations (233, 433, 806, 1500 ppm for tebufenozide; 500, 965, 1200 ppm for flupyradifurone and 8, 12, 19, 30 ppm for clothianidin) were provided. For each concentration, 10 first instar larvae at five replicates were used. The mortality rate for clothianidine and flupyradifurone were counted after 48 h while for tebufenozide, it was recorded after 72 h. First instar larvae were stimulated with a soft brush and immobile larvae were considered dead. Toxicological bioassays were performed onfirst instar larvae using the contact method. After preparation of the solutions, 2 ml from each concentration bypotter spray tower was poured into both petri-dishes (10 cm diameter). After desiccation of Petri-dishes, 20 first instar (< one-day-old) larvae were introduced into each Petri-dish. To avoid egg cannibalism by neonate larvae in exclusion interval, some eggs of E. kuehniella were added to Petri-dishes. Abbott formula was used to correct of mortality value (Abbott 1925). The LC50 values were compared based on the method of Robertson et al. 2007.

Sublethal effects

The sublethal concentration (LC30) for clothianidin, tebufenozide and flupyradifurone were determined to be 77.83, 20.43 and 12.89 ppm, respectively. The control was treated by distilled water only. There were five replicates per treatment. Sublethal effects of each of the three insecticides (LC30) on life table parameters of C. carnea were evaluated using contact method. For this purpose, 100 first instar larvae were used for each insecticide. The mortality rate for clothianidine and flupyradifurone were counted after 48 h while for tebufenozide, it was recorded after 72 h. The first instar larvae were stimulated with a soft brush and immobile larvae were considered dead. Life status of larvae were recorded until adult emergence. After oviposition of adults, the Petri-dishes were replaced daily and this trend continued until the death of the last individual. All experiments were conducted at controllable conditions of 25 ± 1 °C, 60 ± 5% and a photoperiod 16: 8 h (L: D). Next, biological characteristics of C. carnea treated by the three insecticides such as, the means of male and female development times, the means of reproductive periods consisting of oviposition period, APOP (adult pre-oviposition period), TPOP (total pre-oviposition period) and total fecundity were calculated. The total effects of insecticides were calculated as follows (Overmeer and van Zon 1982):

$$ \mathrm{E}=100\%-\left(100\%-\mathrm{M}\right)\times \mathrm{R} $$

Where: M is the value of mortality, R is mean number of eggs and E is total effect of insecticides. Afterwards, according to the total effects, the treated insecticides were categorized using IOBC classification (Sterk et al. 1999).

Demographic toxicology

The life table data of green lacewing, C. carnea treated by three insecticides, flupyradifurone, tebufenozide and clothianidin were analyzed using age-stage, two-sex life table method (Chi 2018). In order to study the demographic toxicology, the fecundity life table was formed. Life table parameters of C. carnea such as: the intrinsic rate of increase (rm), the finite rate of increase (\( \lambda ={e}^{\gamma_m} \)), the net reproductive rate (R0), the gross reproductive rate (GRR), the mean generation (T) and the doubling time (DT) were calculated. Moreover, the age-stage survival rate, lx (where x = age and j = stage) and the age specific fecundity, mx were determined and their curves were plotted.

Analyses of data

Bioassay data were analyzed by SPSS software vr. 24 (SPSS Institute 2017). Life table of C. carnea were studied using Age-Stage, Two-Sex Life Table Analysis software by TWOSEX-MSChart, available at http://140.120.197.173/Ecology/prod02.htm (Chung Hsing University). The Jackknife method was utilized for the means and standard errors (SEs) of the life table parameters (Chi 2018). The SE was estimatedusing 100,000 bootstraps. Before analysis of variance, for normalize of data obtained sublethal effects were utilized from logarithmic transformation. Analysis of variance data was performed using the SAS software vr.6 (SAS Institute 1990). Moreover, the means comparison was carried out using Duncan’s Multiple Range Test at 0.05 confidence level.

Results and discussion

Insecticides toxicity

The category and toxicity of tested insecticides on the first instars larvae of green lacewing, C. carnea has been shown in Table 1. The coefficient R2 shows that there is a high correlation between insecticide concentration and tested population response, indicating that the populations were homogeneous. According to the results, LC30 of clothianidin was much less than tebufenozide and flupyradifurone (Table 1). Moreover, the LC50 and LC90 values of the three tested insecticides are presented in Table 1. These findings revealed that there was significant difference among LC50 and LC30 of the insecticides. LC90 of tebufenozide and flupyradifurone were not statistically significantly different (Table 1).

Table 1 Toxicity of three insecticides applied to first instars larvae of green lacewing, Chrysoperla carnea
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Life table parameters

The means (±SE) values of life table parameters of C. carnea treated by LC25 of three insecticides, clothianidin, tebufenozide and flupyradifurone are presented in Table 5. The lowest value of intrinsic rate of increase (rm) was calculated for clothianidin and tebufenozide which were significantly different from that of the control, whereas flupyradifurone treatment did not differ from the control statistically (df = 2, F = 11.32, P = < 0.05). One of the most important parameters utilized to compare and reveal the potential of what insect populations undergo under different treatments is the intrinsic rate of increase (Farhadi et al. 2011). On the other hand, the rm is the sole statistical parameter that presents a brief synopsis of the physiological quality for an animal associated with its capacity (Andrewartha and Birch 1954). Razaei et al., 2006 stated that imidacloprid and propargite had no significant effects on the rm of C. carnea but pymetrozine caused a reduction on the rm.

The means followed by the same letter are not significantly different using paired bootstraps test at the 5% significance level.

The finite rate of increase (λ) in flupyradifurone treatment had no statistically significant difference with control (df = 2, F = 41.06, P = 0.003), while LC30 of clothianidin and tebufenozide caused decline of the finite rate of increase in 30 C. carnea (1.14 ± 0.004 days−1). Also, effect of LC of clothianidin and tebufenozide on the net reproductive rate (R0) and gross reproductive rate (GRR) were significant (df = 2, F = 10.1, P = 0.012). The lowest and highest values of R0 were calculated for clothianidin (95.72 ± 10.79 offspring per individual) and control (143.26 ± 16.07 offspring per individual). However, the lowest GRR value associated to tebufenozide and was equal to 100.39 ± 10.79 offspring /individual (Table 2). The R0 (basic reproductive rate) indicates the number of times that a population will multiply during generation, while the GRR means total number of eggs produced by females, over their life span (Jervis 2007).

Table 2 Effect of LC30 of three insecticides on life table parameters (mean ± SE) of Chrysoperla carnea
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Previous studies on sublethal effects of imidacloprid, endosulfan and indoxacarb on the first instars larvae of C. carnea were evaluated by Golmohammadi et al. 2009 stating that none of the treatments on the life table parameters had statistically significant differences. In another study, effects of imidacloprid, propargite andpymetrozin on population growth parameters were assessed and revealed that between imidacloprid and control, there was no significant difference (Rezaei et al. 2007). The mean generation time (T) of C. carnea treated by LC30 of clothianidin, tebufenozide, flupyradifurone and control were 34.97, 34.31, 31.78 and 32.93 days, respectively (Table 2). Furthermore, there were significant differences between insecticidal treatments with control, of course the clothianidin and tebufenozide were not significantly different (df = 2, F = 1.66, P = 0.68). Furthermore, the doubling time (DT = ln2/rm) for C. carnea exposed to LC30 of clothianidin, tebufenozide, flupyradifurone and control treatment was 5.33, 5.33, 4.62 and 4.62 days, respectively. Accordingly, the doubling time of flupyradifurone did not differ to that of the control, while for clothianidin and tebufenozide, there were significant differences compared to the control.

The age-stage survival curves (lx) of C. carnea, treated by three insecticides; clothianidin, tebufenozide and flupyradifurone are presented in Fig. 1. According to the Fig. 1, mortality trend of different stages of C. carnea by LC30 of flupyradifurone was similar to control while in the case of clothianidin and tebufenozide there were differences compared to control. In all treatments, the survival of both male and female in 27th to 41st day was the same and fixed and after that, the survival was decreased (Fig. 1). Based on the survival curves, the highest and lowest survival rate of C. carnea was observed in tebufenozide (54 days) and clothianidin treatment (46 days), respectively, while in the control, the survival rate was 58 days. Therefore, use of tebufenozide on target insect pests can have less effect on the survival of green lacewing.

Fig. 1
figure 1

Age-stage survival rates (lx) of green lacewing, Chrysoperla carnea exposed to LC30 of Clothianidin, Tebufenozide and Flupyradifurone compared with the control

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The age-specific fecundity (mx) of C. carnea exposed to three tested insecticides; clothianidin, tebufenozide and flupyradifurone with control are plotted in Fig. 2 (Fig. 2, a-c). The fecundity trend for control was similar to flupyradifurone, while in the cases of clothianidin and tebufenozide, fecundity curve was different with control (Fig. 2). In the control and flupyradifurone treatments, the fecundity was started on the 19th day but in other treatments (clothianidin and tebufenozide) the fecundity was began later. The highest fecundity was observed in clothianidin on the 44th day and after that, the fecundity decreased until on 46th day which achieved zero. Moreover, the fecundity in control, tebufenozide and flupyradifurone on the 55th, 48th and 49th days was zero (Fig. 2). Thus, based on obtained results, the LC30 of clothianidin and tebufenozide significantly decreased the fecundity of green lacewing, C. carnea compared to the control. Based on the values of the net reproductive rate (R0), together with the intrinsic rate of increase (rm), oviposition period, IOBC classification of insecticides and survival curves, it can be concluded that flupyradifurone was less hazardous, than clothianidin and tebufenozide to green lacewing.

Fig. 2
figure 2

Age-specific fecundity (mx) of green lacewing, Chrysoperla carnea exposed to LC30 of three insecticides compared with the control

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IOBC classification

Based on the results of this study, each insecticide was classified according to IOBC criteria which is presented in Table 3. Comparison of the total effects indicated that clothianidin is classified as a slightly harmful, as it has the lowest fecundity rate (Tables 1 and 2). Nevertheless, based on the IOBC classification; tebufenozide and flupyradifurone were harmless compounds that categorized into class 1. Based on the IOBC classification, among the tested insecticides, the flupyradifurone had the least toxicity on C. carnea, thus it had the highest fecundity rate (Tables 1 and 3). The tebufenozide was classified as harmless insecticide (class 1). Overall, clothianidin was classified as slightly harmful to C. carnea, whiles the two other insecticides were classified as harmless.

Table 3 Total effects of tested insecticides for C. carnea based on IOBC method
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Investigating the activity of esterase enzymes in 1st instar larvae of C. carnea

Measurement of esterase activity was done, using the standard curve of alpha-naphthol and beta-naphthol (50–250 μm), which are reactive products, expressed as micromol / min per milligram protein. Study of alpha-esterase activity in 1st larvae with the End Point method, showed that the alpha-esterase activity of the control treatment with other treatment was in the same statistical group, which did not differ statistically significantly (Table. 4).

Table 4 The value of alpha-esterase activity in the L1 of the Chrysoperla carnea was investigated for clothianidin tebufenozide and flupyradifurone insecticides and control treatment
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Comparison of the mean of the beta-esterase activity, indicated four statistical groups which have a significant difference to each other. The highest value of beta-esterase activity is related to clothianidin treatment and the lowest value is for treatment with tebufenozide (Table 5).

Table 5 The value of beta-esterase activity in the L1 of the C. carnea was investigated for clothianidin tebufenozide and flupyradifurone insecticides and control treatment
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Matowo et al. 2010 reported that both mixed function oxidases and non-specific esterases contributions to the permethrin resistance in Anopheles arabiensis. Also, Ahmadi 2009 showed that the esterase enzymes in the body of a Coccinella montrozieri Mulsant (Coleoptera: Coccinellidae) were significantly reduced after the use of and imidacloprid insecticides. Comparison of alpha and beta-esterase activity in treatment with clot bamectin hianidin, tebufenozide and flupyradifurone insecticides showed that beta-esterase activity in L1 of C. carnea is higher than that of alpha-esterase (Fig. 3).

Fig. 3
figure 3

Comparison of alpha and beta esterase of 1st larvae of green lacewing, Chrysoperla carnea

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To sum up, the results showed that alpha and beta-esterase activity in treated insects with clothianidin and flupyradifurone was increased (compared to the control ones), while the enzymatic activity was reduced in insects with tebufenozide treatment. So, it seems that esterase activity can be stimulated by clothianidin and flupyradifurone treatments by some pathways and tebufenozide can inhibit esterase activity.

Developmental rates

The means of developmental times for male and female of C. carnea were reported in Table 6. The results showed that in larval stage of male, there was no statistically significant differences between flupyradifurone and tebufenozide with the control (df = 2, F = 41.39, P = 0.15) whereas significant difference was observed in clothianidin treatment. In the larval stage of females, statistically significant difference was observed in clothianidin and tebufenozide in comparison with control (df = 2, F = 198.63, P = 0.001). In pupal stage of both male and female there was statistically significant difference in clothianidin and tebufenozide compared to control while no difference was observed between flupyradifurone and control.

Table 6 Mean (±SE) of the developmental times (days) in female and male of C. carnea treated by three insecticides
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Longevity and life span of both sexes in three treatments; clothianidin, tebufenozide and flupyradifurone were different with that of control. The lowest life span in both sexes was in clothianidin (43.00 ± 0.29 male and 45.00 ± 0.28 female).

The smaller body size in males, being that there is more fat in females than males (Croft 1990), and mode of action of tested insecticides are probable reasons for these differences. For instance, high sensitivity of male Coccinellaseptempunctata to Malathion than female due to less fat in males was reported by (Olszak et al. 2004).

Moreover, sublethal effect of endosulfan on biocontrol agent females of Catolaccusgrandis (Burks) was significantly less toxic than male (Elzen et al. 2000). Generally, sublethal effects on the biological agents happen at multiple levels such as developmental and malformation rates when emerging from pupae (Desneux et al. 2007).

Furthermore, positive sublethal effects of insecticides on orientation behaviors of natural enemies have been reported (Delpuech et al. 2005; Komeza et al. 2001).

Reproductive periods

Reproductive periods and total fecundity of female C. carnea treated by LC30 of three selected insecticides presented in Table 7. Three selected insecticides compared to control had statistical significant difference on oviposition period (df = 2, F = 25.56, P = 0.01). The most impact of LC30 on oviposition period was associated with clothianidin (16.52 ± 0.24 days). No difference was observed in three treatments for APOP parameter (df = 2, F = 102.01, P = 0.002) while in TPOP, clothianidin and tebufenozide were differing with control (df = 2, F = 98.16, P = <0.001). Following the control group, the highest fecundity was related to flupyradifurone (272.55 offspring per female) but clothianidin and tebufenozide had significant difference compared with control. Reductions in fecundity by insecticides may be due to behavioral and physiological effects (Desneux et al. 2007). Many researchers have presented universal effects on natural enemy’s fecundity (Brunner et al. 2001; Corrales and Campos 2004). When the parasitoid, Trichogramma pretiosum was exposed to tebufenozide and teflubenzuron, a reduction in its fecundity was observed (Consoli et al. 1998). Impact of tebufenozide on larvae of C. carnea reported by Medina et al. 2003 had no significant effect on oviposition rate and egg fertility and this matches our results. Moreover, fecundity of C. carnea exposed to LC25of indoxacarb, endosulfan and imidacloprid were reported by Golmohammadi et al. 2009 and their results demonstrated that indoxacarb and endosulfan significantly differ with control but with imidacloprid, there was no difference.

Table 7 Mean (±SE) reproductive periods and total fecundity of offspring from females of C. carnea for three insecticides
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