Introduction

Mirid bugs, including Apolygus lucorum, have become the key pests of cotton and fruit trees owing to the major reduction in broad-spectrum insecticide use associated with the wide-scale adoption of Bt cotton in China since 19971,2. The nymphs and adults of these bugs mainly feed on the young tissues of crops, including the leaves, bolls, fruits, terminal meristems, and other tissues, resulting in the stunting of plants and dropping of bolls and fruits, and in turn causing serious yield loss3. Broad-spectrum insecticides, such as organophosphates and pyrethroids, have been extensively applied to reduce the infestation by these insects4,5,6. However, many environmental risks are related to the use of these conventional insecticides, e.g., insect resistance7,8, with negative effects to natural enemies9,10 and on human health. Thus, it is crucial that insecticides with highly effective to pests and low-toxicity to mammals should be adopted to manage mirid bugs to reduce the environmental risks.

Neonicotinoid insecticides are among the most widely used pesticides globally, with the advantages of favourable toxicological properties, flexible use, and systemic activity11,12. They selectively act as agonists of insect nicotinic acetylcholine receptors (nAChRs), preventing signal transduction and in turn resulting in a lasting impairment of the nervous system and the death of insect12,13, and such insecticides have been used to control aphids, whiteflies and thrips in many agricultural crops14. In addition to kill the pests directly, these insecticides also exhibited sublethal effects on the physiological and behavioural traits of insect pests, such as survival15,16, development duration17,18,19, fecundity16,18,20,21,22, and feeding behaviour23,24,25,26,27. For example, Pan et al.18 observed reduction in adult longevity, oviposition period, fecundity, and egg hatching rate but an increase in the pre-oviposition period of A. lucorum at either LD10 or LD40 of cycloxaprid. Daniels et al.27 reported that a sublethal dose of thiamethoxam (0.8 mg L−1) reduced xylem feeding by Rhopalosiphum padi. Thus, the sublethal effects caused by these insecticides should be rigorously assessed to determine the response characteristics of insect pests.

Electrical penetration graphs (EPGs) are an effective tool to study the feeding behaviour of sap-sucking pests such as aphids23,24,25,27. However, phytophagous mirids produce no salivary sheath28 and perform macerating or lacerating behaviour28,29,30,31. As a consequence, the feeding behaviour of these bugs observed on the EPGs recording may be different from those of other sap-sucking pests. Fortunately, the biological meaning of the feeding waveforms in a few species of mirid bugs was recently clarified in several studies29,32,33. Additionally, EPGs were also applied to reveal the impacts of neonicotinoid insecticides on the feeding behaviour in Aphis gossypii24,34, Myzus persicae23,35, Sitobion avenae25, and R. padi27,36. Cui et al.25 found that cycloxaprid significantly increased the total length of non-probing periods and inhibited the phloem ingestion of S. avenae. Therefore, EPGs may have the potential to reveal the sublethal effects of insecticides on feeding behavioural traits of A. lucorum.

Dinotefuran, the third generation of neonicotinoid insecticides, was developed by Mitsui Chemicals, Inc. in 2002 and has a characteristic tetrahydro-3-furylmethyl group that differs from that of most other neonicotinoids such as imidacloprid37. Few studies have investigated the sublethal effects of dinotefuran on Bemisia tabaci19 and Halyomorpha halys38. However, its sublethal effects on A. lucorum have not yet been reported. Here, we first assessed the acute toxicity of dinotefuran to 3rd-instar nymphs of A. lucorum. Then, the sublethal effects of dinotefuran on the biological parameters and feeding behaviour of parent (F0) and offspring generations (F1) were determined using the age-stage, two-sex life table and EPGs, respectively. The purpose was to clarify whether the population-level performance and behavioural traits of A. lucorum were influenced by dinotefuran exposure.

Results

Acute toxicity of dinotefuran to Apolygus lucorum

At 48 h, the linear regression equation derived from the concentration-mortality response bioassay was Y = 1.2X − 2.683 (χ2 = 3.44, P = 0.33; X represents the log-transformed concentrations of dinotefuran, and Y represents the probability of A. lucorum mortality). The LC10, LC30, and LC50 of dinotefuran against 3rd-instar nymphs of A. lucorum were 14.72 (95% confidence interval: 2.66–33.62 mg L−1), 62.95 (24.95–102.47 mg L−1), and 172.20 mg L−1 (106.93–243.74 mg L−1), respectively.

Sublethal effects of dinotefuran on the F0 generation of Apolygus lucorum

The development duration and fecundity of F0 generation of A. lucorum are shown in Fig. 1. Compared with the control, the development duration from 3rd-instar nymph to adult was significantly extended by 0.78 and 0.87 days at LC10 and LC30, respectively (F = 6.73, df = 2, 279, P = 0.002). LC30 also significantly increased the oviposition period and male adult longevity compared with the control and the LC10 (oviposition period: F = 6.43, df = 2, 74, P = 0.003; male adult longevity: F = 3.16, df = 2, 163, P = 0.045). In contrast, the nymphal survival rate from 3rd-instar nymph to adult was reduced at LC30 (Control: 66.67 %; LC10: 64.44 %; LC30: 40 %; X2 = 8.04, P = 0.018) and the female adult longevity and fecundity were significantly lower at LC10 than at LC30 (female adult longevity: F = 4.60, df = 2, 105, P = 0.012; fecundity: F = 4.17, df = 2, 105, P = 0.018). However, the pre-oviposition period did not differ between dinotefuran treatments and the control (F = 0.89, df = 2, 74, P = 0.415).

Figure 1
figure 1

Sublethal effects of dinotefuran on development duration (A) and fecundity (B) of the F0 generation of Apolygus lucorum. Nymphal duration represents the development duration from 3rd-instar nymph to adult. Data are mean ± standard errors (SEs). The same lowercase letters within each parameter indicate that treatments are not significantly different from each other based on one-way ANOVA followed by Tukey’s multiple comparisons test at P ≤ 0.05.

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Sublethal effects of dinotefuran on the F1 generation of Apolygus lucorum

The development duration and fecundity of F1 generation of A. lucorum are shown in Fig. 2. Both LC10 and LC30 significantly shortened the egg duration and preadult duration. Compared with the control, the duration of 4th-instar nymph, oviposition period, and female fecundity were markedly decreased by LC30, and the total preoviposition period was also reduced by LC10. However, other biological parameters including the preadult survival rate (Control: 43 %; LC10: 50 %; LC30: 47 %) and the four demographic parameters, namely, R0, r, λ, and T, were not affected by these two concentrations (Table 1).

Figure 2
figure 2

Sublethal effects of dinotefuran on development duration of immature (A) and adult (B) and fecundity (C) of the F1 generation of Apolygus lucorum. The standard errors (SEs) were estimated using bootstrap technique with 100,000 resamplings. The same lowercase letters within each parameter indicate that treatments are not significantly different from each other based on a paired bootstrap test at P ≤ 0.05.

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Table 1 Sublethal effects of dinotefuran on demographic parameters of the F1 generation of Apolygus lucorum.
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The sublethal effects of dinotefuran on the age-specific survival rate (lx), age-specific fecundity (mx), age-specific maternity rate (lxmx), and age-stage-specific fecundity (fxj) of A. lucorum are shown in Fig. 3. The lx gradually decreased with ages and the maximum ages reached 76, 65, and 65 days in the control, LC10, and LC30, respectively. The time spans of female oviposition in the control were longer than that at the LC10 and LC30 (Control: 48 days, LC10: 37 days, LC30: 38 days). The mx has two peaks in each treatment across whole ages, and the fxj also has two peaks at LC10 and LC30, but not in the control. The highest values of lxmx were 0.94, 0.92, and 0.78 for the control, LC10 and LC30, respectively.

Figure 3
figure 3

Sublethal effects of dinotefuran on the age-specific survival rate (lx, A), age-specific fecundity (mx, B), age-specific maternity (lxmx, C), and age-stage specific fecundity (fxj, D) of the F1 generation of Apolygus lucorum.

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Sublethal effects of dinotefuran on the feeding behaviour of Apolygus lucorum

Four main electrical waveforms were identified in A. lucorum fed on Bt cotton plants: stylet probing (P), stylet insertion into cells (I), cell rupturing and salivation (B), and feeding on the cell mixture (S). Neither LC10 nor LC30 significantly affected the number of probes or the duration of each waveform (P, I, B, and S) (Table 2).

Table 2 Sublethal effects of dinotefuran on probing number and probe duration of Apolygus lucorum fed on Bt cotton plants for 6 h.
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Discussion

In this study, we provided the evidence that both LC10 and LC30 of dinotefuran have no transgenerational effects on A. lucorum with respect to the demographic parameters: R0, r, λ, and T (Table 1). This finding was in accord with the previous reports on A. gossypii exposed to LC20 of cycloxaprid39 and B. tabaci treated by LC25 of imidacloprid40. However, significant impacts on the demographic parameters were shown in A. lucorum treated with LD15 of sulfoxaflor41; in M. persicae exposed to imidacloprid17,23 or thiamethoxam20; and in A. gossypii24, Aphis glycines42 and R. padi43 exposed to imidacloprid. Zhen et al.41 showed that the LD15 of sulfoxaflor significantly reduced the r, λ, T, R0, and gross reproduction rate (GRR) of the F1 generation of A. lucorum compared with the control. Thus, the sublethal effects of insecticides on the demographic parameters were affected by multiple factors, e.g., insect species and kinds and amount of insecticides. Since the demographic parameters reflect the performance of insect pests at the population level, the present finding indicates that dinotefuran would not affect the performance of A. lucorum population.

The dinotefuran concentrations tested here also significantly increased the nymphal development duration, oviposition period, and male adult longevity in F0 generation of A. lucorum, while decreased the egg duration, preadult duration, total preoviposition period and oviposition period in F1 generation (Figs. 1 and 2). Interestingly, the oviposition period of A. lucorum exposed to LC30 of dinotefuran in the F0 and F1 generation was inverse. Compensatory effects might exist in A. lucorum to synchronize the developmental rate of various populations. Similarly, Li et al.43 observed a longer oviposition period in R. padi treated by imidacloprid, while a shorter oviposition period was documented in A. lucorum18,41 and A. glycines42. Additionally, imidacloprid significantly extended the nymphal development duration of M. persicae17,23 and R. padi43. In contrast, a reduction in nymphal development duration was reported in A. lucorum41, B. tabaci15, A. gossypii24,34, and A. glycines42. And the reduction in preadult duration may attribute to the lower egg duration at both LC10 and LC30 of dinotefuran in F1 generation of A. lucorum (Fig. 2). The development changes in these stages might be caused by two reasons. First, the antifeedant effects of these insecticides at low concentrations22 negatively affected the nutrition absorption of exposed insects22,24. The other may be related to the disruption of hormone balance42.

We also showed that the nymphal survival rate in F0 generation and fecundity in F1 generation of A. lucorum were decreased at LC30. Many insect species also have lower survival rates in their immature stages, including A. lucorum41, R. padi43, B. tabaci15,44, Euschistus heros16, and A. gossypii45. Additionally, the reduction in fecundity were also observed in A. lucorum with LD40 of cycloxaprid18, A. gossypii exposed to LC10 and LC40 of cycloxaprid34, A. glycine with 0.20 mg L−1 of imidacloprid42, and N. lugens treated with imidacloprid and dinotefuran46. This phenomenon could attribute to the reduction in vitellogenin (Vg) and the expression level of Vg mRNA significantly decreased by 43.8% in F1 generation of A. lucorum whose parents were treated with LD15 of sulfoxaflor41.

EPGs analysis demonstrated that the feeding behaviour of A. lucorum did not differ between dinotefuran treatments and the control, indicating that these two concentrations will not increase crop injury when this pest moves to other plants. In contrast, many studies have reported negative effects of insecticides on the feeding behaviour of A. gossypii24,34, M. persicae23,35, S. avenae25, and R. padi27,36. Cira et al.26 found that H. halys adults that survived from sulfoxaflor exposure produced significantly fewer feeding sites than those in the control. Indeed, A. lucorum performs a macerating or lacerating behaviour, which is different from other sap-feeding pests such as aphids and leafhoppers28,29,30,31, and has a shorter ingestion duration like Adelphocoris suturalis in plants47. The different feeding strategy may led to their different responses towards insecticides. Nevertheless, further experiments are needed to clarify the relationships between the amount of insecticide used and the feeding response of A. lucorum.

In summary, the dinotefuran concentrations tested here showed sublethal effects, but no transgenerational effects on A. lucorum. It implies that dinotefuran would not increase the population size of A. lucorum.

Materials and Methods

Ethics statement

Permission was not required for insect collection, because none of the species used in the study were endangered or protected.

Insects

Overwintering eggs of A. lucorum used in this study were originally collected from a winter jujube orchard in Binzhou, Shandong Province, China, in 2016. After eggs hatched, they were reared on green bean (Phaseolus vulgaris) without exposure to any insecticide in transparent glass jars (10 cm in diameter, 15 cm in height). These jars were maintained in a climate-controlled chamber with a temperature of 25 ± 1 °C, relative humidity (RH) of 65 ± 5%, and photoperiod of L16: D8.

Insecticides

Dinotefuran (95.57% purity) was purchased from Suzhou Aotelai Chemical Group (Suzhou, Jiangsu Province, China) and used in all the following experiments.

Acute toxicity of dinotefuran to Apolygus lucorum

The acute toxicity of dinotefuran to 3rd-instar nymphs of A. lucorum was assessed using the leaf-dipping method. The stock solution of dinotefuran prepared in acetone was diluted into a series of concentrations: 68.75, 137.5, 275, 550, and 1100 mg L−1, using distilled water containing 1‰ (v/v) Tween-80. Distilled water containing 1‰ Tween-80 was used as the control. Fresh green beans with the same size were cut into 2-cm-long sections, dipped into each insecticide solution and the control for 20 min, and air-dried at room temperature for 2 h. These beans were then placed into the transparent plastic containers (6 cm in diameter, 7 cm in height), each of which included three 2-cm-long sections of green bean. After 5 h of starvation, 3rd-instar nymphs of A. lucorum were transferred into these containers. Each treatment was repeated three times with 15 nymphs per replicate. All the containers were then maintained in a climate-controlled chamber with a temperature of 25 ± 1 °C, RH of 65 ± 5%, and photoperiod of L16: D8. After 48 h, the nymphal mortality was recorded, and the nymphs that did not move when touched with a thin brush were regarded as dead.

Sublethal effects of dinotefuran on the F0 generation of Apolygus lucorum

The LC10 (14.72 mg L−1) and LC30 (62.95 mg L−1) were used to evaluate the sublethal effects of dinotefuran on A. lucorum. These two concentrations were prepared as the method described in the “Acute toxicity of dinotefuran to Apolygus lucorum” section above. Distilled water containing 1‰ Tween-80 was used as the control. There were 154, 135, and 225 of 3rd-instar nymphs used for the control, LC10, and LC30, respectively. After 48 h, the survivors of each treatment were individually transferred to the smaller transparent plastic container (1.5 cm in diameter, 2 cm in height) with one 1.5-cm-long green bean section free from insecticide. The development and survival of nymphs were recorded daily. After adults emerged, they were paired (1 male and 1 female) in new transparent plastic containers (1.5 cm in diameter, 2 cm in height) with one 1.5-cm-long section of green bean (as food and oviposition substrate). The old green beans were replaced by new ones daily. The eggs in the old green beans were checked and counted under a stereomicroscope until adult death. If the male adult died during the experiment, it was removed and replaced by a new one from the same treatment. All the experiments were conducted in a climate-controlled chamber with a temperature of 25 ± 1 °C, RH of 65 ± 5%, and photoperiod of L16: D8.

Sublethal effects of dinotefuran on the F1 generation of Apolygus lucorum

When the eggs laid by female adults of the F0 generation peaked, there were 117, 131, and 134 eggs randomly selected and assigned to the control, LC10, and LC30, respectively, to initiate the life table study of the F1 generation. The hatched eggs were recorded daily and the newly born nymphs were individually transferred into a new transparent plastic container (1.5 cm in diameter, 2 cm in height) with one 1.5-cm-long green bean section without exposure to any insecticide. The nymphal stage and survival were checked and recorded daily. Within 24 h of adult emergence, adults were paired (1 male and 1 female) in a transparent plastic container with one 1.5-cm-long section of green bean (as food and oviposition substrate). The old green beans were replaced by new ones daily. The eggs in the old green beans were checked and counted under a stereomicroscope until adult death. If the male adult died during the experiment, it was removed and replaced by a new one from the same treatment. All the experiments were conducted in a climate-controlled chamber with a temperature of 25 ± 1 °C, RH of 65 ± 5%, and photoperiod of L16: D8.

Sublethal effects of dinotefuran on the feeding behaviour of Apolygus lucorum

The feeding behaviour of A. lucorum exposed to dinotefuran on Bt cotton plants at the seedling (variety: Lumianyan 36, developed by Cotton Research Center, Shandong Academy of Agricultural Sciences, Jinan, China) were recorded using a Giga-4 direct-current electrical penetration graph system (DC-EPG, manufactured by WF Tjallingii, Wageningen University, Wageningen, Netherlands). Briefly, the 3rd-instar nymphs reared on green beans were exposed to LC10 and LC30 of dinotefuran and the control prepared as described in the “Acute toxicity of dinotefuran to Apolygus lucorum” section above for 48 h. After 5 h starvation, these nymphs were immobilized on an ice plate and then secured on a vacuum device for the attachment of wires. A gold wire (2 cm in length, 20 μm in diameter) was attached to the pronotum of individual nymph using silver glue under a stereomicroscope. The gold wire allowed the nymphs to move relatively unaffectedly with a radius equal to the length of the wire tether. Then, each nymph was connected to the amplifier before being placed on a cotton leaf. Another copper electrode was inserted into the soil in the pot with one Bt cotton plant. Finally, the entire experimental arena was covered by a Faraday cage to shield external noise and other interference. Recordings were made simultaneously on four individual Bt cotton plants over 6 h using ANA 34 software (Wageningen, The Netherlands). Nymphs and Bt cotton plants were used only once and then discarded. For each treatment, at least 20 nymphs were successfully tested. Electrical signals were identified and characterized based on the descriptions by Song et al.33 and Zhao et al.32.

Data analysis

The LC10, LC30, and LC50 values were determined based on the probit analysis. The development and fecundity of F0 generation and feeding behaviour were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-range test. The nymphal survival rate of F0 generation were compared by Chi-squared test (χ2). All these analyses were conducted in SPSS 19.0 software (IBM Inc., New York, USA).

The life table data for all A. lucorum individuals in the F1 generation were analysed using TWOSEX-MSChart computer program48 according to the age-stage, two-sex life table theory49,50. The demographic parameters, namely, the net reproductive rate (R0), intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T), of the F1 generation were calculated based on the following Eqs. (1–4) via the computer program. The age-specific survival rate (lx), the age-specific fecundity (mx), age-specific maternity rate (lxmx), and the age-stage-specific fecundity (fxj, where x is the age and j is the stage) were also obtained from the computer program.

$${R}_{0}=\mathop{\sum }\limits_{x=0}^{\infty }\,{l}_{x}{m}_{x}$$
(1)
$$\mathop{\sum }\limits_{x}^{\infty }\,{e}^{-r(x+1)}{l}_{x}{m}_{x}=1$$
(2)
$${\rm{\lambda }}={e}^{r}$$
(3)
$$T=\frac{\mathrm{ln}\,{R}_{0}}{r}$$
(4)

The means and standard errors of all the life history traits and demographic parameters were estimated using bootstrap technique with 100,000 resamplings51,52, and the differences among treatments were compared using the paired bootstrap test based on the confidence intervals53. All the figures were created in SigmaPlot 14.0 software (Systat Software Inc., San Jose, CA). For all the test, α = 0.05.