The low-lethal concentrations of rotenone and pyrethrins suppress the population growth of Rhopalosiphum padi

As an important pest on winter wheat, Rhopalosiphum padi (L.) causes damage to the wheat yield by sucking plant nutrients, transmitting plant viruses and producing mildew. R. padi has been reported to develop resistance to pyrethroids and neonicotinoids. To explore potential alternative approaches for R. padi control, the activity of 10 botanical insecticides was evaluated. Results suggested that the toxicity of rotenone and pyrethrins to R. padi were the highest and near to the commonly used chemical insecticides. When exposed to the low-lethal concentrations (LC10, LC30) of rotenone or pyrethrins for 24 h, the lifespan and fecundity of adults in F0 generation decreased significantly compared to control. The negative effect could also be observed in the F1 generation, including the decreased average offspring, longevity of adult, and prolonged nymph period. The population parameters in F1 generation of R. padi were also inhibited by exposing to the low-lethal concentrations of rotenone or pyrethrins, including the decreased net reproductive rate, intrinsic rate of natural increase, finite rate of population increase, and gross reproduction rate. Co-toxocity factor results showed that mixtures of rotenone and thiamethoxam, pyrethrins and thiamethoxam showed synergistic effect. Our work suggested that rotenone and pyrethrins showed negative effect on the population growth under low-lethal concentrations. They are suitable for R. padi control as foliar spraying without causing population resurgence.

A total of 10 botanical pesticides and 2 commonly used chemical insecticides were selected to determine their toxicity to the adults of R. Padi.Among the botanical pesticides, the LC 50 values of rotenone and pyrethrins to R. Padi were the lowest, which were 10.091 and 5.161 mg L −1 , respectively after treated for 24 h, while 7.146 and 3.403 mg L −1 , respectively after treated for 48 h (Table 1).While the LC 50 values of the other botanical pesticides were between 171.597 and 5993.123mg L −1 , indicating their low contact toxicity.The LC 50 values of the chemical insecticides, bifenthrin and thiamethoxam, were 3.028 and 16.447 mg L −1 after treated for 24 h, respectively (Table 1).The contact toxicity of rotenone and pyrethrins was near to that of commonly used chemical insecticides.The low-lethal concentrations of rotenone and pyrethrins to R. Padi were also obtained after treated for 24 h.The LC 10 and LC 30 values of rotenone were 1.424 and 4.529 mg L −1 , respectively, and 0.410 and 1.830 mg L −1 , respectively for pyrethrins (Table 2).

Exposure to the low-lethal concentrations of rotenone decreased the population growth of R. padi and increased the development duration of nymph
After exposed to rotenone at LC 10 or LC 30 for 24 h, the adults of R. padi were kept on fresh wheat leaves until death.Compared with adults exposed to 0.1% Tween 80, the average lifespan and fecundity decreased significantly in adults exposed to LC 30 rotenone, and the decrease rate was 17.74% and 19.54%, respectively (Table 3).There was no significant difference of average lifespan and fecundity between adults exposed to 0.1% Tween 80 and LC 10 rotenone, or LC 10 and LC 30 rotenone (Table 3).
The low-lethal concentrations of rotenone showed significant sublethal negative effect on the development duration and fecundity in the F1 generation of R. padi.Although the F1 generation of R. padi were kept on fresh wheat leaves since they were new-born, R. padi derived from parental generation exposed to LC 30 rotenone showed significantly longer development duration of nymphs and fewer offspring when compared with that derived from parental generation exposed to 0.1% Tween 80 (Table 4).After exposed to rotenone at LC 10 or LC 30 , the longevity of adults in the F1 generation showed no change.Exposure to LC 10 rotenone also prolonged the development duration of nymph significantly than that in control, while the average offspring showed no significant difference with that in control (Table 4).
The population parameters of R. padi in the F1 generation were calculated.The values of r m and λ in LC 10 and LC 30 groups decreased significantly in the F1 generation than that of control, while they showed no significant difference between LC 10 and LC 30 groups.The values of R 0 and GRR in LC 30 group decreased significantly than that of control or LC 10 group, while they showed no significant difference between LC 10 and control groups.The values of T in LC 10 or LC 30 group showed no change with that of control (Table 5).After exposed to the low-lethal concentrations of rotenone, the population dynamics of R. padi were estimated.The l x in the F1 individuals showed CK > LC 10 > LC 30 during 8-11 d and 15-16 d (Fig. 1A).The highest peak of m x in the control group was 4.13 offspring/female occurring at age of 7 d.While the highest peak of m x in LC 10 group was 3.44 offspring/female occurring at age of 7 d.The highest peak of m x in LC 30 group appeared at age of 6 d with 3.1 offspring/female, ahead of control (Fig. 1B).The l x m x curves showed a similar trend with that of m x (Fig. 1C).
The e xj indicate the expected life-span of individuals at age x and stage j after age x.The e xj of immature and adult stages for the aphids treated with rotenone at LC 10 and LC 30 were slightly lower than those of control (Fig. 2).For example, the e xj of the new born nymph in LC 10 and LC 30 rotenone group was 12.92 d and 13.00 d, respectively, lower than control (13.52 d, Fig. 2).While e xj of newly molted adult in LC 10 and LC 30 rotenone was 8.92 d and 9.00 d, respectively, lower than control (9.52 d, Fig. 2).
The V xj curve of female aphids showed a trend of first increasing and then decreasing (Fig. 3).However, the reproductive peak of the offspring in the LC 10 and LC 30 treatment groups reached 7.96 offsprings on the 6th day and 7.23 offsprings on the 4th day post-treatment, respectively, which were lower than those in the control group, reaching a reproductive peak of 8.26 offsprings on the 7th day post-treatment (Fig. 3).

Table 5.
The sublethal effect of rotenone on the population parameters in F1 generation of R. padi.Different lowercase letters in a column indicated significant differences analyzed by the one-way ANOVA with Turkey's HSD test (P < 0.05).

Exposure to the low-lethal concentrations of pyrethrins decreased the population growth of R. padi and increased the development duration of nymph
Exposed to pyrethrins at LC 30 for 24 h significantly decreased the average lifespan and fecundity of adults when compared to the control group.Exposed to pyrethrins at LC 10 for 24 h significantly decreased the average lifespan and showed no influence on the fecundity of adults when compared with that of control.There was no significant difference of lifespan and fecundity between adults exposed to LC 10 and LC 30 pyrethrins (Table 6).
Compared with control, exposed to LC 30 of pyrethrins showed significant increase in the development duration of nymph, and inhibition in the longevity of adults and the fecundity of R. padi in the F1 generation.Exposed to LC 10 of pyrethrins significantly decreased the fecundity compared to the control, while showed no influence on the development duration of nymph or adults (Table 7).There was no significant difference between these parameters of R. padi derived from LC 10 and LC 30 groups.Table 6.The sublethal effect of pyrethrins on the fecundity and lifespan of F0 generation of R. padi.Different lowercase letters in a column indicated significant differences analyzed by the one-way ANOVA with Turkey's HSD (P < 0.05).

Low-lethal concentration
The   The values of R 0 , r m , λ and GRR in LC 10 and LC 30 groups decreased significantly in the F1 generation than that of control.The values of T increased significantly in LC 10 than those in LC 30 and control groups (Table 8).The values of R 0 and GRR in LC 30 group decreased significantly than that in LC 10 group, while r m , λ, T showed no significant change than that in LC 10 group.
The l x in the F1 individuals of control groups was obviously higher than that in LC 10 and LC 30 pyrethrins treatment groups during 8-12 d.After 12 d, the l x showed a trend of CK > LC 10 > LC 30 (Fig. 4A).Compared with the highest peak of m x in the control group (4.13 offspring/female at the 7th d), that in LC 10 and LC 30 groups decreased to 3.64 offspring/female at the 7th d and 2.10 offspring/female at the 8th d (Fig. 4B).The l x m x curves showed a similar trend to that of m x (Fig. 4C).
The e xj of immature and adult stages for the aphids treated by pyrethrins at LC 10 and LC 30 were slightly lower than those of the control (Fig. 5).For example, the e xj of the new born nymph in the LC 10 and LC 30 pyrethrins group were 12.91 d and 12.50 d, respectively, lower than that in the control (13.50 d, Fig. 5).While e xj of newly   molted adult in the LC 10 and LC 30 pyrethrins group were 8.91 d and 8.50 d, respectively, lower than that in the control (9.52 d, Fig. 5).The immature stage in LC 10 and LC 30 group were 6 d, delayed for 1 d than that of control.The adult stage in LC 30 group began from the 2nd d, earlier than the 3 rd d in LC 10 and control groups.
The reproductive peak of the V xj curve of immature aphids in control, LC 10 and LC 30 groups appeared at the 4th, 5th and 4th d, respectively, while 6th, 7th, 4th for that in female aphids.The reproductive peak of the V xj curve of immature aphids in control, LC 10 and LC 30 groups were 5.47, 5.13 and 4.18 offsprings, respectively, while 8.30, 7.87, 6.49 offsprings for female aphids (Fig. 6).

Screening of the synergist mixture containing rotenone or pyrethrins and chemical insecticides to R. padi
The co-toxicity factor method was used to screen the synergist mixtures containing rotenone or pyrethrins and thiamethoxam or bifenthrin.Among the set ratios, synergist mixtures were obtained in all the combinations except for the mixtures of pyrethrin and bifenthrin at 1/2 LC 50 .The combination of rotenone and thiamethoxam Table 9.The synergist mixtures screening using the co-toxicity factor method.showed the best synergist effect, as four out of five ratios (1:5, 1:2, 1:1, 5:1) prepared with LC 50 or 1/2 LC 50 showed synergist (Table 9).The combination of rotenone and bifenthrin prepared with LC 50 and 1/2 LC 50 both showed synergist at the ratio of 1:5.The mixture of pyrethrins and thiamethoxam prepared with LC 50 showed synergist for all the ratios, while synergist was screened out only for ratios 1:5, 1:2, 1:1 prepared with 1/2 LC 50 .The mixture of pyrethrins and bifenthrin prepared with LC 50 showed synergist for ratios 1:5, 1:2, 1:1 (Table 9).

Discussion
Many botanical chemicals with potential insecticide activity have been reported 24 and only a few studies focused on their sublethal assessments 23 .Rotenone, pyrethrins, matrine, azadirachtin, eucalyptol and extract of Veratrum nigrum L., have been registered for aphid control in China.According to our result, the toxicity of rotenone and pyrethrins after treated for 24 and 48 h were near to that of bifenthrin and thiamethoxam, the commonly used chemical insecticides.The acute toxicity of matrine and azadirachtin were much lower than the chemical insecticides.The acute toxicity of eucalyptol, osthole, resveratrol, eugenol, carvacrol, citronellal was the lowest, showing no potential in R. padi control by foliar spraying.Ikbal and Pavela summarized essential oils showing high contact toxicity to aphids with LD 50 lower than 1 µL (µg) mL −1 or more than 90% mortality upon application at 2 µL L −1 .A series essential oils with high toxicity were obtained, including carvacrol to Lipaphis pseudobrassicae (Davis) 25 and Cerataphis lataniae (Boisduval) 26 , eucalyptol to Aphis gossypii (Glover) 27 , citronellal to Aulacorthum solani (Kaltenbach) 28 .The contrastive low toxicity of these botanical insecticides to R. padi might be resulted from different aphid species and the different constituents of essential oils.Besides, essential oils containing eugenol as the major constituents showed low contact toxicity to Phyllaphis fagi (Linnaeus), either 29 .Due to the degradation under sunlight and unbalanced spatiotemporal distribution of botanical insecticides when applied by foliar spraying, they might show sublethal effect to R. padi.Vania et al. found that exposed to the recommended dosage of botanical insecticide, eucalyptus oil, garlic extract, neem oil, and rotenone, decreased the rate of walking activity in adult workers of honey bees 30 .Zhang et al. 31 reported that the sublethal concentrations of beta-cypermethrin showed negative impacts on R. padi fitness by decreasing pre-adult survival rate and delayed the development of some stages.Here, the low-lethal concentrations (LC 10 and LC 30 ) of rotenone or pyrethrins also showed obvious negative effect on the development and fecundity of the F0 generation, which was consist with most sublethal effects of chemical insecticides to R. padi, including dinotefuran 32 , pirimicarb 33 ; thiamethoxam 34 .Although botanical insecticides degraded shortly after application and not persisted in the environment or even insect, the negative effect of rotenone and pyrethrins showed transgenerational effect in the F1 generation.This might be related with the embryonic manner of aphids.An aphid has its own embryo since it is born.The embryos of F0 generation were exposed to the low-lethal concentrations of rotenone or pyrethrin with their parental generation.The development duration of nymph in the F1 generation was increased, but the longevity of adult and fecundity was decreased.This might be a fitness cost of insect resulted from the consumption of energy to manage the insecticide suppression and leading to a shortage of energy for productivity.
The sublethal effects of insecticides to the life history and fecundity of individuals might also affect population dynamics 22 .Here, the R 0 , r m , λ, GRR decreased significantly after exposed to low-lethal concentrations of rotenone and pyrethrins, while the value of T showed no obvious change.These results suggested that the low-lethal concentrations of rotenone or pyrethrins could suppress the population growth.This phenomenon is similar with the sublethal effect of flonicamid to Schizaphis graminum (Rondani) 35 .Exposed to the low-lethal concentrations (LC 5 and LC 10 ) of flonicamid decreased the adult longevity, the fecundity and key demographic parameters (R 0 , r, and λ) in the F1 generation significantly, and also decreased the total duration of feeding on phloem and concurrent salivation in the F0 and F1 generation of S. graminum 35 .However, multiple studies reported a hormesis effect after exposed to the sublethal concentrations of chemical insecticides.Wang et al. 36 reported that exposed to the sublethal concentrations of sulfoxaflor caused hormesis effects on A. gossypii feeding, growth, reproduction behavior, including increasing the fecundity, R 0 in the F1 generation, and phloem feeding.Exposed to the sublethal concentrations of sulfoxaflor also showed positive effect on the population resurgence of R. padi 37 .Similar effect in R. padi was reported after exposed to dinotefuran 38 , beta-cypermethrin to A. glycines 32 , thiamethoxam to A. gossypii 39 , and flupyradifurone to A. craccivora 40 .Our results indicated that botanical insecticide rotenone and pyrethrins were not easy to cause pest resurgence and much appropriate for R. padi control.
To achieve the reduction and enhanced efficiency of chemical pesticides, and prolonged the control period of botanical insecticide to R. padi, synergism combinations containing botanical insecticide and chemical insecticide were screened.Thiamethoxam was suggested to be the best partner to be combined with rotenone or pyrethrins.Mixtures containing rotenone and thiamethoxam or pyrethrins and thiamethoxam both showed synergism effect at most ratios.The co-toxicity factors were also the highest for the mixtures containing thiamethoxam.The LC 50 value of thiamethoxam to R. padi was 16.447 mg/L in this study.According to the susceptibility base line to thiamethoxam (3.6 mg/L) from Gong et al 41 , the R. padi population used in this study have developed low resistance to thiamethoxam, which might be accompanied with the fitness cost of energy consumption.Rotenone is an energy inhibitor.The combined application of rotenone could inhibit the energy production and might thus increase its susceptibility.Besides, the target of thiamethoxam, rotenone and pyrethrins are all different and they are suitable to be combined applied.Mixtures of pyrethrins and bifenthrin showed less synergism ratios than the other mixtures, which might be related to their similar target of Na + channel.These results provided suitable combinations for R. padi control and could delay the resistance to thiamethoxam.
In conclusion, among the 10 tested botanical chemicals, rotenone and pyrethrins were most valuable to control R. padi when applied by foliar spraying.Exposed to the low-lethal concentrations of rotenone or pyrethrins showed negative effect on the development, fecundity, and population growth.Compared with the chemical insecticides with hormesis effect at sublethal concentrations, rotenone and pyrethrins were much more appropriate for R. padi control.Mixtures containing rotenone and thiamethoxam, pyrethrins and thiamethoxam at ratios 1:5, 1:2, 1:1 all showed synergist effect.

Insects and Chemicals
The R. padi population was provided by the pesticide institute of Henan Institute of Science and Technology.They were kept on wheat seedling without exposure to insecticides and reared in insectary at 22-25 °C, 60-70% relative humidity, 14:10 h of light:dark photoperiod.

Bioassay
The toxicity of insecticides to R. padi was determined by leaf-dip method.Briefly, the stock solution (10,000 mg L −1 ) was prepared using the technical grade insecticides dissolved in acetone, except for matrine, which was dissolved in water.Then, a total of 5-8 diluted concentrations of insecticides were prepared using 0.1% Tween 80.A total of 15 adults of R. padi with almost the same size on wheat leaves were dip in the insecticide work solution for 10 s and dried in the air.The treated R. padi were transferred to plastic cup laid with 2% agar solution (10 mL) and covered with cling film to prevent escape.Each concentration was conducted in triplicate.The mortality of R. padi was checked 24 h and 48 h after treatment.Application of 0.1% Tween 80 was used as control.

Sublethal effect determination of rotenone and pyrethrins
A total of 30 R. padi adults (≤ 24 h old) on fresh wheat leaves was treated with rotenone or pyrethrins at LC 10 or LC 30 by dipping in the insecticide solution for 10 s.R. padi treated with 0.1% Tween 80 was used as control.The survived adults of R. padi (the F0 generation) after treated for 24 h were transferred to the wheat leaves without insecticides treatment and reared in petri dish individually.The fresh wheat leaves were replaced every three days and each treatment included 30 R. padi adults.The new born R. padi (the F1 generation) was checked twice (9:00 a.m. and 21:00 p.m.) every day, and removed timely.The fecundity of the F0 generation was recorded until the R. padi adults died naturally.The lifespan of the F0 generation R. padi was calculated.
The F1 generation nymph of R. padi (≤ 24 h old) laid at the same day under the same low-lethal concentrations was selected and transferred on the fresh wheat leaves without insecticides treatment.Each nymph was reared individually and each treatment included 30 R. padi nymphs.The fecundity of the F1 generation and their lifespan were recorded as the description above.

Screening of the synergistic mixture containing botanical insecticide and chemical insecticide
The LC 50 value of each insecticide in bioassay section was calculated using SPSS 16.0 (SPSS, Chicago, IL, U.S.A.).The synergistic mixtures to R. padi were screened using the co-toxicity factor method 42 .The stock solution of insecticides was diluted to LC 50 and 1/2 LC 50 using 0.1% Tween 80. Insecticides were mixed well with the volume ratio at 1:5, 1:2, 1:1, 2:1, 5:1, respectively.The adults of R. padi were treated with the mixture of insecticides as described in bioassay section.The mortality of adults was recorded 24 h after treatment.The co-toxicity factor was calculated using the following formula:

Statistical analysis
The significant difference of life history and fecundity among the LC 10 , LC 30 groups and the control group of R. padi from F0 and F1 generations were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's Honestly Significant Difference (HSD) test using SPSS 16.0 (SPSS, Chicago, IL, U.S.A.).P < 0.05 was regarded to be statistically significant.Raw data of life history and fecundity of the F1 generation R. padi were analyzed according to the age-stage, two-sex life table 43,44 using TWOSEX-MSChart 45 .The life history parameters (the age-specific survival rate (l x ), the age-specific fecundity (m x ), the age-specific maternity (l x m x ), the life expectancy (e xj ), the age-stage specific reproductive value (V xj )), and means and standard error (SE) of population parameters (mean generation time (T), net reproductive rate (R 0 ), intrinsic rates of natural increase (r m ), finite rate of population increase (λ), gross reproduction rate (GRR)) were estimated using 100,000 bootstraps.A paired bootstrap test was performed using TWOSEX-MSChart to compare the differences of population parameters among the low-lethal concentrations (LC 10 and LC 30 ) groups and the control group.The curves of l x , m x , l x m x , e xj and V xj were constructed using SigmaPlot

Figure 1 .
Figure 1.Age-specific survival rate (l x ), fecundity (m x ), net maternity (l x m x ) of R. padi exposed to control (A), LC 10 (B) and LC 30 (C) of rotenone.

Figure 4 .
Figure 4. Age-specific survival rate (l x ), fecundity (m x ), net maternity (l x m x ) of R. padi exposed to control (A), LC 10 (B) and LC 30 (C) of pyrethrins.

Table 1 .
Toxicity of botanical insecticides and chemical insecticides to R. padi.
a Time after treatment.b Indicated data not available.

Table 2 .
The LC 10 and LC 30 values of rotenone and pyrethrins to R. padi.

Table 3 .
The sublethal effect of rotenone on the lifespan and fecundity of F0 generation of R. padi.

Table 4 .
The sublethal effect of rotenone on the longevity and mean fecundity of adults in F1 generation of R. padi.Different lowercase letters in a column indicated significant differences analyzed by the one-way ANOVA with Turkey's HSD (P < 0.05).

Table 7 .
The sublethal effect of pyrethrins on the longevity and mean fecundity of adults in F1 generation of R. padi.Different lowercase letters in a column indicated significant differences analyzed by the one-way ANOVA with Turkey's HSD (P < 0.05).

Table 8 .
The sublethal effect of pyrethrins on the population parameters in F1 generation of R. padi.Different lowercase letters in a column indicated significant differences analyzed by the one-way ANOVA with Turkey's HSD test (P < 0.05).