Sublethal effects of buprofezin on development and reproduction in the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae)

In the present study, the effects of sublethal concentrations of buprofezin on life-table traits of S. furcifera were evaluated for two consecutive generations (F0 and F1). Our results exhibited that the fecundity, life span (longevity) and hatchability of the F0 and F1 generations were significantly decreased at LC30 compared to the control. However, copulation was not significantly affected for the F0 or F1 generations at sublethal concentrations. The female life span was affected negatively at both treatments in F0 and at LC30 in F1, compared to the control. Furthermore, significant effects of the sublethal concentrations were found on the developmental rate of all instars except the 3rd instar of F1. However, the pre-adult period, total pre-oviposition period (TPOP) and adult pre-oviposition period (APOP) significantly increased in F1 individuals at LC30 and LC10 compared to the control. Our findings revealed that demographic characters (survival rate, intrinsic rate of increase (ri), finite rate of increase (λ), net reproductive rate (R0), and gross reproductive rate (GRR)) of the F1 generation (from F0 parents) significantly decreased compared to the untreated group; however, the generation time (T) increased at LC10. Therefore, the results suggested that buprofezin could adversely affect individuals in the successive generation.

Some scientists have suggested that the sublethal concentrations of pesticides might persuade insect outbreaks in field 23,24 . For instance, organophosphorus, pyrethroid, and organochlorine pesticides have been revealed to cause the resurgence of pests when insecticide contaminants degraded to near a low lethal level 23,24 . Some reports have found that sublethal concentrations of insecticides affect growth and increased the productivity and developmental duration in insect, for S. furcifera, the population growth was inhibited by sublethal concentration of triazophos, chlorantraniliprole and imidacloprid 25,26 . In various investigations, increased fecundity and survival time were also observed in M. persicae after treatment with sublethal concentrations of azadirachtin, imidacloprid 27 .
The use of two-sex life tables is one of the most important tools for investigating sublethal effects, particularly in life cycle studies, as it can highlight population effects that may be underestimated at the individual level [28][29][30] . The possible sublethal effects of buprofezin on S. furcifera have not yet been reported. In our study, for data interpretation two-sex life table was used to observe the sublethal effects of buprofezin, with a particular focus on the trans-generational effects on S. furcifera.

Results
Buprofezin toxicity against S. furcifera. The toxicity level of buprofezin to 3 rd instar S. furcifera is presented in Table 1; the estimated the LC 10 , LC 30 , LC 50 , and LC 100 values are 0.173 mg a.i. L −1 (95% CI from 0.0132 to 0.483 mg a.i. L −1 ), 0.847 mg a.i. L −1 (95% CI from 0.217 to 1.526 mg a.i. L −1 ), 2.541 mg a.i. L −1 (95% CI from 0.731 to 6.647 mg a.i. L −1 ), and 332.460 mg a.i. L −1 (95% CI from 81.71 to 12072.15 mg a.i. L −1 ), respectively. Finally, these concentrations LC 10 and LC 30 were used as the sublethal concentrations for further experiments. In order to evaluate the sublethal effects, 3 rd instar S. furcifera nymph were exposed to these sublethal concentrations of buprofezin, the 120-h mortality of nymphs were 6.616 ± 0.925, 12.21 ± 0.845 and 30.11 ± 1.352% for control, LC 10 and LC 30 (Table 2), fecundity, hatchability, emergence and longevity of S. furcifera F1 offspring produced by F0 parents treated with LC 10 and LC 30 buprofezin are shown in Fig. 1A-D. We found significant differences in fecundity, hatchability, longevity of female and emergence, for LC 30 compared to LC 10 and the control, whereas no significant effects on copulation were reported in the F1 offspring. Additionally, the developmental period of all instars (1 st to 5 th instar) of F1 individuals was significantly affected positively or negatively except for the 3 rd instar at LC 30 and LC 10 compared to the control ( Table 2). The overall nymph developmental period significantly affected in the treatments compared to the control. However, the pre-adult period, APOP (adult pre-oviposition period) and TPOP (total pre-oviposition period) in the offspring of the F0 parents was significantly increased compared to the control. The total male and female longevity in F1 offspring significantly decreased at LC 30 .
The trans-generational effects of the sublethal concentrations (LC 10 and LC 30 ) of buprofezin on population dynamics (Table 3) were calculated with bootstrap procedure based on a life cycle. The finite rate of increase (λ), intrinsic rate of increase (r i ), net reproductive rate (R 0 ) and gross reproduction rate (GRR) of F1 individuals significantly decreased in the LC 30 treatment, while the net reproductive rate and gross reproduction rate were not affected by the LC 10 . In contrast to LC 30 , LC 10 caused a significant increase in the mean generation time (T) of the exposed offspring.
The age-stage survival rate (s xj ) indicated the probability that a newly laid egg will survive to age x and stage j (Fig. 2). It represents variation in the developmental rate occurring among individuals, and the coinciding projected curves clearly showed the overlapping between the different stages for buprofezin (LC 30 ) and control. The plotted peaks for each developmental stage under LC 10 and control showed almost the same pattern, with the exceptions that the curves for male adults ended earlier than those for females, while the peaks of the plotted curves at LC 30 were not high as for the control. The peaks also showed that male and female exhibited the same survival rate for LC 10 and the control, while they exhibited a shortened survival period at LC 30 . The curves for the female and male adults showed that sexes emerged after 16 days in the buprofezin treatment but after 15 days in the control. Likewise, the age-specific survival rate (l x ) for the control and treatments is plotted in Fig. 3. It indicates that a similar l x pattern was observed for LC 10 and the control, while the age-specific survival rate (l x ) declined more in the LC 30 -treated populations' offspring at day 8 compared to the control. These results showed Treatment n a LC 10 mg a.i. L −1 (95% CI) b LC 30 mg a.i. L −1 (95% CI) LC 50 mg a.i. L −1 (95% CI) LC 100 mg a.i. L −1 (95% CI) Slope ± SE c X 2 (df) d that the probability of new-born nymphs surviving to the adult stage was 0.80 at LC 30 and 0.83 at LC 10, whereas it was 0.87 in the control. The age-stage reproductive values (v xj ) of the buprofezin treatments showed (Fig. 4) that the v xj of the LC 30 buprofezin treatment was lower compared to LC 10 and the control individuals in the 5 th instar stage. At this stage (5 th instar), the peak is sharper in cases of LC 10 and the control than for LC 30 . However, in case of emerging females, the plotted curve for LC 30 rose slightly higher and declined more rapidly compared to the control and LC 10 but increased in an additional curve at later ages in LC 10 treated individuals; the maximum v xj value 78 d −1 on 23 rd day at LC 10 , whereas it was 60 d −1 on the 22 nd day in the LC 30 group and 60 d −1 on the 19 th day in the control.

Discussion
This is the first study to evaluate the effects of sublethal exposure to buprofezin on the life cycle of S. furcifera. Buprofezin is an insect growth regulator and is highly effective against several sucking pests 31,32 .
Insecticides are usually distributed unequally as well as subjected to degradation after application in the field so the probability of targeted and non-targeted pests to low concentration of insecticides happens very often 11,33,34 , However, studies on the effects of sublethal concentrations of insecticides on target pests are of great importance to increase their rational use [35][36][37][38] . Therefore, buprofezin may also cause a wide range of sublethal effects to pests such as in S. furcifera. A thorough investigation of these possible effects would help to improve IPM in rice crops.
Lethal and sublethal effects of buprofezin have been studied in some arthropods, e.g Encarsia inaron (Hymenoptera: Aphelinidae) 31 , Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) 32 , Bemisia tabaci (Hemiptera: Aleyrodidae) 32 . Sublethal effects such as decreased fecundity, hatchability, longevity, and copulation could result in stimulatory effects on pest population growth 10 . In the present study, we investigated the sublethal effects of buprofezin for two consecutive generations and found a significant decrease in fecundity, hatchability, emergence and longevity of S. furcifera females in the F1 generation at LC 30 but not copulation, whereas in the F0 generation, a significant difference was found in fecundity and female longevity at LC 30 . Our results are in line to Zhou et al. 25 reported that sublethal concentrations of imidacloprid showed significant effects on the fecundity of S. furcifera.
Moreover, trans-generational effects on the F1 individuals of S. furcifera were also found. We found that the exposure to the LC 10 and LC 30 of buprofezin in the parent (F0) population significantly affected the F1 offspring growth rate, especially by increasing the duration of the pre-adult stage, TPOP and APOP and decreased the longevity, total longevity of males and females and fecundity at LC 30 and vice versa at LC 10 . These effects are related to reductions in the intrinsic rate of increase (ri), finite rate of increase (λ), net reproductive rate (R 0 ), gross reproduction rate (GRR) and survival rate. Such effects on offspring growth have also been reported in the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) and the small brown planthopper, Laodelphax striatellus (Homoptera: Delphacidae) through treatment with sublethal concentrations of sulfoxaflor and thiamethoxam 39,40 . Other insecticides, such as imidacloprid, chlorantraniliprole and triazophos have also lead to significant effects on the life cycle of the white-backed planthopper 23,26,41 . Although imidacloprid significantly affects the GRR in various cases, the generation time (T) was not affected in B. tabaci (Hemiptera: Aleyrodidae) 40 . Lashkari et al. 42 observed that sublethal concentrations have an effect on the mean generation time in B. brassicae (Hemiptera: Aphididae) when treated with imidacloprid. Shorabi et al. 32 also pointed out that biological character of B. tabaci have no significant effect at low concentration of imidacloprid and buprofezin. These reports revealed that insecticide concentrations effects life history and physiological state of the species 21,[35][36][37]43,44 .
The analysis of the plotted curves of the age-specific survival rate (lx) showed more decline at LC 30 compared to LC 10 and the control, indicating that the LC 30 of buprofezin is more effective. The age-stage reproductive value (v xj ) indicated that buprofezin at sublethal concentrations may significantly affect the duration of immature stage but have no significant effect on adult nymphs. This result could be interrelated to an invisible effect due to inhibition when treated with a sublethal concentration of buprofezin. Due to different physical and chemical processes, the pre-adult period, APOP, total pre-oviposition period (TPOP) and mean generation time (T) are    36,45 . In this study, we considered that hormesis is not the only important factor in terms of effects of buprofezin on S. furcifera. But all the biological processes may operate simultaneously after the exposure of arthropods to pesticides due to which they may develop, ultimately, hormesis and/or resistance responses against such chemicals 33,34,46 . Therefore, further research using numerous lethal and sublethal concentrations is needed to provide a detail evaluation report of nymphal responses to buprofezin in S. furcifera. In summary, this study found that sublethal concentrations of buprofezin affect the longevity and egg laying of parent (F0) individuals of S. furcifera and also cause some variation in the biological traits of the F1 generation of S. furcifera. However, non-stimulatory effects on copulation and hatchability were found in the F0 generation.
Overall, these observations of the present study under laboratory conditions emphasized the significance of assessing sublethal effects of the buprofezin on WBPH and to also determine how these effects may be interpreted to population dynamics in the field. Our research suggested the need to investigate further possible effects of buprofezin on WBPH in the aim to improve optimized IPM packages including this insecticide.

Materials and Methods
Insects and Insecticide. The WBPH (S. furcifera) population was initially sampled from rice fields in Xiaogan District Hubei Province, in 2014 and has been maintained on rice seedlings for 3 years at a temperature of 27 ± 1 °C, relative humidity (RH) of 70-80% and light/dark cycle of 16:8 h in a growth chamber in a laboratory at Huazhong Agricultural University without exposure to any insecticide. All experiments were performed in the above-mentioned growth chamber. Buprofezin (97.4%, technical grade) was purchased from Jiangsu Anpon Electrochemical Co., Ltd. China.

Bioassay.
A bioassay test was carried out using the rice seedling dipping method with slight modifications 46,47 .
Briefly, a stock solution of buprofezin (97.4%) was prepared in acetone and then serially diluted with water containing 0.1% Triton X-100 for five dilutions (16,8,4,2, and 1 mg a.i. L −1 ). Rice plants were collected at the seedling stage and washed thoroughly with water, and then air dried at room temperature to eliminate excess water. Fifteen rice seedlings were grouped together and dipped into the serially diluted buprofezin solution treatments for 30 seconds 25 . After the treated rice stems were air dried at room temperature, the rice roots were wrapped with moistened cotton. Then, these wrapped stems were placed in 500 ml plastic cup. Forty-five 3 rd instar nymphs were introduced into the plastic cups using a vacuum device. Distilled water containing 0.1% Triton-X was used as a control. Both control and treatments were replicated three times for each serial dilution. The treated and control insects were kept in a plant growth chamber maintained at a temperature of 27 ± 1 °C, RH of 70 ± 1% and a light/ dark cycle (L:D) of 16:8 h. Mortality was recorded after 120 h. Individual nymphs were considered dead if they did not show movement after being slightly pushed with a soft brush.

Evaluation of sublethal effects of buprofezin on life history traits of F0 S. furcifera. The suble-
thal effects of buprofezin on the life cycle parameters of S. furcifera were followed by Liu and Han method with slight modifications 47 . Approximately 800-1000 adult WBPH were transferred to a clean cage with fresh and healthy rice to lay their eggs upon. After 24 h, the rice seedlings were removed and placed in another cage; these rice seedlings were retained for a number of days for nymphs to hatch out of the eggs and until these nymphs had developed into the 3 rd instar. These 3 rd instar nymphs were used as F0 generation individuals. Approximately 200-300 3 rd instars were transferred to and reared separately in a glass tube containing rice seedlings dipped in a sublethal concentration (LC 10 and LC 30 ) of buprofezin. The live pests were collected after 5 days. The control pests were fed rice seedlings not treated insecticide. Each surviving pest was then transferred to a separate clean tube with a healthy rice stem, and each glass tube was numbered. As the nymphs became adult males and females, they were paired (40-60 per treatment) at once in a glass tube containing a single fresh rice seedling and kept under controlled temperature (27 ± 1 °C), RH (70 ± 1%) and light/dark cycle (16:8 h). The rice seedlings in each tube were changed every day during experiment. The fecundity and longevity of the couple was recorded, and measurements continued until the death of the couple. The experiment was repeated three times.  and kept in separate tubes under the control conditions as discussed previously. These offspring were fed rice stems, and the stage and condition of the pests were observed daily. This method was followed for both the buprofezin treated and control groups. When these nymphs become adults, they were paired as described above. The population characteristics, including developmental time, longevity, fecundity and hatchability were checked daily until the couple died. The newly born nymphs were counted and discarded. Then, the rice stems were thoroughly checked using a microscope, and the number of unhatched eggs was recorded. During this study, the following observations were noted; the developmental rate of each instar, the emergence of adults, the duration of the adult stage, mating, fecundity and hatchability. This whole experiment was repeated 3 times.

Effects
Statistical analysis. The Probit-MSChart 48 program was used for probit analysis of the concentration-response data. The raw data of the life table of each S. furcifera individual was analysed using the age-stage, two-sex life table procedure 28,29 . The basic life-table parameters, such as age-stage survival rate (s xj ), age-specific survival rate (l x ), reproductive value (v xj ), intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R 0 ) and mean generation time (T), were analysed using the computer program TWOSEX-MS Chart 49 . The variances and standard errors of the population growth parameters were calculated using the bootstrap technique included in TWOSEX-MS Chart with 100,000 random resampling. Developmental growth, adult longevity, total preoviposition period (TPOP), adult pre-oviposition period (APOP), fecundity and population parameters (r, λ, R 0 , and T) were compared using the paired bootstrap test based on the confidence interval of the differences. Therefore, the finite rate of increase (λ) and intrinsic rate of increase (r) are the most crucial parameters for determining the potential of population growth, and these have been used to represent the fitness of populations [50][51][52] . Survival rate and reproductive value curves were plotted using SigmaPlot 12.0 (Systat Software Inc., San Jose, CA).