Ecotoxicology and Environmental Safety 161 (2018) 208–213
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Influence of lethal and sublethal exposure to clothianidin on the sevenspotted lady beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae) Jiangong Jianga, Zhengqun Zhanga, Xin Yub, Dicheng Maa, Caihong Yuc, Feng Liua, Wei Mua,
T
⁎
a
College of Plant Protection, Key Laboratory of Pesticide Toxicology & Application Technique, Shandong Agricultural University, Tai’an, Shandong 271018, China Research Center of Pesticide Environmental Toxicology, Shandong Agricultural University, Tai'an 271018, Shandong, China c College of Chemistry and Environment Engineering, China University of Mining and Technology (Beijing), 100083 Beijing, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Coccinella septempunctata Clothianidin Sublethal effects Life cycle NOER
The seven-spotted ladybird beetle, Coccinella septempunctata L., as a dominant predator of aphids, has played a crucial role in integrated pest management (IPM) strategies in agricultural ecosystems. To study the risk of insecticides to C. septempunctata, the neonicotinoid clothianidin was selected for evaluation of its influence on C. septempunctata at lethal and sublethal doses. The LR50 (application rate causing 50% mortality) in the exposed larvae decreased from 19.94 to 5.91 g a.i. ha−1, and the daily HQ (hazard quotient) values increased from 3.00 to 10.15, indicating potential intoxication risks. We also determined NOERs (No Observed Effect application Rates) of clothianidin on the total developmental time (10 g a.i. ha−1), survival (2.5 g a.i. ha−1) and pupation (5 g a.i. ha−1). Moreover, clothianidin at a NOER of 2.5 g a.i. ha−1 did not profoundly affect adult emergence, fecundity or egg hatchability. The total effect (E) assessment also showed that clothianidin at 2.5 g a.i. ha−1 was slightly harmful to C. septempunctata. These results suggested that clothianidin would impair C. septempunctata when applied at over 2.5 g a.i. ha−1 in the field. Conservation of this biological control agent in agricultural ecosystems thus requires further measures to decrease the applied dosages of clothianidin.
1. Introduction Within most agricultural ecosystems, insect pest management typically relies on chemical insecticidal sprays and biological control by natural enemies (Desneux et al., 2007). However, the extensive use of pesticides to control pests may seriously harm natural enemies, and pesticides have various sublethal effects on the physiological and behavioral processes in these beneficial arthropods (Desneux et al., 2007). Integrated pest management (IPM) programs suppress the pest population below the economic threshold while conserving limited biological control agents (Cook et al., 2007; Naranjo et al., 2015). Compatibility for the combination of pesticides and biological control agents, such as predators and parasitoids, meets the demands of ecologically and economically sound IPM tactics. The pesticides applied in IPM pest control strategies would be of low risk to a wide variety of natural enemies (Youn et al., 2003; He et al., 2012). Therefore, a precise preliminary assessment of the negative impacts of such pesticides on biological control agents is urgently necessary to develop sustainably effective IPM strategies (Desneux et al., 2006, 2007). The distribution of the seven-spotted ladybird beetle, Coccinella septempunctata L. (Coleoptera: Coccinellidae), is generally
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acknowledged to be widespread over agricultural and natural habitats worldwide (Stark et al., 2007). Both the larvae and adults of C. septempunctata are considered highly omnivorous, feeding on many species of insect pests (e.g., Aphidoidea, Tetranychidae, Psylloidea, and Coccoidea) that infest crops in both greenhouses and fields (Volkl et al., 2007; Lu et al., 2012; Hodek and Michaud, 2013). C. septempunctata has thus been used as a major biological control agent for suppressing various aphids in agroecosystems (Bianchi and Werf, 2004; Landis and Fox, 2004). Furthermore, C. septempunctata can be an ideal model test organism for studying the side effects on non-target natural enemies as a segment of the registration process for pesticides because of the convenience and efficiency of the rearing process (MAPRC, 2017). Biological control is the potential pillar of IPM systems, but chemical controls are still the major and most effective interventions for combating pests in China (Naranjo et al., 2015; Yao et al., 2015). In cotton and wheat crops, neonicotinoid insecticides were the most widely used for inhibiting the aphid population growth rate at seedling stages due to their systemic action, diverse application methods, high efficiency and long persistence (Jeschke et al., 2011; Zhang et al., 2017). However, the potential negative impacts of neonicotinoids on pollinators and other non-target organisms have recently led to great
Correspondence to: College of Plant Protection, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong 271018, China. E-mail address:
[email protected] (W. Mu).
https://doi.org/10.1016/j.ecoenv.2018.05.076 Received 11 January 2018; Received in revised form 22 May 2018; Accepted 25 May 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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ingredient) L−1 and decreasing by halves to the following five concentrations (20, 10, 5, 2.5, 1.25 mg a.i. L−1) to mimic the half-life degradation of pesticides under actual field conditions. Acetone was used for the blank controls. A total of 580 μL of the same clothianidin solution was added to each glass culture tube (12 cm height × 1.5 cm diameter; plugged with cotton balls). Afterwards, these tubes were immediately rotated on a micro-rotator (Verly Co., Ltd., Guangzhou, China) until all the solvent evaporated to dryness. Then, thirty 2ndinstar larvae were introduced to every replicate and fed daily with 30 mixed instars of A. craccivora, with each treatment and blank control consisting of three replicates. The mortality of C. septempunctata was identified and recorded when individuals were unable to respond to a mild touch using a paint brush at daily intervals throughout the entire C. septempunctata generation (He et al., 2012). In the long-term experiment, the detailed periods of different life stages were also recorded at daily intervals. Emerged adults were collected and paired at a 1:1 ratio. Each pair was then removed to a 30 × 20 × 10 cm plastic cage containing sufficient A. craccivora as food, and the data on fecundity (i.e., the number of eggs produced) were recorded every 24 h until the death of the female parent. Dead males were removed immediately after identification. All the collected eggs in each treatment were transferred to another climate-controlled room under the conditions described above. The egg hatchability proportion was calculated by counting the number of larvae derived from each replicate group. The entire generation extended from the 2nd-instar stage of the first generation to the counterparts of the next generation. The development from egg hatching to the next 2nd-instar stage took approximately 5 days in the long-term observation.
concern about their environmental and ecological impacts (Elbert et al., 2008; Cloyd and Bethke, 2011; Tirello et al., 2013). Non-target natural enemies might be negatively affected not only by direct contact with spray droplets and residues of neonicotinoid insecticides but also by ingestion of contaminated plant material and/or prey (Yao et al., 2015). Neonicotinoid exposure could entail both acute toxic effects and sublethal effects on the feeding behavior, development, longevity, and reproduction of natural enemies (Desneux et al., 2007; Cloyd and Bethke, 2011). Sublethal effects can lead to the inability of natural enemies to control pests, resulting in incompatibility between the incorporation of natural enemies and the use of insecticides in IPM strategies, causing more harm than the lethal effects on natural enemies from a demographic perspective (Desneux et al., 2007; Yao et al., 2015). Clothianidin, as a second-generation neonicotinoid insecticide with a substituted chlorothiazolyl-methyl group, has been registered for managing a broad spectrum of insect pests (e.g., Coleoptera, Diptera, Lepidoptera and Hemiptera) on approximately 40 crops in at least 34 countries worldwide (Jeschke et al., 2011; Uneme, 2011). In China, clothianidin is registered mainly against piercing-sucking insect pests such as planthoppers, thrips, whiteflies and aphids (China Pesticide Information Network, 2017). To date, most studies have primarily evaluated the lethal effects of clothianidin on various parasitoids and predators, showing that clothianidin at the recommended label rates was directly harmful to these natural enemies (Cloyd and Dickinson, 2006; Cloyd et al., 2009; Sugiyama and Saito, 2011; Prabhaker et al., 2011). Little information is currently available on the long-term influence of clothianidin on C. septempunctata. Hence, to proceed to the compatible usage of clothianidin and C. septempunctata for pest management under realistic field conditions, an overall risk assessment on the clothianidin exposure on C. septempunctata was critical and necessary. Here, we identified the LR50s (application rates causing 50% mortality) and NOERs (No Observed Effect application Rates) from chronic exposure for the larvae of C. septempunctata in laboratory microcosms. Favorable results would promote the conservation of ladybird beetles and provide available references for optimizing the use of clothianidin as a component of effective IPM strategies in agricultural ecosystems.
The ecological risk of clothianidin to C. septempunctata was determined by the hazard quotient (HQ) method. The HQ value is calculated by dividing the field-recommended label rates of clothianidin by the LR50 of clothianidin to C. septempunctata acquired from the laboratory study (Candolfi et al., 2001). Ratios of greater than or equal to 2 highlight clothianidin as a potential hazard to C. septempunctata. Ratios of less than 2 indicate a low intoxication risk.
2. Materials and methods
2.5. Statistical analysis
2.1. Insecticides
The LR50 was determined by a log-probit regression analysis with SPSS v.17.0 (SPSS Inc., Chicago, IL). The NOER values were estimated by means of a one-way analysis of variance (ANOVA). Means were compared by Tukey's least significant difference (LSD) tests (P < 0.05) (Zar, 1996). The total developmental time and survival rates over different instars were analyzed with a repeated-measures ANOVA to examine differences within the treatment groups. The total effect (E) was estimated using the formula E (%) = 100 (100-Mc) × ER (Overmeer and Zon, 1982), where Mc represents the corrected mortality of C. septempunctata in the group treated by each dosage of clothianidin; ER is the ratio of the mean weekly number of eggs laid by females in each group versus those laid by females in the control. According to the IOBC laboratory scale (Hassan, 1994) and on the basis of their total effects, the different dosages of clothianidin were classified in the following toxicity categories: (1) harmless (< 30%); (2) slightly harmful (30–79%); (3) moderately harmful (80–99%); and (4) harmful (> 99%).
2.4. Risk assessment
Technical-grade clothianidin (98.1% purity) was obtained from Veyong Bio-Chemical Co., Ltd., Hebei, China, with no greater purity available. A 100 mL stock solution (1000 mg/L) was prepared by dissolving 0.1019 g clothianidin with 100 mL HPLC-grade acetone. 2.2. Test species The test organisms for the start of the trial were the 2nd-instar larvae of C. septempunctata from a laboratory-reared colony. The adults and larvae at four different stages were reared on black bean aphids, Aphis craccivora Koch, which were maintained on fresh seedlings of broad beans Vicia faba L. under laboratory conditions of 20 ± 1 °C, 50–70% relative humidity (RH) and a 16:8 (light:dark) photoperiod. The hatchability of the eggs and the growth of the first-instar larvae were evaluated based on growth in a climate-controlled incubator at 25 ± 1 °C, 70% RH and 16:8 (L:D) photoperiod.
3. Results 2.3. Bioassays 3.1. Influence of clothianidin on the survival rate of C. septempunctata The toxicity experiment was performed with a previously designed laboratory method (C. Yu et al., 2014). The six tested concentrations of clothianidin in this study were chosen on the basis of a preliminary 72 h acute toxicity experiment, (Table S1), starting with 40 mg a.i. (active
The effects of six concentrations of clothianidin on the survival percentage of larvae of C. septempunctata are presented in Fig. 1. The survival rate of the larvae in the control averaged 96.7% at the end of 209
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Fig. 1. Survival rates of C. septempunctata larvae at six different dosages (g a.i. ha−1) of clothianidin. Data are expressed as the mean values ± SE (standard error), n = 3. (ANOVA, Tukey's LSD, P < 0.05).
Fig. 2. Estimated LR50 and HQ values for the first-generation larvae of C. septempunctata in response to clothianidin. Error bars correspond to the margins of 95% confidence intervals (95% CI). HQ (Hazard quotient) values were calculated as the recommended field rate (g a.i. ha−1) divided by the LR50 (g a.i. ha−1) of C. septempunctata. In China, the maximum recommended field rate of clothianidin for controlling aphids is 60 g a.i. ha−1.
Table 1 Repeated-measures ANOVA parameters for survival rate and developmental time of C. septempunctata over the instar stages treated with six different concentrations of clothianidin (Tukey's LSD test). Source
Treatment Stage Treatment × stage
df
6 3 18
Survival rate
Developmental time
F
P values
F
P values
106.78 18.14 2.91
0.0001 0.0001 0.0012
13.04 504.48 0.82
0.0001 0.0001 0.0218
periods of C. septempunctata exposed to 20 and 40 g a.i. ha−1 of clothianidin were 32.07 and 30.48 days, respectively, and were higher than the control. The cumulative mortality calculated for the treatments over 1.25 g a.i. ha−1 differed significantly from the control (Table 2). The mean weekly number of eggs laid by individual female C. septempunctata was 222.40. The mean fecundity of females was greatly decreased in the groups treated with clothianidin at dosages above 2.5 g a.i. ha−1. (ANOVA, P < 0.05; NOER = 2.5 g a.i. ha−1). Overall, the results of the total effect (E) in Table 2 indicated that clothianidin at 1.25 g a.i. ha−1 was harmless (IOBC class 1). The doses of 2.5 and 5 g a.i. ha−1 were classified as slightly harmful (IOBC class 2) to C. septempunctata. Additionally, the three highest levels, 10, 20 and 40 g a.i. ha−1, were evaluated as moderately harmful (IOBC class 3).
the observation period. The mortality of C. septempunctata treated with clothianidin at 10, 20 and 40 g a.i. ha−1 rapidly declined from the 2nd day following treatment, which is the end of the 2nd-instar stage of C. septempunctata. After the 3rd day following treatment, the survival rates of C. septempunctata in the three highest concentrations were all below 50%, and all were significantly different from the control. The survival rates in the test treatments of 10, 20 and 40 g a.i. ha−1 ultimately remained 13.3%, 16% and 26%, respectively from the 13th day after treatment to the last day of the experiment. At the tested rate of 5 g a.i. ha−1, the mortalities of C. septempunctata were close to 50% beginning on the 12th day. Dose-response effects of clothianidin were observed on the survival of C. septempunctata at the rate of 2.5 g a.i. ha−1, though statistically significant differences could not be demonstrated (ANOVA, P < 0.05). Significant differences in survival rates were found between the concentrations over a lifetime (Table 1). The estimated LR50 for the first-generation larvae was 15.10 g a.i. ha−1 at 72 h after treatment in the long-term test, and it gradually declined to 5.91 g a.i. ha−1 during the next observation periods of the larval stage. The daily HQs for the larvae individual ranged from 3.00 to 10.15, all greater than 2, which is the level of concern (Fig. 2).
3.3. Influence of clothianidin on pupation, adult emergence and egg hatchability of C. septempunctata Regression analysis revealed significantly negative linear relationships between the dosages of clothianidin and the pupation rate (R2 = 0.97, F = 8.162, df = 6,14, P < 0.001), adult emergence (R2 = 0.94, F = 6.335, df = 6,14, P = 0.0022) and egg hatchability (R2 = 0.96, F = 36, df = 6,14, P < 0.001) of C. septempunctata. As the dosage increased, the pupation rate of C. septempunctata gradually decreased to 61.2%. The pupation rates of C. septempunctata treated with clothianidin at the three highest dosages (10, 20 and 40 g a.i. ha−1) were significantly lower than in the control (ANOVA, P < 0.05; NOER = 5 g a.i. ha−1) (Fig. 3A). The adult emergence rate of C. septempunctata in the untreated groups was significantly higher than in the groups treated with clothianidin at 5, 10, 20 and 40 g a.i. ha−1 (ANOVA, P < 0.05; NOER = 2.5 g a.i. ha−1) (Fig. 3B). The mean egg hatchability of C. septempunctata in the control treatment was 96.1%. The egg hatchability of C. septempunctata treated with clothianidin at 40 g a.i. ha−1 was 59.7%, which was significantly lower than the other clothianidin treatment groups and the control (ANOVA, P < 0.05; NOER = 2.5 g a.i. ha−1) (Fig. 3C).
3.2. Influence of clothianidin on the developmental time, egg production and total effect (E) of C. septempunctata After being treated with clothianidin, the durations of the 2nd- and 3rd-instar larval stages of C. septempunctata were significantly longer in the groups treated at dosages above 1.25 g a.i. ha−1 than in the controls (ANOVA, P < 0.05, NOER = 1.25 g a.i. ha−1). The durations of the 4th-instar larval and pupation stages under the 5, 10, 20 and 40 g a.i. ha−1 treatments were significantly longer than those in the control group (ANOVA, P < 0.05, NOER = 2.5 g a.i. ha−1). Compared with the control group, 10, 20 and 40 g a.i. ha−1 of clothianidin significantly lengthened the adult preoviposition period of C. septempunctata. (ANOVA, P < 0.05, NOER = 2.5 g a.i. ha−1). The oviposition period was significantly shortened at doses above 2.5 g a.i. ha−1 (ANOVA, P < 0.05, NOER = 2.5 g a.i. ha−1). In total, the entire developmental
4. Discussion The LR50 of clothianidin to C. septempunctata eventually decreased from 19.94 to 5.91 g a.i. ha−1 owing to the cumulative larval mortality caused by the potent activity of clothianidin against C. septempunctata throughout the larval stages (Uneme, 2011). C.H. Yu et al. (2014) also found that the LR50 of imidacloprid to C. septempunctata decreased from 210
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One life cycle covered from the 2nd-instar larvae in the first generation to the emergence of the 2nd instar in the subsequent generation. Proportion of dead larvae, pupae and adults that failed to emerge. c IOBC toxicity category for laboratory experiments in accordance with the total effect caused by clothianidin: (1) harmless (< 30%); (2) slightly harmful (30–79%); (3) moderately harmful (80–99%); and (4) harmful (> 99%).
27.16 to 4.07 g a.i. ha−1 during the larval periods, indicating that this neonicotinoid had potential risks to the ladybirds. Therefore, neonicotinoids such as clothianidin and imidacloprid may have equivalent degrees of toxicity and risk to C. septempunctata. Furthermore, in C. septempunctata, clothianidin was more toxic to larvae than to adults, possibly due to variations in enzyme activity and target-site sensitivity in response to insecticides (Cho et al., 2002). Clothianidin at dosages above 10 g a.i. ha−1 extended the duration of larval development of C. septempunctata, which was in accordance with the influence of sublethal concentrations of imidacloprid on some ladybird species (He et al., 2012; C.H. Yu et al., 2014; Xiao et al., 2016). This may occur for two reasons: 1) clothianidin had a negative impact on the feeding behavior of C. septempunctata and 2) larval energy is diverted for detoxification processes rather than being used for growth and development (Desneux et al., 2007; Simelane et al., 2008). Some studies have shown that neonicotinoids such as thiamethoxam and thiacloprid decreased predators’ foraging time and predation rate (Martinou et al., 2014; Yao et al., 2015). It remains to be determined which factor is responsible for prolonging the larval stage. Briefly, clothianidin may cause a decline in survival and delay the larval development of C. septempunctata by direct residual contact. Neonicotinoid insecticides at the recommended field dosages may exhibit high toxicity risks to ladybeetles. For example, Yao et al. (2015) reported that the HQ value yielded 7.52 after a 24-h exposure of thiamethoxam to Serangium japonicum adults, demonstrating high toxicity risks. In terms of our study, the obtained dynamic HQ values increased as the contact duration increased, demonstrating that long-term exposure of clothianidin to C. septempunctata larvae may induce unacceptable risks. In China, the common registered usage of clothianidin spray ranges between 24 and 60 g a.i. ha−1, which is usually used at up to 10–15 applications per season to prevent infestation in serious cases (Zhang et al., 2017). Considering our results, the timing of foliar application should avoid times when substantial numbers of C. septempunctata larvae are present on target crops. In addition, if clothianidin is to be used, dosages below 30 g a.i. ha−1 are recommended because acceptable toxicity risks (HQ) to C. septempunctata can be reached under this condition. The side effects of pesticides on biological control agents can also be evaluated by several relevant ecological endpoints (Desneux et al., 2007). The NOER endpoints represent the safety margins of individual chemical substances on non-target organisms in risk assessment procedures (Min et al., 2012). Clothianidin may decrease the pupation percentage and extend the pupal time of C. septempunctata when the application dosages exceed the NOER of 5 and 2.5 a.i. ha−1 estimated for pupation percentage and time, respectively. Meanwhile, clothianidin above the NOER of 2.5 g a.i. ha−1 could reduce the adult emergence rate of C. septempunctata. Similarly, hexaflumuron exceeding the NOER of 3.04 g a.i. ha−1 also significantly decreased the pupation percentage and lengthened the pupal duration of C. septempunctata (C. Yu et al., 2014). Here, we hypothesized that clothianidin may disrupt neurohormone transmission associated with molting and cuticle formation in C. septempunctata individuals. Additionally, clothianidin also reduced the adult emergence rate of C. septempunctata, a finding that may be attributable to deformities in the organs (e.g., trophi, brain and wings) at the pupal stages caused by the persistent influence of clothianidin (Desneux et al., 2007). Future studies should provide new insights into the basis of these phenomena. Neonicotinoid insecticides such as thiamethoxam, acetamiprid, and imidacloprid can strongly lower the fecundity (number of eggs) and fertility (egg hatchability) of ladybird predators (Papachristos and Milonas, 2008; Fogel et al., 2013; Shima and Ali, 2013; Fernandes et al., 2016). In our study, clothianidin above the tested rate of 2.5 g a.i. ha−1 significantly not only decreased the fecundity and fertility of C. septempunctata but also diminished the oviposition duration of female parents (NOER = 2.5 g a.i. ha−1). Some hypotheses can be proposed to explain this phenomenon. 1) The negative affect of clothianidin on the
b
a ab ab bc c bc bc 17.26 27.47 16.12 14.60 13.55 16.13 17.88 ± ± ± ± ± ± ± 222.40 208.13 204.14 188.85 173.98 180.94 181.88 6110 4.243 0.007 10.11 ± 0.19 f 21.1 ± 1.90 e 35.53 ± 3.86 d 51.1 ± 1.90 c 84.4 ± 3.81 b 89.96 ± 3.35 a 92.2 ± 1.90 a 6,14 476.489 0.0001 c c c c bc b a 1.30 1.15 2.18 1.61 1.90 1.33 1.66 27.66 ± 28.16 ± 29.23 ± 28.75 ± 29.73 ± 30.48 ± 32.07 ± 6220 5.794 0.0001 0.20 0.13 0.19 0.14 0.16 0.22 0.21
d d c c b ab a
2.32 ± 2.42 ± 2.57 ± 2.64 ± 2.67 ± 2.66 ± 3.03 ± 6364 29.757 0.0001
0.11 0.12 0.11 0.16 0.18 0.26 0.29
c c b b b b a
4.03 ± 4.19 ± 4.28 ± 4.64 ± 5.00 ± 5.26 ± 5.37 ± 6310 56.899 0.0001
0.22 0.18 0.23 0.24 0.23 0.36 0.46
d d d c b a a
5.03 ± 5.31 ± 5.19 ± 5.59 ± 5.80 ± 5.93 ± 5.69 ± 6291 12.109 0.0001
0.22 0.25 0.35 0.26 0.60 0.44 0.80
e de e cd ab a bc
8.99 ± 0.97 cd 8.93 ± 0.67 cd 8.48 ± 0.27 d 9.92 ± 0.80 bc 10.74 ± 1.44 ab 11.28 ± 0.96 a 11.69 ± 1.33 a 6110 6.487 0.0007
37.38 ± 36.16 ± 35.30 ± 35.01 ± 31.08 ± 31.90 ± 28.45 ± 6110 22.45 0.0001
1.32 1.30 1.15 1.98 1.82 1.69 2.23
a ab b b c c d
Entire generationa Oviposition Preoviposition Pupation 4th Instar 3rd Instar 2nd Instar
2.13 ± 2.10 ± 2.27 ± 2.31 ± 2.46 ± 2.51 ± 2.55 ± 6411 22.581 0.0001 Control 1.25 2.5 5 10 20 40 df F P
a
– 17.86 34.17 53.81 86.42 90.91 92.90 – – –
(1) (2) (2) (3) (3) (3)
Ec (IOBC Cat.) Fecundity (eggs/week) Cumulative lethalityb (%) Developmental time (d) Treatment (g a.i /ha)
Table 2 Influence of clothianidin on developmental time of different life stages, lethality and the reproduction of per female adult of C. septempunctata in the long-term microcosm test within different treatments. Data are presented as the mean values ± SD (standard deviation), n = 3. Different letters in the same column indicate values with significant differences from the corresponding control and other treatments based on ANOVA and Tukey's LSD test (P < 0.05).
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Fig. 3. Relationship between the concentrations of clothianidin and the pupation rate, emergence rate, and egg hatchability of C. septempunctata. Data are expressed as the mean values ± SE (standard error), n = 3. Different letters indicate significant differences among the treatments (ANOVA, Tukey's LSD, P < 0.05).
neurosecretory system of C. septempunctata may paralyze the muscle tissue of the parental reproductive tract. For instance, Moser and Obrycki (2009) reported that clothianidin induced significant neurotoxic symptoms, such as paralysis, trembling and loss of coordination, on the ladybeetle H. axyridis. 2) Clothianidin inhibiting specific types of nervous activity may also lower the predatory power and feeding efficiency of C. septempunctata, resulting in low reproductive capacity, as described by Xie et al. (2015), who reported that restricted food ingestion could lower the reproduction parameters of ladybeetle Cryptolaemus montrouzieri.
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