Sublethal effect and detoxifying metabolism of metaflumizone and indoxacarb on the fall armyworm, Spodoptera frugiperda

doi:10.21203/rs.3.rs-3312587/v1

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Sublethal effect and detoxifying metabolism of metaflumizone and indoxacarb on the fall armyworm, Spodoptera frugiperda

https://doi.org/10.21203/rs.3.rs-3312587/v1

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published 29 Feb, 2024

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The fall armyworm (FAW), Spodoptera frugiperda, is a highly polyphagous invasive pest that damages on various crops. Pesticide control is the most common and effective strategy to control of FAW. In this study, we evaluated the toxicity of metaflumizone and indoxacarb against third-instar FAW larvae using the insecticide-incorporated artificial diet method under laboratory conditions. Both metaflumizone and indoxacarb exhibited substantial toxicity against FAW, with LC₅₀ values of 2.43 and 14.66 mg/kg at 72 h, respectively. The sublethal effects of metaflumizone and indoxacarb were investigated by exposing FAW third-instar larvae to LC₁₀ and LC₃₀ concentrations of these insecticides. Sublethal exposure to these two insecticides significantly shortened larval and adult developmental times, extended pupal developmental times, and led to reduced pupal weight, pupation rates, and adult fecundity in the treated parental generation at LC₁₀ or LC₃₀ concentrations, in comparison to the control group. We also assessed he transgenerational sublethal effects, and the findings indicated that metaflumizone and indoxacarb had comparable effects on the F0 generation, except for an observed significant increase in larval developmental time in the F1 generation. Furthermore, Larvae exposed to LC₁₀ or LC₃₀ concentrations of indoxacarb exhibited elevated activity levels of Multifunctional oxidase (MFO) and glutathione S-transferase (GST), which coincides with the observed synergistic effect of PBO and DEM. In conclusion, the high toxicity and negative impact of metaflumizone and indoxacarb on FAW provided significant implications for the rational utilization of insecticides against this pest.

Spodoptera frugiperda

fitness costs

sublethal effects

detoxification enzyme

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a polyphagous pest that damages multiple crops, and caused significant yield losses in many countries (Sun et al. 2021). FAW is native to tropical and subtropical regions of the Americas, and has invased to most provinces in China, which covering the major corn farming regions and threatened corn production (Wan et al. 2021). Chemical control, one of the most common and effective strategy to control of FAW, is recommended primarily in China. Unfortunately, the intensive selection pressure of insecticides promotes the development of field resistance of FAW to a variety clasess of synthetic insecticides, namely organophosphates (chlorpyrifos, profenofos) (Yu 1991; Zhang et al. 2021), carbamate (methomyl) (Yu 1991), pyrethroids (deltamethrin and lambda-cyhalothrin) (Belay et al. 2012; Zhao et al. 2020; Zhang et al. 2021), spinosyn (spinetoram and spinosad) (Lira et al. 2020; Zhang et al. 2021), diamides (chlorantraniliprole) (Boaventura et al. 2020), and even the transgenic Bacillus thuringiensis (Bt) corn (Banerjee et al. 2017).

Switching between pesticide classes with distinct modes of action, as well as employing lower insecticide concentrations, is an efficient strategy to manage insecticide resistance. Therefore, it is necessary to evaluate the effect of new chemical insecticides on control of FAW. In this study, we selected two voltage-dependent sodium channel blockers, metaflumizone and indoxacarb for use in FAW management due to their quite distinct chemical structures and potential in integrated pest management schemes. Metaflumizone is a novel semicarbazone insecticide that binds preferentially to the slow-inactivated sodium channel, causing flaccid paralysis and, finally, death in the affected insects (Salgado and Hayashi 2007). Metaflumizone has been effectively used for control of a wide range of pests, including the beet armyworm, Spodoptera exigua (Tian et al. 2014), and the diamondback moth, Plutella xylostella (Shen et al. 2017). Metaflumizone displayed excellent insecticidal efficacy against FAW, and binary combinations of metaflumizone and other insecticides had a synergistic effect on FAW larvae, suggesting that it might be used to manage FAW resistance (Wu et al. 2023). Indoxacarb is a novel oxadiazine insecticide with high toxicity against a wide range of Lepidopteran pests, as well as several Homoptera and Coleoptera (Wing et al. 2000). Indoxacarb can be converted to N-decarbomethoxylated metabolite (DCJW), a more effective sodium channel blocker than indoxacarb, which blocks the transport of sodium ions in nerve cells, resulting in paralysis and death of the target pest species (Wing et al. 2010).

During the pesticide application process, due to the varying sensitivities of different insects to insecticides and factors such as the decline in pesticide concentration over time, insecticides not only exhibit lethal effects on target pests but also show sublethal effects on some resistant target pests and non-target pests (Müller 2018; Müller et al. 2019). The sublethal effect refers to the physiological and behavioral impacts on insects when exposed to certain doses of insecticides, compared to healthy individuals, without causing mortality (Desneux et al. 2007; Guedes et al. 2017). When pests are exposed to sublethal doses of insecticides over an extended period, the offspring of resistant individuals will reproduce in large numbers. Through prolonged accumulation of resistance, the entire pest population will develop resistance to the insecticide, thus leading to a resurgence of certain pests (Müller 2018). For example, Nandihalli et al. found that the use of sublethal doses of imidacloprid and chlorpyrifos resulted in increased resistance in the cotton aphids, Aphis gossypii, leading to their resurgence and even outbreaks (Nandihalli et al. 1992). Sublethal doses of insecticides have a significant impact on the physiological metabolism of insects, primarily by inducing or inhibiting the insect's enzyme systems, which mainly involves detoxifying enzymes such as Multifunctional oxidase (MFO), Glutathione S-transferase (GST) and Carboxylesterase (CarE) (Liu 2015). The enhanced detoxification metabolism of the key metabolic enzymes in insects is an important mechanism for the development of insect resistance to insecticides (Liu 2015).

Understanding of the physiological and biological parameters of FAW following sublethal exposure to metaflumizone and indoxacarb is crucial for the development of long-term fall armyworm management programs. In this study, we examined the lethal and sublethal effects of metaflumizone and indoxacarb on various physiological and biological parameters of FAW, including development time, fecundity, fertility, and alterations in detoxification enzymes.

Insects and insecticides

The strain of fall armyworm (FAW) used in our study was collected from a corn field in Huangshan City, Anhui Province, China, in May 2019. The FAW larvae were reared on an artificial diet at an indoor temperature of 27 ± 1℃, with a relative humidity (RH) of 70 ± 10% and a photoperiod of 16:8 h (light: dark). Subsequently, the emerged adults were transferred to oviposition cages equipped with a reticulate for oviposition and were fed with 10% honey solution for adult nutrition.

Metaflumizone (96%) was supplied by Jingbo Agrochemicals Technology Co., Ltd. (Binzhou, Shandong, China), and indoxacarb (99%) was provided by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

Bioassay of metaflumizone and indoxacarb to FAW

The toxicity of metaflumizone and indoxacarb to FAW larvae was conducted by using an artificial diet-incorporated method as described previously (xxx et al). Metaflumizone and indoxacarb were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentrate of 10000 mg/L. The required serial dilutions of metaflumizone (37.5, 75, 150, 300 and 600 mg/L) and indoxacarb (500, 1000, 2000, 3000, and 4000 mg/L) were further obtained by stock solution serially diluted with DMSO. Then, the serial dilutions of metaflumizone and indoxacarb were thoroughly mixed with the artificial diet with the weight of 1:99 to prepare insecticide incorporated artificial diets. The FAW third-instar larvae were fed with insecticide incorporated diets and reared individually in a 12-well plates. The artificial diet incorporated with the same quality of DMSO solution was set as the control group. Each treatment was conducted in triplicate with 12 larvae per replicate. on the artificial diet treated with the different The mortality of larvae were recoded after 72 h treatment, and insects that unable to move when gently touched with afine brush were counted as dead larvae.

Sublethal effects of metaflumizone and indoxacarb on the treated generation of FAW

We used the artificial diet-incorporated method described above to expose 2 h-starved FAW third-instar larvae with two sublethal doses, LC₁₀ or LC₃₀ of metaflumizone and indoxacarb. The control was an artificial diet mixed with DMSO. There were 360 larvae (30 replicates of 12 larvae each) for each of the two sublethal doses and DMSO control. After 72 h treatment, the surviving larvae of each dose or control were individually transferred to a clean 12-well plates and provided with insecticide-free artificial diet. The survival rate and development time were recorded every 12 h. The female and male pupae were separated and weighed individually. The pupation rates and adult emergence rates were recorded after pupation and adult eclosion, respectively. The duration of pupae and adult were recorded every 12 h. The newly emerged female and male adults were placed in a plastic cup and supplied with a 10% sugar solution for nutrition. Fresh corn leaves were provided in the plastic cup for breeding and egg-laying. The fecundity and longevity of male and female adults were recorded every 12 h. The egg hatching rate was calculated by recording hatched neonate larvae from 100 radomly collected FAW eggs.

Sublethal effects of metaflumizone and indoxacarb on the offspring generation of FAW

To investigate the transgenerational effects of sublethal metaflumizone and indoxacarb, 200 neonates were randomly selected from each treatment of the above experiment. The larvae were raised individually in a 12-well plates and provided with artificial diets daily. The developmental time of larvae, pupae, and adults were recorded every 12 h. The pupal weight, pupation rate, emergence rate, fecundity and egg hatching rate were recorded as above. To develop a life table for FAW, data was collected for every life stage and evaluated using the theory of age-stage two sex life table (Chi 1988). The study included life table parameters such as Net reproductive rate R₀=∑l_x m_x, mean generation time T=∑l_x m_x x/∑l_x m_x, the intrinsic rate of increase R_m= lnR₀/T, finite rate of increase λ = e ^Rm. In the life table, x represents the age of the cohort, l_x represents the proportion of individuals surviving to age x, m_x represents age-specific fecundity (Tuan et al. 2014).

Synergism assay

To analysis the effects of synergists on toxicity of metaflumizone and indoxacarb, a suitable concentration of 100 mg/L synergists (DEM, TPP and PBO) that would have no efect on larval mortality was selected based on preminiraly experiment. Corn leaves treated with that concentration of each synergist were applied to third instars for 12 h, and then larvae were assayed for toxicity against metaflumizone and indoxacarb according to the insecticide-incorporated diet bioassay method as described above. The synergism ratio (SR) was determined by dividing the LC₅₀ of insecticide alone by the LC₅₀ of insecticide with synergist.

Detoxifying enzyme activity assays

The effect of sublethal doses of metaflumizone and indoxacarb on the activities of detoxitying enzymes were evaluated by treating FAW third-instar larvae with LC₁₀ and LC₃₀ of these two insecticides. The larvae treated with DMSO was set as control. Larvae were sampled at four time points (12, 24, 48 and 72 h) after treatment. The crude enzyme solutions were prepared by homonizing larvae using a 0.1 M phosphate buffer with pH 7.6, which containing 1 mM EDTA, 1 mM DTT, 1 mM PTU, 1 mM PMSF and 20% glycerol. The procedure of homonization was generated on ice. The homogenate was subsequently centrifuged at 4 ℃, 10, 000 ×g for 15 min to obtain supernatant, which was served as crude enzyme solution and immediately used for mixed function oxidase (MFO), glutathione-S-transferase (GST), esterase (EST) assays and total protein content assays.

The enzyme activities of MFO, GST, and CarE were measured using an insect MFO ELISA kit (Feiya, Yancheng, Jiangsu, China), a GST activity assay kit (Jiancheng, Nanjing, Jiangsu, China), and a CarE activity kit (Jiancheng), respectively, following the manufacturer’s instructions. Total protein content was determined by the Bradford method using bovine albumin as a standard (Bradford 1976).

Statistical analysis

The LC₅₀ values with corresponding 95% confidence limits (C.I.), chi-square (χ2), and slopes of the log-dosage probit mortality lines were calculated using probit regression analysis with IBM SPSS Statistics 22.0 software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by the Duncan test (P < 0.05) was utilized to analyze differences among treatments.

Toxicity of metaflumizone and indoxacarb to FAW larvae

The toxicity of hexaflumuron and indoxacarb on third-instar larvae of FAW were determined by a diet-incorporated assay. The LC₅₀ of metaflumizone and indoxacarb was 2.43 and 14.66 mg/kg at 72 h, respectively (Table 1), according to the toxicity assay. The estimated LC₁₀ and LC₃₀ values were 0.73 and 1.49 mg/kg for metaflumizone, and were 5.99 and 10.16 for indoxacarb at 72 h, respectively (Table 1). The obtained LC₁₀ and LC₃₀ values were further used to evaluate the sublethal and transgeneration effects of metaflumizone and indoxacarb on the growth parameters of FAW.

Table 1

**Toxicity of metaflumizone and indoxacarb against FAW third-instar larvae.**
Insecticides	LC₁₀ (mg/kg) (95% CL)	LC₃₀ (mg/kg) (95% CL)	LC₅₀ (mg/kg) (95% CL)	Slope ± SE	Chi-square	df
Indoxacarb	5.99 (3.71ཞ7.98)	10.16 (7.52ཞ12.42)	14.66 (11.92–17.37)	3.30 ± 0.47	0.73	3
Notes: CL, SE, and df indicate confidence limits, standard error, and degrees of freedom, respectively.

Sublethal effects of metaflumizone and indoxacarb on biological parameters of the FAW F0 generation

The sublethal concentrations of metaflumizone or indoxacarb (LC₁₀ and LC₃₀) had considerable effects on the longevity and fecundity of test individuals (F0 generation) of FAW (Fig. 1–3). The longevity of FAW larvae, female and male adults were significantly reduced when treated with LC₁₀ and LC₃₀ of metaflumizone, as compared to the control (Fig. 1A, C&D). While for indoxacarb, the longevity of female and male adults were significantly reduced at LC₃₀ treatment, as compared to the control (Fig. 1C&D). However, the duration of pupal stage were significantly prolonged after being treated with LC₁₀ and LC₃₀ doses of metaflumizone or indoxacarb, as compared to the control (Fig. 1B).

The pupation rates were significantly lower in the LC₃₀ treatments, but had no significant differences in the LC₁₀ treatments of metaflumizone and indoxacarb, as compared to the control (Fig. 2A). Moreover, the female and male pupal weights were significantly decreased after being treated with LC₃₀ of metaflumizone or indoxacarb (Fig. 2B&C). Meanwhile, Significant reduction in the male pupal weight was also observed in the LC₁₀ treatment of metaflumizone (Fig. 2C).

There was no statistically significant difference was found in emergence rates of adults when FAW larvae were exposed to sublethal doses of metaflumizone and indoxacarb (Fig. 3A). The mean number of eggs per female and egg hatching rate were significantly affected by sublethal metaflumizone or indoxacarb exposure. LC₁₀ and LC₃₀ doses of metaflumizone treatments reduced the mean number of eggs per female by 45.48% and 56.73% in comparison to controls, respectively. LC₁₀ and LC₃₀ doses of indoxacarb treatments reduced the mean number of eggs per female by 51.27% and 58.36% in comparison to controls, respectively (Fig. 3B). In additional, the egg hatching rate was reduced significanlty after being treated with sublethal doses of metaflumizone indoxacarb, and was decreased as the increased insecticide concentration (Fig. 3C).

Sublethal effects of metaflumizone and indoxacarb on biological parameters of the FAW F1 generation

The sublethal concentrations of metaflumizone or indoxacarb (LC₁₀ and LC₃₀) significantly prolonged the larval and pupal duration time (Fig. 4A&B). The larval duration were prolonged by 1.34 and 1.65 d for metaflumizone and 1.53 and 2.09 d for indoxacarb, after being treated with LC₁₀ and LC₃₀ doses, respectively, as compared to the control (Fig. 4A). Meanwhile, the pupal duration were prolonged by 0.81 and 0.58 d for metaflumizone and 0.47 and 0.65 d for indoxacarb, after being treated with LC₁₀ and LC₃₀ doses, respectively, as compared to the control (Fig. 4B). While the duration of female and male adults were significantly reduced at both LC₁₀ and LC₃₀ treatments of two insecticeides (Fig. 4C&D). Compared to the control, LC₁₀ and LC₃₀ doses of metaflumizone reduced female duration by 1.46 and 1.69 d and reduced male duration by 1.63 and 2.13 d, respectively. For indoxacarb, LC₁₀ and LC₃₀ treatment reduced female duration by 1.12 and 2.06 d and reduced male duration by 1.04 and 1.97 d, respectively.

After being treated with sublethal doses of metaflumizone and indoxacarb, the pupation rate was only significantly inhibited in the LC₃₀ group of metaflumizone, whereas there was no significant difference in other treatments, as compared to the control (Fig. 5A). The weight of female pupae were significantly decreased after being treated with LC₃₀, but not influenced by LC₁₀ of metaflumizone and indoxacarb (Fig. 5B). Significant reduction in the male pupal weight was only observed in the LC₃₀ treatment of metaflumizone (Fig. 5C).

The exposure of FAW F0 larvae to sublethal doses of metaflumizone and indoxacarb had no statistically significant difference to the emergence rates of F1 adults (Fig. 6A). The fecundity of F1 adults and egg hatching rates were significantly reduced by sublethal exposure of FAW F0 larvae to metaflumizone or indoxacarb. LC₁₀ and LC₃₀ doses of metaflumizone treatments reduced the mean number of eggs per F1 female by 57.51% and 65.87% in comparison to controls, respectively. LC₁₀ and LC₃₀ doses of indoxacarb treatments reduced the mean number of eggs per female by 60.15% and 67.57% in comparison to controls, respectively (Fig. 6B). In additional, the egg hatching rate of F1 were 64.22% and 49.98% in the LC₁₀ and LC₃₀ group of metaflumizone, and were 65.3% and 54.7% in the LC₁₀ and LC₃₀ group of indoxacarb, respectively, which were reduced significanlty as compared to 89.98% of the control (Fig. 6C).

Sublethal effects of metaflumizone and indoxacarb on population growth parameters of the FAW F1 generation

The population growth parameters of the FAW F1 generation were significantly affected by the treatment of F0 third-instar larvae with subletal doses of metaflumizone and indoxacarb (Table 2). The mean generation time (T) was significantly prolonged, while the Net reproductive rate (R₀), Intrinsic rate of increase (r_m) and Finite rate of increas (λ) were significantly decreased at both sublethal concentrations of metaflumizone and indoxacarb. Noteblely, The R₀ of the control group was 3.08 and 4.16 times to that of metaflumizone group, and was 3.21 and 4.32 times to that of of indoxacarb group, after treated with LC₁₀ and LC₃₀ dose, respectively.

Table 2

Effects of sublethal concentrations of metaflumizone and indoxacarb on life table parameters of FAW F1 generation
Parameters	Treatments
Parameters	CK		Metaflumizone LC₁₀		Metaflumizone LC₃₀	Indoxacarb LC₁₀	Indoxacarb LC₃₀
Net reproductive rate (R₀)		1065.44 ± 25.09 a		346.39 ± 13.24 b	256.01 ± 45.10 b	332.00 ± 50.34 b	246.70 ± 30.00 b
Mean generation time (T)		28.33 ± 0.36 b		29.69 ± 0.16 a	29.51 ± 0.24 a	29.48 ± 0.35 a	29.74 ± 0.06 a
Intrinsic rate of increase (r_m)		0.25 ± 0.004 a		0.197 ± 0.002 b	0.187 ± 0.008 b	0.196 ± 0.005 b	0.185 ± 0.004 b
Finite rate of increas (λ)		0.67 ± 0.01 a		0.54 ± 0.006 b	0.51 ± 0.02 b	0.53 ± 0.02 b	0.502 ± 0.01 b

Effects of synergists on the toxicity of metaflumizone and indoxacarb to FAW

The effects of three representative synergists, PBO, DEM, and TPP on activities of metaflumizone and indoxacarb against FAW third-instar larvae was shown in Table 3. The synergism tests showed that PBO, DEM, and TPP had minor synergism effects on metaflumizone with synergistic ratio (SR) values of 1.18, 1.12 and 1.01, respectively. There was a weak synergism effect of PBO and DEM on indoxacarb toxicity with SR values of 1.73 and 1.56, respectively.

Table 3

Toxicity of metaflumizone and indoxacarb with synergists against FAW third-instar larvae
Insecticides	Synergist	LC₅₀ (mg/kg) (95% confidence limits)	Slope ± SE	χ²(df)	Synergistic ratio (SR) ^a
Metaflumizone	PBO	2.06 (1.13–2.73)	4.36 ± 0.67	5.50 (3)	1.18
	DEM	2.17 (1.37–2.81)	3.97 ± 0.62	4.34 (3)	1.12
	TPP	2.40 (1.31–3.35)	3.65 ± 0.59	5.62 (3)	1.01
Indoxacarb	PBO	8.47 (6.11–10.66)	2.43 ± 0.38	2.62 (3)	1.73
	DEM	9.38 (7.14–11.52)	2.74 ± 0.40	2.28 (3)	1.56
	TPP	21.12 (14.48–28.72)	3.38 ± 0.55	3.47 (3)	0.69

^a Synergist ratio (SR) = LC₅₀ of insecticide to FAW third-instar larvae divided by LC₅₀ of insecticide to FAW third-instar larvae after leaf treatment with feeding synergist.

Detoxification enzyme activities of FAW response to metaflumizone and indoxacarb treatment

The activities of mixed function oxidase (MFO), glutathione S-transferase (GST), and carboxylsterase (CarE) were determined after treated with subulethal dose of both insecticides at several time points (12, 24, 48, and 72 h) (Fig. 7). The MFO activities were significantly inhibited following exposure to LC₃₀ of metaflumizone for 12, 24 and 72 h, while increased significantly for 48 h. The LC₁₀ of metaflumizone inhibited MFO activities at only 72 h (Fig. 7A). For the indoxacarb treatment, both LC₁₀ and LC₃₀ dose significantly increased MFO activities at almost all tested time points, except for LC₁₀ dose reduced MFO activities at 12 h (Fig. 7D). The GST activities were increased significantly following exposure to both LC₁₀ and LC₃₀ dose of metaflumizone or indoxacarb at almost all tested time points, in comparison to controls, except for no changes of GST activities observed at metaflumizone exposure for 12 h (Fig. 7B&E). Statistically significant change in CarE activities were only observed in insects that were exposed to metaflumizone for 24 h, which were reduced in both LC₁₀ and LC₃₀ treatments, as compared to the control (Fig. 7C&F).

This study assessed the toxicity of metaflumizone and indoxacarb to third-instar larvae of FAW. The results revealed that metaflumizone exhibited higher toxicity against FAW compared to indoxacarb, with respective LC₅₀ values of 0.73 and 5.99 mg/kg, respectively (Table 1). In India, indoxacarb is not so effective control FAW compared with other insecticides according to the field experiment (Worku and Ebabuye 2019). In China, susceptibility of FAW to indoxacarb showing 10.0-fold difference, with LC₅₀ values varied from 3.952 mg/L (Yichang) to 40.148 mg/L (Yuhang), across the various geographic populations (Zhao et al. 2020). Although metaflumizone and indoxacarb belong to the voltage-dependent sodium channel blockers, while a field populations of P. xylostella, with various levels of resistance to indoxacarb, did not have cross-resistance to metaflumizone (Khakame et al. 2013). Similarly, one field population (HZ11) of S. exigua developed 942-fold resistance to metaflumizone, but only 16-fold resistance to indoxacarb (Su and Sun 2014). Collectively, these research findings indicate that metaflumizone exhibits a higher level of toxicity against FAW compared to indoxacarb, and was a potential candidate strategy for management of FAW.

Insecticides at different sublethal concentrations can leading to distinct impacts on the growth, development, and reproduction of insects, thus resulting in fluctuations in population numbers. These effects are typically manifested in changes in parameters such as larval developmental period, male and female pupal weights, pupation rate, pupal duration, eclosion rate, male and female adult lifespans, and egg production (Desneux et al. 2007; Guedes et al. 2016). In the present study, both sublethal doses of metaflumizone and indoxacarb had considerable effects on the development and fecundity of FAW. We observed a significant reduction in the duration of larval stage and adult longevity, and lower in pupation rate, pupal weight, egglaying number, and hatching rate of FAW after treated with LC₁₀ or LC₃₀ of metaflumizone and indoxacarb (Fig. 1–3). These findings suggest that sublethal concentrations of metaflumizone and indoxacarb can effectively suppress the population growth and reproduction potential of FAW. Similarly, in Plutella xylostella, the pupation rate, pupal weight and adult emergence rate was significantly decreased and pupal period extended at LC₁₅ and LC₂₅ of metaflumizone (Zhang et al. 2012). At the same time, in Helicoverpa amigera, sublethal concentrations of indoxacarb results in a significant reduction in pupal weight, pupation rate, adult longevity, and fecundity in comparison to controls (Vojoudi et al. 2017). The negative effect of sublethal doses of metaflumizone or indoxacarb on insect growth and reproduction have reported in various pests (Ling et al. 2013; Wanumen et al. 2016; Rajab et al. 2022), as well as benefit natural enemies and non-target aquatic invertebrates (Galvan et al. 2005; Garzón et al. 2015; You et al. 2016; Wang et al. 2023).

In addition to sublethal effects of treated larvae, the LC₁₀ and LC₃₀ concentrations of indoxacarb or metaflumizone had negative transgenerational effects to the F1 progeny of FAW. Sublethal doses of indoxacarb and metaflumizone can significantly prolonged larval and pupal duration time, decrease adult life span, reduce pual weight and pupation rate, and heavily suppress adult fecundity of FAW in F1 progeny (Fig. 4–6). The negative impact of sublethal concentrations of insecticides to F1 generation has been documented in various insects, such as cyantraniliprole to Helicoverpa assulta (Dong et al. 2017), fluxametamide and spinetoram to Plutella xylostella (Tamilselvan et al. 2021; Gope et al. 2022), flupyradifurone to Aphis gossypii (Liang et al. 2019), chlorantraniliprole and beta-cypermethrin to FAW (Wu et al. 2022). The alterations in biological characteristics offer insight into how sublethal amounts of insecticides negatively impact individual insects. Meanwhile, using demographic parameters that rely on life history factors and other indicators of population growth rate has been suggested as a preferable method for assessing the overall impact of pesticides on insect populations (Stark and Banks 2003). In the present study, the demographic parameterssuch as intrinsic rate of increase (r_m), finite rate of increase (λ) and net reproductive rate (R₀) of the F1 generation in the sublethal treatments were significantly lower, while Mean generation time (T) was significantly increased, as compared to the control group (Table 2). The findings suggest that sublethal doses of metaflumizone and indoxacarb can decelerate the population dynamics of FAW by decreasing their ability to reproduce and survive. The population parameters of FAW F1 generation, such as R₀, λ, and r_m were also found to be decreased in the sublethal exposure of emamectin benzoate (Zhang et al. 2023) and chlorantraniliprole (Akhtar et al. 2022), are consistent with the present findings.

The development of insecticide resistance is attributable to long-term exposurse of insects to low doses of pesticides, with the increased activation of detoxifying enzymes, such as P450s, GSTs, and CarEs, standing out as one of the key factor driving this resistance mechanism (Ishaaya 1993). Our results show that the activities of P450s and GSTs are increased after being expoursed to sublethal doses of metaflumizone and indoxacarb at assessed time points (Fig. 7). Noteblely, for indoxacarb treatments, the activities of P450s and GSTs are significantly upregulated at almost all time points after treated with LC₁₀ and LC₃₀ concentrations (Fig. 7D&E), indicating that both P450s and GSTs are playing possible roles in detoxifying indoxacarb. Our findings also demonstrate that the synergists PBO and DEM, with SR values of 1.73 and 1.56 respectively, substantially enhanced the effectiveness of indoxacarb (Table 3). On the other hand, concerning CarEs, the enzyme activities remained unaffected by both metaflumizone and indoxacarb treatments, aligning with the loss of synergistic effects (Table 3). This provides additional evidence that P450s and GSTs are potential enzymes engaged in the detoxification of indoxacarb. The maximum synergistic effects of PBO to indoxacarb were found to have a synergistic ratios at 4.9-fold among 18 Hainan populations in China (Liu et al. 2022). In previous studies, it was reported that the increased activity of the P450 enzyme conferred indoxacarb resistance in H. armigera (Cui et al. 2018). Additionally, PBO decreased resistance in the strain selected for indoxacarb, implying that metabolic detoxification enzymes likely played a role in indoxacarb resistance in H. armigera (Bird 2017). Collectively, these results provide a valunable information for understanding the possible indoxacarb resistance mechanisms in FAW.

In conclusion, both metaflumizone and indoxacarb exhibited high acute toxicity against third-instar larvae of FAW. Exposure to sublethal doses of these insecticides had adverse effects on reproductive parameters and biological traits in both the parental and F1 generations. Additionally, the activities of detoxification enzymes such as P450s and GSTs were notably stimulated following exposure to sublethal doses of indoxacarb. This aligns with the observed synergistic impact of PBO and DEM, indicating the potential involvement of P450s and GSTs in the detoxification process of indoxacarb. The cumulative sublethal impacts of metaflumizone and indoxacarb on biological and biochemical factors offer valuable insights into FAW management. With the observed decrease in offspring population under sublethal stress from metaflumizone or indoxacarb, it can be deduced that these two insecticides hold substantial promise as a pesticide rotation strategy for FAW control.

Acknowledgements

This work was supported by National Natural Science Foundation of China (grant No. 32001931 and 32102223), the Natural Science Foundation of Anhui Province (grant No. 2308085MC89), and the Natural Science Foundation of Jiangxi Province (grant No. 20224BAB215019).

Author contributions

Conceived and designed the experiments: CW-S YC-P and HQ-C. Performed the experiments: L-Z and HZ-W performed toxicity determination and enzyme activity assays, PY-H and WN-Z analyzed the data. Wrote and revised the manuscript: CW-S and YC-P.

Conflict of interest declaration

The authors declare that there is no conflict of interests regarding the publication of this article.

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Sublethal effect and detoxifying metabolism of metaflumizone and indoxacarb on the fall armyworm, Spodoptera frugiperda

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Abstract

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Introduction

Materials and methods

Insects and insecticides

Bioassay of metaflumizone and indoxacarb to FAW

Sublethal effects of metaflumizone and indoxacarb on the treated generation of FAW

Sublethal effects of metaflumizone and indoxacarb on the offspring generation of FAW

Synergism assay

Detoxifying enzyme activity assays

Statistical analysis

Results

Toxicity of metaflumizone and indoxacarb to FAW larvae

Sublethal effects of metaflumizone and indoxacarb on biological parameters of the FAW F0 generation

Sublethal effects of metaflumizone and indoxacarb on biological parameters of the FAW F1 generation

Sublethal effects of metaflumizone and indoxacarb on population growth parameters of the FAW F1 generation

Effects of synergists on the toxicity of metaflumizone and indoxacarb to FAW

Detoxification enzyme activities of FAW response to metaflumizone and indoxacarb treatment

Discussion

Conclusion

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References

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