Toxicity and Sublethal Effects of Fluxametamide on the Key Biological Parameters and Life History Traits of Diamondback Moth Plutella xylostella (L.)

Abstract

Fluxametamide, a novel isoxazoline insecticide, is newly registered for the control of various lepidopteran, coleopteran and thysanopteran insect pests on lethal and sublethal levels. In the present study, the toxicity and sublethal effects of fluxametamide on diamondback moth Plutella xylostella (L.), an invasive lepidopteran foliage feeder of cruciferous vegetables, were assessed to explore its bio-ecological impact on pest populations. The toxicity of fluxametamide to the third instar larvae of P. xylostella was 0.18 mg L⁻¹ (LC₅₀) at 72 h bioassay. After treatment with LC₁₀ and LC₃₀ concentrations of fluxametamide, the fourth instar larval duration, the rate of deformed pupa and adults, and the adult pre-oviposition period were significantly increased, whereas the pupation rate and pupal weight were significantly decreased in the F₀ generation. In the F₁ generation, sublethal effects of fluxametamide were indicated by a reduced fecundity, rate of pupation and adult emergence, pupal weight, and adult longevity, however a significant increase in eggs and total larval duration, deformed adults rate, and total longevity and pre-oviposition period was observed in the offspring. The intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R₀) of sublethal treatments were significantly lower than the control. The relative fitness of F₁ was 0.68 and 0.64 in LC₁₀ and LC₃₀ treatments, respectively. The LC₃₀ fluxametamide treatment exhibited increased glutathione S-transferase activities (elevated 1.433-fold) in P. xylostella. Our results suggest that in addition to its high lethal toxicity, the sublethal concentrations of fluxametamide might suppress the reproduction, development and survival of the P. xylostella population and its progeny, which can help to optimize integrated pest management program.

1. Introduction

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a widely distributed and economically important oligophagous pest of cruciferous vegetable crops [1,2]. Presently the control of P. xylostella is mainly dependent on chemical insecticides. However, the injudicious application of insecticides has caused P. xylostella to rapidly develop resistance to almost all classes of old fashioned insecticides [3,4,5] as well as varying degrees of resistance to novel chemicals, such as chlorantraniliprole [6], spinosad [7], emamectin benzoate [8] and spinetoram [9]. Besides, owing to its high fecundity and short life span, very few insecticides are currently available that remain effective in controlling P. xylostella in India [9]. Therefore, new active compounds with unique mechanisms of action are urgently required to participate in insecticide resistance management programmes.

Fluxametamide is a novel isoxazoline insecticide that differs chemically from fiproles, avermectins and diamides. It has a new target site for arthropods, interfering with GABA Cl⁻ and Glu Cl⁻ channels [10,11], and is listed in group 30 in the IRAC mode of action classification [12]. Fluxametamide has an exceptional insecticidal activity on a range of insect pest species, such as lepidoptera, thysanoptera, coleoptera and diptera [13] and the advantage of negligible non-target toxicity, including pollinators [10,14]. The unique binding site of fluxametamide in GABA-gated chloride channels, different from those for existing antagonists, makes this molecule effective against fipronil-resistant pest populations [15]. After Japan, Australia and China, fluxametamide has been launched in India very recently and is expected to be used effectively to control various lepidopteran insects. Besides lethal effects, the sublethal effects must be considered while making an insecticide selection [16,17]. The effect of sublethal exposure to an insecticide could impair the growth, developmental rate, feeding, fecundity, and various physiological and biochemical processes of an insect pest, and sometimes causes the rapid evolution of insecticide resistance [18]. The extensive application of fluxametamide in the coming years may cause sublethal effects on P. xylostella gradually. Therefore, estimation of the toxicity and sublethal effects of fluxametamide on P. xylostella is very important for coordinating chemical and biological control strategies in an integrated pest management program. However, there is no report on the sublethal effects of fluxametamide on P. xylostella, and very little literature is available for their comparison. Previous studies reported that fluxametamide has excellent efficacy against Musca domestica, Laodelphax striatellus, Drosophila melanogaster, Tetranichus urticae and Cylas formicarius [10,19]. On the other hand, the sublethal concentrations of various insecticides such as chlorantraniliprole, metaflumizone, fenoxycarb, spinosad, spinetoram and broflanilide significantly reduced the longevity and fecundity of P. xylostella [20,21,22,23,24]. The life-table bioassay is a precise tool to estimate the effect of sublethal exposure to an insecticide on major biological factors and demographic traits of an insect population in the laboratory [25,26]. In addition to the pest’s biology, the sublethal effect of a new chemical on the biochemistry of a target insect must be considered for a comprehensive study [27]. These pieces of information can be crucial in decision-making on pest management by predicting the possible impacts that are likely to occur in field strains.

The present study aimed to evaluate the toxicity and baseline susceptibility of a novel isoxazoline insecticide fluxametamide against third instar larvae of a susceptible population of P. xylostella. Additionally, the age-stage and two-sex life table methods were used to analyze the demographic parameters of P. xylostella treated by sublethal concentrations (LC₁₀ and LC₃₀) of fluxametamide, with a particular focus on the potential toxicological and possible transgenerational effects in the subsequent generations. The activities of cytochrome P450 monooxygenase, glutathione S-transferase and esterases were also quantified to assess the biochemical responses and metabolic enzyme-mediated detoxification mechanisms to sublethal concentrations of fluxametamide which could provide crucial data for the field application guidelines of this insecticide to control P. xylostella in the future.

2. Materials and Methods

2.1. Insect Population and Insecticide

A P. xylostella strain with no history of insecticide exposure for more than 60 generations was initially purchased from the ICAR-NBAIR (National Bureau of Agricultural Insect Resources), Bangalore, Karnataka, India in 2016 and was maintained in the laboratory for 5 years in insecticide-free conditions. Larvae were fed cabbage leaves (Brassica oleracea var. capitata L.), while adult moths were provided with 10% w/v honey solution and kept at 25 ± 1 °C, 70–80% RH and a photoperiod of 16:8 h light: dark. Fluxametamide (purity > 98%) used in this study was obtained from Med Chem Express, Suite Q, Monmouth Junction, NJ 08852, USA through Allianz BioInnovation, Mumbai, India.

2.2. Toxicity Bioassay

The standard leaf-dip bioassay method [28] was adopted by taking newly molted third instar larvae for the toxicity bioassay of fluxametamide. The stock solution of fluxametamide was prepared by dissolving in dimethyl sulfoxide (DMSO) and the working solutions were made through serial dilutions in double-distilled water containing 0.1% Triton X-100. Bioassays consisted of four replicates per concentration with nine different concentrations (0.0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, and 1.0 mg L⁻¹). Fresh cabbage leaves were clipped into 6 cm diameter spherical discs and dipped in different test concentrations for 20 s. Then, the treated leaf discs were allowed to dry naturally at room temperature for 1 h and placed individually in a Petri dish (9-cm-diameter) lined with moistened filter paper (Whatman no. 1). Leaf discs that were emerged in sterile solutions only were served as controls. Twenty early third-instar larvae for each concentration were placed in a Petri dish and sealed with a nylon mesh net. Thus, a total of 720 larvae were used in the concentration-mortality bioassay. Mortality was recorded after 72 h of insecticide exposure. Individuals that were 30% shorter than those of the control treatment or larvae that did not show any coordinated movement after probing with a soft camel-hair brush were assumed to be dead [29].

2.3. Sublethal Effects of Fluxametamide on Development of P. xylostella F₀

Based on the toxicity bioassay, LC₁₀ and LC₃₀ concentrations were selected to investigate the sublethal effects of fluxametamide on P. xylostella. A total of one hundred and twenty newly molted 3rd instar larvae were treated with sublethal concentrations of fluxametamide (LC₁₀ or LC₃₀) or the sterile diluents only (control) on cabbage leaf discs for 72 h, using the standard leaf-dip bioassay method described previously. Fifteen larvae were defined as one replicate with eight replicates per concentration. After 72 h, larval mortality was recorded and the surviving larvae of each treatment were shifted individually to a plastic container (10-cm-height; 2.5-cm-diameter) containing fresh cabbage leaves. The larvae were monitored daily until pupation and the cabbage leaves were replaced when required. The number of larvae that pupated and deformed pupae were recorded regularly. The pupation and deformed pupa rate were enumerated as well. Additionally, the pupae were weighed individually within 24 h. On the first day of adult emergence, twenty mating pairs for each treatment were respectively released into a glass bottle (12-cm-depth; 6.5-cm-diameter) containing 10% honey solution, which was changed at an interval of 48 h. Subsequently, the rate of adult emergence, deformed adults and female rates were calculated. The eggs laid on the cabbage leaves were recorded daily and replaced at 24 h intervals until the death of the female moth. The longevity of adult males and females, and the pre-oviposition period (APOP) were also recorded.

2.4. Transgenerational Effects of Sublethal Dose of Fluxametamide on P. xylostella F₁

To examine whether fluxametamide has carryover activity on the F₁ generation, 100 eggs from each treatment (LC₁₀, LC₃₀ and control) of the previous experiment were used to study the life table parameters. For easy observation, five newly hatched F₁ larvae were transferred to each plastic container (10-cm-height; 2.5-cm-diameter) containing fresh cabbage leaves and kept under the aforesaid controlled conditions. Cabbage leaves were replaced every 24 h. The developmental duration of F₁ larvae and subsequent stages, and their related survivorship, were recorded. Pupal weight was taken individually and the pupation rate, deformed pupa rate and adult emergence rate were recorded as above. Upon emergence, newly emerged F₁ adult pairs were placed in a glass bottle as described earlier. The fecundity and survival rate of F₁ adults were recorded daily and the pupal sex was differentiated after the adult emergence.

2.5. Detoxification Enzyme Assay

2.5.1. Cytochrome P450 Monooxygenase

Cytochrome P450 monooxygenase (P450) activity was assayed according to the method demonstrated by Aitio [30] using 7-ethoxycoumarin O-demethylase (7-ECOD) as the substrate with slight modification. Ten randomly collected fourth-instar P. xylostella larvae from F₁ generation were homogenized with phosphate-buffered saline (PBS) (0.1 mmol L⁻¹, pH 7.5) containing 1 mmol L⁻¹ EDTA, 1 mmol L⁻¹ PMSF, 1 mmol L⁻¹ DTT, and 15% glycerol. The homogenates were centrifuged at 10,000× g at 4 °C for 15 min, and the supernatants were treated at 10,000× g for 30 min. The supernatant was used as the crude enzyme source. Each treatment had four replicates with 10 larvae per replicate. Each reaction solution contained 2 μL of 7-ECOD (0.32 mmol L⁻¹), 70 μL of PBS (0.1 mmol L⁻¹, pH 7.5), 2 μL of NADPH (0.8 mmol L⁻¹), and 50 μL of the crude enzyme solution. After incubation of the reaction mixture at 30 °C for 30 min with shaking at 50 r min⁻¹, a 60 μL solution of 15% trichloroacetic acid was added to terminate the reaction. The mixture was then centrifuged, and the supernatant was extracted. A 100 μL aliquot of the resulting extract was mixed with 90 μL of 1.6 mmol L⁻¹ glycine-NaOH buffer (pH 10.5). Wells without microsomal pellets served as controls. The fluorescence intensity was measured using a spectrofluorometer (Edinburgh Instruments Ltd., 2 Bain Square, Kirkton Campus, UK) at 358 and 465 nm wavelengths for excitation and emission, respectively. The P450 activity was calculated by converting the fluorescence intensity into picomoles of the product using a 7-hydroxycoumarin standard curve.

2.5.2. Glutathione S-Transferase

The glutathione S-transferase (GST) activity was determined using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate according to Habig et al. [31]. Ten F₁ fourth-instar larvae of P. xylostella were homogenized in 0.1 mol L⁻¹ PBS (pH 6.5) and centrifuged at 4 °C and 1000× g for 10 min. The extracted supernatant was used as the crude enzyme sample. Each reaction mixture (300 μL) consisted of 20 mmol L⁻¹ reduced glutathione (60 μL), 0.1 mol L⁻¹ PBS (136 μL), 20 mmol L⁻¹ CDNB (4 μL), and supernatant (100 μL), whereas the nonenzymatic reaction without crude enzyme solution served as control. Absorbance values were continuously recorded at 340 nm wavelength for 2 min using a spectrophotometer (Shimadzu, UV-1900, Albert-Hahn-Str. 6-10, Duisburg, Germany). An extinction coefficient of 9.6 mmol⁻¹ L⁻¹ cm⁻¹ was used to calculate the conjugated CDNB concentration. Each treatment was assayed using four replicates with 10 larvae per replicate.

2.5.3. Esterase

The esterase (EST) activity was determined using α-naphthol as substrate according to the method of Asperen [32] and Zhu and He [33]. Ten fourth-instar P. xylostella larvae of F₁ generation were homogenized with PBS (0.04 mol L⁻¹, pH 7.0). The homogenate was centrifuged at 4 °C and 10,000× g for 20 min, and the obtained supernatant was used as an enzyme sample. The reaction mixture (150 μL) in each well consisted of 135 μL α-naphthol (0.3 mmol L⁻¹) and 15 μL supernatant solution and was incubated at 37 °C for 30 min. The reaction was stopped with the addition of 50 μL of a dye reagent, containing the mixture of fast blue B and sodium dodecyl sulfate (2:5 v/v). Wells with buffer instead of supernatant served as controls. Absorbance values were continuously measured at 600 nm wavelength for 5 min using a spectrophotometer (Shimadzu, UV-1900, Albert-Hahn-Str. 6-10, Duisburg, Germany). Each treatment group was replicated four times with 10 individuals per replicate. The quantity of free α-naphthol was computed from the α-naphthol standard curve.

The protein content in the enzyme source was quantified using bovine serum albumin as a standard by following the method of Bradford [34].

2.6. Data Analyses

Bioassay data were corrected for control mortality using Abbott’s formula where required [35]. Data were analyzed using PoloPlus statistical software version 2.0 (LeOra Software Company, Beverly Hills, CA, USA) for probit analysis, and the LC values (LC₁₀, LC₃₀ and LC₅₀) and their 95% confidence limits were calculated. The analysis of variance (ANOVA) (Tables S1 and S2) followed by the Tukey’s HSD test for multiple comparison (p < 0.05) was done using SPSS software (version 18.0: Inc., Chicago, IL, USA) to analyze the significance of statistics among different treatments.

According to the theory of age-stage and the two-sex life table [36,37], raw data on the F₁ life table were analyzed using the TWOSEX-MS chart program [38]. The demographic traits at age x and stage j, such as age-stage specific survival rate (s_xj), age-specific survival rate (l_x), age-specific fecundity (m_x), age-specific maternity (l_xm_x), age-stage specific life expectancy (e_xj), intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R₀) and mean generation time (T) were calculated (Supplementary Material S1). The standard errors (SEs) and mean values of the life table parameters were calculated with 10000 bootstrap replicates in a non-parametric method, and the significant differences among the three treatments were preciously computed by the paired bootstrap test (p < 0.05) using the TWOSEX-MS chart program [39].

3. Results

3.1. Toxicity of Fluxametamide

Based on the concentration–mortality data, the estimated LC₅₀ value of fluxametamide for the third instar larvae of P. xylostella was 0.18 mg L⁻¹ (

Table 1. Lethal toxicity of fluxametamide to third-instar larvae of P. xylostella.

Insecticide	n	LC10 (95% CL) (mg L−1)	LC30 (95% CL) (mg L−1)	LC50 (95% CL) (mg L−1)	Slope ± SE	p-Value
Fluxametamide	720	0.06 (0.03–0.08)	0.11 (0.09–0.14)	0.18 (0.15–0.23)	2.34 ± 0.41	0.82

n: Total number of larvae used. CL: Confidence limits.

). Also, the LC₁₀ and LC₃₀ values were determined to be 0.06 and 0.11 mg L⁻¹, respectively. These two concentrations were used in the study of the sublethal effect.

3.2. Sublethal Effects of Fluxametamide on F₀ Generation of P. xylostella

When compared with the control treatment, the pupation rates of F₀ larvae treated with LC₁₀ and LC₃₀ concentrations of fluxametamide were significantly decreased by 22.45 and 35.63% (F = 479.513; p < 0.0001; df = 2, 21), respectively; their deformed pupa rates were significantly increased by 60.28 and 67.18% (F = 152.118; p < 0.001; df = 2, 21), respectively (

Table 2. Sublethal effects of fluxametamide on larval and pupal parameters of P. xylostella F₀ generation treated at 3rd larval instar.

Treatments	Parameters
	4th LD (Day)	PD (Day)	PR (%)	PW (mg)	DPR (%)
Control	1.72 ± 0.06 c	3.62 ± 0.02 c	92.16 ± 0.73 a	5.21 ± 0.05 a	0.85 ± 0.02 c
LC10	2.16 ± 0.07 b	3.69 ± 0.02 b	71.47 ± 0.43 b	4.53 ± 0.02 b	2.14 ± 0.03 b
LC30	2.43 ± 0.04 a	3.84 ± 0.02 a	59.32 ± 0.99 c	4.01 ± 0.04 c	2.59 ± 0.03 a

Values are means ± SE. LD: Larval duration; PD: Pupal duration; PR: Pupation rate; PW: Pupal weight; DPR: Deformed pupa rate. The means indicated by different letters in a column are significantly different at p < 0.05 by Tukey’ HSD test.

). The pupal weight was significantly decreased by 0.68 and 1.20 mg (F = 223.651; p < 0.0001; df = 2, 21) in LC₁₀ and LC₃₀ fluxametamide-treated groups of F₀ larvae, respectively. The duration of fourth instar larvae was also significantly higher than the untreated control (F = 32.602; p < 0.0001; df = 2, 21) (Table 2).

Besides, the deformed adults rate was significantly increased by 42.33 and 58.59% (F = 156.549; p < 0.0001; df = 2, 21), and the mean APOP was also increased by 0.25 and 0.32 days (F = 28.994; p = 0.0073; df = 2, 21) in the LC₁₀ and LC₃₀ concentrations of fluxametamide, respectively (

Table 3. Sublethal effects of fluxametamide on adult parameters of P. xylostella F₀ generation treated at 3rd larval instar.

Treatments	Parameters
	AE (%)	DA (%)	AML (Day)	AFL (Day)	FR (%)	APOP (Day)
Control	93.84 ± 1.15 a	3.57 ± 0.02 c	14.29 ± 0.12 a	13.15 ± 0.10 a	54.26 ± 2.79 a	1.22 ± 0.03 c
LC10	86.20 ± 1.55 b	6.19 ± 0.09 b	13.38 ± 0.12 a	13.27 ± 0.08 a	51.43 ± 2.22 a	1.47 ± 0.03 b
LC30	77.13 ± 1.52 c	8.62 ± 0.06 a	13.33 ± 0.11 a	10.94 ± 0.08 b	49.17 ± 1.72 a	1.54 ± 0.03 a

Values are means ± SE. AE: Adult emergence; DA: Deformed adults; AML: Adult male longevity; AFL: Adult female longevity; FR: Female ratio; APOP: Adult pre-oviposition period (time between adult emergence and first oviposition). The means indicated by different letters in a column are significantly different at p < 0.05 by Tukey’ HSD test.

). However, the biological parameters, including the adult male longevity and female ratio were not significantly different among the three concentration treatments (Table 3).

3.3. Sublethal Effects of Fluxametamide on F₁ Generation of P. xylostella

The sublethal effects of fluxametamide on the biological parameters of F₁ from the third instar larvae of F₀ are depicted in

Table 4. Sublethal effects of fluxametamide-treated F₀ generation of P. xylostella on egg and larval parameters of F₁ generation.

Treatments	Parameters
	ED (Day)	1st LD (Day)	2nd LD (Day)	3rd LD (Day)	4th LD (Day)	TLD (Day)
Control	2.28 ± 0.03 c	3.01 ± 0.03 a	1.89 ± 0.03 b	1.42 ± 0.02 b	1.76 ± 0.02 c	8.07 ± 0.05 c
LC10	2.92 ± 0.03 b	2.98 ± 0.05 a	1.94 ± 0.03 b	1.53 ± 0.03 a	2.29 ± 0.04 b	8.71 ± 0.03 b
LC30	3.43 ± 0.03 a	2.92 ± 0.05 a	2.11 ± 0.04 a	1.45 ± 0.03 ab	2.38 ± 0.02 a	8.86 ± 0.04 a

Values are means ± SE. ED: Egg duration; LD: Larval duration; TLD: Total larval duration. The means indicated by different letters in a column are significantly different at p < 0.05 by Tukey’ HSD test.

. At the LC₁₀ and LC₃₀ concentrations, the egg duration was significantly prolonged by 0.64 and 1.15 days (F = 399.513; p < 0.0001; df = 2, 297), respectively. Similarly, the larval duration was also significantly prolonged by 0.64 and 0.79 days in the LC₁₀ and LC₃₀ concentrations (F = 7.351; p = 0.004; df = 2, 274), respectively, because the mean duration of second instar larvae was significantly extended by 0.22 days in LC₃₀, and third and fourth instars were significantly extended by 0.11 and 0.03 days and 0.53 and 0.62 days in LC₁₀ and LC₃₀ concentrations, respectively (Table 4). Both the pupation rate (F = 44.561; p = 0.024; df = 2, 144) and pupal weight (F = 187.408; p = 0.0031; df = 2, 144) in the LC₃₀ concentration were significantly lower than that of the control treatment (

Table 5. Sublethal effects of fluxametamide-treated F₀ generation of P. xylostella on pupal parameters of F₁ generation.

Treatments	Parameters
	PD (Day)	PR (%)	PW (mg)	DPR (%)
Control	3.31 ± 0.04 a	85.13 ± 2.58 a	5.64 ± 0.05 a	0.00 ± 0.00 b
LC10	3.34 ± 0.04 a	79.56 ± 2.36 a	5.12 ± 0.05 b	0.63 ± 0.19 a
LC30	3.46 ± 0.04 a	53.07 ± 2.87 b	4.41 ± 0.03 c	0.39 ± 0.10 a

Values are means ± SE. PD: Pupal duration; PR: Pupation rate; PW: Pupal weight; DPR: Deformed pupa rate. The means indicated by different letters in a column are significantly different at p < 0.05 by Tukey’ HSD test.

).

The adult emergence rate was significantly decreased by 19.94 and 28.29% (F = 14.726; p = 0.0029; df = 2, 144), whereas the deformed adults rate was significantly increased by 60.85 and 67.51% (F = 64.395; p < 0.0001; df = 2, 144) in the LC₁₀ and LC₃₀, respectively (

Table 6. Sublethal effects of fluxametamide-treated F₀ generation of P. xylostella on adult parameters of F₁ generation.

Treatments	Parameters
	AE (%)	DA (%)	AML (Day)	AFL (Day)	APOP (Day)	F (Egg Female−1)
Control	79.24 ± 3.62 a	3.10 ± 0.04 c	11.49 ± 0.07 a	10.35 ± 0.05 a	1.15 ± 0.03 a	154.19 ± 2.54 a
LC10	63.44 ± 3.04 b	7.92 ± 0.34 b	10.67 ± 0.08 b	8.72 ± 0.05 c	0.96 ± 0.02 a	139.56 ± 2.71 b
LC30	56.82 ± 2.18 b	9.54 ± 0.61 a	9.31 ± 0.05 c	9.08 ± 0.06 b	1.08 ± 0.03 a	112.37 ± 2.38 c

Values are means ± SE. AE: Adult emergence; DA: Deformed adults; AML: Adult male longevity; AFL: Adult female longevity; APOP: Adult pre-oviposition period (time between adult emergence and first oviposition); F: Fecundity. The means indicated by different letters in a column are significantly different at p < 0.05 by Tukey’ HSD test.

). Fluxametamide at sublethal doses extended the total pre-oviposition period (TPOP) (F = 30.694; p = 0.0032; df = 2, 144), and the total longevity of males (F = 11.296; p < 0.0001; df = 2, 111) and females (F = 32.958; p = 0.0082; df = 2, 144) significantly (

Table 7. Sublethal effects of fluxametamide-treated F₀ generation of P. xylostella on total biological parameters of F₁ generation.

Treatments	Parameters
	TML (Day)	TFL (Day)	FR (%)	TPOP (Day)
Control	25.42 ± 0.13 b	23.90 ± 0.08 c	52.70 ± 2.89 a	14.37 ± 0.10 a
LC10	26.15 ± 0.11 a	24.37 ± 0.12 b	55.61 ± 2.21 a	16.82 ± 0.07 b
LC30	25.87 ± 0.09 a	24.98 ± 0.08 a	47.39 ± 1.80 a	17.15 ± 0.12 b

Values are means ± SE. TML: Total male longevity; TFL: Total female longevity; FR: Female ratio; TPOP: Total pre-oviposition period (time from birth to first reproduction in female). The means indicated by different letters in a column are significantly different at p < 0.05 by Tukey’ HSD test.

), but reduced the fecundity (F = 43.910; p < 0.0001; df = 2, 145), longevity of adult males (F = 269.402; p = 0.031; df = 2, 111) and adult females (F = 275.531; p = 0.0026; df = 2, 144) (Table 6). However, no significant differences (p > 0.05) were observed between the control and the sublethal treatments concerning the first instar duration, or the pupal duration, or APOP or female ratio.

3.4. Fitness Costs of Fluxametamide on F₁ Generation of P. xylostella

The age-stage-specific survival rate (s_xj) of F₁ generation exhibited distinct overlaps between sublethal concentrations of fluxametamide and control treatments (Figure 1). The age-specific survival rate (l_x) showed a higher survival duration for LC₁₀ (31 days) and LC₃₀ (30 days) treatments than the control (29 days) (Figure 2). The tendency of sublethal curves was steeper than the control due to the increased mortality rate in LC₁₀ and LC₃₀ treatments of fluxametamide. The age-specific fecundity (m_x) and maternity (l_xm_x) had the decreasing trend as control > LC₃₀ > LC₁₀ and control > LC₁₀ > LC₃₀, respectively. The life expectancy (e_xj) curves of the F₁ generation indicated a decreased life span with the increase of age irrespective of treatments, and a longer life in sublethal concentrations when compared to the control (Figure 3). However, the maximum e_xj appeared at the egg stage, which in the LC₁₀ and LC₃₀ treatments was at 24.1 and 22.3 days, respectively, whereas in the control group it was 23.5 days.

3.5. Sublethal Effects of Fluxametamide on Life Table Parameters of P. xylostella

The demographic traits of the F₁ generation were compared and significant differences were observed between the fluxametamide-treated and the control groups (

Table 8. Transgenerational effects of fluxametamide on life table parameters of F₁ generation of P. xylostella.

Treatments	Demographic Traits
	r (Day−1)	λ (Day−1)	R0 (Offspring/Individual)	T (Day)	Rf
Control	0.213 ± 0.01 a	1.245 ± 0.01 a	76.429 ± 5.34 a	20.316 ± 0.19 b	-
LC10	0.189 ± 0.01 b	1.238 ± 0.003 a	51.681 ± 4.82 b	21.074 ± 0.17 a	0.68
LC30	0.191 ± 0.01 b	1.231 ± 0.01 ab	49.133 ± 4.94 b	20.673 ± 0.21 a	0.64

Values are means ± SE. Different letters in a column indicate significant differences (p < 0.05) by a paired bootstrap test using TWOSEX-MS chart program.

). The intrinsic rate of increase (r) (p < 0.0001) and the finite rate of increase (λ) (p < 0.0001) were significantly decreased by LC₁₀ and LC₃₀, and only LC₃₀, respectively, whereas the net reproductive rate (R₀) (p = 0.0026) was significantly decreased by both LC₁₀ and LC₃₀ concentrations. However, fluxametamide significantly extended the T (p = 0.0175) for about 0.76 days in LC₁₀ and 0.36 days in LC₃₀ concentrations when compared to the control. The R_f calculated from the R₀ values for the LC₁₀ and LC₃₀ concentrations relative to the control treatment were 0.68 and 0.64, respectively.

3.6. Detoxification Enzyme Activities

When compared to the control treatment, GST activities were significantly higher in the LC₃₀ (elevated 1.433-fold) group (p < 0.0001), whereas no significant difference was observed for the LC₁₀-treated P. xylostella population (

Table 9. Activities of three detoxification enzymes in LC₁₀ and LC₃₀-fluxametamide treated P. xylostella.

Treatments	P450 Activity (pmol min−1 mg−1 Protein)	Ratio †	Glutathione S-Transferase Activity (μmol min−1 mg−1 Protein)	Ratio	Esterase Activity (μmol min−1 mg−1 Protein)	Ratio
Control	0.597 ± 0.042 a	-	1.169 ± 0.078 b	-	0.171 ± 0.018 a	-
LC10	0.604 ± 0.039 a	1.012	1.233 ± 0.104 b	1.055	0.168 ± 0.021 a	0.983
LC30	0.632 ± 0.041 a	1.059	1.675 ± 0.092 a	1.433	0.176 ± 0.019 a	1.029

Values are means ± SE. Significant differences (p < 0.05) in the mean activity values in the same column are designated with different alphabets. ^† Ratio = Value of the LC₁₀ or LC₃₀ activity/Control activity.

). However, P450 and EST activities were not significantly different between the sublethal treatments of fluxametamide.

4. Discussion

Fluxametamide is a novel isoxazoline insecticide that can effectively control various lepidopteran and coleopteran insect pests [19,40]. In the present study, the authors tried to explore the potential influence of the sublethal concentrations of fluxametamide on P. xylostella for the first time. A high insecticidal activity of fluxametamide was observed against the third-instar larvae of P. xylostella with an LC₅₀ value of 0.18 mg L⁻¹. The result indicated that the fluxametamide toxicity to P. xylostella was higher than that of abamectin, spinosad, indoxacarb, chlorantraniliprole and fenoxycarb [21,41,42]. In addition, the second instar larvae of P. xylostella exhibited an LC₅₀ value of 56.41 mg L⁻¹ when treated with farnesyl acetate, was higher than the present results with a lower toxicity [43]. However, the LC₅₀ of the technical grade fluxametamide, obtained from the present study, was also lower than that of rac-fluxametamide (1.90 mg L⁻¹) and its enantiomers; S-(+)-fluxametamide (0.62 mg L⁻¹), or R-(-)-fluxametamide (34.18 mg L⁻¹) at 48 h [40].

Considering that most of the long-lasting insecticides could degrade gradually after their initial field application, turning lethal dosages into sublethal levels, we determined the effects of the sublethal concentrations of fluxametamide on P. xylostella. Very few workers have reported the laboratory and field efficacy of fluxametamide against Tetranichus urticae, Laodelphax striatellus and sweet potato weevil [10,19]. In addition to larval toxicity, the present study demonstrated a variety of sublethal effects of fluxametamide on key biological traits of P. xylostella. The LC₁₀ and LC₃₀ concentrations of fluxametamide not only prolonged the fourth larval duration of F₀ of P. xylostella but also extended the larval period of F₁ progeny in a dose-dependent manner. This observation was corroborated by the previous reports on the sublethal effects of spinosad [20] or chlorantraniliprole [21] or spinetoram [23] on P. xylostella larvae. Prolongation of the larval duration in fluxametamide treatments could be attributed to retarded growth due to a reduced appetite, feeding disruption, abnormal metabolism, starvation stress, or the imbalance between metabolic detoxification and physiological development [44,45]. However, it is important to note that the extended larval duration could make a significant contribution to the field management of P. xylostella by increasing the probability of natural parasitism or predation [46] and by forcing immature larvae to feed on suboptimal nutritional foliage for completing their life cycle, which leads to reduced fecundity and survival [47]. In contrast, the sublethal concentrations of cyantraniliprole and beta-cypermethrin did not show any significant effect on the life history of Helicoverpa assulta [48] and P. xylostella [49], respectively. Besides, da Silva et al. [50] reported that chlorantraniliprole (<10% mortality) and methomyl (0% mortality) registered very negligible toxicity to P. xylostella at their sublethal levels. This phenomenon might be attributable to the typical symptoms of positive fitness effects of sublethal insecticides exposure to insect pests [51].

Fluxametamide at sublethal concentrations could persistently affect the metamorphosis and biology of treated P. xylostella. For example, the LC₁₀ and LC₃₀ fluxametamide treatments significantly decreased the pupation rate in both F₀ and F₁ generations, which could, in turn, reduce the probability that the treated strain completed the developmental process. In addition, a significant increase in the deformed adult rate was observed at sublethal doses of fluxametamide in both generations. The adverse effect of chlorantraniliprole, spinosad and tebufenozide on the pupation rate of Spodoptera exigua was already reported [52]. Tamilselvan et al. [23] also observed a significant reduction and increase in the pupation rate and the adults with wings deformity, respectively, in F₀ generation of P. xylostella when third instar larvae were treated with sublethal doses of spinetoram. In the present study, exposure to LC₁₀ and LC₃₀ concentrations of fluxametamide significantly reduced the mean pupal weight of P. xylostella in the F₀ and F₁ generations. This result was consistent with the previous report on sublethal doses of metaflumizone or spinetoram on P. xylostella [22,23] and broflanilide on Spodoptera litura [53]. The reduction in pupal weight might be due to the decrease in food ingestion for optimal larval growth and the poor recovery efficiency of fluxametamide-treated larvae.

The adult male and female longevity of P. xylostella F₀ generation were not significantly differed in fluxametamide treatments, but the adult longevity and total longevity of both males and females and the fecundity of gravid females were affected in the F₁ generation. Similar results were obtained by Yin et al. [54], Su and Xia [55] and Tamilselvan et al. [23] who reported that sublethal concentrations of chlorantraniliprole, methylthio-diafenthiuron and spinetoram reduced the fecundity of P. xylostella in F₁ progeny. We hypothesized that the production of viable female eggs and male sperms, required for normal fertilization, was less in the fluxametamide-treated P. xylostella adults due to the cessation of feeding and lower food intake by the larval instars. The involvement of similar mechanisms in reduced fecundity has been reported for other lepidopteran pests such as Platynota idaeusalis [56] and S. litura [57]. Although the copulation and mating behavior of the adults that emerge after sublethal exposure is unknown, our findings indicate the potential for sublethal effects of fluxametamide on the reproductive physiology of P. xylostella. On the other hand, it should be noted that endocrine signaling pathways regulate the fecundity of insects [53]. Unlike the present study, Hu [58] observed that two GABA-receptor-targeting insecticides, endosulfan and butane-fipronil, enhanced the fecundity of Drosophila melanogaster by upregulation of the signaling pathway-related genes of juvenile hormone (JHATT), ecdysone (CYP307A1) and insulin-like (ILP2, ILP5 and ILP7), and of the insulin-like (ILP2 and ILP5), respectively. In addition, the non-significant increase in the mean fecundity of S. litura was observed at the sublethal concentrations of a meta-diamide insecticide broflanilide, but the exact reason has yet to be confirmed [53]. However, neither any positive effect nor insecticide hormoligosis was reported in the fecundity of broflanilide-treated Tetranychus urticae [59], and this substantiated the present results. Besides, the sublethal effects of spinosad [60] and spinetoram [23] on the pre-oviposition period of P. xylostella have been investigated. In the current study, exposure to LC₁₀ and LC₃₀ concentrations of fluxametamide also increased the TPOP of P. xylostella F₁ generation, which is consistent with the previous authors.

Demographic traits have been recommended as a more suitable approach for examining the total effects of an active ingredient on an insect population [17,61]. In addition, the effects of an insecticide on an insect population level could be predicted better by combining toxicological and ecological parameters. However, it is essential to study the effects of sublethal insecticide exposure on the fitness costs associated with insecticide resistance for the development of potential resistance management strategies [62,63]. In the present study, the mean values of r, λ and R₀ tended to be lower in the sublethal treatments of fluxametamide than in the control, while T was significantly increased. The results indicate that the sublethal doses of fluxametamide can slow down the population dynamics of P. xylostella through the reduction in their reproduction and survival. Furthermore, after the sublethal exposure, the significant changes in development times and fecundity caused a decrease in the r and R₀ and resulted in a lower R_f and longer T, which was identical to the results seen with of P. xylostella treated with chlorantraniliprole [21], spinetoram [23] and broflanilide [24]. The population parameters of P. xylostella, such as intrinsic rate of increase (r), finite rate of increase (λ), age-specific fecundity (m_x) and life expectancy (e_xj) were also found to be lower in the sublethal exposure of indoxacarb [64], pyriproxyfen [65] and teflubenzuron [66], and are consistent with the present findings.

The sublethal effects of an active ingredient on the fecundity and life stages of insects resulted from biochemical responses of various metabolic enzymes [67,68]. In the present study, the increased activities of GST may be contributing to the biochemical effects of the LC₃₀ treatment of fluxametamide in P. xylostella. Based on the nature, dosage, and timing of chemical exposure, fluctuating activities of GST are possible in chemically stressed insects [69,70]. Similarly, in Haemaphysalis longicornis and Spodoptera litura, upregulation and expression of GST genes were detected after sublethal treatments with flumethrin [71] and chlorpyrifos [72], respectively, indicating a possible role in insect biochemical responses under sublethal insecticide exposure.

5. Conclusions

In conclusion, fluxametamide has excellent insecticidal activity against P. xylostella. Besides the direct lethal effects, fluxametamide at sublethal doses has a negative influence on different development stages of P. xylostella F₀ generation. The reduced female fecundity and delayed larval development indicated that fluxametamide can suppress the F₁ progeny due to its transgenerational sublethal effect on the P. xylostella. However, under field and semi-field conditions, insects may be subjected to continuous sublethal exposure to insecticides for multiple generations, which may lead to polygenic or multifactorial insecticide resistance. Therefore, based on the present results, field studies should be conducted to obtain more reliable information for the use of fluxametamide at the farm level as an alternative to existing molecules in the sustainable management of P. xylostella. Additionally, laboratory studies should also be undertaken to understand the mechanisms of fluxametamide-induced effects in P. xylostella.