Bifenthrin at Sublethal Concentrations Suppresses Mating and Laying of Female Conogethes punctiferalis by Regulating Sex Pheromone Biosynthesis and JH Signals

Abstract

Conogethes punctiferalis, a polyphagous pest in Asia, infests various crops, causing severe economic losses. Its larvae feed inside plants, making management challenging, with conventional insecticides. This study examines sublethal bifenthrin effects on the reproductive capabilities of adult females. Findings show sublethal bifenthrin concentrations (LC₁, LC₁₀, LC₂₀, and LC₃₀) significantly reduce sex pheromone production and mating success in a dose-dependent manner. Furthermore, these sublethal exposures influence the expression of pheromone biosynthesis activating neuropeptide and key juvenile hormone signaling genes, including methoprene-tolerant and Krüppel-homologue 1. Enzyme activity assays and metabolite measurements indicated that sublethal bifenthrin exposure decreases trehalose and pyruvic acid levels, suppressing the enzyme activities required for sex pheromone biosynthesis. Additionally, bifenthrin exposure delays ovarian development, reduces ovary size, and decreases egg production and hatchability. These results suggest bifenthrin’s potential in attract-and-kill strategies by disrupting essential pathways for pest control, offering insights for improved insecticide use and innovative pest management for C. punctiferalis.

1. Introduction

Conogethes punctiferalis, commonly known as the yellow peach moth, belongs to the family Crambidae. This species is primarily prevalent as a pest in Asia and has been observed in numerous countries within the region, including China, India, Japan, Korea, and Thailand, among others in the region.¹ As a polyphagous pest, this insect feeds on a wide variety of host plants, which includes, but is not limited to, peaches, citrus fruits, maize, durian, chestnut, and chickpeas.² Its adaptability to various plants makes it a significant threat to a wide range of agricultural and horticultural crops, allowing it to thrive under different farming conditions. Most importantly, larval feeding disrupts normal fruit development, causing extensive damage to numerous plant species and leading to significant financial losses for farmers.³ The polyphagous feeding habits of the yellow peach moth and the larvae feeding in protected sites within plant tissues make effective management of its infestations challenging. An integrated pest management (IPM) approach is recommended to effectively control the pest population. This approach combines agricultural, biological, and chemical strategies.⁴ It is essential to rotate insecticides with different modes of action to prevent the development of resistance, including the widely used pesticides chlorantraniliprole, emamectin benzoate, and bifenthrin.^5,6

Bifenthrin, a synthetic pyrethroid insecticide structurally analogous to natural pyrethrin extracted from chrysanthemum flowers, has become a crucial tool in modern pest management practices.⁷ This pesticide is widely used for its high efficacy, versatility, and relative safety compared to more toxic alternatives, effectively controlling a variety of pests, including those from the Hemiptera, Lepidoptera, and Homoptera orders.⁸ Despite its numerous advantages, the use of bifenthrin requires careful consideration of its environmental impact. Therefore, its applications are closely regulated, and best practices call for precise targeting and judicious timing of treatments to align with periods of pest vulnerability while limiting exposure to nontarget organisms. Consequently, bifenthrin serves as a crucial pesticide in IPM, where it is combined with other sustainable and eco-friendly practices.

The management of pests such as C. punctiferalis, however, poses a significant challenge for agricultural practices. After hatching from their eggs, the larvae initiate their invasion by tunneling into various parts of the host plant, including shoots, fruits, or other parts, where they feed on the internal tissues. They are effectively shielded from conventional pest control methods.⁹ Pyrethroid insecticides, with bifenthrin as a prime example, are limited in their effectiveness, as their mechanism requires direct contact with the pest, which is unlikely when the larvae are hidden inside plant tissues. As the larvae penetrate deeper and establish more extensively within the plant’s interior, control becomes increasingly difficult. They become even more insulated from surface treatments with insecticides.¹⁰ The plant’s own defenses, coupled with the larvae’s elusive behavior, create additional barriers that impede chemical penetration, clearly indicating the need for more innovative approaches in pest management. Considering these challenges in targeting larvae, a shift has been observed toward strategies that focus on the adult stage of pests. Food attractants have emerged as highly promising tools against adult insects due to their efficacy and targeted nature.¹¹ Originating from plant-derived volatile compounds, these attractants exert a powerful lure on various pests, including moths, flies, and beetles, drawing them toward traps or areas where they can be more effectively managed or eliminated.¹² In China, the development and utilization of highly effective food attractants have made a significant impact on the management of pest moth populations across various crops.¹³ These attractants often mimic the scent or flavor of the pests’ preferred food sources, exploiting their innate feeding behaviors for improved control measures. These food attractants, when synergistically combined with insecticides, form the basis of an effective “attract-and-kill” strategy.¹³ This approach capitalizes on the attractants’ ability to lure pests to a specific location where the insecticides can then be applied more precisely and efficiently, thereby reducing the amount of chemicals released into the environment.¹⁴ By integrating these attract-and-kill systems into a broader IPM program, it is possible to achieve a more sustainable and environmentally conscientious approach to pest control. Based on this attract-and-kill system, our study aims to explore an innovative approach to pest control focusing on adult stages of C. punctiferalis. Specifically, this research investigates the effects of sublethal bifenthrin exposure on reproductive capabilities. By selecting C. punctiferalis as a model, we aim to understand how sublethal doses of bifenthrin impact sex pheromone production, mating efficiency, gene expression related to juvenile hormone (JH) signaling, and overall reproductive success. These insights will contribute to the development of more sustainable and effective pest management strategies, ultimately improving control measures for this economically significant pest.

2. Materials and Methods

2.1. Insect, Insecticide, and Chemicals

C. punctiferalis larvae were collected in Zhengzhou, Henan Province, China, and have been reared with freshly prepared maize for over 20 generations without exposure to any pesticides under the conditions of 27 ± 1 °C, 60 ± 10% relative humidity, and a 16L/8D photoperiod at Henan Agricultural University. Newly emerged adults were supplemented with 5% sugar water.¹⁴

Bifenthrin (with a purity of 95%) was acquired from Hebei Yanxi Chemical Co., Ltd. (Xingtai, Hebei, China).

2.2. Bioassay

Bifenthrin was dissolved in acetone to obtain a stock solution with a concentration of 62.5 mg/mL. This stock solution was further diluted with a 5% sugar solution to obtain a range of final concentrations of the active ingredient (0, 3.125, 6.25, 12.5, 25, 50, and 100 mg/L). In all studies, a 5% sugar water solution without any pesticide was used as the control. Newly emerged females were fed a 5% sugar solution with various bifenthrin concentrations for toxicity testing. This bifenthrin-infused sugar solution was refreshed daily. For each concentration tested, 24 females were employed, and their mortality was assessed after 3 days of feeding (the criterion for death was a lack of movement upon stimulation). Three biologic replicates were used for each dose. A control group, which received an acetone and 5% sugar solution mix without any pesticide, was also maintained for comparison. Lethal concentrations for 1, 10, 20, and 30% mortality (LC₁, LC₁₀, LC₂₀, and LC₃₀) were calculated through Probit analysis in the SPSS 20 software.

2.3. Impact of Sublethal Bifenthrin Exposure on Sex Pheromone Synthesis and Mating Efficacy

Following the aforementioned toxicity evaluation, doses corresponding to LC₁, LC₁₀, LC₂₀, and LC₃₀ were selected as sublethal concentrations for further experimentation. Newly emerged females were placed in a box (750 mL) covered with gauze. They were then fed with 5% sugar water containing these specific concentrations. To prevent the degradation of the pesticide, the sugar solutions laced with bifenthrin were refreshed daily. Following a 3-day period of exposure to the varied treatments, the females were administered an injection of 5 μL (10 pmol) of PBAN, a neuropeptide activating pheromone biosynthesis, which was diluted in normal saline. After waiting for 1 h, the PGs were collected and soaked in 200 μL hexane. The resulting sample was then analyzed using gas chromatography (GC) to assess sex pheromone production. Each treatment group contained three biological replicates, with each replicate comprising no fewer than 20 females.

Newly emerged females and males were paired in a 1:1.5 ratio (20 females and 30 males) in a 750 mL box covered with gauze. Subsequently, they were provided with 5% sugar water containing each of these specific concentrations to evaluate their impact on mating success. To prevent the degradation of the pesticide, sugar solutions laced with bifenthrin were replaced each day. After the 3-day exposure to these treatments, the proportion of successful mating in females was determined by detecting a spermatophore in the female bursa copulatrix following the previous method.¹⁵ Three biological replicates were employed for each group of experiments.

2.4. Impact of Sublethal Bifenthrin Exposure on PBAN Expression and Its Secondary Messenger

Newly emerged females were transferred into a 750 mL box covered with gauze. They were then provided with 5% sugar water containing different concentrations of bifenthrin. In order to minimize pesticide degradation, the sugar solutions with different bifenthrins were replaced daily. After 3 days of exposure to the treatments, the brain-subesophageal ganglion complex was collected from the treated females. Total proteins were extracted by triturating, lysing, and centrifuging the dissected tissues. These proteins were then subjected to Western blot analysis to assess the expression levels of PBAN protein. Quantification of PBAN protein based on grayscale analysis was carried out using ImageJ software (version 1.54). For each concentration group, three biological replicates were employed, with each replicate including no fewer than 50 females. The actin was used as the control.

Newly emerged females were placed in a 750 mL box covered with gauze. They were then fed with 5% sugar water containing predetermined concentrations of bifenthrin. To prevent the bifenthrin in the sugar solutions from degrading, the sugar solutions laced with bifenthrin were refreshed daily. After a 3-day exposure to these treatments, the females were injected with 5 μL (10 pmol) of PBAN. Following the 30 min waiting time, PGs were collected from the treated females and then used to measure Ca²⁺ level by using the Calcium Assay Kit (Beyotime, Jiangsu, China) in accordance with the manufacturer’s instructions following the description of the previous study.^14,16 Every sample contained three biological replicates, and each replicate contained at least 50 PGs.

2.5. Quantitative Real-Time PCR

PGs were collected from the females after a 3-day exposure to the aforementioned treatments, and total RNA extraction was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s protocol. Every sample contained a minimum of 50 PGs. First-strand cDNA was synthesized from 1 μg of total RNA extracted from the PGs using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Beijing, China). This cDNA was then served as a template for quantitative PCR. The primers for qPCR are listed in Table S1. qPCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI, Carlsbad, CA, USA) using ChamQ Universal SYBR qPCR Master Mix (Q711-02-03) (Vazyme, Nanjing, China). The qPCR cycling conditions began with an initial denaturation at 95 °C for 5 min, followed by 40 cycles consisting of 15 s of denaturation at 95 °C and 20 s for annealing and extension at 60 °C. The amplification specificity of the SYBR Green PCR was confirmed by performing both melting curve analysis and agarose gel electrophoresis. To ensure the reliability and reproducibility of the qPCR results, each sample was tested with three biological replicates, and each replicate included at least 50 PGs. Gene expression normalization was conducted using two reference genes: ribosomal protein 49 (PCR efficiency: E = 0.9575) and ribosomal protein L49 (PCR efficiency: E = 1.0183). The normalized expression level of the target gene is calculated by dividing the target gene’s expression level by the square of the expression levels of two reference genes. Analysis of the qPCR data was conducted using the DPS software version 9.5.¹⁴

2.6. Measurement of Enzymatic Activities

Following a 3-day exposure to the specified treatments, the treated females were injected with 5 μL (10 pmol) of PBAN, which was diluted in normal saline. After a 30 min waiting period, PGs were collected from the treated females and subjected to enzyme activity measurement. A variety of kits were utilized to measure specific enzyme activities according to the manufacturers’ protocols, which included the Calcineurin Activity Assay Kit (Jiancheng, Nanjing, China), acetyl-CoA carboxylase (ACC) Assay Kit (Grace Biotechnology, Suzhou, China), Trehalase Enzymatic Activity Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), hexokinase (HK) Activity Assay Kit (Grace Biotechnology, Suzhou, China), and pyruvate kinase (PYK) Activity Kit (Abcam, Cambridge, UK). Each treatment included three biological replicates, with each replicate comprising a minimum of 100 PGs.

2.7. Measurement of Trehalose, Glucose, and Pyruvic Acid Contents

Following a 3-day exposure to the aforementioned treatments, the treated females were injected with 5 μL (10 pmol) of PBAN that was diluted in normal saline. After a waiting period of 30 min,¹⁴ PGs were collected from these females and analyzed for trehalose, glucose, and pyruvic acid content. The analyses were performed using a Trehalose Content Assay Kit (Solarbio, Beijing, China), a Glucose Assay Kit (Beyotime Biotechnology, Shanghai, China), and a Pyruvic Acid Assay Kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. For each treatment group, there were three biological replicates, with each replicate containing at least 100 PGs.

2.8. Impact of Sublethal Bifenthrin Exposure on Ovary Development

After a 3-day exposure to the aforementioned treatments, the ovaries were dissected and imaged using a digital microscope (M205A, Leica, Wetzlar, Germany). Investigations were carried out to determine the number of eggs produced as well as the length and weight of the ovaries. Each treatment contained three biological replicates, with every replicate containing at least 20 ovaries.

2.9. Impact of Sublethal Bifenthrin Exposure on Egg Production and Hatching Rate

After a 3-day exposure to the aforementioned treatments, the treated females (n = 15) were transferred to box and with males at a 1:1.5 female-to-male ratio. Following a 24 h waiting time, the number of eggs laid by the females was counted, and the corresponding hatchability of the eggs was assessed.

2.10. Effects of Sublethal Bifenthrin Exposure on the Expression of JH Signaling Genes

Following a 3-day exposure to the previously described treatments, the ovaries were harvested for qPCR analysis. The primers for the methoprene-tolerant (Met) and Kruppel-homologue 1 (Kr-h1) genes are provided in Table S2. The qPCR analysis was performed following the description mentioned above. Each sample was tested with three biological replicates, and each replicate included at least 50 ovaries. The qPCR data were analyzed using DPS software version 9.5.¹⁴

2.11. Preparation of Antibodies and Western Blot Analysis

PBAN peptide (add a cysteine at the C-terminus) (DQEMYRQDPEQIDSRTKYC) was biosynthesized by Sangon Biotech Company (Shanghai, China). The obtained PBAN peptide was conjugated to KLH as an antigen and injected into rabbits for animal immunization. Briefly, rabbits were initially injected with the KLH-PBAN conjugate emulsified in a complete adjuvant. Fourteen days after the first injection, a second immunization using the KLH-PBAN conjugate emulsified in an incomplete adjuvant was administered. Seven days later, a third immunization, also using the KLH-PBAN conjugate and incomplete adjuvant, was given. A booster shot using just the KLH-PBAN protein followed 7 days after the third immunization. To monitor the production of antibodies, blood samples were collected regularly from the rabbits. Once an adequate immune response was confirmed, larger quantities of blood were drawn to obtain the serum. The isolated serum, which contained anti-PBAN antibodies, was then separated from the blood.

Following a 3-day exposure to the previously described treatments, the brain–subesophageal ganglion (SOG) complex was collected from the treated females. Protein was extracted from treated samples and separated by SDS-polyacrylamide gel electrophoresis (Zhonghui Hecai, Xian, China, PE008) followed by transferring to a PVDF membrane (Roche, Basel, Switzerland, 3010040001). The membrane was incubated with TBST containing 5% skim milk for 1 h at room temperature and reacted with the PBAN polyclonal antibody for 1 h. The membrane was incubated with HRP-labeled secondary antibody. The signal was detected using the superlumia ECL plus HRP substrate kit (Abbkine, Wuhan, China, K22030) according to the manufacturer’s instruction.

2.12. Sex Pheromone Measurement

The concentrations of E-10–16: Ald in the PGs of adult females subjected to various treatments were quantified using a GC system (Agilent 7890B) equipped with an HP-5MS column, following the previous study.¹⁷ Helium served as a carrier gas. The standard sample of E-10–16: Ald (Weikeqi-Biotech, Sichuan, China) was used as an external standard to quantify the pheromone titer. The PGs of female adults from different treatments were collected. The GC operating conditions were programmed as follows: the initial temperature was set at 150 °C and maintained for 5 min, followed by a first gradient heating rate of 30 °C/min until reaching 220 °C. Subsequently, a second gradient heating rate of 1 °C/min was applied until reaching 230 °C, followed by maintaining at 260 °C for 2 min. A solvent delay of 2 min was also incorporated into the protocol. The column was then heated to 245 °C at 20 °C/min and held at this temperature for 15 min to clean the column before the next analysis. The FID was held at 250 °C. The extract was collected with a pipet and transferred to a GC sample vials. A 1 μL aliquot of the extract for every sample was sequentially uploaded to the GC instrument. The FID was held at 250 °C. Every sample produced a peak corresponding to the retention time of 8.02 min for E-10–16: Ald. The quantities of E-10–16: Ald in each sample were calculated by comparing their peak areas to those of the standards. Each treatment group consisted of three biological replicates, and each replicate contained at least 20 females.

2.13. Statistical Analysis

The data were shown as the means of all biological replicates with the standard error (SE). Mortality data were analyzed using probit analysis on SPSS 20 software (IBM Software, USA). Multiple comparisons were analyzed for significant differences using LSD test on DPS software version 9.5. Graphs were generated using GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, CA).

3. Results

3.1. Insecticidal Activity of Bifenthrin against Adults of C. punctiferalis Females

The insecticidal activity of bifenthrin against adults was evaluated. The result showed that LC₅₀ of bifenthrin was 44.3773 and 29.2762 mg/L in females and males, respectively. LC₉₅ of bifenthrin was 251.035 and 502.6124 mg/L in females and males, respectively. The dose–response lines of bifenthrin were y = 2.1856x + 1.3999, R² = 0.9457 (for females) and y = 1.3308x + 3.0460, R² = 0.9376 (for males) (Table 1). Based on the dose–response line of bifenthrin, corresponding values of LC₁, LC₁₀, LC₂₀, and LC₃₀ (95% confidence limits (CL)) were 3.8263, 11.5030, 18.2849, and 25.5406 mg/L in females after 5-day exposure to bifenthrin, respectively. The values of LC₁, LC₁₀, LC₂₀, and LC₃₀ (95% CL) were 0.4552, 2.9692, 6.5396, and 11.5564 mg/L in males after 5-day exposure to bifenthrin, respectively (Table S2). Consequently, the derived concentrations for LC₁, LC₁₀, LC₂₀, and LC₃₀ against adults were used in subsequent experiments.

Table 1. Determination of Bifenthrin Toxicity to C. punctiferalis Adults.

	LC50 (95% CLb) (mg/L)	LC95 (95% CL) (mg/L)	R2	slope	intercept
femalea	44.3773	251.035	0.9457	2.1856	1.3999
malea	29.2762	502.6124	0.9376	1.3308	3.0460

^aThe results obtained on the 5th day after the newly emergence adults were fed with bifenthrin.

^bConcentration killing 50% of insects with 95% CL in parentheses.

3.2. Impact of Sublethal Bifenthrin Exposure on Sex Pheromone Biosynthesis and Mating Efficacy in Adult Female C. punctiferalis

Sex pheromone production was investigated following exposure to sublethal concentrations of bifenthrin. The results revealed that sex pheromone production exhibited a negative correlation with increasing concentrations of bifenthrin. The decrement in sex pheromone production due to sublethal bifenthrin exposure displayed a dose-dependent pattern. Treatments with four different doses (LC₁, LC₁₀, LC₂₀, and LC₃₀) significantly decreased sex pheromone production when compared with the control group (Figure 1A). Additionally, there was a significant difference in sex pheromone production among the treatments with LC₁, LC₁₀, and LC₂₀, though no significant difference was detected between the LC₂₀ and LC₃₀ treatments.

Figure 1.

Sublethal bifenthrin exposure inhibits mating behavior by reducing sex pheromone production. (A) Effect of sublethal concentrations of bifenthrin on sex pheromone production. (B) Effect of sublethal concentrations of bifenthrin on successfully mating proportion. Bars represent the mean (±SE) of three biological replicates. Significant differences were indicated with lowercase letters P < 0.05 level (LSD multiple comparison test, DPS 9.50).

The proportion of successful matings was also investigated following exposure to sublethal concentrations of bifenthrin. The findings indicated a negative correlation between the proportion of successful matings and increasing concentrations of bifenthrin, illustrating a dose–response effect. Treatments with three different doses (LC₁₀, LC₂₀, and LC₃₀) significantly reduced the proportion of successful matings compared to the control group (Figure 1B). However, no significant difference was found between the LC₁ treatment and the control group.

3.3. Effects of Sublethal Bifenthrin Exposure on PBAN Expression and Its Secondary Messenger

Effects of sublethal bifenthrin exposure on PBAN signaling were examined. The results demonstrated that exposure to the LC₁, LC₁₀, and LC₃₀ concentrations significantly lowered PBAN protein levels when compared with the control group (Figure 2A,B). However, the LC₂₀ treatment was associated with an increase in PBAN protein levels compared with the control group.

Figure 2.

Effect of sublethal bifenthrin exposure on expression level of PBAN protein and its second messenger. (A) Content of PBAN protein. (B) Quantification of PBAN protein using grayscale analysis. (C) Concentration of second messenger Ca²⁺. Bars represent the mean (±SE) of three biological replicates. Significant differences were indicated with lowercase letters P < 0.05 level (LSD multiple comparison test, DPS 9.50).

Similarly, Ca²⁺ levels, the secondary messenger in PBAN signal transduction, were also measured (Figure 2C). The results reflected the same pattern observed for PBAN protein expression. The LC₁ and LC₃₀ treatments reduced Ca²⁺ levels compared to the control group. The LC₁₀ treatments did not yield a significant change in Ca²⁺ levels relative to the control, while the LC₂₀ treatment resulted in a significant increase in Ca²⁺ levels when compared with those of the control group.

3.4. Impact of Sublethal Bifenthrin Exposure on Gene Expression Associated with Sex Pheromone Biosynthesis

Given that exposure to sublethal bifenthrin significantly affected PBAN signal and sex pheromone production, further investigation into the gene expression associated with sex pheromone biosynthesis was performed (Figure 3). The results demonstrated that sublethal bifenthrin exposure has no significant effect on the transcripts of sex pheromone biosynthesis-associated genes, such as PBANR, CaN, ACC, HK, Far, and Des.

Figure 3.

Effect of sublethal concentrations of bifenthrin on the mRNA expression levels of PBANR, CaN, ACC, HK, Far, and Des. Bars represent the mean (±SE) of three biological replicates. Different letters indicate significant differences at P < 0.05 level by LSD multiple comparison test, DPS 9.50 software.

3.5. Impact of Sublethal Bifenthrin Exposure on Trehalose, Glucose, and Pyruvic Acid Contents and Enzyme Activities Associated with Sex Pheromone Biosynthesis in PGs

The effects of sublethal bifenthrin exposure on the glycolysis process were examined (Figure 4). The results revealed that exposure to a low concentration of bifenthrin (LC₁) significantly elevated trehalose levels in PGs. Meanwhile, LC₁₀ exposure did not alter the trehalose levels within the PGs. On the other hand, higher concentrations (LC₂₀ and LC₃₀) significantly reduced the trehalose content in the PGs (Figure 4A). The glucose levels in PGs showed a significant reduction after exposure to three varying doses (LC₁, LC₁₀, and LC₂₀). However, the LC₃₀ treatment did not impact the glucose levels in PGs (Figure 4C). Intriguingly, treatment with all four doses (LC₁, LC₁₀, LC₂₀, and LC₃₀) resulted in a significant decrease in the pyruvic acid levels in the PGs (Figure 4F).

Figure 4.

Effects of sublethal bifenthrin exposure on sugar metabolism pathway in PGs. (A,C,F) Effect of sublethal bifenthrin exposure on the contents of trehalose, glucose, and pyruvic acid in female PGs. (B,D,E,G,H) Effect of sublethal bifenthrin exposure on the enzymatic activities of trehalase, HK, PYK, ACC, and CaN in female PGs. Error bars indicated the mean ± SE of independent biological experiments. Different letters indicate significant differences at P < 0.05 level by LSD multiple comparison test, DPS 9.50 software.

The effects of sublethal bifenthrin exposure on the activities of enzymes involved in sex pheromone biosynthesis were further investigated. The results demonstrated that all four treatment concentrations (LC₁, LC₁₀, LC₂₀, and LC₃₀) significantly decreased the activity of trehalase (Figure 4B). In the case of HK activity, low-dose exposure (LC₁) did not cause any change when compared with that of the control group. However, the exposure to three higher doses (LC₁₀, LC₂₀, and LC₃₀) led to a significant increase in HK activity within the PGs (Figure 4D). Interestingly, the activities of three enzymes (PYK, ACC, and CaN) displayed a similar pattern following sublethal bifenthrin exposure, with low-dose exposure (LC₁ and LC₁₀) inducing increased activities of these three enzymes, whereas higher dose exposure (LC₂₀ and LC₃₀) resulted in significant decreases in the activities of these enzymes (Figure 4E,G,H).

3.6. Impact of Sublethal Bifenthrin Exposure on Ovarian Development and Female Fecundity

Further investigation into the effects of sublethal bifenthrin exposure on ovarian development revealed a dose-dependent adverse effect (Figure 5). Increasing concentrations of bifenthrin directly led to developmental delays in the maturation of the ovarian tubes (Figure 5A). Moreover, as the doses of bifenthrin increased, there was a significant decrease in the overall size, indicated by the reduced length and weight of the ovaries (Figure 5B,C). Alongside these morphological changes, fecundity also significantly declined, as evidenced by the reduced number of eggs conceived by individuals exposed to higher levels of bifenthrin (Figure 5D). Furthermore, as sublethal doses of bifenthrin increased, there was a corresponding and progressive decrease in the number of eggs laid by the females, as well as the viability of the laid eggs (Figure 5E,F).

Figure 5.

Effect of sublethal bifenthrin exposure on ovarian development and female fecundity. (A) Morphology of the insect’s ovaries. (B) Length of ovary (N ≥ 26). (C) Weight of ovary (N ≥ 15). (D) Number of eggs conceived (N ≥ 23). (E) Number of eggs laid by female. (F) Hatching rate of eggs. Error bars indicated the mean ± SE of independent biological experiments. Different letters indicate significant differences at P < 0.05 level by LSD multiple comparison test, DPS 9.50 software.

3.7. Impact of Sublethal Bifenthrin Exposure on the Expression of JH Signaling Genes

Given the substantial influence of sublethal bifenthrin exposure on ovarian development, the investigation was extended to examine its influence on JH signaling pathways, which are crucial for ovarian development. The results indicated a significant decline in the transcriptional level of key genes involved in the JH signaling pathway (Figure 6). Specifically, when compared with the control group, sublethal bifenthrin exposure led to a decrease in the expression levels of Met and Kr-h1. These results suggest that sublethal bifenthrin exposure influence with normal hormonal signaling, thereby potentially leading to adverse effects on insect reproductive capabilities.

Figure 6.

Effect of sublethal bifenthrin exposure on the mRNA expression levels of Met and Kr-h1. Error bars indicated the mean ± SE of independent biological experiments. Different letters indicate significant differences at P < 0.05 level by LSD multiple comparison test, DPS 9.50 software.

4. Discussion

Traditionally, chemical pesticides have been the most effective means for controlling crop pests, primarily targeting larvae. However, the extensive use of these insecticides poses significant ecological concerns. They can pollute soil and water sources, affecting the broader ecosystem, including aquatic life and human drinking water supplies. Additionally, insecticides disrupt nontarget organisms like pollinators and natural predators, leading to ecological imbalances and increasing pest resistance, necessitating stronger chemicals.¹³ Given these issues, there is an urgent need for alternative, sustainable pest control methods. Preventive control of adult pests can form the basis of an effective regional IPM strategy, reducing adult populations and the likelihood of the emergence of next-generation larvae. One promising method is the attract-and-kill strategy, which uses food attractants combined with insecticides to lure and kill pests.¹⁸ Investigating the impact of chemical pesticides on adult insects is crucial to this strategy. Corresponding studies have been conducted to evaluate the toxicity of various insecticides on certain moth species. For example, chlorantraniliprole, emamectin benzoate, spinetoram, spinosad, and methomyl exhibited high levels of toxicity to Helicoverpa armigera moths, resulting in mortality rates of 86.67, 91.11, 73.33, 57.78, and 80.00%, respectively, after 24 h exposure to a concentration of 1 mg/L.¹³ Among these insecticides, Agrotis ipsilon and Spodoptera litura moths were more susceptible to chlorantraniliprole, emamectin benzoate, and methomyl. Our current investigation also demonstrates a significant impact of bifenthrin on the mating and oviposition of C. punctiferalis females. Sublethal doses of bifenthrin suppress mating and oviposition in C. punctiferalis by regulating sex pheromone biosynthesis and ovarian development, leading to a marked reduction in egg production and egg viability. These findings suggest that bifenthrin can be effectively used as a potential adulticide to control C. punctiferalis.

Sex pheromones are crucial for interactions between females and males, and the mechanisms underlying sex pheromone biosynthesis have been well elucidated. Studies have revealed that sugars from supplemental nutrition are converted into pyruvates through glycolysis and then decarboxylated to form acetyl-CoA. This process involves a series of enzymes, including trehalase, HK, and PYK. Acetyl CoA then serves as the precursor for sex pheromone production, where it is used to generate fatty acids through the catalytic actions of ACC and fatty acid synthase. These fatty acids undergo a series of enzyme modifications to form the final sex pheromone compounds under the regulation of PBAN.¹⁹ Consequently, the biosynthesis of sexual pheromones is a complex and intricate process. Our results demonstrated a significant decrease in sex pheromone production and successful mating rates in females following treatments with sublethal doses of bifenthrin (LC₁, LC₁₀, LC₂₀, and LC₃₀). This indicated that sublethal doses of bifenthrin suppress female mating by affecting the sex pheromone biosynthesis. Similar observations were reported in other studies. For example, sublethal doses of thiacloprid (LC₁₀ and LC₂₀) led to a significant reduction in the amount of the sex pheromone component in Cydia pomonella.²⁰ Also, exposure to sublethal doses of deltamethrin (1 ng/moth) during the larval stage resulted in decreased sex pheromone titers in Ostrinia furnacalis.²¹ These findings suggested that these pesticides can influence sex pheromone biosynthesis and can potentially be used to manage adult populations. Further investigation revealed that the decrease in sex pheromone production caused by sublethal doses of bifenthrin is due to a reduction in the activities of associated enzymes and catalyzed products in the sex pheromone biosynthesis pathway. For example, while LC₁ and LC₁₀ treatments lead to increased enzyme activities of PYK, CaN, and ACC, as well as higher trehalose content in the PGs, the decreased activity of trehalase and reduced glucose content in both treatments undoubtedly result in a decline in sex pheromone biosynthesis. Most importantly, the pyruvate content in PGs significantly decreased after exposure to the four treatments, causing reduced sex pheromone production due to a lack of necessary materials for sex pheromone biosynthesis. Overall, sex pheromone biosynthesis involves a series of enzymes and their catalyzed products, making it a complex process. Any decrease in enzyme activities or their associated products inevitably leads to a reduction in sex pheromone production, as demonstrated by our present results.

In agricultural ecosystems, the application of insecticides can result in both lethal and sublethal effects on pest populations. Sublethal exposure to these chemicals, while not immediately fatal, can profoundly impact the reproductive capabilities of surviving pests. For instance, when A. ipsilon and Agrotis segetum were exposed to sublethal doses of the insecticide chlorantraniliprole, their longevity and fecundity were significantly diminished.²² Similar effects were observed in H. armigera, where the fecundity of adults that exposed to low concentrations of chlorantraniliprole, was markedly lower compared to control groups.²³ Likewise, when Spodoptera cosmioides larvae were exposed to low concentrations of chlorantraniliprole, adult fecundity was significantly reduced than that of the control group.²⁴ Our study also mirrors these findings, demonstrating that fecundity of female adults was significantly decreased upon exposure to sublethal bifenthrin (LC₁, LC₁₀, LC₂₀, and LC₃₀), as shown by the reduced number of eggs deposited by the females as well as the lowered viability of the laid eggs. Our findings highlight the promising potential of bifenthrin as a key component in pest management strategies, especially when combined with food attractants. This combination can be utilized in an attract-and-kill strategy, specifically targeting pest populations such as C. punctiferalis.

Further investigation was conducted to explore the mechanism underlying the decreased fecundity of female adults after exposure to sublethal bifenthrin. It is well-known that JH is a critical endocrine hormone that controls the molting and metamorphosis in insects. Additionally, JH is essential for adult maturation, including ovary and egg development, in female adults. The mechanisms underlying JH signaling have been well-studied in some insects. For example, Met has been identified as a JH receptor in Drosophila melanogaster.²⁵ The binding of JH to its receptor Met activates the Kr-h1 gene, which is an early JH-responsive gene that functions as a regulator of the JH signaling pathway. In species such as Locusta migratoria and Nilaparvata lugens, disrupting the JH signaling pathway by silencing the Kr-h1 gene leads to blocked oocyte maturation and impaired ovarian development.^26,27 Similarly, in Diploptera punctata and Pyrrhocoris apterus, knocking down Met suppressed ovarian development.^28,29 In the present study, developmental delays in the maturation of ovarian tubes, reduced length and weight of the ovaries, and fewer eggs produced by individuals after exposure to sublethal bifenthrin suggest that this exposure likely affects the JH signaling pathway. Further investigation confirmed this speculation: exposure to sublethal bifenthrin caused a significant decrease in mRNA levels of Kr-h1 and Met, suggesting the sublethal bifenthrin suppresses the expression of these genes. This suppression inhibits JH signaling, leading to decreased fecundity in female adults. Similar findings have been reported in other insects; for example, in Plutella xylostella, sublethal concentrations of spinosad led to decreased fecundity, smaller egg size, and reduced hatchability.³⁰ In S. litura, sublethal doses of methoxyfenozide caused a significant decrease in adult reproduction.³¹ Similar results were also found in Spodoptera littoralis, in which, spinosad and methoxyfenozide reduced the fecundity and fertility of adults.³² These results suggest that these pesticides affect adult fecundity by influencing the JH signaling pathway. Based on the current findings, we propose a model for the effect of sublethal bifenthrin exposure. In this model, on the one hand, the exposure to sublethal bifenthrin led to decreased sex pheromone production and mating efficiency by influencing the enzyme activities and metabolism products involving sex pheromone biosynthesis. On the other hand, sublethal bifenthrin exposure suppresses the JH signaling pathway by reducing the expression level of the Met and Kr-h1, resulting to developmental delays in ovarian maturation, reduced ovary size, and a significant decrease in egg production and hatchability (Figure 7).

Figure 7.

Model of sublethal bifenthrin exposure on the mating and laying of female C. punctiferalis. Exposure to sublethal bifenthrin led to decreased sex pheromone production and mating efficiency by influencing the enzyme activities and metabolism product involving sex pheromone biosynthesis. Furthermore, sublethal bifenthrin exposure suppresses the JH signaling pathway by reducing the expression level of the Met and Kr-h1, resulting to developmental delays in ovarian maturation, reduced ovary size, and a significant decrease in egg production and hatchability.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c06445.

• Primer sequence of real-time PCR in C. punctiferalis and bifenthrin toxicity to C. punctiferalis female and male adults (PDF)

The authors declare no competing financial interest.