Dissecting the manipulation of lufenuron on chitin metabolism, and the sublethal effects on Helicoverpa armigera development

doi:10.21203/rs.3.rs-3870071/v1

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Dissecting the manipulation of lufenuron on chitin metabolism, and the sublethal effects on Helicoverpa armigera development

https://doi.org/10.21203/rs.3.rs-3870071/v1

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Lufenuron, acting as a chitin synthesis inhibitor of benzoylureas class, is effective against many insect pests, particularly detrimental to their immature stages. However, the insecticidal activity of lufenuron has not been completely elucidated, nor do its disturbing effect on chitin metabolism genes. In this study, our bioassay demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC₂₅ lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument in a concentration-dependent manner, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that 7 of 10 chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increases in larval susceptibilities to LC₂₅ lufenuron by 75.48%, 65.0%, 68.42% and 28.0%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and highlighted the combined administration of insect growth regulators (IRGs) and the RNAi of specific genes would be developed as a promising strategy to efficiently manage insect pests.

Helicoverpa armigera

Lufenuron

sublethal effects

Chitin synthesis

integument

Chitin is a polymer of N-acetyl glucosamine (GlcNAc), contributing substantially to barrier and shaping function of exoskeletons in many invertebrates (Cohen, 2010; Liu et al., 2019; Chen et al., 2022). In insects, the biosynthesis of chitin is critical for their survival and reproduction due to the fact that chitin is an essential constituent of epidermis, trachea, midgut and ovary (Merzendorfer et al., 2006; Beverly et al., 2020). Chitin biosynthesis begins with the disaccharide trehalose, and culminates in the polymerization of GlcNAc subunits to produce chitin microfbrils (Chen et al., 2022), which involves eight key regulatory enzymes, including trehalase (TRE), hexokinase (HK), glucose-6-phosphate isomerase (G6PI), glutamine fructose-6-phosphate aminotransferase (GFAT), glucosamine 6-phosphate N-acetyl-transferase (GNPAT), phosphoacetyl glucosamine mutase (PGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and chitin synthase (CHS). TRE, the first enzyme in chitin biosynthesis pathway, is responsible for the conversion of trehalose to glucose (Liu et al., 2019). The last step is executed by CHS, a membrane-integrated hexosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain (Yang et al., 2021; Chen et al., 2022). Insects appear to possess two chitin synthase genes (CHS1 and CHS2). Previous studies in Tribolium castaneum and many other insect species have documented that CHS1 is exclusively expressed in the ectoderm-derived epidermal cells such as epidermis and trachea, and is responsible for chitin synthesis in these tissues, whereas CHS2 is specialized in producing chitin in the midgut peritrophic matrix (Hogenkamp et al., 2005; Zhang et al., 2006; Chen et al., 2007; Arakane et al., 2008; Kelkenberg et al., 2015; Jiang et al., 2021). Besides, two chitin-degrading enzymes, chitinases (CHIs) and N-acetyl-glucosaminidases (NAGs), cooperate with chitin synthases in remodeling chitinous structures, playing a crucial role in larval molt and pupation. Considering the irreplaceable role of chitin in insect growth and the absence of chitin biosynthesis in mammals, disruption of chitin metabolism has been recognized as an attractive target for developing insecticides with high efficiency and low eco-toxicity.

In insects, exposure to insect growth regulators (IGRs) interfere with specific developmental processes, causing perturbations in insect development, such as growth obstruction, molting failure and metamorphosis abnormality, and representing a novel insecticidal mode of action (Douris et al., 2016; Catchot et al., 2020). Lufenuron, an insect growth-regulating insecticide functioning as chitin synthesis inhibitor, was developed as the newest generation of benzoylureas. To date, the outstanding insecticidal performance of lufenuron has been documented in Spodoptera frugiperda (Lv et al., 2022), Spodoptera exigua (Chen et al., 2019), Rhynchophorus ferrugineus (Milosavljević et al., 2019), and etc. At high concentration, lufenuron exhibits outstanding toxicity against eggs and larvae, whereas the sublethal doses of lufenuron would extend larval duration, disrupt pupation and eclosion, and suppress oviposition and egg hatching (Moreira et al., 2007; Mansur et al., 2010; Aliabadi et al., 2016). As previously reported, the action mode of lufenuron is that this chemical mainly blocks chitin synthesis, breaks cuticle formation, and causes ecdysis failure (Martínez et al., 2022). However, the precise biochemical explanation of the insecticidal activity of lufenuron has not been completely defined, nor do its disturbing effect on chitin synthesis pathway genes.

Previous studies have proposed that benzoylureas insecticides may interact with chitinases or chitin synthases to manipulate their functions. For example, when housefly larvae received the dietary treatment of chitin synthesis inhibitor TH6040, the cuticle chitinase activity was significantly enhanced, and simultaneously the amount of cuticle chitin was progressively reduced with the increasing concentrations of TH6040 treatment (Ishaaya et al., 1974). In mosquito affected by diflubenzuron, a significant increase in chitin synthase (AqCHS1) expression was coupled with a reduced chitin synthesis (Zhang et al., 2006). In S. frugiperda, exposures of the 3rd-instar larvae to lufenuron significantly decreased the expression of chitinase gene (SfCHT), while the expression of chitin synthase gene (SfCHS) remained relatively stable (Lv et al., 2023). However, the relationship between reduced chitin content and disturbed chitin biosynthesis pathway has not been resolved in insect response to benzoylureas insecticides. In this work, the toxicity of lufenuron to Helicoverpa armigera larvae was first determined. To clarify the perturbation of lufenuron on chitin synthesis, we explored the adverse effect of lufenuron on cuticle formation, investigated the effect of lufenuron on the chitin synthesis in epidermis and the trehalose content in hemolymph, analyzed the lufenuron-mediated changes of chitin metabolism genes, and further explored the impact of RNAi on the larval susceptibility to lufenuron. These results would help to deepen the knowledge of the action mechanism of benzoylureas chitin synthesis inhibitors.

2.1 Insect rearing and samples collection

The H. armigera individuals used in this work was collected from Jiujiang county, Jiangxi province, China. This strain was maintained under a phytotron of 27 ± 2°C, 75 ± 10% relative humidity, and a photoperiod of 14 h:10 h (light: dark). Larvae were reared on artificial diet in 24-well plates, and transferred into 25 mL glass tubes at 5th-instar for pupation (one larva per tube). After emergence, adults were placed in cages for oviposition and supplied with 10% honey solution.

For trehalose content analysis, 3rd-instar larvae were anesthetized on ice, and their hemolymph was immediately collected from a small cut made on the first proleg. Next, epidermis was dissected separately from larvae on ice, washed in ice-cold phosphate-buffered saline (PBS), and drained by blotting paper. These detached tissues of epidermis were sampled for chitin content assay. At least ten larvae were included in each replicate, and four replicates were performed for each treatment.

2.2 Bioassays

Bioassays were conducted with 3rd- and 4th-instar larvae following a leaf-dipping method with slight improvements. Lufenuron dissolved in acetone containing 0.1% Triton X-100 was prepared as the stock solution (100 µg mL^− 1). Then the stock solution was diluted to seven serials of working concentrations (8, 4, 2, 1, 0.5, 0.25 and 0.125 µg mL^− 1) using an aqueous solution of 0.1% (w/v) Triton X-100. A water solution containing 1% acetone and 0.1% Triton X-100 was set as the blank control. Small squares of artificial diet (approximately 1 cm³) were dipped with the diluted insecticide at each working concentration for 5 s, then left to air-dry for 2 h at room temperature. Each piece of insecticide-treated diet was placed in a single well of 24-well plate, into which one larva was transferred. All plates were kept under laboratory conditions as mentioned. Larvae mortality was determined at three days post treatment, as well as the lethal concentrations of LC₁₀, LC₂₅ and LC₅₀. Larvae showing no sign of movement when gently touched with a brush were scored as dead. Each concentration was conducted with three replicates, and at least 24 individuals were included in each replicate.

2.3 Sublethal effects of lufenuron on larval development

To analyze the sublethal effects of lufenuron on larval development, the 3rd- and 4th-instar H. armigera larvae were initially starved for 2 h, then subjected to LC₁₀ and LC₂₅ lufenuron treatment following a modified leaf-dipping method, respectively. Larvae treated with water agent containing 0.1% Triton X-100 and 1% acetone served as the control. Each treatment was performed four times, with at least 30 larvae per replicate (a total of 120 larvae per treatment). After 72 h treatment, the surviving individuals were transferred to diet without insecticide, and reared until pupation. Then pupae were sexed and weighed individually, and each pupa was placed in a single plastic cup. After emergence, adults were fed with 10% honey solution. The biological parameters, including larval duration, pupal duration, pupal weight, and adult longevity, were recorded. Besides, the rates of pupation and adult emergence were calculated.

2.4 Paraffin section and hematoxylin & eosin staining

The detached tissues of larval epidermis were fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M PBS (pH 7.4), rinsed in distilled water, dehydrated in an ethanol series (with ascending concentrations from 30–100%), and then embedded in paraffin. After dewaxing with xylene, histological sections of 10 µm thickness were stained with hematoxylin, thoroughly washed with distilled water, and further stained with 1% eosin solution. After sealing, the sections were observed under a stereomicroscope (BX61, Olympus, Tokyo, Japan).

2.5 Quantification of trehalose content in hemolymph

The trehalose content in hemolymph was determined using an Insect Trehalose ELISA Kit (item number: YS04657B; Yaji Biotechnology, Shanghai, China). For trehalose content quantification, 10 µL of hemolymph was 5-fold diluted and used for ELISA following the product manual. In colorimetric assay, a six-point calibration curve was constructed by standard trehalose solutions at 0, 1, 2, 4, 8 and 16 µmol L^− 1. The absorbance of sample solution was measured at 450 nm via a microplate spectrophotometer (Spectramax 190, Molecular Devices Corporation, Sunnyvale, USA), and the trehalose content in each sample was calculated based on the established standard curve.

2.6 Quantification of chitin content in integument

After vacuum freeze-dry, the detached tissues of larval integument were used for chitin content quantification. According to the method of chitin extraction (Shi et al., 2016a), samples were first homogenized in 0.5 mL of 6% KOH, and then incubated at 80°C for 90 min. After centrifugation at 12,000×g for 10 min, the resultant supernatant was discarded and the pellet was repeatedly washed by PBS. Next, each pellet was dissolved in 200 µL Mcllvaine’s buffer (0.1 M citric acid, 0.2 M NaH₂PO₄, pH 6). After addition of Streptomyces griseus chitinase (item number: C6137; Sigma-Aldrich, Darmstadt, Germany), the sample solutions were incubated at 37°C for 40 h to hydrolyze chitin to GlcNAc.

The chitin content was determined following a modified Morgan-Elson assay (Reissig et al., 1955). Briefly, 10 µL of sample solutions was mixed with the same volume of 0.27 M sodium borate, and incubated at 99°C for 10 min. After cooling to 20°C, the reaction solution was further mixed with 100 µL of the diluted 4-(Dimethylamino)-benzaldehyde solution, followed by incubation at 37°C for 20 min. For colorimetric assay, an eight-point calibration curve were first constructed by serial dilutions of standard GlcNAc solutions (0, 20, 40, 60, 80, 100, 120, 240 µg mL^− 1). Then the absorbance of sample solution was measured at 585 nm by a microplate spectrophotometer (Spectramax 190), and the chitin content in each sample was calculated based on the standard curve.

2.7 RNA isolation and cDNA synthesis

Total RNA was extracted from samples using Trizol reagent (Sangon Biotech, Shanghai, China). The RNA integrity was checked by 1% agarose gel electrophoresis, and the quantity of RNA was detected by a microplate spectrophotometer (Spectramax 190). After removal of residual genomic DNA, 2 µg of purified RNA was applied to synthesize first-strand cDNA with a FastKing RT kit (TianGen, Beijing, China).

2.8 cDNA Library Construction and RNA Sequencing

The cDNA library was constructed using the total RNA from 3rd-instar H. armigera larvae after LC₅₀ lufenuron treatment for 24 and 48 h, respectively. Briefly, each treatment was carried out in 24 larvae, and three biological replicates were processed for lufenuron treatment or control, serving as three independent replicates in each treatment for RNA sequencing. Next, 2 µg of qualified total RNA was used for constructing cDNA library. The tasks of mRNA enrichment, mRNA fragment digestion, cDNA synthesis, adapter ligation, PCR amplification and cDNA library sequencing were delegated to Sangon Biotech (Shanghai, China). The cDNA libraries were sequenced using the Illumina HiSeq X ten platform. After omission of adapters and low-quality reads, the clean data were de novo assembled by Trinity. For functional annotation, the resultant contigs were further aligned with the databases of NR, NT, SwissProt, COG, and KEGG through a BLASTx research. To screen the differential expression of genes between samples, transcript abundances were normalized by FPKM metric (fragment per kilobase of exon per million fragments mapped). Transcripts were identified as differentially expressed at significant levels when the false discovery rate (FDR) ≤ 0.05 and the absolute value of log₂(fold change) ≥ 1. To validate the reliability of the transcriptome sequencing data, the expressions of chitin metabolic genes were further investigated by qRT-PCR analysis (Table S1).

2.9 qRT-PCR analysis

The expression patterns of chitin biosynthesis and chitin degradation genes were analyzed by qRT-PCR. qRT-PCR was performed using SYBR Green SuperReal PreMix Plus (Tiangen, Beijing, China) through a CFX96 Touch Real-time PCR detection system (Bio-Rad). Amplifications were conducted under the following conditions, initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 5 s and 60°C for 32 s, followed by a melting curve stage (60 to 95°C) to confirm the gene-specific amplification. Two house-keeping genes, β-actin (accession no. EU527017) and Ef-1α (accession no. FJ768770.1), were selected as the internal controls. The amplification efficiencies of the targets and reference genes were calculated using a gradient dilution of templates. Each reaction was carried out in triplicate, and negative controls without template were included in each reaction. Relative expression levels normalized to reference genes were calculated by the comparative Ct (2^−ΔΔCt) method. The primer pairs for target and reference genes are listed in Table S1. Then a linear regression analysis of the correlation between RNA-Seq data and qRT-PCR analysis was performed, and the results showed an R² value of 0.913 and a corresponding slope of 1.182, suggesting a strong positive correlation between qRT-PCR analysis and transcriptome sequencing (Figure S2).

2.10 RNAi and Bioassays

After introduction of T7 sequences in both strands, a linear DNA containing a segment of HaChA (325 bp) was constructed as template for dsRNA synthesis, and simultaneous a partial sequence of GFP (green fluorescent protein) was amplified to produce dsRNA for GFP (dsGFP) as control (Table S1). Then dsRNAs were synthesized using T7 RiboMAX™ Express RNAi System (item number: P1700; Promega, Madison, WI, USA). After the transcription reaction, the DNA template was removed by digestion with DNase, and the synthesized dsRNA was purified by 3M sodium acetate and isopropanol precipitation. Finally, the purity and yield of dsRNA was checked on 1.0% agarose gel and a microplate spectrophotometer (Spectramax 190), respectively. Primers used for dsRNA synthesis were listed in Table S1.

In each RNAi experiment, 30 individuals of 3rd-instar larvae were first cold-anesthetized, then 1 µL dsRNA (2 µg µL^− 1) targeting HaChA was injected into the abdomen segmacoria of each larva using a microsyringe (Hamilton, Bonaduz, Switzerland), and the control individuals were treated with equal doses of dsGFP. The injection point was sealed with geoline immediately. To investigate the RNAi efficiency, 10 specimens were randomly selected from each treatment at 24, 48 and 72 h post-injection for qRT-PCR analysis. Moreover, dsRNAs targeting HaChS1, HaUAP, HaGNPAT, and HaPGM were synthesized separately, and their RNAi experiments were performed following the protocols as mentioned above.

To examine the effect of HaChA RNAi on larval susceptibility to lufenuron, the treatments of (i) dsGFP, (ii) dsHaChA, (iii) LUF + dsGFP, and (iv) LUF + dsHaChA were performed on the newly molted 3rd-instar larvae, separately. In brief, RNAi experiments of HaChA and GFP were operated as described above. For the treatments of (iii) LUF + dsGFP and (iv) LUF + dsHaChA, larvae injected with dsRNA were further subjected to the lufenuron-treated diet at LC₂₅ concentration following a modified leaf-dipping method as described. Larval mortality was detected at 48 h after treatment. Each treatment was carried out in 24 larvae and repeated thrice. In addition, the changes in lufenuron susceptibility were also evaluated in RNAi larvae of HaChS1, HaUAP, HaGNPAT, and HaPGM, respectively, in the same manner.

2.11 Data analysis

All data were presented as means ± SE. Bioassay data were analyzed by probit analysis using POLO-PC (LeOra Software, Berkeley, CA). Significant differences were determined by Student's t-test or one-way ANOVA followed by a least significant difference test for mean comparison. All statistical analyses were performed using SPSS 18.0 (SPSS, Chicago, IL, USA).

3.1 Toxicity of lufenuron

Toxicity assays on 3rd-instar larvae showed that the values of LC₁₀, LC₂₅ and LC₅₀ were 0.822, 2.024 and 5.506 µg mL^− 1 at 72 h after lufenuron treatment, respectively (Table 1). The toxicity regression equation is y = 0.674x-1.150 (R² = 0.947). When the 4th-instar larvae were treated with lufenuron, the resulting values of LC₁₀, LC₂₅ and LC₅₀ were 1.773, 2.710 and 6.036 µg mL^− 1 at 72 h after treatment, respectively, and the corresponding toxicity regression equation is y = 1.431x–2.101 (R² = 0.986).

Table 1

Toxicity of lufenuron against *H. armigera* larvae at 72 h after treatment
Larval instar	df	Slope ± SE	LC₁₀ (95% CL) (µg/ml)	LC₂₅ (95% CL) (µg/ml)	LC₅₀ (95% CL) (µg/ml)	χ²
3rd-instar	5	0.674 ± 0.116	0.822 (0.415 ~ 1.254)	2.024 (1.340 ~ 2.970)	5.506 (3.666 ~ 10.410)	7.267
4th-instar	4	1.431 ± 0.31	1.773 (1.161 ~ 2.319)	2.710 (2.017 ~ 3.366)	6.063 (4.681 ~ 8.121)	3.056
CL, confidence limit

Phenotypic observations revealed that the LC₅₀-treated larvae encountered severe ecdysis failure at 72 h post treatment, and only the head carapace completed molting (Fig. 1). The body of lufenuron-treated larvae become swollen, and their cuticle became darkened and wrinkled. Hematoxylin and eosin staining showed the severe separation of epidermis and subcutaneous tissues in lufenuron-treated larvae, and their cuticle become significantly thinner compared with the control (Fig. 2).

3.2 Sublethal effects of lufenuron treatment on larval development

The sublethal effects of lufenuron on larval development were shown in Table 2 and Table 3, and significant differences were observed in various life traits after exposure to sublethal doses of lufenuron. When the 3rd-instar larvae were treated with lufenuron, our results showed that the larval duration had a marked extension of 0.74 and 1.82 days in LC₁₀ and LC₂₅ treatment groups (P < 0.0001), respectively, compared with the control; and meanwhile their pupal durations were extended by 0.54 and 1.29 days (P < 0.001). Moreover, the pupation rate of LC₂₅ treatment was decreased by 24.08% relative to the control. Likewise, the emergence rate decreased significantly after LC₁₀ and LC₂₅ treatments (P = 0.012). Additionally, the mean weight of male and female pupae was significantly decreased after LC₁₀ and LC₂₅ treatments relative to the control group (P < 0.0001).

Table 2

Sublethal effects of lufenuron on the development of 3rd-instar larvae of *H. armigera*
Treatment	Larval duration (days)	Pupation rate (%)	Pupal weight (mg)		Pupal duration (days)	Emergence rate (%)
Treatment	Larval duration (days)	Pupation rate (%)	Male	Female	Pupal duration (days)	Emergence rate (%)
CK	15.21 ± 0.10c	91.36 ± 2.56a	249.08 ± 7.68a	279.13 ± 14.61a	9.95 ± 0.10c	90.73 ± 2.42a
LC₁₀	15.95 ± 0.31b	84.74 ± 2.90a	202.81 ± 13.18b	216.45 ± 13.23b	10.49 ± 0.15b	81.94 ± 0.69 b
LC₂₅	17.03 ± 0.31a	69.36 ± 3.66b	173.06 ± 7.86c	200.25 ± 11.86b	11.24 ± 1.40a	73.89 ± 3.89b
P	< 0.0001	0.008	0.0001	< 0.0001	< 0.001	0.012
F	14.13	11.83	16.75	9.37	24.001	10.183
df	2, 167	2, 6	2, 60	2, 55	2, 118	2, 6

Table 3

Sublethal effects of lufenuron on the development of 4th-instar larvae of *H. armigera*
Treatment	Larval duration (days)	Pupation rate (%)	Pupal weight (mg)		Pupal duration (days)	Emergence rate (%)
Treatment	Larval duration (days)	Pupation rate (%)	Male	Female	Pupal duration (days)	Emergence rate (%)
CK	15.08 ± 0.11c	91.25 ± 2.29a	211.8 ± 4.03a	240.22 ± 3.16a	10.10 ± 0.10c	92.83 ± 0.30a
LC₁₀	15.97 ± 0.26b	77.38 ± 4.29a	197.03 ± 4.26ab	187.42 ± 4.37b	11.18 ± 0.15b	74.89 ± 3.89b
LC₂₅	16.82 ± 0.21a	53.68 ± 4.44b	169.83 ± 1.85b	178.38 ± 3.95b	12.32 ± 0.20a	69.44 ± 2.78b
P	< 0.0001	0.001	0.074	0.0001	< 0.001	0.001
F	18.04	24.21	2.77	12.43	54.84	31.285
df	2, 198	2, 6	2, 84	2, 80	2, 162	2, 6

In 4th-instar larvae subjected to lufenuron treatments, the LC₁₀- and LC₂₅-treated individuals significantly prolonged their larval duration by 0.89 and 1.74 days, respectively (P < 0.0001), and similarly their pupal durations were extended by 1.08 and 2.22 days (P < 0.001). It was notable that the emergence rate decreased significantly after LC₁₀ and LC₂₅ treatments (P = 0.001). Similarly, the pupation rate of LC₂₅ treatment decreased by 41.17% compared with that in the control, however, no significant difference was observed in pupation rate between CK and LC₁₀ group. Moreover, the LC₁₀- and LC₂₅-treated individuals showed significant decrease in female pupal weight, compared with that in the control. On the other hand, the average weight of male pupae was also significantly declined in LC₂₅ group, but no significant difference was detected in that between CK and LC₁₀ group.

3.3 Effects of lufenuron treatment on trehalose content in hemolymph

As the starting substrate for chitin biosynthesis, the trehalose content in hemolymph was investigated in 3rd-instar larvae after lufenuron treatment (Fig. 3). It was notable that the LC₅₀-treated larvae showed a reduction in trehalose content in their hemolymph throughout the whole period of treatment, and significant differences were detected at 12, 36, 48, 72, 96 h after treatment between groups (P < 0.05), decreasing by 25.06%, 30.82%, 19.12%, 28.31% and 30.03%, respectively. Moreover, our results showed that exposure of larvae to lufenuron at 2.0, 4.0, 8.0 and 10.0 µg/mL for 72 h resulted in a decrease of trehalose content in hemolymph by 66.10%, 53.11%, 45.42% and 17.92%, respectively, relative to the control.

3.4 Effects of lufenuron treatment on chitin content in integument

In 3rd-instar larvae after LC₅₀ lufenuron treatment, it was notable that the chitin contents were greatly reduced in their integument throughout the treatment, compared with those in control individuals, and significant differences were detected at 24, 36, 48, 72, 96 h after treatment (P < 0.05), decreasing by 41.29%, 35.76%, 58.44%, 69.67% and 38.10%, respectively (Fig. 4). Additionally, our results showed that exposure of larvae to lufenuron at 2.0, 4.0, 8.0 and 10.0 µg/mL for 72 h resulted in a decrease of chitin content by 47.70%, 54.31%, 56.56% and 76.63% in integument, respectively, relative to the control.

3.5 Expressions of chitin biosynthesis and degradation genes after lufenuron treatment

To investigate the effects of lufenuron on chitin metabolic genes, a comparative transcriptome analysis was performed between control and the lufenuron-treated larvae (Fig. 5). The results showed that 998 unigenes were identified as differentially expressed genes (DEGs) after LC₅₀ lufenuron treatment for 24 h; of these, 595 were up-regulated and 403 were down-regulated relative to the control. According to GO enrichment analysis, the down-regulated DEGs were significantly enriched to the pathways of cuticle development, chitin-based cuticle development, and extracellular region. After lufenuron treatment for 48 h, a total of 6,187 DEGs were detected after comparative transcriptome analysis between control and the lufenuron-treated group, among which 4,248 were up-regulated and 1,939 were down-regulated.

Subsequently, ten and three genes were screened out as chitin biosynthesis and chitin degradation genes, respectively, and their expressions were investigated after lufenuron treatment. The results showed that the chitin synthesis genes of HaG6PI, HaPGM, HaUAP, HaCHS1, and HaCHS2 were down-regulated by 16.17%, 22.20%, 10.95%, 26.0%, and 15.0% in lufenuron-treated larvae for 24 h, but significant difference was only detected in the expressions of HaCHS1 and HaPGM (P < 0.05); while the significantly up-regulated genes were HaHK and HaGNPAT. Moreover, two chitin degradation genes, HaCHI1 and HaCHI2, were significantly overexpressed by 131.74% and 130.58%. When larvae were exposed to LC₅₀ lufenuron for 48 h, three chitin synthesis genes, HaTreh2, HaHK, and HaPGM, were significantly overexpressed, while the significant down-regulation were detected in the expressions of HaGNPAT and HaUAP (P < 0.05).

3.6 The effect of RNAi on larval susceptibility to lufenuron

To evaluate the effect of RNAi on larval susceptibility to lufenuron, four chitin synthesis genes (HaCHS1, HaUAP, HaG6pa, and HaPGM) and one chitin degradation gene (HaCHA) were chosen for RNAi experiments, and their expressions were examined via qRT-PCR (Fig. 6). Our results showed that the transcripts of HaCHI, HaCHS1, HaUAP, HaGNPAT, and HaPGM were significantly attenuated from 24 to 72 h after RNAi, with silencing effects of 79.20%, 66.33%, 72.77%, 50.26%, and 61.95% at 72 h, separately, relative to the control.

Larval mortality was checked at 72h after RNAi treatment or the combined treatment of RNAi and lufenuron exposure (Fig. 7). Our results revealed that separate RNAi of HaCHI, HaCHS1, HaUAP, HaGNPAT, and HaPGM caused no significant changes in larval mortality compared with that in dsGFP-treated group. Moreover, it was notable that the larval susceptibilities to LC₃₀ lufenuron were significantly enhanced in dsHaCHS1, dsHaUAP, dsHaGNPAT, and dsHaPGM treated individuals (P < 0.05), and their mortalities were increased by 75.48%, 65.0%, 28.0%, and 68.42%, respectively, relative to that in dsGFP-treated larvae. Among these, knockdown of HaCHS1 significantly increased larval mortality from 31.25–54.83% when the larvae were further exposed to lufenuron, and a marked increase in larval mortality from 31.25–51.56% were observed following lufenuron administration after HaUAP knockdown. Besides, the susceptibility to lufenuron rose from 31.25–52.63% in HaPGM-RNAi larvae, compared with that in dsGFP-treated group. However, lufenuron treatment caused no significant change in mortality between HaCHI-RNAi larvae and dsGFP-treated larvae (P > 0.05).

Lufenuron, acting as a chitin synthesis inhibitor whose main mode of action is stomach toxicity, is effective against many economically important insect pests, particularly detrimental to their immature stages (Chen et al., 2019; Milosavljević et al., 2019; Lv et al., 2022). Our results of bioassay demonstrated an outstanding toxicity of lufenuron against 3rd and 4th instar larvae of H. armigera, and the treated larvae died from abortive molting and metamorphosis abnormality. Likewise in S. frugiperda and S. exigua, lufenuron treatment caused serious molting failure of the larvae, and their cuticle became thinner and easily ruptured (Chen et al., 2019; Lv et al., 2022). Moreover, our results revealed the severe separation of epidermis and subcutaneous tissues in lufenuron-treated larvae of H. armigera, and their cuticle become noticeably thinner (Fig. 2). The detrimental effects of lufenuron on cuticle architecture was also documented in Drosophila, where the larval cuticle showed undistinguishable and uneven functional layers and has lost regular thickness, compared with the wild-type cuticle (Gangishetti et al., 2009). Besides, our results revealed that lufenuron can severely reduce chitin content in H. armigera larvae in a concentration-dependent manner, wherein the cuticular chitin content was decreased by 76.63% in exposure to 10.0 µg/mL for 72 h (Fig. 4). These results generally followed a similar pattern for the toxicity of lufenuron as indicated by mortality (Table 1). Overall, these findings suggested that the cuticle organization were disrupted after lufenuron exposure.

After field application of insecticides, insecticide degrades over time reducing the initial (lethal) deposit to a (sublethal) residue, and insect survivors suffer from insecticide residue over long periods (Guedes et al., 2017). In our study of H. armigera, treatment of 3rd- and 4th-instar larvae with LC₂₅ of lufenuron significantly extended their duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Likewise in 3rd-instar larvae of S. frugiperda, the sublethal doses of lufenuron prolonged larval duration, but reduced the pupation rate and emergence rate (Lv et al., 2023). In regards to another benzoylurea pesticide hexaflumuron, it was documented that the treatment at LC₃₀ concentration decreased larval weigh by 55.5%, and conversely increased the larval and pupal developmental time in H. armigera (Vojoudi et al., 2017). Previous studies have proposed that benzoylureas insecticides might affect hormonal balance in insects, such as the crosstalk between juvenile hormone (JH) and 20-hydroxyecdysone (20E), thereby resulting in physiological disturbances such as the retarded development in larval and pupal stage (Zhang et al., 2006). Overall, our determination of the sublethal effects of lufenuron would provide guidance for prolonging its long-term efficacy as a successful pest management tool.

Although the adverse effects of lufenuron on larval development and cuticle formation have been documented, the action mechanisms of lufenuron remain largely unknown. To explore the effects of lufenuron on chitin metabolism, comparative analysis of the transcriptomes from the lufenuron-treated and the control H. armigera larvae at 24 h revealed that the chitin synthesis genes of HaG6PI, HaPGM, HaUAP, HaCHS1, and HaCHS2 were down-regulated, whereas the expressions of two chitin degradation genes, HaCHI1 and HaCHI2, were significantly enhanced. Likewise, oral administration of lufenuron in S. exigua caused the down-regulation of PGM, UAP, GFAT, and CHS1, but showed an inductive effect on chitinase genes (Zhang et al., 2022). Collectively, these finding provides compelling evidence that the in vivo chitin metabolism pathways were manipulated by lufenuron treatment.

To further investigate the involvement of chitin metabolism genes in the insecticidal activity of lufenuron, toxicity bioassays combined with RNAi were operated in five differentially expressed chitin metabolism genes. Our results showed that the RNAi-mediated knockdown of HaCHS1, HaUAP, HaPGM, and HaGNPAT led to significant increases in larval susceptibilities to LC₃₀ lufenuron by 75.48%, 65.0%, 68.42% and 28.0%, respectively. Likewise, knockdown of UAP and CHS1 increased the insecticidal efficiency of lufenuron to 3rd-instar larvae of S. exigua, leading to 49.9% and 63.9% increase in larval mortality, separately (Zhang et al., 2022). As a key enzyme in chitin biosynthesis, CHS is essential for the polymerization and deposition of chitin chains in insect chitinous structures (Araujo et al., 2005); depletion of CHS would inhibit chitin production and lower chitin content in insect body and integument in many insect species (Shi et al., 2016b; Jiang et al., 2021; Yang et al., 2021). Remarkably, functional study by genome-editing technology has proved that a single mutation in CHS1 resulted in high resistance to benzoylureas insecticides, providing compelling evidence that benzoylureas directly interact with CHS (Douris et al., 2016). In the present study, our RNAi largely consumed the expression of HaCHS1; combined with previous documents, we speculated that the depletion of HaCHI could seriously weakened the chitin synthesis in H. armigera, and consequently contributed to the susceptibilities to lufenuron in HaCHI-RNAi larvae. All in all, our result supported that the combined administration of IGRs and the RNAi of specific genes would be developed as a promising strategy to efficiently manage insect pests.

Previous studies have documented that knockdown of any of CHS, UAP and PGM led to severe developmental deformities and significant mortality, wherein the HvCHS1-RNAi larvae finally died within ten days in Henosepilachna vigintioctopunctata (Jiang et al., 2021), the cumulative mortalities in HaPGM or HaUAP-RNAi larvae reached more than 65.61% recorded from RNAi treatment until adult emergence in H. armigera (Das et al., 2022), and the 4th-instar larvae fed with dsLdUAP1 were wrapped in larval cuticle within 12 days and finally died in Leptinotarsa decemlineata (Shi et al., 2016a). In insects during ecdysis, chitin synthesis is necessary to construct a new cuticle with sufficient mechanical strength to break through the old exoskeleton (Arakane et al., 2011). Besides, knockdown of any of CHS1, PGM and UAP could result in chitin deficiency and impair the formation of new cuticle, and consequently the treated larvae died from molting failure (Arakane et al., 2011; Shi et al., 2016a; 2016b; Jiang et al., 2021; Das et al., 2022). However, our RNAi revealed that separate knockdown of HaCHI, HaCHS1, HaUAP, HaGNPAT, and HaPGM caused no significant changes in larval mortality at three days post RNAi, and we supposed that this may be attributed to the duration after RNAi treatment, in which our duration (72 h) was apparently shorter than those in previous documents, and only after the prolonged chitin deficiency could cause larval molting failure and death.

Taken together, our findings revealed the adverse impacts of lufenuron on H. armigera development, elucidated the perturbations on chitin metabolism pathways after lufenuron exposure, revealed a decline in trehalose content in hemolymph, and detected impaired chitin synthesis in epidermis of lufenuron-treated larvae. Currently, it has been documented that the combination of bio-pesticide (Bt toxin) and RNAi suppression (interfering with JH synthesis) in one transgenic cotton plant conferred excellent insecticidal capacity against H. armigera (Ma et al., 2019), and our results also highlighted that the joint action of RNAi and lufenuron provided a new perspective in effective controlling this menacing pest. Our findings therefore provide a deep insight into the action mechanisms of lufenuron, and our assessment of the sublethal effects of lufenuron informs practical guidance for its long-term usage.

Author Contribution

WNZ and LM designed research. LM, ZWZ, RHY, YCP, and XFS conducted the experiments. WNZ and LM analyzed the data and wrote the manuscript.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (31960543, 32060642, 32260714), Natural Science Foundation of Jiangxi Province (20224BAB215018, 20232BAB215037, 20232BCJ23048, 20223BBF61017), and Top Young Talents Fund Project of Jiangxi Science & Technology Normal University (2022QNBJRC002).

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Dissecting the manipulation of lufenuron on chitin metabolism, and the sublethal effects on Helicoverpa armigera development

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Abstract

Figures

1 Introduction

2 Material and Methods

2.1 Insect rearing and samples collection

2.2 Bioassays

2.3 Sublethal effects of lufenuron on larval development

2.4 Paraffin section and hematoxylin & eosin staining

2.5 Quantification of trehalose content in hemolymph

2.6 Quantification of chitin content in integument

2.7 RNA isolation and cDNA synthesis

2.8 cDNA Library Construction and RNA Sequencing

2.9 qRT-PCR analysis

2.10 RNAi and Bioassays

2.11 Data analysis

3 Results

3.1 Toxicity of lufenuron

3.2 Sublethal effects of lufenuron treatment on larval development

3.3 Effects of lufenuron treatment on trehalose content in hemolymph

3.4 Effects of lufenuron treatment on chitin content in integument

3.5 Expressions of chitin biosynthesis and degradation genes after lufenuron treatment

3.6 The effect of RNAi on larval susceptibility to lufenuron

4 Discussion

Declarations

Author Contribution

Acknowledgments

References

Additional Declarations

Supplementary Files

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