Novel compounds ZK-PI-5 and ZK-PI-9 regulate the reproduction of Spodoptera frugiperda (Lepidoptera: Noctuidae), with insecticide potential

Abstract

Trehalase inhibitors prevent trehalase from breaking down trehalose to provide energy. Chitinase inhibitors inhibit chitinase activity affecting insect growth and development. This is an important tool for the investigation of regulation of trehalose metabolism and chitin metabolism in insect reproduction. There are few studies on trehalase or chitinase inhibitors’ regulation of insect reproduction. In this study, ZK-PI-5 and ZK-PI-9 were shown to have a significant inhibitory effect on the trehalase, and ZK-PI-9 significantly inhibited chitinase activity in female pupae. We investigated the reproduction regulation of Spodoptera frugiperda using these new inhibitors and evaluated their potential as new insecticides. Compounds ZK-PI-5 and ZK-PI-9 were injected into the female pupae, and the control group was injected with solvent (2% DMSO). The results showed that the emergence failure rate for pupae treated with inhibitors increased dramatically and aberrant phenotypes such as difficulty in wings spreading occurred. The oviposition period and longevity of female adults in the treated group were significantly shorter than those in the control group, and the ovaries developed more slowly and shrank earlier. The egg hatching rate was significantly reduced by treatment with the inhibitor. These results showed that the two new compounds had a significant impact on the physiological indicators related to reproduction of S. frugiperda, and have pest control potential. This study investigated the effect of trehalase and chitin inhibitors on insect reproduction and should promote the development of green and efficient insecticides.

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Introduction

Some insect pests cause serious agricultural production losses due to their relatively high fecundity. Controlling aspects of their reproduction through biochemical means is one way to help mitigate these production losses. Vitellogenin (Vg), as a precursor to vitellin (Vtn) and the most important insect yolk protein, is one of the key factors for insect reproduction (Tufail and Takeda 2008). It is widely found in the hemolymph, fat body, and ovaries of oviparous females and is the yolk protein precursor of almost all oviparous animals (Sappington and Raikhel 1998). After synthesis in the fat body of female insects, Vg is released into the hemolymph and transported to the oocyte, combining the vitellogenin receptor (VgR) on the oocyte membrane. It provides a variety of nutritional and functional substances for embryonic development and plays an important role in the development of the insect ovary (Tufail and Takeda 2009, Ge and Wu 2010, Wu et al. 2018).

Trehalose is a nonreducing disaccharide consisting of 2 glucose molecules, commonly found in bacteria, plants, insects, and other organisms (Elbein et al. 2003). As an important energy substance in the hemolymph of insects, trehalose metabolism is beneficial to the development of oocytes and participates in the uptake of Vg by oocytes (Santos et al. 2008, Wang et al. 2020). Trehalase (TRE) is the only enzyme known to break down trehalose in all organisms participating in the energy metabolism process of the organism and regulating physiological processes such as development and reproduction, molting, and metamorphosis (Elbein et al. 2003, Tang et al. 2018, Wang et al.2021, Yu et al. 2022). Inhibition of trehalose metabolism causes delayed ovarian development and abnormal oogenesis in Bombyx mori (Katagiri et al. 1998). TRE is the first enzyme in the chitin synthesis pathway in insects and can regulate also the expression of chitin biosynthesis-related genes in insects (Tang et al. 2017), so trehalose metabolism and chitin metabolism are closely linked. Chitinase catalyzes the degradation of chitinin to chitooligosaccharides and plays a role in the growth and development of pests such as insects, fungi, and nematodes (Adrangi and Faramarzi 2013, Arakane and Muthukrishnan 2010). The indispensability of trehalase and chitinase in insects and the absence of trehalose and chitin synthesis in mammals make trehalase and chitinase an important target for pest control (Argüelles 2014, Zhu et al. 2016).

Most trehalase inhibitors bind to the amino acids at the active site of trehalase through a hydrogen-bonded tight complex and competitively inhibit the enzyme activity, thus hindering the breakdown of trehalose and affecting the metabolism of sugar and chitin in insects (Iwasa et al. 1997, Wang and Liu 2009, Wegener et al. 2010). However, no trehalase inhibitors have been developed as commercial insecticides due to their high hydrophilicity (Matassini et al. 2020). Chitinase inhibitors are a class of compound molecules that specifically bind to chitinase and inhibit chitinase activity (Saguez et al. 2008). Most of the chitinase inhibitors reported have been discovered through virtual screening or guided by natural products, and lack of artificial synthesis. Some of these compounds have high inhibitory activity, but their insecticidal activity still needs to be explored and improved (Zhang et al. 2021). Therefore, the design and development of new agricultural inhibitors targeting trehalase and chitinase must continue. Studied by Jiang et al., ZK-PI-5 (C₂₀H₁₉NO₄) and ZK-PI-9 (C₁₉H₁₆ClNO₃) were rationally designed based on the conserved aromatic residues of 3 chitinases of Asian corn borer (Ostrinia furnacalis, OfChtI, OfChtII, and OfChi-h) and then synthesized (2022). The main structure of piperine consists of 3 parts: the benzo[d][1,3]dioxole ring, the bridge chain, and the piperine ring (Chavarria et al. 2016). Piperine (Ki = 43.78–83.03 μM) was first shown to exhibit inhibitory activities against OfChtI, OfChtII, and OfChi-h (Jiang et al. 2022). Some Studies also have shown that piperine compounds act as a potential inhibitor backbone for trehalase (Shi et al.2022, Wang et al. 2022a). The new compounds ZK-PI-5 (C₂₀H₁₉NO₄) and ZK-PI-9 (C₁₉H₁₆ClNO₃) are novel piperine derivatives. In the early stages of this study, it was shown that microinjection of ZK-PI-5 and ZK-PI-9 into Spodoptera frugiperda larvae can significantly inhibit trehalase activity, disrupting its chitin metabolism and glucose metabolism pathways and eventually leading to death due to difficulties in molting and abnormal development (Zhong et al. 2023). Similarly, we believe that ZK-PI-5 and ZK-PI-9 have potential as novel trehalase inhibitors. Inhibitors targeting trehalase or chitinase have been studied mainly in terms of the effects on insect growth and development, while there are few studies on insect reproductive regulation. To further explore the new compounds’ insecticide potential, the regulatory effect on pest reproduction must be studied.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a native crop pest in South and North America (Kenis et al. 2023). As an invasive species, it has quickly become a major pest in China. The larvae of S. frugiperda feed on gramineous plants, developing and reproducing rapidly, and causing significant damage (Liu et al. 2022, Wang et al. 2022b, Kenis et al. 2023). The moth has a wide host range, as well as high reproductive and dispersal capabilities, making it difficult to control and posing the risk of spreading in one step (Guo et al. 2019, Shi et al. 2022, Kenis et al. 2023). Agricultural pest control relies heavily on chemicals, however heavy use of insecticides can lead to insecticide resistance in pests (Yu 1991, Day et al. 2017, Pires Paula et al. 2021, Li et al. 2022). It also leads in a series of problems such as environmental pollution and side effects on nontarget organisms (Desneux et al. 2007). Spodoptera frugiperda is extremely harmful and prompts for the development of novel management methods (Hou et al. 2022), and potential bioinsecticides able to manage it are currently being studied (Jia et al. 2022, Luo et al. 2022, Kenis et al. 2023). Therefore, this study investigated the effectiveness of the new novel compounds in targeting S. frugiperda reproduction, providing new ideas and effective methods for environmentally friendly plant protection.

Materials and Methods

Insect Source and Feeding Method

Spodoptera frugiperda was provided by the Zhejiang Academy of Agricultural Sciences and reared in the laboratory of Hangzhou Normal University (Hangzhou, China). The larvae were fed with an artificial diet (Supplementary Table 1), and the adults were fed with 10% honey water in an artificial climate chamber. The environmental conditions were set as follows: temperature 26 ± 1 °C, relative humidity 60 ± 10%, and photoperiod 16L:8D. Considering that S. frugiperda emergency on the sixth day of pupation at the earliest, female pupae were selected for experimental treatment on the fifth day of pupation.

Microinjection of S. frugiperda Female Pupae

Two novel compounds (ZK-PI-5 and ZK-PI-9) were provided by the PMDD Laboratory of China Agricultural University (Table 1, Fig. 1, Beijing, China). The female pupae were collected on the fifth day of pupation to identify the sexes according to Dong et al (2019). In the early experiment, 3 injection single dose of Validamycin (2 × 10⁻³ μmol, 5 × 10⁻³ μmol, and 1 × 10⁻² μmol) were set to determine the optimum dose among semilethality rate in S. frugiperda pupa on the fifth day, LC₅₀(μg) = 0.841. The final injection amount of the novel compound was calculated by relative molecular weight conversion and determined to be 2 × 10⁻³ μmol (2 × 10⁻² μmol/μL, 100 nL). Female pupae on fifth day of pupation were placed in an agarose gel table, ventral side up, and 2 × 10⁻³ μmol of novel compound was injected into the ventral cavity of the pupae through the junction of ventral segments 5 and 6 of the female pupae using a 3.5 Drummond needle and Transferman 4r (Eppendorf, Hamburg, German). Female pupae of S. frugiperda injected with the solvent 2% DMSO were the control group (2% DMSO group). 2% DMSO was diluted with ddH₂O. Samples were examined for viability after injection, and experimental insects that were injured or leaked most of the tissue fluid and inhibitors were discarded. Female pupae were reared singly after injection and live pupae were taken 48 h after treatment for subsequent experiments.

Determination of Trehalase and Chitinase Activity

For trehalase activity test, female pupae were selected 24 h after injection: 3 individual insects per group, with 3 biological replicates. Female pupae were homogenized in liquid nitrogen. Weigh approximately 0.1 g of tissue to 1 ml of extract and homogenize in an ice bath. Suspension was centrifuged at 8,000 g at 4 cc for 10 min once and the supernatant was used to determine trehalase activity with Trehalase Assay Kit (Solarbio, Bejing, China) according the manufacturer’s instruction. In addition, the protein concentration in the sample was detected according to the Bradford protein concentration determination kit (Beyotime, Shanghai, China). Finally, trehalase activity was calculated according to protein concentration. Chitinase activity (U/mg prot) = 100 × y/Cpr (y, sample concentration; Cpr, sample protein concentration).

The activity of chitinase in S. frugiperda was determined according to the instructions of the chitinase kit (Comin, Suzhou, China). Chitin broken down into N-Acetyl glucosamine by chitinase, furthering reacted with DNS (3,5-dinitrosalicylic acid) reagent to produce a brown-red compound, which has a characteristic absorption peak in 540 nm. Chitinase activity (mg/h/g fresh weight) = 3.119 × (∆A + 0.2753)/W (A, absorbance value; ∆A = A assay − A control; W, sample weight).

Determination of Carbohydrate Content

Female nymphs were selected 24 h after injection. Firstly, after homogenization via grinding with liquid nitrogen and a mortar, they were mixed with 1 ml phosphate-buffered saline (PBS, pH 7.0), followed by 30 min sonication (VCX 130PB, Sonics, Newtown, CT, USA). The samples were centrifuged at 1,000 × g at 4 °C for 20 min. The supernatant was used to measure the contents of trehalose and glycogen. Subsequently, 350 μl supernatant was collected and centrifuged again for 60 min at 20,800 × g and 4 °C. Next, 300 μl supernatant was collected and used to determine the glucose content and protein concentration. Trehalose content was detected by anthrone method, glucose and glycogen were detected by Glucose Assay Kit (Sigma, MO, USA). The protein contents of samples were determined using the BCA Protein Assay Kit (Beyotime, China). The specific experimental steps can be found in the study of Zhang et al. (2017). Finally, the absorbance value was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Spodoptera frugiperda Female Pupae Emergence Rate and Emergence Phenotype

The pupae were observed and recorded every 24 h. If the pupae did not emerge for 5 days and did not respond when their heads were touched with a brush, they were considered dead and failed to emerge. Phenotypes such as abnormal body shape and inability to completely break the pupae were photographed and recorded.

Oviposition Duration, Egg Production, Egg Hatching Rate, and Longevity of Female S. frugiperda

Adult females in the control group, ZK-PI-5 group, and ZK-PI-9 group were paired with males that emerged on the same day. After pairing, the insects were placed in individual adult rearing boxes and fed with 10% honey water. Each treatment group was replicated with 10–30 pairs. The preoviposition period, oviposition period, egg production, and longevity of the female adults were recorded until they died. Meanwhile, 4 egg masses (100–200 eggs per egg mass) were randomly selected from each group at the same time of day in the rearing box for a total of 10 groups for 7 days to record the hatching rate. In addition, the treatments set up 30 groups individually, each containing one male and one female in each group after their emergence. Nine groups were randomly selected per treatment and females’ ovaries were subsequently dissected and photographed (8×) using a Leica EZ4 HD stereomicroscope and LAS EZ software on day 1, 3, 5, and 7, and the same for control. Finally, the ovarian development was judged according to Zhao et al. (2019).

Determination of S. frugiperda Vitellogenin and Vitellogenin Receptor Gene Expression

Female adults on days 1, 3, 5, and 7 after emergence were randomly selected from each group, and the whole abdominal tissues of 3 female adults were used as one biological replicate. Three biological replicates were performed in each group. Total RNA was extracted using a Trizol kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality and concentration were determined by agarose gel electrophoresis and NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the cDNA synthesis was performed according to the instructions of PrimeScript RT reagent Kit With gDNA Eraser (TaKaRa, Kyoto, Japan).

The sequences of primers were designed using primer 3 (https://primer3.ut.ee/) shown in Table 2. The expression of SfVg (GenBank ID:MT505383) and SfVgR (CenBank ID: XM_035595777.1) genes were detected separately using qRT-PCR. PCR reaction system (10.0 μl): SYBR Green master mix (SYBR Green Premix Ex Taq, Takara, Japan) 5 μl, forward primer (10 pmol) 0.4 μl, reverse primer (10 pmol) 0.4 μl, template cDNA 1 μl, RNase Free ddH₂O 3.2 μl. The reaction procedure comprised predenaturation at 95 °C for 2 s; denaturation at 95 °C for 30 s, and annealing extension at 59 °C for 30 s (35 cycles). The final plotted melting curve was in a range of 65–95 °C. The ribosomal protein L10 (RPL10, GenBank ID: OK319023.1) was used as an internal reference gene. The amplicon from qPCR was unique and the relative expression was calculated by the 2^−ΔΔCT method (Livak and Schmittgen 2001).

Data Analysis

The data were collated and analyzed using Excel. Significance analysis and graphing were performed using SPSS Statistics 20 and GraphPad Prism 9, and the data in the graphs are expressed as mean ± standard deviation (SD). An independent samples t-test was used to analyze the significance of differences. This article will use 0.05 as an alpha cutoff (P< 0.05 for significant differences, denoted by *; P > 0.05 indicates no significance, denoted by ns). The chi-squared (χ²) test was used to analyze the effect of inhibitors on the emergence rate.

Results and Analysis

Changes in Trehalase and Chitinase Activity of S. frugiperda after Injection

The trehalase activity in the female pupae 24 h after ZK-PI treatments was significantly different than that in the control group. After injecting ZK-PI-5and ZK-PI-9 into the female pupae of S. frugiperda, we found that the activities of trehalase were significantly inhibited, and the enzyme activities were 62.9% (t = 4.647, df = 4, P = 0.010) and 36.0% (t = 3.226, df = 4, P = 0.032) lower than the 2% DMSO group, respectively (Fig. 2A). At the same time, the chitinase activity in the ZK-PI-9 group decreased significantly compared with the control group (t = 2.666, df = 5, P = 0.045, Fig. 2B). The results showed that these 2 types of ZK-PI can be used as a potential trehalase inhibitor for follow-up experiments.

Contents of Trehalose, Glucose, and Glycogen after Injection

The contents of trehalose, glucose, and glycogen in the female pupae 24 h after ZK-PI treatments were no significant difference than that in the control group (Fig. 2C–E).

Effect of Compound Injection on the Emergence Failure Rate and Emergence Phenotype of Adult Female

After the injection of the two ZK-PIs into the female pupae of S. frugiperda, the emergence failure rate of female pupae increased sharply compared with the control group (2% DMSO group), for which the emergence failure rate was 9.6%. For the ZK-PI-9 group, it was as high as 39.5% (χ² = 22.43, P < 0.001), while for the ZK-PI-5 group, it was as high as 33.0% (χ² = 16.35, P < 0.001). This was an increase of 30.0% and 23.5% (Fig. 3A, not including the phenotypes shown in Fig. 3B), respectively, compared with the control group. The emergence phenotypes revealed that the female adults in the treatment group showed emergence deformities during the emergence process, such as the adults being unable to shed their pupal shells normally or fully spread their wings (Fig. 3B). The survival time of the adults with deformed wings was less than 24 h.

Effect of Compound Injection on Female Adult Lifespan, Prelaying, and Laying Period

The S. frugiperda adult females were observed and analyzed for longevity, pre-oviposition, and oviposition after treatment with 2 compounds. It was found that the longevity of the normally emerging adult females treated with ZK-PI-5was 7.0 d (t = 5.245, df = 38, P < 0.001), and the longevity of ZK-PI-9 group was 7.7 d (t = 4.090, df = 39, P < 0.001). These periods were extremely significantly shorter than those of the control group (Table 3). The preoviposition period of the female adults in the inhibitor-treated group was not significantly different from that of the control group, but the oviposition period of the female adults in the inhibitor-treated group was significantly different, with the oviposition period of ZK-PI-5 being 3.7 d. This was significantly shorter than that of the control group.

Effect of Compound Injection on Egg Laying, Egg Hatching Rate, and Number of Viable Larvae of S. frugiperda

Interestingly, the daily egg production of female adults in the treatment groups initially showed the same upward and downward trend, while the daily egg production of the control group gradually decreased with the increase in longevity. The egg production of female adults treated with ZK-PI-9 was significantly higher than that of the control group on the second (t = −2.234, df = 35, P = 0.032), third (t = −5.423, df = 49, P < 0.001), and fourth (t = −2.580, df = 26, P = 0.016) days (Fig. 4B). The egg production of female adults treated with ZK-PI-5 was significantly higher than that of the control group on the second day (Fig. 4A). However, the egg production of female adults in ZK-PI-5 (t = 2.836, df = 12, P = 0.015) and ZK-PI-9 (t = 2.317, df = 8, P = 0.049) group was significantly lower than that of the control group on the seventh day (Fig. 4A and B). There was no difference in total egg production between groups. However, the hatching rate of eggs laid by the female adults treated with ZK-PI-5 and ZK-PI-9 was as low as 55.7% (t = 3.461, df = 4, P = 0.026) and 42.4% (t = 0.216, df = 4, P= 0.003), respectively, which was significantly different from the control group (Table 4). The overall number of viable larvae emerged in the experimental group was significantly lower than that in the control group (Table 4, ZK-PI-5 group: t = 0.540, df = 4, P = 0.048, ZK-PI-9 group: t = 0.055, df = 4, P = 0.009).

Effect of Compound Injection on Ovarian Development and Vg and VgR Gene Expression in Female Adult

The ovary grades of female adults of each group on the first day after emergence were mainly in stage I (milky transparent stage) to II (yolk deposition stage), among which some ovaries in stage I after inhibitor treatment displayed delayed development compared with the control group (Figs. 5 and 6). The ovarian tubes were short and thin, and the fat bodies were also low. On the third day of emergence, the ovary grade was mainly in classes II–III (mature for laying), with full and clearly visible eggs and little difference between groups. On the fifth day of emergence, the ovaries were mainly in grade IV (peak oviposition stage) and the density of fat bodies was reduced. On the seventh day of emergence, the control ovaries were mainly in class IV (peak oviposition stage) with fuller eggs than the treated group, while the treated group was mainly in class V (late oviposition stage) with significant atrophy of the ovarian canal (Fig. 5, ZK-PI-9 group: t = −2.513, df = 9, P = 0.033). Most of the eggs had been expelled, leaving only a few leftover eggs.

The mRNA expression compounds ZK-PI-9 and ZK-PI-5 did not change significantly compared to the control. The mRNA expression levels of SfVg in ZK-PI-9-treated female adults increased significantly on the fifth day of emergence (t = −2.282, df = 4, P = 0.048), and those of the ZK-PI-5-treated female adults also decreased significantly on the seventh day of emergence (t = 3.544, df = 4, P = 0.024, Fig. 7A). On the first day of emergence, the mRNA expression levels of SfVgR in females treated with the inhibitor ZK-PI-5 increased significantly (t = −3.187, df = 4, P = 0.033); the mRNA expression levels of SfVgR in females treated with ZK-PI-9 showed no significant difference. There was no significant difference in the mRNA expression levels of SfVgR in each group on the third day and the seventh day, and the mRNA expression levels of SfVgR in females treated with ZK-PI-5 (t = 3.786, df = 4, P = 0.019) and ZK-PI-9 (t = 3.018, df = 4, P = 0.049) decreased significantly on the fifth day of emergence (Fig. 7B).

Discussion

Lepidoptera has the morphology and behavior to mate and reproduce only through emergence into adults, and emergence can reflect the population size of S. frugiperda that reproduces normally. A study reported that when the last instar larvae of Spodoptera litura were injected with the effective Avalidoxylamine A and Avalidamycin A, high rates of prepupal mortality and abnormal pupation occurred (Asano et al. 1990). In addition, silencing the trehalase gene of Spodoptera exigua also resulted in high mortality in the pupal–adult stage and deformed phenotypes (Chen et al. 2010). This concurred with our results. After the injection of a new inhibitor, the emergence rate of the female pupae decreased significantly, and the emergence of wing deformity and other abnormal emergence phenotypes was repeated (Fig. 3A and B); this is related to the influence of trehalase and chitinase in the body. TRE can regulate genes related to wing development, resulting in abnormal insect wing phenotypes (Zhang et al. 2017). It can also regulate chitin synthesis by reducing the expression of some key enzymes in the chitin synthesis pathway (Chen et al. 2010, Zhao et al. 2016). For example, reduced transcriptional and translational levels of TRE led to reduced expression of chitinase, which results in difficulty in molting and higher mortality (Tang et al. 2017). Specific knockdown of ChtII transcripts in the coleopteran Tribolium castaneum prevented larval–, larval–pupal, and pupal–adult molting (Qu et al. 2021). The process of emergence is highly energy consuming, and the energy limitation makes it difficult to break the pupae and emerge. Meanwhile, the obstruction of chitinous decomposition and synthesis affects the fading of the old epidermis and the formation of the adult epidermis, resulting in emergence deformity. The reduced emergence rate of female pupae reduces the number of adult females in the population. The flight ability and mating rate of deformed females reduced, and their fecundity was affected.

Some studies have shown that insecticides affect insect reproduction via stimulation or inhibition by regulating Vg gene expression and synthesis and its transfer to the ovary, which is similar to our results (Zhou et al. 2020). The fecundity of insects injected by 2 novel compounds was strongly affected by the availability of energetic substances. Inhibition of Periplaneta americana trehalase activity leads to a decrease in oocyte accumulation of Vg by the ovary as the energy supply is not sufficient for Vg uptake (Kono et al. 2001). In this study, after inhibition of TRE in female adults, this will lead to enhanced expression of SfVgR gene (Fig. 7B), allowing normal development of their ovaries in the early stage by increasing the protein level of VgR. The results showed that the egg production of female adults treated with ZK-PI-5 increased significantly in the early stage of egg laying (Fig. 4B). This coincides with the previous finding that the expression level of the VgR gene of Spodoptera exigua was significantly and linearly positively correlated with the egg production of female moths (Zhao et al. 2018). The results of this experiment revealed that the mRNA expression levels of SfVg and SfVgR in adult females treated with inhibitor ZK-PI-9 on the fifth day increased and decreased, respectively (Fig. 7A and B). Gomaa et al. fed the queen honey bee Apis mellifera L. with the Bacterium Paenibacillus larvae larvae and found that the amount of extracted ovarian protein increased significantly, while the Vg expression increased significantly (Gomaa et al. 2021). The new compound may have caused the excitation effect of pests and stimulated their expression of SfVg gene(Fig. 7A). Trehalose, a hemolymph carbohydrate that affects insect reproduction, can provide energy for oocytes to absorb Vg through VgR, and related studies have also shown that only when Vg and VgR exist together can the normal reproduction of insects be guaranteed and the inhibition of VgR expression can cause a large amount of Vg deposition in the hemolymph; thus, it cannot enter the ovaries smoothly. This eventually causes the obstruction of ovarian tube development, a reduction in egg size and color mismatch, and a significant decrease in egg production (Chen et al. 2010, Huang and Lee 2011, Shu et al. 2011, Cong et al. 2015, Lu et al. 2015, Zhang et al. 2017, Zhou et al. 2020). It was hypothesized that the late stage of the ZK-PI-9-treated and ZK-PI-5-treate group were not able to transport Vg to the oocyte to provide nutrition for embryonic development due to insufficient energy supply and the lack of receptors on the ovarian cell membrane that specifically mediate Vg endocytosis, resulting in the accumulation of Vg in the hemolymph, which affected the subsequent development of the ovary and egg production.

Other studies demonstrated that the decrease in VgR expression resulted in a prolonged preoviposition period and shortened reproductive period in brown orange aphid Aphis citricida (Shang et al. 2018). This was similar to the situation in the present study (Table 3). At the same time, the mRNA expression level of SfVg treaded with ZK-PI-5 decreased significantly on the seventh day (Fig. 7B). Vg and VgR gene expression also decreased significantly after 200 Gy beam radiation treatment of adult S. litura, resulting in inhibition of ovarian development (Koo et al. 2019). In addition, injection of the trehalase inhibitor validoxylamine A (VAA) inhibited Vg synthesis in the fat body and its uptake by mature oocytes (Tanaka et al. 1998; Kono et al. 2001, Lu et al. 2019). The expression of Vg decreases, affecting the supply of nutrients required for embryonic development. Combined with the significant detrimental effects of both longevity and oviposition period of female adults after inhibitor treatment (Table 3), it is hypothesized that there is a survival–reproduction trade-off in S. frugiperda populations, that is, an antagonistic effect between early reproduction and late survival, with females with higher early fecundity having shorter longevity and vice versa (Silbermann and Tatar 2000, Okada et al. 2014). This S. frugiperda population fits the former scenario. In order to ensure smooth reproductive behavior, the oviposition period and longevity were shortened to cope with energy limitations.

The egg hatching rate was significantly reduced in the inhibitor-treated group (Table 4). Chitinase expressed in the body wall is mainly related to insect embryo development and hatching of eggs (Zhu et al. 2008, Zhang et al. 2018). Relevant studies have shown Nematode chitinase is a key enzyme during nematode egg hatching (Adrangi and Faramarzi 2013). Specific knockdown of ChtII transcripts also prevented egg hatching in the coleopteran Tribolium castaneum (Qu et al. 2021). In Aedes albopictus, the expression of trehalase was downregulated under the stress of a low concentration of cadmium, and the hatching rate of offspring was reduced (Yu et al. 2020). Therefore, novel inhibitors may control the number of offspring of S. frugiperda by inhibiting the trehalase and chitinase activity of eggs. It limits the energy available for the offspring or lacks chitinase to break through the egg’s shell, thereby reducing the hatching rate of eggs.

The new compound was evaluated in advance by injecting compounds to accurately inhibit enzyme activity in insects. ZK-PI-5 and ZK-PI-9, have shown good inhibitory effects on the physiological indexes related to reproduction of S. frugiperda. However, the practical application of these compounds needs to be explored further. Thus, it is necessary to explore more combinations of joint pest control; for example, RNA insecticides (Nandety et al. 2015, Zhu and Palli 2020, Yan et al. 2022), microbes (Liu et al. 2022), natural enemy insects or the combination of these with biological pesticides, among other methods (Kunte et al. 2020, Li et al. 2021, Wang et al. 2022).

Funding

This work was supported by the National Key Research and Development Program of China (Grant No. 2022YFD1700200), the Sixth Batch of Guizhou Province High-level Innovative Talent Training Program[2022], the Guiyang Science and Technology Personnel Training Project [2022]43-16, the Program for Natural Science Research in Guizhou Education Department QJJ[2023]024, Hangzhou Normal University’s Starlight Plan in 2022, Hangzhou Normal University Undergraduate Innovation Ability Improvement Project and Key projects of education departement of GuiZhou Province KY[2020]047.

Author contributions

Xinyi Jiang (Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing – review & editing-Equal), Fan Zhong (Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing – review & editing-Equal), Yan Chen (Formal analysis-Equal, Writing – review & editing-Equal), Dongmei Shi (Formal analysis-Equal, Writing – review & editing-Equal), Lei Chao (Data curation-Equal, Investigation-Equal), Liuhe Yu (Investigation-Equal, Methodology-Equal, Visualization-Equal), Biner He (Investigation-Equal, Methodology-Equal, Visualization-Equal), Caidi Xu (Supervision-Equal, Visualization-Equal), Yan Wu (Supervision-Equal, Visualization-Equal), Bin Tang (Conceptualization-Equal, Project administration-Equal, Supervision-Equal, Writing – review & editing-Equal), Hongxia Duan (Formal analysis-Equal, Supervision-Equal), Shigui Wang (Conceptualization-Equal, Project administration-Equal, Supervision-Equal, Writing – review & editing-Equal)

References

Author notes