Finding new untapped resources with high potential is crucial because of the negative impacts associated with chemical pesticides used in pest management (Gade and Goldsworthy, 2003). Insect neuropeptides represent a novel class of potentially effective pest control chemicals that are both highly selective and safe for the environment (Stay, 2000;Van Hiel et al., 2010). As an insect neuropeptide, AST has been extensively studied and modified to develop novel insecticides. In this study, we investigated the physiological activity of Ⅲ-2 in S. frugiperda. Our results demonstrate, for the first time, that AST can achieve specific pest control by interfering with JH titers in insects, providing a new approach for realizing effective control of target organisms through AST interference.
Insect growth regulators (IGRs), which include many subcategories of JHAs, chitin synthesis inhibitors, and ecdysone analogs, are a class of specialized insecticides that can obstruct and disrupt the normal growth and development of insect larvae, causing them to perish. They exhibit excellent control over lepidopterans, mosquitoes, whiteflies, and aphids (Bacci et al., 2007;Gogi et al., 2021;Noelia et al., 2023). Through experiments on the physiological activity of Ⅲ-2 against S. frugiperda, we found that Ⅲ-2 could reduce the food intake of larvae, inhibit the growth rate, shrink the body, blacontrolen, cause molt failure, and cause death, similar to the effect of IGR. As a type of JHA, FC can inhibit Chrysomya megacephala during pupal-adult transformations. FC can produce sublethal effects in Plutella xylostella (Mahmoudvand and Moharramipour, 2015). The LC50 was 93.6 mg L− 1. However, our research indicates that FC may only have minor effects on S. frugiperda growth and development and does not appear to have a deadly effect on this lepidopteran. The LC50 of Ⅲ-2 against the second instar larvae of S. frugiperda calculated at 96 h was 18.7 mg L− 1, which seems to be more efficient than the sublethal effect of FC on Plutella xylostella.
The types of JH synthesized in different insects differ, and different JH have different important physiological functions (Ando et al., 2020;Bendena et al., 2011;Kodama et al., 2023;Kotaki,Shinada,Kaihara,Ohfune and Numata, 2009). AST can affect subsequent JH synthesis by acting on HMG-CoA of the mevalerate pathway in the JH synthesis pathway, further regulating insect growth, development, metamorphosis, reproduction, and other physiological activities. The specific synthesis of JH Ⅲ in different insects can be divided into two pathways. One is catalyzed by CYP15C1 and JHAMT, such as Orthoptera and Dictyoptera. The other is catalyzed by JHAMT and CYP15A1, such as Lepidoptera(Daimon et al., 2012;Daimon and Shinoda, 2013). In this study, we found that Ⅲ-2 treatment significantly increased the expressions of SfCYP15C1 and SfJHAMT, decreased the expressions of SfJHE and SfJHEH, and increased the titers of JH III, whereas the titers of JH I and JH II remained unchanged. These results indicate that Ⅲ-2 can increase the titer of JH III, specifically in S. frugiperda larvae, by promoting SfCYP15C1 and SfJHAMT and inhibiting the expression of SfJHE and SfJHEH. Meanwhile, the excessive presence of JH III in insects over a short period of time may lead to disease and even death of insects, which makes it possible to achieve effective control of different kinds of pests through specific interference of JH and provides a new approach for the control of target pests containing JH III. JH and 20E regulate insect development and metamorphosis via their interactions. In Bombyx mori, interactions between JH and 20E regulate the transcription of fibroin modulator-binding protein-1 (Jia et al., 2017;Liu L. et al., 2019). We found that the Ⅲ-2 treatment reduced 20E titers in S. frugiperda larvae, suggesting that 20E and JH Ⅰ and JH Ⅱ may jointly regulate the growth and development of S. frugiperda and have antagonistic effects. Exogenous JH can induce BrZ7 phosphorylation by inhibiting 20E-related gene transcription to prevent metamorphosis (Cai et al., 2014), which is consistent with our findings. Our findings may serve as a theoretical foundation for the development of novel, environmentally friendly pesticides that target the synthesis and metabolism of JH III, particularly in insect species, to safeguard beneficial insects and selectively eradicate pests.
The capacity of insects to adapt to their environment and develop resistance to insecticides depends on the stratum corneum; therefore, the cuticle protein gene is highly expressed in insects (Futahashi et al., 2008;Yan et al., 2022). The dynamic changes in chitin are consistent with the ecdysis process (Wang Y. et al., 2022), and its biosynthesis involves the expression of Tre, Tret and other enzymes (Kanamori et al., 2010;Kikawada et al., 2007;Takiguchi et al., 1992). As a result of III-2 treatment the expression of LCP1, LCP16/17, LCP17, LCP22, and other LCP genes was inhibited, according to the qPCR results. Tret and Tre genes also differed substantially from those in the control. The epicuticle, exocuticle, endocuticle, and epidermal cell layer are all parts of the insect epidermis (Liao et al., 2018). The endocuticle and epidermal cell layers create an ecdysis membrane between them during the insect’s ecdysis process, which causes the endocuticle and skin cell layer to split. Paraffin sectioning revealed that while in the control group, the inner epidermis and skin cell layer were separated at 24 and 96 h, this was not the case in the III-2 treated larvae. This phenomenon may be explained by the effect of III-2 affected the titers of JH III and 20E in S. frugiperda larvae, which further impacted the production of cuticle proteins and chitin and prevented ecdysis.
AST-A mainly targets cockroaches and inhibits the synthesis of juvenile hormone in D. punctataz (Woodhead,Stay and Seidel, 1989). Related studies have shown that in Drosophila, AST-A is involved in assigning value to nutrients to coordinate metabolic and feeding decisions. It also inhibits feeding behavior in adult flies (Hentze et al., 2015;Hergarden,Tayler and Anderson, 2012). But so far, there does not seem to be any research showing that AST-A can inhibit juvenile hormone synthesis in other insects. In our study, III-2 promoted the synthesis of JH Ⅲ in S. frugiperda, which seems to contradict the effect of AST-A on inhibiting the synthesis of juvenile hormone. However, we speculate that the reason for the difference is the different types of JH in different insects and their synthesis pathways. For example, only JH Ⅲ exists in D. punctataz, and its terminal synthesis is catalyzed by CYP15A1. However, there are JH I, JH II and JH III in S. frugiperda, and their terminal synthesis is catalyzed by CYP15C1(Daimon and Shinoda, 2013;Yi et al., 2023). Ⅲ-2 increases JH Ⅲ titer by promoting the expression of CYP15C1 in S. frugiperda. Then, will the expression of CYP15A1 in D. punctataz be inhibited to inhibit the synthesis of juvenile hormone? This results in this contradiction, which obviously has to be investigated further. Additionally, different insects may have various AST to control the synthesis of various JH. As a novel AST-A analogue, Ⅲ-2 acts on insects with unique targets. For the first time, we conducted a study on the control of lepidopteran insects based on the AST interference mechanism and identified its effect on the JH and 20E of S. frugiperda. This provides a new strategy for the development of a new generation of green pesticides based on AST and its targets, and for the implementation of pest control strategies based on JH-specific interference. However, why Ⅲ-2 specifically interfered with the titer of JH III but did not affect the synthesis of JH I and JH II in S. frugiperda is unclear. How JH Ⅲ and 20E regulate the metamorphosis development of S. frugiperda. And if JH Ⅰ and JH Ⅱ do not participate in the metamorphosis development of S. frugiperda, what is the significance of their synthesis in S. frugiperda. These issues require further in-depth research.
In summary, we found that feeding AST analog Ⅲ-2 can specifically promote the formation of JH Ⅲ and inhibit the biosynthesis of 20E in S. frugiperda larvae. The poisoned larvae eventually die. Further studies showed that Ⅲ-2 treatment promoted SfCYP15C1 and SfJHAMT, the key genes of JH biosynthesis, and inhibited the expressions of SfJHE and SfJHEH in the JH metabolic pathway. Significant differences were also observed in the expression of genes related to cuticle protein formation. The epidermal changes revealed by histopathological examination were consistent with 20E titers and genetic changes in the stratum corneum. Our study revealed that the AST analog interfered with the growth and development of insects by interfering with the synthesis of JH and 20E, eventually leading to insect death. It is also revealed that effective prevention and control of pests can be achieved through the interference of AST. These results provide a theoretical basis for the development of new green insecticides that specifically affect JH III synthesis and metabolism in insects.