The interaction between herbivorous pests and their host plants is frequently cited as a textbook example of co-evolution (Vandenhole et al., 2021). Feeding on plants is complicated because plants have developed a wide range of morphological and chemical defensive line tactics. To avoid or diminish feeding injury, most plants induce complex chemical defense combinations to withstand insect attack (Büchel et al., 2016; Lackus et al., 2018). These chemical compounds may serve as defensive due to their unpleasant flavors and odors, or they may be toxic or mitigate the digestibility of plant tissues (Biere et al., 2004; Rehman et al., 2012). Nonetheless, identification and response to phytochemicals (allelochemicals) released by plants to defend themselves against herbivores is an important aspect of arthropod-plant interactions (Janz, 2011; Suchan and Alvarez, 2015; Vandenhole et al., 2021). The study of the interaction between plant secondary chemistry and insect herbivores is essential in the development of a successfully integrated pest management plan.

To survive, different tactics have been evolved by adapted herbivores to deal with the defense compound present in their host plants (Heidel-Fischer and Vogel, 2015a; Rashid War et al., 2018; Yang et al., 2022). Similarly, some herbivores insects metabolize and excrete plant defense compounds they consume, but others store them in their bodies to defend themselves against all-natural enemies (Petschenka and Agrawal, 2016; Heckel, 2018). Metabolic detoxification of plant toxins is the primary strategy of herbivores occurring in three phases (solubilization), phase II (conjunction) and phase III (excretion), each with its own enzymes (Krempl et al., 2016; Stahl et al., 2018; Lu et al., 2021). For example, cytochrome P450 monooxygenases (P450s) and carboxylesterases (CarE) carry out phase I, glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs) phase II, and ATP-binding cassette transporters (ABC) phase III (Nauen et al., 2022; Kennedy and Tierney, 2013; Jin et al., 2019; Ullah et al., 2020). These enzymes also work on endogenous substrates like hormones and lipids to carry out additional physiological processes and housekeeping functions in insects (Ketterman et al., 2011; Feyereisen, 2012). Host plant utilization and dietary diversity have both been linked to elevating detoxification enzyme activity and mRNA expression levels of related genes (Adesanya et al., 2016; Jin et al., 2019; Israni et al., 2020). In previous research, the increased P450 and GST activity have been observed in lepidopteran larvae and plant-feeding hemipterans feeding on non-preferred or less compatible plant species (Krieger et al., 1971; Yu, 1983; Mullin, 1986; Adesanya et al., 2016; Hafeez et al., 2021a). Similarly, higher P450 enzyme activity was observed in the generalist caterpillar Spodoptera eridania after feeding on the carrot, a non-preferred host plant compared to the lima bean, which is a more favored host (Brattsten, 2012). In addition, a significant variation in P450, CoE and GST activities in Bemisia tabaci and Popillia japonica have been reported among different host plants that are diverse in suitability (Xie et al., 2011; Adesanya et al., 2016). More recent research suggests that a large number of P450 genes related to detoxification enzymes from insects have been isolated and characterized (Schuler, 2011; Berenbaum and Calla, 2021; Berenbaum et al., 2021; Calla, 2021). For example, gossypol-induced P450s genes CYP9A12, CYP9A14, and CYP9A98 showed high divergence in the mRNA level of Helicoverpa armigera and Spodoptera exigua larvae (Tao et al., 2012; Hafeez et al., 2019). CYP6B8 and CYP321A1 in the corn earworm, Helicoverpa zea, can metabolize xanthotoxin, flavone, quercetin as well as a variety of other phytochemicals, indicating that this insect species has evolved systems for phytochemical detoxification (Sasabe et al., 2004; Rupasinghe et al., 2007). Additionally, CYP6B1 and CYP6B3 in Pailio polyxenes, which specializes on Rutaceae and Apiaceae, show high efficiency in metabolism of furanocoumarins in its host plants and CYP6AS could metabolize the flavonoid quercetin in Apis mellifera (Mao et al., 2009). However, it is important to investigate how selective or inducible enzyme systems could provide generalist herbivores with an ecological opportunity to utilize secondary plant compounds before expending metabolic costs for detoxification.

The fall armyworm (FAW), Spodoptera frugiperda is a damaging insect that feeds on a variety of essential food crops, including maize and rice (Machado et al., 2008; Gouin et al., 2017; Hafeez et al., 2021a). Since 2016, this invasive pest has spread throughout Sub-Saharan Africa, resulting in significant agricultural losses (Goergen et al., 2016; Day et al., 2017; Kenis et al., 2022) and it has also made its way into South Asia, including China, where it is also dispersing rapidly across the region (Swamy et al., 2018; Li et al., 2020). Two ecological strains of FAW have been recognized from natural populations, the so-called corn and rice strains (Pashley, 1986; Nagoshi et al., 2019). Corn strain insects are prevalent on grasses such as maize and sorghum, while, insects belonging to the rice strain appear to predominate on small grasses such as rice and Bermuda grass. Although the two strains are identical physically in the field, they do have distinct preferences for host plants and show signs of reproductive isolation (Groot et al., 2010; Dumas et al., 2015). The recent genome sequencing of this species has provided new insights into how P450s function in vivo and how these enzymes and related genes may be involved in the pest insect’s adaptive mechanism (Gouin et al., 2017). Yet, the P450 enzymes and related genes induced by this polyphagous pest insect for host plants adaptation have not been characterized.

In this study, molecular characterization and functional analysis of cytochrome P450-mediated detoxification gene involved in host plant adaptation in S. frugiperda was examined after feeding on rice and corn host plants for consecutive 33 generations. Tissues and stage expression patterns of the CYP302A1 gene were also evaluated. Additionally, we investigated how cytochrome P450-specific detoxification enzyme led to larval mortality by PBO inhibitor followed by feeding on rice and corn host plants. To determine if the CYP302A1 gene functions in S. frugiperda host plant adaptation, a functional study of the gene was carried out using RNA interference.

During August 2019, larvae of S. frugiprda populations were collected from two different corn fields in Ping Hu, Zhejiang Province and maintained on corn seedlings in a climate control chamber at 25 ± 2°C with a 14: 10 h light: dark photoperiod at Zhejiang Academy of Agricultural Sciences, Hangzhou, China according to (Hafeez et al., 2021a).

Two populations were established to avoid any homogenization effect. According to our previous study, the population was reared on corn plants for 33 generations (Hafeez et al., 2021a). Larvae for control treatment were reared on an artificial diet (Poitout and Bues, 1974). Both colonies were maintained in climate chambers at 27 ± 2°C and 70%–75% relative humidity (R.H) under a 14: 10 light: dark photoperiod until adult emergence. Each population was assigned a code denoting its host plant (corn or rice).

Herbivorous insects and their host plants use signals from each other to intensify reciprocal responses (Rashid War et al., 2018; Yactayo-Chang et al., 2020a). Upon attack by herbivorous insects, plants increase the synthesis of defensive compounds such as phytochemicals and proteins to fend them off (Zhu-Salzman and Zeng, 2015; Yactayo-Chang et al., 2020b). In response, herbivores have to evolve diverse strategies to overcome several challenges, to thrive on chemically defense compounds present in their host plant tissues by increasing the activity of their counteroffensive digestive and detoxification mechanism (Heidel-Fischer and Vogel, 2015b; Rashid War et al., 2018; Hafeez et al., 2021b).

In this study, we reported the characterization and functional analysis of the detoxification gene CYP302A1 and how S. frugieprda larvae use a detoxification mechanism to adapt host plants. The functional and evolutionary diversification of insect P450s was likely a key factor in accelerating the unprecedented success of insects (Feyereisen, 2006b; Zhu et al., 2018). The deduced amino acid sequence of S. frugiperda CYP302A1 aligned with other insect CYP321 family members showed that it has conserved motifs and domains (Wang et al., 2017). In previous studies, it has been shown that P450 expression profiles vary dramatically during different developmental stages in most insects (Feyereisen, 2006b; Wang et al., 2018a). Similarly, it has been reported that the midgut fat bodies and Malpighian tubules are frequently associated with higher P450 activity (Hu et al., 2014;). In addition, the midgut is a highly crucial organ in the process of detoxification, and the genes that code for the detoxification enzymes involved in this process are frequently highly regulated in this organ (Feyereisen, 2006a; Hafeez et al., 2022). In this study, we investigated the expression profile of CYP302A1 in different tissues and developmental stages of S. frugiperda by quantitative RT-qPCR. Similar to the expression patterns of CYP6B48, CYP658, and CYP321B1 in S. litura larvae, which may potentially be involved in the plant allelochemicals metabolism, our findings demonstrated that CYP302A1 expression levels were much higher in the midgut tissue and the older S. frugiperda larvae. Similarly, the elevated expression level of CYP321E, CYP321A8, CYP321A9, and CYP321B1 genes was reported in midguts and fat bodies of P. xylostella and S. frugiperda (Bai-Zhong et al., 2020). The enhanced expression level observed in midgut tissue and late larval instars could be attributed to a greater need for xenobiotic detoxification at this stage due to increased feeding activity (Feyereisen, 2006a; Wang et al., 2015; Israni et al., 2020). Tissue-specific expression levels of the CYP302A1 gene in S. frugiperda further suggest that CYP302A1 could be involved to adapt the host plant by detoxifying the plant xenobiotics.

Utilization of a large diversity of host plants and diet variability increase detoxifying enzyme activity in insect herbivores (Ahmad, 1983; Adesanya et al., 2016; Erb and Reymond, 2019). Elevated enzyme activities may be induced by the variety of phytochemicals present across the host pant range (Feyereisen, 2005). Our current studies revealed the enhanced P450 activity in the midguts of S. frugiperda larvae after exposure to rice plants as compared to corn plants and an artificial diet. Our findings are consistent with those of earlier studies; for instance, consumption of a non-preferred host plant by caterpillars of the Spodoptera eridania species and adult Japanese beetles induced higher P450 enzyme activities in comparison to the consumption of a more preferred host plant (Brattsten, 2012; Adesanya et al., 2016). Similarly, variation in P450 activities among five host plants that varied in suitability has been reported in Bemisia tabaci (B-biotype), a generalist whitefly (Xie et al., 2011). To further confirm the possible role of metabolic detoxification enzyme in S. frugiperda larvae to host plant adaptation, higher mortality was observed in pretreated larvae with PBO followed by the exposure of rice plants as compared to the corn plants. Our results are consistent with (Wu et al., 2021) who reported higher mortality of H. armigera larvae pretreated with PBO after exposure to plant volatile than control. Though, selective or inducible enzyme systems could provide generalist herbivores with an ecological opportunity to adapt to diverse host plants. This could be accomplished by generalist herbivores using secondary compounds present in their host plants before expending metabolic resources on detoxification.

Functional analysis of important genes has extensively been studied in insects using the RNAi technique (Kim et al., 2015; Choi and Vander Meer, 2019; Adeyinka et al., 2020; Hafeez et al., 2021b; Ullah et al., 2022). To further investigate if the host-plant-induced gene CYP302A1 is involved in host-plant adaptation, we fed dsCYP302A1 to S. fugiperda larvae to study the knockdown effects of the target gene on mortality and growth parameters. The dsRNA of target gene-fed larvae significantly showed reduced CYP302A1 mRNA expression level in the midgut followed by the exposure of rice plant as compared to the corn plant and DEPC-water treated plants as a control. Similar to our findings, RNAi-triggered CYPAB14, CYPA98, CYP321A7 and CYP6B8 genes downregulation through uptake of dsRNAs have been documented for other insect species (Li et al., 2000; Mao et al., 2011; Hafeez et al., 2019; Hafeez et al., 2022). Present results indicate that the dsRNA-mediated knockdown of CYP302A1 in the S. frugieprda larvae lead to higher mortality after feeding rice plant as compared to the corn plant and DEPC-water treated plants as control at 72 and 96 h. Our results provide the advocacy of previous findings by (Hafeez et al., 2019) who reported that silencing of CYP6AB14 and CYP9A98 genes in S. exigua larvae followed by feeding on 0.1% gossypol caused larval mortality. Similarly, silencing of HaAK gene in H. armigera using RNAi-mediated transgenic plant increased larval mortality when larvae were fed on the leaves of the transgenic plant (Liu et al., 2015). In previous reports, it has been documented that silencing of the CYP6AE14 gene in H. armigera larvae and CYP6AB14 and CYP9A98 genes in S. frugiperda larvae by transgenic plant-mediated RNAi retarded larval growth and weight (2011; Mao et al., 2007; Tao et al., 2012; Hafeez et al., 2022). Similarly, the results obtained in the present work also indicated that the RNAi-mediated knockdown of the CYP302A1 gene increased larval mortality, reduced the larval weight and developmental time after exposure to a dsRNAs-supplemented diet with subsequent feeding on host plants as compared to the control.

In this study, we provide evidence that the insect P450 monooxygenases play a key role in host plant adaptation by detoxifying plant defense compounds. In the current study, tissue-specific expression levels of the CYP302A1 gene in S. frugiperda further advocate that CYP302A1 might be involved to adapt host plants. We revealed the enhanced P450 activity in the midguts of S. frugiperda larvae after exposure to rice plants as compared to corn plants and an artificial diet. These results concluded that the inducible enzyme system and related genes, however, could provide herbivores with an ecological opportunity to adapt diverse host plants by utilizing phytotoxins present in their host plants. We reported that the dsCYP302A1 caused mortality and had harmful effects on the growth and development of S. frugiperda larvae before exposure to dsRNA followed by the feeding on host pants. The harmful effects would be magnified if RNAi targeted multiple genes involved in the P450 complex system. Further studies are needed to explore more P450 genes using RNAi-based approaches against insect pests for crop protection based on a recently developed genetic tool.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The original study design was made by MH, XL, YGL and YL and discussed with the other authors and approved the manuscript All authors contributed to the article and approved the submitted version.