Mapk-dependent hormonal signaling plasticity contributes to overcoming bacillus thuringiensis toxin action in an insect host

ABSTRACT The arms race between entomopathogenic bacteria and their insect hosts is an excellent model for decoding the intricate coevolutionary processes of host-pathogen interaction. Here,

we demonstrate that the MAPK signaling pathway is a general switch to _trans_-regulate differential expression of aminopeptidase N and other midgut genes in an insect host, diamondback moth

(_Plutella xylostella_), thereby countering the virulence effect of _Bacillus thuringiensis_ (Bt) toxins. Moreover, the MAPK cascade is activated and fine-tuned by the crosstalk between two

major insect hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH) to elicit an important physiological response (i.e. Bt resistance) without incurring the significant fitness costs

often associated with pathogen resistance. Hormones are well known to orchestrate physiological trade-offs in a wide variety of organisms, and our work decodes a hitherto undescribed

function of these classic hormones and suggests that hormonal signaling plasticity is a general cross-kingdom strategy to fend off pathogens. SIMILAR CONTENT BEING VIEWED BY OTHERS A SINGLE

TRANSCRIPTION FACTOR FACILITATES AN INSECT HOST COMBATING _BACILLUS THURINGIENSIS_ INFECTION WHILE MAINTAINING FITNESS Article Open access 12 October 2022 JNK SIGNALING REGULATES OVIPOSITION

IN THE MALARIA VECTOR _ANOPHELES GAMBIAE_ Article Open access 01 September 2020 INTERACTIONS OF JUVENILE HORMONE, 20-HYDROXYECDYSONE, DEVELOPMENTAL GENES, AND MIRNAS DURING PUPAL

DEVELOPMENT IN _APIS MELLIFERA_ Article Open access 26 March 2025 INTRODUCTION Insects are the most speciose group of animals, with approximately 5.5 million species estimated to exist on

earth1. Many insects are agricultural pests regularly causing 10–20% crop losses and with on-going global warming, they post a real threat to world food security2. The gram-positive

bacterium _Bacillus thuringiensis_ (Bt) can produce protein toxins to kill different insects with high host specificity and environmental safety3, which makes it the most successful

biopesticide for the last few decades4. Transgenic crops expressing Bt toxins (Bt crops) have become the cornerstone of bioinspired pest control technology, with >100 million hectares

planted globally in 20185. Although Bt products have provided unprecedented economic, environmental, and social benefits3,6,7,8,9,10, the rapid evolution of Bt resistance in at least nine

insect species in the field has seriously eroded their potential4,11,12,13,14. Unraveling the molecular mechanisms of Bt resistance has important implications for the sustainable utilization

of Bt-based technology15,16,17. Bt Cry toxins exert toxicity in insect larval midguts via a multi-step process requiring protoxin activation, toxin–receptor interaction, toxin

oligomerization, membrane insertion, and pore formation18,19. Alterations of midgut receptors such as cadherin (CAD), aminopeptidase N (APN), alkaline phosphatase (ALP), and ABC transporters

(e.g., ABCC2) disrupt toxin binding and are generally associated with high-level resistance to Bt Cry toxins in insects20,21. The diamondback moth, _Plutella xylostella_ (L.), is one of the

most devastating and cosmopolitan agricultural pests22. It was the first insect to develop field-evolved resistance to Bt biopesticides23, and the availability of complete whole genome

information24 renders it an excellent model to probe how insect hosts withstand Bt infection during host–pathogen interaction. Previously, field-evolved resistance to Bt Cry1Ac toxin in _P.

xylostella_ has been linked to both a _cis_-mutation in the _PxABCC2_ gene25 and MAPK-mediated differential expression of _PxmALP_, _PxABCB1_, _PxABCC1-3_, and _PxABCG1_ genes26,27,28.

Although we found that the MAPK signaling pathway can alter the expression of multiple midgut genes related to Cry1Ac resistance in _P. xylostella_, its downstream response gene repertoires

and upstream activation signals remained mysterious29. APN proteins are a class of endoproteases catalyzing the cleavage of neutral amino acids from the N-terminus of protein or polypeptide

substrates30. They belong to the M1 family (metallo-type) of zinc-dependent aminopeptidases that are implicated in many physiological processes of diverse organisms31. In the insect midgut,

besides their important role in food digestion, APNs were the first identified functional receptors of Bt Cry toxins20,30. Moreover, we had previously detected several differentially

expressed midgut PxAPN genes in Bt Cry1Ac-resistant _P. xylostella_32,33. However, whether the differential expression of these PxAPN genes associated with Cry1Ac resistance was also

_trans_-regulated by the MAPK cascade in _P. xylostella_ was unclear. Insect endocrinologists have studied insect hormones for more than a century, and they have discovered that two major

insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), act antagonistically with each other to coordinately orchestrate insect life-history traits including growth,

development, and reproduction34,35,36,37. Moreover, JH and 20E are multifunctional players that can also participate in insect immune defense to pathogenic infection38,39, and the MAPK

signaling pathway is involved in this pleiotropic hormone signaling network35,40. Since exogenous hormone treatments can alter APN gene expression in insects41, we also wanted to test

whether altered levels of insect hormones can activate the MAPK cascades thereby _trans_-regulating the differential expression of PxAPN and potentially other midgut genes to confer Cry1Ac

resistance in _P. xylostella_. In this study, we further probe the mechanism of Bt Cry toxin resistance in _P. xylostella_, starting by looking at the potential role of APN genes, and we

confirm that MAPK-mediated differential expression of APN and other midgut genes does lead to Cry1Ac resistance in _P. xylostella_. More importantly, we uncover that the MAPK cascade is

activated and modulated by an enhanced pleiotropic hormone signaling pathway. This study provides a model for understanding how intracellular signaling networks shape the expression

landscape of midgut genes causing Bt resistance/tolerance in insects within the context of balancing growth-defense tradeoffs. RESULTS GENOME-WIDE ANALYSIS OF APN GENE FAMILY IN _P.

XYLOSTELLA_ Based on currently available transcriptome and genome databases of _P. xylostella_, 18 M1 aminopeptidase genes including 15 APN genes were identified in silico, and their

full-length cDNA sequences were successfully cloned except for _PxAPN3b_ (Supplementary Table 1 and Supplementary Fig. 1a). A representative lepidopteran APN protein contains six common

features (Supplementary Fig. 1b), including the characteristic gluzincin aminopeptidase motif GAMEN and the zinc-binding/gluzincin motif HEX2HX18E located in the peptidase_M1 domain which

are conserved in nearly all of these M1 aminopeptidases (Supplementary Table 1 and Supplementary Fig. 1c). We found that the APN1-12 gene cluster possesses highly conserved synteny in both

gene order and orientation in different lepidopteran insects, indicating that it has undergone tandem gene duplication during insect genome evolution (Fig. 1a). Although the paralogous

PxAPN1-12 genes show similar features including exon number, size, and intron phase (Supplementary Fig. 1d), they share relatively low protein sequence similarity (Supplementary Fig. 1e),

implying their evolutionary and functional diversity. A model-based phylogenetic analysis demonstrates that lepidopteran APN proteins cluster into 13 classes and are evolutionarily conserved

in each class. Sister phylogenetic relationships were also observed between APN1 and APN3 and between APN5 and APN6, suggesting close protein structure and functional similarities within

these pairs (Supplementary Table 2 and Fig. 1b). ALTERED EXPRESSION OF FOUR PXAPN GENES IN RESISTANT STRAINS A previous study showed that _cis_-mutations in PxAPN genes are not linked to

Cry1Ac resistance in _P. xylostella_42. Likewise, we did not detect mutations in PxAPN genes in any of our resistant _P. xylostella_ strains. Nevertheless, considering that our previous _P.

xylostella_ midgut transcriptome and RNA-Seq studies had identified differentially expressed PxAPN genes in the resistant strains32,33, the transcription profiles of these genes were further

investigated. Unsurprisingly, given their function as digestive enzymes, the APN genes were found to be most highly expressed in the midgut and during the larval stages. Some (PxAPN1-9)

were expressed to a greater extent than the others (Supplementary Fig. 2a). These data were compared with our previous midgut transcriptome32 and RNA-Seq33 data (Supplementary Fig. 2b),

which showed that the PxAPN1-9 genes (except for _PxAPN7_) were expressed in midgut tissues of third-instar Bt susceptible larvae. When the constitutive midgut transcription profiles of

these genes in fourth-instar larvae from both Bt-susceptible and -resistant strains were compared, it was revealed that both _PxAPN1_ and _PxAPN3a_ genes were substantially decreased in all

of the Bt-resistant strains, whereas _PxAPN5_ and _PxAPN6_ genes were increased (Fig. 1c). Assessment of protein levels confirmed that PxAPN1 and PxAPN3a were both reduced in midgut samples

from all the Bt-resistant fourth-instar larvae (Supplementary Table 3 and Fig. 1d). PXAPN1 AND PXAPN3A ARE FUNCTIONAL MIDGUT CRY1AC RECEPTORS In order to ascertain the role, if any, of the

differentially expressed PxAPN genes in the mechanism of action of Bt toxins, the _PxAPN1_, _PxAPN3a_, _PxAPN5_, and _PxAPN6_ genes were transiently expressed in Sf9 cells. These

insect-derived cells are not naturally susceptible to the Bt Cry1Ac toxin and as a result have been used to test putative toxin receptors43. Specific APN activity assays showed that high

levels of APN activity were detected in all the PxAPN-expressing Sf9 cells, indicating that these recombinant PxAPN proteins were biologically active (Supplementary Fig. 3a). The successful

ectopic expression of these GFP-fused PxAPN proteins was further verified by Western blots (Supplementary Fig. 3b). Immunofluorescent localization detection illustrated that all four PxAPN

proteins were located on the cell surface, but that only PxAPN1 and PxAPN3a proteins could bind Cry1Ac toxin (Fig. 2a). Furthermore, Cry1Ac toxin induced a time and concentration-dependent

cell cytotoxicity in cells expressing PxAPN1 or PxAPN3a but not in those expressing PxAPN5 or PxAPN6 (Fig. 2b and Supplementary Fig. 3c). These observations led to the conclusion that PxAPN1

and PxAPN3a are functional receptors of the Bt Cry1Ac toxin in _P. xylostella_. _PXAPN1_ AND _PXAPN3A_ KNOCKDOWN/OUT CONFERS CRY1AC RESISTANCE Having established that PxAPN1 and PxAPN3a

could act as receptors for Cry1Ac, we then intended to establish whether their loss could induce resistance to the toxin. RNAi-induced knockdown of _PxAPN1_, _PxAPN3a_, _PxAPN5_, and

_PxAPN6_ gene expression in the Bt-susceptible strain of _P. xylostella_ was undertaken. The transcript levels for these four PxAPN genes were significantly reduced at 24 h post-RNAi, with

the lowest expression at 48 h and lasting for about 72 h in both cases. The observed silencing was specific to each targeted gene (Fig. 2c and Supplementary Fig. 4a). Subsequent bioassays

conducted at 48 h post-RNAi for 72 h revealed a remarkable reduction in the susceptibility to Cry1Ac protoxin at both 1 mg/L (LC50 value) and 2 mg/L (LC90 value) in both dsPxAPN1- and

dsPxAPN3a-treated groups compared to the controls. Furthermore, the use of combinational RNAi to silence both PxAPN genes simultaneously by microinjection of a combination of dsPxAPN1 and

dsPxAPN3a (dsMultigenes) caused incremental increases in Cry1Ac tolerance at both doses (Fig. 2d). In contrast, no susceptibility changes to Cry1Ac protoxin were detected in dsPxAPN5- and/or

dsPxAPN6-treated groups compared to the controls (Supplementary Fig. 4b). CRISPR/Cas9-mediated knockout of both _PxAPN1_ and _PxAPN3a_ genes was further utilized in vivo to validate their

roles in Cry1Ac resistance. We used an optimized germline transformation and mutation screening strategy to successfully construct stable homozygous mutant strains of _PxAPN1_ and _PxAPN3a_

genes designated APN1KO and APN3aKO, respectively (Supplementary Table 4 and Supplementary Fig. 5). Subsequent bioassays showed that APN1KO and APN3aKO larvae exhibited approximately

463-fold and 346-fold levels of resistance to Cry1Ac protoxin, respectively (Fig. 2e), confirming that the disruption of either PxAPN1 or PxAPN3a can confer high-level Cry1Ac resistance in

_P. xylostella_. Genetic complementation tests between the knockout strains and the NIL-R strain found that F1 progeny from mass crosses between pairs of strains had high larval mortality at

the discriminating concentration (10 mg/L) of Cry1Ac protoxin. This indicated that resistance was effectively recessive for each allele but also that resistance in NIL-R was not due to a

mutation in PxAPN1 or PxAPN3a (Fig. 2f). REDUCED _PXAPN1_ AND _APN3A_ EXPRESSION IS LINKED TO RESISTANCE Genetic linkage analysis was conducted to test the cosegregation of differential

expression of _PxAPN1_, _PxAPN3a_, _PxAPN5_, and _PxAPN6_ genes with Cry1Ac resistance in the NIL-R strain. qPCR analysis showed that the transcript levels of _PxAPN1_ and _PxAPN3a_ genes in

both Cry1Ac-unselected F2 backcross families exhibited two distinct groups: one group displayed reduced expression of _PxAPN1_ and _PxAPN3a_ genes, whereas the other group showed transcript

levels resembling larvae from the susceptible parental strain or the F1 progeny (Fig. 3a, b). Moreover, the ratio between the numbers of individuals in each group was 9:9 in both backcross

families for the _PxAPN1_ and _PxAPN3a_ genes, which was a perfect fit to the theoretically random assortment ratio 1:1 (_p_ = 1.0; _χ_2 test). In stark contrast, all of the survivors from

Cry1Ac-treated F2 backcross families had reduced _PxAPN1_ and _PxAPN3a_ transcript levels compared to larvae from the DBM1Ac-S strain or the F1 progeny, indicating tight linkage

(cosegregation) with Cry1Ac resistance in NIL-R (_p_ < 0.001, _χ_2 test). The transcript levels for _PxAPN5_ and _PxAPN6_ genes in both Cry1Ac-treated and non-treated groups remained

significantly up-regulated and did not associate with Cry1Ac resistance (Fig. 3c, d). MAPK-INDUCED DIFFERENTIAL EXPRESSION OF DIVERSE MIDGUT GENES We previously established that an activated

MAPK cascade _trans_-regulates differential expression of _PxmALP_, _PxABCC1_, _PxABCC2_, _PxABCC3_, and possibly other midgut genes involved in Cry1Ac resistance in _P.

xylostella_26,27,28. Hence, silencing of _PxMAP4K4_ expression in resistant NIL-R larvae was performed to ascertain whether this MAPK-mediated mechanism also accounts for the differential

expression of PxAPN and other midgut genes to cause Cry1Ac resistance. After dsRNA injection in the Bt-resistant strain, the transcript levels of _PxMAP4K4_ were substantially reduced at 48 

h post-RNAi (Fig. 4a). Correspondingly, the transcript levels of _PxmALP_, _PxABCB1_, _PxABCC2_, _PxABCC3_, _PxABCG1_, _PxAPN1_, and _PxAPN3a_ were increased, while the transcript levels of

_PxABCC1_, _PxAPN5_, and _PxAPN6_ decreased at 48 h post-injection (Fig. 4a). Bioassays conducted at 48 h post-RNAi for 72 h indicated that silencing of _PxMAP4K4_ expression led to

significantly increased larval susceptibility to Cry1Ac protoxin compared to the controls (Fig. 4b). Therefore, _PxMAP4K4_ gene silencing recovered the expression of PxAPN1, PxAPN3a and

other midgut proteins and restored Cry1Ac susceptibility in NIL-R larvae. INCREASED TITERS OF INSECT HORMONES IN RESISTANT STRAINS Previous work had shown that exogenous hormone treatments

can alter the transcript levels of two isoforms of _AjAPN1_ genes in _Achaea janata_41, so we decided to investigate whether insect hormones might be the upstream activation signals of the

MAPK cascade to _trans_-regulate differential expression of PxAPN and other midgut proteins thereby mediating Cry1Ac resistance in _P. xylostella_. We initially quantified hormone titers in

_P. xylostella_ by optimized multiple reaction monitoring (MRM)-based ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Hormone detection showed specific single

peaks for JHs, 20E and their respective analogs methoprene and 22S, 23S-homobrassinolide in the MRM mass chromatograms (Fig. 5a). As expected, the hormone titers fluctuated during different

developmental stages with clear JH II peaks observed around larval molting, and the sensitivity of this initial assay only resulted in changes in 20E levels being observed during pupation

(Fig. 5b). A more detailed analysis of hormone titers during the third- and fourth-instar stages showed sharp drops in 20E and small peaks in JH II during the molting stage, in contrast, the

levels of both hormones did not fluctuate significantly during the feeding intermolt stage (Fig. 5c, d). When the titers of JH II and 20E in feeding third/fourth stage larvae from the

resistant and susceptible strains were compared, it was found that the levels were higher in the NIL-R strain than the DBM1Ac-S strain (Fig. 5c, d). Furthermore, it was found that they were

also higher in fourth-instar feeding larvae of all the resistant strains, which correlated well with their resistance ratios (Fig. 5e, f), and hinted at the potential involvement of insect

hormones in Cry1Ac resistance in _P. xylostella_. THE MAPK CASCADE IS MODULATED BY INSECT HORMONAL CROSSTALK To investigate a potential causal link between hormone titer and resistance,

exogenous JH analog (JHA) methoprene (200 ng), 20E (2 ng), or a mixture of the two, were microinjected into newly molted third-instar DBM1Ac-S larvae. qPCR analysis showed that the

expression of _PxMAP4K4_ was significantly decreased at 48 h after methoprene treatment, whereas it had increased at 48 h after 20E treatment (Fig. 6a). The transcript levels of four PxAPN

and other midgut genes were increased at 48 h after methoprene treatments, whereas their levels were notably decreased at 48 h after 20E treatments. However, when larvae were synchronously

treated with both methoprene and 20E, while the transcript levels of _PxMAP4K4_, _PxAPN5_, _PxAPN6_, and _PxABCC1_ were increased at 48 h post-microinjection, those of _PxAPN1_, _PxAPN3a_,

_PxmALP_, _PxABCB1_, _PxABCC2_, _PxABCC3_, and _PxABCG1_ had decreased (Fig. 6a). Intriguingly, this pattern of differential expression exactly, but inversely, mimicked that seen in the

NIL-R strain following _PxMAP4K4_ knockdown (Fig. 4a). To investigate whether downstream elements of the MAP4K cascade (notably p38, ERK, and JNK) might be involved in the control of

expression of the midgut proteins, a combination of methoprene, 20E, and a MAPK inhibitor cocktail targeting the above three kinases was microinjected into DBM1Ac-S larvae. Despite the

expected increase in expression of _PxMAP4K4_ following this treatment, the expression levels of all of the tested midgut genes either remained the same as the control or only showed modest

changes (Fig. 6a). To confirm the downstream effects of hormone treatment on the MAPK cascade, Western blots were performed to measure the levels of total and phosphorylated p38, ERK, and

JNK (Fig. 6b). These data showed a significant increase in phosphorylated protein following treatment with 20E or a combination of 20E and methoprene. These increases were not observed when

the MAPK inhibitor cocktail was included alongside the hormone mixture, results consistent with the qPCR data in suggesting that the hormones are exerting their effect via the MAP4K cascade.

Bioassays performed at 48 h post-microinjection demonstrated that methoprene treatment resulted in significantly increased larval susceptibility to Cry1Ac protoxin compared to the controls,

in contrast, 20E treatment caused significantly reduced susceptibility (Fig. 6c). Treatment with both methoprene and 20E simultaneously led to a more greatly reduced susceptibility to

Cry1Ac protoxin than 20E treatment alone, and this effect was negated by simultaneous treatment with the MAPK inhibitor cocktail (Fig. 6c). To further probe the interplay between hormone

exposure and the MAPK cascade, we studied the effect of hormone treatment on insects in which _PxMAP4K4_ expression had been reduced by RNAi. In particular, we were interested in whether 20E

induced loss of susceptibility could be reversed by silencing of _PxMAP4K4_. Figure 6d, e shows that the effect of 20E on the silenced strain was similar to the effect on the non-silenced

strain. A similar observation was made with the dual hormone treatment. Given that the data presented in Fig. 6a, c clearly indicate an association between hormone treatment and _PxMAP4K4_

expression, we surmise that the 20E-induced increase in _PxMAP4K4_ transcription can overcome the effect of RNAi-induced reduction in transcript levels. ASSOCIATIONS AMONG HORMONE TITER, BT

RESISTANCE, AND FITNESS Based on the above observation that exposure to methoprene and 20E could directly affect resistance levels to Bt, and induce the same changes in midgut proteins as

seen in the resistant strains, we proposed a model. In this model, elevated levels of 20E can induce resistance to Bt through blocking the expression of many midgut proteins, in particular,

those which act as functional Bt toxin receptors. Fitness costs related to life-history traits frequently occur during insect resistance to Bt infection44, therefore, resistance evolution

without growth penalty is extremely important for successfully overcoming Bt pathogenesis. Thus, we suggest that fitness costs associated with this 20E-induced physiological change are

modulated by a corresponding increase in JH which results in the restoration of midgut physiology through the expression of non-receptor paralogs. To test this model, we exposed larvae to

methoprene and 20E either individually or in combination and measured various life-history traits (Fig. 7a–i). These data support our model in that exposure to the individual hormones

resulted in significantly reduced pupation rates, eclosion rates, adult longevity, oviposition time, female fecundity, and egg hatchability, whereas those in the hormone combination were

indistinguishable from the buffer control (Fig. 7b, e–i). Methoprene resulted in larger pupae, but which stayed in that state for longer, whereas 20E gave smaller pupae and a shorter time in

this stage, once again, the combination treatment was indistinguishable from the control (Fig. 7a, c, d). Moreover, we observed that the 20E-treated third-instar larvae molted early to the

fourth-instar and pupal stages with reduced appetite and food intake, while methoprene-treated third-instar larvae behaved completely opposite, i.e., molted late to the fourth-instar and

pupal stages with increased appetite and food intake. In contrast, the development time and feeding behavior of third-instar larvae treated with both hormones were comparable to those in the

control group. Thus, these differences might attribute to the observed variation in the resultant pupal size (Fig. 7a). Since it is known that exposure to Bt can induce tolerance to Bt45,

and that toxin can induce the expression of _PxMAP4K4_ gene in the DBM1Ac-S strain26, we further examined the effect of Bt toxin exposure on hormone levels and fitness. Both newly molted

third-instar DBM1Ac-S and NIL-R larvae were exposed to LC50 levels of Cry1Ac protoxin for 72 h. Surviving fourth-instar larvae showed two quite distinct groups in larval weight, some were

significantly lighter than the others and were termed “weak”, whereas the others were indistinguishable from untreated larvae and were termed “robust”. For the robust larvae, it was found

that while exposure of the DBM1Ac-S larvae resulted in significant increases in the levels of both hormones, there were no significant increases seen in the NIL-R larvae (Fig. 6f, g). For

the weak larvae, while an increase in 20E in DBM1Ac-S was observed that was comparable to that seen in the robust larvae, the increase in JH II was significantly less. The levels of 20E in

weak NIL-R specimens were equivalent to the robust and untreated control, however, a lower level of JH II was found (Fig. 6f, g). As expected, when the life-history traits of the surviving

insects were measured, it was found that the robust individuals were indistinguishable from the untreated controls, whereas the weak ones were significantly affected in all the tested

parameters (Fig. 7j–r). LINKAGE OF INCREASED 20E TITERS WITH CRY1AC RESISTANCE The data presented above suggest a causal association between increased titers of insect hormones and

receptor-mediated resistance to Bt Cry toxin, a link which involves the MAPK signaling cascade. To further strengthen this association, we used genetic linkage analysis to determine

co-segregation of increased titers of JH II and 20E with Cry1Ac resistance in the NIL-R strain. UPLC–MS/MS detection showed that JH II titers in both Cry1Ac-treated and non-treated backcross

groups had the same level of up-regulation as the resistant parental strain, indicating a lack of association with Cry1Ac resistance (Fig. 8a). In stark contrast, 20E titers in both

Cry1Ac-unselected F2 backcross families exhibited two distinct groups: one group displayed increased 20E titers, whereas the other group showed 20E levels resembling larvae from the

susceptible parental DBM1Ac-S strain or the F1 progeny (Fig. 8b). Moreover, the ratio between the numbers of individuals in each group, 10:8 in backcross family a and 8:10 in backcross

family b, followed the 1:1 random assortment ratio (_p_ > 0.10; _χ_2 test). Crucially, all of the survivors from the Cry1Ac-treated F2 backcross families had increased 20E titers,

indicating tight linkage (cosegregation) with Cry1Ac resistance in NIL-R (_p_ < 0.001, _χ_2 test). DISCUSSION The proteinaceous Cry toxins produced by Bt represent highly potent virulence

factors which when presented to an insect at a high dose, such as in Bt crops, overwhelm the insect’s immune system leading to death3. Through modification of the toxin binding protein

(receptor) on the surface of its midgut epithelium, an insect can effectively block the action of the toxin leading to high levels of resistance19. The first Cry toxin receptor identified in

an insect was an APN protein in _Manduca sexta_20,30. In this work, we show that two isoforms of this enzyme can act as functional receptors in _P. xylostella_ and that reduction in their

expression leads to loss of susceptibility towards Cry1Ac. We have also conducted the genome-wide characterization of the APN gene family and unified their nomenclature and classification in

insects. Interestingly, we discovered a syntenic APN gene cluster, containing 12 or more APN genes in different lepidopteran insects, is estimated to have been present since the Jurassic

period about 150 million years ago46. Our previous studies have shown that silencing of other potential _P. xylostella_ Cry toxin receptor genes including _PxmALP_, _PxABCB1_, _PxABCC2_,

_PxABCC3_, and _PxABCG1_ also results in decreased Cry1Ac susceptibility26,27,28. One model for Cry toxin mechanism of action involves sequential binding of the toxin to different

receptors19, whereas others suggest that multiple receptors in a susceptible host represent alternative receptors rather than members of a sequential pathway47. In a sequential model, one

would predict that blocking any of the steps would lead to a similar level of resistance. In this work, we show that knocking out either APN receptor, whilst leading to high levels of

resistance, does not match the level of resistance seen in the parent strain, suggesting at least some degree of receptor redundancy. The high level of resistance in the parent strain can be

associated with the simultaneous down-regulation of multiple Cry toxin receptors. Whilst the loss of physiologically important proteins might be expected to impart a fitness cost to the

insect, it is noteworthy that the down-regulation of proteins known to act as toxin receptors is accompanied by the up-regulation of paralogs that have no receptor function. Gene duplication

and subsequent transcriptional plasticity is a major driving force for adaptive evolution of insect response to environmental stress48,49. Specifically, up-regulation of _PxAPN5_ and

_PxAPN6_ might compensate for the down-regulation of _PxAPN1_ and _PxAPN3a_ to diminish the fitness costs of Cry1Ac resistance and perhaps resembling the differential alteration of _TnAPN1_

and _TnAPN6_ in resistant _Trichoplusia ni_50. It is noteworthy that the total midgut APN enzyme activities were similar in our susceptible and resistant strains26 and we did not detect any

fitness costs in our near-isogenic Cry1Ac-resistant NIL-R strain51. Previously, we found that a MAPK signaling pathway can _trans_-regulate the differential expression of _PxmALP_,

_PxABCC1-3_, and possibly _PxABCB1_ and _PxABCG1_ genes thereby resulting in Cry1Ac resistance in four different _P. xylostella_ strains26,27,28. We have now identified that this pathway can

also _trans_-regulate the differential expression of four PxAPN genes to mediate Cry1Ac resistance in the same four resistant _P. xylostella_ strains. Although the mechanism underlying the

altered MAPK signaling pathway is unknown, in this work, we discovered an intriguing causal link to the insect hormones 20E and JH. Levels of both insect hormones were significantly

increased in all of our resistant strains of _P. xylostella_, but more relevantly when susceptible insects were exposed to both hormones: differences in gene expression of _PxMAP4K4_,

_PxAPN1,3a,5,6_, _PxABCB1_, _PxABCC1,2,3_, _PxABCG1_ and _PxmALP_ all mirrored those seen in the resistant strain, and a decrease in toxin susceptibility was observed. The MAPK inhibitor

assays indicated that insect hormones can orchestrate the expression of these resistance-related midgut genes via the MAPK signaling cascade, which strengthens the case for a causal link

between insect hormones and MAPK signaling cascade. We confirmed that increased 20E, but not JH II, titers were genetically linked to Cry1Ac resistance in _P. xylostella_, which provided

more direct evidence that increased titers of insect hormones in resistant _P. xylostella_ strains is actually a cause rather than an effect of resistance. In this regard, we can ask: why JH

II titers were also increased when only increased 20E titers were tightly linked to Cry1Ac resistance? Besides modulating life-history traits including growth, development, and

reproduction, JH and 20E can also participate in insect host immune defense and resistance to pathogenic infection: JH suppresses stress resistance and immunity, whereas 20E activates these

processes38. The insect immune system is like a double-edged sword: on the one side, its activation can effectively fight against infections from a broad range of pathogens, but on the

other, its activation has a negative impact on insect growth and development52. Hence, maintaining both immune defense and life-history traits is physiologically and energetically costly,

and the tradeoff between them is crucial for limiting resource allocation in diverse insects39. A similar phenomenon is well established in plants where the growth-defense tradeoff is a fine

balance and one which is modulated by MAPK-dependent hormonal signaling pathways53,54,55,56. Similar tradeoffs have also been identified in vertebrates57,58, indicating that hormonal

signaling plasticity is a general cross-kingdom strategy for thwarting pathogens. As mentioned above, an imbalance in the ratio of the two insect hormones can shift the insect’s physiology

towards either growth/reproduction or defense with a corresponding cost on the other process, and our data in which fitness costs were associated with individual hormone exposure support

that. We are proposing a fine-tuning of this process in which a balanced increase in the two hormones affects the insect in such a way that it loses susceptibility to the Cry toxin whilst

maintaining gut homeostasis through the expression of paralogous genes. We demonstrated that a balanced rise in both hormones is induced by sub-lethal exposure to Bt toxin and also found,

that via some unknown genetic changes, a constitutive and balanced rise has occurred in the resistant strains. A similar case was documented in _Drosophila virilis_, in which increased

titers of 20E and JH were identified as an induced adaptive response to the unfavorable environmental heat stress by decreasing egg production and oviposition. Moreover, in a heat stress

mutant strain of _D. virilis_, elevated JH and 20E levels caused by an as yet uncharacterized gene mutation in chromosome 6 resulted in the same reduced fertility phenotype as observed for

the wild type strain under heat stress59,60. In _P. xylostella_, we hypothesize that a balanced rise in the levels of the two hormones pushes the insect towards a growth-defense tradeoff

instead of a full-scale defense response to successfully evolve a Bt resistance phenotype, and we present a model of this proposed system in Fig. 9. Although Bt Cry toxin has been shown to

induce various aspects of an immune response45, our previous comparisons of transcript levels between susceptible and resistant _P. xylostella_ strains did not find any examples of

immune-related genes such as peptidoglycan recognition proteins (PGRPs), antimicrobial peptides (AMPs), or phenoloxidases (POs) in the list of genes that had significantly increased

expression in the resistant strain32,33,61. A previous study using Bt-resistant _Helicoverpa armigera_ larvae found that there was an increased titer of JH in the resistant strain62.

Although that study indicated that this hormone was directly associated with fitness costs in the resistant strain, it did not look at levels of 20E nor establish any causal link between JH

titer and Bt-resistance. Detailed work on Bt tolerance in _Ephestia kuhniella_ found that non-genetically inherited tolerance could be induced by exposure to sub-lethal concentrations of Bt

and vertically transmitted through maternal effect45. This work had noted that while the initial elevated immune response in this insect carried significant developmental penalties,

continued exposure to Bt led to a reduced developmental penalty while tolerance was maintained63. Our results are consistent with a model in which tolerance and resistance to Bt are linked

and both associated with hormonal regulation. The mechanistic basis of the interplay between hormone levels, MAPK signaling pathway and Bt resistance remains unclear. While there is evidence

for altered protein phosphorylation levels of p38, JNK, and ERK in the MAPK signaling cascade triggering the differential expression of the midgut genes, it is less clear how the hormones

feed into this process. Our data here indicate that changes in JH and 20E concentrations can affect MAP4K4, p38, JNK, and ERK, which suggests that they are acting upstream of the MAPK

signaling cascade. However, in the resistant strains, the causative mutation maps to the _MAP4K4_ locus and most likely directly affects the expression of this gene26. The fact that JH and

20E levels are also increased in these strains might suggest that their levels are tightly coordinated with MAP4K4 levels in some way. Although the MAPK signaling pathway is strongly linked

to down-regulation of the Bt toxin receptors, it is quite possible that the two hormones are affecting other aspects of the insect’s response via other routes. Initial exposure to Bt can

trigger an immediate defense response, that may come with fitness costs, but then a fine-tuning between 20E and JH can balance that tolerance while reducing those costs. Where that balance

is not maintained, e.g., in some of our larvae exposed to Bt toxins, severe fitness costs prevail. Differences between the weak and robust larvae may reflect different levels of intoxication

and a more extreme, and costly, response to deal with excessive exposure. Continued exposure can result in genetic mutation that leads to constitutive expression of the defense response now

manifested as resistance. In other cases of acquired resistance, the phenotype can be as a result of less subtle changes, such as functional deletions of a receptor. As suggested in a

recent review64, we should be looking to better integrate work on immunity, toxicology, and endocrinology to better understand the host–pathogen interactions between insects and Bt. Our

current work linking endocrine associated growth/defense tradeoffs with Bt tolerance/resistance should greatly facilitate future research in this emerging field. METHODS INSECT STRAINS Five

_P. xylostella_ strains including one susceptible strain DBM1Ac-S and four resistant strains DBM1Ac-R, NIL-R, SZ-R, and SH-R were used in this study26,65,66. The DBM1Ac-R, NIL-R, and SZ-R

strains have developed approximately 3500-, 4000-, and 450-fold resistance to Cry1Ac protoxin compared to the susceptible DBM1Ac-S strain, while SH-R larvae have developed about 1900-fold

resistance to Bt var. _kurstaki_ (Btk) formulation compared to the DBM1Ac-S strain. The field-evolved or laboratory-selected resistance to Bt Cry1Ac toxin or Btk formulation in these four

independent _P. xylostella_ strains has a similar mechanism involving MAPK-mediated differential expression of _PxmALP_, _PxABCB1_, _PxABCC1-3_, and _PxABCG1_ genes26,27,28. In addition, two

new _P. xylostella_ strains designated APN1KO and APN3aKO were established by knocking out _PxAPN1_ and _PxAPN3a_ from DBM1Ac-S strain by CRISPR/Cas9 genome editing system, respectively.

All _P. xylostella_ strains were reared _en masse_ on Jing Feng No. 1 cabbage (_Brassica oleracea_ var. _capitata_) at 25 °C with 65% relative humidity (RH) and a 16:8 (light:dark)

photoperiod, and adults were supplied with a 10% honey/water solution. TOXIN PREPARATION AND BIOASSAY Cry1Ac protoxin was prepared from Btk strain HD-73 and both purified Cry1Ac protoxin and

trypsin-activated Cry1Ac toxin were quantified using Bradford’s method with bovine serum albumin (BSA) as a standard67. Toxicity of Cry1Ac protoxin in 72 h bioassays with _P. xylostella_

larvae was assessed using a leaf-dip method65. Briefly, 10 third-instar larvae were tested on each of seven toxin concentrations and bioassays were replicated in quadruplicate. The median

lethal concentrations (LC50) values and 95% confidence limits (CL) were calculated by probit analysis using POLO Plus 2.0 statistical software (LeOra Software). LC50 values without

overlapping among 95% CL were considered as significantly different. RNA EXTRACTION, CDNA SYNTHESIS, AND GDNA ISOLATION Different _P. xylostella_ samples were collected and homogenized in

TRIzol reagent (Invitrogen), and the total RNA was extracted, purified, and quantified according to the manufacturer’s protocol. First-strand cDNA was then prepared either with the

PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa) for gene cloning or with the PrimeScript RT kit (containing gDNA Eraser, Perfect Real Time) (TaKaRa) for qPCR detection following the

manufacturer’s recommendations. Genomic DNA (gDNA) samples of fourth-instar DBM1Ac-S larvae were isolated for gene cloning using a TIANamp Genomic DNA Kit (TIANGEN), and the individuals were

fully homogenized with an electric pestle in 1.5 ml centrifuge tubes before gDNA extraction according to the manufacturer’s instructions. Finally, the prepared cDNA and gDNA samples were

used immediately or stored at −20 °C until used. GENE IDENTIFICATION, CLONING, AND SEQUENCING To identify all the PxAPN genes in _P. xylostella_ genome, the amino acid sequences of the

N-terminal conserved peptidase_M1 domain of M1 family aminopeptidases (Pfam ID: PF01433) in four known PxAPN proteins retrieved from GenBank database (PxAPN1, AAB70755; PxAPN2, CAA66467;

PxAPN3b, AAF01259; PxAPN5, CAA10950) were used as queries for a blastp search against the Diamondback moth Genome Database (DBM-DB, http://59.79.254.1/DBM/). Then, the putative CDS sequences

of acquired genes were further in silico corrected with blastn search against our previous _P. xylostella_ midgut transcriptome databases32. To obtain the full-length cDNA or partial gDNA

sequences of all the M1 aminopeptidase genes, the PCR cloning strategies and the gene-specific primers designed by the Primer Premier 5.0 software (Premier Biosoft) are shown in

Supplementary Table 6. The PCR reactions (25 μl) using cDNA or gDNA as template were conducted for 35 cycles with the following parameters: denaturing at 94 °C for 30 s, annealing at

optimized 50–60 °C (depending on the primers) for 45 s, and extension at 72 °C for 1–4 min based on the PCR product size; and a final extension of 10 min at 72 °C using LA Taq polymerase

with general or high GC buffers (TaKaRa). The obtained amplicons were purified, subcloned, and sequenced. After large-scale cloning and sequencing (20 clones from two independent cDNA

batches), the full-length cDNA sequences of differential expressed PxAPN genes from DBM1Ac-S and NIL-R were compared to detect potential sequence variations. BIOINFORMATIC ANALYSIS Gene

sequence assembly, multiple sequence alignment, and exon–intron analysis were conducted using DNAMAN 9.0 (Lynnon BioSoft). The deduced protein sequences were obtained by Translate

(https://web.expasy.org/translate/), and the calculated isoelectric point (pI) and molecular weight (Mw) were predicted with Compute pI/Mw (http://ca.expasy.org/tools/pi_tool.html). The

N-terminal signal peptide was identified using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). Two site prediction servers (big-PI Predictor:

http://mendel.imp.ac.at/sat/gpi/gpi_server.html and GPI-SOM: http://gpi.unibe.ch/) were used to predict potential GPI-modification sites. Transmembrane helices were predicted using the TMHMM

Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The presence of N- and O-glycosylation sites on the protein sequences were respectively predicted with the NetNGlyc 1.0

(http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc 4.0 (http://www.cbs.dtu.dk/services/NetOGlyc/) servers. The conversed protein motifs were displayed by WebLogo 3 program

(http://weblogo.threeplusone.com/create.cgi). PHYLOGENETIC ANALYSIS Full-length protein sequences of all the current available lepidopteran PxAPN and other M1 aminopeptidase genes were

retrieved and corrected from GenBank or other genome databases (Supplementary Table 2). To determine the phylogenetic relationship of these genes, sequence alignments were conducted with

Clustal W in MEGA 7.0 (http://www.megasoftware.net/). The high-quality unrooted phylogenetic tree was then constructed using the maximum likelihood (ML) method based on the model optimized

using the Bayes Information Criterion with “complete deletion” as the gaps/missing data treatment and 1000 bootstrap replications. QPCR ANALYSIS The gene transcript levels were quantified by

real-time quantitative PCR (qPCR) detection processed in the QuantStudio 3 Real-Time PCR System (Applied Biosystems)27. Briefly, gene-specific primers of the PxAPN genes (Supplementary

Table 6) with high amplification efficiencies (95–100%) were selected and used in qPCR reactions with 2.5× SYBR Green MasterMix Kit (TIANGEN) following the manufacturer’s instructions. The

qPCR program included an initial denaturation at 94 °C for 6 min, followed by 40 cycles of amplification at 94 °C for 30 s, 50–60 °C (depending on the primers) for 30 s, and 72 °C for 35 s.

Relative expression levels were calculated using the 2−ΔΔCt method and normalized to the ribosomal protein _L32_ (_RPL32_) gene (GenBank accession no. AB180441). BBMV PREPARATION

Approximately 2000 fourth-instar larval midgut tissues from each _P. xylostella_ strain were dissected in cold MET buffer [17 mM Tris–HCl (pH 7.5), 5 mM EGTA, 300 mM mannitol] plus protease

inhibitors (1 mM PMSF). Midgut brush border membrane vesicles (BBMV) were prepared by the differential magnesium precipitation method and quantified using Bradford’s method with BSA as a

standard26, and then flash-frozen and kept in aliquots at −80 °C until used. Between 5- and 8-fold enrichment in specific APN activity using l-leucine-_p_-nitroanilide (Leu-_p_-NA) (Sigma)

as substrate was detected compared to initial midgut homogenates. WESTERN BLOTS Antibodies of PxAPN1 and PxAPN3a proteins used for Western blots were generated from synthetic peptides

(Jiaxuan Biotech) derived from respective specific amino acid sequences 952PPVDTTPDMTPPQP965 and 977DPPPEVDTETTPQP990, and other specific antibodies targeting p38, JNK, ERK, and β-actin were

commercially purchased (Supplementary Table 3). The protein and protein phosphorylation levels of target proteins were detected by Western blots using β-actin as an internal control. Midgut

proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto Immobilon-P membranes (Merck Millipore). The membranes were then blocked

with blocking buffer containing BSA (CWBIO) at 25 °C for 1 h and incubated with the appropriate primary antibody (Supplementary Table 3) at 4 °C overnight, followed by incubation with goat

anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000, CWBIO) (Supplementary Table 3). The protein bands were visualized using the SuperSignal West Pico Chemiluminescent

Substrate (Thermo Fisher Scientific), and the images were captured by the Tanon-5200 Chemiluminescent Imaging System (Tanon). Densitometric analysis of the protein bands was performed using

ImageJ v.1.51 software (http://rsbweb.nih.gov/ij/), and the relative band intensities were calculated based on densitometric ratios between target proteins and β-actin with the band

intensities in the DBM1Ac-S BBMV samples considered as 1. HETEROLOGOUS EXPRESSION The recombinant PxAPN-GFP fusion proteins were in vitro transiently expressed in Sf9 cells26. The Sf9 cells

were harvested, lysed and quantified, and specific APN activity assays and Western blots were performed. Enzymatic activity was detected using Leu-_p_-NA (Sigma) as substrate, with one

enzymatic unit (U) being defined as the amount of the enzyme that would catalyze the production of the chromogenic product from 1 µmol specific substrate per min and per mg of total Sf9 cell

proteins at 37 °C. Western blots were performed as mentioned above with mouse monoclonal anti-GFP antibody (1:1000, Abcam) using β-actin as an internal control. For immunolocalization of

Cry1Ac toxin binding to Sf9 cells expressing PxAPN proteins, the transfected Sf9 cells were incubated with Cry1Ac toxin and fixed in ice-cold 4% paraformaldehyde. After blocking with 1% BSA,

the cells were probed with rabbit polyclonal anti-Cry1Ac primary antibody (1:1000) and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 555 (1:1000, Abcam). The treated cells

were examined under a LSM 700 confocal laser scanning microscope (Carl Zeiss). For cytotoxicity assays, WST-8 in the Cell Counting Kit-8 (CCK-8) (Dojindo) was used to perform a CCK-8 assay

following the manufacturer’s recommendations. The absorbance was measured at 450 nm after 24 h incubation with Cry1Ac toxin, and the relative cell viability was calculated in relation to

untreated Sf9 cells, which were defined as 100%. In addition, the morphological changes of Cry1Ac-treated Sf9 cells were also observed under a Zeiss LSM 700 microscope. RNA INTERFERENCE

RNAi-induced gene silencing and subsequent functional assays were performed in vivo in _P. xylostella_26,27,28,65. Briefly, specific dsRNA was generated in vitro using the T7 Ribomax Express

RNAi System (Promega) and subsequently mixed with an equal volume of Metafectene PRO transfection reagent (Biontex) before microinjection. Microinjection of dsRNA into newly molted

third-instar _P. xylostella_ larvae was conducted using the Nanoliter 2000 microinjection system (World Precision Instruments). Silencing effects were tested by qPCR and leaf-dip bioassays

were performed to detect the larval toxic response to Cry1Ac protoxin post-RNAi. CRISPR/CAS9 EXPERIMENT The CRISPR/Cas9 genome editing system has been recently successfully applied to

efficiently probe gene functions in diverse insects68, so CRISPR/Cas9-mediated gene knockout and subsequent functional validation were performed in vivo in _P. xylostella_69. Briefly,

gene-specific sgRNA target sequences were designed in exon 12 of _PxAPN1_ and exon 13 of _PxAPN3a_ (Supplementary Table 6). In vitro transcription was conducted to generate sgRNAs with the

MEGAscript Transcription Kit (Ambion) following the manufacturer’s instruction. The mixtures of sgRNA (150 ng/μl) and Cas9 (300 ng/μl) were microinjected into fresh preblastoderm-stage _P.

xylostella_ eggs using FemtoJet 4i and InjectMan 4 Microinjection System (Eppendorf). Nondestructive genotyping of CRISPR/Cas9-induced indel mutations around the sgRNA target site was

performed using PCR amplification with gDNA samples prepared from exuviates of individual fourth-instar larvae as templates, followed by direct sequencing and TA cloning. The germline

transformation and mutation screening strategy was developed and optimized to construct stable homozygous mutant strains of both the _PxAPN1_ and _PxAPN3a_ genes designated APN1KO and

APN3aKO, respectively (Supplementary Table 6). Leaf-dip bioassays were conducted to determine whether knockout of _PxAPN1_ and _PxAPN3a_ genes affects susceptibility to Bt Cry1Ac protoxin,

and genetic complementation tests were further performed to characterize the Cry1Ac resistance phenotype in both knockout strains. GENETIC LINKAGE ANALYSIS The near-isogenic resistant NIL-R

and susceptible DBM1Ac-S strains were used for genetic linkage analysis26. Briefly, a single-pair cross was conducted between a NIL-R male and a DBM1Ac-S female to generate the F1 progeny.

Reciprocal crosses between single-pair F1 and NIL-R moths were performed to acquire two different backcross families. The resulting progenies from both backcross families were reared on

control (cabbage) or experimental (cabbage with 20 mg/L of diagnostic Cry1Ac protoxin dose) diets, and single robust survivors were used for RNA extraction and cDNA synthesis as mentioned

above. Linkage between gene expression levels or hormone titers and Cry1Ac resistance trait was tested using qPCR detection as described above. HORMONE EXTRACTION AND DETECTION For JH and

20E extraction, _P. xylostella_ samples were weighed and homogenized in glass homogenizers with 2 ml of ice-cold methanol/ether (1:1, v/v) or 75% methanol respective containing 0.1 ng JH

analog methoprene (Dr. Ehrenstorfer GmbH; 98.5%) or 22S, 23S-homobrassinolide (Yuanye Biotech; 95%) as internal standards. The resulting homogenates were then vortexed vigorously and

centrifuged three times for 10 min at 4500×_g_ at 4 °C with 2 ml of _n_-hexane or 75% methanol to collect the hexane (upper) phase or the supernatant in brown siliconized glass tubes.

Subsequently, the combined extracts were dried completely under a pressure blowing concentrator and dissolved in 500 µl mobile phase (70% methanol for JH and 50% methanol for 20E). The final

solution was filtered with 0.22 μm membrane before being subjected to chromatographic analysis. Mixed calibration standards of 0.01, 0.1, 0.5, 1, 2, 5, and 10 ng/ml concentrations were

prepared with 70% methanol for JHs [JH II (SciTech; 78%); JH III (Sigma; ≥65%)] and 50% methanol for 20E (Selleck Chemicals; 99.25%). Each solution contained 50 ng methoprene or 0.1 ng 22S,

23S-homobrassinolide as internal standards, respectively. Calibration curves were constructed by plotting the relative peak area ratios (the peak area of hormones/the peak area of their

internal controls) versus their concentrations, and the regression lines were calculated using a weighted factor (1/_y_) least-squares linear regression model. The final constructed

calibration curves of JH II, JH III, and 20E were linear over concentration ranges with perfect regression correlation coefficients (_R_2 = 1), indicating the specificity of the constructed

hormone detection methods (Supplementary Fig. 6). To accurately quantify the JH and 20E titers, UPLC–MS/MS analysis was conducted using the ACQUITY UPLC I-Class/Xevo TQ-S micro System

(Waters) equipped with an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size). The chromatographic separation of JH and 20E was carried out using binary gradient program

between methanol (mobile phase A) and water containing 0.1% formic acid (mobile phase B). For 20E detection, the gradient started with 70% of component B: water (0.1% formic acid) for 1 min

and then decreased to 0% of component B within 5 min. This gradient was kept for 8 min, and then increased to 70% of component B within 0.5 min. The total run time was 10 min, and an

equilibration step of 1.5 min was included. For JH detection, the gradient started with 30% of component B: water (with 0.1% formic acid) for 2 min and then decreased to 15% of component B

within 2 min. This gradient was kept for 2 min, and then increased to 30% of component B within 2 min. The total run time was 9 min, and an equilibration step of 1 min was included. The flow

rate of the mobile phase was constant at 0.2 ml/min (injection volume: 10 μl) and the column temperature was 40 °C. To enhance the selectivity and sensitivity of the MS detection, the mass

spectrometer was processed under electrospray ionization positive ion mode with MRM of two transitions per compound (precursor ions/product ions). The source parameters under MRM detection

were optimized for JH and 20E: source temperature 150 °C, desolvation temperature 400 °C, cone gas flow 50 L/h, desolvation gas flow 800 L/h, source voltage 1760 V, core voltage 62 V. To

enhance the selectivity and sensitivity of the MS detection, the MRM transition parameters applied for JH and 20E and their respective internal controls methoprene and 22S,

23S-homobrassinolide with regard to the transitions from precursor to product ions were also optimized and shown in Supplementary Table 5. The system operation, data acquisition, and

analysis were performed using the MassLynx V4.1 software (Waters). During the UPLC–MS/MS detection of JH and 20E titers, the limits of detection (LOD), defined as the lowest concentration

that the analytical process can differentiate from background levels, were estimated for a signal-to-noise ratio (SNR, also called the _S_/_N_ ratio) of 3 from the chromatograms of the

detected samples, while limits of quantification (LOQ), defined as the lowest concentration that the analytical process can quantify from background levels, were estimated for a SNR of 10

from the chromatograms of the detected samples. Of particular note, the LOD and LOQ are very important threshold values to consider when measuring 20E titers since the 20E levels in the

larval molting stages are very low (Supplementary Fig. 7). HORMONE ASSAYS Hormone treatments were performed using newly molted third-instar _P. xylostella_ larvae. 20E and methoprene were

dissolved in ethanol (20 mg/ml) and diluted with ice-cold insect Ringer’s solution (130 mM NaCl, 0.5 mM KCl, 0.1 mM CaCl2) before microinjection. The MAPK inhibitors SB203580 (specific p38

inhibitor, Merck Millipore), SP600125 (specific JNK inhibitor, Merck Millipore) and PD0325901 (specific MEK/ERK inhibitor, TargetMol) were dissolved in dimethyl sulfoxide (DMSO, Sigma

Aldrich). Specific dsRNA targeting _PxMAP4K4_ (dsPxMAP4K4) was prepared as described in the RNAi section. The detection time of hormone-induced effects on gene expression and the quantities

of hormones injected was optimized in preliminary microinjection experiments (Supplementary Fig. 8). Subsequently, newly molted third-instar DBM1Ac-S larvae were starved for 6 h and

anesthetized for 30 min on ice, 70 nl solutions containing 20E (2 ng), methoprene (200 ng), mixture of 20E (2 ng), and methoprene (200 ng) or the hormone mixture with 100 μM MAPK inhibitor

cocktail and dsRNA combined with hormone treatments were respectively microinjected into some of these larvae using the Nanoliter 2000 microinjection system, whereas the remaining control

larvae were injected with the equal volumes of buffer (diluted ethanol and DMSO), with the final concentration of ethanol not exceeding 0.05%. More than 30 larvae were microinjected for each

treatment, and these injected larvae were allowed to recover for about 3 h at room temperature and were then returned to normal rearing conditions, the final survivors from each control and

treatment groups were used for the subsequent midgut dissection, qPCR analysis, and Western blots as described above. Meanwhile, leaf-dip bioassays were performed for 72 h using larvae at

48 h after hormone, MAPK inhibitors or dsRNA injection and Cry1Ac protoxin concentrations representing approximately the LC50 (1 mg/L) or LC20 (0.3 mg/L) value for non-injected DBM1Ac-S

larvae. Bioassays were performed with 20 larvae in each group, and each bioassay replicated three times. Mortality in control treatments was below 5% and bioassay data processing was as

described above. TOXIN INDUCTION ASSAYS Toxin induction of insect hormones was conducted using newly molted third-instar DBM1Ac-S and NIL-R larvae26. The DBM1Ac-S or NIL-R larvae were

selected with 1 or 3500 mg/L Cry1Ac protoxin (respective LC50 values for each strain) for 72 h as for the leaf-dip bioassay mentioned above, unselected larvae from both strains were used as

control groups. After 72 h, sample preparation from both robust and weak survivors to detect hormone titers was undertaken as described above. Three independent assays were performed, and

one-way ANOVA with Holm–Sidak’s test (overall significance level = 0.05) was used to determine the significant statistical difference between control and treatment groups. FITNESS COST

ANALYSIS The effects of exogenous hormone and Cry1Ac protoxin treatments on subsequent developmental stages of surviving larvae to determine potential fitness costs were analyzed by

comparing biological parameters including pupation percentage, pupal weight, pupation duration, eclosion percentage, adult longevity, oviposition duration, female fecundity, and egg

hatchability. Larvae injected with buffer and unselected with toxin were used as negative controls in the hormone treatment and toxin induction experiments, respectively. All the larvae used

in the test were fed on fresh cabbage leaves. Each treatment was replicated three times with 10 larvae per replicate. One-way ANOVA with Holm–Sidak’s test (overall significance level = 

0.05) was used to assess statistical significance of differences in these biological parameters related to fitness costs between control and treated groups. REPORTING SUMMARY Further

information on research design is available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The full-length cDNA sequences of all the cloned genes in this

study have been deposited in the GenBank database (Accession Nos. MG873047–MG873063 and MH213067–MH213068). The authors declare that the data supporting the findings of this study are

available within the paper and its Supplementary Information. The source data underlying Figs. 1c, d, 2b–f, 3, 4, 5b–f, 6, 7b–i, k–r, and 8 and Supplementary Figs. 1a, 2, 3a, b, 4, 6, and 8

are provided as a Source Data file. REFERENCES * Stork, N. E. How many species of insects and other terrestrial arthropods are there on Earth? _Annu. Rev. Entomol._ 63, 31–45 (2018). Article

  CAS  PubMed  Google Scholar  * Deutsch, C. A. et al. Increase in crop losses to insect pests in a warming climate. _Science_ 361, 916–919 (2018). Article  ADS  CAS  PubMed  Google Scholar

  * Sanahuja, G., Banakar, R., Twyman, R. M., Capell, T. & Christou, P. _Bacillus thuringiensis_: a century of research, development and commercial applications. _Plant Biotechnol. J._

9, 283–300 (2011). Article  CAS  PubMed  Google Scholar  * Bravo, A., Likitvivatanavong, S., Gill, S. S. & Soberón, M. _Bacillus thuringiensis_: a story of a successful bioinsecticide.

_Insect Biochem. Mol. Biol._ 41, 423–431 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * ISAAA. _Global Status of Commercialized Biotech/GM Crops in 2018: Biotech Crops

Continue to Help Meet the Challenges of Increased Population and Climate Change_. ISAAA Brief No. 54 (ISAAA, Ithaca, NY, 2018). * Wu, K. M., Lu, Y. H., Feng, H. Q., Jiang, Y. Y. & Zhao,

J. Z. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton. _Science_ 321, 1676–1678 (2008). Article  ADS  CAS  PubMed  Google Scholar  *

Hutchison, W. D. et al. Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers. _Science_ 330, 222–225 (2010). Article  ADS  CAS  PubMed  Google

Scholar  * Lu, Y., Wu, K., Jiang, Y., Guo, Y. & Desneux, N. Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. _Nature_ 487, 362–365 (2012). Article

  ADS  CAS  PubMed  Google Scholar  * Carrière, Y., Crickmore, N. & Tabashnik, B. E. Optimizing pyramided transgenic Bt crops for sustainable pest management. _Nat. Biotechnol._ 33,

161–168 (2015). Article  PubMed  CAS  Google Scholar  * Dively, G. P. et al. Regional pest suppression associated with widespread Bt maize adoption benefits vegetable growers. _Proc. Natl.

Acad. Sci. U.S.A._ 115, 3320–3325 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tabashnik, B. E., Brévault, T. & Carrière, Y. Insect resistance to Bt crops: lessons

from the first billion acres. _Nat. Biotechnol._ 31, 510–521 (2013). Article  CAS  PubMed  Google Scholar  * Tabashnik, B. E. & Carrière, Y. Surge in insect resistance to transgenic

crops and prospects for sustainability. _Nat. Biotechnol._ 35, 926–935 (2017). Article  CAS  PubMed  Google Scholar  * de Bortoli, C. P. & Jurat-Fuentes, J. L. Mechanisms of resistance

to commercially relevant entomopathogenic bacteria. _Curr. Opin. Insect Sci._ 33, 56–62 (2019). Article  PubMed  Google Scholar  * Xiao, Y. & Wu, K. Recent progress on the interaction

between insects and _Bacillus thuringiensis_ crops. _Philos. Trans. R. Soc. B_ 374, 20180316 (2019). Article  CAS  Google Scholar  * Tabashnik, B. E. et al. Efficacy of genetically modified

Bt toxins against insects with different genetic mechanisms of resistance. _Nat. Biotechnol._ 29, 1128–1131 (2011). Article  CAS  PubMed  Google Scholar  * Gould, F., Brown, Z. S. &

Kuzma, J. Wicked evolution: can we address the sociobiological dilemma of pesticide resistance? _Science_ 360, 728–732 (2018). Article  CAS  PubMed  Google Scholar  * Badran, A. H. et al.

Continuous evolution of _Bacillus thuringiensis_ toxins overcomes insect resistance. _Nature_ 533, 58–63 (2016). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Nielsen-LeRoux,

C., Gaudriault, S., Ramarao, N., Lereclus, D. & Givaudan, A. How the insect pathogen bacteria _Bacillus thuringiensis_ and Xenorhabdus/Photorhabdus occupy their hosts. _Curr. Opin.

Microbiol._ 15, 220–231 (2012). Article  PubMed  Google Scholar  * Pardo-López, L., Soberón, M. & Bravo, A. _Bacillus thuringiensis_ insecticidal three-domain Cry toxins: mode of action,

insect resistance and consequences for crop protection. _FEMS Microbiol. Rev._ 37, 3–22 (2013). Article  PubMed  CAS  Google Scholar  * Adang, M. J., Crickmore, N. & Jurat-Fuentes, J.

L. Diversity of _Bacillus thuringiensis_ crystal toxins and mechanism of action. _Adv. Insect Physiol._ 47, 39–87 (2014). Article  Google Scholar  * Wu, Y. Detection and mechanisms of

resistance evolved in insects to Cry toxins from _Bacillus thuringiensis_. _Adv. Insect Physiol._ 47, 297–342 (2014). Article  Google Scholar  * Furlong, M. J., Wright, D. J. & Dosdall,

L. M. Diamondback moth ecology and management: problems, progress and prospects. _Annu. Rev. Entomol._ 58, 517–541 (2013). Article  CAS  PubMed  Google Scholar  * Tabashnik, B. E., Cushing,

N. L., Finson, N. & Johnson, M. W. Field development of resistance to _Bacillus thuringiensis_ in diamondback moth (Lepidoptera: Plutellidae). _J. Econ. Entomol._ 83, 1671–1676 (1990).

Article  Google Scholar  * You, M. et al. A heterozygous moth genome provides insights into herbivory and detoxification. _Nat. Genet._ 45, 220–225 (2013). Article  CAS  PubMed  Google

Scholar  * Baxter, S. W. et al. Parallel evolution of _Bacillus thuringiensis_ toxin resistance in Lepidoptera. _Genetics_ 189, 675–679 (2011). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Guo, Z. et al. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to _Bacillus thuringiensis_ Cry1Ac toxin in diamondback moth. _PLoS

Genet._ 11, e1005124 (2015). Article  PubMed  PubMed Central  CAS  Google Scholar  * Guo, Z. et al. Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac

resistance in the diamondback moth, _Plutella xylostella_ (L.). _Insect Biochem. Mol. Biol._ 59, 30–40 (2015). Article  CAS  PubMed  Google Scholar  * Zhou, J. et al. Reduced expression of

the P-glycoprotein gene PxABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin in _Plutella xylostella_ (L.). _Pest Manag. Sci._ 76, 712–720 (2020). Article  CAS  PubMed 

Google Scholar  * Crickmore, N. _Bacillus thuringiensis_ resistance in Plutella—too many trees? _Curr. Opin. Insect Sci._ 15, 84–88 (2016). Article  PubMed  Google Scholar  * Pigott, C. R.

& Ellar, D. J. Role of receptors in _Bacillus thuringiensis_ crystal toxin activity. _Microbiol. Mol. Biol. Rev._ 71, 255–281 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar

  * Albiston, A. L., Ye, S. Y. & Chai, S. Y. Membrane bound members of the M1 family: more than aminopeptidases. _Protein Pept. Lett._ 11, 491–500 (2004). Article  CAS  PubMed  Google

Scholar  * Xie, W. et al. Tissue-specific transcriptome profiling of _Plutella xylostella_ third instar larval midgut. Int. _J. Biol. Sci._ 8, 1142–1155 (2012). Google Scholar  * Lei, Y. et

al. Midgut transcriptome response to a Cry toxin in the diamondback moth, _Plutella xylostella_ (Lepidoptera: Plutellidae). _Gene_ 533, 180–187 (2014). Article  CAS  PubMed  Google Scholar 

* Jindra, M., Palli, S. R. & Riddiford, L. M. The juvenile hormone signaling pathway in insect development. _Annu. Rev. Entomol._ 58, 181–204 (2013). Article  CAS  PubMed  Google Scholar

  * Yamanaka, N., Rewitz, K. F. & O’Connor, M. B. Ecdysone control of developmental transitions: lessons from Drosophila research. _Annu. Rev. Entomol._ 58, 497–516 (2013). Article  CAS

  PubMed  Google Scholar  * Roy, S., Saha, T. T., Zou, Z. & Raikhel, A. S. Regulatory pathways controlling female insect reproduction. _Annu. Rev. Entomol._ 63, 489–511 (2018). Article 

CAS  PubMed  Google Scholar  * Liu, S. et al. Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila. _Proc.

Natl. Acad. Sci. U.S.A._ 115, 139–144 (2018). Article  CAS  PubMed  Google Scholar  * Flatt, T., Tu, M. P. & Tatar, M. Hormonal pleiotropy and the juvenile hormone regulation of

Drosophila development and life history. _BioEssays_ 27, 999–1010 (2005). Article  CAS  PubMed  Google Scholar  * Schwenke, R. A., Lazzaro, B. P. & Wolfner, M. F. Reproduction-immunity

trade-offs in insects. _Annu. Rev. Entomol._ 61, 239–256 (2016). Article  CAS  PubMed  Google Scholar  * Wheeler, D. E. & Nijhout, H. F. A perspective for understanding the modes of

juvenile hormone action as a lipid signaling system. _BioEssays_ 25, 994–1001 (2003). Article  CAS  PubMed  Google Scholar  * Ningshen, T. J., Chaitanya, R. K., Hari, P. P., Vimala Devi, P.

S. & Dutta-Gupta, A. Characterization and regulation of _Bacillus thuringiensis_ Cry toxin binding aminopeptidases N (APNs) from non-gut visceral tissues, Malpighian tubule and salivary

gland: comparison with midgut-specific APN in the moth _Achaea janata_. _Comp. Biochem. Physiol._ 166B, 194–202 (2013). Article  CAS  Google Scholar  * Baxter, S. W., Zhao, J. Z., Shelton,

A. M., Vogel, H. & Heckel, D. G. Genetic mapping of Bt-toxin binding proteins in a Cry1A-toxin resistant strain of diamondback moth _Plutella xylostella_. _Insect Biochem. Mol. Biol._

38, 125–135 (2008). Article  CAS  PubMed  Google Scholar  * Bretschneider, A., Heckel, D. G. & Pauchet, Y. Three toxins, two receptors, one mechanism: mode of action of Cry1A toxins from

_Bacillus thuringiensis_ in _Heliothis virescens_. _Insect Biochem. Mol. Biol._ 76, 109–117 (2016). Article  CAS  PubMed  Google Scholar  * Gassmann, A. J., Carrière, Y. & Tabashnik, B.

E. Fitness costs of insect resistance to _Bacillus thuringiensis_. _Annu. Rev. Entomol._ 54, 147–163 (2009). Article  CAS  PubMed  Google Scholar  * Rahman, M. M., Roberts, H. L. S.,

Sarjan, M., Asgari, S. & Schmidt, O. Induction and transmission of _Bacillus thuringiensis_ tolerance in the flour moth _Ephestia kuehniella_. _Proc. Natl. Acad. Sci. U.S.A._ 101,

2696–2699 (2004). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * d’Alençon, E. et al. Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high

rates of local genome rearrangements. _Proc. Natl. Acad. Sci. U.S.A._ 107, 7680–7685 (2010). Article  ADS  PubMed  PubMed Central  Google Scholar  * Vachon, V., Laprade, R. & Schwartz,

J. L. Current models of the mode of action of _Bacillus thuringiensis_ insecticidal crystal proteins: a critical review. _J. Invertebr. Pathol._ 111, 1–12 (2012). Article  CAS  PubMed 

Google Scholar  * Li, X., Schuler, M. A. & Berenbaum, M. R. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. _Annu. Rev. Entomol._ 52, 231–253 (2007).

Article  PubMed  CAS  Google Scholar  * Mathers, T. C. et al. Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant

species. _Genome Biol._ 18, 27 (2017). Article  PubMed  PubMed Central  CAS  Google Scholar  * Tiewsiri, K. & Wang, P. Differential alteration of two aminopeptidases N associated with

resistance to _Bacillus thuringiensis_ toxin Cry1Ac in cabbage looper. _Proc. Natl. Acad. Sci. U.S.A._ 108, 14037–14042 (2011). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  *

Zhu, X. et al. Lack of fitness costs and inheritance of resistance to _Bacillus thuringiensis_ Cry1Ac toxin in a near-isogenic strain of _Plutella xylostella_ (Lepidoptera: Plutellidae).

_Pest Manag. Sci._ 72, 289–297 (2016). Article  CAS  PubMed  Google Scholar  * Cory, J. S. Evolution of host resistance to insect pathogens. _Curr. Opin. Insect Sci._ 21, 54–59 (2017).

Article  PubMed  Google Scholar  * Pieterse, C. M. J., Van der Does, D., Zamioudis, C., Leon-Reyes, A. & Van Wees, S. C. M. Hormonal modulation of plant immunity. _Annu. Rev. Cell Dev.

Biol._ 28, 489–521 (2012). Article  CAS  PubMed  Google Scholar  * Denancé, N., Sánchez-Vallet, A., Goffner, D. & Molina, A. Disease resistance or growth: the role of plant hormones in

balancing immune responses and fitness costs. _Front. Plant Sci._ 4, 155 (2013). Article  PubMed  PubMed Central  Google Scholar  * Huot, B., Yao, J., Montgomery, B. L. & He, S. Y.

Growth-defense tradeoffs in plants: a balancing act to optimize fitness. _Mol. Plant_ 7, 1267–1287 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ning, Y., Liu, W. &

Wang, G. L. Balancing immunity and yield in crop plants. _Trends Plant Sci._ 22, 1069–1079 (2017). Article  CAS  PubMed  Google Scholar  * Martin, L. B., Weil, Z. M. & Nelson, R. J.

Seasonal changes in vertebrate immune activity: mediation by physiological trade-offs. _Philos. Trans. R. Soc. B_ 363, 321–339 (2008). Article  Google Scholar  * Klein, S. L. & Roberts,

C. W. _Sex Hormones and Immunity to Infection_ 1–315 (Springer, Heidelberg, 2010). * Gruntenko, N. E., Bownes, M., Terashima, J., Sukhanova, M. Z. & Raushenbach, I. Y. Heat stress

affects oogenesis differently in wild-type _Drosophila virilis_ and a mutant with altered juvenile hormone and 20-hydroxyecdysone levels. _Insect Mol. Biol._ 12, 393–404 (2003). Article  CAS

  PubMed  Google Scholar  * Gruntenko, N. E. & Rauschenbach, I. Y. Interplay of JH, 20E and biogenic amines under normal and stress conditions and its effect on reproduction. _J. Insect

Physiol._ 54, 902–908 (2008). Article  CAS  PubMed  Google Scholar  * Liu, A. et al. Characterization of immune-related PGRP gene expression and phenoloxidase activity in Cry1Ac-susceptible

and -resistant _Plutella xylostella_ (L.). _Pestic. Biochem. Physiol._ 160, 79–86 (2019). Article  CAS  PubMed  Google Scholar  * Zhang, W. N. et al. Reproductive cost associated with

juvenile hormone in Bt-resistant strains of _Helicoverpa armigera_ (Lepidoptera: Noctuidae). _J. Econ. Entomol._ 109, 2534–2542 (2016). Article  CAS  PubMed  Google Scholar  * Rahman, M.,

Glatz, R., Roush, R. & Schmidt, O. Developmental penalties associated with inducible tolerance in _Helicoverpa armigera_ to insecticidal toxins from _Bacillus thuringiensis_. _Appl.

Environ. Microbiol._ 77, 1443–1448 (2011). Article  CAS  PubMed  Google Scholar  * Tetreau, G. Interaction between insects, toxins, and bacteria: have we been wrong so far? _Toxins_ 10, 281

(2018). Article  PubMed Central  CAS  Google Scholar  * Guo, Z. et al. The midgut cadherin-like gene is not associated with resistance to _Bacillus thuringiensis_ toxin Cry1Ac in _Plutella

xylostella_ (L.). _J. Invertebr. Pathol._ 126, 21–30 (2015). Article  CAS  PubMed  Google Scholar  * Zhu, X. et al. Construction and characterisation of near-isogenic _Plutella xylostella_

(Lepidoptera: Plutellidae) strains resistant to Cry1Ac toxin. _Pest Manag. Sci._ 71, 225–233 (2015). Article  CAS  PubMed  Google Scholar  * Guo, Z. et al. Comprehensive analysis of Cry1Ac

protoxin activation mediated by midgut proteases in susceptible and resistant _Plutella xylostella_ (L.). _Pestic. Biochem. Physiol._ 163, 23–30 (2020). Article  CAS  PubMed  Google Scholar

  * Sun, D., Guo, Z., Liu, Y. & Zhang, Y. Progress and prospects of CRISPR/Cas systems in insects and other arthropods. _Front. Physiol._ 8, 608 (2017). Article  PubMed  PubMed Central 

Google Scholar  * Guo, Z. et al. CRISPR/Cas9-mediated knockout of both the PxABCC2 and PxABCC3 genes confers high-level resistance to _Bacillus thuringiensis_ Cry1Ac toxin in the diamondback

moth, _Plutella xylostella_ (L.). _Insect Biochem. Mol. Biol._ 107, 31–38 (2019). Article  CAS  PubMed  Google Scholar  * Guo, Z., Qin, J., Zhou, X. & Zhang, Y. Insect transcription

factors: a landscape of their structures and biological functions in Drosophila and beyond. _Int. J. Mol. Sci._ 19, 3691 (2018). Article  PubMed Central  CAS  Google Scholar  Download

references ACKNOWLEDGEMENTS We thank Prof. Kaiyu Liu from the Central China Normal University for providing the pie2-GFP-N1 expression vector, and we also thank Prof. Xia Cui and Dr. Haijing

Wang from our institute for the assistance with the UPLC–MS/MS experiment. This work was supported by the National Natural Science Foundation of China (31630059 and 31701813), the Central

Public-interest Scientific Institution Basal Research Fund (IVF-BRF2020015), the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables, and the Science and

Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS). AUTHOR INFORMATION Author notes * These authors contributed equally: Zhaojiang Guo, Shi

Kang, Dan Sun. AUTHORS AND AFFILIATIONS * Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China Zhaojiang Guo,

 Shi Kang, Dan Sun, Lijun Gong, Junlei Zhou, Jianying Qin, Le Guo, Liuhong Zhu, Yang Bai, Fan Ye, Qingjun Wu, Shaoli Wang & Youjun Zhang * School of Life Sciences, University of Sussex,

Brighton, BN1 9QE, UK Neil Crickmore * Department of Entomology, University of Kentucky, Lexington, KY, 40546-0091, USA Xuguo Zhou Authors * Zhaojiang Guo View author publications You can

also search for this author inPubMed Google Scholar * Shi Kang View author publications You can also search for this author inPubMed Google Scholar * Dan Sun View author publications You can

also search for this author inPubMed Google Scholar * Lijun Gong View author publications You can also search for this author inPubMed Google Scholar * Junlei Zhou View author publications

You can also search for this author inPubMed Google Scholar * Jianying Qin View author publications You can also search for this author inPubMed Google Scholar * Le Guo View author

publications You can also search for this author inPubMed Google Scholar * Liuhong Zhu View author publications You can also search for this author inPubMed Google Scholar * Yang Bai View

author publications You can also search for this author inPubMed Google Scholar * Fan Ye View author publications You can also search for this author inPubMed Google Scholar * Qingjun Wu

View author publications You can also search for this author inPubMed Google Scholar * Shaoli Wang View author publications You can also search for this author inPubMed Google Scholar * Neil

Crickmore View author publications You can also search for this author inPubMed Google Scholar * Xuguo Zhou View author publications You can also search for this author inPubMed Google

Scholar * Youjun Zhang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Z.G., X.Z., N.C., and Y.Z. designed the research. Z.G., S.K., D.S.,

L. Gong, J.Z., J.Q., L. Guo, L.Z., Y.B., F.Y., Q.W., and S.W. performed the experiments. Z.G. analyzed the data. Z.G., N.C., X.Z., and Y.Z. wrote and revised the manuscript. CORRESPONDING

AUTHORS Correspondence to Zhaojiang Guo or Youjun Zhang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION

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CITE THIS ARTICLE Guo, Z., Kang, S., Sun, D. _et al._ MAPK-dependent hormonal signaling plasticity contributes to overcoming _Bacillus thuringiensis_ toxin action in an insect host. _Nat

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