Bacillus thuringiensis (Bt), a class of spore-forming gram-positive bacterium that can produce different insecticidal crystal proteins (Cry and Cyt toxins), has been widely used as an entomopathogen for pest control (Wu et al., 2008; Raymond et al., 2010; Palma et al., 2014). The application of Bt toxins cannot only increase crop yields and bring substantial economic benefits, but also reduce environmental pollution caused by the abuse of chemical insecticides (Bravo et al., 2011). Bt Cry toxin has specific control efficiency on lepidopteran pests, and the mode of action involves toxin solubilization, proteolytic activation, interaction with midgut proteins of insects, formation of a pre-pore oligomeric structure, facilitation of the insertion into cell membrane, and creation of an ionic pore that kills midgut cells (Bravo et al., 2011). There are several resistance strategies taken by insects to counter Bt toxins, including mutations in receptor [e.g., cadherin, aminopeptidase N (APN) or ATP-binding cassette (ABC) transporters], alteration in Cry toxin activation and binding ability to gut membrane, sequestration of toxins by glycolipid moieties or esterases, and the elevation of insects immune responses (Pardo-Lopez et al., 2013). So far, more than 22 cases of field-evolved Bt resistance in several insect species have been documented, potentially reducing the control efficacy of Bt toxins toward agricultural pests (Janmaat and Myers, 2003; Tabashnik and Carrière, 2017, 2019; Calles-Torrez et al., 2019). Therefore, to delay the resistance evolution, it is urgent to understand the mechanisms causing Bt resistance and provide novel targets for pest control.

The diamondback moth (DBM), Plutella xylostella, a notorious pest of cruciferous crops, has caused severe economic losses globally. One of the main challenges of DBM control is its rapid evolution of resistance against a wide range of insecticides, including Bt-based products (Furlong et al., 2013). Considering its severe insecticide-resistance, short life cycle and host range expansion (Li et al., 2016), microRNA (miRNA)-mediated efficient and environmentally friendly approaches have been proposed to combat DBM (Vaschetto and Beccacece, 2019).

MiRNAs are a class of endogenous non-coding RNAs ranged from 19 to 24 nt in length and play crucial roles in the post-transcriptional regulation (Bartel, 2004; Cullen, 2004; Asgari, 2013). MiRNAs regulate gene expression by partially or completely binding their seed sequence region with the 3′ untranslated region (3′UTR) of corresponding target genes (Bartel, 2004). A large number of miRNAs have been identified from multiple insect species, participating in insect development (Liu et al., 2018, 2020b; Xu et al., 2019; Lim et al., 2020), reproduction (Lucas et al., 2015; He et al., 2016; Ling et al., 2017), behavior (Cristino et al., 2014; Chen and Rosbash, 2016; Niu et al., 2019), and host-pathogen interaction (Singh et al., 2014; Dubey et al., 2019; Wu et al., 2019). Recently, a growing body of evidence has illustrated the pivotal role of miRNAs in responses to environmental chemical and pathogen exposures, which attracts great interests in the field of insect toxicology. Insecticide resistance is predominantly caused by the rising metabolic detoxification of insecticides and declining in the target sites sensitivity of the insecticides (Liu, 2015). MiRNAs enhance insecticide resistance by negatively regulating the expression of detoxification-related genes or altering insect susceptibility to various kinds of insecticides. In Culex pipiens pallens, the pyrethroid-resistance is modulated by miR-92a via suppressing CpCPR4 expression (Ma et al., 2017), miR-278-3p and miR-932 are also involved in the pyrethroid resistance by targeting CYP6AG11 and CpCPR5, respectively (Lei et al., 2014; Liu et al., 2016). In addition, it has been reported that miR-13664 could interact with CpCYP314A1 and subsequently regulate deltamethrin resistance (Sun et al., 2019). Another study confirms the significant role of miR-2∼13–71 cluster in deltamethrin resistance by regulating the expression of CYP9J35 and CYP325BG3 (Guo et al., 2017). In DBM, miR-7a and miR-8519 regulate chlorantraniliprole resistance via overexpressing ryanodine receptor (RyR) (Li X. et al., 2015). MiR-2b-3p and miR-14-5p are proposed to inhibit the detoxification pathways by suppressing the transcript levels of CYP9F2 and CYP307a1, respectively (Etebari et al., 2018). MiR-189942 directly targets the ecdysone receptor (EcR) isoform B and increased the tolerance to fufenozide (Li X. et al., 2020). Besides, miR-998-3p is implicated in Bt resistance through targeting ABCC2, a putative receptor for Bt Cry1Ac toxin in three lepidopterans P. xylostella, Helicoverpa armigera, and Spodoptera exigua (Zhu et al., 2019). While these studies provide insight into the critical roles of miRNAs in regulating metabolic resistance to insecticides, most of them are focused on the chemical insecticide. The mechanism of miRNA-mediated Bt resistance still remains limited.

A previous study has profiled the dynamic expression patterns of miRNAs in response to Bt at different time courses using the whole body of DBM, indicating the potential role of miR-2b-3p in regulating insect immunity (Li S. et al., 2019). In the present study, we aimed to expand the numbers of identified miRNAs in specific tissues, i.e., midguts of the Bt resistant and susceptible DBM stains and explore the potential mechanisms of miRNA-mediated regulation in Bt resistance. The differentially expressed miRNAs in midguts of both strains were putatively linked to Bt resistance. One of the differentially expressed miRNAs, novel-miR-240, which showed an inverse correlation with its target genes, was then selected for dual luciferase assays to validate the interaction with its target genes. Our work is important for further understanding the potential roles of miRNAs in the evolution of Bt resistance in DBM field populations, providing novel insights for their putative applications in integrated pest management.

MiRNAs, a class of endogenous non-coding RNAs, are considered to be modulators of gene expression at the post-transcriptional level (Lee et al., 2004). In recent years, with the development of the in silico miRNA identification platform such as mirMachine (Cagirici et al., 2021), miRanda (Rehmsmeier et al., 2004) and RNAhybrid (Mohebbi et al., 2021), many miRNAs have been characterized from multiple organisms including plants, vertebrates and invertebrates following the integration of various tools in bioinformatics, genetics, molecular biology and biochemistry. In insects, miRNAs played indispensable roles in development (Shen et al., 2020), behavior (Niu et al., 2019), immunity (Li R. et al., 2019), host-virus interaction (Singh, 2020), and resistance to multiple insecticides (Zhu et al., 2019; Li X. et al., 2020). In addition, the high-quality genome offers the opportunity to explore the putative biological function of identified miRNAs (Robertson et al., 2018). DBM is a notorious agriculture pest, understanding the roles played by miRNAs in insecticides resistance can provide a significant theoretical basis for finding novel targets for pest control.

Here, we successfully constructed sRNA libraries from Bt resistant and susceptible strains, and identified a total of 437 miRNAs including 76 known and 361 novel miRNAs belonging to 91 miRNA families. Importantly, 12 differentially expressed miRNAs (three up and nine down) were revealed in the Cry1S1000 strain compared to the G88 strain. We also predicted the putative target genes for each of these 12 differentially expressed miRNAs, and the target genes were enriched in 43 GO terms based on GO and KEGG pathway analyses. Furthermore, the expression patterns of these differentially expressed miRNAs and their potential target genes were investigated with RT-qPCR. Finally, the dual luciferase assay was carried out to confirm the interaction between novel-miR-240 and its target genes Px017590 and Px007885.

From the identified miRNAs, a member of the miR-279 family, miR-279b-3p, was listed as the most abundant miRNA in our sRNA libraries. Previously, it has been found that miR-279, another member of the miR-279 family, was also listed as an abundantly expressed miRNA in DBM larvae after destruxin A injection and predicted to regulate the immunity-related genes (Shakeel et al., 2018). MiR-306, a common abundantly expressed miRNA identified in our study, has been shown to associated with Bt Cry1Ab resistance in Ostrinia furnacallis (Xu et al., 2015).

Additionally, a total of 78 known and novel miRNAs were classified into 91 miRNA families. MiRNAs sharing the same seed region were classified as a miRNA family, which likely indicated that they targeted the same genes and performed similar biological functions. In the current study, novel-miR-133 and novel-miR-250 were classified into the let-7 family, which is functionally conserved from insects to humans and participated in multiple biological processes. In insects, modulating the abundance of let-7 altered the tolerance of Myzus persicae nicotianae to nicotine (Peng et al., 2016). Let-7 was also required in many developmental processes, such as sleep homeostasis (Goodwin et al., 2018), age-dependent behavioral changes (Behura and Whitfield, 2010), developmental transitions of egg hatching, molting, pupation, adult eclosion (Zhang et al., 2020) and the regulation of ecdysone levels (Liu et al., 2007).

Novel-miR-93 and novel-miR-125 were classified into the miR-14 family, which is conserved in insects and is closely related to olfactory and chemoreception (Shan et al., 2020). Another study showed that miR-14-3p, another member of the miR-14 family, could tightly control the ecdysone signaling pathway by regulating EcR and E75 (Luo et al., 2020). A total of five miRNAs (novel-miR-142, novel-miR-214, novel-miR-219, novel-miR-222, and novel-miR-253) identified in our study belong to the miR-2 family. The conserved miR-2 family was an invertebrate-specific family of miRNAs and involved in insect metamorphosis (Lozano et al., 2015), oogenesis (Song et al., 2019) neural development (Marco et al., 2012), as well as deltamethrin resistance in Cx. pipiens pallens (Guo et al., 2017).

Pxy-miR-252 belonged to the miR-252 family. It has been found that miR-252 could directly repress the mbt expression to control the developmental growth of Drosophila (Lim et al., 2019) and was also involved in dengue virus replication in Aedes albopictus (Yan et al., 2014). Pxy-miR-274 was classified into the miR-274 family. Inhibition of miR-274-3p could facilitate B. mori cytoplasmic polyhedrosis virus (BmCPV) replication (Wu et al., 2017). Novel-miR-78 was classified into the miR-276 family. The miR-276 family might be involved in the spirotetramat resistance of Aphis gossypii Glover (Wei et al., 2016) and the dengue virus (DENV) replication in C6/36 cells (Su et al., 2019). Pxy-miR-277 belonged to the miR-277 family, which was conserved in insects and participated in multiple physiological processes such as restoring immune homeostasis of the IMD pathway (Li R. et al., 2020), controlling metamorphosis (Shen et al., 2020), lipid metabolism and reproduction (Ling et al., 2017). MiR-277-3p was also a part of the molecular toolkit regulating reproductive diapause in Culex pipiens (Meuti et al., 2018) and Helicoverpa zea (Reynolds et al., 2019). Hence, we speculate that the pxy-miR-277 might be invloved in diverse biological processes in DBM and indirectly affect detoxification of Bt Cry toxin. Pxy-miR-279a, pxy-279b-5p, and pxy-279-3p were classified to the miR-279 family. It has been reported that miR-279 was essential to maintain circadian rhythm (Sun et al., 2015) and reproductive responses by females to male sex peptide in D. melanogaster (Fricke et al., 2014). So, pxy-miR-279a, pxy-279b-5p, and pxy-279-3p might play the similar roles with miR-279 in insects. Pxy-miR-306 and pxy-miR-308 belonged to miR-306 and miR-308 families, respectively. MiR-279, miR-306, and miR-308 were also predicted to be involved in the Destruxin A-responsive immunity process in DBM (Shakeel et al., 2018), indicating that these miRNA families might be closely associated with Bt resistance. Novel-miR-21 was classified into the miR-317 family, and miR-317 was found to be involved in the process of pupation in B. dorsalis (Zhang et al., 2020). In Drosophila, miR-87 was an important regulator of dendrite regeneration (Kitatani et al., 2020), so we speculated that novel-miR-199 as a member of the miR-87 family, might paly the similar roles in DBM. In summary, these miRNA families identified in the current study were widely involved in diverse biological processes in insects, some of the miRNA families also took part in insect immunity. Investigating the functions of these conserved miRNA families and their representative members will provide a better understanding of miRNA-mediate post-transcriptional regulation in insects.

It has been previously shown that the differentially expressed miRNAs among the susceptible and resistant strains might be related to insecticides resistance in DBM. For instance, the expression of miR-276-5p and miR-8530-5p were 2–3 times higher in the chlorantraniliprole resistant DBM strain compared to the susceptible strain, indicating that these two miRNAs likely participated in the response of DBM to chlorantraniliprole (Zhu et al., 2017). The expression level of miR-278-3p was 4.2 fold lower in the deltamethrin-resistant strain (DR-strain), which induced the pyrethroid resistance in Cx. pipiens pallens (Lei et al., 2014). The other two conserved miRNAs, miR-932 and miR-285 were estimated to be 1.8- and 2.8-fold higher, respectively, in the DR-strain and were involved in pyrethroid resistance (Liu et al., 2016; Tian et al., 2016). In this study, the expression levels of novel-miR-240, novel-miR-270 and novel-miR-116 in the resistant Cry1S1000 strain were significantly lower than that in the susceptible G88 strain, other miRNAs such as novel-miR-210, novel-miR-274, novel-miR-288, novel-miR-25, and pxy-miR-8522 represented significantly higher expression levels in the resistant Cry1S1000 strain, and novel-miR-48, novel-miR-97, novel-miR-237, and novel-miR-225 exhibited no significant difference between these two strains. Moreover, the differentially expressed novel-miR-240, novel-miR-270, and novel-miR-116 did not belong to any known miRNA family, indicating that there was no miRNA shared the same seed regions or derived from the same arm of the stem-loop with these two miRNAs (Meyers et al., 2008). While novel-miR-270 had a similar sequence with ame-miR-9864-5p, suggesting that they may be the same miRNA in different species and play similar roles in insects. Our results suggested that the expression of miRNAs varies among different strains, and these differentially expressed miRNAs may regulate detoxification to Bt Cry1Ac toxin in a complicated manner by targeting detoxification-related genes.

It should be noted that novel-miR-240 represents down-regulated expression in the Cry1S1000 strain relative to the G88 strain, whereas its target genes Px017590 and Px007885 were up-regulated in the Cry1S1000 strain. The DLR assay results implied the involvement of novel-miR-240 in the regulation of Px017590 and Px007885, indicating that this miRNA might modulate Bt resistance of DBM via negatively regulating its target genes.

Furthermore, GO annotation exhibited that most of the predicted target genes of differentially expressed miRNAs were enriched in biological process (cellular process, metabolism process, single-organism process, and biological regulation), cellular component (membrane, cell, cell part, and membrane part) and molecular function (binding and catalytic activity), which might be related to the insect immunity and the metabolism to the Bt Cry1Ac toxin. The target genes of differentially expressed miRNAs were mostly enriched in metabolic-related pathways such as Hippo signaling pathway, minoacyl-tRNA biosynthesis and MAPK signaling pathway. And in a previous study, MAPK signaling pathway was found to lead to Bt Cry1Ac resistance by mediating differential expression of APN and other midgut genes in DBM (Guo et al., 2020). The DBM-DB database was used for the annotation of target genes. Px015953 and Px000596, the target genes of novel-miR-25, were predicted to be associated with chitinase and cuticle protein, which may be involved in the process of DBM combating the exogenous Bt Cry1Ac toxin.

Chitinase, an insect molting enzyme, is a potential biopesticide that degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. Previous research has shown that chitinases could degrade the vital structures of insects such as their peritrophic membranes and cuticles, thus facilitating the entry of the pathogens into the tissues of susceptible insects (Brandt et al., 1978). Chitinases also facilitated the penetration of host cuticle by entomopathogenic fungi. Analysis of the top 20 differentially expressed genes showed that the chitinase ChiA2 was up-regulated after Beauveria bassiana infected non-natural hosts such as H. armigera and Clanis bilineata (Liu et al., 2021). These chitinase-related genes indicated that when entomopathogenic fungi transfer to non-original hosts, the fungi changed the metabolic response of hosts and used the novel infection strategies to break the barrier of different cuticle chitin components to better adaption to new hosts. Therefore, the chitin based bioformulation may act as an effective pesticide against Lepidopterous pests. A prior work revealed that the expression of cuticle proteins were siginificantly induced at 6 h post-treatment of Cry1Ac toxin (Qin et al., 2021). Genes encoding cuticle proteins were induced during B. mori bidensovirus (BmBDV) infection (Sun et al., 2020). The down-regulation of some Cardinium-responsive miRNAs enchanced the cuticle proteins in whitefly Bemisia tabaci (Gennadius) biotype Q and modulated insect reproduction and growth (Li et al., 2018). In addition, RNA-seq of Cryptolestes ferrugineus individuals from phosphine susceptible and resistant populations revealed the significantly increased expression levels of nine cuticular protein genes in the resistance population, demonstrating that the cuticular protein genes might be related to phosphine resistance (Chen et al., 2021). In the current study, the target genes of identified differentially expressed miRNAs, especially the chitinase and cuticle protein, might involved in the insects immune response and the detoxification of xenobiotics including the Bt Cry1Ac toxin. Although the RT-qPCR results showed that some of the identified differentially expressed miRNAs exhibited positively correlate with their potential targets, the target genes of these differentially expressed miRNAs were still worthy for further investigation, where functional characterizations of the target genes will provide novel insights of insects immunity and detoxification mechanisms.

In the present study, we identified 76 known and 361 novel miRNAs from midguts of the Bt resistant and susceptible DBM strains. Among the identified miRNAs, 178 were classified into 91 miRNA families, and the potential functions of these miRNA families have also been illustrated. Expression of 12 differentially expressed miRNAs and their potetial target genes were profiled, revealing that these miRNAs may play vital roles in detoxicating Bt Cry1Ac toxins by regulating their corresponding target genes. Moreover, the interaction between novel-miR-240 and its target genes Px017590 and Px007885 have been confirmed by the DLR assay. However, further experiments are still required to demonstrate the functional pathways which the differentially expressed miRNAs and their target genes are involved in. Our study contributes to the field for further investigation of the miRNA-mediated gene regulation at the post-transcriptional level in insect resistance and the RNAi-based pest management.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI SRA; PRJNA754494.

MX and MY conceived and designed the study. JY performed the experiments and analyzed the data with the help of GL, SC, and JB. JY wrote the first draft of the manuscript. XX, SL, MX, MY, and QS participated in manuscript drafted and modification. All authors reviewed and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.