Dual roles of α1,4-galactosyltransferase 1 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the development of Drosophila melanogaster; however, their role in spermatogenesis is still unclear. In this study, we found that α1,4-galactosyltransferase 1 (α4GT1) was expressed at a significantly higher level in the testis than in the ovary of Drosophila. Importantly, the hatching rate was significantly decreased when α4GT1 RNA interference (RNAi) males were crossed with w1118 females, with only a few mature sperm being present in the seminal vesicle of α4GT1 RNAi flies. Immunofluorescence staining further revealed that the individualization complex (IC) in the testes from α4GT1 RNAi flies was scattered and did not move synchronically, compared with the clustered IC observed in the control flies. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay showed that apoptosis signals in the sperm bundles of α4GT1 RNAi flies were significantly increased. Moreover, the expression of several individualization-related genes, such as Shrub, Obp44a and Hanabi, was significantly decreased, whereas the expression of several apoptosis-related genes, including Dronc and Drice, was significantly increased in the testes of α4GT1 RNAi flies. Together, these results suggest that α4GT1 may play dual roles in Drosophila spermatogenesis by regulating the sperm individualization process and maintaining the survival of sperm bundles.

Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival.

Macroautophagy/autophagy is a conserved process in eukaryotic cells to degrade and recycle damaged intracellular components. Higher level of autophagy in the brain has been observed, and autophagy dysfunction has an impact on neuronal health, but the molecular mechanism is unclear. In this study, we showed that overexpression of Toll-1 and Toll-7 receptors, as well as active Spätzle proteins in Drosophila S2 cells enhanced autophagy, and Toll-1/Toll-7 activated autophagy was dependent on Tube-Pelle-PP2A. Interestingly, Toll-1 but not Toll-7 mediated autophagy was dMyd88 dependent. Importantly, we observed that loss of functions in Toll-1 and Toll-7 receptors and PP2A activity in flies decreased autophagy level, resulting in the loss of dopamine (DA) neurons and reduced fly motion. Our results indicated that proper activation of Toll-1 and Toll-7 pathways and PP2A activity in the brain are necessary to sustain autophagy level for DA neuron survival.

Expression profiles of lncRNAs, miRNAs, and mRNAs and interaction analysis indicate their potential involvement during testicular fusion in Spodoptera litura.

Testicular fusion of Spodoptera litura occures during metamorphosis, which benefits sperms development. Previous research identified involvement of ECM-integrin interaction pathways, MMPs in testicular fusion, but the regulatory mechanism remains unclear. RNA-seq was performed to analyze long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in testes, aiming to uncover potential regulatory mechanisms of testicular fusion. 2150 lncRNAs, 2742 targeted mRNAs, and 347 miRNAs were identified in testes at three different developmental stages. Up-regulated DElncRNAs and DEmRNAs, as well as down-regulated DEmiRNAs, were observed during testicular fusion, while the opposite expression pattern was observed after fusion. Enrichment analysis of DEmRNAs revealed that cAMP signal pathway, ECM remodeling enzymes, ECM-integrin interaction pathways, and cell adhesion molecules were potentially associated with testicular fusion. The identified DElncRNA-DEmiRNA-DEmRNA regulatory network related to cAMP signal pathway, ECM remodeling enzymes suggests their roles during testicular fusion. Our research will provide new targets for studying the mechanism of testicular fusion.

Identification and functional analysis of CG3526 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2 H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.

Eurycomanone (EN) Activates Transcription Factor FoxO by Inhibiting the Insulin Signaling Pathway to Suppress the Development of Spodoptera frugiperda.

The insulin-like signaling (IIS) pathway is essential for insect growth and development. In this study, we showed that eurycomanone (EN) is an active compound with growth inhibitory activity against Spodoptera frugiperda larvae. Experiments in cells and RNA-seq analysis in the midgut showed that EN targeted the IIS pathway in S. frugiperda to activate the transcription factor SfFoxO (S. frugiperda forkhead boxO) to regulate mRNA levels associated with nutrient catabolism. Additionally, mass spectrometry imaging revealed that EN was distributed in the larval gut and enriched in the inner membrane of the gut. Immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed that EN induced program cell death (PCD) in the larvae midgut. Thus, EN targeted the insulin receptor to inhibit the IIS signaling pathway, exerting inhibitory activity on the growth and development of S. frugiperda larvae. Our results suggest that EN has great potential as a botanical pesticide, and the IIS signaling pathway may be an effective target for botanical pesticides.

Transcriptome-wide analysis of mRNA N6 -methyladenosine modification in the embryonic development of Spodoptera frugiperda.

N6 -methyladenosine (m6 A) RNA is the most abundant modification of mRNA, and has been demonstrated in regulating various post-transcriptional processes. Many studies have shown that m6 A methylation plays key roles in sex determination, neuronal functions, and embryonic development in Drosophila and mammals. Here, we analyzed transcriptome-wide profile of m6 A modification in the embryonic development of the destructive agricultural pest Spodoptera frugiperda. We found that the 2 key mRNA m6 A methyltransferases SfrMETTL3 and SfrMETTL14 have high homologies with other insects and mammals, suggesting that SfrMETTL3 and SfrMETTL14 may have conserved function among different species. From methylated RNA immunoprecipitation sequencing analysis, we obtained 46 869 m6 A peaks representing 8 587 transcripts in the 2-h embryos after oviposition, and 41 389 m6 A peaks representing 9 230 transcripts in the 24-h embryos. In addition, 5 995 m6 A peaks were differentially expressed including 3 752 upregulated and 2243 downregulated peaks. Functional analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes suggested that differentially expressed m6 A peak-modified genes were enriched in cell and organ development between the 2- and 24-h embryos. By conjoint analysis of methylated RNA immunoprecipitation-seq and RNA-seq data, we found that RNA m6 A methylation may regulate the transcriptional levels of genes related to tissue and organ development from 2- to 24-h embryos. Our study reveals the role of RNA m6 A epigenetic regulation in the embryonic development of S. frugiperda, and provides new insights for the embryonic development of insects.

The mechanism of Cry41-related toxin against Myzus persicae based on its interaction with Buchnera-derived ATP-dependent 6-phosphofructokinase.

BACKGROUND: Myzus persicae (Hemiptera: Aphididae) is one of the most notorious pests of many crops worldwide. Most Cry toxins produced by Bacillus thuringiensis show very low toxicity to M. persicae; however, a study showed that Cry41-related toxin had moderate toxic activity against M. persicae. In our previous work, potential Cry41-related toxin-binding proteins in M. persicae were identified, including cathepsin B, calcium-transporting ATPase, and Buchnera-derived ATP-dependent 6-phosphofructokinase (PFKA). Buchnera is an endosymbiont present in almost all aphids and it provides necessary nutrients for aphid growth. This study investigated the role of Buchnera-derived PFKA in Cry41-related toxicity against M. persicae. RESULTS: In this study, recombinant PFKA was expressed and purified, and in vitro assays revealed that PFKA bound to Cry41-related toxin, and Cry41-related toxin at 25 µg ml-1 significantly inhibited the activity of PFKA. In addition, when M. persicae was treated with 30 µg ml-1 of Cry41-related toxin for 24 h, the expression of dnak, a single-copy gene in Buchnera, was significantly decreased, indicating a decrease in the number of Buchnera. CONCLUSION: Our results suggest that Cry41-related toxin interacts with Buchnera-derived PFKA to inhibit its enzymatic activity and likely impair cell viability, resulting in a decrease in the number of Buchnera, and finally leading to M. persicae death. These findings open up new perspectives in our understanding of the mode of action of Cry toxins and are useful in helping improve Cry toxicity for aphid control. © 2023 Society of Chemical Industry.

BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis.

20E-hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E-regulated insect immunity is unknown. Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD-2A is also positively regulated by 20E, and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD-2A in larval metamorphosis, implying that B. mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.

Sublethal exposure to spinetoram impacts life history traits and dengue virus replication in Aedes aegypti.

Insecticides are anthropogenic environmental stressors and also a common stressor for mosquito vectors. However, the use of insecticides is often guided by short-term efficacy, and the sublethal effect on their target or nontarget species has long been ignored. Here, we analyzed how sublethal exposure of the promising vector-control bioinsecticide spinetoram to Aedes aegypti larvae alter adult performance and susceptibility to dengue virus (DENV) infection. We found that the surviving adult mosquitoes were significantly smaller and exhibited weaker blood-feeding capacity than control females, apart from the extended immature development period. In terms of reproductive potential, although the F0 generation produced a similar number of eggs and offspring during the first gonotrophic cycle, the survival rates of the F1 generations were significantly lower as compared to the control group, suggesting transgenerational sublethal effects on the F1 generation. Notably, surviving adult females had higher DENV-2 viral loads than the control group after spinetoram sublethal exposure. Mechanistically, transcriptomic analysis showed that inhibition of oxidative phosphorylation may function in stimulating DENV production in adult Ae. aegypti. In Aag2 cells, significant accumulation of apoptosis, mitochondrial reactive oxygen species production, and DENV-2 replication by spinetoram exposure consistently support our conclusion. Our study highlights the threat of sublethal spinetoram exposure on outbreaks of mosquito-borne viruses.

A dual role of lola in Drosophila ovary development: regulating stem cell niche establishment and repressing apoptosis.

In Drosophila ovary, niche is composed of somatic cells, including terminal filament cells (TFCs), cap cells (CCs) and escort cells (ECs), which provide extrinsic signals to maintain stem cell renewal or initiate cell differentiation. Niche establishment begins in larval stages when terminal filaments (TFs) are formed, but the underlying mechanism for the development of TFs remains largely unknown. Here we report that transcription factor longitudinals lacking (Lola) is essential for ovary morphogenesis. We showed that Lola protein was expressed abundantly in TFCs and CCs, although also in other cells, and lola was required for the establishment of niche during larval stage. Importantly, we found that knockdown expression of lola induced apoptosis in adult ovary, and that lola affected adult ovary morphogenesis by suppressing expression of Regulator of cullins 1b (Roc1b), an apoptosis-related gene that regulates caspase activation during spermatogenesis. These findings significantly expand our understanding of the mechanisms controlling niche establishment and adult oogenesis in Drosophila.

A possible mechanism of Cry7Ab4 protein in delaying pupation of Plutella xylostella larvae.

Cry toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activities against Lepidopteran, Dipteran, and Coleopteran species. In our previous work, we showed that trypsin-digested full-length Cry7Ab4 protoxin did not have insecticidal activity against Plutella xylostella larvae but strongly inhibited their growth. In this paper, we expressed and purified recombinant active Cry7Ab4 toxic core from Escherichia coli for bioassay and identified its binding proteins. Interestingly, Cry7Ab4 toxic core exhibited activity to delay the pupation of P. xylostella larvae. Using protein pull-down assay, several proteins, including basic juvenile hormone-suppressible protein 1-like (BJSP-1), were identified from the midgut juice of P. xylostella larvae as putative Cry7Ab4-binding proteins. We showed that feeding P. xylostella larval Cry7Ab4 toxic core upregulated the level of BJSP-1 mRNA in the hemocytes and fat body and decreased the free juvenile hormone (JH) level in larvae. BJSP-1 interacted with Cry7Ab4 and bound to free JH in vitro. A possible mechanism of Cry7Ab4 in delaying the pupation of P. xylostella larvae was proposed.

Genome-wide identification and characterization of basic helix-loop-helix transcription factors in Spodoptera litura upon pathogen infection.

Basic helix-loop-helix (bHLH) transcription factors play an important role in a wide range of metabolic and developmental processes in eukaryotes, and bHLH proteins also participate in immune responses, especially in plants. However, their roles in insects upon entomopathogen infection are unknown. In this study, 54 bHLH genes in 41 families were identified in a polyphagous pest, Spodoptera litura, including a new bHLH gene in group B, which is specifically present in Lepidoptera and was thus named Lep. The conserved amino acids in the bHLH domain, structural architecture, and chromosomal distribution of bHLH genes in S. litura were analyzed. The bHLH genes in Plutella xylostella and Apis mellifera were also updated, and genome-wide comparison and phylogenetic analysis of bHLH members in 5 holometabolous insects were performed. The expression profiles of S. litura bHLH (SlbHLH) genes in 3 tissues at different developmental stages and their responses to S. litura nucleopolyhedrovirus (SpltNPV), Nomuraea rileyi (Nr), and Bacillus thuringiensis (Bt) infection were investigated. More SlbHLHs in group B were expressed and differentially expressed during pathogen infections, and SlbHLHs tended to be downregulated in the midgut of S. litura larvae after B. thuringiensis treatment. Our study provides an overview of bHLH family members in S. litura and their responses to different pathogens used for pest biocontrol. These findings on bHLH members may contribute to uncovering the mechanism of host-pathogen interaction.

Genome-wide identification and comparative analysis of Cry toxin receptor families in 7 insect species with a focus on Spodoptera litura.

Cadherin, aminopeptidase N (APN) and alkaline phosphatase (ALP) have been characterized as Cry receptors. In this study, comparative genomic analysis of the 3 receptor families was performed in 7 insects. ALPs and APNs are divided into three and eight clades in phylogenetic trees, respectively. ALPs in clade 3 and APNs in clade 1 contain multiple paralogs within each species and most paralogs are located closely in chromosomes. Drosophila melanogaster has expanded APNs in clade 5 and were lowly expressed in midgut. Cadherins are divided into 16 clades; they may diverge before holometabolous insect speciation except for BtR and Cad89D-like clades. Eight insects from different orders containing BtR orthologs are sensitive to Cry1A or Cry3A, while five species without BtR are insensitive to both toxins. Most APNs in clade 1, several ALPs in clade 3, BtR and Cad89D-like genes were highly or moderately expressed in larval midgut of Spodoptera litura and the other six species, and several members in these clades have been identified as Cry receptors. Expressions of putative S. litura Cry receptors in the midgut after exposing to Bt toxins were also analyzed.

Gut Microbiota Dysbiosis Influences Metabolic Homeostasis in Spodoptera frugiperda.

Insect gut microbiota plays important roles in acquiring nutrition, preventing pathogens infection, modulating immune responses, and communicating with environment. Gut microbiota can be affected by external factors such as foods and antibiotics. Spodoptera frugiperda (Lepidoptera: Noctuidae) is an important destructive pest of grain crops worldwide. The function of gut microbiota in S. frugiperda remains to be investigated. In this study, we fed S. frugiperda larvae with artificial diet with antibiotic mixture (penicillin, gentamicin, rifampicin, and streptomycin) to perturb gut microbiota, and then examined the effect of gut microbiota dysbiosis on S. frugiperda gene expression by RNA sequencing. Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were the most dominant phyla in S. frugiperda. We found that the composition and diversity of gut bacterial community were changed in S. frugiperda after antibiotics treatment. Firmicutes was decreased, and abundance of Enterococcus and Weissella genera was dramatically reduced. Transcriptome analysis showed that 1,394 differentially expressed transcripts (DETs) were found between the control and antibiotics-treated group. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that antibiotics-induced dysbiosis affected many biological processes, such as energy production, metabolism, and the autophagy-lysosome signal pathway. Our results indicated that dysbiosis of gut microbiota by antibiotics exposure affects energy and metabolic homeostasis in S. frugiperda, which help better understand the role of gut microbiota in insects.

Toll9 from Bombyx mori functions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

Comparative genomic analysis of C-type lectin-domain genes in seven holometabolous insect species.

C-type lectins (CTLs) recognize various glycoconjugates through carbohydrate recognition domains (CRDs) and they play important roles in immune responses. In this study, comparative genomic analysis of CTLs were performed in 7 holometabolous species. CTL-S1 to S8 and CTL-X1 to X4 orthologous groups existed in the 7 species, while CTL-X5 group with dual-CRD, CTL-S11 group with triple-CRD, CTL-S9 group with a long C-terminus and Lepidopteran specific CTL-S10 group were not conserved. SliCTL-S12 to S14 cluster was only present in Spodoptera litura, and CTL-S genes were expanded on chromosomes 2 L and 2 R in Drosophila melanogaster. Most IMLs were clustered into three groups and the numbers of IMLs vary among species due to gene duplications. D. melanogaster specific CTLs and Lepidopteran IMLs within each of the three groups evolved more rapidly with higher dN/dS ratios. Two CRDs in IMLs clustered into two clades, with conserved Cys4-Cys5 and Cys1-Cys2 bonds in the first and second CRDs, respectively. The CTL-S and CTL-X family members in S. litura were mainly expressed in the fat body of 5th but not 6th instar larvae, and responded differently to S. litura nucleopolyhedrovirus (SpltNPV) and Nomuraea rileyi infection. The transcription levels of SliCTLs that expressed in fat body but not highly expressed in hemocytes were decreased at the middle and late stages of SpltNPV infection, and the mRNA levels of SliCTLs highly or specifically expressed in hemocytes were mainly decreased by SpltlNPV, N. rileyi and Bacillus thuringiensis infection. These results provide valuable information for further exploration of CTL functions in host-pathogen interaction.

The novel lncRNA PTTG3P is downregulated and predicts poor prognosis in non-small cell lung cancer.

INTRODUCTION: Lung cancer is the leading cause of cancer-associated mortality worldwide. Recently, long non-coding RNAs (lncRNAs) have been studied as key regulators in some biological processes. Of note, the molecular mechanism and prognostic value of lncRNAs in non-small cell lung cancer (NSCLC) have largely remained unclear. MATERIAL AND METHODS: In this study, we compared the PTTG3P expression levels between lung cancer and normal lung samples by analyzing 5 public datasets (GSE18842, GSE19804, GSE27262, GSE30219, and GSE19188). Next, pentose phosphate pathway and co-expression networks were constructed to identify key targets of lncRNA PTTG3P. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to explore the potential roles of lncRNA PTTG3P. Moreover, we constructed PTTG3P-mediated ceRNA networks in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). RESULTS: In the present study, our analysis showed that PTTG3P expression was higher in high T stage LUAD and LUSC samples, as well as high N stage NSCLC tissues. Of note, we found that higher PTTG3P expression is correlated with shorter survival time in NSCLC patients by analyzing Kaplan-Meier plotter datasets. We found that PTTG3P was significantly associated with NSCLC cell proliferation regulation by affecting a series of cell cycle related biological processes. CONCLUSIONS: Bioinformatics analysis showed that PTTG3P was associated with NSCLC cell proliferation. These results suggested that PTTG3P could serve as a new therapeutic and prognostic target for NSCLC.

Possible Insecticidal Mechanism of Cry41-Related Toxin against Myzus persicae by Enhancing Cathepsin B Activity.

Cry toxins produced by Bacillus thuringiensis are well known for their high insecticidal activities against Lepidoptera, Diptera, and Coleoptera; however, their activities against Aphididae are very low. Recently, it has been reported that a Cry41-related toxin exhibited moderate activity against the aphid Myzus persicae, and thus, it is highly desirable to uncover its unique mechanism. In this paper, we report that Cathepsin B, calcium-transporting ATPase, and symbiotic bacterial-associated protein ATP-dependent-6-phosphofructokinase were pulled down from the homogenate of M. persicae as unique proteins that possibly bound to Cry41-related toxin. Cathepsin B has been reported to cleave and inactivate antiapoptotic proteins and plays a role in caspase-initiated apoptotic cascades. In this study, Cathepsin B was expressed in Escherichia coli and purified, and in vitro interaction between recombinant Cathepsin B and Cry41-related toxin was demonstrated. Interestingly, we found that addition of Cry41-related toxin obviously enhanced Cathepsin B activity. We propose a model for the mechanism of Cry41-related toxin as follows: Cry41-related toxin enters the aphid cells and enhances Cathepsin B activity, resulting in acceleration of apoptosis of aphid cells.

Immune responses to Bacillus thuringiensis in the midgut of the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella, is the first insect to develop resistance to Bacillus thuringiensis (Bt) in the field. To date, little is known about the molecular mechanism of the interaction between Bt and midgut immunity in P. xylostella. Here, we report immune responses in the P. xylostella midgut to Bt strain Bt8010 using a combined approach of transcriptomics and quantitative proteomics. Many genes in the Toll, IMD, JNK and JAK-STAT pathways and antimicrobial peptide genes were activated at 18 h post-infection. In the prophenoloxidase (PPO) cascade, four serpin genes were activated, and the PPO1 gene was suppressed by Bt8010. Inhibition of the two PPO proteins was observed at 18 h post-infection. Feeding Bt8010-infected larvae recombinant PPOs enhanced their survival. These results revealed that the Toll, IMD, JNK and JAK-STAT pathways were triggered and participated in the immune defence of the midgut against Bt8010, while the PPO cascade was inhibited and played an important role in this process.