A Toll-Spätzle Pathway in the Immune Response of Bombyx mori

Simple Summary The Toll-Spätzle pathway is a crucial defense mechanism in insect innate immunity through inducing the expression of antimicrobial peptides (AMPs) against pathogens. As an important economical insect, Bombyx mori (B. mori) has been used as an insect model of Lepidoptera. However, the recognition of the Toll-Spätzle pathway in silkworm is very limited. In this study, we reported that B. mori Spätzle2 (BmSpz2) could be activated by pathogens. The activated BmSpz2 could bind with B. mori Toll11 (BmToll11) or B. mori Toll9-1 (BmToll9–1). Further investigations showed that overexpression of BmToll11 or BmToll9–1 could significantly upregulate AMPs expression. The findings of this study confirmed that a Toll-Spz pathway regulated immune response to microbial infection in B. mori and may help improve the understanding of the signaling pathways involved in the innate immune responses of Lepidoptera insect. Abstract The Toll-Spätzle pathway is a crucial defense mechanism in insect innate immunity, it plays an important role in fighting against pathogens through the regulation of antimicrobial peptide gene expression. Although Toll and Spätzle (Spz) genes have been identified in Bombyx mori, little is known regarding the specific Spz and Toll genes members involved in innate immunity. There is also limited direct evidence of the interaction between Spz and Toll. In this study, the dual-luciferase reporter assay results showed that BmToll11 and BmToll9–1 could activate both drosomycin and diptericin promoters in S2 cells. Furthermore, BmToll11, BmToll9–1, and five BmSpzs genes were found to be significantly upregulated in B. mori infected by Escherichia coli and Staphylococcus aureus. Additionally, the yeast two-hybrid assay results confirmed that BmSpz2, but not other BmSpzs, could interact with both BmToll11 and BmToll9–1. These findings suggest that the activated BmSpz2 can bind with BmToll11 and BmToll9–1 to induce the expression of AMPs after the silkworm is infected by pathogens.


Introduction
Innate immunity works as an evolutionarily conserved system, which provides the first defense line in animals against a wide variety of pathogens [1]. Antimicrobial peptides (AMPs) are an important family of innate immunity effectors in insects, they are involved in the three main signaling transduction pathways-Toll, JAK/STAT, and immune deficiency (IMD) [2]. The critical step of innate immune responses in the Toll signaling pathway is that pathogen-associated molecular patterns (PAMPs) are Insects 2020, 11, 586 3 of 13 silkworms injected with 0.85% sterilized NaCl were used as controls. The fat body was harvested at 3, 6, 12, and 24 h after injection. The collected samples were stored at −80 • C and later used to total RNA extraction.

RNA Isolation and cDNA Synthesis
Total RNA was extracted using a total RNA extraction kit (OMEGA, Doraville, GA, USA), and the contaminating genomic DNA was digested with RNase-free DNase I (Takara, Tokyo, Japan) for 15 min at 37 • C. Then, total RNA was used to reverse transcribe the first-strand cDNA using a commercial kit (Promega, Madison, WI, USA). The cDNA samples were stored at −80 • C.

Gene Cloning
All 14 BmToll gene sequences were obtained from the NCBI GenBank (https://www.ncbi.nlm.nih. gov/genbank), SilkDB (https://silkdb.bioinfotoolkits.net/main/species-info/-1) and KAIKObase (http: //sgp.dna.affrc.go.jp/KAIKObase/) databases. The TIR domains, signal peptides, and transmembrane regions were predicted by SMART [43] (Table S1). All 14 BmTIRs were amplified by polymerase chain reaction (PCR) from cDNA samples and cloned into the pMT/BiP/V5-His A vector (V413020, Invitrogen, Waltham, MA, USA) for dual-luciferase reporter assays. Forward and reverse primers were listed in Table S2. PCR reactions were performed with the following conditions: 94 • C for 3 min, 35 cycles of 94 • C for 30 s, Tm−5 • C for 30 s and 72 • C for 1 min, then a final extension at 72 • C for 10 min. The PCR products were purified using a gel purification kit (OMEGA, Doraville, GA, USA). The purified PCR products were first inserted into the pMD-19T vector (Takara, Tokyo, Japan). Recombinant plasmids were purified using a Pure Yield™ Plasmid Miniprep System (A1222, Promega, Madison, WI, USA) and digested with respective restriction enzymes. The purified DNA fragments were inserted into the pMT/BiP/V5-His A vector using T4 DNA ligase (M0202L, NEB, Beverly, MA, USA). Positive clones were sequenced by the Sangon Company (Shanghai, China) for further experiments.
The construction of recombinant plasmids for the study of BmToll11 and Toll9-1 involved in the immune response and yeast two-hybrid assays was performed as described above. For the analysis of immune responses, BmTIR11, BmTIR9-1, BmSpz2 (the active domain), and DsRed (as a control) genes were inserted into the pIZT/V5-His vector (V8010, Invitrogen, Waltham, MA, USA). For the yeast two-hybrid assay, BmToll ecto 11 and BmToll ecto 9-1 were cloned into the pGBKT7 vector (Takara, Tokyo, Japan), while the active domain of BmSpzs was cloned into the pGADT7 vector (Takara, Tokyo, Japan). The mature active C-terminal domain of BmSpzs was cleaved by pro-Spätzle (proSpz), and the predicted cleavage sites were listed in Figure S2. The forward and reverse primers were listed in Table S2.

Immune Response Analysis
For immune response analysis, the B. mori cell line BmN-SWU1 was plated in 6-well culture plates (10 5 cells/well) and incubated overnight in serum-free medium. Then, BmN-SWU1 cells were transiently transfected with the recombinant expression plasmids pIZT/BmTIR11, pIZT/BmTIR9-1 and pIZT/BmSpz2 (2 µg), while cells cotransfected with pIZT/DsRed served as the control. After 72 h, the cells were collected for RNA isolation and cDNA synthesis, as described above.
For RNAi, the B. mori cell line BmN-SWU1 was plated in 6-well culture plates (10 5 cells/well) and incubated overnight in serum-free medium. Then, BmN-SWU1 cells were transfected with the interference fragments of BmToll11, BmToll9-1, BmSpz2, or EGFP (3 µg), while cells were challenged by inactivated E. coli or S. aureus (PBS buffer were used as a control). After 48 h, the cells were collected for RNA isolation and cDNA synthesis, as described above.  Table S3. The relative gene expression levels were estimated according to the 2 −∆∆Ct method [45]. All samples were run in triplicate.

Yeast Two-Hybrid Assay
Briefly, BmTolls and BmSpzs genes were fused to the yeast two-hybrid vectors pGADT7 and pGBKT7. Yeast competent cells were transformed simultaneously with the bait and prey constructs pGBKT7-BmTolls/pGADT7-BmSpzs. Yeast cells were plated on synthetic defined premix (SD) agar base plates that did not contain leucine (Leu) or tryptophan (Trp). Then, the positive clones containing the constructs pGADT7-prey/pGBKT7-bait were grown on SD plates in the absence of Leu, Trp, histidine (His), and adenine (Ade) but the presence of 5-bromo-4-chloro-3-indoxyl-D-gal (X-α-Gal).

Statistical Analysis
One representative set of data was used to generate figures with GraphPad Prism software (GraphPad Prism 8). All statistical analyses were conducted using SPSS software (IBM SPSS v. 22), and cumulative survival was analyzed by Tukey's multiple comparison test. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01. All results are shown as means ± SD of triplicate samples. All data presented are representative of a minimum of three independent experiments.

Overexpression of Recombinant BmTIRs in S2 Cells Activates Drosomycin and Diptericin Reporter Genes
The function of the Toll-Spz signaling pathway in regulating innate immunity in D. melanogaster is well understood. However, in other insect species, it is not well studied. In B. mori, Toll and Spz genes have been identified [30,31,39], but few were known that the specific Tolls and Spzs implicated in the innate immune system. To explore the Toll-Spz pathway in B. mori, we constructed recombinant vectors and expressed the TIR of Toll receptors (BmTIRs) in the S2 cell line. Immunoblotting results showed that recombinant BmTIR proteins were detected in S2 cells ( Figure 1A,B).
The function of the Toll-Spz signaling pathway in regulating innate immunity in D. melanogaster is well understood. However, in other insect species, it is not well studied. In B. mori, Toll and Spz genes have been identified [30,31,39], but few were known that the specific Tolls and Spzs implicated in the innate immune system. To explore the Toll-Spz pathway in B. mori, we constructed recombinant vectors and expressed the TIR of Toll receptors (BmTIRs) in the S2 cell line. Immunoblotting results showed that recombinant BmTIR proteins were detected in S2 cells ( Figure  1A,B).
We next cotransfected the different combination vectors of BmTIR proteins with the vector containing the D. melanogaster drosomycin or diptericin promoter in S2 cells, then the activity of drosomycin or diptericin promoter was measured using dual-luciferase reporter assays. The results showed that BmTIR9-1 overexpression significantly improved the levels of the relative luciferase activity (RLA) of the drosomycin reporter. In addition, the overexpression of BmTIR11 remarkably increased the RLA of drosomycin and diptericin reporters ( Figure 1C). These results showed that BmTIR11 and BmTIR9-1 could activate drosomycin and diptericin genes in S2 cells, which suggested that they may be involved in B. mori AMP gene regulation. The cells were cotransfected with recombinant BmTIR plasmids and luciferase plasmids with or without different promoter fragments (pGL3B, pGL3B-drosomycin or pGL3Bdiptericin) for 48 h, and then the relative luciferase activity (RLA) was measured by dual luciferase assay. The statistical analysis used Tukey's multiple comparison test. Bars represent the mean of three individual measurements ± SD. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01. The cells were cotransfected with recombinant BmTIR plasmids and luciferase plasmids with or without different promoter fragments (pGL3B, pGL3B-drosomycin or pGL3B-diptericin) for 48 h, and then the relative luciferase activity (RLA) was measured by dual luciferase assay. The statistical analysis used Tukey's multiple comparison test. Bars represent the mean of three individual measurements ± SD. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01. We next cotransfected the different combination vectors of BmTIR proteins with the vector containing the D. melanogaster drosomycin or diptericin promoter in S2 cells, then the activity of drosomycin or diptericin promoter was measured using dual-luciferase reporter assays. The results showed that BmTIR9-1 overexpression significantly improved the levels of the relative luciferase activity (RLA) of the drosomycin reporter. In addition, the overexpression of BmTIR11 gene remarkably increased the RLA of drosomycin and diptericin reporters ( Figure 1C). These results showed that BmTIR11 and BmTIR9-1 could activate drosomycin and diptericin genes in S2 cells, which suggested that they may be involved in B. mori AMP gene regulation.

BmToll11 and BmToll9-1 are Involved in the Immune Response
To investigate whether B. mori Toll11 and Toll9-1 genes were involved in the immune response in silkworms, the mRNA expression levels of B. mori AMP genes were analyzed by RT-qPCR in BmN-SWU1 cells overexpressing BmTIR11 or BmTIR9-1. First of all, overexpression levels of BmTIR11 or BmTIR9-1 genes were tested, which results in a significant up-regulation in BmN-SWU1 cells overexpressing BmTIRs ( Figure S3A). Cecropin A1 and gloverin-like protein 3 genes were upregulated in BmN-SWU1 cells overexpressing BmTIR11 (Figure 2A). Gloverin-like protein 3 and lysozyme genes were upregulated in BmN-SWU1 cells overexpressing BmTIR9-1 ( Figure 2B). Then, the expression levels of B. mori AMP genes were also analyzed in BmN-SWU1 cells knocking down BmTIR11 or BmTIR9-1 genes. The expressions of AMPs were inhibited in BmToll11 or BmToll9-1 gene knockdown cells ( Figure  S4A,B). These results showed that BmToll11 and BmToll9-1 could activate the immune response in the BmN-SWU1 cell line. Furthermore, an experiment was performed using silkworms infected with E. coli (Gram-negative bacteria) or S. aureus (Gram-positive bacteria). Then, the transcriptional levels of B. mori Toll11 and Toll9-1 genes were analyzed by RT-qPCR at different times after infection. The RT-qPCR results showed that the Toll11 and Toll9-1 genes of B. mori were activated by microbes at 3 h and 6 h compared with saline water but downregulated after 12 h ( Figure 2C,D). The Myd88 was also activated by the two microbes ( Figure 2E). These results suggested that both Gram-negative and Gram-positive bacteria could induce the expression of BmToll11 and BmToll9-1 genes.  The Toll signaling pathway was activated by Escherichia coli or Staphylococcus aureus. The fifth instar larvae were injected with E. coli or S. aureus, while the 0.85% sterilized NaCl was used as control. The fat body was harvested for the cDNA synthesis at 3, 6, 12, and 24 h after injection. Then BmToll11, BmToll9-1, Myd88, and AMPs were detected by RT-qPCR. For all RT-qPCR, RPL3 was used as an internal control. Bars represent the mean of three individual measurements ± SD. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01, which is determined by one-way ANOVA followed by a Tukey's multiple comparison test.

BmToll ecto 9-1 and BmToll ecto 11 Interact with BmSpz2 but not Other BmSpzs
The mechanism that the activation of the Toll signaling pathway in D. melanogaster and M. sexta is relatively clear. In Drosophila, the Toll pathway is activated after the activated Spz binding with the Toll receptor [6]. We demonstrated that BmToll11 and BmToll9-1 function in innate immune responses in silkworms. However, it is unclear which Spzs may be involved in innate immune responses and interact with BmTolls. The amino acid sequence of Spz contains a cystine knot domain (Spz CK domain) ( Figure S2). Furthermore, phylogenetic analysis was conducted using the CK domain sequence of Spz genes, the results showed that there was a highly conserved monophyly among different insect species ( Figure S1). To identify which of the five BmSpzs may be involved in The total RNAs were prepared after transfected for 72 h, cDNA was synthesized with olig (dT)-primer. Then the AMPs were determined by RT-qPCR. (C-K): The Toll signaling pathway was activated by Escherichia coli or Staphylococcus aureus. The fifth instar larvae were injected with E. coli or S. aureus, while the 0.85% sterilized NaCl was used as control. The fat body was harvested for the cDNA synthesis at 3, 6, 12, and 24 h after injection. Then BmToll11, BmToll9-1, Myd88, and AMPs were detected by RT-qPCR. For all RT-qPCR, RPL3 was used as an internal control. Bars represent the mean of three individual measurements ± SD. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01, which is determined by one-way ANOVA followed by a Tukey's multiple comparison test.
In D. melanogaster, some AMP genes expression was regulated through the Toll pathway activated by fungi or Gram-positive bacteria, such as drosomycins, metchnikowin, and defensins. Another AMP genes expression was regulated by the IMD pathway activated by Gram-negative bacteria, such as drosocin, diptericin, and attacins. Several AMP genes in silkworms, including cecropin A1 (CecA1), lebocin (Leb), defensins (Def), gloverin-like protein 3 (Glo3), lysozyme (Lys), and moricin 1 (Mor) were activated by the two microbes ( Figure 2F-K). These results suggest that both Gram-negative and Gram-positive bacteria could activate the Toll signaling pathways and cause the increased expression of BmToll11, BmToll9-1, and AMPs in silkworms.

BmToll ecto 9-1 and BmToll ecto 11 Interact with BmSpz2 but not Other BmSpzs
The mechanism that the activation of the Toll signaling pathway in D. melanogaster and M. sexta is relatively clear. In Drosophila, the Toll pathway is activated after the activated Spz binding with the Toll receptor [6]. We demonstrated that BmToll11 and BmToll9-1 genes function in innate immune responses in silkworms. However, it is unclear which Spzs may be involved in innate immune responses and interact with BmTolls. The amino acid sequence of Spz contains a cystine knot domain (Spz CK domain) ( Figure S2). Furthermore, phylogenetic analysis was conducted using the CK domain sequence of Spz genes, the results showed that there was a highly conserved monophyly among different insect species ( Figure S1). To identify which of the five BmSpzs genes may be involved in innate immune responses, RT-qPCR was performed. The results showed that all five BmSpzs genes were upregulated after the injection of inactivated bacteria for 6 h (Figure 3).  RPL3 was used as an internal control. Bars represent the mean of three individual measurements ± SD. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01, which is determined by one-way ANOVA followed by a Tukey's multiple comparison test.
To determine which Spzs interact with Toll11 and Toll9-1, a yeast two-hybrid analysis was conducted. The results confirmed that BmSpz2 could interact with both BmToll ecto 11 and BmToll ecto 9-1, whereas other BmSpzs failed to interact with them ( Figure 4). Furthermore, the mRNA expression levels of B. mori AMP genes were analyzed by RT-qPCR in BmN-SWU1 cells overexpressing BmSpz2. Cecropin A1 and gloverin-like protein 3 genes were upregulated in BmN-SWU1 cells overexpressing BmSpz2 ( Figure 3F). The expression levels of B. mori AMP genes were also analyzed in BmSpz2 knocking down BmN-SWU1 cells. The expressions of AMPs were inhibited in BmSpz2 knocking down cells ( Figure S4C). These results suggested that BmToll11 and BmToll9-1 could bind to BmSpz2 and activate the expression of AMPs in B. mori. The AMPs were detected by RT-qPCR in BmSpz2 overexpressed cells. RPL3 was used as an internal control. Bars represent the mean of three individual measurements ± SD. Significant differences are indicated with * for p < 0.05 and ** for p < 0.01, which is determined by one-way ANOVA followed by a Tukey's multiple comparison test.
To determine which Spzs interact with Toll11 and Toll9-1, a yeast two-hybrid analysis was conducted. The results confirmed that BmSpz2 could interact with both BmToll ecto 11 and BmToll ecto 9-1, whereas other BmSpzs failed to interact with them ( Figure 4). Furthermore, the mRNA expression levels of B. mori AMP genes were analyzed by RT-qPCR in BmN-SWU1 cells overexpressing BmSpz2. Cecropin A1 and gloverin-like protein 3 genes were upregulated in BmN-SWU1 cells overexpressing BmSpz2 ( Figure 3F). The expression levels of B. mori AMP genes were also analyzed in BmSpz2 knocking down BmN-SWU1 cells. The expressions of AMPs were inhibited in BmSpz2 gene knocking down cells ( Figure S4C). These results suggested that BmToll11 and BmToll9-1 could bind to BmSpz2 and activate the expression of AMPs in B. mori.

Discussion
In insects, the Toll-Spätzle pathway works as an evolutionarily conserved system, which plays a key role in their innate immunity against pathogens. In D. melanogaster, Toll1 and Spz1 were implicated in regulating AMP genes and other immune-related functions [9,46]. In B. mori, BmSpz1, BmSpz4 and BmToll9-1 have been confirmed to activate the Toll signaling pathway and function in innate immunity [37,38,40,41]. In addition to Spz1, Spz4, and BmToll9-1, whether the other Tolls and Spzs can activate innate immunity pathways are unknown. The results of our dual-luciferase reporter assays showed that BmTIR11 and BmTIR9-1 were strongly elicited AMP genes ( Figure 1C). The findings also confirmed that BmToll11 and BmToll9-1 were upregulated when silkworms were challenged with E. coli or S. aureus ( Figure 2C,D) and that they could activate the transcription of AMP genes (Figure 2). In vivo experiments showed that the transcriptional expression levels of five Spzs were all increased in the silkworm (Figure 3). These results showed that BmToll9-1, BmToll11, and BmSpz1-5 were involved in the innate immunity in silkworms.
Although the interactions between Tolls and Spzs were well studied in D. melanogaster and M. sexta [6,33,46], it remains unclear whether the Tolls can participate in the innate immune response in the silkworm through binding with BmSpz1 or other Spzs. The yeast two-hybrid assay results showed that BmToll11 and BmToll9-1 could interact with BmSpz2 and activate the expression of AMP genes (Figures 2 and 4). Additionally, the results indicated that one BmSpz could combine with multiple BmTolls, which is consistent with the reported binding of Spzs to multiple Tolls in D. melanogaster [6]. In our study, we also found that other Tolls, except for BmToll9-1 and BmToll11, Yeast two-hybrid assay for determination of BmToll ecto 11 and BmToll ecto 9-1 with BmSpz2. Yeast cells were transformed simultaneously with the bait (pGBKT7-BmToll ecto s) and prey constructs (pGADT7-BmSpzs). On the up line, yeast cells containing the bait and prey constructs were plated on synthetic defined premix (SD) agar base plates that did not contain leucine (Leu) or tryptophan (Trp). Howere, on the down line, only the clones containing the constructs pGBKT7-53/pGADT7-T (PC), pGADT7-Toll11 ecto /pGBKT7-Spz2 and pGADT7-Toll9-1 ecto /pGBKT7-Spz2 could grow on SD plates in the absence of Leu, Trp, histidine (His), and adenine (Ade). These clones also formed blue colonies in the presence of 5-bromo-4-chloro-3-indoxyl-D-gal (X-α-Gal). (B): The table summarized the information given above A. BmToll ecto 11 and BmToll ecto 9-1 interact with BmSpz2, while other BmSpzs were not. Positive control pGBKT7-53/pGADT7-T (PC) and negative control pGBKT7-lam/pGADT7-T (NC) reactions were provided for each group.

Discussion
In insects, the Toll-Spätzle pathway works as an evolutionarily conserved system, which plays a key role in their innate immunity against pathogens. In D. melanogaster, Toll1 and Spz1 were implicated in regulating AMP genes and other immune-related functions [9,46]. In B. mori, BmSpz1, BmSpz4 and BmToll9-1 have been confirmed to activate the Toll signaling pathway and function in innate immunity [37,38,40,41]. In addition to Spz1, Spz4, and BmToll9-1, whether the other Tolls and Spzs can activate innate immunity pathways are unknown. The results of our dual-luciferase reporter assays showed that BmTIR11 and BmTIR9-1 were strongly elicited AMP genes ( Figure 1C). The findings also confirmed that BmToll11 and BmToll9-1 genes were upregulated when silkworms were challenged with E. coli or S. aureus ( Figure 2C,D) and that they could activate the transcription of AMP genes (Figure 2). In vivo experiments showed that the transcriptional expression levels of five Spzs were all increased in the silkworm (Figure 3). These results showed that BmToll9-1, BmToll11, and BmSpz1-5 were involved in the innate immunity in silkworms.
Although the interactions between Tolls and Spzs were well studied in D. melanogaster and M. sexta [6,33,46], it remains unclear whether the Tolls can participate in the innate immune response in the silkworm through binding with BmSpz1 or other Spzs. The yeast two-hybrid assay results showed that BmToll11 and BmToll9-1 could interact with BmSpz2 and activate the expression of AMP genes (Figures 2 and 4). Additionally, the results indicated that one BmSpz could combine with multiple BmTolls, which is consistent with the reported binding of Spzs to multiple Tolls in D. melanogaster [6]. In our study, we also found that other Tolls, except for BmToll9-1 and BmToll11, participated in immune regulation, including BmToll 1, BmToll 3, BmToll 5, and others ( Figure 1B). Five BmSpzs genes were also upregulated when the silkworm was challenged by two microbes (Figure 3). Therefore, we speculate that except for BmSpz2, the other Spzs can also bind to other BmTolls. Furthermore, it might be possible that multiple BmSpzs can bind to one or more BmTolls. However, further studies are required to confirm these predictions.
Insects lack an acquired immune system and mainly rely on innate immunity to resist pathogens infection. AMPs play an important defense role in insect innate immunity that are regulated by many signals, such as the Toll and IMD signal transduction pathways [3,7,47,48]. In D. melanogaster, seven kinds of AMPs are synthesized. Some of the AMPs genes expression was regulated by the Toll pathway activated by fungi or Gram-positive bacteria, such as drosomycins, metchnikowin, and defensins, whereas another AMPs expression regulated by the IMD pathway activated by Gram-negative bacteria, such as cecropins, drosocin, diptericin, and attacins. In M. sexta, AMP genes, including attacin, cecropin, lysozyme gloverin, lebocin, and moricin have been identified [49,50]. Among these genes, attacin and cecropin are widely found in most insect species, whereas lebocin, moricin, and gloverin were conserved in Lepidoptera species [49,[51][52][53]. These genes can be activated by the host infected by different bacterial [54]. The overexpression of MsTIR and DmTIR in S2 cells can activate drosomycin (a target gene of the Toll pathway) but not diptericin (a target gene of the IMD pathway) [33]. At present, the members of B. mori AMPs can be classified into six different families, including cecropin, attacin, moricin, gloverin, lebocin, and defensin [35,51,[55][56][57][58][59]. The activation of AMPs is different depending on the pathogen used to challenge silkworms [38,41,60]. Our studies showed that the Toll pathways were both activated by Gram-positive and Gram-negative bacteria in B. mori (Figures 1 and 2), unlike in M. sexta or D. melanogaster [6,33], which is consistent with a recent study in Tenebrio molitor [61]. In addition, the activation of AMP genes was not absolutely specific following the challenge with E. coli or S. aureus (Figure 2). Our results indicated that the activation of the Toll receptor signaling pathway and the regulation of AMP gene expression are different between B. mori and D. melanogaster.

Conclusions
In summary, B. mori infected by pathogens could activate BmSpz2 to bind with BmToll11 and BmToll9-1, and then induce the expression of AMPs. In this study, we identified a Toll-Spz pathway involved in the immune response to bacteria in B. mori. The findings of this study may help improve the understanding of the signaling pathways involved in Lepidoptera insect innate immune responses.