Bombyx mori Ecdysone Receptor B1 May Inhibit BmNPV Infection by Triggering Apoptosis

Simple Summary Ecdysone is an essential factor affecting host immunity, in addition to regulating insect metamorphosis. There are two isoforms of ecdysone receptors (EcR) in silkworm, BmEcR-B1, and BmEcR-A. In this study, BmEcR-B1 was found to be more critical than BmEcR-A in responding to BmNPV infection. Further studies confirmed that BmEcR-B1 played an anti-BmNPV role in the participation of 20E, which was associated with 20E-induced apoptosis. This study provides theoretical support for elucidating the resistant mechanism of silkworm to BmNPV and the control of BmNPV disease in sericulture. Abstract Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious threat to sericulture. Nevertheless, no effective control strategy is currently available. The innate immunity of silkworm is critical in the antiviral process. Exploring its molecular mechanism provides theoretical support for the prevention and treatment of BmNPV. Insect hormone receptors play an essential role in regulating host immunity. We found a correlation between Bombyx mori ecdysone receptor B1 (BmEcR-B1) and BmNPV infection, whereas the underlying mechanism remains unclear. In this study, the expression patterns and sequence characteristics of BmEcR-B1 and its isoform, BmEcR-A, were initially analyzed. BmEcR-B1 was found to be more critical than BmEcR-A in silkworm development and responses to BmNPV. Moreover, RNAi and an overexpression in BmN cells showed BmEcR-B1 had antiviral effects in the presence of 20-hydroxyecdysone (20E); Otherwise, it had no antiviral activity. Furthermore, BmEcR-B1 was required for 20E-induced apoptosis, which significantly suppressed virus infection. Finally, feeding 20E had no significant negative impacts on larval growth and the cocoon shell, suggesting the regulation of this pathway has practical value in controlling BmNPV in sericulture. The findings of this study provide important theoretical support for understanding the mechanism of the silkworm innate immune system in response to BmNPV infection.


Introduction
Silkworm is not only a kind of economic insect, but it is also widely used in scientific studies as a model organism of Lepidoptera. Sericulture is constantly threatened by BmNPV, which generates huge economic losses each year [1]. However, there is still no effective way to solve this problem. One essential method to solving the current problem is understanding the antiviral molecular mechanism of silkworms. This will provide a theoretical basis for breeding resistant silkworm strains and solving the viral disease in sericulture [2]. However, the antiviral mechanism of silkworm against BmNPV is quite complex, and the current Insects 2023, 14, 505 3 of 16 hemolymph, and the purified ODV was dissolved in double distilled water (ddH 2 O). The density of ODV was determined by a hemocytometer.

Sample Preparation for Expression Pattern Analysis
To determine the spatio-temporal expression pattern of BmEcR-A and BmEcR-B1, eggs at different stages, tissues on the third day of fifth instar larvae, and the whole body at first, second, third, fourth, fifth, pupa, and adult of p50 were collected. To analyze the immune response of BmEcR-A and BmEcR-B1 to BmNPV, different tissues of YeA and YeB on the third day of fifth instar were collected after being fed with 5 µL of ODV (1.0 × 10 5 OB/mL) for 48 h, including the Malpighian tube, hemolymph, fat body, and midgut. The ddH 2 O treatment was used as a blank control. Thirty larvae or tissues were mixed in each group to minimize individual genetic differences, and each group was repeated three times. All samples were quickly powdered with liquid nitrogen and stored at −80 • C until use.

Bioinformatics Analysis
BmEcR-A (ID: NM_001173377.1), BmEcR-B1 (ID: NM_001173375.1), and their homologs in other species (Tables S1 and S2) were obtained from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/, accessed on 23 October 2022). The functional domain was predicted using the online SMART server (http://smart.embl-heidelberg.de/, accessed on 15 February 2020). The sequences homology of EcR-A and EcR-B1 amino acid sequence in different species were analyzed using DNAMAN 8.0 software. MEGA 7.0 software was used to generate the evolutionary relationship of EcR-A and EcR-B1 in different species using the Neighbor-Joining method with 1000 bootstrap replications and an optimal DNA/protein model of LG + G. The evolutionary distances were computed using the Maximum Composite Likelihood method.

RNA Extraction and the First Strand cDNA Synthesis
The total RNA was extracted using RNAiso Plus (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. RNA precipitate was dissolved in RNase-free ddH 2 O after cleaning with 75% ethanol. The concentration and purity of RNA were measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, New York, NY, USA). The integrity of RNA was determined by 1% agarose gel denaturation electrophoresis. The qualified RNA was stored at −80 • C until use. The cDNA was generated using the PrimeScript TM RT Reagent Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China; with gDNA Eraser) following the manufacturer's instructions. The qualified cDNA was stored at −20 • C.

Real-Time Quantitative PCR (RT-qPCR)
The relative expression levels of genes were determined using RT-qPCR. Table 1 shows all primers that were generated on the NCBI website. A total of 10 µL of RT-qPCR reaction system consisted of 5.0 µL of 2 × NovoStart ® SYBR qPCR SuperMix Plus (Novoprotein, Suzhou, China), 0.5 µL of upstream and downstream primers, 1.0 µL of template, and 3.0 µL of ddH 2 O. The reaction was performed on the LightCycler ® 96 system (Roche, Basel, Switzerland) using pre-denaturation at 95 • C for 30 s, followed by 40 cycles at 95 • C for 20 s and 60 • C for 60 s. The raw data were processed with the LightCyler 96 SW 1.1 software (Roche, Basel, Switzerland). All samples were replicated three times, and the relative expression levels were calculated using the 2 −∆∆CT method. The optimal reference gene of Bombyx mori glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) was selected based on a previous report [19]. To knock down the expression of BmEcR-B1 in BmN cells, two targets located in the function domain of BmEcR-B1 were designed on the online website (https://www. genscript.com/tools/sirna-target-finder accessed on 23 October 2022). siRNAs targeting BmEcR-B1 functional domain are named siEcR-B1. siRNA targeting red fluorescent protein (RFP) is named siRFP. The siRNA used in this study was short RNA of 19 bp. Table 2 shows primers with target DNA sequences placed behind the T7 promoter. The siRNA was synthesized using oligo primers by the In Vitro Transcription T7 Kit (for siRNA synthesis, TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. Briefly, Olig-1 to 4 were used to synthesize one siRNA product. Olig-1/2 and Olig-3/4 were used to synthesize the forward and reverse templates required to transcribe siRNA, and the two strands were annealed separately and then hybridized. The quality of siRNA was confirmed by 3% agarose gel electrophoresis. The purity and concentration were determined by the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, New York, NY, USA). The qualified siRNA was stored at −80 • C until use. Table 2. The list of primer sequences used to synthesize siRNA.

Primer Names Sequences (5 -3 )
The transfection complex was prepared using a Neofect TM DNA transfection reagent (NEOFECT, Beijing, China) according to the manufacturer's instructions. Briefly, 4 µg of each siRNA was gently mixed with 200 µL of serum-free TC-100 medium, followed by 4 µL of Neofect TM Transfection reagent that were thoroughly mixed with 200 µL of serum-free TC-100 medium and then maintained at room temperature for 30 min. The transfection complex was then applied to the prepared BmN cells and cultured at 28 • C, and the effect of siRNA on target genes was determined after 24 h of transfection using RT-qPCR.

Construction of the Overexpression Vector
The functional domain of BmEcR-B1 was amplified with the primes BmEcR-B1-OP (Table 1; the underlined portions indicate the EcoRI and XbaI restriction sites, respectively). The purified PCR products were cloned into the pMD19-T vector for sequencing. The confirmed pMD-19T-BmEcR-B1 and the pIZT/V5-His-mCherry were digested with EcoRI and XbaI and then ligated for 12 h at 16 • C using T4 DNA ligase (TaKaRa Biotechnology Co., Ltd., Dalian, China). The recombinant plasmid pIZT/V5-His-mCherry-BmEcR-B1 was validated by the two restriction enzymes and then sequenced at Sangong Biotech (Shanghai, China) to ensure its accuracy. The original vector of pIZT/V5-His-mCherry was set as a negative control. Transfection of overexpression vectors is the same as for siRNA.

The Treatment of 20E on Silkworm Larvae
Five series concentrations of 20E dissolved in ddH 2 O were used to identify the optimal dose for activating BmEcR-B1, including 0, 13, 45, 130, 260, and 390 µg/mL. To detect the effect of 20E on BmEcR-B1 expression and BmNPV reproduction, 4th instar larvae were given the optimal dose of 20E after being inoculated with 2 µL of BV-eGFP for 24 h, 48 h, and 72 h.
To determine the effect of 20E on silkworm resistance to BmNPV, 540 larvae of p50 on the first day of second instar were divided into six groups, each with three equal repetitions. Each group was fed 1 × 10 6 OB/mL ODV-treated mulberry leaves, and then the larvae were fed with the optimal dose of 20E at 0.5 d, 1.0 d, 1.5 d, and 2.0 d for half a day after BmNPV infection. Fresh mulberry leaves (1 cm 2 ) were soaked in different concentrations of 20E solution, dried naturally, and fed to silkworm larvae until the leaves were consumed, and then replaced with fresh mulberry leaves. The mortality of third instar silkworm larvae was recorded every 12 h after infection.
To study the effects of 20E on the growth of silkworms, the weight of silkworm larvae at different developmental stages, as well as the weight of cocoon shells, were counted after being fed with the optimal dose of 20E at 0.5 d, 1.0 d, 1.5 d, and 2.0 d after the first day of second instar. Thirty silkworm larvae at second, third, fourth, and fifth instar were weighed independently in each treatment group, and 30 cocoons of each sex were weighed independently. Then, the average weight of each larva or cocoon shell was calculated.

The Effect of 20E Treatment on BmEcR-B1 Expression and BmNPV Infection in BmN Cells
The optimal dose of 20E was coupled with 4 µg of siEcR-B1, which was subsequently introduced into BmN cells. siRFP was set as the control. BmEcR-B1 expression and BmNPV infection were detected after 48 h of treatment using RT-qPCR and fluorescence microscope. The same treatment was repeated in overexpression group, with the difference that BmN cells contained pIZT/V5-His-mCherry-BmEcR-B1 was treated directly with 20E.

Caspase Activity Assay and Apoptotic Bodies Detection
The 20E/siEcR-B1-treated group was set as the recovery experiment of the 20E-treated group. The control was 20E/siRFP. The changes in caspase activity were detected using the caspase-3 activity assay test kit (Njjcbio, Nanjing, China) according to the manufacturer's instructions. BmN cells were treated with lysis buffer containing DTT on ice for 30 min. The supernatant was collected and incubated with the reaction solution containing DTT and Ac-DEVD-pNA at 37 • C overnight. The absorbance value was detected by Spectramax i3 multifunctional microplate reader (Molecular Devices, CA, USA), and the degree of caspase activation was generated based on the absorbance value. Apoptotic bodies were detected by the TUNEL apoptosis detection kit (YEASEN, Shanghai, China) according to the manufacturer's instructions. BmN cells were collected and resuspended in 1 × phosphate-buffered saline (PBS). Each cell smear was incubated with an equilibration buffer at room temperature for 30 min and then mixed with terminal deoxynucleotidyl transferase (TdT) incubation buffer at 37 • C for 1 h in the dark. DAPI (Sangon Biotech, Shanghai, China) was used to stain the cell nucleus. The fluorescence signal was detected by the OLYMPUS IX3 inverted fluorescence microscope (OLYMPUS, Tokyo, Japan), and the captured data of the control group setup is used as the background.

Statistics Analysis
Data statistical analysis and charting were completed in GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). The SPSS Statistics 20 software (IBM, Endicott, New York, NY, USA) was used to assess differences between samples using one-way ANOVA. The Kruskal Wallis test was performed to analyze the data that did not match the Insects 2023, 14, 505 6 of 16 normality in SPSS software. Student's t-test was performed to detect the significance of the difference between the two data sets that follow the normal distribution. p < 0.05 was considered statistically significant.

Bioinformatics Analysis
The amino acid sequences of BmEcR-A and BmEcR-B1 and their homologs in other species were aligned using DNAMAN 8.0 software ( Figure S1). The results showed that the amino acid sequence of the ZnF C4 domain in both BmEcR-A and BmEcR-B1 are conserved, while the HOLI domain is absent in BmEcR-A. Moreover, the evolutionary relationship of EcR-A and EcR-B1 in different species was clustered into two separate groups ( Figure S2). These results suggested that the function of BmEcR-B1 might be different from that of BmEcR-A. Furthermore, BmEcR-B1 showed a closer evolutionary relationship with Bombyx mandarina, Galleria mellonella, Manduca sexta, and Antherae pemyi than other Lepidopterans, suggesting that EcR-B1 might be genetically distinct between Lepidopterans.

The Temporal and Spatial Expression Profile of BmEcR-B1 and BmEcR-A
The p50 strain has been used to build the silkworm genome [20]; detecting expression patterns in p50 would provide a better data reference. Thus, the expression profiles of BmEcR-A and BmEcR-B1 in different developmental stages and tissues of p50 were analyzed using RT-qPCR. The results showed that both the expression of BmEcR-A and BmEcR-B1 were higher in the early developmental stages of eggs than that in the late stages ( Figure 1A). Their expression levels in the testis and ovary were the highest among various tissues ( Figure 1B). Relatively high expression levels of BmEcR-A and BmEcR-B1 were found during metamorphosis, including before molting of fourth instar, pupa, and adult ( Figure 1C). Although BmEcR-A shared a similar expression pattern with BmEcR-B1 in different developmental stages and different tissues, the expression level of BmEcR-A was significantly lower than that of BmEcR-B1. Although BmEcR-B1 and BmEcR-A shared a similar expression pattern in the spatio-temporal expression spectrums, the levels of BmEcR-A are statistically much lower than that of BmEcR-B1. All the data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001. . Although BmEcR-B1 and BmEcR-A shared a similar expression pattern in the spatio-temporal expression spectrums, the levels of BmEcR-A are statistically much lower than that of BmEcR-B1. All the data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

BmEcR-B1 Showed a Significant Response to BmNPV Infection
To preliminarily compare the role of BmEcR-B1 and BmEcR-A in response to BmNPV, their relative expression levels in four immune-related tissues of YeA (resistant strain, LC 50 > 10 9 OB/mL) and YeB (susceptible strain, LC 50 = 10 5 OB/mL) after BmNPV infection at 48 h were analyzed using RT-qPCR. The results showed that the immune response of BmEcR-B1 to BmNPV was much more significant than that of BmEcR-A in four tissues, except in the hemolymph (Figure 2A). In addition, BmEcR-B1 expression was significantly higher in YeB tissues after BmNPV infection, except for the Malpighian tube ( Figure 2D) and markedly lower in YeA tissues after infection, except for the midgut ( Figure 2C). The significantly different expression levels of BmEcR-B1 in the two strains after BmNPV infection indicated that it might be the key gene in response to viral infection. Results showed that BmEcR-B1 was more important than BmEcR-A in response to BmNPV infection. YeA+ and YeB+ were infected with BmNPV. YeA− and YeB− were not infected with BmNPV. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

E activates BmEcR-B1 Expression and Inhibits BmNPV Infection In Vivo
EcR is a 20E receptor that mediates 20E-regulated host immune response [21], while its role in silkworm remains unclear. To analyze the role of 20E on BmEcR-B1 expression and BmNPV infection, the relative expression levels of BmEcR-B1 and viral capsid gene vp39 were determined after 24 h, 36 h, and 48 h of 20E treatment, as well as the survival rate of infected larvae. The results showed that 45 μg/mL of 20E were the most effective for activating BmEcR-B1 expression ( Figure 3A). BmEcR-B1 expression was significantly Results showed that BmEcR-B1 was more important than BmEcR-A in response to BmNPV infection. YeA+ and YeB+ were infected with BmNPV. YeA− and YeB− were not infected with BmNPV. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

E activates BmEcR-B1 Expression and Inhibits BmNPV Infection In Vivo
EcR is a 20E receptor that mediates 20E-regulated host immune response [21], while its role in silkworm remains unclear. To analyze the role of 20E on BmEcR-B1 expression and BmNPV infection, the relative expression levels of BmEcR-B1 and viral capsid gene vp39 were determined after 24 h, 36 h, and 48 h of 20E treatment, as well as the survival rate of infected larvae. The results showed that 45 µg/mL of 20E were the most effective for activating BmEcR-B1 expression ( Figure 3A). BmEcR-B1 expression was significantly increased at 36 h and 48 h after 45 µg/mL of 20E treatment ( Figure 3B). The survival rate of the third instar larvae was significantly increased in the 20E-treated groups, and the effect was most pronounced after 0.5 days of 20E treatment compared with other time points ( Figure 3C). The expression of vp39 decreased significantly at 36 h and 48 h after 20E treatment ( Figure 3D). Therefore, 20E has an important role in regulating BmEcR-B1 expression and inhibiting BmNPV infection.  The data were presented as mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

BmEcR-B1 Plays a Vital Role in 20E Inhibition of BmNPV In Vitro
To further analyze the relationship of 20E, BmEcR-B1, BmNPV, and BmN cells were selected for in-depth analysis. The regulation effects of 20E on BmEcR-B1 expression and BmNPV replication were analyzed again in BmN cells, and the results ( Figure 4A-C) were consistent with the above in-vivo results (Figure 3), confirming that 20E plays an important role in activating BmEcR-B1 expression and inhibiting viral infection. On this basis, two experiments were designed to assess the involvement of BmEcR-B1 in the inhibition of BmNPV infection by 20E. The first was the knockdown of BmEcR-B1 with siEcR-B1 in the presence of 20E ( Figure 4D-F), and the other one was in the absence of 20E ( Figure 4G-H). During the presence of 20E, the inhibition of 20E on BmNPV titer and capsid gene vp39 was eliminated after the knockdown of BmEcR-B1 ( Figure 4D-F), while  A,B,D) were analyzed using Student's t-test, and (C) was the Kruskal Wallis test. NC, negative control. The data were presented as mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

BmEcR-B1 Plays a Vital Role in 20E Inhibition of BmNPV In Vitro
To further analyze the relationship of 20E, BmEcR-B1, BmNPV, and BmN cells were selected for in-depth analysis. The regulation effects of 20E on BmEcR-B1 expression and BmNPV replication were analyzed again in BmN cells, and the results ( Figure 4A-C) were consistent with the above in-vivo results (Figure 3), confirming that 20E plays an important role in activating BmEcR-B1 expression and inhibiting viral infection. On this basis, two experiments were designed to assess the involvement of BmEcR-B1 in the inhibition of BmNPV infection by 20E. The first was the knockdown of BmEcR-B1 with siEcR-B1 in the presence of 20E ( Figure 4D-F), and the other one was in the absence of 20E ( Figure 4G-H). During the presence of 20E, the inhibition of 20E on BmNPV titer and capsid gene vp39 was eliminated after the knockdown of BmEcR-B1 ( Figure 4D-F), while no significant difference was observed in the absence of 20E ( Figure 4G-H). The results showed that BmEcR-B1 is vital for 20E to inhibit viral proliferation, and 20E is also required for BmEcR-B1 resistance to BmNPV. The results showed that BmEcR-B1 is vital for 20E to inhibit virus proliferation. All the data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences compared to the control and are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Overexpression of BmEcR-B1 Inhibits BmNPV Infection in BmN Cells
To further validate the RNAi results that knockdown BmEcR-B1 benefits BmNPV infection, BmEcR-B1 was overexpressed in BmN cells using pIZT-mCherry-BmEcR-B1. The results of correct BmEcR-B1 fragmentation ( Figure 5D), red fluorescent signals of mCherry ( Figure 5A-C), and significantly upregulated levels of BmEcR-B1 indicated that it was overexpressed in BmN cells. Moreover, an extremely high expression in the 20Etreated group revealed that it could be regulated by 20E ( Figure 5E). The results showed that BmEcR-B1 is vital for 20E to inhibit virus proliferation. All the data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences compared to the control and are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Overexpression of BmEcR-B1 Inhibits BmNPV Infection in BmN Cells
To further validate the RNAi results that knockdown BmEcR-B1 benefits BmNPV infection, BmEcR-B1 was overexpressed in BmN cells using pIZT-mCherry-BmEcR-B1. The results of correct BmEcR-B1 fragmentation ( Figure 5D), red fluorescent signals of mCherry ( Figure 5A-C), and significantly upregulated levels of BmEcR-B1 indicated that it was overexpressed in BmN cells. Moreover, an extremely high expression in the 20E-treated group revealed that it could be regulated by 20E ( Figure 5E). Insects 2023, 14, x FOR PEER REVIEW 11 of 17 The results showed that BmEcR-B1 was successfully overexpressed in BmN cells using pIZT/V5-His-mCherry. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences compared to the control and are indicated by different letters (a, b, c, d).
To analyze the effect of BmEcR-B1 overexpression on viral infection, eGFP intensity, vp39 expression, and virus titer were analyzed in different groups at 24 h, 48 h, and 72 h ( Figure 6). The results showed that BmNPV proliferation was significantly decreased in BmEcR-B1 overexpression group ( Figure 6A,B,D), and it was even lower after 20E treatment ( Figure 6C,E). When these findings are combined with the results in Figure 4A-C, it is reasonable to assume that BmEcR-B1 plays an important role in limiting viral replication in the presence of 20E. The results showed that BmEcR-B1 was successfully overexpressed in BmN cells using pIZT/V5-His-mCherry. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences compared to the control and are indicated by different letters (a, b, c, d).
To analyze the effect of BmEcR-B1 overexpression on viral infection, eGFP intensity, vp39 expression, and virus titer were analyzed in different groups at 24 h, 48 h, and 72 h ( Figure 6). The results showed that BmNPV proliferation was significantly decreased in BmEcR-B1 overexpression group ( Figure 6A,B,D), and it was even lower after 20E treatment ( Figure 6C,E). When these findings are combined with the results in Figure 4A-C, it is reasonable to assume that BmEcR-B1 plays an important role in limiting viral replication in the presence of 20E.

Inhibition of Viral Infection by BmEcR-B1 Is Associated with the Activation of Apoptosis
To analyze whether BmEcR-B1 inhibits BV-eGFP infection via the activation of apoptosis, apoptosis bodies and caspase activity were detected in the 20E-treated and 20E/siEcR-B1-treated groups. The 20E/siEcR-B1-treated group was set as the recovery experiment of the 20E-treated group. The results showed that the quantity of apoptotic bodies was increased after 20E induction, while it was recovered by siEcR-B1 ( Figure 7A, B). Moreover, Caspase activities were increased in the 20E-treated group ( Figure 7C), while it also reduced significantly after siEcR-B1 transfection ( Figure 7D). The results suggested the inhibition of BmNPV infection by BmEcR-B1 is associated with the activation of apoptosis. The results showed that overexpression of BmEcR-B1 inhibits viral replication. The data were analyzed using Student's t-test and were presented as the mean ±standard error of three independent replicates. Significant differences compared to the control and are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Inhibition of Viral Infection by BmEcR-B1 Is Associated with the Activation of Apoptosis
To analyze whether BmEcR-B1 inhibits BV-eGFP infection via the activation of apoptosis, apoptosis bodies and caspase activity were detected in the 20E-treated and 20E/siEcR-B1-treated groups. The 20E/siEcR-B1-treated group was set as the recovery experiment of the 20E-treated group. The results showed that the quantity of apoptotic bodies was increased after 20E induction, while it was recovered by siEcR-B1 ( Figure 7A, B). Moreover, Caspase activities were increased in the 20E-treated group ( Figure 7C), while it also reduced significantly after siEcR-B1 transfection ( Figure 7D). The results suggested the inhibition of BmNPV infection by BmEcR-B1 is associated with the activation of apoptosis. The results showed that overexpression of BmEcR-B1 inhibits viral replication. The data were analyzed using Student's t-test and were presented as the mean ±standard error of three independent replicates. Significant differences compared to the control and are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001. The results showed that BmEcR-B1 inhibits viral infection related to the activation of apoptosis. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; *** p < 0.001.

The Analysis of the Effect of 20E on Silkworm Larvae Development and Cocoon Shell
To analyze whether 20E can be used to control BmNPV in sericulture, the effect of 45 μg/mL of 20E on silkworm larvae development and cocoon score was evaluated. Silkworm larvae on the second instar were fed with 20E at 0.5 d, 1.0 d, 1.5 d, and 2.0 d after ecdysis, respectively. The larval weight of the silkworm was recorded at second, The results showed that BmEcR-B1 inhibits viral infection related to the activation of apoptosis. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; *** p < 0.001.

The Analysis of the Effect of 20E on Silkworm Larvae Development and Cocoon Shell
To analyze whether 20E can be used to control BmNPV in sericulture, the effect of 45 µg/mL of 20E on silkworm larvae development and cocoon score was evaluated. Silkworm larvae on the second instar were fed with 20E at 0.5 d, 1.0 d, 1.5 d, and 2.0 d after ecdysis, respectively. The larval weight of the silkworm was recorded at second, third, fourth, and fifth instars, as well as the cocoon shell weight. The results showed that feeding 20E reduced larval weight at different development stages and cocoon shell weights compared to the control group, but the differences were not statistically significant ( Figure 8A,B).
bodies after treatment with 20E (A) and 20E/siEcR-B1 (B). DAPI (blue), nuclear dye. FITC (green), apoptotic bodies. Scale bar = 200 μm. The analysis of caspase activities after treatment with 20E (C) and 20E/siEcR-B1 (D). The results showed that BmEcR-B1 inhibits viral infection related to the activation of apoptosis. The data were analyzed using Student's t-test and were presented as the mean ± standard error of three independent replicates. Significant differences are indicated by asterisks (p < 0.05). ns, no significant; * p < 0.05; *** p < 0.001.

The Analysis of the Effect of 20E on Silkworm Larvae Development and Cocoon Shell
To analyze whether 20E can be used to control BmNPV in sericulture, the effect of 45 μg/mL of 20E on silkworm larvae development and cocoon score was evaluated. Silkworm larvae on the second instar were fed with 20E at 0.5 d, 1.0 d, 1.5 d, and 2.0 d after ecdysis, respectively. The larval weight of the silkworm was recorded at second, third, fourth, and fifth instars, as well as the cocoon shell weight. The results showed that feeding 20E reduced larval weight at different development stages and cocoon shell weights compared to the control group, but the differences were not statistically significant ( Figure 8A,B).  The results showed that 20E treatment did not significantly affect silkworm larvae development and cocoon shell. The data were analyzed using the Kruskal-Wallis test and presented as mean ±standard error of three independent replicates. ns, no significant.

Discussion
BmEcR-B1 was found to be significantly differentially expressed following BmNPV infection in our earlier study [22]. Related studies have revealed that EcR contributes to insect immune response [23], but it is uncertain whether BmEcR-B1 also has an immunological function in silkworms. In this study, the importance of BmEcR-B1 and BmEcR-A was first compared, and then the function of BmEcR-B1 was determined in vivo and in vitro.

BmEcR-B1 Is More Critical Than BmEcR-A in Silkworm
There are two kinds of isoforms of EcR in silkworms, BmEcR-B1, and BmEcR-A. Although the expression patterns of BmEcR-B1 and BmEcR-A have been reported [15], their sequence characteristics are still unclear. In this study, the differences in the expression between BmEcR-A and BmEcR-B1 were analyzed in different developmental stages and tissues. Although BmEcR-A shared a similar expression pattern with BmEcR-B1, the relative expression level of BmEcR-A was significantly lower than BmEcR-B1 ( Figure 1). Moreover, BmEcR-A lacked the HOLI domain compared to BmEcR-B1 ( Figure S1). HOLI domain is a ligand-binding domain. It has been reported that EcR-A, which lacks the HOLI domain in Drosophila, completely loses its activation of ecdysone-induced genes even in the transgenic expression of EcR-A [24]. Thus, the higher expression of BmEcR-B1 suggested that it has broader functions than BmEcR-A during silkworm development. Moreover, the immune response of BmEcR-B1 was more significant than that of BmEcR-A after BmNPV infection (Figure 2), suggesting that BmEcR-B1 might be a key gene in silkworm in response to BmNPV infection but not BmEcR-A.

BmEcR-B1 Plays a Vital Role in Inhibiting BmNPV, Which Needs the Presence of 20E
It was reported that EcR belongs to the nuclear receptor superfamily of 20E, which requires the activation of 20E [25], but its role in silkworm has not been reported yet. In this study, the relationship between 20E and BmEcR-B1 was determined. We found that 20E could activate the expression of BmEcR-B1, and the optimal concentration was 45 µg/mL ( Figure 3A,B). One of the previous studies used 10 mg/mL of 20E to inject silkworms to regulate gene expression [26]. Thus, it should be reasonable that 45 mg/mL of 20E was the optimal concentration to upregulate BmEcR-B1 expression. Moreover, 20E has been reported to regulate the silkworm innate immune [27], with the exception of BmNPV. This study found that feeding 20E enhanced silkworm resistance to BmNPV infection and inhibited vp39 expression ( Figure 3C,D), suggesting that 20E had an important role in silkworm immunity of silkworm to BmNPV. To determine the role of BmEcR-B1 in 20E-induced inhibition against BmNPV, siRNA targeting on BmEcR-B1 was used to knock it down. We found that the knockdown of BmEcR-B1 increased BmNPV infection in the presence of 20E ( Figure 4D-F), but without effect in the absence of 20E ( Figure 4G-I). EcR was reported to be silent in the absence of 20E [26], which may be the reason why the knockdown of BmEcR-B1 also did not affect viral infection. To further confirm the function of BmEcR-B1 in BmNPV infection, it was overexpressed using pIZT/V5-mCherry vector in BmN cells ( Figure 5). It was found that BmNPV infection was significantly inhibited after overexpression of BmEcR-B1 ( Figure 6D,F), and the effect was better in the presence of 20E ( Figure 6E,G). Thus, BmEcR-B1 was proved to play a vital role in the resistance to BmNPV infection, and this requires the participation of 20E. When the function of EcR in Dengue Virus 2 [28] and Helicoverpa armigera SNPV [29] is considered, it is plausible to conclude that EcR is a vital gene for host immunity to the virus.

BmEcR-B1 Likely Inhibits Virus Infection in BmN Cells via the Activation of Apoptosis
Although BmEcR-B1 has been shown to have an anti-BmNPV effect, the underlying mechanism remains unclear. Studies have shown that 20E and BmEcR-B1 can activate apoptosis [30][31][32]. To analyze whether the anti-BmNPV role of BmEcR-B1 is related to this, the present study analyzed the apoptotic bodies and caspase activity in 20E treated-group after RNAi of BmEcR-B1. The significant increase in apoptotic bodies and caspase activities in the 20E-treated group ( Figure 7B,C), whereas this activation effect was eliminated after siEcR-B1 treatment ( Figure 7D), suggesting that BmEcR-B1 inhibited BmNPV via the activation of apoptosis. The results provide another example of the association between BmEcR-B1 and apoptosis. Moreover, it was reported that 20E-induced apoptosis was inhibited when the binding of USP and EcR-B1 was blocked by silkworm 30K proteins [33]. Thus, the inhibition of BmNPV by BmEcR-B1 is related to the activation of apoptosis, which provides a vital supplement for EcR-mediated 20E triggering apoptosis in response to viral infection. Finally, to evaluate whether 20E can enhance silkworm antivirus levels, the larval weight and cocoon shell weight of silkworms at different developmental stages were determined after feeding with 20E at different times, and the results showed insignificant impacts, making it feasible (Figure 8).
In conclusion, the activation of BmEcR-B1 plays an important role in inhibiting BmNPV infection by activating apoptosis, which needs the existence of 20E (Figure 9). Moreover, as 20E has an insignificant effect on silkworm development and cocoons, it can be used in sericulture to reduce the losses caused by BmNPV. determined after feeding with 20E at different times, and the results showed insignificant impacts, making it feasible (Figure 8).
In conclusion, the activation of BmEcR-B1 plays an important role in inhibiting BmNPV infection by activating apoptosis, which needs the existence of 20E (Figure 9). Moreover, as 20E has an insignificant effect on silkworm development and cocoons, it can be used in sericulture to reduce the losses caused by BmNPV. Figure 9. The schematic diagram of the mechanism of BmEcR-B1 in response to BmNPV infection.

Supplementary Materials:
The following supporting information can be downloaded at www.mdpi.com/xxx/s1, Figure S1, homologous alignment of the EcR-A and EcR-B1 amino acid sequences in different species. The amino acid sequence of the ZnF C4 domain is conserved in BmEcR-A and BmEcR-B1, while the HOLI domain is absent in BmEcR-A. The conserved amino acid sequence is highlighted in navy blue. The differential sequence is highlighted in pink, yellow, and light blue. The red underline denotes the ZnF C4 domain, and the green underline denotes the HOLI domain. ★, Bombyx mori. Figure S2, the phylogenetic tree of EcR-A and EcR-B1 in different species The evolutionary relationship of EcR-A and EcR-B1 in different species was clustered into two separate groups, suggesting the function of BmEcR-B1 might be different from that of BmEcR-A. The star denotes EcR-A and EcR-B1 in Bombyx mori. BmEcR-B1 and its homologs are in blue, and BmEcR-A and its homologs are in red. Figure S3, analysis of BmEcR-B1 expression in BmN cells following 24 h, 48 h, and 72 h of siEcR-B1 transfection. Table S1, the list of accession numbers of BmEcR-B1 homologous in other species. Table S2, the list of accession numbers of BmEcR-A homologous in other species.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects14060505/s1, Figure S1, homologous alignment of the EcR-A and EcR-B1 amino acid sequences in different species. The amino acid sequence of the ZnF C4 domain is conserved in BmEcR-A and BmEcR-B1, while the HOLI domain is absent in BmEcR-A. The conserved amino acid sequence is highlighted in navy blue. The differential sequence is highlighted in pink, yellow, and light blue. The red underline denotes the ZnF C4 domain, and the green underline denotes the HOLI domain. , Bombyx mori. Figure S2, the phylogenetic tree of EcR-A and EcR-B1 in different species. The evolutionary relationship of EcR-A and EcR-B1 in different species was clustered into two separate groups, suggesting the function of BmEcR-B1 might be different from that of BmEcR-A. The star denotes EcR-A and EcR-B1 in Bombyx mori. BmEcR-B1 and its homologs are in blue, and BmEcR-A and its homologs are in red. Figure S3, analysis of BmEcR-B1 expression in BmN cells following 24 h, 48 h, and 72 h of siEcR-B1 transfection. Table S1, the list of accession numbers of BmEcR-B1 homologous in other species. Table S2, the list of accession numbers of BmEcR-A homologous in other species.

Data Availability Statement:
All the datasets in this study can be provided relay on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.