The p50 and C108 silkworm strain was bred at the Key Laboratory of Sericulture within the School of Life Science at Jiangsu University of Science and Technology. p50 is a susceptive strain of AcMNPV with a half lethal concentration (LC₅₀) of 4.5×10⁴ pfu/mL, and C108 is a resistant strain of AcMNPV with a LC₅₀ more than 6.2×10⁸ pfu/mL. The larvae were provided with fresh mulberry leaves and kept at 26 ± 1°C, 75% ± 5% relative humidity, and a 12-h photoperiod. The temperature was reduced to 24 ± 1°C during the final two instars, while the other conditions remained unchanged.

Budded AcMNPV with enhanced green fluorescent protein (BV-eGFP) is maintained in our laboratory. The titer of BV-eGFP was calculated as described by Li et al. (17).

On the 1st day of 5th instar, each silkworm larva was injected with 2.0 μL of culture medium containing BV-eGFP at a concentration of 1.0 × 10⁸ plaque-forming units (pfu)/μL. The control group was injected with 2.0 μL of culture medium. Various tissues, including the midgut, fat body, hemolymph, and the Malpighian tubule, were collected 36 h after BV-eGFP injection. The samples were rapidly frozen in liquid nitrogen, immediately ground into a powder, and stored at -80°C until use. BmN cells were transfected with 2 ng of DNA/RNA or treated with 5.0 μL of culture medium containing BV-eGFP (1.0× 10⁸ pfu/μL) per well, and then collected at 24, 48, and 72 h post-transfection or BV-eGFP treatment. After centrifugation at low speed for 5 min and discarding the culture medium, phosphate-buffered saline (PBS) was added to wash the cells. Then, the cells were centrifuged at low speed for 5 min. The supernatant was discarded, and the cells were washed again. After the final wash, the cell pellet was frozen in liquid nitrogen, immediately ground into a powder, and then stored at -80°C until use.

The BmElmo nucleic acid sequence (GenBank ID: LOC101744642) and its homologous sequences in other species were retrieved from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). The functional domain of BmElmo was predicted using the online SMART tool (http://smart.embl-heidelberg.de/). The homologous nucleic acid sequence of BmElmo was analyzed using the DNAMAN 8.0 software, and a neighbor-joining tree was constructed using the MEGA-X software.

BmN cells and silkworm tissues were lysed with the TransZol reagent (Transgen, Beijing, China). Then, RNA was isolated by chloroform extraction, purified by the isopropanol precipitation, and dissolved in diethyl pyrocarbonate (DEPC)-treated water. The purity of the isolated RNA was assessed based on the optical density (OD) 260/280 ratio. The RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo, Waltham, USA), and its integrity was verified by 1% agarose gel electrophoresis. A total of 1.0 µg of RNA was subjected to reverse transcription with the PrimeScript™ RT reagent kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions.

The relative expression of the genes of interest was detected using RT-qPCR. The specific primers were designed using the NCBI website and are presented in Table 1 . Each reaction comprised 1 µL of cDNA, 1 µL of primer mix, 3 µL of double-distilled water (ddH₂O), and 5 µL of 2× NovoStart^® SYBR qPCR SuperMix Plus. The standard cycling protocol was 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 10 s and annealing at 60°C for 30 s, and finally a melting curve was generated at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. To reduce experimental error, all samples underwent three repeats. The expression level of each gene was calculated using the 2−ΔΔCT method. Bombyx mori glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) was used as internal references to normalize the gene expression.

Using p50 testis cDNA as a template, the open reading frame (ORF) fragment of BmElmo was amplified using BmElmo primers ( Table 1 ). The purified ORF was ligated into the pMD-19T vector and subsequently sent to Sangon (Shanghai, China) for sequencing. The pMD-19T-BmElmo and pIZT/V5-His-mCherry vectors were digested with KpnI and EcoRI (Takara), and the correctly sequenced ORF was ligated into the overexpression vector using T4 DNA ligase (Takara). After sequencing validation, the pIZT/V5-His-mCherry-BmElmo vector was stored at -80°C until use.

siRNA targeting the functional domain of BmElmo was designed using the BLOCK-iT™ RNAi designer website (https://rnaidesigner.thermofisher.com/), which was used to knock down the expression of BmElmo in BmN cells. siRFP served as a negative control. The primer sequences are shown in Table 2 . siRNAs were synthesized using the In Vitro Transcription T7 Kit (Takara) according to the manufacturer’s instructions. Following purification, the high-quality siRNA was stored at -80°C for future use.

BmN cells were cultured at 27°C. Each 50 mL of cell culture medium comprised 44.5 mL of TC-100 (AppliChem, Gatersleben, Germany), 5 mL of fetal bovine serum (FBS; Thermo Fisher Scientific, New York, NY, USA), and 0.5 mL of dual antibiotics (penicillin and streptomycin). The culture medium of Sf9 cells was Sf-900TM (Thermo Fisher Scientific, New York, NY, USA). BmN cells were transfected with siRNA and the overexpression vector using a transfection reagent (NEOFECT, Beijing, China) according to the manufacturer’s instructions. Each 60 mm dish was transfected with 4.0 µg of plasmid or siRNA. The transfection solution for 2 mL of culture medium comprised 2 μg of DNAor RNA, 2 μL of transfection reagent (NEOFECT), and 100 μL of TC-100 solution; it was prepared 30 min before transfection. The transfection efficiency was measured at different time points. The expression level of BmElmo was detected at 24, 48, and 72 h after transfection.

All statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA, USA). The data are presented as the mean ± standard error of the mean of three biological replicates, and each biological replicate was derived from samples of different individuals. Student’s t-test was used to compare two groups of normally distributed data. The Kruskal–Wallis test was used to analyze the data that did not meet the normality requirement. Statistically significant differences between samples were analyzed using one-way ANOVA test. Significance is indicated by an asterisk: * P < 0.05, ** P < 0.01, and *** P < 0.001; ns means not significant.

BmElmo (GenBank ID XM_004923980.4) has a full length of 3,725 base pairs (bp) and a coding sequencing (CDS) length of 936 bp, encoding a protein of 313 amino acids. Based on functional domain analysis of BmElmo using the SMART website, there is a CED-12 domain spanning amino acids 125 to 286. Additionally, protein sequence homology alignment analysis showed that BmElmo has a sequence conservation of 86.43% ( Figure 1 ).

An evolutionary tree has been constructed by neighbor joining method in the MEGA-X software with the identified homologous Elmo protein sequences from various species, including B. mori, Drosophila melanogaster, Manduca sexta, and Melitaea cinxia ( Figure 2 ).

The relative expression of BmElmo across various developmental stages of the p50 strain and in different tissues of 5th instar larvae have been examined by RT qPCR. BmElmo expression was highest during the pupal stage, and there was notable expression for the 1st instar and adult stages. The egg stage presented the lowest expression ( Figure 3A ). Among the various tissues, BmElmo expression was highest in the testis, followed by the ovary and silk gland, while the epidermis presented the lowest expression ( Figure 3B ).

The relationship between BmElmo and AcMNPV has been analyzed based on BmElmo expression in four immune-related tissues (hemolymph, midgut, Malpighian tubules, and fat body) from a susceptible strain (p50) and a resistant strain (C108) 36 h after AcMNPV infection. The silkworms in the control group were injected with same volume of SF-900tm insect culture medium. BmElmo expression in the midgut, hemolymph, and Malpighian tubule of the C108 strain was significantly elevated compared with the control group, while in the p50 strain, BmElmo expression in the Malpighian tubule, hemolymph, and fat body was notably decreased relative to the control group ( Figure 4 ).

The expression level of BmElmo has been knocked down in BmN cells by transfecting siRNA which target to its functional domain CED-12 (siBmElmo). Subsequently, these cells were infected with AcMNPV 24 h after transfection. BmElmo expression was significantly decreased in the siBmElmo group 24, 48, and 72 h after transfection ( Figure 5A ). AcMNPV infection was observed by fluorescence microscopy at 24, 48, and 72 h post-infection. The virus fluorescence signal rose significantly at 48 and 72 h after infection ( Figure 5C ). To validate the above results, the relative expression of lef3, a viral single-strand DNA-binding protein, was detected at 24, 48, and 72 h after AcMNPV infection of BmN cells. lef3 relative expression was markedly higher in the siBmElmo group compared with the control group ( Figure 5B ).

The recombinant plasmid pIZT/V5-His-mCherry-BmElmo was constructed to further investigate BmElmo’s role in the AcMNPV response ( Figure 6A ) and transfected BmN cells with it to overexpress BmElmo ( Figure 6B ). BmElmo expression was detected by RT-qPCR following transfection with the recombinant plasmid. BmElmo expression increased significantly at different times after transfection ( Figure 6C ), indicating BmElmo was overexpressed successfully in BmN cells. Moreover, the green fluorescence signal of AcMNPV was lower 48 and 72 h after BmElmo overexpression compared with the control group ( Figure 6E ). Additionally, lef3 expression was significantly lower after BmElmo expression compared with the control group ( Figure 6D ).

The pathway by BmElmo in BmN cells mediates the response to AcMNPV infection was investigated. Specifically, the expression of genes that encode JNK pathway members and that participate in phagocytosis was examined 48 h after knockdown and overexpression of BmElmo. The JNK pathway–related genes BmJun, BmJnks, and BmFosS showed significantly reduced expression after BmElmo knockdown ( Figures 7A–C ) and significantly increased expression after BmElmo overexpression ( Figures 7D–F ). However, expression of the phagocytosis-related genes BmActinA1 and BmTet did not change significantly after BmElmo knockdown or overexpression of ( Supplementary Figure S1 ).

The Elmo protein sequences from different species was compared and it was found that the BmElmo amino acid sequence has been highly conserved, especially its functional domain CED-12 ( Figure 1 ). Moreover, CED-12 has been highly phylogenetically conserved among Lepidoptera ( Figure 2 ). CED-12 is involved in phagocytosis and apoptosis (21, 22), which provides a valuable clue for detecting the mechanism of BmElmo in response to AcMNPV infection. Silkworm larvae at 5th instar is the fastest period of weight gain that body weight increases more than 15-time compared with 1st instar (23). The very high BmElmo expression in 1st and 5th instars indicates that it plays a key role in silkworm development ( Figure 3A ). The tissue expression profiles showed that it is highly expressed in ovaries and testis ( Figure 3B ), indicating that it is involved in gonad development, which also corresponds to its high expression during the pupal stage ( Figure 3A ). Of note, the gonads of silkworms are in a primitive stage during the larval period and undergo several maturation steps during the pupal stage. Additionally, BmElmo expression was markedly higher compared with the control group in most of the analyzed tissues ( Figure 4 ), indicating that BmElmo is an important gene in silkworm immunity.

The role of BmElmo in the response of BmN cells to AcMNPV infection was further investigated by knocking down and overexpressing this gene. Compared with the control, virus replication increased significantly following BmElmo knockdown ( Figure 5 ), but decreased significantly after BmElmo overexpression ( Figure 6 ). These findings demonstrate that BmElmo plays a critical inhibitory role in AcMNPV infection of BmN cells.

The Elmo protein participates in cellular processes such as cell migration, immunity, and phagocytosis by regulating the downstream RhoA/Rac1 (14), JNK (24) and phagocytosis pathways (6). However, prior to the present study, there had been no reports on which pathways Elmo regulates in the silkworm. In insects, the JNK pathway is closely related to infection of hosts by viruses and other microorganisms (25, 26). In silkworms, reactive oxygen species–mediated JNK phosphorylation is involved in the regulation of Bombyx mori nucleopolyhedrovirus (BmNPV) proliferation (27). Phagocytosis is a conserved evolutionary process wherein hemocytes identify, engulf, and ultimately dispose of apoptotic cells and invading microbial pathogens (28, 29). The relative expression of key genes involved in these processes was examined after BmElmo knockdown and overexpression. The relative expression of JNK pathway members, BmJun, BmJnks, and BmFosS, changed significantly after BmElmo knockdown or overexpression ( Figure 7 ). However, the relative expression of the phagocytosis-related genes BmActinA1 and BmTet did not change when BmElmo was knocked down or overexpressed ( Supplementary Figure S1 ). Hence, the BmElmo protein exerts its effects through the JNK pathway, but it is not clear whether the JNK pathway directly affects AcMNPV infection.

Our results indicate that the expression of BmElmo can enhance the ability of silkworms to baculoviruses infection, which might be related to the activation of JNK phosphorylation (27, 30), yet this point still needs to be further addressed.