was evaluated by 12% SDS-PAGE and the concentration of fusion protein was quantified using the Bradford method (Bradford, 1976).

The New Zealand white rabbit was injected intraperitoneally with 1.0 mL mixture of 1.0 mg purified fusion protein and Freund’s complete adjuvant (Sigma, USA). The immunity was boosted three times with 0.5 mg fusion protein in Freund’s incomplete adjuvant at one week intervals. Serum was collected after the last injection. The elution buffer (0.09 mM NaHCO3, 0.3 mM NaCl, 0.25 mM C3H4N2, pH 8.0) was used as a negative control. Indirect ELISA was used to detect the titer of polyclonal antibodies in the serum. The remaining serum was stored at -80 °C for later use.

Tissue protein extraction

All tissues were ground to powder in liquid nitrogen and suspended in extraction buffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% NP-40, 1.0 mM PMSF), then kept on ice for 30 min. The homogenates were centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatants were collected and total protein was quantified by the Bradford method.

Western blotting

The protein samples were lysed in 5 × protein loading buffer (50 mM Tris-HCl pH 8.0, 250 mM DTT, 5% SDS, 50% glycerol, 0.04% Bromophenol Blue) and then boiled for 10 min. The samples were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Switzerland). Subsequently, the membranes were blocked with 5% nonfat milk in 0.01 M phosphate-buffered saline (PBS; 10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl pH 7.4) at room temperature (RT) for 1 h, then incubated with rabbit serum (1:10,000) at RT for 1 h. After washing with PBST (PBS + 0.05% Tween-20) three times, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000; TransGen biotech, China) at RT for 1 h, washed three times in PBST and visualized using a diaminobenzidine (DAB) Kit (TIANGEN, China) according to the manufacturer’s instructions. β-actin was used as a loading control. Anti-β-actin mouse monoclonal antibody and HRP-conjugated goat anti-mouse IgG (TransGen biotech, China) were diluted as per the manufacturer’s instructions.

Immunofluorescence

All the tissues fixed in 4% paraformaldehyde were dehydrated and embedded in paraffin. Tissue sections (4 μm thick) were transferred to microscope slides and then deparaffined and rehydrated. Subsequently, sections were treated with transparent buffer (PBS containing 0.3% hydrogen peroxide and 0.3% Triton X-100) at RT for 30 min, washed three times in PBS, and then incubated with 0.01M citrate buffer (5 mM C6H8O7, 24 mM Na3C6H5O7·2H2O, pH 6.0) at 96 °C for 15 min. After washing three times with PBS, sections were blocked with 10% goat serum for 30 min, then incubated with rabbit serum (1:100) at RT for 1 h, then at 4 °C overnight followed by 37 °C for 45 min. Following washing five times in PBST, sections were incubated with anti-rabbit IgG conjugated with FITC (1:100; TransGen biotech, China) at 37 °C for 30 min, washed three times in PBST, and then incubated with 4,6-diamidino-2-phenylindole (DAPI, 1:1000; FanBo, China) at RT for 2 min. After washing with PBST three times, images were obtained using a fluorescence microscope (Olympus BX51, Japan).

Results

Sequence analysis of BmARM-like

(KOB71817.1), Athalia rosae (XP_012263420.1), Aedes aegypti (XP_001650042.1), Culex quinquefasciatus (XP_001843480.1), Anopheles gambiae (XP_319205.3), Anopheles sinensis (KFB52334.1), and Habropoda laboriosa (KOC68595.1). Comparison of the BmARM-like amino acid sequence with the published sequences of ARM-repeat proteins from others species showed that BmARM-like protein shared 69.2% sequence homology with both Papilio machaon and Papilio xuthus, 67.6% with Amyelois transitella, 66.7% with Plutella xylostella, 66.2% with Danaus plexippus, 65.7% with Operophtera brumata and more than 32% with other species (Fig. 2).

To better elucidate the evolutionary relationships between BmARM-like protein and other sequenced proteins, the whole sequence of BmARM-like and other species was used to determine the evolutionary relationships. A phylogenetic tree consisting of BmARM-like protein and 13 other homologs was constructed. These proteins were clearly classified into three groups: Lepidoptera, Hymenoptera, and Diptera. BmARM-like

protein and its homologs from 6 other insects, including Operophtera brumata, Amyelois transitella, Plutella xylostella, Danaus plexippus, Papilio machaon, and Papilio xuthus were clustered together into a Lepidoptera group. Whereas, Athalia rosae and Habropoda laboriosa were clustered into the Hymenoptera group and Aedes aegypti, Culex quinquefasciatus, Anopheles gambiae and Anopheles sinensis were clustered into the Diptera group (Fig. 3).

Expression patterns of BmARM-like in different tissues and developmental stages

To elucidate the molecular biological function of BmARM-like, the relative expression levels of BmARM-like mRNA were detected in different silkworm larval tissues and at developmental stages. In different larval tissues, the highest expression of BmARM-like mRNA was observed in hemolymph, followed by testis and ovary, and the lowest expression was observed in the head (Fig. 4A). Comparing developmental stages, the relative high expression levels were observed in 4th instar, moth, and eggs, with decreased expression in 5th instar, and the lowest expression in 1st instar (Fig. 4B). During embryonic development, BmARM-like kept a stable high expression level in the early stages of embryonic development (1st – 6th day of egg development), then gradually decreased until the embryo developed into the head bluing period (the last two days) (Fig. 4C). During pupal development, BmARM-like was up-regulated on the 1st day and then down-regulated over the following three days, with expression again increasing on the 5st day and then decreasing again on the last two days (Fig. 4D). Additionally, BmARM-like showed the highest expression level during the molting stage of 4th instar while other stages of molting show significant low expression as compared to 4th instar (Fig. 4E).

Pattern of BmARM-like expression in different resistant silkworm strains following BmNPV infection

To clarify the relationship between BmARM-like and BmNPV, the expression patterns of BmARM-like mRNA in the midgut and hemolymph of different resistant silkworm strains were analyzed using RT-qPCR before and after BmNPV infection (Fig. 4). In midgut, the highest expression of BmARM-like mRNA was observed in BC9 (near-isogenic line). Following BmNPV infection, BmARM-like was up-regulated in all three strains, while relatively higher expression levels were observed in BC9 and A35 (resistant strain) as compared with P50 (susceptible strain) with the up-regulation in A35 being statistically significant (Fig. 4F). In hemolymph, the highest BmARM-like mRNA expression was also observed in the BC9 strain. However, the expression level of BmARM-like was up-regulated in P50, but down-regulated in BC9 and A35 (Fig. 4G).

Polyclonal antibody preparation

The functional domain of BmARM-like was successfully cloned and ligated into PET-28a to express the fusion proteins (Supplementary Fig. S1). The fusion protein that was confirmed with the antibody to histidine using Western blot was purified using high affinity Ni-NTA resin (Supplementary Fig. S2) and used to produce polyclonal antibodies. Indirect ELISA was adopted to determine the titer of polyclonal antibodies. The titer of the polyclonal antibodies was greater than 1:384,000 (Supplementary Fig. S3) which met the requirement for later experiments.

The polyclonal antibodies to BmARM-like protein trapped a protein from the total lysates of E.coli with a band approximately 31.1 kDa in size. Additionally, it also crossreacted with the extracts of silkworm tissues, producing a band about 71.2 kDa in size. These results demonstrate the high specificity of the polyclonal antibodies required for later experiments (Fig. 5).

Tissue localization of BmARM-like protein

To determine the tissue-specific localization of BmARM-like protein, head, midgut, fat body, testis and ovary from the 3rd day of 5th instar were removed and subjected to immunofluorescence analysis with the polyclonal antibodies to BmARM-like protein. As illustrated in Figure 6, BmARM-like protein was found to be distributed uniformly in all selected tissues and did not show significant tissue specificity.

Expression pattern of BmARM-like protein in the midgut of different silkworm strains following BmNPV infection

Western blot was used to further investigate the expression patterns of BmARM-like protein in the midguts of three different silkworm strains following BmNPV infection. The highest expression level of BmARM-like protein was identified in BC9 (near-isogenic strain) as compared with P50 (susceptible strain) and A359 (resistant strain). In addition, the expression of BmARM-like protein was higher in A35 and BC9 following BmNPV infection as compared with P50 (Fig. 7). The expression patterns of BmARM-like protein in different silkworm strains following BmNPV infection were basically consistent with the expression patterns of BmARM-like mRNA in the transcriptome levels (Fig. 4).

Discussion

In this study, the molecular characterization and functional analysis of a novel silkworm gene, BmARM-like, was described. As far as we know, this is the first report of tissue–specific expression and subcellular localization of BmARM-like in silkworms.

Venkiteswaran et al. (2000) reported that VE-cadherin and the armadillo family protein plakoglobin regulated vascular endothelial permeability and growth. In this study, BmARM-like protein was observed in all selected tissues, but the highest expression of BmARM-like mRNA was found in hemolymph (Fig. 4A). Therefore, we speculated that BmARM-like protein is related to silkworm hemocoel endothelial permeability and growth.

Some reports have shown that ARM repeat–containing proteins play an important role in silkworm growth process (Xiao et al., 2001). In this study, BmARM-like showed significantly high expression in 4th instar, moth, and eggs (Fig. 4B). Additionally, BmARM-like showed relatively high expression levels on the 1st day and 5th day of pupa, and down-regulation within the last few days of this stage (Fig. 4D). These results indicate its role in the growth and development processes of silkworms.

It has been reported that armadillo motif–containing protein possesses a developmentally regulated function during mouse embryonic development (Smith et al., 2005). Knockdown of armadillo motifs containing KPNA7 in the early embryo of silkworm resulted in a decreased

proportion of embryos developing to the blastocyst stage (Shen et al., 2009). In this study, we observed a consistently high expression of BmARM-like in silkworm eggs and during the early embryonic developmental stages (Fig. 4B, C); therefore, we speculated that BmARM-like was involved in regulating embryonic development during the early developmental stages of silkworm embryo.

Insect metamorphosis is triggered by a pulse of steroid hormones, which can regulate the expression of target genes in the body and thus orchestrate drastic biological changes (Niwa and Niwa, 2016). In the silkworm larvae molting stage, the highest expression of BmARM-like was observed in the 4th instar (Fig. 4E), indicating that the expression of BmARM-like was potentially regulated by molting hormones.

Silencing of sterile alpha and armadillo motif–containing (SARM) protein in Litopenaeus vannamei using dsRNA–mediated RNA interference increased the expression of Penaeidins and antilipopolysaccharide factors and increased the mortality rate after Vibrio alginolyticus infection (Wang et al., 2013). Liu et al. (2014) reported that the increased SARM expression could repress respiratory syncytial virus (RSV) immunopathology caused by deregulation of the toll-like receptor–mediated immune response. In this study, relatively high expression level of BmARM-like were observed in the midgut of BC9 (near-isogenic line) following BmNPV infection as compared to P50 (susceptible strain), which was also further validated in A35 (resistant strain). Specifically, the up-regulation of BmARM-like in A35 was statistically significant following BmNPV infection (Figs. 4F, 7). These results indicated that BmARM-like was potentially involved in activating the silkworm immune response in an attempt to repress BmNPV infection. In hemolymph, the expression levels of BmARM-like were up-regulated in P50 but down-regulated in BC9 and A35 (Fig. 4G), which might be due to the activation of the host immune system to suppress the virus infection.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [31472148] and Seed Engineering Fund of Anhui Academy of Agricultural Sciences [16D0608].

Conflict of interest statement

The authors declare that there is no conflict of interest with any one about this manuscript.