BSFL used in this study were obtained from the Department of Agricultural Biology at the National Institute of Agricultural Sciences. The larvae were reared at 26±1℃ and 60% relative humidity for 2 weeks. They were fed an organic waste mixture containing rotten vegetables, fruits, and meat until they reached the fifth instar stage.

To compare the antimicrobial activity of the hemolymph from E. coli-challenged and unchallenged BSFL, the two experimental groups were formed: E. coli-challenged and unchallenged BSFL. The E. coli-challenged larvae were injected with a 20 µL of E. coli inoculum containing approx. 1.0-2.0×10⁵ CFU/mL. The inoculum concentration was refered from Bruno et al. (2021), the optimal concentration for BSFL challenge without high mortality during the 72 h required to induce immune responses and AMP expression in E. coli. While the unchallenged larvae were injected with 20 µL of PBS as an experimental control.

Hemolymph was collected by puncturing and inserting 1mL syringe needle into the abdomen of two hundred larvae from both unchallenged and E. coli-challenged BSFL. To prevent melanization, a small amount of phenylthiourea (Sigma-Aldrich, USA) was added to a 1.5 mL conical tube on ice, and the hemolymph sample was immediately transferred to the tube. To obtain cell-free hemolymph containing AMPs, the cellular components and debris were removed by centrifuging the sample tubes at 13,000 RPM at 4℃ for 5 min. The recovered supernatant (plasma) was mixed with 10 % acetic acid at a 1:1 ratio at 4℃, and incubated with shaking at 100 RPM overnight. After removing the pellets, the supernatant was collected and stored at -80℃ until used.

Protein samples extracted with 10 % acetic acid were quantified using SIGMA Bradford Assay Reagent (Sigma-Aldrich). Briefly, a standard curve was prepared within the range of 0, 0.25, 0.5, 1.0, and 1.4 mg/mL using BSA (Thermo Scientific, USA). To bring the sample concentrations within the standard curve range, our samples were diluted 1:10, 1:20, and 1:40, then used in the following steps. The 250 µL of Bradford reagent was added to 5 µL of the BSA standard and the samples. Sample concentrations were calculated using a linear regression equation derived from a standard curve. The protein concentrations of pooled hemolymph samples were 15.1 mg/mL and 13.1 mg/mL for the E. coli-challenged and unchallenged larvae, respectively.

Six different animal pathogenic bacteria, common causative agents of intestinal infection and septicemia, were selected to evaluate the antimicrobial activities of the BSFL hemolymph. A. salmonicida subsp. salmonicida strain KACC 14791, L. garviae strain KACC 13444, V. anguillarum strain KCTC 2711, and V. alginolyticus strain 14902 were obtained from KACC, and S. aureus and S. Typhimurium type strains were obtained from the Korean Food and Drug Administration (KFDA). The lyophilized bacterial stocks were recovered and activated by culturing them four times. A. salmonicida, S. aureus, S. Typhimurium, V. alginolyticus, and V. anguillarum were cultured on tryptic soy agar (TSA), whereas L. garviae was cultured on TSA supplemented with 0.5% yeast extract. Bacterial cultures were incubated at 35±2℃ for 16±2 h, except for L. garviae and V. anguillarum, which were incubated for 20±2 h.

To evaluate the antimicrobial activity of BSFL hemolymph, the two experimental groups were tested against six animal pathogenic bacteria, including the common causative agents of intestinal infection and/or septicemia. The agar well diffusion method was used to assess the antibacterial activity of the BSFL hemolymph. After a few single colonies were collected from each bacterial TSA culture, they were suspended and diluted to an OD₆₀₀ =0.1±0.02 in 10 mL PBS. The culture diluents were spread onto TSA plates using sterile cotton swabs. Six 8-mm holes were made on the TSA plates using sterile stainless steel cylinders. Each well was filled with 100 or 200 mg/mL of hemolymph from unchallenged and E. coli-challenged BSFL. Ampicillin (128 µg/mL) and PBS were used as the positive and the negative control, respectively. The plates were then cultured at 35℃ for 24 h, and the zone of inhibition was measured.

All experiments were performed in triplicate. Statistical analyses were performed using SPSS ver. 29.0 S/W (IBM, USA). The inhibition zone diameters generated by the BSFL hemolymph extract was compared by using one-way analysis of variance (ANOVA). Tukey’s HSD or Games-Howell post-hoc tests were performed to test the equality of variances. Spearman’s correlation and linear regression analyses were performed to assess the relationships among antimicrobial activity, hemolymph concentration, and E. coli challenge. A p≤0.05 was considered statistically significant for both ANOVA and Spearman’s correlation.

The E. coli inoculum concentration used for the challenge was 10⁵ CFU/mL. A previous study (Bruno et al., 2021) suggested that 10⁵ CFU/mL was the optimal concentration for BSFL challenge without high mortality during the 72 h required to induce immune responses and AMP expression in E. coli.

The antimicrobial activity of the hemolymph extracted from E. coli-challenged and unchallenged BSFL was evaluated using agar well diffusion tests as shown in Table 1. Compared to the 100 and 200 mg/mL unchallenged BSFL hemolymph, the 200 mg/mL E. coli-challenged BSFL hemolymph showed significantly greater inhibition against five bacteria: S. Typhimurium (p=0.004 and p=0.038, respectively), A. salmonicida (p=0.000 and p=0.000), V. alginolyticus (p=0.010 and p=0.007), L. garvaie (p=0.010 and p=0.007), and S. aureus (p=0.050 and p=0.050). In addition, compared to the 100 and 200 mg/mL unchallenged BSFL hemolymph, 100 mg/mL of E. coli-challenged BSFL hemolymph significantly inhibited the growth of the two bacterial species, A. salmonicida (p=0.000 and p=0.000) and L. garviae (p=0.016 and p=0.016, respectively). The hemolymph of BSFL contains AMPs responsible for antimicrobial activity that are activated and released when infection occurs (Park & Yoe, 2017; Li et al., 2019). However, the AMPs in BSFL hemolymph was not quantified in the present study. Based on our result, higher concentration of the E. coli-challenged BSFL hemolymph presented higher antimicrobial activities. Therefore, the antimicrobial activities is supposed to increase by the amount of AMPs contained in hemolymph in dose-dependent manner.

The hemolymph from unchallenged BSFL also formed inhibition zones against some bacterial species, including S. Typhimurium, V. alginolyticus, A. salmonicida, and S. aureus. However, its antimicrobial effect was relatively weaker than that of the E. coli-challenged ones. V. alginolyticus (p=0.028) and A. salmonicida (p=0.046) showed significantly larger growth inhibition zones with 200 mg/mL unchallenged BSFL hemolymph than with 100 mg/mL hemolymph. When the insect immune system encounters an infection, signaling cascades are serially activated by immune pathways, leading to the production of AMPs and inhibition of pathogen growth in the host (Buchon et al., 2014). Compared to 100 mg/mL of the unchallenged BSFL hemolymph, 200 mg/mL significantly inhibited the growth of S. Typhimurium (p=0.007). Previous studies have suggested that feeding substrates and their dietary components, such as proteins, lipids, and carbohydrates, can affect the production of AMPs and activate immune signaling pathways (Marusich et al., 2020; Guiné et al., 2021). One limitation of this study is that we did not analyze the nutritional composition of the diets fed to BSFL. Several factors, such as metabolic changes, aging, and stressors, can also lead to the expression of AMPs in BSFL (Park & Yoe, 2017; Li et al., 2019; Stączek et al., 2023). In addition, Alvarez et al. (2019) demonstrated that BSFL hemolymph had three to five times greater antimicrobial activity against H. pylori when challenged with E. coli than the hemolymph of unchallenged BSFL. Without immunization, AMP could not be consistently produced in BSFL. Immunization is essential to ensure a certain amount of AMPs.

Positive correlations between the hemolymph concentration and the inhibition zone diameter were found in five of the six bacteria used in our study, except V. anguillarum (Figure 1): A. salmonicida (R²=0.968, p<0.001), S. Typhimurium (R²=0.901, p<0.001), L. garviae (R²=0.850, p<0.001), V. alginolyticus (R²=0.821, p<0.001), and S. aureus (R²=0.619, p<0.005). All regression lines of hemolymph from E. coli-challenged BSFL were much steeper than those from unchallenged BSFL. This suggests that E. coli immunization is an important factor in antimicrobial activity of BSFL hemolymph.

Electrophoretic analysis of hemolymph protein expression in both E. coli-challenged and unchallenged larvae was performed. The results showed the differential expression of low-molecular-weight bands around 15 kDa (Figure 2). A Jin et al. (2022) showed that reconstitution of acetic acid extracts resulted in protein ranging 0 to 40 kDa and the AMPs from BSFL mainly consisted of small proteins such as cecropin (≋7 kDa), attacin (≋20 kDa), defensin (≋10 kDa), and lysozyme (≋15 kDa). Additionally, according to Azmiera et al. (2023), E. coli infection increases the bacteriolytic activity of insect hemolymph, attributed to increased lysozyme production. The highly expressed ≋15-kD band in our study is assumed to be a lysozyme. But the limitation of our study is that the identification of the ≋15-kD overexpressed protein band was not conducted. Future study is needed to identify the protein band expressed in BSFL hemolymph by E. coli immunization using mass spectrometry, N-terminal sequencing, western blotting. The present study found that hemolymph isolated from both E.coli-challenged and unchallenged BSFL exhibited relatively more potent antimicrobial activity against Gram-negative bacteria than against Gram-positive bacteria. The BSFL hemolymph may be used as feed additive to control pathogenic bacteria infection in animal, as well as can be developed as animal health product and/or veterinary drug such as therapeutic pet shampoo and topical pet serum.