Laboratory BSFL was provided by the culturing center of Henan Agricultural University. BSFL was reared until 3rd instar larvae at 30°C with 70% moisture content using wheat bran, and then continuously reared for 10 days using food waste mixed with ∼10% peanut shell powder (25). Then, the food waste conversion rate, food waste consumption rate, and survival rate of BSFL were determined as follows:

W₁ and W₂ are the dry weight of BSFL before and after rearing, respectively; W₃ and W₄ are the dry weight of the substrate before and after BSFL conversion, respectively. N₁ and N₂ are the BSFL survival number before and after rearing, respectively.

After 10 days of rearing, one part of BSFL was dried at 105°C in an oven for 48 h until the weight was stable for the following analysis. The dry weight of BSFL was measured with a thermogravimetric method (26). Then, they were grounded and passed through a 100-mesh sieve for nutrient determination. The crude fat and protein contents of BSFL were measured referencing the national standard of GB/T6433-20 and GB/T6432-1994, respectively (27). Another part of BSFL was dissected to collect their midgut tissues. Then, they were mixed with 1 ml of Tris-HC1 buffers (pH 7.5) and sterile glass beads. The mixture was shaken to grind the intestine thoroughly. After being centrifuged (8,000 r/min, 10 min), the supernatant was used to detect the activity of lipase, cellulase, and amylase, respectively, by the Lipase Activity Assay Kit (Solarbio, China), CL Activity Assay Kit (Solarbio, China), and AL Activity Assay Kit (Solarbio, China), according to manufacturer’s instruction (28). In addition, the protease enzyme activity of the midgut was determined using the Folin-phenol method (29).

Natural BSFLs were first reared on wheat bran until 3rd instar larvae as mentioned in the “Insect Husbandry and Determination of Bio-Physiological Indicators” section. Then, they were divided into two groups, one of which was reared with non-sterile food waste (CK), and the other was reared with non-sterile food waste and 10B (CK + 10B). After 10 days of incubation, BSFL gut and frass were collected separately in both groups, consisting of CK-gut, CK-frass, 10B-gut, and 10B-frass sets, which were subjected to extracting gDNA using the E.Z.N.A. Soil Kit (Omega Bio-Tek, United States) following the instructions. The primers 806R (5′-GGACTACHVGGGTWTCTAAT-3′) and 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) were used to amplify the bacterial V3–V4 region of 16S rRNA genes. PCR reactions were performed as previously described (30). Purified PCR products were analyzed using the Illumina Miseq PE300 sequencing platform (Illumina, United States) (31). All sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA781802. The raw reads were processed according to the standard procedure of Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) (9).

In a 10-ml sterile centrifuge tube, 0.4 g of fresh BSFL eggs were weighed. To disperse the eggs, 1 ml of 2.7% NaClO solution was added by shaking for 1 min to aspirate them thoroughly. After that, 1 ml of Sporgon (Beijing Mingyangkehua Bio-Technology, China) was added. The mixture was shaken for 2 min to aspirate it completely. After leaving for 2 min, the BSFL eggs were washed two times with sterile water. Finally, they were placed on the ultraclean bench for 30 min for drying. Disinfected eggs were inoculated into the brain heart infusion (BHI) medium for further incubation for 24 h at 37°C. The culture was subsequently spread on the agar plates to verify the disinfection effect (32).

Subsequently, 30 sterile BSFLs were inoculated into 45 g autoclaved food wastes without or with 1 ml of 10B suspension (OD₆₀₀ = 1.0) in which the germ-free and monobacterial intestinal BSFL were obtained, respectively. To verify the construction effect of the germ-free and monobacterial intestinal BSFL models, after 10 days of rearing at 37°C, germ-free and monobacterial intestinal BSFLs were dissected. On the one hand, 0.2 g of BSFL midgut and frass were suspended in 1 ml of sterile 0.85% NaCl, respectively, and 50 μl of suspension was spread on the BHI agar medium to verify the disinfection effect. On the other hand, 10 germ-free and monobacterial intestinal BSFLs were homogenized. The QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) was used to extract the gDNA in both sets. The 16S rRNA gene was amplified using universal 1492R/27F primers (33).

To verify the effect of 10B on the host, 3rd instar BSFL were divided into three groups, namely, CK (natural BSFL fed with sterile food waste), GF (germ-free BSFL fed with sterile food waste), and GF + 10B (germ-free BSFL fed with sterile food waste and 10B). After 10 days of rearing, BSFLs in each group were used to determine bio-physiological parameters, such as survival rate, average dry weight of BSFL, substrate consumption rate, and substrate conversion rate as described in the “Insect Husbandry and Determination of Bio-Physiological Indicators” section. Furthermore, the metabolites of BSFL in CK, GF, and GF + 10B groups were all determined by a broadly targeted metabolism strategy. Wuhan MetWare Biotechnology Co., Ltd.¹ assisted with the metabolite extraction, identification, detection, and quantification following the forementioned protocol (34). Each biological sample was checked in three replicates. All sample extracts were mixed and used as the quality control (QC). The metabolites were annotated by the Metware database (MWDB) and other publicly available databases (35).

Principal component analysis (PCA) was carried out to uncover the relationships among the samples based on the identified metabolites. The differentially accumulated metabolites (DAMs) between samples were distinguished by orthogonal partial least squares discriminant analysis (OPLS−DA) using the criteria of variable importance in the project (VIP) ≥ 1 and log₂ (fold change) > 1. DAMs were posted to the corresponding metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. KEGG enrichment analysis was then performed using a clusterProfiler (36). All the data analyses were performed in the R environment.²

Food waste (45 g) was autoclaved in glass bottles. By adding different vitamins or 10B alone, five experimental groups were set up, as follows: (i) GF: 30 germ-free BSFLs; (ii) GF + 10B: 30 germ-free BSFLs and 1 ml 10B (OD600 = 1.0); (iii) thiamine: 30 germ-free BSFLs and 1 ml thiamine (5 mg/ml); (iv) riboflavin: 30 germ-free BSFLs and 1 ml riboflavin (5 mg/ml); (v) biotin: 30 germ-free BSFLs and 1 ml biotin (5 mg/ml). The experiments were performed for 10 days in an incubator at 37°C. Then, BSFLs in each group were used to determine bio-physiological parameters, such as the survival rate, average dry weight of BSFL, substrate consumption rate, and substrate conversion rate, as described in the “Insect Husbandry and Determination of Bio-Physiological Indicators” section.

All experiments were performed with three replications. Data were exhibited as the mean ± SD. Tukey’s test was used to examine the results of bio-physiological indicators, colony counting, and microbial diversity indices. The significant correlation between the bio-physiological indicators and the relative abundance of dominant microbes was determined by Spearman’s analysis. SPSS 16.0 for Windows and the free online platform of Majorbio I-Sanger Cloud Platform³ were used to analyze the statistical differences. Spearman’s rank correlation analysis was used to determine the significant correlation among the relative abundance of dominant microbes.

To evaluate the influence of 10B on the growth and substrate conversion efficiency of BSFL, they were inoculated into food waste. The larval survival rate and substrate consumption rate of BSFL were almost the same between the CK and CK + 10B groups (Figures 1A,B), indicating that the addition of 10B did not affect the survival of BSFL. In addition, the substrate conversion rate of BSFL in the CK + 10B group was improved by ∼5% compared to the CK group (Figure 1B), and thus the average dry weight of BSFL in the CK + 10B group was also increased (Figure 1C). The above results demonstrated that 10B greatly influences the substrate conversion process of BSFL. To further verify the influence of 10B on the nutritional value of BSFL, the nutrient composition of BSFL was presented. The crude protein content of BSFL in the CK + 10B group was increased ∼8% compared to the CK group, while there was no significant difference in crude fat content between them (Figure 1D). This result stated that 10B improved the protein synthesis process in BSFL. Moreover, the activities of the four intestinal digestive enzymes, namely, protease, amylase, cellulase, and lipase, increased by 65.17, 10.11, 37.62, and 31.61%, respectively in the CK + 10B group compared to the CK group (Supplementary Figure 1). This result further demonstrated that the addition of 10B enhanced the activity of the digestive enzymes, especially the protease activity, in the gut of BSFL, and thus elevated substance uptake and protein conversion ability of BSFL.

To explore the effect of 10B on the gut microbes of BSFL, the microbial composition of the gut and frass was characterized using Illumina Miseq sequencing after rearing for 10 days. After trimming and quality filtering, a total of 886,845 sequences with > 99.9% coverage for all samples were generated (Supplementary Figure 2A). The rarefaction curves further showed that all samples were almost approaching the saturation plateau, indicating that microbial communities were represented well (Supplementary Figure 2B). In general, the Shannon index value of frass was higher than that of the gut, indicating that microbial diversity of frass was higher than that of the gut. The Shannon index in both the CK_frass and CK + 10B_frass groups did not differ, while it was much lower in the CK + 10B_gut group compared to the CK_gut group (Figure 2). This result demonstrated that the addition of 10B altered the gut’s microbial diversity instead of the frass in BSFL.

Dissimilarities of the above four groups were investigated using the principal coordinate analysis (PCoA). The first two axes totally explained 86.34% variance of species. In general, three categories were clustered, namely, (i) CK_frass and CK + 10B_frass, (ii) CK_gut, (iii) CK + 10B_gut (Figure 3A). The above results further demonstrated that the addition of 10B greatly influenced the gut microbial composition rather than the frass microbial composition of BSFL. The microbial composition analysis illustrated that the intestinal microbial composition differed from the frass at the genus level. In general, with the addition of 10B, the microbial composition has a massive change in the gut instead of frass. At the genus level, the relative abundance of unclassified_of_Bacillaceae and Graclibacillus in frass was significantly elevated, while the relative abundance of Mohebacter and Corynebacterium was markedly reduced. In addition, the relative abundance of Bacillus, unclassified_of_Caloramatoraceae, and Gracilibacillus was increased in the CK_gut group compared to the CK + 10B_gut group, whereas the relative abundance of Cerasibacillus, Ureibacillus, and Sinibacillus was decreased (Figure 3B). Notably, the relative abundance of Bacillus in the CK_gut group reached 48.39%, which was 12% higher than that in the 10B_gut group (Figure 3B), which indicated the colonization of 10B in the gut.

Spearman’s correlation analysis showed that Bacillus displayed positive correlation coefficients with unclassified_of_Caloramatoraceae and Gracilibacillus, whereas it presented negative correlations with Cerasibacillus, Ureibacillus, and Sinibacillus (p < 0.05). This result indicated that 10B might also affect host metabolism by influencing the relative abundance of these mentioned microbes (Table 1).

The relationship between the microbial community structure and physiological indexes, including the survival rate, substrate conversion rate, dry weight, crude fat content, and crude protein content of BSFL, was revealed by redundancy analysis (RDA). As shown in Figure 4A, the survival rate, substrate conversion rate, dry weight, and crude protein content were significantly and positively correlated to the bacterial community structure of the 10B + CK_gut group. In contrast, crude fat content presented a significantly negative correlation with the microbial community structure of the CK_gut group (Figure 4A). This result demonstrated that 10B influenced the metabolism of BSFL by affecting the structure of the microbial community. To further understand the specific genera’s contribution to the BSFL’s metabolism, the relationship between the 10 most dominant microbes and physiological indexes was analyzed using Pearson’s correlation analysis. Notably, there was a high positive correlation between unclassified_of_Caloramatoraceae, Bacillus, and Gracillibacillus and the survival rate of BSFL, with correlation coefficients of 0.825, 0.872, 0.861, respectively (p ≤ 0.05). In addition, Gracillibacillus also had significant correlations with the substrate conversion rate of BSFL (correlation coefficients = 0.883, p ≤ 0.05). Unclassified_of_Caloramatoraceae showed significant positive correlations with the crude protein content and substrate conversion rate, and the correlation coefficients were up to 0.963 (p ≤ 0.01) and 0.814 (p ≤ 0.05), respectively (Figure 4B). The above findings suggested that 10B directly impacted the host’s survival and influenced BSFL’s metabolism by altering the abundance of other intestinal microbes.

To further evaluate the impact of 10B on BSFL growth and substrate conversion efficiency, it was inoculated into the sterile BSFL system to construct a monobacterial intestinal BSFL model. The germ-free BSFL failed to develop normally, with a survival rate of 34%, whereas the larval survival rate of BSFL reared with 10B only recovered to 100% after 10 days of rearing (Figure 5A). Besides, the substrate consumption rate of BSFL in the GF + 10B and CK groups was increased to 54.35% and 63.67%, respectively, while it only reached 11.91% in the GF group (Figure 5B). The substrate conversion rate of BSFL in the GF + 10B group was elevated to 15.64%, which was only ∼5% lower than BSFL in the CK group (Figure 5B). As a result, the average dry weight of BSFL was increased from 0.01 g (GF) to 0.67 g (GF + 10B) (Figure 5C). In addition, the crude protein and crude fat contents of BSFL in the GF + 10B group reached 20.19% and 43.02%, respectively, while they could not be detected in germ-free BSFL (Figure 5D). Moreover, the activities of four intestinal digestive enzymes, namely, protease, amylase, cellulase, and lipase, were significantly higher in the GF + 10B group than those in the GF group (p < 0.05), but lower than those in the CK group (Supplementary Figure 4). Based on the above results, we hypothesized that 10B could provide BSFL with the nutrients required for survival and facilitate the substrate synthesis and conversion process of BSFL.

To further explore the effect of 10B on BSFL, systematic metabolic profiling of BSFLs in the GF, GF + 10B, and CK groups was carried out. A total of 917 metabolites were identified in the three groups (Supplementary Sheet 1). PCA analysis of the detected metabolites showed that three biological replicates of each sample tended to group together, indicating that the generated metabolic data were highly reproducible. Meanwhile, in a two-dimensional plot, the dispersion pattern of the three groups showed significant differences among metabolite profiles (Figure 6A). To investigate the metabolic differences of BSFL under different rearing conditions, pair-wise comparisons of the metabolites were used to identify the DAMs (Figure 6B), the details of which were listed in Supplementary Sheets 2–4. A similar number of DAMs were identified when comparing the GF_vs._GF + 10B (394) and CK_vs._GF + 10B groups (401). A total of 451 DAMs were discovered in the comparison of the GF_vs._CK group. There are more upregulated DAMS than the downregulated DAMs in the comparison of GF vs. CK groups. In contrast, the number of downregulated DAMs was greater than that of upregulated in the comparison of the GF_vs._GF + 10B and CK_vs._GF + 10B groups. The DAMs identified in the pair−wise comparisons accounted for 42.97–49.18% of all detected metabolites, verifying the rich diversity of metabolites exhibited in BSFL rearing under the three conditions described above. In addition, the highest number of DAMs was detected in the comparison of the GF_vs._CK groups (Figure 6B), indicating a significant influence of intestinal microbes on BSFL.

The KEGG classification analysis revealed that a large proportion of identified DAMs was involved in critical biological pathways, such as vitamin metabolism, vitamin synthesis, protein metabolism, glucose metabolism, fatty acid metabolism, amino acid synthesis, and amino acid metabolism (Figure 7). Notably, the GF_vs._CK group had 23.32% DAMs in the vitamin synthesis and metabolism classification (Figure 7A), indicating vitamin synthetic and metabolic differences between the GF and CK groups. Although 24.99% DAMs in the GF_vs._GF + 10B group were related to the synthesis and metabolism of vitamins (Figure 7B), 10B influenced vitamin synthesis and metabolism in BSFL. Moreover, the CK_vs._GF + 10B group was enriched in fewer DAMs (20.89%) than the other two groups (Figure 7C), implying that 10B plays an important role in BSFL via vitamins. Besides, DAMs were enriched in 60% of amino acid synthesis metabolism classification in the GF_vs._CK group (Figure 7A), indicating that gut microbes strongly affected the amino acid synthesis and metabolism of BSFL. In addition, 74.29% of DAMs were clustered with the amino acid synthesis and metabolism in the GF_vs._GF + 10B group (Figure 7B), suggesting that 10B had a more significant influence on BSFL amino acid synthesis and metabolism in the GF_vs._GF + 10B group. Furthermore, 52% of DAMs were found to be related to the amino acid synthetic and metabolic process in comparison to the CK_vs._GF + 10B group (Figure 7C), indicating that 10B mitigates to some extent the differences in the amino acid synthesis and metabolism of the host. These results also demonstrated that 10B played a vital role in BSFL by affecting the amino acid synthesis and metabolism process. Meanwhile, there were differences in the classification of glucose metabolism, fatty acid metabolism, and protein metabolism among these three groups (Figure 7). This result also implied that 10B might also play a role in BSFL by affecting the above metabolic processes. Furthermore, a total of 12 classes of differentially accumulated vitamins were identified, of which biotin, riboflavin, and thiamine were downregulated in the GF_vs._GF + 10B and GF_vs._CK groups and upregulated in the CK_vs._GF + 10B group (Table 2). This result suggested that 10B may increase the survival of BSFL by altering the uptake and metabolism of the three vitamins mentioned above.

To further verify our hypothesis, 10B, thiamine, riboflavin, and biotin were added to the food waste, respectively. With the addition of 10B or riboflavin alone, the survival rate of BSFL reached 100% and 98%, respectively, which amounted to that of the CK group. This result confirmed that 10B improved the BSFL survival rate through the provision of riboflavin to the host. Although the substrate consumption rate of BSFL reached 50% after the supplement of riboflavin, which was only 10% lower than that of the CK and GF + 10B groups, the substrate conversion rate was much lower than that of BSFL in the CK and GF + 10B groups, resulting in a much lower dry weight of BSFL in the riboflavin-added group than in the CK and GF + 10B groups. This result indicates that 10B also plays a critical role in converting substrate to nutrients.