Damage and recovery of artificial diet rearing silkworms (Bombyx mori L.) exposed to high-concentration florfenicol as well as the dynamic changes of midgut ‌flora after functional bacterium supplementation

Damage and recovery of artificial diet rearing silkworms (Bombyx mori L.) exposed to high-concentration florfenicol as well as the dynamic changes of midgut ‌flora after functional bacterium supplementation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Damage and recovery of artificial diet rearing silkworms (Bombyx mori L.) exposed to high-concentration florfenicol as well as the dynamic changes of midgut ‌flora after functional bacterium supplementation Yating Liu, Zhongwen Liu, Chunjiu Ren, Huiju Gao, Shuangxin Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8174585/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Mar, 2026 Read the published version in BMC Microbiology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Florfenicol (FF) is widely used in animal husbandry due to its broad-spectrum bactericidal activity, while there is little research focused on its toxic effects on the silkworm( Bombyx mori L.), a model organism. Result It was demonstrated in this paper that high-concentration florfenicol exposure significantly inhibited the activities of digestive and antioxidant enzymes, reduced the body weight and cocooning ability of silkworms, prolonged the instar duration, and simultaneously led to organelle swelling and vacuolization in the midgut, a large number of autophagosomes, and nuclear condensation. Meanwhile, it was found that exposure to FF reduced the α diversity and richness of the midgut flora, leading to a severe loss of core microbiota. The functional bacterium, Bacillus velezensis LY5, which was demonstrated that it significantly also improved the body weight and cocoon quality of silkworms exposed to FF in this study, accelerated the repair of midgut tissue damage, helped recover the abundance of core midgut flora, increased the proportion of potentially beneficial bacteria, and reduced the excessive proliferation of opportunistic pathogens. Conclusion This study reveals the toxicological mechanism by which high concentrations of antibiotics (FF) disrupt the midgut microbiota balance in silkworms, induce tissue damage, and subsequently impede their growth and development. It also demonstrates for the first time that functional probiotics can effectively reverse this damage by restoring the silkworm midgut microbiota and intestinal tissue Florfenicol Bombyx mori L. artificial diet rearing damage and recovery midgut flora Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Florfenicol (FF), as a new generation of broad-spectrum antibacterial drug belonging to the chloramphenicol class, has been gradually introduced into the field of bacterial disease prevention and control in livestock farming in recent years, to a large extent due to its advantages of high efficacy, low drug resistance and safety. Although FF can deal with the bacterial disease, it may also lead to other issues, such as drug residues and microbial ecological disturbance. FF exerts its antibacterial effect by targeting the 50s subunit of the 70s ribosome in bacteria, and finally prevents protein synthesis. Its broad-spectrum bactericidal activity covered a lot of common pathogens [ 1 ]. Despite its significant advantages in pathogenic control in animal farming༻2༽, the toxic effects of FF should not be neglected. For instance, FF induced embryonic malformations in sea urchins and exerted a direct impact on the gonad ༻3༽, caused liver inflammation and oxidative stress damage in chickens ༻4༽. The glucose metabolism of zebrafish was disrupted, leading to abnormal blood glucose and glycogen levels, after being exposed to a water environment containing FF for 28 days ༻5༽. It was reported that the damage to the symbiotic bacterial flora, as well as the loss of core microbiota and a significant reduction in diversity was observed due to the broad-spectrum bactericidal activity of FF [ 6 ]. Gut microbiota was a complex microbial community colonizing the intestines of the host, comprising various microorganisms such as bacteria and fungi, which formed a symbiotic relationship with the host and played a crucial role in host health༻7༽. The imbalance of gut microbiota might further amplify the physiological stress of the host. Exposure of high-concentration FF significantly reduced the diversity and richness of zebrafish gut microbiota ༻8༽. The intestinal dysfunction and dysbiosis of the gut microbiota were observed when the Chinese mitten crab was exposed to FF in water ༻9༽. Bombyx mori L. is an important economic insect and at the same time, a model organism. Bacterial infections caused by Staphylococcus , Enterococcus , and other pathogenic bacteria often led to large-scale mortality of silkworms, resulting in a reduction of cocoon production by up to 30% to 50%, which posed a serious threat to cocoon yield and quality [ 10 ]. Although the toxicity of FF to various organisms was extensively studied, its impact on lepidopteran insects has been less explored. Previous studies had shown that high concentrations of FF altered the gut microbiota structure of silkworms, delayed their growth and development, and affected their midgut tissue structure and immune response༻11༽. Current research has primarily focused on the changes in the microbiota during the medication period, while it was also crucial to analyze the influencing factors of microbiota recovery after drug withdrawal. Many probiotics, as a beneficial external intervention, could recover microbial ecological disorders caused by external factors, thereby reestablishing a new intestinal microbial homeostasis. Luo K [ 12 ] observed the recovery of intestinal microbiota communities in shrimp after being exposed to FF through various recovery methods (adding probiotics and natural recovery), and found that the abundance of potentially pathogenic bacteria reduced and at the same time, the abundance of potentially beneficial bacteria increased when probiotics were used. It was also reported that fecal microbiota transplantation accelerated the recovery of intestinal microbiota communities in koi fish exposed to FF, repaired intestinal damage, and normalized key metabolic pathways ༻13༽. The antibiotic-associated diarrhea (AAD) in mice alleviated, at the same time the intestinal barrier integrity improved, and the immune response enhanced after Bacillus DU-001 was used as an additive agent༻14༽. So far, there were few reports focused on the recovery of intestinal microbiota disruption caused by FF exposure in silkworms. This study was for the first time designed to systematically explore the damage and the recovery of the midgut of silkworms reared on artificial diet under conditions of sterile control, natural recovery, and functional bacterial intervention after exposure to high concentrations of FF by means of activity detection of digestive enzyme and antioxidant enzyme, TEM observation of midgut cell ultrastructure, dynamic changes analysis of midgut flora, whole-genome sequencing and functional prediction. The aim of this research was to explore the possible mechanisms of damage and recovery in model organism Bombyx mori L. exposed to FF, provide a basis for the effective application of FF in animal husbandry, especially in silkworm rearing, and to pave the way for the large-scale application of probiotic supplementation for silkworm gut health. Materials and Methods Bacterial strains and culture conditions The NCBI accession number for B. velezensis LY5 used in this paper was PRJNA1265109. The strain was cultured in LB liquid medium at 37°C for 12 hours. The precipitate was collected by centrifugation (5000 rpm, 10 min), and rinsed 3 times with normal saline, then suspended at a concentration of 10⁷ CFU mL⁻¹ for later use. Test silkworms and diet The test silkworm variety was Youshi No. 1, which exhibited high adaptability to artificial diet. Silkworm larvae were reared under conditions of 70–80% humidity, a 12-hour dark/12-hour light cycle, and a temperature of 27 ± 1°C. The artificial diet used in this study was M20 (composed of mulberry leaf powder, soybean meal powder, corn flour, vitamins, etc., heated at 100°C for 60 minutes). All-age diet rearing was adopted. Treatment Silkworms at the 4th instar were divided into CK, CY, and FF groups. The basic artificial diet was the M20. The CK group was fed on M20 diet, the CY group was fed on M20 supplemented with B. velezensis LY5 (10⁷ CFU/10g), and the FF group was fed on M20 mixed with high-concentration FF (0.15 g/100 g, Shanghai McLean Biochemical Technology Co., Ltd.). After rearing the silkworms to the 5th instar, the FF group was randomly divided into three groups: FFw (sterile group, maintained a sterile state as much as possible), FFz (natural recovery group), and FFy (10⁷ CFU/10g of B. velezensis LY5). Each group had three replicates, with 150 silkworms per replicate. Measurement of the body weight and cocoon quality Silkworms were randomly selected at the beginning of the 5th instar and on 3rd day of the 5th instar for weighing. Each group was replicated 3 times, with 20 individuals per replicate (3n, n = 20). On the 7th day after cocooning, cocoon quality surveys were conducted with 3 replicates per group, each comprising 10 silkworm cocoons (3n, n = 10). Measurement of the enzyme activity 5 silkworms were randomly selected per group, with 3 replicates. After 12 hours of starvation, the silkworms were placed on ice until anesthetized. After dissection, midgut fluid was collected and centrifuged at 4000 rpm for 5 minutes. The activities of α-amylase and trypsin were determined using assay kits from Nanjing Jiancheng Bioengineering Institute. Lipase activity was measured employing an assay kit from Puyinte Biotechnology Co., Ltd. 15 silkworms per group with 3 replicates were pretreated as described above. Hemolymph was collected and stored on ice. Enzyme activities for POD, SOD, and CAT were immediately measured using commercially available kits (all purchased from Beijing Solarbio Technology Co., Ltd.). Ultrastructural analysis of midgut tissue The midgut tissue fragments were dissected as illustrated above, further cut into small pieces, placed in the fixative for 2 hours, then stored at 4°C. Tissue cell samples were resin-embedded and sectioned to prepare tissue slides. Following staining with heavy metals lead and uranium, the specimens were observed under TEM (Wuhan Savier Biotechnology Co., Ltd.). Dynamic Analysis of Gut Microbiota The 5th instar stage larvae (1d, 2d, 3d and 5d) of CK, FF group and FFy group, were subjected to 12 hours of starvation. Midguts were dissected and isolated, washed twice in PBS, rapidly frozen in liquid nitrogen, and stored at -80°C for future use. Sequencing libraries were prepared using the TruSeq Nano DNA LT Library Prep Kit (Lianchuan Biotechnology Co., Ltd.). Sequence denoising was performed using QIIME2 D2DA2. This yielded representative, biologically accurate Amplicon Sequence Variants (ASVs) and ASV abundance tables for subsequent midgut flora analysis. Whole-genome sequencing B. velezensis LY5 was cultured in LB 12 hours, centrifugated at 5000 rpm for 3 min. DNA was extracted using a bacterial DNA extraction kit (TianGen Bio-Technology Co., Ltd.) and sent to LianChuan Bio-Technology Co., Ltd. for sequencing. Raw data obtained after sequencing were filtered using fastp (Version: 0.23.2) to obtain cleanData, which was then processed for genome assembly and annotation. Statistical Analysis Statistical analyses were conducted in the GraphPad Prism 9.0 program. Independent samples t-test was used to analyze the data between different groups. Data from all experiments are presented as mean values ± SD. Statistical significance is indicated as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Results FF Significantly Inhibited the Growth and Development of Silkworms Significant growth and development inhibition was exhibited in the silkworms of FF group after 4 days (all ages of 4th instar) of exposure to high-concentration FF. Compared to CK, the average larvae weight in FF group at the initial stage of 5th instar was 10.9g, a significant decrease of 8.53% ( P = 0.001, Fig. 1 a). Enzyme activity assays showed that the activities of SOD, CAT, and POD enzymes respectively decreased by 20.21%, 60.33%, and 39.05% ( P < 0.001). Meanwhile, the activities of amylase, lipase, and trypsin related to digestive function decreased by 47.78%, 56.52%, and 19.83%, respectively ( P < 0.001). Both digestive and antioxidant enzyme activities showed significant reduction (Fig. 1 b-g). Additionally, the 4th instar stage of the FF group was extended by 2 days, significantly prolonging the developmental duration (Fig. 1 h). It was revealed that significant changes of the ultrastructure of midgut tissue occurred in the FF group compared to the normal structure in CK by transmission electron microscopy (TEM) (Fig. 2 CK). The prominent changes included karyopyknosis, mitochondrial swelling, Golgi and endoplasmic reticulum swelling and vacuolization, disruption of tight junctions between cells, and the formation of numerous autophagosomes (Fig. 2 FF). Environmental Microorganisms Played a Crucial Role in the Recovery of Silkworms from Damage to FF Exposure A comparison between the natural recovery group (FFz) and the sterile rearing group (FFw) revealed that the average body weight of 20 individuals of 5th-instar in FFz group was 40.3 g, which was significantly higher by 15.09% than that of the FFw group, still significantly lower than that of the CK group ( P < 0.01; P < 0.001; Fig. 3 a). Investigation into cocoon quality indicated that the FFz group exhibited significant improvements in whole cocoon weight, cocoon shell weight, and cocoon shell rate ( P 0.05; Fig. 3 b-d), compared to the FFw group. The recovery effect of FF damage in the natural recovery group was significantly better than that in the sterile rearing group, indicating that microorganisms in the environment played a crucial role in facilitating the recovery of FF damage. Dynamic Changes of the Midgut Flora in Silworms during Natural Recovery Process 16S sequencing technology was used to investigate the dynamic shifts in the midgut flora of the natural recovery silkworms on 1, 2, 3 and 5 days of 5th instar after FF damage in 4th instar. The Simpson index indicated that the diversity of the midgut flora in CK fluctuated slightly due to the feeding of 5th instar silkworms, while the diversity in the FFz group significantly decreased. The Chao1 index showed that the bacterial richness in CK remained relatively stable, while after exposure to FF, the bacterial richness in the FFz group experienced a steep decline and then significantly increased to a level much higher than that of the CK group, clearly influenced by the antibiotic FF (Fig. 4 a). It was revealed by the PCoA plot that significant differences in the midgut flora between the FFz group and CK (R = 0.6103, P = 0.001;Fig. 4 b upper image), while the flora differences were not significant on 5d among the FFz group (R = 0.1259, P = 0.103; Fig. 4 b lower image). Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, and Latescibacteria were the dominant phyla in both CK and FFz groups by dynamic analysis of the relative abundance of the top 30 phyla at the phylum level. Firmicutes was the absolutely dominant phylum in CK, however, it was severely absent in the FFz group. The relative abundance of Firmicutes in the FFz group increased significantly from 2d, while the relative abundances of other dominant phyla such as Proteobacteria continued to decrease with the recovery time (Fig. 4 c). As for the relative abundance of the top 30 genera, Enterococcus was the absolutely dominant genus in CK, with its quantity slightly fluctuating due to the large amounts of food intake during the peak feeding period. Enterococcus was commonly found in the gut and included more than 30 beneficial and harmful bacterial species. In the FFz group, Enterococcus did not even appear in the top 30 dominant genera on 1d. It showed a significant increase on day 5, although it still did not reach the CK level (Fig. 4 d). This indicated that the recovery of the midgut flora did not complete within the limited 5 days, and the recovery from FF damage was a relatively lengthy process. The Venn diagram showed that the number of ASVs in the FFz group surged, increasing by twice compared to CK on 5d (Fig. S1 ). It was found that the differential marker bacterial genus were group-specific with different treatments and different times, by LDA analysis and LefSe analysis (Fig. S2). The top 15 characteristic bacterial genera in each group were screened out using the random forest algorithm. It was found that the composition of characteristic bacterial genera in FFz group was significantly differentiated compared to CK. There were only 6 overlapping bacterial communities between the two groups. The data indicated that the Mean Decrease Accuracy (MDA) value of Bacillus was the highest in CK, indicating the highest importance of Bacillus in CK. In the FFz group, the top-ranked genus was the infectious pathogenic bacterium Escherichia-Shigella , demonstrating that the colonization of conditional pathogenic bacteria significantly increased during the recovery period after FF exposure (Fig. 4 e). The Functional Bacterium B. velezensis LY5 Significantly Improved the Body Weight and Cocoon Quality of Silkworms Exposed to FF Since Bacillus occupied the most important position in silkworm midgut based on the biomarker results in CK, we chose B. velezensis LY5 in our laboratory as an exogenous functional bacterium to intervene and mitigate the negative effects on silkworms after high-concentration FF exposure. The FFy group, which was fed with the functional bacterium B. velezensis LY5, exhibited a significant growth-promoting effect during the recovery period. The body weight of the FFy group on 3d in the 5th instar was significantly increased by 6.13%, compared to CK ( P 0.05; Fig. 5 b-d). Functional Bacterium B. velezensis LY5 Accelerated the Repairation of Damaged Midgut Cell Structure The midgut tissue of the FFz group still contained a large number of autophagosomes, with swollen and vacuolated mitochondria, and nuclear condensation by TEM observation on 5d after exposure to FF. While the cell structure in the FFy group was intact, with tight junctions between cells, and no autophagosomes were observed in the field of view (Fig. 6 ). This indicated that the structural damage to the midgut caused by high concentrations FF continued to affect physiological functions during the recovery period, and natural recovery could not repair the intestinal tissue damage in a short period of time. However, the functional bacterium B. velezensis LY5 significantly promoted the repairation of the ultrastructure in the silkworm midgut. The Functional Bacterium B. velezensis LY5 Enhanced the Enzyme Activity of Silkworms The activity of key antioxidant enzymes (CAT, POD, SOD) and digestive enzymes (Lip, Amy, Try) were determined. The activities of CAT, POD and SOD in both FFz group and FFy group continued to increase with the extension of recovery time, and the FFy group increased significantly higher than that in the FFz group ( P < 0.01; P < 0.001; Fig. 7 a-c). The addition of the functional bacterium B. velezensis LY5 significantly reduced the number of deaths during the cocooning period of silkworms (Fig. 7 d), which presented the conjecture that there was a close relationship between the increased activity of antioxidant enzymes and the reduction in mortality rate. There were no significant differences among the three digestive enzymes on 1d and 2d of recovery, indicating a lag effect in the recovery of digestive function. Significant differences were observed in amylase and lipase on 3d and 5d, and the FFy group showed a significant increase ( P < 0.01; P < 0.001; Fig. 7 e,g). Meanwhile, trypsin activity in FFz group significantly increased on 3d and 5d, significantly higher than that in the FFy group (Fig. 7 f). It was the addition of B. velezensis LY5 which significantly improved antioxidant capacity and regulated digestive function. The Functional Bacterium B. velezensis LY5 Improved the Disrupted Midgut Flora of Silkworms Injured by FF The Simpson index indicated that the diversity of midgut flora in FFy group remained at a low level as recovery time passed. The Chao1 index revealed that after a dramatic decrease, the richness of the midgut flora in group FFy increased from low to high and gradually approached the CK level on day 5, however, the midgut flora richness in group FFz still remained in a radical change state (Fig. 8 a). This suggested that the supplementation of B. velezensis LY5 contributed to the recovery of the midgut flora in silkworms. PCoA analysis revealed a significant segregation between CK and FFy group (R = 0.6593, P = 0.001; Fig. 8 b upper image). However, unlike the FFz group, the FFy group also exhibited segregation over time (R = 0.4724, P = 0.002; Fig. 8 b lower image). Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria and Latescibacteria were also the dominant phyla in the FFy group, while they exhibited significant inter-group differences over time. The relative abundance of the core phylum Firmicutes in the FFy group increased rapidly from 3d, and by 5d, both the core phylum Firmicutes and the sub-core phylum Proteobacteria had essentially returned to the CK level. It was speculated that functional bacterium might assist in the recovery of the midgut symbiotic flora through competitive inhibition or metabolic regulation. From the relative abundance changes in the top 30 genera, we discovered a close correlation between key functional bacterial genera and external interventions. B. velezensis LY5 intervention in FFy group significantly activated specific functional bacterial genera in the midgut. The core genus Enterococcus rapidly increased from 3d and was already close to CK level by 5d. During this period of time, the sub-core genus Lactiplantibacillus was also increased, which was very close to the CK level (Fig. 8 c). We draw a conclusion that the supplementation of the functional bacterium B. velezensis LY5 significantly accelerated the recovery rate of the core bacterial community in the midgut of silkworms. The differential marker bacterial genus were group-specific with different treatment and different time by LefSe analysis (Fig. S3). Screening of biomarkers in FFy group using the random forest algorithm revealed that, compared to CK, only 3 of the top 15 bacterial genera were shared between the two groups (Fig. 8 d). This indicated that the exogenous addition of B. velezensis LY5 altered the midgut flora distribution after FF exposure. The genus stenotrophomonas , which mostly consisted of harmless bacterial species, had the highest MDA value. Through the analysis of the flora correlation heatmap based on top 30 bacterial genera, it was found that the important bacterial genus Bacillus in the midgut of silkworms had significant interactions with multiple bacterial genera (Fig. 8 e). Methyloversatilis and Acinetobacter were positively correlated with Bacillus , while Acinetobacter showed a significant positive correlation. In fact, Acinetobacter was a conditional pathogen. We supposed that there might be a functional association of co-metabolism or synergistic degradation of organic matter between Bacillus and Acinetobacter , which controlled the associated activities of these conditional pathogens. Meanwhile, Levilactobacillus was significantly negatively correlated with Bacillus , which played an important role in regulating the gut microbiota. The Impact of Functional Bacterium B. velezensis LY5 on Physiological Indicators and Midgut Flora of Normal Silkworms To investigate the effects of B. velezensis LY5 on healthy silkworms, we conducted a two-day short-term feeding test from 1d of the 5th instar (referred to as the CY group). The results showed that the body weight of silkworms in CY group increased by 37.6% compared to CK, with the total cocoon weight and the cocoon shell weight increasing ( P > 0.05; P < 0.05; P < 0.01; P < 0.001; Fig. 9 a). However, there was no significant improvement in the cocoon shell rate. It was demonstrated that there was no significant difference in the midgut flora in α diversity between CK and CY group by high-throughput identification at 3d of the 5th instar (Fig. 9 b). The Venn diagram indicated that CK group had 101 unique genera, while CY group had 116 unique genera, with a total of 185 genera shared between the two groups (Fig. S4). The PCoA diagram revealed that there were no significant differences in the overall midgut flora structure between CY group and CK (R = 0.125, P = 0.772; Fig. 9 c). Further analysis of the differences in gut bacterial composition between the two groups was carried out. Compared to CK, there were no significant differences in the main core bacterial genera in CY group, with only slight changes in the relative proportions of a few genera (Fig. 8 d). The LEfSe method was used in order to identify the differential abundance biomarkers between the two groups (Fig. 9 e). The results showed that specific bacterial taxa were enriched in both CY and CK. The significantly different bacterial group in CY was Acetobacter , while the significantly different bacterial group in CK was Weissella , both possessing probiotic potential. Therefore, although short-term supplementation with B. velezensis LY5 triggered changes in the abundance of some genera, it did not significantly alter the overall structure of the silkworm midgut flora. Whole-genome Sequencing and Functional Prediction of B. velezensis LY5 Sequencing was conducted using the Nanopore sequencing technology platform to elucidate the molecular basis of the vitality and potential metabolites of B. velezensis LY5, with the explanation of how B. velezensis LY5 worked in the repair of FF damage. A total of 1,364,847,346 bp of effective sequencing data (Clean data) was obtained, and the genome contained one circular chromosome and one unclassified plasmid. The chromosome was 4,283,909 bp in length, with a GC content of 45.81%. Through isDDH and ANI analysis, the taxonomic status of the functional strain LY5 was confirmed as B. Velezensis (Fig. 10 a). Gene prediction indicated that strain B. velezensis LY5 had a total of 4409 genes, accounting for 89.57% of the genome, with 4205 CDS, 86 tRNA, 1 tmRNA, 9 16sRNA, and 90 misc_rna. Gene annotation revealed that 3057 genes were annotated in KEGG, 2800 genes in GO, and 3173 genes in COG (Fig. 10 b). According to the annotation results, B. velezensis LY5 could synthesize α-amylase, lipase, and cellulase, and possessed multiple vitamin synthesis pathways. This suggested that B. velezensis LY5 had the potential acting on digestion and promote absorption in the host. The CAZy database showed 46 glycosyl hydrolases (GHS), 34 glycosyltransferases (GTs), 3 polylactones (PLs), 16 chitinases (Ces), 6 acetylases (Aas), and 4 carboxylesterases (CBMs). Secondary metabolism gene cluster analysis revealed that the strain contained 10 secondary metabolites, among which six, namely difficilein, bacilysin, surfactin, butirosin A/butirosin B, macrolactin H and fengycin, exhibited antibacterial and antiviral activities. Therefore, B. Velezensis LY5 also owned the potential to assist the host in resisting potential pathogens. Discussion FF significantly inhibited the growth and development of silkworms Antibiotics, as an important tool for preventing and treating bacterial diseases, have attracted more attention due to their broad-spectrum antibacterial properties, which can also cause side effects in organisms. Florfenicol (FF) is a broad-spectrum antibacterial antibiotic widely used in animal husbandry [ 15 ]. Former studies have shown that antibiotics could interfere with animal metabolism and had a negative impact on organism quality ༻16༽. This study demonstrated that high-concentration FF exposure significantly reduced the body weight of silkworms rearing on artificial diet at the 5th instar stage, and delayed their instar duration by up to 2 days. In the natural recovery group from FF damage, both female and male, their silkworm cocoon shell weight and total cocoon weight were significantly lower than those in the control group (CK), while the difference in cocoon shell ratio was not significant. Electron microscopy results revealed that the midgut cell structure of silkworms in CK was tightly packed and intact, with no abnormal changes. However, in the FF-treated group, the midgut tissue of silkworms exhibited mitochondrial swelling, endoplasmic reticulum and Golgi apparatus swelling, dense vacuolization, and the formation of numerous autophagosomes, indicating significant changes in the midgut structure. Autophagy was a ubiquitous process in eukaryotes. The emergence of a large number of autophagosomes could severely affect cellular function [ 17 ]. Mitochondria were often damaged due to oxidative stress ༻18༽. Similar pathological damage was observed in mice༻19༽, zebrafish༻20༽, and laying hens༻21༽ exposed to high concentrations FF, suggesting that FF had tissue-damaging capabilities across multiple species. FF reduced immune response and induced intestinal inflammation by inhibiting the proliferation of immune cells [ 22 ]. As an important model insect, the antioxidant enzyme system of Bombyx mori L. was highly susceptible to external interference, and the key enzymes of this system such as CAT, POD, SOD, effectively resisted intestinal oxidative stress༻23༽. It was reported that FF led to the accumulation of a large amount of reactive oxygen species (ROS) by inhibiting the activity of antioxidant enzymes, thereby causing tissue oxidative damage༻20༽. Our study also confirmed that FF treatment significantly reduced the activity of CAT, POD and SOD in the midgut of Bombyx mori L., which in turn led to a decrease in body weight and an increase in mortality. Some studies reported that the decline of antioxidant enzymes in the midgut of Bombyx mori L.caused mitochondrial damage, further leading to apoptosis༻24༽. The changes in midgut ultrastructure, especially the mitochondrial swelling and deformation caused by FF in our study, gave further support to this conclusion. Digestive enzymes played a crucial role in the metabolism and absorption of nutrients during the larval stage of silkworm [ 25 ]. In this study, the enzyme activities of three major digestive enzymes, α-amylase, lipase and trypsin in FF group, were significantly reduced. α-amylase promoted the enzymatic hydrolysis of carbohydrates in the intestine༻26༽, lipase broke down dietary lipids into free fatty acids and glycerol༻27༽, and protease was highly expressed in the midgut and was crucial for protein digestion༻28༽. Therefore, we believed that the inhibition of digestive enzyme activity by FF might be a key factor leading to slow growth and developmental delay in silkworms. The functional bacterium B. velezensis LY5 accelerated the recovery of FF damage in silkworms rearing on artificial diet Currently, there are few studies focused on the damage recovery in the silkworm, a model organism, despite numerous works on the effects of FF on various organisms. It was mentioned that piglets required a recovery time of more than approximately 1 week after antibiotic treatment [ 29 ]. Similarly, mice required over 6 weeks for recovery༻30༽. However, our study discovered that after 5 days of recovery in the natural recovery group (FFz), there were still numerous autophagosomes and mitochondrial vacuolization in the midgut tissue. The results indicated that the body weight of the FFz group was still significantly lower than that of CK. The cocoon quality survey found that both the cocoon shell weight and total cocoon weight were still significantly lower than those of CK. Therefore, it was fully demonstrated that the natural recovery of silkworms after FF damage required a relatively long time. This study found that during the recovery period after FF exposure, environmental microorganisms were a key factor affecting the reconstruction of the midgut flora as well as the recovery of growth and development in silkworms. The recovery of the organism after antibiotic treatment often depended on diet and environmental microorganisms [ 31 ], and environmental microorganisms were often considered to have a close interaction with the gut microbiota༻32༽. We set up a sterile rearing group (FFw) as a control and found that although the growth and development indicators of FFz group were still significantly lower than those of CK, its recovery effect was significantly better than that of the FFw group. This indicated that during the recovery period after FF exposure, the microbial communities present in feed and the environment was a key factor in promoting the later growth and development of silkworms. Although functional bacteria have been extensively studied in the field of silkworm [ 33 ], there were no relevant reports on antibiotic damage recovery. Functional bacterium B. velezensis LY5 in our laboratory was used as exogenous additives. It was found that this functional bacterium effectively promoted the growth, development, and cocooning ability of silkworms exposed to FF. The activities of amylase and lipase in both FFz and FFy groups continued to increase with the extension of recovery time. Except for trypsin, the increase in the FFy group was significantly higher than that in the FFz group. The three antioxidant enzyme indicators, CAT, POD and SOD, were significantly elevated in the FFy group. Therefore, the addition of functional bacterium B. velezensis LY5 significantly improved the antioxidant capacity and regulated digestive function of silkworms after FF exposure. At the same time, the midgut tissue of the FFy group with added B. velezensis LY5 was closer to that of CK, with a compact structure and no abnormal vacuoles. Thus, the exogenous addition of B. velezensis LY5 effectively promoted the repair of the silkworm midgut and significantly shortened its damage recovery time. Previous studies have shown that the addition of probiotics effectively enhanced antioxidant enzymes, and Bacillus licheniformis repaired intestinal damage in antibiotic-treated mice༻34༽, which was consistent with the results of our study. Further study in our laboratory found that short-term feeding of B. velezensis LY5 to ordinary silkworms also promoted their growth and development. High-throughput results showed that brief feeding of functional bacterium did not significantly alter the midgut flora structure of silkworms. Compared to CK, the FFy group, ordinary silkworms fed with B. velezensis LY5, exhibited different bacterial composition at the genus level. Specific bacterial taxa were enriched in both FFy and CK by using the LEfSe method to identify differential abundance biomarkers between the two groups. The significantly different bacterial group in FFy was Acetobacter , while the significantly different bacterial group in CK was Weissella . Previous studies have shown that microbial metabolites and derivatives affected organisms themselves [ 35 , 36 ]. For example, Lactobacillus metabolites affected the viability of colon cancer cells༻37༽. Therefore, we speculated that it was the metabolites of B. velezensis LY5 which might be one of the reasons for promoting silkworm growth. The analysis of the whole genome, especially the ANI and isDDH analysis results, further confirmed that the functional bacterium LY5 belongs taxonomically to B. velezensis . Meanwhile, multiple vitamin synthesis pathways and various digestive enzyme genes were presented in the genome of B. velezensis LY5, which was consistent with the findings that the addition of B. velezensis LY5 exogenously enhanced digestive enzymes and significantly increased the body weight of silkworms. Antimicrobial peptides played a crucial role in maintaining the homeostasis of insect gut microbiota and the immune system. Former studies have shown that the addition of probiotics improved the antioxidant capacity and antimicrobial activity of silkworms [ 38 ]. The whole genome indicated that B. velezensis LY5 produced secondary metabolites such as difficilein, bacilysin, surfactin, butirosin A/butirosin B, macrolactin H, and fengycin. Therefore, B. velezensis LY5 had antibacterial and antiviral effects and helped the host inhibit potential pathogenic bacteria in the gut. This indicated that B. velezensis LY5 might synthesize antimicrobial substances to protect the gut microbiota and inhibit the excessive proliferation of harmful bacteria such as enteric conditional pathogens. Dynamic changes of midgut flora during the recovery period of FF damage in silkworms reared on artificial diet The gut microbiota of silkworms reared on artificial diet was relatively stable during the early stage of the 5th instar [ 39 ]. However, antibiotics disrupted the gut microbiota, leading to ecological imbalance and functional disorder, and altering the diversity of the microbiota༻40,41༽. Short-term exposure to antibiotics had long-term effects on gut microorganisms༻42༽. Previous studies have reported that the addition of antibiotics increased diversity༻43༽, while some studies suggested that antibiotics did not alter or reduce the diversity of the gut microbiota༻44༽. In our study, it was found that the diversity of the midgut flora in CK showed a slight downward trend followed by a recovery during the 5th instar period. We speculated that this change might be closely related to the feeding behavior of silkworms during the instar stage. Influenced by environmental microorganisms, the dominant midgut flora proportion slightly decreased and the diversity of the flora declined as the silkworms took abundant food from the 5th instar. After the peak feeding period, the silkworms reduced their food intake, and the proportion of the original dominant flora increased, leading to a recovery of the diversity of the flora to its original level. However, in the FFz and FFy groups, the diversity of the midgut flora significantly decreased during the recovery period after FF exposure compared to CK and remained at a low level. The proportion of the dominant flora was greatly reduced, which was consistent with the results reflected by the top 30 phyla and top 30 genera data. The core genus Enterococcus did not even appear on the top 30 genera list on 1d of FF damage. As for the richness of the flora, CK group remained very stable, while the FFz and FFy groups showed a steep decline after exposure to FF, indicating that the impact of antibiotics on the gut microbiota was devastating. The FFz group showed a continuous increase in richness during the recovery period, while the FFy group rapidly increased to the CK level on 5d. It was the intervention of functional bacterium who kept the richness of the midgut flora in the FFy group at a lower level on days 1, 2 and 3. As for the genus level, compared to CK, the recovery of the core genus Enterococcus was significantly better in the FFy group than that in the FFz group. The Venn diagram showed that the number of ASV in FFz and FFy groups surged on 5d, increasing twice compared to CK, which was consistent with the previous research result that the addition of antibiotics led to excessive growth of the microbiota. Additionally, the exogenous addition of probiotics significantly altered the diversity and community structure of the gut microbiota of organisms [ 45 , 46 ]. Our β diversity analysis revealed a clear separation between FFz group and FFy group in terms of community structure compared to CK. The community structure of FFz group did not exhibit significant changes within 5 days, and remained nearly stagnant during this period, indicating a prolonged recovery time after disruption of FF. Conversely, FFy group exhibited significant changes in flora structure from 2d onwards, with a pronounced separation between the 5d and 1d flora structures. It was speculated that the addition of the functional bacterium B. velezensis LY5 accelerated the recovery and reconstruction process of the tested silkworm midgut flora. The Firmicutes, Proteobacteria, Actinobacteria, and Cyanobacteria were the common dominant phyla in all three groups in this paper, while their relative proportions varied significantly. In CK group, Firmicutes had always been the absolutely dominant phylum; in FFy and FFz groups, FF exposure led to a severe depletion of the core phylum, Firmicutes. And the relative abundance of Firmicutes began to show a significant upward trend from 3d and 2d, respectively. At the same time, the relative abundance of other dominant phyla (Proteobacteria, Actinobacteria, etc.) decreased simultaneously, giving way to the Firmicutes phylum. Firmicutes possessed a variety of functional strains that played an important role in maintaining the microbiota homeostasis [ 47 ], containing various drug-resistant strains. As for the genus level, CK group maintained a relatively stable flora structure, with Enterococcus as the core dominant genus. However, its relative abundance slightly decreased from 2d to 3d, possibly related to a large amount of food intake during this period of time. In FFz and FFy groups, the relative abundance of the core genus Enterococcus was on an upward trend. The core genus Enterococcus in FFy group was closer to that in CK by 5d, indicating better recovery of the midgut flora. The top 15 characteristic genera in each group was screened out using the random forest algorithm. Compared to CK, the characteristic genera was significantly differentiated, and Escherichia-Shigella became the most dominant characteristic genus in FFz group. This genus was often considered a pathogenic bacterium that caused various infectious diseases༻48༽. Bacillus was the most influential genus in normal silkworms—this was also the reason why we chose B. velezensis LY5 as an exogenous functional bacterium in this paper. Bacillus was an important gut constituent bacterium in animals༻49༽, and played a role in inhibiting pathogenic bacteria in the silkworm gut༻50༽. The most influential genus in FFy group was Sphingomonas by random forest analysis. Some strains of this genus contained key enzymes for degrading aromatic compounds and were often used in the biodegradation of aromatic compounds, regarding them as beneficial bacteria. Therefore, the exogenous addition of B. velezensis LY5 effectively reduced the excessive proliferation of harmful bacteria and positively promoted the proliferation of beneficial bacteria during the recovery period after FF exposure. There existed a significant positive correlation between Bacillus and Acidibacter , Acinetobacter by heatmap analysis. Meanwhile, there was a significant negative correlation between Bacillus and Pelomonas , Corynebacterium (some of them was pathogenic), Lactiplantibacillus , and Levilactobacillus . This suggested that the intestinal flora possessed a complex metabolic network, where beneficial and harmful bacteria engaged in intense competition. It exerted inhibitory or promotive effects on the growth, development, spinning, and cocooning of silkworms. Further research was needed to reveal the truth. This study reveals the toxic effects of high-concentration FF on domestic silkworms, for the first time, investigates the dynamic changes in the midgut microbiota during the natural recovery process of FF-damaged silkworms. It also identifies a functional probiotic strain, B. velezensis LY5, which significantly promotes the recovery of growth and development in damaged silkworms, accelerates the restoration of midgut microbiota homeostasis, and effectively repairs intestinal tissue damage. Abbreviations FF Florfenicol LB Luria-Bertani medium CFUs Colony Forming Units ASVs Amplicon Sequence Variants ANI Average Nucleotide Identity Declarations Clinical trial number No applicable Ethics approval and consent to participate The Animal Welfare and Ethics Committee of Shandong Agricultural University approved all protocols of this study (Approval No. SDAUA-2025-271), which were conducted in accordance with the approved guidelines. In this study, informed consent was obtained from the owners to allow the use of these animals in the research. Consent for publication Not applicable Availability of data and materials The raw data in this study have been deposited in the NCBI BioProject under PRJNA1265109 and PRJNA1371349. Competing interests The authors declare that they have no competing interests Funding This work was supported by the Independent Innovation Project of Henan Academy of Agricultural Sciences (2025ZC115), the earmarked fund for CARS-18, and the Modern Agricultural Technology System of Shandong Province (No. SDAIT-18). Authors’ Contributions Yating Liu: investigation, methodology, and writing—original draft; Zhongwen Liu: investigation; Chunjiu Ren: investigation; Shuangxin Wu: investigation; Huiju Gao: investigation; Hongxia Zhang: investigation; Shengxiang Zhang: conceptualization, supervision; Bing Wang: conceptualization, supervision, and writing—review and editing. References Kehrenberg C, Schwarz S, Jacobsen L, Hansen LH, Vester B. A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. Mol Microbiol. 2005;57(4):1064–73. Zhang X, Huo Y, Kong Y, Zhou W, Qin F, Hu X. Effects of short-term florfenicol exposure on the gene expression pattern, midgut microbiota, and metabolome in the lepidopteran model silkworm (Bombyx mori). Sci Total Environ. 2024;912:169099. El Ayari T, Ben Ahmed R, Bouriga N, Gravato C, Chelbi E, Nechi S, El Menif NT. Florfenicol induces malformations of embryos and causes altered lipid profile, oxidative damage, neurotoxicity, and histological effects on gonads of adult sea urchin, Paracentrotus lividus. Environ Toxicol Pharmacol. 2024;110:104533. Wang X, Liu W, Zhang D, Jiao Y, Zhao Q, Liu Y, et al. Salvia miltiorrhiza polysaccharides alleviate florfenicol-induced inflammation and oxidative stress in chick livers by regulating phagosome signaling pathway. Ecotoxicol Environ Saf. 2023;249:11442. Zhao F, Gong Z, Yang Y, Li X, Chen D, Shi X, et al. Effects of environmentally relevant concentrations of florfenicol on the glucose metabolism system, intestinal microbiome, and liver metabolome of zebrafish. Sci Total Environ. 2024;938:173417. Ma Z, Gao X, Yang X, Lin L, Wei X, Wang S, et al. Low-dose florfenicol and copper combined exposure during early life induced health risks by affecting gut microbiota and metabolome in SD rats. Ecotoxicol Environ Saf. 2022;245:114120. Dogra SK, Doré J, Damak S. Gut Microbiota Resilience: Definition, Link to Health and Strategies for Intervention. Front Microbiol. 2020;11:572921. Yun X, Zhou J, Wang J, Li Q, Wang Y, Zhang W, Fan Z. Biological toxicity effects of florfenicol on antioxidant, immunity and intestinal flora of zebrafish (Danio rerio). Ecotoxicol Environ Saf. 2023;265:115520. Guo X, Qian Z, Pan Q, Hu Y, Mei W, Xing X, et al. Effects of Florfenicol on Intestinal Histology, Apoptosis and Gut Microbiota of Chinese Mitten Crab (Eriocheir sinensis). Int J Mol Sci. 2023;24(5):4412. Qin L, Qi J, Shen G, Qin D, Wu J, Song Y, et al. Effects of Microbial Transfer during Food-Gut-Feces Circulation on the Health of Bombyx mori. Microbiol Spectr. 2022;10(6):e0235722. Tian C, Cheng J, Gu H, Zou H, Peng P, Li B. Effects of florfenicol on the midgut physiological function of Bombyx mori, based on the diversity of intestinal microbiota. Entomol exp appl. 2024;172:953–63. Luo K, Yang Z, Wen X, Wang D, Liu J, Wang L, et al. Recovery of intestinal microbial community in Penaeus vannamei after florfenicol perturbation. J Hazard Mater. 2024;480:136158. Han Z, Sun J, Jiang B, Chen K, Ge L, Sun Z, et al. Fecal microbiota transplantation accelerates restoration of florfenicol-disturbed intestinal microbiota in a fish model. Commun Biol. 2024;7(1):1006. Huang D, Chen Y, Chen H, Deng X, Huang J, Lu S, et al. Supplementation of Bacillus sp. DU-106 Alleviates Antibiotic-Associated Diarrhea in Association with the Regulation of Intestinal Microbiota in Mice. Probiotics Antimicrob Proteins. 2022;14(2):372–83. Trif E, Cerbu C, Olah D, Zăblău SD, Spînu M, Potârniche AV, et al. Old Antibiotics Can Learn New Ways: A Systematic Review of Florfenicol Use in Veterinary Medicine and Future Perspectives Using Nanotechnology. Anim (Basel). 2023;13(10):1695. Tulstrup MV, Roager HM, Thaarup IC, Frandsen HL, Frøkiær H, Licht TR, et al. Antibiotic treatment of rat dams affects bacterial colonization and causes decreased weight gain in pups. Commun Biol. 2018;1:145. Ye W, Bian D, Mao T, Dai M, Feng P, Zhu Q, et al. Cloning and functional analysis of autophagy-related gene 7 in Bombyx mori, silkworm. Arch Insect Biochem Physiol. 2021;107(4):e21827. Plotnikov EY, Pevzner IB, Zorova LD, Chernikov VP, Prusov AN, Kireev II, et al. Mitochondrial Damage and Mitochondria-Targeted Antioxidant Protection in LPS-Induced Acute Kidney Injury. Antioxid (Basel). 2019;8(6):176. Yun S, Guo Y, Yang L, Zhang X, Shen W, Wang Z, et al. Effects of oral florfenicol on intestinal structure, function and microbiota in mice. Arch Microbiol. 2020;202(1):161–9. Yun X, Zhou J, Wang J, Li Q, Wang Y, Zhang W, et al. Biological toxicity effects of florfenicol on antioxidant, immunity and intestinal flora of zebrafish (Danio rerio). Ecotoxicol Environ Saf. 2023;265:115520. Yu K, Choi I, Kim M, Pyung YJ, Lee JS, Choi Y, et al. Florfenicol-induced dysbiosis impairs intestinal homeostasis and host immune system in laying hens. J Anim Sci Biotechnol. 2025;16(1):56. Shuang G, Yu S, Weixiao G, Dacheng W, Zhichao Z, Jing L, et al. Immunosuppressive activity of florfenicol on the immune responses in mice. Immunol Invest. 2011;40(4):356–66. Micheal AS, Subramanyam MV. Antioxidant enzymes as defense mechanism against oxidative stress in midgut tissue and hemocytes of Bombyx mori larvae subjected to various stressors. Arch Insect Biochem Physiol. 2013;84(4):222–34. Tian C, Cheng J, Gu H, Zou H, Peng P, Li B. The mechanism of high-concentration florfenicol exposure inducing apoptosis in the midgut of Bombyx mori (Lepidoptera: Bombycidae). J Asia-Pac Entomol. 2025;28(2):102399. Gu Z, Li M, Xia S, Mao T, Lu Z, Chen J, et al. Effects of sublethal phoxim exposure and lower food intake on nutrient metabolism in the midguts of Bombyx mori. Pestic Biochem Physiol. 2020;167:104593. Aderibigbe A, Cowieson A, Sorbara JO, Adeola O. Intestinal starch and energy digestibility in broiler chickens fed diets supplemented with α-amylase. Poult Sci. 2020;99(11):5907–14. Shen Y, Chen G, Zhao S, Wu X. Genome-wide identification of lipases in silkworm (Bombyx mori) and their spatio-temporal expression in larval midgut. Gene. 2022;813:146121. Cao HH, Zhang SZ, Zhu LB, Wang J, Liu YX, Wang YL, et al. The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori). Dev Comp Immunol. 2011;119:104035. Zhang S, Tang S, Liu Z, Lv H, Cai X, Zhong R, et al. Baicalin restore intestinal damage after early-life antibiotic therapy: the role of the MAPK signaling pathway. Pharmacol Res. 2024;204:107194. Romick-Rosendale LE, Legomarcino A, Patel NB, Morrow AL, Kennedy MA. Prolonged antibiotic use induces intestinal injury in mice that is repaired after removing antibiotic pressure: implications for empiric antibiotic therapy. Metabolomics. 2014;10(1):8–20. Ng KM, Aranda-Díaz A, Tropini C, Frankel MR, Van Treuren W, O'Loughlin CT, et al. Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs. Cell Host Microbe. 2019;26(5):650–e6654. Haberecht S, Bajagai YS, Moore RJ, Van TTH, Stanley D. Poultry feeds carry diverse microbial communities that influence chicken intestinal microbiota colonisation and maturation. AMB Express. 2020;10(1):143. Zeng Z, Tong X, Yang Y, Zhang Y, Deng S, Zhang G, et al. Pediococcus pentosaceus ZZ61 enhances growth performance and pathogenic resistance of silkworm Bombyx mori by regulating gut microbiota and metabolites. Bioresour Technol. 2024;402:130821. Zeng Z, Gong S, Quan C, Zhou S, Kulyar MF, Iqbal M, et al. Impact of Bacillus licheniformis from yaks following antibiotic therapy in mouse model. Appl Microbiol Biotechnol. 2024;108(1):139. Sadeghi A, Ebrahimi M, Kharazmi MS, Jafari SM. Effects of microbial-derived biotics (meta/pharma/post-biotics) on the modulation of gut microbiome and metabolome; general aspects and emerging trends. Food Chem. 2023;411:135478. Liu Q, Yu Z, Tian F, Zhao J, Zhang H, Zhai Q, et al. Surface components and metabolites of probiotics for regulation of intestinal epithelial barrier. Microb Cell Fact. 2020;19(1):23. Peng M, Lee SH, Rahaman SO, Biswas D. Dietary probiotic and metabolites improve intestinal homeostasis and prevent colorectal cancer. Food Funct. 2020;11(12):10724–35. Mojgani N, Bagheri M, Ashique S, Islam A, Moharrami M, Modirrousta H, et al. Honeybee defense mechanisms: Role of honeybee gut microbiota and antimicrobial peptides in maintaining colony health and preventing diseases. Microb Pathog. 2025;198:107161. Chen C, Li M, Li F, Liang X, Zhang H, Gu Y, et al. Dynamic Alterations of the Intestinal Microbiota of Fifth-Instar Silkworms ( Bombyx mori ) Fed an Artificial Diet or Mulberry Leaves. Insects. 2024;15(12):970. Taitz JJ, Tan J, Ni D, Potier-Villette C, Grau G, Nanan R, et al. Antibiotic-mediated dysbiosis leads to activation of inflammatory pathways. Front Immunol. 2025;15:1493991. Duan H, Yu L, Tian F, Zhai Q, Fan L, Chen W. Antibiotic-induced gut dysbiosis and barrier disruption and the potential protective strategies. Crit Rev Food Sci Nutr. 2022;62(6):1427–52. Hong Y, Li H, Chen L, Su H, Zhang B, Luo Y, et al. Short-term exposure to antibiotics begets long-term disturbance in gut microbial metabolism and molecular ecological networks. Microbiome. 2024;12(1):80. Gupta S, Fernandes J, Kiron V. Antibiotic-Induced Perturbations Are Manifested in the Dominant Intestinal Bacterial Phyla of Atlantic Salmon. Microorganisms. 2019;7(8):233. de Nies L, Kobras CM, Stracy M. Antibiotic-induced collateral damage to the microbiota and associated infections. Nat Rev Microbiol. 2023;21(12):789–804. Yao G, Cao C, Zhang M, Kwok LY, Zhang H, Zhang W. Lactobacillus casei Zhang exerts probiotic effects to antibiotic-treated rats. Comput Struct Biotechnol J. 2021;19:5888–97. Liang W, Gao Y, Zhao Y, Gao L, Zhao Z, He Z, et al. Lactiplantibacillus plantarum ELF051 Alleviates Antibiotic-Associated Diarrhea by Regulating Intestinal Inflammation and Gut Microbiota. Probiotics Antimicrob Proteins. 2024;16(6):1996–2006. Sun Y, Zhang S, Nie Q, He H, Tan H, Geng F, et al. Gut firmicutes: Relationship with dietary fiber and role in host homeostasis. Crit Rev Food Sci Nutr. 2023;63(33):12073–88. Li S, Guo J, Liu R, Zhang F, Wen S, Liu Y, et al. Predominance of Escherichia-Shigella in Gut Microbiome and Its Potential Correlation with Elevated Level of Plasma Tumor Necrosis Factor Alpha in Patients with Tuberculous Meningitis. Microbiol Spectr. 2022;10(6):e0192622. Tang X, Zeng Y, Xiong K, Zhong J. Bacillus spp. as potential probiotics: promoting piglet growth by improving intestinal health. Front Vet Sci. 2024;11:1429233. Liu R, Wang W, Liu X, Lu Y, Xiang T, Zhou W et al. Characterization of a Lipase From the Silkworm Intestinal Bacterium Bacillus pumilus With Antiviral Activity Against Bombyx mori (Lepidoptera: Bombycidae) Nucleopolyhedrovirus In Vitro. J Insect Sci 201818(6):3. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 18 Dec, 2025 Reviews received at journal 15 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers invited by journal 04 Dec, 2025 Editor assigned by journal 04 Dec, 2025 Editor invited by journal 02 Dec, 2025 Submission checks completed at journal 01 Dec, 2025 First submitted to journal 01 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8174585","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":556361070,"identity":"152e1c7b-c791-4ee6-bbc3-fbd03da10cf8","order_by":0,"name":"Yating Liu","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yating","middleName":"","lastName":"Liu","suffix":""},{"id":556361071,"identity":"ea13638b-a6da-4e22-930c-18fbf4be4785","order_by":1,"name":"Zhongwen Liu","email":"","orcid":"","institution":"Henan Sericultural Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Zhongwen","middleName":"","lastName":"Liu","suffix":""},{"id":556361072,"identity":"52d25037-a021-40cc-b23d-70ac42de2c5d","order_by":2,"name":"Chunjiu Ren","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chunjiu","middleName":"","lastName":"Ren","suffix":""},{"id":556361073,"identity":"befe16b6-21bb-4133-b05d-a85157ef3369","order_by":3,"name":"Huiju Gao","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huiju","middleName":"","lastName":"Gao","suffix":""},{"id":556361074,"identity":"8496830c-9ce5-4465-ba6b-5eba43d5c762","order_by":4,"name":"Shuangxin Wu","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shuangxin","middleName":"","lastName":"Wu","suffix":""},{"id":556361075,"identity":"bcc425d8-432d-4caf-8e8a-6c2a6d597cf0","order_by":5,"name":"Hongxia Zhang","email":"","orcid":"","institution":"Shandong Institute of Sericulture","correspondingAuthor":false,"prefix":"","firstName":"Hongxia","middleName":"","lastName":"Zhang","suffix":""},{"id":556361076,"identity":"79c6694e-b4b8-4f38-959d-e900faa3d848","order_by":6,"name":"Shengxiang Zhang","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shengxiang","middleName":"","lastName":"Zhang","suffix":""},{"id":556361077,"identity":"016a846e-b306-49a3-b79a-e4dcc20f0a2f","order_by":7,"name":"Bing Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACAzBZwcDYAKJ5iNdyhmQtjG2kaDGXSH728Ou8Otn+GQmMD962McibE9JiOSPN3Fh222HjGTcSmA3ntjEY7mwg5LDbCWbSktsOJDbcSGCT5m1jSDA4QFBL+jdpyTl1ifNvJLD/JlJLjpnkxwbmxA1AW5iJ0mI5/02ZNMOxw8YbzzxslpxzTsJwAyEt5jzHt0n+qKmTnXc8+eCHN2U28gRtAQFmSHSAo0aCCPUgtT+IUzcKRsEoGAUjFQAA+t1CXVHMtlEAAAAASUVORK5CYII=","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-11-21 14:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8174585/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8174585/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-026-04953-5","type":"published","date":"2026-03-30T15:59:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":97894812,"identity":"2adc6e8c-4887-4e26-a455-d74e9270ca86","added_by":"auto","created_at":"2025-12-10 15:33:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80823737,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/9be476b233d84ecd3629c2b3.docx"},{"id":97703432,"identity":"a8328298-ab9b-45a3-84c3-3f5e085c8cb5","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8864,"visible":true,"origin":"","legend":"","description":"","filename":"24cab6ca35894d9b9c2003eac94e3bd3.json","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/adcded11ac12989af326800a.json"},{"id":97703440,"identity":"ee1c3c07-6af7-4638-a450-fe5b7813bd81","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":646247,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/27732ae061e8e03b15ebbca2.docx"},{"id":97703422,"identity":"f4e5f07c-276d-409e-b471-52ba113a84f7","added_by":"auto","created_at":"2025-12-08 12:39:02","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152153,"visible":true,"origin":"","legend":"","description":"","filename":"24cab6ca35894d9b9c2003eac94e3bd31enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/7c9f41c829239cb32e14c8ec.xml"},{"id":97703439,"identity":"eeb89665-982a-4736-9a2f-211b0163d0c1","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7897988,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/5799888d1ae52715cffad03b.jpeg"},{"id":97703411,"identity":"656bb5f2-861b-4056-a2a7-5157d2d43e8e","added_by":"auto","created_at":"2025-12-08 12:39:01","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5850844,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/3d5dd62126349777db2a3a46.jpeg"},{"id":97703450,"identity":"27712ae4-b1dc-42f4-968d-8ffbb427783f","added_by":"auto","created_at":"2025-12-08 12:39:04","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19599686,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/3b438189c1e53a4fbd9aef74.jpeg"},{"id":97703435,"identity":"226efb67-ecc6-40dc-bdd2-e2730bfae168","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1147704,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/ae3e693896500c862269e1d3.jpeg"},{"id":97703425,"identity":"ad96a955-822b-434d-954e-ecd0f5c1d7b3","added_by":"auto","created_at":"2025-12-08 12:39:02","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10192128,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/43d3655d1785196a80607df1.jpeg"},{"id":97895048,"identity":"f3d03cea-fc30-493b-a46a-938a2c2ae1ad","added_by":"auto","created_at":"2025-12-10 15:33:26","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":647092,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/d9746bf37ad18f0ebcdd3f5f.jpeg"},{"id":97703442,"identity":"541cb8e0-ae51-4d51-8cc1-ef8e38879a07","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":21577960,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/7f2a3426441c1ec42b708cca.jpeg"},{"id":97703404,"identity":"728764e7-f012-45b6-96b9-e87b2f6f2972","added_by":"auto","created_at":"2025-12-08 12:38:58","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7454312,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/120685b42ddac461db48a6a9.jpeg"},{"id":97703427,"identity":"da34c5dc-a462-4333-9c39-9f2ec65a325f","added_by":"auto","created_at":"2025-12-08 12:39:02","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7287164,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/a3d1e4f65ad7702feeb1534f.jpeg"},{"id":97703455,"identity":"4ac9c96e-fe7c-476d-b53e-a843bd8c583b","added_by":"auto","created_at":"2025-12-08 12:39:04","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2719260,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/dacf0e9d7e396c8a0e4d2c0a.jpeg"},{"id":97894000,"identity":"d46a7cfc-4d71-4b0a-b218-2f0392f5549d","added_by":"auto","created_at":"2025-12-10 15:31:47","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":460399,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/15edd121b20ac2d5843392d1.png"},{"id":97703446,"identity":"9097fd82-605b-4511-93fa-12555354839e","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":674621,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/386b53e7d713c61aa840e585.png"},{"id":97703428,"identity":"108fd2d2-96db-4be8-8373-24754a9280be","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1846475,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/765b59992b18631f545ff0b0.png"},{"id":97703448,"identity":"62758dd4-c4e5-4175-9c85-5f52e4830e1d","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":254862,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/d6b6807f1bfbc5e06cdcb952.png"},{"id":97703444,"identity":"194915d7-a58e-45ca-be90-3ba92ba54134","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1167512,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/6b2842d740e417fb8b829189.png"},{"id":97703410,"identity":"73f1639a-5d0a-4210-a937-bbdf765ba5a8","added_by":"auto","created_at":"2025-12-08 12:39:01","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168539,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/c9713ae45cae89618f2059b6.png"},{"id":97703438,"identity":"ca449e99-56c3-42e2-88e3-920126e4f524","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3423060,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/1aa55926d33dafc690808b09.png"},{"id":97703431,"identity":"3f4cd2bd-ec2e-45f2-a0ca-efbb6da68c2a","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":708496,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/2d0cf084e51285c873f278c4.png"},{"id":97894614,"identity":"db59e894-e05b-400b-ada9-09bfd5853426","added_by":"auto","created_at":"2025-12-10 15:32:49","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":918996,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/f388b66a3ee77def56f39700.png"},{"id":97703426,"identity":"9099cda7-0a09-4f40-9749-ad852915d0a8","added_by":"auto","created_at":"2025-12-08 12:39:02","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":358507,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/70a6dfa62d8ffcefa0330362.png"},{"id":97893717,"identity":"dc18dbe1-a27a-4ea6-9b68-683861d6c3d0","added_by":"auto","created_at":"2025-12-10 15:30:58","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147787,"visible":true,"origin":"","legend":"","description":"","filename":"24cab6ca35894d9b9c2003eac94e3bd31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/7288b8c91f4cd67e19b32a75.xml"},{"id":97703424,"identity":"57704ea6-e93c-4aab-b21a-44a1cf75fa0d","added_by":"auto","created_at":"2025-12-08 12:39:02","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166083,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/481eb752356b6bebb8d0ab55.html"},{"id":97894615,"identity":"0c03f377-75cf-4ac9-9d5c-64c4a5b2080c","added_by":"auto","created_at":"2025-12-10 15:32:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2739925,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of FF exposure on the body weight and enzyme activities of the initial stage of the 5th instar of silkworms. \u003cstrong\u003ea\u003c/strong\u003e Body weight (n=20). \u003cstrong\u003eb\u003c/strong\u003e CAT enzyme activity. \u003cstrong\u003ec\u003c/strong\u003e POD enzyme activity. \u003cstrong\u003ed\u003c/strong\u003e SOD enzyme activity. \u003cstrong\u003ee\u003c/strong\u003eAmylase enzyme activity (f) Trypsin enzyme activity. \u003cstrong\u003eg\u003c/strong\u003e Lipase enzyme activit. \u003cstrong\u003eh\u003c/strong\u003e Differences in the body appearance. Data are presented as mean ± SD. Statistical significance is indicated as follows: \u0026nbsp;**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0. 001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/cdd10feca5832b135cb2c4f0.png"},{"id":97703445,"identity":"9e626012-ebdf-4d4b-85de-a109be8808bc","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5456217,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high-concentration FF on the midgut tissue structure of silkworms. \u003cstrong\u003eCK\u003c/strong\u003e Epithelial cells of midgut tissue in the CK group ( Magnified view of the boxed area on the left was showed on the right). \u003cstrong\u003eFF\u003c/strong\u003eEpithelial cells of midgut tissue in the FF group (Magnified view of the boxed area on the left was showed on the right). Arrows indicate swollen Golgi apparatus(red), swollen endoplasmic reticulum (yellow), pycnotic cell nucleus (blue), swollen mitochondria (orange), intercellular space (green), and autophagosomes with double-membrane structure (purple). mv, microvillus; Nu, chromatin; Cy, cytoplasm\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/f764a49bb3dc42e4fe00f11f.png"},{"id":97703452,"identity":"d420ad80-c62d-4cb5-bb1f-a7497acc8f0d","added_by":"auto","created_at":"2025-12-08 12:39:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2463599,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of natural recovery and aseptic treatment on the growth, development, and silk - producing ability of silkworms. \u003cstrong\u003ea\u003c/strong\u003e Body weight on the 3rd day of the 5th instar (n = 20). \u003cstrong\u003eb\u003c/strong\u003eCocoon shell weight (n = 10).\u003cstrong\u003e c\u003c/strong\u003e Whole cocoon weight (n = 10). \u003cstrong\u003ed \u003c/strong\u003eCocoon shell ratio (n = 10). Data are presented as mean ± SD. Statistical significance is indicated as follows: **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/ae5be302610ac05853f7f757.png"},{"id":97703436,"identity":"6bb88127-9011-43fb-b0bb-a483f0a5bc33","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6190029,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of natural recovery on the midgut flora of silkworms exposed to FF. \u003cstrong\u003ea \u003c/strong\u003eDynamic changes in α-diversity of the CK group and FFz group on day1, 2, 3 and 5. \u003cstrong\u003eb\u003c/strong\u003e PCoA analysis of differences in bacterial composition structure between the CK group and the FFz group (R = 0.6103, P = 0.001) and differences in midgut flora composition structure among the FFz group on day1, 2, 3 and 5 (R = 0.1259, P = 0.103).\u003cstrong\u003e c\u003c/strong\u003e Relative abundance of midgut flora at the phylum levels between the CK and FFz group. \u003cstrong\u003ed\u003c/strong\u003eRelative abundance of midgut flora at the genus levels between the CK and FFz group. \u003cstrong\u003ee\u003c/strong\u003e Random forest analysis of the CK and FFz group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/607221cf4e821e73fc5a034c.png"},{"id":97703409,"identity":"d7454963-3f3e-4406-982a-f3000ba85881","added_by":"auto","created_at":"2025-12-08 12:39:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2147400,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of natural recovery on the midgut flora of silkworms exposed to FF. \u003cstrong\u003ea \u003c/strong\u003eDynamic changes in α-diversity of the CK group and FFz group on day1, 2, 3 and 5. \u003cstrong\u003eb\u003c/strong\u003e PCoA analysis of differences in bacterial composition structure between the CK group and the FFz group (R = 0.6103, P = 0.001) and differences in midgut flora composition structure among the FFz group on day1, 2, 3 and 5 (R = 0.1259, P = 0.103).\u003cstrong\u003e c\u003c/strong\u003e Relative abundance of midgut flora at the phylum levels between the CK and FFz group. \u003cstrong\u003ed\u003c/strong\u003eRelative abundance of midgut flora at the genus levels between the CK and FFz group. \u003cstrong\u003ee\u003c/strong\u003e Random forest analysis of the CK and FFz group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/528d80b401144de1866dae24.png"},{"id":97703433,"identity":"76cd6fcf-11d3-435e-8f41-28992523325a","added_by":"auto","created_at":"2025-12-08 12:39:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7385880,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of natural recovery (FFz) and supplementary feeding with \u003cem\u003eB. velezensis\u003c/em\u003e LY5 (FFy) on the midgut tissue structure of silkworms on the 5th day of the recovery period. \u003cstrong\u003eFFz\u003c/strong\u003eUltrastructural diagram of midgut epithelial cells in the FFz group (Magnified view of the boxed area on the left was showed on the right picture). \u003cstrong\u003eFFy\u003c/strong\u003e Ultrastructural diagram of midgut epithelial cells in the FFy group (Magnified view of the boxed area on the left was showed on the right picture in the FFy group.\u003cstrong\u003e \u003c/strong\u003eArrows indicated nuclear pyknosis (blue), mitochondrial swelling and vacuolation (orange), and autophagosomes (purple).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/832db46f46df98a73209a856.png"},{"id":97894687,"identity":"e3fa93a0-7e94-414f-baab-82c326c61d2b","added_by":"auto","created_at":"2025-12-10 15:32:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2929406,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of antioxidant enzymes and digestive enzymes in silkworms. \u003cstrong\u003ea\u003c/strong\u003e CAT enzyme activity. \u003cstrong\u003eb\u003c/strong\u003e POD enzyme activity. \u003cstrong\u003ec\u003c/strong\u003e SOD enzyme activity. \u003cstrong\u003ed\u003c/strong\u003e Mortality rate of FFz and FFw. \u003cstrong\u003ee\u003c/strong\u003e Amylase enzyme activity. \u003cstrong\u003ef\u003c/strong\u003e tryptic enzyme activity. \u003cstrong\u003eg\u003c/strong\u003elipase enzyme activity. Data are presented as mean ± SD. Statistical significance is indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/9fbc079eb9c1c2a336a3ab7b.png"},{"id":97703423,"identity":"feb184aa-6b2a-41b6-95db-a6c84ee2ad9e","added_by":"auto","created_at":"2025-12-08 12:39:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6241823,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of the functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 on the midgut flora of silkworms exposed to FF. \u003cstrong\u003ea\u003c/strong\u003e Dynamic changes in α-diversity of the CK group and the FFy group on days 1, 2, 3 and 5. \u003cstrong\u003eb\u003c/strong\u003e PCoA analysis of differences in microbial composition structure between the CK group and the FFy group (R = 0.6593, P = 0.001) and differences in midgut flora composition structure within the FFy group on days 1, 2, 3 \u0026nbsp;and 5 (R = 0.4724, \u003cem\u003eP\u003c/em\u003e = 0.002). \u003cstrong\u003ec\u003c/strong\u003eRelative abundance of midgut flora at the phylum (left) and genus (right) levels in the FFy group. \u003cstrong\u003ed\u003c/strong\u003e Random forest analysis of the FFy group. \u003cstrong\u003ee\u003c/strong\u003e Heatmap visualization of the top 30 bacterial genera selected based on the criteria of VIP \u0026gt; 1 and P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/a8a7652b8a09c69771cee326.png"},{"id":97703453,"identity":"86b624e8-34e5-4c1f-9e1a-fc9e99561542","added_by":"auto","created_at":"2025-12-08 12:39:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3201836,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eB. velezensis\u003c/em\u003eLY5 on the growth and midgut flora of Non-FF-exposed Silkworms. \u003cstrong\u003ea\u003c/strong\u003e Body weight on the 3rd day of the 5th instar (n = 10); total cocoon weight (n = 10); cocoon shell weight (n = 10); cocoon shell ratio (n = 10). \u003cstrong\u003eb\u003c/strong\u003e Dynamic changes in α-diversity of the CK group and the CY group on 3 day.\u003cstrong\u003e c\u003c/strong\u003e PCoA analysis of differences in midgut flora composition structure between the CK group and the CY group (R = 0.125, \u003cem\u003eP\u003c/em\u003e= 0.772). \u003cstrong\u003ed\u003c/strong\u003e Relative abundance of midgut flora at the genus levels in the CY group. \u003cstrong\u003ee\u003c/strong\u003e LEfSe analysis identifying differentially abundant taxa between the CK group and the CY group. Data are presented as mean ± SD. Statistical significance is indicated as follows: *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/0cf16f56b7cedf0f8bdebdf3.png"},{"id":97895021,"identity":"be80eafc-5191-47c2-94ca-fb8f6db73d35","added_by":"auto","created_at":"2025-12-10 15:33:23","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2977521,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-genome analysis of \u003cem\u003eB. velezensis\u003c/em\u003e LY5. \u003cstrong\u003ea\u003c/strong\u003e Analysis using Average Nucleotide Identity (ANI) and Digital DNA-DNA Hybridization (isDDH) methods. \u003cstrong\u003eb\u003c/strong\u003e Complete genome map.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/92f91e7fec247b33e0222927.png"},{"id":106343836,"identity":"eec7ad44-2927-4177-a5b3-2c51a106063a","added_by":"auto","created_at":"2026-04-07 16:09:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":43045040,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/1b0d0a80-7314-45a5-8041-ce5b221cb124.pdf"},{"id":97703454,"identity":"489dd477-7004-4af5-b28d-10eaafd45c33","added_by":"auto","created_at":"2025-12-08 12:39:04","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":646247,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8174585/v1/85caefd6cf7ea160a37baf64.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Damage and recovery of artificial diet rearing silkworms (Bombyx mori L.) exposed to high-concentration florfenicol as well as the dynamic changes of midgut ‌flora after functional bacterium supplementation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFlorfenicol (FF), as a new generation of broad-spectrum antibacterial drug belonging to the chloramphenicol class, has been gradually introduced into the field of bacterial disease prevention and control in livestock farming in recent years, to a large extent due to its advantages of high efficacy, low drug resistance and safety. Although FF can deal with the bacterial disease, it may also lead to other issues, such as drug residues and microbial ecological disturbance. FF exerts its antibacterial effect by targeting the 50s subunit of the 70s ribosome in bacteria, and finally prevents protein synthesis. Its broad-spectrum bactericidal activity covered a lot of common pathogens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite its significant advantages in pathogenic control in animal farming༻2༽, the toxic effects of FF should not be neglected. For instance, FF induced embryonic malformations in sea urchins and exerted a direct impact on the gonad ༻3༽, caused liver inflammation and oxidative stress damage in chickens ༻4༽. The glucose metabolism of zebrafish was disrupted, leading to abnormal blood glucose and glycogen levels, after being exposed to a water environment containing FF for 28 days ༻5༽.\u003c/p\u003e\u003cp\u003eIt was reported that the damage to the symbiotic bacterial flora, as well as the loss of core microbiota and a significant reduction in diversity was observed due to the broad-spectrum bactericidal activity of FF [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Gut microbiota was a complex microbial community colonizing the intestines of the host, comprising various microorganisms such as bacteria and fungi, which formed a symbiotic relationship with the host and played a crucial role in host health༻7༽. The imbalance of gut microbiota might further amplify the physiological stress of the host. Exposure of high-concentration FF significantly reduced the diversity and richness of zebrafish gut microbiota ༻8༽. The intestinal dysfunction and dysbiosis of the gut microbiota were observed when the Chinese mitten crab was exposed to FF in water ༻9༽.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBombyx mori\u003c/em\u003e L. is an important economic insect and at the same time, a model organism. Bacterial infections caused by \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, and other pathogenic bacteria often led to large-scale mortality of silkworms, resulting in a reduction of cocoon production by up to 30% to 50%, which posed a serious threat to cocoon yield and quality [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although the toxicity of FF to various organisms was extensively studied, its impact on lepidopteran insects has been less explored. Previous studies had shown that high concentrations of FF altered the gut microbiota structure of silkworms, delayed their growth and development, and affected their midgut tissue structure and immune response༻11༽.\u003c/p\u003e\u003cp\u003eCurrent research has primarily focused on the changes in the microbiota during the medication period, while it was also crucial to analyze the influencing factors of microbiota recovery after drug withdrawal. Many probiotics, as a beneficial external intervention, could recover microbial ecological disorders caused by external factors, thereby reestablishing a new intestinal microbial homeostasis. Luo K [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] observed the recovery of intestinal microbiota communities in shrimp after being exposed to FF through various recovery methods (adding probiotics and natural recovery), and found that the abundance of potentially pathogenic bacteria reduced and at the same time, the abundance of potentially beneficial bacteria increased when probiotics were used. It was also reported that fecal microbiota transplantation accelerated the recovery of intestinal microbiota communities in koi fish exposed to FF, repaired intestinal damage, and normalized key metabolic pathways ༻13༽. The antibiotic-associated diarrhea (AAD) in mice alleviated, at the same time the intestinal barrier integrity improved, and the immune response enhanced after \u003cem\u003eBacillus\u003c/em\u003e DU-001 was used as an additive agent༻14༽. So far, there were few reports focused on the recovery of intestinal microbiota disruption caused by FF exposure in silkworms.\u003c/p\u003e\u003cp\u003eThis study was for the first time designed to systematically explore the damage and the recovery of the midgut of silkworms reared on artificial diet under conditions of sterile control, natural recovery, and functional bacterial intervention after exposure to high concentrations of FF by means of activity detection of digestive enzyme and antioxidant enzyme, TEM observation of midgut cell ultrastructure, dynamic changes analysis of midgut flora, whole-genome sequencing and functional prediction. The aim of this research was to explore the possible mechanisms of damage and recovery in model organism \u003cem\u003eBombyx mori\u003c/em\u003e L. exposed to FF, provide a basis for the effective application of FF in animal husbandry, especially in silkworm rearing, and to pave the way for the large-scale application of probiotic supplementation for silkworm gut health.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains and culture conditions\u003c/h2\u003e\u003cp\u003eThe NCBI accession number for \u003cem\u003eB. velezensis\u003c/em\u003e LY5 used in this paper was PRJNA1265109. The strain was cultured in LB liquid medium at 37\u0026deg;C for 12 hours. The precipitate was collected by centrifugation (5000 rpm, 10 min), and rinsed 3 times with normal saline, then suspended at a concentration of 10⁷ CFU mL⁻\u0026sup1; for later use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTest silkworms and diet\u003c/h3\u003e\n\u003cp\u003eThe test silkworm variety was Youshi No. 1, which exhibited high adaptability to artificial diet. Silkworm larvae were reared under conditions of 70\u0026ndash;80% humidity, a 12-hour dark/12-hour light cycle, and a temperature of 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The artificial diet used in this study was M20 (composed of mulberry leaf powder, soybean meal powder, corn flour, vitamins, etc., heated at 100\u0026deg;C for 60 minutes). All-age diet rearing was adopted.\u003c/p\u003e\n\u003ch3\u003eTreatment\u003c/h3\u003e\n\u003cp\u003eSilkworms at the 4th instar were divided into CK, CY, and FF groups. The basic artificial diet was the M20. The CK group was fed on M20 diet, the CY group was fed on M20 supplemented with \u003cem\u003eB. velezensis\u003c/em\u003e LY5 (10⁷ CFU/10g), and the FF group was fed on M20 mixed with high-concentration FF (0.15 g/100 g, Shanghai McLean Biochemical Technology Co., Ltd.). After rearing the silkworms to the 5th instar, the FF group was randomly divided into three groups: FFw (sterile group, maintained a sterile state as much as possible), FFz (natural recovery group), and FFy (10⁷ CFU/10g of \u003cem\u003eB. velezensis\u003c/em\u003e LY5). Each group had three replicates, with 150 silkworms per replicate.\u003c/p\u003e\n\u003ch3\u003eMeasurement of the body weight and cocoon quality\u003c/h3\u003e\n\u003cp\u003eSilkworms were randomly selected at the beginning of the 5th instar and on 3rd day of the 5th instar for weighing. Each group was replicated 3 times, with 20 individuals per replicate (3n, n\u0026thinsp;=\u0026thinsp;20). On the 7th day after cocooning, cocoon quality surveys were conducted with 3 replicates per group, each comprising 10 silkworm cocoons (3n, n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e\n\u003ch3\u003eMeasurement of the enzyme activity\u003c/h3\u003e\n\u003cp\u003e5 silkworms were randomly selected per group, with 3 replicates. After 12 hours of starvation, the silkworms were placed on ice until anesthetized. After dissection, midgut fluid was collected and centrifuged at 4000 rpm for 5 minutes. The activities of α-amylase and trypsin were determined using assay kits from Nanjing Jiancheng Bioengineering Institute. Lipase activity was measured employing an assay kit from Puyinte Biotechnology Co., Ltd.\u003c/p\u003e\u003cp\u003e15 silkworms per group with 3 replicates were pretreated as described above. Hemolymph was collected and stored on ice. Enzyme activities for POD, SOD, and CAT were immediately measured using commercially available kits (all purchased from Beijing Solarbio Technology Co., Ltd.).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eUltrastructural analysis of midgut tissue\u003c/h2\u003e\u003cp\u003eThe midgut tissue fragments were dissected as illustrated above, further cut into small pieces, placed in the fixative for 2 hours, then stored at 4\u0026deg;C. Tissue cell samples were resin-embedded and sectioned to prepare tissue slides. Following staining with heavy metals lead and uranium, the specimens were observed under TEM (Wuhan Savier Biotechnology Co., Ltd.).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDynamic Analysis of Gut Microbiota\u003c/h3\u003e\n\u003cp\u003eThe 5th instar stage larvae (1d, 2d, 3d and 5d) of CK, FF group and FFy group, were subjected to 12 hours of starvation. Midguts were dissected and isolated, washed twice in PBS, rapidly frozen in liquid nitrogen, and stored at -80\u0026deg;C for future use. Sequencing libraries were prepared using the TruSeq Nano DNA LT Library Prep Kit (Lianchuan Biotechnology Co., Ltd.). Sequence denoising was performed using QIIME2 D2DA2. This yielded representative, biologically accurate Amplicon Sequence Variants (ASVs) and ASV abundance tables for subsequent midgut flora analysis.\u003c/p\u003e\n\u003ch3\u003eWhole-genome sequencing\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eB. velezensis\u003c/em\u003e LY5 was cultured in LB 12 hours, centrifugated at 5000 rpm for 3 min. DNA was extracted using a bacterial DNA extraction kit (TianGen Bio-Technology Co., Ltd.) and sent to LianChuan Bio-Technology Co., Ltd. for sequencing. Raw data obtained after sequencing were filtered using fastp (Version: 0.23.2) to obtain cleanData, which was then processed for genome assembly and annotation.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted in the GraphPad Prism 9.0 program. Independent samples t-test was used to analyze the data between different groups. Data from all experiments are presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical significance is indicated as follows: *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns, not significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFF Significantly Inhibited the Growth and Development of Silkworms\u003c/h2\u003e\u003cp\u003eSignificant growth and development inhibition was exhibited in the silkworms of FF group after 4 days (all ages of 4th instar) of exposure to high-concentration FF. Compared to CK, the average larvae weight in FF group at the initial stage of 5th instar was 10.9g, a significant decrease of 8.53% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Enzyme activity assays showed that the activities of SOD, CAT, and POD enzymes respectively decreased by 20.21%, 60.33%, and 39.05% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Meanwhile, the activities of amylase, lipase, and trypsin related to digestive function decreased by 47.78%, 56.52%, and 19.83%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Both digestive and antioxidant enzyme activities showed significant reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-g). Additionally, the 4th instar stage of the FF group was extended by 2 days, significantly prolonging the developmental duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e\u003cp\u003eIt was revealed that significant changes of the ultrastructure of midgut tissue occurred in the FF group compared to the normal structure in CK by transmission electron microscopy (TEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e CK). The prominent changes included karyopyknosis, mitochondrial swelling, Golgi and endoplasmic reticulum swelling and vacuolization, disruption of tight junctions between cells, and the formation of numerous autophagosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e FF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnvironmental Microorganisms Played a Crucial Role in the Recovery of Silkworms from Damage to FF Exposure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA comparison between the natural recovery group (FFz) and the sterile rearing group (FFw) revealed that the average body weight of 20 individuals of 5th-instar in FFz group was 40.3 g, which was significantly higher by 15.09% than that of the FFw group, still significantly lower than that of the CK group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Investigation into cocoon quality indicated that the FFz group exhibited significant improvements in whole cocoon weight, cocoon shell weight, and cocoon shell rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d), compared to the FFw group. The recovery effect of FF damage in the natural recovery group was significantly better than that in the sterile rearing group, indicating that microorganisms in the environment played a crucial role in facilitating the recovery of FF damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eDynamic Changes of the Midgut Flora in Silworms during Natural Recovery Process\u003c/h2\u003e\u003cp\u003e16S sequencing technology was used to investigate the dynamic shifts in the midgut flora of the natural recovery silkworms on 1, 2, 3 and 5 days of 5th instar after FF damage in 4th instar. The Simpson index indicated that the diversity of the midgut flora in CK fluctuated slightly due to the feeding of 5th instar silkworms, while the diversity in the FFz group significantly decreased. The Chao1 index showed that the bacterial richness in CK remained relatively stable, while after exposure to FF, the bacterial richness in the FFz group experienced a steep decline and then significantly increased to a level much higher than that of the CK group, clearly influenced by the antibiotic FF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eIt was revealed by the PCoA plot that significant differences in the midgut flora between the FFz group and CK (R\u0026thinsp;=\u0026thinsp;0.6103, P\u0026thinsp;=\u0026thinsp;0.001;Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb upper image), while the flora differences were not significant on 5d among the FFz group (R\u0026thinsp;=\u0026thinsp;0.1259, P\u0026thinsp;=\u0026thinsp;0.103; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb lower image). Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, and Latescibacteria were the dominant phyla in both CK and FFz groups by dynamic analysis of the relative abundance of the top 30 phyla at the phylum level. Firmicutes was the absolutely dominant phylum in CK, however, it was severely absent in the FFz group. The relative abundance of Firmicutes in the FFz group increased significantly from 2d, while the relative abundances of other dominant phyla such as Proteobacteria continued to decrease with the recovery time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). As for the relative abundance of the top 30 genera, \u003cem\u003eEnterococcus\u003c/em\u003e was the absolutely dominant genus in CK, with its quantity slightly fluctuating due to the large amounts of food intake during the peak feeding period. \u003cem\u003eEnterococcus\u003c/em\u003e was commonly found in the gut and included more than 30 beneficial and harmful bacterial species. In the FFz group, \u003cem\u003eEnterococcus\u003c/em\u003e did not even appear in the top 30 dominant genera on 1d. It showed a significant increase on day 5, although it still did not reach the CK level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). This indicated that the recovery of the midgut flora did not complete within the limited 5 days, and the recovery from FF damage was a relatively lengthy process. The Venn diagram showed that the number of ASVs in the FFz group surged, increasing by twice compared to CK on 5d (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIt was found that the differential marker bacterial genus were group-specific with different treatments and different times, by LDA analysis and LefSe analysis (Fig. S2). The top 15 characteristic bacterial genera in each group were screened out using the random forest algorithm. It was found that the composition of characteristic bacterial genera in FFz group was significantly differentiated compared to CK. There were only 6 overlapping bacterial communities between the two groups. The data indicated that the Mean Decrease Accuracy (MDA) value of \u003cem\u003eBacillus\u003c/em\u003e was the highest in CK, indicating the highest importance of \u003cem\u003eBacillus\u003c/em\u003e in CK. In the FFz group, the top-ranked genus was the infectious pathogenic bacterium \u003cem\u003eEscherichia-Shigella\u003c/em\u003e, demonstrating that the colonization of conditional pathogenic bacteria significantly increased during the recovery period after FF exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe Functional Bacterium\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5 Significantly Improved the Body Weight and Cocoon Quality of Silkworms Exposed to FF\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSince \u003cem\u003eBacillus\u003c/em\u003e occupied the most important position in silkworm midgut based on the biomarker results in CK, we chose \u003cem\u003eB. velezensis\u003c/em\u003e LY5 in our laboratory as an exogenous functional bacterium to intervene and mitigate the negative effects on silkworms after high-concentration FF exposure. The FFy group, which was fed with the functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5, exhibited a significant growth-promoting effect during the recovery period. The body weight of the FFy group on 3d in the 5th instar was significantly increased by 6.13%, compared to CK (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), and the cocoon quality parameters (whole cocoon weight, cocoon shell weight and cocoon shell ratio) all returned to the normal levels of CK (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional Bacterium\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5 Accelerated the Repairation of Damaged Midgut Cell Structure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe midgut tissue of the FFz group still contained a large number of autophagosomes, with swollen and vacuolated mitochondria, and nuclear condensation by TEM observation on 5d after exposure to FF. While the cell structure in the FFy group was intact, with tight junctions between cells, and no autophagosomes were observed in the field of view (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This indicated that the structural damage to the midgut caused by high concentrations FF continued to affect physiological functions during the recovery period, and natural recovery could not repair the intestinal tissue damage in a short period of time. However, the functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 significantly promoted the repairation of the ultrastructure in the silkworm midgut.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe Functional Bacterium\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5 Enhanced the Enzyme Activity of Silkworms\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe activity of key antioxidant enzymes (CAT, POD, SOD) and digestive enzymes (Lip, Amy, Try) were determined. The activities of CAT, POD and SOD in both FFz group and FFy group continued to increase with the extension of recovery time, and the FFy group increased significantly higher than that in the FFz group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). The addition of the functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 significantly reduced the number of deaths during the cocooning period of silkworms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), which presented the conjecture that there was a close relationship between the increased activity of antioxidant enzymes and the reduction in mortality rate. There were no significant differences among the three digestive enzymes on 1d and 2d of recovery, indicating a lag effect in the recovery of digestive function. Significant differences were observed in amylase and lipase on 3d and 5d, and the FFy group showed a significant increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee,g). Meanwhile, trypsin activity in FFz group significantly increased on 3d and 5d, significantly higher than that in the FFy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). It was the addition of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 which significantly improved antioxidant capacity and regulated digestive function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe Functional Bacterium\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5 Improved the Disrupted Midgut Flora of Silkworms Injured by FF\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Simpson index indicated that the diversity of midgut flora in FFy group remained at a low level as recovery time passed. The Chao1 index revealed that after a dramatic decrease, the richness of the midgut flora in group FFy increased from low to high and gradually approached the CK level on day 5, however, the midgut flora richness in group FFz still remained in a radical change state (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). This suggested that the supplementation of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 contributed to the recovery of the midgut flora in silkworms. PCoA analysis revealed a significant segregation between CK and FFy group (R\u0026thinsp;=\u0026thinsp;0.6593, P\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb upper image). However, unlike the FFz group, the FFy group also exhibited segregation over time (R\u0026thinsp;=\u0026thinsp;0.4724, P\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb lower image). Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria and Latescibacteria were also the dominant phyla in the FFy group, while they exhibited significant inter-group differences over time. The relative abundance of the core phylum Firmicutes in the FFy group increased rapidly from 3d, and by 5d, both the core phylum Firmicutes and the sub-core phylum Proteobacteria had essentially returned to the CK level. It was speculated that functional bacterium might assist in the recovery of the midgut symbiotic flora through competitive inhibition or metabolic regulation. From the relative abundance changes in the top 30 genera, we discovered a close correlation between key functional bacterial genera and external interventions. \u003cem\u003eB. velezensis\u003c/em\u003e LY5 intervention in FFy group significantly activated specific functional bacterial genera in the midgut. The core genus \u003cem\u003eEnterococcus\u003c/em\u003e rapidly increased from 3d and was already close to CK level by 5d. During this period of time, the sub-core genus \u003cem\u003eLactiplantibacillus\u003c/em\u003e was also increased, which was very close to the CK level (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). We draw a conclusion that the supplementation of the functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 significantly accelerated the recovery rate of the core bacterial community in the midgut of silkworms.\u003c/p\u003e\u003cp\u003eThe differential marker bacterial genus were group-specific with different treatment and different time by LefSe analysis (Fig. S3). Screening of biomarkers in FFy group using the random forest algorithm revealed that, compared to CK, only 3 of the top 15 bacterial genera were shared between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). This indicated that the exogenous addition of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 altered the midgut flora distribution after FF exposure. The genus \u003cem\u003estenotrophomonas\u003c/em\u003e, which mostly consisted of harmless bacterial species, had the highest MDA value.\u003c/p\u003e\u003cp\u003eThrough the analysis of the flora correlation heatmap based on top 30 bacterial genera, it was found that the important bacterial genus \u003cem\u003eBacillus\u003c/em\u003e in the midgut of silkworms had significant interactions with multiple bacterial genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). \u003cem\u003eMethyloversatilis\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e were positively correlated with \u003cem\u003eBacillus\u003c/em\u003e, while \u003cem\u003eAcinetobacter\u003c/em\u003e showed a significant positive correlation. In fact, \u003cem\u003eAcinetobacter\u003c/em\u003e was a conditional pathogen. We supposed that there might be a functional association of co-metabolism or synergistic degradation of organic matter between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e, which controlled the associated activities of these conditional pathogens. Meanwhile, \u003cem\u003eLevilactobacillus\u003c/em\u003e was significantly negatively correlated with \u003cem\u003eBacillus\u003c/em\u003e, which played an important role in regulating the gut microbiota.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe Impact of Functional Bacterium\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5 on Physiological Indicators and Midgut Flora of Normal Silkworms\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effects of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 on healthy silkworms, we conducted a two-day short-term feeding test from 1d of the 5th instar (referred to as the CY group). The results showed that the body weight of silkworms in CY group increased by 37.6% compared to CK, with the total cocoon weight and the cocoon shell weight increasing (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). However, there was no significant improvement in the cocoon shell rate. It was demonstrated that there was no significant difference in the midgut flora in α diversity between CK and CY group by high-throughput identification at 3d of the 5th instar (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). The Venn diagram indicated that CK group had 101 unique genera, while CY group had 116 unique genera, with a total of 185 genera shared between the two groups (Fig. S4). The PCoA diagram revealed that there were no significant differences in the overall midgut flora structure between CY group and CK (R\u0026thinsp;=\u0026thinsp;0.125, P\u0026thinsp;=\u0026thinsp;0.772; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eFurther analysis of the differences in gut bacterial composition between the two groups was carried out. Compared to CK, there were no significant differences in the main core bacterial genera in CY group, with only slight changes in the relative proportions of a few genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). The LEfSe method was used in order to identify the differential abundance biomarkers between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee). The results showed that specific bacterial taxa were enriched in both CY and CK. The significantly different bacterial group in CY was \u003cem\u003eAcetobacter\u003c/em\u003e, while the significantly different bacterial group in CK was \u003cem\u003eWeissella\u003c/em\u003e, both possessing probiotic potential. Therefore, although short-term supplementation with \u003cem\u003eB. velezensis\u003c/em\u003e LY5 triggered changes in the abundance of some genera, it did not significantly alter the overall structure of the silkworm midgut flora.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eWhole-genome Sequencing and Functional Prediction of\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSequencing was conducted using the Nanopore sequencing technology platform to elucidate the molecular basis of the vitality and potential metabolites of \u003cem\u003eB. velezensis\u003c/em\u003e LY5, with the explanation of how \u003cem\u003eB. velezensis\u003c/em\u003e LY5 worked in the repair of FF damage. A total of 1,364,847,346 bp of effective sequencing data (Clean data) was obtained, and the genome contained one circular chromosome and one unclassified plasmid. The chromosome was 4,283,909 bp in length, with a GC content of 45.81%. Through isDDH and ANI analysis, the taxonomic status of the functional strain LY5 was confirmed as \u003cem\u003eB. Velezensis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eGene prediction indicated that strain \u003cem\u003eB. velezensis\u003c/em\u003e LY5 had a total of 4409 genes, accounting for 89.57% of the genome, with 4205 CDS, 86 tRNA, 1 tmRNA, 9 16sRNA, and 90 misc_rna. Gene annotation revealed that 3057 genes were annotated in KEGG, 2800 genes in GO, and 3173 genes in COG (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb). According to the annotation results, \u003cem\u003eB. velezensis\u003c/em\u003e LY5 could synthesize α-amylase, lipase, and cellulase, and possessed multiple vitamin synthesis pathways. This suggested that \u003cem\u003eB. velezensis\u003c/em\u003e LY5 had the potential acting on digestion and promote absorption in the host. The CAZy database showed 46 glycosyl hydrolases (GHS), 34 glycosyltransferases (GTs), 3 polylactones (PLs), 16 chitinases (Ces), 6 acetylases (Aas), and 4 carboxylesterases (CBMs). Secondary metabolism gene cluster analysis revealed that the strain contained 10 secondary metabolites, among which six, namely difficilein, bacilysin, surfactin, butirosin A/butirosin B, macrolactin H and fengycin, exhibited antibacterial and antiviral activities. Therefore, \u003cem\u003eB. Velezensis\u003c/em\u003e LY5 also owned the potential to assist the host in resisting potential pathogens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eFF significantly inhibited the growth and development of silkworms\u003c/h2\u003e\u003cp\u003eAntibiotics, as an important tool for preventing and treating bacterial diseases, have attracted more attention due to their broad-spectrum antibacterial properties, which can also cause side effects in organisms. Florfenicol (FF) is a broad-spectrum antibacterial antibiotic widely used in animal husbandry [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Former studies have shown that antibiotics could interfere with animal metabolism and had a negative impact on organism quality ༻16༽. This study demonstrated that high-concentration FF exposure significantly reduced the body weight of silkworms rearing on artificial diet at the 5th instar stage, and delayed their instar duration by up to 2 days. In the natural recovery group from FF damage, both female and male, their silkworm cocoon shell weight and total cocoon weight were significantly lower than those in the control group (CK), while the difference in cocoon shell ratio was not significant.\u003c/p\u003e\u003cp\u003eElectron microscopy results revealed that the midgut cell structure of silkworms in CK was tightly packed and intact, with no abnormal changes. However, in the FF-treated group, the midgut tissue of silkworms exhibited mitochondrial swelling, endoplasmic reticulum and Golgi apparatus swelling, dense vacuolization, and the formation of numerous autophagosomes, indicating significant changes in the midgut structure. Autophagy was a ubiquitous process in eukaryotes. The emergence of a large number of autophagosomes could severely affect cellular function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Mitochondria were often damaged due to oxidative stress ༻18༽. Similar pathological damage was observed in mice༻19༽, zebrafish༻20༽, and laying hens༻21༽ exposed to high concentrations FF, suggesting that FF had tissue-damaging capabilities across multiple species.\u003c/p\u003e\u003cp\u003eFF reduced immune response and induced intestinal inflammation by inhibiting the proliferation of immune cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As an important model insect, the antioxidant enzyme system of \u003cem\u003eBombyx mori\u003c/em\u003e L. was highly susceptible to external interference, and the key enzymes of this system such as CAT, POD, SOD, effectively resisted intestinal oxidative stress༻23༽. It was reported that FF led to the accumulation of a large amount of reactive oxygen species (ROS) by inhibiting the activity of antioxidant enzymes, thereby causing tissue oxidative damage༻20༽. Our study also confirmed that FF treatment significantly reduced the activity of CAT, POD and SOD in the midgut of \u003cem\u003eBombyx mori\u003c/em\u003e L., which in turn led to a decrease in body weight and an increase in mortality. Some studies reported that the decline of antioxidant enzymes in the midgut of \u003cem\u003eBombyx mori\u003c/em\u003e L.caused mitochondrial damage, further leading to apoptosis༻24༽. The changes in midgut ultrastructure, especially the mitochondrial swelling and deformation caused by FF in our study, gave further support to this conclusion.\u003c/p\u003e\u003cp\u003eDigestive enzymes played a crucial role in the metabolism and absorption of nutrients during the larval stage of silkworm [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, the enzyme activities of three major digestive enzymes, α-amylase, lipase and trypsin in FF group, were significantly reduced. α-amylase promoted the enzymatic hydrolysis of carbohydrates in the intestine༻26༽, lipase broke down dietary lipids into free fatty acids and glycerol༻27༽, and protease was highly expressed in the midgut and was crucial for protein digestion༻28༽. Therefore, we believed that the inhibition of digestive enzyme activity by FF might be a key factor leading to slow growth and developmental delay in silkworms.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe functional bacterium\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eLY5 accelerated the recovery of FF damage in silkworms rearing on artificial diet\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCurrently, there are few studies focused on the damage recovery in the silkworm, a model organism, despite numerous works on the effects of FF on various organisms. It was mentioned that piglets required a recovery time of more than approximately 1 week after antibiotic treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, mice required over 6 weeks for recovery༻30༽. However, our study discovered that after 5 days of recovery in the natural recovery group (FFz), there were still numerous autophagosomes and mitochondrial vacuolization in the midgut tissue. The results indicated that the body weight of the FFz group was still significantly lower than that of CK. The cocoon quality survey found that both the cocoon shell weight and total cocoon weight were still significantly lower than those of CK. Therefore, it was fully demonstrated that the natural recovery of silkworms after FF damage required a relatively long time.\u003c/p\u003e\u003cp\u003eThis study found that during the recovery period after FF exposure, environmental microorganisms were a key factor affecting the reconstruction of the midgut flora as well as the recovery of growth and development in silkworms. The recovery of the organism after antibiotic treatment often depended on diet and environmental microorganisms [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and environmental microorganisms were often considered to have a close interaction with the gut microbiota༻32༽. We set up a sterile rearing group (FFw) as a control and found that although the growth and development indicators of FFz group were still significantly lower than those of CK, its recovery effect was significantly better than that of the FFw group. This indicated that during the recovery period after FF exposure, the microbial communities present in feed and the environment was a key factor in promoting the later growth and development of silkworms.\u003c/p\u003e\u003cp\u003eAlthough functional bacteria have been extensively studied in the field of silkworm [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], there were no relevant reports on antibiotic damage recovery. Functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 in our laboratory was used as exogenous additives. It was found that this functional bacterium effectively promoted the growth, development, and cocooning ability of silkworms exposed to FF. The activities of amylase and lipase in both FFz and FFy groups continued to increase with the extension of recovery time. Except for trypsin, the increase in the FFy group was significantly higher than that in the FFz group. The three antioxidant enzyme indicators, CAT, POD and SOD, were significantly elevated in the FFy group. Therefore, the addition of functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 significantly improved the antioxidant capacity and regulated digestive function of silkworms after FF exposure. At the same time, the midgut tissue of the FFy group with added \u003cem\u003eB. velezensis\u003c/em\u003e LY5 was closer to that of CK, with a compact structure and no abnormal vacuoles. Thus, the exogenous addition of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 effectively promoted the repair of the silkworm midgut and significantly shortened its damage recovery time. Previous studies have shown that the addition of probiotics effectively enhanced antioxidant enzymes, and \u003cem\u003eBacillus licheniformis\u003c/em\u003e repaired intestinal damage in antibiotic-treated mice༻34༽, which was consistent with the results of our study.\u003c/p\u003e\u003cp\u003eFurther study in our laboratory found that short-term feeding of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 to ordinary silkworms also promoted their growth and development. High-throughput results showed that brief feeding of functional bacterium did not significantly alter the midgut flora structure of silkworms.\u003c/p\u003e\u003cp\u003eCompared to CK, the FFy group, ordinary silkworms fed with \u003cem\u003eB. velezensis\u003c/em\u003e LY5, exhibited different bacterial composition at the genus level. Specific bacterial taxa were enriched in both FFy and CK by using the LEfSe method to identify differential abundance biomarkers between the two groups. The significantly different bacterial group in FFy was \u003cem\u003eAcetobacter\u003c/em\u003e, while the significantly different bacterial group in CK was \u003cem\u003eWeissella\u003c/em\u003e. Previous studies have shown that microbial metabolites and derivatives affected organisms themselves [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For example, \u003cem\u003eLactobacillus\u003c/em\u003e metabolites affected the viability of colon cancer cells༻37༽. Therefore, we speculated that it was the metabolites of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 which might be one of the reasons for promoting silkworm growth.\u003c/p\u003e\u003cp\u003eThe analysis of the whole genome, especially the ANI and isDDH analysis results, further confirmed that the functional bacterium LY5 belongs taxonomically to \u003cem\u003eB. velezensis\u003c/em\u003e. Meanwhile, multiple vitamin synthesis pathways and various digestive enzyme genes were presented in the genome of \u003cem\u003eB. velezensis\u003c/em\u003e LY5, which was consistent with the findings that the addition of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 exogenously enhanced digestive enzymes and significantly increased the body weight of silkworms. Antimicrobial peptides played a crucial role in maintaining the homeostasis of insect gut microbiota and the immune system. Former studies have shown that the addition of probiotics improved the antioxidant capacity and antimicrobial activity of silkworms [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The whole genome indicated that \u003cem\u003eB. velezensis\u003c/em\u003e LY5 produced secondary metabolites such as difficilein, bacilysin, surfactin, butirosin A/butirosin B, macrolactin H, and fengycin. Therefore, \u003cem\u003eB. velezensis\u003c/em\u003e LY5 had antibacterial and antiviral effects and helped the host inhibit potential pathogenic bacteria in the gut. This indicated that \u003cem\u003eB. velezensis\u003c/em\u003e LY5 might synthesize antimicrobial substances to protect the gut microbiota and inhibit the excessive proliferation of harmful bacteria such as enteric conditional pathogens.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDynamic changes of midgut flora during the recovery period of FF damage in silkworms reared on artificial diet\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe gut microbiota of silkworms reared on artificial diet was relatively stable during the early stage of the 5th instar [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, antibiotics disrupted the gut microbiota, leading to ecological imbalance and functional disorder, and altering the diversity of the microbiota༻40,41༽. Short-term exposure to antibiotics had long-term effects on gut microorganisms༻42༽. Previous studies have reported that the addition of antibiotics increased diversity༻43༽, while some studies suggested that antibiotics did not alter or reduce the diversity of the gut microbiota༻44༽. In our study, it was found that the diversity of the midgut flora in CK showed a slight downward trend followed by a recovery during the 5th instar period. We speculated that this change might be closely related to the feeding behavior of silkworms during the instar stage. Influenced by environmental microorganisms, the dominant midgut flora proportion slightly decreased and the diversity of the flora declined as the silkworms took abundant food from the 5th instar. After the peak feeding period, the silkworms reduced their food intake, and the proportion of the original dominant flora increased, leading to a recovery of the diversity of the flora to its original level. However, in the FFz and FFy groups, the diversity of the midgut flora significantly decreased during the recovery period after FF exposure compared to CK and remained at a low level. The proportion of the dominant flora was greatly reduced, which was consistent with the results reflected by the top 30 phyla and top 30 genera data. The core genus \u003cem\u003eEnterococcus\u003c/em\u003e did not even appear on the top 30 genera list on 1d of FF damage. As for the richness of the flora, CK group remained very stable, while the FFz and FFy groups showed a steep decline after exposure to FF, indicating that the impact of antibiotics on the gut microbiota was devastating. The FFz group showed a continuous increase in richness during the recovery period, while the FFy group rapidly increased to the CK level on 5d. It was the intervention of functional bacterium who kept the richness of the midgut flora in the FFy group at a lower level on days 1, 2 and 3. As for the genus level, compared to CK, the recovery of the core genus \u003cem\u003eEnterococcus\u003c/em\u003e was significantly better in the FFy group than that in the FFz group. The Venn diagram showed that the number of ASV in FFz and FFy groups surged on 5d, increasing twice compared to CK, which was consistent with the previous research result that the addition of antibiotics led to excessive growth of the microbiota.\u003c/p\u003e\u003cp\u003eAdditionally, the exogenous addition of probiotics significantly altered the diversity and community structure of the gut microbiota of organisms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our β diversity analysis revealed a clear separation between FFz group and FFy group in terms of community structure compared to CK. The community structure of FFz group did not exhibit significant changes within 5 days, and remained nearly stagnant during this period, indicating a prolonged recovery time after disruption of FF. Conversely, FFy group exhibited significant changes in flora structure from 2d onwards, with a pronounced separation between the 5d and 1d flora structures. It was speculated that the addition of the functional bacterium \u003cem\u003eB. velezensis\u003c/em\u003e LY5 accelerated the recovery and reconstruction process of the tested silkworm midgut flora.\u003c/p\u003e\u003cp\u003eThe Firmicutes, Proteobacteria, Actinobacteria, and Cyanobacteria were the common dominant phyla in all three groups in this paper, while their relative proportions varied significantly. In CK group, Firmicutes had always been the absolutely dominant phylum; in FFy and FFz groups, FF exposure led to a severe depletion of the core phylum, Firmicutes. And the relative abundance of Firmicutes began to show a significant upward trend from 3d and 2d, respectively. At the same time, the relative abundance of other dominant phyla (Proteobacteria, Actinobacteria, etc.) decreased simultaneously, giving way to the Firmicutes phylum. Firmicutes possessed a variety of functional strains that played an important role in maintaining the microbiota homeostasis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], containing various drug-resistant strains. As for the genus level, CK group maintained a relatively stable flora structure, with \u003cem\u003eEnterococcus\u003c/em\u003e as the core dominant genus. However, its relative abundance slightly decreased from 2d to 3d, possibly related to a large amount of food intake during this period of time. In FFz and FFy groups, the relative abundance of the core genus \u003cem\u003eEnterococcus\u003c/em\u003e was on an upward trend. The core genus \u003cem\u003eEnterococcus\u003c/em\u003e in FFy group was closer to that in CK by 5d, indicating better recovery of the midgut flora. The top 15 characteristic genera in each group was screened out using the random forest algorithm. Compared to CK, the characteristic genera was significantly differentiated, and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e became the most dominant characteristic genus in FFz group. This genus was often considered a pathogenic bacterium that caused various infectious diseases༻48༽. \u003cem\u003eBacillus\u003c/em\u003e was the most influential genus in normal silkworms\u0026mdash;this was also the reason why we chose \u003cem\u003eB. velezensis\u003c/em\u003e LY5 as an exogenous functional bacterium in this paper. \u003cem\u003eBacillus\u003c/em\u003e was an important gut constituent bacterium in animals༻49༽, and played a role in inhibiting pathogenic bacteria in the silkworm gut༻50༽. The most influential genus in FFy group was \u003cem\u003eSphingomonas\u003c/em\u003e by random forest analysis. Some strains of this genus contained key enzymes for degrading aromatic compounds and were often used in the biodegradation of aromatic compounds, regarding them as beneficial bacteria. Therefore, the exogenous addition of \u003cem\u003eB. velezensis\u003c/em\u003e LY5 effectively reduced the excessive proliferation of harmful bacteria and positively promoted the proliferation of beneficial bacteria during the recovery period after FF exposure.\u003c/p\u003e\u003cp\u003eThere existed a significant positive correlation between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eAcidibacter\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e by heatmap analysis. Meanwhile, there was a significant negative correlation between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003ePelomonas\u003c/em\u003e, \u003cem\u003eCorynebacterium\u003c/em\u003e (some of them was pathogenic), \u003cem\u003eLactiplantibacillus\u003c/em\u003e, and \u003cem\u003eLevilactobacillus\u003c/em\u003e. This suggested that the intestinal flora possessed a complex metabolic network, where beneficial and harmful bacteria engaged in intense competition. It exerted inhibitory or promotive effects on the growth, development, spinning, and cocooning of silkworms. Further research was needed to reveal the truth.\u003c/p\u003e\u003cp\u003eThis study reveals the toxic effects of high-concentration FF on domestic silkworms, for the first time, investigates the dynamic changes in the midgut microbiota during the natural recovery process of FF-damaged silkworms. It also identifies a functional probiotic strain, \u003cem\u003eB. velezensis\u003c/em\u003e LY5, which significantly promotes the recovery of growth and development in damaged silkworms, accelerates the restoration of midgut microbiota homeostasis, and effectively repairs intestinal tissue damage.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Florfenicol\u003c/p\u003e\n\u003cp\u003eLB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Luria-Bertani medium\u003c/p\u003e\n\u003cp\u003eCFUs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Colony Forming Units\u003c/p\u003e\n\u003cp\u003eASVs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Amplicon Sequence Variants\u003c/p\u003e\n\u003cp\u003eANI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Average Nucleotide Identity\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Animal Welfare and Ethics Committee of Shandong Agricultural University approved all protocols of this study (Approval No. SDAUA-2025-271), which were conducted in accordance with the approved guidelines. In this study, informed consent was obtained from the owners to allow the use of these animals in the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data in this study have been deposited in the NCBI BioProject under PRJNA1265109 and PRJNA1371349.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by the Independent Innovation Project of Henan Academy of Agricultural Sciences (2025ZC115), the earmarked fund for CARS-18, and the Modern Agricultural Technology System of Shandong Province\u0026nbsp;(No. SDAIT-18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u0026nbsp;\u003c/strong\u003eYating Liu:\u0026nbsp;investigation, methodology, and writing—original draft; Zhongwen Liu: investigation; Chunjiu Ren: investigation; Shuangxin Wu: investigation; Huiju Gao: investigation; Hongxia Zhang: investigation; Shengxiang Zhang: conceptualization, supervision; Bing Wang: conceptualization, supervision, and writing—review and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKehrenberg C, Schwarz S, Jacobsen L, Hansen LH, Vester B. A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. Mol Microbiol. 2005;57(4):1064\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang X, Huo Y, Kong Y, Zhou W, Qin F, Hu X. Effects of short-term florfenicol exposure on the gene expression pattern, midgut microbiota, and metabolome in the lepidopteran model silkworm (Bombyx mori). Sci Total Environ. 2024;912:169099.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl Ayari T, Ben Ahmed R, Bouriga N, Gravato C, Chelbi E, Nechi S, El Menif NT. Florfenicol induces malformations of embryos and causes altered lipid profile, oxidative damage, neurotoxicity, and histological effects on gonads of adult sea urchin, Paracentrotus lividus. Environ Toxicol Pharmacol. 2024;110:104533.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang X, Liu W, Zhang D, Jiao Y, Zhao Q, Liu Y, et al. Salvia miltiorrhiza polysaccharides alleviate florfenicol-induced inflammation and oxidative stress in chick livers by regulating phagosome signaling pathway. Ecotoxicol Environ Saf. 2023;249:11442.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao F, Gong Z, Yang Y, Li X, Chen D, Shi X, et al. Effects of environmentally relevant concentrations of florfenicol on the glucose metabolism system, intestinal microbiome, and liver metabolome of zebrafish. Sci Total Environ. 2024;938:173417.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa Z, Gao X, Yang X, Lin L, Wei X, Wang S, et al. Low-dose florfenicol and copper combined exposure during early life induced health risks by affecting gut microbiota and metabolome in SD rats. Ecotoxicol Environ Saf. 2022;245:114120.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDogra SK, Dor\u0026eacute; J, Damak S. Gut Microbiota Resilience: Definition, Link to Health and Strategies for Intervention. Front Microbiol. 2020;11:572921.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYun X, Zhou J, Wang J, Li Q, Wang Y, Zhang W, Fan Z. Biological toxicity effects of florfenicol on antioxidant, immunity and intestinal flora of zebrafish (Danio rerio). Ecotoxicol Environ Saf. 2023;265:115520.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuo X, Qian Z, Pan Q, Hu Y, Mei W, Xing X, et al. Effects of Florfenicol on Intestinal Histology, Apoptosis and Gut Microbiota of Chinese Mitten Crab (Eriocheir sinensis). Int J Mol Sci. 2023;24(5):4412.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin L, Qi J, Shen G, Qin D, Wu J, Song Y, et al. Effects of Microbial Transfer during Food-Gut-Feces Circulation on the Health of Bombyx mori. Microbiol Spectr. 2022;10(6):e0235722.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTian C, Cheng J, Gu H, Zou H, Peng P, Li B. Effects of florfenicol on the midgut physiological function of Bombyx mori, based on the diversity of intestinal microbiota. Entomol exp appl. 2024;172:953\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuo K, Yang Z, Wen X, Wang D, Liu J, Wang L, et al. Recovery of intestinal microbial community in Penaeus vannamei after florfenicol perturbation. J Hazard Mater. 2024;480:136158.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan Z, Sun J, Jiang B, Chen K, Ge L, Sun Z, et al. Fecal microbiota transplantation accelerates restoration of florfenicol-disturbed intestinal microbiota in a fish model. Commun Biol. 2024;7(1):1006.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang D, Chen Y, Chen H, Deng X, Huang J, Lu S, et al. Supplementation of Bacillus sp. DU-106 Alleviates Antibiotic-Associated Diarrhea in Association with the Regulation of Intestinal Microbiota in Mice. Probiotics Antimicrob Proteins. 2022;14(2):372\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrif E, Cerbu C, Olah D, Zăblău SD, Sp\u0026icirc;nu M, Pot\u0026acirc;rniche AV, et al. Old Antibiotics Can Learn New Ways: A Systematic Review of Florfenicol Use in Veterinary Medicine and Future Perspectives Using Nanotechnology. Anim (Basel). 2023;13(10):1695.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTulstrup MV, Roager HM, Thaarup IC, Frandsen HL, Fr\u0026oslash;ki\u0026aelig;r H, Licht TR, et al. Antibiotic treatment of rat dams affects bacterial colonization and causes decreased weight gain in pups. Commun Biol. 2018;1:145.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYe W, Bian D, Mao T, Dai M, Feng P, Zhu Q, et al. Cloning and functional analysis of autophagy-related gene 7 in Bombyx mori, silkworm. Arch Insect Biochem Physiol. 2021;107(4):e21827.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlotnikov EY, Pevzner IB, Zorova LD, Chernikov VP, Prusov AN, Kireev II, et al. Mitochondrial Damage and Mitochondria-Targeted Antioxidant Protection in LPS-Induced Acute Kidney Injury. Antioxid (Basel). 2019;8(6):176.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYun S, Guo Y, Yang L, Zhang X, Shen W, Wang Z, et al. Effects of oral florfenicol on intestinal structure, function and microbiota in mice. Arch Microbiol. 2020;202(1):161\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYun X, Zhou J, Wang J, Li Q, Wang Y, Zhang W, et al. Biological toxicity effects of florfenicol on antioxidant, immunity and intestinal flora of zebrafish (Danio rerio). Ecotoxicol Environ Saf. 2023;265:115520.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu K, Choi I, Kim M, Pyung YJ, Lee JS, Choi Y, et al. Florfenicol-induced dysbiosis impairs intestinal homeostasis and host immune system in laying hens. J Anim Sci Biotechnol. 2025;16(1):56.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShuang G, Yu S, Weixiao G, Dacheng W, Zhichao Z, Jing L, et al. Immunosuppressive activity of florfenicol on the immune responses in mice. Immunol Invest. 2011;40(4):356\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMicheal AS, Subramanyam MV. Antioxidant enzymes as defense mechanism against oxidative stress in midgut tissue and hemocytes of Bombyx mori larvae subjected to various stressors. Arch Insect Biochem Physiol. 2013;84(4):222\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTian C, Cheng J, Gu H, Zou H, Peng P, Li B. The mechanism of high-concentration florfenicol exposure inducing apoptosis in the midgut of Bombyx mori (Lepidoptera: Bombycidae). J Asia-Pac Entomol. 2025;28(2):102399.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu Z, Li M, Xia S, Mao T, Lu Z, Chen J, et al. Effects of sublethal phoxim exposure and lower food intake on nutrient metabolism in the midguts of Bombyx mori. Pestic Biochem Physiol. 2020;167:104593.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAderibigbe A, Cowieson A, Sorbara JO, Adeola O. Intestinal starch and energy digestibility in broiler chickens fed diets supplemented with α-amylase. Poult Sci. 2020;99(11):5907\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen Y, Chen G, Zhao S, Wu X. Genome-wide identification of lipases in silkworm (Bombyx mori) and their spatio-temporal expression in larval midgut. Gene. 2022;813:146121.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao HH, Zhang SZ, Zhu LB, Wang J, Liu YX, Wang YL, et al. The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori). Dev Comp Immunol. 2011;119:104035.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang S, Tang S, Liu Z, Lv H, Cai X, Zhong R, et al. Baicalin restore intestinal damage after early-life antibiotic therapy: the role of the MAPK signaling pathway. Pharmacol Res. 2024;204:107194.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRomick-Rosendale LE, Legomarcino A, Patel NB, Morrow AL, Kennedy MA. Prolonged antibiotic use induces intestinal injury in mice that is repaired after removing antibiotic pressure: implications for empiric antibiotic therapy. Metabolomics. 2014;10(1):8\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNg KM, Aranda-D\u0026iacute;az A, Tropini C, Frankel MR, Van Treuren W, O'Loughlin CT, et al. Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs. Cell Host Microbe. 2019;26(5):650\u0026ndash;e6654.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHaberecht S, Bajagai YS, Moore RJ, Van TTH, Stanley D. Poultry feeds carry diverse microbial communities that influence chicken intestinal microbiota colonisation and maturation. AMB Express. 2020;10(1):143.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng Z, Tong X, Yang Y, Zhang Y, Deng S, Zhang G, et al. Pediococcus pentosaceus ZZ61 enhances growth performance and pathogenic resistance of silkworm Bombyx mori by regulating gut microbiota and metabolites. Bioresour Technol. 2024;402:130821.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng Z, Gong S, Quan C, Zhou S, Kulyar MF, Iqbal M, et al. Impact of Bacillus licheniformis from yaks following antibiotic therapy in mouse model. Appl Microbiol Biotechnol. 2024;108(1):139.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSadeghi A, Ebrahimi M, Kharazmi MS, Jafari SM. Effects of microbial-derived biotics (meta/pharma/post-biotics) on the modulation of gut microbiome and metabolome; general aspects and emerging trends. Food Chem. 2023;411:135478.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Q, Yu Z, Tian F, Zhao J, Zhang H, Zhai Q, et al. Surface components and metabolites of probiotics for regulation of intestinal epithelial barrier. Microb Cell Fact. 2020;19(1):23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng M, Lee SH, Rahaman SO, Biswas D. Dietary probiotic and metabolites improve intestinal homeostasis and prevent colorectal cancer. Food Funct. 2020;11(12):10724\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMojgani N, Bagheri M, Ashique S, Islam A, Moharrami M, Modirrousta H, et al. Honeybee defense mechanisms: Role of honeybee gut microbiota and antimicrobial peptides in maintaining colony health and preventing diseases. Microb Pathog. 2025;198:107161.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen C, Li M, Li F, Liang X, Zhang H, Gu Y, et al. Dynamic Alterations of the Intestinal Microbiota of Fifth-Instar Silkworms (\u003cem\u003eBombyx mori\u003c/em\u003e) Fed an Artificial Diet or Mulberry Leaves. Insects. 2024;15(12):970.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaitz JJ, Tan J, Ni D, Potier-Villette C, Grau G, Nanan R, et al. Antibiotic-mediated dysbiosis leads to activation of inflammatory pathways. Front Immunol. 2025;15:1493991.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuan H, Yu L, Tian F, Zhai Q, Fan L, Chen W. Antibiotic-induced gut dysbiosis and barrier disruption and the potential protective strategies. Crit Rev Food Sci Nutr. 2022;62(6):1427\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHong Y, Li H, Chen L, Su H, Zhang B, Luo Y, et al. Short-term exposure to antibiotics begets long-term disturbance in gut microbial metabolism and molecular ecological networks. Microbiome. 2024;12(1):80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta S, Fernandes J, Kiron V. Antibiotic-Induced Perturbations Are Manifested in the Dominant Intestinal Bacterial Phyla of Atlantic Salmon. Microorganisms. 2019;7(8):233.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Nies L, Kobras CM, Stracy M. Antibiotic-induced collateral damage to the microbiota and associated infections. Nat Rev Microbiol. 2023;21(12):789\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYao G, Cao C, Zhang M, Kwok LY, Zhang H, Zhang W. \u003cem\u003eLactobacillus casei\u003c/em\u003e Zhang exerts probiotic effects to antibiotic-treated rats. Comput Struct Biotechnol J. 2021;19:5888\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiang W, Gao Y, Zhao Y, Gao L, Zhao Z, He Z, et al. Lactiplantibacillus plantarum ELF051 Alleviates Antibiotic-Associated Diarrhea by Regulating Intestinal Inflammation and Gut Microbiota. Probiotics Antimicrob Proteins. 2024;16(6):1996\u0026ndash;2006.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun Y, Zhang S, Nie Q, He H, Tan H, Geng F, et al. Gut firmicutes: Relationship with dietary fiber and role in host homeostasis. Crit Rev Food Sci Nutr. 2023;63(33):12073\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S, Guo J, Liu R, Zhang F, Wen S, Liu Y, et al. Predominance of Escherichia-Shigella in Gut Microbiome and Its Potential Correlation with Elevated Level of Plasma Tumor Necrosis Factor Alpha in Patients with Tuberculous Meningitis. Microbiol Spectr. 2022;10(6):e0192622.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang X, Zeng Y, Xiong K, Zhong J. \u003cem\u003eBacillus\u003c/em\u003e spp. as potential probiotics: promoting piglet growth by improving intestinal health. Front Vet Sci. 2024;11:1429233.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu R, Wang W, Liu X, Lu Y, Xiang T, Zhou W et al. Characterization of a Lipase From the Silkworm Intestinal Bacterium Bacillus pumilus With Antiviral Activity Against Bombyx mori (Lepidoptera: Bombycidae) Nucleopolyhedrovirus In Vitro. J Insect Sci 201818(6):3.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Florfenicol, Bombyx mori L., artificial diet rearing, damage and recovery, midgut flora","lastPublishedDoi":"10.21203/rs.3.rs-8174585/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8174585/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eFlorfenicol (FF) is widely used in animal husbandry due to its broad-spectrum bactericidal activity, while there is little research focused on its toxic effects on the silkworm(\u003cem\u003eBombyx mori\u003c/em\u003e L.), a model organism.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e\u003cp\u003eIt was demonstrated in this paper that high-concentration florfenicol exposure significantly inhibited the activities of digestive and antioxidant enzymes, reduced the body weight and cocooning ability of silkworms, prolonged the instar duration, and simultaneously led to organelle swelling and vacuolization in the midgut, a large number of autophagosomes, and nuclear condensation. Meanwhile, it was found that exposure to FF reduced the α diversity and richness of the midgut flora, leading to a severe loss of core microbiota. The functional bacterium, \u003cem\u003eBacillus velezensis\u003c/em\u003e LY5, which was demonstrated that it significantly also improved the body weight and cocoon quality of silkworms exposed to FF in this study, accelerated the repair of midgut tissue damage, helped recover the abundance of core midgut flora, increased the proportion of potentially beneficial bacteria, and reduced the excessive proliferation of opportunistic pathogens.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study reveals the toxicological mechanism by which high concentrations of antibiotics (FF) disrupt the midgut microbiota balance in silkworms, induce tissue damage, and subsequently impede their growth and development. It also demonstrates for the first time that functional probiotics can effectively reverse this damage by restoring the silkworm midgut microbiota and intestinal tissue\u003c/p\u003e","manuscriptTitle":"Damage and recovery of artificial diet rearing silkworms (Bombyx mori L.) exposed to high-concentration florfenicol as well as the dynamic changes of midgut ‌flora after functional bacterium supplementation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 12:38:31","doi":"10.21203/rs.3.rs-8174585/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T05:46:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-18T16:48:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T19:11:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78929912945519202833461509411235198710","date":"2025-12-09T14:53:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122674782412807655055498200852412834981","date":"2025-12-09T12:11:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317748022933438491096764897316190100140","date":"2025-12-04T19:06:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198138834464050709496687538756622640159","date":"2025-12-04T10:20:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-04T10:01:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-04T09:14:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-02T05:15:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T13:54:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-12-01T13:16:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c219abc-9bd6-41fe-a33e-63c8cfc7471e","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:06:04+00:00","versionOfRecord":{"articleIdentity":"rs-8174585","link":"https://doi.org/10.1186/s12866-026-04953-5","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-03-30 15:59:00","publishedOnDateReadable":"March 30th, 2026"},"versionCreatedAt":"2025-12-08 12:38:31","video":"","vorDoi":"10.1186/s12866-026-04953-5","vorDoiUrl":"https://doi.org/10.1186/s12866-026-04953-5","workflowStages":[]},"version":"v1","identity":"rs-8174585","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8174585","identity":"rs-8174585","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}