The silkworm (*Bombyx mori*) is a valuable insect, providing both silk and nutritious pupae used in food, pharmaceuticals, and animal feed. Traditional sericulture faces limitations due to mulberry leaf availability and labor shortages, prompting the development of artificial diets for silkworm rearing. However, these nutrient-rich, moisture-laden diets are prone to bacterial and mold contamination, leading to spoilage and impacting silkworm health. To address this, preservatives are needed to maintain feed quality and extend shelf life. This study investigates the efficacy and safety of two preservatives: ethylparaben (EP), a synthetic chemical preservative, and medium-chain fatty acids (MCFA), a natural alternative. EP's antimicrobial mechanism involves disrupting mitochondrial function, while MCFA's action is attributed to membrane destabilization and inhibition of bacterial lipases. Given the growing consumer preference for natural ingredients, this research compares the efficacy of EP and MCFA in controlling bacterial growth in silkworm artificial diets while assessing their impact on silkworm fitness and gut microbiota.

Existing literature highlights the nutritional value and diverse applications of silkworm pupae. Studies demonstrate its protein content, essential amino acids profile, and bioactive components with potential health benefits. The use of silkworm pupae as a food source and animal feed is well-documented, including successful replacements for fishmeal and soymeal in various animal diets. However, the challenge of maintaining the quality of artificial silkworm diets due to microbial contamination has been identified, emphasizing the need for effective and safe preservatives. Previous research has explored the antimicrobial properties of both synthetic and natural preservatives in various food applications, but their specific effects on silkworms and their gut microbiota remain understudied. The antimicrobial mechanisms of EP and MCFA are understood to some extent but detailed studies on their impact within a silkworm context are lacking.

This study evaluated the antibacterial activity of EP and MCFA against three common silkworm pathogens: *Serratia marcescens* (Sm), *Bacillus thuringiensis* (Bt), and *Lactobacillus plantarum* (Lp). The most effective concentration (0.1%) of each preservative was determined through preliminary agar plate assays. Subsequently, the antimicrobial efficacy was assessed in artificial silkworm diets inoculated with the three pathogens. Colony-forming units (CFU) were counted at 0, 12, and 48 hours post-inoculation to determine the effectiveness of the preservatives in inhibiting bacterial growth. The impact of EP and MCFA on silkworm fitness was assessed by monitoring larval and pupal weight over five days. Gut microbiota analysis involved CFU counting of gut contents at days 1, 3, and 5 and 16S rRNA gene high-throughput sequencing on day 5. Transcriptomic analysis of silkworm gut tissue was performed to examine the effects of preservatives on gene expression. Differentially expressed genes (DEGs) were identified and analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Finally, nine key genes were selected for validation using quantitative real-time PCR (qRT-PCR).

Both EP and MCFA effectively inhibited the growth of Sm, Bt, and Lp in both agar plates and artificial diets. The 0.1% concentration demonstrated the best antimicrobial activity. There were no significant negative effects on silkworm larval body weight or pupal weight when the artificial diet was supplemented with either EP or MCFA. Gut microbiota analysis revealed no significant differences in bacterial load or community composition between the control and treatment groups. Transcriptomic analysis revealed that while both EP and MCFA had minimal impact on the microbiome, EP resulted in the upregulation of detoxification genes such as *Ugt2* and immune response genes such as *Cecropin B*, indicating a potential stress response from the silkworm. MCFA had fewer DEGs. Notably, the number of DEGs was significantly higher in the EP group compared to the MCFA group, suggesting a greater impact on gene expression. GO enrichment analysis showed that EP treatment affected metabolic and cellular processes, while MCFA had a lesser impact. KEGG pathway analysis highlighted the involvement of detoxification pathways (cytochrome P450, UGTs) in the EP treatment and protein digestion and absorption pathways in the MCFA treatment. GSEA analysis showed a significant upregulation of gene sets involved in ribosome biogenesis, oxidative phosphorylation, and retinol metabolism in EP-treated silkworms, and upregulation of ECM-receptor interaction and proteasome activity in MCFA-treated silkworms. Protein-protein interaction (PPI) network analysis indicated significant interactions between DEGs in EP-fed silkworms, with key hub genes identified. qRT-PCR analysis confirmed the findings from the RNA-Seq data, showing a positive correlation between the two methods. Notably, the expression of genes related to antioxidant activity was downregulated by both treatments, while digestive enzyme genes were upregulated only in MCFA treated group.

The findings demonstrate that both EP and MCFA are effective preservatives for silkworm artificial diets, inhibiting the growth of pathogenic bacteria without significantly affecting silkworm growth or gut microbiota homeostasis. However, the upregulation of detoxification and immune response genes in EP-treated silkworms suggests a degree of toxicity, underscoring the potential benefits of using natural preservatives such as MCFA. The differences in gene expression patterns between EP and MCFA groups highlight the importance of considering the safety and biocompatibility of preservatives in insect diets. The results suggest that MCFA, being a natural preservative, is more suitable than EP for use in silkworm artificial diets due to its biocompatibility and minimal impact on silkworm physiology. Further research is needed to investigate the long-term effects of these preservatives on silkworm health and productivity and to explore their application in other insect rearing systems.

This study successfully demonstrated the effectiveness of both EP and MCFA as preservatives in silkworm artificial diets. While both suppressed pathogen growth, MCFA showed superior safety, as indicated by transcriptomic analyses. The minimal impact of MCFA on silkworm gene expression and gut microbiome presents a promising avenue for enhancing the sustainability and safety of silkworm production. Future studies should focus on optimizing the concentration of MCFA in artificial diets, investigating other natural preservative options, and evaluating the long-term effects of these preservatives on silkworm health and reproductive success. The findings are relevant to both improving silkworm production and broader efforts in sustainable insect farming for food and feed.

This study focused on a specific silkworm strain (*Bombyx mori* Jingsong Haoyue) and a particular artificial diet formulation. The results may not be directly generalizable to other silkworm strains or different artificial diet compositions. The study period was relatively short (five days), and long-term effects of preservative exposure on silkworm health remain to be fully explored. Additionally, detailed investigation of the specific mechanisms underlying the observed gene expression changes requires further study. Finally, the high inter-individual variation among silkworms led to relatively large error bars in the qRT-PCR data analysis, which may limit the precision of the results.