Silkworms (Bombyx mori) are insects belonging to the order Lepidoptera, which includes butterflies and moths. They undergo metamorphosis with four stages of development, i.e., egg, larva, pupa, and adult, within 2 months (Dong et al., 2022). Silkworms were domesticated approximately 5,000 to 10,000 years ago through the breeding of wild silkworms (Bombyx mandarina) for the development of the silk industry (Li et al., 2010). The silkworm is the only domesticated insect whose survival is dependent on humans, and is used as an industrial resource in a wide range of fields, including sericulture, and the production of animal proteins as a bioreactor (Zabelina et al., 2015; Marzoli et al., 2022). More recently, silkworm larvae have been considered to be useful as an ingredient in entomophagy. As a food resource, many different types of insects have been shown to have a positive influence on the host gut environment (Stull et al., 2018; Cunha et al., 2023). However, more studies are required to better understand the mechanisms behind these effects as well as the potential health impacts of different types of insects.

Silkworms have several unique characteristics that may make them a particularly useful food resource. For instance, the primary food source of silkworms is mulberry leaves from the family Moraceae; mulberry leaves contain many nutrients, such as sugars, lipids, proteins, minerals, and vitamins, which are essential for silkworm growth (Dong et al., 2017; Qin et al., 2020). Silkworms also have a silk gland lumen in which silk fiber protein is synthesized, secreted, and stored; this silk fiber protein is used in the pupa stage for protecting the silkworm from predators and pathogenic microorganisms (Dong et al., 2016, 2022). Silk fibers, which are essential for the growth of silkworms, are composed mainly of fibroin and sericin, which are non-digestible proteins, i.e., resistant proteins (RPs) (Yuan et al., 2012; Wang et al., 2019; Dong et al., 2020). RPs have physiological effects that help maintain health throughout the digestive tract, and they are broken down by intestinal bacteria (Chen et al., 2016).

Since the whole body of silkworms contains various nutrients, including those in organs (such as silk glands containing RPs), we hypothesized that the consumption of whole silkworm bodies may have beneficial effects on the host gut environment via effects on the intestinal bacteria. Thus, in the present study, we investigated the effects of the oral administration of silkworm larval powder (SLP) on the intestinal microbiota in mice.

Silkworm larvae, which are rich in nutrients, are expected to be useful as an ingredient in entomophagy (Mahanta et al., 2023). Some unique characteristics of silkworm larvae are that they eat mulberry leaves, and they have silk glands. It is well-known that RPs, which are found in silk fibers, can be utilized by gut microorganisms, and they may have several beneficial effects, e.g., they may protect and help maintain the intestinal environment (Warman et al., 2022). There have been a few reports on the effects of fibroin and sericin derived from the cocoons of silkworm larvae on the gut microbiota and some diseases (Park et al., 2020; Xu et al., 2021; Zhou et al., 2023). However, until now, there have been no studies on the effects of the ingestion of the entire body of silkworm larvae, which contain RPs.

The SLP that we independently prepared contained the entire body of the silkworm larvae, and it thus included all of the components of the silk glands. Silk glands produce and secrete liquid silk, which becomes silk threads (Radtke, 2016). In our study, the SLP diet had no influence on the body weight of the mice, or their food and water intake. However, the cecal weight and n-butyric acid level in the cecal contents were higher in the SLP group than in the Ctrl group, indicating that the SLP diet exerted significant effects ( Figure 1 ). N-butyric acid is an SCFA, and SCFAs are known to be metabolites of RPs in gut microorganisms. SCFAs function in maintaining host gut homeostasis (Liu et al., 2021). Ohira et al. (Ohira et al., 2013) found that butyrate can inhibit the production of the inflammatory cytokines; tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6 in macrophages. In addition, there have been many reports of the protective effect of butyrate on intestinal barrier function (Ma et al., 2012; Wang et al., 2012; Zheng et al., 2017). The SLP diet likely regulates the intestinal environment by increasing the level of n-butyric acid.

To investigate how the gut microbiota changed and which bacteria were involved, we conducted a Qiime2 analysis based on the 16sRNA V3-V4 sequences in the cecal and fecal contents. No differences were seen in the Shannon index between the two groups, but the observed ASVs were significantly higher in the SLP group than in the Ctrl group, and the β-diversity analysis revealed different clusters between the SLP and Ctrl groups. Although the SLP diet had little influence on the species diversity within a group, it modified the structure of the bacterial clusters between two groups ( Figure 2 ). The bar plot in Figure 3A shows the composition of the gut microbiota at the phylum level, revealing that in both groups, the gut microbiota mainly consisted of bacteria from four phyla, i.e., Firmicutes, Bacteroidota, Desulfobacterota, and Deferribacterota. Of note, Campylobacter and Helicobacter, which belong to the phylum Campylobacterota (previously called Epsilonproteobacteria), are known to be pathogenic in humans and to inhabit the digestive tract (Beeby, 2015; van der Stel and Wosten, 2019), and our results indicated that the administration of an SLP diet may decrease the amount of Campylobacterota species in the gut ( Figure 3 ). In addition, the LDA scores at the genus level revealed that Helicobacter and Anaerotruncus were among the bacteria that markedly declined in the fecal and cecal contents of mice in the SLP group ( Figures 4 , 5 ). Helicobacter infection is closely associated with the gut microbiota and various diseases, such as obesity, inflammatory bowel disease, and diabetes (Pellicano et al., 2020). Additionally, Helicobacter has a reverse relationship with Alistipes (Chen et al., 2021), which was also found at a high frequency in the cecal and fecal contents ( Figures 4 , 5 ). According to previous reports, Anaerotruncus has a strong positive correlation with obesity-related indices (Liu et al., 2022); in contrast, Alistipes is negatively correlated with obesity indices (Dai et al., 2022). Zhao et al. (2022) showed that the administration of adzuki beans effectively decreased the abundance of Helicobacter, and altered the gut microbiota to a composition that suppressed high-fat diet-induced body weight gain. These findings strongly suggest that SLP may have the potential to not only suppress the growth of pathogenic bacteria, but also to promote the growth of obesity-suppressing bacteria in the gut microbiota.

Some of the other highly modified bacteria were Clostridia UCG 014, Lachnospiraceae FCS020 group, RF39, and Lachnospiraceae A2. Clostridia UCG 014 and Lachnospiraceae have been reported to be associated with the production of SCFAs (Vacca et al., 2020; Liu et al., 2023). Similarly, we also found that Clostridia UCG 014 and Lachnospiraceae FCS020 group were significantly correlated with n-butyric acid, while Lachnospiraceae A2 was not ( Figure 6 ). Of note, Lachnospiraceae A2 has been reported to be correlated with an increase in anti-inflammatory regulatory T cells in the mesenteric lymph nodes that was associated with obesity (Cao et al., 2021; van de Wouw et al., 2021). In other words, Lachnospiraceae A2 might improve obesity by regulating inflammation via the induction of regulatory T cells, and by cooperating with the abovementioned obesity-suppressing bacteria. In a study by Dai et al (Dai et al., 2023), the abundance of RF39 was increased by the administration of sodium butyrate to broiler chickens. In our study, the amount of n-butyric acid was increased due to some SCFA-producing bacteria ( Figures 1 , 6 ). Namely, the abundance of RF39 may have increased due to stimulation by the n-butyric acid produced by Clostridia UCG 014 and Lachnospiraceae FCS020 group. Furthermore, Wang et al. (Wang et al., 2020) found that RF39 was prevalent in the gut microbiota, and that it might produce metabolites, such as acetate and hydrogen, that affect the amount of acetic acid, which is an SCFA, in the gut.

In conclusion, by analyzing the changes in the gut microbiota that resulted from the feeding of the SLP-containing diet in mice, we found that the administration of the SLP diet may have several benefits. The SLP diet changed the composition of the gut microbiota; in particular, it reduced the amounts of Helicobacter and Anaerotruncus, which are associated with obesity, and increased the amounts of Alistipes, which are associated with the prevention of obesity and the production of SCFAs. Clostridia UCG 014 and Lachnospiraceae FCS020 group showed a close association with n-butyric acid, the levels of which were increased in the cecal contents of the SLP group, and the SCFAs produced by these bacteria might promote the growth of RF39. Moreover, the proportion of Lachnospiraceae A2, which may potentially reduce inflammation, was high in the SLP group, and it was thought that it might regulate obesity by maintaining the intestinal environment. These results suggested that SLP might help prevent obesity. Nonetheless, more studies are needed in the future to examine its association with obesity, the underlying mechanisms, and other related factors, including studies on the liver, adipose tissues, and responses to insulin and glucose in animal models of obesity. We hope SLP will be useful as a food ingredient that can promote our health by promoting the maintenance of a healthy gut environment.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ddbj.nig.ac.jp/, PRJDB17364.

All experimental procedures were carried out in accordance with the Regulations for Animal Experimentation of Shinshu University. All experimental methods were reviewed by the Committee for Animal Experiments of Shinshu University, and complied with national regulations and guidelines, as specified by Law No. 105 and Notification No. 6 in Japan. The Committee for Animal Experiments of Shinshu University approved the animal protocol (Approval No. 021082). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

AM: Writing – original draft, Writing – review & editing, Investigation, Data curation, Validation, Visualization. HY: Writing – original draft, Investigation, Data curation, Validation. FN: Writing – original draft, Writing – review & editing, Investigation, Data curation. TSa: Writing – original draft, Writing – review & editing, Methodology. MY: Writing – review & editing, Resources. HU: Writing – review & editing, Resources. TF: Writing – review & editing, Investigation, Data curation. TT: Writing – review & editing, Investigation, Data curation. KS: Writing – original draft, Writing – review & editing, Project administration, Conceptualization, Resources. TSh: Writing – original draft, Writing – review & editing, Visualization, Methodology, Funding acquisition, Project administration, Supervision.