Tormentic Acid Alters the Gut Microbiota of the Silkworm (Bombyx mori)

Tormentic Acid Alters the Gut Microbiota of the Silkworm (Bombyx mori) | 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 Article Tormentic Acid Alters the Gut Microbiota of the Silkworm (Bombyx mori) Veysel Bay, Seray Gür, Oğuz Bayraktar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1576191/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract In recent years, phytochemicals have started to attract more attention due to their contribution to health and bioactivity. Microorganisms in the intestines of organisms contribute to the processing, function, and biotransformation of these substances. The silkworm ( Bombyx mori ) is one of the organisms used for the biotransformation of phytochemicals due to its controlled reproduction and liability to microbial manipulation. In this study, a bioactive compound, tormentic acid (TA), extracted from Sarcopoterium spinosum was used in the diet of silkworm and the alterations of intestinal microbiota of the silkworm were assessed. To do this, silkworms were fed on diet with various tormentic acid content, and 16S metagenomic analysis was performed to determine and the alterations in the gut microbiota profile of these organisms. Diet with different TA content did not cause a change in bacterial diversity of the samples. Comprehensive comparisons between different feeding groups indicated increased abundance of bacteria associated with health, i.e., Intestinibacter spp., Flavonifractor spp., Senegalimassilia spp., through the utilization of bioactive substances such as flavonoids. In conclusion, it might be said that use of TA as a supplementary product might help ameliorating the infected gut and promoting the healthy gut and relieving the undesirable effects of medicines to gastrointestinal system. tormentic acid silkworm 16S amplicon sequencing phytochemicals Sarcopoterium spinosum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Herbal natural compounds include phenolic compounds and secondary plant metabolites, generally known as phytochemicals. These compounds have started to gain great importance with their positive effects against many diseases due to their bioactivity 1 , 2 .Most of the natural compounds undergo microbial changes, namely biotransformation, in the small intestine before being transformed in the liver 3 . Biotransformation processes using mammalian, microorganism, plant cells and insect cell cultures or enzymes isolated from these play an important role in the discovery and development of new drug molecules 4 – 6 . The silkworm ( Bombyx mori ), which feeds on mulberry ( Morus alba ) leaves, has been domesticated for thousands of years to produce economically important silk fibre 7 . Silkworm has provided a livelihood for humans for centuries and has been a highly productive source of biological and chemical substances 8 , 9 . In addition to the benefits of silkworm itself, many compounds are produced from its excrement, which are sources of treatment and food for other organisms(Liu J. et al. 2013, Liu X. et al. 2019; Li et al. 2019). Besides, it is possible to form bioactive compounds by feeding the silkworm with mulberry leaves enriched with different natural compounds by making use of the biotransformation ability of the silkworm 13 , 14 . Compounds that prevent liver damage, trigger tissue regeneration, or reduce the activity of toxic substances are called hepatoprotective agents. Some of the natural herbal compounds have antimicrobial, antidiabetic, antifungal and hepatoprotective effects 15 , 16 . It is stated that tormentic acid (TA), one of the terpenes found in the content of Sarcopoterium spinosum , has the potential to be a hepatoprotective agent, as well as its effectiveness in type 2 diabetes 17 , 18 . Many microorganisms live in the body of the silkworm. However, the microbiota of this insect model has not been well characterized to date 7 . With the advent of metagenomics technology, microbial communities of various species have been characterized and listed by genome studies on vertebrates and invertebrates 19 . The silkworm gut has less microbial diversity compared to the other vertebrates due to controlled microenvironment for growth and development and shorter lifespan. In addition, the silkworm microbiota must be able to tolerate the alkalinity up to pH 11–12, which adversely affects the microbiota of most vertebrates, and the change of the peritrophic matrix during moulting also limits the microbial growth. In addition, the silkworm gut has low O2 levels, thus it allows only the growth of facultative anaerobic microorganisms 20 . Diet has a great impact on gut microbiota profile of the organisms, and it is significantly associated with health and disease 21 , 22 . It is also possible to manipulate gut microbiota with using different diet 23 .Tormentic acid has been shown to take part in the treatment of bacterial disorders such as ulcerative colitis 24 , and periodontal disease 25 . Therefore, it could be a good candidate molecule for manipulation of the gut microbiota. In the present study, our aim was to investigate the effect of feeding silkworms with diet, consisting of different tormentic acid content, on the microbiota profile of silkworm gut. Results Intestine samples were collected from silkworms fed on diet with various tormentic acid content. The number of samples in each group is shown in Table-1. Shannon and Chao1 alpha-diversity indexes of the samples are presented in Table 2. ClustVis webtool 26 was used to calculate principal components (PCs) and PC values were charted in 3D scatterplots in JMP Pro 13. Microbiota composition of the samples did not indicate any clear clustering (Fig. 1 ). In total, 20 phyla were detected, and relative abundances of these phyla were charted in Fig. 2 . Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria constitutes more than 90% of all phyla for all the samples. Figure 3 shows the relative abundances of the 15 most prevalent genera in all groups. Staphylococcus spp. constituted approximately 30% of all genera in Control, RawExtract and TA_rich feeding groups. TA_poor and RawExtract groups had relatively more Bacillus spp. compared to TA_rich and Control groups. The TA_poor group also had relatively more Enterococcus spp. compared to the other groups. The response screening analysis resulted in a more detailed analysis of the differences at genus level between the groups. Based on the response screening analysis, Intestinibacter spp. were significantly more abundant in the gut microbiota of silkworm fed on TA_rich diet compared to the ones fed on raw extract (Fig. 4 ). TA_rich group samples also had significantly more prevalent Intestinibacter spp. when compared to the TA_poor group samples. Besides that, Flavonifractor spp. and some members of Veillonella spp. were significantly more prevalent in TA_rich group samples compared to the TA_poor group samples (Fig. 5 ). TA_rich group samples had significantly more abundant Senegalimassilia spp. compared to the Control group samples (Fig. 6 ). The silkworms fed on raw extract had significantly more abundant Flavonifractor spp. and Leuconostoc spp. in their gut compared to the ones fed on TA_poor diet. However, TA_poor group samples had significantly more prevalent Coprobacter spp. compared to the Raw Extract group samples (Fig. 7 ). There was no significant difference between TA_poor group and Control group samples (Supplementary Fig. 2). Raw Extract group samples had significantly more prevalent Hungatella spp., Senegalimassilia spp., Turicimonas spp., Catenibacterium spp. compared to the Control group samples (Fig. 8 ). Discussion In the present study, we have investigated the alterations in the gut microbiota profile of silkworms fed on diet comprising different TA content using 16S rRNA gene sequencing. TA, a terpene, is a bioactive molecule with the potential to be a hepatoprotective agent, and it is also effective in type 2 diabetes. We have chosen the silkworm because it is an advantageous model organism due to its low gut microbial diversity compared to other organisms. Feeding silkworms with TA and evaluating changes in the gut microbiota profile of silkworms may provide insight into the biotransformation process of terpenes in the gut. Although there was a certain difference in the microbiota profiles of the feeding groups, there was no significant difference in bacterial diversity of these groups. This may be due to the controlled microenvironment in which silkworms are grown and the slight microbial changes that occur after feeding. In this study, fragments extracted from Sarcopoterium spinosum with different TA content were used in the diet of silkworms. The silkworm fed on TA_rich diet had significantly more Intestinibacter spp. compared to the ones fed on RawExtract and TA_poor diet. TA_rich group samples also had significantly more Flavonifractor spp. and some members of Veillonella spp. compared to TA_poor group samples. It was previously reported that the abundance of Intestinibacter spp. decreases and the abundance of Escherichia spp. increases in type 2 diabetes patients treated with metformin 27 – 29 . Metformin is an effective medicine widely used for type 2 diabetes, and one of the adverse effects of metformin is gastrointestinal disorders 30 ,31 which could be associated with increased abundance of Escherichia spp. Furthermore, current knowledge on the role of Intestinibacter spp. is scarce, but functional annotations suggest a role for these bacteria in mucus production through consumption of mucins 28 , 32 , which are helpful for immune and metabolic responses. Flavonifractor spp. are known for their role in degradation of flavonoids 33 which are bioactive molecules like terpenes 34 . Veillonella spp. are known as producers of propionate which supports health in the human gut 35 . TA_rich group samples had significantly more prevalent Senegalimassilia spp. compared to the control group. Like Intestinibacter spp., the abundance of Senegalimassilia spp. has been shown to decrease just after the metformin treatment. In conclusion, it can be said that TA, which is currently used in the treatment of type 2 diabetes, may have the potential to reverse the side effects of the widely used medication metformin. TA could also be given with metformin as a supplementary material to type 2 diabetes patients. To be able to assert this more precisely, further studies with larger sample size and appropriate experimental design are required. Relative abundance of Flavonifractor spp. was significantly higher in RawExtract samples compared to TA_poor samples. On the other hand, TA_poor samples had higher abundance of Coprobacter spp., and Leuconostoc spp. than RawExtract samples. Coprobacter spp. were shown to be positively associated with dietary uptake of polyphenols 36 , and Leuconostoc spp., members of lactic acid bacteria, have been shown to be associated with the production of extracellular polysaccharides 37 . RawExtract group samples had relatively higher abundance of Hungatella spp., Senegalimassilia spp., Turicimonas spp., Catenibacterium spp. compared to the Control group samples. Hungatella spp. play role in the conversion of carbohydrates into acetic acid which is involved in ATP synthesis pathways 38 . Catenibacterium spp. were shown to be associated with the fermentation of fibre and production of short chain fatty acids which play promote the gut health 39 . To date, there is no reported association between Turicimonas spp. and bioactive molecules. In conclusion, microbiota profile of Bombyx mori could be manipulated with the diet containing the terpen, TA. Our results indicated that there was an increase in the relative abundance of bacteria associated with healthy gut in TA-rich diets. Hence, it might be said that TA could be used as a supplementary product to ameliorate and stabilize the healthy gut. It could also be used as a reversal agent to alleviate the adverse effects of medicines. On the other hand, the sample size is an important limitation for this study, but the results provide an insight into the microbiota changes in the case of TA supplementation to the silkworm diet. In brief, the results we report in this study, albeit on a small scale, may lead to potential future studies. Methods Sampling Sarcopoterium spinosum plant was incubated at 60 ºC for 6 hours in 70% ethanol at a solid-liquid ratio of 1:20. The solvent was removed in the evaporator and the extract was obtained in the water phase. A part of this extract was used in the diet of silkworm in RawExtract group. The remaining extract was taken into a separatory funnel and butanol was added. It was incubated until phase formation was observed and then the water phase and butanol phase were separated. The butanol phase was taken into a 50 ml tube as TA_poor fraction and the remaining extract was evaporated to remove the butanol. The remaining phase was subjected to liquid-liquid extraction using ethyl acetate. The ethyl acetate phase was used as TA_rich fraction. Silkworm was reared at 26±2 °C with 75-85% humidity and normal daylight photoperiod. Silkworm in 5 th instar were fed on mulberry leaves incubated with different fractions of S. spinosum extraction; RawExtract, TA_rich, TA_poor, and control. The silkworm larva was fixed with the help of a needle from the head and tail and the digestive system was opened by carefully cutting the shell. Silk glands were separated and placed in a sterile 15 ml tube for gastrointestinal microbiota analysis. DNA Extraction Microbial DNA was extracted using the EurXGeneMATRIX Tissue and Bacterial DNA purification kit (EurX Ltd., Poland) and following the manufacturer’s instructions. The Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Fair Lawn, NJ, USA) was used to measure DNA concentrations before PCR. 16S rRNA gene amplification, and sequencing The 341F (Illumina_16S_341F 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WG CAG), and 805R (Illumina_16S_805R 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C) universal primers with adapter sequences were used 40 for amplification of the V3-V4 hypervariable region of 16S rRNA gene. For the first step PCR, 5 μl of amplicon PCR forward primer (1 μM), 5 μl of amplicon PCR reverse primer (1 μM), 2.5 μl of microbial DNA (5ng/μl) and 12.5 μl of 2X KAPA HotStart PCR Mix (Roche, Switzerland) were used at 95 °C initial denaturation for 3 min, followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. PCR products were cleaned up with AgencourtAMPure XP beads (Beckman Coulter Genomics, Fullerton, CA, USA) following the manufacturer’s protocol. In a second PCR step, dual indices and Illumina sequencing adapters were attached using 5 μl of amplicon PCR product DNA, 5 μl of Illumina Nextera XT Index Primer 1 (N7xx), 5 μl of Nextera XT Index Primer 2 (S5xx), 25 μl of 2X KAPA HotStart PCR Mix, and 10 μl of nuclease free water with thermocycling at 95 °C for 3 min, followed by 8 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. The final PCR products were cleaned with AgencourtAMPure XP beads, and final concentrations of samples were measured with Qubit™ dsDNA HS Assay Kit. Amplicon libraries was sequenced on a lane of the Illumina® MiSeq platform. Bioinformatics Sequenced raw data was converted to FASTA format. Quality control of reads were performed using QIIME 2 software 41 . Reads with Phred scores lower than 20, primer and barcode sequences, and chimeric sequences were filtered out using DADA2 software 42 . Taxonomic assignment of each cluster was carried out using QIIME2 software to match a representative sequence from each OTU to a sequence from the GreenGenes database. Statistical analyses. Richness and evenness of the samples were analysed using Chao1 and Shannon diversity indexes. The indexes of groups were compared to each other using Student’s t-test. Beta-diversity of the samples were analysed and compared using the first three principal components. Mean relative abundances of the twenty phyla and fifteen genera were charted compared for each pair of groups. Microbiota profiles of each group were compared to each other as previously described 43 . Briefly, Logfold changes (Log10) in relative abundance of the genera were calculated for each group, and pairwise comparisons between groups were performed. Robust response screening analysis was performed in JMP Pro 13 (SAS Institute Inc., Cary, NC) in order to evaluate the differences in genera relative abundance between pair of groups. A false discovery rate (FDR) correction was applied, and statistical significance was declared at FDR LogWorth of 1.3 (equivalent of a p -value of 0.05), and 2 (equivalent of a p -value of 0.01). Subsequently, the log fold change was plotted versus the Robust FDR LogWorth value using bubble plot graphs in JMPPro 13. Genera mean relative abundance defined the bubbles’ size, and effect size was indicated by the bubbles’ colouring. Declarations Author contributions V.B. assisted supervision of the laboratory work, analysed the data, and wrote the manuscript. S.G. collected the samples, performed laboratory work, and wrote part of the manuscript. O. B. obtained the funding, designed, and supervised the study, and critically evaluated the manuscript. 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Group Shannon Chao1 TA_rich 3.89±0.57 201.72±46.85 TA_poor 3.36±1.95 136.07±83.79 RawExtract 2.75±1.84 185.86±22.83 Control 4.36±2.28 157.67±25.13 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresBayetal.TormenticAcidAlterstheGutMicrobiotaoftheSilkwormBombyxmori.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 06 Jun, 2022 Reviews received at journal 30 May, 2022 Reviews received at journal 27 May, 2022 Reviewers agreed at journal 17 May, 2022 Reviewers agreed at journal 16 May, 2022 Reviewers invited by journal 16 May, 2022 Editor assigned by journal 16 May, 2022 Editor invited by journal 16 May, 2022 Submission checks completed at journal 16 May, 2022 First submitted to journal 20 Apr, 2022 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. 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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-1576191","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":106413501,"identity":"1fc66208-d760-4eac-b09c-bab89dac0360","order_by":0,"name":"Veysel Bay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYNCCAoYEBgbmAxDOAaK0GIC0sCXAtDA2EKmFx4A4LQbHe4w/fDBgyOOfkfPx4ZcKBjm+GwnsjyvwaTlzxkxyhgFDscSN3M3GMmcYjCVvJDA2nsGjRXJGjhkz0EmJDTdyt0lLtjEkbgBpwecyyflvjD//AWqZfyPn+W/Jfwz1BLXwS/AYSAO9DzQ8h43xYwNDggFBLTxpZZI9BhLFhmeeGUszHJMwnHnmYeNMfFrY2A9v/vCjwiZP7njyw48/amzk+Y4nH/iITwsDAwcoOiQYGAQSGJh5QAzCMcn+AOpEYBT+IKB2FIyCUTAKRiYAALRRUfNjjzyDAAAAAElFTkSuQmCC","orcid":"","institution":"Ege University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Veysel","middleName":"","lastName":"Bay","suffix":""},{"id":106413503,"identity":"afa94392-bbf2-4609-91a2-4d649813bebb","order_by":1,"name":"Seray Gür","email":"","orcid":"","institution":"Ege University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seray","middleName":"","lastName":"Gür","suffix":""},{"id":106413505,"identity":"07c250ed-8a24-4fca-8614-25736b1d9425","order_by":2,"name":"Oğuz Bayraktar","email":"","orcid":"","institution":"Ege University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Oğuz","middleName":"","lastName":"Bayraktar","suffix":""}],"badges":[],"createdAt":"2022-04-20 11:14:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1576191/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1576191/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21634791,"identity":"4f981356-ab49-42c9-bd30-42a0051103b3","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48143,"visible":true,"origin":"","legend":"\u003cp\u003e3D scatterplot of the first 3 PCs calculated for the microbiota composition of the samples.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/cf2e4ead8b5aa1f3c3d1fd49.png"},{"id":21635111,"identity":"1d8cc858-a616-4e94-8ef4-aec23ca6ebaf","added_by":"auto","created_at":"2022-05-18 19:21:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45876,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of bacterial phyla in all feeding groups.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/0eb0d73af9d4eea1ce1dbb80.png"},{"id":21634793,"identity":"2e8ad578-b1db-48d4-8113-118e4dc1c582","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46163,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of fifteen most prevalent genera in all feeding groups\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/aaa6eaa0c991fd7fa4859d17.png"},{"id":21634794,"identity":"c096b3e2-9ec3-4d69-9da2-98b4d75fec6f","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97248,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the microbiota profiles ofTA_rich vs RawExtract feeding groups (line at 1.3 (- - -) = \u003cem\u003ep\u003c/em\u003e-value 0.05, line at 2 (----) = \u003cem\u003ep\u003c/em\u003e-value 0.01 adjusted for FDR) The log fold change in genera relative abundances in samples from TA_rich group comparing to RawExtract group samples is plotted versus the corrected robust false discovery rate (FDR) LogWorth (i.e., log10P). Size of the circles represents the mean relative abundance of each genus, and colour represents the effect size.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/d4e6d6f6f05d65d1e4757f6f.png"},{"id":21634800,"identity":"7ae31ff3-3914-4e50-aa89-483d1944b9c3","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107861,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the microbiota profiles of TA_rich vs TA_poor feeding groups (line at 1.3 (- - -) = p-value 0.05, line at 2 (----) = p-value 0.01 adjusted for FDR) The log fold change in genera relative abundances in samples from TA_rich group comparing to TA_poor group samples is plotted versus the corrected robust false discovery rate (FDR) LogWorth (i.e., log10P). Size of the circles represents the mean relative abundance of each genus, and colour represents the effect size.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/171f2191a7f81bde0280adc4.png"},{"id":21634795,"identity":"3b89dad5-417d-4077-87e9-7b1c8405cf8f","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":101067,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the microbiota profiles of TA_rich vs Control feeding groups (line at 1.3 (- - -) = p-value 0.05, line at 2 (----) = p-value 0.01 adjusted for FDR) The log fold change in genera relative abundances in samples from TA_rich group comparing to Control group samples is plotted versus the corrected robust false discovery rate (FDR) LogWorth (i.e., log10P). Size of the circles represents the mean relative abundance of each genus, and colour represents the effect size.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures6.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/419974ecc5832a93f96a2739.png"},{"id":21634797,"identity":"83bbddc5-872c-4ecd-9805-7156b9570af0","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":101059,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the microbiota profiles of TA_poor vs RawExtract feeding groups (line at 1.3 (- - -) = p-value 0.05, line at 2 (----) = p-value 0.01 adjusted for FDR) The log fold change in genera relative abundances in samples from TA_poor group comparing to RawExtract group samples is plotted versus the corrected robust false discovery rate (FDR) LogWorth (i.e., log10P). Size of the circles represents the mean relative abundance of each genus, and colour represents the effect size.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figures7.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/05f720ff941dbde51e5628cf.png"},{"id":21635112,"identity":"d1372213-b49b-4cf2-b320-0ea3f8bf4b45","added_by":"auto","created_at":"2022-05-18 19:21:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":216822,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the microbiota profiles of RawExtract vs Control feeding groups (line at 2 (----) = p-value 0.01 adjusted for FDR) The log fold change in genera relative abundances in samples from RawExtract group comparing to Control group samples is plotted versus the corrected robust false discovery rate (FDR) LogWorth (i.e., log10P). Size of the circles represents the mean relative abundance of each genus, and colour represents the effect size.\u003c/p\u003e","description":"","filename":"Figures8.png","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/232cab9f9051525dc3b265ad.png"},{"id":21635113,"identity":"c9a7afa5-0222-4019-b2b3-1d3372449aa4","added_by":"auto","created_at":"2022-05-18 19:21:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":259567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/d98765cc-656e-4f4e-8abd-78e9c6eb6e3f.pdf"},{"id":21634799,"identity":"972aa720-a540-4c92-9da1-dd6b8a50f9d8","added_by":"auto","created_at":"2022-05-18 19:16:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1449303,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresBayetal.TormenticAcidAlterstheGutMicrobiotaoftheSilkwormBombyxmori.docx","url":"https://assets-eu.researchsquare.com/files/rs-1576191/v1/71e200100c1df3dd3931994f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tormentic Acid Alters the Gut Microbiota of the Silkworm (Bombyx mori)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHerbal natural compounds include phenolic compounds and secondary plant metabolites, generally known as phytochemicals. These compounds have started to gain great importance with their positive effects against many diseases due to their bioactivity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.Most of the natural compounds undergo microbial changes, namely biotransformation, in the small intestine before being transformed in the liver\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Biotransformation processes using mammalian, microorganism, plant cells and insect cell cultures or enzymes isolated from these play an important role in the discovery and development of new drug molecules\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe silkworm (\u003cem\u003eBombyx mori\u003c/em\u003e), which feeds on mulberry (\u003cem\u003eMorus alba\u003c/em\u003e) leaves, has been domesticated for thousands of years to produce economically important silk fibre \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Silkworm has provided a livelihood for humans for centuries and has been a highly productive source of biological and chemical substances\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to the benefits of silkworm itself, many compounds are produced from its excrement, which are sources of treatment and food for other organisms(Liu J. et al. 2013, Liu X. et al. 2019; Li et al. 2019). Besides, it is possible to form bioactive compounds by feeding the silkworm with mulberry leaves enriched with different natural compounds by making use of the biotransformation ability of the silkworm \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCompounds that prevent liver damage, trigger tissue regeneration, or reduce the activity of toxic substances are called hepatoprotective agents. Some of the natural herbal compounds have antimicrobial, antidiabetic, antifungal and hepatoprotective effects \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is stated that tormentic acid (TA), one of the terpenes found in the content of \u003cem\u003eSarcopoterium spinosum\u003c/em\u003e, has the potential to be a hepatoprotective agent, as well as its effectiveness in type 2 diabetes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMany microorganisms live in the body of the silkworm. However, the microbiota of this insect model has not been well characterized to date \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. With the advent of metagenomics technology, microbial communities of various species have been characterized and listed by genome studies on vertebrates and invertebrates \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe silkworm gut has less microbial diversity compared to the other vertebrates due to controlled microenvironment for growth and development and shorter lifespan. In addition, the silkworm microbiota must be able to tolerate the alkalinity up to pH 11\u0026ndash;12, which adversely affects the microbiota of most vertebrates, and the change of the peritrophic matrix during moulting also limits the microbial growth. In addition, the silkworm gut has low O2 levels, thus it allows only the growth of facultative anaerobic microorganisms\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDiet has a great impact on gut microbiota profile of the organisms, and it is significantly associated with health and disease \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It is also possible to manipulate gut microbiota with using different diet \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.Tormentic acid has been shown to take part in the treatment of bacterial disorders such as ulcerative colitis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and periodontal disease \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, it could be a good candidate molecule for manipulation of the gut microbiota. In the present study, our aim was to investigate the effect of feeding silkworms with diet, consisting of different tormentic acid content, on the microbiota profile of silkworm gut.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIntestine samples were collected from silkworms fed on diet with various tormentic acid content. The number of samples in each group is shown in Table-1.\u003c/p\u003e \u003cp\u003eShannon and Chao1 alpha-diversity indexes of the samples are presented in Table\u0026nbsp;2. ClustVis webtool \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e was used to calculate principal components (PCs) and PC values were charted in 3D scatterplots in JMP Pro 13. Microbiota composition of the samples did not indicate any clear clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn total, 20 phyla were detected, and relative abundances of these phyla were charted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria constitutes more than 90% of all phyla for all the samples.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the relative abundances of the 15 most prevalent genera in all groups. \u003cem\u003eStaphylococcus\u003c/em\u003e spp. constituted approximately 30% of all genera in Control, RawExtract and TA_rich feeding groups. TA_poor and RawExtract groups had relatively more \u003cem\u003eBacillus\u003c/em\u003e spp. compared to TA_rich and Control groups. The TA_poor group also had relatively more \u003cem\u003eEnterococcus\u003c/em\u003e spp. compared to the other groups.\u003c/p\u003e \u003cp\u003eThe response screening analysis resulted in a more detailed analysis of the differences at genus level between the groups. Based on the response screening analysis, \u003cem\u003eIntestinibacter\u003c/em\u003e spp. were significantly more abundant in the gut microbiota of silkworm fed on TA_rich diet compared to the ones fed on raw extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). TA_rich group samples also had significantly more prevalent \u003cem\u003eIntestinibacter\u003c/em\u003e spp. when compared to the TA_poor group samples. Besides that, \u003cem\u003eFlavonifractor\u003c/em\u003e spp. and some members of \u003cem\u003eVeillonella\u003c/em\u003e spp. were significantly more prevalent in TA_rich group samples compared to the TA_poor group samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTA_rich group samples had significantly more abundant \u003cem\u003eSenegalimassilia\u003c/em\u003e spp. compared to the Control group samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe silkworms fed on raw extract had significantly more abundant \u003cem\u003eFlavonifractor\u003c/em\u003e spp. and \u003cem\u003eLeuconostoc\u003c/em\u003e spp. in their gut compared to the ones fed on TA_poor diet. However, TA_poor group samples had significantly more prevalent \u003cem\u003eCoprobacter\u003c/em\u003e spp. compared to the Raw Extract group samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). There was no significant difference between TA_poor group and Control group samples (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eRaw Extract group samples had significantly more prevalent \u003cem\u003eHungatella\u003c/em\u003e spp., \u003cem\u003eSenegalimassilia\u003c/em\u003e spp., \u003cem\u003eTuricimonas\u003c/em\u003e spp., \u003cem\u003eCatenibacterium\u003c/em\u003e spp. compared to the Control group samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we have investigated the alterations in the gut microbiota profile of silkworms fed on diet comprising different TA content using 16S rRNA gene sequencing. TA, a terpene, is a bioactive molecule with the potential to be a hepatoprotective agent, and it is also effective in type 2 diabetes. We have chosen the silkworm because it is an advantageous model organism due to its low gut microbial diversity compared to other organisms. Feeding silkworms with TA and evaluating changes in the gut microbiota profile of silkworms may provide insight into the biotransformation process of terpenes in the gut. Although there was a certain difference in the microbiota profiles of the feeding groups, there was no significant difference in bacterial diversity of these groups. This may be due to the controlled microenvironment in which silkworms are grown and the slight microbial changes that occur after feeding. In this study, fragments extracted from \u003cem\u003eSarcopoterium spinosum\u003c/em\u003e with different TA content were used in the diet of silkworms. The silkworm fed on TA_rich diet had significantly more \u003cem\u003eIntestinibacter\u003c/em\u003e spp. compared to the ones fed on RawExtract and TA_poor diet. TA_rich group samples also had significantly more \u003cem\u003eFlavonifractor\u003c/em\u003e spp. and some members of \u003cem\u003eVeillonella\u003c/em\u003e spp. compared to TA_poor group samples. It was previously reported that the abundance of \u003cem\u003eIntestinibacter\u003c/em\u003e spp. decreases and the abundance of \u003cem\u003eEscherichia\u003c/em\u003e spp. increases in type 2 diabetes patients treated with metformin\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Metformin is an effective medicine widely used for type 2 diabetes, and one of the adverse effects of metformin is gastrointestinal disorders\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,31\u003c/sup\u003ewhich could be associated with increased abundance of \u003cem\u003eEscherichia\u003c/em\u003e spp. Furthermore, current knowledge on the role of \u003cem\u003eIntestinibacter\u003c/em\u003e spp. is scarce, but functional annotations suggest a role for these bacteria in mucus production through consumption of mucins\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, which are helpful for immune and metabolic responses. \u003cem\u003eFlavonifractor\u003c/em\u003e spp. are known for their role in degradation of flavonoids\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e which are bioactive molecules like terpenes\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eVeillonella\u003c/em\u003e spp. are known as producers of propionate which supports health in the human gut \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. TA_rich group samples had significantly more prevalent \u003cem\u003eSenegalimassilia\u003c/em\u003e spp. compared to the control group. Like \u003cem\u003eIntestinibacter\u003c/em\u003e spp., the abundance of \u003cem\u003eSenegalimassilia\u003c/em\u003e spp. has been shown to decrease just after the metformin treatment. In conclusion, it can be said that TA, which is currently used in the treatment of type 2 diabetes, may have the potential to reverse the side effects of the widely used medication metformin. TA could also be given with metformin as a supplementary material to type 2 diabetes patients. To be able to assert this more precisely, further studies with larger sample size and appropriate experimental design are required.\u003c/p\u003e \u003cp\u003eRelative abundance of \u003cem\u003eFlavonifractor\u003c/em\u003e spp. was significantly higher in RawExtract samples compared to TA_poor samples. On the other hand, TA_poor samples had higher abundance of \u003cem\u003eCoprobacter\u003c/em\u003e spp., and \u003cem\u003eLeuconostoc\u003c/em\u003e spp. than RawExtract samples. \u003cem\u003eCoprobacter\u003c/em\u003e spp. were shown to be positively associated with dietary uptake of polyphenols\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eLeuconostoc\u003c/em\u003e spp., members of lactic acid bacteria, have been shown to be associated with the production of extracellular polysaccharides \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRawExtract group samples had relatively higher abundance of \u003cem\u003eHungatella\u003c/em\u003e spp., \u003cem\u003eSenegalimassilia\u003c/em\u003e spp., \u003cem\u003eTuricimonas\u003c/em\u003e spp., \u003cem\u003eCatenibacterium\u003c/em\u003e spp. compared to the Control group samples. Hungatella spp. play role in the conversion of carbohydrates into acetic acid which is involved in ATP synthesis pathways\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCatenibacterium\u003c/em\u003e spp. were shown to be associated with the fermentation of fibre and production of short chain fatty acids which play promote the gut health\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. To date, there is no reported association between \u003cem\u003eTuricimonas\u003c/em\u003e spp. and bioactive molecules.\u003c/p\u003e \u003cp\u003eIn conclusion, microbiota profile of \u003cem\u003eBombyx mori\u003c/em\u003e could be manipulated with the diet containing the terpen, TA. Our results indicated that there was an increase in the relative abundance of bacteria associated with healthy gut in TA-rich diets. Hence, it might be said that TA could be used as a supplementary product to ameliorate and stabilize the healthy gut. It could also be used as a reversal agent to alleviate the adverse effects of medicines. On the other hand, the sample size is an important limitation for this study, but the results provide an insight into the microbiota changes in the case of TA supplementation to the silkworm diet. In brief, the results we report in this study, albeit on a small scale, may lead to potential future studies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eSampling \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSarcopoterium\u0026nbsp;spinosum\u0026nbsp;\u003c/em\u003eplant was incubated at 60 \u0026ordm;C for 6 hours in 70% ethanol at a solid-liquid ratio of 1:20. The solvent was removed in the evaporator and the extract was obtained in the water phase. A part of this extract was used in the diet of silkworm in RawExtract group. The remaining extract was taken into a separatory funnel and butanol was added. It was incubated until phase formation was observed and then the water phase and butanol phase were separated. The butanol phase was taken into a 50 ml tube as TA_poor fraction and the remaining extract was evaporated to remove the butanol. The remaining phase was subjected to liquid-liquid extraction using ethyl acetate. The ethyl acetate phase was used as TA_rich fraction.\u003c/p\u003e\n\u003cp\u003eSilkworm was reared at 26\u0026plusmn;2 \u0026deg;C with 75-85% humidity and normal daylight photoperiod. Silkworm in 5\u003csup\u003eth\u003c/sup\u003e instar were fed on mulberry leaves incubated with different fractions of \u003cem\u003eS. spinosum\u003c/em\u003e extraction; RawExtract, TA_rich, TA_poor, and control. The silkworm larva was fixed with the help of a needle from the head and tail and the digestive system was opened by carefully cutting the shell. Silk glands were separated and placed in a sterile 15 ml tube for gastrointestinal microbiota analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA Extraction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMicrobial DNA was extracted using the EurXGeneMATRIX Tissue and Bacterial DNA purification kit (EurX Ltd., Poland) and following the manufacturer\u0026rsquo;s instructions. The Qubit\u0026trade; dsDNA HS Assay Kit (Thermo Fisher Scientific, Fair Lawn, NJ, USA) was used to measure DNA concentrations before PCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e16S rRNA gene amplification, and sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 341F (Illumina_16S_341F 5\u0026prime;-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WG CAG), and 805R (Illumina_16S_805R 5\u0026prime;-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C) universal primers with adapter sequences were used\u0026nbsp;\u003csup\u003e40\u003c/sup\u003e for amplification of the V3-V4 hypervariable region of 16S rRNA gene.\u003c/p\u003e\n\u003cp\u003eFor the first step PCR, 5 \u0026mu;l of amplicon PCR forward primer (1 \u0026mu;M), 5 \u0026mu;l of amplicon PCR reverse primer (1 \u0026mu;M), 2.5 \u0026mu;l of microbial DNA (5ng/\u0026mu;l) and 12.5 \u0026mu;l of\u0026nbsp;2X KAPA HotStart PCR Mix (Roche, Switzerland) were used at 95 \u0026deg;C initial denaturation for 3 min, followed by 25 cycles of 95 \u0026deg;C for 30 s, 55 \u0026deg;C for 30 s, and 72 \u0026deg;C for 30 s, and a final extension at 72 \u0026deg;C for 5 min. PCR products were cleaned up with AgencourtAMPure XP beads (Beckman Coulter Genomics, Fullerton, CA, USA) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003eIn a second PCR step, dual indices and Illumina sequencing adapters were attached using 5 \u0026mu;l of amplicon PCR product DNA, 5 \u0026mu;l of Illumina Nextera XT Index Primer 1 (N7xx), 5 \u0026mu;l of Nextera XT Index Primer 2 (S5xx), 25 \u0026mu;l of\u0026nbsp;2X KAPA HotStart PCR Mix, and 10 \u0026mu;l of nuclease free water with thermocycling at 95 \u0026deg;C for 3 min, followed by 8 cycles of 95 \u0026deg;C for 30 s, 55 \u0026deg;C for 30 s, and 72 \u0026deg;C for 30 s, and a final extension at 72 \u0026deg;C for 5 min. The final PCR products were cleaned with AgencourtAMPure XP beads, and final concentrations of samples were measured with Qubit\u0026trade; dsDNA HS Assay Kit. Amplicon libraries was sequenced on a lane of the Illumina\u0026reg; MiSeq platform.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBioinformatics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSequenced raw data was converted to FASTA format. Quality control of reads were performed using QIIME 2 software\u0026nbsp;\u003csup\u003e41\u003c/sup\u003e. Reads with Phred scores lower than 20, primer and barcode sequences, and chimeric sequences were filtered out using DADA2 software\u0026nbsp;\u003csup\u003e42\u003c/sup\u003e. Taxonomic assignment of each cluster was carried out using QIIME2 software to match a representative sequence from each OTU to a sequence from the GreenGenes database.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analyses.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRichness and evenness of the samples were analysed using Chao1 and Shannon diversity indexes. The indexes of groups were compared to each other using Student\u0026rsquo;s t-test. Beta-diversity of the samples were analysed and compared using the first three principal components. Mean relative abundances of the twenty phyla and fifteen genera were charted compared for each pair of groups. Microbiota profiles of each group were compared to each other as previously described\u0026nbsp;\u003csup\u003e43\u003c/sup\u003e. Briefly, Logfold changes (Log10) in relative abundance of the genera were calculated for each group, and pairwise comparisons between groups were performed. Robust response screening analysis was performed in JMP Pro 13 (SAS Institute Inc., Cary, NC) in order to evaluate the differences in genera relative abundance between pair of groups. A false discovery rate (FDR) correction was applied, and statistical significance was declared at FDR LogWorth of 1.3 (equivalent of a \u003cem\u003ep\u003c/em\u003e-value of 0.05), and 2 (equivalent of a \u003cem\u003ep\u003c/em\u003e-value of 0.01). Subsequently, the log fold change was plotted versus the Robust FDR LogWorth value using bubble plot graphs in JMPPro 13. Genera mean relative abundance defined the bubbles\u0026rsquo; size, and effect size was indicated by the bubbles\u0026rsquo; colouring.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV.B. assisted supervision of the laboratory work, analysed the data, and wrote the manuscript. S.G. collected the samples, performed laboratory work, and wrote part of the manuscript. O. B. obtained the funding, designed, and supervised the study, and critically evaluated the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequences are available on the MG-RAST metagenomics analysis server at https://www.mg-rast.org/mgmain.html?mgpage=project\u0026amp;project=mgp101832.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests or other competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eUpadhyaya, S. Screening of phytochemicals, nutritional status, antioxidant and antimicrobial activity of Paederia foetida Linn. from different localities of Assam, India. 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The number of samples in each group\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eTA_rich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eTA_poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eRawExtract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;Table 2. Shannon and Chao1 alpha-diversity indexes of the samples (Mean \u0026plusmn; s. d.).\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.04597701149425%\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.022988505747126%\"\u003e\n \u003cp\u003eShannon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.93103448275862%\"\u003e\n \u003cp\u003eChao1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.04597701149425%\"\u003e\n \u003cp\u003eTA_rich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.022988505747126%\"\u003e\n \u003cp\u003e3.89\u0026plusmn;0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.93103448275862%\"\u003e\n \u003cp\u003e201.72\u0026plusmn;46.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.04597701149425%\"\u003e\n \u003cp\u003eTA_poor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.022988505747126%\"\u003e\n \u003cp\u003e3.36\u0026plusmn;1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.93103448275862%\"\u003e\n \u003cp\u003e136.07\u0026plusmn;83.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.04597701149425%\"\u003e\n \u003cp\u003eRawExtract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.022988505747126%\"\u003e\n \u003cp\u003e2.75\u0026plusmn;1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.93103448275862%\"\u003e\n \u003cp\u003e185.86\u0026plusmn;22.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.04597701149425%\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.022988505747126%\"\u003e\n \u003cp\u003e4.36\u0026plusmn;2.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.93103448275862%\"\u003e\n \u003cp\u003e157.67\u0026plusmn;25.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n"}],"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"tormentic acid, silkworm, 16S amplicon sequencing, phytochemicals, Sarcopoterium spinosum","lastPublishedDoi":"10.21203/rs.3.rs-1576191/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1576191/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent years, phytochemicals have started to attract more attention due to their contribution to health and bioactivity. Microorganisms in the intestines of organisms contribute to the processing, function, and biotransformation of these substances. The silkworm (\u003cem\u003eBombyx mori\u003c/em\u003e) is one of the organisms used for the biotransformation of phytochemicals due to its controlled reproduction and liability to microbial manipulation. In this study, a bioactive compound, tormentic acid (TA), extracted from \u003cem\u003eSarcopoterium spinosum\u003c/em\u003e was used in the diet of silkworm and the alterations of intestinal microbiota of the silkworm were assessed. To do this, silkworms were fed on diet with various tormentic acid content, and 16S metagenomic analysis was performed to determine and the alterations in the gut microbiota profile of these organisms. Diet with different TA content did not cause a change in bacterial diversity of the samples. Comprehensive comparisons between different feeding groups indicated increased abundance of bacteria associated with health, i.e., \u003cem\u003eIntestinibacter\u003c/em\u003e spp., \u003cem\u003eFlavonifractor\u003c/em\u003e spp., \u003cem\u003eSenegalimassilia\u003c/em\u003e spp., through the utilization of bioactive substances such as flavonoids. In conclusion, it might be said that use of TA as a supplementary product might help ameliorating the infected gut and promoting the healthy gut and relieving the undesirable effects of medicines to gastrointestinal system.\u003c/p\u003e","manuscriptTitle":"Tormentic Acid Alters the Gut Microbiota of the Silkworm (Bombyx mori)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-18 19:16:14","doi":"10.21203/rs.3.rs-1576191/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-06-06T10:18:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-31T02:43:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-27T07:13:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0bac6990-e7e9-4e80-a4ca-5154a3785062","date":"2022-05-17T12:42:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142369a7-5d4c-4caf-9405-148c2ff13d81","date":"2022-05-17T00:12:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-16T15:38:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-16T15:34:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-05-16T15:30:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-05-16T15:28:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-04-20T11:11:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"430041cb-2bfb-49f0-9467-33f48f3515c9","owner":[],"postedDate":"May 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-07-26T07:14:21+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-18 19:16:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1576191","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1576191","identity":"rs-1576191","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}