16S rRNA sequence data of Bombyx mori gut bacteriome after spermidine supplementation
BMC Research Notes volume 13, Article number: 94 (2020)
The silkworm Bombyx mori (B. mori) is an important domesticated lepidopteran model for basic and applied research. They produce silk fibres that have great economic value. The gut microbiome plays an important role in the growth of organisms. Spermidine (Spd) is shown to be important for the growth of all living cells. The effect of spermidine feeding on the gut microbiome of 5th instar B. mori larvae was checked. The B. mori gut samples from control and spermidine fed larvae were subjected to next-generation sequencing analysis to unravel changes in the bacterial community upon spermidine supplementation.
The changes in gut bacteriota after spermidine feeding is not studied before. B. mori larvae were divided into two groups of 50 worms each and were fed with normal mulberry leaves and mulberry leaves fortified with 50 µM spermidine. The gut tissues were isolated aseptically and total genomic DNA was extracted, 16S rRNA region amplified and sequenced using Illumina platform. The spermidine fed gut samples were shown to have abundance and diversity of the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria.
B. mori is an economically important and domesticated silkworm from family Bombycidae [1, 2]. B. mori feeds on mulberry leaves and produces silk which has been used as a fabric, biomaterial, and in cosmetics [3, 4]. The quality and quantity of the silk fibres produced depend on the food consumed . The growth, absorption, and utilization of nutrients are influenced by the gut microbiota [6,7,8]. Bacterial flora associated with B. mori gut help them in degrading various otherwise inaccessible polysaccharides from the diet . These complex communities are essential for the environmental adaptation and development of the host [10,11,12,13,14,15]. Polyamines are biogenic amines found in all eukaryotic cells, which perform distinct cellular functions . Spd is a biologically important polyamine that serves as a key regulator of processes like DNA stability, protein synthesis, cell proliferation, differentiation, and apoptosis [17,18,19]. The potential effect of Spd supplementation to B. mori efficiently enhanced the larval weights, silk gland weights, silk quality and quantity, and mechanical and structural properties of the silk fibres [20, 21].
We aimed to understand changes in the gut microbiota post Spd supplementation when compared to control. The role of polyamines in biological functions, diseases, drug targeting, growth, free radical scavenging and cell viability are known. Our data could be of potential support to the researchers to understand the effect of Spd from a different perspective on gut bacteriota and foster a challenging and exciting research further. This study offers an improved diet with enhancement in beneficial microbes of the gut. This data was collected as part of finding the influence of Spd on the nutrition of B. mori. The detailed influence of Spd on the nutrition of B. mori 5th instar silkworms is unpublished research data.
Silk worm rearing and DNA isolation
The high silk yielding bivoltine breed, CSR2 × CSR4 B. mori larvae in 4th moult were procured from Andhra Pradesh state sericulture farm. Larvae were reared in a cleaned and disinfected room at 26 ± 2 °C with relative humidity of 65–85%. 50 µM Spd solution was applied to the mulberry leaves based on established protocol  from day 1 of 5th instar stage, and continued till the pupation (about 6 days) as during this stage larvae consume high quantity of mulberry leaf and produce maximum silk. The whole gut was extracted aseptically on day 5 and gut contents were flushed out for total genomic DNA isolation from both control and Spd fed groups (n = 1) using the QIAamp DNA Kit. The concentration and purity were determined by spectrophotometry.
Library preparation and 16S rRNA sequencing
The 16S rRNA gene V3–V4 hypervariable regions were amplified using region-specific primers from KAPA HiFi HotStart PCR Kit (KAPA Biosystems Inc., Boston, MA USA). A negative control was maintained without the template DNA. A second-round indexing PCR was performed with the multiplexed amplicons which were amplified for 10 cycles with Illumina sequencing barcoded adaptors using Nextera XT v2 Index Kit, Illumina, U.S.A. The PCR products were analysed on 1.2% agarose gel after each round. The normalized, pooled and quantified libraries were used for Illumina MiSeq sequencing. 5% PhiX was spiked into introduce nucleotide diversity. The metagenome sequencing was carried out at Genotypic Technologies Pvt. Ltd., Bangalore, India.
Analysis of metagenome sequence data
The Illumina paired-end reads were demultiplexed using bcl2fastqtool (“bcl2fastq” n.d.) and quality checked by FastQC . The high- quality raw reads were stitched using Fastq-join  and further analysed using QIIMEpipeline . The query sequences were clustered using the UCLUST5 method  against a curated chimera free 16S rRNA database (Greengenes v 13.8) . The taxonomies were assigned using the RDP classifier  to these clusters at ≥ 50% sequence similarity against the reference database and generated of a biom file for further advanced analysis and visualization. The raw sequence files of control (Data file 1, Table 1) and Spd treated (Data file 2, Table 1) were deposited in NCBI, SRA database. 16S rRNA sequences were used to pick operational taxonomic units (OTUs) at 97% similarity threshold from the Greengenes database. To determine sampling depth and species richness, rarefaction curves were plotted (Data file 3, Table 1). The read details were mentioned in Data file 4, in Table 1. The taxonomic profile analysis of control and Spd fed gut tissues were indicated in stacked bar plots in Datafile 5 Table 1.
The Proteobacteria provide nutrients to their host, while Firmicutes increases the energy harvest from the diet . Spd feeding in the mouse model increased the abundance of Prevotella and Clostridium . Another major bacterial symbiont in Spd treated gut, Halomonas were reported to produce extracellular polysaccharides that help in adhesion and create microenvironments which favour, interaction and a cellular association between microorganisms .
Current data is based on only one biological replicate for single strain of B. mori gut metagenome sequencing.
Availability of data materials
The data described here is available in the NCBI, SRA database under the accession (https://www.ncbi.nlm.nih.gov/sra/?term=SRP126130). Please refer data file 1 and 2 for links to the sequence data.
Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, et al. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science. 2004;306(5703):1937–40.
Yang S-Y, Han M-J, Kang L-F, Li Z-W, Shen Y-H, Zhang Z. Demographic history and gene flow during silkworm domestication. BMC Evol Biol. 2014;14(14):185.
Song J, Che J, You Z, Ye L, Li J, Zhang Y, et al. Phosphoproteomic analysis of the posterior silk gland of Bombyx mori provides novel insight into phosphorylation regulating the silk production. J Proteomics. 2016;04(148):194–201.
Wang H, Wang Y, Wu C, Tao H, Chen X, Yin W, et al. Changes in 30K protein synthesis during delayed degeneration of the silk gland by a caspase-dependent pathway in a Bombyx (silkworm) mutant. J Comp Physiol B Biochem Syst Environ Physiol. 2016;186(6):689–700.
Maribashetty VG. Food and water utilization patterns in new bivoltine races of silkworm, Bombyx mori L. Bull Indian Acad Sericulture. 1991;3:83–90.
Adams AS, Jordan MS, Adams SM, Suen G, Goodwin LA, Davenport KW, et al. Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio. ISME J. 2011;5(8):1323–31.
McFall-Ngai M, Hadfield MG, Bosch TCG, Carey HV, Domazet-Lošo T, Douglas AE, et al. Animals in a bacterial world, a new imperative for the life sciences. Proc Natl Acad Sci USA. 2013;110(9):3229–36.
Senderovich Y, Halpern M. The protective role of endogenous bacterial communities in chironomid egg masses and larvae. ISME J. 2013;7(11):2147–58.
Anand AAP, Vennison SJ, Sankar SG, Prabhu DIG, Vasan PT, Raghuraman T, et al. Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion. J Insect Sci. 2010;10:107.
Chen B, Du K, Sun C, Vimalanathan A, Liang X, Li Y, et al. Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives. ISME J. 2018;12(9):2252–62.
Xiang H. Bacterial community in midguts of the silkworm larvae estimated by PCR/DGGE and 16S rDNA gene library analysis. Acta Entomol Sin. 2007;50:222–33.
Subramanian S. Use of 16S rRNA probes for characterization of gut microflora of silkworm (Bombyx mori L.) breeds. Karnataka J Agric Sci. 2009;22:476–8.
Vitthalrao BK, Rajendra MM. Diversity of bacterial flora in the midgut of fifth instar larvae of silkworm Bombyx mori (L.) (Race: PM X CSR2). GJBB. 2012;1(2):191–200.
Sun Z, Lu Y, Zhang H, Kumar D, Liu B, Gong Y, et al. Effects of BmCPV infection on silkworm Bombyx mori intestinal bacteria. PLoS ONE. 2016;11(1):e0146313.
Sun Z, Kumar D, Cao G, Zhu L, Liu B, Zhu M, et al. Effects of transient high temperature treatment on the intestinal flora of the silkworm Bombyx mori. Sci Rep. 2017;7(1):3349.
Bachrach U. Functions of naturally occurring Polyamines. New York: Academic Press Inc; 1973.
Eisenberg T, Knauer H, Schauer A, Büttner S, Ruckenstuhl C, Carmona-Gutierrez D, et al. Induction of autophagy by spermidine promotes longevity. Nat Cell Biol. 2009;11(11):1305–14.
Minois N, Rockenfeller P, Smith TK, Carmona-Gutierrez D. Spermidine feeding decreases age-related locomotor activity loss and induces changes in lipid composition. PLoS ONE. 2014;9(7):e102435.
Morselli E, Mariño G, Bennetzen MV, Eisenberg T, Megalou E, Schroeder S, et al. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. J Cell Biol. 2011;192(4):615–29.
Yerra A, Mysarla DK, Siripurapu P, Jha A, Valluri SV, Mamillapalli A. Effect of polyamines on mechanical and structural properties of Bombyx mori silk. Biopolymers. 2017;107(1):20–7.
Lattala GM, Kandukuru K, Gangupantula S, Mamillapalli A. Spermidine enhances the silk production by mulberry silkworm. J Insect Sci. 2014;14:207.
Andrews S. Babraham bioinformatics—FastQC a quality control tool for high throughput sequence data. [cited 2019 Aug 22]. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
Erik A. Comparison of sequencing utility programs. Open Bioinforma J. 2013;7:1–8.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7(5):335–6.
Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26(19):2460–1.
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006;72(7):5069–72.
Gaujoux R, Seoighe C. A flexible R package for nonnegative matrix factorization. BMC Bioinformatics. 2010;11:367.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444(7122):1027–31.
Gómez-Gallego C, Collado MC, Ilo T, Jaakkola U-M, Bernal MJ, Periago MJ, et al. Infant formula supplemented with polyamines alters the intestinal microbiota in neonatal BALB/cOlaHsd mice. J Nutr Biochem. 2012;23(11):1508–13.
Llamas I. The potential biotechnological application of the exopolysaccharide produced by the halophilic bacterium Halomonas almeriansis. Molecules. 2012;17:7103–20.
The authors wish to thank Mr. K. Satya Rao (Asst. Director) of Department of Sericulture, Srikakulam, Government of Andhra Pradesh for providing silkworms and the Department of Biotechnology (DBT), New Delhi, India for their financial support.
This study was funded by DBT, New Delhi, India under the project sanction order number: BT/PR15319/TDS/121/12/2015.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Rajan, R., Chunduri, A.R., Lima, A. et al. 16S rRNA sequence data of Bombyx mori gut bacteriome after spermidine supplementation. BMC Res Notes 13, 94 (2020). https://doi.org/10.1186/s13104-020-04958-x