Abstract
Background:
Bombyx mori is a key species in the sericulture industry and depends on mulberry leaves for growth and silk production.

Aim:
The present study characterized the gut microbiota of B. mori fed on two mulberry species using 16S rRNA amplicon sequencing

Methods:
A total of ten healthy fifth instar larvae were randomly selected from each treatment group. The larvae were dissected under sterile conditions to extract the gut tissues to make a pooled sample. DNA was extracted using QIAamp DNA Microbiome kit and primer set 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492-R (5-GGTTACCTTGTTACGACTT-3) was used to amplify variable regions V3–V4 of 16S rRNA gene using Illumina sequencing. OTU data was used to estimate the bacterial species richness and evenness using the EzBioCloud platform. The microbial communities’ phylum to genus level were grouped. Raw paired end reads (FASTQ format) were imported in Qiime2 v2021.4 software and Krona plot was constructed.

Results:
The gut microbiota of B. mori larvae fed on Morus alba was predominantly composed of Proteobacteria (36.46%), Bacteroidota (22.98%), Firmicutes (20.31%), and Actinobacteriota (20.15%). All of these play critical roles in nitrogen fixation, carbohydrate breakdown, and immune modulation, thereby supporting optimal gut health and nutrient absorption. In contrast, silkworms fed on Morus nigra exhibited a microbiota dominated by Proteobacteria (31.74%), Verrucomicrobiota (23.35%), Bacteroidota (12.58%), and Bdellovibrionota (12.30%). The Bdellovibrionota, a predatory bacterium which may disrupt the balance and potentially affect silkworm health and growth. The differences in microbiota composition of M. alba and M. nigra was further supported by proximate analysis. M. alba had significantly higher levels of crude protein (26.16%) and crude fat (4.53%) as compared to M. nigra (19.50% and 3.20%, respectively). Additionally, silkworms fed with M. alba exhibited superior growth across all molting stages compared to those fed M. nigra.

Conclusion:
Future studies should focus on the effects of probiotic supplementation to evaluate the microbial diversity, growth performance and cocoon production. It is also recommended that increasing sample size would provide more comprehensive results and individual level variations in gut microbiota. These insights will contribute towards optimizing sericulture strategies for silkworm growth and silk yield.

Key words: Silkworms; NGS; Gut microbiota; Proteobacteria; Firmicutes.