Phylogenetic Affiliation of 16S rRNA
We analysed the 16S rRNA sequences for the phylogenetic affiliation of both unidentified and identified strains by neighbour-joining analysis with the optimal criteria set for distance in MEGA 6. 16S rRNA sequences were compared with sequence data deposited in GenBank using the BLAST search program. Comparative analysis with GenBank sequences revealed that most of the intestinal bacteria were showing 99% similarity and a few were showing 98% similarity to their closest relatives retrieved from the Gene bank database. The 16SrRNA sequences of gut bacterial isolates generated from our study were submitted into GenBank vide accession nos.MT416410.1 to MT416415.1 and the details are presented in Table 2.
Bacterial isolates obtained from the midguts of A. proylei were screened based on their colony characteristics and identified on the basis of 16S rRNA gene sequences. Based on 16S rRNA gene sequence analysis, tasar silkworm, A. proylei showed Firmicutes as the dominant group forming a major clade with Bacillus as the dominant genus with ten different species.
Bacillaceae was found to be abundantly represented in the gut of both healthy and diseased A. proylei (Table 3). Bacillus was found to be the predominant genera in our study from cultivable gut bacterial isolates of A. proylei of North Eastern India. The predominant Bacillus genus was represented by species such as Bacillus toyonensis, Bacillus thuringiensis, Bacillus pacificus, Bacillus mobilis and Bacillus mycoides in healthy worms. The diseased group on the other hand are represented by species such asBacillus megaterium, Bacillus aryabhattai, Bacillus zanthoxyli, Bacillus flexus and Bacillus simplex.
Phylogenetic analysis revealed that gut microflora diversity of diseased and normal tasar silkworms is almost similar with only 6 % differences between the groups. The evolutionary distances were computed using the Maximum Composite Likelihood method(Tamura, Nei, & Kumar, 2004). A phylogenetic tree was constructed (Fig. 4) using the 16S rRNA sequences by neighbour-joining method 1000 bootstraps, to better understand the evolutionary relationship of the intestinal bacteria.
In the diseased silkworms, B. aryabhattai seems to be the most abundant genera found in all the diseased worms. In healthy silkworms, microflora is affiliated with the species B. toyonensis andB. thuringiensis , showing that these species share high sequence similarity. This showed more than 99% identity with the sequences deposited in the GenBank database.
Generally, Bacillus species are widely used as biopesticides and biofertilizers in agricultural practice (Pérez-García, Romero, & De Vicente, 2011), as potential probiotics for both animals and humans (Cutting, 2011; Elshaghabee, Rokana, Gulhane, Sharma, & Panwar, 2017). Hence our results also might help in potential application ofBacillus species against a variety of pathogenic gut microflora.