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.