DISCUSSION
Many Lepidopteran insects including silkworms are described as
beneficial insects in the past two millennia. Gut microflora of most of
the insects comprises a wide variety ranging from obligate endosymbionts
to facultative bacteria and others (Dillon & Dillon, 2004).
Comprehensive analysis of bacterial diversity within the gut of insects
from different geographical regions will enable us to better understand
their ecological roles and their interactions with the insect host. Very
little information is available on the gut bacterial diversity of tasar
silkworm, A. proylei which is exclusively present in the world in
North Eastern regions of India. Culture-dependent 16s rRNA gene sequence
based approaches in this study was used to assess the cultivable gut
bacterial communities associated with these tasar silkworm populations
in Manipur, North Eastern state of India.
Our analysis shows that the tasar silkworm harboured unique bacterial
flora in its gut with Firmicutes group as the major one and Bacillus
being the predominant genera constituting about 100 per cent of total
gut bacterial isolates from A. proylei. Earlier studies have also
documented the presence of Pseudomonas and Bacillus species as the
dominant bacterial communities in the gut of gypsy moth, L.
dispar (Lepidoptera)(Broderick et al., 2004). These results were in
agreement with that of Bombyx mandarina and Bombyx morigut bacterial diversity where phylum Firmicutes was found to be the
predominant bacteria and Enterococcus to the predominant bacterial genus
through 16S rRNA gene sequencing(D. Kumar et al., 2019). Bacillus was
also the most dominant bacterial genus in an earlier study, accounting
for 18% of the total gut community in 21 different insect species (Yun
et al., 2014).
Gut morphology was early being described as a potential phylogenetically
informative character in a study on Passalids (Bess
beetles)(Reyes-Castillo, 1970). A limited diversity was revealed in 16S
rRNA gene analysis of gut bacteria of P. xylostella larvae
(Indiragandhi et al., 2007). The phylogenetic analysis of gut bacterial
isolates from tasar silkworm indicates that tasar silkworm has limited
gut bacterial diversity.
We also investigated the histopathological importance of healthy and
diseased silkworm larvae to understand in-depth pathogenic effects a
disease imposes on the cells, tissues and organs. As infected silkworms
fail to spin cocoons analysis of cytological damages in the silk gland
is very essential. The digestive system in silkworm larvae needs much
attention as oral entry of pathogenic microbes are quite common. Apart
from dual function of digestive and absorptive, the midgut region also
provides a barrier to invading parasites too. Here we report for the
first time the histopathological effects in A. proylei silkworm.
Our results show that silk glands that contains tissue of infected
larvae were ruptured and deformed along with the formation of lump cells
compared with healthy larvae. In disease infected worm, the silk mass
shows a notable destruction and vacuolation of the fat body cells and
the fat tissues became soft and compactness as compared to those of the
healthy insect. Also the IV instar larvae with disease stop feeding and
colour change appears in the integument. The abnormal swelling, cell
hypertrophy and other cytotoxic effects are the result of loss of
silkworm ability to maintain homeostasis.
Histopathological changes in midgut by bacterial or viral infections
have been documented in other silkworms (Choudhury et al., 2004;
Jurat-Fuentes & Jackson, 2012; Mohanta et al., 2015; Ponnuvel et al.,
2003). The midgut portion of nuclear poly hydrosis infected silkworm
larvae showed hyper trophism of nuclei. Hypertrophic nuclei were swollen
compared to normal nuclei. We observed notable hypertrophy, hyperplasia
and multi-layered epithelial cells of the midgut.
Also, phylogenetic tree analysis of the gut bacterial 16S rRNA gene
sequences in our study (Fig. 4) showed Firmicutes as the dominant family
forming a major clade with Bacillus as the dominant genus with ten
different species.
It appears that the histopathological effects with respect to the Tiger
band disease are mainly localized in the silk glands, midgut, and
muscles surrounding the alimentary canal and body fat. The most common
pathological changes observed include hypertrophy, vacuolation
ultimately leading to insect death. Molecular phylogenetic analysis
further reveals that Firmicutes and Bacillus being the predominant clade
and genera in both healthy and diseased silkworms. Detailed
characterization and further investigation from different geographical
locations in the North eastern region of India would advance our
understanding of diversity and composition of gut bacteria of oak tasar
silkworms and its disease management.