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.