Activation of autophagic programmed cell death and innate immune gene expression reveals immuno-competence of integumental epithelium in Bombyx mori infected by a dipteran parasitoid

Abstract

In insects, the integument forms the primary barrier between the environment and internal milieu, but cellular and immune responses of the integumental epithelium to infection by micro- and macro-parasites are mostly unknown. We elucidated cellular and immune responses of the epithelium induced through infection by a dipteran endoparasitoid, Exorista bombycis in the economically important silkworm Bombyx mori. Degradative autophagic vacuoles, lamella-like bodies, a network of cytoplasmic channels with cellular cargo, and an RER network that opened to vacuoles were observed sequentially with increase in age after infection. This temporal sequence culminated in apoptosis, accompanied by the upregulation of the caspase gene and fragmentation of DNA. The infection significantly enhanced the tyrosine level and phenol oxidase activity in the integument. Proteomic analysis revealed enhanced expression of innate immunity components of toll and melanization pathways, cytokines, signaling molecules, chaperones, and proteolytic enzymes demonstrating diverse host responses. qPCR analysis revealed the upregulation of spatzle, BmToll, and NF kappa B transcription factors Dorsal and BmRel. NF kappa B inhibitor cactus showed diminished expression when Dorsal and BmRel were upregulated, revealing a negative correlation (R = (-)0.612). During melanization, prophenol oxidase 2 was expressed, a novel finding in integumental epithelium. The integument showed a low level of melanin metabolism and localized melanism in order to prevent the spreading of cytotoxic quinones. The gene-encoding proteolytic enzyme, beta-N-acetylglucosaminidase, was activated at 24 h post-infection, whereas chitinase, was activated at 96 h post-infection; however, most of the immune genes enhanced their expression in the early stages of infection. Thus the integument contributes to humoral immune responses that enhance resistance against macroparasite invasion.

Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution

“…Rearing of the silkworm, B. mori, infestation by E. bombycis and collection of integument, haemolymph and hemocyte samples were carried out as described earlier [11][12][13] and briefed here. Protein samples were extracted from integument using total protein extraction reagent (T-PER; Pierce).…”

“…The protein samples were denatured by 2M urea and reduced by 1M DTT before resolving on 10% SDS-PAGE. For 2D electrophoresis analysis, the protein was extracted in 2D buffer [23,24], followed by focussing on 11 cm strip (pH 3-10) on Protean IEF cell (Biorad), reduced in DTT and alkylated in iodoacetamide as described earlier [11]. The second dimension was resolved on a 12% precasted PAGE gel (Biorad).…”

“…It is interesting to investigate whether these genes undergo recombination together or not. In B. mori, upregulation of Dorsal is accompanied by down regulation of Cactus at 48 h after the infestation by E. bombycis whereas the reverse trend is noticed at 72 h after infestation indicating a negative correlation in expression pattern of these genes [11,12]. Under normal conditions, Cactus binds with Dorsal to prevent the transcription of antimicrobial proteins (AMP).…”

“…In B. mori, genes encoding the host-response proteins showed differential expression after the parasitic invasion [11][12][13]. Few immune genes showed regulated expression in control larvae which was deregulated due to the parasitic maggot infestation (Table 5).…”

“…On hatching, maggot invade the body and live as an endoparasitoid that induced organismal variations like loss of biomass, less production of silk protein, biochemical changes etc., [10]. Our recent studies showed differential production of host-response proteins, upregulation of associated genes and activation of immunocompetence in B. mori induced by the infestation [11]. The host-response proteins comprised of stress proteins, cytokines, Toll-pathway components, proteolytic enzymes, apoptosis -associated proteins and melanisation pathway components [12,13].…”