Casati, Barbara (2014) Molecular and morpho-functional effects of starvation on Bombyx mori tissues. [Tesi di dottorato]

Preview

Text casati_barbara_25.pdf Download (7MB) | Preview

Collection description

The silkworm, Bombyx mori, is a good model organism among Lepidoptera, a large order of holometabolous insects of valuable economic and agronomic interest. Thus a greater knowledge of morphological events, and their regulation at molecular level, occurring in the silkworm during metamorphosis and under stress conditions, such as complete food withdrawal, could boost studies on several biological areas. Three main larval organs, the alimentary canal, the fat body, and the silk glands, undergo a drastic remodeling, or even disappear, during metamorphosis through programmed cell death (PCD) processes. Autophagy is an evolutionarily conserved physiological process that can intervene in PCD during animal development, is activated during insect metamorphosis, and is rapidly induced when cells need to generate intracellular nutrients and energy, for example during starvation. During the autophagic process, a portion of cytoplasm and organelles, sequestered into autophagosomes, are degraded to small molecules, which are then recycled for macromolecular synthesis and/or used for generating energy. Autophagosome formation involves a series of Autophagy-related (Atg) proteins encoded by ATG genes; among these factors, Atg1, whose kinase activity is regulated by the nutrient status, plays a pivotal role. Even if it is known that, in B. mori, autophagy intervenes during metamorphosis, a clear evidence for the involvement of this process in silkworms subject to starvation, as well as a complete coding sequence for ATG1, are not yet available. In order to evaluate the effects of starvation on tissues development, a morphological and functional characterization of midgut, fat body, and silk gland was carried out during last larval instar and spinning phase in larvae both fed and subjected to complete food withdrawal. In addition, the complete coding sequence for BmATG1 was obtained and in silico analyses of its transcript and protein were performed. Moreover, BmATG1 expression was evaluated, by quantitative real-time RT-PCR, in pre-spinning phase larvae, as well as in silkworms subjected to food deprivation during last larval instar, to gain insight into the involvement of this gene in the response to starvation. According to our morphological and functional analyses, starvation induced anticipation in midgut development, and possibly delayed silk gland degeneration. Even if reduction in fat body mass resulted in a total lipid content decrease, the rapid and massive consumption of glycogen indicated that this is a major energy source during food deprivation. Our morphological and histochemical data also confirmed the involvement of autophagic process in tissues remodelling during silkworm metamorphosis. The complete coding sequence for BmATG1 was obtained, revealing the existence of two transcripts variants differing by a 96 nucleotide-long insert in the variant B. Molecular analyses demonstrated that BmATG1 is an evolutionarily conserved gene coding for a serine/threonine protein kinase whose N-terminus, containing the kinase domain and ATP- and Mg-binding sites, was the most conserved part of Atg1 proteins during evolution, thus confirming its fundamental role in function and regulation of this key autophagic protein. The BmATG1 expression profiles confirmed the involvement of this gene in the starvation-induced autophagy, especially in an energy storing tissue such as fat body.

Downloads

Actions (login required)

View Item