Silkworm larval midgut: a striking example of tissue remodeling.

Bombyx mori is a pivotal model organism among Lepidoptera, a group of insects that is important for both commercial and agronomic purposes. The study of midgut development during larval period and metamorphosis is helpful to obtain a background useful for many applications concerning these contexts. In these insects, metamorphosis involves a series of highly ordered mechanisms and passes through a well-defined sequence of events to eliminate tissues and organs that are functional only in larval stages (Meléndez and Neufeld, 2008). In Lepidoptera, midgut tissue undergoes extensive remodelling (Vilaplana et al., 2007) during the development and these modifications lead to cell death of the larval epithelium and to its replacement with a new pupal epithelium, which becomes the adult midgut epithelium. For this reason midgut remodelling has been chosen as preferential model to study cell death mechanisms and regeneration processes, and their regulation. In particular, although features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two cell death processes and the nature of their relationship are still cryptic. In order to analyze the remodelling processes in B. mori midgut tissues during larvaladult transformation, we performed a morpho-functional analysis at different developmental stages. In addition, we accomplished a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut, with the aim to analyze autophagy and apoptosis in cells that dye under physiological conditions. We found that stem cells proliferate actively since the wandering stage, leading to the formation of a new pupal midgut which is progressively remodelled until adult ecdysis. Larval midgut cells undergo progressive degradation, forming a compact mass called yellow body, that progressively degenerates inside the lumen of the new pupal midgut and finally disappears. Through histochemical analysis we showed marked changes in metabolic activity in both larval and pupal epithelium at different stages of the midgut renewal process. We demonstrated that larval midgut degradation is a gradual process due to the concerted action of autophagic and apoptotic mechanisms, which occur at different times and have different functions. In particular, autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages. Our data showed also that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells.