Autophagy precedes apoptosis during the remodeling of silkworm larval midgut

Abstract

Although several 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 processes and the nature of their relationship are still cryptic. In this study, we perform a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut of Bombyx mori during larval-adult transformation, with the aim to analyze autophagy and apoptosis in cells that die under physiological conditions. We demonstrate that larval midgut degradation is due to the concerted action of the two mechanisms, which occur at different times and have different functions. Autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages, as demonstrated by specific autophagic markers. Our data show 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.

BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20-hydroxyecdysone or starvation

“…31 In a previous study, we have found that autophagy precedes apoptosis in the midgut during Bombyx metamorphosis, but the core mechanism is not illustrated. 40 Here, we found that starvation and 20E addition caused significant cell death after 4 to 16 h treatment, with autophagy proceeding apoptosis at least by 3 h in Bombyx Bm-12 cells (Figs. 1 to 4), which was similar to the observation in midgut during metamorphosis.…”

“…28,29,39 Importantly, we found that autophagy precedes apoptosis in the midgut. 40 In this study, we discovered that being cleaved into BmATG5-tN and BmATG6-C, BmATG5 and BmATG6 mediate apoptosis following autophagy induced by 20E or starvation in Bombyx Bm-12 cells, reflecting that autophagy precedes apoptosis in the midgut during Bombyx metamorphosis.…”

“…1 to 4), which was similar to the observation in midgut during metamorphosis. 40 In 2 other lepidopteran insect cell lines, glucose starvation in Sl-1 cells (from Spodoptera litura) and amino acid starvation in SL-ZSU-1cells (S. litura) induces autophagy preceding apoptosis at least by 48 h. 41,42 In this work, we used PBS starvation and 20E treatment to mimic the conditions during larval-pupal transition, when the animals are severely starved and the 20E titer is extremely high. 31,43 Together, in certain circumstances, it is a common feature that autophagy precedes apoptosis induced by the common stimuli such as 20E or starvation.…”

“…These phenomena could explain why both 20E and starvation causes massive cell death in the larval midgut during Bombyx metamorphosis, with autophagy preceding apoptosis. 31,40 However, it is currently unclear whether 20E induces starvation-like conditions in cultured cells. Our preliminary experiments suggest that 20E might inhibit the PI3K-AMPK-TORC1 interactions to initiate autophagosome formation in cultured cells, but the underlying molecular mechanism requires further investigation.…”

The fine structure of the midgut epithelium in Xerobiotus pseudohufelandi (Iharos, 1966) (Tardigrada, Eutardigrada, Macrobiotidae)

“…However, other potential mechanisms which might be involved in the maintenance of homeostasis in invertebrates, such as apoptosis, self-regeneration of tissues and organs, accumulation of spherites, synthesis of methallothioneins or antioxidants (Parthasarathy and Palli, 2007;Park and Takeda, 2008;Hakim et al, 2010;Rost-Roszkowska et al, 2010a, 2010bChajec et al, 2012), have been studied only in some tardigrade species (Rebecchi et al, 2009a;Rizzo et al, 2010;Bonifacio et al, 2012). In many invertebrates the regenerative properties of the midgut epithelium are also considered as adaptive mechanisms for coping with external stress factors (Wilczek, 2005;Tettamanti et al, 2007;Park and Takeda, 2008;Malagoli et al, 2010;Rost-Roszkowska et al, 2011b;Franzetti et al, 2012).…”