Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis

Abstract

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

Figure 1. Autophagy is activated at spinning stage.

(a) qRT-PCR analysis of BmATG8 mRNA levels in midgut tissues; (b) Western blot analysis of BmATG8–PE; (c,d) immunofluorescence staining of BmAtg8 showing the presence of autophagosomes (arrows) in midgut tissues at SD2 (d) compared to L5D5 (c) stage; (e) TEM images of autophagosomes in midgut cells at SD2 stage (A: autophagosome; arrows: autophagosome membranes); (f) analysis of acid phosphatase activity in midgut cells. Values represent mean ± s.e.m. (*p < 0.05; **p < 0.01 compared to L5D2 using ANOVA followed by Tukey’s HSD test).

Figure 2. Apoptosis is activated after autophagy.

(a,b) qRT-PCR analysis of BmCASPASE-5 (a) and BmCASPASE-1 (b); (c,d) Western blot analysis of uncleaved (c) and cleaved (d) BmCaspase-1 during metamorphosis. Values represent mean ± s.e.m. (*p < 0.05; **p < 0.01 compared to L5D2 using ANOVA followed by Tukey’s HSD test).

Figure 3. Autophagy is activated by 20E.

(a,b) qRT-PCR analysis of BmATG8 (a) and BmATG1 (b) in midgut cells after administration of 20E; (c,d) Western blot analysis of BmAtg8–PE (c) and acid phosphatase activity (d) in midgut of larvae injected with the hormone; (e) Western blot analysis of p-Bm4ebp1 in hormone-treated larvae. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).

Figure 4. Rapamycin fails to activate a full autophagic response.

(a) Western blot analysis of p-Bm4ebp1 in midgut of larvae injected with rapamycin; (b,c) qRT-PCR analysis of BmATG8 (b) and BmATG1 (c) expression after rapamycin treatment; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) after administration of rapamycin. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).

Figure 5. Apoptosis is activated by 20E.

(a,b) qRT-PCR analysis of BmCASPASE-5 (a) and BmCASPASE-1 (b) in midgut of larvae injected with 20E; (c,d) Western blot analysis of cleaved BmCaspase-1 after single (c) or double (d) administration of 20E. Values represent mean ± s.e.m. (**p < 0.01 using Student’s t-test).

Figure 6. Autophagy inhibition by chloroquine determines an increased degeneration of the larval midgut.

(a,b) TEM analysis of midgut cells in control (a) and chloroquine-treated (b) insects showing a significant accumulation of vesicles (arrows) after inhibition of autophagy; (c) detail of autophagic compartments in midgut cells of treated larvae; (d,e) Western blot analysis of BmAtg8–PE (d) and acid phosphatase activity (e) demonstrating the inhibition of autophagy in midgut cells by chloroquine; (f,g) morphology of the larval and pupal epithelium in control (f) and treated (g) pupae; (h) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with chloroquine. A: autophagosomes; Lm: larval midgut epithelium; N: nucleus; Pm: pupal midgut epithelium. Values represent mean ± s.e.m. (*p < 0.05 using Student’s t-test).

Figure 7. Wortmannin impairs autophagy and leads to increased degeneration of the midgut epithelium.

(a) Western blot analysis of BmAtg8–PE demonstrating autophagy inhibition in midgut cells treated with wortmannin; (b) acid phosphatase activity; (c,d) morphology of larval and pupal epithelium in control (c) and treated (d) pupae; (e) Western blot analysis of cleaved BmCaspase-1 in midgut cells of larvae treated with wortmannin. Lm: larval midgut epithelium; Pm: pupal midgut epithelium. Values represent mean ± s.e.m.

Figure 8. Caspase inhibition delays the degeneration of the midgut epithelium.

(a) Western blot analysis of cleaved BmCaspase-1 in midgut of larvae treated with z.vad.fmk. The absence of activated caspase expression can be appreciated at PP, P1, and P3 stages following the administration of the inhibitor at SD2 stage; (b,c) TEM analysis of midgut in control (b) and treated (c) larvae at PP stage; (d,e) morphology of larval and pupal epithelium in control (d) and treated (e) P3 pupae; (f,g) morphology of larval and pupal epithelium in control (f) and treated (g) P9 pupae; (h) Western blot analysis of BmAtg8–PE in the midgut of z.vad.fmk-treated larvae. Lm: larval midgut epithelium; N: nucleus; Pm: pupal midgut epithelium; arrows: apoptotic nuclei.