Figure 1.
Different morphology of silkworm moth midgut compared with that of a butterfly.
(A–C) Morphology of a butterfly (Pachliopta aristolochiae) and its adult midgut; (D–F) Morphology of a silkworm moth (Bombyx mori) and its adult midgut. The silkworm moth’s foregut (FG) is not easily dissected with the midgut. In (B and E), the arrow-indicated part of midgut (MG) was sampled for histological study as shown in (C and F). The butterfly (A) ingests nectar and the midgut has many layers of cells (C) and appears in good condition. However, the midgut of silkworm moth is full of yellow body debris that cannot be excreted (E) and appears in weak condition due to one layer of cells (F). The silkworm moth (D) does not ingest anything and dies after egg-laying. FG: foregut; MG: midgut; HG: hindgut. (G) Three immunity-related proteins were significant expressed in the midgut during the wandering stage. Some proteins, such as lysozyme, βGRP2 (antibody against M. sexta βGRP2; 31% similarity to B. mori βGRP2), and TAK1 (antibody against mouse TAK1; 70% similarity to B. mori TAK1) were detected in the midgut during the wandering stage. Plasma (P) was from larvae injected with E. coli. For each lane, approximately 10 µg cell lysate was loaded. Bars: (B and E) 4 mm; (C and F): 50 µm.
Figure 2.
General statistics on the genes regulated between the feeding stage (V-3:12 h) and wandering stage (W:3 h).
(A) The times of different sampling. We selected larvae at 12 h on day 3 of the fifth larval stage (V-3:12 h), or 3 h after the initiation of the wandering stage (W: 3 h), which were dissected for the microarray. The time points for Western blot and qRT-PCR were determined according to the preliminary work with lysozyme. (B) Volcano plots depicting estimated fold change (log2, X-axis) and statistical significance (−log10 P value, Y-axis). Each point represents a gene, and colors correspond to the range of negative log10 P and log2 fold-change values. (C) GO categories of differentially transcribed genes between the feeding and wandering stages. (D) The numbers of up- and down-regulated genes associated with various metabolic events and innate immunity.
Figure 3.
Genes concerned with regulation of hormones were differentially transcribed in the midgut.
(A, B) Transcriptional changes of two JHBP genes, two 3-DE 3α-reductase genes, ecdysone oxidase and JHEH1 were different during development. The two JHBP genes and ecdysone oxidase were up-regulated, but the two 3-DE 3α-reductase genes and JHEH1 were down-regulated in the midgut during the wandering stages. (C–H) Influence of 20-E injection on the transcription of the above genes. JHEH1 and 3-DE 3α-reductase were down-regulated in the midgut when the larvae were injected with 20-E. However, the remaining genes responded to 20-E inconsistently. *p<0.05; **p<0.001.
Figure 4.
Ecdysone receptors (EcR) and ultraspiracle (USP) proteins are under the control of 20-E.
(A) EcR and USP genes were quickly up-regulated when entering the wandering stage. The transcription levels of these genes were low during the feeding stage and the end of the wandering stage. (B–E) 20-E injection induced the transcription of EcR1, EcR3, and USP during the first 12 h compared with naive or buffer injection. EcR2 was up-regulated at 24 h after 20-E injection. *p<0.05; **p<0.001.
Figure 5.
Regulation of HSPs in the midgut during the wandering stage.
(A, B) The transcription levels of HSP 22.6, HSP 19.9, HSP 20.4, HSP 25.4, and HSP 70 were up-regulated in the midgut during development. Only HSP 75 was down-regulated. (C–H) Effect of 20-E on HSPs. HSP 19.9, HSP 20.4, HSP 22.6, HSP 25.4, and HSP 70 were up-regulated at 24 h after 20-E injection as compared to the naïve and buffer injection. HSP 75 was down-regulated after 20-E injection. *p<0.05.
Figure 6.
Silkworm midguts produce a cocktail of antimicrobial proteins during the wandering stage.
A time-course assay of the transcriptional changes of specific immunity-related genes (A–D). Several antimicrobial peptides and proteins were up-regulated during the wandering stage.
Figure 7.
Transcription of immunity-related proteins in the midgut positively respond to 20-E injection.
All genes as indicated were significantly up-regulated at different time points after 20-E injection. *p<0.05; **p<0.001.
Figure 8.
The immune deficiency (Imd) pathway may regulate antimicrobial peptides (AMPs) production in the midgut during the wandering stage.
(A, B) All genes of the Imd pathway were up-regulated. (C) A few genes of the Toll pathway were also up-regulated. All others were not changed. Therefore, the Imd pathway might be the main pathway for regulating the production of AMP in the midgut during the wandering stage.
Figure 9.
Morphological changes of silkworm midguts during the wandering stage.
(A–C) Comparison of the morphology of silkworm guts on the 3rd day of the fifth larval stage (A; V-3: 12 h), and 6 h (B; W: 6 h) and 24 h (C; W: 24 h) after the initiation of the wandering stage. (A–a, B–a and C–a) The whole gut is divided into foregut (FG), midgut (MG), and hindgut (HG). The arrowhead-indicated part of each midgut was sampled for histological study with haematoxylin and eosin which are shown in (b and c) of each panel. Each (b) is a picture with low magnification, and the white-dot-lined area is shown in (c) with high magnification. In (B–c), the arrow indicates a cell full of vesicles probably due to apoptosis. In (C–c), the arrow shows the detached midgut from the basement membrane. Bars: A–a, B–a and C–a: 4 mm; A–b, B–b and C–b: 100 µm; A–c, B–c and C–c: 50 µm.
Figure 10.
Apoptotic cells in the midgut. Midguts from larvae during the feeding (V-3: 12 h) and wandering (W: 6 h and W: 24 h) stages were sampled.
Very few TUNEL-positive (red) cells were found in the midgut during the feeding stage (A, B). However, many cells were undergoing apoptosis in the midgut during the wandering stage (C, D, E, F). Before pupation (W: 24 h), old midguts were observed to slough off from the outer layer of basement membrane. DAPI was used for nuclei counter-staining. All images were merged from pictures taken using red and blue filters or using red and DIC (Nomarski) filters. Bars: 20 µm.
Figure 11.
Cell proliferation in the midguts of larvae during the feeding and wandering stages.
Green labeling indicates a BrdU-incorporating cell. In the normal feeding stage midgut (V-3: 12 h), very few cells incorporated BrdU (A, B). The midgut at 6 h after initiation of wandering (W: 6 h) had more dividing cells (C, D). At the end of wandering stage (W: 24 h), no cells in the midgut incorporated BrdU (E, F), indicating that cell division there stopped. However, hemocytes from the BrdU injected larvae (W: 24 h) were still stained positively (G, H). Images were taken using a green filter using a fluorescent microscope (A, C, E, G) or under DIC (Nomarski) filter (B, D, F, H). Control experiments performed without primary antibody (anti-BrdU) showed no staining (data not shown). Bars: 50 µm.