Transcriptional Profiling of Midgut Immunity Response and Degeneration in the Wandering Silkworm, <em>Bombyx mori</em>

<div><h3>Background</h3><p>Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the <em>Bombyx mori</em> silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear.</p> <h3>Principal Findings</h3><p>We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (<em>Pachliopta aristolochiae</em>), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage.</p> <h3>Conclusions</h3><p>This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway probably regulates the production of antimicrobial peptides in the midgut during the wandering stage.</p> </div

Different morphology of silkworm moth midgut compared with that of a butterfly.

<p>(A–C) Morphology of a butterfly (<i>Pachliopta aristolochiae</i>) and its adult midgut; (D–F) Morphology of a silkworm moth (<i>Bombyx mori</i>) 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 <i>M. sexta</i> βGRP2; 31% similarity to <i>B. mori</i> βGRP2), and TAK1 (antibody against mouse TAK1; 70% similarity to <i>B. mori</i> TAK1) were detected in the midgut during the wandering stage. Plasma (P) was from larvae injected with <i>E. coli</i>. For each lane, approximately 10 µg cell lysate was loaded. Bars: (B and E) 4 mm; (C and F): 50 µm.</p

Silkworm midguts produce a cocktail of antimicrobial proteins during the wandering stage.

<p>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.</p

Genes concerned with regulation of hormones were differentially transcribed in the midgut.

<p>(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.</p

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.

<p>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.</p

Morphological changes of silkworm midguts during the wandering stage.

<p>(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.</p

Cell proliferation in the midguts of larvae during the feeding and wandering stages.

<p>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.</p

MET Is Required for the Maximal Action of 20-Hydroxyecdysone during <em>Bombyx</em> Metamorphosis

<div><p>Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, <em>Bombyx mori</em>, participate in 20E signal transduction. <em>Met</em> is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of <em>EcR</em>-<em>USP</em> (the ecdysone receptor genes), RNAi knockdown of <em>Met</em> at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during <em>Bombyx</em> metamorphosis.</p> </div

Transcription of immunity-related proteins in the midgut positively respond to 20-E injection.

<p>All genes as indicated were significantly up-regulated at different time points after 20-E injection. *p<0.05; **p<0.001.</p

Regulation of HSPs in the midgut during the wandering stage.

<p>(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.</p