Transcriptional Profiling of Midgut Immunity 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. doi:10.1371/journal.pone.0043769.g006 silkworm, autophagy is active during the spinning and pre-pupal stages [15,16]. However, genes concerned with autophagy were also up-regulated, but the fold changes were lower than the stated threshold for selection (data not shown). Thirdly, when 59-bromo- 2-deoxyuridine (BrdU) was injected to label dividing cells in the midgut, a proportion of cells in the midgut in the feeding stage were positively labeled (Fig. 11A and 11B). We observed that few cells incorporated BrdU after the initiation of the wandering stage (data not shown). The number of positively labeled cells increased at 6 h (Fig. 11C and 11D), but no cells were labeled at 24 h (Fig. 11E and 11F). However, many circulating hemocytes from the same larvae (W: 24 h) had incorporated BrdU (Fig. 11G and 11H). This is probably induced by the decreased capacity of transport because the down-regulation of many transport-related genes happened in the wandering stage (Table S1). It appears that cell apoptosis was continuous in the midgut, but cell division was terminated by the decreased transportation activity of the midgut. However, in the tobacco budworm H. virescens (another type of Lepidoptera insect), cell division and cell apoptosis are balanced, and eventually a new fully-functioning epithelium appears during metamorphosis [8]. The midgut of adult butterfly has many layers of cells (Fig. 1C), indicating normal cell replacement during the pupal stage in this species of insect. However, the midgut of the silkworm moth is full of un-excreted yellow bodies that are covered by a layer of cells (Fig. 1F). Therefore, normal cell division to produce a new epithelium with normal absorption and digestion functions would appear to be a prerequisite to stop the degeneration of the midgut during metamorphosis. The degeneration of the midgut during the wandering stage seems to require certain changes to the transcriptome, as indicated by transcriptional profiling. First, 65 genes concerned with transportation functions changed significantly between feeding and wandering stages (Table S1). Among them, 36 genes are involved in ion transport and 29 are involved in transporting other molecules, such as amino acids, lipids, and sugars. Only six of the ion transport genes were up-regulated; all the others were down- regulated in the wandering stage. This may affect the midgut to have normal functions of ion and other small molecules transport in the wandering stage. Secondly, 19 genes involved in apoptosis showed altered transcript levels between the two developmental stages; 16 were up-regulated during the wandering stage. Growth arrest and DNA damage-inducible gene 45, which is involved in DNA repair, cell cycle control, and apoptosis [44,45], had an up- regulated level of transcription in the wandering stage. In addition, the transcription levels of eight cuticle proteins also changed (Table S1), among which the levels of seven proteins decreased in the wandering stage (Table S1). Cuticle proteins have been found in the insect midgut, suggesting that this group of proteins may contribute to the growth of the midgut [46]. Conclusions The insect midgut is an important organ for food digestion and nutrition absorption. The insect midgut innate immunity has become a research focus because of its heightened immunity against pathogens ingested with foods [21]. During metamorpho- sis, old tissues are replaced by new ones that might have a different PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43769