Transcriptional Profiling of Midgut Immunity 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 na¨ıve and buffer injection. HSP 75 was down-regulated after 20-E injection. *p,0.05. doi:10.1371/journal.pone.0043769.g005 (Fig. 6C). Various genes belonging to the Imd pathway as identified in Drosophila melanogaster [20] were up-regulated during the wandering stage (Fig. 8A and 8B). However, only a few genes belonging to the Toll pathway were up-regulated (Fig. 8C), and others related to Toll pathway showed no obvious change by microarray or qRT-PCR assays (data not shown). Thus, the Imd pathway may be responsible for producing AMPs in the midgut during the wandering stage. Hormones also affect the expression of insect innate immunity- related proteins. In Drosophila, 20-E controls the transcription of various genes in the fat body [5]. Treatment with 20-E induced the Drosophila stable cell line l(2)mbn to express AMP [42]. However, JH and JH homologs, such as methoprene and pyriproxyfen, counteracted the immunity response induced by 20-E in these cells [43]. Therefore, in Drosophila, 20-E has a stimulatory effect on AMP production that can be counteracted by JH. In the silkworm fat body, JH has a stimulatory effect, but 20-E has an antagonistic effect, on AMP production [26]. However, in the midgut of the silkworm, the transcription of different AMP genes was up- regulated during the wandering stage (Fig. 6B–6D). 20-E injection significantly induced the transcription of all AMPs at 24 hours (Fig. 7A–7H), which is consistent with the developmental change. In addition, when 20-E was injected into the larvae during the feeding stage, all other genes, pattern recognition receptors (CTL10, CTL21, PGRP-L2, PGRP-S6, bGRP2) were up- regulated (Fig. 7I–7M). The above results indicate that many antimicrobial proteins or peptides are produced when the concentration of 20-E increases in the hemolymph. Midgut Degeneration The midgut of silkworm moth is under weak condition due to incomplete development if compared with that of a butterfly (Fig. 1A–1F). To date, the morphologies of silkworm midgut during the wandering stage change a lot (Fig. 9). On the feeding stage, the midgut has a normal morphology (Fig. 9A). When entering the wandering stage, the old midgut becomes smaller and is undergoing degeneration (Fig. 9B). The midgut of the feeding stage, as shown in Fig. 9A, is approximately 35 mm in length. But the midgut from an adult, as shown in Fig. 1E, is approximately 4 mm. Therefore, the silkworm midgut is shortened considerably after changing from the feeding stage to the adult stage. Before pupation, the larval midgut is slough off from the outer basement membrane (Fig. 9C). TUNEL staining showed that apoptosis in the midgut started at the beginning of the wandering stage (data not shown) and became intensive at the 6 and 24 h time points (Fig. 9B–9C and Fig. 10C–10F). In the midgut during the feeding stage, there were very few apoptotic cells (Fig. 10A and 10B). In addition to the continuous apoptosis observed in other species of PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43769