adult. Most insects show complete metamorphosis with obvious morphological changes between the egg, larva, pupa, and adult stages [6]. For each stage of complete metamorphosis, the internal tissues and organs undergo great change. During metamorphosis, the insect midgut also undergoes many morphological changes. The midgut stem cells differentiate into a simple cuboidal epithelium, which separates the remnant epithe- lium from the basement membrane and releases it into the lumen as a yellow body [7,8]. The yellow body then undergoes apoptosis and autophagy to re-utilize and absorb nutrients [7,8]. Finally, the pupal epithelial cells differentiate and develop into the adult midgut. In Drosophila, the intestinal stem cells are located in the basal membrane and distribute evenly along the midgut [9,10,11]. Under the control of the Delta-Notch signaling pathway, these stem cells divide and differentiate to become the adult midgut, which performs the functions of food digestion and nutrient absorption during the adult stage [11,12]. Interestingly, there are several layers (the larval midgut inside, transient pupal midgut in the middle, and the new emerged adult midgut outside) of pupal midgut observed in Drosophila [13]. In some insects, like Tenebrio molitor, many pouches appear in the adult midgut, which is very different from the larval midgut [14]. In Bombyx mori, one typical Lepidoptera insect, the midgut continues to degenerate after entering the wandering and pupal stages [15,16]. After egg laying, silkworm moths do not eat anything and soon die. However, many Lepidoptera adults need functional midguts for nectar ingestion and digestion [17]. Interestingly, Manduca sexta produces a cocktail of potent antimicrobial proteins and peptides (AMPs), such as hemolin, lysozyme, and phenoloxidase, in the lumen of wandering stage larvae [18]. AMPs produced in the midguts of wandering stage larvae are believed to be involved in removing midgut bacteria before metamorphosis. However, little is known about the mechanism or its regulation. Here, we performed a microarray assay of gene transcriptional changes in the midgut between the feeding stage (12 h on day 3 of fifth larval stage, V-3:12 h) and the wandering stage (3 h after the initiation of wandering, W:3 h) to determine why silkworm midguts continue to degenerate and if they produce AMPs during the wandering stage. Our results indicate many genes associated with metabolism, digestion, and ion and small molecule transpor- tation are down-regulated during the wandering stage when ecdysteroid is high in the hemolymph. These changes may cause abnormal absorption in the midgut. In addition, cell division in the midgut ceases, but extensive cellular apoptosis was observed at the end of the wandering stage. Thus, the imbalance of cell division and apoptosis eventually drives the degeneration of the midgut. Microarray profiling, qRT-PCR, and Western blot proved the production of antimicrobial proteins/peptides during the wander- ing stage. Some key genes belonging to the Toll pathway showed no obvious changes, whereas genes of the immune deficiency (Imd) pathway were up-regulated. This suggests that the Imd pathway regulates the production of AMPs in the wandering stage midgut. Results and Discussion Transcriptional Changes between the Feeding Stage and Wandering Stage Butterfly and silkworm belong to Lepidoptera insects. The adult butterfly (Pachliopta aristolochiae) living on nectar (Fig. 1A), has a midgut with many layers of cells (Fig. 1B and 1C) for nectar ingestion and digestion [17]. The butterfly midgut is covered by many unknown tissues that contain yellow pigments. No midgut content was visible in the midgut (Fig. 1C) likely because the fluid nectar was easily lost during the process of dissection. The Transcriptional Profiling of Midgut Immunity silkworm moth midgut is composed of one layer of cells in most regions of the midgut and is still full of yellow bodies (Fig. 1E and 1F), and the midgut is not suitable food digestion. Obviously, the midgut of silkworm moth has a poor progress of development since the wandering stage (Fig. 1C and 1F). Beside morphological changes in the midguts of Lepidoptera insects, many immunity- related proteins are produced during the wandering stage in M. sexta [18,19]. Several immunity related proteins were specifically detected in the midguts of silkworm during the wandering stages (Fig. 1G). For example, lysozyme and bGRP2 had the highest protein levels in the midgut at 24 h after the initiation of wandering stage (W:24 h). TAK1, a very important component in the Imd pathway [20,21], was obvious expressed at 6 h after the initiation of wandering stage (W:6 h). Obviously, just like M. sexta, there are also many immunity related proteins expressed in the silkworm midgut during the wandering stage. To date, there has been no explanation why the midguts of silkworms degenerate or whether silkworms produce antimicrobial proteins in their midguts after entering the wandering stage. For these reasons, we did a microarray to analyze gene transcription between the feeding stage and wandering stage (Fig. 2A). The results show a volcano plot that indicates that the transcript levels of many genes changed dramatically (Fig. 2B). Totally there are 399 genes that were differentially regulated. Among them, 155 genes (39%) were up- regulated and all others were down-regulated during the wandering stage (Table S1). These genes were classified into 13 families according to GO analysis (Fig. 2C). Metabolism and transport genes were the two largest families, with 126 (32%) and 65 (16%) genes whose transcription levels changed. Other categories included genes concerned with immunity (6%), cell morphology (5%), apoptosis (5%), and transcription (4%). In addition, there were 49 genes of unknown functions, among which 21 genes were down-regulated and 28 were up-regulated. Metabolism Thirty-two percent of the differentially regulated genes were associated with metabolism, such as the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation, carbohydrate metabo- lism, lipid metabolism, nucleic acid metabolism, amino acid metabolism, and vitamin metabolism (Fig. 2D). Most of those genes (113 genes) were down-regulated after entering the wandering stage, particularly those genes associated with carbo- hydrate metabolism, the TCA cycle, and oxidative phosphoryla- tion (Fig. 2D). The results indicate that many metabolic activities of the midgut were probably adversely affected during the wandering stage by the down-regulation of transcription of these genes. Hormones Insect development differs from advanced vertebrate animals by the three or four rounds of metamorphosis that occur during their life cycle. These metamorphic events are coordinately controlled by JH and 20-E [4,5]. The microarray assay showed that the transcript levels of several genes associated with hormone degradation were reduced during the wandering stage (Table S1). For example, genes encoding 3-dehydroecdysone 3a-reduc- tase (3-DE 3a-reductase 1 and 3-DE 3a-reductase 2) and ecdysone oxidase, which are responsible for ecdysteroid degradation, changed considerably during development [22] (Fig. 3A). Another gene encoding juvenile hormone epoxide hydrolase-like protein 1 (JHEH1), which can convert JH into juvenile hormone diol [23], was also down-regulated (Fig. 3B). JH binding protein (JHBP) can bind with JH to form a JH-JHBP complex [24]. When there is no juvenile hormone esterase (JHE), JHBP can protect JH by forming PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43769