Midgut Remodeling Research Articles

Autophagy genes AMBRA1 and ATG8 play key roles in midgut remodeling of the yellow fever mosquito, Aedes aegypti.

The function of two autophagy genes, an activating molecule BECN1 regulated autophagy (AMBRA1) and autophagy-related gene 8 (ATG8) in the midgut remodeling of Aedes aegypti was investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of RNA samples collected from the last instar larvae and pupae showed that these two genes are predominantly expressed during the last 12h and first 24h of the last larval and pupal stages, respectively. Stable ecdysteroid analog induced and juvenile hormone (JH) analog suppressed these genes. RNA interference (RNAi) studies showed that the ecdysone-induced transcription factor E93 is required for the expression of these genes. JH-induced transcription factor krüppel homolog 1 (Kr-h1) suppressed the expression of these genes. RNAi-mediated silencing of AMBRA1 and ATG8 blocked midgut remodeling. Histological studies of midguts from insects at 48h after ecdysis to the final larval stage and 12h after ecdysis to the pupal stage showed that ATG gene knockdown blocked midgut remodeling. AMBRA1 and ATG8 double-stranded (dsRNA)-treated insects retained larval midgut cells and died during the pupal stage. Together, these results demonstrate that ecdysteroid induction of ATG genes initiates autophagy programmed cell death during midgut remodeling. JH inhibits midgut remodeling during metamorphosis by interfering with the expression of ATG genes.

Juvenile hormone-induced histone deacetylase 3 suppresses apoptosis to maintain larval midgut in the yellow fever mosquito

SignificanceJuvenile hormone (JH), a sesquiterpenoid, regulates many aspects of insect development, including maintenance of the larval stage by preventing metamorphosis. In contrast, ecdysteroids promote metamorphosis by inducing the E93 transcription factor, which triggers apoptosis of larval cells and remodeling of the larval midgut. We discovered that JH suppresses precocious larval midgut-remodeling by inducing an epigenetic modifier, histone deacetylase 3 (HDAC3). JH-induced HDAC3 deacetylates the histone H4 localized at the promoters of proapoptotic genes, resulting in the suppression of these genes. This eventually prevents programmed cell death of midgut cells and midgut-remodeling during larval stages. These studies identified a previously unknown mechanism of JH action in blocking premature remodeling of the midgut during larval feeding stages.

Midgut remodeling during the metamorphosis of Chironomus riparius, Meigen (1804)

The holometabolous insects go through a complete metamorphosis that includes four life stages: egg, larva, pupa, and imago (adult). Chironomus riparius is a suggested model organism by the Organization for Economic Cooperation and Development (OECD) that is used in acute and chronic tests of chemicals. Tissue morphology of healthy non-biting midge larval stage was already described but the faith of the midgut digestive cells and tissue organization during the metamorphosis is unknown. We here described histological alterations of the midgut during ecdysis to distinguish them from the ones caused by toxins’ negative effects. The present study showed differences in tissue architecture of the midgut in the larval, prepupal, and pupal stages of development of C. riparius. During ecdysis, larval digestive cells detached from the midgut epithelium and moved to the lumen. In the pupa, the larval midgut layer was replaced with an adult midgut that had considerably reduced width. These changes in the midgut tissue morphology and organization probably follow changes in the environment and feeding behavior of C. riparius at different stages of development.

The steroid hormone 20-hydroxyecdysone induces phosphorylation and aggregation of stromal interacting molecule 1 for store-operated calcium entry

Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.

Insulin and 20-hydroxyecdysone oppose each other in the regulation of phosphoinositide-dependent kinase-1 expression during insect pupation

Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, the PDK1 knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation.

Cathepsin L participates in the remodeling of the midgut through dissociation of midgut cells and activation of apoptosis via caspase-1

The larval midgut in holometabolous insects must undergo a remodeling process during metamorphosis to form the pupal-adult midgut. However, the molecular mechanism of larval midgut cell dissociation remains unknown. Here, we show that the expression and activity of Helicoverpa armigera cathepsin L (Har-CatL) are high in the midgut at the mid-late stage of the 6th-instar larvae and are responsive to the upstream hormone ecdysone. Immunocytochemistry shows that signals for Har-CatL-like are localized in midgut cells, and an inhibitor experiment demonstrates that Har-CatL functions in the dissociation of midgut epithelial cells. Mechanistically, Har-CatL can cleave pro-caspase-1 into the mature peptide, thereby increasing the activity of caspase-1, which plays a key role in apoptosis, indicating that Har-CatL is also involved in the apoptosis of midgut cells by activating caspase-1. We believe that this is the first report that Har-CatL regulates the dissociation and apoptosis of the larval midgut epithelium for midgut remodeling.

Proteomic analysis of the silkworm midgut during larval-pupal transition

Metamorphosis is a key process in holometabolous insects since specific larval tissues, such as the midgut, the silk gland and the fat body, are remodeled into adult organs, or even disintegrate. However, the role of proteins and factors involved in the remodeling of these organs is still unclear. For this reason we undertook a proteomic study on the larval midgut of Bombyx mori to identify proteins whose expression significantly changes during larval-pupal metamorphosis and thus can have a role in this process. 2D-PAGE analysis showed upregulation or new expression of midgut proteins at 45 (pI 5.2), 29.8 (pI 6.11) and 20-kDa (pI 6-7) during the pupal stage. In addition zymogram analysis demonstrated the occurrence of protease activity at 37-kDa at the same developmental stage. By using MALDI-TOF-MS analysis the protein spots from 2D gel were identified as Hemolin, low molecular weight 30-kDa lipoproteins, HSP 20.8 and HSP 20.4, while the proteolytic band was identified as 37-kDa serine protease. The evidence herein presented suggests that the identified proteins could be involved in the immune protection during the remodeling of silkworm midgut that occurs at larval-pupal transition and represents a platform of knowledge necessary for future functional studies.

Aedes aegypti midgut remodeling during metamorphosis

The Aedes aegypti midgut is restructured during metamorphosis; its epithelium is renewed by replacing the digestive and endocrine cells through stem or regenerative cell differentiation. Shortly after pupation (white pupae) begins, the larval digestive cells are histolized and show signs of degeneration, such as autophagic vacuoles and disintegrating microvilli. Simultaneously, differentiating cells derived from larval stem cells form an electron-dense layer that is visible 24h after pupation begins. Forty-eight hours after pupation onset, the differentiating cells yield an electron-lucent cytoplasm rich in microvilli and organelles. Dividing stem cells were observed in the fourth instar larvae and during the first 24h of pupation, which suggests that stem cells proliferate at the end of the larval period and during pupation. This study discusses various aspects of the changes during midgut remodeling for pupating A. aegypti.

The steroid hormone 20-hydroxyecdysone upregulated the protein phosphatase 6 for the programmed cell death in the insect midgut

Programmed cell death (PCD) plays an important role in insect midgut remodeling during metamorphosis. Insect midgut PCD is triggered by the steroid hormone 20-hydroxyecdysone (20E) and it is mediated by a series of genes. However, the mechanism by which 20E triggers midgut PCD is still unclear. Here, we report a protein phosphatase 6 (PP6) from Helicoverpa armigera playing roles in midgut PCD. PP6 was expressed in the midgut during larval growth and it is significantly increased during metamorphosis. The increase was proven to be regulated by 20E. The juvenile hormone analog methoprene has no effect on PP6 expression. RNA interference analysis suggests that 20E upregulated the PP6 transcript levels through the ecdysone receptor EcRB1. PP6 knockdown by larval feeding or PP6 dsRNA injection resulted in the repression of the midgut PCD during the metamorphic stage. The mechanism was demonstrated to be through the suppression of genes such as Broad (Br), E74a, E75b, HR3, E93, rpr, and caspase, which are involved in 20E signaling pathway or midgut PCD. These findings suggest that PP6 is involved in the 20E signal transduction pathway and participates in the PCD in midgut.

Rab32 and the remodeling of the imaginal midgut in Helicoverpa armigera

Midgut remodeling is a complex physiological process in holometabolous insects. During midgut remodeling, the larval midgut is decomposed by apoptosis or autophagy during metamorphosis, and the degraded larval midgut is partially absorbed as nutrients by the imaginal midgut for its formation. The molecular mechanism involved in this process is not clear. Here, we found that a Rab protein, which we have named HaRab32, is related to the organogenesis of insect imaginal midgut. Results show that HaRab32 is up-regulated in epidermis and midgut during metamorphosis. Its expression could be up-regulated by 20E. Immunohistochemistry shows Rab32 is distributed in the epithelium of the imaginal midgut during metamorphosis. Knockdown of HaRab32 by RNA interference disturbs the formation of the imaginal midgut. These data imply HaRab32 plays important roles in midgut remodeling by participating in the imaginal midgut formation.

A serine protease in the midgut of the silkworm, Bombyx mori: Protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form and activation during midgut remodeling

We identified a serine protease with a molecular mass of 37 kDa in the midgut of the silkworm, Bombyx mori. The activity of this protease (37-kDa protease: p37k) appears after pupation, when the metamorphic remodeling of the midgut is under progress. The sequence analysis of the purified protease and its cDNA revealed that p37k is a trypsin-type serine protease, which is highly similar to serine proteases of other insects, including CG4386 of Drosophila melanogaster. In our molecular phylogenetic analysis, these proteases are grouped together with CG4386-like serine proteases of other insects to form an isolated cluster. The p37k protein and its putative orthologs present in this cluster have two unique sequence motifs, CxxCxC and FIDWLxxLLG, in the N-terminal side of the catalytic region. The gene for p37k is expressed in the midgut on day 2 of the silk-spinning larva, and the p37k polypeptide becomes detectable with a specific antibody at this stage of the midgut. On the other hand, p37k activity is not detectable until pupation, indicating that p37k is present in the larval midgut as an inactive precursor, which then is activated after pupation. A recombinant p37k produced using a baculovirus system is also inactive in its intact form. However, the recombinant p37k can be converted to an active protease when incubated in the homogenate of the midgut, suggesting that some unidentified midgut factor(s) are involved in the activation of p37k.

Molecular analysis of juvenile hormone analog action in controlling the metamorphosis of the red flour beetle, Tribolium castaneum

The juvenile hormone analogs (JHA) are known to disrupt insect development but the molecular mechanisms of their action have been studied only in a few model insects belonging to orders Diptera and Lepidoptera. Here, we investigated the mechanisms of JHA action in red flour beetle, Tribolium castaneum, belonging to the order Coleoptera. Application of JHA during penultimate and final instar larval stages blocked larval-pupal metamorphosis and induced supernumerary larval molts. When compared to the control insects undergoing larval-pupal molt, down-regulation of expression of transcription factor, Broad, and up-regulation of other genes involved in 20-hydroxyecdysone (20E) action (FTZ-F1, E74) were observed in JHA-treated larvae undergoing supernumerary larval molts. The presence of JHA during the final instar larval stage blocked the midgut remodeling wherein programmed cell death (PCD) of larval cells and proliferation and differentiation of imaginal cells to pupal gut epithelium were impaired. The comparative analysis of 20E-induced gene expression in the midguts of JHA-treated and control insects revealed that JHA suppressed the expression of EcRA, EcRB, Broad, E74, E75A, and E75B, resulting in a block in PCD as well as proliferation and differentiation of imaginal cells.

Proliferation and differentiation of intestinal stem cells during metamorphosis of the red flour beetle, Tribolium castaneum

The insect midgut epithelium is remodeled during larval-pupal metamorphosis when larval polyploid cells (LPCs) are replaced by the daughters of intestinal stem cells (ISCs). We characterized the proliferation of ISCs during midgut remodeling in the red flour beetle, Tribolium castaneum. Midgut remodeling is initiated at 96 hr after ecdysis into the final instar larval stage. Immunocytochemistry with bromodeoxyuridine and phospho-histone H3 antibodies showed that the ISCs are the progenitors of the pupal/adult midgut epithelium and they undergo proliferation and differentiation to form new midgut epithelium. In vitro midgut culture experiments revealed that 20-hydroxyecdysone (20E) in the absence of juvenile hormone induces proliferation of ISCs. RNA interference (RNAi) mediated silencing of ecdysone receptors (EcRA and EcRB) and ultraspiracle (USP) identified EcRA and USP but not EcRB as the proteins involved in 20E regulation of ISCs proliferation. These data show that the proliferation of ISCs is under both developmental and endocrine regulation.

Transcription factor broad suppresses precocious development of adult structures during larval–pupal metamorphosis in the red flour beetle, Tribolium castaneum

Broad (br), a transcription factor containing the Broad-Tramtrack-Bric-a-brac (BTB) and zinc finger domains was shown to mediate 20-hydroxyecdysone (20E) action and pupal development in Drosophila melanogaster and Manduca sexta. We determined the key roles of br during larval–pupal metamorphosis using RNA interference (RNAi) in a coleopteran insect, Tribolium castaneum. Two major peaks of T. castaneum broad (Tcbr) mRNA, one peak at the end of feeding stage prior to the larvae entering the quiescent stage and another peak during the quiescent stage were detected in the whole body and midgut tissue dissected from staged insects. Expression of br during the final instar larval stage is essential for successful larval–pupal metamorphosis, because, RNAi-mediated knock-down of Tcbr during this stage derailed larval–pupal metamorphosis and produced insects that showed larval, pupal and adult structures. Tcbr dsRNA injected into the final instar larvae caused reduction in the mRNA levels of genes known to be involved in 20E action (EcRA, E74 and E75B). Tcbr dsRNA injected into the final instar larvae also caused an increase in the mRNA levels of JH-response genes (JHE and Kr-h1b). Knock-down of Tcbr expression also affected 20E-mediated remodeling of midgut during larval–pupal metamorphosis. These data suggest that the expression of Tcbr during the final instar larval stage promotes pupal program while suppressing the larval and adult programs ensuring a transitory pupal stage in holometabolous insects.

Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval–pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.

Stage- and cell-specific expression of ecdysone receptors and ecdysone-induced transcription factors during midgut remodeling in the yellow fever mosquito, Aedes aegypti

In insects, especially in mosquitoes that are adult blood feeders, midgut remodeling is an important event during metamorphosis. It involves two processes viz., programmed cell death (PCD) of larval cells, and proliferation and differentiation of imaginal cells to form pupal/adult midgut. These processes are regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH), but the signaling mechanisms, which trigger specific changes remain poorly understood. Here, we report stage- and cell-specific expression of ecydone receptor (EcR), ultraspiracle (USP), broad (Br), E75B and hormone receptor 3 (HR3) during midgut remodeling in Aedes aegypti. In Ae. aegypti both EcR and USP genes code for two isoforms each and the expression of mRNA for these isoforms showed both stage- and cell-specific regulation. In general, EcR-B and USP-A mRNAs were detected during larval stages in larval cells, and EcR-A and USP-B mRNAs were detected during pupal stages in imaginal cells. These data suggest that EcR-B/USP-A heterodimer is important for PCD of larval cells and EcR-A/USP-B heterodimer is important for formation of pupal/adult midgut. Broad Z1 mRNA was detected only in the larval cells suggesting its primary role in PCD. It is likely that E75B and HR3 are probably involved in both PCD and imaginal cell proliferation and differentiation as their mRNAs were expressed in the larval as well as in imaginal cells. Application of JH analog, methoprene, lowered or delayed the expression of all the genes studied. These data suggest that 20E plays a major role in midgut remodeling and coordinates this process through stage- and cell-specific expression of different isoforms of nuclear receptors and transcription factors in the target larval and imaginal cells.

Mechanisms of midgut remodeling: Juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36 h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12 h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36 h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.

Expression of nuclear receptor-transcription factor genes during Aedes aegypti midgut metamorphosis and the effect of methoprene on expression

Exposure of mosquito 4th instars to the juvenile hormone analogue methoprene prevents the emergence of adults by interfering with metamorphosis. One metamorphic processes that is disrupted is midgut remodeling. To investigate the molecular mechanisms by which this occurs, the pattern of transcription factor gene expression during the Aedes aegypti (L.) 4th instar was investigated by the method of real time PCR. The results indicate that in untreated larvae, expression of transcription factors genes AHR3 and AaE75B increases within 24 h after the last larval–larval molt, transcription of AaEcR-B, AaUSP-a and AaßFTZ -F1 increases approximately 24 h later, and transcription of AaE75A increases just before the larval–pupal molt. There is uniform expression of AaUSP-b throughout the 4th instar. The effect of methoprene exposure on transcription factor gene expression during midgut remodeling was investigated. The results indicate that, in a dose and stage dependent manner, methoprene affects increases in expression that normally occur during midgut remodeling. The coincident effects of methoprene on metamorphic midgut remodeling and on transcription factor gene expression suggests that the two processes are related.

Methoprene interferes with mosquito midgut remodeling during metamorphosis.

Many juvenile hormone analogs interfere with insect metamorphosis, a property that makes them useful in insect control and as investigatorial tools with which to study metamorphic mechanisms. We report that one such analog, methoprene, interferes with mosquito metamorphic midgut remodeling. Methoprene treated Aedes aegypti (L.), Aedes albopictus (Skuse), and Culex quinquefasciatus (Say) fourth instars pupate, but the pupal midguts are morphologically similar to larval midguts. The degree of midgut remodeling is affected by the dose of methoprene applied and the extent of fourth instar development when methoprene exposure is initiated. DNA staining indicates that high methoprene concentrations interfere with diploid cell division and programmed death of polytene cells. Lower methoprene concentrations do not interfere with diploid cell division but the removal of polytene cells is incomplete. The effect of methoprene dose on the extent of midgut remodeling correlates well with its effect on pupal and adult survival. Metamorphic midgut remodeling offers a mosquito system with which to study the effect of ecdysone, juvenile hormone and juvenile hormone analogs on metamorphosis. An understanding of the molecular mechanisms by which methoprene affects mosquito metamorphosis might lead to the development of more effective mosquito control chemicals having fewer deleterious environmental effects and to genetic strategies by which mosquito populations might be controlled.

Coordinated Morphological Changes in Midgut, Imaginal Discs, and Respiratory Trumpets During Metamorphosis of <I>Aedes aegypti</I> (Diptera: Culicidae)

Abstract The study of mosquito metamorphosis has relevance to understanding the mosquitocidal mechanisms of juvenile hormone analogues and may provide information concerning dipteran evolution. This report details the chronology and coordination of morphological changes in imaginal disc growth and differentiation, respiratory trumpet morphogenesis, and midgut remodeling that occur during Aedes aegypti (L.) metamorphosis. These observations were facilitated by the transparent A. aegypti larval cuticle and the relatively synchronous development of individually grown larvae. The results indicate that within 12 h of the last larval-larval ecdysis, leg imaginal discs start to grow and differentiate, pupal respiratory trumpets start their morphogenesis, and midgut remodeling starts in that programmed death of midgut polytene cells and division of midgut diploid cells are initiated. Morphological changes in all of these structures continue throughout the fourth stadium and are concomitant with substantial larval...