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 24 h 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 24 h 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.     

A novel tissue in an established model system: the <Emphasis Type="Italic">Drosophila</Emphasis> pupal midgut

The Drosophila larval and adult midguts are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact “yellow body.” The formation of a pupal midgut has been reported from several other species and may represent a general feature of intestinal metamorphosis in insects.     

Identification of adult midgut precursors in Drosophila

The adult Drosophila midgut is thought to arise from an endodermal rudiment specified during embryogenesis. Previous studies have reported the presence of individual cells termed adult midgut precursors (AMPs) as well as “midgut islands” or “islets” in embryonic and larval midgut tissue. Yet the precise relationship between progenitor cell populations and the cells of the adult midgut has not been characterized. Using a combination of molecular markers and directed cell lineage tracing, we provide evidence that the adult midgut arises from a molecularly distinct population of single cells present by the embryonic/larval transition. AMPs reside in a distinct basal position in the larval midgut where they remain through all subsequent larval and pupal stages and into adulthood. At least five phases of AMP activity are associated with the stepwise process of midgut formation. Our data shows that during larval stages AMPs give rise to the presumptive adult epithelium; during pupal stages AMPs contribute to the final size, cell number and form. Finally, a genetic screen has led to the identification of the Ecdysone receptor as a regulator of AMP expansion.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

The larval alimentary canal of the Antarctic insect, Belgica antarctica

On the Antarctica continent the wingless midge, Belgica antarctica (Diptera, Chironomidae) occurs further south than any other insect. The digestive tract of the larval stage of Belgica that inhabits this extreme environment and feeds in detritus of penguin rookeries has been described for the first time. Ingested food passes through a foregut lumen and into a stomodeal valve representing an intussusception of the foregut into the midgut. A sharp discontinuity in microvillar length occurs at an interface separating relatively long microvilli of the stomodeal midgut region, the site where peritrophic membrane originates, from the midgut epithelium lying posterior to this stomodeal region. Although shapes of cells along the length of this non-stomodeal midgut epithelium are similar, the lengths of their microvilli increase over two orders of magnitude from anterior midgut to posterior midgut. Infoldings of the basal membranes also account for a greatly expanded interface between midgut cells and the hemocoel. The epithelial cells of the hindgut seem to be specialized for exchange of water with their environment, with the anterior two-thirds of the hindgut showing highly convoluted luminal membranes and the posterior third having a highly convoluted basal surface. The lumen of the middle third of the hindgut has a dense population of resident bacteria. Regenerative cells are scattered throughout the larval midgut epithelium. These presumably represent stem cells for the adult midgut, while a ring of cells, marked by a discontinuity in nuclear size at the midgut-hindgut interface, presumably represents stem cells for the adult hindgut.     

Lysozyme in the midgut of Manduca sexta during metamorphosis.

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval-pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.     

Replacement of midgut epithelium in the greater wax moth,Galleria mellonela,during larval-pupal moult

The epithelium of larval midgut of the greater wax moth, Galleria mellonela, was replaced during the larval-pupal moult. The development of this moth was tentatively divided into 11 stages, from the full-grown larva of last instar to the 4-day-old pupa. The midgut at each stage was observed for (1) overall structure, (2) the position of goblet cells, and (3) the appearance of the yellow body. Light microscopy revealed that cell death in the midgut began in a cocoon-spinning larva (stage II), when pigments in the stemmata started to migrate. Before drastic remodeling started to occur, cytoplasmic projections in the goblet cavities were transformed. The larval midgut changed markedly at stage III, when the pigments left the stemmata. The epithelium of the larval midgut dropped as a whole into the lumen, transforming into the yellow body. Simultaneously, a pupal midgut epithelium developed. Electron microscopy of the columnar cells of a stage III larva showed that microvilli and mitochondria looked normal even though the nucleus with condensed heterochromatin resembled an apoptotic nucleus of vertebrate and higher plant cells. Caspase-3-like protease activity was restricted to the larval midgut and increased in parallel with the formation of the yellow body. The results indicate that the replacement of the larval midgut is facilitated by a typical apoptotic process.     

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.     

Comparative midgut ultrastructure of unfed larvae and adult mites of Platytrombidium fasciatum (C.L. Koch, 1836) and Camerotrombidium pexatum (C.L. Koch, 1837) (Acariformes: Microtrombidiidae)

The midgut of unfed larvae and adult mites of Platytrombidium fasciatum (C.L. Koch, 1836) and Camerotrombidium pexatum (C.L. Koch, 1937) (Acariformes: Microtrombidiidae) was investigated by electron microscopy. The sac-like midgut occupies the entire body volume, ends blindly and is not divided into functionally differentiated diverticula or caeca. The midgut walls are composed of one type of digestive cell that greatly varies in shape and size. In larvae, the lumen of the midgut is poorly recognizable and its epithelium is loosely organized, although yolk granules are already utilized. In adults, the midgut forms compartments as a result of deep folds of the midgut walls, and the lumen is well distinguished. The epithelium is composed of flat, prismatic or club-like cells, which may contain nutritional vacuoles and residual bodies in various proportions that depend on digestive stages. In both larvae and adult mites, parts of cells may detach from the epithelium and float within the lumen. The cells contain a system of tubules and vesicles of a trans-Golgi network, whereas the apical surface forms microvilli as well as pinocytotic pits and vesicles. Lysosome-like bodies, lipid inclusions and some amount of glycogen particles are also present in the digestive cells. Spherites (concretions) are not found to be a constant component of the digestive cells and in adult mites occur for the most parts in the midgut lumen.     

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.     

Lepidopteran larval midgut during prepupal instar: digestion or self-digestion?

Programmed cell death (PCD) is crucial in body restructuring during metamorphosis of holometabolous insects (those that have a pupal stage between the final larval and adult stages). Besides apoptosis, an increasing body of evidence indicates that in several insect species programmed autophagy also plays a key role in these developmental processes. We have recently characterized the midgut replacement process in Heliothis virescens larva, during the prepupal phase, responsible for the formation of a new pupal midgut. We found that the elimination of the old larval midgut epithelium is obtained by a combination of apoptotic and autophagic events. In particular, autophagic PCD completely digests decaying tissues, and provides nutrients that are rapidly absorbed by the newly formed epithelium, which is apparently functional at this early stage. The presence of both apoptosis and autophagy in the replacement of midgut cells in Lepidoptera offers the opportunity to investigate the functional peculiarities of these PCD modalities and if they share any molecular mechanism, which may account for possible cross-talk between them.     

Ultrastructural studies of midgut epithelium formation in Lepisma saccharina L. (Insecta, Zygentoma)

At the end of embryogenesis of Lepisma saccharina L. (Insecta, Zygentoma), when the stomodaeum and proctodaeum are completely formed, the midgut epithelium is replaced by the primary midgut, a yolk mass is surrounded by a cell membrane. Midgut epithelium formation begins in the 1st larval stage. Energids migrate toward the yolk periphery and aggregate just beneath the cell membrane. They are gradually enclosed by cell membrane folds of the primary midgut. Single cells are formed. Succeeding energids join just formed cells. Thus, groups of cells, regenerative cell groups, are formed. Their number gradually increases. The external cells of the regenerative cell groups transform into epithelial cells and their basal regions spread toward the next regenerative cell groups. Epithelial cells of neighboring regenerative cell groups join each other to form the epithelium. At the end of the 2nd larval stage, just before molting, degeneration of newly the formed epithelium begins. Remains of organelles and basal membrane occur between the regenerative cell groups. The new epithelium is formed from the regenerative cell groups, which are now termed stem cells of the midgut epithelium.     

Salivary gland development in the blowfly,Calliphora erythrocephala

The salivary gland of adult Calliphora erythrocephala is a tubular structure composed of secretory, reabsorptive, and duct regions. Development of these structures has been followed during the six days of larval and ten days of pupal growth. Two small groups of imaginal cells located at the junction between larval gland and duct give rise to the adult gland. These presumptive adult cells divide during all larval stages and appear to be functional components of the larval gland. Shortly after pupation, the larval gland breaks down and the imaginal cells proliferate rapidly, forming sequentially the duct, reabsorptive and secretory regions. Proliferating regions of the developing gland are frequently encrusted with haemocytes. As it elongates the gland establishes intimate contacts first with the basement membrane of the degenerating larval gland, later with an epithelial layer surrounding the main dorsal tracheal trunks, and then with the gut. Cell division continues until about five days after pupation, bu t the gland is unable to secrete fluid in response to 5-hydroxytryptamine stimulation until two hours after the adult fly emerges. The Golgi complex appears to be involved in forming the highly folded membranes of the canaliculi in the secretory region. Presumptive adult salivary gland cells appear to increase in number logarithmically from the time of hatching of the larva until five days after pupation. This contrasts with the development of classical imaginal discs, in which cell division ceases prior to 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.     

The fine structure of the digestive system of Daphnia pulex (Crustacea: Cladocera).

The alimentary canal of Daphnia pulex consists of a tube-shaped foregut, a midgut (mesenteron) with an anterior pair of small diverticula, and a short hindgut. The foregut and hindgut are structurally similar. Each is formed by a low cuboidal epithelium 5 mum tall and lined with a chitinous intima. The midgut wall consists of a simple epithelium resting on a thick beaded basal lamina which is surrounded by a spiraling muscularis. Anteriorly the midgut cells are columnar in shape being 30 mum in height each having a basal nucleus, anteriorly concentrated mitochondria and in apical border of long thin microvilli. Posteriorly the midgut cells become progressively shorter so that in the posteriormost region of the midgut the cells are 5 mum tall and cuboidal in shape. The microvilli concomitantly become shorter and thicker. All mesenteron cells contain the usual cytoplasmic organelles. The paired digestive diverticula are simple evaginations of the midgut. The wall of each consists of a simple epithelium of cuboidal cells 25 mum in height, each with a brushed border of long thin microvilli. Enzyme secretion appears to be holocrine in mode and not confined to any one region of the mesenteron though definitely polarized anteriorly. The thin gut muscularis encircles the entire length of the midgut and caeca. Thick and thin filaments appear to be in a 6:1 ratio.     

L-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius

From apolysis until pupal ecdysis, the pharate pupa of the Brazilian Skipper (Calpodes ethlius) lies wrapped in a prepupal shell composed of the larval cuticle and an ecdysial space (ES) filled with enzyme-rich moulting fluid (MF). In the 4h before ecdysis the pharate pupa drinks the moulting fluid through its mouth and anus, and transfers the cuticular degradation products to its midgut (MG). At the same time, extra fluid passes across the body wall of the pharate pupa and flushes out the ES. The MF is recovered at an overall rate of 70μl/h and reabsorbed across the pharate pupal midgut at about 26μl/h. L-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850nmol about 8h before pupal ecdysis (P-8), but by ecdysis it had dropped to nearly zero as the MF became diluted with new fluid and was consumed. The drop in glutamate in the ES coincided with a rise in the glutamine content of the fluid in the midgut lumen. The highest rate of glutamine synthesis occurred in midguts isolated from pharate pupae actively drinking MF (P相似文献   

Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.

The homeobox gene tinman and the nuclear receptor gene seven-up are expressed in mutually exclusive dorsal vessel cells in Drosophila, however, the physiological reason for this distinction is not known. We demonstrate that tin and svp-lacZ expression persists through the larval stage to the adult stage in the same pattern of cells expressing these genes in the embryo. In the larva, six pairs of Svp-expressing cells form muscular ostia, which permit hemolymph to enter the heart for circulation, however, more anterior Svp-expressing cells form the wall of the dorsal vessel. During pupation, the adult heart forms from a chimera of larval and imaginal muscle fibers. The portion of the dorsal vessel containing the larval ostia is histolyzed and the anterior Svp-expressing cells metamorphose into imaginal ostia. This is the first demonstration that the significant molecular diversity of cardial cells identified in the embryonic heart correlates with the formation of physiologically and functionally distinct muscle cells in the animal. Furthermore, our experiments define the cellular changes that occur as the larval heart is remodeled into an imaginal structure in an important model organism.     

Programmed cell death in the wing of Orgyia leucostigma (Lepidoptera: Lymantriidae)

Programmed cell death is an integral and ubiquitous phenomenon of development that is responsible for the reduction of wing size in female moths of Orgyia leucostigma (Lymantriidae). Throughout larval and pupal life, cells of the wing epithelium proliferate and interact to form normal imaginal discs and pupal wings in both sexes. But at the onset of adult development, most cells in female O. leucostigma wings degenerate over a brief, 2-day period. Lysosomes and autophagic vacuoles appear in cells of the wing epithelium shortly after it retracts from the pupal cuticle. Hemocytes actively participate in removing the resulting cellular debris. By contrast, epithelial cells in wings of developing adult males of O. leucostigma do not undergo massive cell death. Wing epithelium of female pupae transferred to male pupal hosts behaves autonomously in this foreign environment. By pupation, cells of the female wing apparently are committed to self-destruct even in a male pupal environment. Normal interactions among epithelial cells within the plane of a wing monolayer as well as between the upper and lower monolayers of the wing are disrupted in female O. leucostigma by massive cell degeneration. Despite this disruption, the remaining cells of the wing contribute to the formation of a diminutive, but reasonably proportioned, adult wing with scales and veins.     

Replication of the endosymbiotic bacterium Blochmannia floridanus is correlated with the developmental and reproductive stages of its ant host

The dynamics of replication of the intracellular endosymbiotic bacterium Blochmannia floridanus was determined during the larval development of its host ant Camponotus floridanus by real-time quantitative PCR. The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants). In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly. In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal. An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species C. herculeanus and C. sericeiventris, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes.