Growth and differentiation of the larval midgut epithelium during molting in the moth, Manduca sexta

The number of epithelial cells comprising larval midgut of the tobacco hornworm moth, Manduca sexta increases 200-fold in development from the first to the fifth instar. We have examined larvae periodically before and during molting to follow epithelial cell proliferation and differentiation. The midgut epithelium in Manduca sexta consists predominantly of columnar and goblet cells. These are arranged in a characteristic pattern with each goblet cell surrounded by a single layer of 4-6 columnar cells (Hakim et al., (1988)). While undifferentiated basal stem cells are infrequently seen in intermolt larvae, just prior to the period when external signs of molting are visible, their number increases and mitotic figures become common. Proliferation continues for several hours and then these stem cells differentiate following a pattern similar to that seen during embryogenesis (Hakim et al., (1988)). Here, however, the newly differentiating cells become intercalated among the mature differentiated cells already present in the epithelium. Since the pattern of individual goblet cells surrounded by a reticulum of columnar cells is maintained after the addition of new cells, the midgut epithelium of molting larvae appears to be a useful model for studying pattern formation in development.     

Cockroach midgut peptides that regulate cell proliferation, differentiation, and death in vitro

Summary The number of insect midgut cells is maintained homeostatically in vivo and in vitro. However, during starvation, the midgut shrinks and the rate of cell replacement appears to be suppressed. When they undergo metamorphosis, the internal organs of insects are drastically remodeled by cell proliferation, differentiation, and apoptotic processes, and the net number of cells usually increases. An extract of 1650 midguts ofPeriplaneta americana was fractionated by highperformance liquid chromatography (HPLC) to obtain the peptides that regulate these processes. The HPLC fractions were tested for myotropic activity in the foregut and for effects on cell proliferation or loss in primary cultures of larvalHeliothis virescens midgut cells and in a cell line derived from the last-instar larval fat body ofMamestra brassicae. Some fractions stimulated midgut stem cell proliferation and differentiation, while others caused loss of differentiated columnar and goblet cells. Other fractions stimulated cell proliferation in the larval fat body cells. Mention of products in this article does not imply endorsement by the U.S. Department of Agriculture.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Altering the fate of stem cells from midgut of Heliothis virescens:the effect of calcium ions

Cultured stem cells from larval midgut tissue of the lepidopteran Heliothis virescens respond to alterations in external calcium ion concentration (Ca(2+) (out)) by changing the rate of stem cell proliferation and by differentiating to larval or non-larval phenotypes. Decreasing the external concentration of Ca(2+) with the Ca(2+) chelating agent EGTA increased proliferation of stem cells in culture, and doubled the proportion of cells differentiating to columnar and goblet cells typical of larval midgut compared to controls. In contrast, increasing inward transport of Ca(2+) into the cells by increasing the concentration of external calcium ion concentration, or by incubation with the Ca(2+) ionophore A23187 (which tends to open inward plasma membrane Ca(2+) channels), induced dose-dependent differentiation to non-midgut cell types such as squamous and scale-like cells. However, the latter treatments did not significantly alter stem cell proliferation or differentiation to normal larval midgut epithelium.     

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

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.     

Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae)

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.     

Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens

Summary Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin β₁, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of culturedHeliothis virescens midgut cells; clusters of immunostained integrin β₁-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor-like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containingManduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells toBacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150–200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered. Products mentioned in this article are not endorsed by the U.S. Department of Agriculture.     

Programmed cell death and stem cell differentiation are responsible for midgut replacement in <Emphasis Type="Italic">Heliothis virescens</Emphasis> during prepupal instar

We have analyzed midgut development during the fifth larval instar in the tobacco budworm Heliothis virescens. In prepupae, the midgut formed during larval instars undergoes a complete renewal process. This drastic remodeling of the alimentary canal involves the destruction of the old cells by programmed cell-death mechanisms (autophagy and apoptosis). Massive proliferation and differentiation of regenerative stem cells take place at the end of the fifth instar and give rise to a new fully functioning epithelium that is capable of digesting and absorbing nutrients and that is maintained throughout the subsequent pupal stage. Midgut replacement in H. virescens is achieved by a balance between this active proliferation process and cell-death mechanisms and is different from similar processes characterized in other insects. This work was supported by FAR 2006 (University of Insubria) to G.T., by a MIUR-FIRB-COFIN grant (no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).     

Morphometry of the midgut epithelium of Diatraea saccharalis Fabricius, 1794 (Lepidoptera) parasitized by Cotesia flavipes Cameron, 1891 (Hymenoptera)

The morphometric study of the midgut in Diatraea saccharalis (Lepidoptera) larvae parasitized by the Cotesia flavipes (Hymenoptera) showed that there was significant increase in the columnar, goblet and regenerative cells and their nuclei; the midgut lumen diameter and the epithelial height were also increased in the parasitized larvae. The multivariate analysis showed that parasitism affected the columnar cell only in the posterior region, and the goblet cells along the midgut length (anterior and posterior regions).     

ACTIVE TRANSPORT BY THE CECROPIA MIDGUT : II. Fine Structure of the Midgut Epithelium

A morphological basis for transcellular potassium transport in the midgut of the mature fifth instar larvae of Hyalophora cecropia has been established through studies with the light and electron microscopes. The single-layered epithelium consists of two distinct cell types, the columnar cell and the goblet cell. No regenerative cells are present. Both columnar and goblet cells rest on a well developed basement lamina. The basal portion of the columnar cell is incompletely divided into compartments by deep infoldings of the plasma membrane, whereas the apical end consists of numerous cytoplasmic projections, each of which is covered with a fine fuzzy or filamentous material. The cytoplasm of this cell contains large amounts of rough endoplasmic reticulum, microtubules, and mitochondria. In the basal region of the cell the mitochondria are oriented parallel to the long axes of the folded plasma-lemma, but in the intermediate and apical portions they are randomly scattered within the cytoplasmic matrix. Compared to the columnar cell, the goblet cell has relatively little endoplasmic reticulum. On the other hand, the plications of the plasma membrane of the goblet cell greatly exceed those of the columnar cell. One can distinguish at least four characteristic types of folding: (a) basal podocytelike extensions, (b) lateral evaginations, (c) apical microvilli, and (d) specialized cytoplasmic projections which line the goblet chamber. Apically, the projections are large and branch to form villus-like units, whereas in the major portion of the cavity each projection appears to contain an elongate mitochondrion. Junctional complexes of similar kind and position appear between neighboring columnar cells and between adjacent columnar and goblet cells as follows: a zonula adherens is found near the luminal surface and is followed by one or more zonulae occludentes. The morphological data obtained in this study and the physiological information on ion transport through the midgut epithelium have encouraged us to suggest that the goblet cell may be the principal unit of active potassium transport from the hemolymph to the lumen of the midgut. We have postulated that ion accumulation by mitochondria in close association with plicated plasma membranes may play a role in the active movement of potassium across the midgut.     

Regeneration of midgut epithelial cell in the silkworm, Bombyx mori, infected with viruses

In the larvae of the silkworm, Bombyx mori, the regeneration of midgut cells infected with a cytoplasmic polyhedrosis virus (CPV), a flacherie virus (FV), and a small DNA virus (SDV) was studied. Large numbers of newly developed cells appeared in the CPV-infected part of the midgut epithelium just before larval molt, and along with their development, the CPV-infected old columnar cells were discharged into the midgut lumen during the molt. On the other hand, in the uninfected portion of the midgut only a few cells developed, and no columnar cells were discharged. Similarly, the marked replacement of midgut epithelial cells during larval molt was also observed in larvae infected with CPV + FV. In the larvae infected with CPV + SDV, the columnar cells lost their regenerative ability, and because of the exfoliation of infected columnar cells, the midgut epithelium consisted mainly of uninfected goblet cells at a late stage of infection. The degree of epithelial regeneration varied with the silkworm strain and the dosage of the virus.     

Implications for the functions of the four known midgut differentiation factors: An immunohistologic study of Heliothis virescens midgut

Antibodies to the peptides that induce differentiation of midgut larval stem cells, the midgut differentiating factors MDF-2, MDF-3, and MDF-4, bind to columnar cells in midgut cultures and in intact midgut of Heliothis virescens, in manners similar to the binding of anti- MDF-1 to those tissues. Antibodies to MDF-2 and MDF-3 also stained droplets in the midgut lumen, suggesting that columnar cells may also release MDF-2- and MDF-3-like cytokines to the lumen. Antibody to MDF-4 exhibited similar staining patterns but also recognized stem and differentiating cells, the presumed targets of peptides that regulate stem cell differentiation. Antibody to MDF-4 also bound to one type of endocrine cell in midgut cultures and in sections of midgut, as well as to the endocrine secretion released both to the midgut lumen and the hemolymph. Antibodies to the MDFs 1, 2, and 3, incubated with cultures of midgut cells, did not appear to prevent differentiation of the stem cells in the cultures but affected viability of mature cells, reflected in increased apoptosis and doubling of the number of differentiating cells compared to controls. Only antibody to MDF-4 induced temporary necrosis and inhibition of population recovery, indicating that MDF4 may be the true differentiation factor. The other MDFs may have additional functions beyond regulation of midgut stem cell differentiation in vivo.     

Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/μl AA 1-9 or 0.06 pg/μl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140-200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.     

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.     

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.     

Differentiation of regenerative cells in the midgut epithelium of Epilachna cf nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae)

Differentiation of regenerative cells in the midgut epithelium of Epilachna cf nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae), a consumer of the Ni-hyperaccumulator Berkheya coddii (Asteracae) from South Africa, has been monitored and described. Adult specimens in various developmental phases were studied with the use of light microscopy and transmission electron microscopy. All degenerated epithelial cells are replaced by newly differentiated cells. They originate from regenerative cells which act as stem cells in the midgut epithelium. Just after pupal-adult transformation, the midgut epithelium of E. nylanderi is composed of columnar epithelial cells and isolated regenerative cells distributed among them. The regenerative cells proliferate intensively and form regenerative cell groups. In each regenerative cell group the majority of cells differentiate into new epithelial cells, while some of them still act as stem cells and persist as a reservoir of cells capable for proliferation and differentiation. Because this species is an obligate monophage of plants which accumulate nickel, proliferation and differentiation of midgut stem cells follow degeneration intensively and in a typical manner.     

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.     

Autophagocytosis in the larval midgut cells of Pieris brassicae during metamorphosis

Summary We observed three types of cells in the epithelial layer of the midgut of last instars of Pieris brassicae. The columnar and goblet cells degenerate during the second part of the last larval stage while the undifferentiated basal cells proliferate during this period and create the epithelium of the pupal midgut. The first morphological sign of involution is the formation of autophagic vacuoles and dense bodies in the cytoplasm of columnar and goblet cells which begins on day 4 of the stage. The number and size of autophagic vacuoles and dense bodies increase during the spinning period (85–96 h). Finally, at the end of the stage, the columnar and goblet cells become displaced by the growing pupal epithelium and reach the lumen where they disintegrate.Autophagocytosis was not seen in the cells during the feeding period (0–72 h). However, we observed many autophagic vacuoles in the columnar and goblet cells of 50-h-old instars 3 h after the administration of 30 g/g body weight of 20-hydroxyecdysone. The hormone treatment elevated by 100% the incorporation of ³H-leucine into the proteins of the midgut. Inhibitors of protein synthesis, cycloheximide and puromycin, in doses that supressed the incorporation of the amino acid by 60–70% either in hormone treated or untreated larvae, exerted diverse effects on the autophagic process. Puromycin did not block the hormone-induced formation of autophagic vacuoles while cycloheximide prevented it. Possible explanations for this diversity are discussed.