Cell renewal in adjoining intestinal and tracheal epithelia of Manduca

Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects’ responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells.     

Subtype-specific neuronal remodeling during Drosophila metamorphosis

During metamorphosis in holometabolous insects, the nervous system undergoes dramatic remodeling as it transitions from its larval to its adult form. Many neurons are generated through post-embryonic neurogenesis to have adult-specific roles, but perhaps more striking is the dramatic remodeling that occurs to transition neurons from functioning in the larval to the adult nervous system. These neurons exhibit a remarkable degree of plasticity during this transition; many subsets undergo programmed cell death, others remodel their axonal and dendritic arbors extensively, whereas others undergo trans-differentiation to alter their terminal differentiation gene expression profiles. Yet other neurons appear to be developmentally frozen in an immature state throughout larval life, to be awakened at metamorphosis by a process we term temporally-tuned differentiation. These multiple forms of remodeling arise from subtype-specific responses to a single metamorphic trigger, ecdysone. Here, we discuss recent progress in Drosophila melanogaster that is shedding light on how subtype-specific programs of neuronal remodeling are generated during metamorphosis.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called “regenerative” cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

Scalariform junctions in the malpighian tubules of the insects Rhodnius prolixus (Hemiptera: Reduviidae) and Aedes taeniorhynchus (Diptera: Culicidae)

The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

Evolutionary contrasts in insects: nutritional advantages of holometabolous development

ABSTRACT. Two insects, one holometabolous and one hemimetabolous, are compared and contrasted in respect to feeding efficiency, growth and respiration. An examination of cuticle production in these and other species leads to the conclusion that the nutritional burden of cuticle production in hemimetabolous forms has a significant effect on efficiency, and it is speculated that greater nutritional efficiency in holometabolous forms has been an important evolutionary advantage.     

Morphological and functional characterization of isolated stem cells from the midgut of Chilo suppressalis larvae

The proliferation and differentiation of stem cell populations allow the midgut to grow/regenerate in lepidopteran insect. Basic epithelial regenerative functions can be assessed in vitro by purifying these stem and mature cell populations. Therefore, we isolated and purified stem and mature cells from the midgut of C. suppressalis larvae by density gradient centrifugation and observed the morphologies of these cells. A flow cytometry method was used to monitor C. suppressalis stem cell proliferation and differentiation under different cell culture conditions. We observed high proportions of the stem and differentiating cells in third- and fourth-instar larvae, respectively, indicating that, in larvae, stem cells rapidly proliferate early in development and are strongly differentiated at late stages. Incubation in medium supplemented with fat body extract and ecdysone resulted in a significantly increased proportion of stem cells, not of the differentiating cells, indicating that co-culture with fat body extract and ecdysone stimulates the proliferation of C. suppressalis stem cells. Viability bioassays showed that Cry1Ab displayed significant cytotoxic effects on the midgut cell culture of C. suppressalis. The proportion of differentiating cells was significantly increased after a 48-h exposure to sublethal doses of Cry1Ab toxin, and peaked at the Cry1Ab concentration of 0.3 μg/ml, demonstrating that epithelial cells with strong regenerative capacity via the differentiation of stem cells. These results improve our understanding of C. suppressalis stem cell biology and illustrate the potential role of the enhanced midgut regeneration induced by stem cell proliferation or differentiation as a reparation mechanism to Bt toxin.     

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 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h 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.     

Larval cells become imaginal cells under the control of homothorax prior to metamorphosis in the Drosophila tracheal system

In Drosophila melanogaster, one of the most derived species among holometabolous insects, undifferentiated imaginal cells that are set-aside during larval development are thought to proliferate and replace terminally differentiated larval cells to constitute adult structures. Essentially all tissues that undergo extensive proliferation and drastic morphological changes during metamorphosis are thought to derive from these imaginal cells and not from differentiated larval cells. The results of studies on metamorphosis of the Drosophila tracheal system suggested that large larval tracheal cells that are thought to be terminally differentiated may be eliminated via apoptosis and rapidly replaced by small imaginal cells that go on to form the adult tracheal system. However, the origin of the small imaginal tracheal cells has not been clear. Here, we show that large larval cells in tracheal metamere 2 (Tr2) divide and produce small imaginal cells prior to metamorphosis. In the absence of homothorax gene activity, larval cells in Tr2 become non-proliferative and small imaginal cells are not produced, indicating that homothorax is necessary for proliferation of Tr2 larval cells. These unexpected results suggest that larval cells can become imaginal cells and directly contribute to the adult tissue in the Drosophila tracheal system. During metamorphosis of less derived species of holometabolous insects, adult structures are known to be formed via cells constituting larval structures. Thus, the Drosophila tracheal system may utilize ancestral mode of metamorphosis.     

Cell death and regeneration in the midgut of the mosquito, Culex quinquefasciatus

Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.     

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.

Addendum to:

Programmed Cell Death and Stem Cell Differentiation are Responsible for Midgut Replacement in Heliothis virescens During Prepupal Instar

G. Tettamanti, A. Grimaldi, M. Casartelli, E. Ambrosetti, B. Ponti, T. Congiu, R. Ferrarese, M.L. Rivas-Pena, F. Pennacchio and M.D. Eguileor

Cell Tissue Res 2007; In press     

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.     

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.     

Cell death and tissue reorganization in Rhynchosciara americana (Sciaridae: Diptera) metamorphosis and their relation to molting hormone titers

Programmed cell death (PCD) is a focal topic for understanding processes underlying metamorphosis in insects, especially so in holometabolous orders. During adult morphogenesis it allows for the elimination of larva-specific tissues and the reorganization of others for their functionalities in adult life. In Rhynchosciara, this PCD process could be classified as autophagic cell death, yet the expression of apoptosis-related genes and certain morphological aspects suggest that processes, autophagy and apoptosis may be involved. Aiming to reveal the morphological changes that salivary gland and fat body cells undergo during metamorphosis we conducted microscopy analyses to detect chromatin condensation and fragmentation, as well as alterations in the cytoplasm of late pupal tissues of Rhynchosciara americana. Transmission electron microscopy and confocal microscopy revealed cells in variable stages of death. By analyzing the morphological structure of the salivary gland we observed the presence of cells with autophagic vacuoles and apoptotic bodies and DNA fragmentation was confirmed with the TUNEL assay in salivary gland. The reorganization of fat body occurs with discrete detection of cell death by TUNEL assay. However, both salivary gland histolysis and fat body reorganization occur under control of the hormone ecdysone.     

Common and distinct roles of juvenile hormone signaling genes in metamorphosis of holometabolous and hemimetabolous insects

Insect larvae metamorphose to winged and reproductive adults either directly (hemimetaboly) or through an intermediary pupal stage (holometaboly). In either case juvenile hormone (JH) prevents metamorphosis until a larva has attained an appropriate phase of development. In holometabolous insects, JH acts through its putative receptor Methoprene-tolerant (Met) to regulate Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) genes. While Met and Kr-h1 prevent precocious metamorphosis in pre-final larval instars, BR-C specifies the pupal stage. How JH signaling operates in hemimetabolous insects is poorly understood. Here, we compare the function of Met, Kr-h1 and BR-C genes in the two types of insects. Using systemic RNAi in the hemimetabolous true bug, Pyrrhocoris apterus, we show that Met conveys the JH signal to prevent premature metamorphosis by maintaining high expression of Kr-h1. Knockdown of either Met or Kr-h1 (but not of BR-C) in penultimate-instar Pyrrhocoris larvae causes precocious development of adult color pattern, wings and genitalia. A natural fall of Kr-h1 expression in the last larval instar normally permits adult development, and treatment with an exogenous JH mimic methoprene at this time requires both Met and Kr-h1 to block the adult program and induce an extra larval instar. Met and Kr-h1 therefore serve as JH-dependent repressors of deleterious precocious metamorphic changes in both hemimetabolous and holometabolous juveniles, whereas BR-C has been recruited for a new role in specifying the holometabolous pupa. These results show that despite considerable evolutionary distance, insects with diverse developmental strategies employ a common-core JH signaling pathway to commit to adult morphogenesis.     

The ultrastructure of the midgut epithelium in millipedes (Myriapoda,Diplopoda)

The midgut epithelia of the millipedes Polyxenus lagurus, Archispirostreptus gigas and Julus scandinavius were analyzed under light and transmission electron microscopies. In order to detect the proliferation of regenerative cells, labeling with BrdU and antibodies against phosphohistone H3 were employed. A tube-shaped midgut of three millipedes examined spreads along the entire length of the middle region of the body. The epithelium is composed of digestive, secretory and regenerative cells. The digestive cells are responsible for the accumulation of metals and the reserve material as well as the synthesis of substances, which are then secreted into the midgut lumen. The secretions are of three types – merocrine, apocrine and microapocrine. The oval or pear-like shaped secretory cells do not come into contact with the midgut lumen and represent the closed type of secretory cells. They possess many electron-dense granules (J. scandinavius) or electron-dense granules and electron-lucent vesicles (A. gigas, P. lagurus), which are accompanied by cisterns of the rough endoplasmic reticulum. The regenerative cells are distributed individually among the basal regions of the digestive cells. The proliferation and differentiation of regenerative cells into the digestive cells occurred in J. scandinavius and A. gigas, while these processes were not observed in P. lagurus. As a result of the mitotic division of regenerative cells, one of the newly formed cells fulfills the role of a regenerative cell, while the second one differentiates into a digestive cell. We concluded that regenerative cells play the role of unipotent midgut stem cells.     

The effect of manipulating ecdysteroid signaling on embryonic eye development in the locust<Emphasis Type="Italic"> Schistocerca americana</Emphasis>

Adult body plan differentiation in holometabolous insects depends on global induction and control by ecdysteroid hormones during the final phase of postembryogenesis. Studies in Drosophila melanogaster and Manduca sexta have shown that this pertains also to the development of the compound eye retina. It is unclear whether the hormonal control of postembryonic eye development in holometabolous insects represents evolutionary novelty or heritage from hemimetabolous insects, which develop compound eyes during embryogenesis. We therefore investigated the effect of manipulating ecdysteroid signaling in cultured embryonic eye primordia of the American desert locust Schistocerca americana, in which ecdysteroid level changes are known to induce three rounds of embryonic molt. Although at a considerably reduced rate compared to in vivo development, early differentiation and terminal maturation of the embryonic retina was observed in culture even if challenged with the ecdysteroid antagonist cucurbitacin B. Supplementing cultures with 20-hydroxyecdysone (20E) accelerated differentiation and maturation, and enhanced cell proliferation. Considering these results, and the relation between retina differentiation and ecdysteroid level changes during locust embryogenesis, we conclude that ecdysteroids are not an essential but possibly a modulatory component of embryonic retina development in S. americana. We furthermore found evidence that 20E initiated precocious epithelial morphogenesis of the posterior retinal margin indicating a more general role of ecdysteroids in insect embryogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by C. Desplan     

Midgut epithelium formation in Thermobia domestica (Packard) (Insecta, Zygentoma)

The origin of midgut epithelium may begin either from yolk cells (energids), tips of stomo- and proctodaeum (ectoderm), inner layer (endoderm) or from both kinds of the above mentioned cells. The origin of the midgut epithelium in wingless insects (Apterygota) has still not been determined. In Thermobia domestica the formation of midgut is much delayed, and it completes in the post-embryonic stage, while the stomo- and the proctodaeum are well-developed in the embryonic period. The energids, which remain inside the yolk, start to migrate to its periphery, where they arrange singly close to cell membrane. The yolk mass with the energids at the 14th day of embryogenesis are referred to as the primary midgut. During the first instar larval stage more and more energids migrate to the yolk periphery and the cell membrane starts to form numerous foldings surrounding the groups of energids, which in turn lead to formation of isolated regenerative cell groups. Eventually the cell membrane invaginations reach the center of the yolk mass. Large cells of the primary epithelium, surrounding the newly formed midgut lumen are formed. The cells of the primary epithelium are filled with yolk and are equipped with microvilli pointing to the midgut lumen. As the yolk is being digested, the process of the primary epithelium cells degeneration begins. The cells are getting shorter and start to degenerate. The definitive midgut epithelium is formed from proliferating regenerative cells. It consists of regularly spaced regenerative cell groups as well as the epithelial cells. The ultrastructure of both these cell groups has been described.