Autophagy as the cell survival in response to a microsporidian infection of the midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada: Hypsibiidae)

The midgut epithelial cells of many invertebrates may possess microorganisms which act as symbionts or pathogens (bacteria, microsporidia, viruses). During our previous studies on Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada, Eutardigrada), which examined alterations of the midgut epithelium during oogenesis, we found that some of the specimens were infected with microsporidia. All stages of pathogens occurred in the cytoplasm of the digestive cells in the midgut epithelium of I. g. granulifer that were infected with microsporidia: meronts, sporonts, sporoblasts, and spores. The cytoplasm of the digestive cells was rich in mitochondria, cisterns of rough endoplasmic reticulum (RER), and Golgi complexes. Autophagy in the digestive cells of the dorsal midgut was much more intensive in comparison with noninfected specimens. Membranes of phagophores surrounded the pathogens forming autophagosomes. These latter structures fused with lysosomes forming autolysosomes and residual bodies appeared. Neither glycogen granules nor droplets of varying electron density, which accumulated in digestive cells during vitellogenesis and choriogenesis, appeared in individuals with microsporidia. While the midgut epithelium in noninfected specimens takes part in vitellogenesis and choriogenesis, in infected specimens, midgut cells are involved in the process of autophagy as a survival strategy.     

The fine structure of the midgut epithelium in a centipede,Scolopendra cingulata (Chilopoda,Scolopendridae), with the special emphasis on epithelial regeneration

Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner.Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods.     

Morphology and ultrastructure of the midgut in Piscicola geometra (Annelida, Hirudinea)

This paper presents information on the organization of the midgut and its epithelium ultrastructure in juvenile and adult specimens of Piscicola geometra (Annelida, Hirudinea), a species which is a widespread ectoparasite found on the body and gills and in the mouth of many types of fish. The analysis of juvenile nonfeeding specimens helped in the explanation of all alterations in the midgut epithelium which are connected with digestion. The endodermal portion (midgut) of the digestive system is composed of four regions: the esophagus, the crop, the posterior crop caecum, and the intestine. Their epithelia are formed by flat, cuboidal, or columnar digestive cells; however, single small cells which do not contact the midgut lumen were also observed. The ultrastructure of all of the regions of the midgut are described and discussed with a special emphasis on their functions in the digestion of blood. In P. geometra, the part of the midgut that is devoid of microvilli is responsible for the accumulation of blood, while the epithelium of the remaining part of the midgut, which has a distinct regionalization in the distribution of organelles, plays a role in its absorption and secretion. Glycogen granules in the intestinal epithelium indicate its role in the accumulation of sugar. The comparison of the ultrastructure of midgut epithelium in juvenile and adult specimens suggests that electron-dense granules observed in the apical cytoplasm of digestive cells take part in enzyme accumulation. Numerous microorganisms were observed in the mycetome, which is composed of two large oval diverticles that connect with the esophagus via thin ducts. Similar microorganisms also occurred in the cytoplasm of the epithelium in the esophagus, the crop, the intestine, and in their lumen. Microorganisms were observed both in fed adult and unfed juvenile specimens of P. geometra, which strongly suggests that vertical transmission occurs from parent to offspring.     

Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta), in relation to its degeneration and regeneration

In two archaeognathans, Lepismachilis notata and Machilis hrabei, the midgut epithelium and processes of its regeneration and degeneration have been described at the ultrastructural level. In both analysed species, the midgut epithelium is composed of epithelial and regenerative cells (regenerative nests). The epithelial cells show distinct regionalization in organelles distribution with the basal, perinuclear, and apical regions being distinguished. Degeneration of epithelial cells proceeds in a necrotic way (continuous degeneration) during the entire life of adult specimens, but just before each moult degeneration intensifies. Apoptosis has been observed. Regenerative cells fulfil the role of midgut stem cells. Some of them proliferate, while the others differentiate into epithelial cells. We compared the organisation of the midgut epithelium of M. hrabei and L. notata with zygentoman species, which have just been described.     

Ultrastructural changes of the midgut epithelium in Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada: Eutardigrada) during oogenesis

The midgut epithelium of Isohypsibius granulifer granulifer (Eutardigrada) is composed of columnar digestive cells. At its anterior end, a group of cells with cytoplasm which differs from the cytoplasm of digestive cells is present. Probably, those cells respond to crescent-like cells (midgut regenerative cells) described for some tardigrade species. Their mitotic divisions have not been observed. We analyzed the ultrastructure of midgut digestive cells in relation to five different stages of oogenesis (previtellogenesis, beginning of the vitellogenesis, vitellogenesis—early choriogenesis, vitellogenesis—middle choriogenesis, late choriogenesis). In the midgut epithelium cells, the gradual accumulation of glycogen granules, lipid droplets and structures of varying electron density occurs. During vitellogenesis and choriogenesis, in the cytoplasm of midgut cells we observed the increasing number of organelles which are responsible for the intensive synthesis of lipids, proteins and saccharides such as cisterns of endoplasmic reticulum and Golgi complexes. At the end of oogenesis, autophagy also intensifies in midgut epithelial cells, which is probably caused by the great amount of reserve material. Midgut epithelium of analyzed species takes part in the yolk precursor synthesis.     

The role of autophagy in the midgut epithelium of Eubranchipus grubii (Crustacea,Branchiopoda, Anostraca)

Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca) is an omnivorous filter feeder whose life span lasts no more than 12 weeks. Adult males and females of E. grubii were used for ultrastructural studies of the midgut epithelium and an analysis of autophagy. The midgut epithelium is formed by columnar digestive cells and no regenerative cells were observed. A distinct regionalization in the distribution of organelles appears – basal, perinuclear and apical regions were distinguished. No differences in the ultrastructure of digestive cells were observed between males and females. Autophagic disintegration of organelles occurs throughout the midgut epithelium. Degenerated organelles accumulate in the neighborhood of Golgi complexes, and these complexes presumably take part in phagophore and autophagosome formation. In some cases, the phagophore also surrounds small autophagosomes, which had appeared earlier. Fusion of autophagosomes and lysosomes was not observed, but lysosomes are enclosed during autophagosome formation. Autophagosomes and autolysosomes are discharged into the midgut lumen due to apocrine secretion. Autophagy plays a role in cell survival by protecting the cell from cell death.     

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.     

Fine structure of the midgut of Sinopanorpa tincta (Navás) (Mecoptera: Panorpidae)

Fine structure of the midgut and degeneration of the midgut epithelium of the scorpionfly Sinopanorpa tincta (Navás) adults were investigated using light microscopy and scanning and transmission electron microscopy. The results show that the tubular midgut lacks gastric caeca and is composed of an outer longitudinal and an inner circular muscle layer, a basal lamina, an epithelium and a lumen from the outside to inside. A peritrophic membrane was not found in the lumen. A mass of nodules was observed on the surface of the basal lamina. Three types of cells were recognized in the epithelium: digestive, secretory, and regenerative cells. The digestive cells contain irregular-shaped infoldings in the basal membrane and two types of microvilli in the apical membrane. The secretory cells are characterized by irregular shape and large quantities of secretory granules in the basal cytoplasm. The regenerative cells are triangular in shape and distributed only in the nodules. The epithelial cells are degenerated through programmed cell-death mechanisms (apoptosis and necrosis). The type, function, and degeneration of the epithelial cells of the midgut are briefly discussed.     

Ultrastructure and regeneration of midgut epithelial cells in Lithobius forficatus (Chilopoda,Lithobiidae)

Lithobius forficatus (Myriapoda, Chilopoda, Lithobiidae) is a widespread species of centipede that is common across Europe. Its midgut epithelial cells are an important line of defense against toxic substances that originate in food, such as pathogens and metals. Despite this important role, the biology of the midgut epithelium is not well known. Here we describe the ultrastructure of the midgut epithelium, as well as the replacement of degenerated midgut epithelial cells. The midgut epithelium of L. forficatus is composed of digestive, secretory, and regenerative cells. The cytoplasm of digestive cells shows regionalization in organelle distribution, which is consistent with the role of these cells in secretion of enzymes, absorption of nutrients, and accumulation of lipids and glycogen. Secretory cells, which do not reach the luminal surface of the midgut epithelium, possess numerous electron‐dense and electron‐lucent granules and may have an endocrine function. Hemidesmosomes anchor secretory cells to the basal lamina. Regenerative cells play the role of midgut stem cells, as they are able to proliferate and differentiate. Their proliferation occurs in a continuous manner, and their progeny differentiate only into digestive cells. The regeneration of secretory cells was not observed. Mitotic divisions of regenerative cells were confirmed using immunolabeling against BrdU and phosphohistone H3. Hemocytes associate with the midgut epithelium, accumulating between the visceral muscles and beneath the basal lamina of the midgut epithelium. Hemocytes also occur among the digestive cells of the midgut epithelium in animals infected with Rickettsia‐like microorganisms. These hemocytes presumably have an immunoprotective function in the midgut.     

The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari,Trombidiformes: Syringophilidae)

The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (=posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 μm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut.     

Autophagic activity in the midgut gland of the overwintering harvestmen Gyas annulatus (Phalangiidae,Opiliones)

Juvenile harvestmen Gyas annulatus overwinter in dormancy in hypogean habitats for 4–5 months. The ultrastructure of the autophagic structures in their midgut epithelium cells was studied by light microscopy, transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) during this non-feeding period. Before overwintering (November), autophagic structures were scarce. In the middle (January) and at the end of overwintering (March), phagophores, autophagosomes and autolysosomes were present in the cytoplasm of both the secretory and the digestive midgut epithelium cells, gradually increasing their abundance during overwintering. In addition, vacuolization of the cytoplasm intensified. Both processes are induced by starvation. Autophagic structures and cytoplasm vacuolization enable the reuse of the cell's own components required for the maintenance of vital processes during dormancy. While TEM is a much more convenient method for recognition of the autophagic structure types and their ultrastructure, IFM enables exact counting of these structures.     

Degeneration of the midgut epithelium in Allacma fusca L. (Insecta, Collembola, Symphypleona): apoptosis and necrosis

Apoptotic and necrotic changes in the midgut epithelium cells of Allacma fusca (Collembola, Symphypleona) are described at the ultrastructural level. The morphological sign indicating the beginning of the apoptotic process in these cells is their shrinkage and the transformation of their mitochondria. The nucleus assumes a lobular shape and finally undergoes fragmentation. The intercellular junctions between an apoptotic cell and adjacent epithelial cells gradually disappear. Apoptotic cells are discharged into the midgut lumen just beneath the peritrophic membrane, where they are initially distributed singly but ultimately form a single layer. No phagocytosis was observed, so no apoptotic bodies are formed. Only young midgut epithelium shows apoptosis; as cells age, necrosis accompanies apoptosis, and necrosis finally completely replaces apoptosis.     

Ultrastructural changes in the midgut epithelium in Podura aquatica L. (Insecta, Collembola, Arthropleona) during regeneration

Data considering the degeneration and regeneration of the midgut epithelium in the primitive wingless insects, such as Collembola, are rather poor. Also information, which treats the regenerative cells as the primordial cells, is poorly known. The midgut epithelium of Podura aquatica L. (Insecta, Collembola, Arthropleona) is formed by the epithelial and regenerative cells. The epithelial cells show distinct regionalisation in the organelles distribution. The ultrastructure of the basal, perinuclear and apical regions of the epithelial cells is described. As in insects without Malpighian tubules, structures which resemble urospherites occur in the cytoplasm of the epithelial cells. After degeneration of the entire midgut epithelium, a new epithelium is formed from regenerative cells. During the process of regeneration, the degenerated epithelium gradually is separated from the basal lamina by the newly formed one. Finally, the detached epithelium is moved into the midgut lumen. Regenerative cells play a role of primordial cells during epithelial regeneration.     

Fine structure of the midgut epithelium of Nicoletia phytophila Gervais, 1844 (Zygentoma: Nicoletiidae: Nicoletiinae) with special emphasis on its degeneration

The midgut epithelium of Nicoletia phytophila is composed of columnar digestive cells and regenerative cells that form regenerative nests. The cytoplasm of midgut epithelial cells shows typical regionalization in organelle distribution. Two types of regenerative cells have been distinguished: cells which are able to divide intensively and cells which differentiate. Spot desmosomes have been observed between neighboring regenerative cells. The occurrence of intercellular junctions is discussed. The midgut epithelium degenerates both in an apoptotic and necrotic way. Necrosis proceeds during each molting period (cyclic manner), while apoptosis occurs between each molting, when the midgut epithelium is responsible for e.g. digestion. These processes of epithelium degeneration are described at the ultrastructural level. Our studies not only add new information about fine structure of the midgut epithelium of N. phytophila, but contribute to resolving the relationships within the Zygentoma. There are no doubts about the very close sister position of Nicoletiidae and Ateluridae. The midgut epithelium characters confirm their close relationship. However we do not recommend classifying the atelurid genera only within Nicoletiidae: Nicoletiinae.     

Ultrastructural changes in the midgut epithelium of the first larva of Allacma fusca (Insecta, Collembola, Symphypleona)

Abstract. In the newly hatched larva in Allacma fusca , the midgut epithelium was fully developed and formed by flattened epithelial cells surrounding the yolk mass in the midgut lumen. Immediately after hatching, the first larva began to feed; the migut lumen was filled with the yolk mass and food (mainly algae). Regenerative cells typical of the developing midgut epithelium of many insects were not observed. Initially, midgut cells of the larva were cuboidal but became columnar in shape with distinct regionalization in the distribution of cell organelles. Furthermore, urospherites appeared in the midgut cell cytoplasm, i.e., structures characteristic for the midgut epithelium of insects having no Malpighian tubules. As a result, cells with the capacity for digestion, absorption, and excretion were observed to be completely formed in the first larval stage.     

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.     

Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum.

We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exists to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.     

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.     

Light and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot,Gasterophilus intestinalis

A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double‐layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages.     

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.