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.     

X-ray microanalysis of elements in frozen-hydrated sections of an electrogenic K+ transport system: The posterior midgut of tobacco hornworm (Manduca sexta) in vivo andin vitro

Summary The lepidopteran midgut is a model for the oxygendependent, electrogenic K⁺ transport found in both alimentary and sensory tissues of many economically important insects. Structural and biochemical evidence places the K⁺ pump on the portasome-studded apical plasma membrane which borders the extracellular goblet cavity. However, electrochemical evidence implies that the goblet cell K⁺ concentration is less than 50mm. We used electron probe X-ray microanalysis of frozenhydrated cryosections to measure the concentration of Na, Mg, P, S, Cl, K, Ca and H₂O in several subcellular sites in the larval midgut ofManduca sexta under several experimental regimes. Na is undetectable at any site. K is at least 100mm in the cytoplasm of all cells. Typicalin vivo values (mm) for K were: blood, 25; goblet and columnar cytoplasm, 120; goblet cavity, 190; and gut lumen, 180. The high K concentration in the apically located goblet cavity declined by 100mm under anoxia. Both cavity and gut fluid are Cl deficient, but fixed negative charges may be present in the cavity. We conclude that the K⁺ pump is sited on the goblet cell apical membrane and that K⁺ follows a nonmixing pathway via only part of the goblet cell cytoplasm. The cavity appears to be electrically isolated in alimentary tissues, as it is in sensory sensilla, thereby allowing a PD exceeding 180 mV (lumen positive) to develop across the apical plasma membrane. This PD appears to couple K⁺ pump energy to nutrient absorption and pH regulation.     

Functional ultrastructure of the midgut of the fire ant Solenopsis saevissima Forel 1904 (Formicidae: Myrmicinae)

Solenopsis saevissima has a midgut composed of columnar, regenerative, and goblet cells. The midgut epithelium was covered by a basal lamina. Outside the basal lamina, layers of inner oblique, circular, and outer longitudinal muscles were present. Columnar cells showed a basal plasma membrane containing numerous folds, mitochondria, and the nucleus. Rough endoplasmic reticulum, Golgi bodies, membrane bounded vacuoles, and spherocrystals were found in this region. The apical plasma membrane was constituted by microvilli, which were above a region rich in mitochondria. Regenerative cells were found in groups lying by the basal lamina. Goblet cells were associated with an ion-transporting mechanism between the haemolymph and the midgut epithelium. These cells were lying by the midgut lumen and large microvilli were evident, but the cytoplasmic features were similar to the columnar 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.     

斑蝥素对粘虫和小菜蛾幼虫中肠组织的影响

粘虫 Mythimna separata (Walker) 和小菜蛾Plutella xylostella L.的幼虫经斑蝥素处理后,中肠组织发生明显的病变。粘虫幼虫中肠柱状细胞微绒毛脱落,杯状细胞变大,质膜消失;线粒体不规则肿胀,内嵴排列紊乱、溶解、断裂;粗面内质网扩张明显,核糖体脱落;细胞核肿胀,染色质浓缩,偏移。小菜蛾幼虫中肠组织病变更为明显。表明斑蝥素主要作用于中肠的膜系统,为进一步探讨其毒理机制提供了理论依据。     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Ultrastructure and immunocytochemistry of endocrine cells in the midgut of the desert locust,Schistocerca gregaria (Forskal)

Summary The endocrine cells of the midgut epithelium of the desert locust are found dispersed among the digestive cells and are similar to those of the vertebrate gut. According to their reactivity to silver impregnation techniques and the ultrastructural features of the secretory granules (shape, electron-density, size, and structure) 10 types of endocrine cell have been identified, of which seven are located in the main segment of the midgut or in the enteric caeca, and the other three seem to be present only in the ampullae through which the Malpighian tubules drain into the gut. The endocrine cells have a slender cytoplasmic process that reaches the gut lumen, a feature that supports the receptosecretory nature postulated for this cellular type in insects as well as vertebrates. Antisera directed against mammalian gastrin, CCK, insulin, pancreatic polypeptide and bombesin reacted with some of the endocrine cells. This is the first time that insulin- and bombesin-like immunoreactive cells have been described in the midgut of an insect.     

Microfilarial perforation of the midgut of a mosquito

To determine whether the midgut envelope of mosquitoes is disrupted by the passage of microfilariae, ultrastructural changes induced by microfilariae of Brugia malayi were observed in midguts of Aedes aegypti mosquitoes. Basal and apical plasma membranes were destroyed, disrupting the full depth of the midgut wall. Ingested ferritin lay against the gut wall, suggesting absence of the peritrophic membrane during penetration. Exsheathment of microfilariae appears to be enhanced by movement against the constricting midgut wall. It was concluded that particles present in the lumen of the gut may be disseminated passively to the hemocoel.     

The morphology and fine structure of the larval midgut of a moth (Manduca sexta) in relation to active ion transport.

Light and electron microscopic examination of the midgut of Manduca sexta has shown that the organization of this tissue is more complex than was originally believed. The midgut can be divided into anterior, middle and posterior regions on the basis of the pattern of folding of the epithelial sheet, and variations in the structure of goblet and columnar cells which occur along its length. The columnar cells show gradual structural changes form the anterior to the posterior end of the midgut. For example, the microvilli in the anterior region form a dense, interconnecting network from which vesicles break off. This organization becomes less obvious through the middle region, until by the posterior region each microvillus is unconnected to adjacent microvilli along its entire length and vesicles are no longer produced. Two distinct types of goblet cells are found. In the anterior and middle regions the goblet cells have a large basally located cavity, but in the posterior region the cavity occupies only the apical half of the cell. In both cases the cavity is formed by invagination of the apical membrane, which is studded with small particles implicated in active ion transport. In the anterior and middle regions this membrane is closely associated with mitochondria, but not in the posterior region. The significance of the observed structural differences is discussed in relation to active ion transport.     

Appearance of apoptotic cells and granular cells in the silkworm midgut lumen during larval-pupal ecdysis

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.     

A study of the concentrically laminated concretions, 'spherites' in the regenerative cells of the midgut of Lepidopterous larvae

Concentrically laminated granules, spherites, are sometimes found in the regenerative cells of midgut of some species of lepidopterous larvae. The spherites are formed in cytoplasmic vesicles just before ecdysis and disappear during the differentiation of the regenerative cells to columnar and goblet cells. They function as intracellular stores of compounds used in the growth of the cell. Phosphates of magnesium and perhaps calcium are probable constituents. Spherites are sometimes also found in the degenerating columnar cells where they are excreted into the lumen with the exfoliating epithelium. The phenomenon of periodic precipitation which is the physical-chemical basis of the formation of spherites is discussed.     

The gut structure of Sinentomon erythanum yin (Protura : Sinentomidae)

The fine structure of the midgut, pyloric region, Malpighian papillae, and hindgut of Sinentomon erythranum (Protura : Sinentomidae) is described. Midgut cells are rich in mineral concretions and are presumably involved in excretory activity; the pyloric chamber, a cavity in the proturan intestine behind the midgut, is formed by cells with microvilli pointing anteriorly; the secretion from 6 Malpighian papillae flows into this cavity. The hindgut consists of 2 regions; the anterior of the 2 has a series of specializations typical of cells engaged in active water reabsorption. Long infoldings of the apical plasma membrane reach deep into the cells. The findings are compared with the gut organization of other genera of Protura examined to date.     

Absorption of albumin by the midgut of a lepidopteran larva

In the last decade, the study of peptide and protein absorption by the insect gut has received increasing attention because of the considerable impact this information may have on the development of new delivery strategies for insecticide macromolecules targeting haemocoelic receptors. Available experimental evidence in vivo suggests that, in insects, peptides and proteins can cross the intestinal barrier reaching the haemocoel, but the functional bases of this absorption pathway have not yet been thoroughly investigated. The current knowledge of the mechanisms involved in protein and polypeptide absorption in animals derives from the extensive studies performed in mammalian polarised epithelial cells, where the transcellular transport of proteins by transcytosis has been demonstrated. In this process, proteins are internalised at one pole of the cell and transported by cytoplasmic vesicular traffic to the opposite plasma membrane domain, where they are released with unchanged biological activity. Here we report data on albumin translocation across the isolated midgut of Bombyx mori caterpillars perfused in vitro. The functional properties of the transepithelial transport of this protein are described and, since absorption prevails over secretion, its lumen-to-haemolymph flux is characterised. Low-temperature incubations nearly abolish the transepithelial transport, while the peculiar physiological features of the larval midgut, i.e. the high lumen positive transepithelial voltage and the luminal alkaline pH, do not affect the flux. The obtained results indicate that albumin crosses B. mori larval midgut by transcytosis.     

Ultrastructure of the digestive system of the Le fhopper Euscelidius variegatus Kirshbaum (Homoptera : Cicadellidae), with and without congenital bacterial infections

In the digestive system of Euscelidius variegatus Kirshbaum (Homoptera : (Cicadellidae), the close apposition of the anterior midgut with its posterior tabular midgut forms a filter chamber, which shunts excess water in the imbibed plant sap to the hindgut. Leafhoppers congenitally infected with a parasitic enteroform bacterium (designated BEV) had slightly atrophied digestive systems. There were numerous bacteria within the cells of the filter chamber, conical segment, and tubular midgut. Bacteria within the epithelium cells were usually enclosed within lysosomes. Epithelium cells swollen with large numbers of bacteria, had deteriorated cell membranes, and bacteria had erupted into the gut lumen. Leafhoppers not infected by BEV, harbored bacteria in the gut lumen, but not intracellularly within gut cells.     

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.     

Larval anatomy and structure of absorbing epithelia in the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae)

The present work describes Aphidius ervi Haliday (Hymenoptera, Braconidae) larval anatomy and development, focusing on time-related changes of body structure and cell ultrastructure, especially of the epithelial layers involved in nutrient absorption. Newly hatched 1st instar larvae of A. ervi are characterised by gut absence and a compact cluster of cells makes up their body. As the parasitoid larva develops, the central undifferentiated cell mass becomes hollowed out, leading to the formation of gut anlage. This suggests that absorption of nutrients at that stage may take place through the body surface, as more directly demonstrated by the occurrence on the epidermis of proteins associated with transepithelial transport, such as Na(+)/K(+)-ATPase and alkaline phosphatase (ALP). Second instar larvae show the presence of the gut with a well-differentiated brush border and a peritrophic membrane. Gut cells are filled by masses of glycogen granules and lipid droplets. The tracheal system starts to be visible. The haemocoel becomes evident in late 2nd instar, and contains large silk glands. Mature 3rd instar larvae are typically hymenopteriform. The midgut accounts for most of the body volume and is actively involved in nutrient absorption, as indicated by the well developed brush border and by the presence of Na(+)/K(+)-ATPase and ALP on the basolateral and luminal membrane respectively. At this stage, large lipid droplets have gradually replaced the cellular glycogen stores in the midgut cells. The tracheae are completely differentiated, but their internal lumen still contains fibrillar material, suggesting that they are not functional as long as host fluids bath the parasitoid larva. In late 3rd instar larvae, silk glands, structurally similar to Malpighian tubules, show a very intense vesicular traffic toward the internal lumen, which, eventually, results in being filled by secretion products, suggesting the possible recycling of metabolic waste products during mummy formation.     

取食Cry2Ab蛋白后棉铃虫幼虫中肠组织的病理变化

为了明确Cry2Ab杀虫蛋白的作用机制, 利用透射电镜观察了棉铃虫Helicoverpa armigera (Hübner)3龄末幼虫取食含Cry2Ab蛋白（8 μg/g）饲料后中肠的组织病理变化, 并与分别取食含Cry1Ac蛋白（0.97 μg/g）饲料和正常饲料的棉铃虫进行了比较。结果表明： 棉铃 虫取食Cry2Ab蛋白后中肠细胞及其细胞器均发生了明显的病变, 主要表现为： 中肠杯状细胞的杯腔肿胀或拉长, 部分柱状细胞被杯状细 胞挤压出来, 微绒毛脱落, 细胞核皱缩, 质膜和核膜不清晰, 染色质凝聚, 线粒体拉伸变形, 内质网肿胀断裂; 并且随着取食时间 的延长病变越来越明显。与取食Cry1Ac蛋白的棉铃虫相比, Cry2Ab引起棉铃虫中肠组织发生病变的速度较慢。本研究可为Cry2Ab作为转基 因棉花的重要杀虫蛋白在将来更好地发挥作用提供理论依据。     

胡氏边白蚁消化系统的微细构造

胡氏边白蚁Marginitermes hubbardi(Banks)消化系统可分为前肠、中肠及后肠三大段.前肠包括葡萄状唾腺、口、咽喉、食道、前胃及贲门瓣;从贲门瓣开始到马氏管着生处为止这一段为中肠;后肠则分为葫芦形胃、结肠、直肠和肛门.其消化系统的特点:在前、后肠有几丁内膜、细胞层上还有一层微绒毛;上皮细胞底膜内陷很深,折叠中夹着许多线粒体;中肠围食膜表面有几丁层一直延伸到后肠;后肠前端膨大的葫芦胃中共生了很多种细菌及原生动物,共生的细菌、动物分泌纤维素酶帮助它消化木质纤维.     

昆虫围食膜的研究进展

围食膜是大多数昆虫中肠内的半透性薄膜 ,主要由几丁质、蛋白质构成。依据其形成的方式分 :Ⅰ型围食膜 ,由整个中肠细胞分泌形成多层管状膜 ;Ⅱ型围食膜由中肠前端特殊的细胞分泌成连续的套筒管状膜。由于位于食物与中肠上皮细胞间而在中肠生理中起重要作用 ,围食膜保护中肠上皮免于机械损伤以及病原菌、毒素的入侵 ;作为半透膜以及将中肠分为不同的区室而在营养物质的消化和吸收中具有重要作用。该文综述了有关围食膜结构、组分、功能、通透性以及与害虫防治的关系等方面的研究进展。