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).     

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.     

Ultrastructure of the excretory duct in the silk gland of the sugarcane borer Diatraea saccharalis (Lepidoptera: Pyralidae)

The excretory duct in the silk gland of the sugarcane borer Diatraea saccharalis consists of two morphologically distinct regions, recognized by scanning and transmission electron microscopy. The thin posterior region, adjacent to the glandular region, presents a regular surface. Secretory vesicles containing either electron-dense or fibrillar cuticular-like materials are observed in their apical cytoplasm; the same cuticular materials were detected as extracellular deposits among the microvilli. The short anterior region, near the common duct, exhibits surface protrusions; there are no secretory vesicles in their apical cytoplasm. These results show that only the duct cells at the posterior region are involved in the secretion of the cuticular intima elements. Desmosome-like structures were visualized linking together adjacent microvillar membranes only in the cells of anterior duct region, with unknown function. The transition between the duct and the glandular region is abrupt; the cells of the glandular and posterior duct regions present large amounts of microtubules. Nerve fibers can be observed between the duct cells in their two regions, suggesting that control of silk secretion may occur in the excretory duct via neurotransmitter liberation.     

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta)

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.     

The larval midgut of the cassava hornworm (Erinnyis ello)

Summary Columnar cells of the larval midgut of the cassava hornworm, Erinnyis ello, display microvilli with vesicles pinching off from their tips (anterior and middle midgut) or with a large number of double membrane spheres budding along their length (posterior midgut). Basal infoldings in columnar cells occur in a parallel array with many openings to the underlying space (posterior midgut) or are less organized with few openings (anterior and middle midgut). Goblet cells have a cavity, which is formed by invagination of the apical membrane and which occupies most of the cell (anterior and middle midgut) or only its upper part (posterior midgut). The infolded apical membrane shows modified microvilli, which sometimes (posterior midgut) or always (anterior and middle midgut) contain mitochondria. The cytoplasmic side of the membrane of the microvilli that contain mitochondria are studded with small particles. The results suggest that the anterior and middle region of the midgut absorbs water, whereas the posterior region secretes it. This results in a countercurrent flux of fluid, which is responsible for the enzyme recovery from undigested food before it is expelled. Intermediary and final digestion of food probably occur in the columnar cells under the action of plasma membrane-bound and glycocalix-associated enzymes.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

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.     

Fine Structure of the Midgut and Hindgut in Lepidocampa weberi (Insecta,Diplura)

The fine structure of the alimentary canal, especially the midgut and hindgut of Lepidocampa weberi (Diplura: Campodeidae) is described. The general organization of the canal is similar to that of Campodea. The midgut epithelium is composed of columnar apical microvillated cells. Each nucleus contains a single intranuclear crystal. Close to the pyloric region, the posterior midgut cells are devoid of microvilli and intranuclear crystals. There is no special pyloric chamber as in Protura or pyloric cuticular ring as in Collembola but a morphological transformation from midgut to hindgut cells. Eight globular Malpighian papillae, consisting of distal microvillated cells and flat proximal cells, open into the gut lumen via ducts formed by hindgut cells. The structure of the hindgut is complicated and can be divided into three segments. The anterior hindgut cells have an irregular shape and compact cytoplasm. A striking interdigitation between the large bottle-shaped epithelial cells and longitudinal muscle cells occurs in the middle segment of the hindgut. The thick cuticle gives rise to long spikes projecting into the gut lumen. The posterior hindgut cells possess the morphological features for water reabsorption. Some hypotheses are advanced about the function of the different regions of the gut.     

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.     

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.     

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.     

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.     

A new enveloped helical virus from the blue crab,Callinectes sapidus

Quite different ultrastructural changes were observed in the columnar cell and the goblet cell of the silkworm midgut after administration of the crystalline toxin of Bacillus thuringiensis. Shortly after the ingestion of the toxin, the deep infoldings of the basal cell membrane of some columnar cells became very irregular in shape and the mitochondria near the basal region were transformed into a condensed form. A few goblet cells showed relatively high electron density in the cytoplasm. The earliest pathological changes were slight and located in a region lying between the first and second thirds of the midgut. With the passage of time, they spread anteriorly and posteriorly to include the entire anterior two thirds of the midgut and became more profound. The cytoplasm of columnar cells became very electron transparent. Most mitochondria were transformed into a condensed form and the endoplasmic reticulum assumed a vacuole-like configuration. The basal infoldings of the cell membrane almost disappeared. On the other hand, the cytoplasm of the goblet cells became very electron dense and granular. The clear basal infoldings of the cell membrane were enlarged making a striking contrast with the dense cytoplasm. However, the mitochondria and the endoplasmic reticulum did not show any pathological deformation.     

A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

In this investigation, the anterior and posterior regions of the midgut of resistant (RL) and non-resistant (SL) Anticarsia gemmatalis larvae were analyzed morphometrically to characterize different regions along their length. Also, this investigation compares the results between SL and RL to improve the understanding of the resistance mechanisms to the virus. Histological sections were analyzed in a computerized system and the data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. The midguts are morphometrically different in the two larval populations; we observed higher values in RL. The morphometric analysis of the epithelial cells showed that only columnar and goblet cells were distinct along the midgut, in both larvae, with the higher values found in the anterior region. Comparing the results between the two larval populations, all the epithelial cells presented significant differences, with RL showing the higher morphometric values. We concluded that there are regional differences along the length of midgut in SL and RL that confirm the idea of two morpho-functional distinct regions. The consistently morphometric superior values in RL indicate that this variability can be related with the resistance of A. gemmatalis to its AgMNPV.     

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.     

The vas deferens of the spider crab,Libinia emarginata

Sperm enter the anterior vas deferens individually in the spider crab male. There they become surrounded by secretion products from the cells of the vas deferens, and are compartmentalized into spermatophores of varying size. The anterior vas deferens can be divided into three regions. The epithelium of the anterior vas deferens varies regionally from low to high columnar. The cytoplasm contains vast arrays of rough endoplasmic reticulum and Golgi complexes but few mitochondria. Intercellular spaces contain septate junctions, gap junctions and vesicles. Once the spermatophores have been formed in the anterior vas deferens, they are moved posteriorly to the middle vas deferens where they are stored and surrounded by seminal fluids. The epithelial cells of the middle vas deferens contain large amounts of rough endoplasmic reticulum and Golgi complexes. Numerous micropinocytotic vesicles appear, forming at the cell surface and within the apical cytoplasm. Their suggested function is the resorption of secretion products of the anterior vas deferens which initiated compartmentalization of the spermatozoa into spermatophores. The posterior vas deferens functions primarily as a storage center for spermatophores until they are released at the time of copulation. Seminal fluid surrounding the spermatophores is produced in this region as well as in the middle vas deferens. The cells of this region contain vast arrays of vesicular rough endoplasmic reticulum and Golgi complexes. The cells are multinucleate. Microtubules are numerous throughout the length of the cells and appear to insert on the plasma membrane.     

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.     

Histochemical and ultrastructural evidence of lipid secretion by the silk gland of the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae)

The silk gland in Lepidoptera larvae is responsible for the silk production used for shelter or cocoon construction. The secretion of fibroin and sericin by the different silk gland regions are well established. There are few attempts to detect lipid components in the insect silk secretion, although the presence of such element may contribute to the resistance of the shelter to wet environment. This study characterizes the glandular region and detects the presence of lipid components in the secretion of the silk gland of Diatraea saccharalis(Fabricius). The silk gland was submitted to histochemical procedure for lipid detection or conventionally prepared for ultrastructural analyses. Lipid droplets were histochemically detected in both the apical cytoplasm of cell of the anterior region and in the lumen among the microvilli. Ultrastructural analyses of the anterior region showed lipid material, visualized as myelin-like structures within the vesicular Golgi complex and in the apical secretory globules, mixed up with the sericin; similar material was observed into the lumen, adjacent to the microvilli. Lipids were not detected in the cells neither in the lumen of the posterior region. Our results suggest that the silk produced by D. saccharalis has a minor lipid content that is secreted by the anterior region together with the sericin.     

Electron microscopic study of the anterior midgut in Cenocorixa bifida Hung. (Hemiptera: Corixidae) with reference to its secretory function

The epithelium of anterior midgut of adult Cenocorixa bifida was examined with light and electron microscopy. The folded epithelium is composed of tall columnar cells extending to the lumen, differentiating dark and light cells with interdigitating apices and regenerative basal cells in the nidi surrounded by villiform ridges that penetrate deeply into the epithelium. The columnar cells display microvilli at their luminal surface. Microvilli lined intercellular spaces and basal plasma membrane infoldings are associated with mitochondria. These ultrastructural features suggest their role in absorption of electrolytes and nutrients from the midgut lumen. The columnar cells contain large oval nuclei with prominent nucleoli. Their cytoplasm is rich in rough endoplasmic reticulum, Golgi complexes and electron-dense secretory granules indicating that they are also engaged in synthesis of digestive enzymes. The presence of secretory granules in close proximity of the apical plasma membrane suggests the release of secretion is by exocytosis. The presence of degenerating cells containing secretory granules at the luminal surface and the occurance of empty vesicles and cell fragments in the lumen are consistent with the holocrine secretion of digestive enzymes. Apical extrusions of columnar cells filled with fine granular material are most likely formed in response to the lack of food in the midgut. The presence of laminated concretions in the cytoplasm is indicative of storageexcretion of surplus minerals. The peritrophic membrane is absent from the midgut of C. bifida.