FMRFamide- and pancreatic polypeptide-like immunoreactivity of endocrine cells in the midgut of a mosquito

Immunocytochemical surveys of midguts from female mosquitoes, Aedes aegypti, reveal that half of the estimated 500 endocrine cells in a midgut contain a substance recognized by antisera to bovine pancreatic polypeptide and a molluscan peptide, FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide). With light microscopy the cells resemble an endocrine type because of their basal position in the epithelium, conical shape, and, in some instances, apical extensions to the lumen. At the ultrastructural level, the immunoreactive substance is contained specifically within the secretory granules of such cells. Immunoreactive cells are distributed exclusively in the midgut region where blood is stored, and ingestion of vertebrate blood reduces the number of such cells and the intensity of reaction in others. These two facts suggest that a blood meal stimulates release of the immunoreactive substance from the cells. Since the immunocytochemical localization is supplemented by a demonstrated secretory response, the cells are considered to be peptidergic endocrine cells.     

Biotinylation of Bacillus thuringiensis Insecticidal Crystal Proteins

Biotinylation of Bacillus thuringiensis insecticidal crystal proteins (ICPs) was evaluated for its potential use in an alternative ICP screening method and in the characterization of ICP receptors. In vivo biological activity of CryIA(b), as inferred from bioassays with Manduca sexta and Ostrinia nubilalis and from histopathological effects on O. nubilalis midgut cells induced by force feeding, was not affected by biotinylation at moderate biotinylation ratios. A competitive radioreceptor assay showed that there was only a minor reduction in binding affinity of biotin-labeled CryIA(b) for M. sexta brush border membrane vesicles. On midgut tissue sections, the binding pattern along the midgut epithelium and the staining intensity of biotinylated ICPs detected with streptavidin-enzyme conjugate were virtually identical to the binding pattern and staining intensity of native CryIA(b) detected with antibodies. The specificity of biotinylated ICP binding to larval midgut tissue was demonstrated by performing homologous competition experiments. The relationship between different ICP receptor types in Plutella xylostella, as inferred from radioligand binding studies, was confirmed by the results of heterologous competition experiments performed with biotinylated and native ICPs.     

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.     

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.     

Regulatory peptides in fruit fly midgut

Regulatory peptides were immunolocalized in the midgut of the fruit fly Drosophila melanogaster. Endocrine cells were found to produce six different peptides: allatostatins A, B and C, neuropeptide F, diuretic hormone 31, and the tachykinins. Small neuropeptide-F (sNPF) was found in neurons in the hypocerebral ganglion innervating the anterior midgut, whereas pigment-dispersing factor was found in nerves on the most posterior part of the posterior midgut. Neuropeptide-F (NPF)-producing endocrine cells were located in the anterior and middle midgut and in the very first part of the posterior midgut. All NPF endocrine cells also produced tachykinins. Endocrine cells containing diuretic hormone 31 were found in the caudal half of the posterior midgut; these cells also produced tachykinins. Other endocrine cells produced exclusively tachykinins in the anterior and posterior extemities of the midgut. Allatostatin-immunoreactive endocrine cells were present throughout the midgut. Those in the caudal half of the posterior midgut produced allatostatins A, whereas those in the anterior, middle, and first half of the posterior midgut produced allatostatin C. In the middle of the posterior midgut, some endocrine cells produced both allatostatins A and C. Allatostatin-C-immunoreactive endocrine cells were particularly prominent in the first half of the posterior midgut. Allatostatin B/MIP-immunoreactive cells were not consistently found and, when present, were only weakly immunoreactive, forming a subgroup of the allatostatin-C-immunoreactive cells in the posterior midgut. Previous work on Drosophila and other insect species suggested that (FM)RFamide-immunoreactive endocrine cells in the insect midgut could produce NPF, sNPF, myosuppressin, and/or sulfakinins. Using a combination of specific antisera to these peptides and transgenic fly models, we showed that the endocrine cells in the adult Drosophila midgut produced exclusively NPF. Although the Drosophila insulin gene Ilp3 was abundantly expressed in the midgut, Ilp3 was not expressed in endocrine cells, but in midgut muscle.     

The distribution and myotropic activity of locustatachykinin-like peptides in locust midgut

The midgut of the African migratory locust, Locusta migratoria, was found to contain endocrine-like cells that stained positively for locustatachykinin I (Lom TK I)-like immunoreactivity. These cells were distributed in an unequal manner throughout the midgut of the locust, with a greater density of Lom TK I-like immunoreactive endocrine-like cells occurring in the posterior region of the midgut. These singly occurring cells appear elongate with an apical extension projecting toward the midgut lumen and a smaller projection extending towards the midgut basal lamina. No immunoreactive neuronal processes were detected along the midgut wall. Radioimmunoassays revealed that the female midgut contained two to three times more Lom TK I-like material than the male midgut, and radioimmunoassay coupled to high-performance liquid chromatography analysis revealed that at least five locustatachykinin isoforms appear to be present in the midgut. This distribution of Lom TK I-like material suggests possible functional differences in the various regions of the midgut. The role that these cells may play in locust midgut secretory activity and motility remains unknown. However, the addition of synthetic Lom TK I through IV to a ring type midgut muscle preparation stimulated contraction of midgut circular muscles, suggesting a possible physiological role for these peptides. Dose-response curves constructed for Lom TK I-IV revealed that the peptide-induced contractions increased in a dose-dependent manner.     

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.     

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

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

The functional morphology of the alimentary canal of larval stages of the parasitic copepod Lepeophtheirus salmonis

The functional morphology of the alimentary canal of copepodite and chalimus stages of Lepeophtheirus salmonis (Krøyer, 1837) is described and compared with that found in other copepods studied to date.
The buccal cavity passes into a gut comprising three major regions: foregut (oesophagus), midgut and hindgut. The foregut and hindgut both posscss a cuticular lining whereas the midgut is lined with specialized epithelial cells. The midgut is divided into three recognizable zones, namely anterior midgut caecum, anterior midgut and posterior midgut. Three main types of epithelial cell are recognizable in the midgut: vesicular cells, microvillous cells and basal cells which correspond to the cell types normally described in other parasitic and free-living copepod species.
Digestion is thought to occur in the midgut and be mediated by the epithelial cells that line it. Although several glands appear to discharge into the area of the buccal cavity, none was seen to interface to any other area of the gut. There was no evidence for the involvement of commensal gut bacteria in food digestion.     

Immunohistological localization of regulatory peptides in the midgut of the female mosquitoAedes aegypti

The midgut of the female mosquitoAedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatotropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.     

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.     

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.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

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

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

A novel tissue in an established model system: the <Emphasis Type="Italic">Drosophila</Emphasis> pupal midgut

The Drosophila larval and adult midguts are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact “yellow body.” The formation of a pupal midgut has been reported from several other species and may represent a general feature of intestinal metamorphosis in insects.     

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.     

Metamorphosis of midgut epithelial cells in the silkworm (Bombyx Mori L.) with special regard to the calcium salt deposits in the cytoplasm. I. light microscopy

The morphological changes of the metamorphosing midgut cell in the silkworm were traced light-microscopically. The regenerative cells of the larval midgut proliferate rapidly during larval-pupal molt and finally replace the larval midgut, establishing new pupal midgut tissue composed of only one cell type. Pupal midgut cells contain numerous basophilic granules which are believed on histological grounds to be the deposits of calcium salts. Calcium seems to be transported from hemolymph to the pupal midgut cells and stored there temporarily as insoluble salts such as phosphate or carbonate, and then finally discharged into the lumen in a merocrine fashion. The midgut cells of the adult no longer contain calcium deposits.     

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.     

Ultrastructure of the midgut and hindgut of Derocheilocaris remanei (Crustacea, Mystacocarida)

Ultrastructural features and structure of the midgut and hindgut of Derocheilocaris remanei were studied. The large endodermal midgut is differentiated into an anterior midgut and a posterior midgut separated by a conspicuous constriction. Both circular and longitudinal striated muscle bands surround the midgut, while the hindgut only presents longitudinal muscles. The limit between the midgut and the cuticle-lined hindgut is marked by a rectal valve. In cross-section, the short hindgut is triradiate and has a distinct Y-shaped lumen. The hindgut cuticular lining appears interrupted at the tip of every branch of the Y. Three different cell types are found in the midgut epithelium: basally located undifferentiated cells that give rise to the other two specialized cell types; secretory zymogen-like cells responsible for extracellular digestion and located mainly in the anterior midgut; and vacuolated cells, distributed all along the midgut and appearing to have several functions, including absorption, intracellular digestion, and nutrient transport. A single basic cell type forms the hindgut epithelium. The suggested function for the hindgut is the transport and ejection of waste products.