<Emphasis Type="Italic">Toxoneuron nigriceps</Emphasis> parasitization delays midgut replacement in fifth-instar <Emphasis Type="Italic">Heliothis virescens</Emphasis> larvae

We have analyzed the effects of Toxoneuron nigriceps parasitization on the midgut development of its host Heliothis virescens. In parasitized H. virescens larvae, the midgut epithelium undergoes a complete replacement, which is qualitatively not different to that observed in synchronous unparasitized larvae, with similar temporal profiles of cell death and metabolic activity. However, the whole gut replacement process is significantly delayed in parasitized larvae, with complete differentiation of the new gut epithelium being observed 4 days later than in unparasitized controls. The administration of juvenile hormone before commitment and of 20-hydroxyecdysone (20E) after commitment delays and fosters, respectively, the replacement process of the midgut epithelium; moreover, the injection of 20E into developmentally arrested and 20E-deficient host last-instar larvae parasitized by T. nigriceps immediately triggers regular gut development. These hormone-based experiments suggest that endocrine alterations in the larval host, induced by T. nigriceps parasitism, are responsible for the temporal alterations in the gut replacement process. The role of this parasitoid-induced developmental change in the host regulation process is discussed. This work was partially supported by FAR 2006–2007 (University of Insubria) to G.T., by MIUR-FIRB-COFIN (grant no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).     

The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Cockroach midgut peptides that regulate cell proliferation, differentiation, and death in vitro

Summary The number of insect midgut cells is maintained homeostatically in vivo and in vitro. However, during starvation, the midgut shrinks and the rate of cell replacement appears to be suppressed. When they undergo metamorphosis, the internal organs of insects are drastically remodeled by cell proliferation, differentiation, and apoptotic processes, and the net number of cells usually increases. An extract of 1650 midguts ofPeriplaneta americana was fractionated by highperformance liquid chromatography (HPLC) to obtain the peptides that regulate these processes. The HPLC fractions were tested for myotropic activity in the foregut and for effects on cell proliferation or loss in primary cultures of larvalHeliothis virescens midgut cells and in a cell line derived from the last-instar larval fat body ofMamestra brassicae. Some fractions stimulated midgut stem cell proliferation and differentiation, while others caused loss of differentiated columnar and goblet cells. Other fractions stimulated cell proliferation in the larval fat body cells. Mention of products in this article does not imply endorsement by the U.S. Department of Agriculture.     

Ductin, a component of the V-ATPase, is developmentally regulated in Heliothis virescens midgut, and anti-ductin antibodies label lateral membranes

We previously cloned from Heliothis virescens a 16-kDa protein that is homologous to other ductin sequences. We also reported its immunolocalization with a specific affinity-purified anti-peptide antibody in the midgut and Malpighian tubule of feeding larvae, and concluded that the cloned proteolipid encodes the V-ATPase proton-transporting subunit c from the V₀ sector. We now present the immunolocalization of this protein in the midgut during the L₄-L₅ larval molt and early post-ecdysis into the fifth instar in H. virescens. The results show that the spatial expression of the 16-kDa protein is developmentally regulated. Labeling by anti-peptide antibody varies during the molt in the midgut goblet cell apical plasma membrane and the goblet cell apical valve. Epifluorescence and confocal microscopy revealed strong anti-ductin labeling in areas of cell-to-cell contact during the molt, and during early post-ecdysis into the fifth larval instar. The characteristic labeling pattern observed in areas of cell-to-cell contact is consistent with the claimed involvement of ductins in gap junctions. Conclusive evidence for the presence of the 16-kDa protein in areas of cell-to-cell contact in the midgut of feeding larvae is, however, lacking. V-ATPase regulation during the molt was also investigated by simultaneous immunohistochemistry with an anti-B subunit antiserum, a probe for the V₁ sector. Received: 2 April 1996 / Accepted: 14 November 1996     

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.     

Bombyxin stimulates proliferation of cultured stem cells derived from Heliothis virescens and Mamestra brassicae larvae

Summary Bombyxin stimulated proliferation of cultured midgut stem cells that were derived from two noctuiid moth larvae, Heliothis virescens and Mamestra brassicae. Bombyxin exhibited the highest activity at 10⁻¹² M. The number of cells increased for 3 d after the addition of bombyxin. Although a single addition of bombyxin did not maintain proliferation, a second addition, made 3 d after the first treatment, retained the effect. Results suggest that the decline of effect after the first addition was not due to the loss of sensitivity of the cultured cells but to the loss of effect of the growth factor added. Addition of bombyxin at more than 10⁻¹⁰ M was less effective. Bombyxin did not affect the number of cultured midgut cells without pupal fat body extract (FBX). The data suggest that FBX contains the factors that maintain sensitivity of midgut cells to proliferate in the presence of bombyxin. Bombyxin must be a unique growth factor that stimulates proliferation of midgut stem cells in vitro from lepidopteran larvae. Materials listed here are not endorsed by the U.S. Department of Agriculture.     

Expression analysis of chlorophyllid a binding protein a secretory,red fluorescence protein in the midgut of silkworm,Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.     

CLONING AND CHARACTERIZATION OF A MIDGUT‐SPECIFIC FATTY ACID BINDING PROTEIN IN Spodoptera litura

A fatty acid binding protein (FABP) gene (Slfabp1) was cloned from the midgut of Spodoptera litura larvae. The gene consists of four exons and three introns and encodes a peptide of 134 amino acid residues with a predicted molecular mass of 14.7 kDa, which was confirmed by in vitro protein expression. Northern blot and Western blot analyses indicated that both of Slfabp1 mRNA and protein were highly and specifically expressed in the midgut during the fifth and sixth instar feeding larval stages. In situ hybridization and immunohistochemistry analyses confirmed the midgut‐specific localization of Slfabp1 mRNA and protein. The result of Western blot showed that expression of the protein was downregulated by starvation and upregulated by refeeding in sixth instar larvae. All of the results taken together suggest that the SlFABP1 plays important role(s) in FA uptake and transport in the midgut during the larval feeding stages of the insect.     

Mechanisms of programmed cell death in the midgut and salivary glands from Bradysia hygida (Diptera: Sciaridae) during pupal–adult metamorphosis

Programmed cell death is involved with the degeneration/remodeling of larval tissues and organs during holometabolous development. The midgut is a model to study the types of programmed cell death associated with metamorphosis because its structure while degenerating is a substrate for the formation of the adult organ. Another model is the salivary glands from dipteran because their elimination involves different cell death modes. This study aimed to investigate the models of programmed cell death operating during midgut replacement and salivary gland histolysis in Bradysia hygida. We carried out experiments of real‐time observations, morphological analysis, glycogen detection, filamentous‐actin localization, and nuclear acridine orange staining. Our findings allow us to establish that an intact actin cytoskeleton is required for midgut replacement in B. hygida and nuclear condensation and acridine orange staining precede the death of the larval cells. Salivary glands in histolysis present cytoplasmic blebbing, nuclear retraction, and acridine orange staining. This process can be partially reproduced in vitro. We propose that the larval midgut death involves autophagic and apoptotic features and apoptosis is a mechanism involved with salivary gland histolysis.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway

In this paper we have investigated the developmental–genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells.     

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.     

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.     

Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (adenosine triphosphate‐binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baseline cytotoxicity of activated Cry1Ac and Cry2Ab against the midgut and fat body cell lines of Helicoverpa zea and the ovary cell line of Spodoptera frugiperda (SF9). As expected, the descending order of cytotoxicity of Cry1Ac against the three cell lines in terms of 50% lethal concetration (LC₅₀) was midgut (31.0 μg/mL) > fat body (59.0 μg/mL) and SF9 cell (99.6 μg/mL). By contrast, the fat body cell line (LC₅₀ = 7.55 μg/mL) was about twice more susceptible to Cry2Ab than the midgut cell line (16.0 μg/mL), the susceptibility of which was not significantly greater than that of SF9 cells (27.0 μg/mL). Further, ligand blot showed the binding differences between Cry1Ac and Cry2Ab in the three cell lines. These results indicated that the receptors of Cry2Ab were enriched in fat body cells and thus largely different from the receptors of Cry1Ac, which were enriched in midgut cells.     

Proteolytic profile in the larval midgut of Chilo suppressalis Walker (Lepidoptera: Crambidae)

Chilo suppressalis is a key constraint on production of rice. The current research was conducted to study the types of digestive proteases in the larval midgut of C. suppressalis. It was found that activity of total digestive proteases increased from the first to the fifth larval instars, which showed different nutritional requirements. Four types of proteinases and two types of exopeptidase were identified so that their activities from the highest to the lowest activities is trypsin‐like, chymotrypsin‐like and elastase for proteinases, and amino and carboxypeptidases for exopeptidases. Meanwhile, just one type of cysteine protease, cathepsin D, was determined in the fourth and fifth instar larvae. The optimal pH for activity of total protease was found to be pH 9–10 and optimal temperature was observed to be 35–40°C, where there was the highest proteolytic activity. Some specific inhibitors of proteases including PMSF, TLCK, TPCK, DTT, E‐64, cystatin, phenanthroline and EDTA were used to confirm the types of proteases in the midgut of C. suppressalis.     

Toxicity and disruption of midgut physiology in larvae of the European corn borer,Ostrinia nubilalis,by anion transporter blockers

In this study, four blockers of anion transporters (ATs) belonging to four different classes of organic acids, including DIDS (4, 4'‐diisothiocyanatostilbene‐2, 2'‐ disulfonic acid; a stilbene disulfonic acid), NPPB [(5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid; an anthranilic acid)], 9‐AC (anthracene‐9‐carboxylic acid; an aromatic carboxylic acid), and IAA‐94 (indanyloxy acetic acid; an indanyloxy alkanoic acid), were tested for their toxicity against the European corn borer (ECB), Ostrinia nubilalis. All the AT blockers inhibited the growth of larvae, increased the developmental time, and decreased survival compared to controls, when second‐instar ECB larvae were fed for seven days on treated diet. In general, DIDS and NPPB were the most active compounds, with the rank order of activity being DIDS>NPPB>IAA‐94>9‐AC. All the AT blockers decreased the midgut alkalinity in fifth‐instar larvae when fed for 3 h on treated diet. Effective concentrations required for 50% decrease in midgut alkalinity (EC₅₀) ranged between 29.1 and 41.2 ppm and the rank order of activity was NPPB>DIDS>IAA‐94>9‐AC. Similarly, all the tested AT blockers inhibited ³⁶Cl^? uptake from the midgut lumen in fifth‐instar larvae when fed for 3 h on treated diet. Concentrations required for 50% inhibition of ³⁶Cl^? uptake (IC₅₀) ranged between 7.4 and 11.0 ppm and the rank order of activity was DIDS>NPPB>9‐AC >IAA‐94. Modest to highly strong positive correlations observed among growth, midgut alkalinity, and midgut Cl^? ion transport in AT blocker–fed larvae suggested that these effects are causally related to each other. Finally, AT blockers have the potential to become good candidates for development of insecticides with a unique mode of action. © 2009 Wiley Periodicals, Inc.     

Analysis of the functions of the signal peptidase complex in the midgut of Tribolium castaneum

Signal peptidase complexes (SPCs) are conserved from bacteria to human beings, and are typically composed of four to five subunits. There are four genes encoding SPC proteins in the red flour beetle, Tribolium castaneum. To understand their importance to insect development, double‐stranded RNA for each SPC gene was injected into red flour beetles at the early larval and adult stages. Knockdown of all four signal peptidase genes was lethal to larvae. Moreover, larvae had difficulty with old cuticle ecdysis. Knockdown of TcSPC12 alone did not affect pupal or adult development. When TcSPC12, TcSPC18, and TcSPC25 were knocked down in larvae, the melanization of hemocytes and midguts was observed. When knocked down in larvae and adults, TcSPC18 induced severe cell apoptosis in midguts, and the adult midgut lost the ability to maintain crypts after knockdown of TcSPC18, indicating its importance to midgut cell proliferation and differentiation. Knockdown of TcSPC22 or TcSPC25 also resulted in many apoptotic cells in the midguts. However, TcSPC12 appeared to be unimportant for midgut development. We conclude that TcSPC18 is essential for maintaining the adult midgut crypts.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system