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.     

Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/μl AA 1-9 or 0.06 pg/μl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140-200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.     

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.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

Altering the fate of stem cells from midgut of Heliothis virescens:the effect of calcium ions

Cultured stem cells from larval midgut tissue of the lepidopteran Heliothis virescens respond to alterations in external calcium ion concentration (Ca(2+) (out)) by changing the rate of stem cell proliferation and by differentiating to larval or non-larval phenotypes. Decreasing the external concentration of Ca(2+) with the Ca(2+) chelating agent EGTA increased proliferation of stem cells in culture, and doubled the proportion of cells differentiating to columnar and goblet cells typical of larval midgut compared to controls. In contrast, increasing inward transport of Ca(2+) into the cells by increasing the concentration of external calcium ion concentration, or by incubation with the Ca(2+) ionophore A23187 (which tends to open inward plasma membrane Ca(2+) channels), induced dose-dependent differentiation to non-midgut cell types such as squamous and scale-like cells. However, the latter treatments did not significantly alter stem cell proliferation or differentiation to normal larval midgut epithelium.     

Growth and mitogenic effects of arylphorin in vivo and in vitro

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.     

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.     

Expression of Cry1Ac cadherin receptors in insect midgut and cell lines

Cadherin-like proteins have been identified as putative receptors for the Bacillus thuringiensis Cry1A proteins in Heliothis virescens and Manduca sexta. Immunohistochemistry showed the cadherin-like proteins are present in the insect midgut apical membrane, which is the target site of Cry toxins. This subcellular localization is distinct from that of classical cadherins, which are usually present in cell-cell junctions. Immunoreactivity of the cadherin-like protein in the insect midgut was enhanced by Cry1Ac ingestion. We also generated a stable cell line Flp-InT-REX-293/Full-CAD (CAD/293) that expressed the H. virescens cadherin. As expected, the cadherin-like protein was mainly localized in the cell membrane. Interestingly, toxin treatment of CAD/293 cells caused this protein to relocalize to cell membrane subdomains. In addition, expression of H. virescens cadherin-like protein affects cell-cell contact and cell membrane integrity when the cells are exposed to activated Cry1Ab/Cry1Ac.     

Light microscope immunolocation of Bacillus thuringiensis kurstaki delta-endotoxin in the midgut and Malpighian tubules of the tobacco budworm, Heliothis virescens

Light microscope immunofluorescence was used to localize the membrane binding of Bacillus thuringiensis kurstaki 63-kDa delta-endotoxin in Heliothis virescens midgut and Malpighian tubules. Staining was observed along all exposed mucosal (apical microvillar) plasma membranes. Interpretation of the serosal (basal) plasma membrane staining was complicated because the basal lamina also stained. The results suggest that the toxin binds to all exposed plasma membranes without apparent specificity for particular membrane domains.     

Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.     

Identification and characterization of Heliothis virescens midgut membrane proteins binding Bacillus thuringiensis delta-endotoxins.

To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins.     

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.     

A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae

Binding of the Bacillus thuringiensis Cry1Ac toxin to specific receptors in the midgut brush border membrane is required for toxicity. Alteration of these receptors is the most reported mechanism of resistance. We used a proteomic approach to identify Cry1Ac binding proteins from intestinal brush border membrane (BBM) prepared from Heliothis virescens larvae. Cry1Ac binding BBM proteins were detected in 2D blots and identified using peptide mass fingerprinting (PMF) or de novo sequencing. Among other proteins, the membrane bound alkaline phosphatase (HvALP), and a novel phosphatase, were identified as Cry1Ac binding proteins. Reduction of HvALP expression levels correlated directly with resistance to Cry1Ac in the YHD2-B strain of H. virescens. To study additional proteomic alterations in resistant H. virescens larvae, we used two-dimensional differential in-gel electrophoresis (2D-DIGE) to compare three independent resistant strains with a susceptible strain. Our results validate the use of proteomic approaches to identify toxin binding proteins and proteome alterations in resistant insects.     

N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bacillus thuringiensis.

Proteins synthesized by the bacterium Bacillus thuringiensis are potent insecticides. When ingested by susceptible larvae they rapidly lyse epithelial cells lining the midgut. In vitro the toxins lyse certain insect cell lines and show saturable, high-affinity binding to brush-border membrane vesicles (BBMVs) prepared from insect midguts. We observed that the sugar N-acetyl galactosamine (GalNAc) specifically decreased the cytolytic activity of a CryIA(c) toxin towards Choristoneura fumiferana CF1 cells, completely abolished toxin binding to Manduca sexia BBMVs, partially inhibited binding to Heliothis virescens BBMVs and had no apparent effect on binding to Pieris brassicae BBMVs. In ligand blotting experiments the toxin bound proteins of 120 kDa in M. sexta, 125 kDa in P. brassicae and numerous proteins in H. zea. Toxin binding to these proteins was specifically inhibited by GalNAc. The toxin binding proteins of M. sexta and H. zea also bound the lectin soybean agglutinin. Taken together these findings suggest that N-acetyl galactosamine might be a component of a CryIA(c) toxin receptor of CF1 cells and of at least two of the insects tested.     

Stimulation of midgut stem cell proliferation by Manduca sexta alpha-arylphorin

Extracts of the green-colored perivisceral fat body of newly ecdysed Manduca sexta pupae stimulate mitosis in midgut stem cells of Heliothis virescens cultured in vitro. Using a combination of cation- and anion-exchange chromatography, we have isolated a protein from these fat body extracts that accounts for the observed stem cell proliferation. SDS-PAGE analysis of the protein results in a single band of 77 kDa. Sequences of tryptic peptides from this protein are identical to internal sequences of the storage hexamer alpha-arylphorin. The alpha-arylphorin isolated by our procedure represents a small fraction of the total arylphorin present in the fat body extract. However, it alone seems responsible for the stimulation of mitotic activity in H. virescens midgut stem cells.     

The Heliothis virescens cadherin protein expressed in Drosophila S2 cells functions as a receptor for Bacillus thuringiensis Cry1A but not Cry1Fa toxins

Genetic knockout of the BtR4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to resistance against Cry1Ac toxin from Bacillus thuringiensis. However, the functional Cry1Ac receptor role of this protein has not been established. We previously proposed HevCaLP as a shared binding site for B. thuringiensis (Bt) Cry1A and Cry1Fa toxins in the midgut epithelium of H. virescens larvae. Considering that Cry1Ac and Cry1Fa are coexpressed in second-generation transgenic cotton for enhanced control of Heliothine and Spodoptera species, our model suggests the possibility of evolution of cross resistance via alteration of HevCaLP. To test whether HevCaLP is a Cry1Ac and Cry1Fa receptor, HevCaLP was transiently expressed on the surface of Drosophila melanogaster Schneider 2 (S2) cells. Expressed HevCaLP bound [(125)I]Cry1A toxins under native (dot blot) and denaturing (ligand blot) conditions. Affinity pull-down assays demonstrated that Cry1Fa does not bind to HevCaLP expressed in S2 cells or in solubilized brush border membrane proteins. Using a fluorescence-based approach, we tested the ability of expressed HevCaLP to mediate toxicity of Cry1A and Cry1Fa toxins. Cry1A toxins killed S2 cells expressing HevCaLP, whereas Cry1Fa toxin did not. Our results demonstrate that HevCaLP is a functional Cry1A but not Cry1Fa receptor.     

Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae.

We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [125I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.     

Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.     

Histopathological Effects and Growth Reduction in a Susceptible and a Resistant Strain of Heliothis virescens (Lepidoptera: Noctuidae) Caused by Sublethal Doses of Pure Cry1A Crystal Proteins from Bacillus thuringiensis

Two strains of the tobacco budworm Heliothis virescens, one selected in the laboratory for resistance to Cry1Ac crystal protein from Bacillus thuringiensis (for which the mechanism of resistance was not associated with reduced binding) and an unselected control strain, were exposed to sublethal doses of pure Cry1A crystal proteins. A histopathological study was conducted to determine the epithelial damage caused by ingestion of Cry1Ac. Tissue sections of the midgut were obtained after 20, 40 and 60 min of toxin ingestion. Histopathological changes were observed primarily in columnar cells and were time-dependent. However, essentially the same level of damage was observed in the two strains. Toxin feeding tests with Cry1Ac and Cry1Ab, indicated that the toxins retarded growth and inhibited food intake of susceptible larvae, but did not significantly affect larvae of the resistant strain. Since the histopathological damage was similar in both strains, it appears that resistant larvae could repair (or substitute) more readily the damaged cells.