Midgut Cell Cultures Research Articles

Anaplasma marginale Infection of Dermacentor andersoni Primary Midgut Cell Culture Is Dependent on Fucosylated Glycans.

Tick midgut is the primary infection site required by tick-borne pathogens to initiate their development for transmission. Despite the biological significance of this organ, cell cultures derived exclusively from tick midgut tissues are unavailable and protocols for generating primary midgut cell cultures have not been described. To study the mechanism of Anaplasma marginale-tick cell interactions, we successfully developed an in vitro Dermacentor andersoni primary midgut cell culture system. Midgut cells were maintained for up to 120 days. We demonstrated the infection of in vitro midgut cells by using an A. marginale omp10::himar1 mutant with continued replication for up to 10 days post-infection. Anaplasma marginale infection of midgut cells regulated the differential expression of tick α-(1,3)-fucosyltransferases A1 and A2. Silencing of α-(1,3)-fucosyltransferase A2 in uninfected midgut cells reduced the display of fucosylated glycans and significantly lowered the susceptibility of midgut cells to A. marginale infection, suggesting that the pathogen utilized core α-(1,3)-fucose of N-glycans to infect tick midgut cells. This is the first report using in vitro primary D. andersoni midgut cells to study A. marginale-tick cell interactions at the molecular level. The primary midgut cell culture system will further facilitate the investigation of tick-pathogen interactions, leading to the development of novel intervention strategies for tick-borne diseases.

Distribution of Glycan Motifs at the Surface of Midgut Cells in the Cotton Leafworm (Spodoptera littoralis) Demonstrated by Lectin Binding.

Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA), GalNAc/Gal (RSA and SSA), GlcNAc (WGA and Nictaba) and Neu5Ac(α-2,6)Gal/GalNAc (SNA-I)], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and Nictaba bound more strongly to the membrane of these undifferentiated cells compared to the microvillar pole of the columnar cells, while SSA, HHA, WGA, and SNA-I showed stronger binding to the microvilli. Our results indicated that polarization of the midgut cells is also reflected by a specific distribution of glycans, especially between the basal and microvillar pole. The data are discussed in relation to the functioning and development of the insect midgut.

Alpha-arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication.

Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we describe the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. The Cry1Ac-induced secretome caused higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified four proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin subunit alpha protein not detected in the control secretome. Feeding of purified alpha-arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify alpha-arylphorin as a protein with a new putative role in the midgut regeneration process in response to Cry1Ac intoxication and possibly pathogen/abiotic stress, identifying alpha-arylphorin as a potential gene to target with insecticidal gene silencing for pest control.

Larval Mid-Gut Responses to Sub-Lethal Dose of Cry Toxin in Lepidopteran Pest Achaea janata.

The lack of homogeneity in field application of Bacillus thuringiensis formulation often results in ingestion of sub-lethal doses of the biopesticide by a fraction of pest population and there by promotes the toxin tolerance and resistance in long term. Gut regeneration seems to be one of the possible mechanism by which this is accomplished. However, the existing information is primarily derived from in vitro studies using mid-gut cell cultures. Present study illustrates cellular and molecular changes in mid-gut epithelium of a Bt-susceptible polyphagous insect pest castor semilooper, Achaea janata in response to a Cry toxin formulation. The present report showed that prolonged exposure to sub-lethal doses of Cry toxin formulation has deleterious effect on larval growth and development. Histological analysis of mid-gut tissue exhibits epithelial cell degeneration, which is due to necrotic form of cell death followed by regeneration through enhanced proliferation of mid-gut stem cells. Cell death is demonstrated by confocal microscopy, flow-cytometry, and DNA fragmentation analysis. Cell proliferation in control vs. toxin-exposed larvae is evaluated by bromodeoxyuridine (BrdU) labeling and toluidine blue staining. Intriguingly, in situ mRNA analysis detected the presence of arylphorin transcripts in larval mid-gut epithelial cells. Quantitative PCR analysis further demonstrates altered expression of arylphorin gene in toxin-exposed larvae when compared with the control. The coincidence of enhanced mid-gut cell proliferation coincides with the elevated arylphorin expression upon Cry intoxication suggests that it might play a role in the regeneration of mid-gut epithelial cells.

Saponins show high entomotoxicity by cell membrane permeation in Lepidoptera

In this study, the effects of three saponins and one sapogenin with a triterpenoid or steroid structure in two lepidopteran insect cell lines, ovarian Bm5 and midgut CF-203 cells, were analysed with regard to cell viability, cell membrane permeation, EcR responsiveness and DNA fragmentation. In addition, the entomotoxic action of Q. saponaria saponin with primary midgut cell cultures and larval stages of the cotton leafworm Spodoptera littoralis was tested. Both lepidopteran cell lines show a high sensitivity to all four sapo(ge)nins, with a concentration-dependent viability loss and EC₅₀ values of 25-100 µM in MTT bioassays. A trypan blue assay with Q. saponaria saponin confirmed rapid cell membrane permeation to be a cause of cytotoxicity. Saponins caused no EcR activation in Bm5 cells, but a loss of ecdysteroid signalling was observed with IC₅₀ values of 5-10 µM. Lower saponin concentrations induced DNA fragmentation, confirming their potential to induce apoptosis. Finally, Q. saponaria saponin caused cytotoxicity in primary midgut cell cultures of S. littoralis (EC(50) = 4.7 µM) and killed 70-84% of S. littoralis larvae at pupation at 30-70 mg g(-1) , while lower concentrations retarded larval weight gain and development. The data obtained provide evidence that saponins exert a strong activity on lepidopteran cells, presumably based on a cytotoxic action due to permeation of the cell membrane. Primary midgut cell cultures and larvae of S. littoralis showed high sensitivity to Q. saponaria saponin, indicating the insect midgut as a primary target for entomotoxicity and the potential use of saponins in the control of pest Lepidoptera.

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.

Penetration of polyhedrosis viruses into the cultured midgut cells of the silkworm, Bombyx moli

Penetration of polyhedrosis viruses into the cultured midgut cells of the silkworm, Bombyx moli

PROTEASES DETECTION OF IN VITRO CULTURE OF MIDGUT CELLS FROM HYALOMMA ANATOLICUM ANATOLICUM (ACARI: IXODIDAE)

Proteases play a key role in protein digestion in ticks and other haematophagous insects. Our understanding of blood meal digestion in digestive system of ticks can be very useful for better understanding of basic rules for control of ticks. Cells of the midgut endocytose blood components. Blood proteins uptake by midgut cells, suggesting the presence of proteases in the midgut cells. In this study, proteases which may be present in midgut cells of engorged female ticks (Hyalomma anatolicum anatolicum) have been studied. The midgut tissue from tick was dissociated to cells. The cells placed in a culture plate with special medium. Then cell extract was obtained from the in vitro cultured midgut cells. Then cells were rinsed in hypotonic solution. The cellular suspension was centrifuged and supernatant removed from cell membrane plette. Cell membrane proteins were isolated by Solution 2% of Triton X-100. Proteases assay performed by specific substrates for cytoplasmic proteins and Cell membrane bound proteins. Then, enzyme like trypsin, enzyme like chymotrypsin, carboxypeptidase B, cathepsin C, cathepsin B and cathepsin D were detected. Our results show that enzyme like trypsin is a membrane bound protein, but carboxypeptidase B, cathepsin C, cathepsin B and cathepsin D are included in the cytoplasm of mid-gut cells. There is no enzyme like chymotrypsin in mid-gut cells of tick.

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.

Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells in vitro

Fluorescent brighteners significantly lower the LC50 and LT50 in a variety of nucleopolyhedrovirus–insect host systems. In larvae of the gypsy moth, Lymantria dispar (L.), a European NPV strain of virus (LdMNPV) does not normally replicate in the midgut, but addition of a fluorescent brightener (Calcofluor M2R) to the virus suspension results in productive infections. In the current study, we show that LdMNPV also does not replicate in a larval midgut primary cell culture system unless a fluorescent brightener (Blankophor P167) is added. Morphological and cellular changes characteristic of apoptotic cell death were noted in infected midgut cells in vitro. We used the TUNEL assay to measure apoptosis in virus-challenged midgut cell cultures at 24–48 h post-inoculation. A significant decrease in apoptotic midgut cells was noted in the presence of 0.01 M brightener. The inhibition of apoptosis and presumptive inhibition of shedding of infected midgut cells in the presence of fluorescent brightener in the insect midgut appeared to promote virus replication and are likely to be partly responsible for enhancement of LdMNPV activity that is observed in gypsy moth larvae.

Role of integrin β1‐like protein in proliferation and differentiation of cultured stem cells from midgut of Heliothis virescens

Cultured midgut cells from Heliothis virescens larvae were incubated with anti-human integrin beta1 made in rabbit and then passed over a column of magnetic beads bound to anti-rabbit IgG (MACS, Miltenyi Bergisch Gladbach, Germany). Cells bound to integrin beta1 antibody also bound to the anti-rabbit IgG on the magnetic beads (MACS) and were retained in the column while it remained in the magnetic field. Non-bound cells were eluted at this time. They did not stain with anti-integrin antibody just after elution. Removing the column from the magnetic field allowed cells bound to the beads-integrin beta1 antibody to be eluted. All of these cells stained with human anti-integrin beta1 upon elution. Each cell fraction was cultured in medium for 3 days. During this time, the populations of cells tended to return to heterogeneous staining patterns characteristic of control populations. However, cells that did not stain immediately with anti-integrin beta1 antibody exhibited double the rate of multiplication and 8 times more differentiation than the integrin-antibody positive cells that eluted later, as well as the non-treated control cells. In a second experiment, midgut cells were incubated for 4 days with various titers of human anti-integrin beta1 to block surface integrin beta1-like reactive sites. Stem cells blocked with anti-integrin beta1 antibody during incubation exhibited double the rate of differentiation than non-treated control cells and those showing anti-integrin beta1-positive stain upon elution.

BOMBYXIN STIMULATES PROLIFERATION OF CULTURED STEM CELLS DERIVED FROM HELIOTHIS VIRESCENS AND MAMESTRA BRASSICAE LARVAE1

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.

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

Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin beta1, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of cultured Heliothis virescens midgut cells; clusters of immunostained integrin beta1-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 containing Manduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells to Bacillus 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.

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.

Apoptosis in cultured midgut cells from heliothis virescens larvae exposed to various conditions.

We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H. virescens midgut cells, and to toxin from Bacillus thuringiensis. A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium. Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B. thuringiensis toxin. Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures. Approximately 1% of goblet, stem, and differentiating cells were apoptotic. However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium. B. thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods. Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei. Stem and differentiating cells normally replace dying mature cells in the midgut. Thus, exposure of cultures of H. virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.

Cultured midgut cells of Heliothis virescens (Lepidoptera): fibronectin and integrin β1, immunoreactivity during differentiation in vitro

Summary The midgut epithelium of larval Lepidoptera consists of a monolayer of mature columnar and goblet cells in vivo with loosely bound stem cells at its base. Cultures of Heliothis virescens midgut contained quiescent and dividing stem cells, differentiating and mature cells as well as small rows of attached cells, randomly distributed in the culture vessel. Fixed cultures were immunostained with polyclonal antibodies to fibronectin and integrin β1. Many stem cells stained darkly; lighter staining stem cells imply that some stem cells can become less adhesive. Developing cells were pale, lacking integrin and fibronectin epitopes on their surfaces, and were probably poorly adhesive. During a molt in vivo, differentiating stem cells insert between existing epithelial cells, increasing gut size; lack of stickiness would enable them to do so. As pre-columnar cells differentiated in vitro, stainability reappeared. Mature columnar cells were decorated with a pattern of intense surface immunostaining material. Small immature columnar and goblet cells adhered to the distal ends of mature columnar cells by their proximal areas near a fibronectin positive area. Goblet cells, seldom found together in vivo or in vitro, lacked prominent fibronectin or integrin-like surface proteins. Cells were joined to each other in the same orientation and cell pattern observed in intact midguts. The pattern was achieved in vitro by cell-cell adhesion without basal membrane or existing midgut as guides.

Insect midgut epithelium in vitro: an insect stem cell system

Mixed cell cultures and stem cell cultures were prepared from midguts of Manduca sexta pharate fourth instar and mid-wandering fifth instar larvae. An extract prepared from the fat body was able to promote stem cell proliferation and affect differentiation in a dose-dependent manner. DNA synthesis activity was confirmed by use of [ 3H]thymidine. Immunohistological localization of bromodeoxyuridine (BrdU), a thymidine analog, indicated that dividing stem cells incorporated the label. In many cases, one of the daughter cells incorporated the label while the other did not; often this daughter appeared morphologically different from its sister cell. These results implied that one of the sister stem cells remained as a proliferating stem cell while the other sister was committed to differentiate. Studies strongly suggest that these midgut cell cultures comprise a true stem cell system. Cell-free conditioned medium from cultures of differentiating pharate fourth instar midgut cells induced development of larval columnar cells from mid-wandering fifth instar midgut stem cells. Conversely, conditioned medium from differentiating cultures of mid-wandering fifth instar midgut induced development of mid-wandering fifth instar low columnar cells from midgut stem cells isolated from pharate fourth instar larvae. Therefore, it appears that differentiating cells produce soluble cytokines which direct specific modes of differentiation by M. sexta stem cells.

In vitro differentiation of isolated stem cells from the midgut of Manduca sexta larvae

Isolated spherical stem cells from midguts of pharate fourth-instar larvae of Manduca sexta proliferated in vitro in the presence of 1 ng ml-1 20-hydroxyecdysone and co-cultured fat body tissue or cell-free fat body extract from M. sexta, Lymantria dispar or Heliothis virescens. In this environment, the stem cells were able to undergo mitosis and increase in number. However, stem cells were only able to differentiate to mature goblet and columnar cells when cell-free conditioned medium, taken from midgut cell cultures containing mature cells as well as stem cells and differentiating forms, was introduced into the culture medium. The presence of early and mature goblet cells, lying randomly on their sides, suggested that cell polarity developed in vitro as an intrinsic property of individual cells rather than with reference to an external inductive material. The differentiation factor (or factors) from the conditioned medium appears to include a heat-stable, peptide-like molecule of 10 kDa or less.

Binding and activity of Bacillus thuringiensis delta-endotoxin to invertebrate cells.

Fluorescein isothiocyanate was used as a label to detect delta-endotoxin of Bacillus thuringiensis subsp. thuringiensis and israelensis in binding studies with different in vitro cell systems. Protoxin of the subspecies thuringiensis could be labelled directly whereas the activated toxin had to be traced indirectly with labelled antibodies. Both protoxin and activated toxin bound to primary midgut cell cultures of Pieris brassicae larvae as well as to cells of an established culture of Drosophila melanogaster. No binding with either toxin form could be observed with hemocytes of P. brassicae. Biological activity as shown by the trypan blue viability assay was obtained only with the activated toxin against the midgut cells. Toxin of the subspecies israelensis reacted very unspecifically. Binding followed by rapid destruction was obtained with all the tested cultures.