The insect brain peptides that induce target tissues to secrete steroid hormones, the ecdysones, are quite different from each other in peptide sequences and structures. Therefore, it is probable that related differences in specific receptors for the peptides exist on target organs, allowing for specific patterns of ecdysteroid synthesis by those tissues coordinated with regulation of metamorphosis. We identified and characterized an ecdysiotropic peptide from brains of Lymantria dispar that induced sheaths of testes to secrete ecdysteroid in vitro. The ecdysteroid in turn promoted synthesis of at least one peptide by fat body and/or testis sheaths that promoted sperm maturation and male gonadal imaginal disc development in vitro. We have cultured midgut stem cells and identified two peptidic factors controlling stem cell multiplication: the fast-acting brain peptide Bombyxin and the slower-acting fat body peptide, alpha-arylphorin. It is not known how these factors enter midgut cells, how they are processed, or how they incite stem cells to divide. We have characterized 4 differentiation-promoting growth factors, the MDFs, from culture medium and hydrolyzed hemolymph. They direct differentiation to normal and abnormal midgut cell fates. Internal [Ca2+] may be a second messenger for MDFs. Antibodies to the MDFs have revealed MDFs 1-4 in columnar cells and MDF4 in secretory cells as well. MDF2 appeared in the gut lumen, and MDF 4 is released from midgut secretory cells to both gut lumen and hemolymph.

Midgut stem cells of last instar larvae and pupae of Heliothis virescens, Lymantria dispar and several other Lepidopteran species have been cultured in vitro and have been induced to proliferate using low titers of ecdysteroids and the 77-Kda peptide fragment, alpha-arylphorin, isolated and identified from pupal fat body tissue. The insulin-related hormone, Bombyxin, also induced mitosis in cultured midgut stem cells; it appeared to be fast-acting and quickly inactivated, while alpha-arylphorin was slower to act and had a longer lasting effect in vitro, indicating different functions for these proliferation agents. Changes in Calcium ion concentration within or outside the cells discretely affected stem cell differentiation, indicating a role for second messenger participation in peptide regulation of this process. Four different peptides (MDFs 1-4) that induced midgut stem cells to differentiate to mature midgut cell types in vitro were isolated and characterized from conditioned media and hemolymph of H. virescens and L. dispar. However, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and all-trans retinoic acid (RA) from vertebrate sources induced differentiation to non-midgut cell types as well. MDF1 was located in basal areas of columnar cells of midgut epithelium, although MDF2 was observed in all of the cytoplasm of columnar cells and in droplets of antibody positive material in the midgut lumen, suggesting a digestive function as well for this peptide. Anti-MDF-3 stained the central areas of cultured midgut columnar cells and the bases of columnar cells of midgut epithelium in vivo. Midgut secretory cells stained with anti-MDF-4; streams of MFD-4-positive material were observed extending from secretory cells facing the epithelial lumen, and as a layer on the hemolymph-facing side, suggesting an endocrine or paracrine function for this or an immunologically similar peptide.

This protocol describes the preparation of primary cell cultures from Lepidopteran midgut. These cultures have been used to identify factors that control midgut growth and differentiation, cell responses to these factors, effects of toxins on midgut growth, and the regulation of cell physiology. The protocol is divided into (1) procedures for cell collection, (2) composition of the culture, and (3) assay methods used for cell health, proliferation, and differentiation. Collection and setup require 4–6 h. Once established, a culture can survive several months at 25°C, be kept a year or longer at 4°C, or be frozen for indefinite storage.

There is considerable effort in the area of proteomics to map and catalog the unique proteomic profile of each organelle. Subcellular fractionation enables the simplification of complex protein mixtures and thereby facilitates this type of proteomic analysis. Pierce developed three organelle enrichment kits for lysosomes, peroxisomes, and nuclei that enable enrichment of intact organelles from cells and/or tissue. Mechanical lysis was employed to effectively lyse cells and tissue, and density gradient centrifugation was used as a method to separate each organelle of interest. The procedure was optimized for diverse cell lines, including A431, HeLa, and HepG2 as well as multiple tissues from Sprague-Dawley rats including liver, heart, and kidney. The purity of the isolated organelles was assessed by Western blot analysis using antibodies against specific marker proteins on targeted organelles. Benchmarking was performed against commercially available kits and a higher yield with significantly less contamination was demonstrated with each of the Pierce protocols. Isolated organelles may be used for a number of downstream applications, including 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction, and interaction or localization studies.

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.

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.

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.

A bstract : Stem cells derived from midguts of the caterpillar, Spodoptera littoralis , can be induced to multiply and differentiate in vitro . Ecdysone (E) and 20-hydroxyecdysone (20E) had a concentration-dependent effect: E was more active in cell proliferation and 20E in differentiation. Ecdysteroid receptors in midgut stem cell nuclei were stained with the antibody 9B9. In addition, α-arylphorin and four midgut differentiation factors (MDF) specifically stimulated proliferation and differentiation of stem cells, respectively. The activity of a panel of peptide growth factors and hormones on growth and metamorphosis of the insect midgut is discussed.

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentraton-dependent manner. This effect was compared with those of insect molting hormones, ecdystone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.

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.

Dear Editor: In insects, growth and metamorphosis are governed by ecdysteroids and juvenile hormones, growth factors, and cytokines. When these compounds bind to their receptor s, the resulting gene activation aiad transcription may induce a number of processes such as cell division, tissue repair, chemotaxis, differentiation, or even cell death. Oberlander and Fulco (1967) showed that ecdysteroids stimulated metamorphosis of cultured Galleria mellonella wing imaginal disks. More recently, 20-hydroxyecdysone (20E) has been shown to induce development of imaginal wing disks of various Lepidoptera in vitro (Smagghe et al., 1996, 1999). Imaginal wing disks are masses of cells that give rise to adult wings during metamorphosis in holometabolous insects, triggered by an increase in ecdysteroids. During this process, epithelial cells show numerous mitoses, Golgi bodies, and well-developed endoplasmic reticulum appear, and a new cuticular layer is deposited (Riddiford, 1985). A few yr later, Dutkowski and Oberlander (1974) reported on interactions between ecdysteroids and the fat body during wing disk development. A lowmolecular weight nonpeptidic factor from the fat body (Oberlander and Tomblin, 1972) or a low-molecular weight, heat-stable, peptidic factor isolated from the fat body tissue (Benson et al., 1974), potentiated the action of 20E in inducing tracheation and elongation of isolated wing disks. In continuation of these results, Loeb and Hakim (1991) and Loeb (1994) reported that an extract of fat body (FBX) from the abdomen of newly pupated Manduca sexta promoted the growth and development of the genital tract (Loeb and Hakim, 1991) and mitosis in cultured midgut cells (Sadrud-din et al., 1994). In the present study, we have tested the effects of FBX on imaginal disk development in vitro and toxicity against intact caterpillars of the cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), an economically important pest of cotton, vegetables, and ornamentals. Pesticide-resistant populations of this insect cause severe problems in numerous countries. Previously, we showed that 20E induces evagination in cultured wing disks after 3 d (Smagghe et al., 2000). All developmental stages of a continuous colony of S. littoralis were kept at standard conditions of 23 -+ 2 ~ C, 60 + 5% r.h. and a 16:8-h LD (light:dark) cycle. Larvae were raised on an artificial diet, and adults provided with 15% honey water as food, as described in Smagghe et al. (2000). FBX was obtained as an aqueous extract from the fat body of pupal abdomens of M. sexta as described in Loeb and Hakim (1991), and kept frozen at 2 0 ~ C until application. To evaluate the action of FBX, we used cultured imaginal wing disks that were incubated with 25 txl/ml FBX alone or in combination with different concentrations of 20E (from 10 p~M to 1 nM; Sigma Chemical Co., Bornem, Belgium), and compared with cultures with 20E alone. Individual wing disks were dissected from 3d-old (0-1 h) last-instar larvae in which the natural peak of ecdysteroids has not yet occurred. Before dissection, larvae were wateranesthetized and surface-sterilized in 70% ethanol for 15 min. The imaginal disks were cultured in modified Grace's insect tissue culture medium with heat-inactivated fetal calf serum (9%, Sigma), 30% bovine serum albumin solution (3%, Sigma), and gentamycine sulfate (50 ~g/ml medium, Sigma) at 25 -+ 1 ~ C and 97 -+ 2% r.h., to prevent evaporation (Smagghe et al., 2000). Ten disks were kept in 1-ml culture medium in a 35 • 10-mm plastic tissue culture plate (Falcon 3001; Becton Dickinson and Co., Belgium). After 72 h, wing disks were inspected for treatment-induced evagination. Completion of the first phase of evagination in which epidermal folds are developed inside the epidermal sac was regarded as the minimum requirement for a positive result (Chihara et al., 1972; Mandaron, 1976). Culturing imaginal wing disks with 20E at 10 txM induced evagination and new cuticle deposition in the disks (Fig. 1). Imaginal disks cultured with 25 ~l/ml FBX alone showed no signs of evagination. Figure 2 shows that in all cases of combination with different concentrations of 20E, FBX at 25 M/ml clearly promoted the effect of 20E on the development in imaginal wing disks. With 20E at 1 ~M alone, 58% of the cultured disks showed evagination, while this percentage was 100% with the addition of FBX. About 50% of evagination was already scored in the case of 20E at 0.01 ~M, in combination with 25 txl/ml FBX. This represents an enhancing activity of FBX that reaches nearly 100-fold. Our observation that FBX potentiated the stimulation of disk evagination as well as new cuticle deposition by 20E in S. littoralis imaginal disks clearly indicates an interaction between some component of fat body and ecdysone for the development of the wings disks. Similarly, Oberlander and Tomblin (1972) reported extensive cuticle deposition in disks of Plodia when cultured in medium containing 20E and fat body. In addition, they observed inhibition of cuticle deposition in disks with juvenile hormone in the culture medium containing 2 ~g/ml 20E. As such, the FBX may supply an additional factor promoting disk development, and/or it may have modified the 20E molecules to a more active form, and/or it may potentiate the complex of 20E, ecdysteroid receptor (EcR), and ecdysteroid response element (EcRE). Howevel, further studies are necessary to draw firm conclusions. Nothing was known, until the present study, of the physiological response of intact lepidopteran larvae to FBX factors from pupae. We assayed FBX from M. sexta pupae on intact, newly molted (012 h), last-instar (6th) larvae from a laboratory colony ofS. littoralis. Larvae were treated orally with 75 ~l of aqueous FBX by covering the artificial diet in 5 • 5-well tissue culture plates (Castor, Belgium) uniformly, in a manner similar to Smagghe et al. (2000). Concentrations of FBX were prepared in distilled water, and at least 25 larvae were tested per concentration. Mortality counts were made 7

The Manduca sexta (L.) [Lepidoptera: Sphingidae] and Heliothis 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.