Herbert Oberlander

Hormonal control of growth and differentiation of insect tissues cultured in vitro.

SummaryThis paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0µg/ml); (b) continuous treatment with 0.2µg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0µg/mlβ-ecdysone in medium conditioned with larval fat body.Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs.The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated.

Mode of action of insect growth regulators in lepidopteran tissue culture

Insect growth regulators (IGRs) have been proposed as agents for the control of insect pests. These compounds disrupt the normal development of insects by mimicking juvenile hormone and the molting hormone, 20-hydroxyecdsyone, or by interfering with chitin synthesis. The effectiveness and selectivity of IGRs provide new tools for integrated pest management. The simultaneous advances in the chemistry of IGRs and the ability to study insect tissues in culture, have led to research on the mode of action of IGRs in vitro. Plodia interpunctella and Spodoptera frugiperda have been used to examine the effects of IGRs on wing imaginal discs in organ culture, as well as in hormonally responsive cell lines established from wing imaginal discs of these species. Our research has focused on the action of ecdysteroid mimics, chitin synthesis inhibitors and juvenile hormone mimics. The effects of the IGRs on chitin synthesis, uptake of amino-sugars, and cellular proliferation were studied in tissue culture. The results demonstrate the effectiveness of using organ cultures and hormonally responsive cell lines for investigating IGRs at the cellular and tissue level.

Inhibition of cuticle production in imaginal discs of Plodia interpunctella (cultured in vitro): Effects of colcemid and vinblastine

Abstract The relationship of the microtubular system to the production of cuticle was evaluated by culturing wing imaginal discs from Plodia interpunctella in medium that contained 20-hydroxyecdysone and either colcemid or vinblastine. Examination of the treated tissue with both a dissection stereomicroscope and an electron microscope showed that the microtubule inhibitors prevented the formation of cuticle. The inhibitors also prevented the synthesis of chitin, but did not reduce protein synthesis. These results support the hypothesis that the secretion of cuticular components by insect cells requires the integrity of the microtubular system.

Imaginal Discs and Tissue Cultures as Targets for Insecticide Action

It is a significant challenge to selectively control insect pests that are of agricultural, medical or veterinary importance, while at the same time protecting the environment. In recent decades, insect resistance, concerns for the environmental, and regulatory actions have reduced the availability and/or the effectiveness of classical insecticides. The discovery of insect growth regulators (IGRs), which act on the physiology and development of insects, has provided some new opportunities to develop selective, environmentally acceptable pesticides. Research on IGRs requires bioassays that reflect the selectivity and specific mode of action desired to control pest insects while, at the same time, minimizing any effects on non-target organisms. Thus, it is important to evaluate novel bioassays that show promise for determining the selectivity and mode of action of candidate insecticides. In this chapter, we focus primarily on the use of imaginal discs, in vivo and in vitro, for investigating the action of IGRs.

Fatty acyl pheromone precursors in the sex pheromone gland of female tobacco hornworm moths, Manduca sexta (L.)

The total fatty acyl composition, including free fatty acids and fatty acyl moieties in lipids, of sex pheromone glands and selected other body tissues of Manduca sextra females was determined by capillary gas chromatographic and mass spectroscopic analysis. Fatty acyl analogs of eleven pheromone aldehydes were found in the epidermal cells of the pheromone gland. While the common fatty acyl groups, hexadecanoate, (Z)-9-hexadecenoate, octadecanoate, (Z)-9-octadecenoate, (Z,Z)-9,12-octadecadienoate and (Z,Z,Z)-9,12,15-octadecatrienoate, were detected in other body tissues of females as well as in the epidermal cells and cuticle of the pheromone gland, uncommon fatty acid pheromone analogs were not found in significant amounts in tissues other than the epidermal cells of the pheromone gland. Quantitative analysis of extracts revealed that the fatty acyl analogs of pheromone aldehydes were present continuously in pheromone glands of females from about 3 or 4 days prior to eclosion from the pupal stage until the adults were 8 days old. While injection of pheromone biosynthesis activating neuropeptide into the abdomen of the females at any age, even in the late pupal stage, resulted in pheromone production, the quantities of the bound fatty acyl components in the pheromone gland were not measurably changed.

Induction of vesicle formation in a cell line derived from imaginal discs

SummaryThe ability of insect hemolymph to induce vesicles in a high passage insect cell line, IAL-TND1, is described. The factor responsible, designated VPA for ‘vesicle-promoting activity’, was determined to be heat sensitive, nondialyzable, and protease Type XIV sensitive but insensitive to trypsin digestion. In efforts to determine the source of VPA, hemolymph was collected from different developmental stages ofTrichoplusia ni, and certain tissues fromT. ni were cocultured with IAL-TND1 cells. Hemolymph from every developmental stage tested exhibited VPA although the effect was somewhat reduced in spinning-stage larvae. Additionally, several tissue, including fat body, tess, and imaginal discs, released VPA into the culture medium. Neural tissues and endocrine glands did not induce vesicle formation.

Morphogenesis in Insect Tissue Culture

Publisher Summary The widespread utilization of organ cultures in insects has demonstrated the advantages of the special properties of imaginal discs for the study of morphogenesis. Investigations of imaginal discs from both the Diptera and Lepidoptera show that these primordia are ideally suited for successful maintenance in vitro that retain their ability to respond to hormones during the culture period and that they can form virtually the entire array of pupal and adult structures under appropriate culture conditions. Certainly, imaginal discs cultured in vitro will continue to be important in the study of ecdysteroid-induced morphogenesis. Establishment of a cell line derived from imaginal discs provides an opportunity for examining the morphological and biochemical effects of hormones on cell lines originating from a specific tissue rather than embryos. It would be instructive to determine the localization of ecdysteroid receptors in the imaginal disc cell lines, in view of the difference in cellular localization reported for such receptors in intact imaginal discs versus embryonic cell lines.

PROTEIN SYNTHESIS IN IMAGINAL DISKS OF PLODIA INTERPUNCTELLA DURING DEVELOPMENT IN VIVO AND IN VITRO

SummaryWing imaginal disks were dissected from larvae ofPlodia interpunctella (Hübner) at various stages during the larval-pupal transformation. The wing-disk proteins separated by electrophoresis and scanned with a densitometer changed quantitatively but not qualitatively during development in vivo. Treatment of wing disks in vitro with β-ecdysone resulted in a 2-fold increase in synthesis of proteins after only 2 hr incubation. The maximum rate of protein synthesis was reached 16 hr after treatment with hormone. The pattern of proteins separated by electrophoresis of wing disks that were incubated in vitro with β-ecdysone did not change qualitatively. The major features of protein synthesis during wing-disk development in vivo were similar to those observed during β-ecdysone-induced development in vitro.

Uptake and metabolism of ecdysone and 20-hydroxyecdysone in a lepidopteran cell line derived from wing discs of Plodia interpunctella

Abstract The metabolism of ecdysone (E) and 20-hydroxyecdysone (20E) was studied in an ecdysteroid-sensitive cell line derived from imaginal wing discs of Plodia interpunctella (IAL-PID2). Kinetic studies of ecdysteroid uptake showed that E and 20E accumulated in the cells during a limited period of time, i.e. between 2 and 24 h after hormone addition in the medium. 20E was slightly converted, mostly into 20,26-dihydroxyecdysone (20,26E) which accumulated during the first 24 h within the cells. E was rapidly hydroxylated in 20E. At 10 −7 M, all these ecydsteroids exhibited a low stimulatory effect on uptake of GlcNAc by IAL-PID2 cells. At higher concentrations (10 −6 M and 10 −5 M), activities of E and 20E increased whereas that of 20,26E remained low. 20E was 3 times more potent than E in the assay, but activity of E could not be only due to its conversion to 20E.