Treatment of last-instar larvae of multi-resistant cotton leafworm Spodoptera littoralis with four dibenzoylhydrazines, methoxyfenozide (RH-2485), tebufenozide (RH-5992), halofenozide (RH-0345), and RH-5849, resulted in premature molting leading to death. Methoxyfenozide was the most toxic followed by tebufenozide, halofenozide, and RH-5849. To explain differences in toxicity, especially between multi-resistant and laboratory strains, absorption in the body tissues and oxidative metabolism were tested with 14C-labeled ecdysone agonist and a Lineweaver-Burk assay, respectively. Then to address different compound potencies in multi-resistant strains, the potency of the four ecdysone agonists was measured based on their ability to mimic the natural insect molting hormone, 20-hydroxyecdysone (20E) by inducing evagination in isolated imaginal wing discs. Using monoclonal antibody 9B9, the presence of ecdysteroid receptors in imaginal discs in vitro was confirmed. In parallel, Scatchard plot analysis with whole imaginal wing discs cultured with different concentrations of 3H-labeled ponasterone A indicated no significant difference in affinity and in number of target sites for binding between multi-resistant and susceptible laboratory strains. The four compounds tested caused the effect as agonists of 20E in vitro, and typically the order of their toxicities (LC50s) corresponded with that for evagination-induction with whole imaginal discs.

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The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.

In a first series of experiments, the biological response of a continuous cell line of the beet armyworm, Spodoptera exigua, was tested with different groups of insecticides with different modes of actions: acetylcholinesterase inhibitors, acetycholine receptor agonists, inhibitors and uncouplers of oxidative phosphorylation, site I electron transport inhibitors, gamma-aminobutyric acid receptor inhibitors, chitin synthesis inhibitors, and juvenile hormone analogues. From the concentration response curves, 50% inhibition concentration (IC(50)) values were calculated. The most active compound in vitro was pyridaben with an IC(50) value of 0.0083 ppm. In a second series of experiments, the toxicity of these insecticide groups was determined on third-instar larvae of S. exigua, and lethal concentration with 50% kill (LC(50)) values were used in the evaluation of their in vivo biological activity. Toxicity bioassays showed that lufenuron was the most toxic (LC(50) = 0.098 ppm). To explain the discrepancies in biological responses in vitro with insect cells compared with in vivo conditions with whole third-instar larvae, the significance of different detoxifying enzyme systems was tested. P(450) monooxygenases, esterases, and glutathione S-transferases were measured in third-instar larvae and cells of S. exigua. Data are discussed in terms of the usefulness of insect cell cultures as tools in the screening for novel insecticide actions.