Insect Biochemistry and Molecular Biology 34 (2004) 743–752 www.elsevier.com/locate/ibmb Significance of the sulfonylurea receptor (SUR) as the target of diflubenzuron in chitin synthesis inhibition in Drosophila melanogaster and Blattella germanica Gamal E. Abo-Elghar 1 , Phillip Fujiyoshi, Fumio Matsumura  Department of Environmental Toxicology and Department of Entomology, One Shields Avenue, University of California, Davis, CA 95616, USA Received 4 February 2004; accepted 29 March 2004 Abstract Diflubenzuron (DIMILIN 1 ) is a powerful insecticidal chemical which has been known for many years to inhibit chitin synthesis in vivo in insects and related arthropod species. However, its action mechanism has remained unresolved partly because of its inaction on any of the enzymes involved in chitin synthesis in vitro. Based on our previous work (Diflubenzuron affects gamma-thioGTP stimulated Ca 2+ transport in vitro in intracellular vesicles from the integument of the newly molted American cockroach, Periplaneta americana L. Insect Biochem. Mol. Biol. 24 (1994) 1009) showing that diflubenzuron inhibits Ca 2+ uptake by vesicles obtained from the integument of American cockroach, Periplaneta americana (L.), in vitro, we tested the hypothesis that the action site of diflubenzuron is an ABC (ATP binding cassette) transporter, probably a sulfonylurea-sensitive transporter. Glibenclamide, one of the most commonly used sulfonylureas for type II diabetes treatment, was the positive control. When given to immature insects, glibenclamide clearly caused toxicity, with symptoms indicating molting abnormality comparable to diflu- benzuron. Its LD 50 (0.472 lg/nymph) was approximately 2.8 times the value obtained for diflubenzuron (0.17 lg/nymph, topical) in German cockroach, Blattella germanica (L.). However, in terms of the inhibitory activities on chitin synthesis, in isolated inte- guments glibenclamide showed an identical potency to diflubenzuron in B. germanica nymphs. A competitive binding assay with [ 3 H]-glibenclamide and unlabeled diflubenzuron clearly established that the latter is capable of competitively displacing the former radioligand. The K D values observed for vesicles prepared from fruit fly larvae, Drosophila melanogaster M., were 44.9 nM for glibenclamide and 65.0 nM for diflubenzuron, respectively. Furthermore, glibenclamide was found to affect Ca 2+ uptake by isolated cuticular vesicles from B. germanica in a manner very similar to diflubenzuron. These results support our conclusion that the sulfonylurea receptor (SUR) is the target of diflubenzuron in inhibition of chitin synthesis in these two insect species. # 2004 Elsevier Ltd. All rights reserved. Keywords: Diflubenzuron; Sulfonylurea receptor (SUR); Binding site; Glibenclamide; Chitin synthesis 1. Introduction It has been well established that diflubenzuron (DFB) inhibits chitin synthesis in vivo and that such action constitutes the main insecticidal mechanism of this compound (Marks et al., 1982; Verloop and Ferrell, 1977). However, the precise molecular mech- anism of its action has not been elucidated. The major problem in answering this question has been that so far neither DFB nor any other benzoylurea-type chemicals have shown inhibitory properties toward any of the enzymes associated with chitin synthesis when tested in vitro (Cohen, 2001). Furthermore, DFB does not inhibit chitin synthesis in yeast (Marks et al., 1982), unlike other chitin synthesis inhibitors such as polyoxin D(Endo et al., 1970), which is effective in inhibiting chitin synthesis both in insects and yeast. Clearly DFB- type chemicals act on an arthropod-specific cuticular component that is vital to chitin synthesis in vivo. Apparently this component contributes to chitin synthesis only in intact integument cells, since it has been shown by Nakagawa et al. (1993) in isolated  Corresponding author. Tel.: +1-530-752-4251; fax: +1-530-752- 3394. E-mail address: fmatsumura@ucdavis.edu (F. Matsumura). 1 Present address: Department of Pesticides, Faculty of Agri- culture, Menoufiya University, Shebin El-Kom, Menoufiya 32511, Egypt. 0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.03.009