Insect Biochemistry and Molecular Biology 30 (2000) 559–568 www.elsevier.com/locate/ibmb Inducible P450s of the CYP9 family from larval Manduca sexta midgut Jennitte L. Stevens 1 , Mark J. Snyder 2 , Josette F. Koener, Rene ´ Feyereisen * Department of Entomology, Forbes 410, PO Box 210036, The University of Arizona, Tucson, AZ 85721, USA Received 20 August 1999; received in revised form 18 January 2000; accepted 20 January 2000 Abstract Several related cytochrome P450 cDNAs belonging to the CYP9 family have been cloned from the midgut of larval tobacco hornworms, Manduca sexta. The first P450, CYP9A2, was obtained by RT-PCR using degenerate primers. Northern blot analysis of expression in the midgut using the CYP9A2 probe revealed a significant induction by a variety of chemicals. Diets supplemented with the wild tomato compound 2-undecanone caused a dose-dependent induction which peaked after 48 h. Induction was also observed after addition to the diet of indole-3-carbinol, phenobarbital, 2-tridecanone and xanthotoxin. Neither α-pinene, clofibrate nor nicotine were effective inducers. The CYP9A2 probe hybridized to two mRNA species, one of 2.0 kb and another of 4.2 kb, suggesting cross-hybridization to other P450 mRNAs. Additional P450 clones of the CYP9 family were then obtained and sequenced. Northern hybridization revealed that the 4.2 kb band also hybridized to CYP9A4 whereas the 2.0 kb hybridized to CYP9A5. Despite being 91% identical, CYP9A4 and CYP9A5 were induced differentially by clofibrate and xanthotoxin. Multiple P450 genes from various families are therefore induced in Lepidoptera in response to plant allelochemicals or xenobiotics.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Cytochrome P450; 2-Undecanone; Xanthotoxin; Clofibrate; Xenobiotics; Manduca sexta 1. Introduction Cytochrome P450 monooxygenases are a diverse and widely distributed protein superfamily resulting from numerous gene duplication events from a common ancestor to all present day P450 forms that existed prior to the evolution of eukaryotes (Nelson and Strobel, 1987). Many functions are attributed to cytochrome P450s including the metabolism of a wide variety of both endogenous substrates and xenobiotics. In insects, cytochrome P450s metabolize hormones and phero- mones but are best known for their roles in the metab- olism of insecticides and host plant chemicals (review in Feyereisen, 1999). * Corresponding author. Tel.: + 1-520-621-9598; fax: + 1-520-621- 1150. E-mail address: rfeyer@ag.arizona.edu (R. Feyereisen). 1 Present address: Molecular Biology Institute, University of Cali- fornia at Los Angeles, Los Angeles, CA 90095-1570, USA. 2 Present address: Bodega Marine Laboratory, University of Cali- fornia, Davis, PO Box 247, Bodega Bay, CA 94923, USA. 0965-1748/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII:S0965-1748(00)00024-2 Herbivorous animals encounter a wide variety of sec- ondary products in the plants on which they feed. They must therefore have developed mechanisms to meta- bolically inactivate some of the potentially toxic plant chemicals that they ingest. It has been proposed that multiple duplication of P450 genes in animals represents an adaptation in this “animal–plant warfare” (Gonzalez and Nebert, 1990). Because the majority of phytopha- gous insects have a restricted host range (three or fewer families of plants; Bernays and Graham, 1988), they encounter large amounts of predictable chemistries, and have characteristically high P450-based metabolism towards such chemistries (Berenbaum, 1999). This is clearly the case for CYP6B1, a P450 from the black swallowtail, Papilio polyxenes, that is induced by and metabolizes toxic host plant furanocoumarins (Cohen et al., 1992; Ma et al., 1994; Prapaipong et al., 1994). Induction of P450s from different families has been taken as an indication of their roles in the metabolism of host plant chemicals. For instance, members of the CYP4 family in the tobacco hornworm, Manduca sexta (Snyder et al., 1995), of the CYP6 family in Helicoverpa