Cyp12a4 confers lufenuron resistance in a natural population of Drosophila melanogaster - PubMed (original) (raw)

Cyp12a4 confers lufenuron resistance in a natural population of Drosophila melanogaster

Michael R Bogwitz et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Lufenuron is an insect growth regulator insecticide mainly used for the control of the cat flea. To understand mechanisms of resistance to lufenuron, we have characterized lufenuron resistance in a natural population of Drosophila melanogaster. In this study we have used precise genetic mapping to identify a mechanism of lufenuron resistance: the overexpression of the cytochrome P450 gene Cyp12a4. Cyp12a4 is predicted to encode a mitochondrial cytochrome P450 enzyme. Expression of Cyp12a4 in D. melanogaster third-instar larvae was detected in the midgut and Malpighian tubules of both lufenuron-resistant and wild-type strains. The level of Cyp12a4 expression in the midgut is higher in the lufenuron-resistant strain than in wild-type strains. Driving the expression of Cyp12a4 in the midgut and Malpighian tubules by using the GAL4/UAS gene expression system results in lufenuron resistance, but it does not result in resistance to three other insecticide classes. Transgenic expression of Cyp12a4 in a ubiquitous expression pattern results in late embryonic lethality, suggesting that high-level ectopic expression of Cyp12a4 is detrimental to development.

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Figures

Fig. 1.

Fig. 1.

Characterization of lufenuron resistance in NB16. (A) Dosage–mortality response for the lufenuron-resistant NB16 strain and lufenuron-susceptible strains Canton-S and y; cn bw sp. NB16 is 3.06 and 3.40 times more lufenuron resistant than Canton-S and y; cn bw sp, respectively. (B) Lufenuron resistance in NB16 maps to chromosome III. +/Cy; +/Ser progeny from Cy/bwD; Sb/Ser × NB16 were test-crossed to Cy/bwD; Sb/Ser and reared in the presence or absence of 2 × 10–4% lufenuron. The relative numbers of each class of progeny are shown. + represents a chromosome from NB16. Note the lack of survivors on lufenuron in progeny where both copies of chromosome III are from the mapping strain.

Fig. 2.

Fig. 2.

The location of the lufenuron resistance locus. (A) Fine-scale genetic mapping of lufenuron resistance within chromosome III. Recombinants between the genetic markers glass and ebony were progressively scored for molecular markers. Molecular distances between markers are indicated. Numbers of recombinants between each marker are presented in Fig. 8. Lufenuron resistance was mapped to a region of 30.7 kb. (B) Genes in the region of the lufenuron-resistance locus at 91F3–6. Arrows indicate direction of transcription.

Fig. 3.

Fig. 3.

Level of Cyp12a5 (A) and Cyp12a4 (B) mRNA in third-instar larvae of NB16 compared with susceptible strains Canton-S and y; cn bw sp, as determined by real-time PCR (±SEM, n = 3). All levels are relative to the susceptible strain Canton-S. Also shown are the mRNA levels of other lufenuron-resistant strains, WC2 and Inn5 (23). Compared with Canton-S, 4.2 times more Cyp12a4 mRNA is detected in NB16. Cyp12a4 mRNA levels were also measured in midguts from third-instar larvae, with 3.1 times more Cyp12a4 detected in the midgut of NB16 than in that of y; cn bw sp.

Fig. 4.

Fig. 4.

Dosage–mortality response for transgenic flies overexpressing Cyp12a4 in the midgut and Malpighian tubules by using the _6g1_Cs-GAL4 driver, compared with controls, reared on increasing concentrations of lufenuron. Between 1.5-(UAS-Cyp12a43) and 1.8-(UAS-Cyp12a42) fold lufenuron resistance is seen in flies overexpressing Cyp12a4 in the midgut and Malpighian tubules, compared with flies not overexpressing Cyp12a4.

Fig. 5.

Fig. 5.

Percentage survival at different life cycle stages for individuals overexpressing Cyp12a4 in a ubiquitous expression pattern by using the tubulin-GAL4 driver (UAS-_Cyp12a43/tub_-GAL4), compared with flies not over-expressing Cyp12a4 (UAS-Cyp12a43/TM3, _Act_-GFP). The presence/absence of GFP was used to determine overexpression of Cyp12a4. Overexpression of Cyp12a4 results in early first-instar lethality.

Fig. 6.

Fig. 6.

In situ hybridization of Cyp12a4 in third-instar larvae of the lufenuron-resistant strain NB16 (A) and the susceptible control y; cn bw sp (B). Expression of Cyp12a4 is detected in the midgut and Malpighian tubules in both strains.

References

    1. Roush, R. T. & McKenzie, J. A. (1987) Annu. Rev. Entomol. 32, 361–380. - PubMed
    1. Wilson, T. G. (2001) Annu. Rev. Entomol. 46, 545–571. - PubMed
    1. ffrench-Constant, R. H., Anthony, N., Aronstein, K., Rocheleau, T. & Stilwell, G. (2000) Annu. Rev. Entomol. 45, 449–466. - PubMed
    1. Williamson, M. S., Martinez-Torres, D., Hick, C. A. & Devonshire, A. L. (1996) Mol. Gen. Genet. 252, 51–60. - PubMed
    1. Pittendrigh, B., Reenan, R., ffrench-Constant, R. H. & Ganetzky, B. (1997) Mol. Gen. Genet. 256, 602–610. - PubMed

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