Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila

Abstract

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

Figure 1. Cyp6g1 and Cyp6g2 copy number in twelve Drosophila species.

Cyp6g1 is duplicated in D. willistoni, D. grimshawi and some strains of D. melanogster, and triplicated in D. virilis. The third copy of Cyp6g1 in the strain of D. virilis used for this study has an inactivating mutation, but this mutation is not present in the sequenced strain, so it is not formally a pseudogene. Comparison with the phylogeny of the species suggests that multiple independent duplication events occurred (cladogram inferred from Stark et al. [21]). In contrast, Cyp6g2 has 1∶1 orthologs in all twelve Drosophila species analyzed.

Figure 2. Unrooted neighbour-joining tree of predicted CYP6G1 amino acid sequences from twelve Drosophila species.

The node labels show bootstrap values from 1000 iterations. Paralogs labelled in the same colour are from the same species. The clustering of paralogs from the same species rather than of orthologs between species supports the hypothesis that the duplications and triplications occurred independently in the separate lineages.

Figure 3. Overexpression of different Cyp6g1 orthologs confers resistance to different insecticides.

A) Changes in survival following exposure to three classes of insecticide by expression of Cyp6g1 orthologs from D. melanogaster, D. simulans, D. willistoni and D. virilis in a consistent genetic background. Resistance ratio (RR) was calculated by comparing the concentration of insecticide that killed 50% of insects (the LC₅₀) between the line expressing the ortholog and the background strain, 86Fb, which was genetically identical except for the absence of the Cyp6g1 construct. Results marked with an asterisk were statistically significant (p<0.05). Orthologs from D. melanogaster and D. simulans were functionally identical at a qualitative level, both providing resistance to all three chemicals, but the resistance profile varied between the other three orthologs. B) Comparison of the potential of the CYP6G1 orthologs to cause resistance when overexpressed. D. melanogaster and D. simulans orthologs cause resistance to a range of chemicals, whilst the ortholog from D. willistoni and the two paralogs from D. virilis only conferred resistance to one of the chemicals tested. These results suggest that adaptation of the protein has occurred repeatedly in Drosophila. The scale bar indicates the number of substitutions per four-fold degenerate site in the genomes of the species (inferred from Stark et al. [21]).

Figure 4. RNA in situ hybridization experiments for Cyp6g1 in the midgut, Malpighian tubules and fat body of third instar larvae of D. melanogaster, D. simulans, D. willistoni and D. virilis.

Expression was observed in the midgut in all four species. Fat body expression was observed in all species except D. willistoni, and Malpighian tubule expression was not detected in D. willistoni or D. virilis, the two species from which orthologs only conferred resistance to one of the insecticides tested. The expression of Cyp6g1 in D. melanogaster has been described previously but is included here for comparison with the species tested in this study .