We tested a combined approach of mutagenesis and next generation transcriptomics to study insecticide resistance in the model organism Drosophila melanogaster.
A Minos-based construct was used for genome-wide insertional gene activation mutagenesis. During this screen, an Imidacloprid-resistant D. melanogaster female was retrieved. In the resistant line MiT[w−]3R2/CyO lacking the TREP (Minos) insertion, which was derived from this retrieved mutant female, both lethality and resistance were detected. In a recombination experiment resistance was separated from lethality and line MiT[w−]3R2 homozygous for resistant second chromosome was established. This line was further analyzed for the resistance mechanism.
Cross-resistance of the Imidacloprid-selected MiT[w−]3R2 mutant to DTT () suggests metabolic resistance as the mechanism of resistance in this line. Furthermore, biochemical analysis showed increased P450 activity in the resistant line compared to the susceptible line ().
The Illumina parallel short-sequencing technology was used to obtain total cDNA sequences of the resistant line and of the non-resistant isogenic line iso31 (w1118iso
. This approach was used in order to identify and quantify differences in expression between mutant line MiT[w−
]3R2 and susceptible line iso31, covering nearly all D. melangaster
genes. Out of 357 genes differently expressed in the resistance line, 150 were up-regulated, and 207 genes were down-regulated in comparison to the susceptible line.
Gene ontology functional classification of the sequenced transcripts identified a significantly up-regulated P450 family group and two groups of genes coding for peptidase activity in the resistant line. Significantly overrepresented down-regulated groups of genes were cuticular protein genes and other peptidase genes.
Deep sequencing analysis detected eight members of the P450 family, Cyp4p2, Cyp6a2, Cyp6g1, Cyp6w1, Cyp4e3, Cyp309a2, Cyp6g2 and Cyp4d14, with elevated expression in the resistant line. Genes encoding glutathione-S-transferases, as well as esterases did not show elevated expression in the resistant line. The most highly over-expressed P450 genes as detected with deep sequencing and confirmed by real time PCR, are Cyp4p2, Cyp6a2 and Cyp6g1 (GSE28560_resistant_vs._susceptible_UPREGULATED_GENES.txt.gz). The cytochrome P450 genes play an important role in insecticide resistance, because of their variety and the broad substrate specificity of some P450 genes 
. We report for the first time elevated expression of the Cyp4p2 gene in a D. melanogaster
line resistant to Imidacloprid and DDT, although its role in resistance (if any) remains to be elucidated. The detoxification function of Cyp6a2 and Cyp6g1 in Drosophila
is well documented. Over-expression of Cyp6g1 in Drosophila
confers resistance to DDT and neonicotinoids 
which provides a certain degree of validation in our approach for detecting genes conferring insecticide resistance. The Cyp6a2 is also highly expressed in different insecticide resistant Drosophila
and the CYP6A2 encoded enzyme can metabolize insecticides 
Five other P450 genes (Cyp6w1, Cyp4e3, Cyp309a2, Cyp6g2 and Cyp4d14) detected in the resistant line are over-expressed up to 6-fold. Microarray analysis showed that expression of Cyp6w1 is higher in DDT resistant Drosophila
strain compared to a susceptible line 
. Over-expression of the Cyp6g2 gene confers resistance to diazonin and nitenpyram in transgenic Drosophila
. To date, Cyp4e3, Cyp309a2 and Cyp4d14 have not been implied in insecticide resistance.
A number of cuticular protein genes were down-regulated in the resistant mutant compared to the susceptible line (). This could occur as a result of the general stress response induced by the up-regulated detoxification system. It is not likely that the down-regulation of cuticular protein genes plays a role in the insecticide resistance mechanism. It would be in disaccord with the fact that reduced cuticular penetration of insecticides can contribute to resistance in some insect species 
The identification of a group of 21 up-regulated genes involved in peptidase activity is consistent with the finding that genes coding for peptidase activity are also significantly over-expressed in DDT resistant Drosophila
. The role of the proteolytic genes and genes showing peptidase activity in insecticide resistance is still poorly understood. There is increasing evidence of involvement of protein metabolism in insecticide resistances of different insect species 
. Proteases may be involved in modification of enzyme conformation and protein biosynthesis, in order to meet energy requirements during xenobiotic stress 
Other groups of overrepresented members among the up- or down-regulated genes belong to the following categories: oxidoreductase activity, chromosome establishment, organelle localization and cellular response to DNA damage (up-regulated; ) and nutrient reservoir activity, response to bacteria, biotic stimulus and immune response (down-regulated; ). Down-regulation of genes involved in immune response was not seen in other DDT-resistant Drosophila
. Oxidoreductase activity plays a role in detoxification, while the other biological processes could be an indication of general stress response.
The line MiT[w−
]3R2, homozygous for the resistance chromosome, derives from the mutant line MiT[w−
]3R2/CyO heterozygous for the second chromosome carrying both resistance and lethality. Genetic analysis of the mutant line MiT[w−
]3R2/CyO line placed the lethality locus to the region between 8.5 and 9.9 Mb on the right arm of the second chromosome of D. melanogaster
. Single nucleotide polymorphism analysis between the resistant line homozygous for resistant chromosome MiT[w−
]3R2 and the susceptible line iso31 indicates that the recombination event that separated the lethality from the resistance locus occurred in close vicinity to the lethality locus (). The three highest up-regulated P450 genes Cyp4p2, Cyp6a2 and Cyp6g1 are also located on the right arm of the second chromosome, but they are not closely linked (). Mapping against P element insertions confirmed that the resistance locus lies on the right arm of the second chromosome between 8 Mb and 9.7 Mb. Chromosomal mapping of the resistance in DDT and Imidacloprid resistant Drosophila
placed the DDT resistance locus (Rst (2) DDT
) in an area that overlaps the interval in which the resistance locus of MiT[w−
]3R2 is located. The position of the lethality locus (between 8.5 and 9.9 Mb), together with the SNP analysis and P element mapping, suggests that the resistance locus in MiT[w−
]3R2 lies within an interval of less than ~1 Mb (). Interestingly, the highly up-regulated Cyp6g1 (16.3-fold – deep sequencing analysis; 8.4-fold – real time PCR analysis) gene is located within this range. In the mentioned study of Daborn and colleagues 
the Cyp6g1 is strongly suggested as the main candidate gene responsible for the resistance in DDT and Imidacloprid resistant Drosophila
The mutation event which causes the resistance in MiT[w−
]3R2 remains to be identified. The resistance locus is not linked to an insertion of the transposon used in the screen. It is conceivable that a “hit and run” Minos
insertion effect might be responsible for the mutation, where the transposon first integrated and then re-excised. In Drosophila
often leaves behind upon excision either a characteristic six bp “footprint” or a deletion around the site of insertion 
, both of which can be mutagenic. It has been suggested that mutations of trans
-regulating factor/s, or of cis-acting elements of some of the Cyp genes are responsible for insecticide resistance in Drosophila
. A recent report suggests that a single mutation event in a specific enhancer can modulate Cyp6g1 tissue-specific induction in Drosophila
. One might thus speculate that a single mutation event occurred in a cis
-acting element of the Cyp6g1 gene, increasing the expression of this gene. This in turn could activate a resistance cascade, affecting the expression of other Cyp genes involved in resistance. Alternatively, the mutation might involve a gene encoding a transcription factor or a microRNA which regulates in trans
the Cyp genes involved. We have so far no evidence for the latter assumption, since an in silico
search failed to identify common transcription factor motifs regulating the over-expressed P450 genes. The same is true for common predicted microRNA targets in the 3′UTRs. Additional analyses are required in order to pinpoint the exact cause of resistance in the MiT[w−