====== Drosophila Cyp6g1, the Rst(2)DDT gene at 64.5 on chromosome 2 ======



The power of Drosophila genetics coupled with the tools made possible by the complete genome sequence has provided the most detailed, yet complex molecular genetic detail about a P450-based insecticide resistance mechanism. The resistance gene Rst(2)DDT has been genetically characterized for over 40 years (review in (Daborn et al., 2001, Wilson, 2001). The position of this gene around 64.5cM on the left arm of chromosome 2 has become almost mythical, as a number of phenotypes were linked to this locus, from dominant DDT resistance to phenylthiorurea susceptibility, from organophosphorus to carbamate resistance, from various P450-dependent activities to vinyl chloride activation. EMS mutagenesis of a wildtype stock and selection with imidacloprid led to two strains with moderate imidacloprid resistance and moderate cross-resistance to DDT (Daborn et al., 2001). Conversely, two DDT-resistant strains (Hikone-R and Wisconsin-1) were shown to be cross-resistant to imidacloprid. Fine scale mapping of this dominant resistance localized Rst(2)DDT to a region from 48D5-6 to 48F3-6 on the polytene chromosome map. Of three candidate P450 genes in this region, Cyp6g1, Cyp6g2 and Cyp6t3, only the first showed constitutive overxpression in the DDT and imidacloprid resistant strains tested (Daborn et al., 2001). 

A DNA microarray comprising probes for all the Drosophila P450 genes was addressed with target cDNAs from susceptible strains and from the DDT-resistant Hikone-R strain and the propoxur-resistant WC2 strain. In both cases, Cyp6g1 was the only P450 gene showing constitutive overexpression (Daborn et al., 2002). Overexpression of Cyp6g1 was confirmed by quantitative (RT)PCR in 20 strains, and DDT, imidacloprid, nitenpyram and lufenuron resistances were all independently mapped to the Cyp6g1 locus in the Hikone-R and WC2 strains. The insertion of a terminal direct repeat of the transposable element Accord was systematically found in the 5' UTR of 20 different resistant strains from across the globe.  Phylogenetic analysis of the first intron sequence of the gene showed a unique haplotype in resistant strains vs a large diversity of susceptible haplotypes, suggesting a selective sweep had occurred in global Drosophila populations (Daborn et al., 2002). This was further demonstrated in a survey of 673 lines from 34 populations collected around the world showing perfect correlation between the presence of the Accord insertion and resistance and a reduction in variability measured by microsatellite analysis in a 20kb region downstream of Cyp6g1 (Catania et al., 2004). In some resistant lines, the presence of a P-element insertion into the Accord element was reported in that study. In fact, the Cyp6g1 locus is even more complex, with at least six different alleles found in nature (Schmidt et al., 2010). Beyond the Cyp6g1 ± Accord insertion alleles, Schmidt et al. reported four alleles in which the Cyp6g1 with Accord insertion was duplicated, and in three of them additional insertions of P-element or HMS Beagle elements were found within the original Accord insertion. This allelic succession is adaptive, with higher resistance and Cyp6g1 transcription found for the most complex allele (Cyp6g1-[BP], Schmidt et al., 2010).
 
Transgenic flies producing CYP6G1 under control of a variety of promoters in the GAL4/UAS system (heatshock, tubulin, or midgut/fat body/Malpighian tubules) showed increased survival to acetamiprid, imidacloprid and nitenpyram in larvae and to DDT in adults (Daborn et al., 2002; Le Goff et al., 2003; Chung et al., 2007; Daborn et al., 2007). Significantly, the 491 bp Accord sequence carries enhancer elements itself and can direct expression of reporter GFP in the tissues (gastric caeca, midgut, Malpighian tubules and fat body of larvae) in which Cyp6g1 expression is localized in resistant strains (Chung et al., 2007). Moreover, the Malpighian tubules of adults are critical, as overexpression of Cyp6g1, or its RNAi knockdown in just this tissue can significantly shift the toxicity of DDT to lower or higher levels, respectively (Yang et al., 2007). 

Definitive functional evidence that CYP6G1 metabolizes insecticides was provided by Joussen et al. (2008) who showed that the enzyme produced in tobacco cell suspensions cultures metabolizes imidacloprid and DDT as predicted by the experiments with transgenic flies resulting in resistance and by homology modeling (Jones et al., 2010).
In most field-collected strains, DDT resistance is significant but low compared to that of strains further selected in the laboratory (e.g. 91-R, see below) suggesting that while Cyp6g1 may constitute a first line of defense seen in field populations, further insecticide pressure in the laboratory may select additional mechanisms. In addition, mechanisms other than Cyp6g1 overexpression (including target site resistance) can also be involved in DDT resistance. For instance, while Cyp6g1 overexpression is observed in the DDT-resistant Wisconsin and 91R strains, the Cyp12d1 (or Cyp12d2) gene is overexpressed as well in both strains (Brandt et al., 2002; Festucci-Buselli et al., 2005) and its transgenic overexpression can confer resistance to DDT (Daborn et al., 2007). Moreover, several other genes are overtranscribed in these strains as well (Pedra et al., 2004). Therefore it is hardly surprising that resistance, that has always been a relative term, is not restricted to Cyp6g1 overexpression, especially when compared to strains that are themselves resistant (Festucci-Buselli et al., 2005; Kuruganti et al., 2006). 
In a Brazilian strain of Drosophila simulans resistant to DDT, imidacloprid and malathion, only the Cyp6g1 ortholog is overexpressed (Le Goff et al., 2003). In a California population of D. simulans the 5'-flanking sequence of the Cyp6g1 ortholog is nearly fixed for a Doc transposable element insertion. This insertion is absent from African populations and is associated with increased transcript abundance of Cyp6g1 and resistance in a what appears to be a case of resistance analogous with the Accord case of D. melanogaster Cyp6g1 (Schlenke and Begun, 2004) __**UPDATE NEEDED**__ Contributions are welcome [[arthropodP450@gmail.com]]