Two classes of GST, the Delta and Epsilon classes, are found uniquely in insects. Both of these classes are represented by a minimum of eight genes in the mosquito and fruit fly and the members of these gene families are largely the product of local gene duplications . The independent radiation of these gene families in An. gambiae and D. melanogaster and the functional verification of the role of a subset of these enzymes in xenobiotic detoxification suggest that they are important in the adaptation of insects to environmental selection pressures ( Ranson et al ., 2002 ). Indeed one of the best characterized functions of these insect GST classes is their role in insecticide detoxification. A small subset of the Delta and Epsilon class insect GSTs can catalyse the dehydrochlorination of the organochlorine insecticide DDT ( Tang & Tu, 1994 ; Ranson et al ., 2001 ; Lumjuan et al ., 2005 ) and other members of the insect-specific GST classes metabolize organophosphate insecticides ( Huang et al ., 1998 ; Wei et al ., 2001 ). Whereas other classes of insect GSTs have been proposed to play a secondary role in protection against insecticides, for example by ameliorating the effects of oxidative stress , all those GSTs directly implicated in insecticide metabolism to date belong to the Delta or Epsilon classes. Hence , the complete absence of the Epsilon class in the honeybee and the presence of only a single Delta class GST may partially account for the extreme sensitivity of this species to certain insecticides.
If CYP307A1 is indeed required for ecdysteroid biosynthesis in A. gambiae , knocking down cyp307a1 expression should decrease ecdysteroid production by steroidogenic tissues. To test this hypothesis, we performed transient RNAi on Anopheles females targeting cyp307a1 before measuring in vitro ovarian 20E production 22 h after blood-feeding. As a positive control, we first determined whether knocking-down by transient RNAi a known steroidogenic gene, cyp314a1 , would indeed decrease 20E production in ovaries of BF females. As shown in Figure 3A , expression of cyp314a1 was strongly decreased in ovaries from BF females injected with ds- cyp314a1 compared to controls (ds- gfp -injected BF females). The decrease in cyp314a1 RNA led to a significant reduction of ovarian 20E production in ds- cyp314a1 -injected BF females compared to controls ( Figure 3C ). Therefore, transient RNAi targeting a steroidogenic enzyme gene in mosquito female is a powerful method to characterize steroidogenic genes. Injection of ds- cyp307a1 also strongly decreased cyp307a1 expression in ovaries from ds- cyp307a1 -injected BF females compared to controls (ds- gfp -injected BF females) ( Figure 3B ). As depicted in Figure 3D , ovarian ecdysteroid production of ds- cyp307a1 -injected females was also significantly decreased compared to controls, demonstrating that cyp307a1 is required for ecdysone biosynthesis in Anopheles .