Howlett et al. BMC Genomics 2012, 13:211 http://www.biomedcentral.com/1471-2164/13/211 Page 4 of 10 Figure 2 MsGR2 is a putative sugar receptor. Neighbor-joining tree showing similarity of MsGR2 to GR genes from Bombyx, Drosophila and Heliothis. Bootstrap values from 1000 replicates greater than 50% are shown. MsGR2, along with lepidoteran receptors BmGr6p and HvCr1, are most similar to the Drosophila GR family that includes the trehalose receptor DmGr5a [44]and putative sugar co- receptor DmGR64f[45]. Other putative Bombyx sugar receptors (BmGr7, BmG48R, BmGr4S) are more distantly related. This tree was rooted using putative CO2 receptors (BmGr1N and BmGr2NJ) and theDmGr43a group, for which ligands are unknown. Sources of sequences shown here are listed in the experimental procedures. Scale bar represents number of amino acid substitutions per site. Delivery of dsRNA via feeding induces RNA interference in larval antennae We targeted the M. sexta ortholog of the olfactory co- receptor Orco (previously MsOR2) [24,27] for RNAi. In a preliminary analysis, we observed that Msex\Orco transcripts are developmentally regulated and sexually dimorphic. Expression was highest in adult antennae, and was approximately 25-fold lower in larval antennae and 2500-fold lower in larval maxilla (which contain gustatory as well as olfactory sensilla) (Figure 3). As has been previously reported, expression was higher in adult female antennae than in males (Figure 3 and [24]). In contrast, in larval antennae, expression was approxi- mately 1.3-fold higher in males than in females; this dif- ference, though slight, was reproducible across different experiments (Figure 3 and data not shown). To minimize variation in endogenous expression, subse- quent RNAi experiments were conducted in staged- matched larvae of the same sex. dsRNA corresponding to a fragment of the Msex\Orco coding sequence was fed to larvae on the first day of the fifth instar. Transcript levels were monitored by quanti- tative RT-PCR after 1, 3 or 5 days. For each time-point, three separate samples were prepared from pooled an- tennal tissue of 2–5 larvae each. Msex\Orco expression Figure 3 Expression of Msex\Orco in chemosensory tissues. Expression was measured by quantitative RT-PCR (qPCR) in male (white bars) or female (grey bars) adult antennae, 5th instar larval antennae, or 5th instar larval maxilla. For qPCR, relative expression of Msex\Orco was measured and normalized to an endogenous control (rpS3) as described in the Experimental Procedures. Expression was normalized relative to male larval antennal expression and plotted on a log scale. Error bars represent SEM from three analytical replicates on samples that contained tissue from 2–5 individuals (larval samples) or a single individual (adult samples). was reduced to an average of 80% of expression in paired control samples after 1 day, and to 72% after 3 days. By 5 days, transcript levels were comparable to controls, and even slightly increased (to an average of 110%). None of these differences was statistically signifi- cant. However, this was due in part to variability across trials. Although mean Msex\Orco expression 1 day after dsRNA treatment was 80% of controls, in individual trials it was 61%, 72% and 106%. To better characterize this effect, we performed a total of ten trials on Msex\Orco expression 3 days after dsRNA treatment, when transcript reduction was max- imal. As described above, each sample contained pooled antennal tissue from 2–5 larvae. In half of the trials, Msex\Orco levels were unaffected (ranging from 97%- 114% of expression compared to paired controls; Figure 4 Trials 6–10). In the other half of the trials, transcript levels were reduced following dsRNA treatment, and ranged between 49-75% of control levels (Figure 4; trials 1–5). Despite this variability in success across trials, dsRNA treatment led to a significant overall reduction in Msex\Orco transcript (relative to a value of 100%), according to a one-sample, one-tailed t-test (t- value = 2.14, df = 9; P ≤ 0.03). Reduction in Msex\Orco transcript levels was specific to Msex\Orco dsRNA, and was not a general effect of dsRNA treatment. When lar- vae were fed an unrelated dsRNA, corresponding to the olfactory receptor MsOR-30, Msex\Orco expression was not affected, averaging 106% compared to control sam- ples. In the same samples, the targeted MsOR-30 tran- script was reduced to an average of 44% compared to