Howlett et al. BMC Genomics 2012, 13:211 http://www.biomedcentral.com/1471-2164/13/211 Page 5 of 10 Figure 4 Msex\Orco expression in larval antennae is variably reduced following dsRNA treatment. Relative expression of Msex \Orco was measured by quantitative RT-PCR in male larval antennae 3 days after dsRNA delivery, and here is shown as a percentage of expression compared with paired control samples (dashed line indicates expression in control samples, normalized to 100%). Note that in half of trials, Msex\Orco expression was similar in treated and control animals (trials 6–10) while in the other half, Msex\Orco expression is markedly reduced following dsRNA treatment (trials 1– 5). Cumulatively, dsRNA treatment results in significant reduction of Msex\Orco expression (p = .03, paired, one-tailed t-test). For quantitative RT-PCR, relative expression of Msex\Orco was measured and normalized to an endogenous control (rpS3) as described in the Experimental Procedures and shown in Additional file 3: Table S1. untreated controls (not shown; n = 3 trials, 2–3 larvae per sample). Therefore, as discussed further below, feed- ing of dsRNA can be used to induce RNAi in larval che- mosensory tissues. Discussion Manduca sexta has great potential as a model for explor- ing the functional aspects of chemosensation at the mo- lecular level, due to its agricultural importance and its position as a key model for chemosensory physiology, behavior and ecology. Here, we report identification of OR and GR genes from M. sexta that, along with previ- ously reported genes, means that several dozen chemo- sensory genes are known in this species. Using the conserved olfactory co-receptor Msex\Orco as a test case, we have shown that systemic RNA interference can be induced in M. sexta larvae, and that it can target per- ipheral neurons in larval antennae. Although details of RNAi assays will need to be optimized for each gene tar- geted, together, these results will prove useful for future functional studies of M. sexta chemosensory genes. The 17 putative ORs that we report here bring the total number of M. sexta OR genes to 64 [22–24]. The M. sexta adult antennal lobe contains approximately 70 glomeruli [22], suggesting that this is a likely approxi- mate number of adult-specific odorant receptor genes, including both ORs and IRs [a second a second class of receptors that we did not investigate in this work; 6 IRs were previously identified in reference [22]]. However, since individual chemoreceptor genes are expressed at low levels, and given known variation in their develop- mental expression patterns (corresponding to different ecologies of larvae and adults), there are likely additional receptor genes present in the M. sexta genome. In fact, while several ORs identified in our approach were also previously identified (data not shown) it will be interest- ing to determine whether the 17 ORs unique to our study and reported here are enriched for expression in larvae, because our transcriptome data is derived from both adult and larval tissue, while a previous transcrip- tome analysis focused on adult antennal tissue [22]. Together, the 64 known M. sexta OR genes provide a good set of targets to explore for future functional ana- lyses. Although potential Bombyx orthologs are clear for many of the genes, many also appear to be the result of recent duplications (Figure 1). Characterizing the func- tion for these receptors could allow us to determine whether particular types of ORs have tended to duplicate recently. The identification of a GR similar to Drosoph- ila sugar receptors also has great potential for functional analysis through RNAi since the physiological and be- havioral aspects of sugar response in larval M. sexta is well understood [28]. For RNAi, we targeted the putative ortholog of Dros- ophila Or83b, Msex\Orco (previously MsOR2) [24,27], which encodes an olfactory co-receptor required for localization and function of conventional ORs [29,30]. Consistent with this role, we detected high levels of Msex\Orco in antennal tissue, which has a large number of ORNs, and much lower expression in the maxilla, which primarily mediate gustation. Expression was higher in adult than larval antennae. In agreement with a previous report, we found that relative expression was higher in adult female than male antennae [24]. In con- trast, expression was slightly higher in larval male than female antennae (Figure 3). The functional significance of these differences in expression, if any, is unknown. We observed significant reduction of Msex\Orco tran- scripts in larval antennae after feeding dsRNA, indicat- ing that systemic RNAi can be used to target Manduca chemosensory neurons in vivo. Knockdown was statisti- cally significant three days after feeding dsRNA, with successful knockdown observed in about half of the trials conducted. The effect was transient, and lost by 5 days following dsRNA delivery. Together, our results delineate a time-course over which Msex\Orco RNAi can be induced by feeding in larval antennae. Variation in relative Msex\Orco to rpS3 expression was observed across biological replicates. Among 10 control samples collected at the same stage, expression ratios varied more than three-fold (Additional file 3: