Howlett et al. BMC Genomics 2012, 13:211 http://www.biomedcentral.com/1471-2164/13/211 Table S1). The source of this variation is unknown. Im- portantly, however, we only observed two results in any RNAi trial: expression in paired control vs. treated sam- ples was either approximately equal, or it was markedly lower in dsRNA treated samples. The reverse (markedly higher expression in treated animals) was never observed. This suggests that despite variation in relative Msex\Orco expression, significant differences between dsRNA-treated and control samples are indeed due to RNAi-mediated knockdown. Variation in relative Msex\Orco expression could ac- count for some of the observed variability RNAi trials. In addition, it is likely that RNAi was not successfully induced in all treated animals. The dsRNA dosage was chosen based on previous reports [in particular, [26,31], and reviewed in [32]], but this should be optimized for each future experiment. Further, it was recently found that starvation improves dsRNA stability in the insect gut and thereby the effectiveness of RNAi [33]. Although animals in our assay were starved overnight prior to dsRNA delivery, there could have been variation in how much (untreated) food they consumed following treat- ment, as they were offered ad libitum access to the artifi- cial diet. Regardless of its cause, however, variation in knockdown success can be accounted for in future experiments by quantifying target gene expression in each experimental and control animal following pheno- typic analysis. In successful trials, transcript levels were reduced to approximately 50-75% of control levels (Figure 4). These fall within the wide range of 30-98% knockdown reported in previous feeding experiments in other insects [reviewed in [32]], although it is important to note many differences in dsRNA concentration, tissue, and species targeted in these experiments. Interestingly, the mechanism(s) of systemic RNAi are not well under- stood in insects, and may be somewhat variable. Lepi- dopteran genomes (and those of other insect orders) do contain a putative ortholog of C. elegans sid-1, which encodes a membrane transporter essential for systemic RNAi in worms [34]. Drosophilids lack a sid-1 ortholog and notably, also lack systemic RNAi in most tissues. However, the role of insect sid-1 family genes is unclear; in Tribolium they are apparently dispensable for sys- temic RNAi [35]. Further, there are differences in the complement of RNAi effectors in the genomes of Dros- ophila, Bombyx and Tribolium suggesting that there could be some differences in RNAi mechanisms across insects [35,36]. The degree of RNA knockdown required for detecting a phenotype, and thus for functional analysis of a gene, is expected to vary with the dosage requirement for the targeted gene and with the sensitivity of phenotypic assays. Electrophysiological assays are likely to be able to Page 6 of 10 detect even small differences in taste or olfactory sensi- tivity, while behavioral assays might require more robust knockdown. While this will have to be established on a case-by-case basis for each gene, partial transcript knockdown in the range of 50% has produced inform- ative phenotypes in several recent cases in insects (reviewed in [32]). Thus, we expect this method will be useful for elucidating the functions of targeted genes. Manduca has long been a model for studying insect chemosensation, and a wealth of behavioral, physio- logical and ecological data exists [reviewed in [17,18]]. The ability to pair the identification and functional ana- lysis of chemosensory genes with well-established behav- ioral, electrophysiological, and molecular methods in this species is likely to significantly advance our under- standing of insect chemosensation. Conclusions Chemosensory systems and the gene families that medi- ate chemosensory responses evolve rapidly. A molecular genetic understanding of insect chemosensation, includ- ing an understanding of how evolution of chemosensory genes has contributed to lineage-specific olfactory and gustatory responses, will require both identification and functional analysis of chemosensory genes in non-model insects. In this work, we have identified 18 chemosen- sory receptors (17 ORs and 1 GR) from the tobacco hornworm Manduca sexta. Further, we demonstrate that systemic RNAi can be used to knock-down expression of chemosensory receptor transcripts in larval olfactory neurons. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation. Methods Rearing conditions and chemosensory tissue collection All tissues were obtained from male and female Man- duca sexta in our rearing facility. The insects were derived from eggs donated by the Manduca rearing facil- ity at the University of Arizona, AZ, USA. Caterpillars were reared on a wheat germ-based artificial diet [37], and maintained in an environmental chamber with a 16 hr light: 8 hr dark cycle at 25°C. Upon pupating, adults were maintained in a holding cage (1 x 1 x 1.5 m) under the same environmental conditions. Chemosensory tissues were collected from adults (an- tennae) and 5th instar larvae (antenna and maxilla). Each larval maxilla contained a single lateral styloconic sensil- lum (taste), medial styloconic sensillum (taste) and max- illary palp (taste and olfaction). The distal tip of the maxillary palp had five uniporous taste sensilla and two multi-porous olfactory sensilla. Tissues were removed under a dissecting scope with iridectomy scissors. For the larval dissections, the caterpillar was anesthetized by