Howlett et al. BMC Genomics 2012, 13:211 http://www.biomedcentral.com/1471-2164/13/211 GRNs, in contrast, can express multiple GRs [6]. The overall variety of chemical cues an insect can detect is the result of the diversity of its receptors. There are 120 chemoreceptor genes in the Drosophila melanogaster genome which encode 130 different proteins [6]. Add- itional gene families, encoding Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs) also contrib- ute to taste and olfaction. OBPs are small, water-soluble extracellular proteins within the lymphatic cavity of ol- factory sensilla that facilitate ligand binding to ORs [7]. Similarly, CSPs may help mediate binding between ligands and receptors, but their exact role is unclear and the CSPs appear to have additional non-chemosensory functions [8]. Although the gene families encoding chemosensory proteins are conserved across all insects, both chemo- sensory gene families and the neuroanatomy of chemo- sensory systems evolve rapidly. Correspondingly, insects show an extraordinary diversity of olfactory and gusta- tory responses [reviewed in [9,10]]. The full complement of chemosensory genes is now known in species from several insect orders. Frequent losses and lineage- specific expansions of chemosensory genes appear to be the rule, exemplified by particularly large OR gene expansions observed in honeybee, jewel wasp and flour beetle genomes [11–13]. However, outside of Drosophila melanogaster, virtually nothing is known about the func- tion of chemosensory genes, and very few studies have linked specific receptors or lineage-specific genetic changes to particular chemical responses, behaviors or ecology [but see [14–16]]. Such characterization will re- quire not only chemosensory gene identification, but also development of methods for functional analysis of these genes in non-model insects. The tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) is a major agricultural pest that feeds on so- lanaceous plants. Manduca sexta has long been an im- portant non-genetic model organism for insect chemosensation, particularly from anatomical, neuro- physiological, behavioral and ecological perspectives [17–21]. Recent analyses have identified 48 ORs and 1 GR, as well as multiple OBP and CSP gene sequences from this species [22–24]. Despite this substantial pro- gress, additional receptors remain to be identified, and a method for functional analysis of these genes is lacking. RNA interference (RNAi) is an invaluable tool for loss-of-function analysis in non-genetic organisms, and will be important to establish in this context. Systemic RNAi has been used before in M. sexta, particularly to target genes expressed in gut and hemolymph [reviewed in [25]], but there have been no reports of RNAi target- ing neural tissue in this species. A previous report showed that peripheral chemosensory neurons could be targeted by feeding double-stranded RNA (dsRNA) to Page 2 of 10 larvae of the moth Epiphyas postvittana [26], suggesting that this approach could potentially be effective in M. sexta chemosensory tissue as well. Here, we have taken significant steps to further the de- velopment of M. sexta as a model for functional analysis of insect chemosensation. We used high-throughput cDNA sequencing to characterize the transcriptome of three chemosensory organs (adult antennae and larval antennae and maxilla), and we identified 17 OR genes not previously known for M. sexta as well as a putative trehalose receptor-related GR gene from this organism. Further, we developed a systemic RNA interference (RNAi) method that can be used to knock down expres- sion of a chemoreceptor transcript in larval olfactory neurons. Although this method will need to be opti- mized for each gene tested, and physiological assays developed, these findings will allow exploration of the functional role of specific chemosensory receptor pro- teins in M. sexta. Results Identification and analysis of M. sexta chemoreceptor genes In order to identify chemosensory genes from Manduca, we performed transcriptome sequencing on mixed che- mosensory tissues (adult and larval antenna and larval maxilla). From approximately 52,000 assembled contigs, we identified 84 with significant similarity to insect (Dros- ophila or Bombyx) ORs. After eliminating those with greater than 95% identity at the nucleotide level to previ- ously identified M. sexta ORs, we identified 17 novel puta- tive OR genes (see Additional File 1: Figure S1). These were numbered randomly starting with MsOR49, building upon the 48 ORs previously identified in M. sexta. A neighbor-joining cluster analysis is shown in Figure 1, which allows inference of potential orthology. Of the 17 putative ORs, 11 show greater sequence similarity to an- other M. sexta OR than to a Bombyx OR, suggesting that they are the products of recent duplications. By the same criteria, we identified 4 contigs matching other insect GRs, although 3 contigs were redundant leaving 2 putative GR genes from M. sexta (see Add- itional File 1: Figure S1). One is most closely related to Bombyx Gr6p, both of which show significant similarity to the DmGR5a trehalose (sugar) receptor and the puta- tive sugar co-receptor DmGR64f (Figure 2). The other corresponded to the single bitter GR previously identi- fied [22]. Sequences corresponding to both GR genes were PCR amplified from genomic DNA and cloned to confirm their sequences (data not shown). Of the contigs with significant similarity to CSP and OBP genes, none were unique to this study (data not shown). This suggests that both gene families are essen- tially fully known in M. sexta [22].