BACKGROUND
animals rely on their olfactory and gustatory systems to detect chemicals in their environments.
chemosensation mediates essential behaviors such as locating food and shelter, avoiding predators, locating mates, and selecting appropriate sites for nesting or laying eggs.
the chemosensory organs and the gene families that encode chemosensory proteins are different in vertebrates and insects, but the underlying logic is in some respects quite similar  <cit> .
the molecular basis of insect chemosensation is best understood in drosophila.
responses to gustatory and olfactory cues are mediated by the olfactory  and gustatory  receptors, which together comprise an insect chemoreceptor super-family.
receptor protein complexes  function as ionotropic membrane channels, whose precise mechanism of action is still under investigation .
or and gr receptors are expressed in olfactory receptor neurons  and gustatory receptor neurons  respectively.
most orns express a single conventional or, and the range of compounds to which they respond depends on the tuning of that receptor.
grns, in contrast, can express multiple grs  <cit> .
the overall variety of chemical cues an insect can detect is the result of the diversity of its receptors.
there are  <dig> chemoreceptor genes in the drosophila melanogaster genome which encode  <dig> different proteins  <cit> .
additional gene families, encoding odorant binding proteins  and chemosensory proteins  also contribute to taste and olfaction.
obps are small, water-soluble extracellular proteins within the lymphatic cavity of olfactory sensilla that facilitate ligand binding to ors  <cit> .
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  <cit> .
although the gene families encoding chemosensory proteins are conserved across all insects, both chemosensory gene families and the neuroanatomy of chemosensory systems evolve rapidly.
correspondingly, insects show an extraordinary diversity of olfactory and gustatory responses .
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  <cit> .
however, outside of drosophila melanogaster, virtually nothing is known about the function of chemosensory genes, and very few studies have linked specific receptors or lineage-specific genetic changes to particular chemical responses, behaviors or ecology .
such characterization will require 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  is a major agricultural pest that feeds on solanaceous plants.
manduca sexta has long been an important non-genetic model organism for insect chemosensation, particularly from anatomical, neurophysiological, behavioral and ecological perspectives  <cit> .
recent analyses have identified  <dig> ors and  <dig> gr, as well as multiple obp and csp gene sequences from this species  <cit> .
despite this substantial progress, additional receptors remain to be identified, and a method for functional analysis of these genes is lacking.
rna interference  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 , but there have been no reports of rnai targeting neural tissue in this species.
a previous report showed that peripheral chemosensory neurons could be targeted by feeding double-stranded rna  to larvae of the moth epiphyas postvittana  <cit> , suggesting that this approach could potentially be effective in m. sexta chemosensory tissue as well.
here, we have taken significant steps to further the development 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 , and we identified  <dig> 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  method that can be used to knock down expression of a chemoreceptor transcript in larval olfactory neurons.
although this method will need to be optimized for each gene tested, and physiological assays developed, these findings will allow exploration of the functional role of specific chemosensory receptor proteins 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 chemosensory tissues .
from approximately  <dig>  assembled contigs, we identified  <dig> with significant similarity to insect  ors.
after eliminating those with greater than 95% identity at the nucleotide level to previously identified m. sexta ors, we identified  <dig> novel putative or genes .
these were numbered randomly starting with msor <dig>  building upon the  <dig> ors previously identified in m. sexta.
a neighbor-joining cluster analysis is shown in figure  <dig>  which allows inference of potential orthology.
of the  <dig> putative ors,  <dig> show greater sequence similarity to another m. sexta or than to a bombyx or, suggesting that they are the products of recent duplications.`
by the same criteria, we identified  <dig> contigs matching other insect grs, although  <dig> contigs were redundant leaving  <dig> putative gr genes from m. sexta .
one is most closely related to bombyx gr6p, both of which show significant similarity to the dmgr5a trehalose  receptor and the putative sugar co-receptor dmgr64f .
the other corresponded to the single bitter gr previously identified  <cit> .
sequences corresponding to both gr genes were pcr amplified from genomic dna and cloned to confirm their sequences .
of the contigs with significant similarity to csp and obp genes, none were unique to this study .
this suggests that both gene families are essentially fully known in m. sexta  <cit> .
delivery of dsrna via feeding induces rna interference in larval antennae
we targeted the m. sexta ortholog of the olfactory co-receptor orco   <cit>  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  .
as has been previously reported, expression was higher in adult female antennae than in males .
in contrast, in larval antennae, expression was approximately  <dig> -fold higher in males than in females; this difference, though slight, was reproducible across different experiments .
to minimize variation in endogenous expression, subsequent 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 quantitative rt-pcr after  <dig>   <dig> or 5 days.
for each time-point, three separate samples were prepared from pooled antennal tissue of 2– <dig> larvae each.
msex\orco expression 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 .
none of these differences was statistically significant.
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 maximal.
as described above, each sample contained pooled antennal tissue from 2– <dig> larvae.
in half of the trials, msex\orco levels were unaffected .
in the other half of the trials, transcript levels were reduced following dsrna treatment, and ranged between 49-75% of control levels .
despite this variability in success across trials, dsrna treatment led to a significant overall reduction in msex\orco transcript , according to a one-sample, one-tailed t-test .
reduction in msex\orco transcript levels was specific to msex\orco dsrna, and was not a general effect of dsrna treatment.
when larvae were fed an unrelated dsrna, corresponding to the olfactory receptor msor- <dig>  msex\orco expression was not affected, averaging 106% compared to control samples.
in the same samples, the targeted msor- <dig> transcript was reduced to an average of 44% compared to untreated controls .
therefore, as discussed further below, feeding of dsrna can be used to induce rnai in larval chemosensory tissues.
discussion
manduca sexta has great potential as a model for exploring the functional aspects of chemosensation at the molecular 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 previously reported genes, means that several dozen chemosensory 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 peripheral neurons in larval antennae.
although details of rnai assays will need to be optimized for each gene targeted, together, these results will prove useful for future functional studies of m. sexta chemosensory genes.
the  <dig> putative ors that we report here bring the total number of m. sexta or genes to  <dig>  <cit> .
the m. sexta adult antennal lobe contains approximately  <dig> glomeruli  <cit> , suggesting that this is a likely approximate number of adult-specific odorant receptor genes, including both ors and irs .
however, since individual chemoreceptor genes are expressed at low levels, and given known variation in their developmental expression patterns , 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  it will be interesting to determine whether the  <dig> 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 transcriptome analysis focused on adult antennal tissue  <cit> .
together, the  <dig> known m. sexta or genes provide a good set of targets to explore for future functional analyses.
although potential bombyx orthologs are clear for many of the genes, many also appear to be the result of recent duplications .
characterizing the function 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 drosophila sugar receptors also has great potential for functional analysis through rnai since the physiological and behavioral aspects of sugar response in larval m. sexta is well understood  <cit> .
for rnai, we targeted the putative ortholog of drosophila or83b, msex\orco   <cit> , which encodes an olfactory co-receptor required for localization and function of conventional ors  <cit> .
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  <cit> .
in contrast, expression was slightly higher in larval male than female antennae .
the functional significance of these differences in expression, if any, is unknown.
we observed significant reduction of msex\orco transcripts in larval antennae after feeding dsrna, indicating that systemic rnai can be used to target manduca chemosensory neurons in vivo.
knockdown was statistically 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 rps <dig> expression was observed across biological replicates.
among  <dig> control samples collected at the same stage, expression ratios varied more than three-fold .
the source of this variation is unknown.
importantly, however, we only observed two results in any rnai trial: expression in paired control vs. treated samples was either approximately equal, or it was markedly lower in dsrna treated samples.
the reverse  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 account 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 , 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  <cit> .
although animals in our assay were starved overnight prior to dsrna delivery, there could have been variation in how much  food they consumed following treatment, as they were offered ad libitum access to the artificial 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 phenotypic analysis.
in successful trials, transcript levels were reduced to approximately 50-75% of control levels .
these fall within the wide range of 30-98% knockdown reported in previous feeding experiments in other insects , although it is important to note many differences in dsrna concentration, tissue, and species targeted in these experiments.
interestingly, the mechanism of systemic rnai are not well understood in insects, and may be somewhat variable.
lepidopteran genomes  do contain a putative ortholog of c. elegans sid- <dig>  which encodes a membrane transporter essential for systemic rnai in worms  <cit> .
drosophilids lack a sid- <dig> ortholog and notably, also lack systemic rnai in most tissues.
however, the role of insect sid- <dig> family genes is unclear; in tribolium they are apparently dispensable for systemic rnai  <cit> .
further, there are differences in the complement of rnai effectors in the genomes of drosophila, bombyx and tribolium suggesting that there could be some differences in rnai mechanisms across insects  <cit> .
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 detect even small differences in taste or olfactory sensitivity, 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 informative phenotypes in several recent cases in insects .
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, physiological and ecological data exists .
the ability to pair the identification and functional analysis of chemosensory genes with well-established behavioral, electrophysiological, and molecular methods in this species is likely to significantly advance our understanding of insect chemosensation.
CONCLUSIONS
chemosensory systems and the gene families that mediate chemosensory responses evolve rapidly.
a molecular genetic understanding of insect chemosensation, including 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  <dig> chemosensory receptors  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.

 animals rely on their olfactory and gustatory systems to detect chemicals in their environments. chemosensation mediates essential behaviors such as locating food and shelter, avoiding predators, locating mates, and selecting appropriate sites for nesting or laying eggs. the chemosensory organs and the gene families that encode chemosensory proteins are different in vertebrates and insects, but the underlying logic is in some respects quite similar. the molecular basis of insect chemosensation is best understood in drosophila. responses to gustatory and olfactory cues are mediated by the olfactory and gustatory receptors, which together comprise an insect chemoreceptor super-family. receptor protein complexes function as ionotropic membrane channels, whose precise mechanism of action is still under investigation. or and gr receptors are expressed in olfactory receptor neurons and gustatory receptor neurons respectively. most orns express a single conventional or, and the range of compounds to which they respond depends on the tuning of that receptor. grns, in contrast, can express multiple grs. the overall variety of chemical cues an insect can detect is the result of the diversity of its receptors. there are chemoreceptor genes in the drosophila melanogaster genome which encode different proteins. additional gene families, encoding odorant binding proteins and chemosensory proteins also contribute to taste and olfaction. obps are small, water-soluble extracellular proteins within the lymphatic cavity of olfactory sensilla that facilitate ligand binding to ors. 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. although the gene families encoding chemosensory proteins are conserved across all insects, both chemosensory gene families and the neuroanatomy of chemosensory systems evolve rapidly. correspondingly, insects show an extraordinary diversity of olfactory and gustatory responses. 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. however, outside of drosophila melanogaster, virtually nothing is known about the function of chemosensory genes, and very few studies have linked specific receptors or lineage-specific genetic changes to particular chemical responses, behaviors or ecology. such characterization will require 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 is a major agricultural pest that feeds on solanaceous plants. manduca sexta has long been an important non-genetic model organism for insect chemosensation, particularly from anatomical, neurophysiological, behavioral and ecological perspectives. recent analyses have identified ors and gr, as well as multiple obp and csp gene sequences from this species. despite this substantial progress, additional receptors remain to be identified, and a method for functional analysis of these genes is lacking. rna interference 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, but there have been no reports of rnai targeting neural tissue in this species. a previous report showed that peripheral chemosensory neurons could be targeted by feeding double-stranded rna to larvae of the moth epiphyas postvittana, suggesting that this approach could potentially be effective in m. sexta chemosensory tissue as well. here, we have taken significant steps to further the development 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, and we identified 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 method that can be used to knock down expression of a chemoreceptor transcript in larval olfactory neurons. although this method will need to be optimized for each gene tested, and physiological assays developed, these findings will allow exploration of the functional role of specific chemosensory receptor proteins in m. sexta.  identification and analysis of m. sexta chemoreceptor genes in order to identify chemosensory genes from manduca, we performed transcriptome sequencing on mixed chemosensory tissues. from approximately assembled contigs, we identified with significant similarity to insect ors. after eliminating those with greater than 95% identity at the nucleotide level to previously identified m. sexta ors, we identified novel putative or genes. these were numbered randomly starting with msor building upon the ors previously identified in m. sexta. a neighbor-joining cluster analysis is shown in figure  which allows inference of potential orthology. of the putative ors, show greater sequence similarity to another m. sexta or than to a bombyx or, suggesting that they are the products of recent duplications.` by the same criteria, we identified contigs matching other insect grs, although contigs were redundant leaving putative gr genes from m. sexta. one is most closely related to bombyx gr6p, both of which show significant similarity to the dmgr5a trehalose receptor and the putative sugar co-receptor dmgr64f. the other corresponded to the single bitter gr previously identified. sequences corresponding to both gr genes were pcr amplified from genomic dna and cloned to confirm their sequences. of the contigs with significant similarity to csp and obp genes, none were unique to this study. this suggests that both gene families are essentially fully known in m. sexta. delivery of dsrna via feeding induces rna interference in larval antennae we targeted the m. sexta ortholog of the olfactory co-receptor orco 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. as has been previously reported, expression was higher in adult female antennae than in males. in contrast, in larval antennae, expression was approximately -fold higher in males than in females; this difference, though slight, was reproducible across different experiments. to minimize variation in endogenous expression, subsequent 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 quantitative rt-pcr after or 5 days. for each time-point, three separate samples were prepared from pooled antennal tissue of 2– larvae each. msex\orco expression 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. none of these differences was statistically significant. 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 maximal. as described above, each sample contained pooled antennal tissue from 2– larvae. in half of the trials, msex\orco levels were unaffected. in the other half of the trials, transcript levels were reduced following dsrna treatment, and ranged between 49-75% of control levels. despite this variability in success across trials, dsrna treatment led to a significant overall reduction in msex\orco transcript, according to a one-sample, one-tailed t-test. reduction in msex\orco transcript levels was specific to msex\orco dsrna, and was not a general effect of dsrna treatment. when larvae were fed an unrelated dsrna, corresponding to the olfactory receptor msor- msex\orco expression was not affected, averaging 106% compared to control samples. in the same samples, the targeted msor- transcript was reduced to an average of 44% compared to untreated controls. therefore, as discussed further below, feeding of dsrna can be used to induce rnai in larval chemosensory tissues. discussion manduca sexta has great potential as a model for exploring the functional aspects of chemosensation at the molecular 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 previously reported genes, means that several dozen chemosensory 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 peripheral neurons in larval antennae. although details of rnai assays will need to be optimized for each gene targeted, together, these results will prove useful for future functional studies of m. sexta chemosensory genes. the putative ors that we report here bring the total number of m. sexta or genes to. the m. sexta adult antennal lobe contains approximately glomeruli, suggesting that this is a likely approximate number of adult-specific odorant receptor genes, including both ors and irs. however, since individual chemoreceptor genes are expressed at low levels, and given known variation in their developmental expression patterns, 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 it will be interesting to determine whether the 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 transcriptome analysis focused on adult antennal tissue. together, the known m. sexta or genes provide a good set of targets to explore for future functional analyses. although potential bombyx orthologs are clear for many of the genes, many also appear to be the result of recent duplications. characterizing the function 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 drosophila sugar receptors also has great potential for functional analysis through rnai since the physiological and behavioral aspects of sugar response in larval m. sexta is well understood. for rnai, we targeted the putative ortholog of drosophila or83b, msex\orco, which encodes an olfactory co-receptor required for localization and function of conventional ors. 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. in contrast, expression was slightly higher in larval male than female antennae. the functional significance of these differences in expression, if any, is unknown. we observed significant reduction of msex\orco transcripts in larval antennae after feeding dsrna, indicating that systemic rnai can be used to target manduca chemosensory neurons in vivo. knockdown was statistically 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 rps expression was observed across biological replicates. among control samples collected at the same stage, expression ratios varied more than three-fold. the source of this variation is unknown. importantly, however, we only observed two results in any rnai trial: expression in paired control vs. treated samples was either approximately equal, or it was markedly lower in dsrna treated samples. the reverse 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 account 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, 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. although animals in our assay were starved overnight prior to dsrna delivery, there could have been variation in how much food they consumed following treatment, as they were offered ad libitum access to the artificial 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 phenotypic analysis. in successful trials, transcript levels were reduced to approximately 50-75% of control levels. these fall within the wide range of 30-98% knockdown reported in previous feeding experiments in other insects, although it is important to note many differences in dsrna concentration, tissue, and species targeted in these experiments. interestingly, the mechanism of systemic rnai are not well understood in insects, and may be somewhat variable. lepidopteran genomes do contain a putative ortholog of c. elegans sid- which encodes a membrane transporter essential for systemic rnai in worms. drosophilids lack a sid- ortholog and notably, also lack systemic rnai in most tissues. however, the role of insect sid- family genes is unclear; in tribolium they are apparently dispensable for systemic rnai. further, there are differences in the complement of rnai effectors in the genomes of drosophila, bombyx and tribolium suggesting that there could be some differences in rnai mechanisms across insects. 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 detect even small differences in taste or olfactory sensitivity, 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 informative phenotypes in several recent cases in insects. 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, physiological and ecological data exists. the ability to pair the identification and functional analysis of chemosensory genes with well-established behavioral, electrophysiological, and molecular methods in this species is likely to significantly advance our understanding of insect chemosensation.  chemosensory systems and the gene families that mediate chemosensory responses evolve rapidly. a molecular genetic understanding of insect chemosensation, including 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 chemosensory receptors 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. 
we also used dsrna feeding as a method to induce rna interference in larval chemosensory tissues. further, we demonstrate that systemic rna interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. the tobacco hornworm, manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. together, our results further the development of m. sexta as a model for functional analysis of insect chemosensation. the ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in m. sexta would be greatly beneficial. we report identification of new chemosensory receptor genes including novel odorant receptors and one novel gustatory receptor. it is also a major agricultural pest on solanaceous crops.
 insects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. although our understanding of the molecular genetic basis for drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. the tobacco hornworm, manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. it is also a major agricultural pest on solanaceous crops. however, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. the ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in m. sexta would be greatly beneficial.  here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. we also used dsrna feeding as a method to induce rna interference in larval chemosensory tissues.  we report identification of new chemosensory receptor genes including novel odorant receptors and one novel gustatory receptor. further, we demonstrate that systemic rna interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. together, our results further the development of m. sexta as a model for functional analysis of insect chemosensation. 