BACKGROUND
in insects, volatile chemical cues  provide information about food, reproduction  <cit> , host selection, oviposition   <cit> , and toxic compound avoidance  <cit> .
as with other insects, olfactory-mediated behaviours are central to the ecology of blowflies  <cit> .
characterised by their larvae’s ability to develop on flesh, blowflies have significant roles in forensic entomology  <cit> , agriculture  <cit> , and medicine  <cit> , and rely heavily on the detection of mammalian decomposition odours.
the decomposition process produces a diverse array of inorganic gases and volatile organic compounds .
the vocs produced cover almost all chemical families, including cyclic and non-cyclic hydrocarbons, oxygenated compounds , nitrogen and sulfur containing compounds, acids/esters, halogens, and ethers  <cit> .
it is believed that blowflies are capable of detecting decomposition odour immediately after death  <cit> .
while it is acknowledged and accepted that chemical cues play a significant role in mediating many aspects of blowfly behaviour, the specific odours responsible are still to be elucidated.
behavioural and physiological studies have shown the olfactory attraction of blowflies to whole host samples   <cit>  as well as their ability to detect  individual odour molecules emitted from those samples  <cit> .
of the numerous vocs available for detection, sulfur compounds appear to be some of the most important.
sulfides are consistently identified within the odour space of mammalian decomposition  <cit> , are produced by plants that mimic decomposition odour  <cit> , and have been shown to elicit physiological and behavioural responses in a variety of blowfly species  <cit> .
knowledge regarding the underlying molecular mechanisms regulating blowfly olfaction, however, is severely limited.
insects, including blowflies, sense odours via olfactory receptor neurons  that are housed within chemosensory sensilla located primarily on the antennae and, to a lesser extent, the maxillary palps   <cit> .
olfactory signal transduction – where environmental chemical signals are converted into electrical signals interpreted by the nervous system – starts with the binding of odour molecules by receptor proteins bound to orn dendrites.
there are three antennal receptor protein gene families that bind odour molecules, namely odorant receptors   <cit> , gustatory receptors   <cit> , and ionotropic receptors   <cit> .
several non-receptor protein families have also been identified to be involved in invertebrate olfaction, including: sensory neuron membrane proteins   <cit> ; odorant binding proteins   <cit> ; chemosensory proteins   <cit> ; and odorant degrading enzymes   <cit> .
extensive information regarding the genes involved in olfaction, including their characteristics and potential roles, are provided by  <cit> .figure  <dig>
scanning electron micrograph of the head of a male
c. stygia
.
the main olfactory organs, the antennae  and maxillary palps , are located between the compound eyes  and the base of the proboscis , respectively.
scale bar: 1 mm.
because of the sequence diversity of olfactory genes, their identification has largely been only possible with insects for which genomic data is available  <cit> .
however, recent advances in rna-seq and computational technologies have opened up such identifications in non-model organisms.
this has resulted in the identification of olfactory genes in a wide range of insects for which no sequenced genome is available  <cit> .
with respect to blowflies, very few chemosensory genes have been identified  <cit> .
for example, the co-receptor crucial for the appropriate function of ligand-binding ors has been identified in various blowfly species  <cit>  along with two candidate ligand-binding ors  <cit> .
candidate genes from the obp, ir, and gr families have also been identified  <cit> .
however, no research regarding the functional characterisation of individual olfactory genes has been published and the ligands for these candidate genes remain unidentified.
this research investigated the antennal chemosensory gene families of the blowfly calliphora stygia   via transcriptomic analysis.
c. stygia is native to australia and is primarily carrion-dependent with respect to its feeding and reproductive behaviour  <cit> .
previous research with c. stygia has focused primarily on factors affecting its growth throughout its lifecycle  <cit> ; there is no information regarding its olfactory abilities.
identification of members of the primary gene families mediating insect olfaction permits a better understanding of the molecular basis of blowfly olfaction.
such knowledge could ultimately lead to the identification of new targets of control strategies  <cit> , an improved understanding of how blowflies recognise, locate, and colonise hosts, as well as improved methods for estimating post-mortem interval  <cit> .
RESULTS
antennal transcriptome
the combined trinity assembly of the male and female c. stygia antennal transcriptomes led to the generation of  <dig>  contigs, from which  <dig>  non-redundant putative transcripts were predicted.
searches against the ncbi non-redundant protein database returned  <dig>  transcripts showing sequence similarity to known proteins .
of these,  <dig>   were assigned at least one go term .
there was no significant difference between the male and female data sets with respect to go annotation therefore the male and female data sets are presented together.
the most abundant go term associations were in relation to basic cell functions; however, go terms associated with olfaction  and enzyme activity  were also represented within the data sets.
the large number of transcripts without associated go terms  potentially represent orphan genes.figure  <dig>
distribution of
c. stygia
antennal transcriptome data in go terms.
go analysis of  <dig>   transcripts for their predicted involvement in molecular functions  and biological processes  or as cellular components .
go categorisation for molecular functions is presented at level  <dig> and at level  <dig> for biological processes and cellular components.
annotated genes are depicted as percentages of the total number of transcripts with go term assignments.
identification of candidate odorant receptors
analysis of the c. stygia antennal transcriptomes identified  <dig> and  <dig> candidate or proteins in the male and female data sets respectively .
additional file 2: table s <dig> summarises transcript name, length, best blastx hit, predicted domains, and male or female specificity.
twenty-four of the putative cstyors likely represent full-length sequences.
the majority of partial length transcripts possess overlapping regions with low amino acid sequence identity, which indicates that they represent separate individual proteins.
however, the possibility that the remaining non-overlapping transcripts represent fragments of individual proteins cannot be excluded; therefore, based on sequence alignments and subsequent fragment location , the total number of cstyors reported could be reduced by two.
consistent with the diversity of the or gene family , full length putative c. stygia ors shared between 9% and 49% amino acid identity .
predictive software also indicated full-length candidate cstyor transcripts possess between three and eight transmembrane domains.
depending on the length of the partial transcripts, the remaining cstyors were predicted to have zero to seven transmembrane domains .
importantly, the highly conserved co-receptor orco was identified in the c. stygia transcriptomes, sharing ~88% to ~99% amino acid sequence identity with orco’s from drosophila melanogaster and other blowfly species.
as expected, greater sequence identity was observed with other blowfly orco’s than with drosophila .
putative d. melanogaster orthologues could be assigned for the majority of the presumably ligand-binding cstyors; however,  <dig> appear to have no d. melanogaster counterpart.
of these  <dig>  ten could be assigned putative orthologues  in other species, including anopheles gambiae bombyx mori, danaus plexippus, and other drosophila species.
interestingly, a putative orthologue of dmelor67d, a pheromone specific receptor, was identified in c. stygia .figure  <dig>
odorant and gustatory receptors.
maximum-likelihood tree of candidate c. stygia ors and grs  with ors and grs  identified in drosophila melanogaster  and various blowfly species, including musca domestica , calliphora vicina , chrysomya megacephala , chrysomya rufifacies , lucilia sericata , and stomoxys calcitrans  .
distinct orco and gr clusters are apparent.
in absolute terms , there were no significant differences in the number of candidate or proteins identified in the respective male and female data sets.
quantitative differences in the relative transcript abundances were observed .
of the identified candidate or transcripts, nine appear to be enriched  in the female data set, while  <dig> are enriched in the male data set.
identification of candidate gustatory receptors
twenty-one candidate gr transcripts were identified in the combined male and female c. stygia transcriptomes  .
the majority of candidate cstygrs were partial fragments , encoding overlapping but distinct sequences.
this establishes the proteins as being fragments of independent genes.
consistent with other insect grs  <cit> , transmembrane domain and topology predictions in full-length transcripts indicated between six and eight domains with an intracellular n-terminus and extracellular c-terminus being the most likely configuration.
the eight candidate cstygr transcripts included in the phylogenetic analysis formed a distinct clade ; none clustered within the or clades thus indicating that the transcripts are more related to grs than ors.
the cstygrs were also observed to group with their presumed drosophila orthologues, which have been shown to have roles in carbon dioxide detection   <cit>  and thermosensation   <cit> , or are members of the candidate sugar gr <dig> receptor subfamily   <cit> .
several of the partial length candidate cstygrs also show high sequence amino acid similarity to known sugar  and bitter  drosophila receptors .
identification of candidate ionotropic receptors
twenty-two candidate irs were identified in both the male and female c. stygia antennal transcriptomes .
structural analysis and amino acid sequence alignments revealed that most candidate cstyirs shared the structural organisation of insect irs and iglurs  .
the most conserved sequence regions were the three transmembrane domains and the ion channel pore   <cit> .
characteristic variability of the glutamate-binding residues located in the ligand-binding s <dig> and s <dig> domains was also present .
only two cstyirs  retain all residues characteristic of iglurs   <cit> ; all other irs have a diversity of amino acids at one or more of these positions indicating variable ligand binding properties.
however, it should be noted that some of the putative cstyirs with incomplete sequences could not be assessed for the presence of these crucial residues.figure  <dig>
ionotropic receptors.
predicted protein domain organisation of irs and iglurs/ir co-receptors illustrated in diagram  and linear  form with associated pfam predicted domains .
mafft amino acid alignment of the ion channel pore  and second transmembrane  domains of candidate c. stygia irs  and drosophila melanogaster irs and iglurs .
ligand-binding s <dig> and s <dig> domains.
important glutamate-interacting residues are lacking in the ligand binding domains of most c. stygia ir candidates.
mafft amino acid alignments of the s <dig>  and part of s <dig>  ligand binding domains of candidate c. stygia irs and drosophila melanogaster irs and iglurs.
the key binding residues in iglurs are boxed.
phylogenetic analysis revealed that the candidate cstyirs were more closely related to irs then iglurs, with all candidate cstyirs assessed clustering with their presumed “antennal” orthologues .
this analysis identified representatives from  <dig> of the  <dig> orthologous “antennal” ir groups conserved across the protostome species analysed by croset et al.
<cit> .
thus, orthologues of the remaining three conserved groups  are either lacking from the c. stygia transcriptome assembly  or are yet to be identified within the putative cstyirs represented by partial sequences .
notably, transcripts putatively encoding ir8a, ir25a, and ir76b – which are thought to function as ir co-receptors  <cit>  – were found in c. stygia antennae.
no candidate cstyirs clustered within the “divergent” clade.figure  <dig>
phylogenetic tree of a selection of dipteran irs.
neighbour-joining tree of candidate c. stygia irs  with irs from anopheles gambiae , all iglurs and irs from drosophila melanogaster  and the single ir identified in musca domestica .
the identified ir candidates of c. stygia cluster with their presumed orthologues within the “antennal ir” clades and the ir25a/8a subgroup.
identification of candidate sensory neuron membrane proteins
analysis of the male and female c. stygia antennal transcriptomes identified three candidate snmps present in both data sets , two of which  likely represent full-length genes.
notably, a putative orthologue to the d. melanogaster protein, snmp <dig>  which has been shown to have an important role in pheromone detection  <cit> , was present in the c. stygia data sets .figure  <dig>
phylogenetic relationships of snmps.
maximum-likelihood tree of candidate c. stygia snmps  with snmps from drosophila melanogaster  and anopheles gambiae .
identification of putative odorant binding proteins
twenty-eight candidate obp transcripts were identified in c. stygia , all of which were present in both the male and female data sets .
of the  <dig> full-length cstyobps,  <dig> exhibited the classic arrangement of conserved six-cysteines,  <dig> was the plus-c gene motif , and  <dig> were minus-c    <cit> .
a further  <dig> classical,  <dig> plus-c , and  <dig> minus-c  type transcripts could be allocated from the partial cstyobp transcripts.
notably, while the cstyobps classified as minus-c do lack one or more of the cysteine residues in the conserved classic locations, additional cysteines  were present in nearby positions that may act as an alternative.figure  <dig>
candidate
c. stygia
obps exhibit all three motif sub-types.
the conserved six cysteines present in the classic motif are boxed.
the potential alternative conserved cysteines in the minus-c sub-group and the additional cysteines present in the plus-c motif are in bold text.
phylogenetic analysis revealed that all candidate cstyobps clustered in accordance with their respective sub-families .
this analysis also indicated that d. melanogaster orthologues were likely to be present for many of the putative cstyobps, although a few small c. stygia specific clades potentially indicate a level of divergence within the blowfly.
the low average amino acid identity exhibited by the candidate cstyobps is consistent with that of other species  <cit>  and aligns with the notion that their role involves interacting with a range of diverse odour molecules.
interestingly, one particular candidate cstyobp shared significant amino acid sequence identity to the d. melanogaster obp lush  , which, in addition to its role in driving the avoidance of high alcohol concentrations  <cit> , has been shown to play a role in pheromone sensitivity  <cit> .figure  <dig>
odorant binding proteins.
phylogenetic tree of candidate cstyobps  with drosophila melanogaster obps .
cstyobps cluster within their respective obp sub-families with classic cstyobps spread throughout various clades.
squares indicate the putative blowfly specific clades related to dmelobp22a and dmelobp19b-d.
identification of candidate chemosensory proteins
four transcripts encoding candidate csps were identified in both the male and female c. stygia transcriptomes , three of which likely represent full-length proteins .
all of the identified amino acid sequences possessed a signal peptide and the highly conserved four-cysteine profile .figure  <dig>
alignment of candidate
c. stygia
csps.
all candidate c. stygia csps exhibit the highly conserved four-cysteine profile.
the four conserved cysteine residues are boxed.
identification of candidate odorant degrading enzymes
go annotation of the transcriptomes indicated an enrichment of proteins involved in catalytic activity.
further analysis of the combined male and female transcripts identified  <dig> candidate cytochrome p450s and esterases .
of the  <dig> candidate esterases ,  <dig> likely represent full-length sequences.
ninety-three of the  <dig> candidate p450s were present in both sexes with  <dig> sequences predicted to be full-length.
phylogenetic analysis revealed that the candidate cstyests clustered within all three of the major functional groups of the esterase gene family  .
this indicates that the cstyests have possible functions in neurodevelopment , detoxification , and hormone and pheromone processing   <cit> .
this analysis also indicated that d. melanogaster orthologues were likely to be present for many of the candidate cstyests, with no apparent c. stygia specific clades or expansions.
candidate cstycyps were also distributed throughout the four major cytochrome p <dig> gene family groups, specifically the cyp <dig>  cyp <dig>  cyp <dig>  and mitochondrial clades  .
genes from the cyp <dig> clade, in which the majority of cstycyps clustered , have been shown to be involved in xenobiotic metabolism and insecticide resistance  <cit> .
additionally, some genes from the cyp <dig> clade have been associated with the metabolism of odorants or pheromones  <cit> .
candidate nadph-cytochrome reductases, proteins required for the reduction of p450s, were also identified.
notably, several of the candidate enzymes shared significant amino acid sequence identity to cyp450s and esterases specifically associated with pesticide resistance and detoxification.
for example, the candidate esterase cstyest <dig> was a best blastx hit to lucilia cuprina’s e <dig> , an enzyme shown to be a prime candidate for pesticide resistance  <cit> .
additionally, cstycyp <dig> was a reciprocal best hit to d. melanogaster’s cyp6g <dig>  which has been associated with ddt resistance  <cit> .
while it is possible that many of the candidate enzyme transcripts are odes, significant biochemical analysis is necessary to identify their specific physiological roles.figure  <dig>
phylogenetic tree of esterases.
maximum-likelihood tree of candidate c. stygia esterases  with esterases from drosophila melanogaster .
the candidate esterases identified in c. stygia are spread throughout all three of the major esterase gene family groups  <cit> , indicating a variety of possible antennal functions.
cytochrome p450s.
maximum-likelihood tree of candidate c. stygia cytochrome p450s  with drosophila melanogaster cytochrome p450s .
the identified cypp <dig> candidates from c. stygia cluster within the four distinct clades .
candidate nadh-cytochrome reductases were also identified.
discussion
this research represents the first comprehensive analysis of a blowfly antennal transcriptome for the purpose of identifying members of the major chemosensory gene families necessary for olfaction.
the reported gene sets therefore represent a significant addition to the data regarding the molecular basis of blowfly olfaction.
due to the importance of olfaction in blowfly behaviour, the identified genes could represent novel targets for future population control methods as well as providing opportunities for improving post-mortem interval estimations through a greater understanding of the odour related factors that favour or inhibit blowfly detection and colonisation.
classification of predicted functions of the male and female c. stygia transcripts via go assignment produced similar results as those obtained for other invertebrates  <cit> .
the number of individual candidate transcripts identified for many of the olfactory gene families is also comparable to those of other dipteran, coleopteran, and lepidopteran species for which the antennal transcriptome has been examined  <cit> .
without considering the potential significance of individual genes to each species studied, the similarity of the different data sets does indicate a certain level of antennal conservation with respect to gene expression.
interestingly, the  <dig> candidate ors identified in c. stygia show greater similarity to the numbers identified in lepitopteran species   <cit>  than to the closer related d. melanogaster   <cit> .
additionally, the number of candidate c. stygia grs  and irs  is greater than those reported for most species  <cit> .
while these studies also analysed antennal transcriptomes, variations in the number of transcripts identified could arise from differences in the sequencing methods, sequencing depth, and/or sample preparation.
the greater number of candidate ligand-binding transcripts annotated in c. stygia could be due to ecological differences; however, further research is required to determine the specific reason such differences may exist.
typically, invertebrates with large numbers of antennal grs use their antennae for tasting purposes as well as for olfaction   <cit> .
however, there are no reports of blowflies exhibiting such behaviour.
interestingly, recent transcriptomic analysis of a. gambiae also identified grs in addition to the previously identified carbon dioxide receptors  <cit> .
while primarily linked to the detection of tastants  <cit> , the c. stygia and a. gambiae transcriptomes, and the increasing range of “non-gustatory” sensory functions being identified for these proteins  <cit> , suggests antennal grs could have far more diverse roles.
functional analysis of d. melanogaster irs has demonstrated their role in the detection of amines and acids  <cit> , which are significant compounds emitted during biological decomposition  <cit> .
candidate c. stygia irs putatively orthologous to d. melanogaster irs shown to respond to individual decomposition compounds   <cit>  were present amongst the male and female c. stygia data sets.
predicting the ligands to which olfactory receptors will respond based on empirical data from other receptors is problematic due to their extensive divergence.
therefore, determining receptor ligands can only be achieved experimentally.
ultimately, the reason for such a large number of ors, grs, and irs in c. stygia is unknown and additional molecular biology and functional experiments are required in order to confirm the expression and role of these genes.
overall, the comparable and/or greater number of genes identified within each of the olfactory gene families suggests that a comprehensive antennal data set has been obtained for c. stygia.
such results also illustrate the sensitivity and value of transcriptomic analysis via next generation rna-seq for non-model organisms.
notably, the c. stygia transcriptome data indicates that the chemosensory gene repertoire is largely similar in the male and female.
this indicates that male and female c. stygia share similar odour-coding capacity.
quantitatively, the range of relative expression levels  of the candidate ligand-binding ors in c. stygia, in relation to orco, are similar to those reported for other dipteran species  <cit> .
however, preliminary data suggests that there is a difference in the relative levels of expression of individual ors between male and female c. stygia .
therefore, while male and female antennae likely perceive similar odour stimuli, their sensitivities, and hence the odour significance to the male and female, may differ.
this is consistent with previous studies, which show that electrophysiological responses can be elicited from males and females by a particular odour  <cit> , while leading to sex-based behavioural differences  <cit> .
additional biological repeats and experimental validation  are required to confirm the expression data.
further research, such as in situ hybridization and single-sensilla recordings, would also be beneficial to determine the distribution and frequency of ors within the antennae.
sexually dimorphic expression of chemosensory genes could indicate roles in sex-specific behaviours, including those mediated by pheromones.
invertebrate pheromone detection mediates various behaviours including aggregation, mate recognition, and sexual behaviour  <cit> .
in d. melanogaster, reception of the male volatile pheromone, cis-vaccenyl acetate, is achieved with lush, or67d, and snmp <dig>  <cit> .
the identification of putative orthologues of these three proteins in c. stygia could indicate potential functional pheromone detection in this species.
proteins sharing high sequence similarity to lush and dmelor67d have also been identified in stomoxys calcitrans   <cit> .
candidate blowfly pheromones have been described for several species  <cit> ; however, all have been non-volatile cuticular hydrocarbons and therefore are more likely to be contact pheromones.
additionally, the mechanisms that allow pheromone reception in blowflies are not yet known.
further characterisation of the candidate c. stygia proteins is required to determine if they exhibit similar chemosensory roles to the d. melanogaster genes or if they have adapted different functions.
CONCLUSIONS
a total of  <dig> transcripts encoding putative chemosensory proteins from the seven major olfactory gene families were annotated in the blowfly c. stygia.
the transcriptomic approach proved to be a highly effective strategy for the identification of divergent blowfly chemosensory receptors for which no genomic data is publically available.
comparative analysis with other species suggests that near-complete information regarding the molecular basis of c. stygia olfaction was obtained.
this research greatly improves the gene inventory for c. stygia and provides a valuable resource for future analysis on blowfly olfaction.
such information will be fundamental for future comparative analyses that could highlight interspecies differences underlying ecological differences and genetic adaption.

 in insects, volatile chemical cues provide information about food, reproduction, host selection, oviposition, and toxic compound avoidance. as with other insects, olfactory-mediated behaviours are central to the ecology of blowflies. characterised by their larvae’s ability to develop on flesh, blowflies have significant roles in forensic entomology, agriculture, and medicine, and rely heavily on the detection of mammalian decomposition odours. the decomposition process produces a diverse array of inorganic gases and volatile organic compounds. the vocs produced cover almost all chemical families, including cyclic and non-cyclic hydrocarbons, oxygenated compounds, nitrogen and sulfur containing compounds, acids/esters, halogens, and ethers. it is believed that blowflies are capable of detecting decomposition odour immediately after death. while it is acknowledged and accepted that chemical cues play a significant role in mediating many aspects of blowfly behaviour, the specific odours responsible are still to be elucidated. behavioural and physiological studies have shown the olfactory attraction of blowflies to whole host samples as well as their ability to detect individual odour molecules emitted from those samples. of the numerous vocs available for detection, sulfur compounds appear to be some of the most important. sulfides are consistently identified within the odour space of mammalian decomposition, are produced by plants that mimic decomposition odour, and have been shown to elicit physiological and behavioural responses in a variety of blowfly species. knowledge regarding the underlying molecular mechanisms regulating blowfly olfaction, however, is severely limited. insects, including blowflies, sense odours via olfactory receptor neurons that are housed within chemosensory sensilla located primarily on the antennae and, to a lesser extent, the maxillary palps. olfactory signal transduction – where environmental chemical signals are converted into electrical signals interpreted by the nervous system – starts with the binding of odour molecules by receptor proteins bound to orn dendrites. there are three antennal receptor protein gene families that bind odour molecules, namely odorant receptors, gustatory receptors, and ionotropic receptors. several non-receptor protein families have also been identified to be involved in invertebrate olfaction, including: sensory neuron membrane proteins; odorant binding proteins; chemosensory proteins; and odorant degrading enzymes. extensive information regarding the genes involved in olfaction, including their characteristics and potential roles, are provided by.figure scanning electron micrograph of the head of a male c. stygia. the main olfactory organs, the antennae and maxillary palps, are located between the compound eyes and the base of the proboscis, respectively. scale bar: 1 mm. because of the sequence diversity of olfactory genes, their identification has largely been only possible with insects for which genomic data is available. however, recent advances in rna-seq and computational technologies have opened up such identifications in non-model organisms. this has resulted in the identification of olfactory genes in a wide range of insects for which no sequenced genome is available. with respect to blowflies, very few chemosensory genes have been identified. for example, the co-receptor crucial for the appropriate function of ligand-binding ors has been identified in various blowfly species along with two candidate ligand-binding ors. candidate genes from the obp, ir, and gr families have also been identified. however, no research regarding the functional characterisation of individual olfactory genes has been published and the ligands for these candidate genes remain unidentified. this research investigated the antennal chemosensory gene families of the blowfly calliphora stygia via transcriptomic analysis. c. stygia is native to australia and is primarily carrion-dependent with respect to its feeding and reproductive behaviour. previous research with c. stygia has focused primarily on factors affecting its growth throughout its lifecycle; there is no information regarding its olfactory abilities. identification of members of the primary gene families mediating insect olfaction permits a better understanding of the molecular basis of blowfly olfaction. such knowledge could ultimately lead to the identification of new targets of control strategies, an improved understanding of how blowflies recognise, locate, and colonise hosts, as well as improved methods for estimating post-mortem interval.  antennal transcriptome the combined trinity assembly of the male and female c. stygia antennal transcriptomes led to the generation of contigs, from which non-redundant putative transcripts were predicted. searches against the ncbi non-redundant protein database returned transcripts showing sequence similarity to known proteins. of these, were assigned at least one go term. there was no significant difference between the male and female data sets with respect to go annotation therefore the male and female data sets are presented together. the most abundant go term associations were in relation to basic cell functions; however, go terms associated with olfaction and enzyme activity were also represented within the data sets. the large number of transcripts without associated go terms potentially represent orphan genes.figure distribution of c. stygia antennal transcriptome data in go terms. go analysis of transcripts for their predicted involvement in molecular functions and biological processes or as cellular components. go categorisation for molecular functions is presented at level and at level for biological processes and cellular components. annotated genes are depicted as percentages of the total number of transcripts with go term assignments. identification of candidate odorant receptors analysis of the c. stygia antennal transcriptomes identified and candidate or proteins in the male and female data sets respectively. additional file 2: table s summarises transcript name, length, best blastx hit, predicted domains, and male or female specificity. twenty-four of the putative cstyors likely represent full-length sequences. the majority of partial length transcripts possess overlapping regions with low amino acid sequence identity, which indicates that they represent separate individual proteins. however, the possibility that the remaining non-overlapping transcripts represent fragments of individual proteins cannot be excluded; therefore, based on sequence alignments and subsequent fragment location, the total number of cstyors reported could be reduced by two. consistent with the diversity of the or gene family, full length putative c. stygia ors shared between 9% and 49% amino acid identity. predictive software also indicated full-length candidate cstyor transcripts possess between three and eight transmembrane domains. depending on the length of the partial transcripts, the remaining cstyors were predicted to have zero to seven transmembrane domains. importantly, the highly conserved co-receptor orco was identified in the c. stygia transcriptomes, sharing ~88% to ~99% amino acid sequence identity with orco’s from drosophila melanogaster and other blowfly species. as expected, greater sequence identity was observed with other blowfly orco’s than with drosophila. putative d. melanogaster orthologues could be assigned for the majority of the presumably ligand-binding cstyors; however, appear to have no d. melanogaster counterpart. of these ten could be assigned putative orthologues in other species, including anopheles gambiae bombyx mori, danaus plexippus, and other drosophila species. interestingly, a putative orthologue of dmelor67d, a pheromone specific receptor, was identified in c. stygia.figure odorant and gustatory receptors. maximum-likelihood tree of candidate c. stygia ors and grs with ors and grs identified in drosophila melanogaster and various blowfly species, including musca domestica, calliphora vicina, chrysomya megacephala, chrysomya rufifacies, lucilia sericata, and stomoxys calcitrans. distinct orco and gr clusters are apparent. in absolute terms, there were no significant differences in the number of candidate or proteins identified in the respective male and female data sets. quantitative differences in the relative transcript abundances were observed. of the identified candidate or transcripts, nine appear to be enriched in the female data set, while are enriched in the male data set. identification of candidate gustatory receptors twenty-one candidate gr transcripts were identified in the combined male and female c. stygia transcriptomes. the majority of candidate cstygrs were partial fragments, encoding overlapping but distinct sequences. this establishes the proteins as being fragments of independent genes. consistent with other insect grs, transmembrane domain and topology predictions in full-length transcripts indicated between six and eight domains with an intracellular n-terminus and extracellular c-terminus being the most likely configuration. the eight candidate cstygr transcripts included in the phylogenetic analysis formed a distinct clade; none clustered within the or clades thus indicating that the transcripts are more related to grs than ors. the cstygrs were also observed to group with their presumed drosophila orthologues, which have been shown to have roles in carbon dioxide detection and thermosensation, or are members of the candidate sugar gr receptor subfamily. several of the partial length candidate cstygrs also show high sequence amino acid similarity to known sugar and bitter drosophila receptors. identification of candidate ionotropic receptors twenty-two candidate irs were identified in both the male and female c. stygia antennal transcriptomes. structural analysis and amino acid sequence alignments revealed that most candidate cstyirs shared the structural organisation of insect irs and iglurs. the most conserved sequence regions were the three transmembrane domains and the ion channel pore. characteristic variability of the glutamate-binding residues located in the ligand-binding s and s domains was also present. only two cstyirs retain all residues characteristic of iglurs; all other irs have a diversity of amino acids at one or more of these positions indicating variable ligand binding properties. however, it should be noted that some of the putative cstyirs with incomplete sequences could not be assessed for the presence of these crucial residues.figure ionotropic receptors. predicted protein domain organisation of irs and iglurs/ir co-receptors illustrated in diagram and linear form with associated pfam predicted domains. mafft amino acid alignment of the ion channel pore and second transmembrane domains of candidate c. stygia irs and drosophila melanogaster irs and iglurs. ligand-binding s and s domains. important glutamate-interacting residues are lacking in the ligand binding domains of most c. stygia ir candidates. mafft amino acid alignments of the s and part of s ligand binding domains of candidate c. stygia irs and drosophila melanogaster irs and iglurs. the key binding residues in iglurs are boxed. phylogenetic analysis revealed that the candidate cstyirs were more closely related to irs then iglurs, with all candidate cstyirs assessed clustering with their presumed “antennal” orthologues. this analysis identified representatives from of the orthologous “antennal” ir groups conserved across the protostome species analysed by croset et al.. thus, orthologues of the remaining three conserved groups are either lacking from the c. stygia transcriptome assembly or are yet to be identified within the putative cstyirs represented by partial sequences. notably, transcripts putatively encoding ir8a, ir25a, and ir76b – which are thought to function as ir co-receptors – were found in c. stygia antennae. no candidate cstyirs clustered within the “divergent” clade.figure phylogenetic tree of a selection of dipteran irs. neighbour-joining tree of candidate c. stygia irs with irs from anopheles gambiae, all iglurs and irs from drosophila melanogaster and the single ir identified in musca domestica. the identified ir candidates of c. stygia cluster with their presumed orthologues within the “antennal ir” clades and the ir25a/8a subgroup. identification of candidate sensory neuron membrane proteins analysis of the male and female c. stygia antennal transcriptomes identified three candidate snmps present in both data sets, two of which likely represent full-length genes. notably, a putative orthologue to the d. melanogaster protein, snmp which has been shown to have an important role in pheromone detection, was present in the c. stygia data sets.figure phylogenetic relationships of snmps. maximum-likelihood tree of candidate c. stygia snmps with snmps from drosophila melanogaster and anopheles gambiae. identification of putative odorant binding proteins twenty-eight candidate obp transcripts were identified in c. stygia, all of which were present in both the male and female data sets. of the full-length cstyobps, exhibited the classic arrangement of conserved six-cysteines, was the plus-c gene motif, and were minus-c. a further classical, plus-c, and minus-c type transcripts could be allocated from the partial cstyobp transcripts. notably, while the cstyobps classified as minus-c do lack one or more of the cysteine residues in the conserved classic locations, additional cysteines were present in nearby positions that may act as an alternative.figure candidate c. stygia obps exhibit all three motif sub-types. the conserved six cysteines present in the classic motif are boxed. the potential alternative conserved cysteines in the minus-c sub-group and the additional cysteines present in the plus-c motif are in bold text. phylogenetic analysis revealed that all candidate cstyobps clustered in accordance with their respective sub-families. this analysis also indicated that d. melanogaster orthologues were likely to be present for many of the putative cstyobps, although a few small c. stygia specific clades potentially indicate a level of divergence within the blowfly. the low average amino acid identity exhibited by the candidate cstyobps is consistent with that of other species and aligns with the notion that their role involves interacting with a range of diverse odour molecules. interestingly, one particular candidate cstyobp shared significant amino acid sequence identity to the d. melanogaster obp lush, which, in addition to its role in driving the avoidance of high alcohol concentrations, has been shown to play a role in pheromone sensitivity.figure odorant binding proteins. phylogenetic tree of candidate cstyobps with drosophila melanogaster obps. cstyobps cluster within their respective obp sub-families with classic cstyobps spread throughout various clades. squares indicate the putative blowfly specific clades related to dmelobp22a and dmelobp19b-d. identification of candidate chemosensory proteins four transcripts encoding candidate csps were identified in both the male and female c. stygia transcriptomes, three of which likely represent full-length proteins. all of the identified amino acid sequences possessed a signal peptide and the highly conserved four-cysteine profile.figure alignment of candidate c. stygia csps. all candidate c. stygia csps exhibit the highly conserved four-cysteine profile. the four conserved cysteine residues are boxed. identification of candidate odorant degrading enzymes go annotation of the transcriptomes indicated an enrichment of proteins involved in catalytic activity. further analysis of the combined male and female transcripts identified candidate cytochrome p450s and esterases. of the candidate esterases, likely represent full-length sequences. ninety-three of the candidate p450s were present in both sexes with sequences predicted to be full-length. phylogenetic analysis revealed that the candidate cstyests clustered within all three of the major functional groups of the esterase gene family. this indicates that the cstyests have possible functions in neurodevelopment, detoxification, and hormone and pheromone processing. this analysis also indicated that d. melanogaster orthologues were likely to be present for many of the candidate cstyests, with no apparent c. stygia specific clades or expansions. candidate cstycyps were also distributed throughout the four major cytochrome p gene family groups, specifically the cyp cyp cyp and mitochondrial clades. genes from the cyp clade, in which the majority of cstycyps clustered, have been shown to be involved in xenobiotic metabolism and insecticide resistance. additionally, some genes from the cyp clade have been associated with the metabolism of odorants or pheromones. candidate nadph-cytochrome reductases, proteins required for the reduction of p450s, were also identified. notably, several of the candidate enzymes shared significant amino acid sequence identity to cyp450s and esterases specifically associated with pesticide resistance and detoxification. for example, the candidate esterase cstyest was a best blastx hit to lucilia cuprina’s e, an enzyme shown to be a prime candidate for pesticide resistance. additionally, cstycyp was a reciprocal best hit to d. melanogaster’s cyp6g which has been associated with ddt resistance. while it is possible that many of the candidate enzyme transcripts are odes, significant biochemical analysis is necessary to identify their specific physiological roles.figure phylogenetic tree of esterases. maximum-likelihood tree of candidate c. stygia esterases with esterases from drosophila melanogaster. the candidate esterases identified in c. stygia are spread throughout all three of the major esterase gene family groups, indicating a variety of possible antennal functions. cytochrome p450s. maximum-likelihood tree of candidate c. stygia cytochrome p450s with drosophila melanogaster cytochrome p450s. the identified cypp candidates from c. stygia cluster within the four distinct clades. candidate nadh-cytochrome reductases were also identified. discussion this research represents the first comprehensive analysis of a blowfly antennal transcriptome for the purpose of identifying members of the major chemosensory gene families necessary for olfaction. the reported gene sets therefore represent a significant addition to the data regarding the molecular basis of blowfly olfaction. due to the importance of olfaction in blowfly behaviour, the identified genes could represent novel targets for future population control methods as well as providing opportunities for improving post-mortem interval estimations through a greater understanding of the odour related factors that favour or inhibit blowfly detection and colonisation. classification of predicted functions of the male and female c. stygia transcripts via go assignment produced similar results as those obtained for other invertebrates. the number of individual candidate transcripts identified for many of the olfactory gene families is also comparable to those of other dipteran, coleopteran, and lepidopteran species for which the antennal transcriptome has been examined. without considering the potential significance of individual genes to each species studied, the similarity of the different data sets does indicate a certain level of antennal conservation with respect to gene expression. interestingly, the candidate ors identified in c. stygia show greater similarity to the numbers identified in lepitopteran species than to the closer related d. melanogaster. additionally, the number of candidate c. stygia grs and irs is greater than those reported for most species. while these studies also analysed antennal transcriptomes, variations in the number of transcripts identified could arise from differences in the sequencing methods, sequencing depth, and/or sample preparation. the greater number of candidate ligand-binding transcripts annotated in c. stygia could be due to ecological differences; however, further research is required to determine the specific reason such differences may exist. typically, invertebrates with large numbers of antennal grs use their antennae for tasting purposes as well as for olfaction. however, there are no reports of blowflies exhibiting such behaviour. interestingly, recent transcriptomic analysis of a. gambiae also identified grs in addition to the previously identified carbon dioxide receptors. while primarily linked to the detection of tastants, the c. stygia and a. gambiae transcriptomes, and the increasing range of “non-gustatory” sensory functions being identified for these proteins, suggests antennal grs could have far more diverse roles. functional analysis of d. melanogaster irs has demonstrated their role in the detection of amines and acids, which are significant compounds emitted during biological decomposition. candidate c. stygia irs putatively orthologous to d. melanogaster irs shown to respond to individual decomposition compounds were present amongst the male and female c. stygia data sets. predicting the ligands to which olfactory receptors will respond based on empirical data from other receptors is problematic due to their extensive divergence. therefore, determining receptor ligands can only be achieved experimentally. ultimately, the reason for such a large number of ors, grs, and irs in c. stygia is unknown and additional molecular biology and functional experiments are required in order to confirm the expression and role of these genes. overall, the comparable and/or greater number of genes identified within each of the olfactory gene families suggests that a comprehensive antennal data set has been obtained for c. stygia. such results also illustrate the sensitivity and value of transcriptomic analysis via next generation rna-seq for non-model organisms. notably, the c. stygia transcriptome data indicates that the chemosensory gene repertoire is largely similar in the male and female. this indicates that male and female c. stygia share similar odour-coding capacity. quantitatively, the range of relative expression levels of the candidate ligand-binding ors in c. stygia, in relation to orco, are similar to those reported for other dipteran species. however, preliminary data suggests that there is a difference in the relative levels of expression of individual ors between male and female c. stygia. therefore, while male and female antennae likely perceive similar odour stimuli, their sensitivities, and hence the odour significance to the male and female, may differ. this is consistent with previous studies, which show that electrophysiological responses can be elicited from males and females by a particular odour, while leading to sex-based behavioural differences. additional biological repeats and experimental validation are required to confirm the expression data. further research, such as in situ hybridization and single-sensilla recordings, would also be beneficial to determine the distribution and frequency of ors within the antennae. sexually dimorphic expression of chemosensory genes could indicate roles in sex-specific behaviours, including those mediated by pheromones. invertebrate pheromone detection mediates various behaviours including aggregation, mate recognition, and sexual behaviour. in d. melanogaster, reception of the male volatile pheromone, cis-vaccenyl acetate, is achieved with lush, or67d, and snmp. the identification of putative orthologues of these three proteins in c. stygia could indicate potential functional pheromone detection in this species. proteins sharing high sequence similarity to lush and dmelor67d have also been identified in stomoxys calcitrans. candidate blowfly pheromones have been described for several species; however, all have been non-volatile cuticular hydrocarbons and therefore are more likely to be contact pheromones. additionally, the mechanisms that allow pheromone reception in blowflies are not yet known. further characterisation of the candidate c. stygia proteins is required to determine if they exhibit similar chemosensory roles to the d. melanogaster genes or if they have adapted different functions.  a total of transcripts encoding putative chemosensory proteins from the seven major olfactory gene families were annotated in the blowfly c. stygia. the transcriptomic approach proved to be a highly effective strategy for the identification of divergent blowfly chemosensory receptors for which no genomic data is publically available. comparative analysis with other species suggests that near-complete information regarding the molecular basis of c. stygia olfaction was obtained. this research greatly improves the gene inventory for c. stygia and provides a valuable resource for future analysis on blowfly olfaction. such information will be fundamental for future comparative analyses that could highlight interspecies differences underlying ecological differences and genetic adaption. 
using next generation sequencing, this research examined the antennal transcriptome of calliphora stygia to identify members of the major chemosensory gene families necessary for olfaction. calliphora stygiablowfly olfactiontranscriptomechemosensory proteinsodorant receptorsissue-copyright-statement© the author 2015 while it is widely accepted that blowflies rely heavily on olfaction for identifying and locating hosts, there is limited research regarding the underlying molecular mechanisms. putative orthologues for the conserved antennal ionotropic receptors and candidate gustatory receptors for carbon dioxide detection were also amongst the identified proteins. such information presents potential benefits to the forensic, pest control, and medical areas, and could assist in the understanding of insecticide resistance and targeted control through cross-species comparisons. representative proteins from all chemosensory gene families essential in insect olfaction were identified in the antennae of the blowfly c. stygia, including odorant receptors, ionotropic receptors, gustatory receptors, odorant binding proteins, chemosensory proteins, and sensory neuron membrane proteins.
 blowflies have relevance in areas of forensic science, agriculture, and medicine, primarily due to the ability of their larvae to develop on flesh. while it is widely accepted that blowflies rely heavily on olfaction for identifying and locating hosts, there is limited research regarding the underlying molecular mechanisms. using next generation sequencing, this research examined the antennal transcriptome of calliphora stygia to identify members of the major chemosensory gene families necessary for olfaction.  representative proteins from all chemosensory gene families essential in insect olfaction were identified in the antennae of the blowfly c. stygia, including odorant receptors, ionotropic receptors, gustatory receptors, odorant binding proteins, chemosensory proteins, and sensory neuron membrane proteins. a total of candidate cytochrome p450s and esterases, some of which may act as odorant degrading enzymes, were also identified. importantly, co-receptors necessary for the proper function of ligand-binding receptors were identified. putative orthologues for the conserved antennal ionotropic receptors and candidate gustatory receptors for carbon dioxide detection were also amongst the identified proteins.  this research provides a comprehensive novel resource that will be fundamental for future studies regarding blowfly olfaction. such information presents potential benefits to the forensic, pest control, and medical areas, and could assist in the understanding of insecticide resistance and targeted control through cross-species comparisons. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords calliphora stygiablowfly olfactiontranscriptomechemosensory proteinsodorant receptorsissue-copyright-statement© the author 2015 