BACKGROUND
the european spruce bark beetle, ips typographus l., and the north american mountain pine beetle, dendroctonus ponderosae hopkins  are serious pests of coniferous forests.
i. typographus mainly attacks norway spruce  in eurasia, whereas d. ponderosae infests several species of pine in western north america.
currently, large-scale d. ponderosae outbreaks have resulted in unprecedented economic losses and turned north american forests into major sources of carbon release  <cit> .
the olfactory sense drives bark beetle behaviors that are important for fitness, such as the localization of suitable hosts and mates  <cit> .
in the search for suitable host material, bark beetles respond to volatiles that emanate from both host and non-host plants  <cit> .
however, most individuals locate trees by means of an aggregation pheromone that is released by beetles that have already attacked the tree.
this signal is responsible for coordinated mass-attacks, which often lead to the death of the host tree  <cit>  and large-scale forest destruction.
due to their ecological and economic impact, an extensive knowledge base on bark beetle chemical ecology and olfactory physiology has been established  <cit> .
however, information on the molecular aspects of odor detection has been lacking until now.
in insects, volatile molecules are detected by olfactory sensory neurons  that are housed within special structures  predominantly on the antennae, and to a lesser extent on the maxillary palps.
the cell membrane of osns contains receptor proteins that bind odor ligands  <cit> .
the binding of a ligand to a receptor protein is the key event in olfactory transduction, as it converts a chemical signal in the environment into an electrical signal that can be interpreted by the insect nervous system  <cit> .
receptors from three large and divergent multigene families are expressed in insect osns  <cit> , namely the odorant receptors , ionotropic receptors , and gustatory receptors , the latter group notably containing carbon dioxide-detecting receptors  <cit> .
however, most grs are expressed in gustatory receptor neurons in taste organs and are involved in contact chemoreception.
these grs typically detect different sugars, bitter compounds, and contact pheromones  <cit> .
insect ors are seven-transmembrane domain proteins  <cit>  with a reversed membrane topology  compared to vertebrate ors, which are g-protein coupled receptors  <cit> .
insect ors and grs are distantly related members of the same superfamily  <cit> .
in general, ors  show little sequence homology to each other and they are unrelated to vertebrate ors.
the conventional exchangeable or that determines ligand specificity  <cit>  forms heteromers of unknown stoichiometry with a conserved co-receptor, known as orco  <cit> .
orco is ubiquitously expressed in osns that express ors  <cit>  and necessary for olfactory responses and for localization of the conventional or in the cell membrane  <cit> .
putative insect ors have been identified mainly in species with sequenced genomes  <cit> .
recently, however, studies on antennal transcriptomes have led to the identification of or sets in several moth species  <cit>  and one beetle  <cit> .
the ors respond to a variety of volatile chemicals  <cit> , including pheromones  <cit>  and plant or microbe-derived compounds  <cit> .
some ors are highly defined in their response specificity  <cit> , whereas others appear more broadly tuned, especially at high stimulus concentrations  <cit> .
irs were recently discovered as another class of receptors involved in chemoreception  <cit> .
they are related to ionotropic glutamate receptors  that function in synapse communication, but have atypical binding domains.
irs have been identified throughout protostome lineages  and, thus, constitute a far more ancient group of receptors than the ors  <cit> .
irs form complexes with up to three subunits, including odor-specific receptors and one or two broadly expressed co-receptors  <cit> .
in insects, the irs are divided into two major groups: the “antennal irs” that have an olfactory function and are conserved across insect orders, and the species-specific “divergent irs”, some of which have been assigned a tentative role in taste  <cit> .
antennal irs in drosophila have different odor specificity compared to the ors and respond to nitrogen-containing compounds , acids, and aromatics   <cit> .
in addition to the receptor genes, other multigene families encode proteins with critical roles in olfaction.
odorant binding proteins  are small soluble proteins  with two or three disulfide bridges  <cit> .
obps are highly abundant in the sensillar lymph of insects and are thought to solubilize hydrophobic molecules and deliver them to the receptors  <cit> .
studies have shown conflicting results whether or not obps affect the response specificity of osns  <cit> .
at least in some studies, the specificity of pheromone receptors was improved by the presence of obps   <cit> .
some evidence suggests that obps might undergo odor-induced conformational changes, with a change in the obp itself triggering the response of the osn  <cit> .
in insects with sequenced genomes, the number of obp coding genes normally ranges from ca.
40– <dig>  <cit> .
chemosensory proteins  constitute another class of small binding proteins .
they are more conserved than obps and are characterized by the presence of  <dig> cysteines that form two disulfide bridges  <cit> .
csps may have shared a common ancestor with the obps near the origin of the arthropods  <cit> .
like obps, csps are present in high concentration in chemosensory sensilla .
however, the majority of them are also expressed in various non-sensory tissues  <cit>  and they seem to play a role in development, moulting  <cit> , and leg regeneration  <cit> .
some csps bind pheromone compounds  <cit> , but their exact role in chemosensory systems remains uncertain  <cit> .
most drosophila genomes contain only  <dig> csp coding genes and t. castaneum has  <dig>  <cit> .
the genome of aedes aegypti mosquitoes contains  <dig> members of this family, the largest number found in insects so far  <cit> .
finally, the sensory neuron membrane proteins  are proteins of the cd <dig> family that associate with pheromone-responding osns  <cit> .
their functional significance is still poorly understood, but snmp is crucial for proper pheromone detection in d. melanogaster and also required for activation of a pheromone receptor in heliothis virescens moths  <cit> .
in contrast, snmp was dispensable for responses of a fly receptor  to fruit-related esters  <cit> .
insects generally have two representatives of snmps , although copy numbers of each orthologue seem to vary somewhat across species  <cit> .
in the largest insect order, coleoptera, ors have been identified from only two species: from the genome of the red flour beetle, tribolium castaneum <cit> , and recently from the antennal transcriptome of the cerambycid beetle, megacyllene caryae <cit> .
members of the other olfactory gene families have been identified only in t. castaneum <cit> .
consequently, additional beetle species need to be investigated to reach a better understanding on the molecular biology of coleopteran and insect olfaction.
in this study, we assembled and analyzed bark beetle antennal transcriptomes from next-generation sequencing.
we report the results from gene ontology  annotation as well as sets of putative obps, csps, snmps, ors, grs, and irs in i. typographus and d. ponderosae.
identification of the chemosensory genes in these devastating insect pests is especially relevant because of their potential as novel targets for pest control.
methods
mountain pine beetle
the source of d. ponderosae  antennal tissue and the method of sequencing have been reported previously in a larger transcriptome study  <cit> .
from this transcriptome dataset originating from several tissue types, sequences originating only from a non-normalized antenna-specific cdna library were re-assembled for the analyses presented in the present study.
this included  <dig>  paired-end sanger reads,  <dig>  single-end sanger reads and  <dig> , <dig> roche  <dig> reads.
newbler  was used for assembly using the "-cdna" switch for transcriptome assemblies and a  <dig> bp cutoff to eliminate short reads.
for identification of obps, csps, and snmps in d. ponderosae we also used a combination of sanger-specific  data and transcriptome assemblies from other tissues and life stages, since these proteins may have sensory or non-sensory functions in non-antennal tissues.
we did not have such assemblies for i. typographus.
european spruce bark beetle
insects, rna extraction and cdna synthesis
i. typographus was reared on norway spruce  logs in an environmental chamber   <cit> , starting from individuals collected from their natural habitat near asa and Älmhult, southern sweden.
emerged adults were kept in a state of low activity in a refrigerator  before being used for rna extraction.
two hundred adult i. typographus were collected in a  <dig> ml plastic tube, approximately two weeks after their emergence.
the tube was submerged in liquid nitrogen, after which it was vigorously shaken using a vortex shaker to separate extremities from the body.
body parts were suspended in -20°c acetone  and passed through meshes that filtered out the antennae.
after removal of the acetone,  <dig>  ml tri reagent  was added to the antennae and the sample was homogenized using a tissue-tearor .
total rna was extracted following the trizol protocol, but using 1-bromo-3-chloropropane instead of chloroform.
<dig>  μg total rna was sent to evrogen  for synthesis of duplex-specific-nuclease normalized cdna  <cit> .
sequencing and assembly
the i. typographus cdna was sequenced at lgc genomics , using  <dig> gs/flx sequencing  with titanium chemistry, to produce  <dig>  reads for a total of  <dig> megabases.
furthermore, illumina sequencing was performed at the max planck institute for molecular genetics in berlin to generate a further  <dig>  million reads for a total of  <dig> megabases.
short or low-quality reads, as well as linker and adapter sequences were removed by the crossmatch program  or by the built-in sequence cleanup of seqman ngen .
the  <dig> reads were assembled using seqman ngen to generate a backbone; subsequently, the illumina reads were mapped onto this backbone using seqman ngen to correct for technology-inherent read errors.
the resultant contigs were annotated using a codequest workstation   <cit> .
annotation
for an initial assessment of the two assembled beetle antennal transcriptomes, gene ontology  annotation was performed using blast2go  <cit> .
blast2go annotation associates genes or transcripts with go terms using hierarchical vocabularies.
genes are described in terms related to molecular function, biological process, or cellular component, allowing for meta-analyses of gene populations  <cit> .
the blast step was performed with a lenient e-value cutoff at  <dig>  to account for the high sequence variability among the olfactory gene families.
the mapping step was done using default settings, whereas a lenient e-value  and lower annotation cut-off  and go-weight  were used in the first annotation step to increase the proportion of annotated transcripts.
annotation was further enhanced by merging annotation with results of interproscan database search at the ebi  <cit> , annex procedure, and the blast2go validation step.
a subsequent go-slim step was not used, as this procedure removed the low frequency odorant protein families from the annotation.
for annotation of ors, irs, grs, obps, csps, and snmps in i. typographus and d. ponderosae, contigs were analyzed with tblastx searches against custom-made databases and the non-redundant nucleotide collection at ncbi.
additionally, hmm-based searches of the panther database of domain family profiles were done.
we identified non-redundant translated proteins with reciprocal blast using the comprehensive datasets available for obps and csps  <cit> , as well as snmps  <cit> .
for contigs/isotigs with hits against genes of interest, open reading frames were identified and the annotation verified by additional blast  searches.
contigs containing suspected sequencing errors  were edited manually after identifying miss-assemblies through manual inspection of the assembly files, ests, or genomic data   <cit> .
the suffix “fix” was added to the gene name of such edited sequences, and also to those extended by race-pcr .
tmhmm  <dig>   was used to predict transmembrane domains of candidate ors, irs, and grs.
for all proteins studied, amino acid sequences were aligned using mafft  <cit> , and maximum-likelihood analysis and dendrogram construction were subsequently performed with fasttree  <cit> .
dendrograms were colored and arranged in figtree .
to ensure that sequences corresponded to unigenes , only those that showed sufficient overlap in multiple sequence alignments were included in the analysis.
in addition, for contigs that shared > <dig> % amino acid identity only one “copy”  was included.
i. typographus 454- and illumina sequences have been submitted to ebi .
the d. ponderosae antennal sanger and  <dig> sequence data have previously been submitted to ncbi .
all bark beetle contigs/isotigs have been submitted to the transcriptome shotgun assembly  sequence database at ncbi  or to genbank  .
race-pcr
the assembled contigs from the 454- and illumina sequencing of the ips transcriptome did not always constitute full-length transcripts.
therefore, for better resolution of phylogenetic analyses, some sequences encoding putative ors were elongated using race-pcr  with a nested protocol following the manufacturer’s instructions.
total rna from  <dig> adult beetle antennae  was used as template to generate race-ready cdna.
primer design was performed manually, but aided with tm-calculations and self-complementarity checks using oligo calc .
amplified and extended dna was cloned  before being sequenced .
RESULTS
assembly
the d. ponderosae antenna-specific assembly resulted in  <dig>  isotigs from  <dig>  isogroups and  <dig>  singletons, of which  <dig> were sanger reads.
the isotigs assembled by newbler were comparable with the contigs generated by other assemblers, with the exception that newbler also considers alternative splice variants when producing the isotigs, and these are grouped into different isogroups.
the n <dig> was  <dig>  bp and the largest isotig was  <dig>  bp.
the i. typographus assembly resulted in  <dig>  contigs with an n <dig> of  <dig> bp.
the largest contig was  <dig>  bp.
gene ontology annotation
go annotation indicated that the analyzed antennal transcriptomes of the two bark beetle species were highly similar with respect to go terms .
in i. typographus,  <dig>  contigs  were associated with go terms.
in d. ponderosae, this number was  <dig>  .
thus, a substantial proportion of contigs in both species was not associated with any go term, and possibly these contigs represent orphan genes.
among the annotated contigs, go terms related to basic cell functions were the most abundant; however, contigs with go terms related to olfaction were also present, such as “odorant binding”, “signal transducer activity” , and “response to stimulus” .
contigs with go terms associated with enzymatic activity were well represented, such as “hydrolase activity” and “transferase activity” .
nonreceptor olfactory gene families
we identified  <dig> transcripts encoding putative obps in i. typographus, and  <dig> transcripts in d. ponderosae.
all but five transcripts  corresponded to full-length genes.
one third of the transcripts identified in d. ponderosae were not found in the antennal cdna library, but rather in the cdna libraries from other body parts .
in general, obps can be classified into different phylogenetic groups.
classic obps are characterized by  <dig> cysteine residues at conserved positions.
the plus-c class has 4– <dig> additional cysteines and one characteristic proline, whereas the minus-c class has lost cysteine residues, generally c <dig> and c <dig>  <cit> .
in our sequence similarity dendrogram, the classic bark beetle obps were spread out on various branches  where they generally formed small subgroups together with obps mostly from t. castaneum.
two obps in i. typographus  and one obp in d. ponderosae  were of the plus-c type and were grouped together with the plus-c obp  from t. castaneum .
itypobp <dig> and dponobp <dig> shared 45% amino acid identity.
members of the minus-c class, i.e.
<dig> dponobps,  <dig> itypobps, and  <dig> tcasobps, formed a large clade .
within this clade, we found a bark beetle-specific expansion, containing itypobp <dig>  itypobp <dig>  dponobp <dig>  dponobp <dig>  dponobp <dig>  dponobp <dig>  dponobp <dig>  dponobp <dig>  and dponobp <dig>  all bark beetle full-length minus-c obps had lost c <dig> and c <dig>
six bark beetle obp orthologous pairs shared >50% amino acid identity between species .
there were several obp pairs with high amino acid identity in d. ponderosae .
we identified  <dig> transcripts encoding putative csps in i. typographus, and  <dig> transcripts in d. ponderosae .
five of the transcripts encoded partial proteins , whereas all the others represented full-length genes.
four of the transcripts identified in d. ponderosae were not found in the antennal cdna library, but rather in the cdna libraries from other body parts .
the bark beetle csps were present on different branches throughout the dendrogram , and no major bark beetle-specific expansion of csp lineages was evident.
amino acid identity among candidate simple orthologues in the two bark beetles was high .
two csp pairs in d. ponderosae  had the highest  amino acid identity.
in each bark beetle species, we found two orthologues of snmp <dig> , and one orthologue of snmp <dig> .
itypsnmp1a was present only as a fragment, whereas transcripts for the others likely represented full-length or very close to full-length genes.
the bark beetle snmps grouped together with orthologues in t. castaneum, with the exception of itypsnmp <dig> that paired up with snmp <dig> in d. melanogaster.
snmp <dig> and snmp1a appeared more conserved across the two bark beetles with 58% and 66% amino acid identity, respectively, compared to the snmp <dig> orthologues that shared 28% identity.
receptor encoding genes
odorant receptors
similar numbers of putative or encoding transcripts were annotated in the two bark beetle species.
we identified  <dig> or candidates in i. typographus.
eleven of these were likely representing full-length genes, encoding proteins with more than  <dig> amino acids.
partial transcripts encoding itypor <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> were extended by 3’-race-pcr.
in d. ponderosae, the number of candidate or transcripts was  <dig>  and the number of full-length candidates was  <dig>  in addition,  <dig> short partial transcripts in i. typographus and  <dig> in d. ponderosae were left unlabeled and excluded from analysis, since unigene identity could not be conclusively confirmed.
the shortest partial or candidate included was itypor <dig> .
two pairs of receptors, i.e.
itypor <dig> and itypor <dig>  as well as itypor <dig> and itypor <dig>  showed the highest amino acid identity .
sequences of the bark beetle ors were compared with those of m. caryae and t. castaneum.
for the latter species we included only those ors with confirmed expression in the adult head  <cit> .
several or subgroups of various size and content could be distinguished .
in order to standardize the numbering of coleopteran or subfamilies, we numbered these subgroups from  <dig> to  <dig> according to previous studies  <cit> .
the majority  of bark beetle ors were present within group  <dig>  which also contained  <dig> ors from m. caryae, but no ors from t. castaneum.
fifty-one of these bark beetle ors formed two subgroups  that were completely devoid of receptors from the other two beetle species.
however, considering only the bark beetle ors, only minor species-specific subgroups  could be seen and they were found within group 7a and 7b .
in addition to the bark beetle specific subgroups, group  <dig> also contained two m. caryae-specific subgroups  that each formed a sister group to either of the two bark beetle specific subgroups.
finally, a fifth subgroup within group  <dig> contained ors from all three species, indicating conservation of some or sequences among the three xylophagous species.
the complete lack of t. castaneum receptors within group  <dig>  and the presence of specific subgroups in the other species, indicate broad expansions of or lineages in bark beetles and cerambycids and/or losses of corresponding or lineages in t. castaneum.
expansions of or lineages were also seen in t. castaneum.
forty-five  tcasors formed a large group that was exclusive to the flour beetle.
within this group, the previously defined coleopteran or subgroups 4– <dig> could be found  <cit> .
these subgroups were collectively rooted by a smaller clade containing receptors from i. typographus and m. caryae.
receptor group  <dig> contained ors only from t. castaneum and m. caryae.
the lack of bark beetle ors in this group suggested that these or lineages have been lost in bark beetles , while retained in cerambycids.
within group  <dig>  subgroups that were specific for t. castaneum or specific for m. caryae could be found.
the dendrogram also contained two groups  with or representatives from all four species .
we found most of the candidate 1: <dig> orthologous relationships among the bark beetle ors within groups  <dig> and  <dig>  .
for these candidate orthologous pairs, amino acid identity was 54–69% .
the orco orthologues rooted group  <dig>
the co-receptor orco was identified in the antenna-specific assembly of d. ponderosae, but surprisingly not in the antennal transcriptome assembly of i. typographus.
however, by using pcr with primers designed from a conserved region close to the c-terminus of the dponorco, we amplified a  <dig> amino acid fragment of orco from i. typographus antennal-specific cdna.
this ityporco fragment shared 97% amino acid identity with dponorco.
as expected, orco in d. ponderosae shared high amino acid identity with orco orthologues in m. caryae  and t. castaneum .
gustatory receptors
six candidate gr-encoding transcripts were identified  in i. typographus, including putative conserved carbon dioxide receptors   <cit> .
two gr candidates  were identified in d. ponderosae.
interestingly, gr <dig> was not found in our d. ponderosae antenna-specific assembly, but was recovered from the draft genome  <cit>  and from larval rnaseq data.
gr <dig> in i. typographus could tentatively be assigned to the trehalose receptor  <dig> in t. castaneum .
since grs and ors are members of the same superfamily, both were included in the same dendrogram analysis, in which grs formed a distinct clade .
all grs except for itypgr <dig> grouped within this clade.
ionotropic receptors
we identified  <dig> transcripts for putative ionotropic receptors in i. typographus, and  <dig> transcripts in d. ponderosae .
we found bark beetle orthologues for all  <dig> conserved antennal irs with representatives in t. castaneum <cit> .
however, we did not find all of them in both species.
in d. ponderosae, we identified candidates for ir21a, ir41a, ir64a, ir76b, ir93a , five members of the ir <dig> group, as well as the co-receptors ir25a and ir8a.
transcripts for dponir25a, dponir8a, dponir75p.1fix, dponir75p.
<dig>  dponir75q, and dponir76b likely corresponded to full-length genes , whereas all the other identified irs were represented as partial genes.
candidate ir fragments located on  <dig> isotigs in d. ponderosae were discarded from our dendrogram analysis, as they were too short to confidently assign them unigene status.
however, among these, two fragments  shared 72%, and 69% amino acid identity with tcasir40a and tcasir68a, respectively.
thus, in d. ponderosae it seems like orthologues for all conserved antennal irs found in t. castaneum <cit>  were present.
in contrast, we identified candidates only for ir25a, ir64a, ir68a, ir76b, and three ir <dig> members in i. typographus.
thus, several orthologues found in d. ponderosae and t. castaneum were lacking in the i. typographus assembly.
ir8a, which is a broadly expressed co-receptor, necessary for odor responses  <cit>  and present in all insects studied to date  <cit> , was one of the receptors lacking in i. typographus.
discussion
the gene sets reported here represent significant additions to the pool of identified olfactory genes in coleoptera.
prior to this study, members of the major chemosensory gene families in coleoptera had been identified only from the genome of t. castaneum .
additionally, as the genes identified here underlie the aggregation behavior that results in tree killing by mass-attack, they represent novel targets for management programs of two of the world’s most destructive forest pests.
in general, we identified somewhat larger numbers of transcripts encoding putative olfactory proteins  in d. ponderosae than in i. typographus.
the greater depth of the 454-sequencing and the access to sanger data for d. ponderosae likely account for this difference.
in addition, duplex-specific nuclease cdna normalization  seems to result in overrepresentation of shorter full-length transcripts, which might explain the lower number of or and ir transcripts identified in i. typographus, and also the absence of orco transcripts in the transcriptome assembly.
however, despite the slight difference in methodology, the go annotation demonstrated a remarkable overall similarity in the types of genes  that are expressed in the antennae of the two species.
go annotation was previously conducted for the antennal transcriptome of manduca sexta moths by grosse-wilde et al.
<cit> , and comparison with their data reveals a striking similarity to the bark beetles analyzed here.
indeed, several go terms with putative relation to olfactory function showed identical  relative abundance, suggesting a kind of across-order conservation of gene expression patterns in antennae, although the data say nothing about expression levels of the individual genes themselves.
odorants are thought to interact with obps or csps in the sensillum lymph prior to the ligand-receptor interaction.
the numbers of obps identified in the bark beetles  are clearly lower than the  <dig> obp-encoding genes reported in the genome of t. castaneum <cit> .
the same is true for the csps, for which we identified  <dig> transcripts in i. typographus and  <dig> in d. ponderosae compared with  <dig> putative csp encoding genes in the t. castaneum genome  <cit> .
however, it might be misleading to compare the number of genes identified in a genome with the number of transcripts in a specific tissue at a specific life stage.
some of the genes might, for instance, be expressed only in the larva  <cit> .
indeed, many of the identified obps and csps  in d. ponderosae were not identified from the antennal library, but seem to be expressed only in non-olfactory tissue.
similar patterns have been found also in other insects, suggesting that these proteins may have physiological functions independent of olfaction  <cit> .
snmps are associated with pheromone-responsive osns in lepidoptera and diptera  <cit> .
in d. melanogaster, snmp <dig> was shown to be necessary for proper osn responses to the pheromone compound cis-vaccenyl acetate, but not for osn responses to food-related fruit esters  <cit> .
benton et al.
<cit>  also demonstrated that snmp was required for activation of heliothis virescens  pheromone receptor hr <dig> by its corresponding ligand when heterologously expressed in drosophila neurons.
it was suggested that the hydrophobic tail of the fatty-acid derived dipteran and lepidopteran pheromone molecules necessitates the presence of snmp.
if so, that raises the question why bark beetles that do not use pheromone compounds with long hydrophobic tails  <cit>  express snmps in their antennae.
the numbers of putative or-encoding transcripts identified in the two bark beetles  are close to the number reported in the antennal transcriptome of m. caryae   <cit> , but lower than the number expressed in the head of adult t. castaneum , and much lower than the number in the t. castaneum genome   <cit> .
in other insects, the number of seemingly intact or-encoding genes identified from genomes is highly variable  <cit> , ranging from only  <dig> in the human body louse, pediculus humanus <cit> , to ca.
<dig> in the fire ant, solenopsis invicta <cit> .
it is not fully understood how the number of ors relates to the ecology of an insect.
in our case, one could expect that the flour beetle might have a less complex sense of smell than the forest dwelling beetles, since it has presumably adapted to an environment with a lower “semiochemical diversity”  <cit> .
this would suggest a lower number of receptors, contrary to our results.
therefore, the chemical ecology of t. castaneum may be more complex than currently understood as also suggested by  <cit> .
however, it is unknown how many of the  <dig> ors that are expressed in the adult head are actually expressed in the olfactory organs of t. castaneum.
in addition, it is likely that some bark beetle ors have been missed in our transcriptome analysis , underestimating the true number of antennal-expressed bark beetle ors.
species -specific expansions of or lineages are seen in most insects studied e.g.
<cit> , and some of the largest expansions have been found in hymenoptera, particularly in the jewel wasp, nasonia vitripennis <cit> .
the pattern of or lineage expansion and conservation observed in the present study likely reflects the evolutionary and ecological relatedness among the four beetle species.
the beetle taxa analysed here all belong to the more derived part of coleoptera   <cit> .
however, the curculionidea  and tenebrionidea  superfamilies are the two furthest separated clades within cucujiformia, sharing a common ancestor ca.
230– <dig> mya.
thus, it may come as no surprise that the ors of these two taxa largely fall into different subgroupings in the tree.
on the other hand, the curculionidea is a sister group to the chrysomeloidea   <cit>  and, likewise, the closer relatedness of these taxa seems to be reflected in the or subgroupings.
within scolytinae, the ips and dendroctonus genera are separated by ca.
<dig> mya  <cit> .
however, despite the fact that culex and aedes mosquitoes are separated by only ca.
<dig> mya  <cit> , they show more distinct species-specific or lineage expansions than the bark beetles  <cit> , indicating that ecological adaptation and life cycle also play important roles in shaping the or repertoire of a species  <cit> .
on this note, the bark beetles and the cerambycid utilize similar types of host material, i.e.
conifer trees and hardwood, respectively  <cit> , whereas t. castaneum has been associated with human populations and stored products, for at least a few thousand years  <cit> .
however, not all ors were grouped in taxon-specific expansions; some subfamilies contained ors from all four species.
this might indicate preservation of ancestral functional patterns within coleoptera, but since non-coleopteran ors were left out from the analysis we are careful to draw any conclusions based on this finding .
the close clustering of or sequences from the two bark beetles raises the question about how similar the semiochemical environment is for i. typographus and d. ponderosae.
they both live in conifers and would thus be expected to share several biologically relevant compounds.
due to their status as very serious forest pests, the plant- and beetle-produced compounds that they respond to are well studied in these two species.
mainly based on a set of review papers  <cit> , we compiled a table of all compounds that have been shown to be physiologically and/or behaviorally active in i. typographus and d. ponderosae .
for  <dig>  of the  <dig> listed compounds, there is evidence of shared bio-activity.
not surprisingly, the host compounds show a large overlap , but there is also a large overlap  among pheromone compounds of beetle origin.
for the non-host volatiles, the overlap is lower .
one might speculate that the extent of this shared “chemosphere” of semiochemicals could account for the low degree of species-specific diversifications among the bark beetle ors and the other proteins studied here.
however, functional data is required to test this hypothesis.
we identified only a small number of putative gr-encoding transcripts  from the antennal transcriptomes.
the identified bark beetle grs included transcripts for carbon dioxide receptors, suggesting that the antennae of bark beetles detect carbon dioxide.
in addition, the presence of gr1- <dig> in i. typographus indicates that carbon dioxide is detected by a heterotrimer receptor, like in mosquitoes, bombyx mori, and t. castaneum <cit> .
however, gr <dig> was not found in the analyzed transcriptome of d. ponderosae.
hence, it is possible that d. ponderosae uses a heterodimer receptor for carbon dioxide detection   <cit> , but it seems unlikely that expression of gr <dig> would have been lost in only one of the bark beetle species analyzed here.
all the conserved antennal irs that previously were found in t. castaneum were also identified in d. ponderosae.
however, some of them were missing in the i. typographus data.
as irs are associated with coeloconic sensilla that are relatively rare on the ips antenna  <cit> , it is possible that the missing ir transcripts are expressed only in a few neurons.
a lower expression level results in a higher probability that these transcripts were missed during the random sequencing of the ips cdna, which had a lesser depth than for d. ponderosae.
generally in insects, the antennal ir subfamily constitutes only a portion of the total number of irs.
the others belong to the divergent irs, a subfamily that shows species-specific expansions that are particularly large in diptera  <cit> .
in d. melanogaster, expression of divergent irs was detected only in gustatory organs  <cit> .
this is consistent with the scarcity of divergent irs in the bark beetle antennal transcriptomes.
CONCLUSIONS
we have carried out comprehensive analyses of the antennal transcriptomes of two major tree-killing bark beetle species.
at an abstract level, the prevalence of transcript types with respect to associated go terms was highly similar in the two beetles.
in addition, we annotated members of six major gene families that encode proteins with crucial roles in chemoreception.
thus, these proteins have now been identified in three coleopteran species .
in combination with the previously published data, the gene sets identified here now allow for improved evolutionary analysis of coleopteran olfaction.
we found clear expanded bark beetle-specific lineages mainly among the ors, suggesting that in comparison to the other analyzed protein families ors are more tightly linked to sensory specialization and adaptation to specific ecological niches and a shared space of semiochemicals.
the results from the present study will also be fundamental for future functional studies.
functional characterization is needed in order to connect the available physiological and ecological knowledge with the molecular information presented here.
identification and de-orphanization of receptor proteins in bark beetles is especially relevant, since they may represent new targets for integrated pest management strategies.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
mna and egw contributed equally to this work, by carrying out lab work  and assemblies  for i. typographus, as well as bioinformatic analysis, sequence alignment, sequence editing, and data analysis for ors, grs, and irs of both species.
mna drafted the manuscript.
egw constructed trees for all gene families.
cik and mmsy performed antenna-specific assembly for d. ponderosae, as well as bioinformatic analysis, sequence alignment, sequence editing, and data analysis for obps, csps, and snmps from both species.
jmb performed lab work, bioinformatic analysis , and data analysis.
ml sequenced representative cdna clones of d. ponderosae genes that appeared to be full-length for submission to ncbi.
fs conducted the go annotations and compilation of shared semiochemicals.
cik, yh, jb, bsh, and fs conceived of the study, coordinated, and designed the study.
all authors contributed to study design and manuscript preparation.
all authors read and approved the final manuscript
supplementary material
additional file 1
accession numbers for genes encoding olfactory proteins that were submitted to genbank and the transcriptome shotgun assembly sequence database  at ncbi.
click here for file
additional file 2
gene ontology results.
gene ontology analyses as in figure  <dig>  but here represented as bar diagrams that have a higher resolution.
a) molecular function level  <dig> in ips typographus, b) molecular function level  <dig> in dendroctonus ponderosae, c) biological process level  <dig> in i. typographus, and d) biological process level  <dig> in d. ponderosae.
click here for file
additional file 3
presence of odorant binding proteins in various tissues of dendroctonus ponderosae.
analyses of sanger-specific data and normalized as well as non-normalized transcriptome assemblies from various body parts of d. ponderosae indicate that large sets of odorant binding proteins were found exclusively in non-antennal tissues.
the numbers in the tables represent reads found.
the clones were sequenced from both 5' and 3' directions.
for further information see keeling et al.
<cit> .
click here for file
additional file 4
presence of chemosensory proteins in various tissues of dendroctonus ponderosae.
analyses of sanger-specific data and normalized as well as non-normalized transcriptome assemblies from various body parts of d. ponderosae indicate that  <dig> of the  <dig> identified chemosensory proteins were found exclusively in non-antennal tissues.
the numbers in the tables represent reads found.
the clones were sequenced from both 5' and 3' directions.
for further information see keeling et al.
<cit> .
click here for file
additional file 5
shared chemosphere of i. typographusandd.
ponderosae.
list of  <dig> semiochemicals that are produced by the two bark beetle species, or present in their host or non-host plants and whether the compounds are active at a physiological and/or behavioral level in each species.
click here for file
acknowledgements
we are thankful to sascha bucks  for invaluable support in early laboratory work, and to bernd timmermann  for next generation sequencing.
we also acknowledge william walker iii  for identifying orco in ips, and liwen song for help with the bioinformatic analysis.
we acknowledge two anonymous reviewers for useful comments on the submitted manuscript.
mna, yh, and fs were funded by the department of plant protection biology, slu, alnarp, and by the linnaeus program, “insect chemical ecology, ethology, and evolution, ice3”.
jmb was supported by the swedish international development agency .
work in jena  was funded by the max planck society.
cik, mmsy, jb, and ml were supported with funding from genome canada, genome british columbia, and genome alberta  and the natural sciences and engineering research council of canada .
during manuscript preparation, mna was funded by the swedish research council .

 the european spruce bark beetle, ips typographus l., and the north american mountain pine beetle, dendroctonus ponderosae hopkins are serious pests of coniferous forests. i. typographus mainly attacks norway spruce in eurasia, whereas d. ponderosae infests several species of pine in western north america. currently, large-scale d. ponderosae outbreaks have resulted in unprecedented economic losses and turned north american forests into major sources of carbon release. the olfactory sense drives bark beetle behaviors that are important for fitness, such as the localization of suitable hosts and mates. in the search for suitable host material, bark beetles respond to volatiles that emanate from both host and non-host plants. however, most individuals locate trees by means of an aggregation pheromone that is released by beetles that have already attacked the tree. this signal is responsible for coordinated mass-attacks, which often lead to the death of the host tree and large-scale forest destruction. due to their ecological and economic impact, an extensive knowledge base on bark beetle chemical ecology and olfactory physiology has been established. however, information on the molecular aspects of odor detection has been lacking until now. in insects, volatile molecules are detected by olfactory sensory neurons that are housed within special structures predominantly on the antennae, and to a lesser extent on the maxillary palps. the cell membrane of osns contains receptor proteins that bind odor ligands. the binding of a ligand to a receptor protein is the key event in olfactory transduction, as it converts a chemical signal in the environment into an electrical signal that can be interpreted by the insect nervous system. receptors from three large and divergent multigene families are expressed in insect osns, namely the odorant receptors, ionotropic receptors, and gustatory receptors, the latter group notably containing carbon dioxide-detecting receptors. however, most grs are expressed in gustatory receptor neurons in taste organs and are involved in contact chemoreception. these grs typically detect different sugars, bitter compounds, and contact pheromones. insect ors are seven-transmembrane domain proteins with a reversed membrane topology compared to vertebrate ors, which are g-protein coupled receptors. insect ors and grs are distantly related members of the same superfamily. in general, ors show little sequence homology to each other and they are unrelated to vertebrate ors. the conventional exchangeable or that determines ligand specificity forms heteromers of unknown stoichiometry with a conserved co-receptor, known as orco. orco is ubiquitously expressed in osns that express ors and necessary for olfactory responses and for localization of the conventional or in the cell membrane. putative insect ors have been identified mainly in species with sequenced genomes. recently, however, studies on antennal transcriptomes have led to the identification of or sets in several moth species and one beetle. the ors respond to a variety of volatile chemicals, including pheromones and plant or microbe-derived compounds. some ors are highly defined in their response specificity, whereas others appear more broadly tuned, especially at high stimulus concentrations. irs were recently discovered as another class of receptors involved in chemoreception. they are related to ionotropic glutamate receptors that function in synapse communication, but have atypical binding domains. irs have been identified throughout protostome lineages and, thus, constitute a far more ancient group of receptors than the ors. irs form complexes with up to three subunits, including odor-specific receptors and one or two broadly expressed co-receptors. in insects, the irs are divided into two major groups: the “antennal irs” that have an olfactory function and are conserved across insect orders, and the species-specific “divergent irs”, some of which have been assigned a tentative role in taste. antennal irs in drosophila have different odor specificity compared to the ors and respond to nitrogen-containing compounds, acids, and aromatics. in addition to the receptor genes, other multigene families encode proteins with critical roles in olfaction. odorant binding proteins are small soluble proteins with two or three disulfide bridges. obps are highly abundant in the sensillar lymph of insects and are thought to solubilize hydrophobic molecules and deliver them to the receptors. studies have shown conflicting results whether or not obps affect the response specificity of osns. at least in some studies, the specificity of pheromone receptors was improved by the presence of obps. some evidence suggests that obps might undergo odor-induced conformational changes, with a change in the obp itself triggering the response of the osn. in insects with sequenced genomes, the number of obp coding genes normally ranges from ca. 40–. chemosensory proteins constitute another class of small binding proteins. they are more conserved than obps and are characterized by the presence of cysteines that form two disulfide bridges. csps may have shared a common ancestor with the obps near the origin of the arthropods. like obps, csps are present in high concentration in chemosensory sensilla. however, the majority of them are also expressed in various non-sensory tissues and they seem to play a role in development, moulting, and leg regeneration. some csps bind pheromone compounds, but their exact role in chemosensory systems remains uncertain. most drosophila genomes contain only csp coding genes and t. castaneum has. the genome of aedes aegypti mosquitoes contains members of this family, the largest number found in insects so far. finally, the sensory neuron membrane proteins are proteins of the cd family that associate with pheromone-responding osns. their functional significance is still poorly understood, but snmp is crucial for proper pheromone detection in d. melanogaster and also required for activation of a pheromone receptor in heliothis virescens moths. in contrast, snmp was dispensable for responses of a fly receptor to fruit-related esters. insects generally have two representatives of snmps, although copy numbers of each orthologue seem to vary somewhat across species. in the largest insect order, coleoptera, ors have been identified from only two species: from the genome of the red flour beetle, tribolium castaneum, and recently from the antennal transcriptome of the cerambycid beetle, megacyllene caryae. members of the other olfactory gene families have been identified only in t. castaneum. consequently, additional beetle species need to be investigated to reach a better understanding on the molecular biology of coleopteran and insect olfaction. in this study, we assembled and analyzed bark beetle antennal transcriptomes from next-generation sequencing. we report the results from gene ontology annotation as well as sets of putative obps, csps, snmps, ors, grs, and irs in i. typographus and d. ponderosae. identification of the chemosensory genes in these devastating insect pests is especially relevant because of their potential as novel targets for pest control. methods mountain pine beetle the source of d. ponderosae antennal tissue and the method of sequencing have been reported previously in a larger transcriptome study. from this transcriptome dataset originating from several tissue types, sequences originating only from a non-normalized antenna-specific cdna library were re-assembled for the analyses presented in the present study. this included paired-end sanger reads, single-end sanger reads and, roche reads. newbler was used for assembly using the "-cdna" switch for transcriptome assemblies and a bp cutoff to eliminate short reads. for identification of obps, csps, and snmps in d. ponderosae we also used a combination of sanger-specific data and transcriptome assemblies from other tissues and life stages, since these proteins may have sensory or non-sensory functions in non-antennal tissues. we did not have such assemblies for i. typographus. european spruce bark beetle insects, rna extraction and cdna synthesis i. typographus was reared on norway spruce logs in an environmental chamber, starting from individuals collected from their natural habitat near asa and Älmhult, southern sweden. emerged adults were kept in a state of low activity in a refrigerator before being used for rna extraction. two hundred adult i. typographus were collected in a ml plastic tube, approximately two weeks after their emergence. the tube was submerged in liquid nitrogen, after which it was vigorously shaken using a vortex shaker to separate extremities from the body. body parts were suspended in -20°c acetone and passed through meshes that filtered out the antennae. after removal of the acetone, ml tri reagent was added to the antennae and the sample was homogenized using a tissue-tearor. total rna was extracted following the trizol protocol, but using 1-bromo-3-chloropropane instead of chloroform. μg total rna was sent to evrogen for synthesis of duplex-specific-nuclease normalized cdna. sequencing and assembly the i. typographus cdna was sequenced at lgc genomics, using gs/flx sequencing with titanium chemistry, to produce reads for a total of megabases. furthermore, illumina sequencing was performed at the max planck institute for molecular genetics in berlin to generate a further million reads for a total of megabases. short or low-quality reads, as well as linker and adapter sequences were removed by the crossmatch program or by the built-in sequence cleanup of seqman ngen. the reads were assembled using seqman ngen to generate a backbone; subsequently, the illumina reads were mapped onto this backbone using seqman ngen to correct for technology-inherent read errors. the resultant contigs were annotated using a codequest workstation. annotation for an initial assessment of the two assembled beetle antennal transcriptomes, gene ontology annotation was performed using blast2go. blast2go annotation associates genes or transcripts with go terms using hierarchical vocabularies. genes are described in terms related to molecular function, biological process, or cellular component, allowing for meta-analyses of gene populations. the blast step was performed with a lenient e-value cutoff at to account for the high sequence variability among the olfactory gene families. the mapping step was done using default settings, whereas a lenient e-value and lower annotation cut-off and go-weight were used in the first annotation step to increase the proportion of annotated transcripts. annotation was further enhanced by merging annotation with results of interproscan database search at the ebi, annex procedure, and the blast2go validation step. a subsequent go-slim step was not used, as this procedure removed the low frequency odorant protein families from the annotation. for annotation of ors, irs, grs, obps, csps, and snmps in i. typographus and d. ponderosae, contigs were analyzed with tblastx searches against custom-made databases and the non-redundant nucleotide collection at ncbi. additionally, hmm-based searches of the panther database of domain family profiles were done. we identified non-redundant translated proteins with reciprocal blast using the comprehensive datasets available for obps and csps, as well as snmps. for contigs/isotigs with hits against genes of interest, open reading frames were identified and the annotation verified by additional blast searches. contigs containing suspected sequencing errors were edited manually after identifying miss-assemblies through manual inspection of the assembly files, ests, or genomic data. the suffix “fix” was added to the gene name of such edited sequences, and also to those extended by race-pcr. tmhmm was used to predict transmembrane domains of candidate ors, irs, and grs. for all proteins studied, amino acid sequences were aligned using mafft, and maximum-likelihood analysis and dendrogram construction were subsequently performed with fasttree. dendrograms were colored and arranged in figtree. to ensure that sequences corresponded to unigenes, only those that showed sufficient overlap in multiple sequence alignments were included in the analysis. in addition, for contigs that shared > % amino acid identity only one “copy” was included. i. typographus 454- and illumina sequences have been submitted to ebi. the d. ponderosae antennal sanger and sequence data have previously been submitted to ncbi. all bark beetle contigs/isotigs have been submitted to the transcriptome shotgun assembly sequence database at ncbi or to genbank. race-pcr the assembled contigs from the 454- and illumina sequencing of the ips transcriptome did not always constitute full-length transcripts. therefore, for better resolution of phylogenetic analyses, some sequences encoding putative ors were elongated using race-pcr with a nested protocol following the manufacturer’s instructions. total rna from adult beetle antennae was used as template to generate race-ready cdna. primer design was performed manually, but aided with tm-calculations and self-complementarity checks using oligo calc. amplified and extended dna was cloned before being sequenced.  assembly the d. ponderosae antenna-specific assembly resulted in isotigs from isogroups and singletons, of which were sanger reads. the isotigs assembled by newbler were comparable with the contigs generated by other assemblers, with the exception that newbler also considers alternative splice variants when producing the isotigs, and these are grouped into different isogroups. the n was bp and the largest isotig was bp. the i. typographus assembly resulted in contigs with an n of bp. the largest contig was bp. gene ontology annotation go annotation indicated that the analyzed antennal transcriptomes of the two bark beetle species were highly similar with respect to go terms. in i. typographus, contigs were associated with go terms. in d. ponderosae, this number was. thus, a substantial proportion of contigs in both species was not associated with any go term, and possibly these contigs represent orphan genes. among the annotated contigs, go terms related to basic cell functions were the most abundant; however, contigs with go terms related to olfaction were also present, such as “odorant binding”, “signal transducer activity”, and “response to stimulus”. contigs with go terms associated with enzymatic activity were well represented, such as “hydrolase activity” and “transferase activity”. nonreceptor olfactory gene families we identified transcripts encoding putative obps in i. typographus, and transcripts in d. ponderosae. all but five transcripts corresponded to full-length genes. one third of the transcripts identified in d. ponderosae were not found in the antennal cdna library, but rather in the cdna libraries from other body parts. in general, obps can be classified into different phylogenetic groups. classic obps are characterized by cysteine residues at conserved positions. the plus-c class has 4– additional cysteines and one characteristic proline, whereas the minus-c class has lost cysteine residues, generally c and c. in our sequence similarity dendrogram, the classic bark beetle obps were spread out on various branches where they generally formed small subgroups together with obps mostly from t. castaneum. two obps in i. typographus and one obp in d. ponderosae were of the plus-c type and were grouped together with the plus-c obp from t. castaneum. itypobp and dponobp shared 45% amino acid identity. members of the minus-c class, i.e. dponobps, itypobps, and tcasobps, formed a large clade. within this clade, we found a bark beetle-specific expansion, containing itypobp itypobp dponobp dponobp dponobp dponobp dponobp dponobp and dponobp all bark beetle full-length minus-c obps had lost c and c six bark beetle obp orthologous pairs shared >50% amino acid identity between species. there were several obp pairs with high amino acid identity in d. ponderosae. we identified transcripts encoding putative csps in i. typographus, and transcripts in d. ponderosae. five of the transcripts encoded partial proteins, whereas all the others represented full-length genes. four of the transcripts identified in d. ponderosae were not found in the antennal cdna library, but rather in the cdna libraries from other body parts. the bark beetle csps were present on different branches throughout the dendrogram, and no major bark beetle-specific expansion of csp lineages was evident. amino acid identity among candidate simple orthologues in the two bark beetles was high. two csp pairs in d. ponderosae had the highest amino acid identity. in each bark beetle species, we found two orthologues of snmp, and one orthologue of snmp. itypsnmp1a was present only as a fragment, whereas transcripts for the others likely represented full-length or very close to full-length genes. the bark beetle snmps grouped together with orthologues in t. castaneum, with the exception of itypsnmp that paired up with snmp in d. melanogaster. snmp and snmp1a appeared more conserved across the two bark beetles with 58% and 66% amino acid identity, respectively, compared to the snmp orthologues that shared 28% identity. receptor encoding genes odorant receptors similar numbers of putative or encoding transcripts were annotated in the two bark beetle species. we identified or candidates in i. typographus. eleven of these were likely representing full-length genes, encoding proteins with more than amino acids. partial transcripts encoding itypor and were extended by 3’-race-pcr. in d. ponderosae, the number of candidate or transcripts was and the number of full-length candidates was in addition, short partial transcripts in i. typographus and in d. ponderosae were left unlabeled and excluded from analysis, since unigene identity could not be conclusively confirmed. the shortest partial or candidate included was itypor. two pairs of receptors, i.e. itypor and itypor as well as itypor and itypor showed the highest amino acid identity. sequences of the bark beetle ors were compared with those of m. caryae and t. castaneum. for the latter species we included only those ors with confirmed expression in the adult head. several or subgroups of various size and content could be distinguished. in order to standardize the numbering of coleopteran or subfamilies, we numbered these subgroups from to according to previous studies. the majority of bark beetle ors were present within group which also contained ors from m. caryae, but no ors from t. castaneum. fifty-one of these bark beetle ors formed two subgroups that were completely devoid of receptors from the other two beetle species. however, considering only the bark beetle ors, only minor species-specific subgroups could be seen and they were found within group 7a and 7b. in addition to the bark beetle specific subgroups, group also contained two m. caryae-specific subgroups that each formed a sister group to either of the two bark beetle specific subgroups. finally, a fifth subgroup within group contained ors from all three species, indicating conservation of some or sequences among the three xylophagous species. the complete lack of t. castaneum receptors within group and the presence of specific subgroups in the other species, indicate broad expansions of or lineages in bark beetles and cerambycids and/or losses of corresponding or lineages in t. castaneum. expansions of or lineages were also seen in t. castaneum. forty-five tcasors formed a large group that was exclusive to the flour beetle. within this group, the previously defined coleopteran or subgroups 4– could be found. these subgroups were collectively rooted by a smaller clade containing receptors from i. typographus and m. caryae. receptor group contained ors only from t. castaneum and m. caryae. the lack of bark beetle ors in this group suggested that these or lineages have been lost in bark beetles, while retained in cerambycids. within group subgroups that were specific for t. castaneum or specific for m. caryae could be found. the dendrogram also contained two groups with or representatives from all four species. we found most of the candidate 1: orthologous relationships among the bark beetle ors within groups and. for these candidate orthologous pairs, amino acid identity was 54–69%. the orco orthologues rooted group the co-receptor orco was identified in the antenna-specific assembly of d. ponderosae, but surprisingly not in the antennal transcriptome assembly of i. typographus. however, by using pcr with primers designed from a conserved region close to the c-terminus of the dponorco, we amplified a amino acid fragment of orco from i. typographus antennal-specific cdna. this ityporco fragment shared 97% amino acid identity with dponorco. as expected, orco in d. ponderosae shared high amino acid identity with orco orthologues in m. caryae and t. castaneum. gustatory receptors six candidate gr-encoding transcripts were identified in i. typographus, including putative conserved carbon dioxide receptors. two gr candidates were identified in d. ponderosae. interestingly, gr was not found in our d. ponderosae antenna-specific assembly, but was recovered from the draft genome and from larval rnaseq data. gr in i. typographus could tentatively be assigned to the trehalose receptor in t. castaneum. since grs and ors are members of the same superfamily, both were included in the same dendrogram analysis, in which grs formed a distinct clade. all grs except for itypgr grouped within this clade. ionotropic receptors we identified transcripts for putative ionotropic receptors in i. typographus, and transcripts in d. ponderosae. we found bark beetle orthologues for all conserved antennal irs with representatives in t. castaneum. however, we did not find all of them in both species. in d. ponderosae, we identified candidates for ir21a, ir41a, ir64a, ir76b, ir93a, five members of the ir group, as well as the co-receptors ir25a and ir8a. transcripts for dponir25a, dponir8a, dponir75p.1fix, dponir75p. dponir75q, and dponir76b likely corresponded to full-length genes, whereas all the other identified irs were represented as partial genes. candidate ir fragments located on isotigs in d. ponderosae were discarded from our dendrogram analysis, as they were too short to confidently assign them unigene status. however, among these, two fragments shared 72%, and 69% amino acid identity with tcasir40a and tcasir68a, respectively. thus, in d. ponderosae it seems like orthologues for all conserved antennal irs found in t. castaneum were present. in contrast, we identified candidates only for ir25a, ir64a, ir68a, ir76b, and three ir members in i. typographus. thus, several orthologues found in d. ponderosae and t. castaneum were lacking in the i. typographus assembly. ir8a, which is a broadly expressed co-receptor, necessary for odor responses and present in all insects studied to date, was one of the receptors lacking in i. typographus. discussion the gene sets reported here represent significant additions to the pool of identified olfactory genes in coleoptera. prior to this study, members of the major chemosensory gene families in coleoptera had been identified only from the genome of t. castaneum. additionally, as the genes identified here underlie the aggregation behavior that results in tree killing by mass-attack, they represent novel targets for management programs of two of the world’s most destructive forest pests. in general, we identified somewhat larger numbers of transcripts encoding putative olfactory proteins in d. ponderosae than in i. typographus. the greater depth of the 454-sequencing and the access to sanger data for d. ponderosae likely account for this difference. in addition, duplex-specific nuclease cdna normalization seems to result in overrepresentation of shorter full-length transcripts, which might explain the lower number of or and ir transcripts identified in i. typographus, and also the absence of orco transcripts in the transcriptome assembly. however, despite the slight difference in methodology, the go annotation demonstrated a remarkable overall similarity in the types of genes that are expressed in the antennae of the two species. go annotation was previously conducted for the antennal transcriptome of manduca sexta moths by grosse-wilde et al., and comparison with their data reveals a striking similarity to the bark beetles analyzed here. indeed, several go terms with putative relation to olfactory function showed identical relative abundance, suggesting a kind of across-order conservation of gene expression patterns in antennae, although the data say nothing about expression levels of the individual genes themselves. odorants are thought to interact with obps or csps in the sensillum lymph prior to the ligand-receptor interaction. the numbers of obps identified in the bark beetles are clearly lower than the obp-encoding genes reported in the genome of t. castaneum. the same is true for the csps, for which we identified transcripts in i. typographus and in d. ponderosae compared with putative csp encoding genes in the t. castaneum genome. however, it might be misleading to compare the number of genes identified in a genome with the number of transcripts in a specific tissue at a specific life stage. some of the genes might, for instance, be expressed only in the larva. indeed, many of the identified obps and csps in d. ponderosae were not identified from the antennal library, but seem to be expressed only in non-olfactory tissue. similar patterns have been found also in other insects, suggesting that these proteins may have physiological functions independent of olfaction. snmps are associated with pheromone-responsive osns in lepidoptera and diptera. in d. melanogaster, snmp was shown to be necessary for proper osn responses to the pheromone compound cis-vaccenyl acetate, but not for osn responses to food-related fruit esters. benton et al. also demonstrated that snmp was required for activation of heliothis virescens pheromone receptor hr by its corresponding ligand when heterologously expressed in drosophila neurons. it was suggested that the hydrophobic tail of the fatty-acid derived dipteran and lepidopteran pheromone molecules necessitates the presence of snmp. if so, that raises the question why bark beetles that do not use pheromone compounds with long hydrophobic tails express snmps in their antennae. the numbers of putative or-encoding transcripts identified in the two bark beetles are close to the number reported in the antennal transcriptome of m. caryae, but lower than the number expressed in the head of adult t. castaneum, and much lower than the number in the t. castaneum genome. in other insects, the number of seemingly intact or-encoding genes identified from genomes is highly variable, ranging from only in the human body louse, pediculus humanus, to ca. in the fire ant, solenopsis invicta. it is not fully understood how the number of ors relates to the ecology of an insect. in our case, one could expect that the flour beetle might have a less complex sense of smell than the forest dwelling beetles, since it has presumably adapted to an environment with a lower “semiochemical diversity”. this would suggest a lower number of receptors, contrary to our results. therefore, the chemical ecology of t. castaneum may be more complex than currently understood as also suggested by. however, it is unknown how many of the ors that are expressed in the adult head are actually expressed in the olfactory organs of t. castaneum. in addition, it is likely that some bark beetle ors have been missed in our transcriptome analysis, underestimating the true number of antennal-expressed bark beetle ors. species -specific expansions of or lineages are seen in most insects studied e.g., and some of the largest expansions have been found in hymenoptera, particularly in the jewel wasp, nasonia vitripennis. the pattern of or lineage expansion and conservation observed in the present study likely reflects the evolutionary and ecological relatedness among the four beetle species. the beetle taxa analysed here all belong to the more derived part of coleoptera. however, the curculionidea and tenebrionidea superfamilies are the two furthest separated clades within cucujiformia, sharing a common ancestor ca. 230– mya. thus, it may come as no surprise that the ors of these two taxa largely fall into different subgroupings in the tree. on the other hand, the curculionidea is a sister group to the chrysomeloidea and, likewise, the closer relatedness of these taxa seems to be reflected in the or subgroupings. within scolytinae, the ips and dendroctonus genera are separated by ca. mya. however, despite the fact that culex and aedes mosquitoes are separated by only ca. mya, they show more distinct species-specific or lineage expansions than the bark beetles, indicating that ecological adaptation and life cycle also play important roles in shaping the or repertoire of a species. on this note, the bark beetles and the cerambycid utilize similar types of host material, i.e. conifer trees and hardwood, respectively, whereas t. castaneum has been associated with human populations and stored products, for at least a few thousand years. however, not all ors were grouped in taxon-specific expansions; some subfamilies contained ors from all four species. this might indicate preservation of ancestral functional patterns within coleoptera, but since non-coleopteran ors were left out from the analysis we are careful to draw any conclusions based on this finding. the close clustering of or sequences from the two bark beetles raises the question about how similar the semiochemical environment is for i. typographus and d. ponderosae. they both live in conifers and would thus be expected to share several biologically relevant compounds. due to their status as very serious forest pests, the plant- and beetle-produced compounds that they respond to are well studied in these two species. mainly based on a set of review papers, we compiled a table of all compounds that have been shown to be physiologically and/or behaviorally active in i. typographus and d. ponderosae. for of the listed compounds, there is evidence of shared bio-activity. not surprisingly, the host compounds show a large overlap, but there is also a large overlap among pheromone compounds of beetle origin. for the non-host volatiles, the overlap is lower. one might speculate that the extent of this shared “chemosphere” of semiochemicals could account for the low degree of species-specific diversifications among the bark beetle ors and the other proteins studied here. however, functional data is required to test this hypothesis. we identified only a small number of putative gr-encoding transcripts from the antennal transcriptomes. the identified bark beetle grs included transcripts for carbon dioxide receptors, suggesting that the antennae of bark beetles detect carbon dioxide. in addition, the presence of gr1- in i. typographus indicates that carbon dioxide is detected by a heterotrimer receptor, like in mosquitoes, bombyx mori, and t. castaneum. however, gr was not found in the analyzed transcriptome of d. ponderosae. hence, it is possible that d. ponderosae uses a heterodimer receptor for carbon dioxide detection, but it seems unlikely that expression of gr would have been lost in only one of the bark beetle species analyzed here. all the conserved antennal irs that previously were found in t. castaneum were also identified in d. ponderosae. however, some of them were missing in the i. typographus data. as irs are associated with coeloconic sensilla that are relatively rare on the ips antenna, it is possible that the missing ir transcripts are expressed only in a few neurons. a lower expression level results in a higher probability that these transcripts were missed during the random sequencing of the ips cdna, which had a lesser depth than for d. ponderosae. generally in insects, the antennal ir subfamily constitutes only a portion of the total number of irs. the others belong to the divergent irs, a subfamily that shows species-specific expansions that are particularly large in diptera. in d. melanogaster, expression of divergent irs was detected only in gustatory organs. this is consistent with the scarcity of divergent irs in the bark beetle antennal transcriptomes.  we have carried out comprehensive analyses of the antennal transcriptomes of two major tree-killing bark beetle species. at an abstract level, the prevalence of transcript types with respect to associated go terms was highly similar in the two beetles. in addition, we annotated members of six major gene families that encode proteins with crucial roles in chemoreception. thus, these proteins have now been identified in three coleopteran species. in combination with the previously published data, the gene sets identified here now allow for improved evolutionary analysis of coleopteran olfaction. we found clear expanded bark beetle-specific lineages mainly among the ors, suggesting that in comparison to the other analyzed protein families ors are more tightly linked to sensory specialization and adaptation to specific ecological niches and a shared space of semiochemicals. the results from the present study will also be fundamental for future functional studies. functional characterization is needed in order to connect the available physiological and ecological knowledge with the molecular information presented here. identification and de-orphanization of receptor proteins in bark beetles is especially relevant, since they may represent new targets for integrated pest management strategies. competing interests the authors declare that they have no competing interests. authors’ contributions mna and egw contributed equally to this work, by carrying out lab work and assemblies for i. typographus, as well as bioinformatic analysis, sequence alignment, sequence editing, and data analysis for ors, grs, and irs of both species. mna drafted the manuscript. egw constructed trees for all gene families. cik and mmsy performed antenna-specific assembly for d. ponderosae, as well as bioinformatic analysis, sequence alignment, sequence editing, and data analysis for obps, csps, and snmps from both species. jmb performed lab work, bioinformatic analysis, and data analysis. ml sequenced representative cdna clones of d. ponderosae genes that appeared to be full-length for submission to ncbi. fs conducted the go annotations and compilation of shared semiochemicals. cik, yh, jb, bsh, and fs conceived of the study, coordinated, and designed the study. all authors contributed to study design and manuscript preparation. all authors read and approved the final manuscript supplementary material additional file 1 accession numbers for genes encoding olfactory proteins that were submitted to genbank and the transcriptome shotgun assembly sequence database at ncbi. click here for file additional file 2 gene ontology results. gene ontology analyses as in figure but here represented as bar diagrams that have a higher resolution. a) molecular function level in ips typographus, b) molecular function level in dendroctonus ponderosae, c) biological process level in i. typographus, and d) biological process level in d. ponderosae. click here for file additional file 3 presence of odorant binding proteins in various tissues of dendroctonus ponderosae. analyses of sanger-specific data and normalized as well as non-normalized transcriptome assemblies from various body parts of d. ponderosae indicate that large sets of odorant binding proteins were found exclusively in non-antennal tissues. the numbers in the tables represent reads found. the clones were sequenced from both 5' and 3' directions. for further information see keeling et al.. click here for file additional file 4 presence of chemosensory proteins in various tissues of dendroctonus ponderosae. analyses of sanger-specific data and normalized as well as non-normalized transcriptome assemblies from various body parts of d. ponderosae indicate that of the identified chemosensory proteins were found exclusively in non-antennal tissues. the numbers in the tables represent reads found. the clones were sequenced from both 5' and 3' directions. for further information see keeling et al.. click here for file additional file 5 shared chemosphere of i. typographusandd. ponderosae. list of semiochemicals that are produced by the two bark beetle species, or present in their host or non-host plants and whether the compounds are active at a physiological and/or behavioral level in each species. click here for file acknowledgements we are thankful to sascha bucks for invaluable support in early laboratory work, and to bernd timmermann for next generation sequencing. we also acknowledge william walker iii for identifying orco in ips, and liwen song for help with the bioinformatic analysis. we acknowledge two anonymous reviewers for useful comments on the submitted manuscript. mna, yh, and fs were funded by the department of plant protection biology, slu, alnarp, and by the linnaeus program, “insect chemical ecology, ethology, and evolution, ice3”. jmb was supported by the swedish international development agency. work in jena was funded by the max planck society. cik, mmsy, jb, and ml were supported with funding from genome canada, genome british columbia, and genome alberta and the natural sciences and engineering research council of canada. during manuscript preparation, mna was funded by the swedish research council.
the protein families important for chemoreception have now been identified in three coleopteran species. predicted protein sequences were compared with counterparts in the flour beetle, tribolium castaneum, the cerambycid beetle, megacyllene caryae, and the fruit fly, drosophila melanogaster. putative grs for carbon dioxide and orthologues for the conserved antennal irs were included in the identified receptor sets. identification of these proteins in two of the most destructive forest pests, sharing many semiochemicals, is especially important as they might represent novel targets for population control. both bark beetle species utilize aggregation pheromones to coordinate mass-attacks on host trees, while odorants from host and non-host trees modulate the pheromone response. however, some clades contained receptors from all four beetle species, indicating a degree of conservation among some coleopteran or lineages. the european spruce bark beetle, ips typographus, and the north american mountain pine beetle, dendroctonus ponderosae, are severe pests of coniferous forests. ips typographusdendroctonus ponderosaegene ontologytranscriptomeodorant receptorionotropic receptorgustatory receptorodorant binding proteinchemosensory proteinsensory neuron membrane protein however, information on the genes underlying olfactory detection has been lacking in bark beetles and is limited in coleoptera. we assembled antennal transcriptomes from next-generation sequencing of i. typographus and d. ponderosae to identify members of the major chemosensory multi-gene families. focusing on the chemosensory gene families, we identified putative odorant binding proteins, chemosensory proteins, sensory neuron membrane proteins, odorant receptors, gustatory receptors, and ionotropic receptors in i. typographus; and putative obps, csps, snmps, ors, grs, and irs in d. ponderosae. transcripts with terms related to olfactory function were found in both species. thus, this study allows for improved evolutionary analyses of coleopteran olfaction.
 the european spruce bark beetle, ips typographus, and the north american mountain pine beetle, dendroctonus ponderosae, are severe pests of coniferous forests. both bark beetle species utilize aggregation pheromones to coordinate mass-attacks on host trees, while odorants from host and non-host trees modulate the pheromone response. thus, the bark beetle olfactory sense is of utmost importance for fitness. however, information on the genes underlying olfactory detection has been lacking in bark beetles and is limited in coleoptera. we assembled antennal transcriptomes from next-generation sequencing of i. typographus and d. ponderosae to identify members of the major chemosensory multi-gene families.  gene ontology annotation indicated that the relative abundance of transcripts associated with specific go terms was highly similar in the two species. transcripts with terms related to olfactory function were found in both species. focusing on the chemosensory gene families, we identified putative odorant binding proteins, chemosensory proteins, sensory neuron membrane proteins, odorant receptors, gustatory receptors, and ionotropic receptors in i. typographus; and putative obps, csps, snmps, ors, grs, and irs in d. ponderosae. predicted protein sequences were compared with counterparts in the flour beetle, tribolium castaneum, the cerambycid beetle, megacyllene caryae, and the fruit fly, drosophila melanogaster. the most notable result was found among the ors, for which large bark beetle-specific expansions were found. however, some clades contained receptors from all four beetle species, indicating a degree of conservation among some coleopteran or lineages. putative grs for carbon dioxide and orthologues for the conserved antennal irs were included in the identified receptor sets.  the protein families important for chemoreception have now been identified in three coleopteran species. thus, this study allows for improved evolutionary analyses of coleopteran olfaction. identification of these proteins in two of the most destructive forest pests, sharing many semiochemicals, is especially important as they might represent novel targets for population control. ips typographusdendroctonus ponderosaegene ontologytranscriptomeodorant receptorionotropic receptorgustatory receptorodorant binding proteinchemosensory proteinsensory neuron membrane protein 