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Genome sequence of the wheat stem sawfly, Cephus cinctus, a primitive hymenopteran and wheat pest, illuminates evolution of hymenopteran chemoreceptors

The wheat stem sawfly, Cephus cinctus, is a major pest of wheat and key ecological player in the grasslands of western North America. It also represents a distinctive lineage of sawflies that appeared early during the hymenopteran radiation, but after the clade of Eusymphyta sawflies that is the sister lineage of all other Hymenoptera. We present a high-quality draft genome assembly of 162 Mbp in 1,976 scaffolds with a scaffold N50 of 622 kbp. Automated gene annotation identified 11,210 protein-coding gene models and 1,307 non-coding RNA models. Thirteen percent of the assembly consists of ~58,000 transposable elements partitioned equally between Class-I and Class-II elements. Orthology analysis reveals that 86% of Cephus proteins have identifiable orthologs in other insects. Phylogenomic analysis of conserved subsets of these proteins supports the placement of the Cephidae between the Eusymphyta and the parasitic woodwasp superfamily Orussoidea. Manual annotation and phylogenetic analysis of families of odorant, gustatory, and ionotropic receptors, plus odorant binding proteins, shows that Cephus has representatives for most conserved and expanded gene lineages in the Apocrita (wasps, ants, and bees). Cephus has also maintained several insect gene lineages that have been lost from the Apocrita, most prominently the carbon dioxide receptor subfamily. Furthermore, Cephus encodes a few small lineage-specific chemoreceptor gene family expansions that might be involved in adaptations to new grasses including wheat. These comparative analyses identify gene family members likely to have been present in the hymenopteran ancestor and provide a new perspective on the evolution of the chemosensory gene repertoire.


Introduction
The wheat stem sawfly, Cephus cinctus Norton, is a major pest of wheat in western North America with a southward expanding geographic range likely driven by localized adaptation to cultivated crops from surrounding wildlands (Beres et al. 2011;Lesieur et al. 2016;Adhikari et al. 2018;Varella et al. 2018). It is a native species that also uses many other grass hosts and hence plays an important role in the ecology of grasslands (Cockrell et al. 2017). Worldwide, as insect burdens on wheat farmers, members of the genus Cephus rival the Hessian fly, Mayetiola destructor, a dipteran for which a genome sequence is available (Zhao et al. 2015). Female sawflies lay eggs in growing wheat stems and the larvae grow within the stem lumen ( Figure 1).
Once in the final instar, the sole survivor of larval cannibalism in each stem retreats to the base of the stem, which it girdles in preparation for pupation 2015). Economic loss is from both the larval presence and the subsequent collapse or lodging of the wheat stem (Bekkerman and Weaver 2018). As a result of this life history, wheat stem sawflies are protected from control using conventional sprayed insecticides. The development of alternative approaches relies on an improved understanding of C. cinctus chemical ecology (Cossé et al. 2002;Weaver et al. 2009) and associated behaviors on host plants Varella et al. 2017), but large-scale molecular biology studies to date remain limited to antennal transcriptomics that examined chemosensory genes (Gress et al. 2013) and analyses of larval and adult RNA sequencing (RNA-seq) data that focused on the identification of non-coding RNA transcripts (Cagirici et al. 2017). A key first step in this direction is to build genomic and additional transcriptomic resources for this sawfly that could, for example, facilitate the development of 5 new resistant wheat strains using expression of RNA interference (RNAi) constructs against essential wheat stem sawfly genes.
An additional significance to this insect is that the genus Cephus represents the sawfly family Cephidae and superfamily Cephoidea, and molecular phylogenomic analysis of the Hymenoptera suggests that this is a distinctive lineage of sawflies that radiated after the more well-known and basal sawflies such as the family Tenthredinidae, now called the Eusymphyta, and before the parasitic woodwasp superfamily of Orussoidea (Peters et al. 2017). It therefore represents an interim step in the origin of the major lineage of the Apocrita, and so provides an important comparator and outgroup for understanding some of the peculiarities of the genomes, gene contents, and biology of the wasps, ants, and bees. Genomic resources for C. cinctus are therefore not only important for pest control but also contribute to augmenting the growing representation of sequenced hymenopteran genomes (Branstetter et al. 2018).
Here we report a draft genome assembly and annotation for C. cinctus, with genome-wide analyses of protein orthology and repeat content, and phylogenomic analysis that supports the placement of Cephoidea between Eusymphyta and Orussoidea. Supported by extensive manual gene annotation efforts, our phylogenetic analyses focused on four major gene families encoding chemosensory proteins of ecological importance. Cephus, as a member of an early radiating lineage, provides a new perspective on the likely ancestral repertoires and subsequent lineagespecific evolution of odorant binding proteins (OBPs), and odorant, gustatory, and ionotropic receptors (ORs, GRs, and IRs), across the Hymenoptera. 6

DNA isolation, genomic library preparation, sequencing, and genome assembly
Genomic DNA was isolated from a single male and separately from pooled females (Table S1).
Briefly, insects were ground in liquid nitrogen before lysing in a SDS solution overnight with Proteinase K. The homogenate was treated with RNaseA, and proteins/debris were collected after high-salt precipitation and centrifugation. After ethanol precipitation, the DNA was resuspended in 10 mM Tris and evaluated on an agarose gel and by Qubit quantification.  (Luo et al. 2012) was employed with K=49 to assemble the 500 bp-insert shotgun library reads followed by scaffolding with iteratively longer-insert shotgun and mate-pair libraries and use of GapCloser v1.12 to close gaps generated in the scaffolding (Luo et al. 2012).

RNA isolation, RNAseq library preparation, sequencing, and transcriptome assembly
We undertook RNAseq studies of whole animals and tissues relevant to chemosensation such as antennae, heads without antennae, abdomen tips, and abdomens without tips (Table S1) microRNA annotation was performed as outlined previously (Cagrici et al., 2017). Highconfidence microRNAs for Hexapoda were downloaded from miRBase.org (Release 21) and a query was formed for a homology-based screen of the genome assembly.

Phylogenomics, orthology, gene set and genome completeness
The maximum likelihood molecular species phylogeny ( Figure 2) was estimated from the concatenated protein sequence superalignment of 852 single-copy orthologs, using the automated gene models of CcinOGSv1.0 extracted from OrthoDB v9.1 ), using the 17 species shown in Table S3. The presence, absence, and copy-numbers of orthologs from 116 insect species were assessed to partition genes from each species into the distribution of categories shown in the bar chart ( Figure 2). For each of the two sawflies and the woodwasp, percent amino acid identities with orthologs from Nasonia vitripennis, Pogonomyrmex barbatus, and Apis mellifera, were calculated from the trimmed multiple sequence alignments of 1,035 single-copy Hymenoptera orthologs ( Figure 2, violin plots). Gene Ontology (GO) terms from Drosophila melanogaster, A. mellifera, Tribolium castaneum, and Anopheles gambiae were compared to identify C. cinctus genes with orthologs associated with GO terms assigned to at least two (i.e. annotations in common), just one (i.e. unique annotations), or none of the four species. Completeness in terms of expected gene content for the genomes and annotated gene sets of each of the eight analysed hymenopteran species was assessed using both Hymenoptera

Transposable and repeat element annotation
Although a repeat analysis for masking purposes is part of the NCBI gene annotation steps, we undertook a dedicated analysis using the REPET package (Quesneville et al. 2005;Flutre et al. 2011). The computational pipeline to predict and classify transposable element (TE) integrations used both de novo-(TEdenovo) and homology-based methods (TEannot). A summary of the REPET pipeline can be found at https://urgi.versailles.inra.fr/Tools/REPET and detailed description of the applications and methods are in the Supplementary Online Material.

Expression Analysis
Levels of gene expression were determined by aligning the trimmed RNAseq reads from each library against the chemoreceptor and OBP transcripts from the i5k Workspace@NAL, occasionally truncated to avoid overlap with the UTRs of well-expressed neighboring genes, using the Burrows-Wheeler Aligner (BWA) ) and Read1 of each read pair.
Samtools ) was used to sort, index, and summarize the BWA. Read counts were standardized as Read counts Per Kilobase transcript per Million mapped reads in each library 1 1

A high quality genome assembly and annotation
The draft genome assembly consists of 10,707 contigs with a N50 of 45 kbp that were connected into 1,976 scaffolds with a N50 scaffold size of 622 kbp and a total of 162 Mbp including 3 Mbp of gaps between contigs within scaffolds. This assembly (v1) is available from the National Center for Biotechnology Information (NCBI) as BioProjects PRJNA297591 and PRJNA168335 (NCBI GCF_000341935.1). It is of a size and quality comparable to those of other Hymenoptera in GenBank (Table S2). We assessed the completeness of this assembly using BUSCO (Benchmarking Universal Single-Copy Orthologs) (Waterhouse et al. 2018), which revealed high completeness scores of 97.6-99.5%, with few duplicated (0.3-0.7%), fragmented (0.3-1.7%), or missing (0.2-0.7%) genes. These assembly completeness estimates are on a par with or marginally better than for the other assessed hymenopteran genomes (Table S4). In addition, 86-94% of the ILLUMINA RNAseq reads described below mapped to the assembly (NCBI C. cinctus Annotation Release 100https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Cephus_cinctus/100/). These high levels of completeness and RNAseq read mapping indicate that this draft genome is a high quality assembly that supports effective automated and manual gene modeling, as demonstrated below.
To support gene modeling and assess gene expression across lifestages and tissues, we performed paired-end ILLUMINA RNAseq on three samples of larvae, two samples each of adult males and females, one sample each of male and female pupae, and one sample each of antennae, heads without antennae, abdomen tips, and abdomens without tips from adult males 1 2 and females (Table S1 -Auxillary file 1). These libraries ranged in size from 3.5-12 Gbp. This was additionally supplemented by an available RNAseq dataset of 717,345 454-pyrosequencing reads from mixed-sex antennae (Gress et al. 2013 complete, and few duplicated (0.4-0.7%), fragmented (0.2-0.7%), or missing (0.4-0.6%) genes.
The close match of BUSCO scores for the assembly and annotations indicate that the annotation strategy was successful. These annotation completeness estimates are on a par with or marginally better than for the other assessed hymenopteran genomes (Table S4). This automated gene set therefore provides a confident base for comparative genomics, as well as subsequent manual gene annotation.

Transposable and repeat element content
The repeat prediction pipeline identified 51,432 simple sequence repeats (SSRs) of unit length ≥ 1 3 repeats were most abundant ( Figure S1). A total of 64,215 TE fragments totaling 20.8 Mbp (~13% of the assembled genome) were resolved into 57,948 predicted copies using the Long_join method to connect disrupted portions of the same integration. These predictions are available as a track at the i5k Workspace@NAL browser for this genome (https://i5k.nal.usda.gov/cephus-cinctus). The PASTEC classification of predicted repeat elements from the REPET TEdenovo pipeline identified 840 unique elements placed into six Class-I families and five Class-II families (Table S5) terminal-repeat retroelements in miniature (TRIM) elements (Table S5). Inverted repeat (TIR) family members are most abundant and encompass the greatest proportion of the genome among Class-II DNA elements. The miniature inverted repeat transposable elements (MITEs) are the second most abundant family, but likely due to being the only fully non-autonomous family of Class-II elements they occupy a smaller proportion of the genome than TIR or Maverick elements.

Phylogenomics and protein orthology
The molecular species phylogeny (Figure 2A), estimated from concatenated protein sequence alignments of single-copy orthologs, supports the current view of sawfly radiations early during  Figure 2B). The two sawflies and the woodwasp have similarly low total gene counts and show similar proportions of genes in each orthology category. In contrast, the other representative hymenopterans have generally higher total gene counts including a fraction of seemingly apocritan-specific orthologs.
The small fraction of sawfly genes with orthologs in outgroup species, but not in other Hymenoptera, highlight potential gene losses in the apocritan ancestor. These include orthologs of D. melanogaster genes TrpA1 (Transient receptor potential cation channel A1) involved in responses to heat and noxious chemicals (Luo et al. 2017), and the G-protein-couple receptor Lgr1 (Leucine-rich repeat-containing G protein-coupled receptor 1) involved in development (Vandersmissen et al. 2014). This fraction also includes a gene encoding a CPCFC family cuticular protein, previously described by Vannini et al. (2015) as being present in C. cinctus but missing from apocritan species. Further examples of putative losses in the Apocrita ancestor, from our detailed analysis of chemoreceptor gene families, are elaborated below.
Examining ortholog sequence conservation between sawfly and Apocrita species showed that C.
cinctus proteins had significantly higher amino acid identities (Wilcoxon signed rank tests, p<1e-39) with the Apocrita than either A. rosae or O. abietinus ( Figure 2C). This apparently slower 1 5 rate of sequence divergence in C. cinctus may at least partially explain the uncertainty of the placement of O. abietinus in the species phylogeny (Figure 2A), which was alternatively placed as sister to C. cinctus in 10% of bootstrap samples. With little detailed functional characterization of C. cinctus genes to date, putative functions can instead be inferred by identifying orthologs from well-studied insects such as the honey bee, malaria mosquito, fruit fly, or flour beetle, to tentatively link Gene Ontology terms to the majority of C. cinctus genes ( Figure 2D).

microRNAs
A total of 36 putative high confidence miRNAs from 22 different miRNA families were identified. Of these, 24 were located on the sense strand while 12 were on the antisense strand (Table 1).

The C. cinctus chemosensory gene repertoire
Insects depend on members of three major gene families for most of the sensitivity and specificity of their senses of smell and taste (Leal 2013;Joseph and Carlson 2015). as the Odorant Binding Protein (OBP) family of secreted small globular proteins, resulting in high-quality gene models from which to build robust inferences of their evolutionary histories.
The OR family consists of a highly conserved Odorant receptor co-receptor (Orco) gene that has 1:1 orthologs throughout the pterygote insects (Ioannidis et al. 2017) and forms dimers with each of the many "specific" ORs (Benton et al. 2006). In Cephus there are 72 OR genes including the expected single highly conserved Orco (Table S6) Phylogenetic analysis using protein sequences from these species, employed because they have undergone similarly intensive manual annotation, reveals that the Cephus ORs are largely present as a single gene, or sometimes small clusters of genes, at the base of numerous small clades and sometimes highly-expanded subfamilies in these other hymenopterans ( Figure S2).
Most remarkably, a single gene (CcinOr69) is at the base of the extremely large 9-exon subfamily, which is particularly highly expanded in ants and mediates perception of their cuticular hydrocarbons of crucial importance to social interactions (Smith et al. 2011;Pask et al. 2017). Similarly, a small species-specific clade consisting of CcinOr1-9 is at the base of the large-tandem-array subfamily of 61 genes in A. mellifera (Robertson and Wanner 2006 The GR family is a highly diverse family of primarily taste receptors, but also some olfactory receptors such as the carbon dioxide receptors of flies and moths, including receptors for sugars and a vast array of "bitter" compounds, as well as light and heat. It is an ancient family within the Metazoa, dating back to basal animals (Saina et al. 2015;Robertson 2015;Eyun et al. 2017), but was lost from vertebrates, which instead employ primarily G-protein-coupled receptors for both olfaction and taste. We identified a total of 35 GR genes in C. cinctus (Table S7) vitripennis for which our manual annotation efforts increased the gene count from just 10 in Croset et al. (2010) to 153 genes, albeit 54 (35%) are pseudogenes (supplement). Cephus has the expected single orthologs of the highly conserved Ir8a, 25a, and 76b genes, which encode proteins that function as co-receptors with other IRs (Rytz et al. 2013). It also has single orthologs for three of the four genes involved in thermo-and hygro-sensation in D. melanogaster (Ir21a, 68a, and 93a), but Ir40a was lost at the base of the Hymenoptera. Ir40a cooperates with the other proteins to sense humidity in D. melanogaster (Knecht et al. 2017), so it would be interesting to discover how this gene loss, and idiosyncratic losses of Ir21a and Ir68a in various Hymenoptera, affect their abilities to perceive temperature and humidity. Cephus has only a 1 9 single relative of the DmelIr75a-c genes that encode olfactory receptors for various acids (Prieto-Godino et al. 2017), a lineage that is expanded in wasps, but of comparable size to flies in ants and bees. There is a single hymenopteran set of orthologs for the DmelIr41a lineage of olfactory receptors that is commonly greatly expanded in other insects (Robertson et al. 2018). Like the ORs and GRs, Cephus has generally single representatives of other hymenopteran IR lineages, but also two Cephus-specific clades, one of several ancient gene lineages that appear to have been lost from Apocrita, and one of very recent gene duplications ( Figure S3). These clades, along with a few bee and ant proteins and the majority of the Nasonia proteins, are related to the DmelIr7a-f/11a proteins and the large DmelIr20a clade implicated in gustation (Rytz et al. 2013;Koh et al. 2014). This analysis suggests that ancestral hymenopterans had approximately 34 IR identified 15 OBP genes and their encoded proteins in C. cinctus (Table S9), which like the chemoreceptors are frequently at the base of a clade of single orthologs or expansions in these other hymenopterans ( Figure S4).

Expression levels of chemoreceptors and OBPs
We examined the levels of expression of the chemoreceptors and OBPs in our 17 diverse RNAseq datasets from larvae, pupae, adults, and adult body parts ( TableS1  perhaps because larvae only have a few olfactory sensilla given their feeding mode within wheat stems. These expected results for Orco provide confidence that our other results are reliable. Most OR genes have appropriately low expression, primarily in antennae ( Figure S5), represented by Or5 in Figure 4, while some of the divergent genes like Or60 and Or72 are barely expressed. There are a few instances of consistent sex bias. For example, Or1 is consistently expressed ~4X higher in males than females, while Or30-32 exhibit 5-7X higher expression in males, although these three might be conflated as they are very similar genes in a tandem array (represented by Or32 in Figure 4). All four are amongst the mostly highly expressed Ors, a result confirmed by prior quantitative PCR that also found Or32 expression 15X higher in male 2 1 antennae (Gress et al. 2013; Table S14), making these candidate receptors for female pheromone.
The expression of Ors 18, 63, 65 and 68 was higher in female antennae, in this study and in prior quantitative PCR experiments (5-15X female bias, Gress et al. 2013; Table S14), making these candidate receptors for host plant volatiles or male-produced pheromones.
Some ORs exhibit unusual expression patterns, for example, the tandemly-arrayed Or11-13 are expressed at levels comparable to Orco in heads without antennae, and Or11 is also extremely specifically their abdomens. The 3' UTR of this gene overlaps that of a neighboring highlyexpressed DNA polymerase gene, hence the analyzed transcript was shortened at the 3' end, so this result has to be treated with caution, nevertheless the reads that map to Or26 are mostly correctly spliced, implying they are from this locus rather than the 3' UTR of the polymerase.
Amongst the GRs, as expected the sugar receptors Gr1/2 are expressed primarily in heads at levels comparable to or higher than Orco, however Gr2 is also expressed in abdomens and abdominal tips, and unusually highly in female abdomens. The candidate fructose receptor Gr3 is the mostly highly expressed GR, and as expected from the expression of its Drosophila ortholog DmelGr43a (Miyamoto et al. 2012), primarily in heads but also in abdomens, larvae, and pupae, and appropriately low in antennae. Most of the other GRs show the expected pattern 2 2 of low levels of expression primarily in heads ( Figure S6), rather than antennae, a pattern exemplified by Gr36 ( Figure 5). Exceptions include Gr12, which is consistently highly expressed in females, primarily in abdomens. The two candidate carbon dioxide receptors, Gr24/25, are also not well expressed in antennae, but rather in heads, and Gr24 is unusually highly expressed in pupae. The related carbon-dioxide receptor subfamily gene Gr26 shows a similar pattern (unfortunately Gr27 overlaps the 3' UTR of a highly-expressed gene, so could not be evaluated).
Amongst the IRs, Ir25a and 76b, two co-receptors, are the most highly expressed, primarily in heads, as are a few others like Ir41a, however Ir76b is extremely highly expressed in male abdominal tips, a clear result with almost entirely spliced reads. The third co-receptor, Ir8a, is expressed at a relatively low level. Ir93a is the most highly expressed of the three genes implicated in perception of temperature and humidity, with Ir68a the lowest of these three. It is noteworthy that these are the more conserved receptors whose orthologs in D. melanogaster are primarily expressed in antennae (Rytz et al. 2013). Amongst the more divergent IRs, the most highly expressed are Ir108 and Ir120, again primarily in heads, while Ir140 is shown as an example of the generally low expression of the IRs with likely gustatory roles ( Figure 6).
Finally, as expected the OBPs commonly exhibited far higher levels of expression than the chemoreceptors ( Figure S8). Perhaps surprisingly, only Obp1, Obp4, Obp6, Obp11 and Obp12 are well expressed in antennae (and male abdominal tips for Obp12) (represented by Obp6 and Obp12 in Figure 7). Obp2 and Obp9 are primarily expressed in heads (and larvae for Obp9), while Obp3, Obp5, Obp8 and Obp15 have unusually low levels of expression for OBPs, and 2 3 mostly in larvae. Obp7 and Obp10 are primarily expressed in male abdomens, especially the tips, while Obp13 is almost exclusively expressed in larvae. Finally, Obp14 is extraordinarily highly expressed in multiple samples, most highly in female abdominal tips. The observation of high expression of several OBPs in abdominal tips implies a non-olfactory role for these proteins.

Discussion
Genome sequencing offers the opportunity to explore the putative genomic or genetic basis of organismal biology that may be widely shared among species or highly lineage-specific. The informed interpretation of observed patterns of conservation and divergence requires a robust understanding of the evolutionary relationships among the considered species, which in the case of sawflies has remained unclear until relatively recently. While the separation of Cephidae from the other symphytan sawflies had been proposed for some time, it was only with the phylogenomics study of Peters et al. (2017), based on protein sequences deduced from transcriptomes, that this placement was put on more solid footing. Our genome-based phylogenomics analysis cements the case for placement of the Cephidae as a secondary radiation after the Eusymphyta, and with considerable confidence agrees with Peters et al. (2017) in the branching of the Cephidae before the orussid woodwasps. The apparently constrained rates of molecular evolution of Cephus genes relative to other sawflies and woodwasps, resulting in higher amino acid identities with their Apocrita orthologs, may partially explain previous difficulties in attempting to resolve these early hymenopteran radiations.

4
The availability of many other hymenopteran and other insect genomes means that the vast majority (86%) of Cephus proteins can be confidently assessed as having orthologs in other species, despite the deep branching of this lineage. Functional information from orthologs in D.
melanogaster and other insects allows assignment of putative biological roles for more than half of the annotated C. cinctus protein-coding genes. These associations reflect the ever-increasing knowledge of insect molecular biology and provide a strong foundation for future functional work on this sawfly.
Our repeat analysis from this 162 Mbp genome assembly revealed that 1% consists of simple sequence repeats, amongst which are just 27 copies of the TTAGG telomeric repeat found to constitute the telomeres of the honey bee (Robertson and Gordon 2006 Our analysis of the three major chemoreceptor families and the OBPs reveals Cephus genes at the base of most apocritan gene lineages, many as single-copy orthologs but also several with remarkably large expansions in wasps, ants, and/or bees. While only a few gene lineages appear In summary, this high-quality draft genome assembly and automated gene annotation will facilitate further molecular biological studies on this sawfly, and offers opportunities to identify genes for engineering of sawfly-resistant wheat strains with RNAi constructs. It also augments the diversity of hymenopterans with sequenced genomes, particularly for the poorly sampled sawflies (Branstetter et al. 2018), making this insect order second only to Diptera for the number of species with genome sequences.   A. After overwintering in diapause in a senesced wheat stem, a pre-pupa metamorphoses in late spring or early summer. The pupal stage lasts 10-12 days, after which the newly-eclosed adult chews a hole in a frass plug left by the larva to emerge from the stem.
B. The adults can mate immediately after emergence, either on the wheat stubble or on new host stems. The short-lived females oviposit 30-40 eggs before dying in 7-10 days.
C. Newly-deposited eggs in the stem lumen, usually from multiple females, hatch in 5-10 days.
The neonate larvae feed on the parenchyma lining the stem interior. As the larvae grow through 4-5 larval instars they forage on the stem lining throughout the stem by boring through the nodes.
The duration of larval feeding can range from 3-8 weeks.
D. Within the stem, cannibalism leads to a single surviving larva that migrates to the base of the stem at plant senescence and ripening. The larva girdles the interior of the ripened stem wall, cutting a groove that encircles the entire stem. Wind or gravity causes the weakened stem to break and lodge, leaving the unharvested wheat head on the soil. Below this groove, the larva makes a frass plug before migrating to the base of the stem near the crown. Within a thin hibernaculum, the pre-pupa remains protected in obligate diapause from late summer through to warming the following spring. This period of inactivity can last more than 9 months. Asterisks indicate statistically significant differences with p-values <1e-108 (**) and <1e-39 (*).
(D) Functional inferences for C. cinctus genes from Gene Ontology (GO) annotations of orthologs from A. mellifera, Anopheles gambiae, Drosophila melanogaster, or Tribolium castaneum. Of the 7,595 C. cinctus genes with orthologs from any of these four species, 57% were in orthologous groups with GO terms shared amongst at least two of the four species (common annotations), 22% with GO terms from only one species (unique annotations), and 21% where no GO terms were associated with any of the orthologs (no annotations).