The genome of the model beetle and pest Tribolium castaneum

Article metrics

Abstract

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell–cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.

Main

By far the most evolutionarily successful metazoans1, beetles (Coleoptera) can luminesce (fireflies), spit defensive liquids (bombardier beetles), visually and behaviourally mimic bees and wasps, or chemically mimic ants that detect intruders by their foreign odour. Many beetles (for example, boll weevil, corn rootworm, Colorado potato beetle and Asian longhorn beetle) are associated with billions of dollars of agricultural and natural resource losses.

The red flour beetle, Tribolium castaneum, found wherever grains or other dried foods are stored, has a highly evolved kidney-like cryptonephridial organ to survive such extremely dry environments. It has demonstrated resistance to all classes of insecticides used against it. Like all beetles, Tribolium has elytra (wing covers) that coordinate precisely with folding wings, allowing flight while providing protection.

Tribolium facilitates genetic analysis with ease of culture, a short life cycle, high fecundity, and facility for genetic crosses (see ref. 2), allowing efficient genetic screens by means of chemical mutagens, radiation and binary transposon systems3. As in Caenorhabditis elegans, RNA interference (RNAi) is systemic in Tribolium, facilitating knockdown of specific gene products in any tissue, developmental stage or offspring of double-stranded (ds)RNA-injected females4,5.

Particularly favoured for developmental studies, Tribolium is much more representative of other insects than is Drosophila6. In contrast to Drosophila, Tribolium larvae display eyes in a fully formed head and three pairs of thoracic legs (Supplementary Fig. 1). In addition, Tribolium develops via short-germ embryogenesis where additional segments are sequentially added from a posterior growth zone (Supplementary Fig. 1). This proliferative mechanism of segmentation differs from the Drosophila model, but resembles that of vertebrates and basal arthropods such as millipedes7.

Genome sequence and organization

Approximately 1.52 million sequence reads (7.3× coverage) were generated from the highly inbred Georgia 2 (GA2) strain and assembled into contigs totalling 152 megabases (Mb) and scaffolds spanning 160 Mb of genomic sequence (Supplementary Tables 1–4 and Supplementary Information). Almost 90% of this sequence was mapped to the ten Tribolium linkage groups using a genetic map of 500 markers generated from the GA2 strain8. Excluding heterochromatic regions dense in highly repetitive sequences, the genome is well represented and of high quality (see Supplementary Data for details).

G+C content

Tribolium, like Apis, has a very (A+T)-rich genome (33% and 34% G+C, respectively), but Tribolium G+C domains lack the extremes of G+C content present in Apis mellifera (Fig. 1 and Supplementary Fig. 3). Despite global G+C similarity to Apis, genes in Tribolium, as in Anopheles and Drosophila but not Apis, show a bias towards occurring in (G+C)-rich regions of the genome (Fig. 1). Whatever mechanism drives the accumulation of A+T nucleotides in Tribolium, it does not affect genes in the manner observed in the honeybee, where perhaps additional mechanisms are present.

Figure 1: Cumulative distribution of genic and genomic G+C-content domains in Apis mellifera, Anopheles gambiae, Drosophila melanogaster and Tribolium castaneum.
figure1

Cumulative distributions show the fraction of genes (thick lines) or of the entire genome (thin lines) occurring in G+C-content domains less than a given percentage G+C (<X). The more (A+T)-rich half of the T. castaneum genome contains only 30.8% of all T. castaneum genes (31.4% and 33% of A. gambiae and D. melanogaster genes, respectively), whereas the more (A+T)-rich half of the A. mellifera genome contains 77.6% of its genes. At every point on the T. castaneum, A. gambiae and D. melanogaster curves there are fewer genes present in the fraction of the genome less than a given percentage G+C than would be expected if the genes were randomly distributed. In contrast, A. mellifera exhibits the opposite distribution.

Repetitive DNA

Fully one-third of the Tribolium genome assembly consists of repetitive DNA, which is also (A+T)-rich. Compared to other insects, there is a paucity of microsatellites (1–6-base-pair (bp) motifs) in Tribolium9. However, Tribolium contains a relative excess of larger satellites, including several with repeat units longer than 100 bp (2.5% of the Tribolium genome compared with 0.7% in Drosophila). Most (83%) of the microsatellites are found in intergenic regions (63%) or introns (20%), but there is strong over-representation of non-frameshift-causing repeats (3- and 6-bp motifs) due to a dearth of dinucleotide repeats (see Supplementary Information). Of 981 randomly chosen microsatellites, 509 (55.2%) are polymorphic in a sample of 11 Tribolium populations from around the world9, providing an extensive collection of markers for population studies. Preliminary efforts to assess global population structure show a shallow but significant correlation between geographic and genetic distance (Supplementary Fig. 4). This suggests that anthropogenic dispersal may maintain a modest level of gene flow across vast distances in this human commensal.

Transposable elements

Transposable elements and other repetitive DNA accumulate in regions along each linkage group that resemble the pericentric blocks of heterochromatin visible in HpaII-banded chromosomes10. These regions are probably composed largely of highly repetitive heterochromatic sequences, and represent most of the 44-Mb difference between the estimated genome size (0.2 pg or 204 Mb11) and the current assembly (160 Mb). Indeed, as much as 17% of the Tribolium genome is composed of a 360-bp satellite12 that constitutes only 0.3% of the assembled genome sequence. Several families of DNA transposons, as well as long terminal repeat (LTR) and non-LTR retrotransposons, constituting approximately 6% of the genome, were identified via encoded protein sequence similarity to previously identified elements using TEPipe or BLAST, and are listed in Supplementary Table 5.

Telomeres

Tribolium has a telomerase and telomeres containing TCAGG repeats13, a variant of the standard arthropod TTAGG telomeric repeat. Manual assembly of the proximal regions of multiple telomeres beyond the ends of the assembled scaffolds (Supplementary Information) reveals TCAGG repeats interrupted by full-length and 5′-truncated non-LTR retrotransposons belonging to the R1 clade, best known for insertions in the rDNA locus14. Tribolium telomeres range in length from 15 kilobases (kb) upwards and probably represent a stage intermediate to the loss of telomeres and telomerase in Diptera compared with the simple canonical structure of the honeybee15 or the more regular insertion of non-LTR retrotransposons into the simple repeats of the silkmoth16.

Gene content and the proteome

Comparative gene content analysis

To understand the consensus set of 16,404 gene models in the context of other available insect and vertebrate genomes, all genes were classified according to their degree of similarity using systematic cross-species analysis. Five insects (Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, T. castaneum, A. mellifera) and five vertebrates (Homo sapiens, Mus musculus, Monodelphis domestica, Gallus gallus, Tetraodon nigroviridis) with similar phylogenetic branching orders were chosen for the comparison. We found the fractions of universal and insect-specific orthologues in Tribolium similar to other insect genomes, as expected, whereas the number of genes without similarity is considerably higher (Fig. 2), possibly attributable to less stringent gene prediction.

Figure 2: Insect gene orthology.
figure2

Comparison of the gene repertoire in five insect and five vertebrate genomes, ranging from the core of metazoan genes (dark blue fraction on the left) to the species-unique sequences (white band on the right). The striped boxes correspond to insect- and vertebrate-specific orthologous genes, where the darker bands correspond to all insects or vertebrates (allowing one loss). N:N:N indicates orthologues present in multiple copies in all species (allowing one loss); patchy indicates ancient orthologues (requiring at least one insect and one vertebrate gene) that have become differentially extinct in some lineages. The species tree on the left (shown in detail in Supplementary Fig. 6) was computed using the maximum-likelihood approach on concatenated sequences of 1,150 universal single-copy orthologues. It shows an accelerated rate of evolution in insects and confirms the basal position of the Hymenoptera within the Holometabola17. Aaeg, Aedes aegypti; Agam, Anopheles gambiae; Amel, Apis mellifera; Dmel, Drosophila melanogaster; Ggal, Gallus gallus; Hsap, Homo sapiens; Mdom, Monodelphis domestica; Mmus, Mus musculus; Tcas, Tribolium castaneum; Tnig, Tetraodon nigroviridis.

Over 47% of Tribolium genes (7,579) are ancient, with traceable orthologous relations between insects and vertebrates including 15% (2,403) universal single-copy orthologues. Another 1,462 Tribolium genes (9%) constitute the core of what are currently insect-specific orthologues. In comparison, 21% (4,937) of human genes have vertebrate-specific orthologues.

Several hundred ancient genes seem to be under limited evolutionary selection and were independently lost in several species studied (the patchy fraction, defined in Fig. 2). Each new genome uncovers previously invisible ancestral relations among genes—for example, as many as 126 orthologous gene groups shared between Tribolium and humans seem to be absent from the other sequenced insect genomes (Fig. 3 and Supplementary Table 10), 44 of which are single-copy genes present in all vertebrates.

Figure 3: Orthologous genes shared between insect and human genomes.
figure3

The Venn diagram shows the number of orthologous groups of genes shared between the insect and human genomes. In addition to the majority of Urbilateria (last common ancestor of the Bilateria) genes shared by all the organisms, there are hundreds of genes that have been lost in some lineages (for example, only retained between human and Tribolium or human and honeybee, but lost in Diptera). Diptera is represented here by Anopheles gambiae, Aedes aegypti and Drosophila melanogaster (with numbers considering only D. melanogaster shown in parentheses).

The evolutionary emergence of many predicted Tribolium genes is not clear. Thousands of genes currently appear to be species-specific as either no sequence similarity to other genes is detectable, or homology but not orthology can be determined. Reassuringly, this fraction is similar in Tribolium and Drosophila.

We quantified the species phylogeny using a maximum likelihood approach with the concatenated multiple alignment of 1,150 universal single-copy orthologues present in all the organisms studied—an ideal genome-wide data set of essential genes evolving under similar constraints (Fig. 2 and Supplementary Fig. 6). This analysis confirmed previous analyses based on expressed sequence tag (EST) sequences that the Hymenoptera are basal within the Holometabola17. The shorter branch length for Tribolium implies that the elevated rate of evolution observed in Drosophila and Anopheles occurred more recently18.

Gene family expansion, frequently associated with a particular adaptation pressure, might reveal physiologically and phenotypically unique features of beetles (Supplementary Table 9 and protein family discussions below). Many duplications shaped the gene content of Tribolium, most notably among odorant-binding proteins and the CYP450 subfamilies CYP6 and CYP9 (Supplementary Fig. 11), some of which are involved in the development of insecticide resistance in the Diptera19. Duplication of genes under copy-number selection in other species is indicative of species-specific neo-functionalization20. At least 152 genes duplicated in Tribolium have single-copy status in all other insects studied, including sevenfold duplication of genes orthologous to Drosophila CG1625, encoding a putative structural constituent of cytoskeleton, and human ENSP00000269392, encoding centrosomal pre-acrosome localization protein 1.

We also analysed the phylogenetic distribution of orthologous gene group members to quantify evolutionary gene losses21. Although least affected, dozens of single-copy orthologues seem to be lost in each lineage. Thirty-eight such genes lost in Tribolium include rather unique genes, encoding phosphotriesterase-related protein and peroxisome assembly factor 1 (peroxin-2), compared to 59 such genes lost in Drosophila. Notably, for the less restricted fractions of orthologues (defined in Fig. 2), several hundred gene orthologues have been lost in each species.

Analysis of specific gene sets

In addition to a global automated analysis of the predicted Tribolium gene set, the consortium manually annotated and analysed 2,000 genes (some additionally subjected to RNAi and expression analysis), focusing on developmental processes and genes of importance for agriculture and pest management.

Development

We identified and analysed homologues of known insect and vertebrate developmental genes to gain novel insights into the molecular basis of developmental differences between Drosophila and Tribolium. Supplementary Table 11 lists selected Tribolium developmental genes and their Drosophila and Apis orthologues.

Oogenesis

Despite profound differences in ovarian architecture—telotrophic versus polytrophic—we identified Tribolium orthologues of most Drosophila genes required for stem cell maintenance, RNA localization and axis formation. Like Apis, however, Tribolium lacks a bag of marbles orthologue, which is essential for the differentiation of cystoblast versus germline stem cells in Drosophila22. Interestingly, an orthologue of the gene gld-1, which fulfils a similar function in C. elegans22, is present in Tribolium.

Anterior–posterior patterning

Analysis of the genome sequence confirmed the absence of a bcd orthologue in Tribolium. Instead, anterior patterning is synergistically organized by otd and hb (ref. 23). However, it is still unclear how the posterior gradient of Tribolium Caudal is shaped in the absence of Bicoid. Notably, Tribolium contains an orthologue of mex-3, a factor that translationally represses the C. elegans cad homologue24. Although the Tribolium genome contains orthologues of the Drosophila segmentation genes, their functions are not entirely conserved25,26,27. Furthermore, the genome reveals the unexpected polycistronic organization of a novel gap gene, mille-pattes28, the transcript of which encodes several short peptides.

In contrast to the classical protostomian model organisms Drosophila and Caenorhabditis, Hox genes in Tribolium map to a single cluster of 750 kb on linkage group 2. Orthologues of all Drosophila Hox genes and the Hox-derived genes ftz and zen are transcribed from the same strand, and we find no evidence for interspersion of other protein-coding genes. Taken together, these results suggest that the evolutionary constraints preserving Hox cluster integrity still function in Tribolium.

Dorso-ventral patterning

As in Drosophila, the dorso-ventral axis of the Tribolium embryo depends on a nuclear gradient of Dl, an NF-κB protein, which is established through ventral activation of a Tl receptor29 (one of four in Tribolium). Factors required for localized Tl activation are also present in Tribolium (potential Tl ligands: six spz-like genes; extracellular proteases: one gd, six snk and four tandem ea genes), suggesting that, as in Drosophila, an extra-embryonic signal induces the embryonic dorso-ventral axis.

Tribolium sog inhibition of Dpp/BMP generates a patterning gradient along the dorso-ventral axis30. Similar chordin/sog function in spiders and a hemichordate suggest that this may represent the ancestral bilaterian condition30. Like Apis, Tribolium lacks an orthologue of Drosophila scw, but knockdown of another ligand, Tribolium gbb1, affected the embryonic Dpp/BMP gradient. Tribolium contains orthologues of all five Drosophila TGF-β receptors; however, Dpp signalling moderators that have duplicated and diverged in Drosophila, such as Tol/tok and Cv/tsg, occur as a single copy in Tribolium. Most strikingly, Tribolium contains homologues of BMP10 as well as bambi, Dan and gremlin BMP inhibitors, which are all known from vertebrates, but are not found in Drosophila.

The growth zone

We identified several members of the Fgf and Wnt signalling pathways. The expression patterns of Tribolium Fgf8, Wnt1, Wnt5 and WntD/8 (refs 31, 32) highlight the dynamic organization of the growth zone and underline its role in axis elongation.

Head patterning

Orthologues of 25 out of 30 key regulators of the vertebrate anterior neural plate are specifically expressed in the Tribolium embryonic head (Supplementary Table 12). Two orthologues are not expressed in the head neuroectoderm (barH, arx) and three do not have Tribolium or Drosophila orthologues (vax, hesx1, atx). Of the canonical Drosophila head gap genes, only the late head-patterning function of otd is conserved. ems function is restricted to parts of the antennal and ocular segments, and knockdown of btd seems to have no phenotypic consequences. Thus, analysis of Tribolium genes defines a set of genes that is highly conserved in bilaterian head development, and underscores the derived mode of Drosophila head patterning.

Leg and wing development

In contrast to Drosophila, ventral appendages in Tribolium develop during embryogenesis from buds that grow continuously along the proximo-distal axis33. Nonetheless, we identified Tribolium orthologues for most of a core set of Drosophila appendage genes (Supplementary Table 13). On the other hand, orthologues of genes not found in Drosophila, such as Wnt11, gremlin, Fgf8 and an F-Box gene, are expressed in the embryonic legs21,31. Although their exact function in Tribolium appendages is not known, Fgf8 is essential to vertebrate limb development.

A major innovation driving the radiation of beetles was the evolution of a highly modified protective forewing. Expression analysis and RNAi experiments revealed a high degree of conservation between Tribolium and Drosophila wing gene networks (Supplementary Table 13), supporting the hypothesis that sclerotized elytra evolved from ancestral membranous wings mainly through new interactions between conserved patterning modules and as yet unknown downstream effector genes.

Eye development

Tribolium has orthologues of nearly all genes currently known to regulate specification and differentiation in the Drosophila retina (Supplementary Table 14). Exceptions are the linker protein Phyllopod and the lens crystallin protein Drosocrystallin, which are restricted to Diptera. Eight of fifty-seven investigated eye developmental genes are duplicated in the Drosophila genome but not Tribolium, and in four cases the Drosophila paralogues have similar function. This suggests a more dynamic evolution of Drosophila retina genes and higher genetic complexity, highlighting the value of Tribolium as a more ancestral and simply organized model of insect eye development.

Genes relevant to pest and Tribolium biology

Tribolium castaneum is a notorious invader of stored grains and grain products. Resultantly, much effort and expense is directed to find better ways to control this and other grain pests. Here we describe established and possible future pesticide targets, as well as genes underlying vision and taste. Finally, we describe genes forming the basis of systemic RNAi in Tribolium.

Established insecticide targets

Cys-loop ligand-gated ion channels

Members of this superfamily mediate chemical synaptic transmission in insects and are targets of successful pest control chemicals with animal health and crop protection applications34. The Tribolium Cys-loop ligand-gated ion channel (Cys-loop LGIC) superfamily contains 24 genes, the largest known so far for insects (Drosophila and Apis superfamilies comprise 23 and 21 genes, respectively), due in part to the additional nicotinic acetylcholine receptor (nAChR) subunits in Tribolium. We also found genes for ion channels gated by γ-aminobutyric acid (γ-aminobutyric acid receptors (GABARs)), glutamate (GluCls) and histamine, as well as orthologues of the Drosophila pH-sensitive chloride channel35. The molecular diversity of the Tribolium Cys-loop LGIC superfamily is broadened by alternative splicing and RNA A-to-I editing, which in some cases generates species-specific receptor isoforms35. The Tribolium Cys-loop LGIC superfamily is the first complete set of genes encoding molecular targets of several insecticides—imidacloprid and other neonicotinoids (nAChRs), fipronil (GABARs) and avermectins (GluCls)—described for an agricultural pest species.

Cytochrome P450 proteins

Most insect cytochrome P450 proteins (CYPs) are thought to be involved in metabolic detoxification of host plant allelochemicals and toxicants, and several are insecticide resistance genes36. Other CYPs act in the synthesis and degradation of lipid signalling molecules, such as ecdysteroids37. Similarly to mosquitoes, especially Aedes, Tribolium has an independently expanded CYP gene family, particularly those involved in environmental response (Supplementary Table 16).

Within the Tribolium P450s, the CYP2 and mitochondrial clans have undergone relatively little gene expansion, lack pseudogenes, and are probably reserved for essential endogenous functions in ecdysteroid metabolism and development. In contrast, expansions via tandem duplication produced 85% of Tribolium P450s clustered in groups of 2–16 genes, with large expansions of CYP3 and CYP4 clans involved in environmental response. In comparison, Apis has only four CYP4 genes, whereas Aedes has relatively similarly sized expansions of CYP3 and CYP4 clans (Supplementary Table 16). We speculate that both mosquito larvae (which are omnivorous scavengers) and Tribolium have adapted to diverse chemical environments in part by expansion of CYP gene families involved in detoxification.

Possible future insect control targets

C1 cysteine peptidase genes

Tribolium castaneum has successfully exploited cereal grains in spite of the arsenal of defensive allelochemicals, including inhibitors of serine peptidase digestive enzymes. In tenebrionid beetles, cathepsins B, L and serine peptidases such as trypsins and chymotrypsins are part of the digestive peptidase complex in the larval gut38.

Comparing potential digestive peptidase genes in Tribolium with those in other sequenced insects (Supplementary Fig. 12) we found more C1 cysteine peptidase genes in T. castaneum. The proliferation of Tribolium C1 cysteine peptidase genes reflects expansions into five gene families, corresponding to four major clusters. This expansion is consistent with a trend seen in some beetles relative to other insects: a shift to a more acidic gut, conducive to cysteine peptidase activity.

Tribolium castaneum C1 cysteine peptidase genes encode B and L cathepsins, and include the first-known insect genes similar to O and K cathepsins (Supplementary Table 17). Most of the cathepsin-B-like peptidases lack conserved residues in functional regions and thus may lack peptidase activity, whereas all but two Tribolium cathepsin L peptidase genes encode potentially functional enzymes. In vertebrates, O and K cathepsins are lysosomal cysteine peptidases, involved in bone remodelling and resorption. Analysis of Tribolium cathepsins may provide insight into this family of proteins whose elevated expression is associated with a significant fraction of human breast cancers and tumour invasiveness.

Neurohormones and G-protein-coupled receptors

Insect neurohormones (neuropeptides, protein hormones and biogenic amines) control development, reproduction, behaviour, feeding and many other physiological processes, often by signalling through G-protein-coupled receptors (GPCRs). We found 20 genes encoding biogenic amine GPCRs in Tribolium (compared to 21 in Drosophila and 19 in Apis) and 52 genes encoding neuropeptide or protein hormone GPCRs (49 in Drosophila, 37 in Apis39). Moreover, we identified the likely ligands for 45 of these 72 Tribolium GPCRs. Furthermore, we annotated 39 neuropeptide and protein hormone genes. We found excellent agreement (95%) between the proposed ligands for the Tribolium neurohormone GPCRs and the independently annotated neuropeptide and protein hormone genes. Interestingly, the Tribolium genome contains a vasopressin-like neuropeptide (TC06626) and a vasopressin-like GPCR gene (TC16363; Supplementary Fig. 14), neither of which has been detected in any other sequenced insect39. Vasopressin in mammals is the major neurohormone stimulating water reabsorption in the kidneys40. Its presence in Tribolium may help the beetle to survive in very dry habitats.

Genes relevant to Tribolium biology

Vision

Most of the 21 investigated genes that participate in the Drosophila photo-transduction network are conserved in Tribolium (Supplementary Table 14). Most notable is the lack of ninaG and inaC, which may be functionally replaced by closely related paralogues in Tribolium.

Tribolium contains only two opsin genes, representing members of the long-wavelength and ultraviolet-sensitivity-facilitating opsin subgroups. In contrast, Drosophila contains seven, and there is evidence for minimally three in most other insects. The lack of a blue-light-sensitive opsin gene in Tribolium is consistent with the unusual expression of long-wavelength opsin in all photoreceptor cells in this species41. The implied reduction in colour discrimination in Tribolium is probably a consequence of the widespread cryptic lifestyle of this species group.

Odorant and gustatory receptors

Odorant and gustatory receptors form the insect chemoreceptor superfamily. Tribolium has a major expansion of both odorant and gustatory receptors relative to Drosophila, Anopheles and Aedes mosquitoes, silkmoth and honeybee (Supplementary Table 19). We identified and annotated 265 apparently functional odorant receptors, 42 full-length pseudogenes and 34 pseudogene fragments. Most of these T. castaneum odorant receptors are in seven species-specific subfamilies, including one containing 150 genes, and most are in tandem gene arrays, created by gene duplication within the Tribolium lineage in the last 300 million years42.

We annotated 220 apparently functional gustatory receptors and 25 pseudogenes (gustatory receptor gene fragments were not assessed). The gustatory receptor families in fruitflies and mosquitoes, but not honeybee, contain several genes that are alternatively spliced, with multiple alternative long first exons encoding at least the amino-terminal 50% of the gustatory receptor spliced into a set of short shared exons encoding the carboxy terminus43,44,45. Most Tribolium gustatory receptors are encoded by single genes; however, T. castaneum Gr214 is a massive alternatively spliced locus with 30 alternative long 5′ exons (six of which are pseudogenic) spliced into three shared 3′ exons encoding the C terminus. Three T. castaneum gustatory receptors are orthologues of highly conserved gustatory receptors in other insects43,44,45, two of which form a heterodimeric carbon dioxide receptor46. The remainder form many species-specific subfamilies, one of which is expanded to 88 genes (Supplementary Information and Supplementary Fig. 16).

Systemic RNAi

In Tribolium, as in C. elegans but not Drosophila, the RNAi effect spreads systemically from the site of injection to other tissues5 and from injected females to their offspring4. Surprisingly, our survey of genes involved in systemic RNA did not reveal much conservation between Tribolium and C. elegans.

The SID-1 multi-transmembrane protein, essential for double-stranded RNA (dsRNA) uptake in C. elegans, is not found in Drosophila, suggesting that the presence or absence of a sid-1 gene is the primary determinant of whether or not systemic RNAi occurs in an organism. We found three genes in Tribolium that encode proteins similar to SID-1. However, their sequences are more similar to another C. elegans protein, TAG-130 (also known as ZK721.1), which is not required for systemic RNAi in C. elegans47. Additionally, the secondary argonaute proteins and RNA-dependent RNA polymerase (RdRP)48,49, essential for the amplification of the initial dsRNA trigger in C. elegans, are absent in Tribolium. Therefore, the molecular basis for systemic RNAi in Tribolium and other insects might differ from that in C. elegans and remains to be elucidated.

Concluding remarks

We observe three trends when comparing Tribolium and other insect genomes. First, phylogenetic trees show shorter branch lengths for Tribolium (and Apis) than Drosophila. The accelerated evolution of the Drosophila lineage in some cases rendered Drosophila atypical for the Insecta. Second, Tribolium retains a different set of ancestral genes that have evolved at a moderate rate (for example, gremlin and cathepsins), and these may provide insights into the function of their vertebrate orthologues. Third, its own evolutionary path has led to beetle- and perhaps Tribolium-specific gene changes (for example, a large increase in odorant receptors).

Expansions of CYP proteins, proteinases, diuretic hormones, a vasopressin hormone and receptor, and chemoreceptors all indicate adaptation to a dry, chemically diverse and toxin-rich microenvironment. Whereas the flour beetle’s drought tolerance probably explains the presence of vasopressin, it is more difficult to rationalize a need for such an unprecedented diversity of chemoreceptors. Functions stemming from the diversity of angiosperm-derived chemicals such as distant detection of food sources and avoidance of toxic host plant defence chemicals suggest that this expansion may be common to the Coleoptera. The expansion of odorant receptors is more intriguing when considered in combination with the reduction of opsin genes. Both trends may reflect the long-term consequences of adaptation to low light biota by Tribolium, enforcing selection for increased discrimination of odour reception but not colour perception41.

Given the chemo-sensing and detoxifying genes described above, it is perhaps no surprise that Tribolium has demonstrated resistance to all insecticides used for its control. Given the association of Tribolium with human food, knowledge of all possible insecticide targets will aid greater selectivity in pesticide design, thereby mitigating possible side effects. Finally, the true value of this sequence may be the entry it provides into the many and richly diverse facets of beetle biology, physiology and behaviour.

Methods Summary

Detailed Methods are described in the Supplementary Information. Resources generated by this project can be found at the following locations: genome assemblies, sequences, and automated and manually curated gene model sequences are available from the BCM-HGSC website and ftp site (http://www.hgsc.bcm.tmc.edu/projects/tribolium/). Browser display of the genome sequence, all gene predictions and available tiling array data are available via http://www.genboree.org and Beetle Base (http://www.bioinformatics.ksu.edu/BeetleBase/), a long-term repository for Tribolium data.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The Tribolium genome sequence, at the NCBI, has project accession AAJJ00000000.

References

  1. 1

    Hunt, T. et al. A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation. Science 318, 1913–1916 (2007)

  2. 2

    Sokoloff, A. The Biology of Tribolium with Special Emphasis on Genetic Aspects I–III (Clarendon Press and Oxford Univ. Press, Oxford, 1972, 1974, 1977)

  3. 3

    Lorenzen, M. D. et al. piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Mol. Biol. 16, 265–275 (2007)

  4. 4

    Bucher, G., Scholten, J. & Klingler, M. Parental RNAi in Tribolium (Coleoptera). Curr. Biol. 12, R85–R86 (2002)

  5. 5

    Tomoyasu, Y. & Denell, R. E. Larval RNAi in Tribolium (Coleoptera) for analyzing adult development. Dev. Genes Evol. 214, 575–578 (2004)

  6. 6

    Tautz, D., Friedrich, M. & Schröder, R. in Development 1994 Supplement (eds Akam, M., Holland, P., Ingham, P. & Wray, G.) 193–199 (The Company of Biologists Limited, Cambridge, 1994)

  7. 7

    Tautz, D. Segmentation. Dev. Cell 7, 301–312 (2004)

  8. 8

    Lorenzen, M. D. et al. Genetic linkage maps of the red flour beetle, Tribolium castaneum, based on bacterial artificial chromosomes and expressed sequence tags. Genetics 170, 741–747 (2005)

  9. 9

    Demuth, J. P. et al. Genome-wide survey of Tribolium castaneum microsatellites and description of 509 polymorphic markers. Mol. Ecol. Notes 7, 1189–1195 (2007)

  10. 10

    Wang, S. & Brown, S. J. Analysis of repetitive DNA distribution patterns in the Tribolium castaneum genome. Genome Biol. (in the press)

  11. 11

    Brown, S. J., Henry, J. K., Black, W. C. & Denell, R. Molecular Genetic manipulation of the red flour beetle: Genome organization and cloning of a ribosomal protein gene. Insect Biochem. 20, 185–193 (1990)

  12. 12

    Ugarkovic, D., Podnar, M. & Plohl, M. Satellite DNA of the red flour beetle Tribolium castaneum—comparative study of satellites from the genus Tribolium . Mol. Biol. Evol. 13, 1059–1066 (1996)

  13. 13

    Osanai, M., Kojima, K. K., Futahashi, R., Yaguchi, S. & Fujiwara, H. Identification and characterization of the telomerase reverse transcriptase of Bombyx mori (silkworm) and Tribolium castaneum (flour beetle). Gene 376, 281–289 (2006)

  14. 14

    Xiong, Y. & Eickbush, T. H. The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons. Mol. Cell. Biol. 8, 114–123 (1988)

  15. 15

    Robertson, H. M. & Gordon, K. H. Canonical TTAGG-repeat telomeres and telomerase in the honey bee, Apis mellifera . Genome Res. 16, 1345–1351 (2006)

  16. 16

    Fujiwara, H., Osanai, M., Matsumoto, T. & Kojima, K. K. Telomere-specific non-LTR retrotransposons and telomere maintenance in the silkworm, Bombyx mori . Chromosome Res. 13, 455–467 (2005)

  17. 17

    Savard, J. et al. Phylogenomic analysis reveals bees and wasps (Hymenoptera) at the base of the radiation of Holometabolous insects. Genome Res. 16, 1334–1338 (2006)

  18. 18

    Savard, J., Tautz, D. & Lercher, M. J. Genome-wide acceleration of protein evolution in flies (Diptera). BMC Evol. Biol. 6, 7 (2006)

  19. 19

    Daborn, P. J. et al. A single p450 allele associated with insecticide resistance in Drosophila . Science 297, 2253–2256 (2002)

  20. 20

    Ciccarelli, F. D. et al. Complex genomic rearrangements lead to novel primate gene function. Genome Res. 15, 343–351 (2005)

  21. 21

    Wyder, S., Kriventseva, E. V., Schröder, R., Kadowaki, T. & Zdobnov, E. M. Quantification of ortholog losses in insects and vertebrates. Genome Biol. 8, R242 (2007)

  22. 22

    Wong, M. D., Jin, Z. & Xie, T. Molecular mechanisms of germline stem cell regulation. Annu. Rev. Genet. 39, 173–195 (2005)

  23. 23

    Schröder, R. The genes orthodenticle and hunchback substitute for bicoid in the beetle Tribolium . Nature 422, 621–625 (2003)

  24. 24

    Draper, B. W., Mello, C. C., Bowerman, B., Hardin, J. & Priess, J. R. MEX-3 is a KH domain protein that regulates blastomere identity in early C. elegans embryos. Cell 87, 205–216 (1996)

  25. 25

    Bucher, G. & Klingler, M. Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function. Development 131, 1729–1740 (2004)

  26. 26

    Cerny, A. C., Bucher, G., Schröder, R. & Klingler, M. Breakdown of abdominal patterning in the Tribolium Kruppel mutant jaws . Development 132, 5353–5363 (2005)

  27. 27

    Choe, C. P., Miller, S. C. & Brown, S. J. A pair-rule gene circuit defines segments sequentially in the short-germ insect Tribolium castaneum . Proc. Natl Acad. Sci. USA 103, 6560–6564 (2006)

  28. 28

    Savard, J., Marques-Souza, H., Aranda, M. & Tautz, D. A segmentation gene in Tribolium produces a polycistronic mRNA that codes for multiple conserved peptides. Cell 126, 559–569 (2006)

  29. 29

    Fonseca, R. N. et al. Self-regulatory circuits in dorsoventral axis formation of the short germ beetle Tribolium castaneum . Dev. Cell (in the press)

  30. 30

    van der Zee, M., Stockhammer, O., von Levetzow, C., Nunes da Fonseca, R. & Roth, S. Sog/Chordin is required for ventral-to-dorsal Dpp/BMP transport and head formation in a short germ insect. Proc. Natl Acad. Sci. USA 103, 16307–16312 (2006)

  31. 31

    Beermann, A. & Schröder, R. Sites of FGF signalling and perception during embryogenesis of the beetle Tribolium castaneum . Dev. Genes Evol. (in the press)

  32. 32

    Bolognesi, R. et al. Tribolium Wnts: evidence for a larger repertoire in insects with overlapping expression patterns that suggest multiple redundant functions in embryogenesis. Dev. Genes Evol. (in the press)

  33. 33

    Beermann, A. et al. The Short antennae gene of Tribolium is required for limb development and encodes the orthologue of the Drosophila Distal-less protein. Development 128, 287–297 (2001)

  34. 34

    Raymond-Delpech, V., Matsuda, K., Sattelle, B. M., Rauh, J. J. & Sattelle, D. B. Ion channels: molecular targets of neuroactive insecticides. Invert. Neurosci. 5, 119–133 (2005)

  35. 35

    Jones, A. K. & Sattelle, D. B. The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum . BMC Genomics 8, 327 (2007)

  36. 36

    Ffrench-Constant, R. H., Daborn, P. J. & Le Goff, G. The genetics and genomics of insecticide resistance. Trends Genet. 20, 163–170 (2004)

  37. 37

    Ono, H. et al. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera. Dev. Biol. 298, 555–570 (2006)

  38. 38

    Vinokurov, K. S. et al. Diversity of digestive proteinases in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 145, 126–137 (2006)

  39. 39

    Hauser, F., Cazzamali, G., Williamson, M., Blenau, W. & Grimmelikhuijzen, C. J. A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera . Prog. Neurobiol. 80, 1–19 (2006)

  40. 40

    Bankir, L. Antidiuretic action of vasopressin: quantitative aspects and interaction between V1a and V2 receptor-mediated effects. Cardiovasc. Res. 51, 372–390 (2001)

  41. 41

    Jackowska, M. et al. Genomic and gene regulatory signatures of cryptozoic adaptation: loss of blue sensitive photoreceptors through expansion of long wavelength-opsin expression in the red flour beetle Tribolium castaneum . Front. Zool. 4, 24 (2007)

  42. 42

    Engsontia, P. et al. The red flour beetle’s large nose: an expanded odorant receptor gene family in Tribolium castaneum . Insect Biochem. Mol. Biol. (in the press)

  43. 43

    Clyne, P. J., Warr, C. G. & Carlson, J. R. Candidate taste receptors in Drosophila . Science 287, 1830–1834 (2000)

  44. 44

    Hill, C. A. et al. G protein-coupled receptors in Anopheles gambiae . Science 298, 176–178 (2002)

  45. 45

    Robertson, H. M., Warr, C. G. & Carlson, J. R. Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster . Proc. Natl Acad. Sci. USA 100 (suppl. 2). 14537–14542 (2003)

  46. 46

    Jones, W. D., Cayirlioglu, P., Kadow, I. G. & Vosshall, L. B. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila . Nature 445, 86–90 (2007)

  47. 47

    Tomoyasu, Y. et al. Exploring systemic RNA interference in insects; a genome-wide survey for RNAi genes in Tribolium . Genome Biol. (in the press)

  48. 48

    Lipardi, C., Wei, Q. & Paterson, B. M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307 (2001)

  49. 49

    Sijen, T. et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465–476 (2001)

Download references

Acknowledgements

Work at the BCM-HGSC was funded by grants from the NHGRI and USDA. FgenesH and FgenesH++ analysis was donated by Softberry Inc. This research was additionally supported in part by the Intramural Research Program of the NIH, National Library of Medicine.

Author information

Correspondence to Stephen Richards.

Supplementary information

Supplementary Information

The file contains extensive Supplementary Information which includes detailed Supplementary Methods with additional references, Supplementary Tables S1-S20 and Supplementary Figures S1-S16 with Legends. (PDF 7490 kb)

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence (http://creativecommons.org/licenses/by-nc-sa/3.0/), which permits distribution, and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation, and derivative works must be licensed under the same or similar licence.

Reprints and Permissions

About this article

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.