The genome of the model beetle and pest Tribolium castaneum.

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.

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ARTICLES
The genome of the model beetle and pest
Tribolium castaneum
Tribolium Genome Sequencing Consortium*
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
expansionsinodorantandgustatoryreceptors,aswellasP450andotherdetoxificationenzymes.DevelopmentinTribolium
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
thegrowthzonecrucialforaxialelongationinshort-germdevelopment.SystemicRNAinterferenceinT.castaneumfunctions
differentlyfromthatinCaenorhabditiselegans,butneverthelessofferssimilarpowerfortheelucidationofgenefunctionand
identification of targets for selective insect control.
By far the most evolutionarily successful metazoans1, beetles
(Coleoptera) can luminesce (fireflies), spit defensive liquids (bom-
bardierbeetles),visually andbehaviourally mimic beesandwasps,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
coordinatepreciselywithfoldingwings,allowingflightwhileprovid-
ing 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 ele-
gans, 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
contrasttoDrosophila,Triboliumlarvaedisplayeyesinafullyformed
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 seg-
mentation 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 (7.33 coverage) were
generated from the highly inbred Georgia 2 (GA2) strain and
assembled into contigs totalling 152megabases (Mb) and scaffolds
spanning,160Mbofgenomicsequence(SupplementaryTables1–4
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 hetero-
chromatic regions dense in highly repetitive sequences, the genome
is well represented and of high quality (see Supplementary Data for
details).
G1C content. Tribolium, like Apis, has a very (A1T)-rich genome
(33% and 34% G1C, respectively), but Tribolium G1C domains
lack the extremes of G1C content present in Apis mellifera (Fig. 1
and Supplementary Fig. 3). Despite global G1C similarity to Apis,
genesinTribolium,asinAnophelesandDrosophilabutnotApis,show
a bias towards occurring in (G1C)-rich regions of the genome
(Fig. 1). Whatever mechanism drives the accumulation of A1T
nucleotides in Tribolium, it does not affect genes in the manner
observed in the honeybee, where perhaps additional mechanisms
are present.
Repetitive DNA. Fully one-third of the Tribolium genome assembly
consists of repetitive DNA, which is also (A1T)-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
100bp (2.5% of the Tribolium genome compared with 0.7% in
Drosophila). Most (83%) of the microsatellites are found in inter-
genic 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 geo-
graphic 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 (0.2pg or
*Lists of participants and affiliations appear at the end of the paper.
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204Mb11) and the current assembly (160 Mb). Indeed, as much as
17%oftheTriboliumgenomeiscomposedofa360-bpsatellite12that
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 telo-
meric repeat. Manual assembly of the proximal regions of multiple
telomeres beyond the ends of the assembled scaffolds (Supplemen-
tary Information) reveals TCAGG repeats interrupted by full-length
and 59-truncated non-LTR retrotransposons belonging to the R1
clade, best known for insertions in the rDNA locus14. Tribolium
telomeres range in length from 15kilobases (kb) upwards and prob-
ably represent a stage intermediate to the loss of telomeres and telo-
merase in Diptera compared with the simple canonical structure of
the honeybee15or the more regular insertion of non-LTR retrotrans-
posons 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
vertebrategenomes,allgeneswereclassifiedaccordingtotheirdegree
of similarity using systematic cross-species analysis. Five insects
(Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, T. cas-
taneum, A. mellifera) and five vertebrates (Homo sapiens, Mus
musculus, Monodelphis domestica, Gallus gallus, Tetraodon nigrovir-
idis) 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.
Over 47% of Tribolium genes (7,579) are ancient, with traceable
orthologous relations betweeninsects andvertebratesincluding 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.
Severalhundredancientgenesseemtobeunderlimitedevolution-
ary selection and were independently lost in several species studied
(the patchy fraction, defined in Fig. 2). Each new genome uncovers
previouslyinvisibleancestralrelationsamonggenes—forexample,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.
The evolutionary emergence of many predicted Tribolium genes is
not clear. Thousands of genes currently appear to be species-specific
aseithernosequencesimilaritytoothergenesisdetectable,orhomo-
logybutnotorthologycanbedetermined. Reassuringly,thisfraction
is similar in Tribolium and Drosophila.
We quantified the species phylogeny using a maximum likeli-
hood 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.
20
0.2
0.4
0.6
0.8
30 40
G+C (%)
5060
A. mellifera
A. gambiae
D. melanogaster
T. castaneum
70
0.0
1.0
Fraction < x
Figure 1 | Cumulative distribution of genic and genomic G1C-content
domainsinApismellifera,Anophelesgambiae,Drosophilamelanogasterand
Tribolium castaneum. Cumulative distributions show the fraction of genes
(thick lines) or of the entire genome (thin lines) occurring in G1C-content
domainslessthanagivenpercentageG1C(,X).Themore(A1T)-richhalf
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 (A1T)-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 G1C than would be expected if the
genes were randomly distributed. In contrast, A. mellifera exhibits the
opposite distribution.
Genes
1:1:1 orthologues
N:N:N orthologues
Patchy orthologues
Vertebrate-specific orthologues
Insect-specific orthologues
Homology
Undetectable similarity
0
5,000 10,000 15,000 20,000 25,000 30,000
Tcas
Amel
Tnig
Ggal
Mdom
Mmus
Hsap
Dmel
Agam
Aaeg
Figure 2 | Insect gene orthology. Comparison of the gene repertoire in five
insectandfivevertebrategenomes,rangingfromthecoreofmetazoangenes
(dark blue fraction on the left) to the species-unique sequences (white band
ontheright).Thestripedboxescorrespondtoinsect-andvertebrate-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(requiringatleastoneinsectandonevertebrategene)thathave
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.
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Gene family expansion, frequently associated with a particular
adaptation pressure, mightrevealphysiologically andphenotypically
uniquefeaturesofbeetles(SupplementaryTable9andproteinfamily
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, encod-
ing 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
Inadditiontoaglobalautomated analysisofthepredictedTribolium
gene set, the consortium manually annotated and analysed ,2,000
genes(someadditionallysubjectedtoRNAiandexpressionanalysis),
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—
telotrophicversuspolytrophic—weidentifiedTriboliumorthologues
of most Drosophila genes required for stem cell maintenance, RNA
localizationandaxisformation.LikeApis,however,Triboliumlacksa
bagofmarblesorthologue, whichisessential forthedifferentiation 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 translation-
allyrepressestheC.eleganscadhomologue24.AlthoughtheTribolium
genome contains orthologues of the Drosophila segmentation genes,
their functions are not entirely conserved25–27. 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 ,750kb 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 inter-
spersion of other protein-coding genes. Taken together, these results
suggest that the evolutionary constraints preserving Hox cluster
integrity still function in Tribolium.
Dorso-ventralpatterning.AsinDrosophila,thedorso-ventralaxisof
theTriboliumembryodependsonanucleargradientofDl,anNF-kB
protein,which isestablished throughventralactivation ofaTlrecep-
tor29(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 tan-
dem 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 gra-
dient along the dorso-ventral axis30. Similar chordin/sog function in
spidersandahemichordatesuggestthatthismayrepresenttheances-
tralbilateriancondition30.LikeApis,Triboliumlacksanorthologueof
Drosophila scw, but knockdown of another ligand, Tribolium gbb1,
affected the embryonic Dpp/BMP gradient. Tribolium contains
orthologues of all five Drosophila TGF-b 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.
Thegrowthzone.Weidentified severalmembersoftheFgfandWnt
signalling pathways. The expression patterns of Tribolium Fgf8,
Wnt1, Wnt5 and WntD/8 (refs 31, 32) highlight the dynamic organ-
ization 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 ortholo-
gues 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
thatgrowcontinuously alongtheproximo-distal axis33.Nonetheless,
we identified Tribolium orthologues for most of a core set of
Drosophilaappendagegenes(SupplementaryTable13).Ontheother
AmelTcas
Hsap
Diptera
(Dmel only)
188
(115)
314
(284)
118
(148)
5,457
(5,235)
158
(380)
393
(238)
126
(181)
Figure 3 | Orthologous genes shared between insect and human genomes.
TheVenndiagramshowsthenumberoforthologousgroupsofgenesshared
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).
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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 evolu-
tion of a highly modified protective forewing. Expression analysis
and RNAi experiments revealed a high degree of conservation
between Tribolium and Drosophila wing gene networks (Supplemen-
tary 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 proteinPhyllopod and
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.
the lenscrystallin protein
Genes relevant to pest and Tribolium biology
Triboliumcastaneumisanotoriousinvaderofstoredgrainsandgrain
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 pro-
tection applications34. The Tribolium Cys-loop ligand-gated ion
channel (Cys-loop LGIC) superfamily contains 24 genes, the largest
knownsofarforinsects(DrosophilaandApissuperfamilies 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 c-aminobutyric acid (c-ami-
nobutyric acid receptors (GABARs)), glutamate (GluCls) and his-
tamine,aswellasorthologuesoftheDrosophilapH-sensitivechloride
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 iso-
forms35. The Tribolium Cys-loop LGIC superfamily is the first com-
pletesetofgenesencodingmoleculartargetsofseveralinsecticides—
imidacloprid and otherneonicotinoids
(GABARs) and avermectins (GluCls)—described for an agricultural
pest species.
Cytochrome P450 proteins. Most insect cytochrome P450 proteins
(CYPs)arethoughttobeinvolvedinmetabolicdetoxificationofhost
plant allelochemicals and toxicants, and several are insecticide resis-
tance genes36. Other CYPs act in the synthesis and degradation of
lipid signalling molecules, such as ecdysteroids37. Similarly to mos-
quitoes, especially Aedes, Tribolium has an independently expanded
CYP gene family, particularly those involved in environmental res-
ponse (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
(nAChRs),fipronil
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 scaven-
gers) 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 inspite ofthe arsenalofdefensive allelochem-
icals, 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 com-
plex 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
ofTribolium C1cysteinepeptidase genes reflectsexpansions intofive
genefamilies,correspondingtofourmajorclusters.Thisexpansionis
consistentwithatrendseeninsomebeetlesrelativetootherinsects: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 cathe-
psin-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 asignificant fraction ofhuman
breast cancers and tumour invasiveness.
Neurohormones and G-protein-coupled receptors. Insect neuro-
hormones (neuropeptides, protein hormones and biogenic amines)
control development, reproduction, behaviour, feeding and many
other physiologicalprocesses,
G-protein-coupled receptors (GPCRs).Wefound 20genes 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 inde-
pendently 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.
oftenbysignallingthrough
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 para-
logues in Tribolium.
Triboliumcontainsonlytwoopsingenes,representingmembersof
the long-wavelength and ultraviolet-sensitivity-facilitating opsin
subgroups. In contrast, Drosophila contains seven, and there is evid-
ence 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 life-
style of this species group.
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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,AnophelesandAedesmosquitoes,silkmothandhoneybee
(Supplementary Table 19). We identified and annotated 265 appar-
ently functional odorant receptors, 42 full-length pseudogenes and
34pseudogenefragments.MostoftheseT.castaneumodorantrecep-
tors are in seven species-specific subfamilies, including one contain-
ing 150 genes, and most are in tandem gene arrays, created by gene
duplication within the Tribolium lineage in the last 300million
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 mosqui-
toes, but not honeybee, contain several genes that are alternatively
spliced,withmultiplealternativelongfirstexonsencodingatleastthe
amino-terminal 50% of the gustatory receptor spliced into a set of
short shared exons encoding the carboxy terminus43–45. Most
Tribolium gustatory receptors are encoded by single genes; however,
T. castaneum Gr214 is a massive alternatively spliced locus with 30
alternative long 59 exons (six of which are pseudogenic) spliced into
three shared 39 exons encoding the C terminus. Three T. castaneum
gustatory receptors are orthologues of highly conserved gustatory
receptors in other insects43–45, two of which form a heterodimeric
carbondioxidereceptor46.Theremainderformmanyspecies-specific
subfamilies, one of which is expanded to 88 genes (Supplementary
Information and Supplementary Fig. 16).
SystemicRNAi.InTribolium,asinC.elegansbutnotDrosophila,the
RNAi effect spreads systemically from the site of injection to other
tissues5and 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 isnotrequiredforsystemic RNAiinC.elegans47.Additionally,
the secondary argonaute proteins and RNA-dependent RNA poly-
merase (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
Weobservethree trendswhencomparing Triboliumandother 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
adaptationtoadry,chemicallydiverseandtoxin-richmicroenviron-
ment.Whereastheflourbeetle’sdroughttoleranceprobablyexplains
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
asdistantdetection offood sourcesandavoidanceoftoxichostplant
defencechemicalssuggestthatthisexpansionmaybecommontothe
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 dis-
crimination of odour reception but not colour perception41.
Giventhechemo-sensinganddetoxifyinggenesdescribedabove,it
is perhaps no surprise that Tribolium has demonstrated resistance to
allinsecticidesusedforitscontrol.GiventheassociationofTribolium
withhumanfood,knowledgeofallpossibleinsecticidetargetswillaid
greaterselectivityinpesticidedesign,therebymitigatingpossibleside
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.
Received 16 July 2007; accepted 6 February 2008.
Published online 23 March 2008.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements Work at the BCM-HGSC was funded by grants from the
NHGRI and USDA. FgenesH and FgenesH11 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 The Tribolium genome sequence, at the NCBI, has project
accession AAJJ00000000. Reprints and permissions information is available at
www.nature.com/reprints. This paper is distributed under the terms of the
CreativeCommonsAttribution-Non-Commercial-ShareAlikelicence,andisfreely
available to all readers at www.nature.com/nature. Correspondence and requests
for materials should be addressed to S.R. (stephenr@bcm.edu).
The Tribolium Genome Sequencing Consortium
Project leader: Stephen Richards1,2
Principal investigators: Richard A. Gibbs1,2, George M. Weinstock1,2
White paper: Susan J. Brown3, Robin Denell3, Richard W. Beeman4, Richard Gibbs1,2
Analysis leaders: Richard W. Beeman4, Susan J. Brown3, Gregor Bucher5, Markus
Friedrich6Cornelis J. P. Grimmelikhuijzen7, Martin Klingler8, Marce Lorenzen3,
Stephen Richards1,2, Siegfried Roth9, Reinhard Schro ¨der10,{, Diethard Tautz11, Evgeny
M. Zdobnov12,13,14
DNA sequence and global analysis: DNA sequencing Donna Muzny (leader)1,2,
Richard A. Gibbs1,2, George M. Weinstock1,2, Tony Attaway1,2, Stephanie Bell1,2,
Christian J. Buhay1,2, Mimi N. Chandrabose1,2, Dean Chavez1,2, Kerstin P.
Clerk-Blankenburg1,2, Andrew Cree1,2, Marvin Dao1,2, Clay Davis1,2, Joseph Chacko1,2,
Huyen Dinh1,2, Shannon Dugan-Rocha1,2, Gerald Fowler1,2, Toni T. Garner1,2, Jeffrey
Garnes16, Andreas Gnirke17, Alica Hawes1,2, Judith Hernandez1,2, Sandra Hines1,2,
Michael Holder1,2, Jennifer Hume1,2, Shalini N. Jhangiani1,2, Vandita Joshi1,2, Ziad
Mohid Khan1,2, LaRonda Jackson1,2, Christie Kovar1,2, Andrea Kowis1,2, Sandra Lee1,2,
Lora R. Lewis1,2, Jon Margolis17, Margaret Morgan1,2, Lynne V. Nazareth (leader)1,2,
NgocNguyen1,2,GeoffreyOkwuonu1,2,DavidParker1,2,StephenRichards1,2,San-Juana
Ruiz1,2, Jireh Santibanez1,2, Joe ¨l Savard11, Steven E. Scherer1,2, Brian Schneider1,2, Erica
Sodergren1,2, Diethard Tautz11, Selina Vattahil1,2, Donna Villasana1,2, Courtney S.
White1,2, Rita Wright1,2; EST sequencing Yoonseong Park18, Richard W. Beeman4, Jeff
Lord4, Brenda Oppert4, Marce Lorenzen4, Susan Brown3, Liangjiang Wang3, Joe ¨l
Savard11, Diethard Tautz11, Stephen Richards1, George Weinstock1,2, Richard A.
Gibbs1,2; genome assembly Yue Liu1,2, Kim Worley1,2, George Weinstock1,2; G1C
content Christine G. Elsik19, Justin T. Reese19, Eran Elhaik20, Giddy Landan20, Dan
Graur20; repetitive DNA, transposons and telomeres Peter Arensburger21, Peter
Atkinson21, Richard W. Beeman4, Jim Beidler22, Susan J. Brown3, Jeffery P. Demuth23,
Douglas W. Drury24, Yu-Zhou Du25, Haruhiko Fujiwara26, Marce Lorenzen3, Vincenza
Maselli27, Mizuko Osanai26, Yoonseong Park18, Hugh M. Robertson28, Zhijian Tu22,
Jian-jun Wang25, Suzhi Wang3; gene prediction and consensus gene set Stephen
Richards1,2, Henry Song1,2, Lan Zhang1,2, Erica Sodergren1,2, Doreen Werner29, Mario
Stanke29, Burkhard Morgenstern29, Victor Solovyev30, Peter Kosarev31, Garth
Brown32, Hsiu-Chuan Chen32, Olga Ermolaeva32, Wratko Hlavina32, Yuri Kapustin32
Boris Kiryutin32, Paul Kitts32, Donna Maglott32, Kim Pruitt32, Victor Sapojnikov32,
Alexandre Souvorov32, Aaron J. Mackey33, Robert M. Waterhouse14, Stefan Wyder12,
Evgeny M. Zdobnov12,13,14; global gene content analysis Evgeny M. Zdobnov12,13,14,
Stefan Wyder12, Evgenia V. Kriventseva12,34, Tatsuhiko Kadowaki35, Peer Bork36,37
Developmental processes and signalling pathways: Manuel Aranda11, Riyue Bao6,
Anke Beermann10, Nicola Berns10, Renata Bolognesi3, Franc ¸ois Bonneton38, Daniel
Bopp39, Susan J. Brown3, Gregor Bucher5, Thomas Butts40, Arnaud Chaumot41, Robin
E. Denell3, David E. K. Ferrier40, Markus Friedrich6, Cassondra M. Gordon3, Marek
Jindra42, Martin Klingler8, Que Lan43, H. Michael G. Lattorff44, Vincent Laudet38,
Cornelia von Levetsow9, Zhenyi Liu45, Rebekka Lutz10, Jeremy A. Lynch9, Rodrigo
Nunes da Fonseca9, Nico Posnien5, Rolf Reuter10, Siegfried Roth9, Joe ¨l Savard11,
Johannes B. Schinko5, Christian Schmitt8, Michael Schoppmeier8, Reinhard
Schro ¨der10, Teresa D. Shippy3, Franck Simonnet5, Henrique Marques-Souza11,
Diethard Tautz11, Yoshinori Tomoyasu3, Jochen Trauner8, Maurijn Van der Zee11,
Michel Vervoort46, Nadine Wittkopp10, Ernst A. Wimmer5, Xiaoyun Yang6
Pestbiology,senses,MedeaandRNAi:ligandgatedionchannelsAndrewK.Jones47,
David B. Sattelle47; oxidative phosphorylation Paul R. Ebert48; P450 genes David
Nelson49, Jeffrey G. Scott50, Richard W. Beeman4; chitin and cuticular proteins
Subbaratnam Muthukrishnan51, Karl J. Kramer4,51, Yasuyuki Arakane4,51, Richard W.
Beeman4, Qingsong Zhu51, David Hogenkamp51, Radhika Dixit51; digestive protein-
ases Brenda Oppert4, Haobo Jiang52, Zhen Zou52, Jeremy Marshall3, Elena Elpidina53,
Konstantin Vinokurov53, Cris Oppert4; immunity Zhen Zou52, Jay Evans54, Zhiqiang
Lu52,PichengZhao52,NiranjiSumathipala52,BoranAltincicek55,AndreasVilcinskas55,
Michael Williams56, Dan Hultmark56, Charles Hetru57, Haobo Jiang52; neurohor-
mones and GPCRs Cornelis J. P. Grimmelikhuijzen7, Frank Hauser7, Giuseppe
Cazzamali7, Michael Williamson7, Yoonseong Park18, Bin Li18, Yoshiaki Tanaka58,
Reinhard Predel59, Susanne Neupert59, Joachim Schachtner60, Peter Verleyen61;
neuropeptide processing enzymes Florian Raible36, Peer Bork36,37; opsins Markus
Friedrich6; odorant receptors and gustatory receptors Kimberly K. O. Walden28,
Hugh M. Robertson28; odorant binding and chemosensory proteins Sergio Angeli62,
Sylvain Fore ˆt63, Gregor Bucher5, Stefan Schuetz5, Ryszard Maleszka63, Ernst A.
Wimmer5; Medea Richard W. Beeman4, Marce Lorenzen4; systemic RNAi Yoshinori
Tomoyasu3, Sherry C. Miller3, Daniela Grossmann5& Gregor Bucher5
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Affiliations for participants:1Human Genome Sequencing Center, and2Department of
Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston,
Texas 77030, USA.3Division of Biology, Ackert Hall, Kansas State University,
Manhattan, Kansas 66506, USA.4Grain Marketing and Production Research Center,
Agricultural Research Service, United States Department of Agriculture, 1515 College
Avenue, Manhattan, Kansas 66502, USA.5Johann Friedrich Blumenbach Institute,
Department of Developmental Biology, Georg August University, von-Liebig-Weg-11,
37077 Go ¨ttingen, Germany.6Department of Biological Sciences, Wayne State
University, Detroit, Michigan 48202, USA.7Center for Functional and Comparative
Insect Genomics, and Department of Cell Biology and Comparative Zoology, Institute of
Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen,
Denmark.8Institute for Biology, Department of Developmental Biology,
Friedrich-Alexander-University Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany.
9Institute for Developmental Biology, University of Cologne, 50674 Cologne, Germany.
10Animal Genetics, Interfaculty Institute for Cell Biology, University of Tu ¨bingen, Auf der
Morgenstelle 28, 72076 Tu ¨bingen, Germany.11Department of Genetics, University of
Cologne, 50674 Cologne, Germany.12Department of Genetic Medicine and
Development, University of Geneva Medical School, 1 rue Michel-Servet, 1211 Geneva,
Switzerland.13Swiss Institute of Bioinformatics, 1 rue Michel-Servet, 1211 Geneva,
Switzerland.14Imperial College London, South Kensington Campus, SW7 2AZ London,
UK.15Children’s Hospital Oakland Research Institute, BACPAC Resources, 747 52nd
Street, Oakland, California 94609, USA.16Broad Institute of MIT and Harvard, 7
Cambridge Center, Cambridge, Massachusetts 02142, USA.17AgraQuest, Inc., 1530
Drew Avenue, Davis, California 95616, USA.18Department of Entomology, Waters Hall,
Kansas State University, Manhattan, Kansas 66506, USA.19Department of Animal
Science, Texas A&M University, College Station, Texas 77843, USA.20Department of
Biology and Biochemistry, University of Houston, Houston, Texas 77204, USA.
21Department ofEntomology, University ofCalifornia,900UniversityAvenue,Riverside,
California92521,USA.22DepartmentofBiochemistry,VirginiaTech,Blacksburg,Virginia
24061, USA.23Department of Biology, University of Texas at Arlington, Arlington, Texas
76019, USA.24Department of Biology, Indiana University, Bloomington, Indiana 47405,
USA.25Department of PlantProtection,YangzhouUniversity,Yangzhou225009,China.
26Department of Integrated Biosciences, Graduate School of Frontier Sciences,
University of Tokyo, Bioscience Building 501, Kashiwa, Chiba 277-8562, Japan.
27European School of Molecular Medicine and Telethon Institute of Genetics and
Medicine, Via Pietro Castellino 111, 80131 Napoli, Italy.28Department of Entomology,
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.29Institute for
Microbiology and Genetics, Department of Bioinformatics, University of Go ¨ttingen,
Goldschmidtstraße 1, 37077 Go ¨ttingen, Germany.30Department of Computer Science,
Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK.31Softberry Inc.,
116 Radio Circle, Suite 400, Mount Kisco, New York 10549, USA.32National Center for
Biotechnology Information, National Library of Medicine, Bethesda, Maryland 20894,
USA.33GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.34Department of
Structural Biology and Bioinformatics, University of Geneva Medical School, 1 rue
Michel-Servet, 1211 Geneva, Switzerland.35Graduate School of Bioagricultural Sciences,
Nagoya University, Chikusa, Nagoya 464-8601, Japan.36European Molecular Biology
Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.37Max-Delbruck-Centre
forMolecularMedicine,Berlin-Buch,Robert-Roessle-Strasse10,13092Berlin,Germany.
38Institut de Genomique Fonctionnelle de Lyon, Equipe de Zoologie Moleculaire, ENS
Lyon, Universite Lyon 1, CNRS UMR5242, INRA, IFR128, 46 Allee d’Italie, 69364 Lyon
cedex 07, France.39Zoological Institute of the University Zu ¨rich, Winterthurerstrasse
190, CH-8057 Zu ¨rich, Switzerland.40Department of Zoology, University of Oxford,
Tinbergen Building, South Parks Road, Oxford OX1 3PS, UK.41CEMAGREF, Laboratoire
d’e ´cotoxicologie, 3bis quai Chauvea,CP220 69336 Lyon cedex 09, France.42Institute of
Entomology ASCR, Branisovka ´ 31, Ceske ´ Budejovice 370 05, Czech Republic.
43Department of Entomology, University of Wisconsin-Madison, 1630 Linden Drive,
Madison, Wisconsin 53706, USA.44Institute of Biology, Molecular Ecology,
Martin-Luther-University Halle-Wittenberg Hoher Weg 4, 06099 Halle (Saale),
Germany.45Department of Molecular Biology and Pharmacology, Washington
University in St Louis School of Medicine, 3600 Cancer Research Building, 660 South
Euclid Avenue, St Louis, Missouri 63110, USA.46Universite ´ Paris 7 – Denis Diderot,
Centre de genetique moleculaire – CNRS UPR 2167, 1 Avenue de la Terrasse, 91198
Gif-sur-Yvette cedex, France.47MRC Functional Genetics Unit, Department of
Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1
3QX, UK.48School of Integrative Biology & School of Molecular and Microbial Sciences,
University of Queensland, St Lucia, Queensland 4072, Australia.49Department of
MolecularSciencesandCenterofExcellenceinGenomicsandBioinformatics, University
of Tennessee, Memphis, Tennessee 38163, USA.50Department of Entomology, Daljit
and Elaine Sarkaria Professor of Insect Physiology and Toxicology, Cornell University,
Ithaca, New York 14853, USA.51Department of Biochemistry, Kansas State University,
Manhattan, Kansas 66506, USA.52Department of Entomology and Plant Pathology,
Oklahoma State University, Stillwater, Oklahoma 74078, USA.53A. N. Belozersky
Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory,
Moscow 119992, Russia.54USDA-ARS Bee Research Laboratory, Beltsville, Maryland
20705, USA.55Institute of Phytopathology and Applied Zoology, Interdisciplinary
Research Center, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32,
D-35392 Giessen, Germany.56Umea Centre for Molecular Pathogenesis, Umea
University, Umea SE-90187, Sweden.57Institut Biol Mole ´c Cell, CNRS, Strasbourg
67084,France.58NationalInstituteofAgrobiologicalScience,DivisionofInsectScience,
Tsukuba, Ibaraki 305-8634, Japan.59Institute of General Zoology, University of Jena,
Erbertstrasse 1, D-07743 Jena, Germany.60Department of Animal Physiology,
University of Marburg, Karl-von-Frisch Strasse 8, D-35032 Marburg, Germany.
61Department of Animal Physiology and Neurobiology, University of Leuven,
Naamsestraat 59, BE-3000 Leuven, Belgium.62Institute for Forest Zoology and Forest
Conservation, Bu ¨sgenweg 3 D-37077 Go ¨ttingen, Germany.63Visual Sciences and ARC
Centre for the Molecular Genetics of Development, Research School of Biological
Sciences, The Australian National University, Canberra, ACT 0200, Australia. {Present
address: Bioscience Institute, University of Rostock, Albert-Einstein-Strasse 3, 18059
Rostock, Germany.
NATURE|Vol 452|24 April 2008
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Nature

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