Emerging Model Organisms

The Red Flour Beetle, Tribolium castaneum (Coleoptera):

A Model for Studies of Development and Pest Biology
Susan J. Brown,1,5 Teresa D. Shippy,1 Sherry Miller,1 Renata Bolognesi,1
Richard W. Beeman,2 Marc D. Lorenzen,2 Gregor Bucher,3
Ernst A. Wimmer,3 and Martin Klingler4
1

Division of Biology, Kansas State University, Manhattan, KS 66506, USA

Grain Marketing and Production Research Center, Agricultural Research Service, United States Department of Agriculture,
Manhattan, KS 66502, USA
3
Johann-Friedrich-Blumenbach Institute, Department of Developmental Biology, Georg-August-University Gttingen,
37077 Gttingen, Germany
4
Department of Biology, Section Developmental Biology, Friedrich-Alexander-University, 91058 Erlangen, Germany
2

INTRODUCTION
Tribolium castaneum is a small, low-maintenance beetle that has emerged as a sophisticated model system for studying the evolution of development and that complements (in some cases, even rivals)
Drosophila for functional genetic analysis of basic biological questions. Although Tribolium and
Drosophila are both holometabolous insects, they differ fundamentally in larval and adult morphology.
Even generally conserved developmental features, such as body segmentation, are achieved by quite
different means. Thus, comparison of developmental mechanisms between these two insects can
address many interesting questions concerning the evolution of morphology and other characters.
Genetic tools available for Tribolium include genetic maps for visible and molecular markers, chromosomal rearrangements that enable lethal mutations to be balanced in true-breeding stocks, transposon-based transformation systems, a completed and annotated genome sequence, and systemic RNA
interference (RNAi), which makes it possible to knock down any given gene and even particular splice
variants in the offspring or in any tissue of the injected animal. Inactivating gene functions at various
developmental stages provides new opportunities to investigate post-embryonic development, as well
as larval and adult physiology, including hormonal control, host-parasite interactions, and pesticide
resistance.

RELATED INFORMATION
There are many Internet resources available from and for Tribolium researchers:
1. BeetleBase (Kansas State University; http://beetlebase.org/) is the portal into the Tribolium genome

database. It includes a genome browser (assembly 3.0) and BLAST servers.

2. The Tribolium castaneum Genome Project (located at Baylor College of Medicine; http://

www.hgsc.bcm.tmc.edu/projects/tribolium/) houses genome sequence files, BLAST servers, and a

Genboree browser (assembly 2.0).
3. The National Center for Biotechnology Information (NCBI) provides a gateway (http://

www.ncbi.nlm.nih.gov/projects/genome/guide/beetle/) to a variety of genome resources on

T. castaneum including sequence, BLAST servers, and Map Viewer browser (assembly 3.0).

Corresponding author (sjbrown@ksu.edu)

Cite as: Cold Spring Harb Protoc; 2009; doi:10.1101/pdb.emo126

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Vol. 4, Issue 8, August 2009

4. The Agricultural Research Service of the United States Department of Agriculture has a website

(http://www.ars.usda.gov/Research/docs.htm?docid=12890) that contains data and articles about

the genetics of T. castaneum and related species. The site includes instructions for maintaining
stocks, descriptions of mutants, classical genetic maps (note: These follow the old chromosome
numbering system), and a description of the Medea phenomenon in Tribolium.
5. The Tribolium Genetics Program at Kansas State University (http://www.k-state.edu/tribolium/)

presents people and projects at KSU, links to other Tribolium laboratories, and miscellaneous
Tribolium information.
6. Beetle Book (written by Gregor Bucher; http://wwwuser.gwdg.de/~gbucher1/tribolium-casta-

neum-beetle-book1.pdf) is an online resource containing protocols for stockkeeping, RNAi, and

expression analysis.
7. The GEKU Database (http://www.geku-base.uni-goettingen.de) is a searchable database of trans-

genic lines created by a USDA-funded project. These lines are available by request.
8. Tribolium.net (http://www.Tribolium.net) provides methods and protocols, a list of Tribolium

developmental genetics labs, and links to additional resources.

9. The website of the Tribolium Group Gttingen (http://wwwuser.gwdg.de/~gbucher1/) contains

protocols, suppliers of beetle equipment in Europe, and links to additional resources.

10. The Arthropod Genomics Center at Kansas State University (http://www.k-state.edu/agc/

index.htm) highlights issues of interest to arthropod researchers, sponsors an annual arthropod

genomics symposium, and includes the KSU Tribolium Research Group.

BACKGROUND INFORMATION
Collectively known as flour beetles, several species of Tribolium and Tenebrio, which belong to the
family Tenebrionidae (darkling beetles) in the order Coleoptera, are ubiquitous pests of stored grain,
flour, and other cereal products. Although little is known of their ecological niche prior to the introduction of human food stores, the entire clade is thought to have adapted to feed on fungi (Hunt
et al. 2007). Their tolerance for hot, dry environments; reduced visual system; and remarkable expansion of odorant and gustatory receptor genes (Abdel-Latief 2007; Engsontia et al. 2008; Richards et al.
2008) suggests an underground lifestyle in arid subtropical regions, possibly infesting animal food
stores. Their popularity as research organisms arose because they are easy to culture and handle and
they reproduce rapidly. As the only beetle for which the genome sequence is now available, the red
flour beetle, T. castaneum, is one of the best-known members of this family. It is an increasingly popular experimental subject in high school biology classrooms as well as university research laboratories.
Several Tribolium species, particularly T. castaneum and T. confusum, have been studied in the laboratory for many decades. Early work focused largely on population ecology and population genetics.
There were extensive analyses of life history traits and interspecific and intraspecific competition
(including cannibalism and conditioning of media), as well as studies involving genetics, nutrition,
behavior, and physiology. These species are also of interest due to their propensity to develop insecticide resistance. Much information about earlier studies is summarized in a three-volume series
authored by Alexander Sokoloff (1972, 1974, 1976) and in an earlier monograph by the same author
(Sokoloff 1966), who strongly promoted using Tribolium for genetic studies.
Hereafter, the term Tribolium specifically means T. castaneum, unless otherwise indicated. Recently,
genetic analysis in Tribolium has been empowered by extremely efficient reverse genetics. In contrast to
Drosophila, Tribolium mounts a robust systemic RNAi response, which makes it an excellent model for
comparative functional genetic studies. Together with forward genetic screens, RNAi technologies facilitate de novo identification of gene functions without relying on previous genetic knowledge from other
model systems. In combination with tools available for transgenic manipulation, these methodologies
have established Tribolium as a powerful invertebrate model system for genetic and molecular studies.

SOURCES AND HUSBANDRY

Flour beetles can be recovered from infested caches of stored grain or obtained from biological supply companies (e.g., Carolina Biological Supply). There are two commonly used wild-type strains of

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Tribolium, Georgia (GA-1) and San Bernardino (SB), as well as several hundred mutants including both
spontaneous and induced variants. The largest collections of Tribolium strains are housed in
Manhattan, KS (R. Denell and S. Brown at Kansas State University and R. Beeman at the U.S.
Department of Agriculture, Agricultural Research Service [ARS], Grain Marketing and Production
Research Center [GMPRC]), and in Germany (M. Klinger at the University of Erlangen; G. Bucher and
E. Wimmer at the Georg-August-University Gttingen).
Tribolium requires minimal maintenance. The beetles are raised in the laboratory on a 95:5 mixture (by weight) of wheat flour and dried brewers yeast. Medium based on whole grain flour is optimal for stockkeeping, but its large particles interfere with the collection of eggs by sieving. Hence, for
egg collection, type 405 white or instant flour (with yeast) is used in European labs, while in the
United States, commercially available all-purpose flour is used. Alternatively, whole-wheat flour can
be triple-sifted (see below) before use to eliminate large particles. Tribolium does not require a water
supply, but humidity below 40% will reduce fecundity. Temperatures >20C are required for reproduction, with embryonic stages being the most sensitive to cold. Experiments are usually performed
at 30C-32Ctemperatures at which generation time is short and survival rate high. At 25C, the
generation time is approximately twice as long, which is useful for general strain maintenance
between experiments. For long-term maintenance of stocks, 23C has proven reliable. Tribolium displays a typical holometabolous life cycle. The duration of embryonic and larval development (with a
variable number of larval molts) and the length of metamorphosis at different temperatures are
described in Table 1.
Adults must be periodically transferred (subcultured) onto fresh culture flour to prevent overcrowding and starvation. When overcrowding does occur, beetle health may be affected by infection
with various protozoans (manifested by the presence of dead, darkened larvae), problems with cannibalism, or malnutrition (slow development). Frequent subculturing (at least once every generation)
prevents or eliminates most problems. Stocks are easily subcultured by using strips of paper to transfer adults to new flour or by sieving to separate various life stages (see below). Large quantities of
beetles can be reared in plastic storage containers (~18 18 12 cm) containing a 1-in. layer of culture flour. Stocks are generally stored in pint or quart mason jars, or in large Drosophila culture vials
(~170 mL) filled to about one-third with culture flour. Single-pair and other small-scale matings are
performed in smaller vials (e.g., Drosophila vials of ~30 mL). Caps and lids must allow some ventilation while retaining the beetles. Tribolium lives for ~1 yr, with female fecundity declining after 3 mo.
Males are fully fertile within 2 d of eclosion, while females need an additional week on culture flour
to achieve maximum fecundity. The sex of Tribolium can be easily determined at the adult and pupal
stages. Adult males are distinguished by glands (which often accumulate flour) on their prothoracic
legs. Externally visible male and female reproductive organs are easily distinguished in pupae (see Fig.
1), allowing isolation of males and females before they reach sexual maturity.
All life stages can be separated from the culture flour by sieving through commercially available
test sieves. Adults and pupae are separated from small larvae and eggs using 710-m (#25) mesh
sieves; early-instar larvae are separated from older larvae, adults, and pupae using a 500-m (#35)
mesh sieve; beetle eggs are collected from all-purpose or triple-sifted whole-wheat flour using a
300-m (#50) sieve.
More information on maintaining Tribolium stocks can be obtained at the U.S. Department of
Agriculture website (http://ars.usda.gov/Research/docs.htm?docid=12892) and from the online
Beetle Book (http://wwwuser.gwdg.de/~gbucher1/tribolium-castaneum-beetle-book1.pdf).

Table 1. Effect of temperature on development time in Tribolium

Temperature (C)

Embryonic
development (days)

Larval
development (days)

Metamorphosis
(days)

Total development
time (days)

37.5
32.5
30
25
22.5

2.6
2.9
3.6
6.8
9.3

13.7
14.6
17.2
31.2
51

3.9
4.6
5.5
10.2
13.4

20
22
27
48
74

Data from Sokoloff (1972).

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FIGURE 1. Female pupae (left) can be distinguished by the posterior tips of

the genitalia (arrows), which are absent in males (right).

RELATED SPECIES
Five Tribolium species groups, originally associated with major geographic regions (the Americas,
Africa, Madagascar, Indo-Australia, and Australia) were recognized by Hinton (1948), who suggested
that these species groups radiated during the Cretaceous period, ~75-130 million years ago. Analysis
of satellite DNA sequences supports this phylogeny (Juan et al. 1993). T. castaneum is a member of
the Indo-Australian species group. The closely related species Tribolium freemani, which is twice the
size of T. castaneum, can intercross with T. castaneum, but such matings produce only a few hybrids,
which are sterile. Tribolium audax and Tribolium madens are more distantly related to T. castaneum
within the same species group. Tribolium confusum, although a representative of a different (African)
species group, is most similar to T. castaneum in general appearance and is often confused with the
latter. Tribolium brevicornis belongs to the most distantly related species group (American). These
Tribolium species provide an excellent basis for comparative genomic studies. For molecular mapping
approaches, crosses among the distantly related T. castaneum strains GA-1 (North American), ab
(South American), and Tiw1 (East Indian) have proven useful because their respective genomes
display a high number of fixed (in addition to nonfixed) polymorphisms.

USES OF THE TRIBOLIUM MODEL SYSTEM

Tribolium species have enjoyed a long history as model organisms. Since Chapman (1924) introduced
the use of Tribolium for population genetics, T. castaneum and T. confusum have been the focus of many
(mainly ecological) population studies. More recently, several species have been used as model systems
for studies of population differentiation and speciation (Demuth and Wade 2007; Angelini and
Jockusch 2008) and host-parasite coevolution (Fialho and Stevens 1997; Zhong et al. 2003; Detwiler
and Janovy 2008; Wegner et al. 2008). In addition, T. castaneum has now emerged as a model organism for comparative evolutionary developmental biology, for understanding metamorphosis and adult
insect diversity, and for studies on pest control and basic physiology.
Evolutionary developmental biology studies using Tribolium focus on the genetic control of development and the implications of Tribolium mechanisms for understanding the genetic basis of morphological evolution, for example, by comparing forewing and hindwing development in beetles and
flies (Tomoyasu et al. 2005). In addition, it is interesting and important to consider the extent to
which developmental mechanisms elucidated in Drosophila are representative of insects and arthropods in general (for a recent review, see Schrder et al. 2008). Drosophila is a highly specialized
insect, particularly with respect to early embryonic patterning and larval morphology. While Tribolium
certainly shows specialized traits of its own, it possesses many ancestral developmental features (e.g.,
its manner of segmentation, the presence of external larval appendages, and a non-involuted larval
head morphology). These features, along with its sequenced genome, its susceptibility to RNAi, and
the sophisticated genetic and transgenic methods that can be used on Tribolium, make this beetle an
excellent choice for comparative studies.
In particular, its competence for systemic RNAi makes Tribolium better suited than Drosophila as a
model system for the study of certain basic biological problems, despite the highly advanced inventory of genetic tools available for the fruit fly. In Tribolium, a large number of genes can be rapidly
screened for specific knockdown phenotypes by injecting the corresponding double-stranded RNA
(dsRNA), and, in contrast to Drosophila, this approach is not restricted to early development. For
maternal-effect genes, fertility genes, and many other post-embryonic processes, genome-wide RNAi
screens in Tribolium should be more efficient than ethyl methane sulfonate (EMS) screens in Drosophila.
In holometabolous insects, adult morphology depends as much on processes occurring during
metamorphosis as on the initial formation of the body plan during embryogenesis. However,

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metamorphosis is much less understood than early development. This is because many genes relevant
to metamorphosis are also essential for embryonic development. Thus, mutations in such genes will
usually be lethal to the embryo, precluding assessment of their effects during metamorphosis. While
candidate genes can be studied in Drosophila using clonal analysis or other sophisticated techniques,
in Tribolium such genes can be knocked down during metamorphosis by injecting dsRNA into larvae,
that is, after they have performed their embryonic functions. Eye, wing, adult leg, and stink gland
development, as well as hormonal control of metamorphosis, have already been investigated by these
means (e.g., Konopova and Jindra 2007).
The ease with which genes can be studied post-embryonically in Tribolium also makes this beetle
a valuable model for genetic interactions in the adult insect. Insecticide resistance studies in Tribolium
will continue to be important not only because of its status as a pest species, but also because it is a
genetic model for the Coleoptera, which includes many devastating crop and forest pests. The remarkable ability of Tribolium to survive on dry food sources while maintaining a state of adequate tissue
hydration makes it a prime model for studies on the physiology of osmoregulation (Aikins et al. 2008;
Park and Beeman 2008). Recent studies in Tribolium have also greatly expanded our understanding of
the structure and metabolism of insect cuticle (e.g., Arakane et al. 2005a; Zhu et al. 2008).

GENETICS
Classical genetic studies in Tribolium began more than 70 years ago. Spontaneous mutations that did
not appear to affect individual fitness, such as the eye-color mutation pearl (Park 1937), were
exploited to more efficiently distinguish between T. castaneum and T. confusum in population ecology experiments. By 1960, there were enough mutations to construct linkage maps for seven of the
nine autosomes and the X-chromosome. A small region on Chromosome 2 became the focus of
intense study after the discovery of a single intact Hox complex (Beeman 1987). The full power of
genetic analysis in T. castaneum was realized in the reversion of dominant phenotypes, construction
of balancer chromosomes (now covering ~30% of the genome), and sophisticated mutagenesis
schemes. In concerted efforts to develop Tribolium as a genetic model organism, linkage maps based
on molecular markers have been developed (Beeman and Brown 1999; Lorenzen et al. 2005) with
the average, genome-wide marker resolution now approaching 1 cM (~300 kb). Targeted, fine-scale
recombinational analysis has been performed at a very high average marker resolution of ~0.007 cM
(2 kb) (Lorenzen et al. 2008).
Older Tribolium mutants have descriptive gene names. For example, the black, sooty, jet, and cola
mutants are darker in body color than the wild-type chestnut. Because of the comparative focus of
more recent studies, most new gene names are derived from the respective orthologs in other model
systems. Although Drosophila gene names predominate, names derived from mouse or nematode
orthologs are also used. A species-specific prefix (Tc, Tc, or Tc-) is often used to clearly indicate the
Tribolium ortholog. To ease communication with non-Tribolium researchers, names of genetically identified genes usually are changed upon molecular identification of the affected gene, while the originally published name is maintained as a superscript. For example, when a Tribolium eye-color mutant,
white, was associated with a lesion in the Tribolium ortholog of the Drosophila vermilion gene, its name
was changed from white to Tc-vermilionwhite.
Approximately 30% of the Tribolium genome is covered by balancer chromosomes. However,
these are concentrated on the larger linkage groups, and not every chromosome is represented. The
availability of molecularly mapped transposon insertions carrying target sites for site-specific recombination should permit the construction of defined chromosomal rearrangements in the future.

GENOMICS AND ASSOCIATED RESOURCES

The genome of a highly inbred strain of T. castaneum (designated GA-2 to indicate its derivation from
the wild-type GA-1 lab strain), has been sequenced (Richards et al. 2008). Genomic data can be
accessed at BeetleBase (http://beetlebase.org/) or NCBI (http://www.ncbi.nlm.nih.gov). The Tribolium
genome is ~200 Mb. Contigs and scaffolds representing 156 Mb of the genome have been assembled, and the remaining portion is likely to be highly repetitive pericentric heterochromatin that is
difficult to assemble. A super-scaffold for each chromosome was constructed by mapping scaffolds
and contigs onto the molecular linkage map. The remaining sequences were arbitrarily concatenated
into a single chimeric chromosome designated unknown. Since there are relatively few X-linked

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markers and the genetic map does not include the Y-chromosome, scaffolds belonging to the Y in
addition to many belonging to the X-chromosome are probably contained within the unknown file.
The first round of annotation (a computational digest of the genomic sequence) produced
approximately 16,000 gene models defined as the GLEAN gene set. Approximately 2000 of these
were further examined by members of the consortium, taking into account other available information including homologous sequences from other insects and vertebrates. These verified or corrected
gene models are defined as the manually curated gene set. A more conservative, independent analysis at NCBI defined a smaller set of only 9000 predicted genes. The molecular linkage maps are available at NCBI (http://www.ncbi.nlm.nih.gov/projects/genome/guide/beetle/) and BeetleBase
(http://beetlebase.org/), and markers used to integrate the linkage map and the genomic sequences
are included as a track in the genome browser at BeetleBase. DNA tiling arrays based on the nonrepetitive genomic sequences are available through NimbleGen (http://www.nimblegen.com/).
In addition to the genomic sequence, approximately 70,000 T. castaneum expressed sequence
tags (ESTs) derived from various life stages and specific adult tissues have been submitted to dbEST
at NCBI. A BAC library of 27,000 clones equaling 10X to 12X coverage of the Tribolium genome,
constructed by Exelixis (San Francisco, CA), is archived and distributed by the BAC/EST Resource
Center at Clemson University Genome Institute (http://www.genome.clemson.edu/capabilities/
bacCenter.shtml). Additional genomic resources will be integrated into BeetleBase as they are
generated.

TECHNICAL APPROACHES
RNAi

RNAi can be performed on any life stage of Tribolium. When female pupae or adults are treated, suppression of gene transcripts is observed not only in the injected females themselves but also in their
progeny, a phenomenon referred to as parental RNAi. By varying the dsRNA dosage, a graded series
of loss-of-function phenotypes can be produced. In the case of Tribolium, the typical RNAi response is
potent, fully penetrant, and systemic (i.e., spreading throughout the animal after local injection, often
producing knockdown phenotypes indistinguishable from those of genetically defined null mutants).
Furthermore, RNAi is splice-variant-specific in Tribolium, facilitating fine-scale functional analysis of
genes with complex splicing patterns (Arakane et al. 2005b). Because there is no robust systemic RNAi
response in Drosophila (Miller et al. 2008), Tribolium is the preferred insect model system for knocking
down specific gene activity at different time points during development.
Injecting dsRNA directly into preblastoderm eggs (embryonic RNAi [eRNAi]) (Brown et al. 1999)
can produce very strong phenotypes, although embryo-to-embryo variation may be high compared
to that following parental RNAi. Injection at subsequent embryonic stages can be used to analyze later
gene function without affecting earlier requirements for the target gene (Schinko et al. 2008). While
useful for the production of embryonic knockdown phenotypes, the process of egg injection is laborintensive, the survival rate is usually only 50%-70%, and manipulation of individual, injected embryos
for staining is time-consuming.
Fortunately, systemic spread of the RNAi response makes it possible to study embryonic gene
functions by injecting dsRNA into female pupae or adults (pupal RNAi [pRNAi] or adult RNAi [aRNAi])
and to survey the offspring for phenotypic effects (Bucher et al. 2002). The ease of pupal injection and
the long duration and lower variability of the RNAi effect (from weeks to months, depending on the
gene) allow efficient collection of RNAi-affected embryos. Also, the fact that the pRNAi effect
decreases over time, producing an ordered series of embryos with diminishing severity of phenotypic
effect after a single set of injections, makes RNAi at the pupal stage the preferred method for studies
of embryogenesis. However, knockdown of certain genes can interfere with pupal eclosion or egg production. Often, these problems can be circumvented by injecting adult females (aRNAi). For the few
genes where lethality or a block in egg production is also observed after adult injection, one must
resort to more laborious eRNAi (Brown et al. 1999).
RNAi in Tribolium is efficient in most, if not all differentiated tissues. Injection of dsRNA into the
larval hemolymph (larval RNAi [lRNAi]) can produce global knockdown of gene expression in epidermal cells. More recently, ubiquitously expressed enhanced green fluorescent protein (EGFP) was
shown to be depleted in all tissues by larval RNAi (Miller et al. 2008). dsRNA injection into pupae or
adults knocks down target gene expression within a few days, and the effects can last for several
months (Tomoyasu and Denell 2004; Konopova and Jindra 2007; Shippy et al. 2008). RNAi in the

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Red Flour Beetle (Tribolium) (Posnien et al. 2009) includes procedures for injecting dsRNA into
pupae, adults, larvae, and embryos.
In Situ Hybridization and Immunohistochemistry

Most in situ and antibody staining protocols used for Tribolium have been adapted from Drosophila
studies. Few antibodies have been raised specifically against Tribolium antigens. The monoclonal antibodies mAbs4D9 and mAbs2B8, raised against the Drosophila proteins Invected and Even-skipped,
respectively, cross-react with orthologous proteins from a wide variety of insects, including Tribolium
(Patel et al. 1992). These two antibodies have been used extensively to define segmentation boundaries during early embryonic development and, in combination with in situ hybridization, to determine the relative segmental register of the expression of newly characterized genes. In general, the
cross-reactivity of a given antibody cannot be predicted, and each must be tested individually.
Procedures for performing in situ hybridizations and immunohistochemistry in Tribolium embryos are
presented in Single and Double Whole-Mount In Situ Hybridization in Red Flour Beetle
(Tribolium) Embryos (Schinko et al. 2009) and Concurrent In Situ Hybridization and Antibody
Staining in Red Flour Beetle (Tribolium) Embryos (Shippy et al. 2009).
Transgenics

The procedure for inserting transgenes into the Tribolium genome is comparable to that used for
Drosophila, and the resulting efficiency of transformation is similar. However, the longer Tribolium life
cycle more than doubles the time from injection to establishment of a stock. Transgenic animals can
be identified by the expression of fluorescent protein, primarily in eye and brain tissues, that is driven
by the artificial 3xP3 promoter (Berghammer et al. 1999). The strongest signal is obtained when using
EGFP or enhanced yellow fluorescent protein (EYFP) as the transformation marker, but DsRed and
enhanced cyan fluorescent protein (ECFP) have also been used successfully. Alternatively, rescue of the
vermilionwhite eye-color mutation is a very efficient way to identify transformants (Lorenzen et al.
2002a). Multiple dominant markers facilitate genetic manipulation, since trans-heterozygotes carrying
two different markers can be directly detected. While piggyBac (Berghammer et al. 1999) and Minos
transposons (Pavlopoulos et al. 2004) integrate with high efficiency (similar to Drosophila P-elements),
Hermes insertions are rare, and retention of flanking plasmid sequence indicates that this element fails
to integrate properly in Tribolium (R Beeman and M Lorenzen, unpubl.).
Misexpression of genes can be performed by using the endogenous heat shock protein 68 (hsp68)
promoter (G Bucher, unpubl.) and the binary Gal4/UAS system (M Klingler, E Wimmer, and G Bucher,
unpubl.). Components typically used in Drosophila (e.g., the Dm-hsp70 promoter or the GAL4/UAS
system combined with Drosophila basal promoters) do not seem to function properly in Tribolium (E
Wimmer, M Klingler, and S Brown, unpubl.). Hence, the use of native Tribolium promoters is essential
for such experiments (Lorenzen et al. 2002b; Siebert et al. 2008).
The function of putative Tribolium regulatory elements can be tested by creating transgenic reporter
constructs (Eckert et al. 2004). Again, the use of native Tribolium promoters should enhance the efficiency of these types of experiments. For details on creating transgenic lines of Tribolium, see Red Flour
Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis (Berghammer et al. 2009).
Insertional Mutagenesis

A two-component mutagenesis system patterned after one commonly used for Drosophila has been
established for Tribolium. The Tribolium system uses a piggyBac-based donor construct as the mutagenic agent and a Minos-based helper construct to provide a temporary source of piggyBac transposase to mobilize the donor element. New transposon insertions can be obtained by crossing donor
and helper strains to remobilize the donor insertion (Lorenzen et al. 2007). Insertions that produce
interesting phenotypes can be characterized at the molecular level and located within the Tribolium
genome sequence, thus allowing quick identification of the gene(s) that are likely to be affected by
insertion of the transposon. Using this system, research groups in Germany and the United States are
collaborating on a large-scale insertional mutagenesis project (the GEKU screen) and have generated
more than 550 lethal and 600 enhancer trap lines. Information on these lines is accessible at
http://www.geku-base.uni-goettingen.de/, and lines are available from the members of the consortium. For details on insertional mutagenesis in Tribolium, see Red Flour Beetle (Tribolium) Germline
Transformation and Insertional Mutagenesis (Berghammer et al. 2009).

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