The red flour beetle, Tribolium castaneum, is an important model insect and agricultural pest. However, many standard genetic tools are lacking or underdeveloped in this system. Here, we present a set of new reagents to augment existing Tribolium genetic tools. We demonstrate a new GAL4 driver line that employs the promoter of a ribosomal protein gene to drive expression of a UAS responder in the fat body. We also present a novel dual fluorescent reporter that labels cell membranes and nuclei with different fluorophores for the analysis of cellular morphology. This approach also demonstrates the functionality of the viral T2A peptide for bicistronic gene expression in Tribolium. To facilitate classical genetic analysis, we created lines with visible genetic markers by CRISPR-mediated disruption of the yellow and ebony body color loci with a cassette carrying an attP site, enabling future φC31-mediated integration. Together, the reagents presented here will facilitate more robust genetic analysis in Tribolium and serve as a blueprint for the further development of this powerful model's genetic toolkit.

Although, beetles of the genus Tribolium first evolved as saprophylic insects, they have adapted to the stored products environment for several thousand years. In this study reproductive strategies are described for eight species of Tribolium that are known to occur in this environment. Experiments were conducted under the same conditions for every species, and several life history traits, including egg mass, adult mass, developmental time and fecundity were examined and compared among these species. Common reproductive strategies were not found among the different species and univariate analysis highlighted strong differences between the species for most of the traits investigated. Some species showed reproductive traits that are likely to give a fitness advantage in the environment of stored products. Multivariate statistical analysis allowed the detection of different sub-groups with respect to their reproductive strategy. Adult mass and egg-to-adult developmental time discriminated between groups. Intraspecific allometric relationships were further investigated but only a few correlations appeared to be significant.

The segment-polarity gene engrailed is required for segmentation in the early Drosophila embryo. Loss of Engrailed function results in segmentation defects that vary in severity from pair-rule phenotypes to a lawn phenotype lacking in obvious of segmentation. During segmentation, Engrailed is expressed in stripes with a single segmental periodicity in Drosophila, which is conserved in all arthropods examined so far. To define segments, the segmental stripes of Engrailed induce the segmental stripes of wingless at each parasegmental boundary. However, segmentation functions of orthologs of engrailed in non-Drosophila arthropods have yet to be reported. Here, we analyzed functions of the Tribolium ortholog of engrailed (Tc-engrailed) during embryonic segmentation. Larval cuticles with Tc-engrailed being knocked down had segmentation phenotypes including incomplete segment formation and loss of a group of segments. In agreement with the cuticle segmentation defects, segments developed incompletely and irregularly or did not form in Tribolium germbands where Tc-engrailed was knocked down. Furthermore, knock-down of Tc-engrailed did not properly express the segmental stripes of wingless in Tribolium germbands. Taken together with the conserved expression patterns of Engrailed in arthropod segmentation, our data suggest that Tc-engrailed is required for embryonic segmentation in Tribolium, and the genetic mechanism of Engrailed inducing wingless expression is conserved at least between Drosophila and Tribolium.

The juvenile hormone analog methoprene, and the chitin synthesis inhibitor novaluron, were evaluated by exposing late-stage larvae of Tribolium castaneum (Herbst) or Tribolium confusum (Jacqueline DuVal) (Coleoptera: Tenebrionidae) to it. The larvae were exposed to it in food material, on concrete, on plywood, and on floor tile. Larvae of T. castaneum were more susceptible than T. confusum larvae to both methoprene and novaluron on all surfaces. A further evaluation was done by exposing adult T. confusum to methoprene and novaluron through food placed on concrete treated with methoprene and novaluron, and then assessing resulting progeny production. The emergence of adults with normal morphology was reduced for both chemicals, with more malformed adults appearing in the methoprene treatment, and fewer adults of any form emerging in the novaluron treatment. The results show direct exposures to larvae, or determining progeny production from exposed adults, are valid methods for assessing the susceptibility of flour beetles to insecticides.

Catmint oil and hydrogenated catmint oil were evaluated as repellents for adult Tribolium casteneum (Herbst) (Coleoptera: Tenebrionidae), the red flour beetle, and T. confusum (DuVal), the confused flour beetle, using both a traditional method of visual assessment of distribution and a video recording method to determine movement patterns of individual insects. Visual assessments of distribution using groups of adults showed that the hydrogenated catmint oil was more effective than the pure catmint oil, but there was no significant difference between species. However, when repellency was measured using single insects and the visual recording system, both oils were significantly more repellent to T. castaneum than T. confusum at the concentrations evaluated in the study. Avoidance movement and change in direction when T. castaneum encountered the repellent were observed. Results indicate that repellents may be more accurately assessed using single insects rather than groups of individuals, and simple visual observations of distribution may be less sensitive in measuring repellent efficacy. Procedures for utilizing a video system are described as models for future evaluations of repellents for stored-product beetles.

BeetleBase (http://www.bioinformatics.ksu.edu/BeetleBase/) is an integrated resource for the Tribolium research community. The red flour beetle (Tribolium castaneum) is an important model organism for genetics, developmental biology, toxicology and comparative genomics, the genome of which has recently been sequenced. BeetleBase is constructed to integrate the genomic sequence data with information about genes, mutants, genetic markers, expressed sequence tags and publications. BeetleBase uses the Chado data model and software components developed by the Generic Model Organism Database (GMOD) project. This strategy not only reduces the time required to develop the database query tools but also makes the data structure of BeetleBase compatible with that of other model organism databases. BeetleBase will be useful to the Tribolium research community for genome annotation as well as comparative genomics.

Tribolium castaneum is a species of Coleoptera, the largest and most diverse order of all eukaryotes. Components of the innate immune system are hardly known in this insect, which is in a key phylogenetic position to inform us about genetic innovations accompanying the evolution of holometabolous insects. We have annotated immunity-related genes and compared them with homologous molecules from other species.

Bursicon is a neuropeptide belonging to the cystine knot family and is composed of burs and partner of burs (pburs) subunits. It can form heterodimers or homodimers to execute different biological functions. Bursicon heterodimers regulate cuticle sclerotization and wing maturation, whereas bursicon homodimers mediate innate immunity and midgut stem cell proliferation. A recent study has shown that bursicon potentially induces the expression of vitellogenin (Vg) in the black tiger shrimp Penaeus monodon; however, the underlying mechanism remains unknown. In this study, we investigated the role of bursicon in the reproductive physiology of the red flour beetle, Tribolium castaneum. The knockdown of burs, pburs, or its receptor T. castaneum rickets (Tcrk) in 2-day pupae significantly downregulated the expression levels of Vg1, Vg2, and Vg receptor (VgR) genes in females 3- and 5-day post-adult emergence, leading to abnormal oocytes with limited Vg content. The silencing of burs repressed the number of eggs laid and completely inhibited egg hatch, whereas the silencing of pburs dramatically decreased the number of eggs laid, hatch rate, and offspring larval size, and this RNA interference (RNAi) effects persisted to the next generation. Furthermore, the knockdown of burs or pburs downregulated the expression of the insulin/insulin-like signaling/target of rapamycin (TOR) signaling genes encoding insulin receptor (InR), protein kinase B (Akt), TOR, and ribosomal protein S6 kinase (S6K). Most importantly, the injection of recombinant pburs (r-pburs) protein was able to upregulate the expression of Vg, VgR, InR, Akt, TOR, S6K, JH synthesis (JHAMT), Methoprene-tolerant (Met), and Taiman (Tai) in normal females and rescue the expression of Vg and VgR in pburs RNAi females but failed to rescue Vg and VgR in Tcrk knockdown females. We infer that bursicon homodimers influence Vg expression via the receptor Tcrk, possibly by mediating the expression of the juvenile hormone (JH) and IIS/TOR pathway genes, thereby regulating reproduction in T. castaneum.

In a morphological ontology the expert's knowledge is represented in terms, which describe morphological structures and how these structures relate to each other. With the assistance of ontologies this expert knowledge is made processable by machines, through a formal and standardized representation of terms and their relations to each other. The red flour beetle Tribolium castaneum, a representative of the most species rich animal taxon on earth (the Coleoptera), is an emerging model organism for development, evolution, physiology, and pest control. In order to foster Tribolium research, we have initiated the Tribolium Ontology (TrOn), which describes the morphology of the red flour beetle. The content of this ontology comprises so far most external morphological structures as well as some internal ones. All modeled structures are consistently annotated for the developmental stages larva, pupa and adult. In TrOn all terms are grouped into three categories: Generic terms represent morphological structures, which are independent of a developmental stage. In contrast, downstream of such terms are concrete terms which stand for a dissectible structure of a beetle at a specific life stage. Finally, there are mixed terms describing structures that are only found at one developmental stage. These terms combine the characteristics of generic and concrete terms with features of both. These annotation principles take into account the changing morphology of the beetle during development and provide generic terms to be used in applications or for cross linking with other ontologies and data resources. We use the ontology for implementing an intuitive search function at the electronic iBeetle-Base, which stores morphological defects found in a genome wide RNA interference (RNAi) screen. The ontology is available for download at http://ibeetle-base.uni-goettingen.de.

MicroRNAs (miRNAs) are small non-coding RNAs mediating post-transcriptional regulation of gene expression in eukaryotes. Addressing their role in regulation of physiological adaptations to environmental stress in insects, we selected the red flour beetle Tribolium castaneum as a model. Beetles were fed with the bacterial entomopathogen Pseudomonas entomophila (to mimic natural infection), injected with peptidoglycan (experimental setting of strong immune responses) or subjected to either mild heat shock or starvation. Differential expression of selected immunity- and stress-related genes was quantified using real-time PCR, and expression and induction of 455 mature arthropod miRNAs were determined using proprietary microarrays. We found that Tribolium exhibits both gender- and stressor-specific adjustment of immune gene and miRNA expression. Strikingly, we discovered that the number of stressor-induced miRNAs in females is remarkably higher than in males. This observation could support the hypothesis called Bateman's principle in immunity that predicts gender-specific immune responses because females gain fitness through increased longevity, whereas males gain fitness by increasing mating rates. Our results suggest that Tribolium males and females display differential regulatory elements, both pre- and post-transcriptional, likely resulting from different investment strategies in life-history traits.

Higher order repeats (HORs) containing tandems of primary and secondary repeat units (head-to-tail "tandem within tandem pattern"), referred to as regular HORs, are typical for primate alpha satellite DNAs and most pronounced in human genome. Regular HORs are known to be a result of recent evolutionary processes. In non-primate genomes mostly so called complex HORs have been found, without head to tail tandem of primary repeat units. In beetle Tribolium castaneum, considered as a model case for genome studies, large tandem repeats have been identified, but no HORs have been reported. Here, using our novel robust repeat finding algorithm Global Repeat Map, we discover two regular and six complex HORs in T. castaneum. In organizational pattern, the integrity and homogeneity of regular HORs in T. castaneum resemble human regular HORs (with T. castaneum monomers different from human alpha satellite monomers), involving a wider range of monomer lengths than in human HORs. Similar regular higher order repeat structures have previously not been found in insects. Some of these novel HORs in T. castaneum appear as most regular among known HORs in non-primate genomes, although with substantial riddling. This is intriguing, in particular from the point of view of role of non-coding repeats in modulation of gene expression.

Sex determination cascade in insects terminates with the production of sex-specific protein, Doublesex (Dsx). We identified the dsx homolog (Tcdsx) in Tribolium castaneum. The pre-mRNA of Tcdsx is sex-specifically spliced into three female (Tcdsxf1, Tcdsxf2 and Tcdsxf3) and one male-specific (Tcdsxm) isoforms. Cis-regulatory elements potentially involved in sex-specific splicing of the Tcdsx pre-mRNA were identified in the female-specific exon and the adjoining intronic sequences. All the three female-specific TcDsx proteins share common OD1 and OD2 domains and differ in their C-terminal sequences. Knockdown of Tcdsx resulted in a reduction in the oocyte development, egg production and hatching of eggs laid. Several genes, including those coding for Vitellogenins and Vitellogenin receptors were identified as targets of TcDsx. RNAi experiments showed an isoform-specific targeting of identified target genes by TcDsx as knockdown in the expression of Tcdsx isoforms individually or in combinations resulted in differential effects on the expression of target genes.

Engrailed (en) and invected (inv) encode paralogous transcription factors found as a closely linked tandem duplication within holometabolous insects. Drosophila en mutants segment normally, then fail to maintain their segments. Loss of Drosophila inv is viable, while loss of both genes results in asegmental larvae. Surprisingly, the knockdown of Oncopeltus inv can result in the loss or fusion of the entire abdomen and en knockdowns in Tribolium show variable degrees of segmental loss. The consequence of losing or knocking down both paralogs on embryogenesis has not been studied beyond Drosophila. To further investigate the relative functions of each paralog and the mechanism behind the segmental loss, Tribolium double and single knockdowns of en and inv were analyzed. The most common cuticular phenotype of the double knockdowns was small, limbless, and open dorsally, with all but a single, segmentally iterated row of bristles. Less severe knockdowns had fused segments and reduced appendages. The Tribolium paralogs appear to act synergistically: the knockdown of either Tribolium gene alone was typically less severe, with all limbs present, whereas the most extreme single knockdowns mimic the most severe double knockdown phenotype. Morphological abnormalities unique to either single gene knockdown were not found. inv expression was not affected in the Tribolium en knockdowns, but hh expression was unexpectedly increased midway through development. Thus, while the segmental expression of en/inv is broadly conserved within insects, the functions of en and inv are evolving independently in different lineages.

Biogenic amines are physiologically neuroactive substances that affect behavioural and physiological traits in invertebrates. In the present study, the effects of dopamine, octopamine, tyramine and serotonin on tonic immobility, or death-feigning, were investigated in Tribolium castaneum. These amines were injected into the abdomens of beetles artificially selected for long or short duration of tonic immobility. In beetles of the long strains, the durations of tonic immobility were shortened by injection of dopamine, octopamine and tyramine, and the effects of these amines were dose-dependent. On the other hand, serotonin injection did not affect the duration of tonic immobility. In the short-strain beetles that rarely feign death, no significant effects of the amines were found on the duration of tonic immobility. Brain expression levels of octopamine, tyramine and serotonin did not differ between long- and short-strain beetles, in contrast to the higher dopamine levels in short strains previously reported. Caffeine decreased the duration of death-feigning in both oral absorption and injection experiments. It is known that caffeine activates dopamine. Therefore, the present results suggest that the duration of tonic immobility is affected by dopamine via the dopamine receptor in T. castaneum.

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) has had a long association with human stored food and can be a major pest in anthropogenic structures used for the processing and storage of grain-based products. Anthropogenic structures are fragmented landscapes characterized by spatially and temporally patchy resources. Here we investigate the ability of female T. castaneum to evaluate the quality of small patches of food and to adjust the number of eggs they lay per patch (i.e., clutch size) to maximize fitness gains. In multiple choice, paired choice and no choice experiments females tended to lay more eggs in larger amounts of flour. The number of eggs that they lay in a patch of flour was consistent with that predicted to optimize production of adults from that patch (i.e., the 'Lack' clutch size). Progeny size was only significantly impacted in the smallest patch sizes.

Chemoperception in invertebrates is mediated by a family of G-protein-coupled receptors (GPCR). To date nothing is known about the molecular mechanisms of chemoperception in coleopteran species. Recently the genome of Tribolium castaneum was sequenced for use as a model species for the Coleoptera. Using blast searches analyses of the T. castaneum genome with previously predicted amino acid sequences of insect chemoreceptor genes, a putative chemoreceptor family consisting of 62 gustatory receptors (Grs) and 26 olfactory receptors (Ors) was identified. The receptors have seven transmembrane domains (7TMs) and all belong to the GPCR receptor family. The expression of the T. castaneum chemoreceptor genes was investigated using quantification real- time RT-PCR and in situ whole mount RT-PCR analysis in the antennae, mouth parts, and prolegs of the adults and larvae. All of the predicted TcasGrs were expressed in the labium, maxillae, and prolegs of the adults but TcasGr13, 19, 28, 47, 62, 98, and 61 were not expressed in the prolegs. The TcasOrs were localized only in the antennae and not in any of the beetles gustatory organs with one exception; the TcasOr16 (like DmelOr83b), which was localized in the antennae, labium, and prolegs of the beetles. A group of six TcasGrs that presents a lineage with the sugar receptors subfamily in Drosophila melanogaster were localized in the lacinia of the Tribolium larvae. TcasGr1, 3, and 39, presented an ortholog to CO(2) receptors in D. melanogaster and Anopheles gambiae was recorded. Low expression of almost all of the predicted chemoreceptor genes was observed in the head tissues that contain the brains and suboesophageal ganglion (SOG). These findings demonstrate the identification of a chemoreceptor family in Tribolium, which is evolutionarily related to other insect species.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

N-acetyl transferase (NAT) is responsible to catalyze the transfer of acetyl groups to arylamines from acetyl-CoA. Aralkylamine Nacetyl transferase (AANAT), which belongs to GCN5-related N-acetyl transferase member, is a globular 23-kDa cytosolic protein that forms a reversible regulatory complex with 14-3-3 proteins, AANAT regulates the daily cycle of melatonin biosynthesis in mammals, making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. There is no evidence available regarding α and β subunits, active site and their ASA value in Dopamine N-acetyl transferase. Therefore, we describe the development of Dopamine N-acetyl transferase model in Tribolium castaneum. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides insight to the predicted active site and ASA values of Dopamine N-acetyl transferase model in Tribolium castaneum.

The glutamate-gated chloride channels (GluCls) play essential roles in signal transduction by regulating fast inhibitory synaptic transmission in the nervous system of invertebrates. While there is only one GluCl subunit in the insect, the diversity of insect GluCls is broadened by alternative splicing. In the present study, three TcGluCl variant genes were cloned from the red flour beetle Tribolium castaneum. Analysis of the characteristics of TcGluCls including sequence features, genomic structures, and alternative splicing revealed that TcGluCls had the typical structural features of GluCls and showed high homologies with the GluCls from other insect orders. The TcGluCl-encoding gene consists of nine exons and three variants (TcGluCl-3a, TcGluCl-3b, and TcGluCl-3c) were generated by the alternative splicing of exon 3, which was a highly conserved alternative splicing site in insect GluCls. Homology modeling of TcGluCl-3a showed that the exon 3 coding protein located at the N-terminal extracellular domain, and there were no steric clashes encountered between the exon 3 coding region and ivermectin/glutamate binding pocket, which indicated that the alternative splicing of exon 3 might have no impact on the binding of GluCls to glutamate and insecticide. In addition to the head tissue, TcGluCl-3a and TcGluCl-3c also had high expressions in the ovary and testis of T. castaneum, whereas TcGluCl-3b showed high expression in the midgut, suggesting the diverse physiological functions of TcGluCl variants in T. castaneum. The total TcGluCl and three variants showed the highest expression levels in the early stage larvae. The expressions of TcGluCl, TcGluCl-3b, and TcGluCl-3c were significantly increased from the late-stage larvae to the early stage pupae and indicated that the TcGluCl might be involved in the growth and development of T. castaneum. These results are helpful to further understand the molecular characteristics of insect GluCls and provide foundations for studying the specific function of the GluCl variant.

In the short-germ beetle Tribolium castaneum, waves of pair-rule gene expression propagate from the posterior end of the embryo towards the anterior and eventually freeze into stable stripes, partitioning the anterior-posterior axis into segments. Similar waves in vertebrates are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. However, neither a molecular candidate nor a functional role has been identified to date for such a frequency gradient, either in vertebrates or elsewhere. Here we provide evidence that the posterior gradient of Tc-caudal expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. We show this by analyzing the spatiotemporal dynamics of Tc-even-skipped expression in strong and mild knockdown of Tc-caudal, and by correlating the extension, level and slope of the Tc-caudal expression gradient to the spatiotemporal dynamics of Tc-even-skipped expression in wild type as well as in different RNAi knockdowns of Tc-caudal regulators. Further, we show that besides its absolute importance for stripe generation in the static phase of the Tribolium blastoderm, a frequency gradient might serve as a buffer against noise during axis elongation phase in Tribolium as well as vertebrates. Our results highlight the role of frequency gradients in pattern formation.