Model Beetle Tribolium Castaneum Research Articles

The Low-Copy-Number Satellite DNAs of the Model Beetle Tribolium castaneum.

The red flour beetle Tribolium castaneum is an important pest of stored agricultural products and the first beetle whose genome was sequenced. So far, one high-copy-number and ten moderate-copy-number satellite DNAs (satDNAs) have been described in the assembled part of its genome. In this work, we aimed to catalog the entire collection of T. castaneum satDNAs. We resequenced the genome using Illumina technology and predicted potential satDNAs via graph-based sequence clustering. In this way, we discovered 46 novel satDNAs that occupied a total of 2.1% of the genome and were, therefore, considered low-copy-number satellites. Their repeat units, preferentially 140-180 bp and 300-340 bp long, showed a high A + T composition ranging from 59.2 to 80.1%. In the current assembly, we annotated the majority of the low-copy-number satDNAs on one or a few chromosomes, discovering mainly transposable elements in their vicinity. The current assembly also revealed that many of the in silico predicted satDNAs were organized into short arrays not much longer than five consecutive repeats, and some of them also had numerous repeat units scattered throughout the genome. Although 20% of the unassembled genome sequence masked the genuine state, the predominance of scattered repeats for some low-copy satDNAs raises the question of whether these are essentially interspersed repeats that occur in tandem only sporadically, with the potential to be satDNA "seeds".

DIPA-CRISPR is a simple and accessible method for insect gene editing

SummaryCurrent approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed “direct parental” CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.

RNA interference-based characterization of Caspar, DREDD and FADD genes in immune signaling pathways of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

We previously demonstrated that two immune signaling pathways, Toll and IMD, were concomitantly activated in the model beetle Tribolium castaneum by challenges to their immune system by several species of microbes, including Gram-positive and -negative bacteria as well as yeast. This contrasts with the Drosophila immune system in which more specific pathway activation depending on the type of microbe is well established. We suggest that the activation of an indiscriminate immune pathway in T. castaneum is due in part to an unselective recognition of pathogen-associated molecular patterns by the extracellular sensing modules of the two pathways. In order to obtain a more detailed understanding of the T. castaneum immune pathway, we investigated whether potential components of the T. castaneum IMD pathway, Caspar, DREDD and FADD, are involved in immune reactions triggered by microbial challenges. A sequence analysis of these three genes with the orthologues of other species, including insects, mouse and human, indicated that T. castaneum Caspar, DREDD and FADD functioned as immune signal transducers, which are usually induced by microbial challenges. However, these genes were not induced by microbial challenges. To establish whether these genes are involved in immune reactions, we used RNA interference-mediated knockdown of these genes to assess the microbial induction levels of the representative read-out antimicrobial peptide genes of the respective classes. The results indicated that these genes encode the canonical constituents of the IMD pathway of this beetle. DREDD and FADD influenced the induction of Toll-dependent antimicrobial peptide genes, providing novel crosstalk points between the two immune pathways, which appears to support indiscriminate pathway activation in T. castaneum. Furthermore, the phenotypes of DREDD or FADD knockdown pupae challenged by the two model bacterial pathogens correlated with AMP gene induction in the respective knockdowns, indicating that these intracellular factors contributed to antibacterial host defenses.

Knockdown of Genes Involved in Transcription and Splicing Reveals Novel RNAi Targets for Pest Control

RNA interference (RNAi) is a promising next generation technology for the development of species-specific pest management. The key to successful RNAi based-plant protection is dependent in part on data-driven target gene selection, a challenging task due to the absence of laboratory strains and the seasonality of most pest species. In this study, we aimed to identify novel target genes by performing a knowledge-based approach in order to expand the spectrum of known potent RNAi targets. Recently, the protein-coding genes ncm, Rop, RPII-140, and dre4 have been identified as sensitive RNAi targets for pest control. Based on these potent RNAi targets, we constructed an interaction network and analyzed a selection of 30 genes in the model beetle Tribolium castaneum via injection of dsRNA synthesized by in vitro transcription. Nineteen of these targets induced significant mortality of over 70%, including six that caused 100% lethality. Orthologs of active T. castaneum RNAi targets were verified in the economically important coleopteran pests Diabrotica virgifera virgifera and Brassicogethes aeneus. Knockdown of D. v. virgifera genes coding for transcription factor Spt5, Spt6, and RNA polymerase II subunit RPII-33 caused over 90% mortality in larval feeding assays. Injection of dsRNA constructs targeting RPII-215 or the pre-mRNA-processing factor Prp19 into adult B. aeneus resulted in high lethality rates of 93 and 87%, respectively. In summary, the demonstrated knowledge-based approaches increased the probability of identifying novel lethal RNAi target genes from 2% (whole genome) to 36% (transcription- and splicing-related genes). In addition, performing RNAi pre-screening in a model insect increased also the probability of the identification essential genes in the difficult-to-work-with pest species D. v. virgifera and B. aeneus.

The mustard leaf beetle, Phaedon cochleariae, as a screening model for exogenous RNAi-based control of coleopteran pests

RNA interference (RNAi) is a promising, selective pest control technology based on the silencing of targeted genes mediated by the degradation of mRNA after the ingestion of double-stranded (ds) RNA. However, the identification of the best target genes remains a challenge, because large scale screening is only feasible in lab model systems and it remains unclear, to what degree such data can be transferred to pest species. Here, we report on our efforts to transfer target genes found in a lab model to the mustard leaf beetle, Phaedon cochleariae. The mustard leaf beetle can be reared easily and resource-efficient in large quantities all year round and is an established chrysomelid pest for higher throughput screening approaches in the crop protection industry. Mustard leaf beetle transcriptome sequencing and assembly revealed genes orthologous to those previously described as highly efficient RNAi targets in the model beetle Tribolium castaneum. First, we observed mortality after injection of dsRNA targeting the respective orthologous genes in 2nd instar mustard beetle larvae. Next, we adopted a robust, automated multi-well plate foliar RNAi screening procedure with 2nd instar larvae of the mustard leaf beetle to assess those genes. Indeed, foliar application and oral uptake of dsRNA targeting the same genes resulted in larval mortality as well. The most effective target genes with a strong (lethal) phenotype - at dsRNA doses as low as 300 ng/leaf disc (equal to 9.6 g/ha) - were srp54k, rop, αSNAP, rpn7 and rpt3. Rather limited effects were observed after application of dsRNA targeting cactus, shibire and PP-α, though they had previously been shown to be highly lethal in red flour beetle. Importantly, our experiments demonstrated that the overall efficacy pattern obtained after oral dsRNA application was well correlated with the results obtained after dsRNA injection. RT-qPCR confirmed significant target gene knock-down after normalization by employing three reference genes shown to be stably expressed across life stages. In summary, several RNAi targeted genes elicited a strong lethal phenotype and significant target gene knock-down after feeding, suggesting P. cochleariae as a potential coleopteran screening model for foliarly applied exogenous RNAi.

CenH3 distribution reveals extended centromeres in the model beetle Tribolium castaneum.

Centromeres are chromosomal domains essential for kinetochore assembly and correct chromosome segregation. Inconsistent in their underlying DNA sequences, centromeres are defined epigenetically by the presence of the centromere-specific histone H3 variant CenH3. Most of the analyzed eukaryotes have monocentric chromosomes in which CenH3 proteins deposit into a single, primary constriction visible at metaphase chromosomes. Contrary to monocentrics, evolutionary sporadic holocentric chromosomes lack a primary constriction and have kinetochore activity distributed along the entire chromosome length. In this work, we identified cCENH3 protein, the centromeric H3 histone of the coleopteran model beetle Tribolium castaneum. By ChIP-seq analysis we disclosed that cCENH3 chromatin assembles upon a repertoire of repetitive DNAs. cCENH3 in situ mapping revealed unusually elongated T. castaneum centromeres that comprise approximately 40% of the chromosome length. Being the longest insect regional centromeres evidenced so far, T. castaneum centromeres are characterized by metapolycentric structure composed of several individual cCENH3-containing domains. We suggest that the model beetle T. castaneum with its metapolycentromeres could represent an excellent model for further studies of non-canonical centromeres in insects.

Polyandry provides reproductive and genetic benefits in colonising populations

Polyandry, when females mate with more than one male, is theorised to play an important role in successful colonisation of new habitats. In addition to possible benefits from sexual selection, even mild polyandry could facilitate colonisation by protecting against inbreeding and reducing the costs of mating with incompatible or infertile males. Here, we measure the importance of mild polyandry for population viability and reproductive fitness following experimental founder events into a higher‐temperature regime. Using colonisation experiments with the model beetle Tribolium castaneum, in which females can produce offspring for up to 140 days following a single mating, we founded more than 100 replicate populations using single females that had been given the opportunity to mate with either one or two males and then tracked their subsequent population dynamics. Following population viability and fitness across 10 generations, we found that extinction rates were significantly lower in populations founded by females given polyandrous opportunities to mate with two males (9%) compared to populations founded by monogamous females (34%). In addition, populations founded by females that had been provided with opportunities to store sperm from two different males showed double the median productivity following colonisation compared to monogamous‐founded populations. Notably, we identified short‐term and longer‐term benefits to post‐colonisation populations from double‐mating, with results suggesting that polyandry acts to both protect against mating with incompatible males through the founder event, and reduce inbreeding depression as the colonisation proceeds for 10 generations. Our results therefore show that even mild polyandry provides both reproductive and genetic benefits for colonising populations.

Evidence of multifaceted functions of codon usage in translation within the model beetle Tribolium castaneum.

Synonymous codon use is non-random. Codons most used in highly transcribed genes, often called optimal codons, typically have high gene counts of matching tRNA genes (tRNA abundance) and promote accurate and/or efficient translation. Non-optimal codons, those least used in highly expressed genes, may also affect translation. In multicellular organisms, codon optimality may vary among tissues. At present, however, tissue specificity of codon use remains poorly understood. Here, we studied codon usage of genes highly transcribed in germ line (testis and ovary) and somatic tissues (gonadectomized males and females) of the beetle Tribolium castaneum. The results demonstrate that: (i) the majority of optimal codons were organism-wide, the same in all tissues, and had numerous matching tRNA gene copies (Opt-codon↑tRNAs), consistent with translational selection; (ii) some optimal codons varied among tissues, suggesting tissue-specific tRNA populations; (iii) wobble tRNA were required for translation of certain optimal codons (Opt-codonwobble), possibly allowing precise translation and/or protein folding; and (iv) remarkably, some non-optimal codons had abundant tRNA genes (Nonopt-codon↑tRNAs), and genes using those codons were tightly linked to ribosomal and stress-response functions. Thus, Nonopt-codon↑tRNAs codons may regulate translation of specific genes. Together, the evidence suggests that codon use and tRNA genes regulate multiple translational processes in T. castaneum.

Involvement of laccase2 and yellow-e genes in antifungal host defense of the model beetle, Tribolium castaneum.

We previously reported that the moderate knockdown of chitin synthase 1 gene of the model beetle Tribolium castaneum impairs the host defense against entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, which infect host insects via the direct penetration of cuticular integuments (Hayakawa et al., 2017). In this study, we focused on the antifungal roles of laccase2 (Lac2) as well as yellow-e (Y-e) genes, both of which are shown to be important to the establishment of stable cuticular structures in this beetle species. The expression profiles of the two genes somewhat resembled each other, peaking in late prepupae and mid to late pupae, while the transcript levels of Lac2 were higher than Y-e throughout. The knockdown of Lac2 gene at the prepupal and pupal peaks with relatively small amounts of dsRNA resulted in pupae with a lighter color and adults with a lighter color and dimpled/wrinkled elytra, respectively. Meanwhile, similar gene knockdown of Y-e but with 10 times more dsRNA compared to Lac2 resulted in pupae having a normal appearance and adults with a darker color. We conducted fungal infection assays with B. bassiana and M. anisopliae using these knockdown animals. The knockdown of Y-e gene had no or limited effects in both pupae and adults in terms of the antifungal host defense. Similarly, the knockdown of Lac2 gene did not change significantly the defense phenotypes of the resulting pupae. By sharp contrast, the host defense of the adult beetles against the two fungal species was almost totally destroyed by the moderate knockdown of Lac2 gene, suggesting its indispensable role in antifungal host defense presumably through the construction of sound cuticles of the adults. Finally, we investigated the maturation of host defense against fungal infection in the Lac2 knockdown adults and found that while the day 10 adults were still susceptible to M. anisopliae infection with some delay of death in comparison with day 1 adults, they exhibited complete refractoriness to B. bassiana.

Evaluation of high-throughput isomiR identification tools: illuminating the early isomiRome of Tribolium castaneum

BackgroundMicroRNAs carry out post-transcriptional gene regulation in animals by binding to the 3' untranslated regions of mRNAs, causing their degradation or translational repression. MicroRNAs influence many biological functions, and dysregulation can therefore disrupt development or even cause death. High-throughput sequencing and the mining of animal small RNA data has shown that microRNA genes can yield differentially expressed isoforms, known as isomiRs. Such isoforms are particularly relevant during early development, and the extension or truncation of the 5' end can change the profile of mRNA targets compared to the original mature sequence. We used the publicly available small RNA dataset of the model beetle Tribolium castaneum to create the first comparative isomiRome of early developmental stages in this species. Standard microRNA analysis software does not specifically account for isomiRs. We therefore carried out the first comparative evaluation of the specialized tools isomiRID, isomiR-SEA and miraligner, which can be downloaded for local use and can handle next generation sequencing data.ResultsWe compared the performance of isomiRID, isomiR-SEA and miraligner using simulated Illumina HiSeq2000 and MiSeq data to test the impact of technical errors. We also created artificial microRNA isoforms to determine the effect of biological variants on the performance of each algorithm. We found that isomiRID achieved the best true positive rate among the three algorithms, but only accounted for one mutation at a time. In contrast, miraligner reported all variations simultaneously but with 78% sensitivity, yielding isomiRs with 3' or 5' deletions. Finally, isomiR-SEA achieved a sensitivity of 25–33% when the seed region was mutated or partly deleted, but was the only tool that could accommodate more than one mismatch. Using the best tool, we performed a complete isomiRome analysis of the early developmental stages of T. castaneum.ConclusionsOur findings will help researchers to select the most suitable isomiR analysis tools for their experiments. We confirmed the dynamic expression of 3′ non-template isomiRs and expanded the isomiRome by all known isomiR modifications during the early development of T. castaneum.

The model beetle Tribolium castaneum can be used as an early warning system for transgenerational epigenetic side effects caused by pharmaceuticals

Pharmaceuticals are not currently tested for transgenerational and epigenetic side effects. The use of vertebrates as preclinical research models is limited by their long generation times, low numbers of progeny and ethical concerns. In contrast, invertebrates such as insects breed rapidly, produce many offspring and are more ethically acceptable, allowing them to be used for high-throughput screening. Here, we established Tribolium castaneum as a model to screen for the effect of drugs on complex fitness parameters and the expression of epigenetic regulatory genes. We tested diets supplemented with the psychoactive drug valproic acid (VPA), which is a histone deacetylase inhibitor, or the antioxidant curcumin, which is a histone acetyltransferase inhibitor. We found that VPA delayed development, reduced longevity, and increased female body weight compared to a control diet. Fertility and fecundity declined and the expression of epigenetic regulatory genes was induced in the untreated F1 generation. In contrast, curcumin did not affect development or body weight, but it increased longevity, caused a significant reduction in fertility, and induced the expression of epigenetic regulatory genes mostly in the treated F0 generation. VPA and curcumin administered to vertebrate models have similar effects to those we observed in T. castaneum, confirming that this beetle is potentially useful as an alternative model to screen for the epigenetic effect of drugs. T. castaneum also provides a valuable early warning system for transgenerational epigenetic risk factors that are difficult to detect in mammals.

A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus.

The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC) of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16–64 μg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl2 respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT) at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 μg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 μg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 μg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.