RNAi-mediated silencing of pgants shows core 1 O-glycans are required for pupation in Tribolium castaneum

Protein glycosylation is one of the most common and most important post-translational modifications. Despite the growing knowledge on N-glycosylation, the research on O-glycosylation is lagging behind. This study investigates the importance of O-glycosylation in the post-embryonic development of insects using the red flour beetle, Tribolium castaneum, as a model. We identified 28 O-glycosylation-related genes (OGRGs) in the genome of the red flour beetle. 14 OGRGs were selected for functional analysis based on their involvement in the initial attachment of the carbohydrate in the different O-glycosylation pathways or the further elongation of the most abundant O-glycans and, in addition, showing severe RNAi-induced phenotypes in Drosophila melanogaster. The expression profile of these OGRGs was mapped throughout the developmental stages of the insect and in the different tissues of the pupa and adult. Subsequently, these genes were silenced using RNA interference (RNAi) to analyze their role in development. A broad spectrum of phenotypes was observed: from subtle effects and disrupted wing formation when silencing the genes involved in O-mannosylation, to blockage of pupation and high mortality after silencing of the genes involved in O-GalNAc and core 1 O-glycan (O-GalNAc-Gal) synthesis. RNAi experiments were also performed to assess the effects of blocking multiple pathways of O-glycosylation. However, the observed phenotypes induced by multiple RNAi were similar to those of the single gene RNAi experiments. The silencing of OGRGs often resulted in high mortality and wing phenotypes, indicating the importance of O-glycosylation for the survival of the insect and the formation of wings during the post-embryonic development of T. castaneum

Infection routes matter in population-specific responses of the red flour beetle to the entomopathogen Bacillus thuringiensis

Background: Pathogens can infect their hosts through different routes. For studying the consequences for host resistance, we here used the entomopathogen Bacillus thuringiensis and the red flour beetle Tribolium castaneum for oral and systemic (i. e. pricking the cuticle) experimental infection. In order to characterize the molecular mechanisms underpinning the two different infection routes, the transcriptomes of beetles of two different T. castaneum populations – one recently collected population (Cro1) and a commonly used laboratory strain (SB) – were analyzed using a next generation RNA sequencing approach. Results: The genetically more diverse population Cro1 showed a significantly larger number of differentially expressed genes. While both populations exhibited similar reactions to pricking, their expression patterns in response to oral infection differed remarkably. In particular, the Cro1 population showed a strong response of cuticular proteins and developmental genes, which might indicate an adaptive developmental flexibility that was lost in the SB population presumably as a result of inbreeding. The immune response of SB was primarily based on antimicrobial peptides, while Cro1 relied on responses mediated by phenoloxidase and reactive oxygen species, which may explain the higher resistance of this strain against oral infection. Conclusions: Our data demonstrate that immunological and physiological processes underpinning the two different routes of infection are clearly distinct, and that host populations particularly differ in responses to oral infection. Furthermore, gene expression upon pricking infection entailed a strong signal of wounding, highlighting the importance of pricking controls in future infection studies

Development of a biotechnologically enhanced sterile insect technique to fight coleopteran pests

Tribolium castaneum commonly referred to as the Red Flour Beetle (RFB) belongs to the class Insecta and order Coleoptera, and it’s the best model organism for coleopterans. About 380,000 coleopteran species, constituting approximately 25% of all the animal species described in the world have been identified, thus making them the most taxonomically described species in the animal kingdom, and they are found occupying different ecosystems due to their extraordinary diversities. Approximately 75% of the beetle described are polyphagous as larvae and adult, and are found to live on plants, wood products and stored products and are of economic importance for agriculture, as well as forestry and other household products. By such activities they cause several and significant damage both in direct and indirect losses. The current predominant control measure employed for controlling coleopteran pests is the injurious application of chemicals which results in the development of resistance over time, and also have negative effect to the environment and human health. Hence, there is a need for more efficient and environmentally friendly control measures that is devoid of the drawbacks identified, hence the development and usage of biotechnological approaches. Transgenic approaches use recombinant DNA via genetic engineering to mimic classical genetic approaches, which is also less expensive, and addresses the drawbacks of the classical genetic approach. However, while these practices have been employed in various insect pests mostly in the order of dipteran, none has been employed to any coleopteran insect pest so far. Biotechnological approaches could improve techniques such as sexing for male alone releases, sperm marking, and female/embryonic specific lethality strategies, where classical genetic approaches use for example radiation for causing sterility, fluorescent dyes for monitoring and phenotypic strategies such as pupal sizes or colour for sexing which have several disadvantages. Tribolium castaneum as a model insect pest has a comprehensive genome database, which can be utilized for both evolutionary and population genetics approaches. The organism is also the most utilized insect for development biology studies aside from Drosophila melanogaster due to the ease of handling the insect in the laboratory, short generation time, high fecundity, etc. Here I provide the first attempt and proof of principle of transgenic approaches for sterility, sperm marking for monitoring, and sexing using recombinant DNA. Firstly, for monitoring, Iused the Promoter/Enhancer (P/E) region of the Tribolium spermatogenesis specific ß2 -tubulin gene to drive DsRed and/or EGFP fluorescent protein in the sperm of male individuals by first integrating the constructs by piggyBac germline transformation to generate transgenic lines. I observed the marked sperm in the testis of the male, and in the spermatheca of the female when a wildtype female is mated with a transgenically sperm-marked male individual. This can solve the drawback that is observed in the bodily application of fluorescent dyes on the released individuals, which will result in either false negative or false positive in the classical SIT in capture and recapture experiments. Also, the generated sperm marked individuals provided us with a tool to answer some reproductive biology question on sperm storage and usage by twice mated females. The result showed that the female store the sperm for a long period, and use the sperm of the second inseminated males first, with the first male inseminated sperms used subsequently. Moreover, we discovered a new early embryonically expressed gene (Tc007675) which was hitherto not reported and used it for functional studies. I used the amplified 1.4Kb or 2.1Kb of the Promoter/Enhancer (P/E) upstream region of the gene to drive the artificial heterologous transactivator tTA in order to generate several driver strains by piggyBac germline transformation. By Whole Mount In Situ Hybridization (WMISH), two driver lines expressing tTA at cellularization stage were identified. In addition, using CRISPR-Cas9 genome editing, I effectively generated a driver line targeting the Tc007675 gene locus by Homology Directed Repair (HDR) strategy to generate a bi-cistronic locus. The effectiveness of the driver line was verified using WMISH, where we detected a transient expression of tTA under the control of the Tc007675 gene. Furthermore, using the spermatogenesis specific ß2t enhancer/promoter to drive tTA, we generated a driver line that expresses tTA in testis of one of the lines generated. With this line, we intend to express Cas9 and respective guide RNAs in the testis as a new approach to cause reproductive sterility. At the same time, we generated a potential Cas9 expressing effector line which we crossed with our driver line but for now we were not able to drive Cas9 in the testis. This idea will be used to mimic the use of ionizing radiation used in classical approach, where it could take care of the drawback arising from fitness loss due to the mutation effect on somatic cells of the treated organism. The CRISPR-Cas9 mediated reproductive sterility system will provide a novel and efficient way, in which males will have no fitness or competitiveness disadvantages to wildtype when released.2022-02-2

Enhanced genome assembly and a new official gene set for Tribolium castaneum

Background. The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. Results. Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. Conclusions. The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis

Exploring morphological innovation and diversification: Analysis of genes involved in gin-trap formation and antenna remodeling during metamorphosis in Tribolium castaneum

How morphological traits originate and diversify is a central question in evolutionary biology. Insects are the most diverse group of animals on the planet and over 80% of insect species belong to the subgroup of holometabola. The shape of a holometabolous insect experiences a striking change during metamorphosis, which allowed the evolution of an overwhelming morphological diversity. Hence, this process provides excellent samples to study the evolution of morphological innovation and diversity. Among insects, the developmental and genetic mechanisms of epidermal patterning are well understood in the model organism, Drosophila melanogaster. However, this highly derived Dipteran species does not show a typical metamorphosis. Drosophila replaces all larval epidermal cells by imaginal cells to form the adult epidermis. Instead, most holometabolous insects re-use larval cells to generate the adult epidermis, In contrast to Drosophila, the red flour beetle, Tribolium castaneum, shows a more typical mode of metamorphosis. Importantly, unbiased large scale RNA interference screening (iBeetle-screen) in Tribolium allows identifying and investigating gene sets involved in the process of morphological innovation and diversification independently from Drosophila knowledge. In the first part of this thesis, the gin-trap was used as a study case to explore how a morphologically novel structure evolved during metamorphosis in Tribolium. Firstly, the wing genes known from Drosophila were investigated for their potential functions in gin-trap formation. The results showed that a large part of the upstream genes but much few downstream genes of the wing gene network were co-opted into gin-trap formation. Secondly, novel genes required for gin-trap development were searched in the iBeetle database. Ten genes were confirmed for their functions in gin-trap formation, most of which were required for wing formation as well. The only gin-trap specific gene, Tc-caspar, which was recruited from another biological context, was required for establishment of the anterior-posterior symmetry of the gin-traps. This is an innovation to this structure. Taken together, these data suggested that gin-traps evolved by co-option of a pruned wing gene regulatory network and a low level of gene recruitment from a distinct biological context. In the second part, novel genes from iBeetle screen were identified and analyzed on antenna metamorphosis in Tribolium. Of the ten confirmed genes, half belonged to the new classifications which were not reported to be associated with antenna patterning in Drosophila. Interestingly, four genes were related to pre-mRNA splicing, indicating the potential role of this process for antenna remodeling. One taxonomically restricted gene was found to affect a specific region of the antenna. And then, I optimized a protocol for whole mount in situ hybridization of pre-pupal antennae and the expression patterns of novel genes showed that the expression patterns were consistent with a role of these genes in antenna remodeling. Finally, I compared the gene sets between antenna and leg development and verified a complex mix of divergence and constraint among these serially homologous appendages. The data obtained in this thesis provide new insight into the morphological innovation and diversification during metamorphosis and are the basis for future studies