Biology subjects

Bucher, G.

Publications and source records attributed to Bucher, G..

15 recordsLinked to original sources

bioRxiv2026

foxQ2 marks fast-acting brain interneurons including a subset of dopaminergic neurons innervating mushroom bodies and central complex in the beetle Tribolium castaneum

The brain is one of the most complex animal organs and the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is involved in brain patterning but their specific contributions mostly remain to be elucidated, including foxQ2II. This transcription factor is conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. The contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use our enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast-activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

neuroscience↗

bioRxiv2025

Genome-wide identification of genes involved in beetle odoriferous defensive stink gland function recognizes Laccase2 as the phenoloxidase responsible for toxic para-benzoquinone synthesis

Exocrine glands have evolved several times independently in Coleoptera to produce defensive chemical compounds with repellent, antimicrobial, or toxic effects. Research on such glands had focused on morphological or chemical ecology methods. However, modern genetic approaches were missing to better understand this biological process. With the rise of the red flour beetle, Tribolium castaneum, as a model for studies of development and pest biology, molecular genetic tools are now available to also study the safe generation of toxic compounds in defensive stink glands. Using the RNA-interference-based, genome-wide, phenotypic screen "iBeetle" and the re-analysis of gland-specific transcriptomics based on a significantly improved genome annotation, we could identify 490 genes being involved in odoriferous stink gland function. In the iBeetle screen, 247 genes were identified, of which we present here 178 genes identified during iBeetles 3rd phase, while the transcriptomics analyses identified 249 genes, with six genes being identified in both functional genomics approaches. Of these 490 genes, only about 40% of these genes have molecularly characterized homologs in the vinegar fly, while for 213 genes no fly homologs were recognized and for 13 genes no gene ontology at all was identified. This highlights the importance of genome-wide gene identification in tissues that have not been previously analyzed to recognize potentially new gene functions. Gene ontology analysis revealed "SNARE interactions in vesicular transport", "Lysosome", "Pancreatic secretion", and "MAPK signaling pathway - fly" as key pathways. Additionally, many of the genes are encoding enzymes, transcription factors, transporters, or are involved in membrane trafficking. As the phenoloxidase responsible for generating the toxic para-benzoquinones in the stink glands of the beetle, we could identify laccase2, which is expressed in the last secretory cell in contact with the cuticule-lined organelle, where the toxic compounds are safely produced before being released into the gland reservoir. Author summaryCertain beetles produce toxic substances to defend themselves against microorganisms or predators. For the generation of such chemicals, the beetles have specialized glands, which are organized in a way that they can produce and store the toxic compounds without harming themselves. The morphology and biochemistry of such glands had been intensively studied. However, the genes that are involved in this process had not been recognized. Here we present the identification of about 500 genes that are involved in the function of beetle defensive glands, which presents a starting point for a detailed molecular understanding of the process of safe production of toxic substances. A key feature for the production is the use of non-harmful precursors that are only finally processed by a specifically secreted enzyme called Laccase2 to become toxic in a cuticle-shielded organelle.

genetics↗

bioRxiv2025

Optimizing dsRNA sequences for RNAi in pest control and research with the dsRIP Web-Platform

BackgroundRNA interference (RNAi) is a tool for studying gene function and has emerged as a promising eco-friendly alternative to chemical pesticides. RNAi relies on delivering double-stranded RNA (dsRNA), which is processed into small interfering RNA (siRNA) to silence genes. However, so far, knowledge and tools for optimizing the dsRNA sequences for maximum efficacy are based on human data, which might not be optimal for insects and pest control. ResultsHere, we systematically tested different siRNA sequences in the red flour beetle Tribolium castaneum to identify sequence features that correlated with high efficacy using pest control as a study case. Thermodynamic asymmetry, the absence of secondary structures, and Adenine at the 10th position in antisense siRNA were most predictive of insecticidal efficacy. Interestingly, we also found that in contrast to results from human data, high, rather than low GC content from the 9th to 14th nucleotides of antisense was associated with high efficacy. Consideration of these features for the design of insecticidal dsRNAs targeting essential genes in three insect species improved the efficacy of the treatment. The improvement was associated with a higher ratio of the antisense, rather than sense, siRNA strand bound to the RNA-induced silencing complex. Finally, we developed a web-platform named dsRIP (https://dsrip.uni-goettingen.de), which offers tools for optimizing dsRNA sequences, identifying effective RNAi target genes for pest control, and minimizing risk to non-target species. ConclusionsThe identified sequence features and the dsRIP web-platform allow optimizing dsRNA sequences for both application of RNAi for pest control and research.

zoology↗

bioRxiv2024

Differences in size and number of embryonic type-II neuroblast lineages are associated with divergent timing of central complex development between beetle and fly

Despite its conserved basic structure, the morphology of the insect brain and the timing of its development underwent evolutionary adaptations. However, little is known about the developmental processes that create this diversity. The central complex is a brain centre required for multimodal information processing and an excellent model to understand neural development and divergence. It is produced in large parts by type-II neuroblasts, which produce intermediate progenitors, another type of cycling precursor, to increase their neural progeny. These neural stem cells are believed to be conserved among insects, but little is known on their molecular characteristics in insects other than flies. Tribolium castaneum has emerged as a valuable new insect model for brain development and evolution. However, type-II neuroblast lineages and their role in central complex development have so far not been studied in this beetle. Using CRISPR-Cas9 we created a fluorescent enhancer trap marking expression of Tribolium fez/earmuff, a key marker for type-II neuroblast derived intermediate progenitors. Using combinatorial labelling of further markers including Tc-pointed, Tc-deadpan, Tc-asense and Tc-prospero we characterized the type-II neuroblast lineages present in the Tribolium embryo and their sub-cell-types. Intriguingly, we found 9 type-II neuroblast lineages per hemisphere in the Tribolium embryo while Drosophila produces only 8 per brain hemisphere. In addition, these lineages are significantly larger at the embryonic stage of Tribolium than they are in Drosophila and contain more intermediate progenitors. Finally, we mapped these lineages to the domains of early expressed head patterning genes. Notably, Tc-otd is absent from all type-II neuroblasts and intermediate progenitors, whereas Tc-six3 marks an anterior subset of the type-II-lineages. The placodal marker Tc-six4 specifically marks the territory where anterior medial type-II neuroblasts differentiate. In conclusion, we identified a conserved pattern of gene expression in holometabolan central complex forming type-II neuroblast lineages, and conserved head patterning genes emerged as new candidates for conferring spatial identity to individual lineages. The higher number and greater lineage size of the embryonic type-II neuroblasts in the beetle correlate with a previously described embryonic phase of central complex formation which is not found in the fly. These findings stipulate further research on the causal link between timing of stem cell activity and temporal and structural differences in central complex development.

developmental biology↗

bioRxiv2024

Effective target genes for RNA interference-based management of the cabbage stem flea beetle

The cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a key pest of oilseed rape. The ban on neonicotinoids in the European Union due to environmental concerns and the emergence of pyrethroid-resistant populations have made the control of CSFB extremely challenging. In search of a solution, we have recently shown that RNA interference (RNAi) has potential in the management of CSFB. However, the previously tested target genes for RNAi-mediated pest control (subsequently called target genes) exhibited moderate and slow-acting lethal effects. In this study, 27 double-stranded RNAs (dsRNAs) were orally delivered to identify highly effective target genes in CSFB adults by leveraging the findings of a genome-wide RNAi screen in Tribolium castaneum. Our screen using 500 ng of dsRNA identified 10 moderately effective (> 50% mortality) and 4 highly effective target genes (100% mortality in 8-13 days). The latter mainly included proteasome subunits. RT-qPCR experiments confirmed target gene silencing and dose-response studies revealed LD50 values as low as [~]20 ng in 14 days following a single exposure to dsRNA. Four highly effective dsRNAs also inhibited leaf damage (up to [~]75%) and one affected locomotion. The sequences of promising target genes were subjected to in silico target prediction in non-target organisms, e.g., beneficials such as honeybees, to design environmentally friendly dsRNAs. Overall, the study provides valuable insights for the development of dsRNA-based insecticides against CSFB.

zoology↗

bioRxiv2024

Superior target genes and pathways for RNAi mediated pest control revealed by genome wide analysis in the red flour beetle Tribolium castaneum

An increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi) based pesticides have emerged as new option with the first products entering the market. Essentially, double stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear, whether RNAi based insecticides should target the same pathways as classic pesticides or whether the different mode of action would favor other processes. Moreover, there is no consensus on the best genes to be targeted. We performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. GO and KEGG analyses of our genome wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides. In summary, our work revealed the best target genes and target processes for RNAi based pest control and we propose a procedure to transfer our short list of superior target genes to other pests.

genomics↗

bioRxiv2023

Temporal restriction of RNAi reveals breakdown of the segmentation clock is reversible after knock down of primary pair rule genes but not Wnt-signaling in the red flour beetle

Animals from all major clades have evolved a segmented trunk, reflected for instance in the repetitive organization of the human spine or the insect segments. These units emerge during embryonic segmentation from a posterior segment addition zone, where repetitive gene activity is regulated in a spatiotemporal dynamic described by the clock and wavefront/speed gradient model. This model has been tested in the red flour beetle Tribolium castaneum and other insects by studying the effect of the RNAi knockdown of segmentation genes. For upstream components such as primary pair rule genes, caudal or Wnt pathway components, this treatment often led to the breakdown of segmentation. However, it has remained untested, how the system would react to a temporally limited interruption of gene function. In order to ask such questions, we established a novel experimental system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi. We show that the T. castaneum segmentation machinery re-established after we blocked an ongoing RNAi response targeting the primary pair rule genes Tc-eve, Tc-odd and Tc-runt. However, we observed no rescue after blocking RNAi responses targeting Wnt pathway components. We conclude that the insect segmentation system contains both, robust feedback-loops that can re-establish and labile feedback loops that can breakdown irreversibly. This combination may reconcile two partially conflicting needs of the embryonic regulation of segmentation: A tightly controlled initiation and maintenance of the SAZ by labile feedback-loops ensures that only one segment addition zone is formed. Conversely, robust feedback-loops confer developmental robustness required for proper segmentation, which may be challenged by internal or external disturbances. Our results ponder the insect segmentation machinery from a different angle and introduce a new experimental tool for temporal control on RNAi. Significance statementThe generation of repetitive body parts during embryonic segmentation has been of key interest to developmental biologists, who usually used permanent knock-down of gene function for their studies. Using a new tool to temporally stop a gene knock-down effect, we find both robust and labile feedback-loops within the segmentation machinery. Thereby, the embryo may ensure that only one trunk is formed but that trunk formation is robust against external disturbance.

developmental biology↗

bioRxiv2021

An atlas of the developing Tribolium castaneum brain reveals conserved anatomy and divergent timing to Drosophila melanogaster

Insect brains are formed by conserved sets of neural lineages whose fibres form cohesive bundles with characteristic projection patterns. Within the brain neuropil these bundles establish a system of fascicles constituting the macrocircuitry of the brain. The overall architecture of the neuropils and the macrocircuitry appear to be conserved. However, variation is observed e.g., in size and shape and timing of development. Unfortunately, the developmental and genetic basis of this variation is poorly understood although the rise of new genetically tractable model organisms such as the red flour beetle Tribolium castaneum allows the possibility to gain mechanistic insights. To facilitate such work, we present an atlas of the developing brain of T. castaneum, covering the first larval instar, the prepupal stage and the adult, by combining wholemount immunohistochemical labelling of fibre bundles (acetylated tubulin) and neuropils (synapsin) with digital 3D reconstruction using the TrakEM2 software package. Upon comparing this anatomical dataset with the published work in D. melanogaster, we confirm an overall high degree of conservation. Fibre tracts and neuropil fascicles, which can be visualized by global neuronal antibodies like anti-acetylated tubulin in all invertebrate brains, create a rich anatomical framework to which individual neurons or other regions of interest can be referred to. The framework of a largely conserved pattern allowed us to describe differences between the two species with respect to parameters such as timing of neuron proliferation and maturation. These features likely reflect adaptive changes in developmental timing that govern the change from larval to adult brain.

neuroscience↗

bioRxiv2021

Identifying essential genes across eukaryotes by machine learning

Identifying essential genes on a genome scale is resource intensive and has been performed for only a few eukaryotes. For less studied organisms essentiality might be predicted by gene homology. However, this approach cannot be applied to non-conserved genes. Additionally, divergent essentiality information is obtained from studying single cells or whole, multi-cellular organisms, and particularly when derived from human cell line screens and human population studies. We employed machine learning across six model eukaryotes and 60,381 genes, using 41,635 features derived from sequence, gene functions and network topology. Within a leave-one-organism-out cross-validation, the classifiers showed a high generalizability with an average accuracy close to 80% in the left-out species. As a case study, we applied the method to Tribolium castaneum and validated predictions experimentally yielding similar performance. Finally, using the classifier based on the studied model organisms enabled linking the essentiality information of human cell line screens and population studies.

bioinformatics↗

bioRxiv2021

Large portion of essential genes is missed by screening either fly or beetle indicating unexpected diversity of insect gene function

Most gene functions were detected by screens in very few model organisms but it has remained unclear how comprehensive these data are. Here, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes and we compared the gene sets involved in several processes between beetle and fly. We find that around 50 % of the gene functions are detected in both species while the rest was found only in fly (~10%) or beetle (~40%) reflecting both technical and biological differences. We conclude that work in complementary model systems is required to gain a comprehensive picture on gene functions documented by the annotation of novel GO terms for 96 genes studied here. The RNAi screening resources developed in this project, the expanding transgenic tool-kit and our large-scale functional data make T. castaneum an excellent model system in that endeavor.

genetics↗

bioRxiv2020

Shaking hands is a putative terminal selector and controls axon outgrowth of central complex neurons in the insect model Tribolium

Individual cell types are specified by transcriptional programs which act during development. Gene regulatory mechanisms which specify subtype identity of central complex (CX) neurons are the subject of intense investigation. The CX is a compartment within the brain common to all insect species. The CX functions as a "command center" by initiating motor actions in response to incoming information. The CX is made up of several thousand neurons with more than 60 morphologically distinct identities. Accordingly, transcriptional programs must effect the specification of at least as many neuronal subtypes. Here we demonstrate a role for the transcription factor Shaking hands (Skh) in the specification of embryonic CX neurons in Tribolium. The developmental dynamics of Tc-skh expression are characteristic for terminal selectors of neuronal subtype identity. In the embryonic brain Tc-skh expression is restricted to a subset of neurons, many of which survive to adulthood and contribute to the mature CX. Tc-skh expression is maintained throughout the lifetime of the respective CX neurons. Tc-skh knock-down results in severe axon outgrowth defects thus preventing the formation of an embryonic CX primordium. The as yet unstudied Drosophila skh shows a similar embryonic expression pattern suggesting that subtype specification of CX neurons may be conserved.

developmental biology↗

bioRxiv2019

Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: a fly-beetle insight

Animal behavior is guided by the brain. Therefore, adaptations of brain structure and function are essential for animal survival, and each species differs in such adaptations. The brain of one individual may even differ between life stages, for instance as adaptation to the divergent needs of larval and adult life of holometabolous insects. All such differences emerge during development but the cellular mechanisms behind the diversification of brains between taxa and life stages remain enigmatic. In this study, we investigated holometabolous insects, where larvae differ dramatically from the adult in both behavior and morphology. As consequence, the central complex, mainly responsible for spatial orientation, is conserved between species at the adult stage, but differs between larvae and adults as well as between larvae of different taxa. We used genome editing and established transgenic lines to visualize cells expressing the conserved transcription factor retinal homeobox, thereby marking homologous genetic neural lineages in both the fly Drosophila melanogaster and the beetle Tribolium castaneum. This approach allowed us for the first time to compare the development of homologous neural cells between taxa from embryo to the adult. We found complex heterochronic changes including shifts of developmental events between embryonic and pupal stages. Further, we provide, to our knowledge, the first example of sequence heterochrony in brain development, where certain developmental steps changed their position within the ontogenetic progression. We show that through this sequence heterochrony, an immature developmental stage of the central complex gains functionality in Tribolium larvae. We discuss the bearing of our results on the evolution of holometabolous larval central complexes by regression to a form present in an ancestor.

evolutionary biology↗

bioRxiv2019

An ancestral apical brain region contributes to the central complex under the control of foxQ2 in the beetle Tribolium castaneum

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.\n\nSummary statementAn ancestral neuroendocrine center contributes to the evolution of the central complex. foxQ2 is a gene required for the development of midline structures of the insect brain, which distinguish protocerebrum from segmental ganglia.

developmental biology↗

bioRxiv2018

A large-scale systemic RNAi screen in the red flour beetle Tribolium castaneum identifies novel genes involved in arthropod muscle development

Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered ~8,500 and ~5,000 genes, respectively, of a total of ~16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.

developmental biology↗

bioRxiv2016

foxQ2 evolved a key role in anterior head and central brain patterning in protostomes

Anterior patterning of animals is based on a set of highly conserved transcription factors but the interactions within the protostome anterior gene regulatory network (aGRN) remain enigmatic. Here, we identify the foxQ2 ortholog of the red flour beetle Tribolium castaneum as novel upstream component of the insect aGRN. It is required for the development of the labrum and higher order brain structures, namely the central complex and the mushroom bodies. We reveal Tc-foxQ2 interactions by RNAi and heat shock-mediated misexpression. Surprisingly, Tc-foxQ2 and Tc-six3 mutually activate each other forming a novel regulatory module at the top of the insect aGRN. Comparisons of our results with those of sea urchins and cnidarians suggest that foxQ2 has acquired functions in head and brain patterning during protostome evolution. Our findings expand the knowledge on foxQ2 gene function to include essential roles in epidermal development and central brain patterning.\n\nAuthor summaryThe development of the anterior most part of any animal embryo - for instance the brain of vertebrates and the head of insects - depends on a very similar set of genes present in all animals. This is true for the two major lineages of bilaterian animals, the deuterostomes (including sea urchin and humans) and protostomes (including annelids and insects) and the cnidarians (e.g. the sea anemone), which are representatives of more ancient animals. However, the interaction of these genes has been studied in deuterostomes and cnidarians but not in protostomes. Here, we present the first study the function of the gene foxQ2 in protostomes. We found that the gene acts at the top level of the genetic network and when its function is knocked down, the labrum (a part of the head) and higher order brain centers do not develop. This is in contrast to the other animal groups where foxQ2 appears to play a less central role. We conclude that foxQ2 has acquired additional functions in the course of evolution of protostomes.

developmental biology↗