Genome-wide identification of genes involved in beetle odoriferous defensive stink gland function recognizes Laccase2 as the phenoloxidase responsible for toxicpara-benzoquinone synthesis
Authors: Atika Bibi , Lehmann Sabrina , Buchberger Elisa , Dan’azumi Isah Musa , Basirkazerouni Zahra , Rostás Michael , Bucher Gregor , Wimmer Ernst A. 
Published: January 2025

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 iBeetle’s 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 toxicpara-benzoquinones in the stink glands of the beetle, we could identifylaccase2,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 summary

Certain 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.