|dc.description.abstracteng||Chemical warfare is a very common defense strategy in the insect world. A broad range of coleopteran beetles react to predators, invaders, and parasitic microbes with the release of toxic and repellent substances, which are synthesized in specialized secretory organs, referred to as odoriferous defensive stink glands. The worldwide known pest beetle Tribolium castaneum (Coleoptera: Tenebrionidae) uses these glands to produce antimicrobial p-benzoquinones and 1-alkenes. The stink gland morphology has been studied in detail in other tenebrionid beetles in the past, but to date only little is known about the genes involved in the biochemical processes of gland secretion production.
Here, I present first transmission electron microscopic pictures of T. castaneum stink gland cells and a subset of genes required for proper benzoquinone production. I selected 59 potential candidates from a genome-wide RNAi knockdown screen named “iBeetle” and 12 genes based on the analysis of 11 stink gland-related Gal4 enhancer trap lines. All 71 candidate genes were functionally characterized by RNAi-mediated gene knockdown. 34 of them were found to cause stink gland phenotypes upon knockdown and were therefore selected for a subsequent gas chromatography-mass spectrometry (GC-MS) analysis of secretion volatiles in respective RNAi knockdown glands. In total, I identified 14 candidate genes that displayed strongly altered glands in terms of secretion color and chemical composition upon knockdown, revealing their involvement in secretion biosynthesis. Three of them play an essential role specifically in benzoquinone biosynthesis. Based on stink gland transcriptome data, I additionally analyzed the function of four glucosidases, eleven peroxidases, as well as four phenoloxidases via RNAi-mediated gene knockdowns. Morphologically altered stink glands were found for one peroxidase (Peroxiredoxin 3, Prxd3) and two phenoloxidases (Tyrosinase 1, Tyr1; Laccase 2, Lac2). Subsequent GC-MS analysis of secretion volatiles revealed reduced benzoquinone levels in Prdx3 and Tyr1 knockdowns and a complete lack of benzoquinones in the knockdown of Lac2. For Lac2, I could detect its expression in a certain subgroup of secretory stink gland cells and confirm its involvement in p-benzoquinone biosynthesis by enzymatic activity assays on stink gland tissue. This indicates a so far unidentified role of the cuticle tanning phenoloxidase Lac2.||de