|dc.description.abstracteng||Defense strategies are common in insects including Coleoptera, the largest animal taxon containing various types of beetles. Stink gland secretion of beetles is playing an important role in their defense against detrimental factors in the environment. These glands produce and release defensive secretions containing several specific varieties of substituted benzoquinone compounds. These defensive chemicals act toxic, repellent and as pesticides. Beetles respond to invaders, parasitic microbes and predators with the secretion of defensive substances. Particularly, these defensive substances of beetle performs several functions such as boiling bombardment or as surfactants against many life-threatening organisms. The defensive compounds of beetles are biosynthesized and stored in a specialized storage and secretory organ called odoriferous defensive stink glands. Tribolium castaneum (Coleoptera: Tenebrionidae) produces p-benzoquinones and 1-alkenes in stink glands. Morphology of tenebrionid beetles stink gland has been characterized in detail in the past, but not much is known about the genes that are important for the defensive substance production in the stink glands. Understanding molecular mechanisms of the stink gland development and unveiling the metabolic pathway, its regulation, and the enzymes participating in synthesis of defensive chemicals are essential to understand the self-protected mechanism in synthesis of toxic compounds. In the present study, identification and characterization of novel genes playing key roles in the protected biosynthesis of quinones in stink glands of the T. castaneum is done by following two genome-wide approaches: 1) tissue specific transcriptomics based on RNA-seq, and 2) genome-wide phenotypic screen based on RNAi-mediated gene silencing. Gland-specifically expressed genes and genes causing a gland-specific knockdown phenotype were analyzed by GC-MS to uncover functions in benzoquinone synthesis. Four such identified candidates genes being part of sulfate metabolism, carbohydrate sulfotransferase 5 (CHST5), arylsulfatase B (ARSB), sulfate modifying factor-1 (SUMF1) and sodium independent sulfate transporter (SLC26A11) were then characterized in detail.
In the first part of the study, the RNAi data of the 1st and 2nd phases of the iBeetle screen were analyzed and 130 genes were identified having a potential role in stink gland biology. However, in the rescreen only 69 genes were confirmed. In transcriptomic data, previously 77 genes were identified to be specifically highly expressed in the stink glands. Functional analysis showed that 29 genes are necessary for stink gland function. Importantly, in comparative analysis of the different functional genomics approaches such as differential expression in transcriptomic data and phenotypic screen only revealed one common candidate gene, which suggests that one approach is not sufficient to uncover the majority of genes that play an essential role in stink gland biology. Our findings suggest that a combination of functional genomic approaches are necessary to uncover genes essential for stink gland development. Particularly, phenotypic screens and transcriptomics approaches complement each other in functional genomics of defensive stink gland physiology.
In the second part of the study, RNAi-mediated gene silencing screening of 4748 genes from the 3rd phase of iBeetle screen was employed to uncover genes essential for development, gland morphological changes and physiology of stink glands. The main purpose of the 3rd phase screen were to identify lethal genes but additional screens were added to identify genes with function in different biological processes. Particularly, I used this screen to uncover further genes essential for morphologyical changes and physiology of stink glands. In this screen, 178 genes were identified to be essential for morphology alterations and changes in gland volatile composition. Gene ontology analysis demonstrated that the majority of these genes encodes for enzymes, regulator/receptor binding, transcription factors, receptors, transporters and 40% with unknown function. From this screen one gene, CHST5, that has been analyzed in this study is involved in sulfate conjugation of toxic compounds in the self-protection mechanism. To get a more comprehensive insight into stink-gland function, we also re-analyzed a gland-specific transcriptomic dataset, which was generated in 2013 by Li et al. The very recently assembled gene set reference of Tribolium (OGS3) allowed us to increase the mapping rates by about 30% compared to the initial analysis. 33 transcripts from the new analysis were not detected previously, since they were only newly annotated in the current version of the T. castaneum genome. Since they are very highly expressed in the Tribolium gland tissue compared to the control sample, it is definitely worth to analyze these genes in more detail on a functional level.
In the third part of the study, a detailed characterization of a set of newly identified genes with a role in protected biosynthesis of benzoquinone in odoriferous stink glands of the red flour beetle were performed. Especially CHST5, ARSB, SUMF1 and SLC26A11 were selected and characterized in depth. ARSB was selected from the study of Li et al. (2013), SUMF1 and SLC26A11 from the 2nd phase and CHST5 from the 3rd phase of iBeetle screen on the basis of strongly altered gland phenotypes and differential expression. Sulfate conjugation is used by many insect for detoxification of phenolic compounds. However, sulfate role in stink gland was not identified before. Sulfonation is used by some insects to neutralize plant defensive substances. On the basis of stink gland transcriptome and iBeetle screen data, we studied the function of CHST5, ARSB, SUMF1 and SLC26A11 via RNAi-mediated gene knockdowns, qPCR, GC-MS, LC-MS and in situ hybridization. LC-MS analysis showed presence of sulfate precursors i.e., sulfated glycosylated phenolic precursors in the knockdown situation of the sulfatase. Put together, these studies suggest that these genes play an impotant role in the self-protected biosynthesis of benzoquinone in the red flour beetle stink glands.||de