Fundamental Efforts to Develop Novel Biotechnological Approaches in Pest Management Applications against Coleoptera: Transcriptomic Exploration of the Chemical Defense Mechanism in the Red Flour Beetle, Tribolium castaneum

by Jianwei Li
Doctoral thesis
Date of Examination:2013-01-24
Date of issue:2013-04-24
Advisor:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Gregor Bucher
Referee:Prof. Dr. Reinhard Schuh
Referee:Prof. Dr. Andreas Stumpner
Referee:Prof. Dr. Ralf Heinrich
Referee:Dr. Roland Dosch
Persistent Address: http://dx.doi.org/10.53846/goediss-3808

 

 

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Abstract

English

Chemical defense is one of the most important traits, which endow insects with the ability to conquer diverse ecological environments. Chemical secretions are used for defense against anything from vertebrate or invertebrate predators to prokaryotic or eukaryotic parasites or food competitors. Tenebrionid beetles are especially prolific in this category, producing several varieties of substituted benzoquinone compounds. Better understanding of the genetic and molecular basis of defensive systems will not only answer fundamental biological questions, but also inspire the development of novel methods for pest control.  To do so, I performed RNA sequencing in a newly emerging insect model, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae). The odoriferous gland tissues that secret defensive chemical compounds were compared to a control tissue, the anterior abdomen, to detect genes that are highly and specifically expressed in the different glands. A total of 511 genes were identified in different subtraction groups. Of these, 77 genes were functionally analyzed by RNA interference (RNAi) to recognize induced gland alterations morphologically or changes in gland volatiles by gas chromatography-mass spectrometry. 29 genes (38%) presented strong visible phenotypes, while 67 genes (87%) showed alterations of at least one gland content. Three of these genes showing quinone-less (ql) phenotypes – Tcas-ql VTGl; Tcas-ql ARSB; Tcas-ql MRP – were isolated, molecularly characterized, their expression identified in both types of the secretory glandular cells, and their function determined by quantification of all main volatile components after RNAi. In addition, microbe inhibition assays revealed that a quinone-free status is unable to impede bacterial or fungal growth. Phylogenetic analyses of these three genes indicate that they have evolved independently and specifically for chemical defense in beetles. Another major content of the glands is represented by alkenes. In order to identify the alkene biosynthetic mechanisms, the fatty acid profile was explored in glands and different developmental stages of Tribolium castaneum with the fatty acid metabolism related genes being annotated, and their relative transcriptomic expression levels being investigated. Further characterization of three candidate genes isolated two desaturases with in vivo tested activities and one novel gene (Tcas-al P450) with alkene-less RNAi phenotype, which are very rare in nature and have the potential to be applied to produce both fuels and chemicals in industry. The alkene-less gene function was confirmed by the quantification of the main volatiles in the glands, its specific and independent evolution by phylogenetic analysis, and its particular expression in only one type of secretory glandular cells by fluorescent in situ hybridization. Additionally, the phenoloxidase activity tests of the quinone-less and alkene-less genes suggested that the chemical defense system might be linked with innate immunity in Tribolium castaneum.  All the data obtained in this thesis bring the chemical defensive secretion in Tribolium castaneum to a molecular level for the first time, which opens a new biological research field and sheds light on many future studies.
Keywords: odoriferous glands; stink glands; chemical defense; defensive secretion; defense mechanism; quinone secretion; alkene secretion; P450; decarboxylase; desaturase; coleopteran; insect; Tribolium
 
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