The red flour beetle as model insect for molecular studies on stink glands

2.3 The red flour beetle as model insect for molecular studies on stink glands The reasons for choosing T. castaneum to study stink glands on a molecular level are numerous and distinct. First, the red flour beetle is a ubiquitous pest of stored grain, flour, and other cereal products and prolific in developing resistances against insecticides (Brown et al., 2009).

Thus, understanding the molecular functioning of its defense mechanism potentially providing a new basis for pest control is of economic-ecological importance. Secondly, its genome has been fully annotated (Richards et al., 2008) and several genetic tools are available, e.g. highly efficient methods for transposon-based genetic transformation (Berghammer et al., 1999; Lorenzen et

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al., 2003, 2007), a GAL4/UAS expression system (Schinko et al., 2010), a heat-shock based misexpression system (Schinko et al., 2012) and, in contrast to the classical model insect Drosophila melanogaster, which does not possess stink glands, reversed genetics based on systemic RNAi response (Bucher et al., 2002; reviewed by Noh et al., 2012). Thus, in the last decade, T. castaneum has been evolved into a most sophisticated genetic model insect besides D. melanogaster.

Recently, the stink gland transcriptome of the red flour beetle has been published (Li et al., 2013) and a genome-wide RNAi knockdown screen called “iBeetle” (Schmitt-Engel et al., 2015) as well as a Gal4 enhancer trap screen based on insertional mutagenesis (Trauner et al., 2009) are current projects to identify gene functions for insect development and physiology as well as to develop cell-type specific markers and drivers for targeted expression of transgenes, respectively. All candidate genes for this study were obtained from these three genome-wide approaches, which are shortly introduced in the following three subsections.

2.3.1 Stink gland transcriptome

First transcriptome data on beetle stink glands were provided by Li et al. (2013). Samples for mRNA sequencing on a next generation sequencing platform were wildtype male prothoracic glands, female prothoracic glands, male abdominal glands, and female abdominal glands. In addition, prothoracic glands of tar mutant displaying melanotic gland secretions were selected (Beeman et al., 1996). As non-gland reference served anterior abdomen tissue including e.g.

muscle, gut, fat body, and cuticle. For each sample, about 28 million reads were obtained and half of them have been mapped to T. castaneum mRNAs of the official gene set in the BeetleBase (Kim et al., 2010; Wang et al., 2007). Comparative analysis of reads in stink gland and reference samples revealed 511 genes with differential expression in terms of gender, gland type and beetle strain (wildtype or mutant). From these, the authors functionally analyzed 77 genes that were at least 64x higher expressed in the glands compared to the reference tissue and identified three genes that are involved in benzoquinone synthesis in the beetle. Total reads for 16,645 official gene set numbers in every stink gland sample and the reference sample as well as corresponding calculated fold changes as index for gland specific differential expression,

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were kindly provided by Dr. Jianwei Li for this study (Li et al., 2013; SRP040606 in the public database Sequence Read Archive (SRA)).

2.3.2 The iBeetle screen

The iBeetle screen is an RNAi-based, genome-wide, large-scale, and unbiased approach to identify novel genes involved in insect developmental and physiological processes (Schmitt-Engel et al., 2015). The first screening phase, which has already been completed, included a pupal injection screen and a larval injection screen. In the pupal screen, female pupae were injected and analyzed for late metamorphosis phenotypes. In addition, their offspring was examined for embryological defects. In contrast, in the larval screen, dsRNA-injected female larvae were observed for defects in early metamorphosis and general morphological abnormalities that occurred during adulthood, in particular alterations in ovaries and stink glands. After the first screening phase, 4480 genes had been analyzed in the larval screen and 5300 genes in the pupal screen, with reliable results for 3400 genes in both injection screens (Schmitt-Engel et al., 2015).

2.3.3 A Gal4-based enhancer trap screen

A Gal4-based enhancer trap screen for the identification of cell-type specific markers and drivers for targeted (over)expression of (trans)genes in T. castaneum is currently performed by Elke Küster in the laboratory of Professor Dr. Gregor Bucher (Georg-August-University Göttingen). Similar to Trauner et al. (2009), a Gal4-piggyBac-transposon is allowed to jump by crossing a mutator strain with a helper line providing active transposase. Subsequent integration of a UAS-turboGFP reporter construct visualizes the establishment of a new enhancer trap. Details are given in Figure 4.

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Figure 4: Crossing scheme to establish Gal4-based enhancer trap lines. Mutator line beetles carry the Gal4-piggyBac construct marked with 3xP3-v (dark eyes) homozygously on autosome “A”. Beetles that have integrated the helper construct (M26 transposase) homozygously on the X-chromosome, are identified by red fluorescing eyes. In the P1 generation, the actual enhancer trap of the mutator line (Bauchbinde) is not visible, as the UAS-reporter is missing. A male mutator is crossed with a female helper. Their male progeny with red fluorescing dark eyes is collected, as they still carry one copy of the mutator transposon and now also one copy of the transposase construct. This is the generation in which the transposon can jump into a new position in the genome. To visualize a hopping event, a reporter is crossed-in by mating the male P1 progeny with virgins that carry an UAS-turboGFP construct on a different autosome “B” (P2). In the F1 generation, new insertions are detected. Beetles that still show the initial enhancer trap (Bauchbinde) are removed. Only male beetles are kept for raising a population, as they have lost the X-linked transposase. Finally, these male beetles are mated again with virgin females that carry the reporter construct to establish a stock. The scheme was kindly provided by Professor Gregor Bucher, Georg-August-University Göttingen.

2.4 Benzoquinones in beetle defensive secretions