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Heat shock-mediated misexpression of genes in the beetle Tribolium castaneum

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Abstract

Insect gene function has mainly been studied in the fruit fly Drosophila melanogaster because in this species many techniques and resources are available for gene knock down and the ectopic activation of gene function. However, in order to study biological aspects that are not represented by the Drosophila model, and in order to test to what degree gene functions are conserved within insects and what changes in gene function accompanied the evolution of novel traits, the establishment of respective tools in other insect species is required. While gene knock down can be induced by RNA interference in many insects, methods to misexpress genes are much less developed. In order to allow misexpression of genes in a timely controlled manner in the red flour beetle Tribolium castaneum, we have established a heat shock-mediated misexpression system. We show that endogenous heat shock elements perform better than artificial heat shock elements derived from vertebrates. We carefully determine the optimal conditions for heat shock and define a core promoter for use in future constructs. Finally, using this system, we study the effects of misexpressing the head patterning gene Tc-orthodenticle1 (Tc-otd1), We show that Tc-otd1 suppresses Tc-wingless (Tc-wg) in the trunk and to some degree in the head.

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Acknowledgments

We thank Dr. Thomas Czerny for the artificial heat shock elements, Dr. Fakrudin Bashasab for identifying the hsp68 promoter. Dr. Michael Schoppmeier and Dr. Jochen Trauner for help with the interpretation of the signal in the ovaries. We also thank Anna Gilles for help with in situ hybridizations and helpful discussions and Dr. Andrew D. Peel for language corrections.

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Authors and Affiliations

  1. Institute of Molecular Biology and Biotechnology, Nik. Plastira 100, 71110, Heraklion, Crete, Greece

    Johannes Benno Schinko

  2. Johann-Friedrich-Blumenbach Institute, Department of Developmental Biology, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany

    Kathrin Hillebrand & Gregor Bucher

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  1. Johannes Benno Schinko
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  2. Kathrin Hillebrand
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  3. Gregor Bucher
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Correspondence to Johannes Benno Schinko.

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Communicated by S. Roth

Johannes Benno Schinko and Kathrin Hillebrand equal contributors.

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ESM 1

Sequences of the constructs shown in Fig. 1 are provided in gb format in this file. (PDF 84 kb)

ESM 2

Heat shock-mediated lethality of pupae (a) and embryos (b). Pupae and embryos were heat shocked at 46°C/115°F and 48°C/118°F for 5, 10, 15, 20, 30, and 60 min. Pupal survival was scored by development into viable adult beetles whereas embryonic development was scored by development of a wildtype larva cuticle. At both developmental stages lethality was increased at the higher temperature. Whereas pupae tolerated a 60-min heat shock at 46°C/115°F with only slight decrease in viability (73% compared to 80% of unshocked), embryos were more sensitive (37% compared to 90% of unshocked). At 48°C/118°F pupal survival was strongly reduced if heat shocks were given for 20 min (53% survival). A heat shock for 60 min at this temperature caused death of all shocked pupae. At the embryonic stage a heat shock of 5 min at 48°C/118°F already decreased the survival by 20%. A 10-min heat shock reduced viability to 65% and longer heat shocks led to death of more than 50% of the embryos. (PDF 80 kb)

ESM 3

Heat shock induced misexpression of Tc-otd1. Heat shocked v w wildtype embryos, as well as heat shocked embryos of the transgenic line M3 (endoHSE::Tc-otd1) were fixed two hours after heat shock. As a control, non-heat shocked transgenic embryos of the same line and same stage were also fixed. Double whole mount in situ hybridization was performed with Tc-wg (blue) and Tc-otd1 (orange) antisense RNA probes. The v w embryos exhibited normal Tc-wg and Tc-otd1 expression (a–b′), as did the non-heat shocked transgenic embryos (c–d′). Two hours after heat shock Tc-otd1 was strongly expressed ubiquitously in transgenic embryos at a level similar to the endogenous expression (e–f). Expression of Tc-wg was still present in the trunk (e,f) 2 h after onset of misexpression of Tc-otd1. (PDF 170 kb)

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Schinko, J.B., Hillebrand, K. & Bucher, G. Heat shock-mediated misexpression of genes in the beetle Tribolium castaneum . Dev Genes Evol 222, 287–298 (2012). https://doi.org/10.1007/s00427-012-0412-x

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