Open Access

Frederic Strobl

• The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones: - Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events. - Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing. - Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context. - Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect. - Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues. - Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically. - Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines. My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably. Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology. During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.