Ziel der vorliegenden Dissertation war es, zum Verständnis der genetischen Grundlagen des akustischen Kommunikationssystems der Grille Gryllus bimaculatus beizutragen (s. auch 1.2). Dazu wurde die Expression eines Orthologs des no-on-transientA-Gens (nonA) mit Hilfe der RNA-Interferenz-Methode spezifisch herunterreguliert. Bei nonA handelt es sich um ein vielversprechendes Kandidatengen, da Punktmutationen in der codierenden Region des Gens die Eigenschaften des männlichen Balzgesangs bei Drosophila melanogaster beeinflussen. Zudem belegen Gentransfer-Experimente bei Drosophila, dass dieses Gen artspezifische Informationen des Balzgesangs enthält. Die Analyse der Gesangsdaten ergab, dass sich die Periodenlänge durch das Herunterregulieren von NONA nicht verändert. Außerdem konnte gezeigt werden, dass nonA-dsRNA-injizierte Tiere seltener 3-silbige Chirps produzieren, dafür aber mehr 4- und 5-silbige Chirps. Die Auswertung der tageszeitlichen Gesangsaktivität zeigte, dass alle Tiere signifikant am häufigsten im ersten Nachtquartal (nach Erlöschen der Beleuchtung) zirpten. Ein Effekt durch das Herunterregulieren von NONA konnte statistisch nicht belegt werden. Allerdings schien es einen Trend bei nonA-dsRNA-injizierten Tieren zu geben, gleichmäßiger über den Tag verteilt Gesangsaktivität zu zeigen. Transgene Drosophila melanogaster, deren arteigenes nonA durch das der Grille ersetzt bzw. ergänzt worden war, zeigten durchweg eine verbesserte Überlebensfähigkeit (Steigerungen zwischen 27 und 340%). Auch das positiv phototaktische Verhalten wurde durch das Grillen-NONA bei allen transformanten Fliegen verstärkt; allerdings fiel dieser Effekt eher marginal aus. Dennoch kann durchaus von einer zumindest teilweisen funktionellen Konservierung des nonA-Gens zwischen Gryllus bimaculatus und Drosophila melanogaster ausgegangen werden. / The present thesis aims to widen our understanding of the genetic background of the acoustic communication system of the cricket Gryllus bimaculatus (see also 1.2). Therefore the expression of an ortholog of the no-on-transientA (nonA) gene was specifically inhibited via RNA-interference. The nonA gene is one of the most interesting candidate genes in this context, as point mutations in the coding region of the gene affect the characteristics of the male’s calling song. Furthermore, gene transfer experiments in Drosophila showed that this gene obviously carries species-specific song information. The analysis of the calling song of nonA-RNAi-treated crickets, revealed that the duration of the syllable period was not influenced by the “knock-down” of the gene, but that the inhibition had a certain impact on the maximum number of syllables per chirp, as nonA-dsRNA-injected crickets produced significantly less 3-syllable chirps and significantly more 4- and 5-syllable chirps. Differences in the daytime calling activity between nonA-dsRNA-injected crickets and control groups could not be verified. The calling activity of all groups reached its peak in the first quarter of the night and significantly differed from the low calling activity during the remaining quarters of the day. Although the activity of all animals reached its peak during the first quarter of the night, there seems to be a trend that this rhythmical behaviour was less pronounced in nonA-dsRNA-injected crickets. Drosophila melanogaster mutants, which had been transformed with the nonA ortholog of Gryllus bimaculatus, increased their survival by 27% to 340%. In addition, the positive phototactic behaviour was slightly increased in all tested animals - this effect, however, remained marginal. Nevertheless, the nonA gene seems to be at least partly functionally conserved between Gryllus bimaculatus and Drosophila melanogaster.

This thesis describes work from both the University of Cambridge in the lab of Berthold Hedwig and from the HHMI Janelia Research Campus in the lab of Michael Reiser. At the University of Cambridge, my work involved the development and demonstration of a method for electrophoretically delivering dyes and tracers for anatomical and functional imaging into animals that are not amenable to genetic labelling techniques. Using this method in locusts and crickets - model systems of particular interest for their acoustic communication - I successfully delivered polar fluorescent dyes and tracers through the sheath covering the auditory nerve, simultaneously staining both the peripheral sensory structures and the central axonal projections without destroying the nerve's function. I could label neurons which extend far from the tracer delivery site on the nerve as well as local neuron populations through the brain's surface. I used the same method to deliver calcium indicators into central neuropils for in vivo optical imaging of sound-evoked activity, as well as calling song-evoked activity in the brain. The work completed at the Janelia Research Campus began with the development of a modern version of a modular LED display and virtual reality control system to enable research on the visual control of complex behaviors in head-fixed animals. The primary advantages of our newly developed LED-based display over other display technologies are its high-speed operation, brightness uniformity and control, precise synchronization with analog inputs and outputs, and its ability to be configured into a variety of display geometries. Utilizing the system's fast display refresh rates, I conducted the first accurate characterization of the upper limits of the speed sensitivity of Drosophila for apparent motion during flight. I also developed a flexible approach to presenting optic flow scenes for functional imaging of motion-sensitive neurons. Finally, through the on-line analysis of behavioral measures, image rendering, and display streaming with low latency to multi-color (UV/Green) LED panels, I demonstrated the ability to create more naturalistic stimuli and interactive virtual visual landscapes. Lastly, I used this new visual display system to explore a newly discovered cell-type that had been implicated in higher-order motion processing from a large genetic screen of visually-guided behavior deficits. Using genetic silencing and activation methods, and by designing stimuli that modeled the optic flow encountered during different types of self-motion, colleagues in the Reiser lab and I showed that this cell-type - named Lobula Plate Columnar 1 (LPC1) - is required for the stopping behavior of walking flies caused by back-to-front translation motion but is not involved in the rotational optomotor response. Using calcium imaging, I found that LPC1 was selectively excited by back-to-front motion on the eye ipsilateral to the neuron population and inhibited by front-to-back motion on the contralateral eye, demonstrating a simple mechanism for its selectivity to translation over rotation. I also examined an anatomically similar cell type - named Lobula-Lobula Plate Columnar type 1 (LLPC1) - and found that its selectivity results from a similar but opposite calculation for the detection of front-to-back translational motion. The detection of back-to-front motion had previously been hypothesized to be useful for collision avoidance, and this work provides a neural mechanism for how this detection could be accomplished, as well as providing a platform from which to explore the larger network for translation optic flow.

This thesis represents a comprehensive examination of the phonotactic behaviour (i.e. attraction to sound) of the female Gryllus bimaculatus under laboratory conditions. Chapter 2 is the first study to analyze the effect of substrate texture on walking performance in crickets. Substrate texture is found to play an essential role in the phonotactic responses of G. bimaculatus. Smooth substrate texture has a detrimental effect due to slipping, whereas a rough texture results in optimal walking performance due to the friction with the walking legs. Chapter 3 represents the first detailed lifetime study analysing phonotaxis in crickets. My results demonstrate that the optimal age to test phonotaxis in G. bimaculatus females is from day 7 to 24 after the final moult. I also found that selectiveness was persistent with age. These findings contradict the female choosiness hypothesis. This study is also the first to describe the effect of senescence on phonotaxis in insects, as responsiveness decreases with age. Chapter 4 compares the phonotactic behaviour of female crickets from different laboratory-bred colonies. From six tested cricket lab colonies, I found three groups statistically different from each other. Females raised under laboratory conditions at the University of Cambridge and Anglia Ruskin University were most reponsive at a frequency of 4.5 kHz, whereas females bred in Tokushima University in Japan were tuned towards a higher frequency of 5 kHz. These results suggest a degree of artificial allopatric speciation. Comparisons with crickets bred under low-quality conditions in a local pet shop demonstrate a loss of responsiveness, indicating that breeding conditions have a direct effect on phonotactic responsivity. Chapter 5 is the first study to report the presence of phonotaxis in males of G. bimaculatus. Previously it was unknown if G. bimaculatus males were able to perform phonotaxis, given that they were only recognised as endurance signal producers. In the present study, only 20% of the studied males (N=70) performed a weak phonotactic response. This finding has potential ecological implications in terms of male cricket territory establishment, and male-male interactions in the wild, which are discussed. Chapter 6 explores the song pattern recognition of the female G. bimaculatus by changing the duration of either the first, second or third pulse of the chirps. A long first pulse decreased the phonotactic response whereas phonotaxis remained strong when the third pulse was long. Chirps with three pulses of increasing duration of 5, 20 and 50 ms elicited phonotaxis, but the chirps were not attractive when played in reverse order. The data are in agreement with a mechanism in which processing of a sound pulse has an effect on the processing of the subsequent pulse, as outlined in the flow of activity in a delay-line and coincidence-detector circuit.