Animals detect, recognize and localize relevant objects in noisy, multi-source environments. Female crickets locate potential mates in choruses of simultaneously calling males using acoustic signals, a behaviour termed phonotaxis. The mechanisms underlying cricket phonotaxis are now understood across multiple levels: biophysical, neurobiological and behavioural. Phonotaxis has, however, rarely been tested in the complex real-world acoustic environments and no attempts have been made to predict acoustic orientation behaviour in these conditions despite our extensive understanding of its underlying mechanisms. In this thesis, I first characterized the acoustic environments faced by female crickets of the species Plebeiogryllus guttiventris in the field. Phonotaxis behaviour of females was then characterized under laboratory conditions using two sound sources. The data obtained were used to develop a simulation that predicted this behaviour. The predictions of the simulation were then tested against the phonotaxis behaviour of females in realistic, multi-source conditions in the field. My field studies of male behaviour showed that males of this species produced complex and variable songs in choruses where multiple males called simultaneously. The acoustic ranges of males in these choruses overlapped extensively and females performing phonotaxis in such choruses would hear multiple males simultaneously. The acoustic interactions of simultaneously calling males were also characterized for their timing relationships with each other and the changes they made to the temporal patterns of their songs. Males did not either synchronise or alternate their chirps, however they made changes to the temporal patterns of song in a way that is likely to make them more attractive to females. I then characterized the closed-loop walking phonotaxis behaviour of P. guttiventris females in the presence of two active sound sources playing conspecific song. Both the baseline and relative SPLs of the two speakers were systematically varied and female phonotactic paths were obtained. Females were found to preferentially approach louder songs. Several aspects of this behaviour were characterized, in particular orientation ability and motor behaviour under varied conditions of stimulus intensity. A stochastic simulation of closed-loop walking phonotaxis behaviour was developed using both current understanding of field cricket physiology and my data on closed-loop walking phonotaxis. The simulation was demonstrated to both qualitatively and quantitatively recapture female behaviour. It was also able to qualitatively recapture female behaviour in two previously published classical experiments in which the hearing of female crickets was disrupted. Female phonotaxis was then tested under real-world multi-source conditions. The behaviour of real females was compared to the predictions of the simulation. The simulation was found to recapture both female preference and phonotactic path forms at the population level. To my knowledge, this is the first study to both examine and successfully predict phonotaxis behaviour in complex real-world acoustic conditions.

Sound source localization depends on the auditory system to identify, recognize, and segregate elements of salient sources over distracting noise. My research investigates sensory mechanisms involved in these auditory processing tasks of an insect hearing specialist, to isolate individual sound sources of interest over noise. I first developed quantitative methods to determine signal features that the acoustic parasitoid fly Ormia ochracea (Diptera: Tachinidae) evaluate for host cricket song recognition. With flies subjected to a no-choice paradigm and forced to track a switch in the broadcast location of test songs, I describe several response features (distance, steering velocity, and angular orientation) that vary with song pulse rate preferences. I incorporate these response measures in a phonotaxis performance index that is sensitive to capturing response variation that may underlie song recognition. I demonstrate that Floridian O. ochracea exhibit phonotaxis to a combination of pulse durations and interpulse intervals that combine to a range of accepted pulse periods. Under complex acoustic conditions of multiple coherent cricket songs that overlap in time and space, O. ochracea may experience a phantom source illusion and localize a direction between actual source locations. By varying the temporal overlap between competing sources, I demonstrate that O. ochracea are able to resolve this illusion via the precedence effect: exploitation of small time differences between competing sources to selectively localize the leading over lagging sources. An increase in spatial separation between cricket song and masking noise does not reduce song detection thresholds nor improve song localization accuracy. Instead, walking responses are diverted away from both song and noise. My findings support the idea that the ears of O. ochracea function as bilateral symmetry detectors to balance sound intensity, sound arrive time differences, and temporal pattern input to both sides of the auditory system. Asymmetric acoustic input result in corrective turning behaviour to re-establish balance for successful source localization.

Sound source localization depends on the auditory system to identify, recognize, and segregate elements of salient sources over distracting noise. My research investigates sensory mechanisms involved in these auditory processing tasks of an insect hearing specialist, to isolate individual sound sources of interest over noise. I first developed quantitative methods to determine signal features that the acoustic parasitoid fly Ormia ochracea (Diptera: Tachinidae) evaluate for host cricket song recognition. With flies subjected to a no-choice paradigm and forced to track a switch in the broadcast location of test songs, I describe several response features (distance, steering velocity, and angular orientation) that vary with song pulse rate preferences. I incorporate these response measures in a phonotaxis performance index that is sensitive to capturing response variation that may underlie song recognition. I demonstrate that Floridian O. ochracea exhibit phonotaxis to a combination of pulse durations and interpulse intervals that combine to a range of accepted pulse periods. Under complex acoustic conditions of multiple coherent cricket songs that overlap in time and space, O. ochracea may experience a phantom source illusion and localize a direction between actual source locations. By varying the temporal overlap between competing sources, I demonstrate that O. ochracea are able to resolve this illusion via the precedence effect: exploitation of small time differences between competing sources to selectively localize the leading over lagging sources. An increase in spatial separation between cricket song and masking noise does not reduce song detection thresholds nor improve song localization accuracy. Instead, walking responses are diverted away from both song and noise. My findings support the idea that the ears of O. ochracea function as bilateral symmetry detectors to balance sound intensity, sound arrive time differences, and temporal pattern input to both sides of the auditory system. Asymmetric acoustic input result in corrective turning behaviour to re-establish balance for successful source localization.

Akustische Signale dienen vielen Tierarten als Mittel zur Partnerfindung. Diese Tiere müssen ihre Entscheidung für den besten Paarungspartner durch die Integration der Parameter verfügbarer Signale treffen. Das Wahlverhalten weiblicher Grillen basiert auf der Attraktivität des zeitlichen Musters des männlichen Gesangs und auf dessen Intensität. Diese Eigenschaften korrelieren nicht zwangsweise positiv, daher wird sich kein Männchen in einer Aggregation von singenden Männchen als überragend abheben und somit wird dem Weibchen die Entscheidung erschwert. Die vorliegende Arbeit beschäftigt sich mit der Frage wie die relevanten Merkmale des männlichen Gesangs während der Entscheidungsfindung des Weibchens integriert werden. 6 Arten von Feldgrillen wurden in nicht-Wahl- und Wahlexperimenten hinsichtlich ihrer Antworten und Präferenzen für männliche Signale, welche sich in Pulsrate, Modulationstiefe, Intensität, Anordnung der Chirps während der Wiedergabe und der zeitlichen Verschiebung zweier Signale zueinander unterschieden, getestet. Des Weiteren wurde durch Transitivitätstests untersucht, ob dem Wahlverhalten weiblicher Grillen rationale oder vergleichende Entscheidungsmechanismen zugrunde liegen. Zusammenfassend zeigen die Ergebnisse, dass die Verarbeitungsmechanismen, welche der Entscheidungsfindung bei weiblichen Feldgrillen zugrunde liegen, in den untersuchten Arten ähnlich sind. Die Lokalisierung des Signals ist nicht unabhängig von der Erkennung. Neben einem generellen Verarbeitungsschema wurden zwischen den einzelnen untersuchten Arten charakteristische Unterschiede in der Mustererkennung, der Verarbeitung der Signalintensität und der Intensitätsgewichtung festgestellt. Jedoch wiesen nah verwandte Arten ähnlichere Präferenzen auf, als nicht nah verwandte Arten. Die Tests zur Transitivität der Präferenzen weisen auf vergleichende Entscheidungs-mechanismen hin und widersprechen einer rationalen Partnerwahl. / In many animals acoustic signals serve for mate attraction. They thus need to make a decision about which is the best mating partner in a choice situation by integrating the cues of the available signals. Choice behaviour in female crickets is based on the attractiveness of the temporal pattern of a male’s calling song and on its intensity. The parameters of a calling song are not necessarily positively correlated and no male will emerge as superior in an aggregation of singing males. The present thesis addresses the question on which cues decision making is based and how these relevant cues of a male’s song are integrated during the decision process of a female. To this aim no-choice and choice experiments with 6 species of field crickets were conducted. Experiments systematically varied pulse rate, modulation depth, intensity, chirp/trill arrangement and temporal shifts of synchronously presented signals. Furthermore, tests for transitivity of preferences examined if female choice behavior is based on rational or comparative decision making mechanisms. In summary, the results reveal that sensory processing underlying female decisions is similar in the 6 species studied here. Incoming signals are analyzed separately in bilaterally paired networks with parallel pathways for signal attractiveness and signal intensity. A gain-control mechanism fuses the outcome of both pathways and signal intensity is weighted by pattern attractiveness. Thus localization is not independent from signal recognition. Despite this general scheme remarkable characteristic differences between species were observed in pattern recognition, processing of signal intensity and weighting of signal intensity. Closely related species exhibited more similar preferences than unrelated species. Furthermore the tests for transitivity of preferences indicated that females use comparative decision making mechanism and contradicted rational mate choice.

Interest in the question of when and how species recognition and mate preferences emerge in animals with strong species-typical predispositions has faded since the time of the classical ethologists. In its place, the role of plasticity has surfaced as a central emphasis in the study of animal behavior. Here, I step back and examine the origin and execution of sexual behavior in a tropical frog for which auditory predispositions are key. These experiments challenge assumptions about behavioral development, auditory perception, and stereotyped behavior. First, I illustrate when and how a sex- and species-typical behavior—conspecific phonotaxis—emerges during development. This study demonstrates that phonotaxis, presumably restricted to mature females, is present in both sexes early in postmetamorphic development—potentially long before such behavior might serve an adaptive function. I place this result in the context of hypotheses regarding the development of learned versus non-learned behaviors, and in light of the potential for perception to be altered by physiological changes occurring concomitantly with ontogeny. Next, I describe a set of dynamic mate choice studies that highlight how decision-making in a relatively simple system is more flexible, and less stereotyped, than was previously assumed. Results here show that frogs temporally update their mate choice decisions in a moment-to-moment fashion as advertisement signals change in real time. By decomposing the decision-making process, I determine the stimulus parameters essential for commitment to an initial phonotactic approach. These studies are followed up by experiments that reveal a high level of individual variation in female choosiness during mate choice. Lastly, I describe a mate choice study that revealed categorical perception in frogs, the first “lower” vertebrate now known to exhibit a perceptual mode previously considered a hallmark of “higher” organisms. Collectively, I make the following arguments: (1) constraints on sensory systems play a larger role in shaping behavior than is generally appreciated, irrespective of the involvement of learning; (2) unstudied sources of variation may contribute significantly to the raw material for sexual selection; and (3) phonotaxis in anurans amphibians is not the simple, stereotyped behavior that has been suggested of it in the past. / text

Male crickets produce a species specific song to attract females which in response move towards the sound source. This behaviour, termed phonotaxis, has been the subject of many morphological, neurophysiological and behavioural studies making it one of the most well studied examples of acoustic communication in the animal kingdom. Despite this fact, the precise leg movements during this behaviour is unknown. This is of specific interest as the cricket’s ears are located on their front legs, meaning that the perception of the sound input might change as the insect moves. This dissertation describes a methodology and an analysis that fills this knowledge gap. I developed a semi-automated tracking system for insect motion based on commercially available high-speed video cameras and freely available software. I used it to collect detailed three dimensional kinematic information from female crickets performing free walking phonotaxis towards a calling song stimulus. I marked the insect’s joints with small dots of paint and recorded the movements from underneath with a pair of cameras following the insect as it walks on the transparent floor of an arena. Tracking is done offline, utilizing a kinematic model to constrain the processing. I obtained, for the first time, the positions and angles of all joints of all legs and six additional body joints, synchronised with stance-swing transitions and the sound pattern, at a 300 Hz frame rate. I then analysed this data based on four categories: The single leg motion analysis revealed the importance of the thoraco-coxal (ThC) and body joints in the movement of the insect. Furthermore the inside middle leg’s tibio-tarsal (TiTa) joint was the centre of the rotation during turning. Certain joints appear to be the most crucial ones for the transition from straight walking to turning. The leg coordination analysis revealed the patterns followed during straight walking and turning. Furthermore, some leg combinations cannot be explained by current coordination rules. The angles relative to the active speaker revealed the deviation of the crickets as they followed a meandering course towards it. The estimation of ears’ input revealed the differences between the two sides as the insect performed phonotaxis by using a simple algorithm. In general, the results reveal both similarities and differences with other cricket studies and other insects such as cockroaches and stick insects. The work presented herein advances the current knowledge on cricket phonotactic behaviour and will be used in the further development of models of neural control of phonotaxis.

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.

The evolution of isolating mechanisms within a species population impedes gene flow. This allows isolated populations to diverge along different trajectories, which may ultimately lead to the formation of new species. Our attempts to understand the evolution of isolating barriers have benefited enormously from studies of divergent populations that are still recognized as members of the same species. The co-occurrence of five acoustically distinct populations of the bushcricket Mecopoda elongata in south India provided us with the opportunity to study one such divergence of sympatric populations of a single species. In sympatric populations that share identical ecology, sexual selection has the potential to play a prominent role in the maintenance of reproductive isolation. Based on a previous traditional morphometric study, Mecopoda elongata in India were thought to be a morphologically indistinguishable cryptic species complex. The lack of morphological divergence suggests a less significant role of ecology in the divergence of the group. One possibility is that songtypes may be maintained by the preference of Mecopoda elongate females for mating with a specific songtype. In this thesis I show that female phonotaxis to their ‘own’ call has the potential to contribute to behavioural isolation among the songtypes and in particular between two songtypes with overlapping temporal call parameters. This finding is supported by an independent no-choice mating experiment utilizing the same two songtypes. To investigate the cues other than song that Mecopoda elongata females’ may use to exercise preference for their own type, I examined the composition of cuticular lipids in the cuticle and the detailed structure of secondary sexual characters. I was able to differentiate all Mecopoda elongata songtypes with high probability based on CHC profiles and geometric morphometrics of the sub genital plate and cerci. My study reveals that divergence in sexual traits other than acoustic signals, although dramatically less obvious in nature, is present among Mecopoda elongata populations. This provides potential mechanisms for premating isolation among Mecopoda elongata songtypes in the wild suggesting that reproductive isolation is maintained by female preferences for male sexual signals. Additionally, I discovered a parasitoid Tachinid fly responsible for infecting three different songtypes of Mecopoda elongata, namely Double Chirper, Two Part and Helicopter. This Tachinid fly appears to have specialized hearing organ to track down calling Mecopoda elongata males throwing light on potential selection pressure and possible mechanism for Mecopoda elongata song divergence.

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.