Investigating Perception Under Dynamic Auditory Conditions in the Acoustic Parasitoid Fly Ormia Ochracea

Investigating Perception Under Dynamic Auditory Conditions in the Acoustic Parasitoid Fly Ormia ochracea

Dean Koucoulas

A thesis submitted in conformity with the requirements for the degree of Master of Science Cell and Systems Biology University of Toronto

the Acoustic Parasitoid Fly Ormia ochracea

Dean Koucoulas

Master of Science

Cell and Systems Biology University of Toronto

2013 Abstract

Behavioural phonotaxis (oriented movement in response to sound) is an effective means to quantify auditory perception in acoustically communicating insects. Previous phonotaxis studies on the acoustic parasitoid fly Ormia ochracea (Diptera: Tachinidae) have described stereotyped, reflex-like responses towards auditory stimuli modeled after their preferred cricket hosts, yet their ability to demonstrate plasticity of responses in the context of dynamically changing auditory cues has not previously been described. Using a behavioural sensitization protocol, I compared phonotaxis towards behaviourally irrelevant (non-attractive) test stimuli presented alone, and when preceded with the natural, response-evoking cricket song (attractive). Results demonstrate the cricket song as a sensitizing stimulus mediating phonotaxis towards otherwise non-attractive sounds, and differential walking patterns depending on temporal delay between song offset and test stimulus onset. My findings suggest an ecological purpose of sensitization, allowing flies to maintain orientation towards a cricket host amidst conditions of signal disruption in the environment.

Acknowledgments

Throughout my academic career, I have had the immense privilege of being surrounded by an amazing support network of family, friends, peers, and colleagues. I would first like to thank my supervisor, Dr. Andrew C. Mason for being a continual source of support for me, and for giving me the opportunity to explore and incorporate a multitude of interests in the lab, especially re- igniting my passion for electronics. With Andrew’s guidance, I was able to grow both personally, and professionally and owe my sincerest gratitude for having first welcomed me into the lab as a summer student volunteer. I would like to thank my thesis advisory committee members, Drs. Mark J. Fitzpatrick, and Kenneth C. Welch for their continual feedback during the progress of my research, and for promoting my ability to develop as a critical thinker, and as an independent scientist. I am extremely grateful to Dr. Patrick O. McGowan for his continual support during the final stages of my thesis, and for always being open to hearing about the progress of my research. When it comes to my fellow lab members, I cannot thank them enough for their daily encouragement and motivation from hearing about my research ideas, to giving me company during long nights in the lab. I had the extreme privilege of overlapping my graduate studies with Dr. Norman Lee, Dr. Paul A. De Luca, Jenn Van Eindhoven, Sen Sivalighem, and the amazing Andrade lab, and I look forward to maintaining our collaborations well into the future. Thank you also to all the undergraduate research assistants and volunteers including Juli Rasanayagam, Steven Susanto, Alisha Patel, Kiran Beera, Paula Tactay, Olivia Murray, and Michelle Leung for their committed dedication to ensuring the well being of our fly population. Thank you also to the University of Toronto Scarborough, and the many Departmental staff for all your help throughout my time as both an undergraduate, and graduate student. I would like to thank my wonderful parents for their continued support, for always encouraging me to achieve my best, and for always believing in me. To my brothers, thank you for always being at my side, and for all the support and motivation you have provided me along the way. To my grandparents, thank you for all that you have taught me, and for giving me the opportunity to freely pursue the aspirations I am striving for now. It is because of you that I am able to say there are no limits to what I may achieve in my lifetime.

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Table of Contents

Abstract……………………………………………………………...……………………ii

Acknowledgements………………………………………………………………………iii

Table of Contents……………………………………………………………………...…iv

List of Tables……………………………………………………………………………..vi

List of Figures…………………………………………………………………………...vii

Chapter 1 General Introduction…………………………………………………….……..1

1.1 Hearing and the role of sound in insects…………………………………………1

1.2 Auditory challenges for insects………...………………………………………...4

1.3 Insect solutions to complex auditory scenes……………………………………..5

1.4 Directional hearing in Ormia ochracea………………………………………….6

Chapter 2 Behavioural plasticity under dynamic auditory conditions in the acoustic parasitoid fly, Ormia ochracea……………………………………………………………………..10

2.1 Abstract…………………………………………………………………………10

2.2 Introduction……………………………………………………………………..11

2.3 Materials and Methods………………………………………………………….15

2.3.1 Animals…………………………………………………………………15

2.3.2 Acoustic Stimuli………………………………………………………...15

2.3.3 Experimental Apparatus………………………………………………...17

2.3.4 Protocol…………………………………………………………………18

2.3.5 Data Analysis……………………………………………..……………...19

2.4 Results……………………………………………………………………………….21

2.4.1 Responses to Noise………………………………………………………21

2.4.2 Responses to Pulse Trains……………………………………………….24

2.5 Discussion…………………………………………………………………………....27

Chapter 3 General Discussion…………………………………………………………...56

References……………………………………………………………………………….59

List of Tables

Table 1. Noise following chirp…………………………………………………………..51

Table 2. Pulse-trains following chirp – long and short IPI…………………………...….52

Table 3. Pulse-trains following chirp – intermediate IPI………………………………...54

List of Figures

Figure 1. Common acoustic definitions in cricket song structure………………………...9

Figure 2. Outline of auditory stimuli used in noise experiment………………...….……36

Figure 3. Outline of auditory stimuli used in pulse train experiment………………...…37

Figure 4. Experimental apparatus used to assess phonotaxis ………………………..…38

Figure 5. Velocity measurements used to quantify phonotaxis…………………………39

Figure 6. Time indeces for velocity calculations in pulse train experiment………….…40

Figure 7. Steering and forward velocities in noise experiment…………………………41

Figure 8. Delta steering and forward velocity in noise experiment……………………..42

Figure 9. 2-D walking paths and lateral deviation in noise experiment………………....43

Figure 10. Steering and forward velocities in pulse train experiment……………...……44

Figure 11. Closer look at steering and forward velocity in pulse train experiment……..45

Figure 12. Regression lines for steering velocity in pulse train experiment.……….…..46

Figure 13. Regression lines for forward velocity in pulse train experiment…………….47

Figure 14. Average velocity per pulse compared to naïve fly responses.…………….....48

Figure 15. 2-D walking paths and lateral deviation in pulse train experiment……….....49

Figure 16. Full 10 s long cricket chirp effects………………………….….……………50

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Chapter 1 General Introduction

Organisms that make use of auditory communication are exposed to a diversity of acoustic signals that together, comprise what is known as their auditory scene (Hulse, 2002). Auditory scene analysis is the process by which the complex mixture of individual sound sources entering the auditory system is segregated and identified as meaningful representations of the surrounding environment (Bregman, 1990; Fay, 2007). Much of the early work in auditory scene analysis focused on understanding the role of sound in human hearing and speech communication (Bregman, 1990; Bee and Micheyl, 2008). However, the challenge of extracting relevant sources of information amidst a complex auditory backdrop is common amongst all acoustically communicating animals, including insects. Despite receiving less attention than their vertebrate counterparts, investigation of auditory processing mechanisms in insect systems offers a unique opportunity to understand the ubiquitous nature of auditory scene analysis across taxa, and is what defines the topic of my thesis.

1.1 Hearing and the role of sound in insects

Among certain insects, maintaining conspecific communication, avoiding predation, and ensuring reproductive success is largely dependent on the auditory system processing of acoustic stimuli (Hedwig, 2006; Virant-Doberlet and Čokl, 2004). The ability to maintain such critical processes is dependent on the effective recognition of relevant auditory cues in the environment, and localization of their sources.

Recognition of conspecific signals is predominantly determined by assessing the spectral (varying across frequency), and temporal (varying across time) features of the incoming sound field (Pollack, 1998). The importance of spectral characteristics in insects is best realized by considering that the auditory receptors of many insect ears exhibit specialized tuning towards a specific range of sound frequencies, such that spectral characteristics of the surrounding acoustic space are represented by the differential activation of individual receptors (Pollack, 1998; Mason and Faure, 2004). Depending on the pattern of receptor activation, different 1

behavioural responses may be elicited that are matched to the appropriate context. As an example, female crickets of the species Gryllus bimaculatus actively seek mates based on the dominant 4.5 kHz calling song of their male suitors, but will only exhibit the mounting response for reproduction upon hearing the 13.5 kHz courtship song (Pollack, 1998). Crickets also rely on frequency discrimination for the purposes of detecting and avoiding the ultrasonic frequencies indicative of bat predators in the area (Moiseff et al., 1978).

The importance of temporal characteristics for song recognition comes from understanding that many insects must decode temporal patterns in order to obtain conspecific information (Hennig, 2003), of which, recognition of a member of the appropriate species is most important (Bush and Schul, 2006; Doolan and Pollack, 1985; Pollack, 2001). This is especially true considering insect songs rarely exhibit frequency modulation (ability to vary instantaneous frequency over time) in the same way that occurs with human speech for example (but see Morris and Pipher, 1972), and thus rely on temporal parameters to perceive changes in acoustic stimuli over time (Pollack, 1998). The general structure of insect songs, which are often composed of sequences of sound bursts known as pulses, provides such features as pulse duration, interpulse interval, and pulse repetition period which all may be used for the purposes of conspecific signal recognition (Figure 1). Many closely related, sympatric species of insects can be differentiated simply by assessing differences in song temporal structure. In the bushcricket genus Tettigonia, for example, T. cantans and T. viridissima both produce songs comprised of a series of pulses organized into long trills while in T. caudata, the song is broken down into individual verses (Gerhardt and Huber, 2002). Upon conducting song preference trials, Schul (1998) determined that all three species possessed different criteria underlying preferences for temporal patterns in their conspecific song over that of the other species, which may serve as a mechanism maintaining species isolation.

In many behavioural contexts (such as mate-searching in crickets), after a relevant acoustic signal is recognized, the next step is determining where the sound source is located. As with vertebrates, insects that possess tympanal ears (see below) exploit cues of interaural intensity difference (IID; differences in sound intensity at the ears), and interaural timing difference (ITD; differences in the time of arrival of sound at the ears) in order to obtain information on sound localization, but employ different means to achieve this (Michelsen, 1998). For example, the distance separating the ears on a human head is large enough, relative to the wavelength of

relevant sound frequencies, such that diffraction of sound can occur. In doing so, the human brain may estimate the direction of sound based on the comparison of intensity and time of arrival differences at the two ears (Michelsen, 1998). For insects, however, the distance across where the ears are located can be orders of magnitude smaller than the wavelengths of sounds to which they are sensitive, and thus normal diffraction does not occur. As a result, the auditory systems of insects have evolved alternative strategies to deal with the inherent constraints of sound localization imposed by their small size (Michelsen, 1979).

Generally, insect auditory organs fall under one of two categories depending on the frequencies of sound attended to, and the distance over which auditory communication occurs. Near-field, or particle displacement ears, generally consist of sensory hairs or antennae that undergo mechanical distortion under the influence of moving air particles from a nearby sound source, that is, the displacement component of sound waves (Michelsen, 1998). Depending on how the hairs/antennae vibrate upon being deflected by the air particles determines if underlying sensory receptors become activated/fire, to then provide information on the directionality of the incoming sound wave. Examples among insects that make use of such ears are mosquitoes and fruit flies which perceive the low wingbeat frequency courtship songs produced by their mates, and caterpillars who respond to the wingbeat frequency of potential wasp predators (Hoy and Robert, 1996; Michelsen, 1979; Robert et al., 1992). The ability to hear over much larger distances, however, (and to detect higher frequency sounds) is accomplished by far field, or pressure sensitive ears which exploit pressure differences of the incoming sound field. Such ears are predominant in insects which communicate through sound over long distances, as is seen with the mating call songs of crickets, grasshoppers, and cicadas. Generally referred to as tympana, or tympanal membranes, the sites of these organs occur in pairs and are often identified as a localized thinning of the external body surface, often leading into an air filled chamber, and innervated by a scolopidial sensory organ (Hoy, 1998). Rather than depend on deflection by moving air particles, insects with tympanal ears exploit the pressure component of sound waves (Michelsen, 1998), translated into differences in tympanal membrane deflections, or vibrations, in order to determine direction of sound sources. Tympanal hearing organs are further subdivided into pressure receivers or pressure difference receivers depending on how sound interacts with the tympanal membrane (Michelsen, 1979; Hoy and Robert, 1996). In pressure receivers, sound waves can only reach the exterior surface of the tympanal membrane since the inner air chamber is closed off from the external environment. Therefore, sound 3

localization would depend on diffraction of sound to obtain interaural pressure differences between both tympana. Normally, the small size of insects would exclude them from having diffraction-dependent pressure receiver ears. However, some with larger relative body sizes such as moths which listen for high frequency (short wavelength) bat echolocation calls, allows diffraction to occur and thus are equipped with pressure receiver ears (Michelsen, 1979). Crickets, katydids, and grasshoppers on the other hand, use pressure difference receivers where the comparison of pressure of low frequency sound occurs on the outer and inner surfaces of the tympanal membrane through the internal air chamber which is open to the environment (Yager, 1999; Michelsen, 1998; Schowalter, 2006). Depending on the orientation of the incoming sound wave, the path taken to reach the inner and outer tympanal surfaces may differ. In turn, this may lead to differential constructive or destructive interference depending on the relative phases of sound pressure on the two sides affecting the net pressure on the tympana. As will be presented later, some insects have evolved further solutions for the purposes of maximizing auditory cues to achieve sound localization.

Based on the aforementioned details, it is clear that insects maintain a vital dependency on not just the physical properties of sound itself, but also how they interact with the anatomic and physiologic properties of the auditory system. In addition to the constraints imposed by their small size, however, insects are subject to the same auditory perceptual challenges faced by humans and other higher vertebrates, and through an investigation of how this occurs, the realization of insects as champions of the auditory world becomes possible.

1.2 Auditory challenges for insects

Much like songbirds and anurans, social aggregations of acoustic choruses are predominant in insects, with members of the same species (conspecifics) and other species (heterospecifics) both contributing to an overall complex auditory scene (Schul, 2006; Hulse, 2002; Bee and Micheyl, 2008; Gerhardt and Huber, 2002). In particular, this may lead to the presence of overlapping sounds which would increase detection thresholds of conspecific signals, and decrease, or mask, the ability to detect variations in them over time and space (Hartbauer et al., 2012; Bee and Micheyl, 2008). In Drosophila montana flies, for example, female responses to male courtship songs was shown to decrease in the presence of masking noise over the same 4

frequency range (Samarra et al., 2009). Such consequences would impose extreme limitations on the ability to recognize signals of interest from masking noise and detect dynamic changes in auditory conditions which may define events vital to the survival of an individual such as a warning call, or detection of a predator (Schul et al., 2012). Aside from the presence of overlapping signals themselves, the ambient environment may also result in reverberant acoustic conditions with reflections and echoes of sound providing conflicting information for perception and localization of sound sources (Litovsky et al., 1999). Such a situation may result in two sounds arriving at a receiver from different directions, but are perceived to emanate from a common sound source thus making the process of localization a difficult task (Marshall and Gerhardt, 2011).

1.3 Insect solutions to complex auditory scenes

As a means to combat such auditory challenges, acoustically communicating insects have adapted to develop a number of sensory processing mechanisms that exploit anatomic and physiologic features of their auditory systems, and the physical properties of sound itself. One example of such a solution is the phenomenon known as spatial release from masking, where signal to noise ratios are improved with spatial separation of the target signal of interest from background noise (Bee and Micheyl, 2008). In two species of tropical crickets, Schmidt and Romer (2011) provided evidence for spatial release from masking, with improved detection of conspecific calling songs amidst nocturnal background noise when they were separated from being completely overlapping, to 180 degrees apart. Another solution has been demonstrated in the katydid Neoconocephalus retusus, where females were shown to segregate the calling song of their conspecific male suitors from bat echolocation pulses in the background (Schul and Sheridan, 2008). Termed auditory stream segregation, this mechanism compares successive and/or simultaneous sound elements and categorizes them into auditory streams based on their perceived source of origin (Moore and Gockel, 2012). In the case of the katydid, for example, females would categorize all sounds perceived to belong to a singing male into one auditory stream, while those perceived to belong to a predatory bat into another. In doing so, it ensures that while females actively search for potential mates, they are still able to maintain attention towards possible threats. Yet another solution identified in insects relates to the problem of

reverberant acoustic conditions, and is regarded as the precedence effect. For two equivalent sound elements arriving at a receiver from different directions, Polynesian field crickets Teleogryllus oceanicus showed neural responses exclusively to the leading stimulus while suppressing those for the subsequent reflection (Wyttenbach and Hoy, 1993). In this regard, the leading stimulus would be used to assess the effective location of the sound source in the environment, and is therefore said to take precedence while the lagging source is simply not perceived. The precedence effect has also been observed in the acoustic parasitoid fly, Ormia ochracea where flies attend to the leading sound pulses of their cricket host songs to obtain directional information amidst competing auditory stimuli (Lee et al., 2009).

It is important to note that the solutions just described are not unique to insects, but rather reflect common solutions that humans and other higher vertebrates have also been shown to use. Spatial release from masking, auditory stream segregation, and the precedence effect have been observed in humans, birds, frogs, and other mammals (Litovsky et al., 1999; Bee and Micheyl, 2008; Schmidt and Romer, 2011). While sharing such functional parallels with vertebrates, insects face unique and complex challenges for processing sound from the perspective of auditory scene analysis, and may offer new insight into the evolution of auditory processing strategies across taxa.

1.4 Directional hearing in Ormia ochracea

In acoustic communication, it is common for acoustic signals to be used in the context of advertisement (e.g., male crickets sing to advertise for females). Female O. ochracea (Diptera: Tachinidae) have the ability to eavesdrop on such songs for the purpose of host recognition. Ormia ochracea are acoustic parasitoid flies. Despite the fact that adults are freely living, larvae from gravid females act as parasites and develop inside the body of cricket hosts (Adamo et al., 1995; Cade 1975). Population distributions of O. ochracea span across regions in the southern United States (Florida, Texas, California) and Hawaii where each population exploits a different species of field crickets to serve as their hosts (Cade, 1975; Walker 1993). In order for the females to deposit their larvae on the crickets, they make the transition from free flight to walking on the ground, using the acoustic cues from male crickets as a guide to their location (phonotaxis) (Mueller and Robert, 2002). As it turns out, male crickets who are singing to attract 6

female mates put their own survival at risk through being targeted by the female O. ochracea (Zuk et al., 1993). Crickets produce sound by rubbing their wings together, and one cycle of opening and closing the wings generates a burst of sound (called a sound pulse) with a frequency of around 5 kHz (Bennet-Clark and Bailey, 2002) that is largely conserved across different cricket species (Gray et al., 2007). The temporal organization of these relatively uniform sound pulses, however, does vary across different cricket species in such parameters as pulse rate (number of pulses/s), pulse period (time separating successive pulses), and duty cycle (duration of active sound in relation to silence) as seen in Figure 1.

Localization and recognition of the appropriate host calling song are the most fundamental challenges faced by O. ochracea in maintaining their role as acoustic parasitoids. It is not surprising, therefore, that despite their relatively small size, O. ochracea have evolved essential anatomic and physiologic adaptations which allow them to extract crucial sensory information from the environment. Prior to research on the auditory system of O. ochracea, conventional understanding of the physics behind sound propagation would have predicted that the 0.5 mm distance separating the eardrums of O. ochracea, is far too small to determine the directionality of a 5 kHz sound source in the external environment. However, the specialized anatomical structure of O. ochracea eardrums makes directional sensitivity possible. This is primarily achieved by the mechanical coupling of the two eardrums via an intertympanal bridge as described by Robert et al. (2008). As a result of this mechanical linkage, acoustic stimulation on one side of the fly leads to mechanical responses for both the ipsilateral (same side as sound), and contralateral (opposite side of sound) eardrums. This allows the flies’ auditory system to exploit the small interaural time-differences that are the only available cue for sound source direction (Miles et al., 1995).

Many organisms with the capability to hear exploit cues of interaural intensity difference and interaural timing difference in order to obtain information on sound localization. For O. ochracea, however, there is negligible diffraction of sound at both sides of the eardrums thus making an IID non-existent. Thus, ITDs are the only external cues O. ochracea have to maintain directional sensitivity and even those are miniscule (1.5 μs maximum ITD for a sound source oriented at 90 degrees azimuth to the fly’s midline axis) (Mason et al., 2001). Despite this seeming limitation, the mechanical coupling of O. ochracea eardrums account for two essential modifications that are made by the flies’ peripheral auditory apparatus: (1) the 1.5 μs ITD

becomes increased to 55 μs, and (2) the ipsilateral eardrum can vibrate with up to 10 dB greater amplitude than the contralateral eardrum. Both increase the interaural difference cues available to the fly (Robert et al., 1996).

In addition to this mechanical processing stage, additional steps are taken within the neural circuitry of O. ochracea auditory receptors that further enhance the ability for directional cues to be realized. This, is turn, is dependent on three characteristics of O. ochracea type 1 afferent auditory receptor physiology: (1) receptors respond to a suitable sound pulse with a single spike regardless of the intensity of the sound pulse (thus a pulse with a greater intensity elicits a single spike in the receptor just as for a pulse with a lower intensity) or its duration, (2) receptors respond with shorter latency to pulses with greater intensity than to pulses with lower intensity, and (3), for any given intensity, there is little variation (low jitter) in the latency of receptor firing (timing of firing is very predictable for different sound intensities) (Mason et al., 2001). From this, it is evident that differences in intensity of sounds in the surrounding environment may be transferred into the auditory system of O. ochracea in the form of different timing (latency) of auditory receptor firing. Coupling this with the processing mechanisms employed by the flies’ mechanically coupled eardrums thus account for how O. ochracea may exploit miniscule ITD cues, and convert these to IID cues, in order to obtain information important for directional sensitivity.

Directional hearing for the purpose of locating the source of a cricket host calling song has been extensively documented in O. ochracea, in terms of the structural anatomy of its acoustic (tympanal) membrane (Miles et al., 1995; Robert et al., 1994b; Robert et al., 1996; Robert et al., 1998), neural coding mechanisms (Mason et al., 2001; Oshinsky and Hoy, 2002), and the ability of O. ochracea to selectively localize a single source amongst many simultaneous signals (Lee et al., 2009). Despite the common 5 kHz pulse produced by crickets, O. ochracea have been shown to prefer the cricket songs of their primary local hosts (Gray et al., 2007) which suggests that temporal song structure may be an important factor affecting recognition of O. ochracea. This has been supported in recent work on Floridian O. ochracea where their underlying preferences for song recognition modeled after potential cricket hosts was most dependent on assessing the temporal feature of pulse period (Lee, 2012).

The primary goal of my thesis is to investigate mechanisms of auditory processing in an acoustic parasitoid fly (Ormia ochracea) whose use of acoustic signals in the environment assist 8

in their recognition and localization of cricket hosts suitable for acting as reproductive surrogates (Adamo et al., 1995; Cade, 1975). I seek to determine how these flies are able to maintain attention and/or orientation to a particular host amidst dynamic acoustic conditions in their environment, and how this relates to the unique role of sound in auditory parasitism.

Figure 1. Temporal definitions used to describe cricket song structure. Species of field crickets may be distinguished simply based on differences in such temporal parameters. Assessment of temporal features are used as a means to maintain species isolation in crickets.

Chapter 2 Behavioural plasticity under dynamic auditory conditions in the acoustic parasitoid fly, Ormia ochracea

2.1 Abstract

Unlike higher forms of learning which depend on animals making learned associations between stimuli and the behviours they evoke, forms of non-associative learning such as sensitization provide a means for animals to elicit rapid responses towards dynamically changing conditions, a feature of upmost importance in the auditory world. In their pursuit of cricket hosts, the acoustic parasitoid fly Ormia ochracea is subject to many circumstances that may be considered dynamic, such as changing locations and densities of their cricket hosts, and the presence of other biotic and abiotic sounds nearby. Despite this, previous studies have not assessed the influence of such interactions on host seeking behaviour in flies. Using a high resolution spherical treadmill system, flies were subjected to a behavioural sensitization protocol where walking phonotaxis towards auditory stimuli that fail to normally elicit host seeking behaviour in O. ochracea (non-attractive) were compared to responses when these same stimuli were preceded with a stimulus of their preferred hosts in Florida, Gryllus rubens (attractive). It was predicted that phonotaxis towards the test stimuli would increase following the chirp, and specific differences would depend on the temporal and/or spatial relationship between cricket chirp offset and test stimulus onset (noise burst and pulse trains). Using measures of steering and forward walking velocity, flies that had been exposed to the initial cricket chirp (experienced) elicited stronger responses towards the test stimuli, compared to naïve flies presented with the test stimuli on their own. Specific differences among test conditions revealed strongest delta steering and forward velocity responses with longer temporal separation between chirp and test stimulus presentation. As a result, flies are able to effectively sustain an oriented walking trajectory towards the active speaker even while playing an otherwise non-attractive stimulus. Despite overall negligible delta responses for short temporal delays, sustained baseline velocities attest to flies responding to the presence of test stimuli, thus supporting the role of the cricket chirp as a sensitizing stimulus. Sensitization is presumed to play a significant role in allowing flies to maintain attention and orientation towards potential cricket hosts amidst unpredictable auditory conditions in their natural environment. 10

2.2 Introduction

Behavioural decisions are often made based on the momentary relevance and significance of sensory stimuli present in the environment at a particular time. However, the ability to demonstrate behavioural plasticity requires such decisions to be made under dynamic conditions. The natural auditory environment is a prime example of this, where sounds from a myriad of biotic and abiotic sources combine and overlap to create a very complex and ever- changing setting from which, animals must be able to extract useful information (e.g., for communication, navigation, localization. etc). Such is the case for the fly, Ormia ochracea, where gravid females act as parasitoids of field cricket hosts (Gryllus spp.), a relationship mediated by their ability to express directional hearing of their hosts’ calling songs (Cade, 1975; Robert, 1996; Mason et al., 2001). Reproductive success in O. ochracea requires flies to be able to recognize the calling song of their correct host species, and subsequently determine its location in the environment. By relying on the calling song of male cricket hosts, O. ochracea are subject to the same auditory challenges faced by their female cricket counterparts who are also seeking out males, but for purposes of reproduction rather than parasitism (Alexander, 1967). Dense aggregations of male crickets singing in choruses often results in temporal overlap of signals from different members which impairs a listener’s ability to identify and locate the sounds from a single male (Gerhardt and Huber, 2002). In fact, the formation of such aggregations is thought to be driven, to some extent, by the threats imposed by predation and parasitization where members would benefit by being less conspicuous when in a large group (Mhatre and Balakrishnan, 2006; Burk, 1982; Greenfield, 1994). In addition to being subject to the auditory constraints imposed by the songs of their hosts themselves, the presence of sounds from other nearby sources also contributes to an overall level of background noise that flies must be able to deal with.

Despite the obvious challenges that such complex environments pose for O. ochracea in their pursuit of potential hosts, prior behavioural studies have been limited in their ability to infer how flies respond under dynamic auditory conditions. Over the course of a fly’s approach to a cricket, the auditory cues being assessed for host localization may change in predictable ways. For example, as the distance between the fly and cricket narrows, flies would be expected to perceive a greater intensity of the incoming cricket song, and perhaps be subjected to greater interference from the songs of other nearby crickets. Previous work has looked at such

predictable changes, where, for example, the auditory receptors in O. ochracea are known to respond to sounds of greater intensity with decreased firing latency. The differential time-coding of receptor firing would then be used as a directional cue for flies to adjust orientation response headings as they continue to track the location of their hosts (Mason et al., 2001). In addition, Lee (2009) provided support for the precedence effect in O. ochracea where flies were shown to maintain phonotactic walking trajectories to the leading stimulus only, while neglecting directional cues carried by lagging sources. This ensures that while flies are bombarded with competing directional information, attention is focused only on the leading stream of sound input entering the auditory system. While such studies certainly touch on dynamic auditory conditions faced by O. ochracea in their natural environment, a more broad understanding of how flies respond to, and update their source information under more unpredictable conditions is still required.

One example of such a scenario potentially faced by O. ochracea is their ability to maintain attention towards a particular host amidst signal degradation or interruption, in the environment. Production of trilling songs by field crickets (characterized by long trains of individual sound pulses) is an energetically expensive process, and as such, periods of silence are often included in between longer bouts of singing (Prestwich and Walker, 1981; Alexander, 1962; Beckers and Wagner, 2012) . Therefore, for the trilling song of G. rubens, for example, Floridian O. ochracea must be expected to sustain their attention towards a particular host even when the input of auditory information dictating localization is momentarily unavailable. Similar situations may arise with the transient change in location of singing males during the course of their calling behaviour as they attempt to maximize the quality of their songs (Shaw et al., 1981). Many species of singing Orthoptera have been identified to change singing sites while they monitor the density of competing males in the area. Perceived high densities may prompt males to seek areas with better access to resources such as plants for feeding and oviposition sites for female mates (Shaw et al., 1981). In addition, the possibility always remains that the calling song of male crickets may be distorted by nearby vegetation, and simply from interference imposed by other sounds in the area (Romer and Bailey, 1990). With so many factors contributing to an overall dynamic auditory scene, it would be imperative for O. ochracea to demonstrate behavioural plasticity in their pursuit of a potential host.

In addition to attempting to describe the phonotactic walking behaviour of O. ochracea under dynamic auditory conditions that flies likely face in nature (naturalistic approach), such an investigation also affords the opportunity to make general conclusions about the relationship between physical stimuli and the behaviours they invoke (psychophysics approach). Based on the inherent difficulties that arise with directly assessing forms of perception in non-human animals (cannot directly ask non human subjects), experiments often rely on manipulating variables of interest and using resulting physiological and/or behavioural responses as indicators of how the stimuli are perceived (Wyttenbach and Farris, 2004). This has been seen from an auditory perspective in insects, where, for example, intensity discrimination is often tested by presenting stimuli of varying intensities from spatially separated speakers and observing which speaker the organism engages in phonotaxis (walking movement in response to sound) towards (Wyttenbach and Farris, 2004). In the process of engaging in such a psychophysics approach, insight is made into the way living organisms form relationships between stimuli and the unique behaviours they evoke.

Aside from higher forms of learning where associations between stimuli and their resulting outcomes (either positive or negative) are made, behavioural plasticity may also occur simply through adaptation to experience, devoid of any form of reinforcement (Hammer et al., 1994; Engel and Hoy, 1999). This is known as non-associative learning, and represents the simplest means for which animals are able to adapt their behaviour based on prior sensory experience, a process known as experience dependent plasticity (Minoli et al., 2012; Engel and Hoy, 1999). This form of behavioural plasticity is thought to occur in either of two directions depending on if the behaviour is mediated by habituation or sensitization. In habituation, repeated presentations of a stimulus may induce a decrease in behavioural responsiveness over time, while in sensitization, responsiveness increases such that the resulting behaviours occur more quickly and with less effort with continued presentation of the stimulus in question (Groves and Thompson, 1970; Davis, 1972; Hammer et al., 1994; Minoli et al., 2012). Such perceptual phenomena are integral to providing animals with a means to respond quickly to stimuli in their environment, a task of upmost importance in the auditory world.

In sensitization, the initial strong stimulus known as the sensitizing stimulus (SS), naturally invokes a particular response in the organism, and it is through this experience that responses to subsequent stimuli are stronger than those elicited before (Groves and Thompson, 1970;

Hammer et al., 1994). This is especially the case where the subsequent stimuli are repeated presentations of the strong SS, as demonstrated for example, in the gill and siphon withdrawal reflex in the marine snail Aplysia, where repeated noxious electric shocks elicits the reflex more easily, and may continue to do so on the order of weeks afterwards (Hawkins, 1984). However, the sensitization effect has also been observed towards otherwise non-attractive/behaviourally irrelevant stimuli that follow presentation of the SS. For example, blowflies have been shown to demonstrate the proboscis extension reflex (PER) indicative of feeding behaviour in response to non-sucrose solutions when preceded with presentation of the natural response evoking sucrose food (Dethier et al., 1965; McGuire, 1983; McGuire and Tully, 1986). As well as facilitating responses towards stimuli of the same modality (e.g., gustatory in the case of the PER), sensitization has also been observed across sensory modalities. For example, honeybees have been shown to elicit the PER towards an odor stimulus when preceded with a sucrose stimulus (Hammer et al., 1994). In addition, upon exposure to a brief bat call, behavioural sensitivity of male moths towards the female produced sex pheromone increased compared to those presented with a behaviourally irrelevant tone (Anton et al., 2011). In fact, the increased level of responsiveness was found to be on the same order as if the male moths had been pre-exposed to the pheromone itself. Based on these examples, it is clear that sensory stimuli which bear great significance in terms of survivorship (such as presence of food and predators) may induce wide scale, general changes in the activity of living organisms through the process of sensitization, and that such effects are not limited to the modality for which the sensitizing stimulus derives from.

Previous work in O. ochracea has suggested that phonotactic responses are highly stereotyped and reflex like behaviours in response to auditory stimuli modeled after their primary local cricket hosts in Florida, Gryllus rubens (Lee, 2012). However, little is known on whether flies are able to demonstrate plasticity of phonotactic responses in the context of dynamically changing auditory cues. The purpose of this study, therefore, is to examine the role of non- associative learning, specifically sensitization, in mediating the relationship of such dynamic interactions, and provide insight into the possible perceptual mechanisms underlying said behaviours. In this study, sensitization in O. ochracea is assessed using an auditory psychophysics approach where responses towards non-attractive/test stimuli (stimuli that deviate from temporal parameters characteristic of host calling song; fail to elicit phonotaxis) are compared with responses to these same stimuli when initially preceded with a cricket 14

chirp/control modeled after the flies’ primary local hosts (attractive; reliably elicit phonotaxis). Based on prior studies in sensitization, it was hypothesized that a) the initial cricket chirp would act as a sensitizing stimulus in mediating responses towards the non-attractive stimuli, and b) these responses would be directly influenced by the temporal and/or spatial relationship between the control and test stimuli. From this, it was predicted that a) if preceded with the cricket chirp, flies would elicit stronger phonotactic walking responses towards non-attractive stimuli compared to without the chirp, and b) sensitization effects, if any, would differ towards test stimuli presented temporally and spatially grouped with the initial cricket chirp, compared to temporally and spatially isolated with the chirp.

2.3 Materials and Methods

2.3.1 Animals

Experiments were conducted on lab-reared gravid female Ormia ochracea derived from a population originally collected in Gainesville FL, USA. Flies were maintained in environmentally controlled chambers (Power Scientific, Inc. Model DROS52503, Pipersville PA, USA) at 25o C and 75% humidity on a 12 hour:12 hour light:dark cycle and fed nectar solution (The Birding Company, Yarmouth MA, USA) ad libitum. Experiments were conducted at 22 - 25 o C in a dark room illuminated with red light to maintain visibility of flies during behavioural experiments. Flies used for experiments were anesthetized in an ice bath for 5 minutes and while incapacitated, tethered with heated wax onto a micromanipulator, with the wax being applied to the dorsal surface of the fly immediately posterior to the neck region. Flies were then fixed into position on the center of the experimental apparatus and given approximately 10 minutes time to acclimate before commencing experiments.

2.3.2 Acoustic Stimuli

Noise Experiments

Dynamic acoustic stimuli consisted of two parts: (1) an initial cricket chirp (control) modeled after the calling song of Gryllus rubens, which consisted of 10 ms duration, 5 kHz tone pulses (1

ms rise/fall times) separated with 10 ms interpulse intervals (IPI’s), and repeated at a rate of 50 pulses/s for a duration of 0.5 s (25 pulses total) and (2) a subsequent 0.5 s burst of unramped band-limited random noise (test), whose onset occurred at varying time delays of 10, 100, 500, and 1000 ms after offset of the initial cricket chirp (Figure 2A). Considering that the noise stimulus lacked any temporal features inherent in cricket song structure, and does not normally elicit phonotaxis in O. ochracea (Lee, 2012), it represented one category of non-attractive stimulus used in this study. The values of 10, 100, 500, and 1000 ms were specifically chosen to implicitly contrast the noise as being temporally grouped with the cricket chirp in the short delay conditions (10 and 100 ms) with being temporally isolated in comparison for the long delay conditions (500 and 1000 ms). In one group of flies, acoustic stimuli were constructed such that the noise burst followed from the same speaker as that which played the cricket chirp (same-speaker condition, Figure 2B). In a second group of flies, the noise was presented from the speaker opposite to that which played the cricket chirp (opposite-speaker condition, Figure 2C). This was done to investigate the effects of noise location on responses elicited by the flies, and in turn, represented another implicit comparison by looking at the effects of noise being spatially grouped with the cricket chirp in the same speaker condition, or spatially isolated in the opposite speaker condition.

Pulse Train Experiments

Dynamic acoustic stimuli consisted of two parts: (1) a 0.5 s initial cricket chirp (control) modeled after the calling song of Gryllus rubens (same parameters as mentioned above) and (2) pulse trains of individual sound pulses (test) identical to those that comprise the cricket chirp, but separated at varying IPI’s (Group 1 flies: 50, 100, 500, and 1000 ms; Group 2 flies: 200, 300, and 400 ms) and repeated for the remaining 9.5 s for a total stimulus duration of 10 s (Figure 3). Considering that these pulse trains had temporal features beyond the range of preferred values to elicit phonotaxis in O. ochracea (Lee, 2012), it represented the second category of non-attractive stimulus used in this study. The 50 and 100 ms IPI stimuli were chosen to implicitly contrast the pulse trains as being temporally grouped with the initial chirp, with being temporally isolated in the 500 and 1000 ms IPI’s. The 200-400 ms IPI’s represented intermediary stimuli between temporally grouped and isolated. Acoustic stimuli were constructed such that the individual pulses followed from the same speaker as the initial cricket chirp. The number of these individual pulses that could fit in the remaining 9.5 s was a direct

function of the pulse period (e.g., for IPI of 50 ms, pulse period = (10 ms pulse + 50 ms IPI) = 60ms; 9.5 s / 60 ms = 158 pulses). The IPI’s also described the time interval between cricket chirp offset and initial target pulse onset.

Stimulus waveforms were synthesized in Matlab (R2009b, The MathWorks Inc. USA) with custom software, and in Adobe Audition (Version 3.0). Digital signals were converted to analog using National Instruments data acquisition hardware (NI USB-6251, 44100 Hz), amplified using Radio Shack Realistic (SA-10 Solid State Amplifier MOD-31-1982B, Taiwan) and broadcast through 75 W square piezo electric tweeters . Stimulus attenuation levels were controlled with software and programmable attenuators (Tucker Davis Technologies System 3 PA5) and calibrated to 76 + 1 dB SPL using a probe microphone (B&K Type 4182, Denmark) powered by B&K Nexus Conditioning Amplifier (Denmark). At this attenuation level, the intensity of the noise burst was measured to be 71.5 + 1 dB SPL rms (B&K Type 2231 Sound Level Meter, Type 4139 ¼” microphone).

2.3.3 Experimental Apparatus

Behavioral phonotaxis measurements were obtained from tethered flies situated on a high- resolution spherical treadmill system located equidistant (23 cm) from two test speakers positioned at + 45o azimuth from the longitudinal axis of the fly (Figure 4). The treadmill system itself was surrounded with acoustic attenuating foam to preserve the structure of sound stimuli being presented from the speakers. Tethered flies were placed on top of a light weight table tennis ball which was held aloft via a constant supply of air current, above a modified optical computer mouse sensor (ADNS 2620, Avago Technologies, USA) (Lott et al., 2007). The treadmill sphere was marked with a random dot pattern to improve contrast during rotation across the sensor. Rotations of the sphere across the optical sensor were transduced as two- dimensional walking responses from the flies comprised of x and y pixel units. Data points were obtained at a sampling rate of 2160 Hz. Using high speed video capture (DRS Lightening RDT, 500 frames per second), pixel units recorded by the treadmill were calibrated to reflect actual walking distances performed by the flies. Data collection by the treadmill system was controlled using custom Matlab software and was linked with the National Instruments data acquisition system to ensure simultaneous presentation of acoustic stimuli from the speakers and data capture of virtual walking traces from the treadmill.

Because flies were fixed in position relative to the speakers, and their movements transduced by the treadmill, their responses were recorded “open-loop” – their responses did not cause any change in the sensory feedback the flies experienced. I could thus measure the flies’ responses to constant sensory conditions throughout the duration of the stimulus, allowing me to separate the flies’ tendency to orient towards a sound source (steering velocity) and their overall walking speed (forward velocity), measured as the change over time along the x- and y-axes, respectively, from the treadmill system.

2.3.4 Protocol

In subsequent sections of my thesis, I use the terms ‘naïve’, and ‘experienced’ to contrast stages in the protocol where flies had not yet been presented with any acoustic stimulation (beginning of experiment), with after having successfully completed the full test sequence (end of experiment), respectively

Responses to Noise

Experiments began by presenting naïve flies with the 500 ms noise burst alone from either the left or right speaker (randomly determined). This was done to determine the baseline response to the noise alone. Flies were then subsequently presented with the 500 ms cricket chirp alone, also, from a randomly assigned speaker. After this, the main experimental protocol began where flies were presented with the paired stimuli consisting of an initial 500 ms cricket chirp followed with the 500 ms noise burst at varying delays afterwards (10, 100, 500, and 1000 ms). Presentation of each of the four different delay conditions were repeated three times for both the L and R speaker (thus 6 repetitions for the different delays in total), and order of stimulus presentation was randomly determined. At the completion of all paired stimuli, experiments concluded by presenting experienced flies with a final single presentation of the 500 ms noise burst alone, and then the 500 ms cricket chirp alone from either the right or left speaker (random). For each stimulus presentation, data was recorded over a 10 s duration. Flies were given at least 1 minute of silence between individual trial presentation.

The above protocol was repeated for the opposite-speaker condition in the second group of flies.

Responses to Pulse Trains

Experiments began by presenting naïve flies with stimuli consisting of pulse trains of varying IPI’s alone (that is, stimuli devoid of the initial cricket chirp). This was done in order to obtain a baseline response towards the test pulses alone. Presentation of each different IPI condition was repeated three times each and presentation from L or R speaker was randomly determined. At the completion of presenting the pulses alone, flies were then presented with the 500 ms cricket chirp alone from a randomly assigned speaker. After this, the main experimental protocol began where flies were presented with the paired stimuli consisting of an initial 500 ms cricket chirp followed with presentation of the individual pulses of varying IPI’s. Presentation of each IPI condition was repeated three times each from both L and R speakers (thus 6 repetitions for each IPI condition). After completion of the paired stimuli, experienced flies were presented with the pulses alone stimuli as in the beginning of the protocol, and experiments ended with a final single presentation of the 500 ms cricket chirp. It should be noted that for the seven different IPI combinations tested in total (50, 100, 200, 300, 400, 500, and 1000 ms), data were collected from two different groups of flies. The first group obtained data for the 50, 100, 500, and 1000 ms IPI’s, while the second group obtained data for the 200, 300, and 400 ms IPI’s. In both cases, the experimental protocol steps were exactly the same. For each stimulus presentation, data was recorded over a 10 s duration. Flies were given at least 1 minute of silence between individual trial presentation.

2.3.5 Data Analysis

All variables were derived from the 2-Dimensional x and y units recorded from the trackball at a sampling rate of 2160 Hz, and were calculated using custom software. Steering velocity was calculated as changes in the x pixel units over time, and thus represented the extent to which flies were turning laterally (towards active speaker = positive x values, away from active speaker = negative x values). Similarly, forward velocity was calculated as changes in the y pixel units over time, representing the strength of steering in the forward (+y values) and backward (-y values) direction.

For responses obtained from equivalent stimuli presented from both L and R speakers, responses from both speakers were averaged to obtain a single measure, for each delay

condition for the noise experiment, and each IPI condition for the pulse train experiment. Data are given as means + SEM.

Responses to Noise

Responses to repeated presentations of similar stimuli were averaged for each fly, such that each fly contributed a single averaged response in each stimulus category. Walking responses were quantified as the change in velocity (steering and forward velocities measured separately) in response to the onset of the noise burst following an attractive phonotactic stimulus (simulated cricket chirp). This was done by measuring the difference (deltaV) between the average steering (or forward) velocity at noise onset (averaged over the subsequent 46 ms time window), and 100 ms after noise onset (average of the subsequent 46 ms, Figure 5A). Results for steering and forward velocity were analyzed separately via two-way ANOVA with noise- onset delay (10, 100, 500 and 1000 ms) and speaker configuration (same vs opposite – see above) as factors, and post hoc comparisons using Tukey’s HSD. Because (i) no noise burst was delivered for control stimuli, and (ii) the timing of the noise onset was different for each delay value, there was no obvious time during control responses at which to determine control values for deltaV. Therefore, control measurements were generated by an iterative procedure as follows. For each fly in the data set (i.e., each averaged trace in one stimulus category) a time index corresponding to one of the noise-onset delay values was selected at random, and deltaV calculated as described above. This was repeated 10,000 times and the average of these values was taken as the control deltaV for that fly. This procedure was repeated for each fly using a Matlab script. Statistical analyses were carried out using R 2.15.3. For a qualitative description of walking behaviour, the lateral distance flies deviated (lateral deviation) from a straight line walking path was also determined over the 10 s time period. In a 2-D walking plot of x coordinates versus y coordinates, lateral deviation represents changes in x units over time.

Responses to Pulse Trains

For pulse-train responses, two measures were calculated for both steering and forward velocity: (deltaV) and baseline velocity (baseV). In these experiments, baseV was measured as the average velocity for the 46 ms time-window just after pulse onset, and deltaV as the maximum velocity during the current pulse period minus the corresponding baseV (Figure 5B). These two measures were used in these experiments because some of the stimulus conditions

resulted in sustained responses (steering and/or forward velocity), such that onset-responses were not isolated from the ongoing response and it was useful to consider both the discrete responses at pulse onset as well as the sustained overall response (see Results). These response measures were taken for successive pulses throughout the course of the 10 s stimulus presentation. Due to the presentation of pulse trains of varying IPI, the different stimulus categories had different numbers of pulses during the 10 s stimulus presentation, ranging from 158 pulses for 50 ms IPI, to 9 pulses for 1000 ms IPI. For statistical analysis, all shorter IPI responses were subsampled by taking response measurements at those pulses corresponding to the timing of 1000 ms IPI stimuli, such that measurements were included for 9 pulses separated by 1000 ms intervals for all stimuli (Figure 6). In this way, response measurements assess the effects of varying IPI over a common timescale. Measurements were taken from control responses at these same time indices. Results were analyzed with four separate ANCOVAs – deltaV and baseV for both steering and forward velocity – with IPI as a categorical factor and time of pulse onset as a covariate. Statistical analyses were carried out using R 2.15.3. Similar to noise experiment, lateral deviation over the 10 s time window was also assessed for descriptive purposes.

2.4 Results

2.4.1 Responses to Noise

A total of 28 walking responses were recorded from each of 13 flies in the same speaker condition, and 17 flies for the opposite speaker condition.

Comparison of responses in ‘naïve’ vs. ‘experienced’ flies

Figure 7A shows averaged responses (for all flies) to the positive control stimulus (synthetic cricket chirp). Steering (lateral) velocity and forward velocity are plotted separately for stimuli presentation from both speaker conditions, and responses are shown for the initial (left) and final (right) stimulus presentations of the experiment (see Methods). General features of O. ochracea walking responses have been described previously (Mason et al., 2005). Flies respond to a suitable cricket stimulus with accelerated walking in the direction of the active sound 21

source, indicated by the rapid rise in both forward and lateral velocity. Flies maintain walking for the duration of the stimulus and decelerate following stimulus offset. Post-stimulus deceleration is more consistent for steering velocity. Flies eventually cease movement in the direction of the laterally located sound source. They do not, however, stop moving altogether, but rather show a continuation (or renewal) of forward walking after stimulus offset. Comparison of responses from before (Figure 7A left) and after (Figure 7A right) flies have experienced a series of stimuli demonstrates differential effects (naïve vs. experienced). The later responses show significantly higher average steering velocities (Same Speaker Group: 0.49 + 0.07 cm/s vs. 0.16 + 0.03 cm/s, paired t-test: t (24)= 4.03, p<0.001, Opposite Speaker Group: 0.42 + 0.09 cm/s vs. 0.17 + 0.03 cm/s, paired t-test: t (32)= 2.70, p<0.001), average forward velocities (Same Speaker Group: 1.05 + 0.13 cm/s vs. 0.39 + 0.08 cm/s, paired t-test: t (24)= 4.34, p<0.01, Opposite Speaker Group: 1.12 + 0.14 cm/s vs. 0.50 + 0.11 cm/s, paired t-test: t (32)= 3.53, p<0.001), more pronounced maintenance of movement following stimulus offset, and significantly shorter initial response latencies (Same Speaker Group: 75.69 + 3.29 ms vs. 220.67 + 30.21 ms, paired t-test: t (24)= 4.77, p<0.001, Opposite Speaker Group: 86.49 + 6.42 ms vs. 174.60 + 12.32 ms, paired t-test: t (32)= 6.34, p<0.0001). All t-tests were one-tailed.

This increased responsiveness post-protocol was more pronounced in response to the negative control stimulus (noise burst alone, Figure 7B). Naïve flies showed no phonotactic response to noise burst (Figure 7B left). After a series of responses to stimuli (main protocol: which includes attractive cricket chirp), however, flies presented with noise alone showed an augmented (though still weak relative to chirp) response that includes both walking (forward velocity) and orientation (steering velocity), therefore constituting phonotaxis (Figure 7B right). Unlike responses to the chirp however, the rise in steering and forward velocity was only transient and decayed well before offset of the noise burst.

Responses to noise burst following chirp

In the main protocol of this experiment, Female O. ochracea were presented with paired acoustic stimuli consisting of the initial cricket chirp followed by subsequent noise burst at varying delays (Figure 7C). For the short delays (10 and 100 ms), forward velocity was sustained through noise duration (grey region), then showed a similar pattern to chirp responses (velocity dropped at stimulus offset, then increased again, and decayed), with no apparent difference between same and opposite speaker conditions. For steering velocity, there was no 22

effect of noise onset for same speaker (steering simply dropped with cricket chirp offset), but there was a delayed (relative to normal phonotactic response) and weak turn towards the “wrong” speaker in the opposite speaker condition - flies made a transient turn toward the initial speaker (chirp source) during noise presentation from the opposite speaker. For the long delays (500 and 1000 ms), there was a clear effect of noise burst on both forward and steering velocity. At noise onset for both speaker conditions, flies showed a walking response oriented to the active speaker with similar characteristics to initial phototactic response (to cricket chirp), but shorter duration (velocities declined before noise offset). These responses were more transient than the maintenance of forward velocity for short delays – flies began to decelerate in both forward and lateral velocity prior to noise offset. Flies also showed the transient renewal of forward velocity after noise offset.

In order to better characterize the velocity trends initiated at noise onset for the different gap delay conditions, the change (delta) in steering and forward velocity for same and opposite speaker conditions were measured (see Methods).

These results are summarized in Figure 8, which shows the mean (+/-SE) values for changes in steering and forward velocity (deltaV) for each stimulus condition. For steering velocity (Figure 8A), there were significant effects of Delay and Speaker Position, as well as a significant interaction between these factors (Table 1). Pair-wise comparisons indicated that short-delay conditions (10 and 100 ms) are not different from control (chirp alone), with deltaV values consistent with continued deceleration following stimulus offset. For longer delays (500 and 1000 ms), flies show a significant orientation in the direction of the speaker for both speaker locations (Table 1).

In forward velocity, there were significant effects of Delay and Speaker Position (Table 1). Pair-wise comparisons showed forward deltaV was not significantly different from control for the 10 ms delay condition. Flies showed a significant forward acceleration at noise onset for 100, 500 and 1000 ms delay conditions, with slightly but significantly stronger responses in the opposite speaker condition (Figure 8B, Table 1).

Overall 2-D walking paths of flies were fairly similar across the various gap delays, and both speaker orientations, with flies deviating approximately 5 cm laterally and 12 cm forward from their initial starting location (Figure 9, left graphs). By the end of the 10 s data capture window,

flies in all gap delay conditions, and both speaker orientations, ended up at a similar location approximately 12 cm away from the speaker which played the initial cricket chirp (Figure 9, right graphs).

2.4.2 Responses to pulse trains

The above results suggest that there are two distinct effects of the relative timing of a noise burst following an attractive stimulus: (1) an elevation of forward walking velocity at all delays relative to chirp offset, and (2) a more transient elevation of steering velocity only for longer delays. That is, flies increase walking speed at the onset of a novel stimulus (noise burst) following a chirp, but only make an oriented response (steer toward the source) for the longer delays tested. I therefore conducted a second set of experiments to probe these temporal effects by presenting periodic test pulses at varying intervals (IPI) to “titrate” the time-course of forward and steering velocity responses. This second set of experiments examined the flies’ responses to stimuli in which an ongoing chirp (attractive phonotactic stimulus) transitioned to a pulse train with a slower repetition rate (outside of O. ochracea phonotactic preferences, (Lee, 2012)). Initially, these experiments tested flies (n=18, 50 walking responses per fly) with pulse trains using 50, 100, 500 and 1000 ms interpulse intervals (IPI’s). A second cohort of flies (n=13, 38 responses per fly) was later tested using stimuli with 200, 300, and 400 ms IPI. Results for these two sets of flies are presented separately.

Comparison of responses in ‘naïve’ vs. ‘experienced’ flies

Figure 10A-B illustrates averaged responses (for all flies) in steering and forward velocity from flies in response to the test pulse-trains of varying IPI alone (i.e. not preceded by a cricket chirp). Responses are plotted separately for naïve (Figure 10A) and experienced flies (Figure 10B) in Group 1 (50, 100, 500, and 1000 ms IPI) and Group 2 (200, 300, and 400 ms IPI). For the shortest IPI tested (50 ms), there was a weak walking response (steering and forward) in naïve flies. This was not affected by experience. For intermediate IPI’s (200 - 400 ms), there was some evidence of sensitization in forward velocity, with small velocity increases corresponding to the timing of pulse onsets, leading to some overall differences in steering and forward velocity. Namely, post-protocol responses in average steering velocity were

significantly increased in response to 400 ms IPI pulse-train (Group 2: 0.32 + 0.18 cm/s vs. - 0.06 + 0.07 cm/s, paired t-test: t (24)= 1.98, p<0.05); and post-protocol responses in average forward velocity were significantly greater for 200 ms IPI (Group 2: 0.32 + 0.17 cm/s vs. 0.01 + 0.01 cm/s, paired t-test: t (24)= 1.80, p<0.05), and 300 ms IPI (Group 2: 0.31 + 0.18 cm/s vs. - 0.02 + 0.03 cm/s, paired t-test: t (24)= 1.86, p<0.05). For long IPI’s (500, 1000 ms), there were no responses in either naïve or experienced flies. All t-tests were one-tailed.

Responses to pulse-trains following a chirp

Two cohorts of female O. ochracea were presented with paired acoustic stimuli consisting of the initial cricket chirp followed by pulse-trains of varying IPI (Figure 10C, see Methods). Responses to pulse-trains were quantified by measuring changes in velocity at pulse onsets (deltaV), for both steering (dSV) and forward velocity (dFV), and the sustained baseline (baseV) for steering (bSV) and forward velocity (bFV) (Figure 5, see Methods).

Steering velocity (Figure 10C top, and Figure 11 left). In control responses (chirp alone), steering velocity decays to baseline within ~300 ms of chirp offset. In some cases there is a brief renewal of steering velocity afterward (similar to the pattern for forward velocity, but weaker). Pulse trains with short IPI’s (50 and 100 ms) following a chirp, slow the decay of steering velocity, such that it doesn’t reach baseline until ~4s after chirp offset. For long IPI’s (500 and 1000 ms), the delay for the initial pulse is long enough for steering velocity to decay to baseline, then each pulse elicits a steering response (spike in steering velocity, Figure 10C, top left). The amplitude of these steering spikes decays slightly over the 10 s stimulus duration. Intermediate IPI’s (200 - 400 ms, Figure 10C, top right) show a pattern similar to long IPI’s: discrete velocity spikes that decay over time and an elevated baseline velocity (with the weakest effect for 200 ms).

Statistical analyses of steering responses at pulse onsets (dSV) showed significant effects of both IPI and time (Figure 12A left, Table 2). Pairwise comparisons indicated that the amplitude of steering responses (dSV) was significantly greater for the longer IPI’s (500 and 1000 ms), and dSV declined with successive pulse presentations for all IPI’s. For short IPI’s (50 and 100 ms), dSV responses were not different from control. For intermediate IPI’s (200, 300 and 400 ms), there was a graded effect with increasing IPI; amplitudes of dSV responses increase with IPI and slope of the decay also of dSV amplitude increases with IPI (Figure 12A right, Table 3).

Average dSV per pulse (combined average for all pulses in respective IPI conditions) increased progressively with longer IPI’s, and became considerably larger than equivalent measures obtained from naïve flies (Figure 14A).

There were also significant effects of IPI and time on baseline steering velocity (bSV), as well as a significant interaction between these factors (Figure 12B left, Table 2). Base steering velocity was significantly greater than control for all IPI’s and decayed over successive pulse presentations. Pairwise comparisons indicated that the significant interaction was due to a steeper slope for 100 ms IPI, that is, bSV decayed most rapidly at this IPI. For intermediate IPI’s, results were similar to dSV, although somewhat weaker: bSV remained above control for all IPI’s but there were no differences among IPI treatments (Figure 12B right, Table, 3). With average bSV, responses were fairly steady across all IPI’s, with the intermediate values (200- 300 ms) appearing slightly larger (Figure 14B).

Forward velocity (Figure 10C bottom, and Figure 11 right). Forward walking was maintained during a pulse train following offset of an attractive chirp at all IPI’s tested. This renewal of walking after chirp offset was most strongly enhanced by shorter pulse rates, and decayed approximately 1.5 s after chirp offset. For the longest IPI’s (500 and 1000 ms), the decay of forward velocity was interrupted by renewed increases of walking speed with each pulse, these responses were maintained throughout the 10 s duration of the stimulus, and the amplitude of pulse responses was greatest for the longest IPI (1000 ms). Intermediate IPI’s (200-400 ms) also show both of these effects – strong enhancement of the post-chirp renewal of forward velocity and velocity spikes in response to each pulse that decay over time (although velocity remains elevated over control levels throughout the 10 s stimulus duration).

There were significant differences in dFV responses for different IPI’s (Figure 13A left, Table 2). While the main effect of time was not significant, pairwise comparisons indicated that at longer IPI’s (500 and 1000 ms), dFV responses were significantly elevated over control, and increased with successive pulse presentations. For short IPI’s (50 and 100 ms), dFV responses were not different from control. Responses to intermediate IPI’s were similar to long IPI’s – significant increases in forward velocity (dFV) at pulse onsets, with dFV amplitude increasing with greater IPI (Figure 13A right, Table 3). Average dFV responses were similar to average dSV – steadily increasing responses with longer IPI’s, and more pronounced compared to equivalent naïve fly responses (Figure 14C). 26

Baseline forward velocity (bFV) remained significantly elevated over control levels for all IPI’s and declined with successive pulse presentations at a similar rate for all IPI’s (Figure 13B left, Table 2). For intermediate IPI’s, bFV was significantly elevated for all IPI’s with no differences among IPI’s (Figure 13B right, Table 3). Average bFV was quite similar across all IPI’s, and above respective responses from naïve flies (Figure 14D).

Lateral and forward deviation was more pronounced in the intermediate and longer IPI’s (lateral: ~10 cm, forward: ~20 cm), compared to the short IPI’s (lateral: ~5 cm, forward: ~17 cm) (Figure 15, left graphs). By the end of the 10 s data capture window, flies ended up at a location approximately between 5-7 cm away from the active speaker for the intermediate and long IPI’s, and approximately 10 cm away for the short IPI’s (Figure 15, right graphs).

2.5 Discussion

In this study, I investigated how the phonotactic walking behaviour of Ormia ochracea was affected by dynamic auditory stimuli. Extending prior work on temporal pattern preferences in O. ochracea (Lee. 2012) in their quest to exploit male cricket hosts, I combined various non- attractive stimuli (noise bursts and pulse trains of varying IPI’s; irrelevant in eliciting phonotaxis) with preceding attractive cricket chirps (response evoking) in order to determine how prior sensory experience would influence responses to subsequent stimuli separated in time and space. For both types of test stimuli used in this study, walking responses (steering and forward velocity) were augmented following the initial cricket chirp compared to when these test stimuli were presented on their own. Furthermore, there was a longer-lasting component of this elevated activity, in that responses to test stimuli alone remained elevated at the end of the experiment. In addition, different temporal and spatial combinations of cricket chirp and the onset of subsequent test stimuli revealed effects (changes in phonotactic responsiveness) with shorter time-courses. The overall sensitization effect evoked by the initial cricket chirp on responses to subsequent test stimuli is discussed first, followed by the specific differences observed for each of the unique noise burst and IPI pulse train combinations tested.

The overall sensitization effect

Using a phonotaxis performance index comprising total distance walked, peak steering velocity, and localization accuracy, Lee (2012) found that O. ochracea showed strongest phonotaxis towards cricket calling songs with pulse periods of 15-20 ms. Based on this, it was not surprising that naïve flies showed no phonotactic responses towards the noise burst (devoid of any temporal structure) , and only a slight response shown for the 50 ms IPI pulse train (i.e. a pulse period of 60 ms, the shortest period of the test pulse-trains, but well above the preferred range). On their own, therefore, it is apparent that presentation of the noise burst and slow (relative to host cricket song) pulse-trains used in this study are normally insufficient to elicit phonotaxis in flies. This is markedly different, however, when these stimuli follow an attractive cricket chirp. Under this condition, flies visibly orient and walk towards the test stimuli. Because of the particular approach of presenting test stimuli with varying temporal delay from chirp offset (and randomizing the order), no associations between the chirp and test stimuli were suggested (i.e. this was a non-classical conditioning protocol). Therefore, the evident responses made by flies towards the otherwise non-attractive test stimuli when following the chirp is best understood through the process of sensitization, with the initial chirp acting as a sensitizing stimulus (SS, Dethier et al., 1965; McGuire, 1983; McGuire and Tully, 1986; Hammer et al., 1994; Anton et al., 2011).

In order to understand the role of the cricket chirp as a SS, insight may be gained from other invertebrate studies. From prior work performed on field cricket phonotaxis, Hedwig and Poulet (2005) found that while female crickets only showed minor walking responses towards a series of various behaviourally irrelevant sound pulses, stimulation with the calling song of conspecific male crickets resulted in clear phonotaxis, with robust steering movements oriented towards the sound source. Subsequently, this increased steering behaviour was momentarily maintained towards the test pulses when inserted at the end of the calling song stimulation. The authors attributed these findings to the role of the male calling song in activating a recognition process, which in turn modulates and facilitates steering responses in female crickets, allowing them to occur more easily, even towards the non-attractive test pulses. Similarly, this was suggested as the basis for the proboscis extension reflex (PER, Dethier et al., 1965; McGuire, 1983; McGuire and Tully, 1986 , also see Introduction) in that initial presentation of the sucrose food activates a recognition process, which in turn modulates a general, unselective behavioural

response (the PER) in blowflies towards non-sucrose food (water) (Hedwig and Poulet, 2005; Dethier et al., 1965; McGuire, 1983; McGuire and Tully, 1986). It seems likely that the 500 ms cricket chirp preceding the noise or pulse-train test stimuli in this study is operating in a similar manner for gravid female O. ochracea, as the male cricket calling song operates for female crickets, and the sucrose food for blowflies. That is, the calling song of a suitable host may activate a recognition process in flies, inducing a heightened excitatory state such that responses towards subsequently presented non-attractive stimuli are facilitated compared to responses from stimuli in the absence of the initial chirp. In each of these examples, stimuli that were otherwise incapable of initiating the described behaviours subsequently were shown to elicit responses when the naturally evoking stimulus preceded their presentation. When attempting to pinpoint the location of the excitatory state mediating the PER in blowflies, Dethier et al. (1965) identified the CNS as the likely source. This was largely based on their accumulation of electrophysiological evidence which suggested that application of the water drop to one labellar hair (preceded by application of sugar drop to another hair), and the resulting PER, could not be explained by receptor activity at the peripheral level.

While difficult to make inferences from strictly behavioural measurements, properties of the auditory system in O. ochracea may help narrow where such an excitatory state may be located. The majority of afferent auditory receptors innervating the tympanal membrane in O. ochracea are of type 1 (Oshinsky and Hoy, 2002). These receptors are characterized to respond to a single suitable sound pulse with a single spike (action potential) irrespective of duration, and intensity of the sound, and possess a refractory period of approximately 4 ms (Mason et al., 2001; Oshinsky and Hoy, 2002). Therefore, any auditory receptor activity elicited by flies towards the pulses of the initial cricket chirp would have ceased firing by the time the test stimuli were presented. From this, it suggests that the increased responsiveness of flies towards the test stimuli cannot be due to the temporal summation of afferent receptor responses elicited by the chirp and test stimuli. That is, there was no residual receptor activity from flies being exposed to the cricket chirp, that would contribute and carry over upon test stimulus presentation that can account for the increased responsiveness observed. What seems more likely, is that the increased responsiveness towards the test stimuli reflects an excitatory change in the CNS of flies, and it would be the job of future experiments to pinpoint precisely the mechanism underlying this activity. The proposed central excitatory state (CES) described in O. ochracea and discussed in blowflies are in contrast with peripheral sensitization effects, where a 29

sensitizing stimulus would act to reduce the threshold, and increase the gain of sensory transduction at the level of primary afferent receptors (Woolf and Walters, 1991). While electrophysiology experiments would be needed to confirm the absence of peripheral sensitization in O. ochracea under this same experimental protocol, the primary features of auditory afferent receptor physiology in flies suggests a central excitatory origin of sensitization.

In addition to increasing the responsiveness of flies towards the test stimuli proceeding the cricket chirp, it is likely that this heightened central activity was maintained towards the end of the main experiment. This is suggested by the fact that O. ochracea demonstrated clear phonotaxis responses towards the noise burst presented alone post-protocol, and maintained significantly greater average steering and forward velocity for a number of the IPI pulse train combinations compared with naïve fly responses.

Combined stimuli and the time-course of sensitization effect

When looking beyond the general increase in responses towards the test stimuli following the chirp compared to without, specific differences between test conditions were observed. Two aspects of the sensitization effect are relevant to interpreting these results: (1) the amplitude, i.e. the initial level of sensitization attained, and (2) the duration for which sensitization effects last. Initiation of behavioural sensitization effects are often dependent on the quality and intensity of the sensitizing stimulus (SS) under consideration (i.e. must be of appropriate type, and sufficient strength to elicit a particular response) (Hammer et al., 1994). In this study, the initial cricket chirp was of the appropriate type (modeled after Floridian O. ochracea local hosts; G. rubens) and intensity (76 dB sound level, 500 ms duration) to elicit reliable phonotactic walking behaviour in flies. Considering that these parameters were held constant in both noise and pulse train experiments, and between individual trials, it may be inferred that flies were sensitized to the same level upon initial chirp presentation (i.e. presentation of the chirp activates a central excitatory state in flies above threshold for phonotaxis to occur). Granted, differences in internal motivational states of flies may contribute to some flies being more/less influenced by sensitization than others, but this could only be addressed by obtaining results from a suitable sample size and averaging responses across flies. Therefore, the differential responses elicited by flies towards the various test stimuli are not likely related to differences in the absolute level of sensitization attained upon hearing the chirp (which we assume to be consistent, option 1), but rather are related to changes in sensitization effects over time (option 2), where in this study, 30

may be attributable to the different temporal (and spatial) relationships between chirp offset and test stimulus onset.

Unlike the initiation of sensitization, parameters that dictate how long these effects remain active are much less straightforward, as this depends on the particular mechanism(s) mediating sensitization. For example, peripheral sensitization mediating the defensive withdrawal reflex in the marine snail Aplysia has been localized to presynaptic connections, where persistence of the reflex is thought to be due to prolonged activity of adenylate cyclase in the synapse (Greenberg et al., 1987; Krasne and Glanzman, 1986). As long as levels of the neurotransmitter remain high, sensitization in Aplysia would be expected to remain active. Similarly, central sensitization mediating feeding behaviour in locusts has been shown to be tied with phagostimulant concentration, where high levels would maintain the central excitatory state above threshold, resulting in sustained feeding behaviour until inhibitory signals brought on by satiation reduce the excitatory state below threshold (Chapman, 2009). These examples suggest that as long as sensory stimuli mediating a specified behaviour are available, and the behaviour meets the immediate needs of an organism at the particular time, then the behaviour would likely be sustained indefinitely.

Based on the aforementioned studies in Aplysia and locusts, one would expect that continued stimulation of O. ochracea with a suitable host calling song would elicit sustained phonotactic walking activity, for as long as the stimulus was presented, and the need to find a host being upheld. Notably, this has been indirectly observed in the lab where cricket song durations of upwards of 10 s have been shown to elicit continuous, and constant levels of walking activity in flies for the entire duration of stimulus presentation (Figure 16, unpublished data). By superimposing the 10 s cricket song steering velocity trace over the pulse train test stimuli used in this study, it is apparent that the test pulses were not able to sustain the same absolute level of steering as the full cricket song. To a certain extent, this reflects a similar condition as was seen in locust feeding behaviour. That is, without continued input of sensory information meditating host seeking behaviour via presentation of non-attractive test stimuli (behaviourally irrelevant for the purpose of seeking out a host), walking activity was not sustained in the presence of the test stimuli, just as satiation signals failed to maintain feeding behaviour in locusts.

In this study, two main measures of phonotaxis were quantified: steering and forward velocity; and for each of these parameters I measured both the velocity level sustained between sound 31

pulses (baseV) and the change in velocity at the onset of sound pulses (deltaV). Two general observations can be made from these results. 1) Some effects of stimulus timing were consistent with test stimuli being either grouped with the preceding chirp (i.e. a continuation of a single continuous stimulus) or segregated from it (i.e. a novel, separate stimulus), depending on the duration of the interval between chirp offset and test stimulus onset. 2) Steering and forward velocity were sometimes affected differentially. In general, forward velocity was more strongly sensitized and remained elevated over control conditions for a wider range of test stimuli than was the case for steering velocity.

Auditory grouping vs source separation

For short delays between chirp and test stimulus, flies’ responses are consistent with auditory grouping, in that phonotaxis declines with the cessation of the chirp temporal pattern similarly to controls (i.e. chirps presented alone) and there is no evidence of a renewed response at the onset of the test stimulus. For the noise experiment, the decay in steering velocity at noise onset for the 10 and 100 ms gap delays was not significantly different from responses in the complete absence of the noise altogether (control trace), and was not affected by the location of the noise source (Figure 8). This would imply that noise presented in close temporal succession with the chirp would not elicit steering in flies, but only sustained, generalized walking in the forward direction (notice positive delta forward velocity responses, Figure 8B). For the pulse train experiment, the average delta steering and forward velocity per pulse for the 50 and 100 ms IPI’s were very similar to responses shown by naïve flies (absence of initial chirp) (Figure 14A, 14C), and over time, were not different than in the absence of the pulses altogether (control trace) (Figure 12A, 13A).

For a longer temporal separation between offset of the chirp and onset of the test stimuli flies responded to the test stimulus (noise burst or pulse train) as a novel stimulus that elicited a renewed phonotactic response. This was seen in the noise experiment, where flies showed significant changes in steering velocity at the onset of noise bursts following 500 and 1000 ms delays, and these were significantly affected by speaker location (Figure 8, Table 1). In the pulse train experiment, for delays of 200 ms or longer, delta steering and forward velocity were significantly above controls, and increased progressively with larger IPI values (Figures 12A, 13A, 14A, 14C, Table 2). Similarly to the rapid rise in steering and forward velocity that occurs towards the initial chirp, flies were performing true, oriented phonotactic responses towards 32

these temporally isolated stimuli. This is further emphasized by the fact that, in the noise experiment, the orientation of delta steering responses changed with the location of the speaker broadcasting the noise burst. Although the negative delta steering in the opposite speaker condition did not represent a full reversal of direction (steering responses were too transient), but they nevertheless show that flies were attending to and orienting towards the noise from the opposite speaker. These findings were particularly important in the pulse train experiment, which demonstrate that with these more isolated pulses, flies demonstrated reactive steering towards each individual pulse as observed with the spike like peaks, beginning with the 200 ms IPI condition, and increasing in amplitude up to the 1000 ms condition (Figures 10C, 14A, 14C).

The absence of discrete responses to short-IPI pulses appears to be due to temporal proximity with preceding chirp, rather than an effect of simply how much time has elapsed, otherwise would expect either responses to grow over time as sufficient time elapses to approach the larger steering amplitudes in longer-IPI treatments, or a single onset-response when sufficient time has elapsed (as in noise experiment) with on-going responses suppressed by absence of attractive temporal pattern (pulse rate). Instead, short-IPI stimuli (and short-delay noise) do not elicit a novel response and do not sustain the on-going phonotactic response because they lack the appropriate temporal pattern. With a longer delay relative to chirp offset (200 ms or greater), flies treat subsequent sounds as novel stimuli, and individual sound pulses each elicit a sensitized steering response. For comparison, Figure 16 shows plots of steering velocity for control and pulse-train stimuli, along with the steering velocity plot for an attractive cricket chirp of the same total duration (10 s). A sustained cricket chirp elicits sustained steering, with no pulse-by-pulse response. For short-IPI pulse-trains, no pulse-locked steering responses are evident, whereas longer-IPI pulse-trains show discrete steering spikes with little diminution over time. This is unlike the case in cricket phonotaxis where females are observed to elicit transient responses to individual pulses of the male song (Hedwig and Poulet, 2004; 2005).

There were some differences in the temporal effects between delta and baseline velocity. Baseline velocity may be considered a measure of the extent to which the original phonotactic response (i.e. to the initial chirp) is maintained during the subsequent pulse-train. Another possibility is that baseline velocity may remain elevated because there is insufficient time between pulses for the flies to decelerate to a complete stop, but some details of the data are

inconsistent with this. Specifically, baseline velocities decay with a similar time-course for all IPI’s (Figures 12B, 13B) including 1000 ms which would clearly allow sufficient time for velocity to fully decay between pulses. This prolongation of the initial phonotactic response may be related to the delay-dependence of steering responses. Sustained phonotaxis, indicated by a slowly decaying walking response after chirp offset, may represent a continued attention to the original stimulus that prevents responses to novel stimuli. As this decays over time, responses to novel stimuli (at longer delays) are released. Figure 14 shows velocity measurements averaged over the entire test stimulus duration. This ignores changes over time and represents a measure of the overall amplitude of steering responses under each stimulus condition. These plots show distinct patterns for baseV and deltaV: baseV is not affected by IPI; deltaV (both forward and steering) increases with IPI. This is consistent with the possibility that baseV represents the underlying time-course of the initial phonotactic response.

From an ecological standpoint, these findings may suggest a mechanism to allow flies to maintain phonotaxis to an intermittent source. Having initially oriented to an attractive sound source (cricket chirp), sustained responsiveness could allow flies to incrementally continue to approach the source despite interruptions in the signal. While this could, under some circumstances, result in responses being made towards non-attractive stimuli as well, it may represent a reliable rule-of-thumb, especially if since flies will likely orient initially to the loudest detectable source. Female crickets are subject to a similar potential constraint based on the elevated state of responsiveness incurred upon pattern recognition of male calling songs (Hedwig and Poulet, 2005). Considering the finding that flies, like female crickets, require sustained auditory input of cricket song to maintain directional information (as observed by progressive decrease in steering towards non-attractive stimuli following chirp offset) (Hedwig and Poulet, 2005), however, ensures that such responses would only be transient in nature, and eventually cease to elicit responses altogether. If, however, such non-attractive test stimuli were replaced with additional presentations of the cricket chirp, a more likely scenario to be expected in nature, then the benefit of flies maintaining a sensitized state towards perception of auditory cues amidst the possibility of signal degradation or interruption in the environment is understood. Specifically, it would allow flies to maintain a heightened state of arousal, and be more receptive to discontinuous host cues caused by interruptions from other sounds, or changes in locations of hosts.

Forward vs steering velocity

Differences in the results for forward vs steering velocity suggest that, overall, forward velocity was more strongly sensitized and remained elevated over control conditions for a wider range of test stimuli than was the case for steering velocity. This is reflected in the stronger overall elevation of baseV for forward relative to steering velocity (compare Figures 14B, 14D). This may suggest that the most robust components of the sensitization response represent a generalized, or non-directed, walking response, and that a stronger stimulus is required to elicit an oriented walking response that includes a change in direction. Again, in the context of sound source localization under uncertain or intermittent conditions, directional information may be less consistent and a stronger tendency to continue walking in the same direction may be a more reliable localization strategy. Such a mechanism could tend to minimize the effect of transient extraneous sound sources.

In conclusion, the findings of my thesis demonstrate that a suitable cricket chirp modeled after the preferred hosts of Floridian O. ochracea induces a state of sensitized excitatory activity in flies (most likely located in the CNS), that mediates improved walking phonotaxis behaviour towards otherwise non-attractive/behaviourally irrelevant test stimuli. With increased temporal separation between chirp offset and test stimulus onset, flies are more likely to elicit responses more similar to the initial chirp, and in turn, sustain a directed trajectory towards a lateral speaker source, even when the sounds are non-attractive. From a psychophysics approach, these findings are important because they illustrate how flies in a sensitized state may be coerced to track non-attractive stimuli in a way very similar to their tracking of a proper host song. From a naturalistic approach, the results suggest sensitization as a means for flies to demonstrate experience dependent plasticity in their pursuit of a cricket host in the environment.

Furthermore, my results suggest that there may be two mechanisms that modify fly behavioural responses as a result of a preceding acoustic stimulus: (1) an attention-like mechanism that reduces responses to novel stimuli closely following an attractive phonotactic stimulus; and (2) a sensitization, or elevation of overall responsiveness. My results also suggest that these two processes operate with distinct time-courses. These results could be the basis for studies of the underlying neural correlates of central auditory processing in O. ochracea.

Figure 2. Overview of auditory stimuli and protocol used in noise sensitization experiment. The main protocol was comprised of paired stimuli including an initial cricket chirp (blue), followed by a subsequent noise burst (red) at varying intervals after chirp offset (A). In one group of flies the noise burst followed from the same speaker as the chirp (B), while in another, the noise was presented from the opposite speaker (C).

Figure 3. Overview of auditory stimuli used in the pulse train sensitization experiment. Main protocol was comprised of paired stimuli including an initial cricket chirp (blue), followed by subsequent pulse trains of varying interpulse intervals (IPI’s, red) for the remainder of the 10 s stimulation time window.

Figure 4. Experimental apparatus used for behavioural phonotaxis experiments in Ormia ochracea. Synthetic cricket songs presented from the speakers located 23 cm and + 45o from the centre of the trackball simulated presence of singing hosts.

Figure 5. Velocity measurements. In the noise experiment (A), delta velocity was found by subtracting the average velocity over a 46 ms window, 100 ms after noise burst onset (b), with the average velocity over a 46 ms window immediately after onset (a) (onset=red line). In the pulse train experiment (B), base velocity was found as the average over a 46 ms window just after pulse onset (a). Delta velocity was found as the maximum over the respective pulse period (b) for each IPI subtracted by the base velocity.

Figure 6. Time indices corresponding to velocity calculations in the pulse train sensitization experiment. Vertical purple lines indicate individual pulse onsets for the 1000 ms IPI condition. All other IPI’s including 200-400 ms (not shown) were subsampled at these equivalent time periods for calculation of delta and baseline velocities to establish a common time scale.

Figure 7. Velocity versus time graphs for the noise sensitization experiment. Compared to responses elicited by naïve flies towards the initial cricket chirp (A, left), average steering and forward velocity were significantly greater following the main protocol (A, right). This was also the case for noise burst presentation (compare B, left with B, right). During the main protocol (C), differential walking patterns were observed depending on the unique temporal (10, 100, 500, and 1000 ms) and spatial (same speaker n=13, opposite speaker n=17) relationships between chirp 41 offset and noise burst onset.

Figure 8. Delta velocity responses in no ise experiment. Flies oriented in the direction of the active speaker presenting noise for the long gap delays (500 and 1000 ms), whereas steering for the short delays were no different compared to control (A). Generally, noise onset elicited significantly improved delta forward responses for all gap delay conditions (B). Error bars indicate SEM.

Figure 9. 2-D walking paths and lateral deviation versus time graphs for the noise sensitization experiment. Speaker symbols indicate approximate lateral distance of speakers from the midline axis of flies (~ 16 cm). From their starting position at the origin (0,0), flies deviated ~ 5 cm laterally, and ~12 cm forward for all delays. Responses are presented separately for stimulus presentations from the L and R speaker (left graphs). Over a 10 s data capture window, flies presented with a single onset of a noise burst test stimulus from the same speaker as an initial 500 ms cricket chirp (A) or opposite (B) will end up at a similar, and distant location away from the initial active speaker. Responses here are averaged from both speakers (right graphs). Control (black) traces represent responses to the 500 ms cricket chirp alone.

Figure 10. Velocity versus time graphs for the pulse train sensitization experiment. Responses from naïve flies (A) was greatest for the 50 ms IPI condition. Post-protocol responses were particularly improved for the intermediate IPI’s. Responses to the long IPI’s were negligible both for naïve and experienced flies (B). Vertical dashed lines indicate precise onset of the first test pulse in each of the pulse train stimuli (C). Differential walking phonotaxis patterns were observed for the different pulse trains used, with steering and forward velocity being sustained longer in the intermediary (200-400 ms) and long (500, 1000 ms) IPI conditions compared to the short IPI’s (50, 100 ms). 44

Figure 11. Close up look at steering and forward velocities beginning from pulse onset for each respective IPI condition, until the end of the 10 s data capture time window.

Figure 12. Delta and baseline steering veloci ty over time. Amplitudes of dSV were significantly greater than control for all IPI’s except 50 and 100 ms (A). Baseline responses were significantly above equivalent control values, and showed little difference among the various IPI’s, except for the 100 ms IPI which had a slightly steeper slope (B). Over the data capture window, dSV and bSV both decreased progressively over time.

Figure 13. Delta and baseline forward velocity over time. Similar to steering responses, amplitudes of dFV were significantly above control for all IPI’s except 50 and 100 ms which were not different than in the absence of test pulses altogether (A). For baseline responses, all IPI’s were above equivalent control values, and were not different between each other (B).

Figure 14. Average delta and baseline velocities (steering and forward) per pulse (averaged across all pulses for each IPI). With longer IPI’s, delta responses increased progressively and became much larger when pulses followed the initial chirp (blue) compared48 to responses from naïve flies (red). Baseline responses were fairly similar across IPI conditions. Error bars indicate SEM.

Figure 15. 2-D walking trajectories and latera l deviation versus time graphs for the pulse train sensitization experiment. Speaker symbols indicate approximate lateral distance of speakers from the midline axis of flies (~ 16 cm). Flies undergo a greater extent of lateral (~10 cm) and forward (~20 cm) deviation with the intermediate and long IPI’s compared to short IPI’s (lateral ~5 cm, forward ~17 cm) (responses shown for both L and R speakers, left graphs). With repeated onset of test pulses following the chirp, flies demonstrate sustained walking activity, most strongly enhanced with increasing IPI. After 10 s, flies end up at a location much closer to the speaker than in the shorter IPI conditions (~ 6 vs 10 cm away, respectively) (averaged responses from both speakers, right graphs). Control (black) traces represent responses to the 500 ms cricket chirp alone. 49

Figure 16. Steering velocity versus time graph. In response to a cricket chirp with duration of 10 s, flies elicit a sustained level of steering velocity over the full interval (gray trace, data obtained from a separate experiment, n=8). The sudden drop in steering occurs only with chirp offset at the 10 s mark. All non- gray traces are the steering velocity responses from the pulse train sensitization experiment that have been superimposed on the same graph. None of the IPI’s tested sustain the same level as steering as the continuous cricket chirp.

Table 1. Noise following chirp.

Delta Steering Velocity

df Sum Sq Mean Sq F P

Delay 4 31.29 7.822 17.616 <0.0001

Speaker 1 12.49 12.491 28.132 <0.0001

Delay*Speaker 4 8.58 2.145 4.831 <0.002

Residuals 139 61.72 0.444

Delta Forward Velocity

df Sum Sq Mean Sq F P

Delay 4 15.91 3.977 9.723 <0.0001

Speaker 1 1.61 1.614 3.946 <0.05

Delay*Speaker 4 0.63 0.157 0.384 0.82

Residuals 139 56.85 0.409

Table 2. Pulse-trains following chirp – long and short IPI.

Delta Steering Velocity

df Sum Sq Mean Sq F P

Time 1 0.162 0.162 29.84 <0.0001

IPI 4 13.572 3.393 625.75 <0.0001

Residuals 39 0.211 0.005

Base Steering Velocity

df Sum Sq Mean Sq F P

Time 1 4.341 4.341 99.316 <0.0001

IPI 4 0.896 0.224 5.123 <0.003

Time*IPI 4 0.721 0.180 4.123 <0.008

Residuals 35 1.530 0.044

Delta Forward Velocity

df Sum Sq Mean Sq F P

Time 1 0.001 0.0006 0.034 0.854

IPI 4 12.483 3.1208 172.626 <0.0001

Time*IPI 4 0.519 0.1297 7.175 <0.003

Residuals 35 0.633 0.0181

Base Forward Velocity

Df Sum Sq Mean Sq F P

Time 1 32.83 32.83 105.668 <0.0001

IPI 4 10.42 2.61 8.385 <0.0001

Time*IPI 4 1.35 0.34 1.088 0.38

Residuals 35 10.88 0.31

Table 3. Pulse-trains following chirp – intermediate IPI.

Delta Steering Velocity

df Sum Sq Mean Sq F P

Time 1 4.242 4.242 156.4 <0.0001

IPI 3 13.873 4.624 170.5 <0.0001

Time*IPI 3 1.171 0.192 14.4 <0.0001

Residuals 131 3.552 0.027

Base Steering Velocity

Df Sum Sq Mean Sq F P

Time 1 5.855 5.855 96.975 <0.0001

IPI 3 14.183 4.728 78.305 <0.003

Time*IPI 3 0.577 0.192 3.184 <0.0001

Residuals 131 7.909 0.040

Delta Forward Velocity

Df Sum Sq Mean Sq F P

Time 1 1.524 1.524 37.67 <0.0001

IPI 3 10.403 3.468 85.73 <0.0001

Time*IPI 3 1.899 0.633 15.65 <0.0001

Residuals 131 5.299 0.040

Base Forward Velocity

Df Sum Sq Mean Sq F P

Time 1 55.01 55.01 296.59 <0.0001

IPI 3 52.50 17.50 94.36 <0.0001

Time*IPI 3 5.66 1.89 10.18 <0.0001

Residuals 131 24.30 0.19

Chapter 3 General Discussion

Ecological implications of findings

Based on the findings obtained in this study, it appears that O. ochracea are subject to similar sensitization mechanisms observed in other model sensory systems, particularly female crickets. The noise burst and pulse trains of varying IPI’s used in this study are not discrete auditory stimuli that O. ochracea would regularly encounter in nature in the same way as they were presented in the laboratory. Rather, they represent an experimental psychophysics manipulation aimed to deduce how flies would respond when initially preceded with a chirp modeled after their primary local hosts. The finding that the flies would respond to such behaviorally irrelevant stimuli following the chirp might, at face value, seem like a detrimental activity. However, the more likely scenario O. ochracea would face in nature, is for the subsequent sounds to be further presentations of host auditory cues. The primary local host of Floridian O. ochracea are the G. rubens, and this species of crickets has a trilling pattern. Also, it is likely that during advertisement calls male crickets change locations as they try to maximize success in attracting a female (Mueller and Robert, 2001; Shaw et al., 1981). With this is mind and considering that there are many other potential sources of auditory cues available in the surroundings, maintaining an active excitatory state brought on by the presence of a singing potential host would serve as a mechanism enabling flies to maintain attention and/or responsiveness towards them amidst changes in location of hosts, signal degradation, and other interfering stimuli in the environment. The process of O. ochracea parasitizing a cricket host usually begins in the air before flies land on the ground, and complete the remainder of the task via walking phonotaxis (Mueller and Robert, 2001; 2002). Therefore, it is evident that a single chirp event from a singing male cricket would likely not be enough for the flies to successfully localize their position in one shot. Rather, O. ochracea are likely engaging in a stepwise process where they depend on sustained auditory input for the purposes of regularly updating their trajectory in targeting a host. It would be extremely detrimental for flies to treat each occurrence of a cricket’s song as if hearing it for the first time. A much more beneficial strategy, and one that O. ochracea are likely using, is to make use of their prior sensory experiences (experience

dependent plasticity) such that subsequent auditory cues from their hosts are greeted by a fly with a heightened level of attention, as mediated via sensitization.

Future work

There are a number of different avenues for which future work on understanding how O. ochracea process dynamic auditory cues, may be directed. This study, while certainly capable in offering insights into the possible perceptual mechanisms underlying processing of dynamic auditory stimuli, can only go so far from a behavioural perspective. As a result, one obvious future direction would be to begin neurophysiology experiments in hopes of identifying the specific neural correlates responsible for the unique behavioural responses observed via phonotaxis. More specifically, it would be interesting to determine the particular mechanism(s) underlying the central excitatory state presumed to exist in O. ochracea, and hence, identify pathways important for the elucidation of sensitization.

An additional avenue of investigation would be to incorporate a behavioural genetics approach into understanding the role of attention and motivation upheld by flies as they try to locate hosts. As inferred previously, different motivational states within individual flies may result in differential predispositions towards sensitization. Aside from certain characteristics of stimuli that appear to elicit predictable, seemingly automatic reflex like behaviours in insects, it is not unreasonable to infer that some forms of behavioural plasticity are dictated by changes in the central nervous system, and a prime candidate to begin testing this in O. ochracea, would be the so called ‘fight or flight’ hormone in insects, octopamine (Roeder, 1999; Adamo et al., 1995). Thought to be involved in modulating nearly all physiological processes that occur in invertebrates, octopamine may be an ideal candidate to quantitatively assess host seeking behavior in O. ochracea (Roeder, 1999). High levels of octopamine have been shown to correspond to activities categorized as stressful, or highly demanding such as the dance language communication system in honeybees, lunging aggressive behaviour in Drosophila, and escape behaviour in crickets (Barron et al., 2006; Hoyer et al., 2008; Adamo et al., 1995). In addition, work by Krasne and Glanzman (1986) found that exogenous application of octopamine may help mediate sensitization in crayfish by increasing the excitability of lateral giant fiber mediated escape responses towards test shocks.

Based on this, one may infer that upon hearing the calling song of a potential host in O. ochracea, octopamine may play an important role in mediating levels of motivation and activity during phonotaxis, as flies navigate towards their target. This may be investigated in future experiments by injecting various concentrations of octopamine into gravid female O. ochracea, and subjecting them to the same behavioural experiments tested in this study. One may predict that flies subjected to higher levels of octopamine may exhibit more sustained responses towards the non-attractive stimuli, even above those maintained by sensitization alone as found in this study. Similarly, aside from investigating the behaviour of gravid females as a whole, analogous experiments may also be conducted on the larvae of gravid females themselves. Very little is known on the activity of larvae upon being released from gravid females and their path to making contact with a potential host. From personal observations in the lab, however, after dissecting larvae from gravid females and separating them from their enclosed protective membrane, larvae are known to migrate away from the center location, possibly a behaviour aimed to maximize success in making host contact. One possible experiment, therefore, may be to compare rates and magnitudes of dispersion in larvae who have been exposed to octopamine compared to untreated larvae. Along with studies on the adult flies themselves, this would be a prime way to begin to understand the natural ecology of O. ochracea, of which, much information is lacking.

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