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

by

Dean Koucoulas

A thesis submitted in conformity with the requirements for the degree of Master of Science

Cell and Systems Biology University of Toronto

© Copyright by Dean Koucoulas 2013

ii

Investigating Perception Under Dynamic Auditory Conditions in

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.

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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

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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

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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

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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

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

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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

5

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

7

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.

9

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

11

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.

12

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;

13

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

15

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

16

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.

17

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.

18

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

19

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

20

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

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,

23

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

24

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

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.

27

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

28

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