Transmission of Chemosensory Information in Drosophila Melanogaster: Behavioural Modification and Evolution

Transmission of chemosensory information in Drosophila melanogaster: Behavioural modification and evolution

A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in Evolutionary Biology in the School of Life Sciences

2014

Rebecca Lockyer

Contents

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List of Figures……………………………………………………………………………….8

List of Tables……………………………………………………………………………....13

Abstract………………………………………………………………………………….…14

Declaration & Copyright Statement……………………………………………………..15

Acknowledgements…………………………………………………………………….…17

Glossary of Terms……………………………………………………………………...…18

Chapter 1 – Introduction 20

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1.1. Introduction……………………………………………….………………….20

1.2. Transmission of information across generations………………………...21

1.3. Social transmission of information………………………………………...24

1.3.1. Social transmission and oviposition behaviour………………..25

1.3.2. The apple maggot fly…………………………………………….28

1.4. Plasticity and its significance……………………………………………....29

1.5. Genomic imprinting and epigenetics………………………………………32

1.6. Olfactory plasticity…………………………………………………………..34

1.7. Factors influencing olfactory plasticity.……………………………………36

1.8. Drosophila as a model……………………………………………………...43

1.9. Drosophila larval olfaction……………………………………………….....44

1.10. Drosophila adult olfaction………………………………………………....46

1.11. OrCo (previously Or83b)………………………………………………….50

1.12. Drosophila nociception…………………………………………………....52

1.12.1. Trp mutants……………………………………………………...52

1.12.2. Painless mutants…………………………………………….….55

1.13. Drosophila larval gustation………………………………………………..56

1.14. Drosophila adult gustation………………………………………………..62

1.15. Chemosensory processing and oviposition behaviour..…………….…64

1.16. Measuring responses to chemostimulants……………………………...66

1.17. Drosophila courtship and mating……………………………...... 70

1.18. Aims and objectives……………………………………………………….72

Chapter 2 – Materials and Methods 76

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2.1. Fly stocks…………………………………………………………………….76

2.2. Chemostimulants…………………………………………………………....77

2.3. Rearing peppermint- and menthol-exposed flies………………………...78

2.3.1. P1 and M1: Flies reared in one generation in peppermint (P1)

or menthol (M1)………………………………………………………….78

2.3.2. P2: Flies reared on 0.1% peppermint medium for two

generations………………………………………………………………78

2.3.3. P1C1: Control food-reared flies, the offspring of flies reared on

0.1% peppermint medium………………………………………………79

2.3.4. P1C2: Control food-reared second generation offspring reared

on 0.1% peppermint medium…………………………………...... 79

2.4. Larval behaviour: Rearing………………………………………………....82

2.5. Larval behaviour: Olfaction……………………………………………..…83

2.6. Adult behaviour: Gustation……………………………………………...…85

2.7. Adult behaviour: Settling (assessment of host-selection behaviour)….87

2.8. Adult behaviour: Oviposition...……………………………………………..88

2.9. ‘No contact’ rearing………………………………………………………….89

2.10. Impact of peppermint on survival to adulthood…………………………90

2.11. Analysis of data…………………………………………………………….91

Chapter 3 – Results: Do rearing conditions influence behaviour? 92

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3.1. Introduction…………………………………………………………………..92

3.2. Responses of control larvae to peppermint………………………………94

3.3. Settling behaviour in control adults………………………………………..99

3.4. Experiments on larvae reared on peppermint food………………….…102

3.5. Settling behaviour in P1 adults…………………………………………...106

3.6. P1 oviposition behaviour………………………………………………….108

3.7. Mating trials……………………………………………………………...…110

3.8. Chapter summary………………………………………………………….112

Chapter 4 – Results: Exploring the effects of environment and rearing conditions on chemosensory behaviours 114

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4.1. Introduction…………………………………………………………….….114

4.2. Necessity of contact…………………………………………………….…115

4.3. The behaviour of individual larvae……………………………………….120

4.4. Effect of rearing conditions on behaviour…………………………….…122

4.5. Effect of peppermint on juvenile survival……………………………..…126

4.6. Chapter Summary…………………………………………………………129

Chapter 5 – Results: Influence over generations 132

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5.1. Introduction…………………………………………………………………132

5.2. Influence of repeated generational exposure…………………………..133

5.3. Effects of exposure to peppermint for a single generation...………….141

5.4. Influence of re-exposure to peppermint…………………………………153

5.5. Chapter summary.…………………………………………………………155

Chapter 6 – Results: Investigation into underlying causes 157

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6.1. Introduction…………………………………………………………………157

6.2. Parental inheritance……………………………………………………….158

6.3. The responses of peripheral receptor mutants…………………………164

6.4. Testing the chemical legacy hypothesis………………………………...173

6.5. Male only and female only assays……………………………………….175

6.6. Menthol assays…………………………………………………………….177

6.7. Chapter Summary…………………………………………………………180

Chapter 7 – Discussion 182

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7.1. Overview……………………………………………………………………182

7.2. Naïve flies find peppermint repellent, but rearing in its presence reduces

this aversion……………………………………………………………………..183

7.3. Parental and larval environments influence behaviour………………..185

7.4. Direct contact with peppermint is not required for behavioural changes

to occur…………………………………………………………………………..187

7.5. Impact of peppermint upon survival.…………………………………….189

7.6. Repeated exposure to peppermint for several generations may result in

a preference for it……………………………………………………………….191

7.7. Transgenerational effects of exposure to peppermint…………………194

7.8. Transmission of preference across life stages…………………………196

7.9. Input from both parents is used to modify offspring behaviour……….198

7.10. Chemosensory mutants do not find peppermint aversive……………201

7.11. Menthol and peppermint evoke similar behavioural responses……..202

7.12. Comparison of single sex and mixed sex settling assays……………203

7.13. Further work………………………………………………………………204

7.14. Overall conclusions………………………………………………………206

References 208

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Word count =49, 784

List of Figures

. Figure 1.1 Schematic of a typical olfactory sensillum housing two OSN’s…41

. Figure 1.2 Anatomy of the dorsal organ in Drosophila melanogaster

larvae………………………………………………………………………………45

. Figure 1.3 Scanning electron micrograph of a fly head, indicating the major

chemosensory organs…………………………………………………………...46

. Figure 1.4 Scanning electron micrograph of the head of an adult Drosophila

melanogaster female with a surface-rendered 3-D reconstruction of the

brain………………………………………………………………………………..48

. Figure 1.5 Comparative circuitry of the adult and larval olfactory systems..49

. Figure 1.6 Anatomical placement of Do and TO on the larval head………..59

. Figure 1.7 Immunofluorescence stain of the fly brain indicates the location of

the AL and SOG in respect to one another, as well as indicating OSNs ….61

. Figure 1.8 Schematic indicating the position of olfactory and gustatory

neurons on the body of the fly……………………………………………...... 62

. Figure 1.9 Locomotor behaviours involved in the olfactory response of

Drosophila melanogaster larvae………………………………………………..67

. Figure 1.10 Courtship behaviour of Drosophila melanogaster ……………..71

. Figure 2.1 Rearing of peppermint (P1) flies………………………………...…80

. Figure 2.2: Rearing of P2 flies…………………………………………………..80

. Figure 2.3: Rearing of P1C1 flies……………………………………………….81

. Figure 2.4: Rearing of P1C2 flies……………………………………………….81

. Figure 2.5: Olfactory behavioural test plate……………………………………83

. Figure 2.6: Gustatory behavioural test plate………………………………….86

. Figure 2.7: Location of oviposition sites within a laying box………………...89

. Figure 3.1: Olfactory behavioural response indices for second instar control

CS larvae in response to increasing concentrations of peppermint ….…….95

. Figure 3.2: Gustatory behavioural response indices for second instar control

CS larvae when presented with a choice between control agar and 0.1%

peppermint agar, or a choice between control agar and 1% peppermint

agar………………………………………………………………………………..97

. Figure 3.3: Gustatory behavioural response indices for second instar control

CS and OrCo- larvae when presented with a choice between control agar

and 0.1% peppermint agar, or a choice between control agar and 1%

peppermint agar…………………………………………………………………..98

. Figure 3.4: Behavioural assay of adult settling behaviour in control CS

flies……………………………………………………………………………….100

. Figure 3.5: Olfactory behavioural response indices for second instar control

and P1 larval offspring………………………………………………………….103

. Figure 3.6: Gustatory behavioural response indices for second instar control

CS and P1 larval offspring when presented with a choice between control

agar and 0.1% peppermint agar, or a choice between control agar and 1%

peppermint agar…………………………………………………………………105

. Figure 3.7: Behavioural assay of settling behaviour using P1 flies in the

presence of control food and 0.1% peppermint food………………………..107

. Figure 3.8: Oviposition behaviour for control flies and P1 flies when

presented with 3 choices – control agar, 0.05% peppermint agar, and 0.1%

peppermint agar………………………………………………………………...108

. Figure 3.9: CC = C♀C♂ mating pair; CP = C♀P1♂ mating pair; PC =

P1♀C♂ mating pair; PP = P1♀P1♂ log courtship duration, log mating

duration, log mating onset time, log mating end time, log courtship latency

for successful and unsuccessful pairs, as well as for the total number of

pairs………………………………………………………………………………111

. Figure 4.1: Behavioural assay of adult settling behaviour using ‘no contact’

flies in the presence of control food and 0.1% peppermint food…………..116

. Figure 4.2: Olfactory behavioural response indices for control, P1 offspring

and ‘no contact’ offspring larvae in response to 5 µl peppermint extract…117

. Figure 4.3: Gustatory behavioural response indices for control larvae, P1

larval offspring and ‘no contact’ larval offspring on petri dishes, two quarters

of which contain 1% peppermint agar, the other two quarters of which

contain control agar…………………………………………………………….119

. Figure 4.4: Comparison of gustatory behavioural response indices for group

and individual control larvae, P1 larval offspring, and ‘no contact’ larval

offspring in response to gustatory plates containing two quarters 1%

peppermint agar and two quarters control agar…………………………..…121

. Figure 4.5: Gustatory locomotion behaviour analysis………………………123

. Figure 4.6: Cumulative eclosion rate in the presence of a control food

medium and a 0.1% peppermint food medium for control CS; P1 offspring

and P9 offspring…………………………………………………………………128

. Figure 5.1: Olfactory behavioural response indices for control larvae and

larvae whose parents have been exposed to peppermint extract for a

number of generations…………………………………………………………134

. Figure 5.2: Gustatory behavioural response indices for control larvae and

larvae whose parents have been exposed to peppermint extract for a

number of generations, P1, P2, P3, and P5…………………………………136

. Figure 5.3: Settling behaviour on control food and 0.1% peppermint food for

CS, P1, P2, P3, P4, P5, P6, and P7 flies…………………………………….139

. Figure 5.4: Settling behaviour on control food and 0.1% peppermint food for

P1C1, P1C2, P1C3, P1C4, P1C5, P1C6, P1C7, P1C8, P1C9, P1C10 and

P1C11 flies………………………………………………………………………143

. Figure 5.5: Mean difference between settling rate on control and 0.1%

peppermint food for control, P1 and P1C1 to P1C11 flies………………….146

. Figure 5.6: Linear regression of mean difference in settling rate on control

and 0.1% peppermint food for control, P1, P1C1, P1C2, P1C3, P1C4, P1C5,

P1C6, P1C7, P1C8, P1C9, P1C10 and P1C11 flies at 30, 60 and 80

minutes………………………………………………………………………..…148

. Figure 5.7: Collated mean difference in settling rate for three set time points

(30, 60 and 80 minutes) for control, P1, and all P1Cn generations tested,

indicating patterns in behaviour……………………………………………….150

. Figure 5.8: Olfactory behavioural response indices for control larvae, P1

larval offspring and P1C4 larval offspring in response to 5 µl peppermint

extract…………………………………………………………………………….151

. Figure 5.9: Gustatory behavioural response indices for control larvae, P1

larval offspring and P1C4 larval offspring to agar plates, two quarters of

which contain control agar and two quarters of which contain 1% peppermint

agar……………………………………………………………………………….152

. Figure 5.10: Settling behaviour on control food and 0.1% peppermint food for

P1C8P1 flies…………………………………………………………………….154

. Figure 6.1: Settling behaviour on control food (yellow) and 0.1% peppermint

food of C♀C♂ offspring, P1♀P1♂ offspring, C♀P1♂ offspring and P1♀C♂

offspring………………………………………………………………………….159

. Figure 6.2: Settling behaviour on control and 0.1% peppermint food of

C♀P1♂ second generation offspring and B. P1♀C♂ second generation

offspring…………………………………………………………………………161

. Figure 6.3: Settling behaviour on control and 0.1% peppermint food of

C♀P1♂ third generation offspring and P1♀C♂ third generation

offspring………………………………………………………………………….162

. Figure 6.4: Settling behaviour on control and 0.1% peppermint food of

C♀P1♂ fourth generation offspring and P1♀C♂ fourth generation

offspring………………………………………………………………………….163

. Figure 6.5: Oviposition preferences by proportion of eggs laid on 0%, 0.05%

and 0.1% peppermint agar for control females, P1 females, OrCo- mutant

females, Trp mutant females, and Painless mutant females………………165

. Figure 6.6: Settling behaviour on control and 0.1% peppermint food of

OrCo-, Trp, and Painless mutants…………………………………………….168

. Figure 6.7: Olfactory behavioural assays of control, P1, OrCo-, Trp and

Painless third instar larvae in response to 5 µl peppermint extract………..170

. Figure 6.8: Gustatory behavioural assays of control, P1, OrCo-, Trp, and

Painless third instar larvae in response to 1% peppermint agar ………….171

. Figure 6.9: Settling behaviour of P1 larvae which have been washed at third

instar and repositioned in a bottle containing control food in response to vials

containing control food and 0.1% peppermint food…………………………174

. Figure 6.10: Settling behaviour in response to control food and 0.1%

peppermint food of female only control flies and male only control flies….176

. Figure 6.11: Settling behaviour in response to control food and 0.1%

menthol food of control flies and P1 flies……………………………………..177

. Figure 6.12: Settling behaviour in response to control food, 0.1%

peppermint food and 0.1% menthol food for flies reared for one generation

on 0.1% menthol food (M1)……………………………………………………179

List of Tables

. Table 3.1: Settling behaviour for Control CS flies…………………………...101

. Table 4.1: Gustatory end point post hoc……………………………………..124

. Table 4.2: Survival comparison table…………………………………………129

. Table 5.1: Olfactory behaviour for control and peppermint generations post

hoc………………………………………………………………………………..135

. Table 5.2: Gustatory behaviour for control and peppermint generations post

hoc………………………………………………………………………………..137

. Table 5.3: Olfactory behaviour for P1C4 larvae post hoc…………………..151

. Table 5.4: Gustatory behaviour for P1C4 larvae post hoc………………….153

. Table 6.1: Chemosensory mutant oviposition post hoc…………………….167

. Table 6.2: Chemosensory mutant post hoc………………………………….171

. Table 6.3: Chemosensory mutant gustatory post hoc………………………172

Abstract

University: The University of Manchester Name: Rebecca Lockyer Degree: Doctor of Philosophy in Evolutionary Biology Thesis title: Transmission of chemosensory information in Drosophila melanogaster: Behavioural modification and evolution Date: 08/01/2014

The ‘modern evolutionary synthesis’ emphasised the role of genetic inheritance in driving natural selection; however, this is not the only means by which biological changes may be passed on to future generations. Information can also be transmitted non-genetically, and this could be an important agent of evolution. Non-genetic information can be acquired in two different ways: it can be inherited from parents (for example, through maternal and paternal effects) or gathered from the environment. Transmission of information in this manner can result in durable changes in behaviour, which allow for adaptation to variable conditions, and might ultimately bring about adaptive divergence. To investigate non-genetic transmission of information between parents and offspring, I studied the effects of being reared in the presence of an aversive stimulus, peppermint extract, on the fruit fly Drosophila melanogaster using a range of behavioural assays. The results demonstrate that naïve flies exposed to peppermint found it aversive, with exposure substantially reducing their survival; however, the offspring of flies reared in the presence of peppermint showed a significantly reduced aversion despite having no previous direct contact with the stimulus. This strongly suggests that a transmission of information (relating to preference for peppermint) has occurred from parents to offspring. This effect was preserved for four generations if the peppermint stimulus was removed from the food source after only one generation, but with continued exposure to peppermint the reduction in aversion was sustained, and a preference for peppermint may even have developed. Mutant flies lacking OrCo, Trp and Painless showed abnormal behavioural responses to peppermint, suggesting that these genes may be involved in detecting and/or responding to this aversive stimulus. These experiments demonstrate that environmental changes (i.e. the introduction of an aversive stimulus) can instigate biological modifications in D. melanogaster that are passed on non-genetically to future generations. This is most likely true for other insects and animals more generally, and further studies of additional model and non-model species will help to demonstrate the importance and prevalence of non-genetic transmission of information as a driver of fundamental evolutionary change.

Declaration

I declare that no portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

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Acknowledgements

Firstly I would like to thank my supervisors Professor Matthew Cobb and Professor Cathy McCrohan for all their help and guidance, and also my advisor Professor Richard Preziosi for frequently letting me pick his statistical brain! In addition, this body of work would have been impossible without the technical expertise and advice of Dr Derek Hoare and Dr Micheline Grillet.

I experienced a variety of excruciating events during the writing of this thesis, including the death of my Dad, Peter Lockyer, my Grandma, Rose Lockyer, and, in a similar, but less heart-breaking, more morphine-aided way, my very own fractured femur. I would like to thank those who I think prevented me from having a complete mental breakdown: B.2201 – Dr Andres Arce, Rachael Antwis, Rob Clay, Chris Pickford, Veronica Guinness, Dr Dave Springate, Dr Paul Johnston, Dr Danny Rozen and Lisa Andrejczak. Dr Lisa Reynolds – for being a proofreading star and for the encouraging texts, calls and emails, even all the way from Canada, and Dr Annya Smyth – even though, or possibly because, you’re a very clever idiot (in the nicest possible way). Zoe Drymoussi, Candy Leary and Mark Richardson – living with you during my PhD was brilliant. Anne and Habib Rahman – the loveliest in- laws a girl could hope for. My brother, Mike Lockyer, and my dog Lola (because the one of you who can read would be horrified to be classified with the other). Last but most definitely not least my Mum, Annette Lockyer.

Finally, I would like to thank Imran Rahman for a world of encouragement, assistance and support – my proof reader, my writing taskmaster and the only person who could make getting to the end worthwhile! No one would be reading any of this without you.

This PhD project was kindly funded by NERC.

Glossary of terms

Hopkins’ Host Selection Principle (HHSP) – according to HHSP, insects will prefer to feed and oviposit on the host species on which they themselves were laid and developed.

Chemical legacy hypothesis – chemicals from the larval or pupal environment of holometabolous insects stay in the body or the immediate surroundings of the individual and influence adult behaviours.

Pre-imaginal conditioning – in holometabolous insects, larval experience affects adult (“imaginal”) behaviour, despite the intense reorganisation of the nervous system during metamorphosis.

Learning – an observable and measurable change in behaviour as a result of individual experience.

Associative learning – for example, classical or operant conditioning.

Learning that occurs as the result of reinforcement. An action is associated

with a positive or negative response. This is what is generally intended

when people talk about “learning”.

Non-associative learning – for example, adaptation. Changes in behaviour

that are brought about in the absence of associated stimuli to reinforce the

behaviour.

Non-genetic transmission of information – the communication of information from one individual to another without the involvement of genetic material. This can

also occur socially via observing other individuals, or environmentally, in which case it is a form of learning.

Olfactory behaviour – behaviour exhibited in response to an olfactory stimulus.

Eusociality – characterised by three key features – cooperative care of young by more individuals than just the mother; they have sterile castes; and there is overlap of generations so that mother, adult offspring and young offspring coexist so adult offspring can rear their younger siblings instead of rearing their own offspring.

Sociality – when organisms exhibit one or two of the three features of

eusociality.

OR – odorant receptor

OSN – olfactory sensory neuron

OBP – odorant binding protein

AL – antennal lobe; primary centre for olfactory organisation in the insect brain.

The equivalent to the mammalian olfactory bulb (OB).

GSN – gustatory sensory neuron

SOG – suboesophageal ganglion

GR – gustatory receptor

Chapter 1

Introduction

1.1. Introduction

Genetic inheritance has long been established as being able to effect evolutionary change, but in recent years the study into the non-genetic methods of inheritance and the possibility of ensuing influence on adaptive change and evolution have increasingly been growing as an area of interest within the scientific community

(Stearns 1989; Pigliucci 2005). Non-genetic inheritance, as many have asserted, is difficult to accurately quantify (Dickens & Rahman 2012) but can be influenced by many factors, leading to phenotypic changes altering an organisms’ physiological, morphological, and behavioural outputs (Kelly et al 2012). Behavioural modifications that occur in response to environmental change are fundamental to evolution because they allow for rapid adaptation to variable conditions (Mery & Kawecki

2003; Pigliucci 2005). Many studies have found that these behavioural modifications can be transmitted from generation to generation (Helfman & Schultz

1984; Lindqvist et al 2007), resulting in durable adjustments in population behaviour.

Over time behavioural modifications may instigate cultural differences and can even lead to speciation (Seehausen et al 2008). Despite this, however, the mechanisms underlying the transmission of non-genetic (epigenetic) information and behavioural modifications across generations are still not fully understood. This thesis will address the transmission of behavioural modifications across generations in response to a chemosensory stimulus by investigating a variety of factors; the manner of stimulus delivery, the influence of repeated transgenerational exposure, whether behaviour is influenced differently dependent on which parent has experienced the stimulus, whether certain genes or protein channels are required to produce uniform responses. By taking into account the workings of the olfactory and gustatory systems it is hoped that a greater level of understanding can be attained with regard to behavioural plasticity, its mechanisms, and how environmental influences are transmitted across generations.

1.2. Transmission of information across generations

Behavioural modifications, in some cases, can be transmitted across generations; part of this transmission occurs as a result of parental investment. Parental investment encompasses all actions carried out by a parent which enhance offspring fitness, whether intentional or not, and which incurs costs to the parent’s reproductive potential (Trivers 1972; Trivers 1974; Hauser 1988). Parental effects encompass, yet differ from, parental care, which applies specifically to behaviours that are carried out after fertilisation and parturition. As such, the transmission of inherited non-genetic information is a form of parental investment, but not parental care. Simply, parental investment is the process by which parental experience is

converted into, ideally, advantageous adjustments in offspring fitness (Mousseau &

Fox 1998).

There are several examples in mammals of parental environment influencing offspring development. For example, in Syrian hamsters, the photoperiod experienced by the mother affects the development of her offspring, preparing the offspring for the season into which they will emerge (Beery et al 2008). In mice, when a pregnant female is housed next to a non-contagious conspecific infected with the protozoan parasite Babesia microti, the female produces offspring which are less aggressive than control offspring, yet have a superior immune response to

B. microti. This prepares the offspring for the environment in which they are set to emerge, and as such is adaptively beneficial to the offspring, but at a cost with regards to aggression (Curno et al 2009).

Cases of parental investment in invertebrates are rarer (perhaps through lack of investigation, or perhaps through lack of incidence), but they do occur. Like the

Syrian hamster, in the ground cricket, Allonemobius socius, maternal environment influences offspring behaviour; the environment in which oviposition occurs, as well as maternal-oviposition environment (i.e. the environment in which the mother was oviposited) affects offspring behaviour by influencing observed diapause incidence patterns (Olvido et al 1998).

Similarly, in the desert locust, Schistocerca gregaria, maternal phase state – which is triggered by changes in population density, and involves modifications in behaviour, physiology, morphology and colouration – is transmitted to offspring. As a result, hatchlings whose mothers had been kept in an environment with a dense population with clumped vegetation exhibit more outgoing behaviour (i.e. they move around more, at greater speeds, and exhibit a higher incidence of exploratory

behaviour), while hatchlings whose mothers came from an environment with sparse vegetation and a lower population density are much more reserved in terms of their behaviour (Despland & Simpson 2000). Additionally, maternal environment influences the offspring behaviour in the locust Locusta migratoria. Females from environments in which resources are limited lay eggs earlier in their life cycle, and invest more in the offspring that they do produce, generating larger eggs (Chapuis et al 2010).

In the fly Telostylinus angusticollis, there is no evident paternal care, yet having a high condition father, reared on a high quality diet, is sufficient to increase the mating success of male offspring and the fecundity of female offspring

(Bonduriansky & Head 2007). In another study on the desert locust, Schistocerca gregaria, it was found that not only do an individual’s rearing conditions influence their behaviour, but in addition the rearing conditions (whether crowded or solitary) of their parents also influence their behaviour. The rearing conditions of both parents are significant, but maternal environment appears to have more of an effect on behaviour than paternal environment (Islam et al 1994).

In holometabolous insects, i.e. those that undergo complete metamorphosis, behavioural modifications may take place against a background of substantial developmental change. A significant reorganisation of the nervous system occurs during metamorphosis. During this process the mushroom bodies (MBs), the areas in the brain responsible for learning and memory consolidation (Ishii et al 2005), are extensively rewired (60% of the axons in the larval MBs degenerate), and the larval sense organs histolyse, with the adult sense organs forming de novo from imaginal discs (Lee et al 1999; Heisenberg 2001). This sequence of events – the radical changes the larval neurons experience, combined with the vast production of new, adult-specific neurons (Lee et al 1999; Heisenberg 2001), and the understood

importance of the MBs in larval learning (Smith et al 2008) – means that any substantial preservation of larval learned experience must reflect a fundamental aspect of the sensory processing of the insect.

1.3. Social transmission of information

It is well documented that information can be communicated between individuals and between groups of individuals; however, social environment also influences the behaviour of an individual (Helfman & Schultz 1984; Whiten et al 2007). For example, in honeybees, Apis mellifera, cuticular hydrocarbon profiles are used for recognition between subfamilies. These cuticular hydrocarbons are partially genetic-based, but also somewhat tempered by chemicals obtained from their environment and from other individuals within that environment. A study on 5-day- old workers found that if they were kept in isolation, their cuticular hydrocarbon profiles became strongly distinct from those of their relatives (also 5-day-old workers) that had been reared in the hive, severely limiting the ability of their family members to be able to identify them (Arnold et al 2000). In the fruit fly, Drosophila melanogaster, it has been noted that social experience manipulates the display of aggressive behaviours in females. If females are kept in isolation, when they encounter another fly they display significantly more aggressive behaviours than females reared in groups (Ueda & Kidokora 2002). Flies use sensory information from other flies to temper their own behaviour in crowded conditions, but those reared in isolation are not able to interpret these behaviours, leading to higher incidences of aggressive behaviour (Ueda & Kidokora 2002).

Transmission of information across life stages is a form of environmental transmission, as learned information from the larval stage is preserved through to the adult life stage. In the tobacco hornworm moth, Manduca sexta, the larval diet shapes adult eclosion time, size and fat content; larvae reared on an artificial diet instead of the natural tobacco diet exhibit retarded growth and performance as adults supported on a tobacco diet. In addition, individuals are capable of associating a specific odour with an averse stimulus (a small electric shock) as a caterpillar, and retain this information through to their adult life stage (Raguso et al

2007; Blackiston et al 2008).

One of the key ways that non-genetic transmission of information is expressed is through habitat choice. This is determined by many different factors (Taylor 1987), and is under strong selection due to its decisive influence on offspring performance and survival (Resetarits 1996; Hora et al 2005). For an insect to integrate a novel host into its diet, females must be able to find and oviposit on this new host, and the offspring must be able to survive and develop on it. In this respect, previous experience can influence the oviposition site preference of females (Richmond &

Gerkling 1979; Jaenike 1982). Experience in the larval environment influences the host acceptance behaviour of individuals when they reach adulthood and commence dispersal (Davis 2008).

1.3.1. Social transmission and oviposition behaviour

Oviposition is an example of host choice behaviour, as well as representing a socially mediated behaviour. For offspring to survive to adulthood and themselves be able to reproduce, the selection of a suitable oviposition site by the mother is essential. It is known that female fruit flies select for egg-laying sites that contain

acetic acid, but the driving forces behind oviposition behaviour are otherwise poorly understood (Joseph et al 2009). Jaenike (1983) found that the oviposition site preference of D. melanogaster is not necessarily influenced by the type of food individuals develop on, but rather depends on the nature of this food substance. If flies are reared on apple or tomato, their preference to oviposit on them is enhanced; however, these substances produce odours that are known to be desirable to fruit flies (Jaenike 1983). In addition, it is thought that oviposition behaviour is regulated not only by the availability of a suitable host, but also by the female’s individual egg load; females with higher egg loads accept lower quality oviposition sites, and deposit larger clutches, yet they search for and visit more oviposition sites than females with lower egg loads. As a result, a female’s preference might change each time she oviposits (Minkenberg et al 1992). The egg-laying behaviour of other flies also influences a female’s oviposition behaviour; if other females have exploited an egg-laying site, they are more likely to follow suit, resulting in aggregation of eggs (del Solar & Palomino 1966).

In 1916, Hopkins put forward Hopkins’ Host Selection Principle (HHSP), which postulates that phytophagous and parasitic insects prefer to feed and oviposit on the same plants that they encountered in their larval environment (Hopkins 1917;

Hewitt 1917; Barron 2001; Rietdorf & Steidle 2002). This principle is supported by several experiments in which insects have been reared on a novel host, and then as adults are offered the choice between the host species they were reared on and their ancestral host species. When presented with this choice, the insects significantly preferred to oviposit on the host on which they were raised (Smith &

Cornell 1979; Hoffmann 1988; Jaenike 1988).

There are three mechanisms proposed which could trigger host selection, and these may work alone or cooperatively to bring about this effect. The first and most clear-

cut of these is genetic variation; however differences in host preference, even if they are the result of genetic inheritance, can also be modified by experience (Barron

2001). The first of the two non-genetic mechanisms through which host selection might come about is termed “preimaginal conditioning”. Preimaginal conditioning describes learning that occurs in holometabolous insects during the larval stage, which is subsequently retained through the extensive rewiring that occurs in metamorphosis and into the adult stage (Barron & Corbet 1999). This phenomenon was first detailed by Thorpe and Jones (1937), who found that the ichneumonid parasite Nemeritis canescens can develop a preference for a novel host, the wax moth Meliphora grisella, when they are forcibly reared on it (Thorpe & Jones 1937).

Preimaginal conditioning has since been shown experimentally in D. melanogaster

(Thorpe 1939; Manning 1967), the leafminers Liriomyza trifolii and Liriomyza huidobredsis (Facknath & Wright 2007), the aphid parasitoid Aphidius ervi (Villagra et al 2007), the carpenter ant Camponotus floridanus (Carlin & Schwartz 1989) and the parasitic wasp Hyssopus pallidus (Gandolfi et al 2003). However, some of these studies have been criticised for neglecting to show that the conditioning effect was indisputably preimaginal, as in some cases there may also have been condition- exposure in the imaginal post-emergence period (Barron & Corbet 1999). To demonstrate that the conditioning is truly preimaginal, instead of due to preimaginal and imaginal influences acting in concert (perhaps even reinforcing one another), all aspects of the larval stimuli must be removed prior to adult emergence. This would help determine whether information learnt in the larval stage can influence adult behaviour, or whether exposure in the early adult environment is essential (Barron &

Corbet 1999; Facknath & Wright 2007).

The second non-genetic mechanism is termed the chemical legacy hypothesis. In this mechanism, the correlation between larval environment and adult host

preference is influenced not by larval experience, but by direct chemical involvement in early adult life; for example, by persisting through metamorphosis inside or on the surface of the organisms’ body, or externally, on the outside of the pupal cocoon (Veltman & Corbet 1991; Barron 2001). In this respect, habitat fidelity arises as a result of early adult experience due to the chemicals encountered shortly after emergence (Hoffmann 1988).

1.3.2. The apple maggot fly

The apple maggot fly, Rhagoletis pomonella, is one of the most renowned examples of habitat shifting and sympatric speciation (speciation without geographical separation of diverging populations). Their ancestral host is the hawthorne,

Crataegus L. spp., but at some point in the 19th century (prior to 1867) a host shift occurred onto the newly introduced domestic apple, Malus pumila L., in the Hudson

Valley region of New York. The two plant species are regularly grown in close proximity, and reproductive isolation has therefore arisen between the two host races as a result of host choice behaviour – individuals chose to mate on their accepted host with individuals who had also chosen that host, and the female then oviposited on that same host, transmitting the host preference to her offspring. This occurred for repeated generations, and in 1998 it was discovered that there was gametic disequilibrium between the two races on six allozyme loci, Malic enzyme

(Me, 1.1.1.40), Aconitase-2 (Acon-2, enzyme classification (EC) 4.2.1.3), Mannose phosphate isomerase (Mpi, 5.3.1.8), Aspartate amino transferase-2 (Aat-2, EC

2.6.1.1), NADH-Diaphorase-2 (Dia-2, EC 1.6.2.2), and β-Hydroxyacid dehydrogenase (Had, EC 1.1.1.30) (Feder 1998). In 2003 it was further revealed that the gametic disequilibrium was not restricted to these six loci, but was in fact more extensive, with significant standardised composite disequilibrium values for 60

of 64 host race populations that could be tested between Aat-2 100 and Dia-2 100 on chromosome one, 37 of 66 for Me 100/Acon-2 95 on chromosome two, and 17 of

22 for Had 100/Pep-2 100 on chromosome three. The haploid number in R. pomonella is six, so this is a substantial level of reorganisation (Feder 1998; Feder et al 2003; Jiggins & Bridle 2004). Following the initial niche exploitation of apple trees it appears that, for the first few generations, the preference for apple trees was largely socially transmitted, as this occurred prior to the genetic changes. The shift allowed the apple maggot fly to expand its range as it adjusted to the novel host and as a result, reproductive isolation has arisen between the two races, apple and hawthorn (Feder & Filchak 1999; Thibert-Plante & Hendry 2011).

1.4. Plasticity and its significance

Plasticity is the ability of an organism to adjust its phenotype to account for environmental changes. The contributions of plasticity towards learning are essential for the key decision-making processes of most organisms – using information taken from the environment or transmitted from conspecifics (whether consciously, or not) to regulate behaviours such as feeding, social interactions, reproduction, predator avoidance and aggression – to enable adaptation to environmental variability (Dukas 2008). This environmental adaptation can lead to the development of differing phenotypes and may eventually, over several generations, produce premating isolation, leading to speciation, even in the absence of a geographical barrier (Seehausen 2008; Dukas 2008; Moczek et al 2011). The benefits of plasticity do not come without costs, there is a continual trade-off; organisms must be equipped to detect change, and sensory and regulatory mechanisms must be maintained which is energetically expensive. Additionally, a

plastic phenotype may be adopted at great expense and may only be beneficial for a relatively short period of time (DeWitt et al 1998).

Changes in behaviour or physiology in response to the environment have long been reported; for example, maternal diet has been found to have a lifelong impact on offspring in humans (Lumey 1992; Roseboom et al 2001). During the Dutch famine of 1944 a formerly well-nourished population underwent a period during which their access to nutrition was extremely limited under Nazi occupation in World War II, meaning that the adult population generally survived on less than 1000 calories a day. Rations for adults from December 1944 to April 1945 were as low as 400 to

800 calories a day (daily rations for children never fell below 1000 calories).

Women who were breast-feeding or pregnant were permitted an extra amount of food, but at the height of the famine access to these additional provisions could not alwaysbe made available. In a study of children born and conceived during the famine (from which premature births were excluded) an effect on the sex ratio of live born babies was found, with a lower proportion of boys surviving in the wake of exposure during late gestation; this was in addition to an increase in perinatal mortality in both genders. Additionally, when looking at live births, babies whose mothers were exposed to famine during mid- or late-gestation weighed less, were shorter and had smaller heads when compared with babies from the same area whose mothers had not suffered with limited nutrition during their gestation (those born pre- and post-famine). Babies whose mothers were exposed during early- gestation weighed more and were longer at birth (Lumey 1992; Roseboom et al

2001).

Individuals whose mothers had undergone famine conditions in mid- or late- gestation also demonstrated reduced glucose tolerance in later life. Individuals exposed to famine during early-gestation on the other hand suffered from a more

atherogenic lipid profile (i.e. more likely to develop atherosclerosis), a higher body mass index (BMI), an increased risk of chronic heart disease, diabetes and breast cancer, as well as other factors contributing to poor health (Ravelli et al 1998;

Roseboom et al 2001; Painter et al 2006). Blood pressure of all famine-exposed babies was found to be related to the protein/carbohydrate ratio of the average ration during the third trimester. The bronchial tree grows most swiftly during mid- gestation, and indeed, babies who were famine exposed during mid-gestation were found to have an increased prevalence to obstructive airways disease. It is clear from these findings that foetal environment, in this case malnutrition, can have a lifelong impact upon the development of individuals, influencing their health, wellbeing and prevalence to disease (Roseboom et al 2001; Painter et al 2008). It has also been demonstrated that the effects of the famine were passed on to the second generation; the offspring of women who were exposed to famine conditions in utero have increased neonatal adiposity and poorer long-term health (Painter et al 2008). This is a form of neuronal plasticity; the environment experienced by mothers in utero has brought about changes at a cellular level in their offspring, resulting in observable physiological changes.

Experience in utero or pre-eclosion (for species who give birth to live young, and species who lay eggs, respectively) is not the only developmental period in which the environment can bring about behavioural or physiological changes that are exhibited throughout an individual’s life; predator-prey relationships can also be influenced by the environment encountered by juveniles of the prey species.

Iphiseius degenerans is a mite species which is predated upon by the fellow mite

Neoseiulus cucumeris. When I. degenerans encounter N. cucumeris whilst the former is in its juvenile stage it is significantly more likely to predate upon N. cucumeris juveniles once it has reached its adult life stage when compared to individuals of the same species who have not been exposed to N.cucumeris in the

juvenile life stage. This indicates that juvenile experience can influence adult predator-prey relationships (Choh et al 2012). In a similar example of behavioural plasticity, D. melanogaster early experience has been found to influence adult behaviour in terms of the ‘rover’ and ‘sitter’ foraging phenotypes; in individuals that have experienced nutritional deprivation in their larval life stages, sitter morphs increase the range of their exploratory behaviour, whereas no effect occurs to the foraging behaviour of rover morphs (Burns et al 2012).

Behaviour develops and evolves to increase an individual’s fitness and survival prospects, and this is dependent on both the organism’s physical and social environments. An ability to modify these behaviours in response to the environment encountered, or the environment experienced by their mother provides an evolutionary advantage and allows for a cost/benefit trade off. It is assumed that all behaviours arise as a result of a trade-off between the aims of reproduction and relative individual longevity/survival. Using cues from the physical and social environments which are weighed against the costs and benefits, the animal uses this information to maximise the benefits of both whilst reducing costs (Stearns

1989; DeWitt et al 1998; Hager & Gini 2012).

1.5. Genomic Imprinting and Epigenetics

Epigenetic effects can impact organismal behaviour, thus influencing plasticity, for example through the process of genomic imprinting. Genomic imprinting brings about differential inheritance of genes – imprinted genes are maternally or paternally linked, and so maternally linked genes are only expressed when they are inherited from the mother, and paternally linked genes are only expressed when

they are inherited from the father. As such, genomic imprinting influences phenotype expression in a way that can impact upon development, and therefore, the later life of an organism, meaning that the environment experienced by one parent of an individual may impact more significantly on that individual in later life than that of the other parent (Chervaud et al 2008; Hager & Gini 2012). Genomic imprinting is thought to have arisen as a result of conflict between maternal and paternal gene copies over maternal investment, that is, mothers aspire towards a trade off that means they provide sufficient maternal investment to their offspring while still maintaining their own fitness to survive and reproduce again in the future.

Fathers have less interest in the survival and future fecundity of mothers, and are instead more focussed on the survival of offspring. As such they desire a greater level of maternal investment than that which mothers seek to provide, leading to the conflict that may produce genomic imprinting (Wolf & Hager 2006), although proof of this theory is scarce (Wolf et al 2008).

Epigenetic effects are changes that occur in genetic material without bringing about modifications to the DNA sequence and are known to occur in a wide range of species, from insects to mammals. Meaning literally, “outside genetics”, epigenetics describes the consequences of changes which are made to the genome without changing the nucleotide sequence, yet are transmissible from cell to cell, becoming inheritable through the cell lineage and are, on occasion, transmitted across generations from parent to offspring to grand-offspring and so on (Franklin &

Mansuy 2010; Faulk & Dolinoy 2011). The environment an organism experiences can bring about modifications in epigenetic structures, adding to the variability brought about by genomic imprinting (Turan et al 2010). Nutrition, behaviour, stress and toxins, along with a range of stochastic processes have been found to influence the developing epigenome (the overall epigenetic state of the cell) of organisms – the effects of the Dutch famine being just one such example, demonstrating the influence of nutrition (see section 1.6.). The impact of toxins has been repeatedly

demonstrated, including studies in which a link was established between the prenatal exposure of both women and female mice to the synthetic oestrogen diethylstilbestrol (DES) – which was erroneously prescribed to reduce the risk of miscarriage in America from the 1940s through to the 1960s – and the occurrence of ovarian cancer in the female offspring of these women and their murine counterparts (Titus-Ernstoff et al 2008). These changes are believed to occur through processes such as DNA methylation and post-translational chemical changes in the chromatin as a result of the five influencing factors outlined above, which act in concert leading to manipulation of life-stage phenotypes and prevalence of disease. Indeed, Vandegehuchte et al (2010) also revealed transgenerational changes in response to 5-azacytidine, which is known to influence

DNA methylation in mammals, when looking at the crustacean Daphnia magna.

Following exposure in one generation a reduction in body length was observed in the generation that was exposed, and this was maintained across the subsequent two generations (Vandegehuchte et al 2010). It is predicted that as epigenetic effects occur so frequently that they are perhaps evolutionarily selected for.

Environmental factors affect the epigenome most strongly during embryogenesis, and DNA methylation is predominant during early development (Franklin & Mansuy

2010; Faulk & Dolinoy 2011).

1.6. Olfactory plasticity

Olfactory behaviour (that being, behaviour that is observed in response to an olfactory stimulus) is a useful model to study when trying to address the non-genetic transmission of information; olfactory memory is dependent on experience, and so familiarity with an odour alters the degree to which an odour can be discriminated

and the subsequent reaction to that odour (Wilson & Stevenson 2003). Olfactory behaviour is also relatively easy to assess, and due to the ancient origin of the olfactory system in metazoans, shares vast anatomical and physiological homology in a large number of organisms, from aphids to zebras (Davis 2004).

Olfactory learning is a key form of behavioural plasticity controlling central decision- making in many species, and its impacts are far-reaching. Monkeys have been shown to use olfactory information to discriminate between edible and inedible fruit

(Hiramatsu et al 2009), and in several other mammals, for example sheep, it is important for offspring and sibling recognition (Davis 2004). This is also true for colony recognition in eusocial insects such as the ant Cataglyphis cursor, where workers less than 24 hours old are able to single out foreign larvae from those of their queen (Isingrini & Lenoir 1988). In eusocial insects (such as the bumblebee,

Bombus terrestris), olfactory information is also important for coordinating more complex behaviours, such as enlisting nest-mates in a hunt to exploit a food source by providing olfactory cues about food availability and quality (Molet et al 2009).

Olfaction is still important in less social animals. Previous olfactory experience is important to phytophagous insects, such as the diamondback moth, Plutella xylostella, for example, when deciding where to oviposit. Increased oviposition can be induced on non-host plants when females have experience of the odour of the novel host, while naïve females will remain repulsed, or at very least retain a preference for the control host plant (Zhang et al 2007; Wang et al 2008). Olfaction is also used for mate choice (Ruebenbauer et al 2008) and recognition by D. melanogaster, and for recognition, which is based on the detection of specific cuticular hydrocarbons (Blows & Allan 1998).

Olfactory experience can bring about conditioned responses to artificial stimuli.

Fruit flies presented with an odour at the same time as a painful electric shock, and then presented with a painful electric shock followed by an odour, associated the first odour with pain and developed an aversion, but perceived the odour that was presented following the electric shock as a relief, and so found it attractive (Yarali et al 2008). In fruit flies, it is essential to have intact mushroom bodies (MBs) to form short-term memories, to associate them with certain stimuli, and to retain these memories for any significant period of time. As such, individuals with ablated MBs cannot form memories about toxic or repellent olfactory stimuli and, therefore, do not learn about them, or how best to respond, meaning that plasticity of behaviour does not occur (Gasque et al 2006; Fiala 2007). In the same vein, A. mellifora can learn to associate a previously neutral odour with a positive outcome, i.e. presentation with a sucrose solution, leading to the proboscis extension reflex

(PER), or a negative outcome, such as an electric shock which promotes the sting extension reflex (SER). When presented with the conditioned odour, bees who had received sucrose solution were attracted to the odour, demonstrating the PER, whilst bees who had received an electric shock were repulsed, bringing about the

SER. Moreover, this also verifies that the SER is a response to an aversive stimulus in honeybees (Carcaud et al 2009). Olfactory behaviour is therefore variable based on individual encounters, and is subject to change based on individual experiences.

1.7. Factors influencing olfactory plasticity

Behavioural changes may arise in an organism’s pattern of responses to an odour if other variables also change. Devaud et al (2001) looked at one of the most simple

of these variables, the effect of repeated exposure. It was found that repeated, long-term exposure to an olfactory stimulus not only causes behavioural adaptation, but also brings about neural changes in the fly brain, leading to a decrease in volume in selected olfactory glomeruli in conjunction with synapse loss; this is an odorant-specific adaptation (Devaud et al 2001). The same team found that responses to odours change throughout adult life, dependent on age. They uncovered that in Drosophila, the usually aversive odorant benzaldehyde brings about behavioural modification in addition to neural changes two to five days after eclosion, but by eight to ten days post-eclosion this no longer happens (Devaud et al 2003).

It has also been discovered that circadian rhythms regulate olfactory learning in

Drosophila. Circadian oscillations in the clock gene period (per) occur in the chemosensory cells of the antennae of adult flies. This is preserved even if the antennae are removed and sustained in an isolated organ culture (Krishnan et al

1999). Additionally, Krishnan et al (1999), using electrophysiology, found that

Drosophila exhibit a strong circadian rhythm in response to two classes of olfactory stimuli. The first of these two stimuli was a food odorant, ethyl acetate; responses were found to peak during the middle of the night under a 12:12 light-dark (LD) cycle. Benzaldehyde, which as stated above is normally an aversive stimulus, promoted a comparable profile – again responses were elevated during the middle of the night (Krishnan et al 1999). The influence of circadian rhythms on olfactory behaviour was reinforced in 2005, when Zhou et al determined that olfactory response rhythms require Clock genes. Again, they found that responses peaked at night and were lower during the day, but these rhythms were obliterated in the clock mutants per or timeless (tim) (Zhou et al 2005). It has also been discovered that in cockroaches (Leucophaea maderae) that are conditioned to an aversive stimulus (in

this case, peppermint extract) the circadian time at which conditioning of the odour occurs influences the strength of their responses and their ability to retain memories about that odour, with the peak time being early in the subjective night (Decker et al

2007).

Between different populations of fruit flies, variations in olfactory behaviour may also arise. Lavagnino et al (2008) generated isofemale lines of D. melanogaster of six populations from different parts of Argentina and measured their responses to benzaldehyde, finding significant variation in both larvae and adults, although the variation in larvae was more extreme than that of adults. Interestingly, temperature does not appear to be an important factor in this experiment; it is believed, instead, that the different chemical environments encountered influence this behaviour.

Different host-plant species produce different chemical environments, resulting in diversity of environmental pressures (Lavagnino et al 2008). However, temperature was found to be a factor on Drosophila olfactory perception in another study; a reduction in olfactory sensitivity was discovered at high temperatures (30˚C), whilst at lower temperatures (15˚C) an increase in olfactory sensitivity was observed, advocating a role for olfaction in adaptation to ambient temperature (Riveron et al

2009).

As it is used to anaesthetise flies for tasks such as separating male and female flies, and isolating virgin flies, another aspect that needs to be considered is the response of Drosophila to carbon dioxide (CO2). CO2 is detected in Drosophila by Gr21a and

Gr63a (Young Kwon et al 2007), and is repellent to both larvae and adult flies, resulting in avoidance behaviour unless it is presented at low concentrations alongside a food odour (Faucher et al 2006). Under further examination, it was determined that some food odours can inhibit CO2-mediated avoidance behaviour, and this inhibition can last for several minutes (Turner & Ray 2009). Sachse et al

(2007) found that exposure to CO2 for a period of two days brought about an increase in the volume of CO2-specific glomeruli, but this is reversible, and volume is restored to control levels five days after they are returned to normal conditions.

Exposure of two days length was required to have any effect on glomeruli volume

(Sachse et al 2007).

There are wider implications to plasticity of chemosensory behaviour that impact upon species evolution; it allows organisms to adapt to changing environments and novel surroundings (Bush 2009) and makes host-plant search behaviour more efficient (Dotterl et al 2011). That does not mean that plasticity is without its fitness costs, for example, the ability to adapt to novel situations leads to a forfeit in competitive ability in D. melanogaster, but this allows for a greater level of survival, in terms of flexibility of behaviour, and the possibility of range expansions to novel hosts or a switch in ecological niche (Toates 1996; Mery & Kawecki 2003; Shimada et al 2010; Wright et al 2010).

1.8. Detection of odours

An organism’s olfactory system enables it to discriminate between thousands of odours so it can identify food, toxins, potential mates and suitable oviposition sites

(Vosshall 2000; Hallem et al 2004). The olfactory system uses the temporal pattern of neural activity and neuronal firing to discriminate information relating to odour quality and quantity (Okada & Sakuma 2009). Natural odours that organisms encounter on a day-to-day basis are rarely composed of just one molecule – more likely they will consist of an assortment of hundreds of different chemical compounds (Dulac 2006). The olfactory system discriminates between these chemical signals to gather information about the environment it inhabits, with which it elicits effects on behaviour (Stockhorst & Pietrowsky 2004).

Odorant binding proteins (OBPs) are small water-soluble proteins found in the extracellular fluid surrounding odorant receptors (ORs). They are thought to boost the aqueous solubility of odorants, which are predominantly hydrophobic, allowing for the transport of odours into the aqueous environment in which ORs reside (Vogt et al 1990; Zarzo 2007; Kopp et al 2008). The requirement for this transport function is thought to be specific to terrestrial organisms, as aquatic animals usually encounter odorants that are water-soluble, and so they can already access the aqueous environs of the ORs. As such, OBPs indicate a key evolutionary adaptation of the olfactory system for life on land (Vogt et al 1990).

ORs are located on olfactory sensory neurons (OSNs) and detect odours in an organism’s environment following odour transportation by OBPs into the aqueous extracellular fluid in which ORs reside (Zarzo 2007). ORs are G protein-coupled receptors (GPCRs), and were first described by Buck and Axel (1991), then fully identified eight years later by Vosshall et al (1999). A diverse family of 7 transmembrane (TM) GPCRs are found in mammals, birds, fish and amphibians, and an independent, yet equally diverse, family of 7TM domain proteins has been discovered in invertebrates (Ache and Young 2005). These were initially also believed to be GPCRs, until it was observed that the N-terminus of these proteins is positioned in the cytosol, whilst their C-terminus is extracellular. This is at odds with the configuration of a GPCR, and as such their origin and derivation is debated

(Benton et al 2009).

An odour interacts with numerous ORs, in a method described as combinatorial coding. The activation and inhibition of discrete groups of ORs leads to the identification of specific odours. The combinatorial pattern of activation and inhibition of ORs generates an intricate spatial odour map, which is then transmitted

to the brain where classification occurs and the odour is distinguished (Hallem et al

2004; Oka et al 2004; Fishilevich et al 2005).

The OSNs on which ORs are found were initially thought to each express only one

Or gene, but research by Goldman et al (2005) has found that it is possible to co- express two OR genes, as shown in Figure 1.1, adding a further level of flexibility for odour coding.

Figure 1.1: Schematic of an adult olfactory sensillum housing two co-expressed

OSN’s (blue and brown). Adapted from Vosshall and Stocker (2007).

In vertebrates, OSNs are located in the nose. In invertebrates, OSNs are located on antennae, and in some insects such as Drosophila, they have also been found on the maxillary palps (Goldman et al 2005). The OSNs, while exposed on these

olfactory accessories, are protected by a unique covering of specialised hairs called sensilla (Vosshall & Stocker 2007), and these sensilla can be divided into three morphological types: basiconic, coeloconic and trichoid (Hallem et al 2004). OSNs are bipolar and extend from the basal end of the neuron until they reach an olfactory glomerulus in the antennal lobe (AL) – the invertebrate homolog to the mammalian olfactory bulb (OB) – of the brain, where they terminate (Vosshall and Stocker

2007).

Glomeruli have been found not only to be the centre of convergence for OSN axons, but it has also been discovered that neurons expressing a given OR gene converge onto topographically fixed glomeruli. This means that alike and identical OR genes come together at glomeruli, in an arrangement that appears to be fixed from fly to fly, creating a map that encodes olfactory stimuli to aid their interpretation by higher- order brain regions (Vickers et al 1998; Gao et al 2000). This would enable more direct transfer, leading to rapid analysis and interpretation of the olfactory stimulus

(Stockhorst & Pietrowsky 2004).

The higher order brain centres relating to olfactory memory (along with other complex behaviours) in insects are the MBs, which are paired segments located in the dorsal protocerebrum (Ishii et al 2005). The mammalian equivalent which the

OB projects to is the amygdala in the temporal lobe (Barton 2006; Buchanan et al

2008). The MBs are highly conserved in all insects, and are made up of Kenyon cells, a collection of neurons whose somata arise from the cortex lying dorsal to the

MB, with their dendrites arranged in a thick neuropile called the MB calyx, which receives the direct olfactory input from the AL (Ishii et al 2005; Gasque et al 2006).

1.8. Drosophila as a model

Drosophila, and D. melanogaster specifically, are widely used as model systems.

They are inexpensive, easy to maintain in the lab and reproduce quickly, meaning that several generations can be reared in a very short amount of time. In addition, the D. melanogaster genome was published in 2000 (Adams et al 2000), thereby greatly increasing the range and ease of use of genetic and molecular analyses and techniques – indeed, it was this that led to the isolation of ORs and GRs, as well as the identification of a range of other structures and functions. Added to the wealth of behavioural research and anatomical knowledge that has been accumulated on

Drosophila in the past 100 years, the genome sequence provided a huge boost to the advancement in biological sciences that has occurred in the past 12 years (Buck

& Axel 1991; Isono & Morita 2010).

In addition, Drosophila are used as a medical tool – their systems and molecular processes are similar to those exhibited in mammals, mimicking drug action, behaviour and gene response, but are much reduced in complexity. Consequently, pharmaceutical and biotechnology companies are increasingly using Drosophila for drug discovery research. Many methods have been developed for genetic manipulation of the fly, including the breeding of transgenic animals, enabling researchers to perform large-scale assessments of mutations and their traits

(Nichols 2006).

The ramifications for the understanding of basic cognitive processes and higher order behaviours are also huge (Nichols 2006). Most importantly for this research,

Drosophila has a very simple olfactory system, even more so in the larva than in the adult, and the parallels of this olfactory system with those of other species (due to

the fact that structures are highly conserved) make it a useful and important model for advancing understanding in olfactory comprehension and processing (Vosshall and Stocker 2007). It may also provide insights into behavioural responses of a variety of insect pest species, providing significant information for scientists developing specialised pest control techniques (Jones et al 2005). Insects make an immense impression as agricultural pests and in terms of global public health as disease vectors; olfaction is strongly correlated with these behaviours, so a greater understanding of how this system operates could aid in reducing crop destruction and disease transmission through exploitation of more effective novel strategies

(Rützler & Zwiebel 2005).

1.9. Drosophila Larval Olfaction

In D. melanogaster, the olfactory organisation in maggots is much more straightforward than that of the adult, yet is strikingly similar (Vosshall and Stocker

2007). The larval chemosensory system is composed of three major organs found on the head, all situated on the cephalic lobe. These are the dorsal organ, terminal organ and ventral organ. The dorsal organ and terminal organ can be seen in

Figure 1.2.

Figure 1.2: Anatomy of the dorsal organ in Drosophila melanogaster larvae. A.

Head of larva showing dorsal organ (D) and terminal organ (T) (scale bar = 100μm);

B. Dorsal organ showing rim and two basal pores (scale bar = 1μm). Taken from

Cobb (1999).

The paired dorsal organ is responsible for olfactory discrimination, and is made up of a central multiporous “dome” which is innervated by 21 OSNs and six sensilla.

The sensilla of the dorsal organ (and those of the terminal organ and the ventral organ) are believed to be predominantly chemosensory sensilla, but are also likely to exhibit thermosensory and mechanosensory roles (Stocker 1993; Davis 2004;

Vosshall & Stocker 2007). The 21 pairs of OSNs of a Drosophila maggot project to

the 21 larval AL glomeruli. The larval MBs are much reduced when compared to those of the adult form, being comprised of only a small number of glomeruli – so far

34 have been identified (Vosshall & Stocker 2007).

1.10. Drosophila Adult Olfaction

The antenna and the maxillary palp of adult Drosophila, which are shown in Figure

1.3, contain ~1200 and ~120 OSNs, respectively (Hallem et al 2004), which can be split into 16 functional classes (Dobritsa et al 2003). This gives a total of ~1300

OSNs in adult Drosophila, strikingly more than the 21 OSNs found in the larvae

(Davis 2005).

Figure 1.3: Scanning electron micrograph of a fly head, indicating the major chemosensory organs. Taken from Stocker and Vosshall (2007).

In adult Drosophila, 43 glomeruli have been identified (Gao et al 2000), and these structures are regarded as the basic functional components for processing olfactory information in the brain (Vickers et al 1998). These 43 glomeruli converge with

~600 MB neurons. Hundreds of glomeruli are present in the adult MB calyx, and these converge with ~2500 MB neurons (Cobb 1999; Vosshall & Stocker 2007).

Both the MBs and the AL can be seen in Figure 1.4, and, as stated above, the comparison between larval and adult olfactory systems is shown in Figure 1.5.

Figure 1.4: A. Scanning electron micrograph of the head of an adult Drosophila melanogaster female with a surface-rendered 3-D reconstruction of the brain. Major neuropil regions are highlighted. B. Same brain viewed from a slightly more lateral direction. Taken from Heisenberg (2001).

The axons of the Kenyon cells create a structure called a peduncle, and all run in parallel. The peduncle splits into subunits of two or more, and these are the principal output areas of the system (Ishii et al 2005). The MBs ‘learn’ patterns of

activity in OSNs, enabling them to identify the nature and value of odour stimuli, and allowing for the initiation of appropriate responses (Smith et al 2008).

Figure 1.5: Comparative circuitry of the adult and larval olfactory systems. Adapted from Vosshall & Stocker (2007).

1.11. OrCo (previously Or83b)

OrCo- mutants are used in the research carried out herein, and in order to explain the role they play in Drosophila, their scientific background is now described. As stated above, each sensillum can house two OSNs, but each OSN expresses between one and four of the 61 ORs, alongside a co-receptor initially termed Or83b; this has recently been rechristened OrCo and is highly conserved across insect species (Benton et al 2006; Sato et al 2008; Vosshall & Hansson 2011). The

Vosshall lab (Larsson et al 2004) discovered that in Drosophila OrCo- mutants (i.e. those lacking the OrCo receptor), the animal’s responses to odours, both behaviourally and electrophysiologically, were abolished in both adults and larvae, and thus revealed that OrCo is essential for unfailing insect olfaction.

OrCo is believed to act in concert with ORs to mediate their responses to odours.

Its expression is seemingly limited to OSNs, and it has not been discerned in any other tissues, including gustatory neurons. It is found in all 21 larval OSNs in the dorsal organ. These OSNs are extinguished during metamorphosis, but 80 hours after the formation of the puparium, OrCo can be perceived in antennal OSNs

(coinciding with the expression of ORs late in pupal development), and in adult flies it is expressed in all 120 adult maxillary palp neurons, and 70–80% of antennal

OSNs. In OrCo- mutants, ORs do not assemble in OSN dendrites in either adults or larvae, and are constrained to the cell body (although OSN morphology and membrane organisation appear unchanged when visualised). This implies that

OrCo is needed for the correct management of the subcellular localisation of ORs.

OrCo and ORs combine through conserved cytoplasmic loops to form a heteromeric receptor complex early in the endomembrane system, without which insect olfaction becomes unattainable. As such, it is extremely likely that OrCo and ORs coevolved

(Larsson et al 2004; Benton et al 2006). Indeed, it has been found that Drosophila larvae that lack OrCo are disadvantaged in terms of survival, failing to locate food resources which they could exploit and being unable to compete with OrCo functional larvae when in high density conditions (Asahina et al 2008).

OrCo was initially thought to belong to the GPCR superfamily with typical 7TM domains, but further research (Benton et al 2006; Lundin et al 2007), as with all the

Or genes, has shown that its N-terminus is positioned in the cytosol, which is at odds with the configuration expected of such a structure. Lundin et al (2007) additionally found that the C-terminus tail and the EC2 loop are extracellular, and that the glycosylation acceptor sites (important for protein folding, oligomerization, quality control, sorting and transport of secretory and membrane proteins) are not modified as would be expected for the proposed 7TM topology (Kowarik et al 2006;

Lundin et al 2007) – essentially, it displays an inverted topology to that which was expected.

In 2008, it was discovered that the OrCo–OR complex could behave in an ionotropic manner (Sato et al 2008; Wicher et al 2008), but one of the studies also found a G protein dependent metabotropic component. There are now two hypotheses proposed to explain these results: insect ORs have mixed ionotropic-metabotropic components, or they are metabotropically modulated ionotropic receptors. It is still unclear which is correct, but further research has determined that the OrCo channel is regulated by the interaction of elements of the IP3/DAG and cAMP transduction cascades (Martin & Alcorta 2011).

1.12. Drosophila Nociception

In the following research, experiments are carried out using Trp and Painless mutants; to explain why this was done, the scientific background of these two mutations is now presented.

1.12.1. Trp mutants

Transient receptor potential (TRP) ion channels were first discovered in Drosophila in 1969 (Cosens & Manning 1969), but were not fully identified as light-gated Ca2+ channels important for photoreception until some 20 years later (Montell & Rubin

1989; Hardie & Minke 1992). They are highly conserved, and have since been found to be present in every metazoan organism that has been subjected to sequence analysis (Montell 2011), and they perform a range of sensory functions: humans use TRP channels to discern sweet, bitter and umami tastes, and also to distinguish heat and cold in food, such as chilli and mint. Yeast use TRP channels to recognise and react to hypertonicity; nematodes use them to sense and respond to noxious chemicals; and male mice use them to discern males from females

(Clapham 2003; Kim 2004).

In Drosophila, recent research has found that TRP channels come from a much wider family than was previously known, and as a result are used in a broader assortment of sensory signalling than just for photoreception, primarily as nociceptors. They are believed to be responsible for feeling pain (Painless; TrpV1;

TrpV2), for mechanosensation (nompC; Painless), for thermosensation (all members of the TRP family have been shown to function to some extent as

temperature receptors, but notably, for heat avoidance: TrpV2, TrpV3, TrpV4 and

Painless; for cold avoidance: Trpl, TrpM8, and all brivido genes; and for both: TrpV1 and TrpA1), and for hearing (nanchung) (Kim 2004; Rosenzweig et al 2008;

Sénatore et al 2010; Gallio et al 2011; Montell 2011). TRP channels have also been found to be concerned with the secretion of salivary fluid, cardiovascular regulation, changes in pressure, inflammatory responses, lysosome function and the homeostasis of Ca2+ and Mg2+ (Minke 2010).

The TRP superfamily is a collection of seven subfamilies which are highly conserved polymodal cationic channels with six transmembrane domains with cytoplasmic N- and C-termini, and it is believed to have evolved to discern an organism’s environment (Kim 2004; Minke 2010; Montell 2011). The seven subfamilies are classed as TRPC, TRPM, TRPV, TRPA, TRPP, TRPML and TRPN

(Voets et al 2005; Minke 2010). There are 13 members of the TRP superfamily encoded in the Drosophila genome, nine of which have been characterised to date and eight of which have been found to display loss-of-function mutations in their absence (Montell 2005).

In Drosophila, the same signalling cascade which activates TRPA1 downstream of a signalling pathway (employing the same trimeric G-protein phospholipase C that functions in fly phototransduction and thermosensation) is also used to identify certain types of odorants (such as the insect repellent citronella) and for discerning a small subset of gustatory stimulants. As such, TRP channels have been isolated for further research in the hope of improving insect repellents, reducing the incidence of diseases carried by insect vectors and lessening the impact of plant pests (Montell 2011).

The primary concern for this research, in relation to TRP channels, is their role in thermosensation and chemical nociception. Hot and cold receptors are functionally separate classes, converging on glomeruli adjacent to one another (but not overlapping) located on the lateral margin of the Proximal Antennal Protocerebrum

(PAP) in the fly brain; their responses to hot and cold stimuli are proportional to their intensity, and each glomerulus responds to their stimulus with a rapid, transient increase in Ca2+ ions (increase in firing rate), whilst they respond to the opposite temperature stimulus with a reduction in Ca2+ ions (decrease in firing rate). This demonstrates that hot and cold stimuli are each characterised by an individual spatial pattern of activity in the PAP (Gallio et al 2011; Montell 2011).

TRP channels can respond to very small temperature changes of < 0.5˚C, and the ideal temperature is fixed by the individual conduct of each receptor system. Each system negotiates the point at which behavioural aversion (either above or below a certain temperature) is activated – in adult fruit flies this is believed to be temperatures below 21˚C and above 28˚C, while in larvae it is considered to be temperatures below 18˚C and above 24˚C, but both these ranges are approximations – converting temperature signals into behavioural responses in a way that means changing one of the functional classes does not affect the behavioural response to the other. This means that creating a line that has a loss of cold receptors would mean that flies are generated that are no longer deterred by temperatures below the projected limit of 21˚C, but find temperatures over 28˚C aversive (Caterina et al 1997; Voets et al 2005; Gallio et al 2011; Montell 2011).

The TRP channels responsible for thermosensation not only show this response to hot and cold temperatures, but also to hot and cold foods – capsaicin, the active ingredient in countless spicy foods, provokes the same response in Drosophila TRP channels as a physically hot temperature, and menthol and icilin, which elicit a

cooling sensation, prompt a similar reaction as a physically cool temperature

(Montell 2003).

1.12.2. Painless mutants

Painless is a member of the TRP superfamily which was discovered in 2003 (Tracey et al 2003) and is found in the labial palps and the leg tarsus in adult flies. It has been found to play a role in detecting aversive stimuli in a number of ways: it is used for thermosensation and mechanosensation in both larvae and adults, and chemical nociception in adults (Hwang et al 2012; Im & Galko 2012). Painless was the first TRP channel to be identified as being involved in thermosensation; however, despite this, little work with cooling agents has been carried out thus far

(Montell 2003; Montell 2005).

Many gustatory neurons also have a nociceptive sensory role, leading to the belief that some of the main nociceptive sensory neurons are fundamentally gustatory neurons. Kang et al (2010) looked at TrpA1 antibody staining in Drosophila and based on this proposed that the sensory neurons innervating sensilla numbers eight and nine in the labral sense organ in the mouthparts work as chemical nociceptors.

Work on nociception and its sensitisation in Drosophila is in its infancy, and a large array of further work is required for the situation to be understood clearly (Im &

Galko 2012).

TRP melastatin 8 (or TRPM8) is the TRP channel activated by low temperatures and cooling agents such as menthol (Bautista et al 2007). As we know that TrpM8 mutants will not respond to Mentha piperita L., carrying out behavioural experiments with this mutant would prove fruitless; Painless mutants were used instead because

Painless is known to be necessary for thermosensory and chemical sensory behaviour, although it should still have functioning olfactory and gustatory systems, albeit they may be impaired to some extent. Study of this mutant may allow us to elucidate whether mint odours elicit more than a cooling response in Drosophila; for example, do they have a wider range of nociceptive properties, such as causing pain or risk of death, which makes Drosophila avoid them (Amrein & Thorne 2005;

Hwang et al 2012).

An organism’s survival is dependent upon its ability to detect when input from its environment indicates hazardous conditions, and to be able to react to them in an appropriate way – whether they be dangerously high or low temperatures, toxic chemicals or mechanical stress. As such, the presence of Trp and Painless are important factors in an organism’s continued survival (Montell 2003), and it is logical to hypothesise that they may have an involvement in the detection of additional aversive stimuli.

1.13. Drosophila Larval Gustation

Gustatory perception is closely linked to the perception of smell; indeed, gustatory receptors (GRs) and ORs together form a large superfamily of 7TM domain receptors (and, as with ORs, it is still unresolved whether GRs are GPCRs or ligand-gated ion channels) that make up the insect chemosensory receptor genes

(Vermehren-Schmaedick et al 2011). They are remotely related to one another

(they share a common amino residue motif in the seventh transmembrane plus C terminal domain), but as a class GRs are much more divergent than ORs, in some cases sharing an overall amino acid identity as low as 8%. In Drosophila, it has

been found that some Grs (gustatory-specific genes) are expressed in olfactory neurons instead of gustatory ones, and some are expressed in both (Scott et al

2001; Python and Stocker 2002; Vosshall & Stocker 2007; Cobb et al 2008; Isono &

Morita 2010). As such, any study into olfaction also needs to consider, on some level, the potential effect of gustatory input; despite this, very little research has been carried out into Drosophila gustation when compared to the glut of research that is available on Drosophila olfaction.

For larvae, the main goal is to consume as much food as possible prior to pupation.

As eggs, they are laid directly onto their food source, and this is where they live until they emerge from their pupal case as an adult fly. As a result, gustation (along with olfaction) and its related behaviours are very important when it comes to larval ecology and survival. Larval olfactory and gustatory experiences have been found to influence adult behaviour, and so these encounters will frame the life of flies and subsequently, their offspring. To understand Drosophila adult behaviour, it is essential to first comprehend their larval background (Cobb et al 2008).

Despite the different peripheral anatomy of the larval and adult taste systems, they are arranged in an extremely similar fashion, yet in the larvae it is much more condensed. In larvae, the gustatory sensory neurons (GSNs) are primarily located in the chemosensory organs in the terminal organ (TO) on the head (as shown in

Figure 1.2), but sensilla have also been reported in the ventral organ (VO), the pharyngeal sensilla and the poly-innervated external sense organ on the thoracic and abdominal cuticle (Scott et al 2001; Cobb et al 2008).

The TO is assembled into two distinct groups of papillary sensilla (termed “distal” and “dorso-lateral”), with a transversal scolopidium stretch receptor lying beneath.

The location and surface morphology of the TO compared to the DO is shown in

Figure 1.6. The distal group is composed of three papilla sensilla (P1, P2 and P3) and five pit sensilla (T1 to T5), each of which have a single central pore; the three papilla sensilla are believed to have a gustatory function, whilst the five pit sensilla are thought to have a chemosensory function. There are also two knob sensilla (K1 and K2, the second of which is larger), which are each innervated by a single bipolar neurone, and contain a granular substance. Again they have a single pore, but this time it is at the base of the knob and is not believed to have any responsibility for chemosensation. The dorso-lateral group contains four sensilla, two of which are papilla sensilla (one of them being modified), one of which is a ‘spot’ sensillum, and the final one is a small scolopidium stretch sensillum. The structure is presumed to have a gustatory function, but the modified papilla sensilla and the ‘spot’ sensilla are not (Chu-Wang and Axtell 1972a; Cobb 1999; Cobb et al 2008).

Figure 1.6: The anatomical placement of the DO and TO on the larval head. The

DO comprises a single sensillum whereas the TO comprises a number of distinct sensilla (from Matthew Kaiser, unpublished, 2007).

The VO is a raised papillum with four small pores, and it consists of four sensilla, three of which are believed to be mechanoreceptors (they connect to the periphery of the animal via a terminal opening). The remaining sensillum is believed to have a gustatory function (Chu-Wag & Axtell 1972b). The aforementioned pharyngeal sensilla are split into three groups: the dorsal and ventral pharyngeal sense organs

(both of which are paired either side of the pharynx), and the posterior pharyngeal sense organ. They are also the only external larval chemosensory formations that survive through metamorphosis, establishing the foundation upon which similar adult structures develop. Consequently, they might denote a location where transmission of larval chemosensory data through to the adult fly occurs (Gendre et al 2004; Isono & Morita 2010). Lastly, the poly-innervated external sense organ consists of two hemi-segments of the larval thorax, each of which bear two poly-

innervated and axonal projections, which are not fully understood, but their anatomical organisation provokes the belief that they act as chemosensory structures. The differentiation in organ structure indicates that although they all carry our similar roles, there is a segregation of function between the arrangements

(Dambly-Chaudière et al 1992; Cobb et al 2008).

There are some neurons in the TO which have been discovered to react to changes in temperature, although they are not themselves recognised as thermoreceptors; this could have a gustatory function for detection and evaluation of compounds such as capsaicin and menthol (Liu et al 2003; Cobb et al 2008).

Central processing of gustatory stimuli is not as highly regulated, anatomically, as it is for olfactory stimuli, so even if they express the same gene, two neurons may not necessarily project to the same region, as is usually the case for olfactory stimuli

(Vosshall & Stocker 2007). Colomb et al (2007) uncovered that most TO neurons project to the suboesophageal ganglion (SOG) via the maxillary nerve, without passing through the larval AL (in olfaction, OSNs from the maxillary palps travel through the SOG on their way to the AL). The arrangement within the brain can be observed in Figure 1.7. Unlike the AL, the SOG does not consist of structures such as glomeruli which have morphologically apparent structural divisions (Vosshall &

Stocker 2007). Despite this, it has been found that sweet and bitter gustatory signals project to anatomically different areas in the SOG, and so it can be concluded that, at least to some extent, gustatory stimuli are processed according to their anatomical location and functional role (Isono & Morita 2010).

Figure 1.7: This immunofluorescence stain of the fly brain indicates the location of the AL and SOG in respect to one another, as well as indicating OSNs (picked out in green), brain neuropil (red), and nuclei (blue). Taken from Vosshall & Stocker

(2007).

The SOG is responsible for locomotion, so this indicates some close ties between gustatory experience and locomotory behaviour in larvae (Sewell et al 1975). It has four main target subregions, two of which are senior to the other two. The first main area is the focus of the projections that originate from the pharyngeal sensilla, while the second is the focal point for the axons from the TO and the other external organs, as well as some axons from the pharyngeal sensilla. The first of the two lesser regions receives input from the two gustatory neurons present in the DO, whilst the other accepts neurons from the dorsal pharyngeal sensilla and the TO dorso-lateral neurons that join the AL. To date, little is known about how the

information that gathers in the SOG is further processed (Colomb et al 2007;

Vosshall & Stocker 2007; Cobb et al 2008).

1.14. Drosophila Adult Gustation

In adult fruit flies, the expression of gustatory receptors (GRs) is surprisingly divergent – they are found on the two labial palps at the distal end of the proboscis

(87% of the 46 GRs identified from the fly genome and analysed for their expression are located here), but gustatory sensilla can also be found on internal mouthpart organs (35%), the legs (37%), wings (15%) and genitalia (markedly the ovipositor in females), as indicated in Figure 1.8. This contrasts with the positioning of adult olfactory receptors, which are located only on the head (Scott et al 2001; Amrein &

Thorne 2005; Vosshall & Stocker 2007; Isono & Morita 2010).

Figure 1.8: Schematic indicating the position of olfactory (pink) and gustatory

(blue) neurons on the body of the fly. Taken from Stocker and Vosshall (2007).

The expression of a specific GR may be confined to one tissue, or may be found in a combination of tissues; this gives a complex slant to the gustatory profile perceived by the fly (Isono & Morita 2010). Adult Drosophila have different gustatory priorities than their larval counterparts; they eat intermittently, and due to their altered physiology, exhibit a much wider variety of gustatory behaviours (Cobb et al 2008). There are ~300 GSNs on the head of an adult fruit fly, compared to ~80 on the head of a maggot, although this difference is not as severe as the increase in number of OSNs between larval and adult stages. In the larva, the number of GSNs is four-fold higher than the number of OSNs, yet in the adult this relationship is reversed. This transpires as a result of the distinct lifestyle differences between the adult and larval life stages and the consequent change in priorities (Python and

Stocker 2002; Vosshall and Stocker 2007; Isono & Morita 2010).

Gustatory sensilla are found on chemosensory bristles and taste pegs. Bristles are found on the labial palps of the proboscis, legs, wings and ovipositor, whilst taste pegs are found on the proboscis and internal mouthparts. The first are innervated by a single mechanosensory neuron in addition to between two and four gustatory neurons, whilst the latter are innervated by multiple neurons. Both labial palps house 31 bristles and about 30 taste pegs. Bristles and taste pegs, to allow direct access for gustatory receptor neurons (GRNs) to external fluids and nutrients, both have a pore at their terminus. Neurons project from the bristles and taste pegs to the brain; the majority transmit data to the SOG, primarily those from the proboscis, internal mouthparts and some leg neurons, whilst the remaining leg neurons and those from the wings project to the peripheral ganglia. The adult gustatory system is structured similarly to the larval one, but in a numerically amplified configuration

(Amrein & Thorne 2005; Vosshall & Stocker 2007; Cobb et al 2008; Isono & Morita

2010).

Processing of the information received by the olfactory and gustatory systems occurs separately, in the AL and SOG, respectively (Python and Stocker 2002;

Vosshall and Stocker 2007; Cobb et al 2008) – although their perception is closely linked, their processing is carried out independently. Due to the fact that GRs are expressed in a selection of organs, their processing is much more complicated than that of ORs; this allows a broader picture to be formed of the gustatory environment, but at a cost to gustatory discrimination (Isono & Morita 2010).

1.15. Chemosensory processing and oviposition behaviour

Drosophila oviposition, as already discussed in section 1.3.1., is a clear example of host choice behaviour; however, an array of chemical processing is also involved when an individual determines where to deposit her eggs, and this is dependent on olfactory and gustatory inputs. Given that the adult olfactory and gustatory neurons are so thoroughly dispersed across the body (as shown in Figure 1.8), the higher order processing that leads to egg-laying decisions can be complex; this serves as an example of how olfactory and gustatory systems integrate to work in concert to produce behavioural outputs (van Loon 1996; Miller et al 2011; Joseph & Heberlein

2012).

The chemosensory mechanisms regulating oviposition are not wholly understood, but several researchers have unearthed fragments of the bigger picture. Joseph &

Heberlein (2012) have found that the D. melanogaster gustatory neurons expressing Gr66a are required for sensory input relating to “positional aversion” and

“egg-laying attraction” in response to lobeline (i.e. lobeline is found to be aversive by individuals with intact Gr66a-expressing neurons, unless they are looking to

oviposit), a bitter-tasting compound which is a naturally-occurring alkaloid. In addition, Gr66a-expressing neurons in different parts of the body project to different areas of the SOG, through different pathways, so that different behavioural outputs are produced. Despite this it has been discovered that they both converge in the

MBs (Joseph & Heberlein 2012). This is identical to what is observed when looking at the responses of D. melanogaster females to the presence of acetic acid in egg- laying sites (Joseph et al 2009).

Additionally, in Drosophila sechellia and Drosophila simulans it has been found that odorant binding proteins 57d and 57e (OBP57d and OBP57e) influence taste perception and consequentially impact upon host selection behaviour. Introducing

D. sechellia and D. simulans genes Obp57d and Obp57e into D. melanogaster brought about a change in behaviour, switching oviposition preferences and initiating a preference for egg-laying on Morinda citrifolia, a plant that is found aversive by all Drosophila species other than D. sechellia and D. simulans.

Furthermore, in Obp57d and Obp57e knock-out flies oviposition behaviour is disrupted, and clearly both OBPs are involved in host selection behaviour (Matsuo et al 2007). Meanwhile, Yang et al (2008) found that a group of neurons termed insulin-like peptide 7 (ILP7)–producing neurons provides D. melanogaster with feedback on the suitability of egg-laying sites, and as such are key in determining oviposition behaviour.

1.16. Measuring responses to chemostimulants

To establish quantitative measures of an insect’s response to odours, behavioural and electrophysiological methods are primarily used. Larval behaviour is easier to assess than adult behaviour in insects due to the obvious morphological differences

– locomotion is the key feature of behavioural tests, and in maggots locomotion is limited, as can be seen in Figure 1.9, whereas the locomotive range of adult flies is obviously much more developed, allowing them to travel in more planes and at greater speeds (Cobb 1999).

Figure 1.9: Locomotor behaviours involved in the olfactory response of Drosophila melanogaster larvae A. 1-4 muscular contractions involved in forward motion; B. rearing; C. turning; D. bending. Taken from Cobb (1999).

Larval olfaction is measured in behavioural assays that take place on an agar plate which is divided into three sections – responds to odour, non-responder, and control. A piece of filter paper is placed on a raised plastic platform on each side of the agar plate. The odour is then loaded onto the filter paper on either the left or the right. The odour is added an equal number of times to each side to prevent any spatial preference influencing the outcome, and the raised plastic platform prevents physical contact with the odour.

Larval gustation can be assessed in a similar way; an agar plate can be divided into quarters, with a central non-responder region. Two opposite quarters contain control agar, whilst the other two have agar containing the chemostimulant. Again, the position of the plate is randomised to prevent any spatial partiality. In both of these larval assays, a behavioural response index (bRI) can be calculated using the following equation:

n – n bRI = stimulus control , ntot

where nstimulus is the number of maggots responding to the chemostimulant, ncontrol is control and ntot is the total number of maggots (Aceves-Piña et al 1979).

Adult olfactory preference can be assessed in a similar manner. By placing equal numbers of male and female flies in a normal food vial which contains either control food or food containing the chemostimulant, acceptance behaviour can be assessed by measuring the number of flies in each vial which will settle on the food source in a given timeframe, and calculating a behavioural response index in the manner described above. If this is done repeatedly an average settling, and therefore acceptance, rate can be calculated.

Assessment of adult oviposition may be a more accurate method through which to measure adult olfactory or gustatory preferences, because adults are more concerned with reproduction and offspring survival, whilst the main priority of maggots is to eat as much as possible. More compellingly, the spatial location of adults is variable, whilst egg number is fixed following oviposition – it is a straightforward and accurate measure of preference. Adults are presented with three potential oviposition sites (circular pucks containing a suitable material on

which to oviposit): one containing control agar, and the other two containing agar with different concentrations of a chemostimulant, or two different chemostimulants.

Spatial preference is avoided by randomising the location and sequence of the pucks.

In larval tests, timing is important. Towards the end of the third instar, the larva’s focus switches from finding food to finding a good location for pupation. As a result, olfactory preferences change towards the end of third instar, and so will not necessarily correlate with other larval preferences (Cobb 1999). Louis et al (2008) presented odours to larvae dissolved in paraffin oil, but experiments carried out in our lab (Hoare, unpublished, 2008) indicate that paraffin itself presents an odour that influences larval behaviour and interacts with odorants altering their scent, so this is not something we have chosen to replicate. In single-odour-source olfactory assays Louis et al (2008) choose to present their odour in the centre and then graded attraction or aversion in distance from odour source, using an infrared beam to monitor locomotion. These methods have benefits compared to this study; they allow for more accurate measures of individual chemotaxis in D. melanogaster larvae and the multiple-odour-source assay allows for detection of individual preferred concentration, but there are also disadvantages. These are expensive methods to assess locomotion, are likely to be much more time consuming than the methods we employ, and do not allow for the analysis of group behaviour. These assays used by Louis et al (2008) are time consuming and appear to be much more labour-intensive, whilst producing fewer replicate data sets and incorporating the behaviour of far fewer individuals. By following our methods larger data sets can be produced and analysed, providing a clearer picture of population level behaviour

(Louis et a 2008; Asahina et al 2009).

Electrophysiology can be used to measure the firing activity of OSNs, providing a quantitative measure of the organism’s reaction to olfactory inputs, which reflect the stimulus, its concentration and quality, as well as temporal arrangement. It is best achieved when looking at mutants with only one functioning OSN, providing a clearer picture of the inner workings of the Drosophila brain (Schulze et al 1995; de

Bruyne et al 2001; Nikonov and Leal 2002; Amrein & Thorne 2005).

1.17. Drosophila courtship and mating

The Drosophila mating system is very highly regulated and so is another good way to model their behaviour. There are five main stages of Drosophila courtship behaviour performed by the males – orientation of the male towards the female, love song (wing vibration), licking of female genitalia, attempted copulation and copulation as shown in Figure 1.10 – which usually lasts between 15 and 20 minutes (Cobb & Ferveur 1996; Savarit et al 1999; Kubli 2003).

Figure 1.10: Courtship behaviour of D. melanogaster A. Orientation of male towards female. B. Male wing vibration. C. Male licks female genitalia. D. Attempted copulation. E. Copulation. F. A rejection response by the female. Taken from Kubli

(2003).

The female induces these courtship behaviours using cuticular hydrocarbons, predominantly, the 27 carbon diene-7, 11-heptacosadiene (7,11-HD). 7,11-HD is a pheromone that elicits reactions in a dose-response manner – differences in 7,11-

HD concentration lead to differentiation in mating speeds between individuals (Cobb

& Ferveur 1996). Pheromones perform an essential function in the courtship

behaviour of Drosophila, playing a comprehensive regulatory role (Savarit et al

1999). Females’ sexual receptivity is also affected by damage to antennae. To properly respond to the male’s display of wing vibration, a female must have an intact arista and funiculus (Manning 1967).

Mating among hybrids and ancestral strains is regulated by females of the individual strains. Derived males may lose courtship behaviours that ancestral males retain.

Consequently, ancestral females will discriminate against derived males as they lack aspects of courtship, but derived females will copulate with ancestral or derived males, as they both perform the full inventory of courtship components expected of derived males (Cobb et al 1990).

1.18. Aims and Objectives

This project, broadly, aims to answer the following questions; do rearing conditions influence behaviour? To what extent do rearing conditions and environment influence chemosensory behaviours? What impact do rearing conditions have on subsequent generations? And what factors bring about the detection of, and the expressed behavioural responses to, an aversive chemostimulant?

Non-genetic transmission of information across generations is expected to occur in

Drosophila (Islam et al 1994; Plotkin 1996; Elsayed 2011; Messina & Jones 2011), and experience in early life, prior to pupation, might bring about changes to adult

behaviour (Jaenike 1982; Hoffman 1985; Caubet et al 1992; Olvido et al 1998;

Barron & Corbet 1999; Kause et al 1999; Ueda & Kidokoro 2002; Blackiston et al

2008; Zhou et al 2009). If a stimulus from larval life is reinforced (through repeated exposure) in adult life, it will be consolidated, strengthening any transmission of information. The impact of a stimulus may be diminished if there is no reinforcement in adult life because the importance of the stimulus to the adult life stage will be of reduced significance.

Over several generations of being reared in the presence of a stimulus, it is anticipated that preference for this stimulus will increase as a result of environmental adaptation and acceptance as a novel host. Experience will bring about defined changes in the sensory nervous system, eliciting alterations in behavioural and electrophysiological responses which will be transmitted to subsequent generations.

By looking at the effect of early experience on adult behaviour in the fruit fly, D. melanogaster, it is intended to discover if and how transmission of information occurs across life stages. This will be investigated by observing olfactory behaviours primarily to varying concentrations of peppermint, which has previously been reported as being repulsive to D. melanogaster (Thorpe 1939; Barron &

Corbet 1999).

In order to achieve these goals, it must first be established whether a stimulus has an effect on behaviour. Subsequent to it being established that the olfactory stimulus modifies behaviour, flies can then be reared in its presence, and any changes in behaviour recorded and quantified. It is hypothesised that experience with a novel stimulus will induce a change in behaviour; experience with an aversive stimulus will reduce repulsion as individuals adapt to the unfamiliar environment. If

a change is observed when the stimulus is no longer present (either in a subsequent life stage or generation), it would indicate that transmission of information has occurred.

Thus, the aims of my research were to: (1) establish the relationship between an odour and the behavioural response of aversion, both at adult and larval life stages; and (2) determine whether experience of that odour lessens aversion, and how it influences those same behaviours. The central objectives of this study were:

1. To ratify the aversion of D. melanogaster to peppermint extract, and then to

investigate whether being reared in the presence of peppermint reduced the

observed level of aversion, and then observing whether this impacted upon

the behaviour of offspring. Are there effects on both adult and larval olfactory

behaviour? Is the effect dose dependent? Are functioning olfactory receptors

required to be able to respond to peppermint? (Chapter 3)

2. To further examine the effect of peppermint – is contact required between

the organism and the stimulus to trigger a change in behaviour, and are

chemosensory behaviours other than olfaction influenced by the presence of

peppermint. Is peppermint aversive for a reason? Does it impact upon

survival, or impair mobility? (Chapter 4)

3. To investigate whether rearing in the presence of an aversive

chemostimulant over repeated generations strengthens the reduction in

aversion, leading to preference, and also to examine whether rearing in the

presence of an aversive chemostimulant for a single generation influences

subsequent generations. Does the change in behaviour become fixed or

does it change over subsequent generations? (Chapter 5)

4. To explore the underlying mechanisms behind the changes in behaviour.

Are changes in behaviour maternally or paternally linked; can aversion be

linked to any one chemosensory “unit” (e.g. olfaction, gustation, TRP

channels); is chemosensory behaviour sexually dimorphic? Also, is different

behaviour expressed when individuals are exposed to the pure odour

menthol, in comparison to the menthol-containing bouquet of peppermint

extract? (Chapter 6)

Chapter 2

Materials and Methods

2.1. Fly stocks

Fly stocks were maintained in standard 165 ml vials and 330 ml bottles with foam stoppers, and were fed on oatmeal and molasses medium (consisting of 360 g organic maize flour, 396.5 g glucose, 250 g yeast and 45 g agar, giving a total of five litres of fly food; after the mixture had been cooked and cooled, 15 ml of propionic acid and 135 ml of nipagen were added). Peppermint- and menthol- exposed flies were reared on the same oatmeal and molasses medium, with the addition of 0.1% peppermint or menthol, respectively, when the medium was heated to a liquid state. Flies were reared at 25˚C on a 12:12 LD cycle. Control and peppermint-exposed stocks were reared in separate incubators to prevent exposure of peppermint to the control stocks.

The Canton-S (CS) strain (from which peppermint- and menthol-exposed stocks were derived) is a long-established laboratory strain. The fly lines OrCo-: 23130

(w[*]; w[+*] OrCo[2]), Trp: 5692 (Trp[1]), and Painless: 27895 (w[*];

P{w[+mC]=EP}pain[EP2451]) were obtained from the Bloomington stock centre.

2.2. Chemostimulants

Peppermint extract and pure menthol were obtained from Sigma-Aldrich. Both were of the highest purity available. Peppermint was chosen as a chemostimulant because it has long been established as an aversive stimulus for Drosophila melanogaster (Thorpe 1937; Manning 1967). The peppermint oil was of a natural source, extracted from the Mentha piperita L. plant. It has a molecular weight of

136.2 g/mol, and a relative density of 0.898 g/cm3. The menthol was chemically derived, and has a molecular weight of 156.27 g/mol, with a relative density of 0.89 g/cm3. Solid crystals of menthol were dissolved in ethanol to make a 200 mg ml-1 menthol solution, volume was adjusted accordingly to ensure the correct concentration of menthol was supplied to correspond with the pure peppermint extract solution.

Pure menthol was used to investigate whether changes in behavioural patterns arose as a result of that one single odour operating as the active compound in the peppermint plant extract, or whether the naturally occurring bouquet of odours found in peppermint plant extract (from which menthol is derived) contribute, in concert, to produce the overall response. It was also useful for comparison with complementary studies being carried out in the Ferveur laboratory (Université de

Bourgogne, France), in which the odorant used was pure menthol. Peppermint and menthol were presented to the peppermint- and menthol-reared generations by adding them to the fly food medium in its molten state. It is then poured into fly food bottles, covered, and allowed to cool and set before the parental generations were introduced to the bottles.

2.3. Rearing peppermint- and menthol-exposed flies

2.3.1. P1 and M1: Flies reared for one generation in peppermint (P1) or menthol

(M1) media

Control CS flies were placed in bottles with 0.1% peppermint- or 0.1% menthol- containing food, allowed to lay eggs and then removed 24 hours later. The offspring were then reared in the 0.1% peppermint or menthol bottle to adulthood, as shown in Figure 2.1, and then either tested or allowed to breed. Flies exposed for one generation to peppermint are termed P1, flies exposed for two are termed P2, and so on. In the same manner, flies exposed to menthol for one generation are termed

M1. If flies were reared in peppermint-containing food for one generation and then reared in control food for one generation they are termed P1C1, if they were reared in peppermint containing food for one generation and then reared in control food for two generations they are termed P1C2, and so on with increasing generations.

These different treatments were adopted in order to reveal the duration of any apparent transmission of preferences through generations following exposure.

2.3.2. P2: Flies reared on 0.1% peppermint medium for two generations

Flies that eclosed from peppermint P1 bottles (i.e. the offspring of flies placed in bottles containing peppermint medium) were removed from their container and placed in new bottles containing fresh 0.1% peppermint medium. After 24 hours,

these flies were removed (Figure 2.2) and the offspring laid in the new bottle were allowed to mature to adulthood. These flies were termed P2 as they and their parents had both been exposed to peppermint. This was repeated for each subsequent peppermint generation that was reared, to a maximum of nine generations (“P9”) to observe the influence of exposure across several generations.

2.3.3. P1C1: Control food-reared flies, the offspring of flies reared on 0.1% peppermint medium

To produce P1C1 flies, once P1 flies had been reared to adulthood, they were moved to a clean bottle containing control food. They were removed after 24 hours and the offspring were allowed to mature in the control food bottle as P1C1 flies

(Figure 2.3) so that the effect of parental environment on behaviour of offspring could be assessed.

2.3.4. P1C2: Control food-reared second generation offspring of flies reared in 0.1% peppermint medium

P1C1 flies were placed in a bottle containing control food for 24 hours and allowed to lay eggs. The flies were then removed and the eggs they had laid were allowed to mature to adulthood as P1C2 flies (Figure 2.4) so that the influence on control reared flies of peppermint exposure in a previous generation can be assessed.

Controls removed after 24 hrs Control CS flies P1 flies

0.1% peppermint

Figure 2.1: Rearing of peppermint (P1) flies.

Controls removed P1 flies removed after 24 hrs after 24 hrs P1 flies moved P2 flies Control flies to new bottle post-hatching

0.1% 0.1% peppermint peppermint

Figure 2.2: Rearing of P2 flies.

Control flies removed P1 flies removed after 24 hrs after 24 hrs P1C1 flies Control

flies P1 flies moved to new bottle post- hatching

0.1% Control peppermint food

Figure 2.3: Rearing of P1C1 flies.

Control flies removed P1 removed P1C1 removed

after 24 hrs after 24 hrs after 24 hrs

Control P1C2 P1 P1C1 flies flies

0.1% Control Control peppermint food food

Figure 2.4: Rearing of P1C2 flies.

For all behavioural assays, larval and adult, repeats were carried out in the same four hour time period (four to eight hours after artificial sunrise) to minimise variation as a result of circadian rhythms. The time frame could not be reduced any further because of the quantity of time required each day to carry out the required number of behavioural assays.

2.4. Larval behaviour: Rearing

Approximately 200 adult females aged one to seven days post-eclosion were allowed to lay eggs for six hours in laying boxes on a medium containing 2.5% agar,

1% acetic acid and 2% ethanol (Joseph et al 2009). The laying boxes measured

140 mm x 80 mm x 75 mm in volume and contained three removable circular wells in the base, 10 ml in volume, which contained the 2.5% agar medium. A thick yeast paste was also provided as food, and to promote oviposition. After six hours, the adult flies were removed, and the 2.5% agar medium, along with any eggs laid, was moved to a clean Petri dish with a thick yeast paste for larvae to feed on. 48 hrs from the middle of the egg-laying period larvae were washed from the paste and starved for one hour in a clean agar dish prior to behavioural assays. This means that in these assays late first instar and second instar behavioural reactions were tested (Sawin-McCormack et al 1995). This is important because the priority of first and second instar larvae is to attain food and grow. Once individuals reach third instar their priorities change as they commence ‘wandering’, a pre-pupation behaviour a location in which to safely and successfully pupate (Sokolowski et al

1984); to include third instar larvae in assays would skew experimental results. First and second instar larval behaviour provides clearer, more distinct results.

2.5. Larval behaviour: Olfaction

A minimum of 50 second instar larvae were placed in the central start zone (Figure

2.5) of a standard (110 mm diameter) Petri dish containing a layer of 2.5% agar. On each side of the dish, a 1 cm2 piece of filter paper (one piece of which was later loaded with odorant) was raised from the agar by being placed on the lid of an

Eppendorf tube. This ensured the experiment was a test of olfaction, rather than a combination of olfaction and gustation, by preventing the larvae from coming into direct physical contact with the odorant. This set up was first used by Cobb,

Bruneau & Jallon (1992), which itself was an adaptation of a method originally demonstrated by Aceves-Piña et al (1979), and has since been established as the standard method for assessing larval olfactory behaviour in D.melanogaster.

Non-responder section (includes central start zone) Location of Eppendorf lids as raised platforms

Central start zone

Figure 2.5: Olfactory behavioural test plate.

Odorant (menthol or peppermint extract; volumes of 0.5, 1.25, 2.5, 5 or 7.5 μl, as required) was deposited onto one of the pieces of raised filter paper and the lid of the dish was replaced immediately. After five minutes, the distribution of the larvae across the two halves (control and stimulus) and the non-responder section (Figure

2.5) was recorded and a behavioural response index (bRI) was calculated as follows:

nstimulus – n bRI = control ntot

where nstimulus is the number of maggots responding to the chemostimulant, ncontrol is the number of control maggots and ntot is the total number of maggots used in the experiment.

To prevent any positional bias, the location of the odour stimulus and the control were changed sequentially from trial to trial (from left-side to right-side), and uniform light was cast on the test area. At least 20 dishes were tested for each experimental group (strain/stimulus/concentration combination). There are limitations to this approach; because the odour diffuses in the air there is no segregation of “odour” and “no odour”, a gradient is formed with concentration being highest on the filter paper containing the chemostimulant. Further away the concentration is clearly weaker, but on the non-stimulus side the chemostimulant is still likely to be detected. Throughout the experiment, as the odour continues to diffuse through the test arena, the gradient will be reduced. How long this takes will depend upon the volatility of the test odour, and as such the sensory stimulation of larvae is constantly changing over the duration of the assay. We did not grade or zone behavioural responses, and they were classified depending on whether they

were the stimulus side or control side; more precision on level of attraction or repulsion could have been ascertained if this was carried out. Conversely, this heuristic technique is simple, provides a large amount of data relatively quickly, is representative of population level behaviour and is easy to analyse. Additionally the assays are relatively inexpensive, straightforward to repeat, and it would be impossible to devise an assay of an olfactory-only stimulus where diffusion of odours does not occur (Kaiser & Cobb 2008; Louis et al 2008).

2.6. Larval behaviour: Gustation

Larvae were reared using the same method as outlined for larval olfactory behavioural assays. Agar plates were prepared with two opposing quarters containing control 2.5% agar, while the remaining two quarters contained 2.5% agar with either 0.1% or 1% stimulus (varying between plates, not within plates) (Figure

2.6) This is an adaptation of the olfactory assay already outlined from Cobb,

Bruneau & Jallon (1992); modifications were initially carried out by Matthew Kaiser, a former member of the Cobb lab, and described in his thesis (2008).

agar + control stimulus agar

control agar + agar stimulus

Figure 2.6: Gustatory behavioural test plate.

A minimum of 50 second instar larvae were placed in the central start zone, and after 10 minutes the numbers of larvae in each of the four quarters of the plate, either control or stimulus, and in the central start zone (non-responders) were recorded. As with olfaction, a behavioural response index was calculated.

To prevent any positional bias, the agar quarter that was in the top left corner (either control agar or agar plus stimulus) was changed from trial to trial, and uniform light was cast on the test area. At least 20 dishes were tested for each experimental group (strain/stimulus/concentration combination). Unlike olfaction there is no issue with a diffusion gradient in gustatory assays, although it is possible that after leaving peppermint or menthol agar segments that larvae will retain some of the chemostimulant on their bodies for a short period whilst on control segments, much like walking through a puddle leaves wet footprints for the following few steps. This could lead to a clearer distinction between gustatory attraction or aversion when compared with the behavioural response indices found in olfactory assays.

2.7. Adult behaviour: Settling (assessment of host-selection behaviour)

10 males and 10 females (total n = 20; mated, aged one to seven days post- eclosion) were placed in a vial containing either control medium or medium with

0.1% peppermint/menthol. This age range was selected because flies under a day old have their mortality significantly reduced, whilst flies over 7 days old begin to enter “old age” and their senses start to become significantly dulled (Perron et al

1972; Devaud et al 2003). Flies were mated to encourage host-selection in females and in an attempt to limit courtship behaviour which could sway settlng result. A minimum of 30 minutes following transfer to the vial was allowed for recovery from the anaesthetic effects of CO2 application (Faucher et al 2006; Turner & Ray 2009).

The recovery time from CO2 anaesthesia has been found to be proportional to the length of exposure (Nilson et al 2006); the flies were never anaesthetised for more than 15 minutes, and based on work by Nilson et al (2006) they should be 80% recovered in under 10 minutes, and so fully recovered by the end of the allowed 30 minute recovery period. Once vials were transferred to the test area – a walk-in incubator maintained at 25˚C to which entry was prohibited to prevent an impact of noise or human activity on settling behaviour – a 10 minute acclimatization period was allowed to overcome the shock of the new environment. The number of flies settled on the medium was then counted every five minutes for 80 minutes. This was repeated a minimum of 20 times for each experimental group. The mean number of flies settling per vial was then calculated for each time point. This is an experiment that was custom designed for the purpose of assessing Drosophila adult chemosensory behaviour in a quantifiable manner, whilst removing the choice aspect involved in the set-up of a T maze, and ensuring that all chemosensory behaviour involved in host-choice were assessed, not just olfaction.

2.8. Adult behaviour: Oviposition

Oviposition site preference is an excellent method to quantitatively assess female

Drosophila behaviour because it is fundamental to the survival and future fitness of offspring, and so represents a strongly selective factor in species persistence. As such, host choice is closely related to evolutionary history (del Solar & Palomino

1966; Fanara & Hasson 2001) and variations in such behaviour can come about as a result of both genetic or environmental influences (Jaenike 1982). Between 100 and 200 flies were placed in a clear plastic laying box (140 mm x 80 mm x 75 mm) for 24 hours. There were three holes in the lid for ‘pucks’, removable circular wells, that were 10 ml in volume. The pucks contained media for oviposition sites, and once the flies have been added to the boxes they were inverted so that the oviposition sites are at the base and the individuals could settle on them to oviposit; this is displayed in Figure 2.7. This is a long established method of egg collection

(Ephrussi & Beadle 1936), but the laying boxes were custom made for our laboratory so that preferences could be assessed.

The number of flies in each box was roughly equal, but no attempt was made to precisely control the number of flies in each box. This was quite deliberate: the experiment was designed to measure relative attraction to the compounds present in the pucks, and the measure taken was the proportion of eggs laid on each puck for each box. All pucks contained 2.5% agar, 1% acetic acid and 2% ethanol. In each laying box, there were three types of puck: (1) a control with no additional modifications, (2) 0.05% peppermint or menthol and (3) 0.1% peppermint or menthol. After 24 hours, the pucks were removed and the number of eggs laid on each puck was counted. The locations of pucks were rotated from box to box to

prevent any positional bias, all six possible combinations were presented ten times.

It was found in tests for positional bias that when all laying sites were equal, flies significantly preferred to lay on the corner sites 1 and 3, rather than site 2 (in experimental trials using 16 laying boxes, each containing control agar in all three laying sites, a mean of 42.685 eggs were laid on site 1, 23.438 eggs on site 2, and

49.312 on site 3; χ2 =9.389, df=2, p=0.00914516). Repeats were spread over several days, in order to avoid any temporal effects.

Site 2

Site 1

Site 3

Figure 2.7: Location of oviposition sites within a laying box lid.

2.9. ‘No contact’ rearing

To test whether contact with peppermint is required to bring about changes in behaviour flies were reared in the presence of peppermint without allowing them to come into direct contact with it. This was achieved by taking an Eppendorf tube and puncturing holes along the sides in a regular pattern. These holes were large

enough for odour to escape, but not large enough for larvae or flies to enter. 20 µl of chemostimulant was then pipetted into the Eppendorf tube, being careful to ensure none was spilt, whilst a thread was looped through the hinge that attaches the tube to its lid. The lid was then sealed and the tube was dangled inside a bottle containing control fly food. The thread was taped to the outside so that the tube could not drop, flies were added and the bottle was sealed with cotton wool. The flies were left in the bottles for 24 hrs and then removed. The eggs that they oviposited overnight were allowed to mature to adulthood in this environment, where peppermint was present but they could not come into contact with it. This is a technique that was developed specifically for this experiment to ensure that a constant source of peppermint extract could be provided without individuals coming into contact with it.

2.10. Impact of peppermint on survival to adulthood

Adult females were allowed to lay eggs overnight in a laying box with three pucks of medium containing 2.5% agar, 1% acetic acid and 2% ethanol. After six hours, adult flies were removed and the number of eggs on each puck was counted.

Pucks were then placed in standard 165 ml vials containing either control food, or the standard oatmeal and molasses medium with the addition of 0.1% peppermint extract. The number of eggs on each puck was noted, and the number of adults that emerged from these eggs, their gender and which day post-oviposition they emerged were recorded. The long-term effects of peppermint on survival through adulthood to death were not investigated as it was not relevant to this study, and to do so would be incredibly labour intensive.

Survival to adulthood was assessed simultaneously in control flies, P1 flies and P9 flies to prevent temporal effects swaying results. P9 flies were included as a measure to investigate whether being reared on peppermint resulted in artificial selection for flies that did not find peppermint aversive; flies which failed to detect peppermint, and flies which were not susceptible to potential peppermint toxicity.

This would mean that they were more likely to survive because they would be more inclined to contentedly oviposit on peppermint medium and consequently rear offspring of the same disposition, which would then successfully adopt the same host medium, proliferating adaptation.

2.11. Analysis of data

Results were analysed using GraphPad Prism 5 (GraphPad Prism version 5.04 for

Windows, GraphPad Software, La Jolla California USA, www.graphpad.com) and

SPSS (IBM SPSS Statistics 21). Data were tested for normality to determine whether they were parametric or non-parametric and appropriate statistical tests were then decided accordingly. Unless otherwise stated, data was found to be normally distributed and parametric tests were then carried out. Where post hoc tests were required for non-parametric data, a Dunn’s multiple comparison test was carried out; when data was parametric and a statistical test such as an ANOVA was carried out either a Tukey’s multiple comparison test or a Bonferroni multiple comparison test were performed – when all groups needed to be compared primarily a Tukey’s was used, when selected groups were to be compared a

Bonferroni was the post hoc of choice.

Chapter 3

Results: Do rearing conditions influence behaviour?

3.1. Introduction

To look at the impact of a novel stimulus during rearing and its influence on behaviour, first the relationship between the fruit fly, Drosophila melanogaster, and the novel stimulus, peppermint extract, must be considered. Hopkins coined the phrase “Hopkins’ Host Selection Principle” to describe the phenomenon whereby individuals prefer to oviposit on the same host species that they themselves developed (Hopkins 1917; Hewitt 1917), but this behaviour has been found to be plastic, and many have found that the environment an individual experiences influences their development and behaviour (Hoffman 1985; del Pino & Godoy-

Herrera 1999; Dahlgaard et al 2001; Badyaev 2005; Bush 2009). Thorpe and Jones

(1937) explored olfactory conditioning in relation to host selection using the ichneumonid parasite Nemeritis conescens and found that a positive olfactory response can be artificially generated on a novel host, and this host acceptance is heritable (Objective 1).

Thorpe (1939) was the first to experiment on the reactions of D. melanogaster to peppermint in the pursuit of understanding preimaginal conditioning by testing the ability to adopt a previously repellent host, and this was subsequently re-examined by Barron & Corbet (1999). Only responses in the adult life stage were observed, and this was done using an olfactometer. An olfactometer was not used the following work; the settling assays employed ensured that equally quantifiable results could be obtained, but in greater magnitude, and at much reduced cost.

Several settling assays can be carried out concurrently and no specialist equipment was required.

D. melanogaster has two distinct life stages, larval and adult (see Chapter 1:

Introduction for details), therefore it is important to consider and address both in an investigation of D. melanogaster behaviour and how information is transmitted across life stages and generations. As such, the reaction towards peppermint was first examined for larvae, and then adult flies. Olfactory and gustatory behaviour in larvae were examined as an indicator of larval preferences because at the age they were tested (late first and second instar) their primary objective is to eat as much as they can, and to do so as quickly as possible (Cobb 1999). Larvae navigate by utilising olfaction and gustation, and so assays that test these responses are an accurate interpretation of behavioural preference or aversion for particular chemostimulants. Following metamorphosis, there is a shift in priorities. As a result, the manner in which behaviour is being assessed must also change; the behaviours measured in adult flies (also outlined in Chapter 2: Materials and

Methods, sections 2.6 and 2.7) were oviposition behaviour and settling behaviour.

Peppermint extract was chosen as a chemostimulant because it has a history of being found to be aversive in insects, including D. melanogaster (Thorpe 1939;

Manning 1967; El Nagar et al 2012). In Callosobruchus maculatus, the cowpea weevil, peppermint was found, when presented as a fumigant, to significantly reduce fecundity, survival and mating frequency (El Nagar et al 2012). In addition it is a compound that is found naturally, and so one which Drosophila may conceivably encounter in their environment in the wild.

To assess whether different concentrations of peppermint elicit different behavioural responses in Drosophila, experiments are carried out at different volumes and responses documented. Additionally, to examine whether the behavioural response obtained is wholly olfactory, or whether other chemosensory components impact upon the behavioural responses to peppermint, gustatory assays are carried out using OrCo- mutants.

3.2. Responses of control larvae to peppermint

Olfactory behavioural assays were carried out on CS second instar larvae to test whether there is a significant relationship between larval behaviour and peppermint extract, and if this relationship varies with peppermint volume. Thorpe (1939), reported that adult D. melanogaster find peppermint aversive; it is predicted that this would also be the case for larvae, and that there would be a dose-dependent response, as for other aversive stimuli (Jaenike 1983; Jaenike 1986).

Five different volumes of peppermint were used in these olfactory assays (0.5 µl,

1.25 µl, 2.5 µl, 5 µl and 7.5 μl). 11,855 larvae were tested in total across 106 dishes

(c. 100 per dish), and the results are represented as the mean behavioural response index (bRI) ± standard error of the means (SEM) (with 18 to 26 replicate dishes performed per volume) as explained in Chapter 2: Materials and Methods

(Figure 2.5).

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-100 5 Behavioural Response Index (bRI) Index Response Behavioural 0.5 2.5 7.5 1.25 Peppermint volume (l)

Figure 3.1: Olfactory behavioural response indices (displaying mean & SEM) for second instar control CS larvae in response to increasing concentrations of peppermint (n: 0.5 µl = 26 dishes; 1.25 µl = 18 dishes; 2.5 µl = 20 dishes; 5 µl = 24 dishes; 7.5 µl = 18 dishes).

Significant aversion to the odour of peppermint was observed at all doses, with significant differences between odour volumes (ANOVA, F4, 101 = 2.9492, p =

0.0237) (Figure 3.1). A Tukey’s post-hoc multiple comparison test was carried out and revealed that only one pairwise comparison between odour volumes was significant, that between 0.5 µl and 7.5 µl (mean diff = 15.41, p = 0.0309).

The finding that the bRI at a peppermint concentration of 0.5 μl is statistically significantly different from that at a concentration of 7.5 μl suggests that there are behavioural differences associated with varying peppermint concentrations.

However, the lack of any significant differences between the other treatment concentrations (although 5 vs. 7.5 μl comes close) indicates that a large change in peppermint concentration (i.e. a 15-fold increase from 0.5 μl to 7.5 μl) is needed to produce a statistically detectable behavioural difference – clearer dose-response effect might be observed at higher volumes.

Gustatory behavioural assays were carried out on second instar control CS larvae, which were provided with a choice between control agar and agar containing peppermint at one of two concentrations (0.1% and 1%). A total of 5398 larvae across 69 dishes were used for these experiments. Again it was expected that D. melanogaster larvae would find peppermint to be aversive, as it has been found to be in the adult. The effect of peppermint on D. melanogaster larvae has not previously been investigated, but Thorpe (1939) found it to be aversive to D. melanogaster adults.

****

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Behavioural Response Index (bRI) Index Response Behavioural -100 0.1 1 Percent peppermint in agar (%)

Figure 3.2: Gustatory behavioural response indices (mean & SEM) of second instar control CS larvae presented with a choice between control agar and agar containing peppermint at 0.1% or 1% peppermint agar (n = 30 and 39 dishes, respectively).

**** = p < 0.0001 following an independent groups t-test.

Larvae showed significantly aversive responses to both doses of peppermint (Figure

3.2), with a highly significant difference between the two responses (t = 5.454, df =

67, p < 0.0001), showing a dose-dependent aversion. It is possible that there was an olfactory element too, as the peppermint-containing agar smelled of peppermint.

This experiment was therefore repeated using anosmic OrCo- chemosensory mutants (Figure 3.3).

20 ** ****

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-80 ****

Behavioural Response Index (bRI) Index Response Behavioural -100 0.1 1 Percent peppermint in agar (%)

Figure 3.3: Gustatory behavioural response indices (mean & SEM) of second instar control CS larvae (red) and second instar OrCo- larvae (orange) presented with a choice between control agar and agar containing either 0.1% peppermint agar or

1% peppermint agar (CS: 0.1% n = 30 dishes, 1% n = 39 dishes; OrCo-: 0.1% n =

19 dishes; 1% n = 29 dishes). **** = p < 0.0001 following an independent groups t- test.

The experiments with OrCo- mutants show that there is some olfactory component in the gustatory behavioural assays as the gustatory aversion to peppermint agar is effectively eliminated (Figure 3.3). A two-way ANOVA was carried out and there was a significant difference for the two strains, CS and OrCo- (ANOVA, F1, 114 =

64.836, p < 0.0001), and also there was a significant effect for the interaction between strain and percent peppermint in agar (ANOVA, F1, 114 = 8.037, p =

0.00545). A Bonferroni multiple comparison post hoc test was carried out to assess

for differences between relevant groups, and it was found that, in addition to the apparent dose dependence in CS larvae, where aversion increases with peppermint percentage increase, there is significant difference between CS larvae and OrCo- larvae at both 0.1% and 1%.

The olfactory behavioural response indices of control larvae to peppermint gave higher values than the gustatory behavioural response indices, but this difference is difficult to interpret. It may be that on gustatory plates larvae find it more difficult to avoid the peppermint segments for example, not being able to locate them as easily as the direction from which peppermint odour is coming from in olfactory plates.

3.3. Settling behaviour in control adults

10 male and 10 female control CS flies were placed in vials containing either control food or 0.1% peppermint food, in the manner described in Chapter 2. Following introduction to the test area they were left for 10 minutes to adjust to their surroundings, and then the number of flies settling on the food in each vial was counted every 5 minutes, for a total period of 80 minutes. 55 repeats on each type of medium were carried out, using a total of 2200 flies. It was hypothesised that significantly more flies would settle on control food than peppermint food, in line with the observed larval aversion and the prior experiments carried out by Thorpe &

Jones (1937), Thorpe (1939) and Manning (1967). The results are shown in Figure

3.4.

settled on food per vial food on settled Average number of flies of number Average

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins)

Figure 3.4: Behavioural assay of adult settling behaviour (mean & SEM) using control CS flies in the presence of control food (red) or food containing 0.1% peppermint oil (blue) (n = 55 vials per food type).

A two-way repeated measures ANOVA was carried out to test for differences in behaviours between the two food types, as well as over time. There is a significant difference between the two food medium types (ANOVA, F1, 108 = 16.758, p <

0.0001) and a significant effect of time indicating that overall, settling increased

(ANOVA, F15, 1620 = 28.189, p < 0.0001). The strong effect of time on the interaction meant that this was not determined to be significantly different (ANOVA, F15, 106 =

1.718, P = 0.0417). A Bonferroni post-hoc test was also carried out to more meticulously examine behaviour at different time points, and from this it was determined that the control flies on control food settled significantly more frequently than the control flies on peppermint food, from the 40 minute time point until the end of the assay at 80 minutes (Table 3.1).

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Table 3.1: Bonferroni post hoc test of settling behaviour for flies reared on control food

Time P value 5 > 0.9999 10 0.2203 15 0.0400 20 0.1343 25 0.0608 30 0.1952 35 0.3138 40 0.0054 45 0.0223 50 0.0075 55 0.0003 60 0.0033 65 0.0046 70 0.0023 75 0.0010 80 0.0011

The results (Figure 3.4) demonstrate that control CS flies were significantly less likely to settle on food containing peppermint than control food, and as a result it is clear that adult D. melanogaster, like their larvae, find peppermint aversive. In both food types, settling increases over time as the individuals adapt to their new environment and reduce energy expenditure (Parsons 2005). In vials where flies settle rapidly and in larger numbers, the flies are thought to be less stressed than in vials where flies are more reluctant to settle.

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3.4. Experiments on larvae reared on peppermint food

Larval olfactory responses to peppermint at 0.5 µl, 1.25 µl, 2.5 µl, 5 µl, and 7.5 µl were tested in CS second instar larvae whose parents had been reared in peppermint food (P1). They themselves were laid on control agar and fed on live yeast with no peppermint present. It was expected that larval offspring of flies reared in the presence of peppermint would show a reduction in aversion to peppermint in both olfactory and gustatory assays. Control larvae were studied in parallel to accurately survey any divergence in behaviour. Experiments for both P1 offspring and control larvae were repeated between 18 and 24 times at each concentration tested, giving a total of 10461 P1 offspring larvae across 104 dishes and 11855 control larvae across 106 dishes assayed. Assays were carried out at the same concentrations as used for control larvae: 0.5 µl, 1.25 µl, 2.5 µl, 5 µl, and

7.5 µl. Results are shown in Figure 3.5.

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20 * ** *** *** 0

-20

-40

-60

-80 Behavioural Response Index (bRI) Index Response Behavioural -100 0.5 1.25 2.5 5 7.5

Peppermint volume (l)

Figure 3.5: Olfactory behavioural response indices (mean & SEM) for control CS

(red) and P1 (blue) larvae in response to five concentrations of peppermint extract

(n = 18 to 24). * = significant (p = 0.01 to 0.05); ** = very significant (p = 0.01 to

0.001); *** = extremely significant (p < 0.001).

A two-way ANOVA was carried out to investigate the interaction between food type and peppermint volume and a significant difference was found (ANOVA, F4, 209 =

8.644, p < 0.0001), meaning that larvae whose parents were reared in the presence of peppermint, and those whose parents are reared in a control environment react differently to increasing concentrations of peppermint. Additionally, there was significant differences between the two food medium types (ANOVA, F1, 209 = 99.86, p < 0.0001) and a significant effect of peppermint volume (ANOVA, F4, 209 = 9.743, p

< 0.0001), indicating that a lower level of aversion to peppermint was found in larvae whose parents had been reared in the presence of peppermint when

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compared with control larvae, and that aversion to peppermint increases as peppermint volume increases for control larvae, but this was not the case for larval offspring of P1 flies. As indicated by the significance of the interaction larval offspring of P1 flies showed a weak trend towards decreasing aversion to peppermint as the volume increased, signifying an unprecedented level of dose- dependence in P1 larval offspring. This was unexpected and indicates that there may have been a complete behavioural switch in P1 offspring larvae, and instead of finding increasing concentrations of peppermint more repellent, they instead become more attractive, perhaps because they associate the odour with nourishment due to their parents rearing conditions.

A least significant differences (LSD) test was carried out which showed that at all peppermint volumes other than 0.5 µl, the smallest volume tested, the level of aversion is significantly lower in larval offspring of P1 flies than in control flies. The significance of reduction in aversion becomes greater as peppermint volume increases. In control flies there is a significant effect of peppermint volume, with increasing volume intensifying aversion which, as stated above, is not observed in

P1 larval offspring. At 5 µl there is the near abolition of olfactory aversion to peppermint in P1 larval offspring, whilst in control larvae the bRI is -53.45.

Gustatory behavioural assays were carried out with larval offspring of P1 flies, which were reared in the same manner as outlined for olfactory experiments. As with olfactory assays, control experiments and experiments on P1 offspring were carried out concurrently. 5022 P1 larval offspring were tested, along with 5398 control larvae.

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*** * *** 0

-20

-40

-60

-80

-100

0.1 1 Behavioural Response Index (bRI) Index Response Behavioural % peppermint in agar

Figure 3.6: Gustatory behavioural response indices (mean & SEM) for control larvae (red) on petri dishes, two quarters of which contain 0.1% peppermint agar (n

= 30 dishes) or 1% peppermint agar (n = 22 dishes); and P1 larval offspring (blue) on petri dishes, two quarters of which contain 0.1% peppermint agar (n = 30 dishes) or 1% peppermint agar (n = 30 dishes). Two way ANOVA: * = significant (p = 0.01 to 0.05); *** = extremely significant (p < 0.001).

The results show that the parental rearing environment has an effect on gustatory behaviour, that is, being reared in the presence of peppermint reduces aversion to it when compared with control larvae (Figure 3.6). A two-way ANOVA was carried out to scrutinise differences between treatment types (0.1% or 1% peppermint in agar), and also between rearing backgrounds (control or P1 larval offspring). The interaction between food type and peppermint concentration was investigated and found to be significantly different (ANOVA, F1, 111 = 6.223, p = 0.0141). There was

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also a significant effect of food type (ANOVA, F1, 111 = 41.3354, p < 0.0001) and peppermint concentration (ANOVA, F1, 111 = 10.647, p = 0.0015).

The gustatory aversion to peppermint is significantly reduced in larvae whose parents had been reared in its presence (P1 larval offspring). In control larvae the aversion is significantly increased when the concentration of peppermint is increased from 0.1% to 1%. However, in P1 offspring there is no significant difference in aversion when the concentration of peppermint is increased from 0.1% to 1%. This indicates that while the gustatory aversion of control larvae to peppermint is dose-dependent, the reduction in aversion demonstrated by P1 offspring is not. This is similar to the very weak decrease in aversion observed in olfactory assays using P1 larval offspring. Comparably, the increasing concentration of peppermint brings about increasing gustatory aversion in control larvae.

3.5. Settling behaviour in P1 adults

Settling experiments were carried out on P1 flies (flies that had been reared in the presence of peppermint for one generation) in the same manner as for control flies.

40 repeats on each type of medium were carried out, using a total of 1600 flies. It was expected that, as a result of being reared to adulthood in the presence of peppermint, these P1 flies would demonstrate a reduced aversion to the presence of peppermint in a food medium.

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settled on food per vial food on settled Average number of flies of number Average

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins)

Figure 3.7: Behavioural assay of settling behaviour (mean & SEM) using P1 flies in the presence of control food (red) and 0.1% peppermint food (blue) (n = 40 vials per food type).

A reduction in aversion is exactly what was observed; in fact, settling on peppermint appeared to be slightly preferable, in this instance, to settling on control food (Figure

3.7). A two-way repeated measures ANOVA was carried out; time was found to have had a significant effect, with more flies settling over time on both food types

(ANOVA, F15, 1170 = 21.56, p < 0.0001). When looking at the effect of food type it was discovered that the preference of P1 flies for peppermint food was also significant (ANOVA, F1, 78 = 4.399, p = 0.0392). As with control flies (Figure 3.3) there was no significant effect for the interaction between time and food type

(ANOVA, F15, 1170 = 0.5847, p = 0.884), and a Bonferroni post hoc test was carried out to investigate the difference between flies on control food and peppermint food at each individual time point found no significant differences. Overall this implies that although settling on peppermint food looks to be preferable to control food for

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P1 flies it is not be significantly so. This is different to what is seen in settling experiments with control flies, indicating that being reared on peppermint has resulted in a change in behaviour, eliminating the aversion to peppermint food.

3.6. Oviposition behaviour

The final comparison with control flies is that of oviposition preferences. 60 repeats were carried out, with P1 flies placed in a laying box overnight that contained three alternative laying sites: control agar, 0.05% peppermint and 0.1% peppermint.

Control experiments were carried out at the same time to check for temporal variability. The numbers of eggs laid over a 24 hour period on each oviposition site were then counted, totalling 4447 eggs laid by P1 flies and 3928 laid by control flies.

The results are shown in Figure 3.8.

t i

s 5 0

d 4 0

g 3 0

f o

2 0

e b

m 1 0

a 0 e

M 0 0 .0 5 0 .1 P e rc e n t (% ) p e p p e rm in t in o v ip o s itio n s ite

Figure 3.8: Oviposition behaviour (mean & SEM) for control flies (red; n = 60) and

P1 flies (blue; n = 60) when presented with 3 choices – control agar, 0.05% peppermint agar, and 0.1% peppermint agar.

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A two-way ANOVA was carried out; the effect of dose is highly significant (ANOVA,

F2, 354 = 12.35, p < 0.0001), with both control flies and P1 flies laying significantly more eggs on control agar than on 0.05% or 0.1% peppermint agar. There was no significant effect of rearing background (control or P1 flies) on oviposition behaviour.

A Tukey’s multiple comparison post hoc test was carried out to assess differences within rearing backgrounds. For control flies significantly more eggs were laid on control agar than 0.05% (p = 0.002) or 0.1% (p < 0.0001) peppermint agar, but there is no significant difference between the number of eggs laid on 0.05% and 0.1% peppermint agar. For peppermint flies there was no significant difference between any of the laying sites, control agar, 0.05% peppermint agar, and 0.1% peppermint agar, and the mean number of eggs laid, indicating that being reared in the presence of peppermint has eliminated aversion to laying eggs on sites that contain it. There is also no significant difference between the two groups at any of the three site peppermint doses, indicating that oviposition preferences of peppermint flies are not vastly different to the oviposition preferences of control flies – the same pattern of preference is displayed, overall more eggs are laid on control agar than on peppermint-containing sites, but for peppermint flies there is not a significant difference in this pattern.

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3.7. Mating trials

To investigate whether being reared in peppermint had an influence on mating behaviour and sexual acceptance, pairs of virgin flies (C♀C♂, C♀P1♂, P1♀C♂, and

P1♀P1♂) were placed together for 30 minutes the results can be seen in Figure 3.9.

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A B

4 4

3 3

2 2

1 1

0 0 Mating duration log time (secs) time log duration Mating

Courtship duration log time (secs) time log duration Courtship CC CP PC PP CC CP PC PP

C D

4 4

3 3

2 2

1 1 Mating end log time (secs) time log end Mating Mating onset log time (secs) time log onset Mating 0 0 CC CP PC PP CC CP PC PP

E 4 Successful Unsuccessful 3 Total

0 Courtship latency log time (secs) time log latency Courtship CC CP PC PP

Figure 3.9: CC = C♀C♂ mating pair (n = 29 dishes; green in A to D); CP = C♀P1♂ mating pair (n = 24 dishes; red in A to D); PC = P1♀C♂ mating pair (n = 15 dishes; yellow in A to D); PP = P1♀P1♂ mating pair (n =45 dishes; blue in A to D). All graphs show mean and SEM. A. Log courtship duration. B. Log mating duration.

C. Log mating onset time. D. Log mating end time. E. Log courtship latency for successful and unsuccessful pairs, as well as for the total number of pairs.

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To explore the influence of peppermint on courtship duration, mating duration, mating onset time, mating end time and courtship latency were each analysed using a one-way ANOVA, followed by a Tukey’s multiple comparison test to compare individual groups; in all cases any differences were found not to be significant. This indicates that rearing in peppermint does not have an effect on courtship and mating behaviour, and does not alter the way in which affiliates who have not been reared in the same environment react to them. A chi square test was carried out for all four mating pairs for the number of successful and unsuccessful mating pairs and no significant difference was found (χ2 = 4.56, p = 0.207).

3.8. Chapter Summary

The results presented herein have shown that D. melanogaster, both as larvae and adults, find the odour and taste of peppermint extract behaviourally aversive. The aversion to peppermint as a gustatory stimulus appears to be dose-dependent, whilst the aversion to peppermint as an olfactory stimulus shows only a weak indication towards dose-dependence. From gustatory assays using OrCo- mutants it is evident that there is a large olfactory aspect to larval chemosensory behaviour in response to peppermint, as their aversion is effectively eliminated without functioning olfactory receptors.

In flies that have been reared in the presence of peppermint, this aversion is eliminated and they in fact settle on peppermint food significantly more frequently than on control food, albeit weakly so. The larval offspring of flies reared in the

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presence of peppermint also demonstrate a lessening of aversion to peppermint, and in larval olfactory assays aversion appears to decrease as concentration of peppermint increases. In terms of oviposition preference, control flies prefer to oviposit on surfaces containing control medium, over medium containing 0.05% or

0.1% peppermint, whilst in peppermint flies the aversion to laying eggs on sites containing peppermint appears to be eliminated. Being reared in the presence of peppermint does not significantly alter mating behaviour or sexual acceptance. This is in agreement to what was found by Thorpe (1939) and Barron and Corbet (1999).

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Chapter 4

Results: Exploring the effects of environment and

rearing conditions on chemosensory behaviours

4.1. Introduction

The data described in the previous chapter showed that rearing conditions significantly altered chemosensory behaviours in both larvae and adult D. melanogaster. In this chapter I explore how these effects occur. In Chapter 3 –

Results: Do rearing conditions influence behaviour?, it was shown that rearing conditions significantly influence behaviour in D. melanogaster. In this Chapter, the environmental factors relating to this effect are examined in an attempt to elucidate how these behavioural changes occur, as well as the resultant events. As stated in

Chapter 1: Introduction, a wide range of variables can influence Drosophila olfactory behaviour, including repeated exposure, circadian rhythms, temperature, geographical location and exposure to CO2. To counteract these influences, experiments were carried out simultaneously in a temperature controlled walk-in incubator (using a 12:12 LD cycle), and control and peppermint stocks were stored in separate incubators to prevent exposure of control stocks to peppermint prior to experimentation. In addition, the stocks used were all aged under seven days (into

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the adult life stage) to limit the influence of age on outcomes, and exposure to CO2 anaesthesia was limited to only where it was strictly required, and lowest quantity possible to achieve sedation was utilised to limit exposure. Despite this, there are a variety of other factors that might influence the response of D. melanogaster to peppermint extract. The way that peppermint is presented to the organisms within food necessitates contact and ingestion of the peppermint extract, so it was not clear whether this contact was necessary to bring about the observed changes in behaviour. It also needed to be ascertained whether larval outputs were influenced by the behaviour of others within the group, and whether different behavioural outputs would be produced when organisms were introduced to the stimulus as an individual (Kaiser & Cobb 2008). Additionally, is there a reason that peppermint is aversive to Drosophila, is it toxic in a way that impacts upon locomotion and mobility, or does it influence survival (Objective 2).

4.2. Necessity of contact

First, it was investigated whether contact with peppermint in the P1 generation was required to bring about the observed change in behaviour. To achieve this, larvae were reared in the presence of peppermint odours, but without being able to come into contact with it due to its containment within Eppendorf tubes with air holes, not large enough for larvae to pass through, punched into the sides (Materials and

Methods section 2.8.1.); these individuals were termed ‘no contact’. In the initial experiment, these larvae were then reared to adulthood and their adult behavioural responses to control and 0.1% peppermint food were assessed using the standard

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settling experiment. Responses were tested with 20 flies in each vial, 10 female and 10 male, and 20 repeats were carried out on each type of medium.

4 settled on food per vial food on settled Average number of flies of number Average 2

0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins)

Figure 4.1: Behavioural assay of adult settling behaviour (mean & SEM) using ‘no contact’ flies in the presence of control food (red) and 0.1% peppermint food (blue)

(n = 20 vial per food type).

The average number of flies settled on food medium per vial indicates that there was no preference shown by ‘no contact’ flies for control or 0.1% peppermint food

(Figure 4.1). This was shown statistically using a two-way repeated measures

ANOVA, which revealed that although time had a significant effect on settling behaviour (ANOVA, F15, 570 = 10.82, p < 0.0001), the impact of the food type is not significant (ANOVA, F1, 38 = 0.2310, p = 0.6335), and there was no significant interaction between time and food type (ANOVA, F15, 570 = 0.6963, p = 0.7893). This

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implies that the reduction in aversion demonstrated by P1 flies is not wholly dependent on having direct contact with the chemostimulant. ‘No contact’ flies also demonstrate a reduction in aversion without having come into contact with it in their food medium.

The larval offspring of ‘no contact’ reared flies were then tested to assess their behavioural responses to peppermint. Olfactory behaviour assays were carried out using 2476 control larvae across 24 dishes, 2048 P1 larval offspring across 24 dishes, and 1574 ‘no contact’ larval offspring across 26 dishes, at a volume of 5 µl of peppermint extract. This dose was chosen as it elicited the lowest level of aversion in P1 offspring larvae, along with a strong level of aversion in control larvae

(Figure 3.5).

*** *** 0

-20

-40

-60

-80

-100

Control P1 No contact Behavioural Response Index (bRI) Index Response Behavioural

Figure 4.2: Olfactory behavioural response indices (mean & SEM) for control (red; n = 24 dishes), P1 offspring (blue; n = 24 dishes) and ‘no contact’ offspring (green; n

= 26 dishes) larvae in response to 5 µl peppermint extract. A Tukey’s multiple comparison test was carried out to analyse differences between groups: *** = extremely significant (P < 0.0001).

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A one-way ANOVA was carried out on the results (Figure 4.2) and showed that there was a significant difference between the groups (ANOVA, F2, 71 = 19.35, p <

0.0001). A Tukey’s multiple comparison test was carried out post hoc to interpret differences between groups; it was found that there are significant differences between control larvae and P1 larval offspring, as expected. There was also a significant difference between P1 offspring larvae and ‘no contact’ offspring larvae, but there is no significant difference between control larvae and the larval offspring of ‘no contact’ flies. This indicates that contact with peppermint extract in the parental generation is required to reduce olfactory aversion to peppermint in the offspring of ‘no contact’ flies.

Gustatory behaviour of the larval offspring of ‘no contact’ flies was next examined.

For this assay, 2155 control larvae across 30 dishes, 2137 P1 larval offspring across 22 dishes, and 1051 ‘no contact’ larval offspring across 18 dishes were tested on dishes with two quarters containing control agar and two quarters containing 1% peppermint agar.

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***

-20

-40

-60

-80

-100

Control P1 No Contact Behavioural Response Index (bRI) Index Response Behavioural

Figure 4.3: Gustatory behavioural response indices (mean & SEM) for control larvae (red; n = 30 dishes), P1 larval offspring (blue; n = 22 dishes) and ‘no contact’ larval offspring (green; n = 18 dishes) on petri dishes, two quarters of which contain

1% peppermint agar, the other two quarters of which contain control agar. One-way

ANOVA: ** = very significant (p = 0.001 to 0.01); *** = extremely significant (p <

0.001)

A one-way ANOVA revealed significant differences between groups (ANOVA, F2, 67

= 15.80, p < 0.0001); Tukey’s multiple comparisons showed there was a significant difference between control larvae and both P1 larval offspring and ‘no contact’ larval offspring (Figure 4.3). However there was no significant difference found between

P1 larval offspring and ‘no contact’ larval offspring. This indicates that for gustation, unlike for olfaction, contact with peppermint is not necessary for aversive behaviour to be reduced – mere contact with peppermint odour is sufficient. This is contrary to

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gustatory assays with OrCo- larvae, which revealed that there is a largely olfactory component to the behaviour of maggots on gustatory plates. It is also observed that it is more difficult for individuals to orient away from a noxious stimulus present in agar once they are on it as it is difficult to locate agar absent of peppermint. As such olfactory larval assays may be a more consistent method for assessing behaviour than gustatory larval assays.

4.3. The behaviour of individual larvae

The studies of larval olfactory and gustatory behaviour described above all involved the mass plate test, in which a large number of larvae are allowed to migrate on an agar plate, and their distribution is recorded after 5 minutes. Although there is no

‘stampede effect’ whereby the behaviour of one maggot or group of maggots affects the response of other individuals, behavioural interactions between larvae do occur in the plate test, affecting the result compared to individual tests (Kaiser & Cobb,

2008).

To investigate whether the behaviour of other individuals altered the behavioural responses of individual larvae, the gustatory choice behaviour and final location of individual maggots were recorded when the insect was tested on its own. The final destination of larvae, either on a control or peppermint section, was recorded after ten minutes and a behavioural response index was calculated. This was then compared with the bRIs of the gustatory behavioural assays carried out in groups.

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-20

-40

-60

-80

Behavioural Response Index (bRI) Index Response Behavioural -100 Group Individual

Figure 4.4: Comparison of gustatory behavioural response indices for group (mean

& SEM) and individual control larvae (red; group n = 39 dishes, individual n = 30 dishes), P1 larval offspring (blue; group n = 30 dishes, individual n = 33 dishes), and

‘no contact’ larval offspring (green; group n = 20 dishes, individual n = 18 dishes) in response to gustatory plates containing two quarters 1% peppermint agar and two quarters control agar. In individual larval assays final destination of a single larva for each plate was used to calculate bRI; it is not a mean and so there is no SEM for this data.

A Kruskal-Wallis test was carried out (the bRI for “individual” data has no mean as each assay evaluates the behaviour of only one individual, so it is not possible to carry out a parametric evaluation; there is no normal distribution with calculable mean and variance for this data set) to test the significance of variations in behaviour; this showed that there was significant difference in responses (Kruskal-

Wallis statistic = 26.16, p < 0.0001), but a Dunn’s multiple comparison test found that the only significant difference was between the grouped data for both control larvae and P1 larval offspring (Figure 4.4). As there is no mean for individual

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larvae, a χ2 test was carried out to more accurately interpret the larval behaviour (χ2

= 9.592, df = 2, p = 0.008) and a significant difference as found in the behaviour of the three rearing types/strains on individual larva gustatory plates, indicating that the reduction in aversion observed in P1 larval offspring is significantly different to the bRIs for individual CS larvae and the ‘no contact’ larval offspring.

4.4. Effect of rearing conditions on behaviour

It was hypothesised that certain observed changes in behaviour might occur as a result of rearing conditions (i.e. are P1 larval offspring behaviourally or developmentally altered due to their parents being reared in the presence of peppermint). To investigate this, the behaviour patterns of individual control larvae,

P1 larval offspring, ‘no contact’ larval offspring and OrCo- larvae were examined on agar plates. The movement of individual larvae was traced on the plate lid; these traces were then scanned and analysed using ImageJ to provide track-length data.

The final position of each larva was also noted, the results are shown in Figure 4.5.

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A B 6 100

80 4

40 2

point (10 mins) (10 point Speed(pixels/second) 20

Proportion of larvae on agar at end at agar on larvae of Proportion 0 0 Control P1 No contact OrCo Control P1 No contact OrCo

C D 2000

60000

) 2 1500

40000 1000

20000

500 Area covered (pixels covered Area 0 0

Control P1

Averagedistance travelled (pixels)

OrCo

Control No contact No

Figure 4.5: Gustatory responses of individual larvae to peppermint. Control = control larvae; P1 = larvae whose parents were reared on peppermint food; ‘no contact’ = larvae whose parents were reared in the presence of peppermint odour, but without direct contact; OrCo = OrCo- mutants A. End point – proportion of larvae on control agar (red) or 1% peppermint agar (blue) after 10 mins. B. Mean speed of movement on each type of agar (pixels/sec; showing SEM) on control agar (red) and 1% peppermint agar (blue) for control, P1 offspring, ‘no contact’ offspring, and

OrCo- larvae. C. Mean distance travelled per larva on each type of agar (pixels; showing SEM) on control agar (red) and 1% peppermint agar (blue) for control, P1 offspring, ‘no contact’ offspring, and OrCo- larvae. D. Mean area covered from grid overlay (each square = 2000 pixels2; measured in pixels2; showing SEM) by control and P1 offspring larvae on control agar (red) and 1% peppermint agar (blue).

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Control and “no-contact” larval offspring both showed a significant preference for control agar, as measured by their location after 10 minutes (Figure 4.5A, Fisher’s exact test Table 4.1); however, both P1 larval offspring and OrCo- larvae showed no significant difference in their response to control and peppermint agar. This shows that the P1 larval offspring have a significant reduction in aversion to peppermint, that this effect requires parents to have physical contact with peppermint (‘no contact’ larval offspring did not show this effect), and that this effect may be mediated by olfactory receptors (OrCo- larvae also showed no effect). However, it should be noted that the OrCo- larvae are on an Oregon-R background, whereas the other larvae studied here are Canton-S. Strain differences therefore cannot be excluded; indeed, there are known differences in the pattern of OR gene expression between these two strains (see Hoare et al, 2008).

Table 4.1: Gustatory end point post hoc

Fisher’s exact test Significant? P < 0.05? P value

Control vs P1 Yes 0.0099

Control vs no contact No 1.0000

Control vs OrCo- No 0.0569

P1 vs no contact Yes 0.0259

P1 vs OrCo- No 0.7725

No contact vs OrCo- No 0.1014

The behaviour that preceded this final choice between control and peppermint agar can be glimpsed in the data in Figures 4.5b-d. The speed travelled on the two

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different agar types (control and 1% peppermint agar) was examined by calculating the distance travelled (measured in pixels, as digital images were used) and dividing it by the time in seconds spent on each agar type (Figure 4.5 B). Overall, there was no significant effect of agar type on locomotor speed (ANOVA, F1, 199 = 3.136, p <

0.078), there were significant differences between rearing background groups

(ANOVA, F3, 199 = 12.63, p < 0.0001), however there was no significant differences when looking at the interaction between rearing background groups and agar type

(ANOVA, F3, 199 = 2.014, p = 0.113). A Bonferroni’s multiple comparison test was carried out to examine specific differences within and between groups, and revealed that P1 and ‘no contact’ larval offspring moved faster than control and OrCo- larvae.

The fact that these differences do not mirror the final choice results in Figure 4.5A suggests that speed of movement is not involved in determining the final choice, and markedly, these experiments were carried out over several days. Additionally, there was a significant difference in the speed travelled by OrCo- larvae on control and peppermint agar – they travelled faster on peppermint than control agar, an occurrence only observed in this group of strain or rearing type.

The average distance travelled on each agar type was also calculated and examined (Figure 4.5 C). It was found that there was no significant effect of agar type on distance travelled (ANOVA, F1, 199 = 0.168, p = 0.682), but there was a significant difference between rearing background/strain groups (ANOVA, F3, 199 =

4.726, p = 0.003), as well as a significant interaction between agar type and rearing background/strain groups (ANOVA, F3, 199 = 4.785, p = 0.003). To investigate how this varied within and between groups, a Bonferroni’s multiple comparison test was carried out. Larvae did not travel significantly shorter or further distances based on agar type when looking within rearing background/strain groups. Between groups

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the only significant difference being that P1 larval offspring travelled significantly further on peppermint agar than control larvae.

Finally, the area covered by control larvae and P1 larval offspring on control and 1% peppermint agar were calculated by applying a grid (area per square = 2000 pixels2) over the plate image in ImageJ, and then counting how many squares were crossed by each individual maggot for both agar types per plate. Mean and SEM were calculated and results are shown in Figure 4.5 D. A two-way ANOVA was carried out and it was found that, overall, significantly greater area was covered on peppermint agar than control agar (ANOVA, F1, 125 = 12.499, p < 0.001), and a significantly greater area was covered by control larvae than P1 larval offspring

(ANOVA, F1, 125 = 5.278, p = 0.023). The interaction between agar type and rearing background was not significant however (ANOVA, F1, 125 = 0.004, p = 0.953), and when a Bonferroni’s multiple comparison test was carried out it was found that within groups there was no significant difference in area covered between control and peppermint agar by either control larvae or P1 larval offspring.

4.5. Effect of peppermint on juvenile survival

It was necessary to evaluate if peppermint was harmful to survival in D. melanogaster, as this may influence behavioural responses. It may be that control flies find peppermint aversive because it is in fact toxic, and additionally, it may be that any decrease in aversion to peppermint following repeated generational exposure to peppermint results from only flies resistant to peppermint toxicity

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surviving to reproduce, meaning that a selection for flies who do not find peppermint aversive has occurred. This was assessed through recordings of juvenile survival

(from egg to adult); eggs were laid on control agar, the agar was cut into sections, the number of eggs on each segment was counted, and then each segment was placed in a vial containing either control food or 0.1% peppermint food. Each vial was labelled with its food type and egg number. The number of flies that eclosed on each day from each vial, and the ex of those flies, were recorded. A total of 3641 eggs were used in this trial. Control CS flies showed a 7.7% reduction in survival on

0.1% peppermint food, indicating that initial aversion to peppermint may stem from its toxicity (Figure 4.6 A). Strikingly, this effect appeared to be constant over the six days that flies eclosed; a difference in the slope of the peppermint medium curve compared to control might have indicated a cumulative effect of exposure to peppermint.

In addition, juvenile survival on control food and 0.1% peppermint food was investigated in P1 offspring (offspring of flies reared on peppermint food for one generation; Figure 4.6 B) and P9 offspring (offspring of flies reared on peppermint food for nine generations; Figure 4.6 C), to examine whether being reared in the presence of peppermint (for one or several generations) had an influence on survival. In P1 offspring and P9 offspring, reductions in survival of 7.21% and

7.67%, respectively, were observed on 0.1% peppermint food. Thus, D. melanogaster reared on peppermint food have the same survival pattern whether or not they have experienced peppermint food for multiple generations. This suggests that exposure to peppermint does not lead to selection for sensitivity to peppermint

– the reduction in survival is consistent, and it is not the case that individuals that do not find peppermint to be aversive or toxic are being selected for.

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100

umulativeeclosion rate

C 20

0 0 5 10 15 Time (days)

100 B

Cumulativeeclosion rate 20

0 0 5 10 15 Time (days)

100 C

40 Cumulativeeclosion rate 20

0 0 5 10 15 Time (days)

Figure 4.6: Cumulative eclosion rates as juvenile survival in the presence of a control food medium (solid line) and a 0.1% peppermint food medium (dashed line) for A. Control CS (red); B. P1 offspring (light blue); C. P9 offspring (dark blue).

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No significant differences were observed for male and female survival in any of the groups (Figure 4.6). It was found that, as indicated below (Table 4.2), there is no significant sex difference in levels of toxicity of peppermint extract on D. melanogaster.

Table 4.2: Survival comparison table

Female Female survival Sig.? Male Male survival in Sig.? survival in in 0.1% survival in 0.1% control food peppermint food control peppermint (%) (%) food (%) food (%)

CS 51.32 47.45 ns 48.68 52.55 ns

P1 52.74 51.05 ns 47.26 48.43 ns

P9 50.00 53.05 ns 50.00 46.95 ns

4.6. Chapter Summary

Flies reared in the presence of peppermint but without direct contact with the chemostimulant demonstrated reduced chemosensory aversion to peppermint as adults, in line with P1 adults, but the behavioural output of their larval offspring were more variable. In olfactory assays they demonstrated aversion on par with control larvae, but gustatory assays were more convoluted, and whilst in groups larvae appeared to display reduced aversion like P1 larval offspring, but in individual assays they behaved similarly to control larvae.

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As such, in adult chemosensory assays it was shown that contact with peppermint is not essential to bring about a reduction in aversion to peppermint, but does appear to be necessary to ensure durability to larval offspring assays. In all cases the reduction in aversion does not appear to be as substantial as when contact is allowed, so it is likely that the presence of peppermint in food media brings about a more marked reduction in aversion than odour alone (Figure 4.1 to Figure 4.4)

“backing up” and reinforcing the olfactory stimulus. This is similar to what was found by Barron and Corbet, although they did not test the behavioural responses of offspring (1999).

When looking at the gustatory behaviour of individuals (Figure 4.5), control larvae and ‘no contact’ larval offspring demonstrate a significant preference for control agar compared to P1 larval offspring. Despite this, control, P1 larval offspring, and ‘no contact’ larvae did not generally travel faster or further depending on the type of agar used in experiments. In general, P1 larval offspring and ‘no contact’ larval offspring travelled faster than CS and OrCo- larvae, but experiments were carried out across several days so this may result from temporal effects. OrCo- larvae travelled faster on peppermint agar than on control agar. This could be a result of their anosmia – perhaps they did not anticipate encountering peppermint agar, but once they were on it their GRs detected it as a stimulus, accelerating the speed of their transit as a result of shock. It could perhaps be painful, and as a result they tried to travel away from the stimulus as quickly as possible. P1 larval offspring travelled faster than control larvae independent of agar type, and travelled significantly further than control larvae on 1% peppermint agar. This doesn’t appear to be noteworthy and may well be temporal. When looking at the area covered by control and P1 larvae, there was no significant difference within or between groups.

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The aversion to peppermint agar by control larvae was expected, and was not unforeseen in ‘no contact’ larval offspring, whilst OrCo- larvae were expected to demonstrate olfactory impartiality, but were expected to behave differently once they encounter peppermint in a gustatory setting. P1 larval offspring were expected to show reduced aversion, when compared with control larvae, in response to peppermint agar. With regard to OrCo- larvae it may be that they do not demonstrate a preference for control agar in a gustatory setting because they cannot detect it by smell and avoid it through locomotor behaviour as control larvae can. This leads to faster speed travelled when they encounter peppermint in a gustatory setting than in the other strains and backgrounds tested. For the endpoint of gustatory assays it would appear that control larvae and ‘no contact’ larval offspring can anticipate and avoid peppermint agar. P1 larval offspring are less concerned about avoiding peppermint agar, and OrCo- larvae are not forewarned about the presence of peppermint agar due to anosmia.

Peppermint was found to reduce juvenile survival by ~7–8% in control CS flies, P1 offspring and P9 offspring, offering a biological reason for the demonstrated aversion in control larvae and adults, and also indicating that selection is not taking place with repeated generational exposure to peppermint – there is apparently no accidental selection for individuals that do not succumb to peppermint toxicity, and therefore do not find it to be behaviourally aversive. The influence of peppermint on

Drosophila survival has not previously been assessed.

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Chapter 5

Results: Influence over generations

5.1. Introduction

The introduction of a novel plant host into a phytophagous insect’s diet can lead to host–plant specialisation, such as demonstrated in the apple maggot fly on apple trees in North America, and D. sechellia on Morinda citrifolia from the Cousin,

Praslin, and Frigate islands of the Seychelles (FlyBase, version FB2012_06). As a result, it is thought to be an instigator of genetic divergence and can lead to speciation given enough time (Jaenike 1980; Jiggins & Bridle 2004; Diegisser et al

2009). D. sechellia demonstrate allopatric speciation, in that there has been enough geographic separation for them to become a distinct sister species; D. sechellia, and its sister species D. mauritiana, are island species which branched off from the mainland species D. simulans as a result of their isolation (Kliman et al

2000). D. sechellia are the only subspecies of Drosophila that can feed on the fruit of the shrub Morinda citrifolia, and do so almost exclusively. To all other Drosophila species it is lethal following a few minutes of contact (Farine et al 1996). The initial host species of the apple maggot fly Rhagoletis pomonella was hawthorne, but following the introduction of apple fruit trees from Europe to North America in the

19th Century there was a host switch (as described in Chapter 1: Introduction). The

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apple and hawthorne races have since become genetically divergent while inhabiting the same geographic location, in a clear demonstration of sympatric speciation, and the apple race is now a common commercial pest (Jiggins & Bridle

2004). Based on this, the influence of peppermint on behaviour over several generations in D. melanogaster was examined in two ways; populations of flies were either reared on peppermint food across multiple generations, or were reared on peppermint for a single generation following which all descendants were reared on control food and then their behavioural responses were assessed (Objective 3).

5.2. Influence of repeated generational exposure

The influence of exposure to peppermint extract over several generations was assessed in both the larval and adult life stages. Groups of flies and larvae were identified according to their exposure to peppermint. P1… P10 flies and larvae had been reared for 1…10 generations on peppermint.

Larval olfactory assays were carried out on P1, P2, P4, P5 and P10 larval offspring to establish how larval olfactory behaviour is influenced over repeated generations of exposure. All generations were presented with 5 µl of peppermint extract pipetted onto filter paper atop an Eppendorf lid in their test plate.

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-20

-40

-60

-80

Behavioural Response Index (bRI) Index Response Behavioural -100 Control P1 P2 P4 P5 P10 Rearing conditions

Figure 5.1: Olfactory behavioural response indices (mean & SEM) for control larvae

(n = 24 dishes; red) and larvae whose parents have been exposed to peppermint extract for a number of generations (blue), P1 (n = 24 dishes), P2 (n = 30 dishes),

P4 (n = 30 dishes), P5 (n = 22 dishes), and P10 (n = 26 dishes).

With repeated exposure to peppermint over generations aversion to this substance decreases (Figure 5.1). A one-way ANOVA revealed a significant difference between groups (ANOVA, F5, 150 = 35.11, p < 0.0001). To further examine the relationship between individual groups, a Tukey’s multiple comparison test was carried out (Table 5.1). As can be seen, one generation in the presence of peppermint (P1) lead to a significant reduction in aversion to peppermint in larval offspring in response to an olfactory test; one subsequent generation (P2) further reduced that aversion to a level that was not significantly different from zero (t =

-0.308, df = 29, p = 0.760). Further generations on peppermint led to no further significant alteration in the olfactory response of their offspring. This shows that after

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two generations reared on peppermint, larvae are either no longer able to discriminate this substance, or no longer find it aversive.

Table 5.1: Olfactory behaviour for control and peppermint generations post hoc

Tukey's Multiple P value Summary Comparison Test Control vs P1 < 0.0001 ***

Control vs P2 < 0.0001 ***

Control vs P4 < 0.0001 ***

Control vs P5 < 0.0001 ***

Control vs P10 < 0.0001 ***

P1 vs P2 0.5711 Ns

P1 vs P4 0.9051 Ns

P1 vs P5 0.2224 Ns

P1 vs P10 0.0164 *

P2 vs P4 0.0564 Ns

P2 vs P5 0.9736 Ns

P2 vs P10 0.4839 Ns

P4 vs P5 0.0119 *

P4 vs P10 0.0002 ***

P5 vs P10 0.9443 Ns

The olfactory bRI of control larvae is extremely significantly different (p < 0.0001) from that of all peppermint offspring generations tested. The bRI’s for P1, P2, and

P4 are not significantly different from one another, although there are significant differences between P4 and P5, as well as P4 and P10. Additionally, there is significant difference between P1 and P10. This lends further support to the finding

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that repeated exposure decreases aversion and may eventually lead to a preference for that odour in D. melanogaster second instar larvae.

A similar but not identical pattern was seen in larval gustatory responses to peppermint (these gustatory assays, as shown in section 4.4. also have an olfactory component so are strictly “combined chemosensory assays”, but for ease of phrase, because the larval olfactory assays are also chemosensory assays, and because the primary chemosensory stimulation remains gustatory, they will remain being called “gustatory assays”). Gustatory assays were carried out on P1, P2, P3, and

P5 larval offspring to determine how larval gustatory behaviour is influenced by repeated generations of exposure to peppermint. All generations were presented with agar plates, two quarters of which contained control agar, with the remaining two quarters containing 1% peppermint agar.

-20

-40

-60

-80

-100

CS P1 P2 P3 P5 Behavioural Response Index (bRI) Index Response Behavioural

Figure 5.2: Gustatory behavioural response indices (mean & SEM) for control larvae (n = 22 dishes; red) and larvae whose parents have been exposed to peppermint extract for a number of generations (blue), P1 (n = 30 dishes), P2 (n =

11 dishes), P3 (n = 6 dishes), and P5 (n = 6 dishes).

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Control larvae demonstrate gustatory aversion to peppermint, but gustatory aversion is practically eliminated in P1, P2, P3 and P5 larval offspring (Figure 5.2).

This was statistically shown by a one-way ANOVA (ANOVA, F4, 70 = 11.27, p <

0.0001) and a Tukey’s multiple comparison test post hoc (Table 5.2).

Table 5.2: Gustatory behaviour for control and peppermint generations post hoc

Tukey's Multiple P value Summary Comparison Test CS vs P1 < 0.0001 ***

CS vs P2 < 0.0001 ***

CS vs P3 0.0101 *

CS vs P5 0.0030 **

P1 vs P2 0.7378 ns

P1 vs P3 > 0.9999 ns

P1 vs P5 0.9948 ns

P2 vs P3 0.9092 ns

P2 vs P5 0.9868 ns

P3 vs P5 0.9978 ns

Control CS larvae demonstrated significantly different gustatory behaviour in response to 1% peppermint agar than the larval offspring of P1, P2, P3 and P5 flies.

The gustatory aversion displayed by control CS larvae appears to have been eliminated in P1, P2, P3 and P5 larval offspring, which do not display any significant differences in their gustatory responses to 1% peppermint agar, implying that either the larval offspring of peppermint reared flies no longer are able to detect peppermint, or do not find it to be aversive.

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In adult flies, settling behaviour was investigated in P1, P2, P3, P4, P5, P6 and P7 generations, and the results are shown in Figure 5.3. 10 females and 10 males were included in each vial, and for each investigated generation, a minimum of 20 vials were tested for each of the two food types.

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Control P1 12 12

10 10

8 8

6 6

4 4

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settled on food per vial per food on settled

Average number of flies of number Average Average number of flies of number Average

0 0

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Average number of flies of number Average flies of number Average

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Average number of flies of number Average Average number of flies of number Average

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Average number of flies of number Average flies of number Average

0 0

5 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

Figure 5.3: Settling behaviour – number of flies settled on food surface over 80 minutes (mean & SEM) on control food (red) and 0.1% peppermint food (blue). For both food types CS (n = 55 vials), P1 (n = 40 vials), P2, P3, P4, P5, P6, and P7 flies

(all n = 20 vials).

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The results apparently suggest that P1 flies prefer peppermint food, although this may just be anomalous as most subsequent peppermint generations do not demonstrate a preference, but do demonstrate an eradication of aversion (Figure

5.3). A two-way repeated measures ANOVA was carried out for each generation: in all generations there was an extremely significant effect of time, and no significance of interaction. As already stated, in CS flies there is a significant preference for control food over 0.1% peppermint food (ANOVA, F1, 108 = 16.7581, p < 0.0001), and in P1 offspring there is a significant preference for 0.1% peppermint food

(ANOVA, F1, 78 = 4.399, p = 0.0392). For all other peppermint generations, there was a significant difference in preference for food type in P2, P3 and P4, but not in

P5, P6 or P7. When a Bonferroni’s multiple comparison test was carried out to analyse differences at each time point there was no significant difference in settling rate at any time points, indicating that there is truly no significant difference in settling rate for these generations – P2 (ANOVA, F1, 38 = 0.6114, p = 0.4391); P3

(ANOVA, F1, 38 = 0.4798, p = 0.4927); P4 (ANOVA, F1, 38 = 0.6920, p = 0.4107); P5

(ANOVA, F1, 38 = 0.1032, p = 0.7497); P6 (ANOVA, F1, 38 = 0.2879, p = 0.5947); P7

(ANOVA, F 1, 38 = 0.3056, p = 0.5836). It would appear that repeated exposure to peppermint via food in preceding generations, for up to seven generations, results in a steady pattern of eliminating the aversive influence that is observed in control flies in response to peppermint extract. No preference for peppermint or aversion to control food is demonstrated.

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5.3. Effects of exposure to peppermint for a single generation

The behaviour of flies with ancestors that had been reared in the presence of peppermint for a single generation was investigated next, starting with P1C1 flies, and continuing all the way through up to P1C11. This was to more thoroughly understand how long the change in behaviour brought about by rearing in peppermint food for one generation was maintained once the stimulus was no longer present. After exposure in the first peppermint generation neither flies nor larvae were exposed to peppermint again before they entered a test environment.

The same settling experiments as outlined for the peppermint generations were carried out, with 10 females and 10 males in each vial, and with a minimum of 20 vials for each food type at each generation. The results are shown below in Figure

5.4.

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Control P1 12 12

10 10

8 8

6 6

4 4

2 2

settled on food per vial per food on settled settled on food per vial food on settled

Average number of flies of number Average Average number of flies of number Average 0 0 5 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

P1C1 P1C2 12 12 10 10 8 8 6 6 4 4

2 settled on food per vial food on settled 2 flies of number Average

settled on food per vial food on settled Average number of flies of number Average 5 0 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

P1C3 P1C4 12 12

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settled on food per vial food on settled

P1C5 P1C6 12 12

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settled on food per vial food on settled per vial food on settled

Average number of flies of number Average flies of number Average 0 0 5 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

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Figure 5.4: Settling behaviour – number of flies settled on food surface over 80 minutes (mean & SEM) on control food (red) and 0.1% peppermint food (blue). For both food types in flies of rearing background control CS (n = 55 vials), P1 (n = 40 vials) [for reference, from Figure 5.3], P1C1 (n = 20 vials), P1C2 (n = 28 vials),

P1C3 (n = 28 vials), P1C4 (n = 20 vials), P1C5 (n = 20 vials), P1C6 (n = 20 vials),

P1C7 (n = 20 vials), P1C8 (n = 20 vials), P1C9 (n = 20 vials), P1C10 (n = 20 vials), and P1C11 (n = 20 vials).

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From Figure 5.4 it can be seen that for the first four generations after being transferred onto control medium (P1C1-P1C4), there was no significant difference in the number of flies settling on control and 0.1% peppermint medium, indicating that these flies were showing exactly the same response as flies that were reared in peppermint for a similar number of time (P2 to P5 – see Figure 5.3) [P1C1 (ANOVA,

F1, 38 = 0.3629, p = 0.5507); P1C2 (ANOVA, F1, 54 = 1.0308, p = 0.3145); P1C3

(ANOVA, F1, 54 = 0.6003, p = 0.4418); P1C4 (ANOVA, F1, 38 = 0.06522, p = 0.7998)], but from P1C5 onwards significant preferences for control food began to appear in

P1C5, and this trend was continued in P1C7, P1C9, P1C10 and P1C11, although this was not consistent (P1C5 (ANOVA, F1, 38 = 4.4420, p = 0.0417); P1C6 (ANOVA,

F1, 38 = 1.9374, p = 0.1720); P1C7 (ANOVA, F1, 38 = 4.641, p = 0.0376; P1C8

(ANOVA, F1, 38 = 0.0188, p = 0.8916); P1C9 (ANOVA, F1, 38 = 3.8508, p = 0.0571);

P1C10 (ANOVA, F1, 38 = 3.7042, p = 0.0618); P1C11 (ANOVA, F1, 38 = 6.0163, p =

0.0189)], with significant preference for control food found in P1C5, P1C7 and

P1C11. Again, there was a significant difference over time for all the generations tested, with more flies settling as time passed.

P1C1 flies to P1C4 flies and P1C6 and P1C8 flies do not demonstrate the same pattern as P1 flies, it is more transitional (as shown in Figure 5.3 in other peppermint reared flies, P2 to P9) but display a significantly different behavioural pattern to control flies, showing no significant difference in behaviour dependent on food type. The average numbers of flies settled are not effective comparisons because this range of experiments was carried out over approximately 12 months, and so there was a high likelihood of temporal effects bringing about changes in behaviour; the key component of comparison and analysis is therefore the difference between settling behaviour on the two food types for each generation studied. P1C5, P1C7, and P1C9 to P1C11 demonstrate the same behavioural

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pattern as control flies, indicating the transition in behaviour reverting to control behaviours is initiated between generations P1C4 and P1C5.

To further explore these results, to more clearly observe behavioural patterns over time, and to see if the conclusions drawn above can be substantiated, the mean difference in settling preference for control or 0.1% peppermint food for control CS,

P1, and P1C1 to P1C11 flies was calculated. To do this the average number of flies settled per vial at each time point of the experiment on peppermint food was calculated and was subtracted from the average number of flies settled on control food at that time point. The difference was summed, the SEM was calculated, and these were plotted, as can be seen in Figure 5.5.

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-1

and 0.1% peppermint settling peppermint 0.1% and

Mean differenceMean betweencontrol

P1C1 P1C2 P1C3 P1C4 P1C5 P1C6 P1C7 P1C8 P1C9

P1C10 P1C11

PnCn Generation

Figure 5.5: Mean difference (with SEM) between the number of flies settled on control and 0.1% peppermint food for control, P1 and P1C1 to P1C11 flies.

It can be seen that from P1C5 onwards, with the exception of P1C8, flies showed a preference to settle on control food (Figure 5.5). This was broken down further still by looking at the mean difference in settling rates at each 5 minute time point for each generation tested, and then applying a linear regression for these time points to establish patterns in behaviour (Figure 5.6).

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Figure 5.6: Linear regression of difference in settling rate on control and 0.1% peppermint food (mean control settling minus mean 0.1% peppermint settling) at each five minute time point for control, P1, P1C1, P1C2, P1C3, P1C4, P1C5, P1C6,

P1C7, P1C8, P1C9, P1C10 and P1C11 flies. Positive results = preference for control food, negative results = preference for peppermint food.

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For P1, P1C1, P1C2 and P1C8, flies settle increasingly on peppermint over time. In all other lines, control (C1), P1C3, P1C4, P1C5, P1C6, P1C7, P1C9, P1C10 and

P1C11, settling increasingly leans towards control food. Generally, (i.e. excluding the anomaly of P1C8) there is a tendency for the slope to change slowly from P1C1 to P1C11, from a negative slope to a positive slope. This indicates a gradual change in preference from peppermint food in the generations whose ancestors have most recently been exposed to peppermint food to a strong preference for control food in generations whose peppermint ancestor is more distantly related.

The behaviour exhibited by P1C8 flies is a behavioural anomaly that may result from temporal effects. This data has been collated for more direct comparisons

(Figure 5.7).

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l 2

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1 1

C C C C C C C C C

C C

1 1 1 1 1 1 1 1 1

1 1

P P P P P P P P P

P P

P n C n G e n e ra tio n

Figure 5.7: Collated difference in mean settling rate for each 5 minute time point for control, P1, and all P1Cn generations tested, indicating patterns in behaviour.

From these analyses, it seems clear that a change in aversion to peppermint occurs after four generations, but is then apparently weakened for a single generation

(P1C8), before the aversion returns. The key changeover in adults from indifference to peppermint to aversion takes place after four successive generations of being reared on control medium (P1C4). The larval olfactory and gustatory responses of

P1C4 larvae were also examined:

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-20

-40

-60

-80

-100

Behavioural Response Index (bRI) Index Response Behavioural Control P1 P1C4

Figure 5.8: Olfactory behavioural response indices for control larvae (n = 35 dishes; red), P1 larval offspring (n = 24 dishes; blue) and P1C4 larval offspring (n = 30 dishes; lilac) in response to 5 µl peppermint extract.

The olfactory behaviour of P1C4 larval offspring indicates that at this stage the reduction in aversion to peppermint shown by P1 larval offspring has disappeared

(Figure 5.8). A one-way ANOVA revealed that P1 larvae were significantly different from control and P1C4 larvae, which in turn were not significantly different from each other (ANOVA, F2, 88 = 50.78, p < 0.0001), as demonstrated in Table 5.3.

Table 5.3: Olfactory behaviour for P1C4 larvae post hoc

Tukey's Multiple P value Summary Comparison Test Control vs P1 < 0.0001 ***

Control vs P1C4 0.2766 ns

P1 vs P1C4 < 0.0001 ***

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P1C4 larval gustatory behaviour was next examined:

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-40

-60

-80

-100

Behavioural Response Index (bRI) Index Response Behavioural Control P1 P1C4

Figure 5.9: Gustatory behavioural response indices for control larvae (n = 27 dishes; red) P1 larval offspring (n = 30 dishes; blue) and P1C4 larval offspring (n =

20 dishes; lilac) to agar plates, two quarters of which contain control agar and two quarters of which contain 1% peppermint agar.

As with olfactory behaviour, gustatory behaviour of P1C4 larvae did not appear to differ from control larvae (Figure 5.9), showing aversion to peppermint agar, whilst

P1 larval offspring displayed significantly reduced aversion in comparison. This was examined using a one-way ANOVA (ANOVA, F2, 76 = 32.17, p < 0.0001). A Tukey’s multiple comparison test (Table 5.4) confirmed what visual inspection suggested, that as for olfaction, control and P1C4 larvae were both significantly different from

P1C1 larvae, and were not significantly different from each other.

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Table 5.4: Gustatory behaviour for P1C4 larvae post hoc

Tukey's Multiple P value Summary Comparison Test Control vs P1 < 0.0001 ***

Control vs P1C4 0.2144 ns

P1 vs P1C4 < 0.0001 ***

The Tukey’s multiple comparison post hoc test displayed similar results to that observed when looking at olfaction: control larvae and P1C4 larval offspring did not behave significantly differently in the presence of 1% peppermint agar, whilst P1 larval offspring showed significantly reduced gustatory aversion compared to the other two groups.

5.4. Influence of re-exposure to peppermint

After at least four generations of being reared on control food, flies seem to have returned to normal in terms of their responses to peppermint. However, it is possible that the exposure to peppermint in the P1 generation has nevertheless left some legacy effects on the fly stock in terms of their ability to respond to a subsequent exposure to peppermint. To test this hypothesis, P1C8 flies were taken and were allowed to lay eggs of peppermint medium, producing P1C8P1 flies. Their settling behaviour was studied.

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4 settled on food pervial food on settled Average number of flies of number Average 2

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins)

Figure 5.10: Settling behaviour on control food (red) and 0.1% peppermint food

(blue) for P1C8P1 flies (mean & SEM).

Re-exposure to peppermint after an interruption of several generations does not appear to evoke a different reaction to novel exposure – in other words P1C8P1 flies behaved in a manner that is indistinguishable to P1 flies (Figure 5.10). It is also similar to the behavioural pattern demonstrated by P1C8 flies during the settling assay. This experiment was initiated before the result of the P1C8 settling experiments were known, and in hindsight this generation may not have been selected for this experiment, but the mean settling rate on both control and peppermint food was much higher than that demonstrated by P1C8 flies, and as such it may be concluded that the temporal or behavioural influences altering the behaviour of P1C8 flies in response to the settling assay did not impact upon the behaviour of P1C8P1 flies. A two-way repeated measures ANOVA was carried out to look at the relationship between behaviour on the two food types (ANOVA, F1, 38 =

0.0239, p = 0.8779) and it was found that there was no significant difference between the behaviour displayed on control food and 0.1% peppermint food, nor a

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significance of interaction (ANOVA, F15, 570 = 0.5811, p = 0.8903). As with all other settling experiments, time had a significant effect (ANOVA, F15, 570 = 30.5028, p <

0.0001), the settling rate increases as time progresses. This suggests that following re-exposure to peppermint, eight generations after the initial exposure, aversion to peppermint returns to the level ascertained by settling experiments with P1 flies, but there is no strengthening of response.

5.5. Chapter Summary

Repeated exposure to peppermint appears to strengthen the reduction in aversion for larvae. In both olfactory and gustatory assays, aversion to peppermint is eliminated, and in olfactory assays it appears that a weak preference for peppermint develops by P10. Adult settling behaviour shows that repeated generational exposure to peppermint also leads to elimination of aversion, but does not develop into preference. It is possible that this could be the beginning stages of sympatric speciation, similar to the adaptation demonstrated by D. sechellia. Morinda citrifolia is both toxic and aversive to all other species of Drosophila, which is similar to the attributes associated with the presence of peppermint in food media. An attractive stimulus would be more readily accepted into the diet of Drosophila, but acceptance of a host that is both repulsive and toxic to competitors exploits a niche which is yet to be utilised (Farine et al 1996).

After exposure to peppermint for one generation, the elimination of aversion is maintained for a further four generations, after which point behaviour returns to the

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pattern observed in control flies. When looking at the mean difference between control and peppermint settling behaviour, and taking into account the linear regression performed across all 5 minute time points, it is clear that a shift in behaviour occurs from P1C3, but this does not develop fully into preference for control food until P1C5. When looking at P1C4 larval offspring they also demonstrate a return to behaviour that is not significantly different from that of control larvae.

If exposure is interrupted, in this case for eight generations, and then re-exposure to

0.1% peppermint food occurs for one generation, this does not bring about a change in behaviour that differs significantly from flies that have been reared in the presence of peppermint for one generation with no exposure in preceding generations. This generation was chosen because it is after the reduction to aversion brought about through ancestral exposure to peppermint had been eliminated, and behavioural output was reverted to the pre-exposure pattern demonstrated by control flies. P1C8P1 flies have been reconditioned with the same stimulus to their ancestors, nine generations previously; the re-exposure did not strengthen the reduction in aversion, but behaviour equivalent to P1 flies was elicited, showing behavioural plasticity towards peppermint has been retained.

Remy (2010) reported similar findings with Caenorhabditis elegans in response to benzaldehyde and citronellol; additionally he found that after four generations of exposure the preference for these odours became fixed in all subsequent generations tested, the change in response being integrated into stable behavioural responses in a pattern similar to the adoption of Morinda citrifolia into the diet of D. sechellia.

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Chapter 6

Results: Investigation into underlying causes

6.1. Introduction

To better comprehend the motives behind the behavioural changes observed in D. melanogaster to peppermint (see Chapters 3 to 5) and how these motives function, whether in concert or unaccompanied several factors were investigated. First, it was investigated whether responses to peppermint were maternally or paternally linked.

Maternal and paternal linkage has been found to be responsible for many behavioural and physiological changes, for example, parental environment influences offspring phase state in the desert locust Schistocerca gregaria

(Despland & Simpson 2000), in the ground cricket Allonemobius socius the maternal environment influences diapause in offspring (Olvido et al 1998), and in the fly Telostylinus angusticollis sizeable, high condition fathers produced larger offspring, the males of which demonstrated improved mating success, and the females exhibited improved fecundity (Bonduriansky & Head 2007). All this happens epigenetically, initially without genetic input or parental care or communication from offspring during larval stages (learning from conspecifics).

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OrCo-, Trp and Painless mutants were studied to determine if the absence of a sense of smell or impairment in detecting chemosensory stimuli strongly influenced behaviours in response to peppermint extract. Whilst parental environment works in an epigenetic manner, contributing to the mosaic of information transmitted to the egg, these mutations function in a more direct manner, bringing about altered sensory modalities, altering if and how a stimulus is detected. It changes the processing of chemosensory stimulants by modifying the detection at peripheral neurons, meaning that they are perceived differently, or not at all, in the MBs and higher order brain structures. In addition, responses to menthol were assessed to examine whether the pure odour is processed differently to the menthol-containing bouquet resulting in a different behavioural output, an whether being reared I nthe presence of the pure odour changes behavioural responses to the bouquet, and vice versa (Objective 4).

6.2. Parental inheritance

To examine whether reduced aversion to peppermint and its transmission across generations is maternally or paternally linked, control CS virgins and P1 virgins were crossed in both reciprocal heterotypic crosses (i.e. control CS female x P1 male and

P1 female x control CS male) and in the two homotypic crosses (control female x control male, P1 female x P1 male) and the behaviour of their offspring was examined. Once mated, females were allowed to oviposit on control medium, and the offspring then grew and developed to adulthood in the control food medium. As adults they were assayed on their settling behaviour to elucidate their chemosensory preferences.

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T im e (m in s ) T im e (m in s )

Figure 6.1: Settling behaviour on control food (yellow) and 0.1% peppermint food

(purple) showing mean and SEM A. C♀C♂ offspring (n = 20 vials per food type); B.

P1♀P1♂ offspring (n = 20 vials per food type); C. C♀P1♂ offspring (n = 38 vials per food type); and D. P1♀C♂ offspring (n = 38 vials per food type).

Figure 6.1 shows that as expected, C♀C♂ offspring showed a significant difference in their behaviour between the two food types (ANOVA, F1, 38 = 8.6683, p < 0.0055), with a preference for control food, a significant difference over time (ANOVA, F15, 570

= 8.0482, p < 0.0001) and a significant interaction effect (ANOVA, F15, 570 = 3.5858, p < 0.0001). The significant interaction effect makes it difficult to interpret, but the

Bonferroni post hoc test shows that there is a significantly more settling demonstrated on the control food than the peppermint food from the 55 minute time point. P1♀P1♂ offspring also demonstrated a significant difference in their

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behaviour between the two food types (ANOVA, F1, 38 = 7.3301, p = 0.0101), but instead they showed a preference for 0.1% peppermint food, although the

Bonferroni post hoc test showed that at each individual time point this was only significant at 20 minutes and 75 minutes, otherwise there was not significantly more setting on peppermint food when compared to control food. There was a significant effect of time (ANOVA, F15, 570 = 6.5022, p < 0.0001), and no significant interaction

(ANOVA, F15, 570 = 0.5999, p = 0.8760).

The results for the two reciprocal heterotypic crosses were reminiscent of P1♀P1♂ behaviour. C♀P1♂ offspring showed, overall, to have a preference for peppermint food over control food (ANOVA, F1, 74 = 9.4299, p = 0.0030), but this preference was only significant at 20, 60 and 75 minutes when looking at the Bonferroni’s multiple comparison test. There was no significant interaction (ANOVA, F15, 1110 = 0.9814, p

= 0.4725), but, as expected, there is significantly more settling as the experiment progresses (ANOVA, F15, 1110 = 18.8912, p < 0.0001). P1♀C♂ offspring also showed an overall preference for peppermint food (ANOVA, F1, 74 = 7.8263, p =

0.0066), yet this was only significant at 70 and 75 minutes. Again there was no significant interaction (ANOVA, F15, 1110 = 1.4559, p = 0.1145) but a significant influence of time (ANOVA, F15, 1110 = 41.5063, p < 0.0001). Both were significantly attracted by peppermint, so it would appear that reduction in aversion to peppermint is neither maternally nor paternally linked – there is no clear “parent-of-origin” effect, meaning there is no clear differential effect of transmitted alleles depending on parental gender heterotypic crosses, but at the same time the pattern of behaviour is not identical, even though the overall preference is the same. To examine whether “parent-of-origin” effects became evident in subsequent generations, the offspring of the heterotypic crosses, reared on control food, were then tested to investigate at what point this reduction in aversion is lost, and how it is comparable

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to flies of the same generational remove from exposure to peppermint (P1Cn flies) where both parents have been reared in the presence of peppermint (Figure 5.4).

A 12 B 12

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settled on food per vial food on settled per vial food on settled

Average number of flies of number Average flies of number Average

0 0

5 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

Figure 6.2: Settling behaviour on control (yellow) and 0.1% peppermint food

(purple) showing mean and SEM A. C♀P1♂ second generation offspring (F2) (n =

22 vials per food type); and B. P1♀C♂ second generation offspring (F2) (n = 21 vials per food type).

For the F2 generation (Figure 6.2), neither C♀P1♂ descendants nor P1♀C♂ descendants show a significant difference in their behaviour dependent on food type

[C♀P1♂ F2 (ANOVA, F1, 48 = 0.0774, p = 0.7822); P1♀C♂ F2 (ANOVA, F1, 48 =

0.0395, p = 0.8435)]. For both there was a significant effect of time, and no significant interaction. This is similar to the behaviour exhibited by P1C1 to P1C4 flies (see Chapter 5 – Results: Influence over generations). It should be noted that they did demonstrate a very low settling rate when compared to the other assays, this could cause a sort of floor effect, where differences in behaviour cannot be discriminated due to the general low level of settling behaviour.

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settled on food per vial food on settled per vial food on settled

Average number of flies of number Average flies of number Average

0 0

5 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

Figure 6.3: Settling behaviour on control (yellow) and 0.1% peppermint food

(purple) showing mean and SEM A. C♀P1♂ third generation offspring (F3) (n = 20 vials per food type); and B. P1♀C♂ third generation offspring (F3) (n = 20 vials per food type).

Similarly, there is no significant difference in the food type preference displayed by

C♀P1♂ (ANOVA, F1, 38 = 0.0464, p = 0.8306) or P1♀C♂ (ANOVA, F1, 38 = 1.6884, p

= 0.2016) descendants in the third generation, although by observing the graphs it can be seen that there is slightly more settling demonstrated on control food, the plots are less entwined when compared with the F2 generation, particularly for the

P1♀C♂ F3 (Figure 6.3). This suggests that the influence of exposure to peppermint might be more strongly maternally linked than paternally linked, but once more the settling rate is very low when compared with other assays, so this could sway assessment. Again, for both strains there was a significant effect of time, and no significant interaction.

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A 12 B 12

10 10

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settled on food per vial food on settled

settled on food per vial on food settled

Average number of flies of number Average Average number of flies of number Average

0 0

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (mins) Time (mins)

Figure 6.4: Settling behaviour on control (yellow) and 0.1% peppermint food

(purple) showing mean and SEM A. C♀P1♂ fourth generation offspring (F4) (n = 20 vials per food type); and B. P1♀C♂ fourth generation offspring (F4) (n = 20 vials per food type).

Finally, by the fourth generation (F4) a significant difference in behaviour between the two food types is again observed (Figure 6.4). C♀P1♂ F4 offspring settled significantly more frequently on control food than 0.1% peppermint food (ANOVA,

F1, 38 = 13.5581, p = 0.0007), as did P1♀C♂ F4 offspring (ANOVA, F1, 38 = 9.9420, p

= 0.0032). Both demonstrated a significant difference in settling behaviour over time. This is similar to the behaviour demonstrated by control flies and so the reduction in aversion has been completely lost, a generation earlier than is seen in

P1Cn flies. Both demonstrated a significant interaction effect [C♀P1♂ F4 (ANOVA,

F15, 570 = 2.7409, p = 0.0004); P1♀C♂ F4 (ANOVA, F15, 570 = 1.9610, p = 0.0.161)] so to more clearly understand the data a Bonferroni post hoc test was performed. For

C♀P1♂ F4 significantly more settling occurred on control food when compared with peppermint food from the 50 minute time point until the end of the experiment at the

80 minute time point, and for P1♀C♂ F4 significantly more setting occurred on

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control food than peppermint food from the 40 minute time point to the end of the experiment, with the exception of the 65 minute time point.

6.3. The responses of peripheral receptor mutants

Flies reared on peppermint food have been exposed to peppermint in both an olfactory and gustatory setting; it delivers a ‘cold’ sensation in addition to the conventional notion of taste. In order to determine the modalities and receptors involved in detecting peppermint in D. melanogaster, the behavioural effects of peppermint in chemosensory mutant flies were studied.

As stated in Chapter 1: Introduction, OrCo- mutants lack the ability to detect odours via olfactory receptors, but may still receive chemosensory information via functional gustatory and ionotropic receptors (Abuin et al 2011). Trp mutants lack some chemosensory function (including pain sensation, light sensation, mechanosensation, the ability to detect humidity, and hearing in Drosophila), but are still able to locate themselves using ORs and the majority of GRs (Harteneck et al

2000; Christensen & Corey 2007; Damaan et al 2008). Painless is a sub-category of Trp receptor involved in sensing noxious hot and cold stimuli (Goodman 2003), and so the Painless mutants cannot detect hot/cold; additionally Painless is also involved in mechanosenation and sexual receptivity in virgin females (Sakai et al

2009; Hwang et al 2012). By looking at mutant behaviour in response to peppermint extract, it may be possible to establish which components are involved

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in the aversive behavioural responses that are displayed in control organisms. The first behaviour to be examined was oviposition.

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20 Proportion of eggs laid persite laid eggs of Proportion

0 0 0.05% 0.1% Percent peppermint in oviposition site

Figure 6.5: Oviposition preferences by proportion of eggs laid on 0%, 0.05% and

0.1% peppermint agar for control females (red), P1 females (blue), OrCo- mutant females (orange), Trp mutant females (yellow), and Painless mutant females

(green). Showing mean and SEM, n = 60 for all groups.

For all groups it appears that as peppermint percentage in oviposition sites increases, the prevalence for oviposition decreases (Figure 6.5). A statistical examination (Kruskal-Wallis test with Dunn’s multiple comparison test post hoc; data was found not to be normally distributed following a Kolgorov-Smirnoff normality test, the results of the dunn’s multiple comparison are displayed in Table

6.1) showed there were significant differences between the groups (Kruskal-Wallis statistic = 256.8, p < 0.0001). When looking at the relationships within and between

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groups, it was found that CS females oviposit significantly more on control agar than

0.05% peppermint agar and 0.1% peppermint agar, whilst oviposition between

0.05% peppermint agar and 0.1% peppermint agar was not significantly different.

This was also true for P1 and Trp females. OrCo- mutants oviposit significantly less as peppermint concentration increases, yet the standard error for this is large.

Painless mutants show significantly lower oviposition on 0.1% peppermint agar when compared with 0% peppermint agar, but there is no significant difference in oviposition proportion between 0% peppermint agar and 0.05% peppermint agar, or between 0.05% peppermint agar and 0.1% peppermint agar (as can be seen in the table below). Between groups, there is no significant difference in the proportion of eggs laid on 0% peppermint agar in CS, P1, OrCo-, Trp or Painless mutants. This is also true for 0.05% peppermint agar and 0.1% peppermint agar.

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Table 6.1: Chemosensory mutant oviposition post hoc

Dunn's Multiple Comparison P value Summary Test CS 0 vs CS 0.05 < 0.0001 *** CS 0 vs Cs 0.1 < 0.0001 *** CS 0 vs OrCo 0 > 0.9999 ns CS 0 vs Trp 0 > 0.9999 ns CS 0 vs Painless 0 > 0.9999 ns CS 0.05 vs Cs 0.1 > 0.9999 ns CS 0.05 vs OrCo 0.05 0.4524 ns CS 0.05 vs Trp 0.05 > 0.9999 ns CS 0.05 vs Painless 0.05 0.7331 ns Cs 0.1 vs OrCo 0.1 > 0.9999 ns Cs 0.1 vs Trp 0.1 > 0.9999 ns Cs 0.1 vs Painless 0.1 > 0.9999 ns P1 0 vs P1 0.05 0.0351 * P1 0 vs P1 0.1 0.0020 ** P1 0 vs OrCo 0 > 0.9999 ns P1 0 vs Trp 0 > 0.9999 ns P1 0 vs Painless 0 > 0.9999 ns P1 0.05 vs P1 0.1 > 0.9999 ns

P1 0.05 vs OrCo 0.05 > 0.9999 ns P1 0.05 vs Trp 0.05 > 0.9999 ns P1 0.05 vs Painless 0.05 > 0.9999 ns P1 0.1 vs OrCo 0.1 > 0.9999 ns P1 0.1 vs Trp 0.1 > 0.9999 ns P1 0.1 vs Painless 0.1 > 0.9999 ns OrCo 0 vs OrCo 0.05 0.0001 *** OrCo 0 vs OrCo 0.1 < 0.0001 *** OrCo 0 vs Trp 0 > 0.9999 ns OrCo 0 vs Painless 0 > 0.9999 ns OrCo 0.05 vs OrCo 0.1 0.0267 *

OrCo 0.05 vs Trp 0.05 > 0.9999 ns OrCo 0.05 vs Painless 0.05 > 0.9999 ns OrCo 0.1 vs Trp 0.1 > 0.9999 ns OrCo 0.1 vs Painless 0.1 > 0.9999 ns Trp 0 vs Trp 0.05 < 0.0001 *** Trp 0 vs Trp 0.1 < 0.0001 *** Trp 0 vs Painless 0 > 0.9999 ns Trp 0.05 vs Trp 0.1 > 0.9999 ns Trp 0.05 vs Painless 0.05 > 0.9999 ns Trp 0.1 vs Painless 0.1 > 0.9999 ns Painless 0 vs Painless 0.05 0.0657 ns Painless 0 vs Painless 0.1 0.0003 *** Painless 0.05 vs Painless 0.1 > 0.9999 ns

Settling behaviour was the next assay carried out to assess adult behaviour in response to 0.1% peppermint food medium in OrCo, Trp and Painless mutants.

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OrCo Trp 12 12

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8 8

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settled on food per vial food on settled per vial food on settled

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Painless 12

settled on food per vial food on settled Average number of flies of number Average

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

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Figure 6.6: Settling behaviour on control (orange) and 0.1% peppermint food (pink) showing mean and SEM for OrCo-, Trp, and Painless mutants (in all cases n = 20 vials per food type).

Two-way repeated measures ANOVAs were carried out to assess the significance of the data shown in Figure 6.6. For OrCo- and Trp mutant lines, there is no significant difference in settling behaviour displayed on control or 0.1% peppermint food [OrCo- (ANOVA, F1, 38 = 0.1194, p = 0.7316); Trp (ANOVA, F1, 38 = 0.0437, p =

0.8355)], behaviour that is similar to P1 flies. In addition, for both time had a significant effect, but there was no significant interaction effect. Painless showed no significant preference for control food or peppermint food (ANOVA, F1, 38 = 0.8382, p

= 0.3657)], there is a significant effect of time, but there is also a significant

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interaction effect. On further investigation, using a Bonferroni’s multiple comparison test, at every time point for all three chemosensory mutants there was no significant difference between the number of flies settling on control food and the number of flies settling on peppermint food. This suggests that all three, when functional, have a level of involvement in discerning the odour and taste of peppermint in adult

Drosophila. The settling results differ from the results of the oviposition assays, in which all three chemosensory mutants significantly preferred to settle on control agar over 0.05% and 0.1% peppermint agar. It could be that the mutations mean that their ability to detect peppermint is impaired but not eliminated, meaning that when there is a choice they can select for control agar, but where there is no choice in media settling rate is not effected. Additionally, in the oviposition assay they did not display behaviour that was significantly different from the behaviour of either CS or P1 flies.

Larval behaviour was then assessed to explore whether responses to peppermint are different depending on life stage.

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-20

-40

-60

-80

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Behavioural Response Index (bRI) Control P1 OrCo Trp Painless

Figure 6.7: Olfactory behavioural assays of control (n = 24 dishes), P1 (n = 24 dishes), OrCo- (n = 8 dishes), Trp (n = 28 dishes) and Painless (n = 32 dishes) third instar larvae in response to 5 µl peppermint extract (mean and SEM).

Olfactory behavioural assays using third instar larvae were carried out using a peppermint concentration of 5 µl in control, P1, OrCo-, Trp and Painless lines

(Figure 6.7). All of the chemosensory mutants had a significantly diminished response to peppermint compared to control larvae. The three mutants were not significantly different from each other or from P1 larvae (one-way ANOVA, F4, 115 =

14.90, p < 0.0001). The Tukey’s multiple comparison test (Table 6.2) shows that 5

µl of peppermint was significantly more aversive to control larvae than it was to P1 larval offspring and OrCo-, Trp and Painless mutants. It also reveals that there was no significant difference between the responses of P1 larval offspring and OrCo-,

Trp and Painless mutants.

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Table 6.2: Chemosensory mutant olfaction post hoc

Tukey's Multiple P value Summary Comparison Test Control vs P1 < 0.0001 *** Control vs OrCo- < 0.0001 *** Control vs Trp < 0.0001 *** Control vs Painless < 0.0001 *** P1 vs OrCo- > 0.9999 Ns P1 vs Trp 0.0836 Ns P1 vs Painless 0.4954 Ns OrCo- vs Trp 0.4790 Ns OrCo- vs Painless 0.8768 Ns Trp vs Painless 0.8157 Ns

The results indicate, as with settling assays, that OrCo-, Trp and Painless all play a role in the detection of and response to peppermint. Gustatory preference was examined to obtain a broader picture.

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-60

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Behavioural Response Index (bRI) CS P1 OrCo Trp Painless

Figure 6.8: Gustatory behavioural assays of control (n = 22 dishes), P1 (n = 30 dishes), OrCo- (n = 6 dishes), Trp (n = 8 dishes), and Painless (n = 8 dishes) third instar larvae in response to 5 µl peppermint extract (mean and SEM).

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As with olfaction, it appears that under gustatory analysis control larvae found 1% peppermint agar to be significantly aversive when compared with P1 larval offspring,

OrCo-, Trp and Painless mutants, which did not appear to find 1% agar to be significantly aversive (Figure 6.8). Again, a one-way ANOVA followed by a Tukey’s multiple comparison test (Table 6.3) was carried out and revealed that the three mutant lines and P1 larvae were not significantly different from each other but were all significantly different from control larvae (ANOVA, F4, 73 = 9.414, p < 0.0001).

Table 6.3: Chemosensory mutant gustatory post hoc

Tukey's Multiple P value Summary Comparison Test CS vs P1 < 0.0001 *** CS vs OrCo- 0.0070 ** CS vs Trp 0.0039 ** CS vs Painless 0.0007 *** P1 vs OrCo- 0.9990 Ns P1 vs Trp > 0.9999 Ns P1 vs Painless 0.9744 Ns OrCo- vs Trp 0.9998 Ns OrCo- vs Painless 0.9994 Ns Trp vs Painless 0.9929 Ns

This suggests that all three genes may be involved in the wild-type response to peppermint. However, the response of the P1 larvae gives no indication as to whether one, all or none of these receptor types are involved in producing the alteration in the response to peppermint following one generation.

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6.4. Testing the chemical legacy hypothesis

Jaenike (1982; 1983) carried out experiments looking at preimaginal conditioning in

Drosophila, and Gandolfi et al (2003) investigated preimaginal conditioning in the parasitic wasp, but as outlined by Barron and Corbet (1999) and Barron (2001), the individuals were in contact with the stimulus as larvae, and so it is not possible to say whether they carried fragments of the stimulus through to their adult stage, either inside the larval body or on the pupal casing, inside or out, that could have influenced their adult behaviour. As such they were never truly assessing preimaginal conditioning, but instead, the chemical legacy hypothesis, in which the chemical environment experienced during the larval life stage is transmitted through to the adult life stage, but with no required absence of contact with peppermint.

Blackiston et al (2008) provided the stimulus in conjunction with an electrical shock to the moth Manduca sexta whilst it was in its larval stage (either at third or fifth instar), and discovered that in adulthood the stimulus (ethyl acetate) was found to be aversive when delivered in absence of the electric shock, showing that memories from larval stages can be sustained through the considerable internal reorganisation of the brain during pupation through to adulthood.

With the above in mind, P1 third instar larvae were removed from bottles containing

0.1% peppermint food medium, washed using distilled water, and then, an hour later, placed in bottles containing control food where they were allowed to pupate and then mature to adulthood, with the intention of investigating whether experience of peppermint as a larva brought could influence adult behaviour. Barron & Corbet

(1999) criticised the use of washed pupae as an exponent of preimaginal conditioning because of the possibility that the stimulus (in this case peppermint)

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could be contained within the pupal casing. Washing prior to pupation allows larvae to fully experience a peppermint environment, but attempts to reduce the likelihood of peppermint being included within the pupal casing, and thus the individual being exposed to peppermint in their early moments of adulthood (although admittedly not certainly eliminating it). The hour between washing and transfer to control food for pupation also allotted time for defecation of food media containing peppermint to limit contamination of the control pupation environment. The behavioural responses to peppermint were assessed when they reached adulthood.

settled on food per vial Average number of flies

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins)

Figure 6.9: Settling behaviour of P1 larvae which have been washed at third instar and repositioned in a bottle containing control food in response to vials containing control food (red) and 0.1% peppermint food (blue) (n = 20), showing mean and

SEM.

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P1 larvae that were washed during third instar and repositioned in bottles containing control food did not display significantly different settling behaviour on control and

0.1% peppermint food (Figure 6.9). A two-way repeated measures ANOVA was carried out to assess the results (ANOVA, F1, 38 = 0.3634, p = 0.5502), and it was found that there was no significant difference in settling behaviour of flies which were washed as larvae, although they did exhibit a low settling rate overall. This could have resulted from handling during the washing process impairing locomotory behaviour.

6.5. Male only and female only assays

Settling experiments were carried out using vials of control male-only and control female-only flies on both control food and 0.1% peppermint food to investigate whether behaviour was driven by sex. The oviposition assays were not clear cut and so there could be a difference in behaviour in response to peppermint based on gender.

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B A 12 12

10 10

8 8

6 6

4 4

2 2

settledon food per vial

Averagenumber of flies Averagenumber of flies

0 0

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins) Time (mins)

Figure 6.10: Settling behaviour in response to control food (red) and 0.1% peppermint food (blue) for A. female only control flies (n = 44 vials per food type) and B. male only control flies (n = 43 vials per food type), both showing mean and

SEM.

A two-way repeated measures ANOVA was carried out for both male only and female only datasets (Figure 6.10). The male only data was similar to the control, in that settling on control food occurs significantly more often than settling on 0.1% peppermint food (ANOVA, F1, 84 = 11.7151, p = 0.0010). There was also a significant effect for time and a significant interaction effect (ANOVA, F15, 1260 = 5.3907, p <

0.0001); the ambiguity caused by this result led to a Bonferroni multiple comparison post hoc test, which showed that there is a significant difference in settling behaviour between control and peppermint food, with significantly more settling occurring on control food from the 45 minute time point, ,which is continued through to the end of the experiment with the exception of the 60 minute time point. For female only settling trials, settling on control food was not significantly different to settling on peppermint food (ANOVA, F1, 86 = 2.6004, p = 0.1105), but the settling rate was very low in comparison with the male only assays carried out at the same

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time, and a large level of immobility, while remaining at the top of the vial, was observed. There was a significant effect of time, but no significant interaction effect, and the Bonferroni multiple comparison post hoc found that there was no significant difference between the settling rate on control and peppermint food at any of the sixteen time points observed.

6.6. Menthol assays

Menthol is the main component that goes into forming the bouquet present in peppermint extract; peppermint itself is extracted from the plant Mentha piperita L, so to investigate whether behavioural changes are an upshot of the bouquet of contributory odours, or the pure, majority odour present in peppermint extract, menthol the responses of control flies and P1 flies to 0.1% menthol food were examined via a settling assay.

A 12 B 12

10 10

8 8

6 6

4 4

2 2

settled on food per vial per food on settled

Average number of flies of number Average Average number of flies of number Average

0 0

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins) Time (mins)

Figure 6.11: Settling behaviour in response to control food (red) and 0.1% menthol food (green) for A. control flies (n = 25 vials per food type) and B. P1 flies (n = 20 vials per food type), both showing mean and SEM.

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In both cases, a two-way repeated measures ANOVA was carried out to examine the relationship between settling on control food and settling on menthol food

(Figure 6.11). Control flies settled significantly more frequently on control food than on 0.1% menthol food (ANOVA, F1, 48 = 0.9937, p < 0.0001) as well as a significant effect of time, but also a significant interaction effect (ANOVA, F15, 720 = 4.134, p <

0.0001). A Bonferroni multiple comparison post hoc test revealed that there is a significant difference in settling rate for control flies on menthol food at the the 75 and 80 minute time points. P1 flies showed no significant difference in their settling behaviour between the two food types (ANOVA, F1, 38 = 1.6645, p = 0.2048), but strangely there was no significant effect of time (ANOVA, F15, 720 = 1.5196, p =

0.0929), perhaps because of the overall low settling rate demonstrated. The responses of control and P1 flies to 0.1% menthol food were largely comparable to the responses of the same lines to 0.1% peppermint food.

The behaviour of flies reared for one generation in menthol food (M1) was then examined on control food, 0.1% peppermint food and 0.1% menthol food.

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settled on food pervial food on settled Average number of flies of number Average

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Time (mins)

Figure 6.12: Settling behaviour in response to control food (red), 0.1% peppermint food (blue) and 0.1% menthol food (green) for flies reared for one generation on

0.1% menthol food (M1). For all n = 20 vials per food type.

A two-way repeated measures ANOVA was carried out with a Tukey’s multiple comparison post hoc to examine the behaviour of M1 flies across the different food types (ANOVA, F2, 57 = 0.3424, p = 0.7115). No significant difference in behaviour across the three food types was found for M1 flies and no significant interaction, although a significant effect of time was still observed. The Tukey’s post hoc test showed that there was no significant differences in settling behaviour observed at any of the time points on any of the three food types. M1 flies react in the same way to peppermint food as they do to control and menthol food, showing that they are perceived in the same way.

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6.7. Chapter Summary

Reduction in aversion to peppermint does not appear to be maternally or paternally linked, and is not sustained over more generations dependent on which of the parents were reared in the presence of peppermint food. Reduction in aversion is not maintained across so many generations if only one of the parents is reared in

0.1% peppermint food (only three generations rather than four) when compared to assay where both parents have been reared in the presence of peppermint. When looking at the behaviour of larvae which have been reared in peppermint food, washed with distilled water, then allowed to mature to adulthood in bottles containing control food, there is no significant difference between their behaviour on control food to peppermint food, although they do display low levels of settling, indicating that they may have been damaged during the washing process.

OrCo-, Trp and Painless all appear to be involved to some extent in the detection and development of behavioural responses to peppermint extract – in their absence, the responses of individuals are impaired in terms of adult settling behaviour and larval olfactory and gustatory behaviour. The oviposition data were inconclusive.

When looking at male-only and female-only adult settling behaviour in control flies, it was found that female-only vials exhibited a very low level of activity, whilst male- only vials exhibited a higher than normal level of activity. Male-only vials showed the same pattern as control flies, settling more on control food than 0.1% peppermint food, whilst female-only vials did not display significantly different patterns of behaviour dependent on food type.

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Control and P1 flies behavioural responses to 0.1% menthol food were tested using settling assays. P1 flies did not behave significantly differently on menthol food than on control or peppermint food, whilst control flies settled consistently more often on control food than on menthol food, similarly to their behaviour on peppermint food.

Flies reared on 0.1% menthol food, M1, had their behavioural responses to control, peppermint and menthol food tested experimentally, and no significant differences in their settling rate were found.

Evidence of larval environment influencing adult behaviour has been found, and the behaviour of chemosensory mutants implies that the detection of and response to peppermint is a multimodal system, involving, smell, taste, and detection of stimuli perceived to have a thermal effect.

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Chapter 7

Discussion

7.1. Overview

A series of experiments using an aversive stimulus, peppermint extract, were conducted to investigate how early experience can affect the behaviour of adult D. melanogaster, and how this effect can be transmitted across generations.

A series of experiments have been conducted to investigate the behavioural reactions elicited in different lines of D. melanogaster in response to an aversive stimulus, peppermint extract, in a variety of settings. The biological imperative and driving forces behind these responses were investigated. Aversive responses to peppermint were modified by repeated exposure to peppermint extract across generations, following which efforts were made to elucidate how behavioural modification is controlled and the mechanisms behind its initiation. By investigating olfactory and gustatory behaviour in the simple model species D. melanogaster, it was hoped that a greater understanding of insect chemosensory behaviour in general would be attained, as well as clearer picture of the means by which

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behaviour is transmitted across generations, be it chemical transmission, epigenetics.

7.2. Naïve flies find peppermint repellent, but rearing in its presence reduces this aversion

In Chapter 3 (Results: Do rearing conditions influence behaviour?; Objective 1), it was established that peppermint is repellent to control CS D. melanogaster

(individuals which had not previously been exposed to peppermint) both at the larval and adult life stages. In larvae, this was demonstrated by strongly negative olfactory and gustatory behavioural response indices in the presence of peppermint, which are both new findings having been investigated here for the first time. Adult behaviour in response to peppermint was similar to that reported by Thorpe (1939) and Barron & Corbet (1999) and was revealed by flies demonstrating significantly reduced settling and oviposition behaviour on peppermint compared to control media. In larval olfaction and gustation assays a level of dose-dependence was found; with regards to olfaction, control larvae showed a weak but significant increase in aversion to peppermint as the volume increased from 0.5 µl to 7.5 µl (a

15-fold increase). This was the first time dose-dependence in response to peppermint extract has been examined in Drosophila. Larval gustation showed a significant increase in aversion as the concentration of peppermint in agar increased from 0.1% to 1% (a 10-fold increase). This increase in aversion occurs as the peppermint concentration in both assays approached what were found to be disabling intensities (resulting in the death or inability to move of several larvae,

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rendering results impractical to analyse), indicating that as concentrations intensified and increasingly threaten health and survival of individuals, they become more repellent.

No significant level of dose-dependence on oviposition was found between agar containing 0.05% peppermint extract and 0.1% peppermint extract – when 0.5% peppermint, as used by Thorpe 1939, was used in oviposition trials it resulted in the death of the flies that were placed in the box. This may be due to differences in the strains used (Thorpe does not define what strain he used; the Canton-S strain used here was first collected in the 1950s), or differences in the purification methods used for the extraction of peppermint in the intervening 70 years. It is possible that modern extraction procedures are more effective, producing a more refined and therefore more concentrated version of the chemostimulus. In all these cases

(dose-dependence in settling assays was not investigated) aversion to peppermint increased with dose/concentration. The toxicity of the highest dose of extract in the oviposition experiment suggests that the aversion is directly related to the toxicity of the substance.

In contrast, the individuals that were reared in the presence of peppermint showed a substantially reduced aversion, as indicated by significantly weaker behavioural response indices in P1 larval offspring and significantly increased settling on peppermint in P1 adults. P1 females, in concurrence with control females, laid fewer eggs on peppermint laying sites than control agar, however there was no significant difference in the number of eggs laid by P1 females on control agar,

0.05% agar or 0.1% agar, indicating that the aversion to ovipositing on a laying site containing peppermint is eradicated as a result of early experience with the

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repulsive stimulus. Again this is similar to the results reported by Thorpe (1939) and

Barron & Corbet (1999), that individuals who had been exposed to peppermint/menthol demonstrated a reduced level of aversion towards it. Exposure to peppermint resulted in a behavioural modification, with the offspring of flies reared in the presence of peppermint becoming more tolerant of it as larvae and adults. Modifying behaviour in response to a chemostimulant has been shown to be of evolutionary advantage; in primates, a social advantage (Barton 2006), the moth,

Manduca sexta, used olfactory learning to avoid painful stimuli and so survive to reproduce (Blackiston et al 2008), in honeybees a positive and a negative response to an odour can be elicited when presented in conjunction with sucrose and an electric shock, respectively, so either host range expansion or increased survival are benefitted (Carcaud et al 2009), and the tephritid fly, Tephritis conura, range expansion was induced to exploit a novel host (Diegisser et al 2009). In general, it seems that this phenomenon enables individuals in a population to rapidly adapt to changing conditions through changing behaviour – plasticity allows for local adaptation, allowing populations to exploit and survive in novel environments

(Crispo 2008; Seehausen et al 2008; Berlucchi & Buchtel 2009).

7.3. Parental and larval environments influence behaviour

Initial aversion in control flies, followed by a reduction in that aversion after being reared in the presence of peppermint for a single generation (P1), was clearly demonstrated through the study of adult settling behaviour, as well as the olfactory and gustatory behaviour of larval offspring of CS and P1 flies (see Chapter 3,

Objective 1). Reduction of aversion to peppermint in the larval offspring of flies that

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have been reared in 0.1% peppermint food demonstrates that parental environment significantly influences offspring behaviour. This has previously been suggested for

Syrian hamsters, in which maternal photoperiod influences offspring development

(Beery et al 2008), mice, where mothers who have encountered the threat of disease produce less aggressive sons with an altered immune response (Curno et al 2009), the fly, Telostylinus angusticollis, in which both maternal and paternal condition and diet influence the development of offspring (Bonduriansky & Head

2007), the desert locust, where maternal and paternal phase and rearing environment influence behaviour and colour of hatchlings (Islam et al 1994), and, finally chickens have been found to demonstrate behavioural changes based on the environment experienced by their parents, with unpredictable access to light and food leading to poorer spatial learning, but improved adaptive responses in feeding behaviour, higher survival and better condition in offspring (Lindqvist et al 2007; Natt et al 2009). In the present studies the behaviour of Pn flies was tested again as adults using settling assays, and it was found that they still exhibited a reduction in aversion to peppermint following metamorphosis, suggesting that however the effect occurs in the nervous system of the insect, it is similar in both larvae and flies.

There were no consistent differences in mating behaviour when flies were mated with other flies that had been reared in peppermint, compared to when they were crossed with individuals from a control background, suggesting that exposure to peppermint was not marking the insects in some way that enabled homotypic (or heterotypic) mating preferences to be expressed. This was not as expected, it was anticipated that courtship duration and mating onset would be shorter for homotypic mating pairs that were already accustomed to one another’s background odour, with a longer mating time. Conversely, it was expected that heterotypic mating pairs would display longer courtship duration and time to mating onset as a result of

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females being unwilling to accept a male whose odour does not appear to be familiar to the environment thy have experienced (Cobb et al 1990; Cobb & Ferveur

1996; Savarit et al 1999). The fact that this was not true may indicate that not enough repeats were carried out for clear behavioural disparities to be revealed, or it may indicate that even though control flies find peppermint to be repulsive in situations relating to feeding and oviposition, the smell of peppermint on a mate is not considered to be aversive, or does not have negative connotations for the fitness of future offspring.

7.4. Direct contact with peppermint is not required for behavioural changes to occur

To rear ‘no contact’ flies, individuals were reared from egg to adulthood in vials of control food containing an Eppendorf tube punched with small holes, enclosing 20

µl of peppermint extract. Once these individuals reached adulthood and settling assays were performed, they showed a comparable reduction in aversion to peppermint as is observed in settling assays using P1 flies which have contact with peppermint through their 0.1% peppermint food source (Chapter 4; Objective 2).

The larval offspring of ‘no contact’ flies delivered less conclusive results: in gustatory assays, they behaved in a manner that was not significantly different to the larval offspring of P1 flies, but in olfactory assays, they behaved more similarly to control larvae. This implies that the behavioural responses of ‘no contact’ flies are similar to those of P1 flies, but the behaviour of their offspring is roughly intermediate between flies raised on control food and those raised on 0.1%

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peppermint food – more accurately it may imply something about the differences between gustatory and olfactory behaviour in larval offspring. Perhaps modifying olfactory behaviour requires physical contact with peppermint, while modifying gustatory behaviour does not; the reason for this is not clear and, intuitively, sounds like the opposite should be true. Further investigation is required to investigate this effect. The essential point is that direct, physical contact with peppermint extract is not necessary to bring about a change in behaviour, but physical contact does strengthen the change in behaviour and also ensures its longevity across subsequent generations. This agrees with the previous study of Manduca sexta by

Blackiston (2008), in which he found that exposure to an odour in third and fifth instar, in conjunction with an electric shock, induced aversion to that odour in adults.

The notion that direct, physical contact with an aversive stimulus is not required to elicit behavioural modifications to can be interpreted from an evolutionary perspective in relatively straightforward terms; there could be a fitness benefit to pass on information to offspring about any chemostimulants that have been experienced in the parents’ environment, leading to an alteration of the innate response. While such effects, based on the stimulation provided by a food source or an integral part of the environment in which an individual will be immersed for an entire life stage, are clearly of major importance to the fitness of an individual, it is possible that stimuli that are less consistently encountered may also provide information that organisms can use to gain a fitness advantage. However, odours that are often encountered are noteworthy, but chemostimulants in which an individual is submerged for an entire life stage can be key to the survival of any future offspring, and as such are vitally more important.

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Studies of individual ‘no contact’ larval offspring on gustatory plates did not provide much of an insight into the behavioural patterns exhibited whilst encountering control and peppermint agar, and therefore into the perceptual mechanisms underlying altered behaviour in response to gustatory stimulation. There were no significant differences in the speed, distance and area covered on control or peppermint agar by ‘no contact’ larvae when compared with any of the other groups studied (control, P1 and OrCo).This suggests that the ‘no contact’ larvae were moving in similar way to other larvae that either showed no discrimination (P1) or were repulsed by the peppermint (control).

7.5. Impact of peppermint upon survival

Some indication as to why peppermint is aversive to naïve flies, even though it is a natural odour that could conceivably be encountered by D. melanogaster in their natural environment, was found when survival was studied (see Chapter 4,

Objective 2). Being reared on food containing 0.1% peppermint food consistently reduced survival by ~7%, independently of whether previous generations had encountered it in their food source. This implies that peppermint has a toxic effect on the development of D. melanogaster, somehow preventing eggs from maturing to adulthood (this was also indicated by the toxicity of the highest dose of peppermint in the oviposition experiment). There is no precedent for this finding:

Barron & Corbet (1999) found no change in survival when flies were reared on a

0.08% menthol medium, while Thorpe (1939) did not look at the effects of peppermint on survival. Neither examined the influence of rearing conditions on development or morphology. In the present study, no obvious morphological

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changes were observed as a result of being reared on peppermint food medium, but this was not the focus of the research, and no precise measurements were taken. In terms of understanding how the 7% lethality occurs, precise measures of survival at various key ages could be taken, along with detailed morphological measures in order to capture sub-lethal effects. It is possible that as peppermint brings about a reduction in survival, that it may alter or suppress the expression of certain developmental genes; as yet the basis of this consistent reduction in survival remains unknown and requires further work.

The fact that the reduction in survival remained constant, even after repeated generational exposure to peppermint (up to P9 offspring), implies that exposure across several generations does not result in selection for individuals that are less vulnerable to peppermint toxicity; any loss of aversion to or even preference for peppermint does not represent increasing numbers of individuals that are suffering less negative fitness effects in response to the chemostimulant. Increased mortality is consistent across generations suggesting that no genetic modifications have arisen to reduce the negative impact of peppermint on D. melanogaster fitness. This finding of no apparent change in the toxicity of peppermint over the course of the experiment is surprising, as it suggests that no selection has taken place, in turn implying that there was no or little genetic variability in this stock of flies for the response to peppermint. It would be interesting to repeat this experiment with an outbred or recently caught population of Drosophila, where genetic variability might be expected, in order to see whether selection could occur. If it did not, this would give some insight into the nature of the toxicity effect, implying that it affected processes that were so fundamental that there was little or no genetic variability upon which selection could operate.

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7.6. Repeated exposure to peppermint for several generations may result in a preference for it

One striking single result of this study, with the implications both for evolutionary biology and for our understanding of genetic control of behaviour, was the observation in larval olfactory assays that after extensive exposure to peppermint medium over several generations, flies actually began to prefer this previously- aversive stimulus.

Flies were reared in bottles containing peppermint food medium from P1 to P10

(see Chapter 5; Objective 3). At each generation, the behavioural responses to peppermint were tested. In all generations reared on peppermint food, no significant differences in behaviour were found when looking at behaviour of adult flies on control and peppermint food during settling assays (P1 to P9), or when looking at larval gustatory behaviour (P1 to P3, and P5). However, when looking at olfactory behaviour (in P1, P2, P4, P5 and P10), by P10 a preference had developed for peppermint over control food, although admittedly this preference was rather weak (not statistically distinguishable from P2 or P5). This suggests that repeated exposure might lead ultimately to acceptance of peppermint food media as a suitable food source, and perhaps even to preference for peppermint food over control food after exposure across tens of generations. This is in spite of the fact that the presence of peppermint still results in increased mortality in all Pn generations studied, suggesting that the experiment has not inadvertently selected for some altered ability to process or detect peppermint. Had there been such a selection effect, then a decline in mortality over time would have been expected.

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Larval olfactory behaviour might be more refined to peppermint dosage than gustatory behaviour, mostly because in olfactory assays the peppermint can be located to one source, whereas in gustatory assays the odour must be perceived to permeate the whole environment, and so stumbling across peppermint agar, and finding it a repulsive surface to encounter, does not provide such clear cut results if the larva cannot find its way away from the peppermint stimulus. From looking at the results of ‘no contact’ larvae it may be that exposure without contact is equivalent to a low dose of peppermint; the behaviour appears to be intermediate between control and P1 behavioural responses, and as outlined above we do see some dose dependence in olfactory behaviour in Pn flies in Chapter 3. It may even be possible that perhaps the preference for peppermint by P10 represents an accumulation of peppermint in larvae through generations; as such the exposure to peppermint in P10 larvae offspring might be much greater than in P1 larval offspring. As a result this may explain why after multiple generations of exposure, even though it is still harmful, a preference for peppermint is developed – plasticity has occurred in response to an environmental change, bringing about an adjustment in behaviour which is then reinforced by repeated environmental cues producing a more stable behavioural output (Jaenike 1982; Hawlena et al 2011).

There are two examples in the literature of similar effects in flies which might shed light on the results reported here. Rohrbough et al (2002) described a cellular basis for behavioural plasticity in Drosophila, but highlighted the need for further research into the regulatory mechanisms controlling plasticity, along with greater understanding of the MBs and the advancement of electrophysiological research.

There is also the well-known example of the apple maggot fly, where genetic variation has evolved between strains of the fly that breed on hawthorne and apple fruit (Jiggins & Bridle 2004). There are six allozyme loci [malic enzyme (Me),

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aconitase-2 (Acon-2), mannose phosphate isomerase (Mpi), NADH-diaphorase-2

(Dia-2), aspartate amino transferase-2 (Aat-2), and hydroxyacid dehydrogenase

(Had)], all of which are associated with development, that differ depending on the host race, and which are based on a series of chromosomal rearrangements. Aat-2 and Dia-2 are on linkage group I, Me, Acon-2, and Mpi are on linkage group II, and

Had maps to linkage group III. This chromosomal rearrangement reduces the level of recombination, favouring adaptations to the two specific host habitats, an adaptation that occurred in just a period of around 50 years (Feder et al 1997;

Jiggins & Bridle 2004).

Plasticity enables organisms to take advantage of their surroundings to best exploit their environment in order to survive and reproduce, but it does come at a cost. It is believed that plasticity can limit the evolution of optimum phenotypes – by being able to adapt the ability to specialise is compromised (Relyea 2002). Plasticity allows a population level adaptation to environmental change, whilst later chromosomal rearrangements allow a sort of “settling down” once a new host has been accepted. In this experiment it could be predicted that there will be a stronger preference for peppermint by P20 as a result of the interactions between environment and behavioural plasticity, following genetic divergence similar to that demonstrated in the apple maggot fly (Feder et al 1997).

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7.7. Transgenerational effects of exposure to peppermint

The behavioural responses of successive generations to peppermint extract were assayed, from P1C1 through to P1C11, to see how the initial effect of being reared in peppermint (P1) changed over subsequent generations of being reared in control medium (C1-C11) (see Chapter 5). These flies retained a strong reduction in aversion towards peppermint compared to control flies until P1C5: the reduction in aversion was maintained for four generations without contact with peppermint before behaviour returned to the pattern demonstrated by naïve flies and larvae.

This striking result is similar to what has been reported for C. elegans in response to benzaldehyde, an aversive stimulus for this species (Remy 2010). In C. elegans, rearing individuals in the presence of benzaldehyde led to an “olfactory imprint”, i.e. olfactory experiences have a permanent impact on behaviour, and this knowledge from the parental environment was transmitted to the next generation and influenced their behaviour. It was additionally found that if four or more generations were continually exposed to benzaldehyde, then the imprint became stable for all subsequent generations (Remy & Hobart 2005; Remy 2010). Exposure was much shorter than in this trial (for only the first of their four juvenile stages), so it is unclear if persistence of behavioural change in C. elegans might be expected to last longer

(without re-exposure in subsequent generations) if exposure was carried out for longer (Remy 2010). Remy suggests an epigenetic explanation for his findings, via the “fixation of an environmentally induced novel phenotype” (Remy 2010), made possible by plasticity. As with this work, he agrees that further research is required to uncover the molecular mechanisms of this process.

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P1C8 flies showed an anomalous response to peppermint; instead of finding it aversive, as did P1C5, P1C6, P1C7, P1C9, P1C10, and P1C11, they displayed an equivalent settling behavioural pattern to flies that had been reared in peppermint for one generation, then reared on control food for one generation (i.e. P1C1 flies).

The settling rate is quite low (on average around 10% of flies settled after 80 minutes in the experimental vials) and so this could be the result of a behavioural fluctuation in response to an unknown temporal shift (that being, an influence of time, minor temperature and humidity fluctuations within the test environment, a change in the external environment that filters in whenever the incubator door is opened, the number of people using the incubator in a certain week etc.). As discussed in Chapter 1 (Introduction) a variety of factors can influence behaviour in

D. melanogaster. As many as possible were eliminated through the experiments being housed in 25˚C incubators, all on the same 12:12 LD cycle. Media was prepared in the same way, flies were allowed to recover following anaesthetic for the same amount of time, people were prevented from entering the walk-in incubator whilst settling experiments were underway so that flies were not startled by any sudden movement or change in temperature, and experiments were carried out at the same time of perspective ‘day’. Settling experiments were usually carried out over a four or five day period in an attempt to prevent any behavioural anomalies that may be exhibited on any one specific day being amplified during analysis and swaying results. In the case of P1C8 settling, due to scheduling difficulties, all 40 vials were examined in a two day period. This may have led to the aforementioned behavioural anomalies being amplified, resulting in a skewed interpretation of behaviour.

Many mechanisms behind transgenerational effects on the exhibited phenotype have been posited (Shea et al 2011) including the influence of genomic imprinting

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(Triggs & Knell 2012), selection-based effects, which are consistently transmitted across many generations, such as metabolic programming (Shea et al 2011;

Buescher et al 2013); detection-based effects, such as information about which season an organism will me emerging into that is transmitted from mother to offspring, such as is demonstrated in the Syrian hamster (Beery et al 2008; Shea et al 2011); and information about cell state (Shea et al 2011), such as the production of a transmissible secreted factor that can be conveyed between cells (Morgan

2003). There is a variety of ways in which information about peppermint environs can be transported across generations as a result of the utilised method of chemosensory delivery; it is possible that some peppermint is transmitted within or on egg casings, meaning that there is a level of exposure which an individual experiences in early life that bring about behaviours that are significantly different to those which are portrayed by control flies. As such, this would mean that there was a chemical legacy, as described in section 1.5. (Oviposition behaviour), in which there is a direct chemical passage of peppermint across generations. It is also possible that there is some change in cell state or a germ line which brings about a change in behaviour which is becomes evident as the individual develops, epigenetically effecting change.

7.8. Transmission of preference across life stages

Experiments on larvae which were reared in the presence of peppermint, washed with distilled water during their third instar, and then allowed to mature to adulthood in control food (termed P1 washed larvae) – show that larval environment influences behaviour (see Chapter 6). Washing was intended to remove the influence of the

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larval peppermint environment, as much as was possible, prior to pupation, so that as few remnants of the peppermint environment as possible were retained through to potentially stimulate the fly as it emerged from the pupa. Jaenike (1983) carried out a similar experiment, but did not wash Drosophila until they reached their pupal stage, washing only the exterior of the pupal casing.

However, unlike the results reported by Jaenike (1982; 1983) the effect of peppermint in the larval environment is retained through metamorphosis and influences adult behaviour, with flies settling equally on control and peppermint food. This is similar to what is found by Remy (2010), Shikano & Isman (2009),

Thorpe & Jones (1937) and Thorpe (1939) and supports Hopkins’ host selection principle (HHSP) and the idea of pre-imaginal conditioning, which is refuted by

Jaenike (1982; 1983), Barron and Corbet (1999) and Barron (2001). It is possible that not all of the peppermint was washed off the larval body, or that the larvae transferred some peppermint to their new, control food environment internally (i.e. through faeces or other waste products). However, any remaining amounts of peppermint would have been substantially lower than those available in the conditioned medium; given that there was a dose-response effect in the non- washed larvae, this suggests strongly that the persistence of the peppermint- induced effect is not caused by traces of peppermint in the pupal case, or in the medium.

This method ensured that larvae were exposed to peppermint during early development, but reduced (and hopefully abolished) exposure in early adulthood.

Thorpe & Jones (1937) endeavoured to deal with this problem by removing

Nemeritus canscens pupae from their pupal casing and wash them before they fully

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emerged as adults; however, this led to substantial mortality, so this procedure was not attempted here. Nevertheless, there is some indication that washing had a detrimental effect on the larvae. Levels of activity in washed larvae were quite low

(i.e. reduced by about two flies per vial, on average, at 80 minutes when compared with control flies) – this might indicate that larvae were damaged during the washing process, and as a result are in some way ‘disabled’, but it was impossible to devise a washing method that would eliminate the risk of damaging larvae during manual manipulation. Whatever the case, the essential point is that washed larvae showed essentially the same response to exposure to peppermint as the non-washed larvae, suggesting that the process involved in altering adult responses to peppermint does not involve contact with peppermint particles at eclosion.

7.9. Input from both parents is used to modify offspring behaviour

The apparent existence of a non-genetic transmission of altered responses to peppermint raised the question of the relative weights of the maternal and paternal contributions to the effect (Chapter 6; Objective 4). Reduction in aversion to peppermint is apparently not maternally or paternally linked, but instead the full level of aversion (and, ultimately, preference) observed in Pn flies is built upon input from both parents. This is shown by the finding that the first generation offspring of

P1♀C♂ and C♀P1♂ pairings do not show a significant preference for peppermint food, in contrast to P1♀P1♂ offspring (see Chapter 6). This means that peppermint does not appear to be some sort of “nuptial gift” given by males to females – such as sodium transmission from male moths (Gluphisia septentrionis) to their offspring via the spermatophore (Smedley & Eisner 1996), and the provision of nutrients,

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again via the spermatophore, from males to females in Photinus fireflies (Lewis et al

2004) – or perhaps something transferred from mothers to their offspring as is found in fall field crickets, Gryllus pennsylvanicus, in which the females convey chemical cues to their offspring to alert them to increased risk of predation (Storm & Lima

2010). Reduction in aversion to peppermint is most likely a result of traces of peppermint being contained in the egg casing as a result of both male and female backgrounds, or through epigenetic effects, similar to genomic imprinting. Genomic imprinting is a form of transference in which the non-expression of a paternally or maternally derived gene in at least some tissues at some point in development

(Spencer et al 1998) that is exclusive to mammals (Kaneko-Ishino et al 2003) and is often considered an extension of mating choice and parental conflict (Isles et al

2006). Epigenetic transference differs in that it occurs independently of genetic material. One of the main forms of epigenetic control that occurs in Drosophila is histone tagging of the genome, which alters gene transcription, and can be a major way in which characters can alter over generations, allowing phenotypic plasticity.

However, these environment-induced changes are not permanent and do not go

‘into’ the genome. If the environmental conditions are removed, then the histone tags disappear after a few generations.

There are a number of examples of profound non-genetic changes that can occur across the generations. For example, mice that are subject to traumatic experiences in their early life retain memories of this and are able to communicate to their own offspring a level of stress. This brings about significant behavioural changes in offspring that themselves have been reared no differently to control mice, making them more erratic and inconsistent in their behavioural patterns (Franklin et al

2010). Probably the best-known such effect took place under natural conditions after the Second World War, when severed food restriction in the Netherlands (‘the

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Dutch famine’) led to reduced fitness not only in the offspring of women who were pregnant at the time, but also in their grandchildren (Lumey 1992; Painter et a

2008).

It is possible that the pattern of responses seen in the P1Cn experiments reflects the induction of histone tagging – or some other epigenetic effect – on certain genes by the presence of peppermint. The disappearance of the behavioural effect after 4-

6 generations without peppermint and the reversion to control behaviour concords with this interpretation, but much more detailed work would be required to test this hypothesis.

The pattern of reduced aversion to peppermint was maintained in subsequent generations of P1♀C♂ and C♀P1♂ offspring, although if only one parent comes from a peppermint background, the reduction in aversion to peppermint is not as strong, nor is it maintained for as many generations, being lost a generation before the offspring of two parents coming from a peppermint background (the fourth generation in P1♀C♂/C♀P1♂ offspring, compared to the fifth generation in P1Cn flies). This again suggests that the effect is more likely to be due to a process that is of equal weight in either parent (such as histone tagging) rather than some humeral effect, in which it could be expected that one parent would make a greater contribution (for example, given the relative size of the egg and the sperm, it might be expected that the female would make more of a contribution to any such effect).

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7.10. Chemosensory mutants do not find peppermint aversive

Experiments that looked at the behaviour of three different chemosensory mutants,

OrCo, Trp and Painless, found that they all three did not find peppermint aversive, suggesting that they apparently lacked some of the components involved in the detection of and subsequent behaviour in response to peppermint (see Chapter 6).

One caveat is that rescue lines in which the precise genetic lesion in each of the mutants was rescued were not tested. It is possible that any differences between the behaviour of the mutants and those of control flies were due to other, unidentified genetic factors that differed between the two lines. However, despite this it is striking that the mutants all demonstrated behaviour that was significantly different to control flies, and was not significantly different from P1 flies and larval offspring, when looking at both settling behaviour in adults and larval olfaction and gustation. This implies that the chemosensory mutants are unable to detect and respond to peppermint in the same way as control flies – peppermint is not only an olfactory stimulus, but also causes a level of pain in D. melanogaster, and may be detected as a cold stimulus as well as activating a range of sensory functions through Trp channels. This raises the possibility that any one – or all – of these sensory genes may have been the target for any epigenetic effects. A relatively simple way of altering a behavioural response to a stimulus would be to change the transcription levels of a particular receptor involved in detecting the stimulus; this could alter the sensitivity of the neurons and thereby change either the threshold for the induction of a response or the interpretation of the activity of the sensory neuron by the brain.

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7.11. Menthol and peppermint evoke similar behavioural responses

The peppermint extract used here is a mixture – a bouquet – containing potentially a great many chemical compounds. Identifying the exact components that induce the response to this extract was not a priority for this study – the effect was considered to be more important and interesting than its precise chemical underpinning.

However, it was possible to explore the response of flies to one of the key components of peppermint extract, menthol (Chapter 6). Control flies found 0.1% menthol food to be aversive, while P1 flies did not behave significantly differently on either food type (control food or 0.1% menthol food), just as when they were presented with a choice of control food and peppermint-adulterated food. M1 flies did not behave significantly differently on control, peppermint or menthol food, showing that they perceived them all to have a similar level of attraction. Control flies did not behave significantly differently on peppermint or menthol food, and a similar conclusion could be drawn for P1 flies, suggesting that the flies did not (or could not) distinguish between menthol and peppermint. This indicates that even though one is a pure odour (menthol), whilst the other is a naturally occurring bouquet (peppermint), the responses to these stimuli were approximately the same.

M1 flies also did not differentiate between peppermint and menthol food. They have experience of one odour (i.e. menthol) and that is enough to alter their behaviour to the other (i.e. peppermint). The use of peppermint extract was quite justified – it is closer to the natural aversive chemostimulant that could be encountered by D. melanogaster, and is comparable with the pure odour that has widely been used in studies into behavioural plasticity in the past (Thorpe 1939; Barron & Corbet 1999;

Riddick et al 2000). However, from the limited experiments carried out here it would appear that the effect reported with peppermint extract was no different to that found with menthol. It is possible that the whole effect observed with peppermint extract is

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due to the menthol component, although further work would be needed to demonstrate this.

7.12. Comparison of single sex and mixed sex settling assays

Male and female only vials exhibited very different behavioural patterns when compared with mixed sex vials (see Chapter 6). In female only vials, flies moved about very little, whilst in male only vials, there were much greater levels of activity, with many attempted copulations and endeavours to reject courtship behaviour.

Behaviour in mixed sex vials falls somewhere in between the two single sex vials, with copulation occasionally occurring, some flies moving very little, and some flies moving more, although because cameras were not used to more closely identify behavioural patterns it is not possible to accurately discuss whether these differences in behavioural patterns are driven by male or female flies, but in observations it appeared more that they result from the interaction of the two, with most activity coming from pairs engaging in the courtship rituals. Movement was not as erratic as in male only vials, making it easier to count the number of individuals settled on the food medium at a specific time point, but there was also more activity than in female only vials.

The use of mixed sex vials, and the time spent ensuring that there were equal numbers of males and females in each vial is justified as the behaviour is roughly an intermediate representation of both male-only and female-only behaviour, and yet there were great disparities between the behaviour in separate gender vials, and

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mixed sex vials are a more accurate representation of the natural environment and the response of the population as a whole. Behavioural changes could be observed and measured in a quantifiable way (which was more difficult in fast moving male- only vials) while still producing usable results from which differences in behaviour patterns could be observed (which may not be achievable using female-only vials).

7.13. Further work

During the course of this project, it was possible to address most of the questions outlined in Chapter 1 – Introduction. However, the project has also raised a number of additional questions. For instance, it would be interesting to evaluate behaviour after exposure in more than 10 generations, to monitor whether behavioural preference for a previously-aversive stimulus does eventually develop and persist.

This question of the permanence of the change observed after P10 generations is of fundamental interest. Does the change in behaviour remain, or, like the indifference observed in P1 offspring, does it disappear after 4-6 generations of being reared on control food? If a permanent change was observed, studies of sequence composition and gene expression could be carried out to determine whether changes are occurring at the genetic level that drive the observed behavioural modifications. In the apple maggot fly, for example, it has been discovered that chromosomal rearrangements have arisen that differentiate the apple and hawthorne strains (Jiggins & Bridle 2004); if this could be identified in another species, it may enable more general conclusions regarding the mechanisms underlying behavioural changes at the population or cultural levels.

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It would also be advantageous to repeat the trial with ‘no contact’ flies, removing the

Eppendorf tube before any adults had emerged from their pupal casings.

Alternatively, a paintbrush could be used to remove the pupal casings from the ‘no contact’ bottles, replacing them in control vials, although this would raise the possibility of maggots being harmed in the process. These ‘no contact’ flies could be reared to adulthood and their behavioural responses to peppermint tested to see if there are any significant differences to flies that were reared in the presence of peppermint and then washed as third instar larvae, as well as ‘no contact’ flies that were exposed through into adult life. Similar experiments to the ‘no contact’ and washed larvae trials were carried out by Blackiston et al (2008) using the moth

Manduca sexta. They found that exposure to an odour in the larval stage, in conjunction with a negative stimulus, resulted in the association with the odour being transmitted through to the adult life stage. Barron & Corbet (1999), conversely, found that exposure in the larval stage had no impact on adult behaviour when looking at D. melanogaster and their responses to menthol. From looking at the earlier results on olfaction it may be that there was a level of dose- dependence in Barron & Corbet’s trial – the dose may have been slightly too low to elicit a change in behaviour, although at 0.08% it was very similar to the concentration used in this body of work at 0.1% the purity may not have been as precise. Barron & Corbet did not attempt to rear ‘no contact’ flies, they only used washed flies that had been reared on peppermint food medium for set lengths of time. The results described in this body of work suggest that if Barron & Corbet had carried out such an experiment, a similar behavioural pattern would be observed in all three treatments (‘no contact’ larvae, washed larvae, and ‘no contact’ larvae where the peppermint source is removed during pupation) in D. melanogaster – there will be a reduction in aversion to peppermint, but the reduction in aversion will not be maintained for as many generations as in flies which have been reared with peppermint contained within their food source,, and it will not be as strong.

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It would also be interesting to measure the average weight and length of flies reared in the presence of peppermint to see if they demonstrate any noticeable physiological differences, when compared with control flies, as a result of their rearing conditions. For example, temperature has been found to have an influence on body size and wing length in D. melanogaster (de Moed et al 1997), and stress

(including cold shocks and poor nutrition) has also been found to increase development time and bring about morphological changes in wings by Hoffmann &

Schiffer (1998). Finally, Remy (2010) exposed C. elegans to benzaldehyde for four generations before behavioural modifications became persistent, and so it would be fascinating to attempt the same experiment with Drosophila and see if the same, or a similar, pattern emerges – i.e. will D. melanogaster retain their reduced aversion to peppermint for longer than when only exposed to it for one generation. It I suspected that this would be the case, based on the evidence demonstrated by C. elegans, the apple maggot fly, and D. sechellia (Kliman et al 2000; Jiggins & Bridle

2004; Remy 2010).

7.14. Overall conclusions

Behavioural work, even when using a simple model organism such as D. melanogater, is pain-staking and time-consuming, but any breakthrough in understanding is hugely rewarding and enlightening for researchers. Here, it has been demonstrated that peppermint is aversive to D. melanogaster, and that it has a negative impact on survival when exposure occurs from egg through all stages of development to adulthood, even at the strength of only 0.1%. This could also be true for other insects, and as such peppermint might find wide use as a ‘green’

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pesticide (El Nagar et al 2012). Because individuals are able to develop and adapt to the presence of peppermint in their diet, it would be necessary to alternate its use with other odours that insects find to be aversive. As such, a wider level of research needs to be carried out to see if other repellent odours, which are not harmful to the natural environment, can be used in this way. If this proves to be the case, then a series of fragrances could be applied periodically to maintain crop acquisition, and so not lose farmers money, while at the same time preserving biodiversity and the natural environment. Electrophysiological results would also provide insight into the inner workings of the insect brain in response to repulsive odours; as such, further research of this nature may help in developing insect repellents, reducing the incidence of diseases spread by insect vectors. These kind of effects might be widespread in terms of epigenetic alteration of gene expression, and their influence deserves greater research to establish a superior level of understanding.

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