The Mating System and Courtship Behaviour of the Queensland Fruit Fly, Bactrocera Tryoni (Froggatt) (Diptera: Tephritidae)

The Mating System and Courtship Behaviour of the Queensland Fruit Fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae)

W M Thilini Darshika Ekanayake B.Sc. (Hons) (Zoology)

Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy 2017

Earth, Environmental and Biological Sciences Science and Engineering Faculty Queensland University of Technology

Keywords

Bactrocera neohumeralis, Bactrocera tryoni, courtship, cuticular compounds, land- marking, leks, mating system, mating behaviour, Queensland fruit fly, Tephritidae

Abstract

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is one of Australia’s most destructive horticultural pests, attacking a wide range of fruit commodities including drupes, pomes and berries; causing between $28.5 million and $100 million loss per annum. Understanding the mating system of B. tryoni remains a key component to resolving its behavioural ecology from both pure and applied perspectives. Despite much of B. tryoni’s mating system having been investigated previously, critical elements of its sexual behaviour remain either completely unknown or in need of further research, such as: how individuals locate mates within the landscape; whether this species leks as is often assumed; the sequence of its courtship behaviour; if mate choice occurs and varies with flies of different physiological state; and whether close range cues (esp., cuticular compounds) may be involved in mate recognition and mate choice. To address this, I carried out large and fine scale experiments to evaluate: 1) the role of plant architectural features in determining mating site; 2) if the presence or absence of host fruit influences mating site selection and whether B. tryoni behaviours conforms to

‘classical lek’ expectations or has a resourced based mating system; 3) what are the fine-scale behaviours involved in courtship and mate recognition; 4) how variation in male quality (age, size, & sexual experience) may drive differential mating success and whether there is evidence of female choice depending on these factors; and 5) whether cuticular chemical compounds (esp. hydrocarbons) play a role in mate recognition.

The first two objectives are addressed in Chapter 2, for which behavioural observations were made on virgin males and females released in large field cages in

comparative studies using tall vs short artificial trees (to test for land-marking behaviour), and fruit vs no fruit-bearing artificial trees (to test for host-fruit resource use in mating). To address the third objective, Chapter 3 focussed on close-range courtship behavioural observations of B. tryoni males and females using Noldus analytical software to construct an ethogram of the courtship sequence, comparing male and female behaviours for successfully vs unsuccessfully mated individuals in separate replicates. In Chapter 4 the potential effects of male physiological attributes

(i.e., young/old, large/small and virgin/mated) were tested in single-tree field cage studies, with further fine-scale courtship analysis to identify possible mechanisms explaining why males of one type were more successful at securing a mate (as demonstrated in Chapter 3). The final research component, Chapter 5, addressed the fifth objective using gas chromatography mass spectrometry (GCMS) studies to identify cuticular compounds, specifically cuticular hydrocarbons, which may be implicated in mate recognition and also mate choice.

Results of this thesis demonstrate B. tryoni as a tephritid species that does not preferentially mate on plants containing fruit vs those without (i.e., is a non- resource based system), and that significantly more mating aggregations form on tall over short trees (i.e., displays land-marking behaviour). Male/female timing of aggregation is approximately the same, demonstrating no evidence of preliminary male occupancy of mating site before females, as predicted under a classical lek hypothesis. Within this selected mating site, both B. tryoni males and females perform sequences of behaviours before they initiate copulations. Among these behaviours, a male directing wing (i.e., extending both wings perpendicular to the thorax and abdomen) was the most significantly different behaviour between successful and unsuccessful copulations. This implies the presence of visual

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signalling in B. tryoni males which they may use to indicate their presence or readiness to mate. Significantly greater mating success was demonstrated by young males over old males, and by large males over small males; there was no effect of male sexual experience with respect to mating success, but no specific fine-scale behavioural differences were observed between successful vs unsuccessful males.

Subsequent GCMS analyses of male and female cuticular compounds revealed evidence of sexual dimorphism in the relative quantities of fourteen cuticular compounds; however, intra-sexual differences were not observed between males that mated vs those that did not.

In conclusion, B. tryoni has a non-resource based, aggregated mating system for which they likely use land-marking when selecting a mating site in the field. Within this mating arena, the most important male behaviour that leads to a successful copulation is whether a male initiates mounting; no other behavioural differences were observed between males and females for successful vs unsuccessful males. Differences in male physiological attributes affect mating success; however, no evidence was found that, this is driven by female choice and is instead driven by intrinsic male qualities that may render them more capable of repeated attempts at copulation or fending off other males. Analysis of cuticular compounds revealed differences between males and females, implying a potential mate recognition role; however, absence of differences among males suggests that, these compounds may not be involved in individual mate acceptance or rejection. Further resolution of the mating system of B. tryoni reinforces the notion that considerable differences may exist among tephritid fruit fly species, despite them often being considered in the literature as having highly similar mating systems. The outcome of this research contributes with information useful for pest management decisions for adopting

approaches that rely on an intimate knowledge of the mating system, such as the

Sterile Insect Technique (SIT).

Table of Contents

Keywords ...... i

Abstract ...... ii

Table of Contents ...... vi

List of Figures ...... xi

List of Tables ...... xvi

List of Appendices ...... xix

List of supplementary material ...... xx

Statement of Original Authorship ...... xxi

Acknowledgements ...... xxii

Chapter 1: General Introduction ...... 1

1.1 General introduction ...... 2

1.2 Tephritid fruit flies ...... 4

1.3 Sexual reproduction and mating systems of tephritids ...... 5 1.3.1 Tephritid “leks” as a mating system ...... 6 1.3.2 Tephritid “swarms” as a mating system ...... 7

1.4 Mating site selection ...... 8 1.4.1 Extrinsic mate location ...... 8 1.4.1.1 Environmental factors as extrinsic mate location mechanisms ...... 8 1.4.1.2 Resources for mating site selection ...... 9 1.4.2 Intrinsic mate location mechanisms ...... 11 1.4.2.1 Pheromones ...... 11 1.4.2.2 Auditory signals ...... 12 1.4.2.3 Visual signals ...... 13

1.5 Male-male competition in tephritids ...... 14

1.6 Tephritid courtship and courtship behaviours ...... 15 1.6.1 Courtship ...... 15

1.6.2 Individual courtship signals ...... 19 1.6.2.1 Pheromones ...... 19 1.6.2.2 Auditory signals ...... 19 1.6.2.3 Visual signals ...... 20 1.6.2.4 Tactile signals ...... 21 1.6.3 Sexual selection ...... 23 1.6.3.1 Effect of male body size ...... 23 1.6.3.2 Effect of male age ...... 24 1.6.3.3 Effect of male pre-mating experience ...... 26 1.6.4 Summary of section ...... 27

1.7 The Queensland fruit fly, Bactrocera tryoni ...... 28 1.7.1 Distribution and economic significance ...... 28 1.7.2 Bactrocera tryoni mating behaviour ...... 31 1.7.2.1 Mating systems and extrinsic mate location mechanisms ...... 31 1.7.2.2 Bactrocera tryoni male-male competition ...... 32 1.7.2.3 Intrinsic mate location and courtship ...... 32

1.8 Thesis Outline ...... 36

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis ...... 39

2.1 Introduction...... 40

2.2 Material and methods ...... 43 2.2.1 Experiment 1: Mating site preference and tree height ...... 44 2.2.2 Experiment 2: Mating site preference with respect to fruit presence ...... 45 2.2.3 Data analysis ...... 46

2.3 Results ...... 48 2.3.1 Experiment 1: Mating site preference and tree height ...... 48 2.3.2 Experiment 2: Mating site preference with respect to fruit presence ...... 55 2.3.3 Experiments 1 & 2 combined: Mating aggregation ...... 56

2.4 Discussion ...... 59 2.4.1 Aim for the top: evidence in support of landmarking ...... 60 2.4.2 Non-resource based mating system ...... 60 2.4.3 The role of males and females with aggregation...... 61

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2.4.4 To lek or not to lek - that is the question ...... 62

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) ………………………………………………………………………. 65

3.1 Introduction ...... 66

3.2 Material and methods ...... 70 3.2.1 Detailed study of B. tryoni close range courtship and defining their specific courtship behaviours ...... 71 3.2.2 Identification of individual behaviours ...... 71 3.2.3 Male and female behaviour analysis (Hypothesis testing) ...... 72

3.3 Results ...... 73 3.3.1 Behaviours ...... 73 3.3.2 Courtship behaviour sequence of Bactrocera tryoni – Ethogram ...... 82 3.3.3 Male and female behaviour analysis (Hypothesis testing) ...... 85

3.4 Discussion ...... 95

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni ...... 101

4.1 Introduction ...... 102

4.2 Material and methods ...... 105 4.2.1 Experiment 1– Field cage mating competitiveness tests ...... 105 4.2.1.1 Measuring wing sizes ...... 109 4.2.1.2 Sexual competitiveness data analysis ...... 110 4.2.1.3 Wing size data analysis ...... 111 4.2.2 Experiment 2- close range video recordings ...... 111 4.2.2.1 Male and female behaviour analysis ...... 113 4.2.2.2 Male and female ethograms ...... 114

4.3 Results ...... 114 4.3.1 Experiment 1– Field cage mating competitiveness tests ...... 114 4.3.1.1 Wing length comparison ...... 117 4.3.2 Experiment 2- Video analysis of mating behaviours ...... 118 4.3.2.1 Male and female behaviour analyses ...... 119 4.3.2.2 Male and female ethograms ...... 135

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4.4 Discussion ...... 138 4.4.1 Summary of results...... 138 4.4.2 Why young and large males are more reproductively successful than old and small males? – Evidence from cage trials ...... 139 4.4.3 Can we explain the reproductive success of young and large males over old and small males from close range behavioural observations? ...... 141 4.4.4 No effect of mating experience for mating success ...... 142 4.4.5 Next chapter ...... 143

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice ...... 145

5.1 Introduction...... 146

5.2 Materials and methods ...... 149 5.2.1 Source of insect material ...... 149 5.2.2 Experimental setup ...... 149 5.2.3 CHC extraction procedure ...... 150 5.2.4 Chemical analysis ...... 150 5.2.5 Statistical analysis ...... 151

5.3 Results ...... 152 5.3.1 Male and female CHC profiles...... 152

5.4 Discussion ...... 167 5.4.1 Summary of results...... 167 5.4.2 Significance of methyl-branched alkanes ...... 168 5.4.3 Significance of saturated fatty acids...... 169 5.4.4 Significance of experimental results ...... 170

Chapter 6: General discussion ...... 173

6.1 Summary of results ...... 174

6.2 Contradictory ideas on tephritid mating systems...... 176 6.2.1 Tephritids with sufficient evidence for lekking ...... 178 6.2.2 Contradictory ideas on B. tryoni mating behaviour from past to present ...... 181

6.2.3 Uncertainty and conflicting ideas about lekking in other tephritids ..... 183 6.2.4 Tephritids with a resource-based mating system ...... 185

6.3 Implications for tephritid pest management ...... 189

6.4 Conclusions and future research ...... 193

Bibliography ...... 197

Appendices ...... 228

Supplementary material ...... 243

List of Figures

Figure 1.1: A simplified diagram of the sequence of a courtship pattern (= the Specific Mate Recognition System of Paterson, 1985 [figure redrawn from that source])...... 16

Figure 1.2: The courtship of Bactrocera oleae. Percentages are the proportion of individuals displaying each behaviour (n = 50 couples) (Benelli et al., 2012)...... 18

Figure 1.3: Long and short range cues for mating site and mate selection in tephritids...... 28

Figure 1.4: Long and short range cues for mating site and mate selection in Bactrocera tryoni. (Question mark indicates the areas with fewer or no studies done) ...... 36

Figure 2.1: Mean (± SE) maximum number of (a) Bactrocera tryoni or (b) Bactrocera neohumeralis individual males, females and mating pairs in two tall or two short trees. (n = 7 for Bactrocera tryoni, n = 8 for Bactrocera neohumeralis; * indicates significant difference at P < 0.01 and ** indicates significant difference at P < 0.001 for the pair- wise ‘short-tall’ comparison.) ...... 49

Figure 2.2: Mean (± SE) maximum number of (a) Bactrocera tryoni or (b) Bactrocera neohumeralis individual males, females and mating pairs in direct sunlight vs shade in tall trees. (n = 7 for Bactrocera tryoni, n = 8 for Bactrocera neohumeralis; * indicates significant difference at P < 0.01for the pair-wise ‘sun-shade’ comparison)...... 50

Figure 2.3: Mean (± SE) maximum number of (a) Bactrocera tryoni or (b) Bactrocera neohumeralis individual males, females and mating pairs in upper or lower half of tall tree canopies. (n = 7 for Bactrocera tryoni, n = 8 for Bactrocera neohumeralis; * indicates significant difference at P < 0.01 and ** indicates significant difference at P < 0.001 for the pair-wise ‘upper/lower’ comparison.) ...... 52

Figure 2.4: Mean (± SE) number of (a) individual Bactrocera tryoni males (all behaviours summed), females and mating pairs in tall trees at five minute intervals before and after official sunset (n = 7); and (b) individual wing-fanning (i.e., ‘calling’) males, non-wing fanning- males, and fighting males...... 54

Figure 2.5: Mean (± SE) number of individual Bactrocera neohumeralis males, females and mating pairs in tall trees from 9.30am to 3.00pm. The vertical line represents the average time of solar noon for the experimental period. (n = 8.) ...... 55

Figure 2.6: Mean (± SE) number of (a) Bactrocera tryoni or (b) Bactrocera neohumeralis individual males, females and mating pairs in trees with or without fruit. (n = 8 for both species; * indicates significant difference at P < 0.01 for the pair-wise ‘with fruit/without fruit’ comparison.)…………………………………………….58 Figure 2.7: Mean (± SE) number of Bactrocera tryoni females on leaves, females ovipositing and females on fruit not ovipositing in fruited trees. (n = 8). The vertical line represents the official time of sunset for the experimental period...... 58

Figure 2.8: Mean/variance plot for the distribution of mating pairs of Bactrocera tryoni on four artificial trees within a large flight cage...... 59

Figure 3.1: Ethogram for (a) Trupanea vicina courtship behaviour (n = 10) (Zunic Kosi et al., 2013), (b) Rhagoletis mendax courtship behaviour (n = 17) (Benelli et al., 2014b) ...... 69

Figure 3.2: Male and female Bactrocera tryoni courtship behaviours: (a) & (b) extending proboscis; (c) & (d) extends proboscis and tapping the substrate; (e) male-female face-to-face touching...... 77

Figure 3.3: Male and female Bactrocera tryoni courtship behaviours: (a) & (b) Female coming towards the male; (c) fore-legs tapping female’s body; (d) rubbing fore-legs together; (e) rubbing fore- and mid-legs together; (f) rubbing hind- and mid-leg together...... 78

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Figure 3.4: Male and female Bactrocera tryoni courtship behaviours: (a) & (b) Rubbing hind-legs on wings; (c) & (d) rubbing hind-legs against abdomen; (e) & (f) rubbing hind-legs all over the body...... 79

Figure 3.5: Male and female Bactrocera tryoni courtship behaviours: (a) & (b) Rubbing fore-legs against head; (c) & (d) fore-legs interacting with mouth parts; (e) & (f) rubbing hind-legs against ovipositor...... 80

Figure 3.6: Male and female Bactrocera tryoni courtship behaviours: (a) Male fore- legs interacting with female genitalia/abdomen; (b), (c) & (d) dismount; (e) female trying to oviposit; (f) directing wings away from body...... 81

Figure 3.7: Male and female Bactrocera tryoni courtship behaviours: (a) Female extending ovipositor; (b) successful copulation...... 82

Figure 3.8: Ethogram for Bactrocera tryoni male and female courtship behaviours. Percentages indicate the proportion of individuals and n values indicate the number of flies displaying each behaviour in each step ...... 84

Figure 4.1: Octagon field cage (2.64 m maximum diameter x 2.1 m high) with an artificial tree inside used for all three Bactrocera tryoni mating comparison experiments...... 108

Figure 4.2: Right-hand wing of male Bactrocera tryoni. A = the basal junction of veins of cell basal medial, B = terminus of the R4+5 vein. Scale bar = 1 mm...... 109

Figure 4.3: Graphical representation of the Relative Sterility Index (RSI) (FAO/IAEA/USDA., 2014)...... 110

Figure 4.4: Mean (± SE) number of Bactrocera tryoni mating pairs with (a) old versus young males, (b) large versus small males, and (c) virgin versus non-virgin males (n = 10 for each column; * indicates significant difference at P < 0.001) ...... 116

Figure 4.5: Relative sterility index for Bactrocera tryoni mating competitiveness between young versus old, large versus small, and virgin versus previously mated males (RSI ± 95% CI). RSI = 0 denotes old/small/mated males are

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more competitive, RSI = 0.5 for equal competitiveness and RSI = 1 denotes young/large/virgin males are more competitive…………………………...117

Figure 4.6: Bactrocera tryoni wing length for large (n = 446) and small (n = 439) males; large mated (n = 122) and large not-mated (n = 323) males; and small mated (n = 32) and small not-mated (n = 407) males. (• indicates 5th and 95th percentiles for each sample)...... 118

Figure 4.7: Total number of Bactrocera tryoni mate-choice test replicates with particular male behaviours present, categorised between (a) young and old males (total n = 9 replicates) and (b) large and small males (total n = 14 replicates). With two males within one replicate, the maximum number of times a behaviour can occur = 2 x n...... 121

Figure 4.8: Total number of Bactrocera tryoni mate-choice test replicates with particular female behaviours directed towards (a) young and old males (total n = 9 replicates), and (b) large and small males (total n = 14 replicates)...... 129

Figure 4.9: General flow of Bactrocera tryoni courtship behavioural sequence for (a) young/old/large/small males combined and (b) females (n = 20). These were constructed combining all recorded videos under “age” treatment and “size” treatment considering all males and females separately...... 137

Figure 5.1: Examples of chromatographs generated from Bactrocera tryoni cuticular hydrocarbon extracts of (a) successful male, (b) female, (c) unsuccessful male (all from replicate number 55) and (d) R1 unsuccessful male (from replicate number 46) obtained by mass- spectrometry. Prominent peak differences are shown at RT 7.10, 8.22, 9.16, 9.25 and 14.35 which are significantly present in the female...... 162

Figure 5.2: Log mean (± SE) peak areas of 10 Bactrocera tryoni putative cuticular hydrocarbon compounds which are significantly different between all males and female in all replicates. ** indicates a significance at P < 0.001 and * indicates a significance at 0.05 > P > 0.001. The retention times (RT number) can be matched to the putative compound in Table 5.1...... 165 xiv

Figure 5.3: Log mean (± SE) peak areas of Bactrocera tryoni putative cuticular hydrocarbon compounds for successful male, unsuccessful male and female in replicates with copulations in peaks from (a) RT 7.09 to 9.24 (b) RT 14.32 to 14.55. Different letters on adjacent bars on any given RT value indicate significant difference (P < 0.05) in log peak areas between successful male, unsuccessful male and female. The retention times (RT number) can be matched to the putative compound in Table 5.1…………………………...167

List of Tables

Table 3.1: Male and female behaviours used with total duration (seconds) and total number of occurrences calculations to compare replicates between couples that did mate and those that did not (see Results for a description of the individual behaviours)...... 73

Table 3.2: Bactrocera tryoni male and female behaviours identified for Petri- dish mating trials. Behaviours are categorised under three groups: mouth part movements; leg movements; and wing movements ...... 74

Table 3.3: Mean, standard deviations (SD) and standard errors (SE) calculated for total duration (seconds) for Bactrocera tryoni male and female courtship behaviours recorded in 10 courtship sequences...... 87

Table 3.4: Mean, standard deviations (SD) and standard errors (SE) calculated for total number of occurrences of male Bactrocera tryoni courtship behaviours recorded in 10 courtship sequences resulting in both successful and unsuccessful copulations...... 89

Table 3.5: Mean, standard deviations (SD) and standard errors (SE) calculated for total number of occurrences of female Bactrocera tryoni courtship behaviours recorded in 10 courtship sequences resulting in both successful and unsuccessful copulations...... 91

Table 3.6: Statistical test results for comparisons of the number and duration of male Bactrocera tryoni courtship behaviours occurring in replicates with and without successful copulations. Blank cells represent the absence of ‘duration’ as the behaviours are point behaviours. It is important to acknowledge that the power of tests is weak because of low sample size...... 93

Table 3.7: Statistical test results for comparisons of the number and duration of female Bactrocera tryoni courtship behaviours occurring in replicates with and without successful copulations. Blank cells represent the absence of ‘duration’ as the behaviours are point

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behaviours. It is important to acknowledge that the power of tests is weak because of low sample size...... 94

Table 4.1: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for male Bactrocera tryoni courtship and interaction behaviours compared between young and old males without considering if eventual mating was successful or unsuccessful...... 122

Table 4.2: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for male Bactrocera tryoni courtship and interaction behaviours compared between large and small males without considering if eventual mating was successful or unsuccessful...... 124

Table 4.3: Statistical test results for comparisons of the number and duration of male Bactrocera tryoni courtship behaviours between young and old males and large and small males. Occurring in replicates with and without successful copulations. Blank cells represent the absence of ‘duration’ as the behaviours are point behaviours...... 126

Table 4.4: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for female Bactrocera tryoni courtship and interaction behaviours compared between young and old males without considering if eventual mating was successful or unsuccessful...... 130

Table 4.5: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for female Bactrocera tryoni courtship and interaction behaviours compared between large and small males without considering if eventual mating was successful or unsuccessful...... 132

Table 4.6: Statistical data including W/t values for Wilcoxon rank sum tests/ 2 sample t tests for all compared female behaviours towards young and old males and large and small males...... 134

Table 5.1: Fourteen possible compounds at their RTs detected in both male and female Bactrocera tryoni flies. Other information includes the

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type of compound (e.g., an alkane, acid, ester, etc.), notes on their common names and previous records in animals and plants...... 154

Table 5.2: Log mean peak areas ± SE under each detected compound for all successful males, unsuccessful males and females in did mated replicates and unsuccessful males and females in didn’t mate replicates ...... 159

Table 5.3: Statistical results (F and P values) of ANOVA for log mean peak areas of Bactrocera tryoni putative cuticular hydrocarbon compounds compared between successful males and unsuccessful males in replicates with copulations, and log mean peak area differences between successful females and unsuccessful females...... 164

Table 6.1: Behaviours common for tephritids with a resource based mating systems; non-resource based mating systems (except swarms and leks), swarms, and leks...... 188

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List of Appendices

Appendix 1: Previously identified cuticular compounds and their significance in tephritids ……………………………………………………………….… 228

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List of supplementary material

Supplementary material 1 Journal publications relevant to this thesis……….. 244

Supplementary material 2 Abstract of oral presentations relevant to this thesis that were presented in scientific conferences ………………………………………… 245

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.

QUT Verified Signature

Signature:

Date: ______05/05/2017

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Acknowledgements

I wish to express my sincere gratitude first to my supervisors Dr. Mark

Schutze and Prof. Tony Clarke for their guidance, encouragement and patience in helping me to complete this project. Thank you so much for always being there for me with all the complications came with conducting the experiments as well as with writings. Without your kind encouragement and guidance, completion of the project would have not been possible.

I extend my appreciation to Dr. Kumaran Nagalingam, Dr. Paul Cunningham and Dr. Matthew Krosch for the advices and assistance they have given during this study period. I am also grateful to Dr. Megan Higgie (James Cook University,

Townsville) for guiding, sharing her expertise knowledge on insect cuticular hydrocarbons and allowing me to work in her lab to develop and conduct my experiments on B. tryoni cuticular compounds.

Special thanks go to QUT technical staff members especially the senior technicians Ms. Amy Carmichael, Ms. Anne-Marie McKinnon and Mr. Vincent

Chand for their technical support and supplying the equipment and chemicals whenever I need them. Analytical Laboratory Technician (Mass Spectrometry) at the

Institute for future environments (IFE) in QUT, Mr Joel Herring, and Mr. Tommaso

Francesco Villa for supporting me with the GCMS analysis. A big thank you goes to

Mr. Marcus Yates for his great helpfulness with conducting all my field cage experiments at Samford Ecological Research Facility, QUT (SERF). I am very thankful for Ms. Thelma Peek and Ms. Linda Clarke for supplying more than enough

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B. tryoni pupae without any hesitation to complete my experiments. Without their help, it would not be possible to carry out this many trials.

Fellow entomologists and all my dear past and present members of our fruit fly group, Prof. Stephen Cameron, A. Prof. Suk Ling Wee, Dr. Katherina Merkel, Dr.

Yuvarin Boontop, Ms. Jaye Newman, Ms. Jacinta McMahon, Ms. Francesca Strutt,

Ms. Anita Mangalam, Mr. Brett Lewis, Owen, Ayed, Kiran, Shirin and Shahrima; I thank you so much for supporting my experiments and sharing the joy of field work as well as through discussions and meetings.

I have to thank my loving husband Harshana, for his enormous support and being with me all the time encouraging me and without him, this would have been more difficult for me. My parents deserve special thanks who share happiness in all my endeavours. Thank you all my friends in Brisbane for encouraging me, sharing hundreds of exciting experiences through our weekend get-togethers and making

Australia feel like home.

At last but not the least, this PhD study would not have been possible without the support of QUT Post graduate Research Award for giving the financial support.

Australian Entomological Society supported with travel grant to attend Australian

Entomological Society Conference 2014, for that I am very grateful.

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Chapter 1: General Introduction

Chapter 1: General Introduction 1

1.1 General introduction

For all bi-sexual terrestrial animals, the location and selection of a suitable mating partner is central to their individual genetic fitness, and in evolutionary terms to the long-term survival of the species to which they belong. Mate location may occur over short to large geographic distances, and when a potential mate is found a series of close-range courtship interactions may follow (Paterson, 1985). The cues used for location and courtship can include visual, chemical, auditory or tactile signals (Wicker-Thomas, 2007). Despite the widespread recognition of the importance of understanding mating systems and mate selection in animal ecology, there remain significant gaps in our knowledge of these processes for many species.

The family Tephritidae, commonly known as the ‘true fruit flies’, is the most important agricultural pest family within the order Diptera (Foote et al., 1993). There are over 4,000 species of tephritids spread across every continent except Antarctica

(Hendrichs, 2000, Benelli et al., 2014a). Among these, there are approximately 250 species that cause economic damage through the laying of eggs into, and the subsequent larval development within, commercial fruits (Qin et al., 2015).

Bactrocera Macquart is one of the most economically significant tephritid genera and contains 651 described species occurring principally in Asia, the Pacific, and

Oceania (Vargas et al., 2015). Pest Bactrocera species damage most commercially important fruit and vegetable species, causing huge economic losses to agriculture.

Losses are not only due to the direct damage they cause fruit, but also due to indirect effects caused by lost market access opportunities because of quarantine regulations

(Dohino et al., 2016).

Current chemical control methods for tephritid fruit flies are effective, but are costly and harmful to both humans and the environment. Because of health concerns

862 Chapter 1: General Introduction

government regulations in many countries are phasing out widespread usage of synthetic insecticides, such as the organophosphate dimethoate (Dominiak and

Ekman, 2013). With the loss of pesticide treatments, greater emphasis needs to be placed on alternative controls built around manipulating biological attributes of the target pest (Clarke et al. 2011). Understanding the courtship and mating system of a target organism is one such biological element which needs to be understood for sustainable pest management (Walter, 2003). Accordingly, this thesis studies the mating system of B. tryoni (Froggatt), the Queensland fruit fly, Australia’s most damaging horticultural pest insect (Sutherst et al., 2000) and a tephritid for which alternative controls are urgently needed. Comparative studies with its close sibling species, B. neohumeralis (Hardy), are also included in one chapter.

While much has already been published on the mating system of B. tryoni (as reviewed later in this chapter), there remain significant gaps in knowledge and, for what is known, there is conflict and uncertainty in literature. For example, some consider B. tryoni to aggregate at dusk in non-resource based mating leks (Tychsen,

1977, Fletcher, 1987, Weldon, 2007), while others argue that the mating system is resource based and centred on fruiting host plants (Drew, 1987).

This thesis aims to fill some of these gaps in our knowledge of B. tryoni’s mating and courtship system. As a preface, this introductory chapter will first focus on tephritids and what we know of how they locate mating partners in the field and their courtship interactions. This overview will range from long distance courtship interactions to short distance communications, including components of tephritids’ specific mate recognition systems. Focusing still further, the next section will specifically address what we know of B. tryoni mating and courtship, what gaps in knowledge remain to be filled, and which ones will be addressed by this thesis.

Chapter 1: General Introduction 3

Finally, the chapter concludes with an overview of the thesis structure and a description of the research chapters.

1.2 Tephritid fruit flies

Like other dipterans, tephritids are holometabolous and have egg, larval, pupal and adult life stages. Many are frugivorous (= fruit feeding), with the larvae being the stage that develops inside fruit (Cavalloro, 1983, Fletcher, 1987). Pupation typically occurs in the soil, with the resultant adults becoming sexually mature within

1-2 weeks of emergence (Christenson and Foote, 1960, Anderson, 1962); however, this time varies depending on the species. For instances, the Caribbean fruit fly,

Anastrepha suspensa (Loew), reaches sexual maturity seven or more days after adult emergence (Teal et al., 2000) and the Carambola fruit fly, B. carambolae Drew &

Hancock, reaches sexual maturity only after 20 days from emergence (Wee and Tan,

2000). Females of most species require a protein and lipid meal (gained from leaf surface bacteria or bird droppings) to reach sexual maturity and to develop eggs

(Drew et al., 1983, Courtice and Drew, 1984, Meats and Leighton, 2004, Yap et al.,

2015). Males also require protein to reach sexual maturity, but their protein needs are considered significantly less than females (Drew, 1987).

Males in the tephritid genus Bactrocera, and some closely related genera, orientate to and feed on plant derived phenyl-propanoids such as methyl eugenol or raspberry ketone (Shelly, 2010). These chemicals increase male mating success

(Metcalf and Kogan, 1987, Metcalf, 1990, Haq et al., 2014, Kumaran et al., 2014b,

Haq et al., 2015, Hee et al., 2015) by improving male competitiveness (Haq et al.,

2014, Haq et al., 2015) and the quality of male sex pheromones by making them more attractive to females (Shelly, 2010, Tan and Nishida, 2012). These plant- derived chemicals are strongly attractive to males of many Bactrocera species (Haq

864 Chapter 1: General Introduction

et al., 2014, Hee et al., 2015) and these chemicals have been used as baits to control economically important species (e.g., B. dorsalis (Hendel)) (Shelly et al., 2011).

Tephritid mating behaviours are highly diverse and the mating systems of some species have been studied in detail, either because of the species’ pest status

(Whittier, 1993, Bo et al., 2014, Dong et al., 2014) or as model organisms for developing general theories on mating behaviour (Hoglund and Alatalo, 1995, Shelly and Whittier, 1997).

1.3 Sexual reproduction and mating systems of tephritids

Sexual reproduction in most land animals, by definition, requires sexually mature males and females coming together to mate. First, males and females must locate each other in the environment. They must then recognise each other as a suitable partner when at close range, leading to copulation and the transfer of physiologically compatible gametes. Although the specific process of mate recognition and courtship is unique to an animal species, across species there are documented common behavioural patterns associated with obtaining suitable mates: these are generally described as ‘mating systems’ (Zhi-Bin, 2003). Examples of different descriptors applied to mating systems include describing the number of mates per male or female

(e.g., a monogamous or polygamous mating system); describing the genetic relationship between mating pairs (e.g., random mating, outbreeding, or inbreeding)

(Shuster and Wade, 2003); or describing where mating occurs (e.g., resource based or non-resourced based) (Jiguet et al., 2000, Fischer and Fiedler, 2001). Theoretical explanations for the evolution of different mating systems (or their component elements) are well documented (Emlen and Oring, 1977, Thornhill and Alcock,

1983), but in general, the type of mating system shown by a certain species depends

Chapter 1: General Introduction 5

on the mating requirements of each sex and the interactions between them

(Wittenberger and Tilson, 1980).

Trying to introduce all concepts associated with mating systems is beyond of this introduction, and the sections below focus on those most pertinent to tephritids fruit flies which are the focus of the thesis.

1.3.1 Tephritid “leks” as a mating system Leks are aggregations of males that females visit only for the purpose of mating and where the females gain no additional benefits such as food, shelter or oviposition substrates (Baker, 1983, Arita and Kaneshiro, 1985, Hendrichs et al.,

1991, Hoglund and Alatalo, 1995). Males in a lek aggregate at places where most of the females are present because it will increase their chance of being selected by a female (Shelly and Whittier, 1997) and, within this lek, females are highly selective when discriminating between males with which to mate (Jiguet et al., 2000).

Many tephritids including Ceratitis capitata (Wiedemann), Zeugodacus cucurbitae (Coquillett) and B. dorsalis are considered to exhibit lek or “lek like” mating behaviours (Arita and Kaneshiro, 1985, Sivinski, 1989, Shelly and Kaneshiro,

1991, Kaneshiro et al., 1993, Shelly and Whittier, 1997, Benelli et al., 2014b). Like other lekking species C. capitata males aggregate at their mating sites and defend territories from which to signal females (de Souza et al., 2015). This signalling process involves release of male sex pheromones, sound production by wing vibrations, and head movements, which are used by females to compare between males (Prokopy and Hendrichs, 1979, Hendrichs et al., 2002, de Souza et al., 2015).

It is not ‘compulsory’ for males to perform wing fanning prior to copulation as seen in B. dorsalis, non-wing fanning males with no other courtship behaviours were successfully mated with females (Shelly and Kaneshiro, 1991, Schutze et al., 2013).

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Most of the time the selected mating sites for leks are on the underside of leaves in the top layers of host-fruit trees (Shelly and Whittier, 1993, Mankin et al.,

2004), but male aggregations on fruit have also been observed (Prokopy and

Hendrichs, 1979). Although B. dorsalis is considered as a lekking species, during their mating period, the number of females at mating sites is greater than the number of males before dusk. Whereas, the number of females remains constant, more males arrive onto trees at dusk (Shelly and Kaneshiro, 1991). It is worth noting in this study that while there were considerable numbers of males and females on the trees during the dusk mating window, only one mating pair was recorded (Shelly and Kaneshiro,

1991). That females are already present and more males arrive is contradictory with the definition of “classical” lek behaviour, where males are predicted to arrive first and defend territories (Hoglund and Alatalo, 1995, Jiguet et al., 2000).

1.3.2 Tephritid “swarms” as a mating system Swarming may involve aggregation for protection, migration, or increasing the size of a colony (Parrish and Edelstein-Keshet, 1999). Male swarming groups will fly as an evenly distributed group over a large area where females are highly available, or they may fly as a strongly clumped group for a long period over areas where females are less abundant (Sullivan, 1981). Swarms are different from leks, because, in a swarm, males aggregate but do not exhibit courtship before they grasp a female for immediate copulation; but in a lek, males do aggregate as well as perform courtship behaviours prior to copulation (Shelly and Whittier, 1997). In most tephritid mating experiments, a group of males prior to copulation have been considered as a ‘swarm’ though they are performing courtship behaviours (Tychsen,

1977, Drew et al., 2008, Benelli, 2014b, Benelli et al., 2015). For instance, B. cacuminata (Hering) male groups were recorded as a loose ‘swarm’ with 10 – 15

Chapter 1: General Introduction 7

males settling on Solanum mauritianum Scopoli leaves during dusk. Within these settled ‘swarms’ males do wing vibrations which produce buzzing sounds until a female arrives (Drew et al., 2008). Male-male combats have also been observed within these ‘swarms’ (Drew et al., 2008) which is another characteristic of a lek.

1.4 Mating site selection

Tephritid mating site selection depends on two types of factors. Environmental factors such as time of day, landscape attributes, hilltops, food or shelter resources as extrinsic mate location mechanisms. Fly physiological attributes such as chemical or acoustic signals are intrinsic mate location mechanisms.

1.4.1 Extrinsic mate location

1.4.1.1 Environmental factors as extrinsic mate location mechanisms

Two environmental factors are important in how tephritids come together for mating: time and weather conditions. Time of mating is typically driven by intrinsic circadian rhythms that are under strong genetic control (Tychsen, 1978, Smith,

1979), but is triggered by changing light intensity. Some species mate during the day at high light intensities, such as C. capitata (Arita and Kaneshiro, 1985, Prokopy and

Hendrichs, 1979, Vera et al., 2003), B. neohumeralis (Pike and Meats, 2002, Meats et al., 2003) and B. tsuneonsis (Miyake) (Fitt, 1981), while other species become sexually active in the late afternoon with intermediate or fading light intensity (e.g.,

B. oleae (Rossi), A. suspensa) (Suzuki and Koyama, 1981, Sivinski et al., 1994); yet others mate over a short period at dusk with very low light intensity such as B. dorsalis (Christenson and Foote, 1960). Having different mating times is considered an important mechanism of reproductive isolation in some species pairs (Drew, 1989,

Miyatake and Shimizu, 1999).

868 Chapter 1: General Introduction

Together with light intensity, temperature and relative humidity also influence tephritid mating behaviour. For example, C. capitata initiates mating at temperatures between 15 °C – 38 °C and relative humidity between 30 – 90% (Myburgh, 1962,

Niyazi et al., 2005), while Z. cucurbitae mates at temperatures below 32.2 °C with a lower developmental threshold of 8.1 °C and relative humidity between 60 – 70%

(Keck, 1951, Dhillon et al., 2005). There is a relationship with elevation and temperature which has also been considered for tephritid mating behaviour. For instance, C. capitata male flies preferably mate at low elevation sites because at these elevations the air temperature is comparatively greater than high elevated areas

(Shelly et al., 2003).

1.4.1.2 Resources for mating site selection

Mating site selection based on resource availability involves the utilisation of sites which offer one or more benefits to females other than access to sexual partners, such as food, shelter, and oviposition sites (e.g., fruit). Males that gather in such sites compete with other males to protect these resources by butting or pushing incoming intruder males (male-male competition; see below) (Benelli, 2014b), so as to maintain a high quality territory to increase their chances of mating with a female

(Scott, 2009, Macedo and Machado, 2013). In these cases, females select their mating site based on the quality of the territory, not on direct male attributes. In apple maggot flies (Rhagoletis pomonella (Walsh)), both sexually mature males and females gather on host apple fruit for mating because host fruit are needed by females for oviposition (Prokopy and Bush, 1973). In B. oleae, mating site selection is also heavily influenced by the availability of host fruit and flies mostly prefer trees with fruit (Fletcher, 1987, Daane and Johnson, 2010, Benelli, 2014b). The Chinese

Chapter 1: General Introduction 9

citrus fruit fly, Bactrocera minax (Enderlein), is yet another host specialist species which mates at the fruit resource (Dong et al., 2014).

Polyphagous tephritids do not face these constraints as they have many fruit types in which they can lay eggs. Therefore there is less imperative for polyphagous tephritids to go to a specific resource to mate. Nevertheless, the larval host plant which has fruit on it is thought to play a role as the mating site for many species of tephritids including B. dorsalis and B. tryoni (Prokopy, 1980, Burk, 1981, Drew and

Lloyd, 1987, Clarke et al., 2005, Balagawi et al., 2012). Since many tephritids have an intimate relationship with fruit as their mating site, Drew and colleagues proposed the concept that the larval host plant is the ‘centre of activity’ for most of the fly’s biological processes, including mating (Drew et al., 1983, Courtice and Drew, 1984,

Drew, 1987, Prokopy et al., 1991, Drew and Romig, 2000).

However, whether it is resource based or not, other characteristics, such as the location of a tree in the landscape, its height, or silhouette, may play an additional role in attracting individuals to a mating site (Figure 1.3) (Kaspi and Yuval, 1999).

Rhagoletis pomonella, for example, first uses the silhouette of an apple tree at a distance in recognising it as a suitable host plant (Moericke et al., 1975), followed by chemical volatiles of ripe fruit as they fly closer (Forbes and Feder, 2006); while the wild tobacco fly, B. cacuminata, preferentially mates on tall wild tobacco (S. mauritianum) plants over short (Raghu et al., 2002).

While landscape characteristics are predicted to influence animal mating behaviour (Lodé, 2011), the ecological process of mating site selection in animals is often poorly known (Diniz- Filho et al., 2007). What is known is that animals frequently select a mating site based on the availability of resources (e.g., food or shelter), to maximise predator avoidance choosing a mating site devoid of resources

1086 Chapter 1: General Introduction

may be the best strategy, to minimise interspecific competition, or a combination of these features (Rosenzweig, 1991, Abramsky et al., 2002). Ultimately, mating site selection is based on increasing reproductive fitness (Martin, 1998).

1.4.2 Intrinsic mate location mechanisms

In contrast to resource based mating site selection, in non-resource based mating site selection the chance of a female entering into the mating arena is often dependent on male long-distance advertisements (Borgia, 1993). In insects, these often take the form of pheromones and acoustic signals, (Burk and Webb, 1983,

Kortet and Hedrick, 2005, Kumaran, 2014). These mechanisms, plus others, are also involved in fly courtship which is a series of signal exchanges by which potential mates recognise each other as suitable mates, and which will be described below in

Section 1.6.

1.4.2.1 Pheromones

Sex pheromones are volatile chemicals which are a mechanism for advertising an individual’s presence, readiness to mate, and to attract mates (Wyatt, 2003). Sex pheromones are produced in specialised glands and when the animal is ready to mate it disperses the pheromone into the air; a behaviour which in male fruit flies is often referred to as ‘calling’ (Gould and Gould, 1996, Bernays and Chapman, 2014).

Potential mating partners have receptors that can detect and interpret the message sent by the signaller (Bradbury and Vehrencamp, 1998).

Sexually active males of many tephritid species release a sex pheromone in order to attract sexually mature females to their mating site (Kuba and Koyama,

1985, Perez-Staples and Aluja, 2006, Kumaran et al., 2013). For example, males of

C. capitata and Platyparea poeciloptera (Schrank) (Asparagus fly) produce

Chapter 1: General Introduction 11

pheromone secretions from their anal gland and evaporate them by wing beats

(Pritchard, 1967, Prokopy and Hendrichs, 1979, Shelly and Whittier, 1993, Mankin et al., 2004). For this, males first rub their hind legs over the anus where the pheromone is excreted, then wipe this pheromone over the wings and finally vibrate the wings to disperse the odour into the air (Fletcher, 1968, Fletcher, 1969, Symonds et al., 2009, Fanson and Taylor, 2012). In some species, such as A. suspensa, males tend to rub the tip of the abdomen with the pheromone droplet on leaf surfaces in order to increase the dispersion of the pheromone (Sivinski et al., 1994). Virgin females, non-virgin females or those that have not mated for a period of time, have been shown to be attracted to these male pheromones in R. cerasi (Linnaeus)

(Katsoyannos, 1976, Heath et al., 1999), B. dorsalis (Tan and Nishida, 1996, Schutze et al., 2013) and Z. cucurbitae (Dhillon et al., 2005).

Seldom appreciated, however, is the fact that while males are the ones that release sex pheromones in most species, female sex pheromones do occur, as found in B. oleae (Economopoulos et al., 1971, Haniotakis, 1977, Benelli et al., 2012,

Benelli et al., 2013); the specific role of female pheromones remains to be explored, but in B. oleae the pheromone attracts males over a distance and is used in monitoring traps.

1.4.2.2 Auditory signals

Auditory signals may be simple one-pitch calls, or more elaborate with frequency variations. Such calls may attract the opposite sex, or be competitive signalling cues with other members of the same sex (Slabbekoorn et al., 2002). By wing vibrations tephritid males disperse sex pheromones (‘call’) and at the same time they produce an acoustic signal that may or may not be important in sexual communication. Characteristics of these male ‘calls’ can be broken down into three

1286 Chapter 1: General Introduction

parts: (i) ‘calling’ over distance, (ii) ‘calling’ during close-range courtship, and (iii)

‘calling’ when males have mounted the female and started copulation (Mankin et al.,

2008). Tephritid calls are mainly produced by wing vibrations; however, additional sounds may result from rubbing the wings over abdominal setae or the legs (e.g., Z. cucurbitae (Kanmiya, 1988, Sofian-Azirun et al., 2011)) or by thoracic vibrations as documented for A. suspensa and C. capitata (Keiser et al., 1973, Sivinski and Webb,

1985a).

The acoustic signals emitted by males influence females in different ways.

Long distance male calling sounds in A. suspensa make virgin females more active and may thus play a role in mate location (Sivinski et al., 1984): it has been found that mechanoreceptors in female aristae (a rod-like appendage arising from the third antennal segment in dipterans (Foelix et al., 1989)) are responsible for detecting the male call (Sivinski and Webb, 1985a, Sofian-Azirun et al., 2011). In contrast, female papaya fruit flies (Toxotrypana curvicauda Gerstacker) reduce their activity after receiving the males’ calling sounds, which benefits males by making the female easier to grasp (Sivinski and Webb, 1985b). Females have a propensity of selecting a male depending on the quality of the acoustic signal that the male produces. In both

C. capitata and A. suspensa, larger sized males produce low frequency, high intense sounds which females prefer (Sivinski et al., 1989, Briceno et al., 2002).

1.4.2.3 Visual signals

Visual signals as long range cues have rarely been observed in tephritids except for the apparent patterns on wings of A. suspensa (Sofian-Azirun et al., 2011) and head nodding of C. capitata when they are in a lek (de Souza et al., 2015) . However tephritid visual cues are important as short range cues when potential mating partners are together in their mating site.

Chapter 1: General Introduction 13

1.5 Male-male competition in tephritids

In both resource based and non-resource based mating systems, males of some tephritid species are known to engage in male-male competition to defend their mating arena. In non-resource based lek mating systems males may fight for their territories, while in resource based mating systems male-male competition is for defending the resources needed by females (e.g., oviposition sites) (Prokopy and

Papaj, 1999, Benelli, 2014a). Zeugodacus cucurbitae (Iwahashi and Majima, 1986,

Haq et al., 2013), C. capitata (Cayol et al., 1999), A. suspensa (Sivinski et al., 1994),

B. oleae (Benelli, 2014b), and B. dorsalis (Shelly and Kaneshiro, 1991) are species where male-male competition is recorded within lek mating systems, while several or all studied species in the genus Rhagoletis (Prokopy and Bush, 1973, Prokopy and

Papaj, 1999, Benelli, 2014a) and species in the genus Phytalmia Gerstaecker

(antlered flies) (Schutze et al., 2007) are species who engage in male-male competition to defend oviposition sites.

If an intruder male enters a territory of another male, either the resident male or the intruder male will chase the other and the ‘winner’ will be decided by the competitiveness of either male (Shelly and Kaneshiro, 1991). For instance, in antlered flies, a resident male will defend a small rotting area under tree bark which is the female oviposition site; if an intruder male arrives, and resident and intruder are off near-equal size, then the two males will push against each other in a head-to- head position until one male moves away (Dodson, 1997). In resource based mating systems, females gain numerous benefits by selecting a male with high competitiveness for copulation. The females do not need to spend extra energy to find an oviposition site after copulating; there is lower possibility of predation, and increased offspring fitness (Shelly, 2000, Benelli, 2014a).

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1.6 Tephritid courtship and courtship behaviours

1.6.1 Courtship

Having located potential mating partners within the landscape, short range interactions then occur between males and females, which may or may not lead to copulation: these interactions are generally referred to as courtship. Courtship communication permits verification of the status of a potential mate prior to copulation, or may even prevent ‘escape’ of a partner during copulation (Tinbergen,

1953). Courtship may be considered as a signal-response chain of interactions between potential mates, forming part of what has also been termed a ‘specific-mate recognition system’ (Paterson, 1985). Signals involved in courtship may be visual, olfactory, auditory, or a combination thereof, and can be as simple as the release of a single sex pheromone, or highly complex using multiple cues within an elaborate, sequential; courtship processes may operating over both long or short distances

(West-Eberhard, 1984, Headrick and Goeden, 1994, Freidberg, et al., 1999).

When explaining courtship as a signal-response chain, the first step of the process is the release of a signal by a reproductively active male or female. This signal is detected by a ‘tuned receiver’ (= the potential partner) which may then respond with a return signal, which is subsequently detected by the original signaller.

Successful completion of a series of signals and responses will result in copulation and fertilization; a negative response at any point will terminate the interaction

(Figure 1.1) (Paterson, 1985, Benelli et al., 2012). For example, if a male doesn’t produce the correct signal at some point in the exchange, the female will not respond and the sexes will cease communication and the courtship will not progress. The sequence of such signal-response chain of interactions varies uniquely between

Chapter 1: General Introduction 15

species, but the quality of signals within a courtship sequence can vary depending on signaller’s attributes including age, size, physical fitness and previous sexual experiences (Candolin, 2003).

Figure 1.1: A simplified diagram of the sequence of a courtship pattern (= the

Specific Mate Recognition System of Paterson, 1985 [figure redrawn from that

source]).

As reported for B. oleae, the first step of the courtship process is the male responding to a specific female based on visual or olfactory cues followed by wing

1686 Chapter 1: General Introduction

vibrations (Benelli et al., 2012) (Figure 1.2). If the female does not reject the male by walking away from him, he will attempt copulation and rub his legs on the female’s thorax and abdomen as a tactile cue. Although this sequence is very similar to the signalling process of B. dorsalis (Poramarcom, 1988, Poramarcom and Boake,

1991), C. capitata males show a much more elaborate series of behaviours including wing buzzing, head rocking and aristae tapping as courtship cues (Miranda, 2000,

Benelli et al., 2012).

Chapter 1: General Introduction 17

Figure 1.2: The courtship of Bactrocera oleae. Percentages are the proportion of

individuals displaying each behaviour (n = 50 couples) (Benelli et al., 2012).

1886 Chapter 1: General Introduction

1.6.2 Individual courtship signals

1.6.2.1 Pheromones

While sex pheromones are often considered to attract females over relatively large distances, other work indicates that the pheromones of some tephritids operate only over much shorter distances within the canopy (Pike and Meats, 2003). Males possess sex pheromones both in their rectal gland secretions and in oral secretions

(Lu and Teal, 2001, Wicker-Thomas, 2007). Although pheromones in rectal glands are evaporated by wing vibrations, males tend to deposit oral gland secretions on leaves which then attract females in species such as A. suspensa (Lu and Teal, 2001) and C. capitata (Shelly, 2004). The production of sex pheromones in some tephritid males varies with their life stage. Anastrepha suspensa mated males produce twice as much as sex pheromones than virgin males and attract more females (Teal et al.,

2000). The quality of sex pheromones also changes in some Bactrocera and

Zeugodacus species when males are exposed to plant derived chemicals such as methyl-eugenol and cue lure, making the pheromone more attractive to females

(Shelly et al., 1996, Shelly and Nishida, 2004, Kumaran et al., 2014a).

1.6.2.2 Auditory signals

Male tephritids may emit short range acoustic signals in two instances; when they are in courtship and after they have mounted a female (Mankin et al., 2008).

Males of T. curvicauda produce both of these signals where the “approach song” makes the female less active before mounting, and the “pre-copulatory song” is made when the male aculeus is being positioned within the female ovipositor (Sivinski and

Webb, 1985b). In B. oleae there is a significant effect of wing vibration on mating success, with males not performing wing vibrations being unsuccessful during

Chapter 1: General Introduction 19

mating (Benelli et al., 2012). The frequencies and pulse durations of successfully copulated males were greater than those of unsuccessful males (Benelli et al., 2012), and this was also positively correlated with male body size (Benelli et al., 2015).

Ceratitis capitata wild males produce calls with low pulse durations and high frequencies which female flies prefer (de Souza et al., 2015), an important consideration in the production and success of mass produce males reared for Sterile

Insect Technique control programs (Mankin, 2012).

1.6.2.3 Visual signals

Visual cues can play a significant role at close range courtship, particularly when species occur in complex environments where potential mates can’t see each other until very close. Visual information may be relayed by elaborate ‘dances’, or simply by assessment of overall size which may indicate overall fitness of a potential mate.

The Caribbean fruit fly males perform a courtship dance by moving their brown marked wings to signal their presence to females (Sofian-Azirun et al., 2011).

It is suggested that to consider a wing movement as a visual signal, it should be performed within the visual range of a female (Alonso-Pimentel et al., 2000), as seen in R. completa Cresson (Bush, 1966). However, males of the walnut fly, R. juglandis

Cresson, also perform wing movements as a visual signal when they are following females or standing near other individuals, although it is not clear whether they do this within the females’ visual range (Alonso-Pimentel et al., 2000).

In antlered flies, the head projections, which are of minimal importance for intra-sexual competition, are advantageous in inter-sexual visual communication

(Sivinski and Wing, 2008). The size of the antlers correlates with male body size

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(Schutze et al., 2007) and males with larger antlers are more successful when copulating with a female. Males without antlers may also be mistaken as females by other individual males (Sivinski and Wing, 2008).

1.6.2.4 Tactile signals

Short range sexual communication may involve tactile behaviours such as grooming, rubbing and gustatory stimuli, in addition to the cues already described

(Bernays and Chapman, 2014). Tactile behaviours may incorporate low or non- volatile chemicals such as salivary gland secretions, cuticular chemicals or short- range pheromone clouds as an additional mechanism of information transfer (Lu and

Teal, 2001).

Cuticular properties are an oft-overlooked close range signal that may be important in sexual communication in insects. The cuticle is the outermost layer of the insect exoskeleton, and it contains a number of compounds, including hydrocarbons, that are secreted with the cuticle deposition during the process of moulting (Singer, 1998), and non-hydrocarbons such as cuticular lipids (Dietemann et al., 2003). Cuticular hydrocarbons (CHCs) are long-chain, non-volatile, complex, fatty-acid derived hydrocarbons which consist of more than 60 carbons and are reported in numerous insect species (Cvačka et al., 2006, Blomquist, 2010).

Cuticular hydrocarbons are best known for their primary protective role of preventing insect desiccation (Singer, 1998): as hydrophobic substances, CHCs prevent water loss from the cuticle. While their principal role is desiccation resistance, the functions of these hydrocarbons may be numerous. Cuticular hydrocarbons play a role in intraspecific communication in some insect species, such as nest mate recognition in eusocial groups, especially in the Hymenoptera (Lucas et al., 2005, Torres et al., 2007, Martin and Drijfhout, 2009).

Chapter 1: General Introduction 21

Studies on CHC variation in individual tephritids and its impact on mating success are absent, but it has been found that there is a sexual dimorphism in CHCs in tephritids. For instance, in A. fraterculus (Wiedemann) (Vanickova et al., 2012) and in Trupanea vicina (Wulp), males and females possess different amounts of methyl-branched alkanes which are important for either sex to identify their potential mate (Zunic Kosi et al., 2013). This sexual dimorphism is found in A. fraterculus males and females when they are more than five days old (Vanickova et al., 2012).

Although the importance of CHC for mate recognition in Tephritidae are scarce, other aspects such as species level variation (Vanickova et al., 2015a), and CHC composition changes with physiological states (Vanickova et al., 2012) have been studied. For example, geographically distinct populations of C. rosa Karsch exhibit different CHC profiles, supporting their status are two distinct, biological species

(Vanickova et al., 2015b). Similarly three morphologically very similar species, C. fasciventris (Bezzi), C. anonae Graham and C. rosa can also be differentiated on their CHC profiles (Vanickova et al., 2014).

While studies on Tephritidae CHCs are rare, their importance in mate recognition has been widely studied in fruit flies belonging to the family

Drosophilidae. Sexually dimorphic CHC profiles have been shown in Drosophila virilise Sturtevant (Bartelt et al., 1986) and CHC composition differences have been found between mated and virgin females of this species (Scott, 1986, Scott et al.,

1988, Scott and Jackson, 1990). Indeed, the additional function of CHCs in

Drosophilidae mate recognition is becoming more widely appreciated, particularly their role in sexual selection and speciation (Higgie et al., 2000, Howard and

Blomquist, 2005). For instance, in Drosophila serrata Malloch, females show a distinctive mate selection process by selecting males with higher relative abundance

2286 Chapter 1: General Introduction

of 2-methyl alkanes within their CHC profile (Howard et al., 2003, Chenoweth and

Blows, 2005). Compositional differences of CHCs in males, due to the diet they feed on and the environment in which they live, also affects mate choice in D. simulans

Sturtevant (Ingleby et al., 2014); while variation in Drosophila CHC profiles have also been linked to fly age and mating status (Jackson and Bartelt, 1986, Everaerts et al., 2010). Drosophila serrata (Howard et al., 2003, Chenoweth and Blows, 2005),

D. subquinaria and D. recens (Dyer et al., 2014), and D. birchii (Howard et al.,

2003) are all species for which it has been experimentally confirmed that female discrimination of male flies depend on variations in male CHC compositions.

1.6.3 Sexual selection

While courtship is consistent within a species and allows mate recognition between the sexes, not all individuals are equally likely to mate in an environment where choice can be made. Variation in the quality of potential partners, typically males, means that females can actively select one individual over another: this is commonly referred to as sexual selection (Darwin, 1871, Bateman, 1948). Sexual selection is documented in tephritids (Burk and Webb, 1983, Arita and Kaneshiro,

1989, Norry et al., 1999, Kumaran et al., 2014b) and this section of the review focuses on male attributes known to influence female choice.

1.6.3.1 Effect of male body size

In tephritids, the effects of variation in male body size on male mating success have been studied in terms of its indirect effect on pheromone production and acoustic signals. In A. suspensa, large males are characterised by having a high calling propensity and short pulse train intervals compared to small males (Burk and

Webb, 1983). More than 70% of females accepted larger males to mate with, and

Chapter 1: General Introduction 23

they also responded more by increasing their movements and flights, to the acoustic sounds produced by large males (Burk and Webb, 1983, Webb et al., 1984). In male

C. capitata, large males copulated more frequently compared to small males (Taylor and Yuval, 1999). In this species protein feeding makes males heavier and they also produce more pheromones and start calling before protein deprived males (Kaspi et al., 2000).

In some tephritids, visual ornamentation has also been studied as a secondary attribute of body size, such as the head projections in antlered flies (Phytalmia) used in courtship displays (Dodson, 1988, Dodson, 1997, Schutze et al., 2007). The size of antlers correlates positively with male body size, i.e. larger males have larger antlers (Schutze et al., 2007). There is no evidence to support female selection based on the size of antlers, however, males with larger antlers have an advantage in intra- sexual competition with other males and gain greater access to females for copulation (Dodson, 1997, Schutze et al., 2007, Sivinski and Wing, 2008)

1.6.3.2 Effect of male age

Older ‘better’

The age of male tephritids may affect their mating success. In other animal systems females may evolve preferences to mate with older males because their ability to survive may be an indirect measure of having ‘good genes’ important for survival, or for the provision of high quality ejaculate (Kokko and Lindstrom, 1996,

Brooks and Kemp, 2001, Bonduriansky and Brassil, 2005). Further, older males may have improved sexual performance through increased male-male competitive ability.

Mating advantages of older over younger males have been documented in both A. ludens (Loew) (Pérez-Staples et al., 2010) and C. capitata (Leido et al., 2002). In

2486 Chapter 1: General Introduction

the first study, Mexican fruit fly males mated more when they were 36 days old compared to 18 days old.

Younger ‘better’

In contrast to mating success in old males, young males may also be successful due to female selection of young males over older males (Bonduriansky and Brassil,

2005). In contrast to the work of Liedo et al. (2002) as mentioned in the above section, Neto et al. (2009) found C. capitata females preferred to mate with younger males rather than older males, as pheromone release was higher in young males. This preference was clear only when the ratio of old to young was not more than 3:1 in laboratory reared flies, suggesting that females would be unable to differentiate younger males when there are more older males in a lek. Though male-male competitiveness is a direct proximate cue that females use to discriminate between old and young males, based on an ultimate evolutionary approach, females may choose younger males due to the reduced risk of obtaining older sperm which may have higher levels of germ-line mutations (Radwan, 2003). In A. suspensa, on a given day, young males are more reproductively successful compared to older ones

(Teal et al., 2000); however this success of younger males is more prominent if the older males are recently mated (Sivinski, 1984, Teal et al., 2000). This suggests that there is an effect of males’ sexual status for their mating success, which will be discussed in the next section.

CHC variation with age

It is known that the chemical composition in insect CHC changes with their age. This has been recorded, for example, in queenless ants Diacamma ceylonense

Emery (Cuvillier-Hot et al., 2001), in the house fly Musca domestica L. (Mpuru et al., 2001), and in the malaria vector Anopheles gambiae Giles (Caputo et al., 2005).

Chapter 1: General Introduction 25

Within tephritids, CHC profiles differ between young (5-7 days) and old male A. fraterculus (20-30 days); older males having different amounts of n-alkanes, methyl- branched alkanes, alkenes and alkadienes than young males (Vanickova et al., 2012).

Despite the CHC differences with male age, it is not known whether that affects female selection during mating.

1.6.3.3 Effect of male pre-mating experience

Virgin males ‘better’

Male premating experience has considerable effect on their subsequent mating success. Females prefer to mate with virgin males due to increased ejaculate quality, which is important for enhanced fecundity in females (Savalli and Fox, 1999, Wedell et al., 2002). As seen in A. suspensa, recently mated males are rejected by virgin females when presented together with virgin males, but the reason for this rejection is unknown (Sivinski, 1984). Although the mated males are as active in courtship display as virgins, they will not succeed in copulation as these males have spent their accessory gland fluids during first copulation which will make them infertile, especially if remating is attempted within two hours (Sivinski, 1984). In

Drosophilidae it is suggested that females can recognise freshly mated males by scent (Markow, 1987), but more research is needed for the Tephritidae to understand the reasons for female rejection (Radhakrishnan and Taylor, 2008). Results can also be contradictory. Liedo et al., (2002) recorded more mating in C. capitata virgin males than previously mated males, but Shelly and Whittier (1993) found C. capitata virgin and mated males were equally successful in matings when presented to females,

Mated males ‘better’

2686 Chapter 1: General Introduction

Even though A. suspensa virgin males are more successful soon after mating, previously mated males become more reproductively successful six to seven days after first copulation (Teal et al., 2000); presumed to be due to the ‘refilling’ of accessory gland fluids (Markow et al., 1978). Also in A. suspensa, and despite the physiological mechanism being unknown, the level of juvenile hormones are higher in mated males than virgin males (Teal et al., 2000). Having greater amounts of juvenile hormone increases sexual signalling by enhanced sex pheromone release

(Schal et al., 1994, Teal et al., 2000), thus mated males attract more females towards them and have greater mating success in comparison to virgin males (Teal et al.,

2000). In A. ludens, mated males also obtain more chances to mate with females due to increased competitiveness built upon previous experience (Pérez-Staples et al.,

2010).

1.6.4 Summary of section

As identified in this review, the mating systems, courtship and sexual selection of tephritids are highly variable across species, but may be considered to involve a sequential series of cues which lead initially from mate location and culminate in successful mating (Figure 1.3). Despite a large existing literature, there remain very significant gaps in our knowledge and contradictory findings of these processes and patterns for many species, including even the most economically important tephritid pests. In the next section I introduce and review the focus animal of my thesis, B. tryoni. Although an extremely well researched fly in many ways, I identify that along with well-studied components of its mating, that there still remain important and significant gaps.

Chapter 1: General Introduction 27

Figure 1.3: Long and short range cues for mating site and mate selection in

tephritids.

1.7 The Queensland fruit fly, Bactrocera tryoni

1.7.1 Distribution and economic significance

Bactrocera tryoni, my thesis study organism, is Australia’s most destructive horticultural insect pest, attacking over 100 species of fruit and vegetables (Clarke et al., 2011, Collins et al., 2012). The species is endemic across coastal tropical and subtropical eastern Australia (Yonow and Sutherst, 1998, Waterhouse and Sands,

2001), but since European settlement it has spread southwards and is now recorded throughout most of eastern Australia, from Cape York Peninsula, Queensland to

Gippsland, Victoria; it remains rare in cool, high altitude areas (Waterhouse and

Sands, 2001, Yu et al., 2000). While native to Australia, B. tryoni has invaded and established in some South Pacific island nations including French Polynesia, New

Caledonia and the Cook Islands (Clarke et al., 2011).

Within Australia, the species’ distribution depends on temperature and soil moisture. The fly is capable of surviving at high temperatures between 29 - 33 °C, while adults also have the ability of facultative diapause (Yonow and Sutherst, 1998,

2886 Chapter 1: General Introduction

Yonow et al., 2004). Pupal emergence does not occur in 100% saturated soils, and is greatly reduced in fully dry soils, but at nearly all intermediate soil moisture levels pupal survival is high (Hulthen and Clarke, 2006). Based on climatic model predictions tropical north Queensland is the most suitable area for B. tryoni within

Australia (Yonow and Sutherst, 1998), but damaging populations now also persist in cold, temperate Australia.

The horticultural damage caused by B. tryoni may be both direct and indirect.

Direct damage to fruit is caused by female oviposition into fruit. Pierced fruit peel allows bacterial and fungal infection to occur, in addition to the damage caused by larval feeding on fruit tissue. Indirect damage is caused through lost market opportunity. The presence of fruit flies negatively impacts market access opportunities because almost all fruit-importing countries set market access protocols that require imported fruit to be free from fruit fly eggs and larvae (Waterhouse and

Sands, 2001). Current chemical control methods for fruit flies are effective but are costly and harmful to the environment, with government regulation phasing out the widespread usage of traditional insecticides such as dimethoate (Dominiak and

Ekman, 2013).

Tephritid control methods to replace insecticides have been extensively studied. One of these, the Sterile Insect Technique (SIT), is a widely used and effective (Hendrichs et al., 2002, Klassen and Curtis, 2005, Pereira et al., 2013). In

SIT, mass-reared sterile males are released into the environment where they compete with wild males to mate with wild females, causing those females to produce infertile eggs which can lead to the reduction of the wild population (Shelly and McInnis,

2016). The SIT is used operationally against several tephritid species, such as B. oleae (Ant et al., 2012) and C. capitata (McInnis et al., 1996, Yuval et al., 2013).

Chapter 1: General Introduction 29

Knowledge on tephritid mating systems is very beneficial for the effectiveness of

SIT because mass reared sterile males must have the ability to compete with wild males in order to copulate with a wild female (Shelly and McInnis, 2016).

Due to the pest status of B. tryoni, various studies have been carried out on aspects of SIT pertinent to this fly (Andrewartha et al., 1967, Dominiak et al., 2000,

Dominiak et al., 2003, Meats et al., 2003, Reynolds et al., 2012, Raphael et al.,

2014; Reynolds and Orchard, 2015), and a successful eradication of B. tryoni from the state of Western Australia was achieved using SIT (Fisher, 1996). However, the bulk of such studies focuses on the mechanics of mass rearing and release of flies, and many fundamental, biological questions relevant to the SIT and B. tryoni remain unknown. Examples pertinent to mating include “do bigger B. tryoni males get more females?” (relevant to optimising mass rearing conditions), and “where do flies mate in the field?” (relevant to optimising field release sites). These are just two of the questions addressed in this thesis.

While this thesis focuses on B. tryoni, this species is very closely related to three others; B. neohumeralis, B. aquilonis (May) and B. melas (Perkins & May), the four species making up the B. tryoni species complex (Morrow et al., 2000, Clarke et al., 2011). Bactrocera neohumeralis, the lesser Queensland fruit fly, is used in my first research chapter with B. tryoni, as it is the sibling species that has been compared most often with B. tryoni (Bellas and Fletcher, 1979, Morrow et al., 2000,

Pike and Meats, 2002, Meats et al., 2003, Gilchrist and Ling, 2006, Meats, 2006).

Bactrocera neohumeralis can be physically distinguished from B. tryoni due to the presence of a brown humeral calli (‘shoulder pads’) in B. neohumeralis, and yellow humeral calli in B. tryoni. However, the most significant difference between these two species is their mating time. Bactrocera tryoni mates at dusk over a short

3086 Chapter 1: General Introduction

window of time at low light intensities (Tychsen, 1977, Meats et al., 2003, Collins et al., 2008, Collins et al., 2012), while B. neohumeralis mates during mid-day under high light intensities over a six to seven hour period (Pike and Meats, 2002).

1.7.2 Bactrocera tryoni mating behaviour

1.7.2.1 Mating systems and extrinsic mate location mechanisms

While much research has been undertaken on mating in B. tryoni, there remain significant gaps in our knowledge of key components of its courtship sequence, and knowledge about its mating system is conflicting. Drew and colleagues (Drew,

1987, Drew and Lloyd, 1987) argue that B. tryoni mating occurs only on the fruiting host fruit plant and, while they do not use the term, this would thus be considered a resource based mating system. However, the fly is also widely considered to have a non-resource based mating system, specifically a lek system (Fletcher, 1987,

Weldon, 2007); while yet other authors have described mate swarming behaviour for

B. tryoni (Bateman, 1972, Tychsen, 1977).

Conflict in the literature may be due to several reasons, but importantly few specific tests have been undertaken to confirm the various hypotheses about B. tryoni’s mating system, and even fewer have been undertaken outside small laboratory cages. Drew and Lloyd recorded mating on a single, fruiting peach tree and from that argued that all mating occurred on fruiting trees; Weldon (2007) tried to create artificial B. tryoni male leks, but failed to attract females to them; while

Tyschen observed swarms of B. tryoni males in a field cage, but he is the only author ever to have formally recorded this behaviour. As females visit and spend more time on trees with fruit searching for an oviposition site, it is considered males may gain an advantage of finding a mate by visiting trees with fruit where the female

Chapter 1: General Introduction 31

population is high (Dalby-Ball and Meats, 2000, Drew et al., 2003), but as mating occurs at dusk (Tychsen, 1977, Meats et al., 2003, Collins et al., 2008, Collins et al.,

2012) and oviposition peaks around noon (Ero et al., 2011), this is difficult to prove.

When considering the effect of plant architecture, there are observations saying that B. tryoni prefer to rest at high places (Balagawi et al., 2012), in open and branched canopies (Balagawi et al., 2014), but studies on plant attributes for mating site selection is absent. Despite their abundance in the field, B. tryoni mating is seldom seen in nature; we simply don’t know where they mate.

1.7.2.2 Bactrocera tryoni male-male competition

Male-male combat is recorded in one study of B. tryoni, which is one of the characteristics of a true lekking species. In a field cage, Tychsen (1977) recorded B. tryoni as a “settled swarm” and recorded male-male combat within this swarm. He noted that if an intruder male moved within 2 cm of a stridulating male, the resident male rushed towards the intruder and both would clash mouth parts until one or both males left the area.

1.7.2.3 Intrinsic mate location and courtship

As observed in the laboratory, the sequence of B. tryoni behaviours while mating is similar to that of other tephritids. First, with sunset both sexes become more active by increasing cleaning and locomotion activities. Males will then initiate wing fanning and stridulation, followed by a pheromone release. Males will jump onto a nearby female with no obvious close range courtship behaviours documented.

Male-male mountings have also been observed (Tychsen, 1977). Bactrocera tryoni females can accept or reject mounted males by pushing off with their hind legs before copulation starts (Barton-Browne, 1957, Kumaran et al., 2014b): males will

3286 Chapter 1: General Introduction

commence copulation if not rejected by the female (Tychsen, 1977, Weldon, 2005).

While this short summary of mating captures existing observational records, the actual sequence of B. tryoni courtship behaviours, the role played by each courtship step (e.g., pheromone release or wing fanning), together with the timing of events is still poorly studied and, as such, a proper understanding of the species mating system remains incomplete. Further details on specific components of B. tryoni mating follow.

Pheromones

Various studies have shown that B. tryoni males produce a sex pheromone in their rectal gland and evaporate them into the air by rapid wing movements (Fletcher,

1968, Tychsen, 1977, Pike and Meats, 2003, Fanson and Taylor, 2012, Kumaran et al., 2013). N-2-methylbutylacetamide, N-(3-methylbutylpropanamide), N-(3- methylbutylacetamide), N-3-methylbutyl-2-methylpropanamide, N-2- methylbutylpropanamide and N-2-methylbutyl-2-methylpropanamide are the identified six amide components of B. tryoni male sex pheromones (Bellas and

Fletcher, 1979, Tan and Nishida, 1995). At dusk, B. tryoni females are considered to be attracted to male pheromones (Fletcher, 1969, Weldon, 2007, Taylor et al., 2013), but the true role of the pheromone is uncertain and it may play more a role in preparing the female to mate than as a distance attraction (Fletcher, 1968, Tychsen,

1977, Bellas and Fletcher, 1979), or it may be an aggregation cue for other males

(Tychsen, 1977). The pheromone cloud produced by a single male has experimentally been found to travel through an area of 127.6 cm2 under still air (Pike and Meats, 2003). Although B. tryoni males have shown an upwind movement when tested in laboratory conditions, there were no significant responses recorded for an upwind movement in the presence of pheromones, suggesting a likely absence of

Chapter 1: General Introduction 33

pheromone effect on either male or female attraction (Pike and Meats, 2003). This upwind movement of flies has increased when cuelure (see below) was present

(Meats and Hartland, 1999).

Males of B. tryoni are attracted to the plant derived phenylpropanoids raspberry ketone (= the natural analogue the better known cue-lure) and zingerone (Meats and

Hartland, 1999) and they have increased mating success following consumption of the lures (Kumaran et al., 2013). Cue-lure fed males store the cue-lure in their rectal glands in its hydrolysed form of raspberry ketone (Tan and Nishida, 1995, Kumaran et al., 2014a), while zingerone is stored in the rectal gland without conversion

(Kumaran et al., 2014a). Within the rectal gland, these chemicals are incorporated into the male sex pheromone and, for cue-lure but not zingerone, the additional chemical enhances the pheromone’s attractive quality to females (Kumaran et al.,

2014b). Mating with a lure-fed male increases female fecundity and decreases female remating (Kumaran et al., 2013), while offspring males sired by a lure-fed father have an increased ability of locating phenylpropanoids (Kumaran and Clarke,

2014).

Acoustic signals

Sounds produced during wing fanning by B. tryoni males, commonly known as

‘calling’, have been recorded as trains of pulses 50 ms or longer (Mankin et al.,

2008). Males stay largely motionless while calling, except if there is any other fly in the near vicinity (Myers, 1952). As previously described, this sound is further categorised into three parts; “calling” at a distance, “courtship sound” when the female is nearby, and “copula sound” when the male is mounted onto the female

(Neale, 1989). The quality of the above sounds may vary depending on the physical attributes of the respective male. Although it has been found that there is a significant

3486 Chapter 1: General Introduction

difference in the frequency of calling and courtship sounds between mass-reared and wild B. tryoni males (Mankin et al., 2008), frequencies of courtship sounds does not differ with the consumption of cue-lure or zingerone (Kumaran, 2014).

Since there are relatively large number of studies already published on B. tryoni sex pheromones and acoustic signals for mating, I do not experimentally address these courtship components in my thesis.

Courtship dance and male physiological attributes

Visual cues, such as courtship dances, have not been recorded for B. tryoni, although it is unclear in the literature if this is because they are entirely absent, or just unstudied. Male and female physiological attributes may, however, affect their copulation success. Males fed on yeast hydrolysate have an enhanced attraction to cue lure (Weldon et al., 2008), which will lead to greater mating success, while protein feeding also enhances male mating success due to general increased male sexual performances (Perez-Staples et al., 2007, Prabhu et al., 2008, Kumaran et al.,

2013). There is no significant effect of males’ premating experience on their later mating success, but young males are more successful than older males (Fay and

Meats, 1983). This latter study was carried out as a no-choice test under small-cage laboratory conditions and a different result might be obtained if it was done as a choice test under more natural conditions. Given this potential shortcoming, a more detailed study of male physiological attributes on mating success is carried out in this thesis.

Based on other tephritids, non-volatile chemicals (e.g., cuticular hydrocarbons) may be used for sex or partner recognition when flies are in close contact. But there are no studies done on the influence of CHCs on B. tryoni mating.

Chapter 1: General Introduction 35

1.8 Thesis Outline

The literature reviewed in this chapter highlights the potential complexity of mating systems and mating behaviour in tephritids. An understanding of courtship behaviour in pest tephritids is fundamental to their sustainable management, particularly with respect to the Sterile Insect Technique which operates through mating interference (Shelly and McInnis, 2016). Despite being well researched in some ways, much of the mating system and courtship of B. tryoni remains unknown

(Figure 1.4) and this potentially impacts on longer term control for this insect. Given this, the overall aim of this thesis is to characterize the mating system of B. tryoni and to categorise the sequence of its courtship behaviours. Following this introductory chapter, there are four research chapters and a final general discussion chapter.

Figure 1.4: Long and short range cues for mating site and mate selection in

Bactrocera tryoni. (Question mark indicates the areas with fewer or no studies done)

Chapter 2 uses a large field cage study to focus on questions of how B. tryoni identifies a potential mating site (e.g., plant height, direction of sunlight or the presence of fruit), specifically asking whether B. tryoni exhibits behaviour consistent

3686 Chapter 1: General Introduction

with land-marking, or uses host plants based on the presence or absence of fruit.

Assessing the timing of arrival of the sexes to the mating site also tests a long-held assumption that B. tryoni has a lek-based mating system. If lek-based, I predict males should arrive first at an aggregated mating area in order to establish territories prior to female arrival.

After finding how B. tryoni locate a suitable mating site, better resolution of their courtship sequence is the objective of Chapter 3. The precise sequence of fine- scale courtship interactions, and their timing, is yet to be fully resolved for B. tryoni; in particular, the exact cues that elicit acceptance or rejection by either male or female. Therefore the third chapter includes the cues and behaviours that they use to identify their potential mating partner. It further describes the exact sequence of short range courtship behaviours.

Numerous signals are important in communication between mating partners.

The quality of these signals emitted by the signaller may become the reason to accept or reject the signaller by the receiver. Elements of these signals may be influenced by factors such as size, age, or previous sexual experience, and the objective of Chapter

4 is to determine if any of these three variables affects B. tryoni male mating success.

This chapter assesses mating competitiveness of: i) old males versus young males; ii) small males versus large males; and iii) virgin males versus mated males. Further, close range behavioural observations on mate choice tests are carried out to determine if there are any differences in the courtship behaviours identified in

Chapter 3 based on different male physiological attributes and/or female preference.

Having identified the fine-scale courtship behaviour of B. tryoni Chapters 3 and 4, in Chapter 5 I probe more deeply into an individual component of the mating system. In contrast to male calls and volatile pheromones, which have been well

Chapter 1: General Introduction 37

studied in B. tryoni and other Bactrocera species, a possible role for non-volatile chemical communication in Bactrocera sexual communication has been largely ignored. Thus in Chapter 5 I investigate the cuticular compounds of B. tryoni to determine if they differ between males and females, and also if they differ between mated and unmated males and females when there were two males and single female in a mate choice arena. A role for cuticular compounds in sexual selection in any

Tephritidae has not been previously investigated, but as reviewed earlier it is known to occur in the drosophilid fruit flies.

The final chapter (Chapter 6) is a general discussion of the findings of other chapters. It highlights the overall key findings and a comprehensive description on how B. tryoni mating systems moves from finding a suitable mating location to identifying a potential mating partner. It then provides a discussion of what these results mean for better understanding mating in Bactrocera fruit flies and more effective pest control methods for these flies.

3886 Chapter 1: General Introduction

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

This chapter has been published as

Ekanayake W.M.T.D., Jayasundara M.S.H., Peek T., Clarke A.R. & Schutze M.K. (2016). The mating system of the true fruit fly Bactrocera tryoni and its sister species, Bactrocera neohumeralis. Insect Science in press. DOI 10.1111/1744- 7917.12337.

It has been reformatted here for continuity of thesis style, but is otherwise unchanged. Jayasundara and Peek provided technical assistance in the experiments, while Clarke and Schutze assisted with experimental design, analysis and writing, at a level commensurate with normal postgraduate supervision.

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 39

2.1 Introduction

Successful mating in most land animals must be preceded by males and

females first finding each other in the landscape (Diniz- Filho et al., 2007; Lodé,

2011). In resource-based mating systems, the mating site provides more resources

than simple access to sexual partners; for example, the sites may provide food,

shelter or oviposition sites, especially for females. For example, in the antlered fruit

flies (Phytalmia spp), males guard highly limited rot sites under the bark of fallen

trees, which are the necessary substrate for larval development. Females arrive after

the males at these sites, are mated by the awaiting males, and then gain access to the

oviposition resource (Dodson, 1997). Male Phytalmia that gather in such sites

compete amongst themselves to protect the available resources, so maintaining high

quality territories and increasing their chances of mating, a common male

behavioural pattern in resource-based mating systems (Scott, 2009; Summers &

Tumulty, 2013). In contrast, non-resource based mating systems offer no rewards

other than mate location. The best known non-resource based mating systems are

leks: aggregations of males that females visit only for the purpose of mating (Baker,

1983; Arita & Kaneshiro, 1985; Hendrichs et al., 1991; Hoglund & Alatalo, 1995).

Lekking species share the following characteristics: (i) no male parental

contribution to offspring other than sperm; (ii) males aggregate in a specific site and

display; (iii) females gain no material benefits by visiting the lek except for access to

a male; and (iv) females are highly selective of a mate (Jiguet et al., 2000). Males

tend to form leks at locations where large number of females will be present, because

it will increase their chance of being selected by a female (Bradbury & Gibson, 1983,

Sivinski, 1989, Shelly & Whittier, 1997). Importantly, lekking is different from

swarming behaviour, with which it may be confused, as lekking is only for the

4086 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

purpose of mating while swarming may involve aggregation for protection, migration, or increasing the size of a colony (Parrish & Edelstein-Keshet, 1999).

Although first described for birds, leks have also been recorded in insects, fish, amphibians, and mammals (Hoglund & Alatalo, 1995).

Among insects, fruit infesting species of the family Tephritidae are one group with mating systems considered to be closest to ‘classical’ leks (Shelly & Whittier,

1997). It is considered that most tephritids, including major pests such as C. capitata, Z. cucurbitae and B. dorsalis have lek or ‘lek like’ mating systems (Arita &

Kaneshiro, 1985, Sivinski, 1989, Shelly & Kaneshiro, 1991; Kaneshiro et al., 1993;

Shelly & Whittier 1997; Benelli et al., 2014b). Generally, males of these tephritids aggregate and defend territories while performing visual, chemical and acoustic courtship behaviours to attract females for the purpose of mating (Arita & Kaneshiro,

1989).

Despite a belief that “Tephritidae are generally lekking species” (Benelli et al.,

2014b), the literature is not unanimous about this point. Mating site location in frugivorous tephritids is often determined by the presence of additional resources that females require, most commonly the presence of fruit (Prokopy, 1980; Burk, 1981;

Drew, 1987; Fletcher, 1987; Messina & Subler, 1995). Other plant characteristics, such as the location of a plant in the landscape, its height, silhouette, or canopy density, may also play a role in attracting fruit flies to a mate rendezvous site (Stark

1995; Kaspi & Yuval, 1999).

The Australian tephritids, B. tryoni and B. neohumeralis are very closely related sibling species pair (Clarke et al., 2011). The species are thought to only differ in their mating behaviour by mating time: B. tryoni mates during a very short window at dusk, while B. neohumeralis mates over a period of several hours either

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 41

side of midday (Pike & Meats, 2002). The pair have been used extensively over

several decades as a focus for comparative mating studies (Birch & Vogt, 1969; Pike

& Meats, 2002; Gilchrist & Ling, 2006), a situation which is likely to continue now

that the draft genomes of both species are available (Gilchrist et al., 2014).

Nevertheless, despite the extremely detailed laboratory studies of some aspects of

their courtship and mating (e.g., Radhakrishnan & Taylor, 2007; Mankin et al., 2008;

Radhakrishnan et al., 2009; Kumaran et al., 2013, 2014a, b), the basic mating system

of both species is yet to be resolved. For B. tryoni, published observations report

swarming (Bateman, 1972), ‘settled swarms’ (Tychsen, 1977), and resource-based

mating (Drew & Lloyd, 1987); while yet others have automatically assumed that it is

a lekking species without testing (Fletcher, 1987, Weldon, 2007). There remains no

published information on the mating system of B. neohumeralis, although Pike and

Meats (2002) very briefly discuss that its mating system may be different to B.

tryoni, given the former species mates over a period of several hours in the middle of

the day, while the latter species over a few minutes at dusk.

Given the need to increase basic biological knowledge of these flies for pest

management (Clarke et al., 2011), especially for a new research thrust into the Sterile

Insect Technique (Horticulture Innovation Australia, 2015), and the likely renewed

interest in the sibling pair following the publication of their genomes (Gilchrist et al.,

2014), the aim of this study was to better determine the mating systems of these two

species; particularly in the context of lekking behaviour. We conducted two

observational experiments (repeated for both species) in large flight cages that

allowed us to determine if B. tryoni and B. neohumeralis: (i) have resource or non-

resource based mating systems (i.e., mate on plants with or without fruit); (ii) choose

between mating sites based on plant architectural traits (tall versus short trees) or

4286 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

position with respect to sunlight (light versus shade); (iii) are aggregated or non- aggregated at mating; and (iv) if aggregated, do males arrive first and ‘call’ females

(as predicted for a ‘lekking’ species). Our results not only increase our knowledge of these two species, important both as pests and as model systems in evolutionary studies, but also allow us to make more detailed statements about mating systems in other Bactrocera species.

2.2 Material and methods

Source of insect material

Bactrocera tryoni and B. neohumeralis pupae were obtained from laboratory cultures (F20- F30 generations) maintained by the Queensland Government

Department of Agriculture and Fisheries based in Brisbane, Australia. Emergent adults of both species were separated by sex within four days of emergence (i.e., before sexual maturity) and held at densities of 125 individuals per cage (30 x 20 x

20 cm) under laboratory conditions of 27 ºC and 70% relative humidity. Flies were supplied sugar, water and protein hydrolysate ad libitum. Flies were held until they reached sexual maturity, from which 12-14 day-old virgin males and females were used in experiments.

Study site

Work was undertaken in large field cages (7m x 7m x 4.8 m) at the Queensland

University of Technology Samford Ecological Research Facility located in subtropical, South-east Queensland (27º 23’ 22”S, 152º 52’ 37”E). The field cages were constructed from a metal frame covered with a very fine, white screening material. Collins et al., (2008) suggest white mesh as the preferred colour for field cage mating studies of B. tryoni, because in trials comparing field cage mesh colours

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 43

they recorded the highest proportions of B. tryoni matings on trees compared to the

cage wall in unshaded white cages.

Experiments

The design and implementation of experiments was near identical for B. tryoni

and B. neohumeralis. Any differences between experiments for each species (e.g.,

replicate number) are noted. Bactrocera tryoni and B. neohumeralis were tested

separately for every experiment: never were both species released simultaneously in

a replicate.

2.2.1 Experiment 1: Mating site preference and tree height The first experiment compared mating site preference with respect to tree

height (tall versus short) and position of sun (light versus shade). Four artificial

‘apple trees’ were placed in a field cage for each replicate; trees were 2 m tall with a

2 m canopy diameter, with polyester leaves, plastic fruit and plastic branches built

upon a natural timber stem (http://www.artificialplantimporters.com.au/

Product/5940/apple-tree). Artificial trees were used to avoid the potentially

confounding effect of green leaf volatiles affecting fly behaviour (Dalby-Ball &

Meats, 2000), and there was a much higher degree of architectural uniformity

between artificial trees than could be achieved using real plants. Artificial trees of

this type have been used previously in fruit fly behavioural studies (Dalby-Ball &

Meats, 2000; Raghu & Clarke, 2003; Balagawi et al., 2012). Two of the four trees

used in each replicate were increased in height by placing them on 0.7 m-high stands

(hereafter ‘tall trees’), such that the two tall trees were 2.7 m in height and two ‘short

trees’ were 2 m in height. The distance between a tree and the cage wall was 2 m,

and the distance between trees was 3 m.

4486 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

One-hundred and twenty-five sexually mature virgin males and 125 sexually mature virgin females were released at the centre of the field cage at the beginning of each replicate trial. Each trial for B. tryoni commenced between 17:00-17:15 (prior to dusk); observations were taken at 5 min intervals until after sunset and once all new pair formation had ceased. The same observations were taken for B. neohumeralis, but at 30 min intervals from 09:30 – 11:30 and from 13:30 – 15:00, and at 5 min intervals from 11:30 – 13:30. The split in time between observations

(i.e., 5 min or 30 min) was in a response to the logistic constraints of recording over the much longer mating time in this species, but with an expectation that most matings would occur either side of midday (Meats et al., 2003).

The following observations were recorded for both the upper and lower halves of each artificial tree canopy: (i) the number of single male flies, (ii) number of single female flies, (iii) number of mating pairs; (iv) number of male/male fights

(face-to-face attacks and body pushing) and (v) number of males wing fanning (rapid wing movements to disperse the pheromone and to produce a courtship song

(Tychsen 1977)). Repeated counts within a canopy are not independent of each other, and we recognise this in our analysis (see further below).

Seven replicates were completed between February and March (2014) for B. tryoni, and eight replicates for B. neohumeralis on May and October (2015). The position of tall and short trees was rotated within the cage between replicates to account for positional effect

2.2.2 Experiment 2: Mating site preference with respect to fruit presence

The objective of this trial was to explicitly test if B. tryoni or B. neohumeralis preferentially mate on trees with fruit, thereby supporting a resource-based mating

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 45

system hypothesis. This experiment followed the same general procedure as for

Experiment 1, except for the following difference: all four trees in each replicate

were the same height (2 m), but two of the four trees each had 10 organic ‘Royal

Gala’ apple fruit attached to the canopy branches, while the other two trees did not

have any fruit. Observations were the same as for Experiment 1; however, female

oviposition was also recorded in this trial. Eight replicates were completed for both

B. tryoni and B. neohumeralis.

2.2.3 Data analysis

The following procedures for data analyses and visualisation were undertaken

because of non-independence between counts within a tree: (i) mean data is plotted

for visualising changing patterns over time (e.g., number of mating pairs over time),

but no formal analysis is undertaken; (ii) only a single count per tree is used where

comparisons of means are formally analysed (e.g., mean number of flies in tall trees

versus short trees), and this count is always the highest count. For example, if six 5

min sequential counts were made of mating pairs in a tree and these were 2, 3, 2, 7, 5

and 6 pairs, then 7 would be the number used for that specific tree in the analysis.

Using the highest count per tree (as explained above), all raw data was

converted to proportional data prior to analysis (e.g., the number of mating couples

observed on a specific tree as a proportion of all couples observed on all trees in the

replicate). Proportional data was arcsine transformed prior to assessment for

significant differences in the mean numbers of individual flies and mating couplies in

short and tall trees, or trees with and without fruit. The conversion of raw data to

proportion data was necessary due to large variation in the total numbers of flies

responding between replicates caused by variable climatic conditions across

treatment days (e.g., differences in temperature or cloud cover). Data were first

4686 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

evaluated for normality using Kolmogorov-Smirnov tests and for equality of variances using a variance ratio test (F) prior to paired t-tests to test for significant differences in fly abundance for each experiment (tree height and fruit availability).

Wilcoxon rank-sum tests were conducted instead of paired t-tests when the data sets were not normally distributed. Analyses were undertaken in the statistical package

Minitab 16.0.

Because sunset is considered critical for the onset of mating in B. tryoni (Pike

& Meats 2002), and sunset time varied between replicates for our experiments, we needed to correct for that variation in analysis and graphical presentation. Therefore, the observation periods from the beginning to the end of the B. tryoni trials were standardised into minutes before and after official sunset. The official time of sunset for each trial day was obtained from the Australian Bureau of Meteorology.

All replicates for Experiments 1 and 2, were combined for each species (i.e., n

=15 and n = 16 for B. tryoni and B. neohumeralis, respectively) to test if mating pairs were aggregated. The mean and variance of the number of mating couples across trees was calculated for each replicate and to this data Taylor’s Power Law was applied: a standard technique for identifying spatial patchiness (Taylor 1961, 1984).

This technique fits a linear regression to modified mean and variance data for each replicate, such that log variance = log a + b log mean. The value ‘a’ is a scaling factor dependent on sample size and occasion, while ‘b’ is an index of aggregation that varies continuously from b < 1 for uniform distributions, through b = 1 for random distributions, to b > 1 for aggregated distributions. The value ‘b’ is generally considered to be constant for a species (Taylor 1984); hence our ability to combine replicates across experiments. All mean and variance data were (n + 1) transformed prior to analysis.

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 47

2.3 Results

2.3.1 Experiment 1: Mating site preference and tree height

Tall versus short trees

For both B. tryoni and B. neohumeralis, significantly more individual males

(B.t., t = 4.6, df= 6, P = 0.002; B.n., t = 6.36, df = 7, P < 0.01), females (B.t., t =

4.24, df = 6, P = 0.004; B.n., t = 6.38, df = 7, P < 0.01) and mating pairs (B.t., t =

5.20, df = 6, P = 0.001; B.n., W (7) = 95, P = 0.0045) were recorded in tall trees over

short trees (Figure 2.1a, b). The height effect was most dramatic for B. tryoni mating

pairs, for which of 84 couples observed across all trees only one was in short trees;

consequently, all subsequent results for B. tryoni mating, report numbers of flies

from tall trees only.

4886 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

18 (a) 16 * * **

10 (b) * * *

Number of individual flies or mating pairs mating or flies individual of Number 6

Males tall trees Pairs tall treesPairs short trees Males short treesFemales tallFemales trees short trees

Figure 2.1: Mean (± SE) maximum number of (a) Bactrocera tryoni or (b)

Bactrocera neohumeralis individual males, females and mating pairs in two tall or

two short trees. (n = 7 for Bactrocera tryoni, n = 8 for Bactrocera neohumeralis; *

indicates significant difference at P < 0.01 and ** indicates significant difference at

P < 0.001 for the pair-wise ‘short-tall’ comparison.)

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 49

12 (a) * * * 10

7 (b) * * 6

Number of individual flies or mating pairs mating or flies individual of Number

Males sun Pairs sun Males shadeFemales sun Pairs shade Females shade

Figure 2.2: Mean (± SE) maximum number of (a) Bactrocera tryoni or (b)

Bactrocera neohumeralis individual males, females and mating pairs in direct

sunlight vs shade in tall trees. (n = 7 for Bactrocera tryoni, n = 8 for Bactrocera

5086 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

neohumeralis; * indicates significant difference at P < 0.01for the pair-wise ‘sun-

shade’ comparison).

There was no significant difference in abundance of individual B. tryoni males (t = 1.16, df = 6, P = 0.159), females (t = 2.05, df = 6, P = 0.086), or mating couples (t = 1.35, df = 6, P = 0.224) in the ‘westerly’ tall tree (i.e., closer to sunset) over the ‘easterly’ tall tree. This comparison was not undertaken for B. neohumeralis as it was not biologically relevant to distinguish a ‘westerly’ and an ‘easterly’ tree with the movement of the midday sun.

There were significantly more individual B. tryoni males (t = 5.22, df = 6, P <

0.01), females (t = 4.74, df = 6, P < 0.01), and couples (W (6) = 77, P = 0.0022) on the side of the canopy facing sunset over the side in shade (i.e., opposite to sunset)

(Figure 2.2a). Bactrocera neohumeralis males were also observed significantly more often in sunny parts of the canopy over shady parts (t = 6.87, df = 7, P < 0.01), as were mating pairs (W (7) = 100, P = 0.0009). In contrast, B. neohumeralis female abundance was not significantly associated with direct sunlight or shade (W (7) =

0.08, P = 0.942) (Figure 2.2b).

There was no significant difference in the abundance of B. tryoni males (t =

1.00, df = 6, P = 0.356) and mating pairs (t = 1.79, df = 6, P = 0.124) at the top of a tall tree canopy relative to the bottom of the canopy; but, females occurred in significantly larger numbers in the top of the canopy (t = 3.83, df = 6, P < 0.001)

(Figure 2.3a). In contrast, individual B. neohumeralis males (t = 17.81, df = 7, P <

0.01), females (W (7) = 100, P = 0.0009) and mating pairs (W (7) = 95, P = 0.0054) were all observed significantly more often in the upper half of tall tree canopies relative to the lower half (Figure 2.3b).

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 51

10 (a) ** 8

10 (b) * ** *

Number of individual flies or mating pairs mating or flies individual of Number 8

Males top Pairs top Females top Pairs bottom Males bottom Females bottom

Figure 2.3: Mean (± SE) maximum number of (a) Bactrocera tryoni or (b)

Bactrocera neohumeralis individual males, females and mating pairs in upper or

lower half of tall tree canopies. (n = 7 for Bactrocera tryoni, n = 8 for Bactrocera

5286 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

neohumeralis; * indicates significant difference at P < 0.01 and ** indicates

significant difference at P < 0.001 for the pair-wise ‘upper/lower’ comparison.)

Behaviour

For B. tryoni, flies were present in the trees before dusk in relatively low numbers, with slightly more females than males. However, no mating pairs were observed before the time of official sunset, nor were any male wing fanning or male- male aggression observed. However, within five minutes of official sunset, male wing fanning, male-male combat and pairing had begun. Most dramatically, immediately after sunset, large numbers of additional male and female flies began arriving at tall trees, from elsewhere in the field cage, and females arriving at the tree paired very rapidly (Figure 2.4a). Male wing fanning peaked at 10 minutes after sunset and continued throughout the mating period: in contrast male-male contests were very rare (Figure 2.4b). Nearly all females in a tree were paired by 45 minutes after official sunset, although by an hour after sunset there were still large numbers of unpaired males (Figure 2.4a).

In contrast, no clear temporal pattern in B. neohumeralis host plant arrival or mating was observed, and mating overall was observed much less frequently. Males and females were both continuously present in trees in relatively low numbers through the observation period, with slightly more males present than females.

Copulations were rare and distributed over an almost three hour period (Figure 2.5).

Wing fanning males were observed in two instances and male-male aggressive behaviour was rarely observed for this species.

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 53

18 (a) 16 Individual females Individual males 14 Mating pairs

0 5

-5

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

-70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 12 (b)

10 Wing fanning males Non-wing fanning males Male-male combat 8

Mean number of individual flies or pairs flies of individual number Mean

0 5

-5

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

-65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 Minutes before and after sunset

Figure 2.4: Mean (± SE) number of (a) individual Bactrocera tryoni males (all

behaviours summed), females and mating pairs in tall trees at five minute intervals

before and after official sunset (n = 7); and (b) individual wing-fanning (i.e.,

‘calling’) males, non-wing fanning-males, and fighting males.

5486 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

8 Individual males Individual females Mating pairs

Number of individual flies or pairs or flies individual of Number

0 10:00 11:00 12:00 13:00 14:00 15:00

Time

Figure 2.5: Mean (± SE) number of individual Bactrocera neohumeralis males,

females and mating pairs in tall trees from 9.30am to 3.00pm. The vertical line

represents the average time of solar noon for the experimental period. (n = 8.)

2.3.2 Experiment 2: Mating site preference with respect to fruit presence

There was no significant difference in the abundance of individual B. tryoni males (t = -1.61, df = 7, P = 0.151), females (t = 1.80, df = 7, P = 0.115) and mating pairs (t = -0.88, P = 0.408) in trees with fruit versus trees without fruit (Figure 2.6a).

In B. neohumeralis, there was no significant difference of individual males (t = 0.61, df = 7, P = 0.564) and mating pairs (W (7) = 71.5, P = 0.75) in trees with fruit and without fruit. Individual B. neohumeralis females occurred in significantly higher numbers in trees with fruit (t = 4.55, df = 7, P = 0.003) (Figure 2.6b), but most females in the trees with fruit were ovipositing. Bactrocera tryoni females also used

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 55

the fruit for oviposition (Figure 2.7), clearly demonstrating that the experimental fruit

could be considered as a resource for both species.

The temporal pattern of mating in B. tryoni in Experiment 2 did not differ from

that described for Experiment 1; hence, it is not redescribed as part of this

experiment.

2.3.3 Experiments 1 & 2 combined: Mating aggregation

Taylor’s Power Law analysis on the combined mating data showed that B.

tryoni mating behaviour was highly aggregated with a ‘b’ value of 1.8 (Figure 2.8).

Power Law analysis was not carried out for B. neohumeralis, as the low numbers of

mating pairs observed in individual replicates (from 0 to 4) made attempts to

quantify mating pair aggregation biologically meaningless.

5686 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

18 (a)

0 10 (b) *

Number of individual flies or mating pairs mating or flies individual of Number

Males no fruit Pairs with fruitPairs no fruit Males with fruit Females with fruitFemales no fruit

Figure 2.6: Mean (± SE) number of (a) Bactrocera tryoni or (b) Bactrocera

neohumeralis individual males, females and mating pairs in trees with or without

fruit. (n = 8 for both species; * indicates significant difference at P < 0.01 for the

pair-wise ‘with fruit/without fruit’ comparison.)

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 57

12 Females on leaves Females ovipositing 10 Females on fruit not ovipositing

Number of flies of Number 4

0 5

-5

10 15 20 25 30 35 40 45 50

-65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 Minutes before and after sunset

Figure 2.7: Mean (± SE) number of Bactrocera tryoni females on leaves, females

ovipositing and females on fruit not ovipositing in fruited trees. (n = 8). The vertical

line represents the official time of sunset for the experimental period.

5886 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

2.6 log (variance + 1) = log (0.127 + 1) + 1.86 log (mean + 1) 2.4 r2 = 0.696, p< 0.001 2.2

2.0

1.8

1.6

1.4

log (varaince +1) (varaince log 1.2

1.0

0.8

0.6 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

log (mean+1)

Figure 2.8: Mean/variance plot for the distribution of mating pairs of Bactrocera

tryoni on four artificial trees within a large flight cage.

2.4 Discussion

In this study I have formally quantified the mating systems of B. tryoni and B. neohumeralis under manipulated semi-field conditions. Numbers of mating B. neohumeralis couples was low across both experiments (only 30 pairs observed in total), and so we are conservative in our comments about that species; however, nearly 250 mating pairs were observed for B. tryoni; thus, we have much greater confidence in our inference. Consequently, we consider B. tryoni exhibits a non- resource-based, but likely landmark-based, aggregated mating system. The system has both lek and non-lek-like attributes, and further research on female choice is needed. The discussion focuses on the key elements of the mating system identified, and concludes with a discussion on leks.

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 59

2.4.1 Aim for the top: evidence in support of landmarking

Bactrocera tryoni mated almost entirely in tall trees over short, and a weaker

but still significant pattern was shown for B. neohumeralis (Figure 2.1). Preferential

selection of tall trees as mate rendezvous sites has been observed for other tephritids:

the apple-maggot fly, R. pomonella, for example, first uses an apple tree silhouette,

at a distance, in its recognition of a suitable host plant (Moericke et al., 1975) before

honing in on ripe fruit chemical volatiles as they fly closer (Forbes & Feder, 2006).

Wild-tobacco fly, B. cacuminata, preferentially mates on tall S. mauritianum plants

over short plants (Raghu et al., 2004). Thus, B. tryoni may use emergent canopy trees

in their natural rainforest environment (Drew & Romig 2000) as an initial land-

marking site to bring the sexes together; however, this remains speculative until

demonstrated in the field with wild flies. Nevertheless, flies using landmarks (such

as tall trees) as mate rendezvous sites in the environment has been proposed an

important feature bringing together male and female fruit flies, particularly as mating

is rarely seen in the field (so presumably is occurring at sites not easily seen by

human observers) and ‘landmarking’ occurs in many insect species (Fletcher &

Prokopy, 1991, Raghu & Clarke, 2003, Raghu et al., 2004).

2.4.2 Non-resource based mating system There has been direct conflict in the literature on whether Bactrocera species

have a resource, or non-resource, based mating system (for supportive papers of

resource-based mating systems see Drew & Lloyd, 1987; Drew et al., 2008; for non-

supportive papers see Raghu et al., 2002; Ero et al., 2011). We found no evidence

for a resource-based mating system in our direct test which simultaneously offered

‘fruiting’ and ‘non-fruiting’ plants. However, we also found no evidence that the

presence of fruit was unfavourable to the formation of mating aggregations. Thus

6086 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

field observations that record mating behaviour on fruiting plants are equally as likely as observations which do not; but where mating is recorded on fruiting hosts it should not be assumed that fruit presence is the reason for the mating aggregation.

2.4.3 The role of males and females with aggregation

The data clearly shows that B. tryoni mating couples aggregate; a result that supports earlier work by Tychsen (1977). Not only were couples aggregated at the level of the tree (Figure 2.8), but they also occurred to one side of the canopy (i.e., the side facing sunset) (Figure 2.2). Mating aggregations have been reported in other frugivorous tephritids (as reviewed by Shelly & Whittier, 1997); and a preference for light over dark areas by B. tryoni at dusk has also been reported (Collins et al.,

2008). However, in contrast to reports by Tychsen (1977), we saw no sign of male

‘swarms’.

While the literature suggests Bactrocera species aggregate to mate, what has not been previously reported is the temporal pattern of aggregation formation. It is assumed under a lek-based mating system that males arrive first at the mating site and wait for females to come to them (Shelly & Whittier, 1997). This has been the basis for previous experimental studies examining female attraction to caged males

(i.e., artificial leks) for B. dorsalis (Shelly, 2001) and B. tryoni (Weldon, 2007).

Interestingly, there was no correlation between increasing male aggregation size and increasing female visitation in both studies (n.b., Shelly did find such a relationship for C. capitata). Male-led aggregations are also at odds with reports from some field observations, in that females have been found to arrive first at a subsequent (non- host) mating site (Stark, 1995).

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 61

Field cage work with B. tryoni contradicts the ideas of both male or female-led

aggregations, and show that both sexes play a role in aggregation. Prior to dusk,

small numbers of both males and females (slightly more females than males)

occurred at the mating site. Male wing-fanning began at dusk and both male and

female flies arrived at the site in much larger numbers after dusk. By the time pair

formation had ceased, there were nearly no unpaired females remaining (Figure

2.4a), but still significant numbers of unpaired males in the mating arena. It may be

possible, therefore, that male wing-fanning caused the aggregation after sunset (see

further below), but there is no evidence that males alone selected the site prior to

sunset.

In contrast to B. tryoni, we saw no evidence of aggregation behaviour in

midday-mating B. neohumeralis. As observed in earlier studies (Pike & Meats

2002), B. neohumeralis mating behaviour occurred over several hours either side of

noon and the total number of calling and mating B. neohumeralis was very low given

the total population in the field cages. It may be that mating occurs infrequently in

this species, or that cage arenas do not provide suitable environments to promote

mating. Any further interpretations of the B. neohumeralis mating system must,

therefore, be considered speculative until increased mating couple numbers are

obtained.

2.4.4 To lek or not to lek - that is the question

Modern definitions of ‘lek mating systems’ have become very broad (see, for

example, Kotrschal & Taborsky, 2010; Wegge et al., 2013; Turner, 2015). In their

seminal review of lek behaviour in insects, Shelly & Whittier (1997) admit “these

discrepancies [in lek definitions]… render any operational definition imprecise and

idiosyncratic.” Nevertheless, there are some requirements we consider necessary if

6286 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

the “spirit” (sensu Shelly & Whittier, 1997) of lek behaviour is to be satisfied. We consider these as listed:

1. Is there evidence of male parental care? (‘no’ in lek-based systems)

2. Is the distribution of males spatially clumped (i.e., non-random distribution)? (‘yes’ in lek-based systems)

3. Do males control resources of value to females (such as oviposition sites or food resources)? (‘no’ in lek-based systems)

4. Do females exhibit a clear choice in selection of mates? (‘yes’ in lek- based systems)

A fifth requirement is inferred in points 2 to 4 above, although it is not explicitly stated, and that is that females arrive at the lekking site after the males have established territories; this assumption is made in many Bactrocera mating studies (Dong et al., 2014, Haq et al., 2014).

We can now answer some of these questions for B. tryoni with a high degree of confidence. For point 1, as for the great majority of insects, there is no evidence of paternal care. For point 2, males (at time of mating) are spatially aggregated. For point three, males are not obviously controlling resources needed by females; mating occurred equally on artificial trees with or without fruit (= an ovipositional resources used by females). All of these points support a lekking system hypothesis. Contrary to expectations under a lekking system, however, is the demonstration that males do not arrive at the mating site noticeably earlier to females; but, there are more males than females at the mating site during time of mating (i.e., dusk), and many non- mating males are calling (i.e., wing-fanning). Also, males do not appear to

Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis 63

aggressively defend territories; behaviour considered contrary to expectations for

traditional lek behaviour (Shelly & Whittier 1996).

Given these points for and against lekking, this leaves point 4 (i.e., active

female selection of males) as the ‘decider’ to determine if B. tryoni should be

regarded as having a lek-based mating system, or a non-resource (probably

landmark-based) aggregated mating system. Sexual selection is known to occur in B.

tryoni (Kumaran et al., 2013); however, it is unclear if this is due to differential male

fitness, female choice, or a combination of the two. A clear role for male fitness has

been demonstrated (Kumaran et al., 2014b), but female choice is yet to be

conclusively demonstrated. Although female choice in mate selection must,

therefore, be conclusively demonstrated to resolve whether B. tryoni should be

considered a classical lekking species, our experimental results are currently

sufficient to describe it as a non-resource based aggregation system, which may well

use landmarking in the field.

6486 Chapter 2: The mating system of the true fruit fly Bactrocera tryoni and its sister-species, Bactrocera neohumeralis

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 65

3.1 Introduction

The initial requirement of a successful copulation is to locate a mating site within the landscape. Based on the findings of Chapter 2, Bactrocera tryoni males and females may use landmarks in the early stage of selecting a mating site. That is, they prefer to mate at the top of tall trees relative to shorter trees. Further, under experimental conditions flies showed no preference for trees with fruit at the mating site, supporting a non-resource based mating hypothesis. Having developed an understanding of how B. tryoni individuals may locate their mating site within the landscape, in this chapter I describe the close-range courtship sequence by which male and female B. tryoni may recognise each other as potential mating partners.

Courtship is the process of exchanging signals and responses between potential mates, where both sender and receiver may use visual, chemical, acoustic and/or tactile signals to attract the opposite sex and indicate their own readiness to mate.

Existing literature describes how B. tryoni uses sex pheromones for mate selection

(see section 1.7.2.3) and, though there is no unequivocal evidence for the use of auditory signals in mate selection, difference in B. tryoni wing-beat frequencies have nevertheless been found between successful and unsuccessful males (Mankin et al.,

2008). However, it is unclear whether B. tryoni use any specific behavioural displays in order to select a mating partner. What is known about courtship for tephritids has been previously described in Chapter 1 in section 1.6, and in section 1.7.2.3 for B. tryoni. To summarise for B. tryoni, there remain a lack of experimental recordings on

B. tryoni specific courtship behaviours and their courtship sequence. In the introduction of this chapter, I explain the importance of documenting the courtship sequence of B. tryoni males and females.

8666 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Synchronisation of positive behaviours is crucial between the signal sender and receiver to achieve successful copulation (Paterson, 1985, Benelli et al., 2012).

Courtship sequences have been examined in a small number of frugivorous tephritids, these being Zeugodacus cucurbitae (Kuba and Koyama, 1985), B. oleae

(Benelli et al., 2012), Ceratitis capitata (Arita and Kaneshiro, 1989, Whittier et al.,

1992), Anastrepha ludens (Robacker and Hart, 1985), Rhagoletis indifferens Curran

(AliNiazee, 1974, Messina and Subler, 1995), R. pomonella (Biggs, 1972), R. completa and R. suavis (Loew) (Boyce, 1934); with little on B. tryoni (Myers, 1952).

During dusk, Z. cucurbitae males occupy a leaf and form leks on the bottom surface of leaves where they initiate wing fanning and release sex pheromones. This behaviour attracts females and selected males will be approached from the front followed by a male copulation attempt (Kuba and Koyama, 1985). This same behavioural sequence has been observed in A. ludens, B. oleae, B. tryoni (references provided above), and C. capitata (Whittier et al., 1992), except that C. capitata males initiate courtship during the middle of the day (more details in section 1.6

Chapter 1). However, although these studies have explained the most prominent, common courtship behavioural sequences, studies on more detailed tephritid courtship behaviours are limited.

The sequence of courtship behaviours are commonly presented as ethograms, visual diagrams that connect both sender’s and receiver’s signals to obtain a continuous flow of behaviours. Courtship ethograms have been presented for several dipterans (e.g., the sand fly Lutzomyia longipalpis (Lutz and Neiva) (Bray and

Hamilton, 2007)), but in only a few tephritids: B. oleae (Benelli et al., 2012) (see section 1.6.1), a Californian non-frugivorous tephritid Trupanea vicina (Zunic Kosi et al., 2013) (Figure 3.1a) and the Blueberry maggot fly Rhagoletis mendax Curran

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 67

(Smith and Prokopy, 1982, Benelli et al., 2014b) (Figure 3.1b). No ethograms exist for B. tryoni in the published literature.

In all described tephritid courtship ethograms (B. oleae (Figure 1.2 in Chapter

1), T. vicina and R. mendax (Figure 3.1a, b)), both males and females have shown resting, walking or grooming behaviours before leading to the phase of mate attraction. As seen in these species, the mate attraction phase mainly includes male wing vibrations, additional aristae (a rod-like appendage arising from the third antennal segment in dipterans) tapping (Miranda, 2000), and head rocking (Briceño et al., 1996) which will attract the female towards them (but a clear mate attraction phase is absent in R. mendax (Figure 3.1b)).

(a)

8668 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

(b)

Figure 3.1: Ethogram for (a) Trupanea vicina courtship behaviour (n = 10) (Zunic

Kosi et al., 2013), (b) Rhagoletis mendax courtship behaviour (n = 17) (Benelli et al.,

2014b)

If the female is receptive, which is indicated by remaining still near a wing fanning male as seen in T. vicina, the male will attempt copulation by mounting the female (Zunic Kosi et al., 2013). In R. mendax, the success of male mounting attempts will also depend on female movements, where mounting onto a stationary female (e.g., ovipositing female) may lead to a successful copulation (Smith and

Prokopy, 1982). Not all mounting attempts will lead to a successful copulation; in some instances the female is unreceptive and walks away, with the courtship sequence ceasing (Figure 3.1).

Since these types of courtship sequences are undocumented for B. tryoni, the objective of this chapter was to resolve their courtship sequence and to construct an ethogram. For that, I have taken close range video recordings of B.

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 69

tryoni courtship during dusk by keeping a virgin male and a virgin female inside a

Petri-dish. These videos were then analysed and categorised separately for both males and females and an ethogram was constructed by combining all positive and negative behaviours to understand their specific courtship interactions, and the potential differences in these behaviours as related to a successful or unsuccessful copulation. Here I have hypothesised that males which exhibit more wing fanning behaviour, either in duration or number of occurrences, will be more successful at securing a mate; as previously recorded for B. oleae and Z. cucurbitae (Benelli et al.,

2012, Kuba & Koyama, 1985). Further, I hypothesise the males who simply attempt more copulations will be more successful in ultimately obtaining a copulation, as observed in Z. cucurbitae (Kuba & Koyama, 1985).

3.2 Material and methods

Source of insect material

Bactrocera tryoni pupae were obtained from laboratory cultures (F20- F30 generations) maintained by the Queensland Government Department of Agriculture and Fisheries, Brisbane, Australia. Emergent adults were held at densities of 125 individuals per cage (30 x 20 x 20 cm) under laboratory conditions of 27 ºC and 70% relative humidity. Flies were supplied with sugar, water and protein hydrolysate ad libitum. Flies were held until they reached sexual maturity, sexes were separated within 2-3 days of emergence into two separate cages, from which virgin males and females were used in experiments at age 12-14 days-old.

8670 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

3.2.1 Detailed study of B. tryoni close range courtship and defining their specific

courtship behaviours

The objective of this set of experiments was to resolve close-range courtship interactions of B. tryoni. For each of 10 replicates, one virgin male and one virgin female were placed inside a Petri-dish (60 X 15 mm) following which video recordings of their sexual behaviour were taken using a Canon EOS 7D Mark II camera under natural dusk conditions. Supplementary light for video recording during sunset was provided by a red LED light source as needed. Fly behaviours were observed and recorded to ensure capture of any potential subtle interactions, specifically noting timing, sequence, and duration of behaviours. Behavioural recordings were undertaken using Noldus: The Observer XT software (Version 11.5)

(method below), with results analysed to construct the sequence of B. tryoni close- range courtship interactions.

3.2.2 Identification of individual behaviours Sequence analysis with Noldus, The Observer XT (version 11.5)

Bactrocera tryoni courtship behaviours were initially identified based on existing literature (Myers, 1952, Tychsen, 1977) and then refined/added to through the observation of recorded videos. The identification and full description of the behaviours are presented in the Results section (Table 3.2). A coding scheme was designed using all the identified behaviours in Noldus; The Observer XT software.

All videos were coded individually, with coding conducted separately for males and females while the video was playing after slowing the speed of the videos to half of their original to more clearly observe fly behaviours. The sequence of behaviours for each male and female was obtained by visualizing the coded behaviours. An ethogram of B. tryoni courtship behaviours was constructed by combining the

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 71

behaviour sequence outputs of males and females of 10 replicates. Total duration

(seconds) and total number of occurrences were calculated for each behaviour for males and females separately to use for hypotheses testing.

Some identified behaviours (as identified in Tables 3.1 and 3.2) were further classified under three categories; fly behaviour group 1, group 2 and group 3, for the coding of behaviours. This is because there were some instances where B. tryoni males and females performed two behaviours together, such as directing wings

(Synchronous supination (Aluja and Norrbom, 1999)) while walking. These types of behaviours cannot code in the Noldus observer software at the same time unless they are under two separate behavioural groups.

3.2.3 Male and female behaviour analysis (Hypothesis testing) Total duration and total number of occurrences were calculated for selected male and female behaviours (Table 3.1) for each replicate and used for a priori hypothesis testing. Data were evaluated for normality using Kolmogorov-Smirnov tests and for equality of variances using a variance ratio test (F). Since the data sets were independent from each other, 2-sample t-tests for normally distributed data or a

Wilcoxon rank-sum tests for non-normally distributed data were conducted to test whether there was a significant difference between behaviours of successful mating couples and unsuccessful mating couples.

7286 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Male behaviours Female behaviours Directing wings (Synchronous supination) Directing wings Coming towards the female Coming towards the male Male-female face-to-face touching Male-female face-to-face touching Quick flights Quick flights Fore-legs tapping female’s body Fore-legs tapping male’s body Wing fanning Extends ovipositor Fore-legs interacting with female abdomen Rubbing hind-legs against ovipositor while on top Attacking female by head Attacking male by head Initial mount Hind-legs interacting with male body while male on top Mounting attempts

3.3 Results

3.3.1 Behaviours Thirty-seven behaviours across three groups were identified from the literature or from replaying videoed behaviour. The behaviours are listed and described in

Table 3.2, illustrated with photos in Figures 3.2 – 3.7, and, if reading an electronic copy of this document, further illustrated with short video clips imbedded within

Figure 3.8. Many behaviours were subtle, involving body orientation and leg movements. As suggested from the literature, there were no overt courtship dances or other type of obvious behaviour as seen in some other tephritid genera (Sivinski et al., 2000).

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 73

Table 3.2: Bactrocera tryoni male and female behaviours identified for Petri-dish mating trials. Behaviours are categorised under three groups: mouth part movements; leg movements; and wing movements

Behaviour group and male/ female behaviours with interpretations Behaviour group 1: mouth part movements Extending proboscis – stretching out the proboscis but does not touch any surface while walking or still (Figure 3.2a, b) Extends proboscis and tapping the substrate - stretching out the proboscis and touch the Petri-dish wall while walking or still (Figure 3.2c, d) Male-female face-to-face touching – a quick touch of male and female’s tip of the proboscis (Figure 3.2e) Coming towards the female – male walking fast or slowly towards the female from any direction Coming towards the male - female walking fast or slowly towards the male from any direction (Figure 3.3a, b) Attacking the female by head pushing – male taps the female by head from any direction Attacking the male by head pushing - female taps the male by head from any direction Proboscis tapping female’s body – male stretching out the proboscis and touching female’s body Proboscis tapping male’s body - female stretching out the proboscis and touching male’s body Behaviour group 2: leg movements Walking – walking without touching any other individual No movement – fly stands stationary without walking or moving legs or wings Fore-legs tapping female’s body - male quickly tap female’s body using fore-legs (Figure 3.3c) Fore-legs tapping male’s body - female quickly taps male’s body by fore-legs Rubbing fore-legs together – male or female rubs fore-legs together while standing on mid- and hind-legs (Figure 3.3d) Rubbing mid-legs together – male or female rubs mid-legs together while standing on fore- and hind-legs Rubbing hind-legs together – male or female rubs hind-legs together while standing on mid-

8674 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

and fore-legs Rubbing fore- and mid-legs together – male or female rubs fore-legs together with a mid-leg while standing on the other mid-leg and hind-legs (Figure 3.3e) Rubbing hind- and mid-legs together – male or female rubs hind-legs together with a mid- leg while standing on the other mid-leg and fore-legs (Figure 3.3f) Rubbing hind-legs on wings – male or female rubs hind-legs starting from the wing base to the tip while not walking (Figure 3.4a, b) Rubbing hind-legs against abdomen – male or female rubs hind-legs over the abdomen while keeping wings vertically or horizontally away from body (Figure 3.4c, d) Rubbing hind-legs all over the body – male or female start rubbing hind-legs from head/ thorax towards the tips of the wings (Figure 3.4e, f) Rubbing fore-legs against head – male or female rubs fore-legs over the head and antennae (Figure 3.5a, b) Fore-legs interacting with mouth parts – stretching out the proboscis and rubbing it by fore- legs (Figure 3.5c, d) Rubbing fore-legs together after touching the other individual – rubbing fore-legs together immediately after tapping other female by fore-legs Rubbing hind-legs against ovipositor – female rubs the extended ovipositor with her hind- legs (Figure 3.5e, f) Initial mount – first attempt of male mounting onto female Mounting –further mounting attempts of a male onto female, after the initial mount Male fore-legs interacting with female genitalia/abdomen – male fore-legs repeatedly touching the female’s or other male’s genitalia or abdomen, while he is mounted and attempting copulation (Figure 3.6a) Female hind-legs interacting with male’s body – female trying to push (or repeatedly touching) the mounted male with her hind-legs Dismount – male and female separate after mounting attempts but before a successful copulation (Figure 3.6b, c, d) Female oviposition behaviour – female bends her abdomen downwards and pushes the extended ovipositor against the Petri-dish (Figure 3.6e) Behaviour group 3: wing movements No action – no wing movements or fanning while walking or not Directing wings away from body (Synchronous supination (specific terminology in

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 75

Headrick and Goeden 1994, Aluja and Norrbom 1999) ) – keeping wings horizontally perpendicular to the body/thorax (keeping wings horizontally away from body while rubbing hind-legs on the abdomen was not taken as directing wings away from body) (Figure 3.6f) Quick flights – rapidly flying from one place to another Wing fanning – rapid wing beating while walking, not walking or while on top of another individual attempting copulation Female extending ovipositor – female extends the ovipositor while motionless (Figure 3.7a) Successful copulation – the distal ends of the male and female abdomens are joined together. Male beats wings regularly and both remains motionless or struggles occasionally (Figure 3.7b)

8676 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

(a) (b)

(e)

Figure 3.2: Male and female Bactrocera tryoni courtship behaviours: (a) & (b)

extending proboscis; (c) & (d) extends proboscis and tapping the substrate; (e) male-

female face-to-face touching.

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 77

(a) (b)

(e) (f)

Figure 3.3: Male and female Bactrocera tryoni courtship behaviours: (a) & (b)

Female coming towards the male; (c) fore-legs tapping female’s body; (d) rubbing

8678 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

fore-legs together; (e) rubbing fore- and mid-legs together; (f) rubbing hind- and

mid-leg together.

(a) (b)

(e) (f)

Figure 3.4: Male and female Bactrocera tryoni courtship behaviours: (a) & (b)

Rubbing hind-legs on wings; (c) & (d) rubbing hind-legs against abdomen; (e) & (f)

rubbing hind-legs all over the body.

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 79

(a) (b)

(e) (f)

Figure 3.5: Male and female Bactrocera tryoni courtship behaviours: (a) & (b)

Rubbing fore-legs against head; (c) & (d) fore-legs interacting with mouth parts; (e)

& (f) rubbing hind-legs against ovipositor.

86 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 80

(a) (b)

(e) (f)

Figure 3.6: Male and female Bactrocera tryoni courtship behaviours: (a) Male fore-

legs interacting with female genitalia/abdomen; (b), (c) & (d) dismount; (e) female

trying to oviposit; (f) directing wings away from body.

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 81

(a) (b)

Figure 3.7: Male and female Bactrocera tryoni courtship behaviours: (a) Female

extending ovipositor; (b) successful copulation.

3.3.2 Courtship behaviour sequence of Bactrocera tryoni – Ethogram Five replicates resulted in successful copulation and five replicates did not. The ethogram was constructed based on all replicates, with samples sizes indicated respectively (Figure 3.8). Both males and females were cleaning at the start of the mating period in the lead up to dusk. Upon the arrival of dusk, 100% of flies approached each other while holding their wings horizontally perpendicular to their body. Among them, 80% of males and females became more active over time, but

20% of males and females first exhibited body tapping behaviour with the other individual as either by face-to-face touching, head pushing or tapping of fore-legs prior becoming more active. Both males and females started rapid, quick flights from one place to another inside the Petri-dish during this time. Male wing fanning (n = 1) and female ovipositor extension (n = 2) occurred but was infrequently observed at this early stage of courtship. This sequence of behaviours was observed several times

(n = 4.8 ± 1.0 of the replicates) before the initial mount. Some males (n = 6) came towards a stationary or walking female before jumping on to her to attempt

8682 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

copulation. Mounting attempts were absent in four replicates and so obviously these replicates did not lead to successful copulation, but in one replicate there was an initial mounting that also did not lead to a successful copulation.

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 83 ♂ ♀ Cleaning

Chapter 3: Chapter 100%, n=10 ♂ ♀ Directing wings

Resolving the courtship Resolvingthe behaviours ofBactrocera Tephritidae)tryoni (Diptera: 100%, n=10

♂ ♀ Coming towards 20%, n=2

Several times ♂ ♀ Face-to-face touching/ n = 4.8 ± 1.03 80%, n=8 attack by head pushing/ tapping by fore-legs 50%,

♂ ♀ increased activity n=1

(walking & quick flights) 100%, n=1 50%, n=1 100%, n=2 ♂wing fanning

40%, n=4 60%, n=6

♀ extending ovipositor 100%, n=2 ♂ coming towards the female

50%, 50%, n=4 n=6

Initial mount No mounting

16.67%, n=1 100%, n=6 100%, n=6 ♀ shakes her ♂wing fanning & fore- ♀ hind-legs abdomen until legs interacting with interacting with male gets off female abdomen male’s body

100%, n=1 gradually reduced reduced activitygradually 100%, n=6 100%, n=5

♂ Dismount

♀ ♂ Dismount

100%, n=1 ♂ ♀ 100%, n=5 Cleaning –

80%, 20%, no successful

n=4 n=1 copulation ♂continuous wing ♀ directing wings & No female fanning walks away movements

100%, Several times n=5 ♂ ♀ Quick flights n = 6 ± 4.19

60%, n=3

♂ ♀ coming 66.7%, n=2 40%, towards n=2 ♂ ♀ Face-to-face touching/ attack by head pushing/ tapping by fore-legs 33.3%, n=1 100%, n=2 Second mount 40%, n=2 60%, n=3

Dismount

60%, n=3

♂ ♀ Face-to-face touching/ attack by head pushing/ Figure 3.8: Ethogram for Bactrocera tryoni male and female tapping by fore-legs courtship behaviours. Percentages indicate the proportion of

40%, n=2 100%, n=3 individuals and n values indicate the number of flies

Successful displaying each behaviour in each step

copulation

86 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

In replicates with initial mounts present (n = 6), all males (100%) showed wing fanning while curling their abdomen downwards and forwards during attempts to insert the aedeagus into the female’s genital opening at the tip of the ovipositor.

During this process, female hind-legs repeatedly touched the male. Not all initial mounts resulted in copulation, with some ultimately successful males requiring multiple mounting attempts. In such cases, after the dismount, male wing fanning, directing wings and coming towards each other (n = 6 ± 4.19) again occurred before the ultimately successful mount. After males had successfully mounted, males regularly beat their wings and both sexes remained either motionless or occasionally moved together.

3.3.3 Male and female behaviour analysis (Hypothesis testing) There was a significant difference in both total duration (W (8) = 40 P = 0.01) and number of occurrences (t = 3.25, df = 8, P = 0.031) in directing wings between successfully copulated males and unsuccessful males; successful males directed their wings more often (48.85 s ± 32.92) compared to unsuccessful males (5.60 s ± 1.92).

There were no significant differences in other compared behaviours between successful males and unsuccessful males (Table 3.3, 3.4 and 3.6). Initial mounts were obviously observed in all replicates with successful copulations (100%), but in only one replicate with unsuccessful copulations (20% ± 0.02). In this replicate with unsuccessful copulations, there were several mounting attempts following the initial mount, but the female exhibited female body-shaking (total number of occurrences =

20), resulting in the male being dislodged. The transition step which involves courtship progressing from pre-mounting to post-mounting thus seems a very important one in B. tryoni mating, but comparison of initial mounting was still statistically non-significant, probably because of low replication size (Table 3.6).

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 85

There were no significant differences in both the total duration and total number of occurrences between all successfully copulated and unsuccessful female behaviours (Table 3.3, 3.5 and 3.7).

86 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Chapter 3: Chapter

Resolving the courtship Resolvingthe behavioursof Bactrocera tryoni Table 3.3: Mean, standard deviations (SD) and standard errors (SE) calculated for total duration (seconds) for Bactrocera tryoni male and female

courtship behaviours recorded in 10 courtship sequences.

Total duration (s) for male Total duration (s) for female behaviours

behaviours

(Diptera: Tephritidae) (Diptera:

legs against ovipositor against legs

egs interacting with female interacting female with egs

legs interacting with abdomen interacting legs male

Directing wings Directing female Comingthe towards fanning Wing Fore abdomen wings Directing male Comingthe towards ovipositor Extends Rubbinghind Hind to oviposit Trying Replicates with copulations Replicate 1 180.04 0.00 109.84 343.24 23.92 1.90 0.00 0.00 337.78 17.38 Replicate 3 13.95 9.20 54.29 44.35 104.04 34.41 33.29 7.74 39.14 0.00 Replicate 4 12.52 17.48 117.28 38.25 634.16 72.72 36.20 20.00 37.16 0.00

Replicate 6 11.04 12.26 718.89 82.29 53.13 0.93 19.12 16.08 48.41 6.71

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 87

Replicate 7 26.69 8.92 13.07 1.50 178.51 2.03 0.00 0.00 1.95 0.00 Mean 48.85 9.57 202.67 101.93 198.75 22.40 17.72 8.76 92.89 4.82 SD 73.60 6.36 291.70 137.91 250.37 31.51 17.42 9.14 138.04 7.60 SE 32.92 2.85 130.45 61.67 111.97 14.09 7.79 4.09 61.73 3.40 Replicates without copulations

Chapter 3: Chapter Replicate 2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Replicate 5 10.54 3.64 145.51 0.00 204.53 24.83 42.27 19.89 0.00 0.00

Resolving the courtship Resolvingthe behavioursof Bactrocera Tephritidae)tryoni (Diptera: Replicate 8 2.60 1.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Replicate 9 6.44 0.00 0.00 0.00 102.73 9.34 0.00 13.26 0.00 174.54 Replicate 10 8.41 32.30 779.83 328.23 459.86 10.35 0.00 0.00 316.55 0.00 Mean 5.60 7.55 185.07 65.65 153.43 8.91 8.45 6.63 63.31 34.91 SD 4.28 13.92 338.40 146.79 191.15 10.18 18.91 9.38 141.57 78.06 SE 1.92 6.22 151.34 65.65 85.49 4.55 8.45 4.19 63.31 34.91

86 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Chapter 3: Chapter

Table 3.4: Mean, standard deviations (SD) and standard errors (SE) calculated for total number of occurrences of male Bactrocera tryoni Resolving the courtship Resolvingthe behavioursof Bactrocera Tephritidae)tryoni (Diptera:

courtship behaviours recorded in 10 courtship sequences resulting in both successful and unsuccessful copulations.

Total number of occurrences

facetouching

Replicate number Replicate

female face female

legs tapping female's tapping legs body female with interacting legs

- -

Directing wings wings Directing female Comingthe towards Male fligh Quick Fore fanning Wing Fore abdomen head by female Attacking mount Initial Mountingattempts Dismount

Replicates with copulations Replicate 1 2 0 6 25 2 10 1 0 1 1 1 Replicate 3 11 15 1 170 0 10 4 0 1 3 3 Replicate 4 7 14 1 114 1 6 1 1 1 1 1 Replicate 6 4 19 0 390 1 5 15 13 1 21 20

Replicate 7 6 4 0 79 8 1 1 1 1 1 1

8 9

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 89

Mean 6.00 10.40 1.60 155.60 2.40 6.40 4.40 3.00 1.00 5.40 5.20 SD 3.39 8.02 2.51 141.25 3.21 3.78 6.07 5.61 0.00 8.76 8.32 SE 1.52 3.59 1.12 63.17 1.44 1.69 2.71 2.51 0.00 3.92 3.72 Replicates without copulations

Chapter 3: Chapter Replicate 2 0 0 0 0 0 0 0 0 0 0 0

Replicate 5 7 5 1 146 6 14 0 0 0 0 0

Resolving the courtship Resolvingthe behavioursof Bactrocera Replicate 8 2 1 1 35 0 0 0 0 0 0 0 Replicate 9 4 0 0 1 0 0 0 4 0 0 0 Replicate 10 3 15 19 35 1 8 18 1 1 19 20 Mean 3.20 4.20 4.20 43.40 1.40 4.40 3.60 1.00 0.20 3.80 4.00 SD 2.59 6.38 8.29 59.89 2.61 6.39 8.05 1.73 0.45 8.50 8.94 SE 1.16 2.85 3.71 26.79 1.17 2.86 3.60 0.77 0.20 3.80 4.00

tryoni (Diptera: Tephritidae)tryoni (Diptera:

86 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Chapter 3: Chapter

Table 3.5: Mean, standard deviations (SD) and standard errors (SE) calculated for total number of occurrences of female Bactrocera tryoni Resolving the courtship Resolvingthe behavioursof Bactrocera Tephritidae)tryoni (Diptera:

courtship behaviours recorded in 10 courtship sequences resulting in both successful and unsuccessful copulations.

Total number of occurrences

facetouching

legs against ovipositor against legs

Replicate number Replicate

female face female

legs interacting with interacting legs male

legs tapping male's male's tapping legs body

Directing wings wings Directing male Comingthe towards Male head by female Attacking flights Quick ovipositor Extends Rubbinghind Fore Hind abdomen to oviposit Trying

Replicates with copulations

Replicate 1 13 3 6 9 0 0 0 0 1 2 Replicate 3 8 8 1 4 1 4 3 0 3 0 Replicate 4 33 20 1 21 8 5 3 1 1 0 Replicate 6 8 1 0 4 100 2 3 0 15 1

Replicate 7 15 1 0 1 0 0 0 0 1 0 91

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 91

Mean 15.40 6.60 1.60 7.80 21.80 2.20 1.80 0.20 4.20 0.60 SD 10.31 8.02 2.51 7.92 43.84 2.28 1.64 0.45 6.10 0.89 SE 4.61 3.59 1.12 3.54 19.61 1.02 0.73 0.20 2.73 0.40 Replicates without copulations

Chapter 3: Chapter Replicate 2 0 0 0 0 0 0 0 0 0 0

Replicate 5 55 7 1 5 4 2 2 1 0 0

Resolving the courtship Resolvingthe behavioursof Bactrocera Tephritidae)tryoni (Diptera: Replicate 8 0 0 1 0 1 0 0 0 0 0 Replicate 9 12 3 0 0 150 0 5 1 0 17 Replicate 10 18 6 19 4 73 0 0 0 18 0 Mean 17.00 3.20 4.20 1.80 45.60 0.40 1.40 0.40 3.60 3.40 SD 22.63 3.27 8.29 2.49 66.05 0.89 2.19 0.55 8.05 7.60 SE 10.12 1.46 3.71 1.11 29.54 0.40 0.98 0.24 3.60 3.40

86 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Table 3.6: Statistical test results for comparisons of the number and duration of male

Bactrocera tryoni courtship behaviours occurring in replicates with and without successful copulations. Blank cells represent the absence of ‘duration’ as the behaviours are point behaviours. It is important to acknowledge that the power of tests is weak because of low sample size.

Male behaviour W/t and p values for Wilcoxon rank sum test/ 2 sample t test Total number of Total duration occurrences Directing wings t = 3.25 df = 8 P = 0.031 W(8) = 40 P = 0.01 Coming towards the female t = 1.17 df = 8 P = 0.306 W(8) = 32 P = 0.3976 Male-female face-to-face W (8) = 27 P = 1.00 - touching Quick flights W (8) = 35 P = 0.1425 - Fore-legs tapping female’s body W (8) = 32.5 P = 0.3258 - Wing fanning W (8) = 32 P = 0.3961 W(8) = 31 P = 0.5258 Fore-legs interacting with female W (8) = 35 P = 0.1264 W(8) = 36 P = abdomen 0.0847 Attacking female by head W (8) = 30 P = 0.6513 - Mounting attempts W (8) = 36 P = 0.0807 -

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 93

Female behaviour W/t and p values for Wilcoxon rank sum test/ 2 sample t test Total number of Total duration occurrences t = -0.12 df = 8 P = Directing wings 0.909 W (8) = 30 P = 0.6752 t = 0.78 df = 8 P = Coming towards the male 0.480 t = 0.89 df = 8 P = 0.425

Male-female face-to-face touching W (8) = 27 P = 1.00 -

Attacking male by head W (8) = 35 P = 0.1339 -

Quick flights W (8) = 24.5 P = 0.5959 -

Extends ovipositor W (8) = 33.5 P = 0.1938 W (8) = 31 P = 0.4802 Rubbing hind-legs against ovipositor W (8) = 30 P = 0.6513 W (8) = 30 P = 0.6558

Fore-legs tapping male’s body W (8) = 25 P = 0.6005 - Hind-legs interacting with male W (8) = 36 P = 0.0847 body W (8) = 35 P = 0.1264

8694 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

3.4 Discussion

In this chapter, I have carried out close range behavioural observations to categorise the B. tryoni courtship sequence to identify potential recognition signals critical to successful copulation. Both males and females increased activity with dusk, as widely known, which was followed by a sequence of behaviours including the directing of wings (synchronous supination), sexes approaching each other, body tapping using legs, male wing fanning, female ovipositor extension, and mounting attempts. Only 10 replicates were analysed in this experiment, with five successfully mating. Under hypotheses testing, the sample sizes for this analysis are small and there is a high likelihood that there is insufficient statistical power to detect real biological differences should they exist. However, according to the data I analysed, directing wings was the only significant difference between successfully copulated males and unsuccessful males, in both total duration and number of occurrences.

Whether the male initiated mountings onto the female or not was the other behavioural difference between successful and unsuccessful males, but this is actually a transition step with the courtship sequence, rather than a courtship behaviour per se. Nevertheless, that four out of five unsuccessful pairs did not progress to this step, suggests important pre-mounting mate discrimination. No other behaviours were different between successful and unsuccessful males.

The directing of male wings has been considered as the initial step of wing fanning in tephritid male courtship (Briceño and Eberhard, 2000, Benelli et al.,

2012). Wing movements have also been observed as a part of courtship dances in tephritids such as A. suspensa (Sofian-Azirun et al., 2011), R. completa (Bush, 1966) and R. juglandis (Alonso-Pimentel et al., 2000) (see section 1.6.2.3). However, as I mentioned in section 1.7.2.3, wing movements as courtship dances of B. tryoni have

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 95

not been previously recorded, but based on my recordings presented here, the directing of male wings should be regarded as an important part of their courtship. In my opinion, the increased amount of wing display in successful males may have caused enhanced recognition by females, which helped lead to successful copulation.

Or, perhaps, that directing of wings is merely due to males being more active. If the former, this is evidence for synchronous supination being a key element in the mating process, because all other behaviours are not significantly different; if the latter, then this is also important as it supports the idea of mating success being directly proportional to male metabolic activity (Kumaran et al., 2014b).

Making the initial mounting attempt was another important (albeit statistically non-significant) difference between successful copulations and unsuccessful copulations. For five replicates with unsuccessful copulations, an initial mount was recorded in only one replicate. For this male individual several subsequent mounting attempts also did not lead to a successful copulation. This same observation was recorded in Z. cucurbitae, where unsuccessfully copulated males performed very few mounting attempts (Kuba & Koyama, 1985).

The data recorded in this chapter suggests that while B. tryoni individuals may locate their mating partner at a distance based on visual, chemical and acoustic signals (as recorded in literature), final mate selection may occur both when they are in very close contact (i.e., pre-mounting) and possibly also direct contact (i.e., post- mounting). In C. capitata, it has been suggested that males are unable to identify the sex of another individual until they have tactile contact (Prokopy and Hendrichs,

1979). Sex confirmation might be based on the chemical composition of male cuticle

(e.g., CHC), where females are identified by repeated tapping by male hind-legs while the male is on top of her. Although it has been recorded in Drosophila that

8696 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

females do accept or reject males due to variation in male cuticular compounds

(Howard et al., 2003), clear behavioural observations of tephritid female selection is rare and further, it remains unknown why some males initially mounted and others did not.

Tephritid female rejection or acceptance of males post-mounting has been observed in wild A. ludens and Toxotrypana curvicauda, where females vigorously shook their abdomens, pushing mounted males off by their hind- and mid-legs to avoid mounting males (Landolt and Hendrichs, 1983; Robacker et al., 1991). In my experiments I also observed female rejection of mounted males by abdomen shaking and pushing with their hind- and mid-legs, suggesting that females may reject or accept a mounted male. In other insects, female body shaking while the male is attempting copulation is considered a behaviour that shows female refusal to mate, or a way of how she is recognising the motivation of males to mate (Rowe et al., 1994,

Weigensberg and Fairbairn, 1994, Blanckenhorn et al., 2000, Teuschl and

Blanckenhorn, 2007). In Aquarius remiges (Say), the common water strider, females may repeatedly reject mounting males by backward leg kicks, and it is suggested that this kind of female behaviour is to assess the motivation of males to mate

(Weigensberg and Fairbairn, 1994). If the male is highly motivated to mate and shows repeated mounting attempts, the female will stop rejection to avoid further

‘harassment’ (Arnqvist, 1992). This kind of female rejection behaviour also common in sepsid flies (Eberhard, 2002, Teuschl and Blanckenhorn, 2007).

In female Drosophila, ovipositor extension has been regarded as a mate rejection behaviour that previously mated females perform at the time when a male is approaching (Kimura et al., 2015). However, ovipositor extension in my trials was observed before a male approached and the females were virgin, so, female

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 97

ovipositor extension should not be regarded as a male rejection cue in B. tryoni.

Ovipositor extension may be performed in B. tryoni as a means to recognise their environment, as recorded in drosophilids (Kimura et al., 2015), since females have neuro-receptors on their ovipositor (Rice, 1989). Alternatively, the females may be depositing some form of recognition marker: female pheromones are recorded from

B. tryoni but their function and mode of release is unknown (Fletcher & Kitching,

1995). Bactrocera tryoni female ovipositor extension was used as a bioassay scoring tool in early work identifying the B. tryoni male pheromone (Giannakakis &

Fletcher, 1978), and so appears in some way to be linked to courtship. In B. minax, females carry out a form of ‘pseudo-oviposition’ (= minor boring with no egg lay) prior to mating on the citrus host fruit (Dong et al., 2014), another example of ovipositor extension being associated with courtship in tephritids.

Although male wing fanning in tephritids is considered to be a behaviour to attract females by evaporating and dispersing sex pheromones, no differences in the amount of male wing fanning by successful and unsuccessful males prior to mounting was observed in my experiments: indeed it was a rare behaviour observed only once (Figure 3.3). The same observation of copulation success of non-wing fanning males has previously reported in B. dorsalis (Shelly and Kaneshiro, 1991,

Schutze et al., 2013). However, observing male wing fanning when the male is on top of the female trying to copulate, and after a dismount (i.e., a failed attempt), suggest a possibility of production of “copula” sounds. Previous recordings on B. tryoni suggest the production of male “copula” songs while the male is mounted on the female and tapping her with his legs (Neale, 1989, Mankin et al., 2008).

This chapter has provided a comprehensive ethogram of B. tryoni male and female courtship sequences which is critical to understanding their mating behaviour.

8698 Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae)

Male synchronous supination, being a highly significant behavioural difference between successful and unsuccessful males despite low replication size, suggests the possibility of either a simplified courtship dance which B. tryoni females use to recognise males, or as an indication of male activeness that affects their mating success. Further, whether a male actually initiates mounting attempts is a critical transition step for subsequent successful mating, with most failed matings not reaching this step. Observing female avoidance (or non-avoidance) of mounting males suggests that females may accept or reject mounting males and males can overcome this avoidance if they are highly motivated to copulate. To further test the hypothesis that active mate selection does occur in B. tryoni, the next chapter of my thesis focuses on differential mating success of males of different physiological quality (big vs small males; old vs young males, virgin vs non-virgin males) and if specific courtship behaviours vary between those categories of male.

Chapter 3: Resolving the courtship behaviours of Bactrocera tryoni (Diptera: Tephritidae) 99

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni101

4.1 Introduction

Close-range interactions between individuals allows not only for specific mate

recognition (sensu Paterson 1985), but is also the stage at which mate choice may

occur; i.e., the selection of one potential partner versus an alternative partner

(Candolin, 2003). The selection of an individual as a potential mate drives the

evolution of characters that are directly, or indirectly, linked with mating success

(Emlen and Oring, 1977). For most animals, mate choice is primarily determined by

female selection of a male (Sivinski, 1993, Paul, 2002), as female physiological

investment in the egg, embryonic development and (if it occurs) postnatal care, is

generally much greater than that of the male, which is typically restricted to sperm

production (Tinbergen, 1953, Rosvall, 2011, Clutton-Brock and Huchard, 2013).

Mate selection cues may include the behaviours that actively advertise an

individual’s presence or readiness to mate; ‘honest’ signals that the sender may not

actively advertise but which nevertheless provide information to the recipient; or

‘hidden’ cues in which the selection process is cryptic but may still influence mate

choice. Signals that individuals use to actively ‘advertise’ to a potential mate may

include acoustic calls and evaporative pheromone chemicals; both of these have been

well studied in tephritids (Fletcher, 1968, Sivinski et al., 1994, Tan and Nishida,

1996, Teal et al., 2000, Lu and Teal, 2001, Pike and Meats, 2003, Kumaran et al.,

2014a). Other signals may include non-evaporative chemicals such as cuticular

hydrocarbons (Vanickova et al., 2012, Zunic Kosi et al., 2013) and visual signals

such as wing or body positioning (Benelli et al., 2015); but less research has been

done on these within the frugivorous tephritids.

Individual body size is an ‘honest’ signal which a potential partner may

directly assess, or which may be indirectly assessed through body size effects on

10286 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

calling or other behaviours. The cryptic attributes that may influence individual mating may include age and previous sexual experience. Male variation in these attributes may affect their mating success due to differences in fitness and/or female preferences (Candolin, 2003). For example changes in male body size, age and sexual experience may result in changes in their acoustic signals or pheromone characteristics, while potentially also affecting intra-sexual competitiveness.

Females may respond directly to male advertisements; i.e., a female may orient positively or negatively to pheromones or acoustic signals; but documented responses by female tephritids to honest and cryptic signals such as body size, age or previous sexual experience are rare (see section 1.6.3 for a review). In that section, I have addressed each of these male ‘non-advertising’ signals (i.e., individual body size, age and previous sexual experience) in turn; showing how they impact on a male’s ‘signalling quality’ for tephritids in general. In section 1.7.2.3 I review what is known about such variation and mating success for B. tryoni.

Given the literature reviewed, it is clear that body size, age and mating experience may impact on individual mate choice in tephritids. While some information is available for B. tryoni, nearly all studies for this species are based on small cage laboratory experiments. Small cage experiments may artificially preclude key steps in the mating process and, in some cases, yield results that are contrary to those that may be obtained under more natural conditions (e.g., B. tryoni can be crossed with other Bactrocera species in small cages, a clearly anomalous result

(Cruickshank et al., 2001, Pike and Meats, 2002)); such results thus need to be interpreted with caution and are prone to misinterpretation. To address this issue, the objective of my study was to evaluate the effect of male B. tryoni body size, age and mating experience on their mating success in field cages under semi-natural

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni103

conditions. Tephritid mating experiments in field cages have a long history of use

and results obtained within them are considered robust (Cayol et al., 1999, Collins et

al., 2008, Diaz-Fleischer and Arredondo, 2011, Haq et al., 2014).

In this chapter I conduct three sets of field cage experiments to test whether

there is an effect of male B. tryoni physiological attributes on mating success. My

first hypothesis is that males of different quality (defined by age, size, and sexual

experience) will have different levels of mating ‘success’ (i.e., ability to secure a

female mate). Secondly, given a difference, I hypothesise that young, large and

virgin males would obtain more mates than old, small and previously mated males,

respectively. Male mating competitiveness indices were calculated using the number

of mating pairs in each male group to quantify this. After determining the effect of

male physiological state on mating success, further fine scale behavioural

observations were made to determine what specific aspects of the male behavioural

repertoire may vary with respect to sexual interactions of B. tryoni males and females

depending on the results of the field cage trials. That is, if small males were not as

successful as large males in the field cage, was there some aspect of their close range

courtship interactions that may explain this reduced competitive success? This was

achieved by holding two males of different physiological state with a female inside a

Petri-dish and making close range video recordings to compare specific behaviours

according to their physiological state. Results from Chapter 3, specifically the

ethogram of sexual behaviours recorded from ‘control’ individuals, were used as the

basis of evaluation for differences among treatments.

10486 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

4.2 Material and methods

4.2.1 Experiment 1– Field cage mating competitiveness tests Source of insect material

Bactrocera tryoni pupae were obtained from laboratory cultures (F20- F30 generations) maintained by the Queensland Government Department of Agriculture and Fisheries (QDAF), Brisbane. Emergent adults were held at densities of approximately 1000 individuals per cage (90 x 60 x 60 cm) under laboratory conditions of 27 ºC, 70% RH and under white fluorescent light for nine hours (0700

– 1600h) and natural light for the rest of the time in an insectary with a window to the outside environment. Flies were supplied with sugar, water and protein hydrolysate ad libitum. Flies were held until they reached sexual maturity (i.e., 12-14 days); sexes were sorted within 2-3 days of emergence into separate cages (30 x 20 x

20 cm) cages at a density of 125 individuals per cage, from which virgin males and females were used in experiments at age 12-14 days-old, or 28 days old for the ‘old’ age group (see experiments below).

Experimental setup

Three mating competitiveness trials of: i) old males versus young males; ii) small males versus large males; and iii) virgin males versus mated (experienced) males, were carried out as follows.

i. Old versus young males

Two colonies of flies were synchronised (according to the method stated above) separately until virgin males reached 14 and 28 days old for mating tests.

ii. Large versus small males

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni105

Differential adult size was obtained by rearing larvae on different quantities of

diet. Egging cups, made from small disposable plastic cups lined inside with a stripe

of blotting paper moistened with natural apple juice and small pin holes on the cup

wall, were kept overnight in the cages with sexually mature mated flies of at least 14

days of age. On the following day, collected eggs were counted onto standard carrot

diet. Small males were obtained via diet restriction by placing approximately 200

eggs on 10 g of standard carrot diet; large males were obtained by placing

approximately 20 eggs on 10 g of carrot diet during development. Subsequent pupae

were kept separately in two cages (90 x 60 x 60 cm) until they become sexually

mature. Both small and large virgin males and females were separated into four small

cages (30 x 20 x 20 cm) within 2-3 days of emergence. Based on wing length (see

methodology below) as an indirect measure of total body size (the two are highly

correlated, see Fitt (1990)) the differential feeding treatment resulted in significant

differences in size between large and small male treatment groups (W [883] =

259587, P < 0.001).

iii. Virgin versus experienced males

Virgin males were obtained by separating them from females within 2-3 days

of emergence from pupae and then kept in small cages (30 x 20 x 20 cm) at a density

of 125 flies per cage. Sexually experienced males were obtained by keeping virgin

males with females in small cages (30 x 20 x 20 cm) and removing them into another

small cage once they had been observed to copulate. Fifty mating couples were

carefully collected into small cages each day, one day before, each one of the ten

replicates during dusk and allowed to complete mating. These mated males were

used for the experiment the next day.

106 86 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Mating experiments

All experiments were carried out in a 2.6m wide x 2.1m high field cage with a brownish screen colour (Figure 4.1) containing a single artificial tree (2 m tall, 0.75 m canopy diameter, with polyester leaves, plastic fruit and plastic branches built upon a natural timber stem

(http://www.artificialplantimporters.com.au/Product/5940/apple-tree)) during dusk under natural conditions and using laboratory reared flies as described above. This cage design is recommended by the International Atomic Energy Agency for fruit fly mating studies (FAO/IAEA/USDA, 2014). Ten replicates were completed for each treatment using a new set of flies for each replicate. Respective treatment flies were marked on the thorax with nail polish for easy identification (preliminary observations revealed that there is no effect of nail polish on male mating success).

Fifty males of each test treatment and 50 virgin females were released into the cage one hour prior to sunset. Flies were continuously observed and numbers of mating pairs were counted at the end of each replicate. Male treatment type, time of mating, and location (i.e., on the cage wall/roof or on the tree) in the field cage was recorded.

All mating pairs and individual flies (i.e., all 150 individual flies in each replicate for all ten replicates) of male size comparison replicates only were collected into vials to measure wing size.

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni107

Figure 4.1: Octagon field cage (2.64 m maximum diameter x 2.1 m high) with an

artificial tree inside used for all three Bactrocera tryoni mating comparison

experiments.

10886 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

4.2.1.1 Measuring wing sizes The right-hand wing of every male (n = 446 large males; n = 439 small males) of male size comparison replicates were removed and secured separately on a slide with transparent tape and labelled appropriately. The left-hand wing was used when the right-hand wing was damaged (32.9% out of 885 sample wings). Photos of each wing were taken using Leica Application Suite (version 3.1.1) under Leica model

MDG41 microscope. Wing length was measured from the basal junction of veins of cell basal medial to the terminus of the R4+5 vein (point A to B (Figure 4.2))

(Schutze et al., 2012) using ImageJ software (Version 1.38) (Fanson et al., 2009).

Figure 4.2: Right-hand wing of male Bactrocera tryoni. A = the basal junction of

veins of cell basal medial, B = terminus of the R4+5 vein. Scale bar = 1 mm.

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni109

4.2.1.2 Sexual competitiveness data analysis Male sexual competitiveness indices were calculated for each of the three

treatments (Figure 4.3; young versus old used as an example). Statistically significant

deviation from random mating was determined via the calculation of 95% confidence

intervals about the mean, based on standardised measures of sexual competitiveness

(FAO/IAEA/USDA, 2014). Values of relative sterility index (RSI) can vary from 0

to 1. In the example provided, 0 indicates all females mated with old males, 1

indicates that they all mated with young males and 0.5 indicates that old and young

males were equally competitive (Figure 4.3).

풀푭 푹푺푰 = RSI = Relative sterility index/ male sexual (풀푭+푶푭) competitiveness YF = Total number of pairs with young males OF = Total number of pairs with old males

Figure 4.3: Graphical representation of the Relative Sterility Index (RSI)

(FAO/IAEA/USDA., 2014).

11086 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

To further test whether there was a significant difference between the numbers of mating pairs of the two types of males in each treatment, paired t-tests were carried out following assessment of normality using Kolmogorov-Smirnov tests and for equality of variances using a variance ratio test (F). Non-parametric Wilcoxon rank-sum tests were conducted when data violated parametric assumptions.

4.2.1.3 Wing size data analysis

The further elucidate possible size effects on mating, analysis was not restricted to simply comparing the wing size of large mated males with small mated males, but also compared mated versus unmated flies within the ‘large male’ and

‘small male’ categories. All data were placed into six groups (i.e., large, small, large mated, large not-mated, small mated, small not-mated males) and evaluated for normality using Kolmogorov-Smirnov tests and for equality of variances using a variance ratio test (F). Since the two male samples (large and small) were drawn independently from two populations and the sample sizes were also not equal, three 2 sample t-tests were carried out if the distributions were normal and with equal variance for comparisons between large versus small, large mated versus large not mated and small mated versus small not mated. Wilcoxon rank-sum tests were conducted instead of 2 sample t-tests when the data sets violated parametric assumptions. Analyses were undertaken using the statistical package Minitab 16.0.

4.2.2 Experiment 2- close range video recordings

Two experimental comparisons were carried out using the following: i) old males versus young males and ii) small males versus large males. No videos were taken for the comparison between virgin and mated males due to practical limitations

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni111

as described below. All treatment flies were maintained according to the methods

stated above.

Respective treatment flies were marked on the thorax with nail polish for easy

identification. Painting was randomised between males among replicates for each

treatment. Two males from the two comparison groups were kept with a single virgin

female (drawn randomly from the stock culture where treatment males came from)

inside a small Petri-dish (60 x 15 mm) for each replicate. The initial objective was to

complete 30 replicates (10 per treatment as carried out under cage trials), but

following extensive experimentation very few successful copulations occurred when

two males were present in the Petri-dish. Although mating occurred in no-choice

replicates (Chapter 3), the introduction of a second male interfered with each other's

attempts to copulate. Ultimately, nine replicates for the age-class treatment and 14

replicates for size-class treatment were carried out and examined and categorised

behavioural variables. Of all these replicates, only one replicate resulted in successful

copulations for the age-class treatment and four for the size-class treatment. No

replicates were carried out for experience-class treatments as the field cage

experiments detected no effect of mating experience on mating success (see Results).

Video recordings and analysis of B. tryoni sexual behaviour used the same

methodology as described in Chapter 3, except that videos were analysed separately

for all three flies considering them as three subjects; male type I (e.g., big, small, old,

young), male type II and female.

11286 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

4.2.2.1 Male and female behaviour analysis

The introduction of a second male into a mating arena led to additional behaviours not reported in Chapter 3. These were identified from the videos and are described as for the behaviours reported in Chapter 3.

The introduction of a second male into a courtship arena interrupted courtship, and there were few replicates for which a successful copulation was recorded; i.e., five out of 23 across both treatments (i.e. small versus large males, and young versus old males) (see Results). This limited the type of qualitative analyses which could be carried out, as many of the courtship behaviours reported in Chapter 3 were not observed, or were rarely observed. To address this issue, for each treatment, the courtship behaviours are simply summarised by the number of replicates for which the behaviour was observed. Where a behaviour was observed in only a few replicates, no further analysis is undertaken. Where a behaviour was recorded in four

(more than half of the total number of replicates recorded in age-class treatment; the treatment with least number of replicates) or more replicates for both fly classes within a treatment, the individual behaviour was further analysed using the total number of occurrences and the total duration between young and old males, large and small males, female behaviours towards young and old males and finally female behaviours towards large and small males. Data were tested for normality using

Kolmogorov-Smirnov tests and for equality of variances using a variance ratio test

(F). Paired t-tests or 2 sample t-tests were conducted appropriately for normally distributed data or Wilcoxon rank-sum tests for non-normally distributed data.

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni113

4.2.2.2 Male and female ethograms Two separate ethograms were constructed for female courtship behaviours and

male courtship behaviours. An ethogram for male courtship behaviours was

constructed combining all behavioural sequences observed for young, old, large and

small males (n = 23), and an ethogram for female behaviours was constructed

combing both female behavioural sequences in age-class treatments and size-class

treatments (n = 23).

4.3 Results

4.3.1 Experiment 1– Field cage mating competitiveness tests All mating pairs (100%) were recorded on the cage wall or roof and no mating

pairs were recorded on the tree inside. At the beginning of the experiment, released

flies were motionless on the cage wall, roof or on the ground, with dusk all of them

became active and started forming couples. This behaviour is common when the field

cages with brownish screen colour are used.

i. Old versus young males

Significantly more females (W [9] = 61.5, P = 0.001) mated with young males

(13.0 ± 0.52 pairs per replicate) than with old males (9.3 ± 0.60 pairs) (Figure 4.4).

The relative sterility index of 0.585 ± 0.018 for mating competitiveness between old

and young males shows that young males are significantly more competitive than old

(t = 4.58, df = 9, P < 0.001) (Figure 4.5).

ii. Large versus small males

Significantly more females (W [9] = 147.5, P < 0.0014) mated with large

males (12.2 ± 1.81 pairs) than with small males (3.2 ± 0.49 pairs) (Figure 4.4).

Relative sterility index of 0.787 ± 0.016 for mating competitiveness between large

86 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni 114

and small males indicated the increased competitiveness of large males (t =17.08, df

= 9, P < 0.001) (Figure 4.5).

iii. Virgin versus experienced males

There was no significant difference (t = 1.02, df = 9, P = 0.330) in the number of females mating with previously mated males (11.6 ± 1.19 pairs) versus virgin males (10.2 ± 0.70 pairs) (Figure 4.4). The RSI value of 0.475 ± 0.031 was also not significant (t = -0.800, df = 9, P = 0.445) (Figure 4.5).

16 (a) * 14

Mean number of mating couples mating of number Mean

0 Couples with old Couples with young

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni115

16 (b) * 14

Mean number of mating couples mating of number Mean

0 Couples with large Couples with small

14 (c)

Mean number of mating couples mating of number Mean

0 Couples with virgin Couples with mated

Figure 4.4: Mean (± SE) number of Bactrocera tryoni mating pairs with (a) old

versus young males, (b) large versus small males, and (c) virgin versus non-virgin

males (n = 10 for each column; * indicates significant difference at P < 0.001)

11686 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Experience Age Size

0.0 0.5 1.0 Old/ Small/ Mated Equal competitiveness Young/ Large/ Virgin RSI value

Figure 4.5: Relative sterility index for Bactrocera tryoni mating competitiveness

between young versus old, large versus small, and virgin versus previously mated

males (RSI ± 95% CI). RSI = 0 denotes old/small/mated males are more competitive,

RSI = 0.5 for equal competitiveness and RSI = 1 denotes young/large/virgin males

are more competitive.

4.3.1.1 Wing length comparison

There was a significant difference in wing length between large (n = 446, mean

= 4.768 ± 0.011) and small males (n = 439, mean = 4.369 ± 0.018) when comparing all males used in the experiment (W [883] = 259587, P < 0.001). Within large and small groups separately, there was a significant difference in wing sizes between large mated (n = 122, mean = 4.825 ± 0.019) and large not mated flies (n = 323, mean = 4.745 ± 0.013) (W [442] = 31124, P < 0.001), but this size difference was not significant between small mated (n = 32, mean = 4.342 ± 0.067) and small unmated flies (n = 407, mean = 4.371 ± 0.018) (t = -0.42, df = 437, P = 0.673) (Figure 4.6).

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni117

5.5

5.0

4.5

4.0

Wign length (mm) (mm) length Wign

3.5

3.0

Large Small Large mated Small mated Large not mated Small not mated

Figure 4.6: Bactrocera tryoni wing length for large (n = 446) and small (n =

439) males; large mated (n = 122) and large not-mated (n = 323) males; and small

mated (n = 32) and small not-mated (n = 407) males. (• indicates 5th and 95th

percentiles for each sample).

4.3.2 Experiment 2- Video analysis of mating behaviours

All behaviours described in Chapter 3 (Table 3.2) were observed when two

males and a female were in the same arena, be they male behaviours directed to the

female, male courtship behaviours directed towards the other male (including male-

male mounting attempts), or female behaviours directed to males (of either

treatment).

11886 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

4.3.2.1 Male and female behaviour analyses Male behaviours between young versus old

Sixteen male behaviours were identified across the nine replicates of the age- class treatments (Figure 4.7a). Among them, directing wings and quick flights were the two most commonly observed behaviours in both young and old males (Figure

4.7a).

Male behaviours between large versus small

Sixteen behaviours were identified between large and small males in 14 replicates of the size-class treatments (Figure 4.7b). Directing wings and quick flights were again the two most commonly observed behaviours in both large and small males, although attacking the other male and female approaches were also common (Figure 4.7b).

For those behaviours with enough replication to be analysed, none were significantly different between young and old males (Table 4.1 and 4.3) or between large and small males (Table 4.2 and 4.3).

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni119

18 Young males (a) Old males 16

25 (b) Large males Small males

Total number of replicates with the behaviour present behaviour the with of Total number replicates

Wing fan

Initial mount Quick flight

Directing wings

Fore legs tap maleMountingMale-male attempts mounts Successful mounts Attack maleFore by legs head tap female Attack female by head

Coming towards the male Coming towards the female

Male-male face-to-face touching Male-female face-to-face touching

Male fore legs interacting with female/male abdomen/genitalia

12086 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Figure 4.7: Total number of Bactrocera tryoni mate-choice test replicates with

particular male behaviours present, categorised between (a) young and old males

(total n = 9 replicates) and (b) large and small males (total n = 14 replicates). With

two males within one replicate, the maximum number of times a behaviour can occur

= 2 x n.

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni121

122

Table 4.1: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for male

Chapter 4: Chapter Bactrocera tryoni courtship and interaction behaviours compared between young and old males without considering if eventual mating was

successful or unsuccessful.

Effectsize,of male body and agepremat

Male-female face-to-face Male-male face-to-face Fore-legs tapping male's Directing wings Quick flights

touching touching body

Replicate number Young Total Total Total Total Total Total Total Total Total Total duration (s) number of duration (s) number of duration (s) number of duration (s) number of duration (s) number of occurrences occurrences occurrences occurrences occurrences 1 - 1 - 0 - 0 331.46 4 - 4

2 - 0 - 1 - 0 0.00 0 - 6 ing experience male ing mating successBactrocera on in tryoni 4 - 10 - 0 - 0 0.00 0 - 2 5 - 7 - 0 - 2 11.72 1 - 40 6 - 0 - 0 - 0 31.53 7 - 15 7 - 0 - 0 - 0 44.88 4 - 10 8 - 0 - 6 - 3 74.71 1 - 59 9 - 0 - 0 - 2 18.35 2 - 9 10 - 0 - 0 - 0 0.00 0 - 10 Mean - 2.00 - 0.78 - 0.78 56.96 2.11 - 17.22 SD - 3.77 - 1.99 - 1.20 105.91 2.42 - 19.28 SE - 1.26 - 0.66 - 0.40 35.30 0.81 - 6.43

86 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Chapter 4: Chapter

Old

Effectsize,of male experience body and agemalepremating mating successBactrocera on in tryoni 1 - 0 - 1 - 1 21.45 6 - 33 2 - 2 - 1 - 0 9.97 2 - 9 4 - 0 - 0 - 0 0.00 0 - 4 5 - 2 - 0 - 0 0.00 0 - 4 6 - 0 - 0 - 0 10.14 5 - 35 7 - 0 - 0 - 0 1.76 1 - 0 8 - 3 - 1 - 6 23.94 3 - 18 9 - 0 - 1 - 1 11.47 3 - 5 10 - 0 - 0 - 0 381.46 7 - 10 Mean - 0.78 - 0.44 - 0.89 51.13 3.00 - 13.11 SD - 1.20 - 0.53 - 1.96 124.17 2.55 - 12.89 SE - 0.40 - 0.18 - 0.65 41.39 0.85 - 4.30

123

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni123

124

Table 4.2: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for male

Bactrocera tryoni courtship and interaction behaviours compared between large and small males without considering if eventual mating was Chapter 4: Chapter

successful or unsuccessful.

Effectsize,of male experience body and agemalepremating mating success on in

Male-male face-to-face Coming towards other Attack other male by Coming towards female Directing wings Quick flights

touching male head

number Replicate

Large Total Total Total Total number Total Total number Total Total number Total Total number Total Total number duration number of duration of duration of duration of duration of duration of occurrences (s) occurrences (s) occurrences (s) occurrences (s) occurrences (s) occurrences (s) 1 - 0 0.00 0 0.00 0 - 0 19.51 4 - 6 2 - 1 10.24 5 2.72 2 - 2 85.42 16 - 1 3 - 2 42.14 7 47.21 6 - 4 6.94 3 - 24 4 - 2 648.58 4 78.31 11 - 4 0.00 0 - 51 5 - 1 0.00 0 2.76 1 - 3 0.28 1 - 0 6 - 0 13.32 2 1.36 1 - 1 0.00 0 - 83 7 - 0 2.28 1 2.72 1 - 3 16.64 8 - 0 8 - 1 0.00 0 0.00 0 - 2 1.80 2 - 2 9 - 5 4.59 1 9.55 4 - 2 103.27 4 - 11 10 - 0 5.56 1 0.00 0 - 0 13.84 2 - 0 11 - 0 2.36 1 7.84 2 - 0 16.16 4 - 3 12 - 0 20.85 3 115.70 6 - 9 0.00 0 - 17

13 - 1 7.42 1 0.00 0 - 2 100.22 7 - 8

Bactroceratryoni 14 - 1 0.00 0 0.00 0 - 0 0.00 0 - 5 Mean - 1.00 54.10 1.86 19.15 2.43 - 2.29 26.01 3.64 - 15.07

86 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Chapter 4: Chapter

SD - 1.36 171.49 2.14 35.94 3.25 - 2.40 38.93 4.38 - 23.91

SE Effectsize,of male experience body and agemalepremating mating successBactrocera on in tryoni - 0.36 45.83 0.57 9.60 0.87 - 0.64 10.40 1.17 - 6.39 Small

1 - 3 0.80 1 7.31 1 - 0 155.32 5 - 1 2 - 1 10.80 1 3.64 1 - 1 71.50 16 - 42 3 - 2 12.15 3 7.67 4 - 1 171.80 16 - 48 4 - 2 0.00 0 3.60 2 - 3 49.35 7 - 24 5 - 1 36.04 10 98.88 4 - 0 105.16 27 - 1 6 - 0 5.20 3 31.92 2 - 2 59.40 14 - 0 7 - 0 0.00 0 0.00 0 - 1 14.88 1 - 2 8 - 0 0.00 0 10.48 5 - 3 188.20 27 - 16 9 - 5 5.30 3 70.00 1 - 10 100.52 27 - 24 10 - 0 0.00 0 0.00 0 - 1 2.92 1 - 0 11 - 1 2.08 1 0.00 0 - 1 277.04 10 - 3 12 - 0 8.57 4 47.67 15 - 13 0.00 0 - 203 13 - 1 1.13 1 0.00 0 - 0 3.67 2 - 24 14 - 1 0.00 0 0.00 0 - 0 0.00 0 - 13 Mean - 1.21 5.86 1.93 20.08 2.50 - 2.57 85.70 10.93 - 28.64 SD - 1.42 9.67 2.70 31.09 3.98 - 3.96 85.70 10.39 - 52.59 SE - 0.38 2.59 0.72 8.31 1.06 - 1.06 22.90 2.78 - 14.06

125

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni125

Table 4.3: Statistical test results for comparisons of the number and duration of male

Bactrocera tryoni courtship behaviours between young and old males and large and

small males. Occurring in replicates with and without successful copulations. Blank

cells represent the absence of ‘duration’ as the behaviours are point behaviours.

Male behaviours W/t and p values for Wilcoxon rank sum test/ 2 sample t test For total number of For total duration Young and old males occurrences Male-female face-to-face touching W (16) = 87 P = 0.9162 - Male-male face-to-face touching W (16) = 78.5 P = 0.4878 - Fore-legs tapping young male's body W (16) = 87 P = 0.9161 - Directing wings t = -0.91 df = 16 P = 0.390 W (16) = 92 P = 0.5924 Quick flights W (16) = 91.5 P = 0.6256 -

Large and small males Male-male face-to-face touching W (26) = 193 P = 0 .6439 - Coming towards female W (26) = 208 P = 0.8303 - Coming towards male W (26) = 205 P = 0.9434 - Attack male by head W (26) = 215.5 P = 0.5734 - Directing wings W (26) = 164.5 P = 0.0785 - Quick flights W (26) = 188.5 P = 0.5180 -

12686 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Female behaviours towards young and old males

Eight behaviours were recorded when displayed by females towards young and old males (Figure 4.8a). Among them, female hind-legs interacting with male’s body was the most commonly observed female behaviour towards both young and old males. Male-female face-to-face touching and coming towards young male were observed more often as compared with old males, while successful mounts with old males were absent (Figure 4.8a). Further, females more frequently attacked old males with their head than they did towards young males (Figure 4.8a).

Female behaviours towards large and small males

The same eight behaviours categorised female activity towards large and small males (Figure 4.8b). The female coming towards large and small males was frequently observed and male-female face-to-face touching and female hind-legs interacting with male’s body were commonly observed more towards large males compared to small males. Females more frequently attacked small males compared to large males (Figure 4.8a, b).

Statistical analysis identified that no female behaviours were significantly different towards young and old males (Table 4.4 and 4.6), as well as towards large and small males (Table 4.5 and 4.6).

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni127

Towards young male 8 (a) Towards old male

20 (b) Towards large male Towards small male

Total number of replicates with the behaviour present behaviour the with of number Total replicates 10

Mountings Initial mount

Fore legs tap male Successful mounts Attack male by head

Coming towards the male

Male-female face-to-face touching

Female hind legs interacting with males body

12886 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Figure 4.8: Total number of Bactrocera tryoni mate-choice test replicates with

particular female behaviours directed towards (a) young and old males (total n = 9

replicates), and (b) large and small males (total n = 14 replicates).

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni129

130

Table 4.4: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for female

Bactrocera tryoni courtship and interaction behaviours compared between young and old males without considering if eventual mating was

Chapter 4: Chapter successful or unsuccessful.

Effectsize,of male experience body and agemalepremating mating successBactrocera on in tryoni

Hind-legs interacting with male Male-female face-to-face touching Coming towards male Attack male by head

body

Replicate number Young Total Total number of Total Total number of Total Total number of Total Total number of duration (s) occurrences duration (s) occurrences duration (s) occurrences duration (s) occurrences 1 - 0 6.54 3 13.43 6 - 0 2 - 0 0.00 0 4.34 5 - 0 4 - 13 3.13 1 0.00 0 - 1 5 - 4 0.00 0 0.00 0 - 0 6 - 0 0.00 0 0.00 0 - 0 7 - 0 0.00 0 2.24 1 - 0 8 - 0 0.00 0 10.30 1 - 0 9 - 0 0.00 0 0.00 0 - 0 10 - 0 0.00 0 0.00 0 - 0 Mean - 1.89 1.07 0.44 3.37 1.44 - 0.11 SD - 4.37 2.30 1.01 5.10 2.35 - 0.33 SE - 1.46 0.77 0.34 1.70 0.78 - 0.11 Old

1 - 1 0.87 2 0.83 1 - 0 2 - 2 4.21 3 30.74 3 - 0 4 - 0 0.00 0 0.00 0 - 1

86 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Chapter 4: Chapter

5 - 0 0.00 0 0.00 0 - 0

Effectof ma 6 - 0 0.00 0 0.00 0 - 0 7 - 0 0.00 0 0.00 0 - 0 8 - 0 0.00 0 20.19 2 - 3 le body size, experience body le and agemalepremating mating successBactrocera on in tryoni 9 - 0 0.00 0 0.00 0 - 1 10 - 0 0.00 0 0.00 0 - 1 Mean - 0.33 0.56 0.56 5.75 0.67 - 0.67 SD - 0.71 1.40 1.13 11.49 1.12 - 1.00 SE - 0.24 0.47 0.38 3.83 0.37 - 0.33

131

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni131

132

Table 4.5: Means, standard deviations (SD) and standard errors (SE) calculated for total durations (s) and total number of occurrences for female

Bactrocera tryoni courtship and interaction behaviours compared between large and small males without considering if eventual mating was Chapter 4: Chapter

successful or unsuccessful.

Effectsize,of male experience body and agemalepremating mating successBactrocera on in tryoni

Hind-legs interacting with male Male-female face-to-face touching Coming towards male Attack male by head

body

Replicate number Large Total Total number of Total Total number of Total Total number of Total Total number of duration (s) occurrences duration (s) occurrences duration (s) occurrences duration (s) occurrences 1 - 0 8.17 2 0.00 0 - 2 2 - 0 0.00 0 0.00 0 - 0 3 - 1 16.45 7 0.00 0 - 1 4 - 2 32.06 4 3.17 1 - 0 5 - 0 2.84 2 0.00 0 - 1 6 - 1 10.80 6 35.04 3 - 6 7 - 1 9.12 3 0.00 0 - 0 8 - 0 0.96 1 0.00 0 - 0 9 - 4 4.09 2 0.00 0 - 0 10 - 0 0.00 0 0.00 0 - 2 11 - 0 4.84 2 0.00 0 - 4 12 - 0 0.00 0 0.00 0 - 0 13 - 1 0.00 0 0.00 0 - 0 14 - 0 12.64 1 0.83 1 - 0 Mean - 0.71 7.28 2.14 2.79 0.36 - 1.14 SD - 1.14 8.91 2.21 9.32 0.84 - 1.83

86 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Chapter 4: Chapter

SE - 0.30 2.38 0.59 2.49 0.23 - 0.49

Effectsize,of male experien body and agepremating Small

1 - 0 17.05 5 0.00 0 - 1 2 - 0 4.54 2 0.00 0 - 1 3 - 0 59.16 6 6.94 2 - 1 4 - 0 7.34 1 1.27 1 - 3 5 - 0 1.12 1 0.00 0 - 3 6 - 0 5.32 3 0.00 0 - 0 7 - 2 0.00 0 0.00 0 - 0 8 - 0 0.00 0 0.00 0 - 2 9 - 4 13.01 3 64.16 1 - 0 10 - 0 0.00 0 0.00 0 - 1 11 - 1 3.16 1 2.96 1 - 0 12

cemale mating successBactrocera on in tryoni - 0 0.00 0 0.00 0 - 1 13 - 2 0.00 0 37.33 1 - 1 14 - 0 2.75 1 18.89 1 - 1 Mean - 0.64 8.10 1.64 9.40 0.50 - 1.07 SD - 1.22 15.60 1.95 18.99 0.65 - 1.00 SE - 0.32 4.17 0.52 5.08 0.17 - 0.27

133

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni133

Table 4.6: Statistical data including W/t values for Wilcoxon rank sum tests/ 2

sample t tests for all compared female behaviours towards young and old males and

large and small males

Female behaviours W/t and p values for Wilcoxon rank sum test/ 2 sample t test Towards young and old males For total number of For total duration occurrences Male-female face-to-face touching W (16) = 87.50 P = 0.86 - Coming towards male W (16) = 85 P = 1.00 W (16) = 86.50 P = 0.96 Hind-legs interacting with male body W (16) = 91 P = 0.61 W (16) = 88 P = 0.84 Attack male by head W (16) = 71.5 P = 0.13 - Towards large and small males

Male-male face-to-face touching W (26) = 212.50 P = 0.63 - Coming towards male W (26) = 217.50 P = 0.51 W (26) = 213 P = 0.66 Hind-legs interacting with male body W (26) = 184 P = 0.30 W (26) = 179 P = 0.19 Attack male by head W (26) = 187 P = 0.45 -

13486 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

4.3.2.2 Male and female ethograms Courtship ethograms, incorporating behaviours seen when two males were simultaneously exposed to a female, are presented in Figure 4.9. All males and females showed the same flow of courtship behaviours as recorded in Chapter 3, except that there were male-male interactions (pre-mounting behaviours and mounting attempts) observed in treatments where two males were present (Figure

4.9a).

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni135

13686 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

Figure 4.9: General flow of Bactrocera tryoni courtship behavioural sequence for

(a) young/old/large/small males combined and (b) females (n = 20). These were

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni137

constructed combining all recorded videos under “age” treatment and “size”

treatment considering all males and females separately.

4.4 Discussion

4.4.1 Summary of results

The first objective of this chapter was to examine whether B. tryoni males of

different physiological states, as tested through age, size and sexual experience, had

any differential success with respect to mating with virgin females when the

experiments were carried out using laboratory reared flies under manipulated, semi-

natural conditions. It was found that young males were significantly more successful

when securing a female than old males. I also found that large males were

significantly more successful at mating than small males. These observations confirm

the alternative hypothesis of there being a difference between treatments of young

versus old males and large versus small males. However, there was no significant

difference between males that had previously mated or were virgin when presented

to virgin females. Studying close range behaviour, the replicates with successful

copulations were low in all compared treatments, presumably because the very small

arena of a Petri-dish (dictated by the need of high resolution video recording) led to

the males interfering with each other. Therefore, applying statistical analysis to

compare behavioural differences between successful copulations and unsuccessful

copulations was not possible. Among all other courtship behaviours within age-class

treatment and size-class treatment, there were no significant behavioural differences

between young versus old or large versus small males in both total durations as well

as total number of occurrences. Based on the number of replicates carried out,

comparing female behaviours towards young, old, large and small males, also

13886 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

revealed an absence of behavioural differences either towards young and old males or towards large and small males.

All the couples that were collected in the field cage experiments occurred on the cage wall or roof and none were on the tree. The guidelines for cage mating tests state that flies must mate on the tree when calculating compatibility and competitiveness (FAO/IAEA/USDA., 2014) but it is not clear upon what basis this guideline is made. As I recorded in my second chapter, B. tryoni prefer to mate on high places within their mating arena, and here in Chapter 4’s experiments 100% of mating pairs occurred on the upper cage wall or roof, reinforcing that it is a certain height, rather than the substrate, that is important for B. tryoni mating.

4.4.2 Why young and large males are more reproductively successful than old

and small males? – Evidence from cage trials

Bactrocera tryoni male mating success can be due to three reasons; male fitness, female selection, or both. Male fitness can be further categorised as the ability of males to overcome female rejection, the physical ability of males to move around and find a female (i.e., intrinsic male motivation to mate), or the greater ability of males to compete with other males when securing a female. In other tephritids there is no difference in mating success between young and old males when they are presented to females separately (i.e., in no choice tests), but as older males may decline in strength (Cremer et al., 2012) and sexual performance their mating likelihood will decrease in the presence of young males due to failure in competition (Papanastasiou et al., 2011, Abraham et al., 2016). Extrapolating to my results, young B. tryoni males might have more drive and physical strength to move around and find a mate and win competitions with other males when securing a female.

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni139

Having competition for food during larval development makes flies physically

small and with lower strength than if raised in non-competitive food environments

(Prabhu et al., 2008, Fanson et al., 2009, Dominiak and Daniels, 2012, Shenoi and

Prasad, 2016, Mason et al., 2016). As shown in C. capitata, small males are more

likely to forage for food than search for mates, as they have lower carbohydrate and

protein reserves as a result of the nutritional stress during larval development (Kaspi

et al., 2000, Yuval et al., 2002). Therefore, when there were both large and small B.

tryoni males in the same cage, the small males may have been less actively searching

for females than the large males, hence they may lack motivation to mate and

strength to compete with large males. Additionally, both the small and old males

may not have been strong enough to avoid female rejection when attempting

copulation.

Females might be able to discriminate male qualities (e.g., acoustic signals,

pheromones, etc.) directly and accept or reject a male as seen in other tephritids

(Webb et al., 1984, Bonduriansky and Brassil, 2005, Pérez‐Staples et al., 2013).

They may select a certain type of male based on various secondary evolutionary

advantages, such as avoidance of genetic mutations or increased female fitness (e.g.,

clutch size, amount of sperm available for storage) (Whittier and Kaneshiro, 1991,

Shelly and Whittier, 1993, Aluja et al., 2008). For example, C. capitata females

mated with large males store more sperm compared to females mated with small

males (Taylor and Yuval, 1999).

The way my field cage tests were carried out does not allow for separation of

alternating hypotheses of male ability, female choice, or both, to explain the sexual

selection observed. While standing in the cages carrying out the trials I noticed no

14086 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

obvious behaviours that might be associated with mate choice. For these reasons the close range behavioural analyses were carried out.

4.4.3 Can we explain the reproductive success of young and large males over old

and small males from close range behavioural observations?

From close range behavioural observations, I recorded a few interesting trends, although none were statistically significant. The absence of significance is arguably due to low replication. However, having differences in male and female behaviours among treatments reveals some effect of male motivation and strength, as well as female acceptance or rejections for male mating success.

As observed in Chapter 3, and in this chapter also, repeated observations of male synchronous supination reaffirm it as a potentially critical element in mate recognition or, perhaps, a behaviour which is associated with intra-sexual communication or competition. Increased male-male interactions such as male-male face-to-face touching and attacking the other male by head pushing, which were obviously absent in no choice tests (Chapter 3) and led to larger numbers of short quick flights, might have resulted in the few successful copulations seen in this chapter. These behaviours provide evidence for the presence of competitive interactions between males.

Although there was no significant difference in female behaviours towards young, old, large or small males, having an apparently increased rate of female aggression towards old and small males (based on the number of replicates in which this behaviour was seen) reveals the possibility of increased rejection by females of small and old males. Further, with face-to-face touching, and female hind-leg interactions suggests that there might be other, possibly contact factors associated

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni141

with mating success or failure. Non-evaporative contact pheromones (e.g., CHCs)

are a likely mechanism by which differential post-mounting mate choice may be

made in Diptera (Howard et al., 2003, Chenoweth and Blows, 2005, Ingleby et al.,

2014). With no existing experimental work on male CHC variation in B. tryoni, I

might hypothesise that obtaining more mating pairs by young males may be due to

female preference of the CHC profile of young males over old males.

4.4.4 No effect of mating experience for mating success A likely explanation for the lack of effect of B. tryoni prior mating experience

on mating success might be the effect of accessory gland fluids. In C. capitata,

although the mechanism of female detection is unknown, it is suggested that the

mating probability differences between virgin and non-virgin males is due to a

depletion of some components of the ejaculate such that the fecundity of females

mating with mated males is lowered (Whittier and Kaneshiro, 1991, Shelly and

Whittier, 1993). Males transfer accessory gland fluids into the female reproductive

tract with sperm at mating (Chen, 1984, Radhakrishnan and Taylor, 2007, Avila et

al., 2011). Females obtain numerous advantages by receiving accessory gland fluids

during copulation, such as enhanced ovulation and egg production rates, modulation

of feeding behaviours, and protection of the female reproductive tract and eggs from

bacterial infections (Gillott, 2003, Avila et al., 2011). Females can detect the absence

of accessory gland fluids in previously mated males and reject them (Markow, 1987),

but the mechanism by which this is achieved is unclear. Production of accessory

gland fluids is positively correlated with the amount of insect juvenile hormone

(Chen, 1984) and mated males with more juvenile hormone (Teal et al., 2000) will

increase the production of accessory gland fluids. Bactrocera tryoni males take 16 -

22 hours to recover their accessory glands into full size from its contracted size after

14286 Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni

a successful copulation (Radhakrishnan and Taylor, 2008). Since B. tryoni males can only mate once per day, as their mating time period is restricted to few minutes at dusk (Chapter 2), there is no difference in the ejaculate between a virgin and a mated male (Fay and Meats, 1983, Radhakrishnan et al., 2009). This might be a reason for not observing any difference in the number of mating pairs with virgin and previously mated B. tryoni males.

4.4.5 Next chapter While making an initial mounting attempt is a key courtship transition step leading to successful mating in B. tryoni, I have also identified failure to proceed from mounting to successful copulation as another likely critical step associated with mating and mate choice in B. tryoni. Other authors have also identified this as a key element in B. tryoni mating (Barton-Browne, 1957; Kumaran et al., 2014b). Based on literature in other Diptera, cuticular hydrocarbons (CHCs) may play an important role in mate choice at this stage of the courtship process (Howard et al., 2003,

Chenoweth and Blows, 2005, Ingleby et al., 2014). Within tephritids, CHCs have been shown to vary between species, sexes, and maturation stages (Vanickova et al.,

2012, Zunic Kosi et al., 2013, Vanickova et al., 2014, Vanickova et al., 2015a,

Vanickova et al., 2015b, Vanickova et al., 2015c) but correlations between differences in CHC profiles and mating success have not been tested. Based on the results of this chapter, and the hypothesis proposed in section 4.4.2, the next and final research chapter of my thesis focuses on the role of CHCs in B. tryoni courtship.

Chapter 4: Effect of male body size, age and premating experience on male mating success in Bactrocera tryoni143

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 145

5.1 Introduction

In previous chapters (Chapter 3), I have demonstrated that there is a clear copulation success variation in Bactrocera tryoni males due to male physiological changes such as body size and age. That is young and large males are more successful in copulations compared to old and small males. It was suggested that, this variation might be due to various mechanisms. For instance, it can be due to behavioural differences or chemical/ acoustic signal differences. Among these aspects, behavioural differences were tested in chapter 4 and it was observed, there is no significant difference in male behaviours due to the physiological state (i.e., young/ old/ large/ small), but there is evidence explaining that the copulation success of a male greatly depend, on whether he has performed copulation attempts onto the female or not. That means, the possibility for having a successful copulation is lower if the male has not performed any mounting attempts (Section 3.3.3). But, male mounting attempts may not be the only reason for a successful copulation, females may also affect male mating success by accepting or rejecting males who have mounted onto them (Section 3.3.3). Therefore it is assumable that, male and female

B. tryoni flies might recognise or confirm their selection based on cuticular contact pheromones (e.g., CHCs) which are present on the outer surface of the insect body, as a suitable mating partner, after mounting onto the other individual.

Except insect contact pheromones, evaporative sex pheromones and acoustic signals might also have an effect for their success. It has been observed that there are differences in sex pheromone production and acoustic signal frequencies due to male physiological state and for mate choice in tephritids as described in sections 1.4.2.,

1.6.2 and 4.1 and in section 1.7.2.3 for B. tryoni. The differences in CHC compositions in tephritids have been studied under limited categories. Except one

14686 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

study under sexual dimorphism and age depended differences of CHCs in

Anastrepha fraterculus (Vanickova et al., 2012), the studies on the effect of CHC for tephritid mate recognition is considerably less. In species under the genus

Bactrocera, there is a study comparing the CHC compositions of two species in the

Malaysian B. dorsalis complex (considered as Bactrocera Malaysia A and B) and which obtained significant differences in the distribution of hydrocarbons between them (Goh et al., 1993), but could not be associated with mate recognition. When considering B. tryoni CHC variations, there are no studies conducted on B. tryoni

CHCs under inter-sexual or intra-sexual communication or at least there are no studies which have documented sexual dimorphism in B. tryoni CHCs. Since among all other insect sexual chemical communication methods, communication via CHCs is one of the areas that has a major gap in knowledge for B. tryoni, this chapter will investigate the effect of B. tryoni inter-sexual CHC differences as well as intra-sexual variations (e.g., any aggregation effect) for their mating success. As described in section 1.6.2.4. for Drosophilid females, B. tryoni females might also have a difference, in their cuticular compound compositions which will affect male acceptance (Since males are the ones who have mounted onto females as observed in previous chapters) followed by their mating success.

In previous studies, the possibility of transferring cuticular compounds between individuals of the same species or different species in many insect taxa such as ants

(Bagneres et al., 1991), cockroaches (Everaerts et al., 1997) and termites (Vauchot et al., 1997) is suggested. This transfer of compounds can occur actively, that is, by producing the compounds as a chemical mimicry or passively by contact. For instance, when young worker males (less than 24 hours old) and pupae of the ant species Manica rubida (Latreille) and Formica selysi Bondroit were reared together,

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 147

the cuticular profile of these individuals have shown a chemically related cuticular profiles compared to the individuals reared separately (Bagneres et al., 1991).

Although the exact mechanism is unknown, it is explained that, an active synthesis of cuticular compounds in both species has occurred when reared heterospecifically

(Bagneres et al., 1991). In contrast, a passive transfer by contact has been observed between two termite species, Reticulitermes santonensis (Feytaud) and R. lucifugus grassei Clement with a significant difference of their cuticular profiles observed within 2 hours after placed together (Vauchot et al., 1997).

Therefore based on the above facts, the focus of this chapter is resolving the cuticular chemical profiles of B. tryoni and the primary objectives are, to determine compound composition of B. tryoni cuticular profiles, whether there is a sexual dimorphism in B. tryoni cuticluar compounds and whether any of these compounds can be implicitly implicated in playing a role in inter/intra sexual communication as documented for other insects. Secondarily, to evaluate for the potential for cuticular compounds to vary between males under a mate choice scenario and to see if indeed different chemical signatures between males may account for mating success.

Since studies on Tephritidae CHC variations for their mating success are rare, the hypotheses made under this objective were mainly based on previous findings.

The mating trials on Drosophilidae were carried out using laboratory reared flies under semi-natural conditions by keeping two sexually mature males together with a sexually mature female of same age and same size. I hypothesised that, there is a sexual dimorphism in B. tryoni cuticular composition and also between successfully mated males/females and unsuccessful males/females. The cuticular compounds of each mated and unmated males and females of laboratory trials were extracted separately and quantified/ analysed using mass-spectrometry and multifactorial

14886 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

statistical analyses first to evaluate the cuticular compound composition and then to assess the above hypotheses.

5.2 Materials and methods

5.2.1 Source of insect material Bactrocera tryoni pupae were obtained from laboratory cultures (F20- F30 generations) maintained by the Queensland Government Department of Agriculture and Fisheries, Brisbane, Australia. Emergent adults of both species were separated by sex within four days of emergence (i.e., before sexual maturity) and held at densities of 125 individuals per cage (30 x 20 x 20 cm) under laboratory conditions of 27 ºC and 70% relative humidity. Flies were supplied with sugar, water and protein hydrolysate ad libitum. Flies were held until they reached sexual maturity, and they were separated within 2-3 days of emergence into two separate cages.

5.2.2 Experimental setup

Sixty replicates were carried out on the same day and, for each of the replicates, two virgin males and one virgin female were placed inside a small Petri- dish (100 x 15 mm) and kept together until the time of mating (i.e., dusk and an hour after) under natural dusk lighting conditions at 27 °C temperature and 56% relative humidity. Flies were sourced from the same stock colony and all males and females were the same age (18 days old after emergence). When they began copulating, each

Petri-dish with the mating pair was placed inside a small white net cage (30 x 20 x 20 cm) in order to prevent flies from escaping, the Petri-dish opened and the mating pair and the remaining single male were carefully collected (i.e., allowing the mating pair to remain in copulation) into two separate diet cups and labelled. In replicates where mating occurred the successful males were labelled as ‘C’ and unsuccessful males as

‘R2’. In replicates where mating did not occur, both unsuccessful males were

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 149

labelled as either ‘R1’ and ‘R2’ (with random assignment to either category): thus

the number of R2 males always equals 60 and the number of R1 males equals the

number of replicates in which mating did not occur. All females in both ‘did mate’

and ‘didn’t mate’ replicates were labelled as ‘F’. All labelled diet cups were put into

plastic zip lock bags and stored in a freezer at -4 °C until the next morning.

5.2.3 CHC extraction procedure

The solvent extraction of fly cuticular compounds was carried out on the

morning following the mating trials. Each fly was transferred into labelled Eppendorf

tubes and kept for 20 min in 400 µl of 99% methanol to extract CHCs. The CHC-

methanol extraction was then filtered into 2 ml GC vials and labelled based on the

replicate number, sex of the fly, and if male if they were mated or unmated. Flies

were kept in their individual Eppendorf tubes and stored in a freezer for any

subsequent analysis. According to the same method, two negative control samples

(i.e., only 99% methanol samples without flies) were also prepared. The GC vials

with the CHC-methanol extractions were kept in the freezer until analysis.

5.2.4 Chemical analysis

The CHC compounds in the samples were qualitatively and quantitatively

identified by GC-MS under liquid extraction method to test for differences between

males, females and successfully mated and not mated males. The analysis was

carried out on a gas chromatograph ISQTM Series single quadrupole GC-MS system

(Thermo Fisher, United Sates) connected to the analysing software Thermo Xcalibur

version 2.1, located at the Central Analytical Research Facility (CARF), Queensland

University of Technology. The injector was operated in the splitless mode at 80 °C.

The column flow rate was 1 ml/min and the ion source temperature was 280 °C. The

15086 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

oven start temperature was 80 °C held for 1 min, 20 °C/min to 280 °C and held for 0 min and 10 °C/min to 320 °C and held for 5 min. Oven maximum temperature was

350 °C. A sample volume of 1 µl was analysed for 20 min with an equilibration time of 0.5 min. Possible compounds of each peak were identified by doing a library search using NIST (National Institute for Standards and Technology) and peak areas were calculated using Thermo Xcalibur version 2.1 software. Putative identified compounds were not compared to pure standards of those compounds, and therefor the compound identifications should be regarded as tentative until further analysis, using standards, can be done.

5.2.5 Statistical analysis From the 60 replicates there were 51 replicates with copulations and nine without. Therefore, there were altogether 51 successful males (C), 51 successful females and nine unsuccessful females, 60 R2 unsuccessful males and nine R1 unsuccessful males. Absolute values of peak areas were exported into Microsoft

Excel 2010 files. Analysis of variance (ANOVA) tests were performed at 95% confidence interval to find peak area differences between;

i. Regardless of mating or otherwise, all males were compared with all

females to find peak area differences between males and females.

Therefore 120 males were compared with 60 females.

ii. Considering replicates with successful copulations only, successfully copulated males (= C males) were compared with unsuccessful males (n = 51).

iii. Successful females in did-mate replicates were compared with unsuccessful females in didn’t-mate replicates.

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 151

A log transformation for all the absolute peak areas was carried out prior analysing. All of the analyses were done using Minitab 16.0.

5.3 Results

5.3.1 Male and female CHC profiles

Fourteen prominent peaks were detected in both male and female CHC profiles

(Table 5.1), derivatives of a few of which have been recorded in other tephritids

(Appendix 1). The 14 recorded compounds were identified as the most likely compounds through the NIST library search. Within the NIST search, the compounds were given at the maximum probability (i.e., a probability factor based on the differences between adjacent hits in an SI ordered list) for each peak for each fly individual extraction. Although all these 14 peaks were present in all males and females, their peak areas were different as described in the next section.

86152 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

i. Comparing all males and all females

Among the 14 prominent peaks, the compound areas of 10 were significantly different between males and females: Dodecanoic acid ethyl ester (F59, 119 = 43.77,

P<0.001), Heptadecanoic acid ethylester (F59, 119 = 66.8, P<0.001), E-11- hexadecenoic acid ethyl ester (F59, 119 = 61.6, P<0.001), Hexadecanoic acid (F59, 119 =

75.28, P<0.001), 13-methylheptacosane (F59, 119 = 6.92, P = 0.009), 2,3- dimethylnonane (F59, 119 = 6.21, P = 0.014), 9-methylnonadecane (F59, 119 = 10.53, P

= 0.001), 7-methylpentadecane (F59, 119 = 6.58, P = 0.011), 2-methyloctacosane (F59,

119 = 7.27, P = 0.008) and 2-methylnonadecane (F59, 119 = 3.95, P = 0.048) were significantly different between all males and all females. Peak areas of Tetradecanoic acid ethyl ester (F59, 119 = 1.28, P = 0.259), Dodecamethyl-cyclohexasiloxane (F59, 119

= 0.87, P = 0.353), Tetradecamethylcylcloheptasiloxane (F59, 119 = 0.03, P = 0.859) and 2, 3-dimethylheptadecane (F59, 119 = 1.67, P = 0.198) were not statistically different between all males and all females (Table 5.2, Figure 5.1 and 5.2).

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 153

154

Table 5.1: Fourteen possible compounds at their RTs detected in both male and female Bactrocera tryoni flies. Other information includes the

type of compound (e.g., an alkane, acid, ester, etc.), notes on their common names and previous records in animals and plants.

RT Possible compound Type of compound Examples for previous records and uses

Chapter 5: Chapter 4.56 Tetradecanoic acid ethyl ester Ester of a saturated fatty acid Known as Myristic acid ethyl ester and

found as a main compound in the waxy

The role The of Bactroceracuticular hydrocarbons tryoniin and mate mating choice covering of Aphids (Giles et al., 2002) (Source: NIST) and in egg extractions of European grape vine moths (Gabel and Thiéry, 1996). 5.34 Dodecamethylcyclohexasiloxane A siloxane -

86 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

Chapter 5: Chapter

(Source: Open chemistry database)

The role The of Bactroceracuticular hydrocarbons tryoniin and mate mating choice 6.47 Tetradecamethyl-cylcloheptasiloxane A siloxane Found as a compound in the mycelia (i.e., the vegetative part of a fungus) of Hirsutella sinensis (Yu et al., 2011).

(Source: Open chemistry database) 7.09 Dodecanoic acid ethyl ester Ester of a fatty acid Acid form has been used as a mosquito repellent (Gupta et al., 2016).

(Source: Open chemistry database) 8.20 Heptadecanoic acid ethylester Ester of a fatty acid -

155

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 155

156

(Source: NIST) 9.15 E-11-hexadecenoic acid ethyl ester – prob.7.35% An ester of fatty acid Found on male butterfly wings as a Hexadecanoic courtship pheromone (Wang et al.,

Chapter 5: Chapter (Source: NIST) 2014)

9.24 Hexadecanoic acid Saturated fatty acid Common name: Palmitic acid and found

The role The of cuticular hy in animals and plants (e.g., Palm oil) (Gunstone et al., 2007)

drocarbons in Bactrocera drocarbonstryoniin and mate mating choice (Source: NIST) 14.32 13-methylheptacosane Methyl branched alkane A contact sex pheromone in many solitary insects (Vanickova et al., 2012,

Oi et al., 2015) (Source: Open chemistry database) 14.36 2,3-dimethylnonane Methyl branched alkane Derivatives of methyl branched compounds were found in flour beetles.

86 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

Chapter 5: Chapter

The role The of Bactroceracuticular hydrocarbons tryoniin and mate mating choice Considered as an aggregation pheromones (Suzuki, 2014)

(Source: Open chemistry database) 14.47 9-methylnonadecane Methyl branched alkane -

(Source: NIST) 14.50 7-methylpentadecane Methyl branched alkane -

(Source: NIST)

14.55 2-methyloctacosane Methyl branched alkane -

(Source: NIST) 14.69 2-methylnonadecane Methyl branched alkane -

157

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 157

158

(Source: NIST) 14.83 2,3-dimethylheptadecane Methyl branched alkane -

(Source: NIST) Chapter 5: Chapter

The role The of Bactroceracuticular hydrocarbons tryoniin and mate mating choice

86 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

Table 5.2: Log mean peak areas ± SE under each detected compound for all successful males, unsuccessful males and females in did mate replicates and unsuccessful males and females in didn’t mate replicates

Compound Log mean peak areas in Log mean peak areas in

did mate reps. (n = 51) didn’t mate reps. (n = 9)

C R2 F R1 R2 F

Tetradecanoic acid ethyl ester 12.65 12.87 13.05 13.32 12.69 12.18

± 0.16 ± 0.19 ± 0.22 ± 0.23 ± 0.43 ± 0.29

Dodecamethylcyclohexasiloxane 14.15 13.87 14.18 13.58 13.66 13.82

± 0.15 ± 0.20 ± 0.13 ± 0.30 ± 0.46 ± 0.42

Tetradecamethylcylcloheptasiloxane 13.66 13.44 13.64 13.80 13.66 13.43

± 0.12 ± 0.15 ± 0.11 ± 0.35 ± 0.19 ± 0.20

Dodecanoic acid ethyl ester 12.94 12.51 14.38 12.60 12.40 14.42

± 0.17 ± 0.17 ± 0.32 ± 0.30 ± 0.50 ± 0.74

Heptadecanoic acid ethylester 13.17 12.77 14.99 12.91 12.94 15.43

± 0.21 ± 0.16 ± 0.31 ± 0.48 ± 0.52 ± 0.64

E-11-hexadecenoic acid ethyl ester 12.99 12.84 14.54 12.83 12.86 14.89

± 0.14 ± 0.12 ± 0.28 ± 0.39 ± 0.28 ± 0.61

Hexadecanoic acid 12.72 12.54 14.40 12.73 12.53 14.56

± 0.14 ± 0.12 ± 0.26 ± 0.39 ± 0.34 ± 0.61

13-methylheptacosane 15.72 15.43 16.39 17.00 15.60 16.95

± 0.26 ± 0.28 ± 0.26 ± 0.47 ± 0.65 ± 0.65

2,3-dimethylnonane 15.04 14.74 15.53 15.35 14.90 15.41

± 0.20 ± 0.20 ± 0.23 ± 0.44 ± 0.43 ± 0.40

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 159

9-methylnonadecane 15.02 14.63 15.65 15.78 14.58 15.74

± 0.21 ± 0.22 ± 0.23 ± 0.49 ± 0.48 ± 0.41

7-methylpentadecane 14.88 14.65 15.43 15.41 14.76 15.31

± 0.20 ± 0.19 ± 0.22 ± 0.40 ± 0.46 ± 0.57

2-methyloctacosane 14.96 14.64 15.45 15.65 14.71 15.61

± 0.19 ± 0.21 ± 0.20 ± 0.39 ± 0.54 ± 0.52

2-methylnonadecane 15.53 15.27 16.02 16.31 15.58 16.21

± 0.25 ± 0.25 ± 0.26 ± 0.53 ± 0.54 ± 0.52

2,3-dimethylheptadecane 15.13 14.96 15.39 15.55 14.94 15.24

± 0.19 ± 0.22 ± 0.20 ± 0.54 ± 0.40 ± 0.43

16086 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

RT: 0.00 - 20.12 18.90 19.61 NL: 100 17.10 17.51 16.30 5.65E8 95 TIC MS (a) 15.68 24032016_ 15.44 90 btryoni_55 C 85 15.22 15.08 80 14.89 75 14.73 70 14.60 65 14.35 60 14.30 55 14.00 50 13.80 45 13.65

RelativeAbundance 40 13.45 13.30 35 12.98 30 12.71 12.46 25 11.99 20 11.72 11.42 15

10 10.68 10.24 9.74 5 3.40 3.58 5.61 6.21 6.63 9.06

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min)

RT: 0.00 - 20.10 19.24 NL: 17.26 19.11 100 16.95 5.57E8 16.19 95 TIC MS (b) 15.67 24032016_ 90 15.50 btryoni_55F

85 15.24

80 14.35

55 14.22

50 13.89

45 13.77 13.64

RelativeAbundance 40 13.43 13.17 35 8.22 30 12.82 12.55 25 12.27 9.16 7.10 11.98 20 11.58 15 9.25 10.96 10 10.57 8.17 5 3.22 3.54 5.60 6.21 8.82 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min)

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 161 RT: 0.00 - 20.10 19.46 19.70 NL: 100 17.69 18.11 5.41E8 16.59 95 TIC MS (c) 15.88 24032016_ 90 15.53 btryoni_55R 2 15.34 85 15.18 80 14.99 75

70 14.73

65 14.51 60 14.35 55 14.16 50 13.94 45 13.66

RelativeAbundance 40 13.50 13.28 35 13.08 30 12.49 25 12.23 11.99 20 11.58 15 11.18 10 10.78 10.35 3.11 9.90 5 3.55 5.36 6.21 6.79 9.17 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min) RT: 0.00 - 20.09 18.40 NL: 100 19.49 5.04E8 17.24 17.34 95 TIC MS 16.08 24032016_ (d) 15.72 90 btryoni_46R 15.57 1 15.41 85 15.27 15.20 80

75 14.88 70 14.35 65

55 14.24 50 14.04

45 13.70

RelativeAbundance 40 13.52 35 13.24 30 12.87 12.52 25 12.28 11.99 20 11.56 15 11.27 10 10.57 9.65 9.97 5 3.31 3.66 5.35 6.49 6.54 8.87 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min)

Figure 5.1: Examples of chromatographs generated from Bactrocera tryoni cuticular

hydrocarbon extracts of (a) successful male, (b) female, (c) unsuccessful male (all

from replicate number 55) and (d) R1 unsuccessful male (from replicate number 46)

16286 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

obtained by mass-spectrometry. Prominent peak differences are shown at RT 7.10,

8.22, 9.16, 9.25 and 14.35 which are significantly present in the female.

ii. Comparing successful males with unsuccessful males in replicates with

copulations

There were no statistically significant differences in peak areas between successful males and unsuccessful males in replicates with copulations (Table 5.3,

Figure 5.3).

iii. Comparing successful females with unsuccessful females

There were no statistically significant differences in peak areas between successful females and unsuccessful females in replicates with and without copulations (Table 5.3).

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 163

Table 5.3: Statistical results (F and P values) of ANOVA for log mean peak areas of

Bactrocera tryoni putative cuticular hydrocarbon compounds compared between

successful males and unsuccessful males in replicates with copulations, and log mean

peak area differences between successful females and unsuccessful females.

Possible compound F and p values for F and p values for

ANOVA compared ANOVA compared

between successful males between successful

and unsuccessful males females and

unsuccessful females

4-trifluroacetoxytridecane F (59, 119) = 1.38, P = 0.242 F (50, 8) = 2.72, P = 0.104 Dodecamethylcyclohexasiloxane F (59, 119) = 0.86, P = 0.355 F (50, 8) = 1.02, P = 0.316 Tetradecamethylcylcloheptasiloxane F (59, 119) = 0.84, P = 0.361 F (50, 8) = 0.53, P = 0.468 Dodecanoic acid ethyl ester F (59, 119) = 3.11, P = 0.081 F (50, 8) = <0.001, P = 0.949 Heptadecanoic acid ethylester F (59, 119) = 2.15, P = 0.145 F (50, 8) = 0.33, P = 0.566 E-11-hexadecenoic acid ethyl ester F (59, 119) = 0.6, P = 0.441 F (50, 8) = 0.24, P = 0.623 Hexadecanoic acid F (59, 119) = 0.8, P = 0.373 F (50, 8) = 0.06, P = 0.807 13-methylheptacosane F (59, 119) = 0.57, P = 0.452 F (50, 8) = 0.73, P = 0.396 2,3-dimethylnonane F (59, 119) = 0.83, P = 0.364 F (50, 8) = 0.04, P = 0.839 9-methylnonadecane F (59, 119) = 1.42, P = 0.237 F (50, 8) = 0.03, P = 0.87 7-methylpentadecane F (59, 119) = 0.37, P = 0.544 F (50, 8) = 0.04, P = 0.844 2-methyloctacosane F (59, 119) = 0.92, P = 0.34 F (50, 8) = 0.1, P 0.751

2-methylnonadecane F (59, 119) = 0.34, P = 0.564 F (50, 8) = 0.09, P = 0.76 2,3-dimethylheptadecane F (59, 119) = 0.21, P = 0.651 F (50, 8) = 0.09, P = 0.764

16486 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

Males 18 Females

** ** ** ** * * * * * * 16

Log mean peak areas peak mean Log

RT 7.08 RT 8.20 RT 9.15 RT 9.24 RT

RT 14.32 RT 14.38 RT 14.47 RT 14.51 RT 14.57 RT 14.69 RT

Figure 5.2: Log mean (± SE) peak areas of 10 Bactrocera tryoni putative cuticular

hydrocarbon compounds which are significantly different between males and female

in all replicates. ** indicates a significance at P < 0.001 and * indicates a

significance at 0.05 > P > 0.001. The retention times (RT number) can be matched to

the putative compound in Table 5.1.

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 165

Successful male Unsuccessful male (a) a Female

15 a a a

14 b b b b b 13 b b b

11 7.09 8.20 9.15 9.24

17 (b) a

Log mean peak areas peak mean Log

ab a 16 b a a a

ab ab ab ab

15 b b b b

13 14.32 14.36 14.47 14.50 14.55 RT (min) Figure 5.3: Log mean (± SE) peak areas of Bactrocera tryoni putative cuticular

hydrocarbon compounds for successful male, unsuccessful male and female in

replicates with copulations in peaks from (a) RT 7.09 to 9.24 (b) RT 14.32 to 14.55.

16686 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

Different letters on adjacent bars on any given RT value indicate significant

difference (P < 0.05) in log peak areas between successful male, unsuccessful male

and female. The retention times (RT number) can be matched to the putative

compound in Table 5.1.

5.4 Discussion

5.4.1 Summary of results The overall aim of this last research chapter was to identify the cuticular compound composition of B. tryoni and the effects (if any) of these compounds on their mating success. From these objectives, the first task was to find whether there is any sexual dimorphism in the composition of B. tryoni cuticular compounds; and the second to determine whether these compounds differ between successfully copulated flies and unsuccessful flies. GC-MS analysis found that there were 14 prominent peaks in the cuticular extractions, common to both male and female flies. All the analyses and comparisons were carried out using these prominent peaks, but understandably these 14 compounds are not the only compounds which B. tryoni flies have, and there were small traces of other undefined compounds as well.

Therefore a full spectrum analysis is needed for complete compound identification.

Among the 14 prominent peaks, peak areas of ten compounds (Dodecanoic acid ethyl ester, Heptadecanoic acid ethylester, E-11-hexadecenoic acid ethyl ester,

Hexadecanoic acid, 13-methylheptacosane, 2,3-dimethylnonane, 9- methylnonadecane, 7-methylpentadecane, 2-methyloctacosane, 2-methylnonadecane) were significantly different between males and females, indicating a clear sexual dimorphism in B. tryoni’s CHC profile. But there were no significant differences in the CHC profiles between successful and unsuccessful males or females in mating trials.

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 167

5.4.2 Significance of methyl-branched alkanes

Out of 14 putative compounds which have been found in this study, derivatives of methyl-branched compounds have previously been identified as aggregation pheromones and contact sex pheromones (Oi et al., 2015) in a few tephritids, as well as in other solitary insects. The tephritids, A. ludens, A. suspensa, C. capitata and C. rosa males, females and larvae, and Z. cucurbitae larvae, have 2-methyloctacosane as a methyl-branched alkane in their cuticular extractions (Carlson and Yocom,

1986). The exact importance of these compounds was not investigated at that time, but clear differences in compound profiles were observed in adults and in larval stages between species (Carlson and Yocom, 1986). Males of the red flour beetle

Tribolium castaneum (Herbst) and the confused flour beetle, T. confusum Jacquelin du Val contain 4,8-dimethyldecan-1-al as a methyl-branched compound which acts as an aggregation pheromone (Odinokov et al., 1989, Suzuki, 2014). As a contact sex pheromone, 13-methylheptacosane plays a significant role in various insect groups.

For instance, males of the leaf beetles, Gastrophysa atrocyanea Motschulsky become sexually excited when they make contact with female elytra extracts: methylheptacosanes are one of the two main compounds in the female cuticular contact pheromones which stimulate the male sexual behaviour (Sugeno et al.,

2006). Females of Pear psylla, Cacopsylla pyricola (Förster), possess 13- methylheptacosane as a major component of their cuticular hydrocarbons which acts as a sex attractant pheromone largely on males (Guedot et al., 2009). The amount of

13-methylheptacosane produced by females is greater than that of males (Guedot et al., 2009), which was observed in my experiment as well. Additional to it’s role in aggregation and sex attractant, 13-methylheptacosane has also been identified in

Formosan subterranean termites, Coptotermes formosanus Shiraki, as a major

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component of the cuticle in all castes; workers, soldiers, nymphs and alates (Haverty et al., 1996).

5.4.3 Significance of saturated fatty acids

Saturated fatty acids (i.e., fatty acids with all carbon atoms connected with single bonds) such as Tetradecanoic acid, Hexadecanoic acid and Dodecanoic acid have also been previously associated with many insect groups. Pea aphids,

Acyrthosiphon pisum Harris, contain Mytristic acid (Syn. Tetradecanoic acid) as a major compound of their saturated fatty acid on their waxy outer covering and the amount of Mytristic acid depends on the host plant they feed upon (Giles et al.,

2002). For females of European grape vine moths, Lobesia botrana (Denis and

Schiffermuller), Myristic acid has been recorded as a compound in their egg extractions and oviposition is deterred when Myristic acid is detected – the chemical may thus act as an oviposition deterring pheromone (Gabel and Thiéry, 1996). Males of the tropical butterfly, Bicyclus martius sanaos (Hewitson), contain ethyl-esters of hexadecanoic acid on the surface of their wings and they act as courtship pheromones (Wang et al., 2014). Although these derivatives of ethyl-esters were sex specific in B. martius sanaos butterfly males, they have also been associated with the biosynthesis of female sex pheromones of the Red banded leaf roller moths,

Argyrotaenia velutinana (Walker) (Bjostad and Roelofs, 1981).

A number of volatile chemicals in human derived saturated fatty acid odours, such as lactic acid and carboxylic acid react with CO2 and produce volatile attractants which are sensitive to mosquito sensory neurones (Njiru et al., 2006). The red pumpkin beetle, Aulacophora foveicollis (Lucas), and the cotton aphid, Aphis gossypii Glover, are attracted to a synthetic mixture of alkanes and fatty acids such as heptacosane and lauric acid, which are related to the surface waxes of Solena

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 169

amplexicaulis (Cucurbitaceae) flowers (Sy Mohamad et al., 2013, Karmakar and

Barik, 2016). Therefore it is suggested that these compounds may be used in baited traps which is important for their population control (Karmakar and Barik, 2016).

5.4.4 Significance of experimental results

Although it is believed that B. tryoni males and females locate their mating partner based on volatile chemical (Tychsen, 1977, Poramarcom, 1988, Weldon,

2007, Taylor et al., 2013) and auditory signals (Mankin et al., 2008), further confirmation of the sex of their partner may occur after they make close, physical contact. In my previous chapter, as well as in past studies (Tychsen, 1977), there is evidence for B. tryoni male-male mounting attempts. Therefore, observing a clear sexual dimorphism in the composition of the CHC profile of B. tryoni may provide a mechanism whereby sex discrimination and/or confirmation may occur once flies are in very close or physical contact.

When considering the peak areas of specific compounds between successful males and unsuccessful males, although the CHC composition difference was not significant, the amounts of compounds were comparatively (but never statistically) higher in successful males than in unsuccessful males, with the log mean peak areas of 13 of the 14 compounds greater in successful than unsuccessful males in replicates where mating occurred (Table 5.2). Such a consistent pattern is unlikely to occur by chance and may be explained in one of two ways. Firstly, the peak area difference between successful and unsuccessful males in replicates with copulations may reveal that CHCs from females were transferred onto successful males while they were in copulation, increasing the males’ total CHC loads. Cross-individual transfer of

CHCs is recorded in numerous insects, and in social and sub-social insects particularly is an important part of kin recognition (Bagneres et al., 1991; Everaerts

17086 Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice

et al., 1997; Vauchot et al., 1997). Alternatively, the CHC differences between successful and unsuccessful males may occur prior to mounting, which would suggest real biological difference between mating “winners and losers”, but for which the experiment had insufficient sensitivity (e.g., through insufficient replication or the physical limits of CHC extraction and GC-MS analysis) to detect what may be an extraordinarily subtle biological effect. Statistically, I can only conclude that, based on the current results, CHCs play no role in B. tryoni male sexual selection. Nevertheless, further research (e.g., increasing replication, or manually altering the CHC load on individual flies) is justified to further explore this hypothesis.

Chapter 5: The role of cuticular hydrocarbons in Bactrocera tryoni mating and mate choice 171

Chapter 6: General discussion

Chapter 6: General discussion 173

6.1 Summary of results

The objective of my thesis was to characterize the mating system of Bactrocera tryoni, from identifying long range mating site selection cues to resolving close range mechanisms that may play a role in mate recognition. While this knowledge provides fundamental insights to the general field of fruit fly mating systems and their evolution, the information also has clear utility in its application to the development of novel control strategies. I acknowledge that all flies used in these experiments were laboratory mass reared, and this might has an effect on their behaviour (Weldon et al., 2010). It was difficult to avoid this issue as more flies were needed than could be easily reared from wild fruit sources, but I do recognize possible limitations of interpretation this may cause.

To achieve my thesis objectives, it was first important to examine the main aspects of the B. tryoni mating system, specifically how they locate a mating site in the landscape and whether, once individuals locate a mating site, they behave as expected for a true lekking species. I found that B. tryoni flies aggregate to mate in tall trees which may, if extended to the broader landscape, reflect preferential selection for emergent canopy trees as natural mate rendezvous sites if we consider their natural habitat as rain forest environments (Drew and Romig, 2000). Regarding lekking behaviour, the initial requirements of a lek mating system were disproved, because no male encounters were observed before females arrived. However, flies did aggregate and the absence of fruit (a female resource) did not negatively affect mating site selection, both of which are requirements of a lekking system. However, the assumption of a presence of a lek mating system could not be rejected or accepted from the field-cage study, since there was no direct evidence for female selection, a core element of a classical lek mating system. Investigating the presence

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of B. tryoni female active selection was therefore an essential goal while documenting the species’ courtship sequence.

For the first time, a comprehensive ethogram for B. tryoni courtship was constructed in this thesis. From this I observed that males that directed their wings

(synchronous supination) more often towards females secure mates at a higher

174proportion that those males that carried out the synchronous supination behaviour less frequently. This observation suggests the presence of a B. tryoni male visual cue by which they signal their presence or readiness to mate when attracting females towards them. Female avoidance of mounting males was also observed, which suggested that females have the ability to accept or reject mounting males. Males who overcome such rejection may have intrinsic properties, such as elevated motivation or fitness, sufficient to overcome female rejection. These assumptions led to female choice tests to investigate the effect of male physiological attributes (age, body size and sexual experience) on their mating success. I observed that young and large males were more successful in securing copulations than old and small males.

However, I detected no behavioural differences between young and old or large and small males, nor did females show detectable behavioural differences towards each male type to explain the differential mating success. The absence of differential mate selection behaviours, the observation that females do have the ability to accept or reject mounted males by kicking them off with their hind legs, plus further observations of male-male mounting attempts, led to the question; “Do B. tryoni males and females use a cuticular contact pheromone (i.e., cuticular hydrocarbons, or

CHCs), in order to recognise or confirm the status of their mating partner during mounting attempts?”.

Chapter 6: General discussion 175

Based on the above hypothesis, a cuticular compound analysis of B. tryoni was carried out. The first goal was to test for sexual dimorphism in B. tryoni cuticular compounds to support the possibility that sex confirmation occurs once flies are in very close or physical contact: sexual dimorphism in the CHC profiles of males and females was subsequently found. Although the differences were never statistically significant, consistently higher amounts of cuticular compounds in successfully mated males over unsuccessful males suggests either that CHC compounds are transferred between individuals during mating, or the presence of a difference between males prior to mounting which may be linked to their mating success.

Further research by increasing the number of replicates, physically manipulating the

CHC profiles of individual male flies, and/or altering the GC-MS procedure is required to further explore the role of CHCs in B. tryoni sexual selection.

Considering all of the thesis findings, this final discussion chapter will integrate many new results into the broader tephritid literature. In my introductory chapter (Chapter 1), I briefly touched on tephritid mating systems, introducing concepts such as resource-based or non-resource-based mating systems, and what we know about tephritid courtship behaviours. But in the following section, I will look more closely at tephritid mating systems in light of my thesis findings and how we can now better understand frugivorous tephritid mating systems. In the last section of this chapter, I discuss how we can use this knowledge to better apply tephritid pest management tools.

6.2 Contradictory ideas on tephritid mating systems

As the Tephritidae is one of the major families of horticultural pest insects, studies on their biological, ecological, physiological and behavioural interactions have been widely published over many decades. Among these areas, studies on

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tephritid mating systems and mating behaviours are fields of major interest to tephritid researchers. The mating behaviour of a number of tephritid species has been studied and there are both consistent and contradictory findings associated with descriptions of their mating systems.

The main area with conflicting ideas in the tephritid mating literature is the assumption of tephritid leks. The concept of leks was initially described for birds

(Lloyd, 1867), and they have subsequently been reported in insects, fish, amphibians and mammals (Hoglund and Alatalo, 1995). The idea of “classical” leks assumes aggregations of displaying males, with the subsequent selection of a particular male by a female based on male fitness qualities, and not based on the supply or control of some material resource by the male (Lack, 1968) (N.B. lekking theory does allow for male provision of indirect material benefits that are transferred to females during copulation (= “seminal gifts”) (Lewis and South, 2012, Shelly and Nishimoto,

2016)). Many tephritid mating studies appear to have assumed that lekking occurs based on the observation of mating aggregations, but without formally testing other components of the system. For example Weldon (2007) conducted an experiment to determine the effect of male aggregation size on female visitation in B. tryoni. The design of the experiment was explicitly based on the assumption that B. tryoni is a lekking species and that males form aggregations that females visit for mating.

Weldon subsequently observed no correlation between male aggregation size and female visitation, a result that he found difficult to explain. But as I showed in

Chapter 2, mating aggregations in B. tryoni occur without males arriving at the mating site first.

When reviewing the tephritid mating literature, Benelli et al. (2014b) made the statement “Tephritidae are generally lekking species”; they also reported B. tryoni as

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a lekking species “…male-male combat has been observed in several lekking species. Good examples are the males of Bactrocera tryoni…” referring to the publication by Tychsen (1977). Benelli et al. are not the only authors to have made such generalisation. Prokopy and Hendrichs (1979), based on research on C. capitata and citing findings from three other tephritids, which again included

Tychsen’s study on B. tryoni, also stated that lek behaviour is common in tephritids.

In my study I have observed behaviours of B. tryoni males and females which while supporting, also contradict the idea that B. tryoni is a classically lekking species; for instance, male-male combat was rarely observed and females did not arrive after males at a mating site. Importantly, Tychsen himself also never referred to B. tryoni as a lekking species, but did refer to it as a swarming species. Therefore Benelli and others’ assumptions of B. tryoni as a lekking species should not be totally regarded as correct, and if this is the case for one reputedly well studied tephritid, might it not also be the case for other species? In the following section I review studies of tephritid mating systems to determine which species have sufficient evidence to be justifiably described as lekking species, and for which the evidence of lekking or otherwise may be questioned.

6.2.1 Tephritids with sufficient evidence for lekking Ceratitis capitata

Prokopy and Hendrichs (1979) described the C. capitata mating system as a lek system, although they did not document all the necessary requirements for it to be regarded as such. They recorded male aggregations on leaves that females visited, but no observations were made of aggressive male behaviours, nor did they supply evidence that supports strong female selection of males. They also mentioned the

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importance of fruit presence on trees for the formation of male leks, which is contradictory to the definition of leks as non-resource based.

In the experiments Arita and Kaneshiro (1985) to describe the lek behaviour of

C. capitata, it was found that only a small proportion of males participated in leks.

Such males controlled territories, and females entered those territories based on the quality of the territory or after quantifying male courtship behaviour (Arita and

178 Kaneshiro, 1985). Although Prokopy and Hendrichs (1979) were the first to mention the mating system of C. capitata as a lek, I consider the work by Arita and Kaneshiro

(1989) to be the first fully descriptive experiment that provides evidence for all the behavioural requirements of a lek system. In this follow-up paper to their 1985 work, the authors conducted field and laboratory experiments that provided further evidence to support males aggressively defending territories, male calling behaviour that attracts females, and active female choice of males. Contrary to lekking,

Hendrichs et al. (Hendrichs et al., 1991) reported C. capitata mating on fruit. All other studies conducted on C. capitata mating behaviour have considered it a lekking species and numerous experiments have been carried out to describe aspects of C. capitata leks (Shelly and Whittier, 1993, Whittier et al., 1994, Shelly and Whittier,

1995, Calcagno et al., 1999, Cayol et al., 1999, Norry et al., 1999, Kaspi et al., 2000,

Yuval et al., 2002, Mankin et al., 2004, Shelly, 2004, de Souza et al., 2015).

Anastrepha suspensa

Behavioural observations were made on A. suspensa on guava trees which suggested it as a lekking species (Burk, 1983). In this study all behaviours associated with a true lekking species were observed, including: male aggregations where males aggressively defended territories; the release of sex pheromone and acoustic signals by males which attracted females; and evidence for female selection where large

Chapter 6: General discussion 179

males were more successful in copulations (Burk, 1983). Numerous other studies have considered A. suspensa a lekking species and further described its lek behaviour

(Burk and Webb, 1983, Sivinski et al., 1984, Burk, 1984, Sivinski, 1984, Sivinski,

1989).

Anastrepha ludens

Lekking behaviour of A. ludens was described in an experiment carried out on a field-caged host tree (Robacker et al., 1991). In this experiment, all requirements needed by a lekking species were observed including male aggregations, calling which attracts females; males defending non-resource based territories; and female selection of males based on male competition (Robacker et al., 1991).

Zeugodacus cucurbitae

Mating behaviour of Z. cucurbitae was initially described as lekking in an experiment carried out on wild melon flies in a field cage by Kuba et al. (1984). In this experiment, although they mentioned the species as a lekking species, they provided on information on the presence of female selection. They demonstrated females were attracted to male aggregations based on released male sex pheromones, but it is not clear whether females used those pheromones to compare among males.

Kuba and Koyama (1985), who also conducted work on melon fly courtship again failed to demonstrate female selection. However, Iwahashi and Majima (Iwahashi and Majima, 1986) did demonstrate all requirements of a lek for melon fly. They observed male aggregations where males competitively defended their territory, male wing vibrations and calling, and also “female choice” due to low rate of male mating success after male-female encounters. The presence of female choice in melon fly was observed in a study by Shelly (Shelly, 2010), where he recorded increased mating success of cue-lure fed melon fly males compared to non-lure-fed males.

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6.2.2 Contradictory ideas on B. tryoni mating behaviour from past to present The first detailed description of the mating behaviour of B. tryoni is presented from cage experiments (Myers, 1952). Although Myers (1952) recorded males as initiators of courtship, by commencing ‘calling’ through wing vibrations which attracted females towards them, he did not categorise their mating system (i.e., as a lek180 or resource-based); whether the ‘calling’ males were aggregated or not; whether males and females use resources when attracting each other; or any other evidence for lek behaviour. Later, Tychsen and Fletcher (1971) studied the mating behaviour of B. tryoni and considered their courtship male aggregations as “loose flying swarms” (Tychsen and Fletcher, 1971). Male swarming groups were reported from the field by Bateman (1972) and this same wording of ‘male swarms’ was used again by Tychsen (1977, 1978).

Independent of the idea of “swarms”, the idea that B. tryoni has a resource- based mating system was advanced by Drew (Drew, 1987, Drew and Lloyd, 1987).

In his experiments, Drew considered that B. tryoni mating always occurred on the host plant, implying the concept of a resource-based mating system; i.e., mating is linked with the fruit that females use as oviposition sites. This is contrary to the concept of a lek system, where females arrive at male mating aggregations only for the purpose of mating and that other resources, such as oviposition sites, are located elsewhere.

The idea of B. tryoni leks was first introduced by Fletcher (1987) in a review of

Dacine biology. Here, Fletcher mentioned all the behaviours of B. tryoni courtship that are required to consider it a lekking species, except for any mention about the presence of female selection (Fletcher, 1987). However, his document was a review, thus referring to the B. tryoni mating system as a lek system based on the

Chapter 6: General discussion 181

publications of earlier researchers, none of whom (including himself) described it as a lekking species. From Fletcher (1987) onwards, several researchers have referred to

B. tryoni as having a lek mating system without any further experimental confirmation (Pike and Meats, 2003, Weldon, 2007, Kumaran et al., 2013, Kumaran et al., 2014b); thus resulting in the now long held assumption of B. tryoni leks. Male competitive advantage and active female selection of a modified male pheromone was observed in B. tryoni following male feeding on cue-lure (Kumaran et al., 2013,

Kumaran et al., 2014a), providing indirect evidence for female selection in the B. tryoni mating system.

Weldon (2005) is the only one of more recent authors to refer to the mating aggregations of B. tryoni as swarms where he observed calling males, female rejection of mounting males by kicking with the hind-legs, walking or flying away, as well as male-male mountings (all of which I also observed in my experiments).

These behaviours are common for lekking species, but Weldon was unable to provide evidence for strong female selection of males (Weldon, 2005). Although

Weldon described the mating behaviour of B. tryoni as swarms in his 2005 paper, he has since referred it as lek-like behaviour in another study on the effect of male aggregation size for female visitation (Weldon, 2007).

Based on the above literature, and the work from my research chapters, the following summation of B. tryoni mating can be made: B. tryoni mating occurs over a short time period following dusk and under low light intensities. As light intensity falls, males and females simultaneously aggregate on a host or non-host tree and males start calling by wing vibrations at the same time as releasing a sex pheromone.

Within the mating aggregation male-male aggression is infrequent, and when observed it is unclear whether the behaviour is to protect their territory, or as a

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competition among males to directly secure a female mate. More females arrive at the mating site at the same time as males arrive and begin calling. Male calling behaviour and release of pheromones may help in aggregation of both females and additional males. When both males and females are in very close proximity, the male behaviour of synchronous supination towards the females is significantly correlated with subsequent mating success, but no other observable behaviours were linked to mating success. Close-range non/low-volatile cuticular compounds may play a role in mate recognition and acceptance, due to sexual dimorphism and differential quantities in compounds among successful and unsuccessful males. Mate choice clearly occurs within the B. tryoni mating system, but whether this selection is due to male-male competitive advantage, or female preference of particular male attributes, or a combination of both, is still difficult to confirm. And also female rejection of a mounted male by vigorous shaking cannot be considered as “active” female choice. Considering all these findings, it is clear that, we can’t consider B. tryoni as a true lekking species, because, though there is evidence for the presence of sexual selection and the mating site selection is not based on resources; males have not arrived into mating sites before females and male aggressively defending territories was unclear. Therefore, B. tryoni mating system is a ‘lek-like’ non resource based mating aggregation with the presence of a mate choice.

6.2.3 Uncertainty and conflicting ideas about lekking in other tephritids Although there are specific characters needed to be fulfilled by a species to be considered as a true lekking species, some scientists have considered their tephritid study organism as a lekking species without considering all of these characteristics.

Some species which are considered as lekking species, but for which evidence might still be considered inconclusive, follow.

Chapter 6: General discussion 183

Bactrocera dorsalis

Bactrocera dorsalis is a species considered to have a ‘lek-like’ mating system which slightly differ from “classical” lek behaviour (Shelly and Kaneshiro, 1991), yet data required to confirm this is ambivalent. Poramarcom and Boake (1991) have described the mating behaviour of B. dorsalis as including male aggression and female selection, but do not specifically refer to this species as lekking or not. Shelly and Kaneshiro (1991) described the B. dorsalis mating system as a lek, but they recorded more females in the mating site than males, which violates the definition of a “classical” lek (see section 1.3.1). Tan and Nishida (1996) provided indirect evidence for the presence of female selection in B. dorsalis with females mating preferentially with methyl eugenol fed males over methyl eugenol non-fed males in large outdoor cages, but this effect was absent in small indoor cages (Tan and

Nishida, 1996). The mating system of B. dorsalis is further confused by records of mating on the host fruit (Prokopy et al., 1996).

Bactrocera oleae

The mating system of the olive fruit fly, Bactrocera oleae, is confusing, but sometimes is referred as lekking. For example, Benelli (2014b) has referred to B. oleae males as the ‘lekking sex’, yet in the same paper concluded that B. oleae mating strongly differs from ‘classical lek’ (Benelli, 2014). Benelli et al. (2015) again, confusingly, considered male B. oleae groups as swarms, as well as leks, in the same paper. Mating site selection of B. oleae is strongly dependent on the volatile α-pinene produced from olive fruit (Gerofotis et al., 2013). That means the mating behaviour of olive fruit fly is based around the host plant where they find olive fruit; the oviposition substrates needed by females, and so should be regarded as resource based. Female olive fly also produce a pheromone that attracts males

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(Mazomenos and Haniotakis, 1981, Wicker-Thomas, 2007), again a behaviour inconsistent with lekking. Thus, while there are studies that have considered the mating system of B. oleae as a lek, the question of whether B. oleae flies show true lekking, or whether they are a swarming species, or a species with a resource-based mating system, remains open.

Anastrepha fraterculus

Morgante et al. (Morgante et al., 1983) described the mating behaviour of A. fraterculus as lekking after describing male stridulation and pheromone release that was used to attract females to the males, but the authors provide no evidence for males defending territories and female selection of males. Female choice in A. fraterculus has been, postulated based on the increased mating success of males which spend more time ‘calling’ and with a greater eye length and a face width (i.e., bigger flies) (Segura et al., 2007); but no mention for this species has been made about the presence of male aggressiveness and the defending of territories.

6.2.4 Tephritids with a resource-based mating system As mentioned in section 1.4.1.2, there are tephritids that have a resource-based mating system. That means the selection of a mating site by a male is dependent on the availability of resources needed by females. Within these sites, males compete with other males to protect their mating territories, as well as to prevent the use of resources by other females who have not mated with them (Emlen and Oring, 1977,

Dodson, 1997). For instance, males in the tephritid genus Phytalmia (the antlered flies) defend as their mating sites on the trunks of fallen trees which are the oviposition sites needed by females (Dodson, 1997). In these situations, the selection of a male by a female as their mating partner will depend on the quality of the

Chapter 6: General discussion 185

oviposition site, but not directly on the male secondary sexual characters (i.e., females readily mate with small-antlered males as well as large-antlered males).

Rhagoletis pomonella males and females also mate on host apple fruit which are the females’ oviposition substrates (Prokopy and Papaj, 1999, Forbes and Feder,

2006). The idea of resource based mating site selection in R. pomonella was first introduced by Lathrop (1955), when he mentioned the possibility of finding mating couples in or near apple orchards. Although Lathrop was unable to present observational data, Prokopy (1968) observed seven mating couples on apple fruit. To confirm this further, Prokopy et al. (1971) conducted experiments in the field as well as in the laboratory using wild and laboratory reared flies where they found both male and female R. pomonella gather and successfully copulate on fruit.

Confirmed resource-based mating behaviour is not restricted to non-Dacinae fruit flies. Dacus longistylus Wiedemann is a tephrittd considered as a pest of cucurbits (Azab and Kira, 1954). It is a monophagous species with a resource based mating system (Hendrichs and Reyes, 1987). During day time, with increasing sunlight, more males arrived on to fruit of the host plant Calotropis procera (Aiton) where they defended 2-4 fruits by attacking and chasing away the intruder males

(Hendrichs and Reyes, 1987). Female flies spent most of their time on foliage and did short visits to fruit for oviposition. While females are on fruit trying to oviposit, the territory controlling males will jump onto the female and grasp her to initiate copulation (Hendrichs and Reyes, 1987). This same behavioural sequence was recorded for B. minax; the Chinese citrus fly (Dong et al., 2014). In the morning both males and females become active with increasing sunlight; males move onto a selected green coloured fruit and defend this fruit as the ‘suitable’ territory for mating from intruder males. This defensive action involves wing waving and head

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strikes until the intruder male gets off. The winning territorial male will then moves onto a nearby leaf and wait for a female to arrive onto the fruit. When a female arrives and initiated ovipositing by inserting her ovipositor into the fruit, the male will jump onto the female and start copulation (Dong et al., 2014). In both these species, the mating success of males does not depend on female selection of male qualities, but on the ability of males to defend fully grown host fruit which are needed by females as oviposition sites.

Considering the above literature on tephritid mating (summarised in Table 6.1) in my opinion I consider that, in the case of tephritid mating systems, many researches have labelled lekking behaviour only from the males’ behavioural perspective; simply referring to any group of aggregated males as a lek. That means that if a group of males aggregate on a host or non-host trees before females arrive, without considering any other resources needed by females, if those males defend their territories while performing wing fanning and release of sex pheromones to attract females, then such tephritids have generally been considered as lekking species. But in most cases there have not been corresponding studies to demonstrate

“active” female selection, as important a characteristic of classical leks as is male aggregation and display. While it is arguable that several studies on tephritid species demonstrate male aggregation behaviour prior to mating, there is less-clear evidence that male territory defence is common, and even less well-demonstrated is active female selection of one mate over another. In such cases; defining the species as lekking needs to be questioned, as use of the term in these contexts does not meet the classical definition of leks.

Chapter 6: General discussion 187

Table 6.1: Behaviours common for tephritids with a resource based mating systems; non-resource based mating systems (except swarms and leks), swarms, and leks.

Resource based Non resource Swarms Leks mating system based mating system (except swarms and leks) • Mate on host fruit • Males aggregate • Males aggregate • Males aggregate or mating site but this is not not only for the into mating sites selection is based based on the purpose of mating before females but on the availability availability of but also to prevent this is not based on of resources needed resources. predation and for the availability of by female such as • Male performs migration. resources. oviposition sites. courtship displays • No male Males defend their • Male aggregations (i.e., calling by courtship territory by are present. wing vibration and behaviours. competing with • Males defend pheromone • No female other males. their territory by release). selection of males. • Male performs competing with • Not necessary • Males simply courtship displays intruder males. for males on the grasp a female for (i.e., calling by • Females arrive mating arena immediate wing vibration and onto mating sites before females copulation. pheromone release) depending on the arrive. Both males which attract quality of the and females can females towards territory not directly arrive at the same them. on male qualities time. • Females arrive • Males with high into male competitiveness aggregations and will mate with a actively select a female (without male based on their evident form of attributes as a courtship). mating partner.

18886 Chapter 6: General discussion

6.3 Implications for tephritid pest management

The results of this thesis have wide relevance to pest management of B. tryoni, a fruit fly that represents Australia’s most important horticultural pest, and for which a range of pest management strategies have been deployed, including: male annihilation technique (MAT); protein bait spraying (PBS); and sterile insect technique (SIT) as part of area-wide integrated pest management approaches (AW-

IPM). Arguably, the SIT is the management approach of more direct relevance to the outcome of my study, as this control method relies on an intimate knowledge of the mating system of the pest species of concern.

The SIT relies on the induced sterilizing mass-reared flies, as achieved by exposing individuals to electron beams, X-rays or gamma rays (Robinson, 2005) which cause chromosomal damage thus rendering individuals sterile (preferably males only). Sterilization suppresses their ability to produce offspring in the next generation when mated with wild flies. While the sterilisation process takes place at the pupal stage, either pupae or young adults may be released into the environment

(Dominiak et al., 2003). When releasing sterile adult males, they must be able to compete with wild males in order to mate with a wild female. Since the sterile males are mass-reared under laboratory conditions, changes in their behaviour or physiology may occur, resulting in less fit individuals as compared to wild males.

For example, mating age, courtship behaviour and quality of pheromones will change following mass-rearing and sterilisation (Cayol, 2000, Meats et al., 2007). Therefore it is crucial to identify and enhance the male qualities of sterile males.

Since my experiments were focussed on the initial stages of how flies find their mating arena to the behaviours and chemicals associated with successful copulation, the implications for improved SIT methods will be discussed in this context (i.e., I

Chapter 6: General discussion 189

will not address rearing methods). The use of SIT to manage B. tryoni in Australia goes back to the 1960s, with research into rearing facilities, irradiation, transporting and release of irradiated pupae on trees that are protected from sun, rain and predators (Monro and Osborn, 1967). Since the main requirement of an effective SIT programme is the competitiveness of males, procedures that enhance male competitiveness greatly increase the effectiveness of the SIT. Mass-reared sterile males need to be at least as physically capable as wild males in order to compete. As

I observed both B. tryoni males and females aggregate on tall trees before copulation, the release of adult sterile males is recommended to be undertaken near tall trees within a selected area to control B. tryoni populations. This will reduce the flight energy needed by sterile males that they spend while finding suitable mating sites. It will help them to conserve the physical energy needed to compete with wild males in securing a wild female to mate, because mass-rearing and irradiation increase the likelihood that B. tryoni flight ability is reduced (Weldon et al., 2010, Fanson et al.,

2014). Further, releasing males at their specific mating sites during dusk will also reduce the risk of predation by fruit fly predators such as ants, beetles, crickets and mantids (Bateman, 1972, Hendrichs and Reyes, 1987, Allwood, 1997) which will increase the effectiveness of the SIT by reducing the mortality of sterile males in the field.

Mass-reared sterile B. tryoni males are relatively inactive most of the time, spending less time in walking and flying as compared to low-energy activities such as grooming (Weldon et al., 2010). Also, mass-rearing and sterilisation alter courtship songs and calling of B. tryoni males by increasing the pulse intervals of their courtship songs compared to wild flies (Mankin et al., 2008). If we consider the findings of my thesis, I observed a positive effect of male age and body size with

86190 Chapter 6: General discussion

mating success; where young and large males were more active during courtship thus implying males may be highly motivated to mate, more physically strong to compete with other males, or that females actively choose them as a mating partners.

Although large males were more successful compared to small males, making them exceptionally large during mass-rearing might make them less prone to fly around and find a female mate or compete with other males; hence further examination of male behaviour in this context is worth considering. Generally, however, large sterile males with an average wing length of at least 4.825 ± 0.019 (as I recorded) may be effective when controlling wild populations via SIT. Further, since I have observed

14 day-old males as the most successful age for mating, and the above method can be used to investigate the effective age of mass-reared sterile males to compete with wild males before releasing them under SIT programmes.

The characteristics of male B. tryoni male courtship dance may also influence male mating success, whereby males with an increased synchronous supination duration (i.e., courtship dance) were more successful in copulations. Studies on the

B. tryoni courtship sequence have not been conducted until now, or at least are not available in the published literature. Therefore, this observation can be further implemented by carrying out mating compatibility tests to find any difference between mass-reared sterile males and wild males. From these experiments, the most effective durations of male synchronous supination and the number of occurrences can be recorded in sterile males which advantageous for a successful copulation.

Similarly, based on these findings, a sample analysis of mass-reared sterile males with the most effective synchronous supination durations might be conducted before releasing them into the wild as part of a quality control process.

Chapter 6: General discussion 191

From CHC analysis in this thesis, there is evidence for the presence of an effect of the cuticular compound composition for male mating success by observing a sexual dimorphism in CHC and a quantitative difference in compounds present between successfully copulated males and unsuccessful males. Based on this finding, further analyses are recommended to resolve the difference of cuticular compounds between mass-reared sterile males and wild males which affect their mating success, as well as the ‘preferred’ quantity of cuticular compounds by wild females. These findings may be further applied to produce commercial products of B. tryoni cuticular extractions. When releasing sterile males into the environment as part of

SIT, investigated quantity of commercially produced cuticular extractions can be applied on the body of sterile males which will increase their mating success compared to wild males.

The findings of my thesis are not only applicable to the development of SIT, however, but may also be applied to other fruit fly control methods such as lure and kill techniques, where chemical lures (typically plant derived chemicals) to attract flies are incorporated into traps which are killed with insecticide (El-Sayed et al.,

2009). Using MAT, male population size decreases by attracting them in large numbers to traps thus reducing fly population size due to decreased amount of female mating (Ghanim, 2013), as effectively used to eradicate B. dorsalis from the

Okinawa Islands (Koyama et al., 1984, Otuka et al., 2016). The effectiveness of these traps will arguably be greater if we can attract more flies towards the traps.

That means, we can place the traps in places where B. tryoni flies have a greater tendency to gather and use chemicals that are more attractive to them. I have observed both B. tryoni males and females aggregate to mate on the top layers of the highest tree within their environment; most probably the emergent canopy trees in

19286 Chapter 6: General discussion

their natural environment (requiring field confirmation). And also, though the number of flies on trees was comparatively less before dusk, large number of flies has arrived onto trees with dusk. This suggests the possibility of trapping more flies by placing the traps on tall trees than on short trees within B. tryoni’s natural environment. Further, I have identified chemicals (i.e., methyl branched alkanes) in the cuticular compounds of B. tryoni males and females, which have previously identified in other insects that act as aggregation pheromones. Derivatives of these compounds such as aggregation pheromone compounds, after mixing with insecticides may also use as chemicals to attract flies towards the traps and require further research.

6.4 Conclusions and future research

The work presented in this thesis has provided answers for a number of gaps in knowledge of B. tryoni and how we can implement these findings to manage the population of B. tryoni in their natural environment. Further, while working on the objectives of this thesis, I have identified few aspects where the B. tryoni mating system requires further research. In summary:

 Bactrocera tryoni is a land-marking species with a non-resource-based

aggregated mating system that possesses attributes both for and against a

lekking species. I recommend that future research be conducted to determine

if land-marking behaviour, in particular, extends to the wider natural

environment via in-field studies. Confirmation of this will have significant

implications for pest management, specifically in the placement of traps to

control this nationally important horticultural pest.

Chapter 6: General discussion 193

 For the first time, a complete ethogram of B. tryoni male and female courtship

behaviours was constructed, revealing male synchronous supination as a

significant behavioural difference between males that did mate compared to

those that did not; this behaviour likely represents a critical component of the

mate recognition system. This suggests a courtship sequence which B. tryoni

females could use to recognise males or as an indication of male activeness

that affects their mating success. Further research is recommended to identify

differences in male synchronous supination behaviour between mass-reared

sterile males and wild males where the results can be applied to release sterile

males with the most effective synchronous supination durations and number

of occurrences which affect their mating success while competing for a wild

female.

 I have found no behavioural evidence of female selection of males prior to

mounting, yet mounting appears a key transition step which many

unsuccessful males do not proceed past. Further studies are needed to

determine whether females do respond to males with a cryptic courtship

signal (e.g., a female pheromone) which signals their willingness to be

approached by a male and mounted.

 Male physiological attributes might be important for their mating success as

young and large males have mated more frequently compared to old and

small males. The reasons for this difference may be that young and large

males are more competitive, highly motivated to mate or actively selected by

females. By further conducting mating competitiveness tests between sterile

and wild males; mass-reared sterile males for SIT programmes can be

19486 Chapter 6: General discussion

manipulated to release more competitive males with most effective body size

and mating age.

 Bactrocera tryoni mating pairs are highly aggregated and have cuticular

compounds that have been previously identified as aggregation pheromones

in other insect species. Experiments are therefore justified in studying the role

of chemicals in mating aggregations that may be further applied with control

programmes such as trap placement with identified aggregations pheromones

that have the potential to attract more flies.

 Although there were no significant differences in cuticular compounds

between successfully copulated males and unsuccessful males, a visual

difference was present. This might be due to low number of replicates.

Therefore further research is needed by increasing the number of replicates to

find any significant difference between above two types of males.

 Observing comparatively higher amounts of cuticular compounds in

successful males led to formulate two hypotheses: the traces of compounds

may have transferred from females to copulated males; alternatively,

copulated males had larger amounts of cuticular compounds before they

achieved copulations with females. That is, further experiments are

recommended to determine whether females actively accepted the copulated

males based on their cuticular compound composition or transfer of

compounds occurred during copulation. Further:

 As young and large males were more successful in copulations, the cuticular

compositions might be quantitatively high in young and large males

compared to old and small males (e.g., quantity of cuticular compounds may

positively increase with the increment of B. tryoni male body size). If females

Chapter 6: General discussion 195

have accepted males with high amounts of cuticular compounds, the above

assumption can be used to produce large and young males with the most

favourable amount of cuticular composition before releasing sterile males in

SIT programes after conducting laboratory experiments.

19686 Chapter 6: General discussion

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228

Appendices

Appendix 1: Previously idetified cuticular compunds and their significance in tephritids

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals 2-methyloctacosane A. ludens, A suspensa, C. Amount is different between species (Carlson and Yocom, 1986) capitata, C. rosa, Z. cucurbitae but no sexual dimorphism larvae A. fraterculus Differ between spatially isolated (Vanickova et al., 2015c) T. vicina species (Zunic Kosi et al., 2013) Present in both males and females 2-methyltriacontane A.ludens, A suspensa, C. Amount is different between species (Carlson and Yocom, 1986) capitata, C. rosa, Z. cucurbitae but no sexual dimorphism (Vanickova et al., 2012) larvae

Appendices A. fraterculus adults

228 Appendices

Appendices

Cuticular compound Tephritid found in Whether it shows any change Reference

between individuals 11-methyl, 13-methyl, 15- Z. cucurbitae adults No sexual dimorphism (Carlson and Yocom, 1986) methylnonacosane A. fraterculus adults (Vanickova et al., 2012) B. dorsalis sp. (Goh et al., 1993) (Vanickova et al., 2015c) T. vicina (Zunic Kosi et al., 2013) 7-methylnonacosane Z. cucurbitae adults No sexual dimorphism (Carlson and Yocom, 1986) A. fraterculus adults (Vanickova et al., 2012) B. dorsalis sp., C. fasciventris, (Vanickova et al., 2014) C. capitate T. vicina Present only in females (Zunic Kosi et al., 2013) 3-methylnonacosane Z. cucurbitae adults No sexual dimorphism (Carlson and Yocom, 1986) A. fraterculus adults, T. vicina (Vanickova et al., 2012) (Vanickova et al., 2014) B. dorsalis sp., C. anonae, C. (Zunic Kosi et al., 2013) rosa Differ between spatially isolated A. (Vanickova et al., 2015c) fraterculus females

4-methylnonacosane C. anonae, C. rosa, C. capitata No sexual dimorphism (Vanickova et al., 2014)

229

Appendices 229

230 Cuticular compound Tephritid found in Whether it shows any change Reference

between individuals 11-methyl, 13-methyl, 15- B. dorsalis adults, B. dorsalis Amount is different between species (Carlson and Yocom, 1986) methylhentriacontane sp. but no sexual dimorphism (Goh et al., 1993) T. vicina Present in both males and females (Zunic Kosi et al., 2013) 3-methylhentriacontane B. dorsalis adults Amount is different between species (Carlson and Yocom, 1986) but no sexual dimorphism 11-methyl, 13-methyl, 15-methyl, 17- B. dorsalis adults Amount is different between species (Carlson and Yocom, 1986) methyltritriacontane B. dorsalis sp. but no sexual dimorphism (Goh et al., 1993) 11,15- dimethyl, 13,17- dimethyl, B. dorsalis adults Amount is different between species (Carlson and Yocom, 1986) 15,19-dimethyltritriacontane B. dorsalis sp. but no sexual dimorphism (Goh et al., 1993) 2-methylnonacosane A. fraterculus Found in all males and female. No age (Vanickova et al., 2012) or gender specific 9-nonacosene A. fraterculus Found in all males and female. No age (Vanickova et al., 2012) or gender specific. Proportions decreased with age in males more than 5 days old Nonacosane A. fraterculus, B. dorsalis sp. Found in all males and female. No age (Vanickova et al., 2012) T. vicina or gender specific (Goh et al., 1993)

(Vanickova et al., 2015c) Appendices

230 Appendices

Appendices

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals (Zunic Kosi et al., 2013) 8, 14-hentriacontadiene A. fraterculus Found in all males and female. No age (Vanickova et al., 2012) or gender specific 9,11,14-hentriacontadiene A. fraterculus Found in all males and female. No age (Vanickova et al., 2012) or gender specific 9,16-pentatriacontadiene A. fraterculus Found in all males and female. No age (Vanickova et al., 2012) or gender specific 8,16-pentatriacontadiene A. fraterculus Found in all males and female. No age (Vanickova et al., 2012) or gender specific 7-henicosene A. fraterculus Found only in mature males. Absent in (Vanickova et al., 2012) females and immature males. (Vanickova et al., 2015c) Proportions increased with age in males more than 5 days old 7-docosene A. fraterculus Found only in mature males. Absent in (Vanickova et al., 2012) females and immature males (Vanickova et al., 2015c) 7-tricosene A. fraterculus Found only in mature males. Absent in (Vanickova et al., 2012) females and immature males. (Vanickova et al., 2015c)

231 Proportions increased with age in

Appendices 231

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals males more than 5 days old 7-pentacosene A. fraterculus Found only in mature males. Absent in (Vanickova et al., 2012) females and immature males (Vanickova et al., 2015c) 7-tritriacontene A. fraterculus Found only in mature males. Absent in (Vanickova et al., 2012) females and immature males 3-methyldodecane A. fraterculus Proportions increased with age in (Vanickova et al., 2012) males more than 5 days old and females between 5-30 days old 9-methylpentadecane A. fraterculus Proportions increased with age in (Vanickova et al., 2012) males more than 5 days old and females between 5-30 days old n-hexadecane A. fraterculus Proportions increased with age in (Vanickova et al., 2012) males more than 5 days old and females between 5-30 days old n-heptadecane A. fraterculus Proportions increased with age in (Vanickova et al., 2012) males and females more than 5 days

Appendices old 7, 19- tritriacontadiene A. fraterculus Proportions decreased with age in (Vanickova et al., 2012)

232 Appendices

Appendices

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals males more than 5 days old 4-methylpentadecane A. fraterculus Proportions increased with age in (Vanickova et al., 2012) females 5-30 days old n-hentriacontane A. fraterculus, B. dorsalis sp. Proportions increased with age in (Vanickova et al., 2012) females 5-30 days old in A. fraterculus (Goh et al., 1993) but no sexual dimorphism in (Vanickova et al., 2015c) Bactrocera sp. 3, 7-dimethyltetradecane A. fraterculus No sexual dimorphism. Found in (Vanickova et al., 2012) males and females more than 3 days old 2-methyl, 7-methylheptadecane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females n-ocatadecane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females 2-methyl, 3-methylnonadecane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females n-icosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females

Appendices 233

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals n-henicosene A. fraterculus No specific difference between males (Vanickova et al., 2012) or females 2-methyl, 3-methylhenicosene A. fraterculus No specific difference between males (Vanickova et al., 2012) or females n-docosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females Differ between spatially isolated (Vanickova et al., 2015c) species n-tricosene A. fraterculus Prominent in males more than 14 days (Vanickova et al., 2012) old 3-methyl, 9-methyltricosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females n-tetracosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females 3, y-dimethyltetracosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females

Appendices n-pentacosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females

234 Appendices

Appendices

Cuticular compound Tephritid found in Whether it shows any change Reference

between individuals n-hexacosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females 2-methylhexacosane A. fraterculus No specific difference between males (Vanickova et al., 2012) or females n-heptacosane A. fraterculus Prominent in males more than 7 days (Vanickova et al., 2012) old and females more than 30 days old 3-methylheptacosane A. fraterculus Less than 0.3% in both males and (Vanickova et al., 2012) females more than 3 days old Differ between reproductively isolated (Vanickova et al., 2015c) species n-octacosane A. fraterculus, B. dorsalis sp. Less than 0.3% in both males and (Vanickova et al., 2012) females more than 3 days old in A. (Goh et al., 1993) fraterculus amounts are less in Bactrocera sp. 11- hentriacontene A. fraterculus Less than 0.3% in both males and (Vanickova et al., 2012) females more than 3 days old 10- hentriacontene A. fraterculus No specific difference between males (Vanickova et al., 2012)

5 or females

Appendices 235

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals 9, 13, 15, 16, 17- tritriacontadiene A. fraterculus Comparatively more in males less than (Vanickova et al., 2012) 5 days old 11-tritriacontene A. fraterculus Comparatively more in males and (Vanickova et al., 2012) females less than 7 days old 7, 18-pentatriacontadiene A. fraterculus No specific difference between males (Vanickova et al., 2012) or females 13-pentatriacontene A. fraterculus No specific difference between males (Vanickova et al., 2012) or females x, y-heptatriacontadiene A. fraterculus Amounts decrease in females more (Vanickova et al., 2012) than 14 days old n-triacontane B. dorsalis sp. No specific difference between males (Goh et al., 1993) or females T. vicina Present only in females (Zunic Kosi et al., 2013) 3-methyltriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference

Appendices between males or females C. fasciventris Sexually dimorphic, present only in (Vanickova et al., 2014)

females

236 Appendices

Appendices

Cuticular compound Tephritid found in Whether it shows any change Reference

between individuals C. anonae Present in both males and females 9,13-dimethyl, 11,15-dimethyl, , 3,7- B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) dimethyl, 3,9-dimethyl, 3,11- species. No specific difference dimethylhentriacontane between males or females 13,17-dimethylhentriacontane C. fasciventris, C. rosa No sexual dimorphism (Vanickova et al., 2014) 12,14-dimethylhentriacontane C. fasciventris, C. rosa No sexual dimorphism (Vanickova et al., 2014) n-docotriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females 2-methyl, 3-methyl, 8-methyl, 10- B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) methyl, 12-methyl, 14-methyl, 16- species. No specific difference methyldocotriacontane between males or females n-tritriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females 5-methyltritriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference

23 between males or females

Appendices 237

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals 3-methyl, 11-methyl, 13-methyl, 15- B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) methyl, 17-methyltetretriacontane species. No specific difference between males or females n-pentatriaconate B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females 11-methyl, 13-methyl, 15-methyl, 17- B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) methylpentatriaconate species. No specific difference between males or females 11,15-dimethyl, 13,17-dimethyl, B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) 15,19-dimethylpentatriacontane species. No specific difference between males or females 3-methylpentatriaconate B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females n-hexatriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993)

Appendices species. No specific difference between males or females

238 Appendices

Appendices

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals n-heptatriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females n-octatriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females n-tetracontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females n-octacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females n-nonacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females 11,15-dimethyl, 11,17-dimethyl, B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) 11,19-dimethyltricontane species. No specific difference

23 between males or females

Appendices 239

240

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals n-tetratriacontane B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) species. No specific difference between males or females 11-methyl, 13-methyl, 15-methyl, 17- B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) methyltetratriacontane species. No specific difference between males or females 11-methyl, 13-methyl, 15-methyl, 17- B. dorsalis sp. Different between two B. dorsalis (Goh et al., 1993) methylpentatriacontane species. No specific difference between males or females 4-methyloctacosane C. fasciventris, C. anonae, C. Present in both males and females (Vanickova et al., 2014) capitate C. rosa Sexual dimorphism in C. rosa, present only in females 11,13-dimethylhentriacontane C. fasciventris Sexually dimorphic, present only in (Vanickova et al., 2014) females C. anonae No sexual dimorphism

Appendices Hexatriacontene C. anonae, C. rosa, C. capitata No sexual dimorphism (Vanickova et al., 2014)

240 Appendices

Appendices Cuticular compound Tephritid found in Whether it shows any change Reference between individuals Heptatriacontatriene C. rosa, C. capitata No sexual dimorphism (Vanickova et al., 2014) Methylheptatriacontane A. fraterculus Differ between spatially isolated (Vanickova et al., 2015c) species n-dodecane A. fraterculus Differ between spatially isolated (Vanickova et al., 2015c) species Tetratriacontadiene A. fraterculus Differ between spatially isolated (Vanickova et al., 2015c) species Nonyl palmitoleate T. vicina Present only in males (Zunic Kosi et al., 2013) 2,14-dimethyl, 2,16-dimethyl, 2,18- T. vicina Present only in females (Zunic Kosi et al., 2013) dimethyloctacosane

2,22-dimethyloctacosane T. vicina Present in both males and females (Zunic Kosi et al., 2013)

nonacosene T. vicina Present in both males and females (Zunic Kosi et al., 2013) Nonyl oleate T. vicina Present only in males (Zunic Kosi et al., 2013) 12-methyltriacontane T. vicina Present only in females (Zunic Kosi et al., 2013)

2,18-dimethyl, 2,20- T. vicina Present only in females (Zunic Kosi et al., 2013)

241

Appendices 241

242

Cuticular compound Tephritid found in Whether it shows any change Reference between individuals dimethyltriacontane hentriacontane T. vicina Present in both males and females (Zunic Kosi et al., 2013) hentriacontene T. vicina Present in both males and females (Zunic Kosi et al., 2013) 11,19-dimethylhentriacontane T. vicina Present only in females (Zunic Kosi et al., 2013) Tritriacontene T. vicina Present only in females (Zunic Kosi et al., 2013) Tritriacontadiene T. vicina Present only in females (Zunic Kosi et al., 2013) 11,21-dimethyltritriacontane T. vicina Present only in females (Zunic Kosi et al., 2013) Pentatriacontadie T. vicina Present in both males and females (Zunic Kosi et al., 2013)

Appendices

242 Appendices

Supplementary material

Supplementary material 1 Journal publications relevant to this thesis

Supplementary material 243

Supplementary material 2 Abstract of oral presentations relevant to this thesis that were presented in scientific conferences

50th Australian Entomological Society Conference

28 September – 1 October 2014

Canberra, Australia

Oral Presentation: What does Queensland fruit fly, Bactrocera tryoni (Froggatt), do

when the sun goes down?

E.W.M.T.D.Ekanayake (1), A. R. Clarke (1, 2) & M. K. Schutze (1, 2)

(1) School of Earth, Environmental and Biological Sciences, Science and

Engineering Faculty, Queensland University of Technology, Brisbane, Australia; (2)

Plant Biosecurity Cooperative Research Centre, Bruce, Australian Capital Territory,

Australia

Bactrocera tryoni, the Queensland fruit fly (Qfly), is Australia’s worst horticultural pest insect. While much is known of its mating system from small cage laboratory studies, little is known about how the sexes come together to mate at dusk. In replicated, large field cage mating studies, we asked: i) does Qfly exhibit

‘landmarking’ behaviour by using tall trees as mate-rendezvous sites; and ii) do males establish lek territories prior to female arrival, as has been routinely inferred for this species. For each of seven replicates, 125 sexually mature virgin males and females were released prior to dusk in a 7 X 7 X 4 m field cage containing two tall

(2.7 m) and two short (2.0 m) artificial trees. Five-minute counts of individual flies and mating pairs on trees were made, along with recording instances of male-male aggression and male calling (wing vibration and pheromone release) behaviour.

Ninety-nine percent of all mating pairs across replicates (n = 85) occurred on tall trees; only 1% of couples were recorded from short trees. The proportion of

244 Supplementary material

individual females in tall trees prior to dusk was greater than that of males (60 ± 3% vs 40 ± 3%), but within ten minutes of dusk males arrived in large numbers such that they dominated females (68 ± 5% vs 32 ± 5%). Among those males, most ‘called’

(88 ± 5%) but few engaged in male-male aggressive behaviour (12 ± 5%). The preference for tall trees is supportive of Qfly using a ‘land-marking’ system to initially bring the sexes together. The greater number of females at mating sites before dusk, with little male-related territorial behaviour prior to courtship, questions the long-held view that Qfly has a lek-based mating system.

Supplementary material 245