The role of learning in the ecology of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae: Opiinae), and implications for tephritid pest management
Aead M Abdelnabi Muhmed
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
2018 School of Earth, Environmental and Biological Sciences Science and Engineering Faculty Queensland University of Technology
Keywords
Diachasmimorpha kraussii, parasitoid, fruit-mimicking bag, learning behaviour,
Bactrocera tryoni, Tephritidae, wasp, odour, host, biological control, inundative release.
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Abstract
Many species of braconid wasp (Hymenoptera: Braconidae) are agriculturally important, having been successfully used in biological control. Members of the braconid subfamily Opiinae have particularly been used as agents against tephritid fruit fly pests (Diptera: Tephritidae). The braconid Diachasmimorpha kraussii
(Fullaway) is a larval endoparasitoid of at least 18 fruit fly species. This wasp is native to Australia and Papua New Guinea, and has been introduced to many countries worldwide as a fruit fly biological control agent. The ability of adult female
D. kraussii to find their host larvae is crucial to their survival and reproduction, and a better understanding of host finding behaviour in this species could improve the effectiveness of wasps released in the field by improved identification of hosts and odour recognition in complex environments.
This PhD studies host finding in D. kraussii, with a specific focus on olfaction and the role of experience (= learning). The first research chapter develops rearing methods for the wasp, comparing a novel method designed by me that presents host larvae in hanging bags of artificial diet, with an existing method of presenting larvae and artificial diet in a petri dish, and with whole fruits (nectarines).
Wild parasitoids showed poor parasitism rates when using the petri-dish method, and parasitism rates using the ‘bag’ method are equal to those in nectarines. Furthermore, because artificial diet rears more fly larvae per 100 fly eggs than nectarines, the bag method cultures significantly more wasps than whole fruits. This methodology therefore provides a new technique for mass culturing wasps and for carrying out behavioural tests in the laboratory and field.
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The aim of the second research chapter is to improve our understanding of how D. kraussii finds its main host, the Queensland fruit fly (Bactrocera tryoni
(Froggatt)), and more explicitly how experience with host-derived olfactory cues may reinforce successful host finding. Y-tube olfactometer studies were used as a behavioural assay to investigate the influence of naïve and experienced female wasps’ orientation to odours of fruits infested with physiologically suitable larval hosts (B. tryoni) compared to fruits infested by non-host larvae (Drosophila melanogaster). The results showed that naïve wasps had significant attraction to odours of uninfested and B. tryoni infested nectarines, with the preference for odours of infested fruits significantly greatly than that for uninfested fruits.Naïve wasps also showed a clear preference toward fruit infested with B. tryoni versus fruit infested with D. melanogaster. There was no significant difference in choices between uninfested fruits and nectarines infested by D. melanogaster.
Experience of wasps on B. tryoni infested fruits significantly increased wasp preference for odours of host infested fruits compared to naïve preferences.
However, prior wasp experience on non-host (i.e. D. melanogaster) infested fruits did notsignificantly changepreferences for the odours of host and non-host infested fruits compared to naïve wasps. Experience of both host and non-host infested fruits significantly increasedwasppreference for odours of host infested fruits compared to naïve wasps, but no more so than experience on host infested fruits alone.
As a continuation of investigating wasp learning, I examined operant learning. In operant learning, an individual learns to carry out a specific behaviour to increase the frequency of an outcome: in my experiments this outcome was the ability of a wasp to successfully locate larvae within a fruit. I trained wasps by
iii exposing them to fruits where the position of larvae was always in a predictable location; either at the top or base of the fruit. The exploratory movement and probing rate of experienced wasps was then compared with naïve wasps. The results showed that wasps displayed operant learning to improve searching in order to better locate fly larvae. To my knowledge, this is one of the first times operant learning has been demonstrated in wasps.
The final research chapter investigated how learning influences parasitoid behaviour at multiple spatial scales: in laboratory, semi-field and open field conditions. Based on this learning capacity, it has been suggested that providing pre- release training to parasitoids reared for inundative release may lead to a subsequent increase in their efficacy as biological control agents. Using the fruit fly parasitoid
Diachasmimorpha kraussii we tested this hypothesis in a series of associative learning experiments which involved the parasitoid, two host fruits (tomatoes and nectarine), and one host fly (Bactrocera tryoni). In sequential Y-tube olfactometer studies, large field-cage studies, and then open field studies, naïve wasps showed a consistent preference for nectarines over tomatoes. In large field-cages, wasps with prior learning on tomato significantly increased orientation to tomato, in comparison to naïve wasps. Prior experience to nectarine did not, however, significantly increase orientation to this fruit in comparison to naïve wasps. In an open orchard, and using my fruit mimicking ‘bags’ modified to provide either nectarine or tomato odours but with the same visual cues, prior experience again significantly increased host location towards tomato, but not towards nectarine. Prior experience did not increase parasitism rates within located bags for either tomato or nectarines. These results demonstrate that learning results developed from laboratory bioassays can be up-
iv scaled to the open field environment, something which is very rarely tested in parasitoid learning studies.
Results are discussed in the context of the adaptive benefits of learning in the ecology of parasitoid wasps, and the implications of prior experience when releasing wasps as inundative biological control agents.
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Table of Contents
Keywords ...... i
Abstract ...... ii
Table of Contents ...... vi
List of Tables ...... x
List of Figures ...... xi
Thesis by publication statement ...... xv
List of supplementary material ...... xvi
Statement of Original Authorship ...... xvii
Acknowledgements ...... xviii
Chapter 1: General Introduction ...... 1
1.1 General Introduction…………………………………………………………2
1.2 Fruit flies and their biological control with Opiinae…………………….5
1.2.1 The Opiinae ...... 6
1.2.2 Fruit fly biological control ...... 7
1.2.3 Culturing and Mass release of parasitoids for biological control ...... 10
1.3 Parasitoid host selection ...... 13
1.3.1 Olfaction ...... 13
1.3.2 Visual cues ...... 17
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1.3.3 Adult feeding sites...... 18
1.4 Learning Behaviour ...... 18
1.4.1 Associative learning (Classical conditioning) ...... 19
1.4.2 Operant learning ...... 20
1.4.3 Learning in parasitoids ...... 21
1.4.4 Olfactory learning in parasitoids and its adaptive significance ...... 23
1.4.5 Visual learning ...... 26
1.4.6 Learning on multiple host cues and other factors ...... 27
1.5 Thesis foci organisms: Diachasmimorpha kraussii and Bactrocera
tryoni...... 28
1.5.1 D. kraussii life cycle ...... 29
1.5.2 Diachasmimorpha kraussii as a biological control agent ...... 30
1.5.3 Behaviour and ecology of Diachasmimorpha kraussii ...... 31
1.5.4 Queensland fruit fly, Bactrocera tryoni ...... 32
1.5.5. Basic biology and ecology of B. tryoni...... 34
1.5.6 Opiinae parasitoids as biocontrol agents against B. tryoni ...... 35
1.6 Structure of the thesis ...... 36
Chapter 2: An improved culturing method for opiine fruit fly parasitoids and its application to parasitoid monitoring in the field ...... 40
2.1 Introduction ...... 41
2.2 Materials and methods ...... 43
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2.2.1 Insects ...... 43
2.2.2 Exposure of host larvae to wasps ...... 44
2.2.3 Field study ...... 46
2.2.4 Statistical analysis ...... 49
2.3 Results ...... 49
2.4 Discussion ...... 56
Chapter 3. Learning influences host versus non-host discrimination and post- alighting searching behaviour in the fruit fly parasitoid Diachasmimorpha kraussii ...... 60
3.1 Introduction ...... 60
3.2 Material and Methods ...... 62
3.2.1 Insect cultures and fruits ...... 62
3.2.2 Experiments ...... 64
3.2.3 Statistical analysis ...... 70
3.3 Results ...... 71
3.3.1: Experiment 1a: Odour preferences of naïve D. kraussii females for host
(B. tryoni) and non-host (D. melanogaster) infested fruits...... 71
3.3.2: Experiment 1b: Odour preferences of experienced D. kraussii females for
host (B. tryoni) and non-host (D. melanogaster) infested fruits...... 71
3.3.3: Experiment 2.The influence of experience on D. kraussii post-alighting
orientation and oviposition behaviour...... 73
3.4 Discussion ...... 77 viii
Chapter 4. From laboratory to the field: consistent effects of experience on host location by the fruit fly parasitoid Diachasmimorpha kraussii (Hymenoptera:
Braconidae) ...... 83
4.1 Introduction ...... 83
4.2 Materials and Methods ...... 86
4.2.1 Insect cultures ...... 86
4.2.2 Experiments ...... 87
4.3 Results ...... 97
4.3.1 Experiment 1: Y-tube olfactometer ...... 97
4.3.2 Experiment 2: Large field cage ...... 98
4.3.3 Experiment 3: Field study ...... 100
4.4 Discussion ...... 103
Chapter 5: General Discussion ...... 106
5.1 Summary of results ...... 106
5.2 Implications of thesis results for wasp host location ...... 109
5.3 Adaptive significance of parasitoid learning behaviour in complex and variable
environments ...... 112
5.4 Application to inundative biocontrol ...... 115
5.5 Further research ...... 118
7. References ...... 120
Supplementary Material: ...... 160
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List of Tables
Table 1: Fruit fly parasitoid species introduced and used for biological control
programs...... 12
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List of Figures
Figure 1.1. Adults of Diachasmimorpha kraussii ...... 30
Figure 1.2. The Queensland fruit fly, Bactrocera tryoni (Froggatt)...... 33
Figure 2.1. Preparation of the culturing ball: (A) carrot medium infested with B.
tryoni eggs, (B) central agar ball, (C) infested carrot medium on agar ball,
(D) D. kraussii on culturing bag within a breeding cage, (E) female D.
kraussii ovipositing on culturing bag, and (F) wild D. kraussii on culturing
bag in mixed stone fruit orchard...... 48
Figure 2.2. Comparison of Diachasmimorpha kraussii culturing methods, which
presented adult wasps with host larvae either in whole nectarines (fruit), or
artificial diet in Petri dishes (Petri dish) or culturing bags (Bag). Trials (n =
15 per treatment) used 5 adul female wasps in cages with host substrate
(fruit or artificial carrot-based media) infested with 100 Bactrocera tryoni
eggs: Mean numbers of adult wasps emerging. Bars with different letters are
significantly different (Kruskal–Wallis/Mann–Whitney)...... 52
Figure 2.3. Mean pupae (includes parasitized and unparasitized hosts). Bars with
different letters are significantly different (ANOVA)...... 53
Figure 2.4. Mean proportion of parasitized hosts. Bars with different letters are
significantly different (Kruskal–Wallis/Mann–Whitney)...... 53
Figure 2.5. Mean hind tibia length (measure of body size). Bars with different letters
are significantly different (ANOVA)...... 54
Figure 2.6. Culturing bags as a tool for evaluating D. kraussii prevalence in fruit
orchards. Box-plots displaying (A) total and (B) percentage parasitism of
larvae in culturing bags, nectarines, and peaches in a mixed fruit orchard.
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*Outliers. (A) Box-plots with different letters are significantly different (P
<0.05). (B) Data were not significantly different (P >0.05)...... 55
Figure 3.1. Y-tube olfactometer used in these experiments...... 66
Figure 3.2. Diachasmimorpha kraussii female lands on a single nectarine that had
been subjected to oviposition by Bactrocera tryoni as described above and
contained eggs in both the top and bottom sections...... 70
Figure 3.3. Y-tube olfactometer trials testing odour preferences of naïve mated
female Diachasmimorpha kraussii (Innate preferences). Odour sources:
clean air, uninfested nectarines, B. tryoni (host) infested nectarines,
D. melanogaster (non-host) infested nectarines. Bars represent percentage
attraction to odours (insect counts at end of bars). Significance determined
by χ2 tests on count data (* P< 0.05. ** P< 0.005)...... 72
Figure 3.4. Y-tube olfactometer trials testing odour preferences of experienced mated
female Diachasmimorpha kraussii (Experience of infested fruits). Odour
sources: B. tryoni (host) infested nectarines, D. melanogaster (non-
host) infested nectarines. Bars represent percentage attraction to odours
(insect counts at end of bars). Significance determined by χ2 tests on count
data (* P< 0.05. ** P< 0.005)...... 73
Figure 3.5. Post-alighting behavioural responses (post alighting orientation) for
Diachasmimoprha kraussii adult females following experience ovipositing either
at the top or base sections of nectarines, or with no experience. Red and blue
bars denote top and base section of fruit respectively. Within treatment
differences, * = significant at P< 0.05. Between treatment differences, bars with
different letters are significantly different at P < 0.05……………………..…75
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Figure 3.6. Post-alighting behavioural responses (Percentage search time) for
Diachasmimoprha kraussii adult females following experience ovipositing
either at the top or base sections of nectarines, or with no experience...... 76
Figure 3.7. Post-alighting behavioural responses (Probing counts) for
Diachasmimoprha kraussii adult females following experience ovipositing
either at the top or base sections of nectarines, or with no experience ...... 77
Figure 4.1. Large field cages at the QUT Samford Ecological Research Facility in
South-east Queensland...... 91
Figure 4.2. Artificial trees inside Samford field cage...... 92
Figure 4.3. Guava orchard where experiments were run...... 96
Figure 4.4. Fruit mimicking bag in the field with ovipositing parasitoid...... 96
Figure 4.5. Percentage responses of female Diachasmimorpha kraussii to odours of
infested nectarines vs infested tomatoes) under different conditioning
treatments (N = 30 per treatment). Significance:* = within treatment
differences, χ2 test), │= between treatment differences (P < 0.05, χ2 test)...... 98
Figure 4.6. Mean (+ 1SE) number of Diachasmimorpha kraussii caught ovipositing
into nectarine or tomato fruit under three conditioning treatments (naïve,
experienced on infested nectarine, or experienced on infested tomato...... 99
Figure 4.7. Proportional visitation of Diachasmimorpha kraussii to two types of fruit
mimic: nectarine and tomato. Wasps were ovipositionally naïve, or had
prior experience on nectarine and tomato. Significantly more tomato-
experienced wasps visited tomato bags than did naïve wasps, but no other
pairwise comparison between naïve and experienced wasps within the same
fruit type are significantly different based on a binomial test...... 101
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Figure 4.8. Mean (+ 1SE) number of parasitoids emerging from nectarine or tomato fruit
once oviposited into by Diachasmimorpha kraussii of three different ovipositional
experience states: ovipositionally naïve, trained on infested nectarine, or trained
on infested tomato…………………………………………………………….102.
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Thesis by publication statement
This thesis is submitted under the Queensland University of Technology rules of “Thesis by Publication”, which requires one accepted paper and two submitted papers at time of examination.
At the time of final submission of the corrected thesis (Feb 2018) these papers are:
Masry A., Furlong M.J., Clarke A.R. & Cunningham J.P. 2018. An improved culturing method for opiine fruit fly parasitoids and its application to parasitoid monitoring in the field. Insect Science, 25, 99-108.
Masry A., Clarke A.R. & Cunningham J.P. Learning influences host versus non-host discrimination and post-alighting searching behaviour in the fruit fly parasitoid Diachasmimorpha kraussii. Journal of Economic Entomology: in press.
Masry A., Cunningham J.P. & Clarke A.R. 2017. From laboratory to the field: consistent effects of experience on host location by the fruit fly parasitoid Diachasmimorpha kraussii (Hymenoptera: Braconidae). Insect Science, in press.
As senior author I was involved in the design of the experiments, completed all experimental work, carried out analysis and wrote the first draft of papers. My co- authors played a role in experimental design, analysis and editing commensurate with that of normal postgraduate supervisory practice.
Papers are reformatted for the thesis, primarily through the use of consecutive numbering for sections and figures, and through the use of a combined reference list at the end of the thesis. Some additional illustrative figures, for example of experimental equipment and field sites, have also been included.
xv
List of supplementary material
Supplementary material: Abstract of oral presentation relevant to this thesis presented at a scientific conferenc…………………………………………………155
xvi
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: February 2018
xvii Acknowledgements
I would like to express my heartfelt gratitude to my supervisors Prof. Tony Clarke and A/Prof. Paul Cunningham who gave me the golden opportunity by their guidance and support throughout the years of this wonderful project and helped me to pass complications related to my works and writings I am really thankful to them.
I thank all the members of the QUT technical staff members in the past and present especially Ms Amy Carmichael, Mr Mark Crase and Anne-Marie McKinnon: for helpfulness and supplying the experimental equipment and facilities.
I am very much thanks to the School EEBS officers: Prof. Stuart Parsons, Dr Prasanna Egodawatta, Sarie Gould and Noelene Davis for assistance.
I acknowledge the Institute for Future Environments, Queensland University of Technology, for allowing me to work at SERF. Special thanks to Mr Marcus Yates, SERF on-site manager. Thanks to Lona Van Delden and Tommaso Francesco Villa for support and advice.
Many thanks go to Ms Thelma Peek and Linda Clarke (Queensland Department of Agriculture and Fisheries) who kindly supplied the B. tryoni pupae to establish insect cultures. This department also allowed me to access parasitoid collections and field experiments at their Redlands Research Facility.
I wish to thank all my dear members of our fruit fly group in the past and present for their support and advice throughout the regular lab meetings and within lab works; Ms Jaye Newman, Dr Yuvarin Boontop, Thilini, Dr Mark Schutze, Dr Matt Kroch, Dr Kumaran, Kiran, Jacinta, Francesca, Melissa, Dr Katharina, Shirin, Shahrima, Prof. Stephen Cameron, Jim, Brett and Owen; I thank you very much.
I would like to thank my family; my wife Salha Hamad and my kids (Hoda, Abdelrhman, Alaa, Maram, Muhamed, Takwa and Lyan) for their support and patience. Special thanks to my mother, brothers and sisters in Libya who share happiness and good wishes. Thank you for trusting in me! Thanks to all my friends in Brisbane and in Libya for encouragement and good wishes. xviii
I was nominated to study a PhD by Omar Al-Mokhtar University and this PhD project was funded by a Libyan Government Postgraduate Scholarship. I am very grateful indeed for this support.
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Chapter 1: General Introduction
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1.1 General Introduction
Tephritid fruit flies (Diptera: Tephritidae) are worldwide horticultural pests, renowned for their destruction of a wide range of fruit and vegetables in commercial crops and home gardens (Aluja and Mangan 2008, Clarke et al., 2011). There are around 4000 described species of tephritid fruit fly, categorized within almost 500 genera, and tephritids are present on every continent except Antarctica (Fletcher
1987). Only a small proportion of tephritid species are pests, but tens of millions of dollars are spent annually on their control and eradication (McPheron and Steck
1996, Clarke et al., 2005, Godefroid et al., 2015). For over four decades, insecticides have widely been used to control pest fruit fly populations (Harris 1989), but a continued reliance on heavy spraying with conventional pesticides has many drawbacks, particularly in terms of insecticide resistance, and public and environmental health (Pimentel et al., 2005, Konradsen 2007, Marrs and Dewhurst
2012, Onstad 2013). The future sustainable management of fruit fly therefore needs to consider an integrated approach, utilising alternative methods for reducing insect populations (Bautista et al., 1999, Barratt et al., 2010a, Clarke et al., 2011, Sivinski and Aluja 2012). The need for alternative strategies in fruit fly control has encouraged the use of nonchemical and biological agents including use of the sterile insect technology (Hendrichs et al. 2005, Rendon et al. 2006, CSIRO, 2014), and natural enemies (Drew et al., 1978, Sivinski et al., 1996, Duan et al., 1997, Barbosa
1998, Purcell 1998).
The most common natural enemies of fruit fly used in biological control
(=biocontrol) programmes are wasps within the sub-family Opiinae of the family
Braconidae (Hymenoptera) (Wong and Ramadan 1992, Wong 1993, Duan and
Messing 1997, Wen et al., 2002, Rousse et al., 2005, Clarke et al., 2011, Canale and 2
Benelli 2012, Zamek et al., 2012). Large numbers of opiine fruit fly parasitoids are used as biocontrol agents in augmentative and classical biological control to reduce fruit fly populations. Many members of the Opiinae, notably species from the genera
Diachasmimorpha and Fopius, have been introduced and released worldwide against fruit fly pests (Messing and Jang 1992, Purcell 1998, Bautista et al., 1999,
Chinajariyawong et al., 2000, Bokonon-Ganta et al., 2013). The main goal of a biological control programme is having a high quality parasitoid population that can be active in reducing pest populations (Wong and Ramadan 1992, Messing et al.,
1993, Zamek et al., 2012). There are many different research components required to achieve this ideal outcome.
The first aspect of producing high quality parasitoids is laboratory rearing.
Poor lab rearing can reduce the quality of parasitoids to be released, either because absolute numbers available for release are low (i.e. simple inability to rear wasps), or because breeding has led to the loss of key behavioural attributes, for example, the ability of released wasps to find hosts in the natural environment (Murdoch et al.,
1985, Wharton and Cavalloro 1989, Barbosa 1998). Measurements used to monitor the quality of mass reared parasitoids is used as an important measurement including: host size, adult emergence, survival, fecundity, flight and searching behaviour
(Zamec et al., 2012).
Assuming that quality wasps can be reared, understanding the behavioural ecology of parasitoids is another critical step. Studying the mechanistic basis of host- parasitoid interactions provides the scientific basis for actively manipulating parasitoids as biocontrol agents (Wajnberg et al., 2016). Understanding insect foraging behaviour and the role of learning in foraging can provide better
3 information about the population dynamics of parasitoids and their hosts, and their distribution in different environments, making this a critical area of research in biological control programes (Hassell 1978, Murdoch et al., 1985, Mills and Getz
1996, Murdoch et al., 1996, Mehrnejad and Copland 2006, Mills and Kean 2010). In order to forage effectively, a parasitoid should concentrate its searching towards patches of high quality hosts, thus maximizing the number and quality of its offspring (Waage 1983, Wang and Keller 2002). Ecological factors and phylogenetic history can influence the range of hosts used by opiine braconids (Stireman and
Singer, 2003).
Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae: Opiinae) is a fruit fly parasitoid widely used in both classical and inundative biological control programs (Wong 1993, Sime et al., 2006, Zamek et al., 2012). This species has been recorded attacking many fruit- infesting Tephritidae and has successfully been introduced to multiple locations around the world for classical biological control of fruit fly (Sime et al., 2006, Argov and Gazit 2008, Bokonon-Ganta et al., 2013) .
Diachasmimorpha kraussii has been also targeted for use in mass-release against endemic fruit flies because of its low risk to non-target hosts and apparent adaptability to variable conditions (Bokonon-Ganta et al., 2013).
Endemic to the east coast of Australia, D. kraussii’s native, and presumably evolutionary hosts, include the Queensland fruit fly, Bactrocera tryoni (Froggatt)
(Diptera: Tephritidae) (Carmichael et al., 2005). This fly is Australia’s primary insect pest of horticulture, and one for which non-pesticide control options are urgently needed (Clarke et al., 2011). While mass-rearing and release of D. kraussii is an option for B. tryoni control, more fundamental and strategic research on the wasp is
4 needed. This thesis provides some of that required knowledge, by researching laboratory rearing techniques (Chapter 2), carrying out fundamental studies on how wasp learning modifies host usage (Chapter 3), and then testing the effects of learning at multiple spatial scales, including the field (Chapter 4).
The remainder of this chapter provides a more detailed literature review to set a framework for assumptions of this thesis, particularly with respect to selected behavioural and ecological aspects of fruit fly parasitoid host location. In section 1.2,
I provide more information about fruit flies and their biological control with opiine braconids. Within this section I introduce the Opiinae and review cases of fruit fly biological control programmes which involve these wasps. Sections 1.3 and 1.4 focus more intensively on parasitoids, reviewing parasitoid host selection and parasitoid learning, respectively. In section 1.5, I introduce the study organisms, the parasitoid D. kraussii and its host fruit fly B. tryoni. I finish this chapter with an outline of the thesis structure.
1.2 Fruit flies and their biological control with Opiinae
Within the tephritids, dacine fruit flies (Diptera: Tephritidae: Dacinae) have a number of important pest species (Drew 1989), particularly those belonging to the genera Ceratitis, Bactrocera and Dacus (Díaz-Fleischer et al., 2001, Krosch et al.,
2012). Of these, the genus Bactrocera is ranked first among economically important fruit flies, with around 40 species considered to be important pests (White and
Elson–Harris 1994), nearly all of which are from the Oriental and Australasian regions (Drew & Romig 2000). Bactrocera cause direct fruit damage from larval feeding within fruit, and indirect damage through loss of potential market access and the need to maintain quarantine barriers (Drew 1997, Homestead 2015, Malheiro et
5 al., 2015). Highly pestiferous not only in their native Oriental and Australasian regions, pest Bactrocera are also introduced and established in nearly all parts of
Africa, in the U.S. states of Hawaii and California, in some Pacific Ocean states and territories, and in parts of South America (Vera et al., 2002, Clarke et al., 2004, De
Meyer et al., 2010, Dominiak and Daniels 2012, Szyniszewska and Tatem 2014,
Schutze et al., 2015).
In classical biological control, the importation of parasitoids from the pest’s original geographic range to a new, invasive geographic target area, presents an opportunity to economically suppress the pest populations in that new location
(Murdoch et al., 1985, Barbosa 1998, Purcell 1998, Messing 1996, Fagan et al.,
2002, Tscharntke et al., 2007). Initially targeting invasive fruit fly populations through classical biological control, but in more recent times targeting both invasive and endemic populations through inundative release of (mass release of parasitoids against a specific target population of pest (Zamek et al., 2012)), opiine braconids
(Hymenoptera: Braconidae: Opiinae) have been used for over 100 years to control fruit flies (Wharton and Gilstrap, 1983).
1.2.1 The Opiinae
The Opiinae is one of the largest subfamilies of the Braconidae, with approximately 1300 species described worldwide, and opiine wasps are the most commonly used fruit fly parasitoids (Carmichael et al., 2005). The Opiinae was classified once into 22 genera (Fischer 1987), but subsequent changes and mergers of generic names (Wharton 1997) have occurred. This has seen some name changes for economically important opiines, which can potentially be confusing when reading older literature. For example the focus insect of this thesis, Diachasmimorpha
6 kraussii, has been previously placed in Opius and Biosteres (Carmichael et al.,
2005). A stable and comprehensive taxonomy of the Opiinae is critical for efficient biological control programmes, but the sheer size of the sub-family has made this difficult. The most comprehensive current taxonomic and diagnostic resource for fruit fly infesting opiines is the web site “Parasitoids of Fruit-Infesting Tephritidae”
(http://www.paroffit.org/public/site/paroffit/home) (accessed 14/03/2017).
Opiinae commonly attack the egg and/or larval stage of their hosts and are mostly described as koinobiont endoparasites. The subsequent parasitoid larva then feeds upon its host, and ultimately kills it (Godfray 1994, Hawkins and Sheehan
1994, Wharton 1997). The opiine braconids are generally egg/larval/pupal, or larval/pupal parasitoids: the egg and/or larval stage of the host fly are initially parasitised, with the fully developed adult parasitoid emerging from the pupa (Ero
2009). The parasitoid offspring uses only one host, which develops normally until just before the parasitoid emerges, at which stage the host is killed. The opine wasps are not restricted to tephritid hosts, and also show great diversity in agromyzid leaf miners (Hawkins et al., 1990, Lambkin et al., 2008), another family of Diptera with larvae that feed concealed within the host plant. These Opiinae host have been used extensively in classical and inundative biological control of pest species in both groups (Sivinski and Aluja 2012).
1.2.2 Fruit fly biological control
The first attempt of using fruit fly parasitoids as biocontrol agents in Australia was in 1902 (Wharton 1989), but the most extensive use of fruit fly parasitoids as classical biological control agents has been in Hawaii. During the biological control campaign against Bactrocera dorsalis (Hendel) on the Hawaiian Islands, more than
7
30 species of parasitic Hymenoptera were introduced from all over the world
(Clausen et al. 1965). However, only a handful of these species still contribute to the biocontrol of fruit flies in Hawaii (Wong et al., 1984). The success of the Hawaiian fruit fly biocontrol program has spurred further research, and the redistribution of these parasitoids to many countries with tephritid pest problems (Wong et al., 1992).
The most commonly used biocontrol agents for release against fruit flies are opiine wasps in the genera Diachasmimorpha, Psyttalia, Fopius, Bracon and Utetes.
Species from these genera have been successfully used and established in many places worldwide for release (Table 1). Species of Diachasmimorpha, particularly, have been used as effective agents in classical biocontrol and within IPM programmes against tephritid fruit flies (Purcell et al., 1998). Diachasmimorpha longicaudata, native to Southeast Asia, is considered a strong candidate in augmentative mass release to suppress tephritid pests. It was successfully introduced as an augmentative release agent to control B. dorsalis and Ceratitis capitata in
Hawaii (Weidemann) (Messing et al., 1993),and also sent to Australia where it is established in far-northern eastern Australia (Zamek et al., 2012). This species has also been introduced into other locations including Florida, Mexico and South
America to control Anastrepha fruit flies (Sivinski et al., 1996, Montoya et al.,
2000). The Australian native fruit fly parasitoid D. kraussii was introduced to Hawaii and the Mediterranean region to control C. capitata and B. latifrons (Hendel), with success reported (Messing and Ramadan 1999, Argov et al., 2011).
Diachasmimorpha tryoni (Cameron) is another Australian fruit fly parasitoid that has been successfully introduced to Hawaii to control B. dorsalis and C. capitate (Wong et al., 1991) and has been used in mass release in Mexico against the Mexican fruit fly, Anastrepha ludens (Loew), within mango and citrus cultivations (Ovruski et al., 8
2000, Cancino et al., 2009). The parasitoidPsyttalia concolor (Szépligeti), originally described from North Africa, was released into California to control olive fruit fly B. oleae (Gmelin) with subsequent parasitism levels ranging 10.6% in coastal areas to
0% in inland areas (Yokoyama et al., 2008). It has been also reported that Bracon spp could be used and inoculated in IPM systems to control leaf-miner pests in tomato plantations and efforts have been made to recover these biocontrol agents within their native areas, including South America and the Mediterranean basin
(Miranda et al., 2005, Zappalà et al., 2013).
Control of fruit fly populations can be enhanced by incorporating biocontrol with sterile insect technique (SIT) (Knipling 1998). The first original attempt of SIT against fruit flies was in Hawaii to control C. capitata in Hawaii and B. dorsalis in the Western Pacific (Harris et al., 1986). In California this procedure is used against
C. capitata (Cunningham et al., 1980), and in Japan to eradicate Zeugodacus cucurbitae (Coquillet) (Koyama et al., 2004). In Australia the SIT is used in the south-east of the continent to control B. tryoni within horticultural areas (Spinner et al., 2011). However, although this technique has been reported to very successful in many areas, there are complications that still limit its large scale use in many areas.
The main problems of the SIT is that it is expensive (Parker and Mehta 2007), and so increasing its efficacy to maximise the benefit received for the cash invested is required. In this case, incorporating the use of the SIT with parasitoids has significant efficacy benefits in the reduction of the target pest population (Gurr and Kvedaras
2010). Using SIT combined with the parasitoid D. tryoni to control Medfly in coffee cultivation in Guatemala and in Hawaii has given
9 better control of fruit fly pest when compared using SIT alone (Sivinski et al., 2000,
Montoya et al., 2012).
1.2.3 Culturing and Mass release of parasitoids for biological control
Mass release of parasitoids necessitates mass culture of adult wasps.
Producing high numbers of hymenopteran parasitoids for biological control programs requires development of high quality diets and efficient mass-rearing techniques in laboratory conditions (Nurullahoglu and Ergin, 2009). Procedures for rearing parasitoids should aim to optimise female wasp production, and ensure that cultured females can successfully find and parasitise target species (Montoya et al., 2012).
The quality of adult wasps through mass rearing depends on the quality (age and size) of host larvae (Messing et al., 1993, López et al., 2009). Artificial diet quality for the fruit fly host maggots is therefore a crucial component in parasitoid cultures and mass rearing programmes (Snowball et al., 1962). In chapter 2 of this thesis I developed and tested a new method for rearing D. kraussii with a view to improving existing culturing techniques for this wasp.
Insect cultures, being closed populations kept under controlled conditions, can suffer inbreeding depression (Van Lenteren and Bueno 2003). Such populations may be susceptible to loss of genetic variation due to genetic drift in small populations and these genetic problems can negatively affect wasp field performance
(Hoy 1990, Woodworth et al., 2002, González-Cabrera et al., 2011). For example, in the haplo-diploid parasitoid Aphidius ervi Haliday, half of the initial genetic diversity was lost after rearing the wasp for 47 generations (Unruh et al., 1983), and in the parasitoid Trichogramma maidis, there is a positive relationship between the activities of females within deffernt strains and their field performance (parasitism
10 rate) for controlling the eggs of the European corn borer, Ostrinia nubilalis (Bigler et al., 1988).
The effectiveness of parasitoids as inundative biological control agents is most strongly affected by their dispersal, host finding and host handling capacities
(Glenister and Hoffmann 1998, Morales-Ramos et al., 2013). It is important for the parasitoid release technique that it supports the establishment of ongoing, self- perpetuating generations throughout a pest habitat with increases in parasitism level without additional releases required (Knipling 1998). It is clear that high quality adult parasitoids with good genetic variability are required to be produced by rearing facilities to achieve these ends.
Independent of the quality of parasitoids produced by a rearing facility, fully understanding the relationship and interactions between the released parasitoids, the release environment, and the targeted host (s) is arguably still the most important component of creating a successful biological control program. The knowledge in parasitoid behaviours in native habitats and new locations before releases is crucial for enhancing biocontrol agents (Wharton and Cavalloro 1989, Austin et al., 2000).
Parasitoids introduced to a new environment may experience difficulties in dispersal and establishment. Even though some mass-released parasitoids have been successfully augmented in the first period of release, the parasitism rates then decreased (Rousse and Quilici 2009). This decline in parasitoid population may be because of weak adaptability, new host associations, or poor host availability
(Ovruski and Schliserman 2012). In Hawaii, for example, when the parasitoid D. tryoni was introduced to control C. capitata, it attacked non-target tephritid flies
11
Table 1: Fruit fly parasitoid species introduced and used for biological control programs.
Wasp species Origin Target fruit fly Region introduced Successful attempts Unsuccessful References Diachasmimorpha Australia- papua Bactrocera tryoni , B. Hawaii – the Meddle-east Guatemala, the Meddle Wang & Ramadan, 1993; kraussii New Guinea dorsalis, B. latifrons – – Guatemala, California East , Hawaii Zamek et al., 2012; Sime Ceratitis capitata et al, 2006; Argov et al., 2011 D. longicaudata Southeast Asia B. dorsalis, B. oleae – C. Australia – Hawaii – Hawaii – Australia – Greece (against Clausen 1978; Messing et capitata – Anastrepha spp Mexico – Florida – South Florida – South olive fruit fly) al., 1993; Sivinski et al., America, Greece. America 1996. D. tryoni Australia B. dorsalis B. latifrons – Western Australia – Fiji – Hawaii - Mexico Western Australia - Wong et al., 1991; C.capitata – Anastrepha Hawaii - Mexico Fiji Cancino et al., 2009. ludens Fopius arisanus Malaysia Bactrocera spp – C. capitata Australia – Hawaii – Hawaii, Australia, Florida, the Rousse et al., 2005; Zamek Florida, the Meddle-east , Costa Rica Meddle-east, et al., 2012 Mexico – South America Mexico, South America
Psyttalia concolor North Africa B. oleae , B. dorsalis, C. Southern Europe, Hawaii, Southern Europe , California Yokoyama et al., 2008; capitate, California Hawaii Wharton 1989; Rendon et al., 2006.
12 when in the presence of other parasitoid species, with competition for suitable hosts thought to lead to a low level of D. tryoni abundance (Duan et al., 2000).
1.3 Parasitoid host selection
Insect host selection has been described as a catenary process (Kennedy
1965): a linked series of behaviours from host finding in the environment to final host acceptance (for example for oviposition or adult feeding). Parasitoids of tephritids use a range of environmental stimuli and cues in their foraging behaviour.
These stimuli may be provided by host fruit, fruit fly larvae, or via by-products of host infestation and decay (such as fungal-derived volatiles) (Stuhl et al., 2012).
There are also other factors which may affect the host finding process of parasitoids, such as the presence of non-hosts in the same location as true-hosts (de Rijk et al.,
2013). The following review covers some aspects of parasitoid host searching behaviour.
1.3.1 Olfaction
Olfactory cues play a major role in parasitoid behavioural ecology (Dicke and
Loon 2000). They are used as both pre-alighting cues, involved in host habitat location and the selection of suitable host plants; and as post-alighting cues, involved in searching for and parasitising the host insect (Eben et al., 2000, Rousse et al.,
2007b, Segura et al., 2012). The use of olfactory cues by foraging parasitoids can thus be thought of as influencing behaviour in four sequential steps: finding the habitat, host location within the habitat, host acceptance, and assessing host suitability for offspring survival (Doutt 1959, Schoonhoven 1968, Vinson &
Iwantsch 1980, Vinson 1998, Vet et al., 1995). Microhabitats can also be important
13 in determining odour attractiveness: for example, D. longicaudata is attracted to odours released from over-late season fallen and fermented fruits that are unattractive many native Neotropical opiines (Sivinski and Aluja 2012).
Host plant factors, both physical and chemical, provides many of the cues used by parasitoids for food and host finding, so is one of the most important ongoing aspects of interactions between parasitoids and their hosts. Plants release volatiles that are attractive to parasitoids, and changes in plant volatile emissions can influence both herbivore and parasitoid attraction (Dutton et al., 2000, Stuhl et al.,
2011, Kaplan 2012). Parasitoids may locate the habitat of their hosts through attraction to volatiles from plants that their preferred hosts consume (Yang et al.,
2008). Such cues can arise from fruit (Rousse et al., 2007a), leaves (e.g. green leaf volatiles) (Reddy et al., 2002, de Rijk et al., 2016), larval frass (Auger et al., 1989) or host eggs (Rousse et al., 2007b). Such volatiles may attract parasitoids as individual chemicals, but attraction can be greatly enhanced when volatiles are presented as a chemical blend (Bleeker et al., 2006).
Parasitoids can perceive their hosts on the basis of plant volatile emissions.
Plant volatiles released by damaged plants as a result of herbivore feeding are frequently used by herbivore natural enemies as signals for the potential presence of hosts (Vet and Dicke 1992, Du et al., 1996, De Moraes et al., 1998, Kessler and
Baldwin 2001; Hare 2011). For instance, maize (Zea mays) and many species of
Acacia, emit volatile compounds that attract parasitoids and predators when they are attacked by herbivorous insects (Turlings and Wäckers 2004). A large number of parasitoids depend on specific volatile blends emitted from damaged plants to locate
14 specific hosts at the appropriate developmental stage (Hilker and McNeil, 2008,
Wajnberg and Colazza 2013).
Larval cues, including vibrations and volatiles from the larvae themselves, play a critical role in wasp attraction (Duan and Messing 2000a, Stuhl et al., 2011,
Dias et al., 2014, Quicke 2015). These volatiles can be used by parasitoid females to discriminate between frass produced by host and non-host species. Disulphides in larval frass influence searching behaviour in the parasitoid Diadromus pulchellus
Wesmael (Auger et al., 1989). The braconid parasitoid Cotesia plutellae
(Kurdjumov) searches more intensely and increases levels of parasitism when larvae of its host, the diamondback moth (DBM), releases silk fibres mixed with damaged leaf volatiles (Reddy et al. 2002); while the braconid Asobara tabida (Nees) responds to volatiles produced by Drosophila larvae, increasing its searching time with host volatile concentration (Van Alphen and Galis 1983). Asobara tabida females are also attracted by fermentation odours linked to yeast development in damaged fruit due to fly infestation (Kaiser et al., 2009). Because the fruit fly's eggs are inserted underneath the fruit's flesh and the larvae feed deep inside the fruit, the immature larvae are frequently protected from most parasitoids (Wang et al., 2009). Many parasitoids can locate hidden host larvae by perceiving vibrations. In D. tryoni and
D. longicaudata, host larval feeding leads to vibration signals that may be used alongside chemical cues from the fermented host substrate (Duan and Messing
2000a). The opiine parasitoid P. concolor responds stronger to the third than the second instar larval hosts because of strong vibrations from large larvae while feeding (Canale and Loni 2006).
15
Herbivore induced plant volatiles (HIPVs) from fruit fly damaged fruit are highly attractive to opiine braconids, and are a major cue by which these wasps find their hosts (Sivinski and Aluja 2012, Benelli et al., 2013b) and parasitoids often show preferences for odours from host infested plants over uninfested plants (Ero et al., 2011a). In D. kraussii, the presence of adult fruit flies and infested fruit influences host searching and host utilisation behaviours (Ero and Clarke 2012).
Volatiles emitted from C. capitata infested apples elicited significant antennal activity in P. concolor females (Benelli et al., 2013b). Similarly, in other braconid parasitoids it has been shown that HIPVs elicit electrophysiological responses. The egg parasitoid Trichogramma pretiosum Riley is innately attracted by volatiles from newly damaged plants, whilst the specialist parasitoid Telenomus remus Nixon responds to volatiles emitted through fresh and old damage, and this response increases with oviposition experience (Peñaflor et al. 2011).
Volatile chemicals have been used in orchards to enhance the efficacy of parasitoids against fruit fly pests. HIPVs, often consisting of blends of volatiles, such as alkanes, ketones and carboxylic acids (Ode 2006, Wajnberg and Colazza 2013), are used in IPM programmes to monitor pest populations (Simpson et al., 2011b,
Wajnberg and Colazza 2013). Simpson et al., (2011a) demonstrated in vineyards in
Australia that several hymenopteran parasitoids responded to HIPV blends of methyl salicylate, methyl jasmonate, benzaldehyde and (Z)-3-hexenyl acetate, and parasitoids abundance was significantly increased on traps near treated plants.
However, knowledge about how parasitoids respond to HIPVs in the absence of hosts is still limited and further research is necessary to improve the efficacy of these volatile blends in the field.
16
1.3.2 Visual cues
Visual information, such as colour, shape and size, can be used by parasitoids during the host selection process. The ability for colour discrimination has been reported in several species of parasitoid (Messing and Jang 1992). Colour plays a major role during host discrimination and oviposition by the wasp Aphidius ervi
(Battaglia et al., 2000), and in several other parasitoid species (Rousse et al., 2007a,
Lucchetta et al., 2008).Benelli and Canale (2012) illustrated that visual cues may be crucial for P. concolor females to locate the host microhabitat. Moreover, they showed that visual cues may interact synergistically with olfactory and physical cues in this selection process. According to Rousse et al. (2007a), the parasitoid Fopius arisanus (Sonan) preferred dark colours, matching the behaviour of some polyphagous Tephritidae. These authors further found a synergistic effect of olfactory and visual stimuli in F. arisanus host location. Fopius arisanus females recognize previously parasitized host eggs and avoid ovipositing in them when the ratio of host eggs to females is high. This avoidance behaviour might be altered when the number of available host eggs for each parasitoid female is low.
In fruit fly opiine parasitoids, ovipositor length is related to the size and shape of the fruit in which host larvae develop (Segura et al., 2007). However, according to
Ovruski et al., (2007), fruit size does not play an important role in parasitism rate of
Anastrepha suspensa (Loew) larvae. Wäckers and Lewis (1999) also found no relationship between fruit size and host choice when artificial fruit spheres of different sizes were offered to D. longicaudata in a choice test. However, Perez et al., (2012) stated that D. longicaudata females may prefer to seek host larvae in
17 larger fruit, which would produce differences in rates of parasitism within a fruit sample containing different fruit sizes.
1.3.3 Adult feeding sites
Adult parasitoids feed on various foods including floral and extra-floral nectar, host fluids and hemipteran honeydew (Jervis and Kidd 1986, Takasu and
Lewis 1995). Carbohydrate resources are essential to provide energy for wasp longevity, fecundity and movement, and opiine adults without a carbohydrate food source will die after 48-72 hours (Sivinski et al., 2006). Although many nutrients are essential for growth and longevity, others appear to be harmful. For instance, the rate of mortality among D. longicaudata adults is significantly higher when provided with guava pulp or juice, or water only, compared to sugar. This may be because of repellent, innutritious or toxic compounds (Stuhl et al., 2011). A low search-cost to adult feeding is for wasps to consume food from the same substrate as the hosts, or liquids from wounded hosts themselves. This is an important and widespread nutritional strategy in a phylogenetically extensive range of hymenopteran parasitoids, including the Braconidae (Thompson 1999, Wäckers et al., 2005). Blatch
(2008) reported that certain types of bacteria can provide the insect host with essential nutrients consistently lacking in the host diet. Obtaining food and hosts in the same microhabitat can benefit foraging behaviour (Narváez et al., 2012).
1.4 Learning Behaviour
Learning, broadly defined as “a change in behaviour with experience” (Papaj and Prokopy 1989), is known to be widespread in insects (Papaj and Lewis 2012), and includes a variety of more specific terms (e.g. habituation, imprinting,
18 associative and operant conditioning) depending on the way learning is expressed behaviourally and physiologically. Papaj and Prokopy (1989) identified certain criteria by which learning can be identified: (1) the individual’s behaviour changes in a repeatable way as a consequence of experience; (2) the behaviour changes gradually with continued experience (repetition); and (3) the change in behaviour accompanying experience wanes in the absence of continued experience of the same type, or as consequence of a novel experience or trauma (extinction). The two most widely studied forms of associative learning are classical (= Pavlovian) conditioning and operant (or instrumental) conditioning (Papaj and Prokopy 1989). Studies on insect learning have focused on model systems in moths, Drosophila, honeybees and parasitoid wasps (Hoedjes et al., 2011). Much of the focus has been on associative learning (see below) of olfactory or visual stimuli. Theoretical studies predict that learning conveys fitness advantages through improved foraging, particularly in unpredictable environments (Cunningham and West 2008), and the application of theory can help predict how foraging animals respond to spatial and temporal distribution of food in the natural surroundings (Krebs and Inman 1992). The genetic basis for learning has been studied in Drosophila, and the ecological and genetic influences on variation in learning behaviour have been studied in hymenopteran insects (Hoedjes and Smid 2014).
1.4.1 Associative learning (Classical conditioning)
Associative learning occurs when an animal is able to associate one stimulus, or action, in relation to another. In classical conditioning, a stimulus such as a visual or olfactory cue (called the conditioned stimulus, or CS) is paired temporally with an innate or unconditioned stimulus (US or ‘reinforcer’) (such as a sucrose reward in
19 positive reinforcement, or a shock / deterrent stimulus in negative reinforcement).
Thus, classical conditioning can be understood as learning about the temporal or causal relationships between external stimuli, which allows for appropriate behaviour in biologically significant events (Brembs and Heisenberg 2000). Honeybees are a model system for associative learning in insects, where this trait has a strong effect on visual and olfactory behaviour (Giurfa and Sandoz 2012). Nectar foraging outside the hive utilises classical conditioning to learn the association between floral odours
(the CS) and food sources such as nectar and pollen (the US or reinforcer) (Menzel and Muller 1996). Parasitoids can learn unconditioned stimuli from hosts when encountering their hosts and then associating these odours with environmental stimuli (CS) to recognise the hosts during their host searching process (Lewis and
Tumlinson 1988).
1.4.2 Operant learning
Despite a wealth of studies on associative learning in parasitoids, operant learning, which influences searching behaviour in bees (Sandoz et al., 2000) and moths (Cunningham et al., 2004, Giurfa 2013), has received little focus. Operant learning comprises learning a sequence of motor skills that improve the chances of acquiring a reward (in bee nectar foraging studies, this is often termed “flower handling” (Sandoz 2011)). In operant conditioning, carrying out a specific behaviour is associated with increasing or decreasing the frequency of an outcome. The animal learns to do something to get a reward, or avoid a negative situation (Brembs 2003,
Weiss 2014). Although classical and operant conditioning are often viewed as separate forms of learning they are ultimately linked, since an animal generally learns to recognise a stimulus whilst also learning how to behave in accordance with
20
it (e.g. to obtain a reward) (Sandoz 2011). Operant conditioning has been studied in
terms of flower handling (time taken to obtain the nectar) in bumblebees and moths
(Laverty 1994, Cunningham et al., 1999), but not in oviposition behaviour.
Investigating whether parasitoids utilise operant learning to improve post-alighting
host finding is researched in this thesis.
1.4.3 Learning in parasitoids
Associative learning has been shown in parasitoid host searching behaviour,
where adult insects learn to associate chemical and/or visual cues paired with the
positive reinforcement of either parasitising a host (Vet et al., 1995, Lucchetta et al.,
2008) or obtaining food, such as nectar and honeydew (Takasu et al., 1996, Rousse et
al., 2007a). Theory predicts that if learning increases the total survival of offspring
over innate recognition then it will have adaptive significance (Dukas and Duan
2000, Cunningham et al., 2001). Associative learning allows parasitoids to recognise
and utilize profitable cues for survival and reproductive success in a complex
environment (Meiners et al., 2003). To date, there are no experiments looking at a
similar “reward quality” learning effects in oviposition behaviour, and this is a part
of the experimental work in my thesis.
1.4.3.1 Oviposition behaviour
Rewarding a host experience with oviposition increases long-term memory
formation in parasitoids (Schurmann et al., 2012, Koppik et al., 2015). Parasitoid
females Nasonia vitripennis (Walker) that had more training sessions associated with
host odours and oviposition had enhanced host recognition for long-term memory,
while the variability of learning was affected by the differences of reward values
21
(e.g. probing + lay eggs into high or low host values) (Hoedjes and Smid 2014).
Braconid female may innately respond to stimuli associated with hosts or host habitats, but many stimuli are learnt through encounters with hosts (Fujiwara et al.,
2000, Takasu and Lewis 2003). For example Cotesia glomerata (L.) parasitoids display plant odour learning in their oviposition behaviour. Females with previous experience parasitizing larvae on plants responded to plant odours more strongly than naïve females and females with an oviposition experience only; with the retention lasting for up to five days (Bleeker et al., 2006).
1.4.3.2 Feeding:
In feeding behaviour, odour learning can be influenced by features of the food reward (= the unconditioned stimulus). Nectar and honeydew consists of many kinds of sugars, which can affect parasitoid learning behaviour (Takasu and Lewis 1995,
Wäckers et al. 2008). Conditioning experiments using the parasitoid Microplitis croceipes (Cresson) show a clear learning response to glucose, fructose and sucrose, whereas conditioning with mannose and galactose resulted in unsuccessful odour acquisition (Wäckers et al., 2006). Sugar composition also mediates the strength of associative odour learning in the parasitoid Microplitismediator Haliday, with prior oviposition experience modifying sensitivity to food against host-associated odours
(Luo et al., 2013). The learning response to a particular odour (= the conditioned stimulus) could be decreased when the unconditioned stimulus is reduced or absent due to the insect’s physiological state (Takasu and Lewis 1993). Naïve parasitoids respond strongly to food odour, but learning associated with egg-laying transforms the response in favour of host odour. In the braconid Apanteles aristoteliae Vierek, host associated plant-odour learning is strongest when females overfeed, whereas
22 starved females orientate toward a food stimulus (Lightle et al., 2010, Segoli and
Rosenheim 2013).
1.4.4 Olfactory learning in parasitoids and its adaptive significance
Olfactory learning in parasitoid foraging behaviour has now been demonstrated for many wasps (Papaj and Vet 1990, Geervliet et al., 1998, Meiners et al. 2003, Olson et al., 2003, Schurmann et al., 2009, Ngumbi et al., 2012, Luo et al.
2013, Canale et al., 2014, Frederickx et al., 2014, Wilson and Woods 2016).
Parasitoids have innate preferences for environmental odours (e.g. host derived stimuli) (Carrasco et al., 2005, Silva et al., 2007, Ero et al., 2011a, Benelli et al.,
2013a, Segura et al., 2016, Wilson and Woods 2016), but their responses can be modified by associative learning and they can use these learned odours for faster host finding (Molck et al., 2000, Blande et al., 2007, Canale et al., 2014). In summary, learning may: (i) allow generalist parasitoids to specialise on particular cues; (ii) improve host finding; (iii) improve odour recognition in complex environments
(focus); (iv) decrease time to find and oviposit (speed); and (v) improve identification of hosts (discrimination) (Papaj and Vet 1990, Takasu and Lewis 1993,
Du et al., 1997, Duan and Messing 1999, Dukas and Duan 2000, Takasu and Lewis
2003, Wang and Messing 2008, Wei et al., 2013, Canale et al., 2014).
Parasitism rates can rise if parasitoids have previous host experience which allows them to adjust their response to environmental information received; so improving host location and acceptance at low host densities (Vet et al., 1995). For example the braconid parasitoid Dolichogenidea tasmanica (Cameron) showed increased searching and host parasitism rates when it was previously ‘rewarded’ with
23 appropriate host larvae, compared to being exposed to ‘non-rewarding’ larvae
(Yazdani and Keller 2016).
Through experience, parasitoids can decrease the time required to recognise and parasitise a host in order to search and locate more unparasitised hosts (Vinson
1976). In P. concolor, when host larvae ( both C. capitata larvae and B. oleae larvae) were offered to naïve females, the proportion levels of searching and probing were very low, whereas ovipositionally experienced females showed significantly more searching and probing, with shorter latency times, on host larvae (Canale and Benelli
2012).
Natural odours encountred by parasitoid females are complex and variability, and associative learning allows wasps to focus on the most reliable odours. Female parasitoid M. croceipes can learn to respond to single compounds after experiencing odour blends, which might imply that the sensory system in the antenna of the wasp has a higher sensitivity for lower volatile compounds (e.g. methyl jasmonate, β - caryophyllene) than to more volatile compounds (e.g. β-ocimene) (Meiners et al.,
2003). The parasitoid Leptopilina heterotoma (Thomson) can adjust its degree of discrimination between similar odours (e.g. apple types) according to the learning behaviour that is connected with the odour. This form of learning, where the wasp is given both positive (successful oviposition) and negative (unrewarding) experiences, is known as differential conditioning (as opposed to absolute conditioning, when the insect is given only a positive or a negative experience) (Turlings et al., 1993).
Oviposition in a host is the most positive experience for a female wasp. Differential conditioning in odour learning has been shown to allow parasitoids to better discriminate between odours (Vet et al., 1998).
24
Because of the strong correlation between successful host location and fitness, parasitoids would be expected to learn stimuli that enhance their probability of finding suitable hosts (Vet et al., 1995). Learning should thus be a species-specific trait, dependent on the parasitoid species environment and that of its hosts (Smid et al., 2007). Females of the generalist parasitoid Cotesia marginiventris (Cresson) exhibit a significant increase in their learning response to four tested compounds
(trans-2-hexanal, α-pinene, cis-3-hexenyl butyrate, and (E,E)-α-farnesene) compared to naïve females, whereas experienced females of the specialist wasp Microplitis croceipes (Cresson) show a significant increase in behavioural response only to α- pinene, and (E,E)-α-farnesene (Ngumbi et al. 2012). Thus C. marginiventris shows a wider learnt response than the species with a more specialized host range. Although the advantages to learning might be viewed as lower in the case of the specialist insect, both parasitoids may be improving their host searching efficiency through associative learning (Ngumbi 2011). Not all wasps have been shown to exhibit learning. The generalist endoparasitoid Campoletis sonorensis (Cameron) showed no learning of HIPVs during rewarding experiences (Tamò et al., 2006).
A decreased response to non-host odours could be expected to improve foraging in a parasitoid by fine-tuning odour responses associated with damage by its host species (Giunti et al., 2015). Microplitis croceipes decreases its response to a previously learned odour if that odour is subsequently associated with a non-host
(Takasu and Lewis 2003). Female Cotesia marginiventris do not change their responses towards HIPVs after contacting the non-host Pieris rapae L (unrewarding experiences), compared to naive wasps, whereas the ichneumonid parasitoid Pimpla luctuosa Smith avoids artificial odours of vanilla or strawberry after experiencing these odours during unsuccessful oviposition attempts (Costa et al., 2010). 25
In nature, host or host-plant related odours encountered by parasitoids occur in complex mixtures (Wajnberg et al., 2008). Associative learning may therefore assist parasitoids by allowing them to focus on the most specific odours
(Cunningham and West 2008). The parasitoid L. heterotoma can learn to respond to single C6-compounds against a complex background of yeast odour (Vet et al.,
1998). The braconid parasitoid M. croceipes is also able to learn to respond to single compounds within odour blends (Luo et al., 2013).
1.4.5 Visual learning
Visual stimuli are important sensory cues used by insects to locate resources.
Segura et al. (2007) demonstrated the importance of visual learning in D. longicaudata on preferences for different plants during host location. Lucchetta et al.
(2008) demonstrated that the parasitoid, Venturia canescens (Graven), learns visual cues during food foraging, and suggested that this parasitoid uses both innate and learned visual stimuli. In P. concolor, naïve females show no innate preference for colours, whereas experienced females develop clear preferences for colours when they are associated with host larvae (Benelli and Canale 2012). Microplitis croceipes, a larval parasitoid of several lepidopteran species, has been used in a number of studies on visual learning (Takasu and Lewis 1996). Females of this species are better at learning to discriminate between shapes than between colours or patterns
(Wäckers and Lewis 1999), and it has been demonstrated that the use of these cues is improved through associative learning (Takasu and Lewis 2003, Desouhant et al.,
2010).
26
1.4.6 Learning on multiple host cues and other factors
Cotesia congregata (Say) has been shown to learn multiple host-associated plant cues (Lentz-Ronning and Kester 2013), and experiments suggest that this helps to shape host foraging. Regardless of the order, sequential experiences with tomato and tobacco resulted in similar searching responses towards each plant species, and similar production of female offspring (six ratio allocations) were found in hosts
Manduca sexta (tobacco hornworm) presented with the plant with which the wasp was experienced (Lentz-Ronning and Kester 2013). The parasitoid D. tryoni develops on lantana gall fly Eutreta xanthochaeta Aldrich (a non-target host), and from C. capitata. The amount of naïve female probing behaviour to C. capitata infested fruit is only slightly different that of experienced females on the same host; but the probing behaviour of the females experienced to E. xanthochaeta galls is two to three times higher than for naïve females, and seven to 11 times higher than for those exposed to C. capitata infested fruit. It is likely that the oviposition response of
D. tryoni is significantly influenced by the complex host-substrate (Duan and
Messing 1999). Thus the potential impact of non-target host and learning effects should be considered when introducing or mass rearing parasitoids as biological control agents against fruit flies in target areas (Giunti et al., 2015).
Age can play a role in the learning ability of wasps. At one day old, most adult female Microplitis croceipes are unsuccessful at learning odours presented immediately after a paired food-odour experience, whereas older females (2 to 10 days) respond to odours they have experienced (Takasu and Lewis 1996). In the egg- larval parasitoid Ascogaster reticulatus Watanabe, the learned response of 0-day-old
27 females to tea leaf extract is very low compared to older females (Honda and Kainoh
1998).
Knowledge of parasitoid learning in the field and how it influences fitness is still limited (Giunti et al., 2015). Dukas and Duan (2000) stated that it is unclear how learning-based changes in parasitoid behaviour relate to fitness, since differences in host finding rate due to learning were not accompanied by differences in oviposition.
In the Opiinae wasp F. arisanus, naïve females readily lay eggs into previously parasitised hosts, but females that have experienced prior oviposition avoid super- parasitism (Wang and Messing 2008). Such experiments provide evidence for fitness benefits from learning under laboratory conditions, but validation in the field is still needed.
1.5 Thesis foci organisms: Diachasmimorpha kraussii and Bactrocera tryoni
Diachasmimorpha kraussii (Fullaway), the study organism in this thesis, is native to Australia and Papua New Guinea, where it has been recorded from 18
Bactrocera species (Carmichael et al., 2005, Ero et al., 2010). In Australia, it is distributed only in eastern parts of the continent, from the far north of Queensland and south into New South Wales. Bactrocera tryoni, a major host of D. kraussii, is the most serious insect pest of horticulture in Australia attacking a large number of wild and cultivated fruits (Clarke et al., 2011). Diachasmimorpha kraussii has been used in both classical and inundative biological control programs (reviewed below), but knowledge of its host location mechanisms, particularly as modified by learning, is limited (again see below). Due to the minimal information of host location by D. kraussii, especially on any role of experience in this process, my thesis focuses on this knowledge gap through experiments in both laboratory and field.
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1.5.1 D. kraussii life cycle
Diachasmimorpha kraussii is a solitary (one wasp produced per fly larva), polyphagous (attacks multiple host fruit fly species), koinobiont endoparasitoid (lives inside the host without killing it) and adult females attack late second to early third instar fly larvae (Wharton 1997, Duan and Messing 2000a). Development within the host (from oviposition to adult eclosion) ranges from 16 days to nearly a year
(Rungrojwanich and Walter, 2000a), with peak emergence at day 20. The very long development period (i.e. up to one year) is the result of a delayed emergence, where the fully developed, pharate adult wasp remains within the fly pupal case: details on any mechanisms of this ‘diapause’ are unknown. D. kraussii females have a life span around 24 to 31 days and obtaining the peak of offspring is between 4 days and 13 days old of adult females. Females will lay eggs into their host during both day and night, although the latter is less common (Rungrojwanich and Walter, 2000a). Virgin females produce haploid offspring (males), whereas the progeny from mated females are frequently females. Population are protandrous, (males emerge one to two days before females), with mating ocuuring 6 - 48 hours after eclosion (Rungrojwanich and Walter, 2000a). Parasitoid females inject one egg into each host larva. When the egg hatches, the wasp larva feeds firstly on the fat tissue of fruit fly larva without attacking important organs. This keeps the fly larvae alive until fly pupation, and then the parasitoid larva consumes the whole fruit fly pupa, emerging as an adult
(Figure 1.1) from the fly pupal case instead of the fly.
29
5 mm
Figure 1.1. Adults of Diachasmimorpha kraussii
1.5.2 Diachasmimorpha kraussii as a biological control agent
Diachasmimorpha kraussii has been considered for release against fruit fly pests worldwide. It has been used for classical biological control in Hawaii,
Guatemala and the Middle East (Messing and Ramadan, 1999; Rendon et al., 2006;
Argov and Gazit 2008). The first release of D. kraussii in Hawaii was against
Bactrocera latifrons (Hendel) in March 2003, and three years later it was confirmed that D. kraussii had become established (Bokonon-Ganta et al., 2013).
Diachasmimorpha kraussii has also been trialled in Australia as an inundative release
30 agent for the control of B. tryoni (Zamek et al., 2012). Although D. kraussii is able to successfully attack E. xanthochaeta, a non-target tephritid in the laboratory, gravid females showed low levels of searching and oviposition behaviours to the host plant odours of non-target tephritids (Duan and Messing, 2000). In Guatemala, introduced
D. kraussii has been used as an important component in an IPM programme for the control of C. capitata, with parasitoid releases combined with a programme of sterile male fruit fly releases (Rendon et al., 2006).
1.5.3 Behaviour and ecology of Diachasmimorpha kraussii
Diachasmimorpha kraussii has been associated with a range of host fruit fly tephritid hosts (Carmichael et al., 2005) across a variety of those flies’ host plants
(Ero et al., 2011a). The parasitoid females can attack, and their offspring successfully, develop on all three larval instars C. capitata and B. latifrons (Messing and Ramadan, 1999), but offspring may be negatively affected by oviposition into mature third instars because of limited development time available before fly pupation occurs (Lawrence et al., 1976; Messing and Ramadan, 1999).
Diachasmimorpha kraussii can be easily reared on 2nd and 3rd instar B. oleae larvae, but eventually its fecundity rates drop due to olive fruit lacking in essential nutritional elements indirectly passed to the wasp (Sime et al., 2006). The wasphas good development and survival between 10º to 30º C and is more tolerant of cool weather compared to its congener, D. longicaudata (Sime et al., 2006).
Pheromones, visual cues, and host plant species play important roles in successful mating of D. kraussii (Rungrojwanich and Walter, 2000b). Cuticular pheromones of females D. kraussii and D. longicaudata attract males of D. kraussii but none attempted to mate with D. longicaudata (Rungrojwanich and Walter,
31
2000b). Mating behaviour frequently occurs on plant leavies which play a role to locate female partners. Males respond to the close proximity of females with a wing- fanning signal which assists successful copulation (Rungrojwanich and Walter,
2000b) and this observation has been proven in the Opiine wasp P. concolor (Benelli et al., 2012).
Ero and colleagues studied D. kraussii behaviour and ecology in the laboratory and field. Fruit type influences the size and development rate of wasps, but has no impact on sex ratio (Ero et al., 2011a). Female D. kraussii foraging for oviposition sites did not respond to uninfested fresh fruit cues, but responded positively to maggot-infested fruits (Ero et al., 2011a). However, Females were unable to discriminate between physiologically suitable (B. tryoni and B. jarvisi) and unsuitable (B. cacuminata, B. cucumis) host flies in the laboratory in the same host fruit (Ero et al., 2011b). In field cages the wasp preferentially foraged for host plants of physiologically suitable hosts, and not to the hosts of physiologically non-suitable hosts, suggesting positive relationship between D. kraussii fruit preference and host fly usage (Ero & Clarke 2012). Ero did not explore any of the cues (e.g. chemical or visual) which D. kraussii used to discriminate between host fruits.
1.5.4 Queensland fruit fly, Bactrocera tryoni
Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (Queensland fruit fly or
Qfly) (Fig. 1.3) is native to coastal Queensland and northern New South Wales and is the most important fruit pest in eastern Australia (Raghu et al., 2000, Dominiak and
Daniels 2012). Originally restricted to tropical and subtropical eastern Australia, the fly now extends southwards into temperate eastern Australia (Clarke et al., 2011).
Bactrocera tryoni belongs to a species complex which includes itself and three other
32 species; B. neohumeralis (Hardy), B. melas (Perkins & May) and B. aquilonis (May)
(Drew 1989). It is probable that of these three taxa, only B. neohumeralis represents a unique biological species different to B. tryoni (Clarke et al., 2011).
5 mm
Figure 1.2.The Queensland fruit fly, Bactrocera tryoni (Froggatt).
The direct impact of B. tryoni on horticulture is through its laying of eggs into sound fruit on-plant. Once the eggs hatch, the larvae burrow inside the host tissue and begin feeding, which promotes fruit infection by decay causing microorganism
(Drew and Lloyd 1989, Behar et al., 2008). Further, because eggs are laid inside otherwise sound fruit, the fly can easily be spread through trade and informal carriage.Bactrocera tryoni has invaded several Pacific island nations and has the potential to spread around the world because of its large host range and broad climatic tolerance (White and Elson-Harris 1994). Thus, B. tryoni is a major risk to
33 domestic and international market access for horticultural commodities produced in eastern Australia (Plant Health Australia, 2008). The fly infests more than 100 native and introduced hosts, including horticultural crops, garden plants, native plants and weeds (Hancock et al., 2000).
1.5.5. Basic biology and ecology of B. tryoni
The biology and ecology of B. tryoni is most recently reviewed by Clarke et al. (2011). The basic biology, as pertinent to understanding the interactions with its parasitoids, is as follows.
Like mostt higher Diptera, the fly has a life cycle consisting of the eggs, three larval instars, a pupa and then a male or female adult (Drew and Romig, 2000). The adults have a relatively long life span (often more than three months), and high potential fecundity (> 1000 eggs per female and multiple generations per year)
(Fletcher 1987). Within a day to two of adult emergence, sexually immature B. tryoni adults leave their breeding area to forage in search of food and water (Bateman
1972). Sexual maturation occurs at around 10 days of age and mating occurs in aggregations at dusk (Ekanayake et al., 2016).
The fly is highly polyphagous, utilising the fruits of over 100 plants as larval hosts (Hancock et al., 2000). Even though B. tryoni is considered to have originally bred in wild fruits of the Queensland rainforests, since European settlement of
Australia, its breeding has expanded into a wide range of imported fruit along the whole of the Australian east coast and sub-coastal areas (Dominiak and Daniels
2012). Indeed, the wide availability of commercial horticultural taxa is considered the conduit for B.tryoni’s host and geographic range expansions (Bateman 1968). For
34 example B. tryoni was recorded in bananas, cherries and apples in the early 1900s in south-eastern Queensland, and it may then have been introduced to New South
Wales by fruit trade. Two-hundred years after European settlement and the subsequent introduction of many exotic fruit and vegetables from all over the world,
B. tryoni is now highly abundant in a wide range of commercial host fruits including citrus, stone fruit and many vegetables which have become major hosts of the fly
(Hancock et al., 2000; Dominiak et al, 2006). Even poor hosts of B. tryoni such as grapes and cucurbits are attacked under laboratory conditions and at high population densities in the field (Loch, 2008; Clarke et al. 2011).
1.5.6 Opiinae parasitoids as biocontrol agents against B. tryoni
Eight native or exotic braconid wasps have been recorded attacking B. tryoni in Australia: Diachasmimorpha kraussii (Fullaway), D. longicaudata (Ashmead), D. tryoni (Cameron), Fopius arisanus, F. schlingeri Wharton, Opius froggatti
(Fullaway), Psyttalia fijiensis (Fullaway) and Utetes perkinsi (Fullaway) (Carmichael et al., 2005). Four of these wasps are used as biological control agents in Australia, including two endemic parasitoids, D. kraussii and D. tryoni, and two exotic parasitoids D. longicaudata and Fopius arisanus. Some opiine parasitoids first utilise
B. tryoni at the egg stage, including F. arisanus, while other species first parasitise the larval stage, such D. kraussii. Large numbers of braconids (Opius humilis
Silvestri, O. fullawayi Silv. and Tetrastichus giffardianus Silv.) were mass released in NSW to control B. tryoni during the 1930s, but the candidate wasps did not establish (Zamek et al., 2012). The last classical biological control program introducing parasitoids against Australian fruit flies was in the late 1950s and early
1960s (Snowball et al., 1962a, Snowball et al., 1962b, Snowball 1966) when F.
35 arisanus was introduced and is now one of the most common B. tryoni parasitoids.
The first introduction of exotic parasitoids to control the exotic C. capitata was early in the 1900s in Western Australia.
Augmentative biocontrol against B. tryoni in Australia has received limited attention (Clarke et al., 2011), although opiine wasps are successful in augmentative biocontrol programs overseas, and some show promise for similar use in Australia.
During an AWM program to control B. tryoni in south-east Queensland, three parasitoids were identified from fly pupae: the two endemic wasps D. kraussii and D. tryoni, and the non-native parasitoid F. arisanus (Lloyd et al., 2010). These wasps achieved an average 7.4% background parasitism level without intervention (Lloyd et al. 2010), while D. kraussii and D. tryoni occurred in most fruit types infested with
B. tryoni in inland NSW (Spinner et al., 2011) and may therefore represent promising candidates for inundative release in Qfly AWM programs (Zamek et al.,
2012). The former of these, D. kraussii, is the focus of my research.
1.6 Structure of the thesis
My PhD investigates the behaviour of Diachasmimorpha kraussii with a view to improving our understanding of how it finds its host, Bactrocera tryoni. There is a particular focus on the role of learning in influencing host foraging in female wasps.
Despite a large body of research on classical conditioning in parasitoids (as reviewed earlier), there is surprisingly little work on how learning influences the way insects search on host plants. The behavioural mechanisms of host/non-host discrimination is unknown in fruit fly parasitoids, while the effects (if any) of experience on olfactory discrimination are also unknown. Learning in general is very poorly studied in opiine braconids. Understanding how intrinsic biological and extrinsic ecological
36 factors influence host use and foraging by D. kraussii will add essential information to improve the management of this important biological control agent, both in
Australia and other parts of world, such as North Africa (my home region) where fruit flies are serious exotic pests. In a broader ecological context, understanding how braconid parasitoids locate their hosts within multiple species of host fruits, and differentiate between potential hosts and non-host fruit fly species, will provide a fundamental insight into insect foraging behaviour and its evolution.
I have three research objectives, each taking the form of a separate experimental chapter. These objectives progress from understanding how to culture and rear wild insects in the laboratory, to laboratory experiments exploring the role of different forms of learning in influencing foraging behaviour, and finally to studying how experience might influence parasitoid foraging outside confined laboratory arenas.
The first experimental chapter (Chapter 2) describes my evaluation methods for culturing D. kraussii in the laboratory. I tested the hypothesis that presenting host larvae in a way that more closely resembles the natural situation of larvae in fruits will increase parasitism rates and help prevent the culture population ‘crashes’ that frequently happen when wild insects are first brought into the lab. The method presented here uses a standard culture medium containing host fruit fly larvae, presented to wild parasitoids as a ‘culturing bag’. I designed a ‘culturing bag’ containing standard culture medium and host fruit fly larvae, and compared its performance with standard Petri-dish culturing methods, and with whole host- infested fruit. Culturing bags yielded similar parasitism levels to whole fruit, but
37 increased B. tryoni larval survival, providing a superior method for rearing parasitoids.
In the second research chapter (Chapter 3), I conducted experiments investigating the influence of associative learning (classical conditioning) on fruit odour choice in ovipositing female wasps. Specifically, I investigated innate and learnt responses of ovipositing females to determine whether female wasps discriminate between fruits infested by its host, B. tryoni, and a non-host, Drosophila melanogaster. D. kraussii females showed an innate preference for odours of infested fruit over uninfested fruit, but experienced females had a stronger preference for host-infested fruit than naïve females. They also had a clear preference toward fruit infested with B. tryoni versus fruit infested with D. melanogaster; suggesting that females can discriminate host-associated odours (Bactrocera’ derived odours) from odours associated with other frugivores (non-Bactrocera).
In a second component of Chapter 3, I carried out further laboratory-based experiments to explore operant learning. Despite a large body of research on classical conditioning in parasitoids, there is surprisingly little work on how learning influences the way insects search on host plants and no information is available on operant conditioning of the parasitoid D. kraussii. I investigated the influence of operant learning on host finding by ovipositing D. kraussii: specifically to see if operant learning modified ‘on-fruit’ searching. Experiments focused on training wasps on hosts that differed in the location of host larvae (i.e. at the top or the bottom), then testing their host finding efficiency in comparison to naïve females.
While laboratory- based learning has been demonstrated for many wasps, and the ecological/evolutionary implications of such learning inferred, there are
38 surprisingly few studies directly test the impact of learning on wasps in very large arenas and the open field. In my third experimental chapter, I ran a series of learning trials using two B. tryoni host fruits, nectarine and tomato. Essentially the same experiment was run at three spatial scales; in a Y-tube olfactometer, in a large outdoor field cage, and in the field. The results showed a very high degree of consistency at all three spatial scales, with the wasp showing a high degree of innate preference for infested nectarines which could not be significantly increased by prior exposure to nectarine. However, prior experience on infested tomato which significantly increased its tomato foraging preference. These results have significant implications for pre-release conditioning of parasitoids being inundatively released.
The final discussion chapter examines the overall results of the thesis, and focuses on the role of experience in wasp ecology, and how training wasps may lead to better biological control outcomes.
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Chapter 2: An improved culturing method for opiine fruit fly parasitoids and its application to parasitoid monitoring in the field
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2.1 Introduction
Hymenopteran parasitoids are important natural enemies used in the biological control of agricultural and horticultural insect pests (Purcell et al., 1998).
The development of good-quality diets and efficient laboratory rearing techniques are crucial for the success of these programmes (Grenier 2009, Morales-Ramos et al.
2013, Vanaclocha et al., 2014), whether they involve the introduction of new natural- enemy species (Wajnberg et al., 2008) or the mass release of wasps to control local pest populations (Messing et al., 1993, Zamek et al., 2012). When rearing parasitoids for such purposes, laboratory selection is always a concern (Gandolfi et al. 2003,
Joyce et al., 2010). If only a few wild collected individuals reproduce in the early stages of culture then the genetic variability of the population can be severely reduced (Van Lenteren and Bueno 2003, Henry et al., 2010), increasing the susceptibility of the laboratory population to inbreeding depression. Inadvertent selection for specific behaviours (e.g., host searching behaviour and parasitism rates
[(Geden et al., 1992)]) and life-history traits (e.g., development time, fecundity
[(Miyatake 1993, Kuriwada et al., 2010)]) can reduce the fitness of the insects when released into the wild. Good initial survival of wild insects over the first fewgenerations in culture is therefore crucial for producing viable adult wasps that can successfully find and parasitize host larvae when released in the field
(Woodworth et al., 2002, Grenier 2009, González-Cabrera et al., 2014). Additional to their applied use as biocontrol agents, parasitoids have for many years been model organisms in the study of insect behavioural ecology(Godfray and Shimada
1999).Where experiments use insects that have been reared for several generations in the laboratory, the interpretation of results relies implicitly on the assumption that laboratory reared insects respond the same way as wild insects. Although the 41 laboratory culturing environment will always impose selection pressure on insects
(Guzmán‐Larralde et al., 2014, Zygouridis et al., 2014), steps can be taken to maintain genetic diversity by minimizing population bottlenecks and maximizing the reproductive output of wild insects (Hopper and Roush 1993).
We have developed a new method for rearing the fruit fly parasitoid,
Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae), which improves the reproduction of wild insects following their introduction into laboratory culture.
D. kraussii is a highly polyphagous endoparasitoid wasp that is native to Australia and Papua New Guinea and has been recorded from 18 tephritid fruit fly host species
(Rungrojwanich and Walter 2000a, Carmichael et al., 2005, Ero et al., 2011b). The wasp has been used as a biological control agent, as it has a low tendency toward nontarget species (Duan and Messing 2000b) and good survival in diverse climatic conditions (Sime et al., 2006). The first release of D. kraussii was against the invasive tephritid fruit fly, Bactrocera latifrons, in March 2003 in Hawaii, and 3 years later it was confirmed to have become established (Bokonon-Ganta et al.,
2013). Since then, D. kraussii has been successfully introduced into Guatemala
(Rendon et al., 2006), and the Middle East (Argov and Gazit 2008), and has been identified as a potential biocontrol agent for medfly (Ceratitis capitata), and olive fly
(B. oleae) in Libya (Wharton 1989). In Australia,mass rearing and release of D. kraussii has been proposed for preharvest fruit fly control (Clarke et al., 2011,
Spinner et al., 2011).
In attempting to establish a culture of D. kraussii from wild, native populations in Australia, we found very low oviposition rates when fruit fly larvae
(Bactrocera tryoni) were exposed to parasitoids in artificial diet held in Petri dishes
42
(e.g., as used by Ero et al., 2010) (A.M. pers. obs). In order to maximize oviposition by wild wasps, so as to minimize laboratory bottlenecking, we explored various novel culturing techniques. The method presented here uses a standard culture medium containing host fruit fly larvae, presented to wild parasitoids as a “culturing bag”. We compared this novel design to a standard laboratory culturing method (host larvae and culture medium within Petri dishes), and also to the use of whole host- infested fruits (nectarines). The success of thismethod is evaluated in terms of parasitoid emergence, percentage parasitism, and adult parasitoid size. We then tested out culturing bags for their ability to attract D. kraussii in the field, with a view to exploring whether they could be used as a tool for collecting and monitoring wild parasitoid populations.
2.2 Materials and methods
2.2.1 Insects
Wild D. kraussii wasps were collected from an unsprayed nectarine orchard in South East Queensland (Redlands Research Station, Cleveland) in November and
December 2013. Wasps were collected as parasitized tephritid fruit fly larvae emerging from infested fruits (around 80% of total wasps), or hand-caught as sexually mature adult females as they visited ripe fruits for oviposition. Field collected, infested fruits were placed in 2 L plastic containers filled 3 cm deep with vermiculite, and housed in an incubator (25 °C, 70% RH, 13: 11 L: D). Tephritid fruit fly larvae emerging from fruits were allowed to pupate in the vermiculite, then transferred to 500 mL plastic pots fitted with a wire mesh lid that was large enough to allow adult wasps through but not fruit flies (which are larger in size). These pots were placed in 30 cm × 30 cm Bugdorm nylon cages (Bugdorm, Taiwan, China) and 43 adult wasps were collected as they emerged and escaped into the cage. Adult female wasps caught while visiting fruits in the field were placed in Bugdorm cages in the insectary (26 ± 1 °C, 70% RH, natural daylight augmented by 10: 14 L: D fluorescent lighting) for culturing and use in experiments. Female and male wasps were housed together in Bugdorm cages in the insectary where they were provided with honey solution (30% w/v in 30 mL plastic cups fitted with cotton wool wicks) ad libitum to ensure mating and promote longevity (Zamek et al., 2013). Mated female parasitoids (5–11 d old) were used in experiments (Dukas and Duan 2000).
Host fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) were held at approximately 300 flies per cage (33 cm × 30 cm × 25 cm) and supplied with water, yeast autolysate and sugar cubes ad libitum. Mating occurred around 7–10 d after emergence, and fly eggs were collected in 30 mL plastic cups (Dart, USA) perforated with holes and coated inside with a thin layer of the artificial carrot medium (Heather and Corcoran 1985)
2.2.2 Exposure of host larvae to wasps
We compared 3 different methods of presenting B. tryoni larvae to adult wasps: (1) within whole fruits;(2) within carrot medium placed in Petri dishes (a standardrearing method [(Ero et al., 2010)]); and (3) using ournovel method of a culturing bag. For presenting larvae inwhole fruits, we used ripe nectarines, as these are favoured hosts for B. tryoni in the field (Hancock et al. 2000). Fruits (70 ± 10 g) were infested with B. tryoni by scoringthe skin with a knife to a depth of approximately 3 mmand inserting 25 fly eggs into each of 4 slits (i.e., 100 eggsper fruit). To infest the medium, we added 100 fly eggs to50 g of standard fruit fly carrot media (Heather and Corcoran 1985). The infested medium was either (i) placed in a
44
Petri dish and covered with white cotton cloth or (ii) usedin our culturing bags.
Culturing bags (Figs. 2.1.A–E) were constructed as follows: a 6 cm central agar ball
(to provide shape and hydrate the media) was prepared from standard quality agar (9 g agar per 1 L of water) and wrapped in1 ply paper wipe (Kimtech, Roswell,
Georgia, USA) (tohold its shape). Fifty gram infested media (containing 100 B. tryoni eggs) was spread on a piece of tissue paper and wrapped around the agar ball, which was then wrapped within a circular (25 cm diameter) piece of white cotton cloth.
Diachasmimorpha kraussii preferentially oviposit intolate second to early third (final) instar larvae (Lawrence 1990, Ero et al., 2011a), with the wasp larvae completing development once the host enters its pupal stage. The infested substrate for each of the different treatments wasthus first incubated to allow larvae to reach second instar (2 d for culturing bags and Petri dishes, and 3 d for nectarines). For each of the 3 methods, we then placed theinfested substrate in a 30 × 30 cm Bugdorm containing five sexually mature female wasps (1 culturing treatmentper cage). Whole fruits and Petri dishes were placed onthe floor of the cage, whereas the culturing bag was hungby a wire from the top of the cage. All experiments were conducted in the insectary (26 ± 1 °C, 70% RH, natural daylight augmented by 10: 14 L: D fluorescent lighting). Wasps were exposed to the infested media for2 d, after which infested substrates were placed on Petri dishes in 1 L plastic containers containing moistened vermiculite (water 10 % volume) with cotton cloth secured around the lid to allow aeration. Insects were incubated for10 d to allow sufficient time for pupation. For the culturing bag method, the bag was opened before placing in the container (the cotton cloth was removed and the infested media placed in a 500 mL plastic container). Pupae werelater sieved from the vermiculite and upon emergence 45 adult wasps were counted, sexed, and hind tibial measurements were taken to determine adult size (Heinz 1991, Zamek et al., 2013). Adult insects were collected for a periodof 7 d after the first wasps enclosed. Dissection of the remaining pupae after this period (to identify parasitismrates in dead or diapausing insects) was not performed. Parasitism rates were estimated as the number of adult D. kraussii that emerged from the total number of pupae per cage. Fifteen trials (5 replicates × 3 treatments) were run on any 1 d, rotating the positions of each treatment avoid positional biases. Each of the 3 treatments was replicated15 times. Adult wasp size and sex ratio is known tobe influenced by the size of the larval host size and hostfeeding substrate (Nicol and Mackauer 1999, Ode and Heinz 2002). To investigate this further, we compared the mean weights of B. tryoni pupae collected from nectarines and culturing bags (8 replicates), using the previous methods of infesting media or fruit with 100 fly eggs, but without parasitism.
2.2.3 Field study
We conducted a small study to assess the efficacy of the culturing bags for collection and monitoring wild D. kraussii populations within fruit orchards. Ten culturing bags each infested with 300 B. tryoni eggs that had been incubated for 2 d
(see earlier methods) were hung in a mixed nectarine and peach orchard at the
Redlands Research Station, Queensland, together with 10 uninfested culturing bags as controls. One infested and one uninfested bag were hung in each of 10 trees. Bags were left in the orchard for 3 d and then removed and placed into separate 500 mL open plastic pots, together with additional carrot diet to ensure larval development.
These pots were placed separately into 2000 mL plastic containers filled with vermiculite and housed in an incubator (25 °C, 13: 11 L: D, 70% RH) until pupation.
46
Pupae were then transferred to emergence cages and percentage parasitism was calculated, together with the number of adult insects (parasitized or unparasitized) that failed to emerge within the 10 d.To compare parasitism rates on culturing bags and orchard collected fruits, 10 fruit fly infested ripe nectarines and 10 infested ripe peaches (infestation identified by the presence of fruit fly puncture sites) were picked from the orchards during the same period, placed into individual 750 mL containers with vermiculate, and reared through to emergence.
47
A B
C D
E F
Figure 2.1. Preparation of the culturing ball: (A) carrot medium infested with B. tryoni eggs, (B) central agar ball, (C) infested carrot medium on agar ball, (D) D. kraussii on culturing bag within a breeding cage, (E) female D. kraussii ovipositing on culturing bag, and (F) wild D. kraussii on culturing bag in mixed stone fruit orchard.
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2.2.4 Statistical analysis
The non parametric Kruskal–Wallis (KW) and Mann– Whitney (MW) tests, were used to analyse wasp data (number of wasps emerging and proportion parasitism), as these data could not be normalized (most probably due to the numbers of zeros in some treatments). Results are presented as box plots. Pupal counts and hind tibia length (an indicator of body size) followed a normal distribution and were analysed using ANOVA and the post hoc Tukey’s test.
2.3 Results
When cages containing five, wild collected female wasps were exposed to B. tryoni larvae we found that the yields of adult wasps were significantly different among the 3 methods (χ2 = 13.7, P <0.005, KW). Using the culturing bag method, we found that significantly more adult wasps were reared per five females compared to the standard Petri dish method (mean culturing bag wasps = 3.85 ± 1.08 SE; mean
Petri dish wasps = 0.15 ± 0.15 SE) (Z = -3.63, P <0.005, MW); with no significant differences between the nectarine (mean nectarine wasps = 2.65 ± 0.95 SE) and Petri dish (Z = -2.48, P >0.05, MW) or nectarine and culturing bag (Z = -1.59 P >0.05,
MW). In 15 replicate cage trials, only 1 cage containing infested media in Petri dishes (see outlier [*] in Figs. 2.2& 2.4) yielded any adult wasps, compared to 11 cages using the culturing bag, and 7 when using whole nectarines. Sex ratios were not significantly different in adult wasps reared from culturing bags (59% female, χ2
=2.92, P>0.05, n=77wasps) or whole nectarines (56% female, χ2 = 0.64, P >0.05, n
= 39 wasps) (insufficient data at n = 3 wasps for analysis of Petri dish data). Survival of the fruit fly larvae (combined parasitized and non-parasitized) to pupation was significantly different among methods (F44 = 20.837, P <0.001, ANOVA). Survival 49 was lower when whole nectarines were used compared to the two methods that used artificial medium, which were not significantly different from each other (Fig. 2.3).
The proportion of parasitized hosts was significantly different among culturing methods (χ2 = 12.11, P < 0.005, KW) (Fig. 2.4). The culturing bag method had parasitism rates that were not significantly different from whole fruits (Z = -0.66,
P >0.05, MW), whereas the Petri dish method yielded parasitism rates that were extremely poor and significantly lower than both the culturing bags (Z = -3.63, P
<0.005, MW) and nectarines (Z = -2.53, P <0.05, MW).
Adult wasp size, as determined by the length of the hind tibia, was significantly different among groups of male and female wasps reared from the nectarines and culturing bags (F124 = 35.58, P <0.001, ANOVA) (too few wasps were reared from the Petri dish method to provide data for this analysis). In both methods, adult female wasps were significantly larger than males (P < 0.001), and females reared using the culturing bag were significantly larger than females reared using nectarines (P <0.05; Fig. 2.5). No significant differences in adult size were found in males. We tested whether increased wasp size was a result of larger host larvae in the carrot based culture medium, and found that in agreement with this, mean pupal weight of unparasitized host larvae reared on the artificial medium
(using the culture bag method) was significantly higher than those reared on nectarines (mean artificial medium=11.38 mg±0.17 SE and mean nectarine = 10.05 ±
0.46 SE; t14 = 2.68, P <0.05). Although host diet has been shown to influence parasitoid wasp sex ratios (Van Alphen and Thunnissen 1982), we did not find any significant differences in sex ratios within (culturing bag: χ2 = 2.9, P >0.05;
50 nectarine: χ2 = 0.5, P >0.05) or between (χ2 = 0.3, P >0.05) the culturing bag and nectarine treatments.
Under field conditions, culture bags, nectarines, and peaches showed significant differences in numbers of D. kraussii adults reared (χ2 = 18.68, P <0.001,
KW) (Fig. 2.6 A). A mean of 21.75 ± 8.42 wasps/bag emerged from 8 culture bags initially infested with 300 B. tryoni eggs (2 bags were lost due to damage). This was significantly more wasps than obtained from 10 nectarines (mean wasps/fruit = 1.7 ±
0.47, Z = -3.36, P <0.001, MW) and 10 peaches (mean wasps/fruit = 0.40 ± 0.22, Z =
-3.66, P <0.001, MW). However, fruits were heavily infested with brown rot
(Monilinia spp.) at the time of picking. Total wasps per culturing bag was highly variable, with 79 wasps reared from 1 bag (outlier, Fig. 2.6 A). When larval infestation rates were accounted for (i.e., percentage parasitism rather than total wasps emerging), mean parasitism was not significantly different between the culturing bags and the fruits (mean % parasitism: bag = 10.10 ± 3.08; nectarine =
9.61 ± 2.74; peach = 3.02 ± 1.69) (χ2 = 5.59, P >0.05, KW) (Fig. 2.6B). Sex ratios were not significantly different in adult wasps reared from culturing bags (52% female, χ2 = 0.37, P >0.05, n = 174 wasps), or nectarines (39% female, χ2 = 0.89, P
>0.05, n = 18 wasps) (insufficient data at n = 4 wasps for analysis of peach data). No wasps were reared from uninfested bags hung in the field, although 3 of the bags contained B. tryoni larvae upon collection (mean B. tryoni larvae/bag=0.75±0.41, N
= 10 bags) and thus had been oviposited into by wild flies.
51
b
ab
a
Figure 2.2: Comparison of Diachasmimorpha kraussii culturing methods, which presented adult wasps with host larvae either in whole nectarines (fruit), or artificial diet in Petri dishes (Petri dish) or culturing bags (Bag). Trials (n = 15 per treatment) used 5 adult female wasps in cages with host substrate (fruit or artificial carrot-based media) infested with 100 Bactrocera tryoni eggs: Mean numbers of adult wasps emerging. Bars with different letters are significantly different (Kruskal– Wallis/Mann–Whitney).
52
b
b
a
Figure 2.3: Mean pupae (includes parasitized and unparasitized hosts). Bars with different letters are significantly different (ANOVA).
a a
b
Figure 2.4: Mean proportion of parasitized hosts. Bars with different letters are significantly different (Kruskal–Wallis/Mann–Whitney).
53
c
a
b
b
Figure 2.5: Mean hind tibia length (measure of body size). Bars with different letters are significantly different (ANOVA).
54
Figure 2.6: Culturing bags as a tool for evaluating D. kraussii prevalence in fruit orchards. Box-plots displaying (A) total and (B) percentage parasitism of larvae in culturing bags, nectarines, and peaches in a mixed fruit orchard. *Outliers. (A) Box- plots with different letters are significantly different (P < 0.05). (B) Data were not significantly different (P > 0.05).
55
2.4 Discussion
We have identified a new method for collecting and rearing wild caught fruit fly parasitoids that combines the advantages of using artificial media (better host survival and quality of adult wasps, cost effectiveness, and practicality) with those of using host-infested fruits (high parasitism rates). Our methods were developed using wild caught D. kraussii, an important natural enemy of a number of tephritid fruit flies (Carmichael et al., 2005) and could be adapted for culturing other fruit fly parasitoids. Exposing field collected D. kraussii wasps to their larval hosts within a culturing bag yielded vastly improved levels of parasitism compared to a common
Petri dish method used to maintain laboratory cultures of this insect (Wong and
Ramadan 1992, Bautista et al., 2000), and the use of whole nectarines. This improvement was measured in terms of an increased total yield of adult wasps, a parasitism rate comparable to that of using whole fruits, and larger adult female wasps (compared to using nectarine fruits). We suspect the extremely low parasitism in Petri dishes might have resulted from disruption to pre- and post-alighting behaviours that insects use to find their hosts (Kennedy 1965) through the absence of important sensory cues. Host fruit shape is likely to be an important factor, as has been shown in other fruit fly parasitoids (Perez et al., 2012): Our culturing bags may be providing important visual cues that increase female alighting on the infested media and trigger their search for larvae. The location of the culturing bag (i.e., hanging compared to the Petri dish on the floor of the cage) could also have played a role in increasing oviposition (although it should be noted that the nectarine was placed on the floor of the cage), and suspending the nectarines (and Petri dishes) could have increased parasitism rates. Choice tests would be useful here in
56 comparing pre- and post-alighting searching behaviours and parasitism rates on natural versus artificial (bag) fruits.
Our culturing bag method produced larger female wasps (as determined by tarsal length) compared to the use of whole nectarines, probably as a result of the diet being a better resource for the developing fruit fly larvae (as evidenced by larger adult flies and better fruit fly larval survival from egg to pupae). The improved fruit fly larval quality in the carrot-based medium compared to whole fruits was perhaps not surprising, given that this media was developed specifically for B. tryoni (Hooper
1978) and that it is fortifiedwith yeast and vitamins and contains antimicrobials that assist in fruit fly larval development (Thompson 1999, Shah et al., 2014). Studies on other fruit fly parasitoids have shown that adult wasps were larger when host larvae were fed on artificial diet compared to plant hosts (Jervis et al., 2008, Cicero et al.,
2011), and larger body size in parasitoids has been shown to increase successful mating and longevity (Saeki and Crowley 2013). Female fecundity in parasitoids has been shown to be higher with increasing body size, and wasps have been shown to deposit female eggs in larger host larvae, whilst producing more male offspring in smaller hosts (Nicol and Mackauer 1999, Ode and Heinz 2002). Having a culturing method that uses a high-quality artificial diet while still promoting levels of oviposition behaviour comparable to the use of natural fruit is highly desirable. It takes around 2 h to produce 20 culturing bags (which can contain up to 750 larvae per bag), and when culturing bags are placed in cages of wasps (rather than just
5wasps per cage as in this study), 20 bags can produce over 10 000 wasps (AM, pers. obs.).
57
In our field study, culturing bags hung in a mixed peach and nectarine orchard proved to be effective “artificial fruits” for the field collection of wild D. kraussii. A similar technique (presenting tephritid larvae within a cloth bag containing media) has been used to detect the presence of braconid parasitoids
(Montoya et al., 2000), but did not evaluate the efficacy of this method.
Unfortunately, disease (brown rot) within the orchard limited the duration and size of this study, but despite this we found significantly more adult wasps emerged from the culturing bags compared to field collected infested fruits. Using these culturing bags to replenish laboratory cultures, combined with the increased oviposition of adult wasps in laboratory culture, could help maintain genetic variability in laboratory populations (Nowak et al., 2007), preventing the selection of traits that reduce the efficacy of cultured insects when released in the wild (Van Lenteren and
Bueno 2003, Wajnberg 2004). This is likely to influence the long term survival of introduced populations by maintaining important behaviours and variability that allows populations to adapt to environmental change (Frankham 2005). As learning is known to be an important behavioural trait in parasitoid foraging, field performance of mass reared wasps could be further improved by conditioning wasps on host fruit visual and olfactory stimuli prior to their release (Canale and Benelli
2012, Giunti et al., 2015). Our field study found similar percentage parasitism rates between the culturing bags and ripe fruits, although the brown rot infestation in our test orchard could have negatively influenced parasitism on fruits in this instance, making the culturing bags appear more attractive to the wasps.
Comparative studies of fruit fly parasitism in the field are very difficult, as tephritid fruit fly species have different host ranges and preferences for host fruit species (Balagawi et al., 2013), the parasitoids themselves have varying preferences 58 for different fruits and host fly species (Spinner et al., 2011, Ero and Clarke 2012), and fruit abundance is highly variable over space and time. When these variables are combined, it means that field parasitism data is largely restricted to particular wasp- host fly-fruit situations (Chinajariyawong et al., 2000), which severely limits the ability to compare data on parasitoid populations in a meaningful way (e.g., among orchards of different fruits at different times of the year). Culturing bags could serve as standardized monitoring tool for parasitoid populations in wild fruit trees and commercial orchards, allowing comparisons across time and space to be accurately made.
59
Chapter 3. Learning influences host versus non-host discrimination and post-alighting searching behaviour in the fruit fly parasitoid Diachasmimorpha kraussii
3.1 Introduction
The olfactory system of parasitoid wasps is capable of recognising specific odours emitted by plants when they are under attack from insect herbivores (Heil
2008; Mumm and Dicke 2010; Wajnberg and Colazza 2013). These herbivore- induced plant volatiles (HIPVs) have been shown to be emitted through the induction of specific metabolic pathways, and act as signals for ovipositing females in search of their host insects (Kessler and Baldwin 2002; Mumm and Dicke 2010). HIPVs can be plant- and insect-species specific (De Moraes et al., 1998; Hare 2011; Hoballah et al., 2002), providing chemical information that enables the parasitoid olfactory system to not only discriminate between insect damaged and undamaged plants, but also plants damaged by their host insect species compared to a non-host species
(Afsheen et al., 2008; De Moraes et al., 1998; Erb et al., 2010; McCormick et al.,
2012).
In addition to these innate, species-specific olfactory adaptations for host location, ovipositing female parasitoids have been frequently shown to demonstrate odour learning, whereby olfactory preferences for plant odours are modified by previous experience (for a recent review, see Giunti et al., 2015)). Where innate mechanisms in olfactory perception convey advantages via “hard-wired” responses to predictable odour cues, theory predicts that learning is advantageous in 60 environments where odour signals are more variable and unpredictable, such as where plant species are patchily distributed in time and space (Cunningham and
West 2008; Dukas and Duan 2000; Stephens 1993). Learning may benefit both pre- and post- alighting searching behaviour (Steidle 1998; Ueno and Ueno 2005). As
HIPVs are released alongside other volatile emissions that may be highly variable, learning may also improve the recognition of host-related HIPVs within the complex bouquet of a plant’s odour emissions (Wilson and Woods 2016), increasing the parasitoid’s ability to discriminate between host and non-host related HIPVs (Costa et al. 2010).
Compared to the extensive body of research on the olfactory behaviour of parasitoids of leaf-feeding insects, less is known about fine-tuning of olfactory behaviour in parasitoids that use fruit feeding insects as hosts. In these species, host eggs and larvae are often concealed within the fruiting tissue of their respective host plant, and larval feeding spreads microbes (such as yeast and bacteria) that contribute strongly to fruit volatile emissions (Leroy et al., 2011; Hamby and Becher 2016).
Olfactory preferences for odours of decaying or larval infested fruit over undamaged fruits have been demonstrated in several parasitoid species attacking tephritid fruit flies (Greany et al., 1977; Messing et al., 1992; Carrasco et al., 2005; Ero and Clarke
2012; Benelli et al., 2013a), with particular volatile emissions acting as signals for the presence of a host (Benelli et al., 2013a; Giunti et al., 2016a, b). Oviposition preferences in adult fruit fly parasitoids is influenced by larval host species and early adult experience (Giunti et al., 2016c). Canale et al., (2014) and Giunti et al., (2016b) provide evidence for associative learning in the olfactory behavior of fruit fly parasitoids, although these studies focussed on adult feeding rather than oviposition behaviour. Research has yet to confirm whether learning in adult wasps can fine-tune 61 olfactory responses of ovipositing females to enable better host and non host discrimination.
Our study set out to investigate host- versus non-host discrimination, and experienced-based changes in host searching behaviour, in the fruit fly parasitoid
Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae).
Diachasmimorpha kraussii parasitises tephritid fruit fly larvae in the genus
Bactrocera Macquart (Diptera: Tephritidae) (Carmichael et al., 2005; Clarke et al.,
2011), which include highly polyphagous species such as the Queensland fruit fly, B. tryoni Froggatt (Clarke et al., 2011). Using Y-tube olfactometer experiments we first investigated whether female wasps were able to discriminate between odours of fruits infested by B.tryoni, compared to non-host larvae, Drosophila melanogaster, and whether hosts- versus non-host discrimination could be improved through prior experience. We then explored the potential for learning to influence post-alighting search behaviour on fruits by testing whether wasps with previous experience of nectarine fruits that had larvae developing in different locations (top versus base of the fruit) exhibited differences in searching and oviposition (probing) responses.
3.2 Methods
3.2.1 Insect cultures and fruits
Parasitoids. Diachasmimorpha kraussii wasps were collected as wild insects from B. tryoni infested nectarines, and from specifically designed culturing bags containing larval infested carrot medium that were hung out in fruit orchards (Masry et al., 2018). Samples were collected from a nectarine orchard at Cleveland (27°31’S,
153°14’E), an outer suburb of Brisbane, Australia. Wasps used in experiments were
62 either wild (i.e. wasps reared from field collected fruit), or at most F2 generation from the wild. Laboratory culturing of wasps used B. tryoni as a host and followed the methods described Masry et al., (2018). Eclosing adult wasps were housed in 300 x 300 x 300 mm cages (BugDorm, referred to as “BugDorm cages” henceforth) in an insectary (26 ± 1 ºC, L:D 10:14, 70 ± 5% RH), and provided ad libitum with honey solution (30% w/v in 30 ml plastic cups fitted with cotton wool wicks) to promote longevity and maturation (Zamek et al., 2013). To obtain sufficient wasps of a similar age for experiments, eclosing wasps were collected over a 3 d period of peak emergence (males emerge 1-2 days before females). All wasps used in experiments were within the 5th generation from wild. Nectarines, a host fruit for B. tryoni, were used in the trials, having been purchased as ripe fruits from a local supermarket, carefully inspected for any previous oviposition, and washed with tap water.
Host larvae.Bactrocera tryoni pupae were obtained from a continuous culture maintained at the Queensland Department of Agriculture, Fisheries, and
Forestry, Ecosciences Precinct, Brisbane. Adult flies were housed at approximately
500 adult flies per cage within 33 x 30 x 25 cm holding cages and supplied with ad libitum water, yeast autolysate (as a paste) and sugar cubes, and left for 7-10 days to ensure mating. Fly eggs used to infest nectarines in experiments were collected in 30 mL plastic cups, perforated with holes and coated inside with a thin layer of the carrot diet, and exposed to mature flies for 6 h.
Non-host larvae.Drosophila melanogaster were used as a non-host species for D. kraussii. Insects were obtained as wild individuals from a commercial supplier
(Southern Biological, Australia) and transferred into 250 mL jars (approx. 30 adult flies per jar) filled to a depth of 3 cm with media based on polenta, golden syrup, and
63 torula yeast. Both adults and larvae fed on this diet. Drosophila larvae were collected at one day of age for experiments, and were the larvae of the wild-type parents or from the F1 generation.
Fruits. Yellow nectarines Prunus persica (var. nucipersica), were selected as fruits to be used in experiments. Nectarines are known hosts of both B. tryoni and D. melanogaster. All fruits were 120 ± 10 g average weight. Fruits were first wiped with a weak antifungal solution (nipagin / methylparaben, 2.5 g/L water) and then washed in tap water to remove chemical residues.
Fruit infestation. In Experiment 1 (see further below), fruits were infested with one day old fruit fly larvae by making four small slits (3 mm depth) into the surface of a fruit and, using a fine paintbrush, inserting 10 to 15 larvae per slit, to infest with 50 fly larvae total per fruit. In Experiment 2, infestation was achieved using adult flies. Infested fruits were then placed into 1 L plastic containers (2 fruits per container, covered with disposable cotton nappy liners for aeration) and incubated in constant condition cabinets at 25 ± 1 ºC, 13:11 L:D, 70% relative humidity, for 3 days (Experiment 1) or 4 days (Experiment 2) before use in experiments. At this stage the larvae were second instars, the preferred host stage for ovipositing D. kraussii (Ero et al., 2011a).
3.2.2 Experiments
3.2.2.1: Experiment 1: Olfactory preferences for host and non-host infested fruits.
General methodology
Mature parasitoid females were tested in a Y-tube olfactometer to evaluate behavioural preferences for odours in a dual-choice scenario. The olfactometer (Fig.
64
3.1) was of a standard design, consisting of a 5 cm diameter glass Y-tube, having a
15 cm stem length and two 10 cm arms (angled at 75º). A clean (charcoal filtered) airstream was pumped at 2.5 L/minute through two sealed glass jars (20 cm depth, 14 cm diameter) into which odour sources could be placed, the air then flowing into each arm of the Y-tube.
Olfactometer trials were conducted in a heated laboratory lit with fluorescent lighting and natural daylight (26 ± 2 ºC, 55±5 % RH). Female wasps were introduced into the stem of Y-tube and observed for a maximum of 10 min, terminating the experiment when the wasp remained for 15 s in a chosen arm. If a wasp did not respond during the time period, it was discarded. Each parasitoid was tested individually, and used only once. After 10 consecutive trials (i.e. 10 wasps), the Y- tube and holding jars were washed in 70% ethanol, rinsed with hot water, and air dried. New fruits were then placed in the glass jars (odour sources). The position of the odour source (arm of the Y-tube) was switched every five wasps to avoid positional bias.
In all experiments, mated female wasps were used at 5-11 days old and a total of 490 responsive wasps was used. In conditioning trials, wasps were moved from rearing cages and released inside a cage containing three B. tryoni infested fruits
(placed on the floor of the cage) for 10 h (8:00 – 18:00) to allow fruit exploration and oviposition. Olfactometer bioassays using these conditioned wasps were performed on the following day between 9:00 and 15:00. Where B. tryoni infested fruits were used, the larvae were second instar, following artificial infestation with eggs 4 days prior to use in experiments. Instar larvae was confirmed through dissection of sacrificial fruit prior to a day’s experimentation.
65
Figure 3.1: Y-tube olfactometer used in these experiments.
Experiment 1a: Odour preferences of naïve D. kraussii females for host- and non-host infested fruits. We first investigated whether female wasps discriminated between odours from uninfested nectarines, and odours from nectarines infested by a known host, the Queensland fruit fly, Bactrocera tryoni, or a non-host fruit fly, Drosophila melanogaster. Five, dual-choice odour preference trials were conducted: (i) uninfested fruit vs clean air, (ii) B. tryoni infested fruit vs clean air, (iii) B. tryoni infested fruit vs uninfested fruit, (iv) D. melanogaster infested fruit vs B. tryoni infested fruit, (v) D. melanogaster infested vs uninfested fruits. Responses of 50 females were individually scored for each treatment.
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Experiment 1b: Influence of experience on odour preferences for host- and non-host infested fruits. Three trials were performed, each testing preferences for odours from B. tryoni (host) vs D. melanogaster (non-host) infested nectarines: (i) wasps previously experienced on B. tryoni infested fruits; (ii) wasps previously experienced on D. melanogaster infested fruits; and (iii) wasps previously experiences on both B. tryoni and D. melanogaster infested fruits (three infested fruits of each in the training cage). The results from Experiment 1a trial iv, were used as base-line data for the preference of naïve wasps for these odours. Responses of 50 females were individually scored for each treatment.
3.2.2.2: Experiment 2.The influence of wasp experience on post-alighting orientation and oviposition behaviour.
In this experiment, female wasps were given oviposition experience on nectarines that differed in the position of host larvae within the fruit. Their subsequent behaviour when alighting on fruits without larvae was then quantified in terms of location on fruit, search time, and ovipositor probing. The position of larvae within fruit was either the ‘top’ (i.e. stalk end) or ‘bottom’ of the fruit. Bactrocera species preferentially lay in top half of fruit (Rattanapun et al., 2010), and D. kraussii forages while fruit is still on the tree (Ero et al., 2011b), so the position of larvae within the fruit is biologically relevant to the wasp in terms of optimising foraging efficiency.
Conditioning procedure. Ripe nectarines were exposed to ovipositing B. tryoni females in such a way as to limit fruit fly oviposition to either the top section
(nearest stalk) or base section (furthest from stalk) of the fruit. Whole fruits were placed inside a 450 mL cylindrical plastic pot with a circular hole (4 cm diameter) cut into the lid, allowing only the required section of fruit to remain uncovered. 67
These partially exposed fruits were then placed in BugDorm cages containing 20 mated B. tryoni until 10 fly oviposition events had occurred, after which they were removed to an incubator (25 ± 1ºC, 13:11 L: D, 70% RH) for 4 days to allow eggs to hatch and develop to 2nd instar (based on laboratory experience and confirmed at the start of each trial through dissection of sacrificial fruit). For conditioning, 25 female wasps were released into a BugDorm cage together with three infested nectarines from one conditioning treatment (i.e. top or base) and left for 19 hrs (start 12:00 to
19:00) to allow fruit exploration and oviposition. Confirmation that wasps were conditioned was achieved through direct observation, with oviposition by all wasps within a cage generally observed within an hour of initia release.
Post alighting behaviours. Female wasps were tested for their orientation and oviposition behaviour on the day after conditioning (or when testing naïve females, wasps of the same age but without exposure to infested fruits were used), between the hours of 11:00 and 17:00. Insects were released individually into BugDorm cages containing a single nectarine that had been subjected to oviposition by B. tryoni as described above and contained eggs in both the top and bottom sections. Females and fruit were never reused across trials.
The logic/need to have eggs at both the top and bottom of the fruit was three- fold. First, this was effectively a choice experiment and so the experimental condition at both ends of the fruit had to be identical for appropriate design. Second, pilot work showed that wasps responded very poorly to fruits that had no prior oviposition or larval infestation, a result previously reported by Ero et al., (2011b) who showed D. kraussii had little or no response to uninfested whole fruit. Third, if I had used larval infestation at both ends then the wasps may have gained experience
68 during the course of the experiment, invalidating the results. Thus we used fly oviposition and eggs, which appeared to provide some orientation cue, but would avoid providing the probing wasp with reinforcer stimuli that might facilitate learning.
During exposure to trained wasps, fruits were placed on a glass tube (25 mm diameter x 100 mm height) in the centre of the cage, and marked with a solvent and odour free liquid chalk marker pen to identify the larval infested sections (Fig. 3.2)
(for conditioning of wasps, fruit was exposed in an identical fashion). The individually released wasps were monitored continuously over a 15 min period, recording the number of visits to and from the fruit, time spent in each section (top or base), and probing behaviour. Experiments were terminated if the wasp had not alighted on the fruit after 10 min, and fruits were changed after every four trials, using wasps from different differnet treatments in rotation. Twenty females were used for each treatment (oviposition experience at top, base, or no experience).
69
Figure 3.2: Diachasmimorpha kraussii female lands on a single nectarine that had been subjected to oviposition by Bactrocera tryoni as described above and contained eggs in both the top and bottom section
3.2.3 Statistical analysis
Proportions of insects selecting each of the two odour sources in all olfactometer trials from Experiment 1 (a total of eight trials across Experiments 1a and 1b, 50 insects per trial) were statistically compared using a chi-squared test (χ2) with one degree of freedom. Comparisons between treatment groups (prior experience on top of fruit, on bottom of fruit, or naïve, n = 20 individuals per group)
2 in Experiment 2 were compared using a 2 x 2 contingency table (χ , 1 d.f.). Post alighting orientation (top vs base) was analysed using a χ2 test, differences in search time and probing counts in the two sections of the fruit were analysed using the
Wilcoxon signed ranks test (WSR) for within treatment differences, and the Mann-
Whitney U test for pair-wise comparisons between treatments.
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3.3 Results
3.3.1: Experiment 1a: Odour preferences of naïve D. kraussii females for host (B. tryoni) and non-host (D. melanogaster) infested fruits.
Naive wasps showed a preference for odors of undamaged nectarines over clean air (χ2 = 13.52, P < 0.001), and for host (B. tryoni) infested fruit over clean air
(χ2 =32.00, P < 0.001). A significant preference for fruits infested by the host larvae over uninfested fruit was found (χ2 252 = 13.52, P < 0.001), whilst no significant differences were found between choices of odors from fruits infested by non-host larvae (D. melanogaster) and uninfested fruits (χ2 254 = 2.00, P > 0.05). In line with this finding, wasps also showed a significant preference for the odors of the B. tryoni infested fruit over odors of D. melanogaster fruit (χ2 256 = 8.00, P < 0.005) (Figure
3.3).
3.3.2: Experiment 1b: Odour preferences of experienced D. kraussii females for host (B. tryoni) and non-host (D. melanogaster) infested fruits.
All treatment groups (i.e. experienced on B. tryoni, experienced on D. melanogaster, experienced on both) showed a significant within-treatment preference for B. tryoni infested fruits over D. melanogaster infested fruits (Fig. 3.4). For between-treatment comparisons, wasps experienced on B. tryoni infested fruits alone, or both B. tryoni and D. melanogaster infested fruits, demonstrated a significantly higher preference for odours of B. tryoni infested fruits compared to naïve wasps
(χ2= 6.25 and χ2 = 7.87 respectively, P < 0.05), with no significant difference in the strength of this preference between the two groups (χ2 = 0.1, P > 0.05). The preferences of wasps experienced on D. melanogaster infested fruits showed no significant differences from naïve wasps (χ2 = 1.3, P > 0.05) (Figure 3.4).
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Figure 3.3: Y-tube olfactometer trials testing odour preferences of naïve mated female Diachasmimorpha kraussii (Innate preferences). Odour sources: clean air, uninfested nectarines, B. tryoni (host) infested nectarines, D. melanogaster (non-host) infested nectarines. Bars represent percentage attraction to odours (insect counts at end of bars). Significance determined by χ2 tests on count data (* P < 0.05. ** P < 0.005).
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Figure 3.4: Y-tube olfactometer trials testing odour preferences of experienced mated female Diachasmimorpha kraussii (Experience of infested fruits). Odour sources: B. tryoni (host) infested nectarines, D. melanogaster (non-host) infested nectarines. Bars represent percentage attraction to odours (insect counts at end of bars). Significance determined by χ2 tests on count data (* P < 0.05. ** P < 0.005).
3.3.3: Experiment 2.The influence of experience on D. kraussii post-alighting orientation and oviposition behaviour.
Alighting behaviour: Here we observed the first section of the fruit utilised by wasps (top vs base) upon alighting on nectarines (Fig. 3.5). Wasps with experience ovipositing at the top of the fruit showed a significant preference for the top of the fruit (75% of wasps to top, χ2= 5.00, N = 20, P < 0.05). No statistically significant differences in preference (top vs base) were found for wasps experienced
73 on the base of the fruit, although 70% chose the base (χ2 = 3.2, N = 20, P > 0.05).
Wasps without experience similarly showed no statistical preference for the top or bottom of the fruit (63% of wasps to top, χ2 = 2.9, N = 19, P > 0.05). Between group comparisons showed that wasps with experience on the base of the fruit showed significant different preferences compared to controls (χ2 = 4.31, N = 39, P < 0.05) and to wasps experienced on the top of the fruit (χ2 = 8.12, N = 40, P < 0.05), whereas those experienced on the top were not significantly different from controls
(χ2= 0.64, N = 39, P > 0.05.
Search time. Upon release into the cage, wasps spent around two thirds of their time searching on fruit (mean search time: experienced on top = 657.6 ± 54 s; experience on base = 618.2 ± 45.21 s; control = 726.1 ± 44 s). Fig. 3.6 displays box plots for percentage time spent within the top and base target areas. Searching times for wasps experienced at the top of the fruit, and also for wasps without experience showed a clear, significant preference for the top (top: Z = -3.70, base: Z = -2.63, P <
0.05, WSR), whereas wasps experienced on the base showed no significant differences in preference for either area (Z = -0.9, P > 0.05, WSR). Proportional comparisons between groups revealed significant differences in preferences between wasps experienced on the top and those experienced on the base (Z = -2.42, P < 0.05,
MWU), but not between top and no experience (Z = 1.59, P > 0.05, MWU), or base and no experience (Z = -0.90, P > 0.05, MWU).
Probing: Not all wasps tested (N = 20 per group) demonstrated probing with their ovipositor, which limited sample sizes for analysis (N = 16, 12, and 11 for experienced top, experience base, and no experience respectively). Fig. 3.7 displays box plots of probing counts across treatments. Wasps with experience ovipositing at
74 the top of the fruit demonstrated significantly more probing at the top of the fruit than the base (W = 12.5, P < 0.05, N = 16, WSR), whilst wasps with experience ovipositing at the base of the fruit, or without experience, showed no significant differences in probing area (W = 20.5 base, W = 19 no experience, P > 0.05, WSR).
When preferences for probing on the top of the fruit (as proportions) were compared among treatments, significant differences in probing site were found between wasps experienced on the top of the fruit versus those experienced on the base (χ2 = 22.73,
P < 0.001), with no significant differences between those experienced on the top versus no experience (χ2 = 0.27, P > 0.05), or experienced on the base versus no experience (χ2 = 2.25, P > 0.05).
Figure 3.5: Post-alighting behavioural responses (post alighting orientation) for Diachasmimorpha kraussii adult females following experience ovipositing either at the top or base sections of nectarines, or with no experience. Red and blue bars denote top and base section of fruit respectively. Within treatment differences (i.e. preferences for top vs base), * = significant at P < 0.05. Between treatment differences, bars with different letters are significantly different at P < 0.05.
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a
Figure 3.6: Post-alighting behavioural responses (Percentage search time in each of the two sections) for Diachasmimorpha kraussii adult females following experience ovipositing either at the top or base sections of nectarines, or with no experience. Red and blue bars denote top and base section of fruit respectively. Within treatment differences (i.e. preferences for top vs base), * = significant at P < 0.05. Between treatment differences, bars with different letters are significantly different at P < 0.05
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a b
Figure 3.7: Post-alighting behavioural responses (Probing counts in each section) for Diachasmimorpha kraussii adult females following experience ovipositing either at the top or base sections of nectarines, or with no experience. Red and blue bars denote top and base section of fruit respectively. Within treatment differences (i.e. preferences for top vs base), * = significant at P < 0.05. Between treatment differences, bars with different letters are significantly different at P < 0.05
3.4 Discussion
Diachasmimorpha kraussii is known to prefer host-infested compared to uninfested fruit (Ero and Clarke 2012; Ero et al., 2011b). We show here that this discrimination can be achieved through odour recognition, and that the olfactory system of D. kraussii is also able to further discriminate between odours of fruits
77 infested with larvae of host fruit flies versus non-host fruit flies (noting that the non- hosts species used here were fly larvae belonging to a different family). Moreover, we demonstrate that prior experience of infested fruits enables more finely-tuned olfactory discrimination in favour of fruits infested by a host species versus a non- host species.
Our results show that female wasps preferred odours of ripe nectarines infested with their host fruit fly, B. tryoni, over infested fruits by the non-host fruit fly D. melanogaster, indicating that the olfactory system of these wasps is finely- tuned to odour emissions specific to the presence of its host. Although we did not test wasp preferences for odours of D. melanogaster against clean air (to look specifically at attraction towards odours of non-host infested fruits), we showed that preferences for odors from fruits infested by D. melanogaster versus uninfested fruits were not significantly different, suggesting that changes in odour composition as a results of infestation by non-host larvae had little (positive or negative) effect on attraction.
Results from our experiments using insects with prior oviposition experience, strongly suggest that wasps were learning odours specific to host infested fruits. We define learning here as a change in behaviour following experience (Papaj &
Prokopy 1989), as our experiments were not designed to characterise the type 332 of learning (e.g. associative conditioning versus sensitization). However, studies on other fruit fly parasitoid species have shown evidence for associative learning in feeding behaviour (Canale et al. 2014; Giunti et al. 2016b), and we believe this is likely to the type of learning that is occurring in our experiments. Laboratory-based studies demonstrating host-associated learning by fruit fly parasitoids have been
78 experimentally confirmed in the field (Masry et al., in press), and optimising foraging via learning is a likely explanation for field observations of rapid responses of fruit fly parasitoids to corresponding increases in host fly abundance (Vargas et al., 2013).
In the present study, females demonstrated an increased ability to discriminate between host and non-host related odors as a result of oviposition experience, but only when they were exposed to host-infested (as opposed to non- host infested) fruit. This implies that a positive oviposition experience may elicit a stronger learning response (attraction) than an unrewarding stimulus (avoidance). In studies on associative learning in bees, insects provided with both the learnt stimuli and a second unrewarded stimuli (termed differential conditioning) have been found to show greater discriminatory learning compared to being exposed to the single positive stimuli alone (termed absolute conditioning) (Giurfa 2004). We did not find this to be the case here, although high innate (70%) and learnt (90% for host- experience) preferences may have prevented the experimental design and sample size used here from achieving a statistically significant effect.
The volatile cues that enabled olfactory discrimination between odors of fruits infested by host and non-host flies were not elucidated in this study, but we speculate that rather han being related directly to plant metabolic pathways (as with
HIPV’s from leaf herbivory) they may be indirectly produced by species-specific microbial infection. There has been an increased focus on the major role that microbes play in the interactions between insects and their host plants, and how microbial volatile emissions may influence insect olfactory behavior (Leroy et al.,
2011; Davis et al., 2013), including attraction to fruits (Scheidler et al., 2015).
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Interestingly, Ero et al., (2010) showed that D. kraussii, at the ovipositor probing stage, is unable to discriminate among host and non-host tephritid larvae within the genus Bactrocera, when the larvae were offered in artificial diet; and more informatively, D. kraussii could still not distinguish between physiologically suitable and unsuitable Bactrocera larvae in the same fruit type (but in different fruit pieces) in an outdoor flight cage (Ero & Clarke 2012). This between family (i.e. Tephritidae versus Drosophilidae) but not within-genera discrimination of odors may imply either that infestation related odor emissions do not differ among the Bactrocera, or that they do differ, but the parasitoids are unable to perceive these differences.
Noting that D. melanogaster is a widespread and common pest of decaying fruits
(Starmer 1981), strong selection pressure may have been placed on the sensory systems of parasitoids attacking tephritid that has led to adaptations in the parasitoid olfactory system to distinguish between odors emitted as a result of infestation by each fly family.
Our post-alighting behavioural trials showed that experience on infested nectarines altered the orientation of wasps as they landed on these fruits. Those with experience ovipositing at the top of the fruit preferentially orientated towards the top upon alighting, whilst those without experience, or experienced at the base showed no significant difference in orientation (but note, sample sizes may have influenced statistical significance here, as 70% of wasps experienced at the base 380 preferentially orientated towards the base). Similar learning-related changes were seen in search time and probing behavior. The preference for searching the top of the fruit may have evolved as an innate response towards areas of the fruit where host flies most frequently oviposit (Rattanapun et al. 2010). Differences in probing counts were only seen when wasps had experience ovipositing at the top of the fruit, 80 however sample sizes and probing counts were low, particularly in the naïve wasp treatment where many wasps did not respond, perhaps as a result of lack of host stimuli—the nectarines used in the test scenario were infested only with host eggs so there would have been an absence of larval cues such as larval odour or vibration
(Duan and Messing 2000a).
The learning mechanism underlying the post-alighting differences among treatments may have been classical associative conditioning (Papaj and Prokopy
1989) of olfactory, gustatory or visual stimuli that differed between the top and the base of the fruit. Alternatively, these results may provide an example of operant
(instrumental) conditioning in a parasitoid, where learning shapes the behavioural movements of the animal through positive (reward) or negative (punishment) reinforcement (Papaj and Prokopy 1989). Operant conditioning has been shown in honeybees (Abramson et al., 2016), bumblebees (Laverty 1994), and moths
(Cunningham et al., 1998), where it may convey advantages in flower handling behaviour and nectar location. In polyphagous parasitoids such as D. kraussii, oviposition sites of host insects may vary among fruits, and this form of learning might enable insects to better orientate towards areas of the fruit where larvae have been located on previous encounters.
Host searching behaviour in insects is often viewed as a linked series of sensory responses and behaviours that brings the insect in contact with its host
(Kennedy 1965). Fruits are likely to vary unpredictably in D. kraussii environment, both geographically and temporally, and learning mechanisms may act on pre- alighting (odour learning / classical condition) and post-alighting (orientation after landing / operant learning) behaviours in this sequence to fine tune the insect towards ecologically relevant cues in its local environment. There is surprisingly little 81 evidence for operant learning and its adaptive significance in parasitoids, and (with the caveat that effects of operant and associate learning may be closely integrated and difficult to separate (Papaj and Prokopy 1989)), we see this a potential area of future study.
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Chapter 4. From laboratory to the field: consistent effects of experience on host location by the fruit fly parasitoid Diachasmimorpha kraussii (Hymenoptera: Braconidae)
4.1 Introduction
The effectiveness and efficiency of foraging behaviour of entomophagous insects within an ecosystem can be affected by many things, including exogenous variables such as the quality and quantity of habitats, and endogenous variables including physiological motivation and learning (Hassell and Southwood, 1978; Vet et al., 1995; Weiss, 2006, Price et al., 2011). In the widely studied and utilised parasitoids, numerous factors are known which may both improve, but also hinder, their foraging and host location ability.
Environmental cues, especially olfactory signals, are used by parasitoids to locate and then search suitable host patches (Sivinksi and Aluja 2012). The odour cues derived from non-infested host plants, host-infested host plants (= herbivore- induced plant volatiles, HIPVs), and surrounding plants can all influence the distribution of parasitic wasps and their host interactions (Marino et al., 2006;
Bukovonszky et al., 2007; Tscharntke et al., 2007; Simpson et al. 2011b, Letourneau et al., 2012). While plant derived cues may aid parasitoid foraging, they may also hinder foraging. Non-target plants, be they plants which can support host insects but are currently uninfested, or simply other plants in the local environment which are
83 never associated with the host insect, can emit cues that are detected by the olfactory system of a parasitoid and negatively impact foraging by masking the signals from the target host (Gingras et al., 2002; Obermaier et al., 2008; Quicke 2015). In applied systems this may lead to released biological control agents dispersing and establishing within non-target hosts, or a decline in female parasitoid fitness because of the requirement for increased searching time (Barratt et al., 2010; Kostenko et al.,
2015).
Learning, which is broadly defined as a change in behaviour as a result of experience (Papaj and Prokopy 1989), is recognised as an important endogenous mechanism for enhancing the reliability of host location cues over space and time, and for increasing foraging efficiency (Vinson et al., 1998; Cunningham et al., 1999).
Hymenopteran parasitoids have been extensively researched with respect to host learning (Froissart et al., 2012; Giunti et al., 2015), and it is clear that prior exposure to a host can lead to more rapid host location by responding preferentially towards host-related cues relevant to the insect’s local environment. Prior experience may not only improve host location, but alter use pattern once a host is located. For example naïve females of the braconid Fopius arisanus (Sonan) readily laid eggs into previously parasitised hosts, while females with prior oviposition experience avoided super-parasitism (Wang and Messing, 2008). However, Dukas and Duan
(2000) point out that it is unclear how learning-based changes in parasitoid behaviour relate to individual fitness, since differences in host finding rate due to learning need not be accompanied by differences in oviposition. Nevertheless, in applied systems, an experienced wasp is likely to be a more effective biological control agent than a naïve wasp because of a greater ability to find the preferred host (Giunti et al., 2015).
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Fruit flies (Diptera: Tephritidae) are among the world’s worst insect pests of horticulture, as they lay their eggs directly into sound fruit where the subsequent larvae feed (White and Elson-Harris 1994). The Sterile Insect Technique (= SIT), where 10s and even 100s of millions of mass-reared, sterilised male flies are released weekly to outcompete wild males for females, is well developed against several fruit fly species (Hendrichs and Pereira 2013). As part of such mass rearing programmes, specialist fruit fly parasitoids (Hymenoptera: Braconidae: Opiinae) are also reared and released (Ovruski and Schliserman 2012; Garcia & Ricalde 2013), and theoretical predictions suggest that joint inundative releases of sterilised male flies and parasitoids gives more effective control than the sum of the two individual parts
(Gurr and Kvedaras 2010).
In SIT programs, it is recognised that male fly preconditioning, through exposure to certain chemicals, enhances the fitness of the released flies (Shelly et al.
2004, 2007). However, we are not aware of any attempt in fruit fly SIT to precondition wasps being released, although a recent review has suggested this as a field worthy of further study (Giunti et al., 2015). Hare and colleagues (Hare 1996;
Hare et al., 1997; Hare & Morgan 1997) have previously demonstrated the operational validity of this approach, demonstrating that the pre-prelease conditioning of mass-reared Aphytis melinus DeBach with kairomones of its scale host, Aonidiella aurantii (Maskell), subsequently led to increased field parasitism of that host.
Diachasmimorpha kraussii (Fullaway) is an Australian endemic opiine braconid which has been used in both classical and inundative fruit fly biological control (Duan & Messing 2000, Rendon et al., 2006, Argov & Gazit 2008). As with
85 other opiines, the wasp orientates to its fruit fly larval hosts primarily through host- fruit derived olfactory cues (Ero & Clarke 2012). The wasp has a learning capacity, and in Y-tube olfactometer tests preference for infested host fruit type can be changed based on prior experience (Masry unpublished). We postulated a scenario that the parasitoids might be released to target its host fruit fly, Bactrocera tryoni
(Froggatt), in a particular crop, and that prior lab-based wasp experience on that crop would increase attack rate in the field. We tested the scenario at three spatial scales, in the laboratory, in large field cages and then in the open field. Our results have implications not only for inundative biological control, but also for the extension of wasp learning experiments from the laboratory to the field.
4.2 Materials and Methods
4.2.1 Insect cultures
A laboratory colony of the parasitoid Diachasmimorpha kraussii was established from infested fruit collected from nectarines at Cleveland (37°25’ S,
122°05’ W), in South-east Queensland, in November 2015; supplemented with additional wasps collected from the same location in April 2016 from guava. The wasps were identified using the key of Carmichael et al. (2005). In the laboratory, wasps were held at 27 °C and 70% RH at a density of approximately 100 individuals per 30 x 30 cm x 30cm nylon cage (Bugdorm, Taiwan), and provided honey and water ad libitum as described in Masry et al., (2018). Wasps were reared on B. tryoni larvae (the wasp is a larval/pupal endoparasitoid, preferentially ovipositing into 2nd instar larvae (Messing and Ramadan 1999)), with the fly larvae reared on a standard laboratory carrot-based diet (Heather & Corcoran 1985). The B. tryoni came from a
86 continuous stock culture maintained at the [Queensland] Department of Agriculture,
Fisheries and Forestry, Ecosciences Precinct, Brisbane.
4.2.2 Experiments
Background and commonalities
We ran near-identical experiments in three different experimental arenas: (i) a
Y-tube olfactometer; (ii) large field cages (7 m x 7 m x 4.8 m height); (iii) and a non- fruiting guava orchard. In these experiments, we tested the host location of B. tryoni by D. kraussii utilising two host fruit: yellow nectarines (Prunus persica var. nucipersica) and tomato (Solanum lycopersicum, var. Gourmet premium). The two fruit types are both major hosts of B. tryoni (Hancock et al. 2000) and the wasp is known to orientate to both (Ero et al., 2011a; Ero & Clarke 2012). All fruits had an average weight of 120g ±10g. Before use, fruit was wiped with a weak antifungal solution of nipagin 2.5g/L water and then washed in tap water.
In each arena we ran three sets of experiments. Host location was tested with naïve wasps to determine any innate preference towards infested nectarine or tomato; and with wasps previously exposed to either infested nectarine, or infested tomato, to determine if prior experience resulted in a changed host usage (i.e. a learning effect).
In the olfactometer and field experiment our design tested olfactory responses alone, whilst in the field-cage trial the olfactory cues were not separated from visual cues.
We note that the prior training of wasps may also have resulted in unbalanced egg load between experimental organisms, i.e. trained wasps had prior oviposition experience and so may have had fewer eggs in their ovaries than naïve wasps. Based on how training was done, we cannot separate the two influences of neural
87 conditioning and egg load. That trained wasps (i.e. with potentially lower egg loads and hence lower drive to oviposit) could still strong a very strong preference for the innately less preferred host (see Results) suggests to us that altered egg load did not significantly alter the neural learning effect.
4.2.2.1 Experiment 1: Y-tube olfactometer
Sexually mature female wasps (likely mated but this was not confirmed for each individual) were used at 5-11 days old. In learning trials, wasps were transferred from general rearing cages to a cage of the same size (i.e. 30 x 30 x 30cm) containing three B. tryoni infested fruits for 10 h (8:00 – 18:00) to allow sensory experience and oviposition on that fruit type. Fruit was artificially infested in the laboratory with 100
B. tryoni eggs placed under a flap of skin, which was then closed over with surgical tape and the infested fruits incubated for three days. Olfactometer bioassays using these conditioned wasps were performed the following day between 9:00 and 15:00.
Mature parasitoid females were tested in a Y-tube olfactometer to evaluate behavioural preferences for odours in a dual-choice set up. The olfactometer was of a standard design, consisting of 5 cm diameter glass Y tube, having a 15 cm stem length and two 10 cm arms (angled at 75º). A clean, charcoal filtered, airstream was pumped at 2.5 L/minute through two sealed glass jars (20 cm depth, 14 cm diameter) into which odour sources could be placed, the air then flowing into each arm of the Y tube.
Trials were conducted in a heated laboratory lit with fluorescent lighting and natural daylight (26 ± 2 ºC, 55±5 % RH). Female wasps were introduced into the stem of Y-tube and observed for a maximum of 10 min, terminating the experiment when the wasp remained for 15 s in a chosen arm. If a wasp did not respond during
88 the time period, it was discarded. Each parasitoid was tested individually, and used only once. After 10 consecutive trials (10 wasps), the Y-tube and holding jars were carefully washed in 70% ethanol, rinsed with hot water, and air dried. New fruits were then placed in the glass jars (odour sources), and the position of the odour source (i.e. arm of the Y-tube) was switched every five wasps to avoid positional biases. Olfactometer trials compared preferences for odours of host infested nectarines vs host-infested tomatoes, with trials of (i) naïve wasps, (ii) wasps experienced on host-infested nectarines and (iii) wasps experienced on host-infested tomatoes. Responses of 30 females were individually scored for each treatment.
Statistical analysis
Proportions of insects selecting each of the two odour sources in all olfactometer trials were statistically compared using a chi-squared test (χ2) with one degree of freedom. Comparisons between treatment groups were compared using a 2
2 x 2 contingency table (χ , 1 d.f.).
4.2.2.2 Experiment 2: Large field-cage
Experiments were run in large field cages at the QUT Samford Ecological
Research Facility (27°231’23”S, 152°52’22”E), South-east Queensland (Fig. 4.1).
Fruit were hung from 12 artificial trees (each 2 m tall, 0.75 m canopy diameter, with polyester leaves and plastic branches built upon a natural timber stem), arrayed in a grid, equidistance from east other (Figure 4.2).
Fruit was artificially infested in the laboratory with 100 B. tryoni eggs placed under a flap of skin, which was then closed over with surgical tape and the infested fruits incubated for three days. Infested fruits were transferred to the field on the day
89 of an experiment and hung in the mid-canopy of the artificial trees at rate of one infested fruit per tree (i.e. six infested nectarines and six infested tomatoes per replicate). Fruit were alternately placed per tree, beginning at the start of one row, working down that row, turning and coming up the next row, etc. The starting fruit
(i.e. nectarine or tomato) altered between replicates.
For parasitoid conditioning, 50 female D. kraussii were collected from the emergence cages and located to a new cage (= training cage). Three B. tryoni infested fruits (nectarines or tomatoes, infested three days previously as per the experimental fruit) were exposed to wasps at 12.00 pm and left overnight. The infested fruits were removed from the training cage the following day at 7.00 am and then the experienced wasps were transferred to the field-cage site. Four hours later
(i.e. at 11.00 am), the experienced parasitoid females were released into the field cage for testing.
Experiments were scored by the number of ovipositing wasps on each individual fruit at 20, 40 and 60 minutes after release. At each time period, any actively ovipositing wasps were collected and removed from the arena and the score for the replicate was the sum of all wasps collected. Three concurrent replicates for each treatment (naïve wasps, wasps trained on nectarine, wasps trained on tomato) were performed.
Analysis
Means within a treatment were statistically compared using independent samples, 2-tailed t-tests. Data for nectarine-experienced and tomato-experienced
90 treatment were square root transformed prior to analysis to equalise variances (as determined by a Levine’s test): this was not required for the naïve wasp data.
Figure 4.1. Large field cages at the QUT Samford Ecological Research Facility in South-east Queensland.
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Figure 4.2. Artificial trees inside Samford field cage.
4.2.2.3 Experiment 3: Field study
The study was run in a small guava orchard located at Cleveland
(27°31’27”S, 153°14’54”E), South-east Queensland, during November 2016 and
February 2017 (Fig. 4.3). The orchard comprised five rows of mature, fully-leafed guava (Psidium guajava) trees, each row consisting of nine trees (4-5 m tall), covering an area of approximately 2000m2. The orchard was non-fruiting and unsprayed, and the role of the guava trees was to provide an orchard environment
(natural meteorological conditions, background plant odours etc.) to hang the experimental fruit in this situation any tree-fruit type might have been used, guava was simply available and convenient. Guava, when fruiting, is a host plant of both B. tryoni and D. kraussii (Ero et al., 2011b). The guava plot is located within an experimental research farm, which in turn is surrounded by encroaching suburban 92 environment. Other fruiting host plants were present in the larger environment, but none within 100 metres of the experimental plot.we cannot exclude that there may have been some wild D. kraussii in the environment, it is a native insect and occurs on the locality (Ero et al., 2011a), but unpublished research (Ero 2009; K. Mahat,
Q.U.T., pers. comm.) shows that D. kraussii does not locate itself in a local environment where fruit are absent, as was the case in the orchard when used for experimentation. The results also suggest that if wild wasps were present, they were of insufficient number to affect results.
In this experiment, fruit mimicking diet bags were used instead of real fruits.
These fruit mimics consist of a layer of carrot-based artificial diet wrapped around an agar-gel centre, all contained within a fine gauze bag (see details in Masry et al.,
2018). Such ‘fruit’, either infested before hanging or naturally infested by wild fruit flies, have been demonstrated by Masry et al. to be very effective for sampling D. kraussii in the field. The benefit of the fruit mimicking bags in this trial are that they were manipulated so that only the odour varied (i.e. nectarine or tomato odour), but any visual cues were identical between treatments.
Odours were obtained from nectarine and tomato in the form of natural juice.
Four individual fruit pieces of the fruit types used in Experiment 1 were blended with
200 ml of distilled water and the juice was kept refrigerated until used. The bags were prepared following the method of Masry et al. (2018), inoculated with 200 B. tryoni eggs per bag, and then left in an incubator for three days in order for the fly maggots to reach second instar. On the experimental day, infested bags were transferred to the field and then each injected with 20 ml of the fruit juice. The fruiting mimicking bags were alternately distributed throughout the orchard,moving
93 down one row and then up the next, by hanging in the centre of the trees about 2 m above the ground (Fig. 4.4). There were 20 bags of each fruit type per pair-wise trial.With 45 trees in the orchard (5 rows x 9 trees/row), and 40 fruit mimics, the end tree of each of the five rows (same end) was not used for hanging fruit.
Releases of naïve, nectarine-experienced and tomato-experienced wasps were made into the field. For a given trial, 200 female wasps were released in the orchard at 10 am, immediately after the placing of fruit-mimicking bags: the total duration of each trial was 96 hours. The bags were collected, transferred to the laboratory and then separately placed in 750 ml containers with additional carrot diet to ensure larval development, and vermiculite on the bottom for pupation. After 10 days incubation the pupae were sieved and counted, and then returned to the incubator in new containers until adult emergence, to record the numbers of wasps and flies from each fruit-mimicking bag.
In this experiment the fruit mimicking bag was treated as the unit of replication, i.e. n= 20 bags per treatment. Trials were run at a minimum of no less than seven days after the completion of the previous trial. Visual searching did not locate any wasps staying within the orchard between trials. In these trials bags were lost (presumably to birds or possums), such that eventual n = 18 to 20 bags per treatment.
Data analysis
Data analysis of this trial was confounded by the fact that experiments had to be run at different times. This was a limitation of the field work as we had no mechanism, other than to split trials in time, of separating wasps with different
94 experimental conditioning (i.e. naïve, nectarine-trained, or tomato-trained). For this reason the three trials are not statistically compared to each other. Rather, the results of the naïve wasp trial inform the nectarine and tomato-learning trials through modification of a binomial analysis (see further below), but the nectarine- experienced and tomato-experienced trials are analysed entirely independently of the other.
Data analysis was undertaken in two parts: (i) host location; and (ii) host utilisation of located fruit. The two-part analysis was to avoid zero-inflated data from the numerous individual fruit which were never located by a wasp. For host location, data were converted to a binomial format. Fruit with no parasitoid emergence was scored as 0; fruit with any parasitism was scored as 1. To control for innate preference of one fruit type over another by the wasp (see Results, Expts 1 & 2), the results from the naïve-wasp treatment were used to set the ‘expected’ proportion in a binomial test. The default expected proportion for a binomial test is 0.5 (i.e. equal distribution between two treatments), but by using the naïve-wasp results we altered this to accurately reflect the innate, disproportionate host usage between fruit types.
This analysis makes the explicit assumption that the innate preference of D. kraussii for the two fruits does not change from one field trial to the next. Given general knowledge of parasitoid biology, and the earlier results from lab and field-cage, we considered a valid assumption to make.
For host utilisation, only fruit which produced at least one parasitoid were used in the analysis. To compare means within treatments, Mann-Witney U-tests were applied.
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Figure 4.3: Guava orchard where experiments were run.
Figure 4.4: Fruit mimicking bag in the field with ovipositing parasitoid.
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4.3 Results
4.3.1 Experiment 1: Y-tube olfactometer
Naïve insects showed a significant preference for infested nectarine (χ2 = 4.8,
P < 0.05), as did wasps previously experienced on infested nectarines (χ2 = 10.8, P <
0.05) (Fig. 4.5). Experience on B. tryoni infested nectarines did not significantly increase the preferences for nectarines compared to naïve wasps (χ2 = 0.8, P > 0.05), which may have been due to the already high innate preference for those fruit and the sample size tested. When wasps had prior experience on B. tryoni infested tomatoes, however, they showed a significant preference for infested tomato odours compared to nectarine odours (χ2 = 4.8, P < 0,05), which was significantly different from choices of naïve insects for these odours (χ2 = 9.6, P < 0.005) (Fig. 4.5).
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Figure 4.5: Percentage responses of female Diachasmimorpha kraussii to odours of infested nectarines vs infested tomatoes) under different conditioning treatments (N = 30 per treatment). Significance: * = within treatment differences,χ2 test), │= between treatment differences (P < 0.05, χ2 test).
4.3.2 Experiment 2: Large field cage
Naïve wasps showed an innate preference for nectarine over tomato (t =
6.364, df = 4, P [2-tailed] = 0.003), and this preference remained after experience on nectarine (t = 93.48, df = 4, P [2-tailed] = 0.001). Training on tomato again altered the preference, with significantly more tomato-experienced wasps ovipositing on tomato (t = -3.612, df = 4, P [2-tailed] = 0.023) (Fig. 4.6). Prior experience on either nectarine or tomato appeared to greatly increase the response of wasps to either fruit type compared to the naïve wasps (Fig. 4.6), but because of the experimental design a formal test of this comparison was not made.
98
25 Nectarine Tomato A 20 B
15
10 A A B 5
B Mean (+SE) number of ovipositing wasps ovipositing of number (+SE) Mean
0 Naive Nectarine-exp'ed Tomato-exp'ed
Wasp experience status
Figure 4.6: Mean (+ 1SE) number of Diachasmimorpha kraussii collected ovipositing into nectarine or tomato fruit after three conditioning treatments (naïve, experienced on infested nectarine, or experienced on infested tomato), 60 minutes after release into a 7 x 7 x 5m high outdoor flight cage. Fifty wasps were released in each of three replicates, for each of the three conditioning treatments. Letters surmounted columns denote statistical differences only within a conditioning treatment group, not differences or similarities across groups.
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4.3.3 Experiment 3: Field study
Host location
For naïve wasps, 50% of nectarine bags and 33% of tomato bags were parasitised. These proportions (i.e. 0.5 and 0.33) were then used in the binomial tests to determine if experience of a host increased encounter rate with that host.
For nectarine-experienced wasps, 72% of nectarine bags were visited; this was not significantly different to the proportion of nectarine bags visited by naïve wasps (one-sample binomial test, expected probability of visitation 0.50, P =
0.096).For tomato-experienced wasps, 60% of tomato bags were visited (n = 20); this was significantly greater than the proportion of tomato bags visited by naïve wasps
(one-sample binomial test, expected probability of visitation 0.33, P = 0.012).
Wasps experienced on nectarine visited 52% of tomato bags (n = 19) which was not significantly different to the proportion of tomato bags visited by naïve wasps (one- sample binomial test, expected probability of visitation 0.33, P = 0.061). Wasps experienced on tomato visited 40% of nectarine bags (n = 20) which was also not significantly different to the proportion of nectarine bags visited by naïve wasps
(one-sample binomial test, expected probability of visitation 0.50, P = 0.503) (Fig.
4.7).
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0.8 Naive wasps Wasps expereinced on nectarine Wasps experienced on tomato
** 0.6
0.4 Proportion visitation Proportion 0.2
0.0 n =18 n =18 n =20 n =18 n =19 n =20 Nectarine Tomato Fruit mimic type
Figure 4.7: Proportional visitation of Diachasmimorpha kraussii to two types of fruit mimic, nectarine and tomato, hung in a non-fruiting guava orchard. Wasps were ovipositionally naïve, or had prior experience on nectarine and tomato. Significantly more tomato-experienced wasps visited tomato bags than did naïve wasps, but no other pairwise comparison between naïve and experienced wasps within the same fruit type are significantly different based on a binomial test. The ‘n’ for each column is the number of fruit mimics for that particular exposure type.
Host utilisation
Having found a piece of fruit, there was no significant difference in the mean number of wasps emerging from nectarine or tomato for naïve wasps (Mann-
Whitney U-test, P = 0.328), nectarine-experienced wasps (Mann-Whitney U-test, P =
0.336), or tomato-experienced wasps (Mann-Whitney U-test, P = 0.967). 101
Approximately twice as many parasitoids emerged from nectarines infested by parasitoids trained on nectarine than did parasitoids from nectarines infested by naïve parasitoids (Fig. 4.8). However, as these experiments were run at different times, it cannot be determined if the difference is due to the parasitoid treatments, or due to some other experimental condition (e.g. weather effects), and this comparison was not formally tested.
20
18 Nectarine Tomato 16
14
12
10
8
6
4 Mean (+ 1SE) parasitoids emerging 1SE) parasitoids (+ Mean 2
0 n=9 n=6 n=13 n=9 n=8 n=12 Naive Nectarine-exp'ed Tomato-exp'ed
Wasp experience status
Figure 4.8: Mean (+ 1SE) number of parasitoids emerging from nectarine or tomato fruit mimics oviposited into by Diachasmimorpha kraussii of three different ovipositional experience states: ovipositionally naïve, trained on infested nectarine, or trained on infested tomato. The experiment was done in a non-fruiting guava orchard. The ‘n’ for each column is the number of infested fruit mimics for that particular exposure type (see Figure 4.7 for total n [parasitised + unparasitised] for each treatment).
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4.4 Discussion
Summary
At three different spatial scales - the laboratory, in field cages, and the open field – there were highly consistent results.In Experiments 1 and 2, naïve D. kraussii showed an innate preference for nectarine over tomato, as did wasps which had prior ovipositional experience with nectarine. In Experiments 1 and 3, where the comparison was statistically tested, prior nectarine experience did not significantly increase the nectarine response, but this may well be due to an inadequate power in the tests (i.e. insufficient replication) to detect a learning effect when the innate preference was already strong. However, in contrast, prior experience to the less preferred tomatoes always led subsequently to a significantly increased response to that fruit. In the field, where wasps were not constrained in any way in their foraging range, this led to increased parasitism levels of the learnt fruit. While we did not control for the presence of visual cues (fruits) in the field-cage study, both the olfactometer and field cage study confirm that prior experience was modifying responses to odour cues.
Opiine learning: from lab to field
Parasitoid associative learning is well documented, and it has been shown that wasps can enhance their foraging capacity by learning olfactory, visual and vibrational cues (Froissart et al., 2012; Giunti et al., 2015). Learning within the
Opiinae (Hymenoptera; Braconidae) is less well documented, but is known and supports what we have found here for D. kraussii. The parasitoid Psyttalia concolor
(Szépligeti) has been demonstrated to learn visual cues (Benelli and Canale 2012), host-induced fruit volatile cues (Canale et al., 2014) and general host cues (Canale 103 and Benelli 2012; Giunti et al., 2016b). Similarly Fopius arisanus has been shown to learn host fruit cues associated with the location of fruit fly eggs (Dukas and Duan
2000); while Diachasmimorpha longicaudata (Ashmead) can learn colour cues
(Segura et al., 2007) and olfactory cues (Segura et al., 2016). However, one important difference between our study and other opiine studies, and indeed with most parasitoid learning studies, is that we have shown that host-fruit odour learning influences parasitoid behaviour in nature: all prior opiine studies have been restricted to the laboratory or small field cages.
Due largely to their small size, the bulk of parasitoid knowledge comes from laboratory studies, and although this weakness was identified over a decade ago
(Casas et al., 2004), parasitoid behavioural studies in the field are still sparse (but not absent, for examples see Heimpel and Casas 2008; Randlkofer et al., 2010; Kostenko et al., 2015). The scarcity of supportive field studies means that it should not be automatically assumed that results generated in laboratory are applicable at the field level. However, when Casas et al., (2004) directly tested how the timing of laboratory host-searching behaviours matched field behaviour for the parasitoid
Aphytis melinus DeBach, they found generally strong predictive capacity from laboratory results. Our results show similar consistency in parasitoid learning behaviour, all the way from highly artificial Y-tube arenas to the open field. At least for the opiine braconids, this should give us confidence when making operational biological control decisions based on laboratory results.
Implications for pest management
Giunti et al., (2015), when reviewing how knowledge of parasitoid learning could be used for biological control, raised the idea of using pre-release associative
104 training to ‘prime’ mass-released wasps so they could better target a particular crop or pest. Giunti et al., (2016b) again raised this concept, following on from laboratory experiments where they demonstrated very early adult learning in P. concolor. In our paper we have directly tested and proved the viability of this concept: certainly for the first time in fruit fly parasitoids and, to the best of my knowledge, only for the second time with parasitoids, the first being the work of Hare and colleagues with
Aphytis (Hare 1996; Hare et al., 1997; Hare & Morgan 1997) referred to in the
Introduction. Fruit fly parasitoid mass rearing for inundative biological control release is an area of ongoing work for tephritid researchers (Spinner et al., 2011;
Ovruski and Schliserman 2012; Garcia and Ricalde 2013) and there is intent to explicitly link parasitoid release with sterile fruit fly release (Gurrand Kvedaras
2010; Cancino et al., 2012). As demonstrated here, and elsewhere in the literature
(Messing and Jang 1992; Ero et al., 2011b; Ero et al., 2012; Segura et al., 2012), the opiine parasitoids are known to show innate preference between host fruits. If the targeted crop is one which laboratory research shows to be of low innate preference to the intended biological control agent, then pre-release conditioning may well lead to increased field parasitism rates.
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Chapter 5: General Discussion
5.1 Summary of results
My research objectives were divided to three experimental parts, each taking the form of a separate chapter. These objectives progress from understanding how to culture and rear wild D. kraussii in the laboratory using the host fly B. tryoni, to laboratory experiments exploring the role of different forms of learning in influencing foraging behaviour, and finally to studying how experience might influence parasitoid foraging in the field.
In my first research chapter (Chapter 2), I tested the hypothesis that obtaining high quality cultured insects relies on initial rearing that more closely resembles the natural situation of larvae in fruits. I hypothesised that this would increase parasitism rates and help prevent the characteristic decline of the culture population that frequently happens when wild insects are first cultured in the laboratory. Therefore, I ran an experiment to evaluate a new method for culturing wild D. kraussii in the laboratory. My new design of the culturing bag was compared with a traditional laboratory rearing method of presenting hosts and substrate in a Petri dish (e.g.
Messing and Ramadan, 1999; Ero, 2009), and with fruit fly infested nectarines. The results of this study showed culturing bagsreared significantlymore wasps compared to the Petri-dish method and that larval survival in bags was higher than in real fruit.
Thus, the data clearly show that the bag is a superior method for rearing D.kraussii on artificial media compared to the Petri dish approach. Although the parasitism rate is equally effective in the artificial bag as in the real fruit, greater B. tryoni larval
106 survival in artificial bags was found compared to nectarines. So this revealed that the bags are a superior method for rearing wild wasps in culture, which may flow-on to influence the success of cultured wasps when released into the field.
The experiments in the next chapter were conducted to investigate the influence of associative learning (classical conditioning) and operant learning on the host finding process. Within associative learning I used olfactometer bioassays to ask whether experience influences olfactory preferences for fruit of different quality in female D. kraussii. The results showed naïve females had significant preference for odours of infested fruits over uninfested fruits and strongly preferred host fruit infested with B. tryoni over fruit infested with D. melanogaster (a physiological non- host). Within the group of experienced treatments, (experienced on B. tryoni, experienced on D. melanogaster, experienced on both), D. kraussii significantly preferred B. tryoni infested fruits (a physiologically suitable host) over D. melanogaster infested fruits (a physiological non-host). For between- treatment comparisions, experience on non-host infested fruits did not significantly change preference for odours of host and non-host compared to naïve females.
Diachasmimorpha kraussii has thus shown to be able to demonstrate associative learning.
In the second half of Chapter 3, I continued the learning theme, but focused on operant learning rather than associative learning. I determined that D. kraussii females with previous experience in foraging on fruits that differed in the location of host larvae (they were experimentally located at either the top or base of fruit) influenced subsequent alighting, on-fruit searching time, and probing behaviour of female wasps. Naïve and experienced parasitoid females upon alighting showed
107 significant preference for the top of the fruit compared to the base fruit. However, wasps trained with larvae located at the base of the fruit subsequently showed significantly different preferences compared to naïve controls. Even in the absence of host larvae, they showed a shift in their behavioural searching to display different responses in alighting, probing and search time with a primary orientation to the base of the fruit. This reflects learning modifying a sequence of behaviours, consistent with the definitions of operant learning.
Building on the earlier works in the Chapters 2 and 3, in the last research chapter (Chapter 4), I evaluated the role of associative learning in host location at three spatial scales: very fine scale in the laboratory, large scale in field cages, and then unrestrained in the open field. Y-tube choice tests showed that D. kraussii has an innate preference for infested nectarines over infested tomatoes, and prior experience of nectarines did not significantly increase the preference for nectarines.
However, experience of tomatoes significantly increased the subsequent preference for tomato odours compared to naïve insects, demonstrating that D. kraussii can learn and modify its response to plant odours associated with host larvae. In the field cage experiments, these results were confirmed showing that D. kraussii has an innate preference for nectarines over tomatoes, but that prior experience with tomato significantly increased the wasp’s ability to locate tomatoes. Prior experience with the preferred nectarines did not significantly increase the response to that host fruit.
In the orchard experiment the same results were again found, but the design of this experiment showed that the learnt response was linked to host odor, as the visual cues were the same. The field experiment is important as: i) learning research in parasitoids rarely extends to field testing and so offers greater confidence when discussing the ecological/evolutionary benefits of parasitoid learning; and ii) it offers 108 insights into how parasitoid learning theory may be directly relevant to improve pest management.
5.2 Implications of thesis results for wasp host location
Doutt (1959) classically described four steps within the parasitoid host location process: i) host habitat location; ii) host location on an infested host plant; iii) host acceptance after finding a host; and iv) host suitability for parasitisation. A fifth step was added by Vinson and Iwantsch (1980) which is host regulation. Steps
(i) and (ii) can be considered as pre-alighting processes and steps (iii) and (iv) as post-alighting. Olfactory cues are critical environmental signals that can be used by parasitoids at multiple stages in the host location process (Lewis et al., 1976, Hassell and Southwood 1978, De Moraes et al., 1998, Benelli et al., 2013a).
Odours from host habitats are considered a reliable source in the first step of wasps’ orientation from a distance (Vinson 1976). Herbivore-induced plant volatiles
(HIPVs) play an important role in helping parasitoids locate their hosts (Turlings et al., 1991, Wajnberg and Colazza 2013). Despite the known value of volatile cues, within complex environments there may be high variability in both the quality and quantity of the odour cues available to a searching parasitoid (Ero et al., 2011a, Hare
2011, de Rijk et al., 2016). In such complex environments, learning may play a critical role in parasitoid orientation and decision making for successful host location
(Papaj and Vet 1990, Turlings et al., 1993, Wackers and Lewis 1994, Vet et al.,
1995, Geervliet et al., 1998, Meiners et al., 2003, Dukas 2013, Luo et al., 2013,
Giunti et al., 2015).
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In my Y-tube study D. kraussii females would orientate to fruit odours without the presence of their hosts, but the response increased for infested fruit with host larvae. The response to cues from uninfested fruits is at odds with findings of
Ero and colleagues (Ero et al., 2011b; Ero & Clarke 2012) who reported no orientation by D. kraussii to uninfested fruit. The difference may lay in the experimental method. Both of the Ero studies were done in environments where air- flow and any subsequent odour movement was not constrained; one study was done in screen-walled laboratory cages, the other in a field cage. In contrast my Y-tube study directed a flow of clean air, or host-odour air, directly over the wasp. This situation resulted in wasp orientation to host fruit odours, although significantly less so than if fruit damage cues were present. Other opiines have been reported as responding to non-damaged fruit (Cheng et al., 1992; Vet and Dicke 1992; Eitman et al., 2003; Benelli et al., 2013a), but again always as a lesser response to damage cues. A low level response to undamaged fruit will clearly be adaptive in an environment where host larvae are rare, as there remains a cue to the local resource where larvae at most likely to be encountered (i.e. the fruit).
Although D. kraussii in my experiments responded to two different sources of plant volatiles (i.e. nectarines, F. Rosaceae, and tomatoes, F. Solanaceae), the data showed that the females could discriminate between them in both laboratory and field experiments, with an inherent preference for nectarine. Differential response to different fruit odours has been well documented among species of the Opiinae
(Carrasco et al., 2005, Rohrig et al., 2008, Benelli et al., 2013b, Dias et al., 2014).
There are two possible evolutionary drivers why wasps should differentiate between fruit hosts. Physiologically, developing within the same species of fruit fly larvae in different fruit has fitness consequences for parasitoids. Ero et al., (2011a) 110 demonstrated that D. kraussii breeding in the same fruit fly species, but in different fruit types, varied significantly in development time and off-spring size. Thus some fruit types are better indirect hosts for the wasps, most likely mediated by how good the host fruit is for fly larval development (Ero et al., 2011b). Alternatively, or additionally, an innate preference for a particular fruit may represent how frequently that fruit is available in the field, i.e. a fruit which is very common may be highly preferred by the wasp as a reliable environmental cue. Diachasmimorpha kraussii has been shown to preferentially use native fruit fly host plants over exotic hosts (Ero et al., 2011b), a pattern seen in other opiine fruit fly systems (Aguiar-Menezes and
Menezes 1997, López et al., 1999, Ovruski et al., 2004, Dutra et al., 2013). This may represent indirect evidence that the wasps preferentially orientate to hosts which, over evolutionary time, have proven to be reliable environmental signals of the presence of host maggots. Obviously this explanation cannot be applied to the nectarine/tomato situation for D. kraussii, as both are evolutionary novel to the wasp, but should be considered for other fully endemic parasitoid-fly-fruit systems.
The results of my Y-tube study indicate that D. kraussii utilises fruit-wound odours associated with host B. tryoni larvae, but does not respond to fruit-wound odours generated by fruit infestation with non-host Drosophila maggots. Chemical cues derived directly from larvae can include larval faeces and larval volatile compounds (Stuhl et al., 2011, Segura et al., 2012) and are signals which can presumably increase host recognition at pre-alighting host location phase. The high attraction of D. kraussii to fruit infested by B. tryoni larvae, but with a response not statistically different to uninfested fruit when the fruit is infested with Drosophila, strongly infers that the positive odour cues being used by the wasp derive, either directly or indirectly, from the Bactrocera maggot, rather from a generic fruit wound 111 response. This finding adds to, but additionally complicates, what we know about D. kraussii host location. When Ero and Clarke (2012) physically moved B. tryoni larvae into host plants that are not normally hosts of the fly (e.g. zucchini) the wasp did not orientate to those plants, despite the presence of the suitable larvae. The wasp did, however, keep responding to the preferred host plant (tomato) even though physiologically unsuitable larvae (B. cacuminata) had been placed in it. These experiments, when considered along with mine, suggest that host location by the wasp relies on multiple cues (for example host larvae + host fruit), which may modify how the wasp responds. My thesis focused almost exclusively on odour cues, and often in the highly artificial arena of a Y-tube olfactometer, and so should be recognised as just one part of a larger story, not a comprehensive answer on their own.
5.3 Adaptive significance of parasitoid learning behaviour in complex and variable environments
The presence of fruit fly larvae within host fruits leads to the production of chemical (olfactory and gustatory), visual, and mechanical (e.g. vibrational) cues for the host-seeking parasitoid. Where these cues are predictable, the sensory system of the foraging wasp would be expected to be adapted to recognising and responding to them, and distinguishing them from other similar and yet uninformative cues within their environment. In this thesis, I have focussed on olfactory cues, demonstrating innate attraction in D. kraussii towards infested compared to uninfested fruits, and furthermore a preference for host (B. tryoni) infested compared to non-host
(Drosophila melanogaster) infested fruits. I have discussed in the relevant chapters
112 how microbial volatiles and larval odours might form the basis of these innate olfactory cues.
However, B. tryoni is a highly polyphagous fruit fly, and thus newly emerged adult D. kraussii wasps are faced with the task of searching for host larvae in a fruiting environment that can be highly variable, both temporally and geographically.
In addition to this, D. kraussii is itself polyphagous on many fruit fly species, further increasing the potential range of host fruits (Geervliet et al., 1998). In these unpredictable environments, learning has adaptive value over innate responses in enabling the parasitoid to modify its behaviour depending on where it has most frequently found larvae on previous encounters (Papaj and Prokopy 1989,
Cunningham & West, 2008, Quicke 2014), and thus focus on cues that are relevant to its local environment. Stephens (1993), demonstrated theoretically how learning was most advantageous in environments that were “unpredictably predictable”, in that they were unpredictable between insect generations (i.e. mothers and offspring are often faced with different environments) and predictable within environments (an insect finds “rewards” in similar places within its lifetime). Under these conditions, insects cannot easily track their hosts through innate responses alone, and predictability within generations allows for learning to occur in the insect CNS, and be advantageous. The fruiting environment fits well with this, as fruits are often available geographically and seasonally in large patches (either as orchards, or clumps of trees, or simply as many fruits on a single tree). An important finding in my thesis was demonstrating that wasps learn only when experienced on host larvae
(as opposed to non-host larvae). Given the wide range of non-host insects that might attack any given fruit, and the unpredictability of these non-hosts amongst fruit species, learning would be of adaptive value in enabling the foraging wasp to fine- 113 tune its olfactory responses to identify volatiles or blends that relate specifically to its host in any given environment.
Other learning traits may also come into play. During oviposition, learning may help wasps avoid superparasitism, as has been shown in the Opiinae wasp F. arisanus (Wang and Messing 2008). Although this study focussed only on olfactory cues, learning would be expected to have evolved for all sensory cues involved in host location, depending on these same factors of environmental predictability and variability of host-specific cues. Visual cues are undoubtedly important, and have been shownto interact synergistically with olfactory and physical cues in the selection of host microhabitats (Benelli and Canale 2012).
My thesis demonstrated post-alighting learning effects in D. kraussii which form, to the best of my knowledge, the first demonstration of this behaviour in any parasitoid. Wasps preferentially foraged on sections of the fruit (top versus base) where they have previously found host larvae. In nature, different fruit species could differ considerably in the location of fruit fly larvae, as a result of within-fruit variation in substrate suitability for larvae (e.g. due to progressive ripening), or parts of the fruit that are more suitable for fruit fly oviposition (e.g. softer). Learning would enable wasps to find larvae more quickly, where larval position is fruit- specific. Innate preferences for foraging at the top of the fruit, as shown in my experiments, have most likely evolved as this is where larvae are most frequently encountered.
Despite a wealth of laboratory studies on parasitoid learning, learning behaviour is rarely studied in the field, which begs the question as to whether learning might in part be an artefact of the laboratory environment, where foraging is
114 limited (e.g. to a glass tube, or cage), odour cues are simple (odour A versus odour
B), and wasps are confined without the complex olfactory and chemosensory backdrop provided in nature. The field study in my thesis shed new light on the strength of learning in shaping wasps host selection behaviour in the nature.
Parasitism rates were lower in the field experiments, as might be expected when insects are free to leave the experimental arena, but my results indicate that, particularly for low ranking host fruits (in this case tomato), positive oviposition experience in early adult life on such hosts can increase foraging preference within an orchard environment. If a low ranking host fruit was the only fruit available in the local environment, and thus subjected to high levels of B. tryoni infestation, learning could enable the foraging wasp to modify its innate behaviour to increase its responsiveness to this fruit.
5.4 Application to inundative biocontrol
Inundatively released parasitoids have been successfully used as biocontrol agents in area-wide management (AWM) (Wong et al., 1991, Purcell 1998, Montoya et al., 2000, Spinner et al., 2011), a modern pest management approach for fruit fly suppression (Gurr and Kvedaras 2010). The aim of AWM, as applied to the control of fruit fly pests, is the suppression of pest populations over an entire production area through the use of multiple control tools including the sterile insect technique (SIT)
(Ant et al., 2012, Pereira et al., 2013), bait sprays and parasitoids (Jessup et al.,
2007). The use of SIT incorporated with inundative parasitoid release has been recommended by entomologists for conservation biocontrol within AWM (Purcell
1998, Jang et al., 2008). Using inundative parasitoid releases jointly with SIT can
115 increase the efficacy of SIT compared to using SIT alone (Gurr and Kvedaras 2010,
Thorpe et al., 2016).
The first effective implementation of AWM in Australia against fruit flies was in the Central Burnett district of Queensland against the endemic Qfly. Even though mass release of parasitoids was not used in this programe, the presence of the native parasitoid D. kraussii and the exotic parasitoid Fopius arisanus in the same area contributed to control with parasitism levels of 7.4% of infested fruit samples
(Lloyd et al., 2010). If this percentage parasitism could be increased through the augmentative release of parasitoids, then it is considered that it would significantly enhance the effectiveness of AWM (Spinner et al., 2011; Zamek et al., 2012).
The goal of augmentative application is to rear and mass release large number of parasitoids to enhance natural enemy induced mortality in the target area (Gurr et al., 2012). Complications which may affect the successful application of inundative release including the production costs of mass rearing parasitoids, and the wasps’ inherent biological attributed which may be negatively affected during mass culturing and release. One study, now over 20 years old, costed the production of one million wasps at around US$2000 (Harris and Bautista 1994); this figure will have only increased since, making parasitoid production an expensive exercise.
In my study I found my novel culturing bag is a superior method for rearing wild wasps in culture. For example, I estimate to produce one million parasitoids would require 1500-2000 bags, which at an estimated direct cost of approximately
$700 (my calculations) is significantly lower than other methods. An additional advantage of the hanging bags is that it is easy to supply target odours to them, which
116 can then be provided to the parasitoids for training on a particular crop in order to improve wasp abundance when released into target orchards.
Based on my investigations D. kraussii would be a very useful parasitoid for mass release because of its ease of culturing and capacity to be trained on particular crops. Other scientists have argued that Diachasmimorpha spp are preferable for mass rearing and release over Fopius spp due to decreased rearing costs and biological robustness to laboratory rearing and release (Rousse et al., 2005). A capacity to learn, making the wasp biologically ‘flexible’ in the field is also deemed as positive attribute in parasitoids being considered for biological control (Giunti et al., 2015).
HIPVs are used in AWM programmes to monitor pest populations (Wajnberg and Colazza 2013), and could also be used in orchards to increase the efficacy of parasitoids against fruit fly pests. Within Australian vineyards, several hymenopteran parasitoids of thrips species responded to HIPV blends of methyl salicylate, methyl jasmonate, benzaldehyde and (Z)-3-hexenyl acetate and parasitoid abundance was significantly increased on traps near treated plants (Simpson et al.,
2011a). If we identified the HIPVs to which D. kraussii responds, then similar field application may be possible to enhance fruit fly control by increasing parasitoid abundance within orchards.
Importantly, and as dealt with in Chapter 4, the results of this thesis also provide evidence that wasps reared for inundative release may, through pre-release training, be ‘modified’ so that they are more likely to stay within a target crop. This concept has been theoretically postulated previously (Giunti et al., 2015), but not tested. My work suggests that for crops towards which the wasp already shows a
117 strong, innate preference then such training is unlikely to be justified. But for poorly preferred crops (tomato in my study, or pears in Ero et al., 2011b) where the wasp may rapidly move away from the target area, then pre-release conditioning may greatly enhance their efficacy as biological control agents.
5.5 Further research
Host versus non-host discrimination
Ero et al (2012) reported that no significant differences were found in D. kraussii oviposition preference between four sympatric fruit fly species:B. tryoni, B. jarvisi, B. cacuminata and B. cucumis. However, D. kraussii offspring were only able to successfully develop in B. tryoni and B. jarvisi, and were completely unsuccessful in developing within the tephritids B. cacuminata and B. cucumis due to wasp egg encapsulation. In my study I made a comparison of host usage and learning using the physiologically suitable host B. tryoni and the non-host Drosophila melanogaster. That D. kraussii failed to recognise Drosophila larvae may be because
Drosophila is systematically far removed from Bactrocera, or because the physiology of Bactrocera and Drosophila is very different. Further investigations are needed on D. kraussii behavioural acceptance of target and non-target hostsand the mechanisms of host locations and usage. However, such work should investigate host discrimination between host and non-host Bactrocera species, as this would be more ecologically and behavioural informative.
Odor research
The chemical ecology is the goal to investigate chemically mediated interactions between organisms and their environment. More investigations on the
118 interactions between Opiinae wasps and host plant volatiles, including volatile chemical analysis, identification and functional roles, in the field are needed.This includes what the actual HIPV volatiles are to which the wasps orientate. In Y-tube studies, my study has shown that D. kraussii can utilise plant odours for orientation even in the absence of its hosts (contrary to earlier work which showed no orientation to uninfested fruit). However, as there are a wide range of volatiles in the environment, parasitoids could utilise many different olfactory signals to orientate towards preferred plants, which may or may not be targets from a biological control perspective. Identification of HIPVs, and their potential utilisation in the field through the deployment of artificial odour sources, is thus an important area of further research in order to increase the abundance and parasitism levels in the field.
Physiological state
The majority of studies about parasitoid behavioural ecology have been conducted under laboratory conditions, commonly using naïve wasps and hosts of a given quality and condition. However, in the field the physiological states of both parasitoids and their hosts will vary greatly between individuals and populations.
How such variation modifies host searching and parasitism is largely unexplored, although my learning chapters show that even this one variable (i.e. naïve versus experienced) can greatly modify behaviour. Further targeted testing of wasps and hosts of varying physiological state should be undertaken, particularly including greatly enlarged field testing of experienced versus naïve wasps within an inundative biological control scenario.
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Supplementary Material: Abstract of presentation read at a scientific conference 51Australian Entomological Society Conference, 27 – 30 September 2015, Cairns, Australia
Oral Presentation: Role of olfactory cues in host finding in the parasitoid Diachasmimorpha kraussii Muhmed, A.M.A. (1) and Cunningham, J.P. (1, 2) (1) Earth, Environmental and Biological Sciences School, Queensland University of Technology (QUT), Brisbane, QLD 4001 (2) Institute for Future Environments, Queensland University of Technology (QUT), Brisbane, QLD 4001
Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) is a polyphagous, koinobiont endoparasitoid of Bactrocera fruit flies and it has been used as a classical biological control agent against these flies. Previous research has demonstrated that the fly uses a range of environmental stimuli in their host-location foraging behaviour. The aim of the current study is to improve our understanding of how D. kraussii finds its hosts and more explicitly how olfactory cues may reinforce successful host finding. Y-tube olfactometer studies were used as a behavioural assay to investigate the influence of the female wasp‘s innate responses to fruit odours (nectarines) with larval host (Bactrocera tryoni) and non-host (Drosophila melanogaster) in female parasitoids. Naïve wasps showed significant positive responses to both un-infested and B. tryoni infested nectarines, and had a clear preference toward fruit infested with B. tryoni versus fruit infested with D. melanogaster. There were no significant difference in the preference to uninfested fruits and nectarines infested by Drosophila. Previously published studies have shown that D. krausii did not orientate differently to hosts infested with physiologically suitable or non-suitable Bactrocera species, but the poor wasp response to D. melanogaster infested fruit in this study does demonstrate that the wasp can recognises ‘Bactrocera’ derived odours over those of other ‘non- Bactrocera’ frugivorous larvae.
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