Apple Cultivar Preference in the European Apple Sawfly, Hoplocampa Testudinea and the Apple Maggot Fly, Rhagoletis Pomonella

Apple Cultivar Preference in the European Apple Sawfly, Hoplocampa testudinea and the Apple Maggot Fly, Rhagoletis pomonella

Christopher Sean Francis Burgart

Thesis submitted in partial fulfillment of the requirements for the Degree of Master of Science (Biology)

Acadia University Spring Convocation 2015

This thesis by Christopher Sean Francis Burgart was defended successfully in an oral examination on 9th, December 2014.

The examining committee for the thesis was:

______Prof. E. Jackson, Chair

______Dr. D. Strongman, External Reader

______Dr. A Walker, Internal Reader

______Dr. N. K. Hillier, Supervisor

______Dr. S. Blatt, Supervisor

______Dr. D. Shutler, Acting Department Head

This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree Master of Science (Biology).

………………………………………….

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I, Christopher Burgart, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis.

______Author

______Supervisor

______Date

Table of Contents: List of Tables ...... v List of Figures ...... vii Abstract ...... xii List of Abbreviations ...... xiii Acknowledgments...... xiv 1. Introduction ...... 1 1.1 Background...... 1 1.2 European Apple Sawfly ...... 1 1.3 Apple Maggot Fly...... 5 1.4 Management and Control ...... 12 1.5 Cultivar Preferences ...... 15 1.6 Hypothesis and Objectives ...... 18 1.6.1 Significance of research ...... 20 2. Overview ...... 21 2.1 Site of field study ...... 21 2.2 Volatile collection and analysis ...... 21 2.3 Gas chromatography and mass spectrometry ...... 26 3. European Apple Sawfly ...... 27 3.1 Materials and Methods ...... 27 3.1.1 Field Observations ...... 27 3.1.2 Y-Tube Bioassays ...... 30 3.1.3 Electroantennography ...... 35 3.1.4 Data analysis ...... 38 3.2 Results ...... 39 3.21 Adult visitation ...... 39 3.2.2 Pre-harvest larval infestation ...... 48 3.2.3 Post-harvest larval infestation ...... 52 3.2.4 Y-Tube Bioassays ...... 55 3.2.5 Electroantennography ...... 58 4. Apple Maggot Fly ...... 66 4.1 Materials and Methods ...... 66 4.1.1 Field Observations ...... 66 4.1.2 Y-Tube Bioassays ...... 69 4.1.3 Calcium Imaging ...... 71 4.1.4 Data analysis ...... 76 4.2 Results ...... 76 4.2.1 Adult visitations ...... 76 4.2.2 Infestation...... 83 4.2.3 Y-Tube Bioassays ...... 86 4.2.4 Calcium Imaging ...... 86

5. Discussion ...... 97 5.1 European Apple Sawfly ...... 97 5.2 Apple Maggot Fly...... 112 5.3 Future Directions ...... 124 5.3.1 European Apple Sawfly ...... 124 5.3.2 Apple Maggot Fly ...... 127 6. Conclusion ...... 129 6.1 European Apple Sawfly ...... 129 6.2 Apple Maggot Fly...... 131 7. References ...... 133 8. Appendix A: European Apple Sawfly ...... 141 8.1 Y-tube Bioassays (2012) ...... 141 8.1.1 Results ...... 141 8.2 Colony development ...... 145 8.2.1 Materials and methods ...... 145 8.2.2 Results ...... 151 8.2.3 Discussion ...... 152 8.3 Enhanced sleeve cage infestation survey ...... 157 8.3.1 Materials and methods ...... 157 8.3.2 Results ...... 159 8.3.3 Discussion ...... 162 9. Appendix B: Apple Maggot Fly ...... 164 9.1 Colony development ...... 164 9.1.1. Materials and methods ...... 164 9.1.2. Results ...... 165 9.1.3 Discussion ...... 166 9.2 Wind tunnel bioassay ...... 166 9.2.1 Materials and methods ...... 166 9.2.2 Results ...... 171 9.2.3 Discussion ...... 171

List of Tables:

Table 2.1 Timing of bloom of apple cultivars in this study. Bloom time is based on comparison to McIntosh varieties ...... 24

Table 2.2 Characteristics of mature fruit from cultivars studied. Maturation times are based on comparison to McIntosh varieties...... 25

Table 3.1 Number of comparisons performed using cut apple blossoms...... 33

Table 3.2 Summary of Kruskal-Wallis analyses for 2012 and 2013 apple sawfly visitation. α = 0.05, df = 10...... 44

Table 3.3 Pairwise comparison of 2012 sawfly visitations using results of z-test from general linear model and level of significance ...... 46

Table 3.4 Pairwise comparison of 2013 sawfly visitations using results of z-test from general linear model and level of significance ...... 47

Table 3.5 Summary of Kruskal-Wallis analyses using 2012 and 2013 apple sawfly pre-harvest infestation...... 51

Table 3.6 Summary of Kruskal-Wallis analyses of 2012 and 2013 apple sawfly post-harvest infestations...... 54

Table 3.7 Results of Chi-square analysis of first apple sawfly responses to volatile blends ...... 56

Table 3.8 Summary of two-way ANOVA comparing apple sawfly response (mV) to odour and sex ...... 59

Table 3.9 Statistical significance of all sawfly responses to volatiles in electroantennograms using generalized linear model. Responses are based against pentane control ...... 64

Table 3.10 Summary of apple cultivars preferred and least-preferred by Hoplocampa testudinea based on field surveys, behavioural bioassays and electrophysiological tests. Preferred cultivars are ranked most preferred to least preferred, and non-preferred cultivars are ranked least preferred to more preferred ...... 65

Table 4.1 Summary of Kruskal-Wallis analyses of 2012 and 2013 apple maggot visitation data...... 78

Table 4.2 Pairwise comparison of statistical significance of 2012 apple maggot visitation between cultivars using results of z-test from general linear model and level of significance...... 81

Table 4.3 Pairwise comparison of statistical significance of 2013 apple maggot visitation between cultivars using results of z-test from general linear model and level of significance...... 82

Table 4.4 Summary of Kruskal-Wallis analyses using 2012 and 2013 apple maggot infestation data ...... 85

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Table 4.5 Results of one-way Chi-squared analysis of 2013 bioassays using first responses of apple maggot flies ...... 88

Table 4.6 Summary of two-way analysis of variance evaluating male maggot fly responses to odour blends across mapped regions of interest. Region of interest 6 excluded. Region of interest 6 excluded as all results were standardized against its responses ...... 92

Table 4.7 Summary of two–way analysis of variances evaluating female maggot fly responses to odour blends across mapped regions of interest. Region of interest 6 excluded ...... 93

Table 4.8 Summary of cultivar preferences (or sensitivity) in Rhagoletis pomonella across all studies. Preferred cultivars are ranked most preferred to least preferred, and non-preferred cultivars are ranked least preferred to more preferred ...... 96

Table 8.1 Chi-square analysis of first-choice apple sawfly responses to apple blossoms ...... 143

Table 8.2 Summary of 2012 development of Hoplocampa testudinea colonies by substrate. 100% mortality was observed in all trials ...... 155

Table 8.3 Summary of Kruskal-Wallis analyses of rates of infestation among sleeve cages ...... 160

List of Figures: Figure 1.1 Adult Hoplocampa testudinea. Adults shown are approximately 1 cm long. (a) posterior view of male apple sawfly on an apple blossom (b) female apple sawfly. Note retracted ovipositor on posterior. (c) dorsal view of male apple sawfly showing colour markings on head ...... 2

Figure 1.2 ‘C-scarring’ on apple fruitlets. (a) & (b) fruitlets that survived infestation by H. testudinea larvae. (c) C-scarring on fruitlet currently infested by H. testudinea larvae (note presence of frass, liquid droplet in lower right corner)...... 4

Figure 1.3 Damage caused by Hoplocampa testudinea infestation. (a) external damage on vacated fruitlet (b) external damage on currently-inhabited fruitlet; frass is moist and fresh (c) internal damage (longitudinal view of fruitlet). (d) internal damage, lateral view of fruitlet ...... 6

Figure 1.4 Life cycle of Hoplocampa testudinea ...... 7

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Figure 1.5 Rhagoletis pomonella (a) adult apple maggot fly on a heavily infested Silken apple.(b) Ovipositing apple maggot female (c) male apple maggot fly searching for mate or food source on apple damaged by H. testudinea larvae ...... 8

Figure 1.6 Damage to apples caused by a Rhagoletis pomonella larva. (a) trails gnawed through apple flesh caused by larvae moving through fruit (b) extensive damage to apple caused by multiple R. pomonella feeding. (c) external damage; sting marks highlighted. (d) apple maggot removed from apple ...... 10

Figure 1.7 Life cycle of Rhagoletis pomonella ...... 11

Figure 2.1 Satellite imagery of Agri-Food and Horticultural Research Centre ...... 22

Figure 2.2 Blossoming test cultivars, May 2013. (a) Close up of Pinova apple blossoms (b) s23-06-153 tree in full bloom ...... 23

Figure 3.1 Collection of insect specimens using an aspirator ...... 29

Figure 3.2 Set up of Y-tube prior to bioassay ...... 32

Figure 3.3 Placement of apple sawfly head with electrodes attached. Sawfly is secured in pipette tip with dental wax and cotton. Ground electrode (left) is inserted into female sawfly’s eye. Recording electrode (right) is connected to distal end of severed antenna ...... 37

Figure 3.4 Distribution of apple sawfly visitations per cultivar across 2012 flight period ...... 40

Figure 3.5 Distribution of apple sawfly visitations per cultivar across 2013 flight period ...... 41

Figure 3.6 Mean sawfly visitations by cultivar in 2012 (p < 0.0001) and 2013 (p < 0.0001) for mean sawfly visitations throughout field survey. Cultivars sharing the same letter were not significantly different in apple sawfly visitations...... 42

Figure 3.7 Comparison of levels of pre-harvest apple sawfly infestation between 2012 (p < 0.0001) and 2013 (p < 0.0001) field surveys. Cultivars sharing the same letter were not significantly different for apple sawfly infestation...... 49

Figure 3.8 Comparison of apple sawfly infestation levels in 2012 (p > 0.05) and 2013 (p > 0.05) post-harvest larval surveys...... 53

Figure 3.9 Proportion of Hoplocampa testudinea responding to volatile blends based upon first responses...... 57

Figure 3.10 Hoplocampa testudinea responses to EAG (N=22 individuals of both sexes). P-value of all responses: 0.24. P-value of amplitude of female responses: 0.2. P-value of male responses: 0.43. Cultivars sharing the same letter are not significantly different...... 60

Figure 3.11 Female sawfly responses to electroantennography (N=17) at α = 0.1. Cultivars sharing the same letter are not significantly different...... 61

Figure 3.12 Male sawfly responses to electroantennography. (N = 5) at α = 0.1. Cultivars sharing the same letter are not significantly different...... 62

Figure 4.1 Damage caused to apples by apple maggot fly oviposition and infestation. (a) Mature apple showing multiple sting marks. (b) Mature apple cut into quarters along X and Y axes. Presence of apple maggots is confirmed via trails burrowed through flesh of apples (brown track marks)...... 68

Figure 4.2 A Rhagoletis pomonella female explores a junction tube during a Y- tube bioassay. (a) central tube, b) lower junction arm with maggot fly, c) upper junction arm with separating screen and filter paper ...... 70

Figure 4.3 Female apple maggot fly secured in guillotine block with dental wax...... 72

Figure 4.4 Set-up of calcium imaging apparatus at Chemical Analysis and Bio- imaging Laboratory ...... 74

Figure 4.5 Cultivar comparison between 2012 (p <0.001) and 2013 (p < 0.0001) for mean apple maggot fly visitations Cultivars sharing the same letter are not significantly different in apple maggot fly visitations at α = 0.05 ...... 79

Figure 4.6 Comparison of rates of apple maggot infestation between cultivars in 2012 (p > 0.05) and 2013 (p < 0.001) years. Cultivars sharing the same letter are not significantly different in apple maggot infestations at α = 0.05...... 84

Figure 4.7 First choice responses of apple maggots to Y-tube behavioural bioassay. Letters on comparisons indicate significance of result of Chi-square analyses...... 87

Figure 4.8 Apple maggot fly brain, proximal anterior view. All surrounding tissues have been removed. Regions containing antennal lobes have been highlighted. Numbered areas indicate regions surveyed by calcium imaging 1) dorsal area, 2) ventral, 3) medial and 4) lateral. Magnification is 100x ...... 89

Figure 4.9 Pattern of region of interest placement on ventral sections of antennal lobes. Numbers correspond to ROI. (a) Dorsal placement, left lobe (b) Ventral placement, left lobe (c) Medial placement, right lobe (d) Lateral placement, right lobe...... 91

Figure 4.10 Figure 4.10: μ responses in ∆ intensity (nm) of female apple maggot flies by region of interest against volatile blend. (a) μ responses in ∆ intensity (nm) at lower left antennal lobe (P = 1.43, N=4). (b): μ responses in ∆ intensity (nm) at upper right antennal lobe (P = 1.511, N=5). (c): μ responses in ∆ intensity (nm) at upper left antennal lobe (P = 1.45, N=9)...... 95

Figure 8.1 Percentage of all preferential apple sawfly responses to apple blossom in 2012 Y-tube bioassay. Refer to table 3.1 for full listing of comparisons performed per cultivar ...... 142

Figure 8.2 Strong preferences of Hoplocampa testudina to cut apple blossoms in 2012 Y-tube bioassay ...... 144

Figure 8.3 Set up of apple sawfly rearing containers. 100% sand substrate pictured. (a) depth of substrate relative to container indicated (b) mesh covering separating fruitlets from substrate ...... 147

Figure 8.4 Apple sawfly rearing pot (50%/50% soil/peat moss mixture)...... 149

Figure 8.5 Apple sawfly rearing containers in growth chamber. Elastic banding securing the upper mesh broke and was replaced repeatedly during incubation ...... 150

Figure 8.6 Hoplocampa testudinea pupae and larvae collected from 50%/50% soil/sand mixture. Adult codling moth in upper right corner ...... 153

Figure 8.7 Desiccated Hoplocampa testudinea larvae taken from a 100% sand substrate container ...... 154

Figure 8.8 Apple tree with sleeve cage over limb ...... 158

Figure 8.9 Infestation levels of Hoplocampa testudinea in enhanced sleeve cage study using data from all sleeve cages (P = 0.009), sleeve cages with adult apple sawflies (P= 0.088) and sleeve cages without adult apple sawflies (P= 0.002). Cultivars are ranked by infestation across all sleeve cages...... 161

Figure 9.1 Flight chamber at the Agri-Food and Horticultural Research Centre ...... 167

Figure 9.2 Set up of red bait spheres with odour sources on top of stands ...... 169

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Abstract:

European apple sawfly Hoplocampa testudinea (Klug) and apple maggot fly

Rhagoletis pomonella (Walsh) are economically important insect pests. Losses to apple crops caused by these insects’ damage can negatively affect growers and sustainability of crops. Eleven commercial and experimental apple cultivars were studied to examine preferences that apple sawfly and apple maggot fly exhibited among host plants.

Preference among host plant cultivars and the mechanisms informing these preferences were determined via field surveys of adult visitation and larval infestation of apples, behavioural bioassays, and electrophysiological tests. Significant differences in visitation and infestation of hosts were observed by apple sawfly and apple maggot fly in each year of study. Apple sawfly preferred Zestar!, s23-06-153 and Pinova for visitation and oviposition, whereas apple maggot fly preferred Silken, Pinova and Zestar! for adult visitation and infestation. Observed cultivar preferences in each insect were supported via the results of Y-tube bioassays and through observations of olfactory insect responses via electroantennography and calcium imaging.

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List of Abbreviations:

AFHRC: Agri-Food and Horticultural Research Centre ANOVA: Analysis of variance BCMA: British Columbia Ministry of Agriculture CABL: Chemical Analysis and Bio-imaging Laboratory EAG: Electroantennography OMAFRA: Ontario Ministry of Agriculture, Farms and Rural Affairs ON: Ontario OR: olfactory receptor ORN: olfactory receptor neuron ROI: Region of interest UM: University of Minnesota

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Acknowledgements:

The author would like to thank the following people: Dr. Elaine Baltzer, Dr.

Suzanne Blatt, Emily Cruickshank, Dr. Neil Kirk Hillier, Cate Little, Rebecca Rizzato and Dr. Brian Wilson for all their time, assistance and guidance in researching and writing this thesis. The author would also like to thank Julie Alcorn, Denise Anderson and Kristy Thornton Filion for their support while I was working and writing.

I would never have been able to do this would the help and effort of everyone here.

1. Introduction

1.1 Background

In North America, economic damage to apple crops caused by invasive insect pests can reach billions of dollars per year. Apple maggot Rhagoletis pomonella (Walsh) alone has the potential to cause upwards of 4 billion dollars/year damage to apple crops if populations are not controlled (Zhao et al., 2007). In Canada, the European apple sawfly,

Hoplocampa testudinea (Klug), is highly destructive (Graf et al., 2001) and can cause losses of up to 14% of potential yield in apple crops (Vincent et al., 2002). In the sawfly’s original home range in Europe, damages caused by H. testudinea to apple crops can be in excess of several hundred million dollars per country per year (Neupane, 2012).

Research into improved methods of trapping, monitoring, and controlling these insect pests can potentially save growers billions of dollars in lost produce (Vincent et al.,

2002; Zhao et al., 2007). Increased public interest in environmentally sustainable food production will shape the face of agricultural practices for the foreseeable future. This thesis examine the preferences these insects exhibit in choice of different cultivars and the host plant cues that inform this preference.

1.2 European Apple Sawfly

The European apple sawfly Hoplocampa testudinea (Fig. 1.1) is an established invasive species in Canada and the United States. Native to Western Europe, it was inadvertently brought to North America in the 1930s, and by the 1980s had spread throughout Western Canada, from British Columbia to Ontario and Quebec (Vincent et al., 2002; Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), 2011).

Figure 1.1: Adult Hoplocampa testudinea. Adults shown are approximately 1 cm long. (a) posterior view of male apple sawfly on an apple blossom (b) female apple sawfly. Note retracted ovipositor on posterior. (c) dorsal view of male apple sawfly showing colour markings on head.

Hoplocampa testudinea’s range has expanded east since its arrival in Canada (Weires,

1991) and it has invaded the Maritime provinces, including the Annapolis Valley of Nova

Scotia (Vincent, 2011). Continued expansion of H. testudinea throughout Canadian regions and the economic threat of this insect to apple crops means research into its selection of host plants and any demonstrated discrimination between cultivars is vital for developing control and management methods.

Adult female apple sawflies emerge from pupation in early-mid May, synchronized with the blossoming of apple trees (Boevé, 1996) and become sexually mature within 4-10 days (Miles, 1932; Downes, 1944; Weires, 1991; OMAFRA, 2011;

Tamošiūnas & Valiuškaitė, 2013). Adult H. testudinea feed on the pollen and/or nectar of open apple blossoms (Miles, 1932; Weires, 1991; OMAFRA, 2011). After mating, female apple sawflies use their saw-like ovipositor to cut through the sepals and lay an egg towards the base of the blossom at the calyx, often close to the stamens (Weires,

1991; Boevé, 1996; OMAFRA, 2011). The egg may be partially exposed and visible to the naked eye, particularly if the female is disturbed during oviposition (Miles, 1932).

Eggs can take 1-2 weeks to develop (Miles, 1932; Weires, 1991), although normal maturation of larvae occurs within 7-10 days (OMAFRA, 2011). At emergence, larvae are approximately 2 mm in length. After hatching, larvae burrow into a developing fruitlet’s ovary, consuming fruitlets from the inside out. Larvae that die at this stage of their development leave a distinctive curved ‘C-scar’ on developing apples (Fig. 1.2).

Fruit that survives this initial feeding and matures is no longer viable for sale. Surviving larvae hollow the fruitlet out from the inside before moulting and migrating to the next fruitlet within the cluster. Apple sawfly larvae do not share host fruit; a single larvae will

a b

Figure 1.2: ‘C-scarring’ on apple fruitlets. (a) & (b) fruitlets that survived infestation by H. testudinea larvae. (c) C-scarring on fruitlet currently infested by H. testudinea larvae (note presence of frass, liquid droplet in lower right corner).

inhabit each infested fruitlet (Weires, 1991; David’yan, 2009) and are shown to be repelled by the presence of living or dead conspecifics within a fruitlet (Roitberg &

Prokopy, 1984).

Generally, an apple sawfly larva will consume one fruitlet per instar (Weires,

1991; Hull et al., 1995) (Fig. 1.3), increasing in size by up to 1.4 times per instar until it reaches 11 mm at its fifth instar (Weires, 1991; OMAFRA, 2011). In its final instar, the sawfly larva will remain within the fruitlet until the fruitlet aborts and falls from the tree.

The larva will then exit the fruitlet and burrow several centimeters into the soil to pupate and overwinter, emerging the next year as an adult (Weires, 1991; Hull et al., 1995;

OMAFRA, 2011) (Fig. 1.4).

1.3 Apple Maggot Fly

The apple maggot fly Rhagoletis pomonella (Fig. 1.5) is a recurring pest species native to North America (Kelly, 2003; Zhao et al., 2007). Originally known as the hawthorne maggot, a subset of this species’ original population began to exhibit oviposition preferences for apple trees after the plants’ introduction into North America

(Brunner & Klaus, 1993; Kelly, 2003). Although sharing the same scientific name, the hawthorne and apple maggot are genetically distinct, with corresponding variation in their life cycle that allow each species to take advantage of their respective host plants’ maturing fruit (Kelly, 2003). High potential for damage to apple crops that R. pomonella causes and existing annual losses to apple maggot infestation means that developing methods for the control, monitoring and management of this pest species is of value to growers within the Annapolis Valley and throughout North America.

aa bb

c d

Figure 1.3: Damage caused by Hoplocampa testudinea infestation. (a) external damage on vacated fruitlet (b) external damage on currently-inhabited fruitlet; frass is moist and fresh. (c) internal damage (longitudinal view of fruitlet). (d) internal damage, lateral view of fruitlet.

Figure 1.4: Life cycle of Hoplocampa testudinea.

a b

Figure 1.5: Adult Rhagoletis pomonella (a) adult apple maggot fly on a heavily infested Silken apple.(b) Ovipositing apple maggot female (c) male apple maggot fly searching for mate or food source on apple damaged by H. testudinea larvae.

Apple maggot fly emergence begins late June to early July, varying by habitat and environment. Maggot flies are sexually immature at emergence and take 7-10 days to become sexually mature. Maggot flies feed on sugar sources, including the honeydew excreted by aphids (Nielson, 1966) and visit apple leaves to seek out this food source.

After reaching maturity, R. pomonella will mate. The female will oviposit in mature or near-mature fruit (Fig. 1.5b), laying a single egg. Females may repeatedly oviposit on the same apple and can lay up to 300 eggs throughout their lifetime (Brunner & Klaus,

1993).

Apple maggots damage fruit in two ways: (1) oviposition and (2) larval feeding.

Ovipositing females damage mature and ripening fruit by using their sharp, needle-like ovipositor to pierce the fruit’s skin and lay eggs within the flesh of the apple, leaving small ‘dimple’ scars in the skin (Figs. 1.5a & 1.6c). The larvae (Fig. 1.6d) hatch and burrow through the apple, feeding on the fruit, leaving brown, discoloured trails of rot throughout the flesh (Reissig, 1991) (Fig. 1.6a & b). Several R. pomonella larvae infesting a single fruit can cause it to become soft to the touch. Infested apples fall from the trees, and the inhabiting larvae leave to burrow into the ground to pupate and overwinter (Reissig, 1991; Brunner & Klaus, 1993; Zhao et al., 2007). Adult maggot flies emerge from the soil the following summer, although delayed emergences can occur, particularly if the season is dry or inhospitable (Brunner & Klaus, 1993, Plant Pest

Surveillance Unit, 2012) (Fig. 1.7).

c d

Figure 1.6: Damage to apples caused by a Rhagoletis pomonella larva. (a) trails gnawed through apple flesh caused by larvae moving through fruit (b) extensive damage to apple caused by multiple R. pomonella feeding. (c) external damage; sting marks highlighted. (d) apple maggot removed from apple.

Figure 1.7: Life cycle of Rhagoletis pomonella.

1.4 Management and Control

Controlling invasive and widespread pest species is of increasing economic importance (Vincent et al.,2002; Zhao et al., 2007). Many countries take steps to reduce or prevent the spread of alien organisms into native ecosystems and food crops. The larvae of both Hoplocampa testudinea and Rhagoletis pomonella remain within the fruitlets/maturing apples as they develop (Weires, 1991; Zhao et al., 2007), making control of these organisms at this life stage difficult. Most control methods of these insects are therefore targeted towards the adults, before oviposition can occur.

In the case of H. testudinea, control of adult apple sawflies has an additional challenge. The adult sawflies emerge from pupation during apple bloom, feeding on the nectar and/or pollen in apple blossoms (Miles, 1932; Weires, 1991) and use the blossoms themselves as oviposition sites (Weires, 1991; Hull et al., 1995). Pesticide sprays must be targeted in a very brief window (Weires, 1991; Vincent et al., 2002; Tamošiūnas &

Valiuškaitė, 2013). Targeting the first larval instar is common as after this point, because larvae are too deep within the fruitlets to be affected by contact sprays (Tamošiūnas &

Valiuškaitė, 2013). Insecticidal agents intended to kill adult H. testudinea can also harm beneficial insects, particularly pollinating bees because both are Hymenopterans (Weires,

1991; Boevé, 1996). The tropical shrub Quassia amara (Linnaeus) can be used as an organic management tool of H. testudinea. Quassia amara wood, roots and bark contain the natural bitter compounds quassin and neoquassin (Psota et al., 2010; Neupane, 2012).

Application of quassinoid sprays can effectively control H. testudinea populations while having little to no adverse effect on beneficial insects (Downes, 1944; Kienzle et al.,

2004; Psota et al., 2010; Neupane, 2012). Mortality from quassinoids comes from

ingestion rather than direct contact (Kienzle et al., 2004; Pstoa et al., 2010; Neupane,

2012). As a result, the treatment is used to target emergent apple sawfly larvae rather than adults (Kienzle et al., 2004; Pstoa et al., 2010).

Apple maggot control can be expensive and time-consuming (Brunner & Klaus,

1993; Zhao et al., 2007). Countries that have enacted quarantine protocols to curtail the spread of apple maggot and prevent it from reaching local crops include Canada, Chile,

Mexico and Taiwan (Zhao et al., 2007). Examples of quarantine and control methods are described in Zhao et al. (2007). All apples shipped to British Columbia must be from known maggot-free regions, or undergo an expensive cold treatment to kill any potential maggots within this fruit. Mexico imposes the same restrictions on all apples shipped from the United States. Measures to control the spread of economically important pest species result in increased costs in bringing a crop to market; the cost of the cold treatment method of controlling R. pomonella is effectively a 20-30% tariff on imported apples (Zhao et al., 2007).

Pesticide sprays are required every 10-14 days after the first apple maggot fly is identified in an orchard (Philip, 2007). As the emergence period of these insects can last approximately three months (Reissig, 1991), a single orchard may need to be treated with pesticides up to 9 times per season. Non-chemical management techniques of apple maggot require the clearing of unsprayed trees and the physical removal and destruction of all infested fruit (Philip, 2007). Rhagoletis pomonella has several known parasitoids including Biosteres melleus (Gahan) (Hymenoptera: Braconidae), Opius downesi (Gahan)

(Hymenoptera: Braconidae) and Diachasma alloeum (Muesebeck) (Hymenoptera:

Braconidae). Although these organisms have significant impacts on R. pomonella

populations using hawthorne as host plants, their effect on apple maggot fly populations using apples is negligible, as the maggots burrow too deeply into the apples to be reached by the parasitoids’ ovipositors (Brunner & Klaus, 1993).

Managing invasive insects can be more difficult than managing native pests, due to the lack of natural predators and disease. Biological control of sawflies within North

America has been largely unexplored; H. testudinea’s natural predators remain in Europe.

The potential to a species-specific predator, the European braconoid wasp Lathrolestes ensator (Brauns), to control sawfly populations in Canada has been the focus of recent research (Vincent, 2011).

Challenges to traditional control methods of H. testudinea and R. pomonella have created a need for alternatives. Study of these insects is necessary to provide insights into the development of more efficient methods of managing of these pest species. Such methods may include improved techniques for the trapping and monitoring of apple sawfly and apple maggot populations in outbreak areas. The development of superior control and management methods will provide more options for growers that use integrated pest management techniques. In order to develop options, this study will examine if cultivar preference exsists in these species. Field observations of H. testudinea and R. pomonella will be conducted to describe the preferences these insects exhibit towards specific cultivars in a mixed apple orchard, coupled with observations of infestation across a variety of apple cultivars, and laboratory trials to determine and confirm these patterns of in host plant preference.

1.5 Cultivar Preferences

Insects rely heavily on chemical signals for the identification of mates, location of food sources and oviposition sites (Hillier & Vickers, 2007; Benton & Dahanukar, 2010).

Insect behavioural responses can be triggered and mediated by the presence of chemical signals within their environment (Harris & Foster, 1995). The degree of sensitivity and attraction an insect has in response to these stimuli is used to discriminate between potential food sources, location of mates and oviposition sites (Harris & Foster, 1995;

Hillier & Vickers, 2007; Benton & Dahanukar, 2010).

Insects exhibit preferences to various cultivars of the same plant, based on gustatory, environmental and/or chemical cues (Boeve et al.,1996; Hogmire & Miller,

2005). These cues may function as attractants or repellents. Olfactory cues include volatile semiochemicals emitted by the plant (Boeve et al.,1996), but may be influenced by other factors, including variation in the host plant’s own chemical profile during its life cycle (e.g. a host plant’s volatiles may change due to the presence of an infesting conspecific and/or unrelated larva), leading to a shift in the insect’s preference for a specific cultivar (Buteler et al.,2009).

Cultivar preference in insects has been documented for a number of species and host plants: Japanese beetle and grapes (Gu & Pomper, 2008), wheat stem sawfly and wheat (Buteler et al.,2009; Weaver et al.,2009), gypsy moth and cranberries (Neto et al.,2010), the Azalea lace bug and azaleas (Kirker et al., 2008) and apple maggot in apples (Prokopy et al., 1973; Carle et al., 1987; Rull & Prokopy 2001a, 2001b; Nojima et al., 2003). The insect species in this study are known to, or are suspected to, have preferences for cultivars of host plants: R. pomonella demonstrates a preference towards

early-developing, soft-fleshed, sweet and/or subacid (fruit with comparatively low acidity) apples (Reissig, 1991; Weems, 2002). Female host preference in apple sawflies is a novel observation, although other species of sawfly exhibit a preference for host plant cultivars (Buteler et al.,2009; Weaver et al.,2009).

Due to the difficulty in raising H. testudinea in laboratory settings, little research has been done on the chemical ecology and cultivar preferences of this species. The exact mechanisms that H. testudinea uses to determine preference among host plants may be purely olfactory, or the insect may additionally rely on visual, environmental and/or gustatory cues in making choices between host cultivars. Identification of semiochemical cues that European apple sawflies use in selecting cultivars could lead to more efficient trapping and monitoring techniques for this species.

Once cultivar preference in a pest insect species has been observed, identifying the specific plant volatiles and their concentration/ratio is the next step to developing alternative monitoring and/or trapping methods, which is of increasing importance due to public interest in pesticide reduction. Apples are a heavily researched plant species and their aroma profiles have been mapped extensively; most apple volatiles are esters or alcohols (Dixon & Hewett, 2000). Although the aromatic profile of apples can vary greatly among cultivars (Dixon & Hewett, 2000), mechanical damage, larval infestation and insect predation (Boeve, et al.,1996; Buteler et al., 2009) can affect the semiochemical profiles emitted by plants. Likewise, variation in rootstock can have an impact on the susceptibility of particular cultivars to pest insects via changes in the host plant’s emitted volatile chemical profile (Buteler et al., 2009; Darjazi, 2011).

Behaviourally active odourants such as sex pheromones or host plant volatiles can be identified using electrophysiological analyses (Hillier & Vickers, 2007). There are two techniques that were utilized in this study: electroantennograms (EAG) and calcium imaging. Electroantennography involves exposing an insect’s antennae directly to a chemical compound. However, a challenge of electroantennography is that there is no method by which to differentiate behavioural variance (e.g. whether a particular odourant is an attractant or a repellent) and all that can be ascertained is that the insect is sensitive to a particular volatile (Beck et al., 2012). Calcium imaging is used to identify the parts of an insect’s brain that respond to an odourant and the specific neuronal pathways used in this response, however as with electroantennography, it only indicates sensitivity to a stimulus and the region of the brain that activates in response to the stimulus and does not indicate attraction or repulsion. In this study, differential responses to odours will be tested at the antennal lobe level (Christensen, 2005).

Insect olfaction is based upon olfactory receptor neurons (ORNs) that communicate with the central nervous system in response to the presence and/or concentration of odourants within the environment (Todd & Baker, 1999). Sensilla are present on sense organs such as antennae, palps or tarsi and are filled with lymph fluid; this liquid covers dendrites from the ORN that express olfactory receptor proteins (Anton

& Homberg, 1999; Sibbering et al., 2012). Interaction between odourants and these OR proteins cause changes in the basal firing rate of the ORNs. Receptor potentials travel through the ORN’s dendrite; cumulative receptor potentials can reach a threshold point, triggering an action potential that passes from the ORN’s axon into the glomeruli within the antennal lobe of the insect’s brain (Todd & Baker, 1999). These electrical responses

can be analyzed and mapped to determine sensitivity to odour sources (Todd & Baker,

1999, Sibbering et al., 2012) and the regions within the antennal lobe that activate in response to stimulation by these sources.

The antennal lobe is divided into glomeruli. Glomeruli are neural structures that contain the synaptic contacts between ORN axons and the interneurons of the antennal lobe. Each glomerulus is innervated by a specific ORN axon from the antennae or mouthparts (Anton & Homberg, 1999). Within the glomerulus, ORN axons synapse to local interneurons or to projection neurons, which convey olfactory information to the protocerebrum (Anton & Homberg, 1999; Sibbering, et al., 2012). Responses, to volatile blends at the antennal and antennal lobe level from specific cultivars will be examined in this study via EAG and calcium imaging.

1.6 Hypothesis and Objectives:

This research project examined the hypothesis that H. testudinea and R. pomonella exhibit cultivar preferences in their choice of host plant cultivars for feeding and oviposition. Cultivar preference may be based on a number of factors: volatile chemical cues emitted by the plants themselves, visual, tactile and/or additional environmental signals. The response of these insects to semiochemicals emitted by host plants will also be described using electrophysiological techniques. This will provide a greater understanding of which cultivars are the most attractive to these pests, but also how these odours are processed and the differential sensitivity and responses of these insects. This primary hypothesis will be tested via several discrete hypotheses for each species.

Hoplocampa testudinea will demonstrate variations in cultivar preference based on timing of bloom, development of fruitlets and host plant cues.

●H1: Distribution of H. testudinea in visitation and infestation will reflect cultivar

preference. Similarities will exist between preferences for visitation and infestation.

●H0: There will be no variation exhibited in cultivars visited or infested by H.

testudinea. Host plant selection for adult visitation and larval infestation will be

random.

●H1: Hoplocampa testudinea will demonstrate a hierarchy of choice in its preference

of volatiles from cultivars in laboratory behavioural choice assays.

●H0: No variation in patterns of preference will be observed.

●H1: Sensitivity to odours from apple blossoms will reflect the preferences observed

in the field and support the role of olfaction in selection of host plants.

●H0: No differential sensitivity to odours will be observed; host selection is not

influenced by olfaction.

Rhagoletis pomonella is known to prefer early-developing, soft-fleshed apples

(Klass, 1972; Reissig, 1979; Brunner & Klaus, 1993) and use olfaction as a means of differentiating between host plants (Fell et al., 1982; Rull & Prokopy, 2005).

●H1: Cultivar preference will be observed in field surveys of the population of R.

pomonella. Distribution of apple maggot flies and larvae will conform to R.

pomonella’s known preferences for early developing, sweet, soft-flesh cultivars.

●H0: Cultivar preference for visitation and infestation levels of R. pomonella will be

random.

●H1: Rhagoletis pomonella will demonstrate a hierarchy of choice in its preference of

volatiles from different cultivars in laboratory behavioural choice assays.

●H0: No variation among preference for host cultivars will be observed.

●H1: Calcium imaging will indicate differential responses to apple volatiles in R.

pomonella at the antennal lobe level.

●H0: No variation in response to volatile blends will be occur within R. pomonella’s

antennal lobe by odour or region of brain.

1.6.1 Significance of research

This research will lead to a better understanding of host plant preferences in H. testudinea and R. pomonella and elucidate if odour cues emitted by hosts modulate these preferences. This will provide options for future research and eventual management of these pests. The outcomes of this research could lead to more efficient trapping/monitoring systems and improved rearing programs for these insects. As a result, commercial producers will have a better understanding of the cultivar choice for use in their orchard stands, leading to reduced pesticide use and greater environmental sustainability.

2. Overview

2.1 Site of field study

All field research was conducted at the Agri-Food and Horticultural Research

Centre (AFHRC) in Kentville, Nova Scotia (Fig. 2.1; 45°04′08″N and 64°28′41″W).

Unsprayed entomological research orchards B137 and B138 containing newly-developed and commercial cultivars, with 20-year old apple trees were selected for study. Within a block, there were no rootstock variations in the examined cultivars. H. testudinea field research was concurrent with the emergence of apple sawflies and was performed in late spring and early summer, from May until July on blossoming trees (Fig. 2.2) and those with developing apple fruitlets. Field surveys for R. pomonella were concurrent with the emergence of apple maggot flies in late summer, from July through September.

Blossoming of the apple cultivars studied and the development times of mature fruit is described below (Tables 2.1 & 2.2).

2.2 Volatile collection and analysis

Volatile collection was carried out at the Atlantic Food and Horticulture Research

Centre (AFHRC) in Kentville, Nova Scotia by Dr. Suzanne Blatt’s entomology program.

Host plant volatiles were collected in 2012 during apple blossom periods and during apple maturation and development using a dynamic headspace system as described in

Casado (2006). These collection times were scheduled to be concurrent with the active periods of adult apple sawflies and apple maggot flies, thereby collecting chemical cues that could serve as attractants to these insects.

Figure 2.1: Satellite imagery of Agri-Food and Horticultural Research Centre. Location of B137 & B138 orchards highlighted. Image from Google Maps™.

Figure 2.2: Blossoming test cultivars, May 2013. (a) Close-up of Pinova apple blossoms (b) s23-06-153 tree in full bloom

Table 2.1: Timing of bloom of apple cultivars in this study. Bloom time is based on comparison to McIntosh varieties. Cultivar Blossom Time Source

8S-27-43 Mid-late Lane, 2006

Ambrosia Mid BCMA, 2013

COOP 39 Early Janick et al., 2006

Jubilee Fugi Mid-late E.C. Brown's Nursery Inc., 2014a

Pinova Mid-late Orange Pippin Ltd., 2014b

Rogers McIntosh - -

Hove & Willem, 1977; Orange Pippin Royal Gala Mid Ltd., 2014a

s23-06-153 Late Hampson et al., 2008

Silken Early Wilson, 2001

Summer McIntosh - -

Zestar! Early UM, 2010

Table 2.2: Characteristics of mature fruit from cultivars studied. Maturation times are based on comparison to McIntosh varieties. Timing of Cultivar Fruit Flesh Skin Flavour Other characteristics Source Maturation 8S-27-43 Mid-season Crisp, hard Thick Sweet Low acidity Lane, 2006

Ambrosia Mid-Late Crisp, juicy Thin Sweet Aromatic, subacid BCMA, 2013

COOP 39 Mid-Early Crisp, hard Medium Sweet, tart Janick et al., 2006

E.C. Brown's Nursery, Inc., Jubilee Fugi Mid-Early Dense, crisp N/A Sweet 2014

Pinova Mid-Late Crisp, juicy N/A Sweet Orange Pippin Ltd., 2014b

Rogers McIntosh - Crisp, juicy Thin, tough Tart Hunter, 1997

Hove & Willem, 1977; Orange Royal Gala Mid-Early Tender, juicy Thin Sweet Pippin Ltd., 2014a

s23-06-153 Late N/A N/A N/A Hampson et al., 2008

Silken Early Juicy Thin Sweet Aromatic, moderate acidity Wilson, 2001

Summer McIntosh - Crisp N/A Tart Hunter, 1997

Zestar! Early Soft, juicy N/A Sweet UM, 2010

Tree limbs from each cultivar of interest were covered with 18” x 22” oven bags

(Extra Packaging Corp, Boca Raton, FL). Samples were collected by passing charcoal- scrubbed air at 0.5L/min across the bagged limb for 2 hours onto a SR86701305 Tenax® trap (Chromatographic Specialities Inc, Brockton, ON) and a Porapak-Q trap made from pipettes, cotton and 35 mg of Porapak™ Porous Polymer Adsorbents (mesh 50-80)

(Sigma-Aldrich, Oakville, ON). Volume of plant material bagged varied across cultivars, but was standardized to 4-5 blossom clusters (20-24 blossoms) per tree.

2.3 Gas chromatography and mass spectrometry

Analysis of cultivar volatiles and identification of specific semiochemicals were performed at AFHRC by Dr. Blatt’s entomology lab.Tenax samples were analysed using a thermo-desorption gas chromatography-mass spectrometry(GC-MS); the samples from

Porapak-Q traps were eluted with 2 mL 95:5 pentane:diethyl ether and concentrated under a slow stream of N2. Samples were concentrated to a total volume of 200 μL. Each microliter of volatile extract was equivalent to material produced from 1.2 blossoms per minute. After concentration, samples were stored at -20°C until use.

3. European Apple Sawfly

3.1 Materials and Methods

Behavioural preference for apple cultivars was determined via field surveys of adult and larval H. testudinea found in fruitlets and through Y-tube bioassays. The role of olfaction in influencing host plant choice was examined via electroantennography and behavioural bioassays using cut apple blossoms and volatile blends. These combined techniques were used to provide insight into the preference for host plants exhibited by

H. testudinea and the potential cues by which these insects choose between cultivars.

3.1.1 Field Observations

Sawfly cultivar preference was evaluated during its flight season and during larval development within fruitlets. The first instance was observation of adult visitation and behaviours on the surveyed cultivars (May 8 – May 27, 2012 and May 5 – June 3, 2013) and the second instance involved evaluation of larval damage and infestation rates of fruitlets/fruit (June 4 – July 10, 2012 and June 6 – July 4, 2013). During emergence and flight period of H. testudinea, the B137 & B138 orchard blocks were surveyed daily. The cultivars used were Zestar!, Pinova, Rogers McIntosh, Ambrosia, Crimson Crisp (COOP

39), Jubilee Fugi, Royal Gala, Summer McIntosh, Silken, s23-06-153 and 8S-27-43.

Field observations of all 11 cultivars were carried out in spring 2012 and 2013 during adult emergence and flight. The methods of observation involved walking a complete circuit around each tree at 1-3 feet from the trunk and visually identifying adult apple sawflies. The number of trees per cultivar varied from 4-5 in the B137 orchard and 7-8 in the B138 orchard. All trees were surveyed and every blossom viewable from the ground

was checked for the presence of sawflies. Number of blossoms varied per tree per year.

In 2013, 3 field surveys of adult apple sawflies were conducted by an undergraduate student from Acadia University.

The number of apple sawflies per tree per cultivar and the behaviours of these sawflies were recorded. In 2012, 191 adult apple sawflies were observed over the survey period: in 2013, observed population was 334. Behavioural observations were divided into ‘feeding’, ‘visiting’, ‘mating’ and ‘browsing’. ‘Feeding’ denoted those sawflies actively harvesting pollen and/or nectar from apple blossoms at the time of observation.

‘Visiting flower/leaf’ indicated the sawfly was present on the tree, but did not carry out any otherwise noteworthy actions. ‘Browsing’ behaviours were described as any airborne sawfly that remained in proximity (within 10-20 cm) to the tree and/or approached blossoming flowers, but did not land. ‘Mating’ referred to insects copulating at the time of observation.

In 2012 and 2013, apple sawflies were collected from B137, B138 and other entomological research orchards at the AFHRC for use in behavioural bioassays and electrophysiological tests. Adult apple sawflies were captured using an aspirator (Fig.

3.1) and placed in a growth chamber at 15°C, 80% RH and 16:8 L:D photoperiod. This regime was used in 2012 and prolonged sawfly life by up to 2 weeks beyond the typical

20-day life expectancy observed in wild sawflies.

Following the flight period of adult sawflies, weekly surveys of the B137/B138 orchard blocks were performed from June 4 to July 10, 2012 and from June 6 to July 4 in

2013. A weekly schedule was used to allow sawfly larvae to develop and move to new fruitlets between each survey. These surveys examined the developing fruitlets on each

Figure 3.1: Collection of insect specimens using an aspirator.

cultivar of interest for signs of larval sawfly infestation. Infested fruitlets were identified based on the distinctive scarring and frass-filled holes (Figs. 1.6 & 1.7) that larval H. testudinea leave as they move throughout and between fruitlets.

Numbers of infested fruitlets per tree per cultivar were used to quantify larval sawfly infestation and percentages of infested fruitlets per tree were calculated using 10,

25, 50 or 100 randomly selected fruitlets per tree. The variation in total fruitlets surveyed was based upon the productivity of each tree on an individual basis: most surveyed trees had sufficient fruitlets for a sample size of 50 – 100. Trees with less than 50 fruitlets had their percentage of infestation calculated based on a correspondingly smaller sample size.

Damage to apple trees by larval infestation in 2012 was based on observation of 24,274 fruitlets; in 2013, 23,660 fruitlets were observed. As individual H. testudinea larva damage multiple fruitlets, infestation data were based on total number of fruitlets damaged per tree per cultivar.

Mature apples from each cultivar were collected prior to harvest in 2012 and

2013. These fruit were stored in a 3.5°C, 80% RH walk-in cooler for preservation until the apples were examined for traces of infestation. Examination involved visual identification of damage caused by developing sawflies (e.g. C-scars). This information was used to determine the likelihood of apple survival by cultivar after sawfly oviposition. In 2012, post-harvest levels of damage were evaluated based on observations from 1,742 mature fruit and in 2013, 561 apples were examined.

3.1.2 Y-tube Bioassays

Y-tube bioassays were carried out in 2012 and 2013 to test sawfly cultivar preference with cut apple blossoms and volatile extracts.

Year 1 –2012

In 2012, fresh blossoms were removed from the trees daily for use in the bioassay.

The Y-tube assembly used a humidified and charcoal-filtered 2.5 l/minute airflow. The

Y-tube was 45 cm in length with a 23-cm central tube and two 22-cm junction arms. The tubes were comprised of glass 2.5 cm wide and 2.5 mm thick (Fig. 3.2). Temperature and humidity in the room were consistent at 21°C and 60-80% RH with an average of 70%

RH. Light levels in the room were measured at the arms and center of the Y-tube assembly. Illumination was consistent between each site at approximately 510 Lux. Light levels were measured using a Sper Scientific Advanced Light Meter 840022 (Sper

Scientific®, Scottsdale, AZ).

Bioassays were performed daily between 9:30 AM and no later than 1:00 PM.

Blossoms from two different apple cultivars were inserted into each junction arm; all 11 cultivars were tested in this series of bioassays. Number of times a given cultivar’s blossoms could be used varied accordingly to availability in the field (Table 3.1). A live sawfly was placed in the central tube of the assembly and monitored for 7 minutes to determine if it selected a blossom by directed movement to a specific arm. This time limit was determined from previous time trials in this study in which the insects in the assembly either made a choice within 7 minutes, or showed no reaction to the blossoms.

Similarly, Bartelt et al. (1982) used a 7-minute time limit for bioassays involving yellowheaded spruce sawflies (Pikonema alaskensis (Rohwer) (Hymenoptera:

Tenthredinidae)) and Piesik et al. (2008) used 7 minutes in Y-tube bioassays involving

Figure 3.2: Set up of Y-tube prior to bioassay

Table 3.1: Number of comparisons performed in 2012 Y-tube bioassays using cut apple blossoms.

COOP Jubilee Royal Rogers s23-06- Summer 8S-27-43 Ambrosia Pinova Silken Zestar! 39 Fugi Gala McIntosh 153 McIntosh

8S-27-43 0 0 5 0 5 0 0 5 0 0

Ambrosia 0 5 5 5 5 0 5 5 0 5

COOP 39 0 5 5 0 5 0 5 5 0 5

Jubilee 5 5 5 5 5 0 5 5 0 0 Fugi

Pinova 0 5 0 5 5 0 5 0 0 5

Royal 5 5 5 5 5 0 5 5 0 0 Gala Rogers 0 0 0 0 0 0 0 5 0 5 McIntosh s23-06- 0 5 5 5 5 5 0 5 0 0 153

Silken 5 5 5 5 0 5 5 5 5 10

Summer 0 0 0 0 0 0 0 0 5 5 McIntosh Zestar! 0 5 5 0 5 0 5 0 10 5

wheat plants and the wheat stem sawfly Cephus cinctus (Norton) (Hymenoptera:

Tenthredinidae).

Sawfly reactions were categorized as ‘strongly preferred’, ‘weakly preferred,’ and

‘no preference’ (defined below). If a sawfly moved more than 50% (6-7 cm) of the distance down a particular junction arm, this was considered a strong preference for the blossom/odour. If the sawfly travelled less than 6 cm (e.g. never moved past the midpoint of a given junction arm), this was a weak preference. If the sawfly never left the central tube and did not enter either junction arm, it was considered unresponsive and ‘no preference’ was indicated. Each trial was repeated with 5 randomly selected individuals of both genders. After each series of tests, the Y-tube assembly was rinsed with 70% ethanol to clear out any remaining volatiles and new blossoms were inserted. Preference patterns were based on first-choice responses. Five individuals were tested per cross.

Year 2 – 2013

A second series of bioassays was performed in 2013. Protocols for the bioassays were similar to those used in 2012, with the following adjustments: the airflow through the assembly was reduced from 2.5 l/min to 1.5 l/min and instead of blossoms from each cultivar, four blends were selected from the available volatile extracts collected from the cultivars in 2012. The cultivars used were selected based on the preferences indicated by the observations made of H. testudinea visitation preferences in the 2012 field season.

The preferences seen in the 2012 Y-tube bioassays were not considered, due to discrepancies between the results of the bioassay and preferences observed in the field.

Two ‘preferred’ cultivars, Zestar! and Pinova, were compared against two ‘less preferred’ cultivars, 8S-27-43 and Ambrosia. Pentane was used as a negative control as it was the solvent used to extract the floral volatile blends and was not expected to elicit a response from the sawflies. This method was used to confirm observed sawfly preferences in the field between most preferred cultivars and those less preferred by apple sawflies. Odour sources were 0.5 cm x 3 cm filter paper strips with 2 μL of volatile or the pentane control micro-pipetted to their surface.

Wild-caught female sawflies were individually tested for 7 minutes per cross.

Only female sawflies were selected for these Y-tube bioassay trials as ovipositional preference in host plants leads to larval infestation and damage (Weires, 1991).

After each cross, the Y-tube assembly was washed with 70% ethanol to remove any trace odours and air-dried. Fresh filter papers were used after each series of crosses was complete. If a series of crosses used the same volatile blend as a previous test, the filter paper was placed in the opposite junction arm (e.g. if comparisons using Pinova were made consecutively, the filter paper would be in the right arm in one test and the left arm in the next). Light levels were monitored at each junction arm using the Sper

Scientific Light Meter and remained consistent at approximately 500 Lux.

3.1.3 Electroantennography

Olfactory responses from apple sawflies of both sexes to volatile blends were tested via electroantennography using a Syntech IDAC-4 system. Blends (2 μL) taken from each of three ‘preferred’ and three ‘non-preferred’ cultivars; a pentane blank served as a negative control. Preferred and non-preferred categories were determined based on

field observations in 2012. Preferred cultivars included Zestar!, Pinova and s23-06-153.

Non-preferred cultivars were Royal Gala, COOP 39 and Ambrosia. The pentane control standardized any stimulation of antennal mechanoreception caused by airflow versus olfactory stimulation by selected blends.

Adult apple sawflies were immobilized by being placed in a freezer at -20°C for 5 minutes. Whole-body mounts were made by inserting the insect into a cut plastic 100-μL micropipette tip, which exposed the head and antennae but restrained bodily movement.

The sawfly’s head was secured in place with dental wax and the distal tip of the right antenna was severed 1-2 segments from the tip to provide optimal contact between the recording electrode and the now-exposed lumen of the antenna. The ground electrode was inserted into the insect’s eye (Fig. 3.3). Signals were amplified and digitally acquired on a PC using AutoSpike® software (Syntech®, Hilversum Netherlands). Responses to odour stimuli were recorded and measured with AutoSpike®. Responses from 22 individuals (17 female and 5 male) were used in analysis.

Pulled glass electrodes were used, made from 1 mm x 6” Borosilicate 51 Exp glass capillaries (World Precision Instruments; Sarasota, FL). Each capillary was nicked with a diamond cutter blade and used to create two electrodes using a Model P-97

Flame/Brown Micropipette Puller (Sutter Instrument Co., CA, USA). After being pulled, the glass electrodes were broken at each tip and filled with saline. Electrodes were coated with a thin layer of Parker’s Signa gel® (electrode gel, Parker Laboratories, Fairfield, NJ) to improve conductivity. Odour sources were prepared as described in Syntech (2004).

Ten μL of each odour blend was applied on a 0.5 x 3 cm strip of filter paper. Each strip was inserted into a 5 ¾” disposable Pasteur pipette (13-678-6G, Allied Fisher-Scientific,

Figure 3.3: Placement of apple sawfly head with electrodes attached. The sawfly is secured in a pipette tip with dental wax and cotton. Ground electrode (left) is inserted into female sawfly’s eye. Recording electrode (right) is connected to distal end of severed antenna.

Washington, DC) which was then connected to the air supply and inserted into the mixing tube 5-10 seconds prior to each stimulus puff. Insects were subjected to a 0.5- second puff of odour, delivered by a stimulus controller and timed by the EAG software.

For each recording, 3 seconds pre- and 7 seconds post-stimulus were recorded for a total time of 10 seconds per stimulation. Time between puffs was between 30 seconds to 1 minute to prevent adaptation and habituation (Judd et al., 2005).

3.1.4 Data Analysis

Cultivar preference in H. testudinea was tested using field observations of apple sawfly visitation and larval infestation analyzed by non-parametric Kruskal-Wallis tests with a Poisson distribution at α = 0.05. Mean levels of preference were calculated by dividing sawfly presence/rate of infestation per tree by cultivar and were ranked via a “1

2.5 2.5 4”/fractional system. This system ranks each value in a dataset and assigns equal values to the average of the ranks they would have had they not been equal (Kruskal &

Wallis, 1952). Individual cultivars were compared against each other to evaluate differences in rates of infestation using a generalized linear model, using a Tukey’s pairwise comparison and/or a Kruskal post-hoc multiple comparison test (de Mendiburu,

2014) to determine if there were significant differences between visitation levels of apple sawflies to each cultivar. Significance in levels of infestation were evaluated using a

Kruskal-Wallis test with a Poisson distribution at α = 0.05.

Statistical evaluations of sawfly responses from the Y-tube bioassays were performed using a Chi-square analysis. First-choice responses and total responses were considered in both analyses at α = 0.05. For each odour, H0 = 0.5/n was assumed, where n

was equal to number of responses to a given cross. Trials in which the insect did not respond to either blossom or odour were excluded from the analysis. Chi-square tests compared responses between each individual cross (e.g. Zestar! v Pentane).

Electroantennogram responses were analyzed within each year using a one-way

ANOVA at α = 0.05 to determine significant differences in fly sensitivity between cultivar blends. Additional one and two-way ANOVAs were performed to test variation in response between sexes and between sexes by cultivar.

3.2 Results

3.2.1 Adult visitation

Based upon 2012 and 2013 field results, a pattern of preference was exhibited by

H. testudinea. Although H. testudinea were observed visiting earlier-blooming cultivars such as Zestar!, COOP 39 and Silken in both 2012 and 2013, this initial pattern of preference was not sustained across the entire apple bloom period in either year. Once later-blooming cultivars began to blossom, sawflies were found more frequently on several of these cultivars rather than the earlier-blooming apple varieties (Figs. 3.4 &

3.5).

The later blooming cultivars of interest included the highly preferred Pinova and s23-06-153. Mean apple sawfly visits to these cultivars exceeded the visitation levels on several earlier-blooming cultivars (Fig. 3.6). This pattern of bloom was observed in both field seasons: once certain varieties of later-blooming cultivars became available, apple sawfly interest in several earlier-blooming cultivars began to wane. Zestar! was the only early-blooming cultivar that remained consistently of interest to adult H. testudinea

Figure 3.4: Distribution of apple sawfly visitations per cultivar across 2012 flight period. See Table 2.1 for cultivar characteristics

Figure 3.5: Distribution of apple sawfly visitations per cultivar across 2013 flight period.

Figure 3.6: Mean sawfly visitations by cultivar in 2012 (p < 0.0001) and 2013 (p < 0.0001) for mean sawfly visitations throughout field survey. Cultivars sharing the same letter were not significantly different in apple sawfly visitations.

throughout its bloom period in each year, whereas sawfly visitations to other cultivars tapered off once other cultivars became available (Figs. 3.4 & 3.5).

Although differences in mean apple sawfly visitation among cultivars were noted between 2012 and 2013 preference patterns were largely the same: the preferred and non- preferred cultivars remained consistent between years (Fig. 3.6). In 2012, Zestar! was the most heavily visited cultivar with 9.6 sawfly visits per tree. Pinova and s23-06-153 were second and third at 8 and 5.25 mean visits, respectively (Fig. 3.6). In 2013, Zestar was once again the most frequently visited cultivar with 13.4 visits per tree; s23-06-153 was second with 10.9 visits per tree, Pinova was third at an average of 9.2 visits. Although the number of sawfly visitations by cultivar varied between years, the general pattern of sawfly preference remained consistent. During each emergence period, a handful of cultivars were noticeably more preferred than others: Zestar!, Pinova and s23-06-153.

Likewise, 1-2 cultivars consistently had low rates of sawfly visitation. Jubilee Fugi was one of the least-preferred cultivars in 2012 and 2013; in 2012, Silken was least preferred, but the number of visitations to this variety increased between years (Fig. 3.6).

Analysis of sawfly visitation data indicated that all cultivars are not equally attractive to H. testudinea in a mixed orchard (Fig. 3.6). A Kruskal-Wallis test revealed a significant effect of cultivar on sawfly visitation for both years: (χ2 (2012) = 32.93, p <

0.0001, χ2 (2013) = 36.83, p < 0.0001) (Table 3.2). Observations indicated several highly-preferred cultivars which were statistically significantly different in level of apple sawfly visitation from all other cultivars, but not each other. These preferred cultivars remained consistent between years (Fig. 3.6).

Table 3.2: Summary of non-parametric Kruskal-Wallis tests for 2012 and 2013 apple sawfly visitation. α = 0.05, df = 10 Total Mean visits Sawflies (N) per cultivar SE H P

Year * Cultivar 525 3.86 0.09 6.5 0.011

Cultivars (2012) 191 2.81 0.07 32.9 5.83E-05

Cultivars (2013) 334 4.91 0.10 36.8 6.06E-05

The pairwise comparisons (Tables 3.3 & 3.4) showed significant differences in mean sawfly visitations between cultivars (Fig. 3.6). As with the field data, a clear pattern of preference can be noticed from this data. In 2012, sawfly visitations on Zestar!, Pinova and s23-06-153 were significantly different from all other apple cultivars (Fig. 3.6 and

Table 3.3). This supports the results of the Kruskal-Wallis analysis and reactions observed in the field. It can also be seen that the mean sawfly visitations upon later- blooming cultivars of s23-06-153 and Pinova were significantly different from such earlier-blooming varieties as COOP 39 and Jubilee Fugi, further supporting the observed preference patterns of H. testudinea.

This was similar to the 2013 results in which Zestar! Pinova and s23-06-153 again showed consistently significant differences in average apple sawfly visitations when compared to other cultivars (Fig. 3.6 & Table 3.4). Jubilee Fugi and Ambrosia, though not significantly different from several other cultivars, were consistently among the least- preferred for visitation. Royal Gala and COOP 39 were significantly different from other cultivars in 2013. In 2012, these cultivars were not significantly different from the less- preferred cultivars, but had higher average visitations. (Fig. 3.6).

Table 3.3: Pairwise comparison of 2012 apple sawfly visitations using results of z-test from general linear model and level of significance. COOP Jubilee Rogers Royal s23-06- Summer 8S-27-43 Ambrosia Pinova Silken 39 Fugi McIntosh Gala 153 McIntosh

Ambrosia 0.338 0.77 -0.63 4.333c 0.07 0.77 3.382a -1.15 0.27

COOP 39 1.21 0.77 -1.35 4.043b -0.65 -0.07 2.91 -1.91 -0.60 Jubilee -0.36 -0.63 -1.35 4.391c 0.67 1.38 3.595a -0.48 0.93 Fugi

Pinova 5.37c 4.33c 4.04b 4.39c -3.913b -4.894c -1.91 -5.032c -5.172c Rogers 0.39 0.07 -0.65 0.67 -3.91b 0.65 3.033a -1.17 0.18 McIntosh Royal 1.26 0.77 -0.07 1.38 -4.89c 0.65 3.56a -1.97 -0.60 Gala s23-06- 4.3c 3.38a 2.91 3.59a -1.91 3.033a 3.56a -4.238b -3.956b 153

Silken -0.91 -1.15 -1.91 -0.48 -5.03c -1.17 -1.97 -4.24b 1.50 Summer 0.69 0.27 -0.60 0.93 -5.17c 0.18 -0.60 -3.96b 1.50 McIntosh

Zestar! 5.92c 4.80c 4.61c 4.78c 0.85 4.34c 5.60c 2.86 5.42c 5.81c a Difference between cultivars significant at p < 0.05 b Difference between cultivars significant at p < 0.01 c Difference between cultivars significant at p < 0.001

Table 3.4: Pairwise comparison of 2013 sawfly visitations using results of z-test from general linear model and level of significance. Jubilee Rogers Royal s23-06- Summer 8S-27-43 Ambrosia COOP 39 Pinova Silken Fugi McIntosh Gala 153 McIntosh

Ambrosia -1.07 2.82 -0.63 4.693d 0.07 3.132a 5.222d 2.85 0.61

COOP 39 2.40 2.82 -3.136a 2.85 -2.54 0.29 3.792c -0.09 -2.85 Jubilee -1.64 -0.63 -3.14a 4.685d 0.67 3.389b 5.103c 3.153a 1.24 Fugi

Pinova 5.29d 4.69d 2.85 4.68d -4.239d -3.016a 0.92 -3.255b -5.519d Rogers -0.92 0.07 -2.54 0.67 -4.24d 2.81 4.704d 2.55 0.50 McIntosh Royal 2.90 3.13a 0.29 3.39b -3.02a 2.81 4.26d -0.42 -3.338b Gala s23-06- 6.22d 5.22d 3.79c 5.10c 0.92 4.70d 4.26d -4.398d -6.393d 153

Silken 2.46 2.85 -0.09 3.15a -3.26b 2.55 -0.42 -4.40d -2.921a Summer -0.56 0.61 -2.85 1.24 -5.52d 0.50 -3.34b -6.39d -2.92a McIntosh

Zestar! 6.84d 5.66d 4.53d 5.48d 1.96 5.12d 5.11d 1.29 5.19d 6.96d a Difference between cultivars significant at p < 0.1 b Difference between cultivars significant at p < 0.05 c Difference between cultivars significant at p < 0.01 d Difference between cultivars significant at p < 0.001

3.2.2 Pre-harvest larval infestation

Sawfly larvae were only occasionally seen on the surface of infested fruitlets, largely remaining within infested fruitlets. Larvae were most visible on cooler, overcast days, with temperatures <15°C, when they would appear close to the exterior of an infested fruitlet, or emerge entirely. Typically, infested fruitlets contained a single larva; multiple larvae in a single cluster were uncommon and multiple larvae within the same fruitlet were never observed.

A strong pattern of insect choice can be observed in the oviposition preference of

H. testudinea in regards to the rate of larval infestation. In both 2012 and 2013, the cultivars Zestar! and COOP 39 were consistently among the most preferred for oviposition while Silken and Ambrosia were the least preferred (Fig. 3.7). In 2012, an average of 28.3% of all COOP 39 fruitlets were infested by apple sawfly larvae, compared with 26.7% for Zestar!. Silken and Ambrosia were the least-preferred cultivars and had fruitlet infestation rates of 5.8% and 7.7%, respectively (Fig. 3.7). Similar oviposition preferences were observed in 2013. In 2013, over the six-week observation period, Zestar! and COOP 39 were the two most preferred cultivars with an average of

21.1% of Zestar! and 18.0% of COOP 39 fruitlets infested. s23-06-153 became the second-most infested cultivar in 2013, up from 4th in 2012, although its mean rate of infestation remained within the same range (19.1 ± 2.36) and (18.7 ± 1.50) (Fig. 3.7).

Silken and Ambrosia again remained the least-infested cultivars with 8.1% and 2.12% of all surveyed cultivars showing indications of sawfly larvae infestation (Fig. 3.7). As with sawfly visitations, the trend in sawfly behaviours was for 1-2 cultivars to be highly preferred, 1-2 to be least preferred and the remainder to be roughly equivalent

Figure 3.7: Comparison of levels of pre-harvest apple sawfly infestation between 2012 (p < 0.0001) and 2013 (p < 0.0001) field surveys. Cultivars sharing the same letter were not significantly different for apple sawfly infestation.

in preference for ovipositing females. Infestation preferences differ from visitation preferences; e.g. COOP 39 is consistently heavily infested by H. testudinea but adult sawflies show only average preference for visiting/feeding from these blossoms (Fig.

3.6). Conversely, Silken remains an unpopular cultivar for oviposition in both field seasons, despite the increased visitation by adults in 2013 (Fig. 3.7).

A significant difference in level of infestation exists between cultivars.

Differences in the rate of infestations between all studied cultivars in 2012 and 2013 were statistically different from each other. There were also significant differences in apple sawfly infestations among cultivars between years, suggesting non-random variation in level of infestation per cultivar between years (Table 3.5).

Results of the Kruskal-Wallis tests reveal a significant effect of cultivar on apple sawfly infestation in each year (χ2 (2012) = 36.4, p < 0.01, χ2 (2013) = 47.6, p = 0.05) and between years (χ2 (2012*2013) = 16.2, p < 0.01) (Table 3.5). Infestation data from 2013 again indicates a non-random distribution in infestation preference. In both years, these data suggests that apple sawfly choice in oviposition among host plants is influenced by some level of preference, rather than random chance (Table 3.5).

Table 3.5: Summary of Kruskal-Wallis tests for 2012 and 2013 apple sawfly pre-harvest infestation data. Total Mean infested Fruitlets fruit per tree Observed (%) SE H P

Year * Cultivar 47934 14.96 1.92 16.2 5.77E-05

Cultivars (2012) 24274 17.65 2.44 36.6 6.52E-05

Cultivars (2013) 23660 12.28 1.41 47.5 7.51E-07

3.2.3 Post-Harvest Larval Infestations

Jubilee Fugi fruitlets have the greatest chance of maturing after sawfly infestation.

Although Jubilee Fugi had average to low rates of infestation (Fig. 3.7), its fruitlets had a higher rate of survival than those from cultivars with comparable rates of infestation, indicating higher mortality among the apple sawfly larvae within these fruitlets. In 2012 pre-harvest larval infestations of Jubilee Fugi were 17.7 ± 2.2 % (Fig. 3.7) and post harvest levels of damage were 10.5 ±1.07 % (Fig. 3.8). 8S-27-43 had a 2012 mean pre- harvest larval infestation level of 17.5 ± 1.84% (Fig. 3.7) and a post-harvest mean infestation level of 7.1 ± 0.25% (Fig. 3.8). Means of pre-harvest larval infestations had overlapping C.I., but post-harvest levels of damage were significantly different from each other. The post-harvest levels of infestation in Jubilee Fugi suggest that a higher percentage of fruitlets infested by apple sawfly did not abort and instead were more likely to survive apple sawfly infestation and remain on the tree through maturation to harvest.

In 2012, more infested Jubilee Fugi apples survived to harvest than any other cultivar followed by Silken (Fig. 3.8). In 2013, Jubilee Fugi fruit infested by apple sawfly larvae again had the greatest chance of surviving to maturity (Fig. 3.8), although these apples would have no economic value. There was no discernible pattern in the level of infestation of apples by apple sawfly larvae in any other cultivar (Fig. 3.8). A Kruskal-

Wallis test revealed no significant effect of cultivar on larval infestation in either year (χ2

(2012) = 14.4, p > 0.1, χ2 (2013) = 18.2, p > 0.05), although significant differences in post-harvest variation between years were observed (χ2 (2012*2013) = 29.0, p < 0.01)

(Table 3.6).

Figure 3.8: Comparison of apple sawfly infestation levels in 2012 (p > 0.05) and 2013 (p > 0.05) post-harvest larval surveys.

Table 3.6: Summary of Kruskal-Wallis tests of 2012 and 2013 apple sawfly post-harvest infestation surveys. Apples Mean Checked (N) infestation (%) SE H P

Year * Cultivar 2303 15.35 0.51 29.0 7.11E-08

Cultivars (2012) 1742 14.86 0.47 14.4 0.16

Cultivars (2013) 561 15.83 0.55 18.2 0.05

3.2.4 Y-tube Bioassays

For results from the 2012 Y-tube bioassays, see Appendix A.

The 2013 Y-bioassays showed a pattern of apple sawfly choice when apple sawflies were tested against Zestar! and Pinova (Table 3.7), confirming observations from field surveys. These results suggest that apple sawfly preferences for visitation and infestation in these Y-tube bioassays are different than what could be expected from random chance (Fig. 3.9). These data supported the results of field and infestation studies. In 2013, 177 bioassays were conducted: 34% of these were noncommittal responses. Values reported indicate total positive responses; non-responding apple sawfly reactions were excluded. Anecdotally, when using volatile blends instead of cut blossoms, apple sawfly reactions to odour objects were quicker and occurred more often.

When the data from the series of 2013 bioassays were reduced to the first choice each test insect made in each trial, the null hypothesis of H0: H1=H2=Hn was accepted

(Table 3.7). The comparison between Zestar! and Ambrosia was statistically significant.

The overall trend in these responses supported field observations, but the lack of significant differences contradicted the pattern of preferences in the field. Zestar! was highly preferred for both oviposition and visitation in both 2012 and 2013 (Figs. 3.6 &

3.7), but Zestar was not chosen significantly more than the pentane control. Similarly, the

Pinova cultivar was one of the most preferred cultivars for adult apple sawfly visitations in the field, but first choice apple sawfly responses in the bioassays showed no significant difference between the less preferred 8S-27-43 cultivar, the pentane control or the more preferred Pinova blend.

Table 3.7: Results of Chi-square analysis of choices of apple sawfly to volatile blends in 2013 bioassays. More preferred/less

Cross N df preferred choices x2 crit p Ambrosia v Control 6 1 3/3 0 1.000 Zestar! v Control 6 1 5/1 2.67 0.102 Zestar! v Ambrosia 7 1 7/0 7a 0.008 Pinova v 8S-27-43 6 1 5/1 2.67 0.102 Pinova v Control 4 1 3/1 1 0.317 8S-27-43 v Control 5 1 4/1 1.8 0.180 a Choice of sawfly responses to volatile blends significantly different (Chi square p < 0.01)

Control N=5 8S-27-43

8S-27-43 N=6 Pinova

Control N=4 Pinova

a Ambrosia N=7 Zestar! p = 0.008

Control N=6 Zestar!

Control N=6 Ambrosia

100% 75% 50% 25% 0% 25% 50% 75% 100% Apple sawfly selecting stimuli

Figure 3.9: Proportion of Hoplocampa testudinea responding to volatile blends based upon first responses. a Choice of sawfly responses to volatile blends significant at p < 0.01

When the data from all responses to the bioassay comparisons were analyzed, the possibility of non-random selection was supported. Zestar! had significant differences in apple sawfly choice among all comparisons with less preferred cultivars and the pentane control, 8S-27-43 was statistically preferred when compared to the pentane control.

Contradicting field results, even when all responses to volatile blends were accounted for,

Pinova again had no significant differences in apple sawfly choice.

3.2.5 Electroantennography

Differences in response of male and female H. testudinea to volatile extracts by sex were observed. Female sawflies had the greater sensitivity than male to apple floral volatile extracts. Across all tested volatile blends including the pentane solvent control, the mean female response to the compliment of odours tested after stimulation was 8.36 mV. The mean response of a tested male was 0.11 mV. The variance in means was compared with a two-way ANOVA and deemed to be significantly different at p = 0.001

(Table 3.8). Male sawfly antennal responses were most sensitive to odour puffs from

Zestar! (Fig. 3.10 & 3.12), whereas females were more sensitive to Pinova volatiles (Figs.

3.10 & 3.11). Both sexes had the second-highest level of response to Ambrosia (Figs.

3.11 & 3.12). All other means were within the 95% confidence intervals of each other.

Female apple sawflies were more sensitive to odours from volatile blends than were males (Fig. 3.10). A two-way ANOVA determined a significant level of difference in the sensitivity to volatiles between sexes (Table 3.8). This suggests that level of sensitivity to a cultivar is influenced by sex.

Table 3.8: Summary of two-way ANOVA comparing apple sawfly response (mV) to odour. df Sum of squares Mean Squares F p Extract 6 1013.5 168.9 1.4 0.2 Sex 1 1951.9 1951.9 16.3 7.6E-05a Extract * Sex 6 300.8 50.1 0.4 0.9 a Response to volatile extract significant at α = 0.001

20 0.35 Female 18 A 0.3 a Male

SE)

14 0.25 -

12 0.2 B 10 b B 0.15 8 B B b B b b B 6 b 0.1

b Mean male response (mV) (+/ (mV) male response Mean

Mean female response (mV) (+/ (mV) response female Mean 4 0.05 2

0 0 Pinova Ambrosia Zestar! Pentane Royal Gala 8S-27-43 COOP 39 Control Volatile Figure 3.10: Hoplocampa testudinea responses to EAG (22 individuals of both sexes). P-value of all responses: 0.24. p-value of amplitude of female responses: 0.2. p-value of male responses: 0.43. Cultivars sharing the same letter are not significantly different.

18 a

SE)

- 14

12 b 10

b 8 b b b

6 b

Mean EAG Response (mV) (+/ (mV) EAG Response Mean 4

0 Pinova Ambrosia Zestar! Pentane Control Royal Gala 8S-27-43 COOP 39 Volatile Extract

Figure 3.11: Female sawfly responses to electroantennography (N=17) (p < 0.10). Cultivars sharing the same letter are not significantly different.

0.35

0.30

SE)

- 0.25

0.20

b 0.15 b

b b 0.10 b

b Mean EAG Response (mV) (+/ (mV) EAG Response Mean

0.05

0.00 Zestar! Ambrosia 8S-27-43 Pinova Royal Gala Pentane Control COOP 39 Volatile Extract

Figure 3.12: Male sawfly responses to electroantennography (N = 5) (p < 0.10). Cultivars sharing the same letter are not significantly different.

At α = 0.05, EAG responses from both genders were significant only with the

Pinova volatile blend (Table 3.9). Female responses to Pinova volatiles were statistically different from other volatile blends (p = 0.04), but the sexes had no significant difference in their sensitivities between any other volatile blends (Table 3.9 & Fig. 3.11). Male sawflies were generally more sensitive to Zestar! extracts than other volatiles. This response was statistically different at α = 0.1 (p = 0.06) when compared to responses to other volatile blends (Table 3.9 and Fig. 3.12). When responses to odour based sex were included in the model, apple sawfly responses to cultivars were not significant at the 95% confidence level (Tables 3.8 & 3.9), but showed a trend of greatest sensitivity towards volatiles from those cultivars that were highly preferred in the field (Figs. 3.6 & 3.7).

Apple sawflies were most sensitive to volatile blends from highly preferred cultivars

(Fig. 3.10). The next cultivar to which the insects were most sensitive was Ambrosia

(Figs. 3.11 & 3.12), one of the least-preferred cultivars for adult visitation (Fig. 3.6).

Results from field surveys, behavioural bioassays and electrophysiological testing suggest Zestar! and Pinova are consistently highly preferred by H. testudinea and produce volatiles with somewhat greater sensitivity for H. testudinea. Ambrosia and

Jubilee Fugi are the least-preferred cultivars studied (Table 3.10).

Table 3.9: Statistical significance of all sawfly responses to volatiles in electroantennograms using a generalized linear model. Responses are compared against pentane control. Cultivar Sex df t P 8S-27-43 All 21 -0.36 0.72 Ambrosia All 21 0.51 0.61 COOP 39 All 21 -0.49 0.63 Pinova All 21 2.09 3.79E-2b Royal Gala All 21 -0.19 0.85 Zestar! All 21 -0.21 0.83 8S-27-43 Female 16 -0.32 0.75 Ambrosia Female 16 0.64 0.52 COOP 39 Female 16 -0.45 0.65 Pinova Female 16 2.19 3.02E-2b Royal Gala Female 16 -0.14 0.89 Zestar! Female 16 -0.23 0.82 8S-27-43 * Male 4 0.34 0.74 Ambrosia Male 4 0.61 0.54 COOP 39 Male 4 -0.50 0.62 Pinova * Male 4 -0.04 0.97 Royal Gala Male 4 0.09 0.93 a Zestar! Male 4 1.89 0.065 a Response to volatile extract significant at α = 0.1 b Response to volatile extract significant at α = 0.05

Table 3.10: Summary of apple cultivars most preferred and least preferred by Hoplocampa testudinea based on field surveys, behavioural bioassays and electrophysiological tests. Preferred cultivars are ranked most preferred to least preferred, and non-preferred cultivars are ranked least preferred to most preferred. Preferrred Non-preferred Method Cultivars Cultivars 1. Zestar! 1. Jubilee Fugi Field Survey 2. s23-06-153 2. Ambrosia 3. Pinova 3. Rogers McIntosh 1. Zestar! 1. Ambrosia Infestation Survey 2. COOP 39 2. Silken 3. Pinova 3. Jubilee Fugi 1. Zestar! 1. Ambrosia Y-tube Bioassay 2. Pinova 2. Pentane Control 3. 8S-27-43 1. Pinova (female) 1. COOP 39 (female) EAG 2. Zestar! (male) 2. COOP 39 (male)

4. Apple Maggot Fly

4.1 Materials and Methods

Discrimination and preference for host plants by the apple maggot fly, R, pomonella, was examined through a combination of field observations and laboratory experiments. Response of R. pomonella for apple cultivars was determined based on observations of adult visitation, oviposition, larval infestation levels and via Y-tube bioassays. Olfactory responses to apple volatiles were evaluated using bioassays and calcium imaging analysis.

4.1.1 Field Observations

Observations of cultivar preference in adult apple maggot flies were conducted during July-August of 2012 and July-September 2013. Each tree from the 11 cultivars of interest to this study was surveyed daily, weather permitting. Surveys were performed visually while walking a 360-degree circuit <1 meter from the trunk. All apples within view of a standing observer were checked for adult R. pomonella. Distance from tree was determined by the extent of the foliage. Numbers and behaviours of adult R. pomonella were noted per tree per cultivar during their emergence and oviposition period in both

2012 and 2013. Four hundred and seventy-one apple maggot flies were observed in 2012 and 2,044 in 2013. In 2013, 3 field surveys of adult apple maggot fly were performed by an undergraduate student.

Data on apple maggot infestation rates amongst the cultivars of this study were gathered in 2012 by associates in Dr. Blatt’s entomology program at the AFHRC in

Kentville. Apples from each tree of the cultivars of interest (8S-27-43, Ambrosia, COOP

39/Crimson Crisp, Jubilee Fugi, Pinova, Rogers McIntosh, Royal Gala, s23-05-153,

Silken, Summer McIntosh and Zestar!) were harvested. Depending on individual tree productivity, 10-27 apples per tree were sampled. Sample fruit were checked for indications of apple maggot oviposition (Fig. 4.1a). If sting marks were discovered, the fruit was cut vertically across the pedicel and the flesh examined for confirmation of R. pomonella infestation. After these halves of the apple were examined for evidence of apple maggot, a second vertical cut perpendicular to the first was made. Confirmation was determined via either the presence of the larvae themselves or the distinctive trails left by their burrowing through the fruit (Fig. 4.1).

Infestation data from 2013 were based on mature fruit harvested from 4 randomly-selected trees of each cultivar; the total number of trees in each cultivar across the orchard blocks varied between 4 and 8. Depending on the productivity of each tree,

10-15 apples were collected to allow for a relevant statistical analysis of infestation of each cultivar and to leave sufficient apples to be analyzed by other programs at the

AFHRC. The presence of apple maggot was determined as described for 2012. In 2012,

1,742 apples were examined for evidence of apple maggot infestation; 561 mature fruit were checked in 2013. Using this data, a mean percentage of R. pomonella infestation per cultivar was developed.

Level of significance in rates of apple maggot infestation across cultivars was evaluated using a Kruskal-Wallis test with a Poisson distribution at α = 0.05. A non- parametric test was used to account for the non-normal distribution of the count data.

Level of maggot infestation between individual cultivars was evaluated using a general linear model at α = 0.05.

Figure 4.1: Damage caused to apples by apple maggot fly oviposition and infestation. (a) Mature apple showing multiple sting marks. (b) Mature apple cut into quarters along X and Y axes. Presence of apple maggots was confirmed by observation of trails burrowed through flesh of apples (brown track marks).

4.1.2 Y-tube Bioassays

To compare the patterns of cultivar choice observed in the field against cultivar discrimination in a laboratory, two-choice behavioural bioassays were performed in 2013 using adult apple maggot fly females (Fig. 4.2). Test insects were collected from trees within entomological research orchards at the AFHRC using an aspirator (Fig. 3.1). Six volatile blends were used in this series of bioassay. Extracts were used in subsequent bioassays and electrophysiological studies.

Volatile blends from the cultivars were categorized as ‘preferred’ and ‘non- preferred’. ‘Preferred’ versus ‘non-preferred’ blends were determined based on 2012 field data of visitation and infestation by R. pomonella. Preferred blends were Zestar!,

Pinova, COOP 39 and Royal Gala. The ‘non-preferred’ blends used were 8S-27-43 and

Ambrosia. Odour sources for the bioassay were created by pipetting 2 μl of volatile extract on 0.5 cm x 3 cm strips of filter paper. A pentane control was used. Fresh collections of strips (Chapter 3) treated with volatile blends were made every 5-7 days and stored at -20°C until use. Unused strips were removed from storage and utilized for each day’s trials. After each trial, the Y-tube assembly was rinsed with 70% ethanol and wiped with KimTech Science* Delicate Task Kimwipes® (Kimberly-Clarkson

International, Roswell, GA) and allowed to air dry. The position of the filter papers was switched after each trial (e.g. in one trial, COOP 39 would be in the right arm and a pentane control in the left; after rinsing, the next trial would occur with COOP 39 in the left and pentane control in the right arm). Each trial was run using a clean set of junction arms and central tube.

a b c

Figure 4.2: A Rhagoletis pomonella female explores a junction tube during a Y-tube bioassay. (a) central tube, b) lower junction arm with maggot fly, c) upper junction arm with separating screen and filter paper.

The Y-tube assembly described in Chapter 3 was used for the trials with R. pomonella. Each apple maggot was given 8 minutes per trial. This number was based on preliminary trials run earlier in 2013 in which female apple maggots were given 15-20 minutes within a Y-tube. Apple maggot flies either reacted within 8 minutes or did not make a choice between volatiles in the observed time period.

Light levels at each junction of the Y-tube assembly were monitored using a Sper

Scientific Advanced Light Meter 840022 (Sper Scientific®, Scottsdale, AZ). Light levels were consistent at approximately 360 Lux and varied by no more than 2 Lux between each arm. Relative humidity in the test room was between 60 and 80% and temperature was consistent at 22-23°C, which is optimal for R. pomonella (Nielson, 1962; Nielson,

1964; Nielson, 1965). Bioassays were carried out during apple maggot active periods: no earlier than 9:00 AM and no later than 4:00 PM.

4.1.3 Calcium Imaging

Olfactory responses of apple maggot flies to host plant volatile blends at the antennal lobe level was evaluated with calcium imaging. Adult apple maggots were chilled at -20°C for 5-15 minutes to incapacitate, but not kill, them prior to preparation.

They were then inserted into guillotine blocks and secured in place with dental wax (Fig.

4.3). A window exposing the brain in the apple maggot’s head was opened between the orbital bristles, from the frontal suture to the ocelli using a scalpel. The brain cavity was treated with a saline solution to prevent dehydration. Obstructing membranous tissue was gently removed with fine forceps to expose the antennal lobes of the brain. Calcium green fluorescent probe (50 μL of the following: 50 μg calcium green dissolved in 50 μL

Figure 4.3: Female apple maggot fly secured in guillotine block with dental wax.

of pluronic detergent from Invitrogen (Product No. P3000MP) and 950 μL of saline) was applied to the brain using a micropipette and the fly was then set into an ice cooler for 45 minutes to 2 hours. After this absorption period, excess calcium green was washed away with fresh saline and a coverslip placed over the insect’s head, taking care not to wet the antennae. A drop of saline was placed between the objective lens and the coverslip to increase image resolution.

The calcium imaging apparatus was set on a Gibraltar mount which itself was on a Kinetic Systems® Vibraplane (Boston, MA) floating table to prevent vibration (Fig.

4.4). The insect was viewed from above through an Olympus (Tokyo, Japan) BX61W1 binocular microscope at 20x. Images were recorded with a Photometrics (Tucson, AZ)

Evolve® 512 camera. The stimulus was provided via humidified airflow controlled with a Dagan (Minneapolis, MN) Cornerstone Valve Mate-2® to divert airflow from the odour port when the insect’s antennal lobes were not being directly stimulated.

MetaFluor® Basic software (Olympus, Tokyo, Japan) was used to collect and analyze the responses of the selected area of the antennal lobes to each stimulus.

Response was measured by the change in fluorescence between ROIs 1-5 and the control

ROI. Fluorescent emission of Calcium green was measured after excitation from a Sutter

Instrument (Novato, CA) Lambda DG-4® polychromator. A Semrock® (Rochester, NY)

GFP-3035B cube was used, filtering for wavelengths of 472/520 nm and a bandwidth of

30/35 nm.

Six volatile blends were chosen for the calcium imaging study, based on apple maggot responses in visitation and infestation: Zestar!, Pinova, Ambrosia, 8S-27-43,

Royal Gala and COOP 39. Butyl hexanoate was used as a positive control, as described

Figure 4.4: Set-up of calcium imaging apparatus at Chemical Analysis and Bio-imaging Laboratory, Acadia.

by Fein et al. (1982), in which this odourant elicited positive responses from R. pomonella. Odour sources were created following the procedures in Syntech (2004), by placing 2 μL of volatile extract on a 3 x 0.5 cm strip of filter paper (Chapter 3). Each strip was inserted into a 5 ¾” disposable Pasteur pipette (13-678-6G, Allied Fisher-Scientific,

Washington, DC) which was then connected to the air supply, with the narrow end of the pipette aimed beneath the coverslip fragment, so that the airflow would pass over the insect’s antennae. The end of the pipette was positioned approximately 0.5 cm from the antennae. A 10-second period of recording was measured with a 0.5 second ‘puff’ of odour occurring at approximately 3 seconds after recording’s start and timed by the

MetaFluor® software. Time between each 10-second recording was 30 seconds to 1 minute to prevent habituation and adaptation.

Six regions of interest (ROI) were measured per imaging process. Regions 1-5 were placed over the exposed antennal lobes. The 6th ROI was a control region – a section of stained nerve tissue that was not connected to the antennal lobe. This region varied in location between individual flies, depending on the amount and location of exposed brain tissue. The placement of the other ROIs was consistent between recordings. Responses were analyzed with the MetaFluor® program and differences in emitted wavelengths from stained brain tissues were compared against the reaction of the

6th ROI. Only regions with enough repetitions to be analyzed statistically were evaluated.

Data from 18 female flies using the right dorsal, left ventral, left dorsal antennal lobe and data from the right dorsal and left ventral sections of the antennal lobes of 6 males were used to compile a pattern of intensity of response to specific volatile blends.

4.1.4 Data Analysis

As the apple maggot fly visitation and maggot infestation information was count data, the data were evaluated with Kruskal-Wallis non-parametric tests to evaluate the level of significance of the apple maggot visitation and infestation rates. Differences between visitations of cultivars were evaluated with a parametric generalized linear model. Levels of apple maggot infestation were evaluated with a Kruskal multiple comparison test. These tests were run in the statistical software program R. Responses in the 2013 series of Y-tube bioassays were analyzed using Chi-square tests to determine the statistical significance of positive responses between volatile blends in these behavioural bioassays, with H0 = 0.5/n. First-choice responses and total responses were evaluated independently. One-way ANOVAs were performed on the level of response to volatile blends in the calcium imaging studies. The null hypothesis for these analysis was that there would be no variation in responses between cultivars: H0 = x1 = x2 = xn. An α of 0.05 was used in all statistical analyses. Sexes were evaluated separately on the assumption that male and female sensitivity to volatile blends may be different based on the additional needs females have of host plants.

4.2 Results

4.2.1 Adult visitations

The pattern of cultivar preference in Rhagoletis pomonella was largely consistent each year, despite higher overall numbers of flies recorded in 2013. In 2012, 471 apple maggot flies were observed throughout the entire survey period. In 2013, observed apple maggots flies totalled 2,044. Because method, number and timing of field surveys were

consistent in each year’s field survey, the increase in apple maggot flies may be the result of a larger population in 2013 than in 2012.

Apple maggot fly behaviours were categorized as follows: visitation, mating and oviposition. Males were observed in higher numbers earlier in the day; females were found in higher numbers once the temperature reached >10°C. Apple maggot flies were not observed feeding. Male behaviours included waiting on the apples for a female to arrive, initiating courtship and attempting to mate. Observations of apple maggot fly copulation indicated that it could take up to 30 min and would occur in the field, or in the collection jars. Females would examine the surface of an apple before locating a suitable site for oviposition and use their ovipositor to pierce the fruit’s skin. They would repeat this procedure several times on a given fruit, or take flight and browse/visit other apples on the same tree. Females were observed attempting additional ovipositing upon/marking fruit that had already been stung.

Average mean visitations by apple maggots increased between 2012 and 2013 as did the total observed apple maggot population, (Table 4.1). Despite a 434% rise in apple maggot fly population, the pattern of R. pomonella preferences remained consistent between years (Fig. 4.5). Silken was the second most-preferred cultivar in 2012 and most preferred in 2013, with 9.43 and 78.7 mean maggot fly visits during the survey period respectively. Summer and Rogers McIntosh were the least preferred cultivars during each year. In 2012, comparatively few apple maggots were observed on Summer McIntosh, with 1.75 mean visitations. An average of 0.75 R. pomonella were noted on Rogers

McIntosh in 2012. In 2013, no apple maggots were observed on Summer McIntosh; mean visitations on Rogers McIntosh trees were 3.75 apple maggot flies (Fig. 4.5).

Table 4.1: Summary of Kruskal-Wallis analysesTotal of 2012 and 2013 apple maggot visitation data. Apple Mean Maggot Visitations Per Flies (N) Cultivar SE H P

Year * Cultivar 2515 17.75 0.09 34.8 3.59E-09

Cultivars (2012) 471 6.48 0.05 28.5 1.53E-03

Cultivars (2013) 2044 29.01 0.13 49.2 3.75E-07

Figure 4.5: Cultivar comparison between 2012 (p <0.001) and 2013 (p < 0.0001) for mean apple maggot fly visitations Cultivars sharing the same letter are not significantly different in apple maggot fly visitations at α = 0.05.

In both years, a small number of cultivars were highly preferred for visitation, with a gradual decrease in number of visitations among the remaining surveyed cultivars

(Fig. 4.5). In this mixed orchard, cultivars were considered to belong to one of three categories, based on the mean number of apple maggots that visited each cultivar: highly preferred, average preferred and least preferred e.g. in 2012, 8S-27-43, Ambrosia, Jubilee

Fugi, Royal Gala and Silken could be considered highly preferred; in 2013, only Silken was demonstrably visited more often than any other cultivar. In both 2012 and 2013,

Summer and Rogers McIntosh were rarely visited by apple maggot flies and were the least attractive to R. pomonella during each field season (Fig. 4.5).

Analysis of the 2012 and 2013 visitation data indicated a non-random pattern to apple maggot fly preferences (Table 4.1). For each field season, the results of Kruskal-

Wallis analysis indicated a statistically significant difference in the mean visitations across all cultivars (χ2 (2012) = 28.5, p <0.01, χ2 (2013) = 49.2, p <0.01) and a significant variation in cultivar visitations between years (χ2 (2012*2013) = 34.9 , p <0.01) (Table

4.1), indicating a significant effect of cultivar on apple maggot fly visitation.

A pairwise comparison of 2012 visitation data showed Pinova, Rogers McIntosh,

Summer McIntosh and 8S-27-43 had different levels of apple maggot fly visitation when compared to other cultivars (Fig. 4.5 & Table 4.2). 8S-27-43 was the most-preferred cultivar in 2012 and its mean apple maggot fly visitations was significantly different from all other cultivars except Silken and Royal Gala (Fig. 4.5). In 2013, differences among mean visitations were significant between a greater number of cultivars, indicating a more clear pattern of apple maggot fly preference among cultivars (Table 4.3).

Table 4.2: Pairwise comparison of statistical significance of 2012 apple maggot visitation between cultivars using results of z-test from general linear model and level of significance. Jubilee Rogers Royal s23-06- Summer 8S-27-43 Ambrosia COOP 39 Fugi Pinova McIntosh Gala 153 Silken McIntosh

Ambrosia -4.7d

COOP 39 -4.17c 0.67 Jubilee -2.45 2.45 1.82 Fugi

Pinova -5.64d -1.56 -2.19 -3.77c Rogers -5.02d -3.10a -3.43b -4.21c -2.19 McIntosh Royal -2.83 2.66 2.00 0.03 3.98c 4.27d Gala s23-06- -4.97d 0.74 0.00 -2.07 2.34 3.49b -2.34 153

Silken -2.61 2.70 2.05 0.13 4.00c 4.29d 0.11 2.38 Summer -7.37d -3.14a -3.81c -5.44d -1.45 1.33 -5.71d -4.06c -5.72d McIntosh

Zestar! -3.07c 1.17 0.50 -1.34 2.64 3.66b -1.48 0.56 -1.55 4.27d a Difference between cultivars significant at p < 0.1 b Difference between cultivars significant at p < 0.05 c Difference between cultivars significant at p < 0.01 d Difference between cultivars significant at p < 0.001

Table 4.3: Pairwise comparison of statistical significance of 2013 apple maggot visitation between cultivars using results of z-test from general linear model and level of significance. Jubilee Rogers Royal s23-06- Summer 8S-27-43 Ambrosia COOP 39 Pinova Silken Fugi McIntosh Gala 153 McIntosh

Ambrosia -2.98a 9.091d -3.058a 7.903d -4.573d 7.378d 7.862d 13.635d -6.041d

COOP 39 8.12d 9.09d -10.56d -1.514 -9.562d -2.945a -2.296 6.44d -13.116d Jubilee -5.78d -3.06a -10.56d 9.647d -2.221 9.23d 9.599d 13.922d -2.877 Fugi

Pinova 6.49a 7.90d -1.51 9.65d -8.981d -1.24 -0.596 7.882d -12.283d Rogers -6.42d -4.57d -9.56d -2.22 -8.98d 8.649d 8.885d 11.632d -0.107 McIntosh Royal 5.81d 7.38d -2.94a 9.23d -1.24 8.65d 0.729 10.475d -11.939d Gala s23-06- 6.49d 7.86d -2.30 9.60d -0.60 8.88d 0.73 9.842d -12.274d 153

Silken 14.96d 13.64d 6.44d 13.92d 7.88d 11.63d 10.48d 9.84d -16.156d Summer -8.77d -6.04d -13.12d -2.88 -12.28d -0.11 -11.94d -12.28d -16.16d McIntosh

Zestar! 1.43 3.96c -5.87d 6.51d -4.45d 6.94d -3.68c -4.27d -11.58d 9.37d a Difference between cultivars significant at p > 0.1 b Difference between cultivars significant at p > 0.05 c Difference between cultivars significant at p > 0.01 d Difference between cultivars significant at p > 0.001

4.2.2 Infestation

A pattern of apple maggot infestation and cultivar preference was observed in this study. As with visitation, Silken was highly preferred for infestation by R. pomonella. In

2012, 77.3% of Silken apples were infested by apple maggots (Fig. 4.6). In 2013, 100% of all surveyed Silken fruit showed indications of infestation by apple maggots.

Numerous sting marks and the presence of internal damage were present in both years

(Fig. 4.6). Other patterns were observed from these data: consistent with their low levels of visitations, both Summer McIntosh and Rogers McIntosh were lightly infested compared with other cultivars in both 2012 and 2013 (Fig. 4.6).

Infested harvested apples were observed with multiple stings – in some cases, individual apples would have >10 sting marks from oviposition attempts by female maggot flies (Fig. 4.1a). Anecdotally, fruits that had been infested by other insect species were found to have apple maggot stings (e.g. apples with C-scars from H. testudinea larvae could also be infested by R. pomonella (Figs. 1.5c & 4.1a)), indicating that infestations by other species and conspecifics were not inhibitory to oviposition by apple maggot flies. Upon dissection, several apples had multiple apple maggots within. A number of these apples were so infested that the flesh had rotted and become soft.

Infestation levels differed significantly among cultivars in each year, indicating a level of preference in R. pomonella’s selection of host plants for oviposition. The

Kruskal-Wallis test revealed a significant effect of cultivar on apple maggot fly infestation in 2013 (χ2 (2013) = 32.6 , p <0.01) and variation in infestations between years (χ2 (2012*2013) = 30.8, p <0.01) (Table 4.4). These results support the observed preferences noted with apple maggot visitations in each year (Fig. 4.5 & Table 4.1).

Figure 4.6: Comparison of rates of apple maggot infestation between cultivars in 2012 (p > 0.05) and 2013 (p < 0.001) years. Cultivars sharing the same letter are not significantly different in apple maggot infestations at α = 0.05.

Table 4.4: Summary of Kruskal-Wallis analyses using 2012 and 2013 apple maggot infestation data from field surveys.

Total Mean Apples infested Surveyed fruit per (N) cultivar (%) SE H P

Year * Cultivar 2303 67.78 1.74 30.8 1.20E-03

Cultivars (2012) 1742 65.74 2.61 17.1 0.072

Cultivars (2013) 561 69.83 0.88 32.6 3.13E-04

4.2.3 Y-tube Bioassays

Most responses to odour sources by R. pomonella were within 1-2 minutes of exposure; no response took longer than 8 minutes. Apple maggot responses to the volatile blends were in line with results from field observations and infestation surveys; the ‘more preferred’ cultivars, Zestar! Pinova, Coop 39 and Royal Gala elicited more positive responses than Ambrosia and 8S-27-43. The pentane control was infrequently chosen when any other odour source from an apple cultivar was present (Fig. 4.7).

Chi-square analyses of apple maggot fly responses to Y-tube bioassays indicate a pattern of preference in insect choice. Female R. pomonella showed a consistent pattern of preference towards Zestar! and Pinova when compared to other odour sources (Fig. 4.7

& Table 4.5). These findings support the observations made in the field; apples collected from Zestar! and Pinova trees were consistently heavily infested (and therefore chosen as oviposition sites by female R. pomonella) (Fig. 4.6).

4.2.4 Calcium imaging

Responses from 18 female R. pomonella and 8 male maggot flies were successfully recorded. Responses were analyzed based upon which antennal lobe and region(s) exposed during dissection (Fig. 4.8). Most sections of the brain had too few individual replications for statistically significant analysis. In male flies, these regions were left ventral and right dorsal antennal lobes. In females, these regions of the antennal lobes were the left dorsal, left ventral and right dorsal sections.

Control N=8 Ambrosia b p = 0.005

Control N=8 Royal Gala

Ambrosia N=10 Royal Gala

Ambrosia N=12 COOP 39

Control N=7 COOP 39

c Control N=11 8S-27-43 p = 0.001

Control N=10 Pinova

a 8S-27-43 N=11 Pinova p = 0.035

a 8S-27-43 N=11 Zestar! p = 0.035 b Control N=10 Zestar! p = 0.002

100% 75% 50% 25% 0% 25% 50% 75% 100%

Apple maggot fly preferring stimuli

Figure 4.7: First choice responses of apple maggots to Y-tube behavioural bioassay. Letters on comparisons indicate significance as determined by Chi-square analysis. a Choice of apple maggot responses to volatile blends significantly different (Chi square p < 0.05) b Choice of apple maggot responses to volatile blends significantly different (Chi square p < 0.01) c Choice of apple maggot responses to volatile blends significantly different (Chi square p <= 0.001)

Table 4.5: Results of one-way Chi-squared analysis of 2013 bioassays using first responses of apple maggot flies Stimulus 1 Stimulus 2

Stimulus 1 Stimulus 2 N df Chosen Chosen χ2 p Pinova Control 10 1 8 2 3.6 0.058 Pinova 8S-27-43 11 1 9 2 4.45a 0.035 Zestar! Control 10 1 10 0 10b 0.002 Zestar! 8S-27-43 11 1 9 2 4.45a 0.035 8S-27-43 Control 11 1 11 0 11c 0.001 COOP 39 Control 7 1 5 2 1.29 0.257 COOP 39 Ambrosia 12 1 9 3 3 0.083 Royal Gala Control 8 1 6 2 2 0.157 Royal Gala Ambrosia 10 1 7 3 1.6 0.206 b Ambrosia Control 8 1 8 0 8 0.005 a Choice of apple maggot responses to volatile blends significantly different (Chi square P < 0.05) b Choice of apple maggot responses to volatile blends significantly different (Chi square P < 0.01) c Choice of apple maggot responses to volatile blends significantly different (Chi square P < 0.001) 89

1 1 4 3 3 4 2 2

Figure 4.8: Apple maggot fly brain, proximal anterior view. All surrounding tissues have been removed. Regions containing antennal lobes have been highlighted. Numbered areas indicate regions surveyed by calcium imaging 1) dorsal, 2) ventral, 3) medial and 4) lateral. Magnification is 100x.

Stimulation of the antennae by the volatile blends resulted in a consistent pattern of positive responses. Responses were observed in targeted antennal lobes in all successful recordings, although individual nerves and glomeruli could not be differentiated at the resolution used. There was no consistent pattern of preference in the apple maggot fly responses to the volatile blends based on region of interest (ROI;

P<0.05). Figure 4.9a-d indicates the pattern of ROI placement on apple maggot antennal lobes (exact location of ROI 6 varied by individual).

Lowered response to butyl hexanoate supports previous research; although attractive to R. pomonella (Zhang et al., 1999; Nojima et al., 2003), individual synthetic chemicals were not found to elicit as strong a behavioural response in R. pomonella as composite odour blends (Zhang et al., 1999; Rull & Prokopy, 2005). This behavioural sensitivity towards entire volatile blends indicates that composite odour sources should elicit stronger reactions in apple maggot fly brain tissue when compared to single odourants. This is consistent with apple maggot fly reactions; responses to butyl hexanoate were consistently less intense than those triggered by exposure to the volatile blends.

Differences in sensitivity to the volatile blends were observed in several regions of R. pomonella’s antennal lobes. In male apple maggot flies, these regions were the ventral left antennal lobe and dorsal right antennal lobe (Table 4.6). No significant differences between volatile blends based on ROI were observed in male maggot flies.

Female R. pomonella only showed significant differences in their sensitivity among volatile blends in the dorsal and ventral sections of the left antennal lobe and the dorsal right antennal lobe (Table 4.7).

a b

c d

Figure 4.9: Pattern of region of interest placement on ventral sections of antennal lobes. Numbers correspond to ROI. (a) Dorsal placement, left lobe (b) Ventral placement, left lobe (c) Medial placement, right lobe (d) Lateral placement, right lobe.

Table 4.6: Summary of two-way analysis of variance evaluating male maggot fly responses to odour blends across mapped regions of interest. Responses from regions of interest (ROI) 1-5 were standardized against region of interest 6. Antennal Section df Sum of F Ratio Prob > F Lobe Squares ROI Left Ventral 4 3462.110 0.062 0.993 Odour Left Ventral 5 118573.290 1.707 0.148 ROI * Odour Left Ventral 20 9283.780 0.033 1.000 ROI Right Dorsal 4 593.882 0.048 0.996 Odour Right Dorsal 5 74288.358 4.778 0.0025* ROI * Odour Right Dorsal 20 1750.290 0.028 1.000

Table 4.7: Summary of two–way analysis of variances evaluating female maggot fly responses to odour blends across mapped regions of interest. Responses from regions of interest (ROI) 1-5 were standardized against region of interest 6. Antennal df Sum of F Ratio Prob > F Section Lobe Squares ROI Left Ventral 4 2927.64 0.20 0.94 Odour Left Ventral 5 25952.00 1.43 0.22 ROI * Odour Left Ventral 20 10166.84 0.14 1.00 ROI Left Dorsal 4 53337.69 0.29 0.88 Odour Left Dorsal 4 264642.94 1.45 0.22 ROI * Odour Left Dorsal 16 14554.57 0.02 1.00 ROI Right Dorsal 4 4621.14 0.13 0.97 Odour Right Dorsal 5 69047.03 1.51 0.19 ROI * Odour Right Dorsal 20 2273.09 0.01 1.00

No differences by cultivar per section were observed across ROIs. In the dorsal right antennal lobe, intensity of response to the volatile blend Ambrosia was significantly

-9 different from other extracts (p = 6.1, pcrit= 6.3E . The ventral left antennal lobe showed

-11 a significant response to Ambrosia (p = 7.4, pcrit=1.08E ). Intensity of response to

-7 Pinova (p= 2.2, pcrit = 0.027) and Ambrosia (p =5.4, pcrit = 1.1 E ) was significantly different from all other odour blends in the dorsal left antennal lobe.

Although no other volatile blends provoked a significant response at p < 0.05 by female R. pomonella, a general trend can be noted in the results. Responses to Pinova were consistently among the highest at each of the three indicated sections and responses to butyl hexanoate were generally lower than those of other volatile extracts (Fig. 4.10).

This is consistent with observed preferences for Pinova for visitation and oviposition by adult apple flies and infestation by maggots.

Based on the results from field surveys, Y-tube bioassays and calcium imaging, the most preferred cultivars for this population of apple maggot fly are Silken, Zestar! and Pinova. The least preferred cultivars are Rogers and Summer McIntosh (Table 4.8).

a b c

Figure 4.10: μ responses in ∆ intensity (nm) of female apple maggot flies by region of interest against volatile blend. (a) μ responses in ∆ intensity (nm) at lower left antennal lobe (P = 1.43, N=4). (b): μ responses in ∆ intensity (nm) at upper right antennal lobe (P = 1.511, N=5). (c): μ responses in ∆ intensity (nm) at upper left antennal lobe (P = 1.45, N=9). 96

Table 4.8: Summary of cultivar preferences (or sensitivity) observed in Rhagoletis pomonella across all studies. Preferred cultivars are ranked most preferred to least preferred, and non-preferred cultivars are ranked least preferred to most preferred. Preferred Non-preferred Study Cultivars Cultivars 1. Silken 1. Summer McIntosh Visitation Survey 2. s23-06-153 2. Rogers McIntosh 3. Pinova 3. Jubilee Fugi 1. Silken 1. 8S-27-43 Infestation Survey 2. Pinova 2. Rogers McIntosh 3. Royal Gala 3. Summer McIntosh 1. Zestar! 1. Ambrosia Y-tube Bioassay 2. Pinova 2. 8S-27-43 3. Royal Gala 3. Pentane Control 1. Pinova 1. Butyl Hexanoate Ca2+ Imaging 2. Zestar! 2. Ambrosia 3. COOP 39

5. Discussion

5.1 European Apple Sawfly

Apple sawflies exhibited a cultivar preference in visitation, larval infestation and sensitivity in electrophysiological tests (Figs. 3.6, 3.7 & 3.9). This suggests that growers planning to avoid damage from apple sawflies may wish to focus on these non-preferred cultivars as a primary crop, using preferred cultivars in a push/pull control strategy, drawing apple sawflies from the less-preferred trees to the preferred cultivars, which then can be targeted with control methods. Investigation into the volatiles of preferred versus non-preferred cultivars can also provide options for growers by isolating the attractant semiochemicals for use in trapping and monitoring these insects.

The results from the visitation and infestation surveys, the behavioural bioassays and EAGs all support the primary hypothesis that H. testudinea exhibits variation in cultivar preference among apple trees for feeding and oviposition and that this preference is influenced by olfactory cues. The differences in H. testudinea’s cultivar preference may reflect temporal factors in addition to other physiological influences, such as the volatile chemicals emitted by developing apple blossoms and fruitlets and the presence and abundance of food sources.

Miles (1932) suggested that while the timing of apple bloom influenced infestation rate, it was less important in determining a cultivar’s attractiveness to H. testudinea than other host plant cues. Data from the 2012 and 2013 field surveys of apple sawfly visitations supports this interpretation. Several early-blossoming cultivars had an initially high number of visiting apple sawflies, but as more apple trees blossomed and

more options were available to feeding/ovipositing apple sawflies, the visitations on these early-blossoming cultivars decreased (Figs. 3.4 & 3.5).

Silken is an early-blossoming cultivar, maturing 2 weeks ahead of McIntosh cultivars (Wilson, 2001). In 2012, this cultivar was rarely visited by adult sawflies, despite being available from the beginning of apple bloom. In 2013, however, Silken trees had a large increase in mean apple sawfly visitations (Fig. 3.6). The difference in visitation rates among Silken is likely the consequence of an increased opportunity to visit in 2013. None of the Silken trees blossomed across all branches in 2012. Several

Silken trees partially blossomed across several branches, but no Silken trees were as productive in 2012 as in 2013. As a consequence, an increase in sawfly visitation was both expected and observed due to the greater prevalence of Silken blossoms (and food sources) within the orchard. Although sawfly visitation to Silken increased in 2013, the cultivar remained of relatively low preference compared to other apple varieties within the orchards studied (Fig. 3.6).

Availability of a cultivar was not the sole determining factor in host plant choice;

Jubilee Fugi blossomed relatively early (Table 2.1) and was consistently among the least- visited cultivars in 2012 and 2013 (Fig. 3.6). Later-blossoming cultivars such as Pinova

(Orange Pippin Ltd., 2014b) and s23-06-153 (Hampson et al., 2008) were among the top three cultivars visited by H. testudinea in both orchards (Fig. 3.6). In 2012, H. testudinea was observed from May 8 to May 25. Sawflies visited trees as soon as blossoms opened.

Zestar! was visited throughout the entire active period. Pinova was visited from May 14 and until May 22. s23-06-153 was first visited on May 16 and was visited until the end of apple bloom. A similar pattern was observed in 2013; apple sawflies were seen between

May 14 and May 30. Zestar! was first visited on May 14 and continued to be throughout apple bloom. Some Pinova bloomed as early as May 17 and were visited until May 30, although peak visitation for this cultivar was May 24. s23-06-153 bloomed late, with the earliest observed visitation on May 22, and was visited until the end of bloom.

A possible confounding issue in this study is the timing of apple bloom; in 2012, all cultivars blossomed within days of each other, providing a wide selection of choices for apple sawflies. In 2013, apple bloom took longer and the time between the bloom of early-blossoming and later-blossoming cultivars was more than a week: as early- blossoming cultivars, Zestar! and Silken, were among a very few cultivars with open blossoms for several days (Fig. 3.5). This resulted in fewer options to apple sawflies and led to variation in preferences between years, e.g. higher levels of visitation on normally less-preferred earlier-blossoming cultivars (Figs. 3.5 & 3.6).

The lack of productivity among certain cultivars may have influenced sawfly choice in 2012 via reduction of options for food sources and oviposition sites for emergent adult sawflies, leading to the apparent increase in preference for Silken. This does not explain the similarity in mean visitations on Jubilee Fugi between 2012 and

2013; in the latter year, Jubilee Fugi trees did not produce many blossoms or fruitlets.

Several Jubilee Fugi trees produced fewer than 10 mature apples. Mean sawfly visitations remained consistent between years, although the percentage of infested Jubilee Fugi fruitlets decreased in 2013 (Fig. 3.6).

Results from the visitation surveys indicate that European apple sawfly chooses to visit less-preferred cultivars if few alternatives are present, but will readily abandon otherwise suitable food sources and oviposition sites on earlier-blossoming cultivars once

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other options become available. Apple sawflies exhibit a consistent preference towards specific apple cultivars, but not to the extent that they will ignore other available host choices, particularly if there is a limited supply of food sources and oviposition sites. This would explain the increased preference for Silken in 2013; at the beginning of apple bloom, fewer food sources were available to the apple sawfly population, leading to increased visitation of the cultivars that were in bloom. As more apple trees blossomed, more choice in feeding and oviposition sites became available, which led to the shift in apple sawfly visitations as blossoming continued (Fig. 3.5). These data supports known and described patterns of apple sawfly behaviours: susceptibility to infestation is not necessarily linked with timing of apple bloom (Miles, 1932). As the time of bloom does not determine apple sawfly preference, these insects must rely on additional host plant characteristics such as olfaction to differentiate between cultivars in determining preference for feeding and oviposition.

Cultivar preference in H. testudinea was observed in infestation across the apple cultivars in this study (Table 3.5). The pattern of apple sawfly responses to each cultivar in this study remained consistent in 2012 and 2013. Cultivars that were heavily infested in 2012 remained preferred in 2013 and apple cultivars that were less infested in 2012 were again relatively lightly damaged (Fig. 3.7) in 2013, establishing an observable and repeatable trend of host plant choice in these field surveys. This confirms observations made by Downes (1944), where sawflies were noticed to consistently avoid less preferred cultivars when provided a choice of host plants, including more preferred cultivars.

The overall trends of oviposition and infestation remained consistent across all cultivars in this study in each year, although there was no discernible explanation for the

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observed pattern of preferences to the cultivars infested by H. testudinea. Early blossoming cultivars exhibited a range of preferences for infestation by H. testudinea larvae, as did late blossoming cultivars (Fig. 3.7). Zestar! is early-blossoming, and at maturity produces sugary-sweet, softer-fleshed fruit (University of Minnesota (UM),

2010). COOP 39 is early developing, with crunchy and crisp-fleshed apples (Janick et al.,

2006) and s23-06-153 is a later-developing cultivar (Hampson et al., 2008). The least- infested cultivars included Silken, which is early blossoming and highly aromatic

(Wilson, 2001) and Ambrosia which is also highly aromatic (British Columbia Ministrry of Agriculture (BCMA), 2013). Based on this information, fruitlet flesh appears not to have had an impact on host plant choice. Sweeter cultivars of apple such as Zestar!,

Pinova, Ambrosia and Silken all showed wide distribution in preference and level of larval infestation (Fig. 3.7). COOP 39 is a harder-fleshed apple (Janick et al., 2006) and was amongst the two most infested cultivars, while varieties with traditionally softer flesh had lower mean infestations. At the fruitlet stage, the variation in texture and flesh between cultivars is not fully developed (Yahia, 1994; Blatt, pers. comm., May, 2014), making differences between the fruit difficult to gauge for their attractiveness and susceptibility to infestation. Minimal research has been conducted in this area and information on the differences, if any at this stage of development, between apple cultivars is not available.

Cultivars selected by H. testudinea for visitation were frequently chosen to the same degree for oviposition and infestation; e.g. Zestar! and s23-06-153 were heavily infested by sawfly larvae and were highly preferred for visitation (Figs. 3.6 & 3.7).

COOP 39 showed the most drastic difference in apple sawfly preference between

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visitation and infestation. Counter to the observed number of visitations, COOP 39 had high rates of infestation by H. testudinea larvae in 2012 and 2013. These shifts in preference suggest that a host plant’s attractiveness to adult apple sawfly visitation influences, but does not solely determine, the degree to which oviposition and infestation occurs. Selection of fruitlets is vital to the survival of apple sawflies as the larvae can suffer high mortality after hatching as they attempt to burrow into the fruitlet’s ovary

(Miles, 1932). Oviposition on fruitlets belonging to less-susceptible cultivars may increase the death rate at this stage in the life cycle. Cultivars that were suitable as food sources for apple sawflies may not have produced fruitlets suitable for the survival of larvae, or vice versa.

Mature fruit collected from Jubilee Fugi trees showed the highest rate of post- infection survival and therefore the highest mortality among sawfly larvae. In 2012 and

2013, more apples showing evidence of failed infestations by H. testudinea were recovered from this cultivar than any other. Heavily damaged fruitlets fall to the ground, while fruit that survives infestation remains on tree (Weires, 1991). There were no significant differences in the rate of survival of infested fruit from other cultivars (Fig.

3.8). Mature Jubilee Fugi apples are a sweet, crisp fruit (E.C. Brown’s Nursery, 2014a).

The acidity, toughness and chemical composition of Jubilee Fugi fruitlet flesh may be factors that contribute to the higher rate of mortality of H. testudinea larvae observed in

Jubilee Fugi fruitlets compared to the other cultivars in this study .

Increased availability of food sources and oviposition sites influences the rate of visitation by H. testudinea. The results show that in the absence of other food sources and oviposition sites, apple sawflies will gravitate towards blossoming cultivars, regardless of

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preference. Female apple sawflies have a limited window in a blossom’s development to lay their eggs (Graf et al., 1996) and must make use of the available food sources and oviposition sites. Once more-preferred cultivars bloom, H. testudinea’s interest shifts to these trees over already-available earlier blossoming cultivars. Number of blossoms may also play a role in rate of visitation by apple sawflies; a minimum number of blossoms may be necessary to attract adult apple sawflies. In 2013, Jubilee Fugi trees with few blossoms attracted few sawflies throughout the entire active period; the two Jubilee Fugi trees with the highest number of blossoms accounted for all the apple sawflies observed.

This was similar to what was indicated in 2012; prior to the bloom of other, more preferred cultivars, only those Silken trees with the most blossoms were visited by apple sawflies. This shift in visitation and infestation rates has been described previously in

Downes (1944) and may indicate the vulnerability of a given cultivar by year. In mixed orchards, apple sawflies show a clear preference for certain cultivars, although no information on a minimum number of blossoms per tree was described.

Depletion of food sources, age of blossoms and/or the presence of conspecifics may contribute to the susceptibility of a cultivar to visitation by apple sawflies (Miles,

1932; Roitberg & Prokopy, 1984). Other environmental factors, such as olfactory, visual and/or gustatory cues likely inform the selection of host cultivars in apple sawfly visitations and influence the susceptibility of each cultivar to H. testudinea. As indicated by the bioassay results, olfaction is used by H. testudinea in selecting host plants and differentiating between cultivars.

The effect of olfactory cues in host plant preference has been noted in species from other sawfly families such as Cephidae with the wheat steam sawfly Cephus cinctus

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(Norton) (Piesik et al., 2008; Weaver et al., 2009) and other Tenthredinidae sawflies, such as the yellowheaded spruce sawfly, Pikonema alaskensis (Rohwer) (Bartelt et al.,

1982; Cossé et al., 2002). These species of sawfly are influenced in their selection of host plants by a combination of volatile chemicals emitted from the host plants and conspecifics (Bartelt et al., 1982; Cossé et al., 2002; Piesik et al., 2008). Craig et al.

(1988) indicated that the tenthredinid stem-galling sawfly Euura lasiolepsi (Smith) avoided ovipositing on naturally and artificially scarred host plants. Female apple sawflies demonstrate similar avoidance mechanisms to host plants showing signs of previous oviposition and infestation (Roitberg & Prokopy, 1984), although the mechanism by which the tenthredinids E. lasiolepi and H. testudinea determine the suitability of host plants is unknown (Roitberg & Prokopy, 1984; Craig et al., 1988).

A combination of cues from infested/uninfested blossoms, female sex pheromones and host plant volatiles may therefore act in conjunction to drive host plant preference in H. testudinea. Sex pheromones are produced in many sawfly species, including those in the Tenthredindae. These are largely produced by female sawflies

(Cossé et al., 2002) and are used to attract mates (Bartelt et al., 1982; Cossé et al., 2002).

No sex pheromone has been identified in H. testudinea, although its preference has been previously inferred by apple sawfly behaviours (Miles, 1932; Boeve, 1999). Male H. testudinea become agitated and extremely active in the presence of sexually mature female apple sawflies (Miles, 1932). Other olfactory cues that may influence apple sawfly visitation include changes to the volatile profile of blossoms/fruilets caused by H. testudinea oviposition (Boeve, 1996; Boeve et al., 1999). The role of host plant volatiles in determining cultivar choice in H. testudinea is indicated by the preferences shown in

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these field surveys and the behavioural and electrophysiological tests administered in this study.

Apple sawfly responses to volatile extracts in the behavioural bioassays suggest a role for olfaction in host plant discrimination in H. testudinea, confirming the preferences seen in the field surveys. Although the null hypothesis was not rejected in individual comparisons and there were no significant differences in the responses between most other blends, this is likely due to the low number of individuals tested. Zestar! and Pinova were chosen in higher proportions when compared against 8S-27-43, Ambrosia and the pentane solvent. These observations were consistent with choices by H. testudinea in the field: Zestar! and Pinova were heavily preferred for visitation and oviposition by adult apple sawflies, and for infestation by apple sawfly larvae. Zestar! exhibited a higher level of attraction to apple sawflies than Ambrosia in both visitation and infestation surveys.

Pinova was preferred more than 8S-27-43 for infestation (Fig. 3.7.) The pattern observed in the 2013 Y-tube bioassays (Fig. 3.9) supports the hypothesis that apple sawflies exhibit some level in choice in the selection of host plants for feeding and oviposition and that when given a choice between more-preferred and less-preferred cultivars, apple sawflies will respond positively to the more-preferred cultivar. Volatile extracts elicited a higher rate of positive reactions from apple sawflies than cut blossoms did (Fig. 3.9). Using cut blossoms, 104 responses out of 175 were noncommittal. When volatile blends were used, only 60 reactions out of 177 trials were nonresponsive.

The lack of significant responses to the 2012 bioassays using apple blossoms may have been the result of changes in the blossoms’ odour upon removal from the tree; mechanical damage to blossoms can change the volatile profile that they emit (Dixon &

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Hewett, 2000). These changes in the blossom’s chemical profile may have affected the apple sawflies’ behaviour within the bioassay and the test insects no longer recognized the profile of a specific cultivar. Their responses were based upon the different volatiles that were presented. This may account for the increased preference for Silken in this bioassay when this cultivar was consistently less preferred in visitation and infestation field surveys when compared to cultivars such as Zestar! and Pinova(Figs. 3.6 & 3.7).

Another factor which may have negatively affected responses may have been the obstruction of the airflow due to blossom size, leading to less of the odour reaching the insect and consequently, less response to the blossom. These factors may have led to the high rate of null responses observed in the 2012 Y-tube bioassays.

Despite the low number of sawflies responding to any given comparison, the volatile extracts collected from preferred cultivars elicited positive responses by apple sawflies in the Y-tube bioassay, supporting the observed behaviours in the field.

Abdomen tapping seen in females during these trials supports the use of tactile evaluation of host cultivars; female apple sawflies exhibited this behaviour during the 2012 and

2013 bioassays when they were exposed to the odours from the apple blossoms and volatile extracts. This evaluation behaviour suggests that apple sawfly feeding and oviposition preferences may be further influenced by a combination of olfactory, visual and tactile cues.

Electrophysiological testing using EAGs further supported the use of olfaction in differentiating between cultivars and indicated a possible sex-based difference in attraction and sensitivity to odours. Female sawflies showed the greatest degree of sensitivity to volatile extracts from the Pinova and Ambrosia cultivars (Fig. 3.11).

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Intensities of responses to all other stimuli including the pentane control were approximately equal. Although an EAG only determines intensity of response to a stimulant and cannot be used to determine the level of preference to particular odourants

(Beck et al., 2012), the data collected from this source can be compared with observed sawfly behaviours in the field. In both 2012 and 2013, Ambrosia was one of the two least-preferred cultivars for infestation (Fig. 3.7) and consistently of less preference for visitation (Fig. 3.6). In addition, in both series of Y-tube bioassays using volatiles from

Ambrosia, the null hypothesis of random chance determining choice was rejected. This information, coupled with the results of the EAG analysis, suggests that the sensitivity to

Ambrosia may indicate a negative response to the odour; Ambrosia was consistently amongst the least preferred cultivars. The demonstrated sensitivity to Pinova may indicate a positive response to this volatile blend as Pinova was preferred for visitation and oviposition/infestation (Figs. 3.6 & 3.7). The weaker response to other blends may be an artefact of the low number of repetitions in this study and may also indicate that responses based upon volatile chemicals are only one discriminatory mechanism that H. testudinea uses in selection of host plants.

The stronger reaction of females to odour sources was consistent with the use of olfaction in the detection of food and oviposition sites among other insects (Boeve et al.,

1996). Both sexes of apple sawfly forage for food sources at apple blossoms, but sexually mature, non-virgin females choose oviposition sites and may have discriminated between volatile chemicals to a greater degree than males or virgin females as a means to evaluate the suitability of these sites (Roitberg & Prokopy, 1984). A greater sensitivity to plant

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volatiles, chemical, environmental and host cues would be useful in evaluating the suitability of blossoms and the developing fruitlets for oviposition.

As seen in the post-harvest infestation surveys (Fig. 3.8), fruitlets belonging to cultivars less susceptible for infestation may lead to higher rates of mortality amongst H. testudinea larvae, further suggesting that fruitlet development and composition influences larval mortality in H. testudinea.

Electroantennography of male and female sawflies showed responses to cultivars preferred for visitation and infestation, supporting the pattern of behaviour established in field surveys and bioassays. Ergo, key volatile components may be within these odourant blends which are important in host discrimination. Strong responses to less-preferred cultivars were also noted. This suggests that key volatiles emitted from less-preferred cultivars may trigger a repellent response in these insects.

Because mating was observed occurring on the blossoms themselves, male apple sawflies may use a combination of olfactory cues from the blossoms and female sex pheromones as indicators of feeding/mating sites, whereas females are drawn to sources of food and potential oviposition sites and away from fruitlets that other females apple sawflies have oviposited within. Cultivar susceptibility to oviposition and infestation is likely related to host cues that are similar to, but not identical to those that influence visitation by adult sawflies. Ovipositing females may respond differently to volatile chemicals that male or virgin females ignore or are repelled by, such as the epideictic pheromones left after oviposition (Roitberg & Prokopy, 1984; Boevé, 1996).

Competition and temperature may affect sawfly visitation preference. Anecdotal observations indicated that trees that had high numbers of visiting apple sawflies earlier

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in the day had a reduction in apple sawfly numbers once bees arrived. This may have been the result of the larger insects keeping sawflies away from the apple blossoms, or a result of shifting temperature preferences; sawflies are most active at a mean temperature of 15°C (Graf et al., 1996) and become less active in the afternoon (Miles, 1932). As the temperature rose, the sawflies may have become less active, reducing the rate of feeding/searching for mates and oviposition sites. Sawfly activity was recorded during 3 afternoon surveys, but this was not enough to establish a pattern of behaviour based on temperature.

Tactile cues may be one method used by H. testudinea in determining the suitability/susceptibility of oviposition sites. The Cephid sawfly Cephus cinctus uses abdomen tapping as a means of assessing potential oviposition sites on wheat stems

(Weaver et al., 2009). During this study, abdomen tapping on substrates was observed in captive female apple sawflies, both in the colony population and in individuals exposed to volatile extracts from apple blossoms.

The results of this study support the hypothesis that olfaction of plant volatiles is used by apple sawflies for host plant choice and cultivar discrimination. Sawfly species from Cephidae and Tenthredinidae use olfaction as a means of differentiating among host plant cultivars and oviposition sites (Craig et al., 1987; Piesik et al., 2008; Bartelt et al.,

2009; Weaver et al., 2009). The specific volatile chemical cues by which H. testudinea differentiates among cultivars for oviposition and larval infestation is unknown at present

(Roitberg & Prokopy, 1984). Fruitlets infested by apple sawfly larvae produce a strong odour (Vincent et al., 2002) with a higher concentration of terpenoids than healthy fruitlets (Boeve, 1996; Boeve et al., 1999). The increase in these volatiles from

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damaged/infested fruitlets may deter oviposition. Additional olfactory and visual cues may also play a role in determining oviposition preferences. Euura lasiolepsi avoids ovipositioning on arroyo willow shoots with natural or artificial oviposition scars (Craig et al., 1988). Hoplocampa testudinea exhibits similar behaviour. Roitberg & Prokopy

(1984) determined that adult apple sawflies showed lower rates of oviposition on blossoms that were previously infested or were artificially scarred to simulate oviposition. Similarly, H. testudinea larvae show a significant preference for uninfested fruitlets when given the option of attacking uninfested fruitlets and those with living and/or dead conspecific larvae inside (Roitberg & Prokopy, 1984). The cues the larvae use in differentiating between host fruitlets is unknown (Roitberg & Prokopy, 1984). As with adult sawflies, H. testudinea larvae may rely on a combination of olfactory cues from damaged fruitlets and/or conspecifics as well as visual/tactile evaluations of host plants.

The avoidance of previously-infested fruitlets by apple sawflies may have led to apparent shifts in oviposition preference among the H. testudinea population. As apple sawflies only oviposit a single egg on each blossom (Weires, 1991) and females produce epideictic pheromones after oviposition to deter conspecifics (Roitberg & Prokopy,

1984), high level of infestation within a single blossoming tree and/or cultivar may have induced ovipositing sawflies to seek alternate host sites, leading to increases in infestation rate within cultivars that would normally be less preferred for or susceptible to oviposition. Differences in attraction between sexes and may also have influenced infestation preferences. Males and sexually immature female apple sawflies would be seeking sources of food from the nectar and pollen within apple blossoms (Downes,

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1944; Weires, 1991) while non-virgin females are searching for oviposition sites; the olfactory cues that signal the suitability of the latter may be similar to, but not identical to, those of preferred apple blossoms. The combination of these factors may explain the shifts in apple sawfly preferences between visitation and infestation. Cultivars that were unsuitable for feeding, either via depletion of food sources, competition from other insects and/or olfactory or gustatory cues may have remained of value for oviposition, larval feeding and infestation. Observer error in the identification of insects and infestation damage may have caused variation in observed visits and level of infestation.

In 2013, an undergraduate student assisted in adult visitation field surveys. As in Chapter

3.1.1, post-harvest larval damage was evaluated in 2012 by Dr. Blatt’s entomology program and in 2013 by the author.

The largest confounding issue in the electrophysiological tests of H. testudinea is the low number of datasets. Software issues with AutoSpike® prevented recording and analysis on the data from over half the original specimens. The low mean responses to the volatile blends may have been due to the age and quantity of the specimens used; increasing the amount of volatiles used in these bioassays from 2 μL to 10 μL and replacing the filter papers after each run would provide for stronger odour sources and a consistent basis for comparison for all future electrophysiological tests.

The European apple sawfly, Hoplocampa testudinea, exhibits a clear and persistent pattern of preferences for the apple cultivars it visits for feeding, oviposition and larval infestation. Apple sawflies respond positively to specific cultivars across field surveys, behavioural bioassays and electrophysiological tests, supporting the observed behaviours and preferences. These preferences conform to previously described literature

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regarding sawfly cultivar choice as timing of bloom and availability of blossoms are not determining factors in cultivar choice. Host plant choice in H. testudinea appears to be based on multiple factors including olfaction. Apple sawfly responses to behavioural bioassays and EAGs support this observation.

5.2 Apple Maggot Fly

Apple maggot flies preferred several cultivars of apples in this study. Patterns of cultivar preference were largely consistent with known apple maggot fly preferences;

Rhagoletis pomonella prefers to infest earlier-developing, softer-fleshed apples (Brunner

& Klaus, 1993). Early-developing cultivars such as Silken, COOP 39 and Zestar! had high visitation levels by apple maggot flies (Fig. 4.5), in keeping with known preferences of R. pomonella towards early-developing varieties of apple (Klass, 1972; Reissig, 1979;

Brunner & Klaus, 1993) and supporting the primary hypothesis. More preferred cultivars have the potential for use in a push/pull strategy to draw apple maggots flies away from larger fields of less-preferred cultivars, where they can be targeted by control measures.

Analysis of the volatiles emitted by preferred versus non-preferred apples may provide additional resources for the trapping and non-chemical control of R. pomonella.

Apple maggot flies primarily use olfactory information to orient themselves towards host plants from a distance (Fell et al., 1982; Rull & Prokopy, 2005). Once R. pomonella arrive at a potential host, visual cues are then used to find food sources and apples for oviposition on the plant itself (Fein et al., 1982; Prokopy, 2011). Host plants without visual indicators of food or oviposition sites are abandoned, whereas R. pomonella spend more time at plants with real or simulated fruit (Prokopy, 2011). This is

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supported by visitation and infestation results: in 2012, Silken trees with few mature apples had very few observed visitations by R. pomonella; the more productive Silken accounted for virtually all visitations. This behaviour was also seen in 2013. Jubilee Fugi trees with <10 apples had no recorded observations of apple maggot flies; the more productive trees from this cultivar accounted for all visitations during the field surveys.

These results support the use of olfaction by R. pomonella in identifying and discriminating between potential host plant sites in informing this preference.

The increase in apple maggot fly populations between 2012 and 2013 may be due to variations in the environment. A temperature range of 21-23°C is optimal for adult emergence (Nielson, 1962; Nielson, 1964; Nielson, 1965). High humidity is similarly vital to apple maggot development during pupation and during emergence as adults

(Nielson, 1964). Pupating R. pomonella can remain in the soil for 2 or more years if environmental conditions are not suitable for emergence (Reissig, 1991; Brunner &

Klaus, 1993). A weather station at the AFHRC (Lat: 45°04'00.000" N Long:

64°29'00.000" W) reported an average temperature of 20.3°C and 22.6 mm total precipitation in July 2012 when the adult apple maggot fly population would have broken diapause, developed from pupae to adults and undergone eclosion. In 2013, the average temperature in July at AFHRC was 21.1°C, with 90.2 mm precipitation (Environment

Canada, 2014). The lower humidity and dryer climate observed in 2012 may have been sufficient to suppress development in these pupae. In 2013, the increase in temperatures and precipitation resulted in more favourable conditions, resulting in pupae from 2012 and the previously-dormant generation emerging together.

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Differences in apple maggot fly visitations between cultivars may be based on not only olfactory information, but additional host cues like visual or gustatory information, such as size and colouration of the apples, the presence of aphids and/or honeydew as a food source (Brunner & Klaus, 1993; Reissig, 1991). Other olfactory cues may influence the rate of visitation by apple maggot flies. As apple trees mature, the blend of volatiles they exude changes (Mattheis et al., 1991). This shift in volatile profile is used by apple maggot flies as a means of determining host plant suitability for oviposition (Zhang et al.,

1999). Variations in volatile emission among the cultivars of study may have led to changes in apple maggot fly visitation as the apples developed.

Silken apples were consistently preferred for visitation by apple maggot flies (Fig.

4.5), even when production of fruit per tree was low. None of the seven Silken trees in this study blossomed fully in 2012 and had few mature apples compared to other cultivars. Despite this low productivity, Silken trees remained the second-most visited cultivar in 2012. It can be inferred that some cue from this variety is highly attractive to

R. pomonella. Silken is strongly aromatic (Quamme et al., 1999; Wilson, 2001) and this intense odour may be a source of attraction for apple maggot flies. Rull & Prokopy

(2005) suggested that apple varieties that produced more volatiles may be more attractive to R. pomonella, which is supported by the preferences observed in this population of apple maggot flies.

Field study of R. pomonella visitation demonstrated several interesting responses.

The s23-06-153 cultivar is a later-developing cultivar (Hampson et al., 2008) and was consistently heavily visited by apple maggot flies in 2012/2013, preferred over earlier- maturing varieties such as Zestar! (Fig. 4.5). This suggests that although R. pomonella

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generally prefers to infest early-blooming apple cultivars (Brunner & Klaus, 1993), the timing of fruit development is not the sole factor in determining cultivar choice for visitation. Fruit characteristics (Table 2.2) such as thickness of apple skin, hardness of flesh and intensity of volatiles may play a role in determining host plant choice. The presence of fruit itself is an important factor in attracting R. pomonella: trees without fruit are visited less frequently and for less time than plants with fruit/fruit-like objects (Green et al., 1994). This behaviour was supported by the 2012/2013 field surveys. In 2013, when Silken trees were more productive, an increase in visitation by R. pomonella was observed. Jubilee Fugi, which produced less fruit in this year, had a decrease in visitation by apple maggot flies (Fig. 4.5). This supports previously described patterns of apple maggot fly behaviours (Green et al., 1994; Prokopy, 2011) in that olfaction is used by R. pomonella to differentiate between host cultivars at a distance, but visual identification of feeding and oviposition sites determines the amount of time the insects spend at a potential host plant.

Development of R. pomonella larvae is slower and is associated with increased risk of mortality in late-developing, thick-skinned, high acidity and hard-fleshed apples

(Reissig, 1979; Weems, 2002). As a result, apple maggot flies preferentially infest sweeter, thinner skinned and early-maturing cultivars (Reissig, 1979; Brunner & Klaus,

1993; Weems, 2002; College of Agricultural Sciences (CAS), 2005). Overall patterns of preference from infestation surveys within this population from the Annapolis Valley followed this trend, with several exceptions. Earlier-developing cultivars such as Silken and Zestar! and later-maturing varieties including s23-06-153 and Pinova were both preferred; a trait shared by all heavily infested varieties was sweet, tender flesh. Cultivars

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with harder, ‘crunchier’ textures such as COOP 39 (Janick et al., 2006) or 8S-27-43

(Lane, 2006) were less preferred for infestation. In this population of R. pomonella, timing of apple development appeared less important in determining cultivar preference than the fruit characteristics.

Observed preferences of infestation in apple maggot flies were largely consistent between years and across cultivars. Some variation did occur; in 2012, Jubilee Fugi was the most infested cultivar. In 2013, the rate of infestation in Jubilee Fugi apples dropped from 81.96 ± 1.86 % to 57.55 ± 0.48 % (Fig. 4.6). This is likely the result of the decreased productivity of Jubilee Fugi trees; some Jubilee Fugi trees had <5 apples.

Accordingly, there were fewer apples available and therefore fewer odour sources and visual cues to attract apple maggot flies for oviposition (Green et al., 1994). Over seventy-seven percent of all examined Silken apples were infested by R. pomonella in

2012 and one hundred percent of were infested in 2013 o (Fig. 4.6), despite the low production of apples within this cultivar. This shift in infestation levels further suggests that increased productivity of cultivars leads to increased infestation by R. pomonella, although the rate of infestation is still influenced by overall cultivar preference.

8S-27-43 was the least-preferred for infestation in 2012 and 2013 (Fig. 4.6), despite possessing ostensibly sweet, low-acid flesh. This may be due to several factors; the skin of the fruit is tougher and thicker than in other cultivars and the flesh is crisp and hard (Lane, 2006). As apple maggot mortality increases in fruit with firmer flesh

(Reissig, 1979; Brunner & Klaus, 1993), this may explain why 8S-27-43 was consistently less preferred for oviposition and larval infestation that the other cultivars in this study.

Thinner-skinned apples are more easily and heavily infested by apple maggot flies (CAS,

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2014). This suggests that tactile evaluation of host plants informs apple maggot fly discrimination in addition to olfactory and visual cues, and that oviposition may be less successful with thicker-skinned fruit. Ovipositing apple maggot flies may pass over otherwise suitable host plants in favour of those with thinner skin that is easier to pierce.

Differences between visitation and infestation rates can be observed in both the

2012 and 2013 data (Figs. 4.5 & 4.6). COOP 39 was relatively preferred for visitation, but not for infestation. In 2012, 8S-27-43 was the most visited cultivar and in 2013, it was of average interest to R. pomonella compared to the other studied cultivars (Fig. 4.5).

In 2012 and 2013 s23-06-153 was, compared to other cultivars, average in attracting apple maggot flies, but was heavily infested. These fluctuations between visitation and infestation may indicate that certain cultivars are attractive to apple maggot flies for purposes other than oviposition, i.e. certain cultivars may have large aphid populations for production of honeydew, but the apples themselves may be ill-suited to larval development/survival (e.g. too acidic (Carle et al., 1987) or the flesh is too hard,).

Olfactory cues may play a large role in this shift in preference: as apples mature, their volatile profile changes (Fein et al., 1982). If the apple maggot flies are responding to differences in such semiochemicals, this may explain the differences between infestation and visitation. Anecdotally, several 8S-27-43 trees had large aphid infestations in 2012, but as the fruit have thick skins and hard flesh, this would explain the decrease in attractiveness between visitation and infestation surveys (Lane, 2006). The plants were useful for procuring food, but unsuited for oviposition.

The primary draw of immature apple maggot flies to apple trees is the presence of food sources, such as the honeydew secreted by aphids (Brunner & Klaus, 1993). As

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B137/B138 were unsprayed entomology orchards, aphids were present on several trees; an increase in aphid presence was observed in 2013 (personal observation). This could explain the increased rates of apple maggot fly visitation on Ambrosia; it was observed that in both 2012 and 2013 aphids noticeably infested this cultivar. The presence of these insects and the honeydew that they secrete was likely an attractant for R. pomonella.

When given a choice of host plants, apple maggot flies rarely visit McIntosh cultivars. Both the McIntosh apple cultivars that were studied were comparatively rarely visited by apple maggots; in both 2012, few apple maggot flies were found on the 8

Summer McIntosh trees across the entire 70+ day survey period. In 2013, no apple maggot flies were observed on this cultivar. Both Rogers and Summer McIntosh were used as oviposition sites, so visitation by R. pomonella occurred on these apples, but was not observed during the field surveys.

The oviposition preferences of R. pomonella fit within expected patterns: sweeter, low-acid fruits with tender flesh are most heavily infested (Reissig, 1979; Weems, 2002;

CAS, 2005). Timing of fruit development plays a less definitive role in host plant choice within the studied apple maggot fly population within Annapolis Valley than suggested by the literature (Reissig, 1979). Apple maggots are expected to preferentially infest earlier-maturing cultivars to maximize larval survival (Reissig, 1991; Weems, 2002;

CAS, 2005) as larvae that infest later-developing varieties suffer increased mortality

(Reissig, 1979). Preferences within the studied population did not match this behaviour; although fruit from early-maturing cultivars such as Silken and Zestar! were heavily infested, later-developing cultivars like Pinova and s23-06-153 also showed high levels of infestation by R. pomonella. When given a choice between later-maturing fruit with

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sweeter, soft flesh such as Pinova and earlier maturing, harder fruit such as COOP 39, R. pomonella preferentially oviposits within this later maturing fruit. The softer flesh of the preferred cultivars may be a factor; the larvae are able to eat more in a shorter time frame and develop much quicker. Development of an apple maggot can be approximately two weeks in a softer-fleshed cultivar and three or more in fruit with harder, crunchier flesh

(Weems, 2002). With the comparatively short growing season of the Annapolis Valley

(Kittlsen, 2014), preference towards softer-fleshed, sweet apples is likely a survival trait for this population of apple maggot flies.

The comparatively low rate of infestation in Rogers and Summerland McIntosh fits with known preference patterns of R. pomonella; these fruit tend to have tart, crisp flesh (Hunter, 1997) and develop relatively early in the season. Rhagoletis pomonella heavily infest McIntosh cultivars, with low rates of larval mortality (Reissing, 1979), but overall McIntosh varieties are generally considered at a lower risk for infestation (Rull &

Prokopy, 2005). This is supported by field survey observations in visitation and infestation; Rogers and Summer McIntosh possessed relatively lower rates of infestation when compared to other cultivars (Fig. 4.6). This indicates that even when given a choice between early-developing, sweet-fleshed cultivars, apple maggot flies demonstrate some level of preference between host plants.

Differences in levels of attraction between unmated and mated apple maggot flies may lead to differences in preference patterns between sexes throughout the life cycle of

R. pomonella. Mated females are searching for oviposition sites as well as food sources.

If a difference in host plants exists based on mated versus-non-mated insects or sexual maturity, this could have influenced the results. Without capture and dissection of each

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insect, it is impossible to determine rates of visitation by virgin and non-females. Other sources of error include variation in the timing of visitations; most field surveys were performed between 8-10 AM; several surveys were carried out in the afternoon. This may have resulted in a pattern of field surveys that did not fully represent the active period of apple maggot flies. Observer error, particularly as a result the different surveyors in 2013, of may had resulted in misidentification of apple maggot flies, leading to miscounts in population.

The strength and production of apple volatiles may also play a deciding factor in the responses of R. pomonella. Some cultivars produce extremely potent odours (Rull &

Prokopy, 2005), potentially overpowering other odour sources within mixed orchards such as B137 and B138. Apple maggot flies may indeed react to certain odours by ignoring nearby fruits and host plants and orienting themselves to more attractive volatiles. Rull and Prokopy (2005) suggested that in a mixed orchard, the presence of strongly fragrant fruit may influence the behaviour of foraging apple maggot flies, causing them to ignore other cultivars. The behaviours observed in the 2012 and 2013 field seasons may support this supposition. The aromatic cultivars such as Silken

(Wilson, 2001) and Ambrosia (BCMA, 2013) may have acted as a pull on the studied apple maggot population, drawing the insects away from other, less aromatic, cultivars, resulting in lower rates of visitation and infestation on these trees than might be observed in agricultural monocultures.

Apple maggot fly responses to Y-tube behavioural bioassays fit the observed cultivar preferences seen in field surveys. Despite traditionally being strong flyers and performing well in wind tunnel bioassays (Zhang et al., 1999; Nojima et al., 2003), R.

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pomonella reacted consistently to the stimuli used in the walking Y-tube bioassay and there were comparatively few null reactions. The responses to the Y-tube bioassay indicate a level of preference to R. pomonella among host plants, supporting the patterns of preference seen in the field visitation and infestation surveys.

Comparisons using preferred Zestar! and Pinova cultivars against ‘non-preferred’ cultivars and the pentane control showed significant response differences (Fig. 4.7), further supporting and providing a potential cause for the results of the field studies.

Zestar! had higher mean visitations by apple maggot flies and infestation of apple maggots than Ambrosia (Figs. 4.5 & 4.6). Compared with 8S-27-43, Pinova had higher mean visitations and rates of infestation (Figs. 4.5 & 4.6). These reactions match observed behavioural responses in the bioassays. The significant differences in responses between each preferred cultivar and the control support the hypothesis that the insect is making a choice based on olfactory cues rather than random selection.

Temperature, relative humidity and illumination in the location where the bioassays were conducted were all within the parameters of previous behavioural bioassays using R. pomonella (Zhang et al, 1999). Host plant and semiochemical preferences in apple maggots are frequently tested using flight tunnels (Zhang et al.,

1999; Nojima et al., 2003) over walking bioassays. No visual cues were used in these trials as apple maggot flies will react to host plant volatiles even in the absence of fruit/fruit-like objects (Fein et al., 1982). Number of null responses may have been increased due to insufficient odour sources caused by depletion of the volatiles on the filter paper. Increasing the frequency of fresh volatiles and filter papers may account for these factors in any subsequent Y-tube bioassays using these insects.

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All flies were taken from the on-site colony of R. pomonella and were assumed mated. This assumption was based on the frequency of mating observed in the captive population. Mating was observed in the colony, in the collection jar after capture in the field and during transport from the colony to the test room. Low availability of flies prevented the sacrifice of each specimen to confirm mating status.

The Y-tube bioassays showed a trend of preferences that matched field surveys of visitation and infestation. Test insects showed a consistent, if not always statistically significant, pattern of positive responses towards more preferred cultivars when given a choice between these and less preferred cultivars or the pentane control.

These results (Fig. 4.7) support the behaviours observed in the field and in behavioural bioassays, although the electrophysiological tests were inconclusive in indicating the effect host plant odours have on the antennal lobe. Female responses to

Pinova, while not statistically significant overall, generally corresponded with observed preferences for that cultivar. These activity patterns suggest a degree of fidelity in the discrimination of host plant cultivar volatiles at the antennal lobe level. These responses are in line with the observed results for visitation and infestation, as this volatile blend and the host plants themselves were consistently preferred by R. pomonella (Figs. 4.5 &

4.6). The consistently high reactions to volatile blends from 8S-27-43 when compared with other volatiles (Fig. 4.7) support observed trends: this cultivar is generally less preferred by apple maggot flies for infestation and the pattern of strong reactions to its volatile blend is likely a repellent response.

The low level of statistically significant responses to volatile blends observed in the calcium imaging study is likely a consequence of low numbers of tested individuals at

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each site in the antennal lobe; R. pomonella are known to respond consistently to apple volatile blends and chemical extracts in both behavioural and electrophysiological tests

(Fein et al., 1982). Other possibilities are that the areas viewed within the antennal lobes may not correlate with key glomeruli either remaining covered/obstructed, or too deep within the neural tissue to be examined at the 20x magnification used. Further studies to identify the composition of volatiles produced by each cultivar is required to identify specific variations and concentrations in odour components which may influence attraction or repulsion by R. pomonella. This will provide an increased range of options for the monitoring and control of these insects.

When provided with a selection of apple cultivars, this population of Annapolis

Valley apple maggot flies discriminated between host plants in field surveys, behavioural analyses and at the antennal lobe level (Table 4.11). Significant differences in cultivar choice were observed in the visitation and infestation field studies and in Y-tube bioassays, but statistically significant variation in responses to volatile odours was not recorded in these electrophysiological tests. The general pattern of responses observed in this study appears to confirm the results seen in the previous behavioural tests and described research (Zhang et al., 1999). Patterns of preference remained consistent between each study. Rhagoletis pomonella used olfactory cues as a means of differentiating between cultivars, preferring to infest softer-fleshed fruit, even if the maturing fruit were of a later-developing cultivar, which is normally not preferred for infestation by these insects. The high level of infestation observed on several cultivars supports the theory of certain aromatic cultivars having a ‘pulling’ effect on R. pomonella

(Rull & Prokopy, 2005). Further development of the cues that R. pomonella uses to

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discriminate among host plant cultivars, including the specific volatiles that apple maggot flies are most sensitive to can potentially provide growers with a method of controlling apple maggot flies by drawing them from less-preferred cultivars to smaller stands of preferred cultivars, which can then be targeted with control measures.

The Annapolis Valley population of R. pomonella shows definite preferences towards sweeter, soft-fleshed fruit, regardless of being early or late-maturing cultivars

(Tables 2.2 & 4.11). These preferences can be observed through field studies of apple maggot fly visitation to cultivars, degree of apple maggot infestation in mature fruit and in behavioural bioassays. Silken, Pinova and Zestar! are highly preferred and elicit significant differences in R. pomonella’s responses towards these cultivars. When provided a range of options, cultivars such as Rogers McIntosh, Summer McIntosh and

8S-27-43 are among the least attractive to apple maggot flies (Table 4.11).

5.3 Further Directions

5.3.1 European Apple Sawfly

Additional research into the infestation preferences of H. testudinea is recommended; particularly studies of the effect that infested blossoms/fruitlets have on sawfly oviposition, larval infestation and development. As with R. pomonella, characterization of the composition of volatiles produced by each cultivar will be critical to identifying behaviour-modifying compounds in this species. Increasing knowledge by which adult apple sawflies and larvae discriminate between potential hosts will provide more information on the environmental factors that influence a cultivar’s susceptibility to damage from these insects. The development of synthetic cultivar and/or pheromone

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blends can be used as repellents to push H. testudinea away from valuable crops or as attractants to pull these insects towards traps and lures for monitoring and control.

Additionally, study into the qualitative differences between the fruitlets of apple cultivars may provide insights into female oviposition and larval infestation choice by examining how characteristics of fruitlets affect larval mortality and success of oviposition.

Further Y-tube bioassays to support the observed trends and compare/contrast these with field visitation and infestation surveys are recommended. These bioassays would improve the statistical power of future data. Initial bioassays tested each individual three times, but only first responses were used. If enough adult sawflies were available, additional bioassays could be based solely on first-responses with a larger sample size.

This would address the comparatively low n values used in the bioassay. Further bioassays coupled with gas chromatography/mass spectrometry: GCMS tests could elucidate the chemicals specific to each preferred cultivar that apple sawflies are most sensitive to, their concentrations and ratios in the volatile profile. Once isolated, sawfly behavioural responses to these odourants could be used to evaluate their usefulness in the development of traps and lures. This could be linked with analyses of infested fruitlets to isolate the specific odour sources and/or cues that deter oviposition/infestation and test the efficacy of these as a control mechanism for H. testudinea populations. Behavioural bioassays conducted using a flight chamber could also be used; although they are not strong flyers, H. testudinea prefer to fly towards odour sources (Miles, 1932) and the relatively small Y-tube assembly used in this study may have been a confounding factor, disorienting and/or restricting the normal behaviours of the apple sawflies. The use of larger Y-tubes and/or flight tunnels for future bioassays using volatile blends and/or cut

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blossoms would reduce issues with airflow, allow for insect flight and prevent further mechanical damage by putting comparatively large apple blossoms in a small-diameter junction tube. A possibility for future bioassays would involve the use of cuttings from the cultivars of interest in a flight chamber to reduce the damage to the blossoms themselves.

These suggested bioassays could result in improved rate of responses to volatile blends and provide additional behavioural information on the reactions of adult apple sawflies to these odour sources. A productive colony and/or intense capture program would be necessary to provide the raw numbers of sawflies for these studies. Further behavioural bioassays could be conducted using live females sawflies and/or extracts from female sawflies to test the possibility of the production and influence of sex pheromones in host plant choice. Further behavioural bioassays could be performed to test sawfly visitation based on the presence/absence of other insects and pollinators such as bees as well as testing the impact of temperature on sawfly activity and level of visitation and oviposition.

To account for the low sample size in the EAGS, further tests using adult apple sawflies and a wider range of volatile extracts are recommended. Additional comparisons between male and female H. testudinea are recommended to determine whether the observed differences in sensitivity between sexes are repeatable and significant within a larger population, or an artefact of the small sample size and/or preparation methods.

Future electroantennogram studies could examine the differences between visitation and infestation of the cultivars in this study by comparing responses of virgin female and non- virgin female sawflies to the volatile blends. If significant differences in intensity of

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response could be confirmed, this would support the idea that H. testudinea host plant preferences change throughout the life cycle of the insect and/or the development of the plant. Further testing, including the use of a GC-EAD, to determine the specific volatile chemicals that the adult apple sawflies are reacting to is also recommended. Data from such a study would provide the specific odourants from each cultivar that each insect reacts to and could be used to build a library of chemical volatiles useful for luring, trapping and monitoring this species. Additional research examining the presence and role of pheromones emitted by adult apple sawflies and larvae is also recommended to determine how these factors can influence the cultivar preferences of these insects and provide additional methods of monitoring, trapping and controlling this economically important pest.

5.3.2 Apple Maggot Fly

To confirm the patterns seen in the 2012/2013 field surveys and in the 2013 Y- tube bioassays and to improve the statistical reliability of the results, additional bioassays are recommended using an increased selection of volatile chemical blends. These behavioural tests could rank the cultivars and establish a direct hierarchy of preference: e.g. Zestar! being more preferred than Ambrosia, but Pinova is more preferred than

Zestar!.

With a sufficiently productive colony, each female apple maggot fly could also be dissected after each trial to confirm mated/non-mated status and compare preferences exhibited by mated against non-mated R. pomonella. Flywalk trials (Steck et al., 2012) and flight tunnel bioassays could be performed for further analysis of apple maggot fly

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responses to cultivar volatiles and to compare responses between the walking bioassays and those performed in a flight tunnel. An analysis of volatile production among the cultivars of study compared with mean rates of infestation may be able to elucidate the degree to which concentration of volatiles plays a role in host plant choice. Further analyses using calcium imaging to build up a statistically significant pool of responses is recommended to confirm the results of this study, combined with confocal microscopy to identify the specific glomeruli within the antennal lobes that correspond to the observed reactions to each odour and volatile blend.

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6. Conclusion

Hoplocampa testudinea is an invasive hymenopteran from Europe that emerges early in the apple growing season to feed from apple blossoms and infests developing fruitlets (Miles, 1932; Weires, 1991). Rhagoletis pomonella is a dipteran native to North

America that feeds on sugar sources and infests mature/near-mature fruit (Kelly, 2003).

These differences in behaviour and life cycle between each species make direct comparisons in cultivar choice problematic. Cultivars that were highly preferred by one species, such as R. pomonella and Silken were among the least preferred for in the other insect. However, Pinova and to a lesser degree Zestar! and s-23-06-153, were consistently preferred cultivars for visitation by adults and larval infestation in both species (Tables 3.10 & 4.8). In an integrated pest management system, one of these cultivars may be used as a ‘pull’ to draw both species away from other less-preferred orchards. Further research into the characteristics that make these cultivars preferred at different stages in their development to two unrelated insect species with such different life cycles will provide further avenues for methods of trapping, monitoring and controlling these pest populations.

6.1 European Apple Sawfly

Cultivar preference in Hoplocampa testudinea occurred in this study and was based on olfactory and temporal cues; timing of apple bloom appeared to influence, but did not determine attractiveness of cultivars. Adult apple sawflies used olfaction as a means to differentiate between volatile blends in Y-tube bioassays and electrophysiological tests. Attempts by female sawflies to use tactile evaluation of

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potential oviposition sites were noted in behavioural bioassays, suggesting additional mechanisms that inform cultivar preference in H. testudinea.

Variation in host plant preference is likely a result of variation in sensitivity to specific odours or individual odourants. Responses to volatile blends in EAGs showed strong sensitivity to preferred and non-preferred cultivars. Volatile blends to which these insects were most sensitive were among the most/least preferred cultivars for visitation and infestation, suggesting that sensitivity to specific odours may lead to differences in host plant selection for visitation and infestation. Differences in sensitivity to plant odours based on sex may be an artefact of the low sample numbers used in this study or may indicate sex-based discrimination of apple cultivars. Females may be sensitive to a wider range of olfactory or tactile cues that allow them to discriminate between food sources and oviposition sites, leading to sex-based differences in cultivar preferences.

Differences between infestation and visitation/feeding in H. testudinea may be based on different host plant cues. Cultivars that are suitable as food sources may not be as preferred for oviposition. Additional environmental, tactile or olfactory cues may lead to differences in host plant preference in apple sawflies between those selected as food/mating sites and those used for oviposition.

This information can be used to control and monitor apple sawfly populations via integrated pest management techniques. Push/pull strategies using primary crops of non- preferred cultivars coupled with ‘pull’ stands of preferred apple cultivars will allow growers to focus control methods on the more preferred cultivars as these will attract apple sawfly away from the main crops. The volatiles from preferred and/or non- preferred cultivars can further be analyzed for the specific semiochemicals to which H.

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testudinea are most sensitive. These volatile blends can then be used as repellents to

‘push’ apple sawflies away from susceptible orchards or as attractants in enhanced trapping. Additional testing using traps containing these volatile blends and any attractive/repellent odourants isolated from the extracts will provide further information on the potential uses of these compounds as control options. This will provide further avenues of research in monitoring and managing populations of this established invasive pest species.

6.2 Apple Maggot Fly:

Cultivar preference was observed in a population of Rhagoletis pomonella within the Annapolis Valley. Softer-fleshed varieties of apple (Table 2.2) were preferred, even when belonging to later-maturing cultivars, contrasting with observed patterns of host plant preference in other populations of apple maggot flies. Apple maggot flies use olfactory cues to differentiate between the different cultivars and show variation in sensitivity to volatile blends. This sensitivity is strongest with preferred cultivars and may contribute to the differences in visitation and infestation rates across all sampled cultivars.

No difference in cultivars preferred for infestation and therefor visitation was observed for apple maggot flies. As apple maggot flies visit apples in search of mating opportunities and oviposition sites, there appears to be no feeding/oviposition divide in host plant preference as was observed in H. testudinea.

As a means controlling apple maggot flies, the use of Silken apples shows potential. Even when this cultivar’s productivity was low, it remained strongly attractive

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to R. pomonella. Coupling Silken trees with orchards of lower-preference cultivars such as Ambrosia or McIntosh could draw adult apple maggot flies from these trees to the available Silken fruit, where the apple maggot flies can be targeted with sprays and other control methods. As with the preferred/non-preferred cultivars of apple sawflies, determining the specific compounds, their concentration and ratios within the preferred and non-preferred cultivars that R. pomonella was most sensitive to can be used to repel these insects from susceptible cultivars towards traps baited with preferred odours or orchard blocks producing preferred cultivars. This research will provide growers and researchers with increased options for both the monitoring of and management of this economically important pest insect.

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Vincent, C. (2011) Management of the European Apple Sawfly (Hoplocampa testudinea) using a parasitic wasp (Lathrolestes ensator). Available from: [September 29, 2012].

Vincent, C., Babendreier, D. & Kuhlmann, U. (2002) Hoplocampa testudinea (Klug). Biological Control Programmes in Canada, 1981-2000. CABI Publishing, UK.

Weaver, D.K., Buteler, M., Hofland, M.L., Runyon, J.B., Nansen, C., Talbert, L.E., Lamb, P. & Carlson, G.R. (2009) Cultivar preferences of ovipositing wheat steam sawflies as influence by the amount of volatile attractant. Journal of Economic Entomology. 102(3):1009-1017.

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Weems, H.V. Jr. (2002) apple maggot – Rhagoletis pomonella (Walsh). Available from: [March 17, 2014].

Weires, R. W. Jr. (1991) European Apple Sawfly. Available from: [July 18, 2012].

Wilson, K. (2001) New Apple Cultivars: Silken Ontario Ministry of Agriculture and Food. Available from: [ February 12, 2014].

Yahia, E. M. (1994) Apple Flavor. Horticultural Reviews, Volume 16. Edited by J. Janick. John Wiley & Sons, United States of America.

Zhang, A., Linn Jr, C., Wright, S., Prokopy, R., Reissig, W. & Roelofs, W. (1999) Identification of a new blend of apple volatiles attractive to the apple maggot, Rhagoletis pomonella. Journal of Chemical Ecology. 25(6): 1221-1232.

Zhao, Z., Wahl, T. & Marsh, T. (2007) Economic Effects of Mitigating Apple Maggot Spread. Canadian Journal of Agricultural Economics. 55(4): 499-514.

Zijp, J.P. & Blommers, L. H. M. (2001) Apple sawfly Hoplocampa testudinea (Hym., Tenthredinidae) and its parasitoid Lathrolestes ensator in Dutch apple orchards (Hym., Ichneumonidae, Ctenopelmatinae) [sic]. Journal of Applied Entomology. 126(6):265-274.

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8. Appendix A: European Apple Sawfly

8.1 Y-tube Bioassays (2012)

8.1.1 Results

Data from the 2012 bioassays using cut apple blossoms contradicted observed patterns of preference in the mixed orchards. Silken blossoms were among the most heavily preferred blossoms in the Y-tube trials (Fig. 8.1 & Table 8.1), despite being the least-preferred cultivar for visitation and infestation (Figs. 3.6 & 3.7). When only strong responses to apple blossoms were compared, Silken was the most preferred cultivar (Fig.

8.2). When all positive responses to apple blossoms were considered, s23-06-153 and

Pinova were preferred in all successful sawfly responses. These results did support observed preferences in the field. Zestar! was less frequently-chosen than Silken in the

2012 Y-tube bioassays, despite being heavily preferred for visitation and infestation

(Figs. 3.6 & 3.7).

There were no statistically significant differences in the preference patterns exhibited by sawflies when presented with cut apple blossoms (Table 8.1). As a consequence of bloom times, only limited trials of Summer McIntosh and Roger

McIntosh were tested. Out of 175 total trials in 2012, 104 were nonresponsive.

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All other Summer Mcintosh N=15 tested cultivars COOP 39 N=25 Primary Stimulus Rogers McIntosh N=10

Ambrosia N=35

Jubilee Fugi N=35

8S-27-43 N=15 Cultivar Zestar! N=35

Royal Gala N=35

Silken N=15

Pinova N=25

s23-06-153 N=30

100% 80% 60% 40% 20% 0% 20% 40% 60% 80% 100% Percentage of responses to apple blossoms

Figure 8.1: Percentage of all preferential apple sawfly responses to apple blossoms in 2012 Y-tube bioassay. Refer to Table 3.1 for full listing of comparisons performed per cultivar. 143

Table 8.1: Chi-square analysis of first-choice apple sawfly responses to apple blossoms. Times tested Primary Primary Blossom against other Blossom

(Arm 1) blossoms (N) Chosen x2 calc x2 crit Silken 24 14 8.42 18.31 Zestar! 16 7 2.96 12.59 Royal Gala 19 9 3.46 14.07 Jubilee Fugi 16 6 5.79 14.07 8S-27-43 10 4 1.47 11.07 s23-06-153 10 8 4.3 12.59 COOP 39 10 3 5.67 15.51 Ambrosia 13 5 2.85 15.51 Pinova 15 11 3.8 12.59

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SE) / % / SE)

5 Mean responses to cultivar (+/ cultivar to responses Mean

153

Silken

Pinova

Zestar!

Rogers Rogers

Summer Summer

Mcintosh

McIntosh

COOP 39COOP

Ambrosia

Royal Gala Royal

s23 Jubilee Fugi Jubilee

Cultivar Figure 8.2: Strong preferences of Hoplocampa testudina to cut apple blossoms in 2012 Y-tube bioassay. 145

8.2 Colony Development

8.2.1 Materials and Methods

Year 1 – 2012

Live adult sawflies were caught in the field and transferred to an 18 x 12 x 12 inch fine-meshed cage. This population was kept at 15°C and 80% relative humidity on a

16:8 L:D photoperiod to promote longevity of the population. This temperature regime was used as it was found in 2012 that Hoplocampa testudinea survived approximately

14-20 days past the observed flight period of wild apple sawflies. Food was provided via cut apple branches with fresh blossoms taken from an untended orchard containing

NovaSpy and NovaMac apples. Soaked cotton cloths in a 500 mL beaker were used as a source of water. Food and water sources were replaced as needed; for blossoms this was approximately every 4-6 days, although some branches were left for fruitlet development.

Fresh water was supplied every 7-9 days. Although mating was observed, no infested fruitlets were produced by this population. Females and males were taken for use in bioassays and electrophysiological tests.

To establish an active colony, fruitlets infested with apple sawfly larvae were collected from an unsprayed orchard block containing NovaSpy and NovaMac beginning mid-June 2012. Fruitlets were determined to be infested with H. testudinea larvae based on the distinctive scarring caused by H. testudinea larvae boring towards the fruit’s ovary and the presences of frass-filled holes. Once an infested fruitlet was identified, the entire cluster was removed for use.

Plastic containers measuring 17 cm in diameter and 10 cm in height were collected and washed. Three separate substrates were used: soil, sand and vermiculite. 146

Soil and sand were used to approximate natural substrates and vermiculate was used as a medium that would retain moisture to prevent desiccation and promote larval development while still preventing/retarding microbial growth on the pupae. The following combinations of these substrates were used; 100% playground-grade sand;

100% ‘sweet’ topsoil; 100% vermiculite; 50% sand and 50% vermiculite; 50% soil and

50% vermiculite; 50% sand and 50% soil and 33% sand, 33% soil and 33% vermiculite.

Percentages of substrate were determined by volume. Three or four containers were used per substrate type. Each substrate was sterilized twice via autoclave over a 24-hour period to prevent microbial contamination. Each container was filled with 7-10 cm of substrate.

In order prevent contamination of the substrate by microbial flora on the fruitlets, mesh screens were anchored to the walls of each container 0.5-1 inches above the substrate

(Fig. 8.3). This allowed mature sawfly larvae to exit the fruitlets, pass through the mesh and burrow into the substrate. A second, thinner mesh with spacing less than 1 mm apart was laid over the fruitlets in order to prevent any organisms from escaping and to prevent the sawfly larvae from being predated or parasitized by other insects during their incubation.

Number of fruitlets per container varied depending on the size and number of fruitlets per of infested cluster, although a screen was covered with approximately 10

147

a b

Figure 8.3: Set up of apple sawfly rearing containers. 100% sand substrate pictured. (a) depth of substrate relative to container indicated (b) mesh covering separating fruitlets from substrate. 148

clusters (Fig. 8.4). New infested fruitlets were added 2-3 times every 3-5 days until the majority of field-infested fruitlets had dropped from the trees.

The pots were originally placed in an indoor glass house to maintain a natural day cycle and temperature variation. On the second day of rearing, temperatures in the greenhouse reached 41-44°C. The larvae were removed to a 21°C, 60% humidity environment and incubated in these conditions for one week before being moved to growth chambers kept at 23°C and 75-81% humidity (Fig. 8.5). New fruitlets were placed in the affected pots to replace mortalities caused by the extreme heat. Heron (1967) notes sawfly larvae suffer high mortality even with brief exposure to temperatures above 40°C.

Fresh food sources and infested fruitlet clusters were introduced to the containers periodically to allow larvae to continue feeding and to replace any mortalities. The fruitlets were given a light misting of water each day to prevent desiccation.

The pots were kept in growth chambers at 23°C for 2-3 months to simulate normal summer conditions. To initiate and then break diapause, the temperature regimes outlined in Bartelt et al. (1981) and Krener (1983) were used. Temperature within the growth chambers was lowered to approximately 2-3°C at the same level of relative humidity for 10 weeks on a 9:15 L:D schedule. This chill period was to prevent apple sawfly larval development, which occurs at 4.5°C (Neupane, 2012). After the chill period, the temperature was raised by 5°C every 2 weeks to a maximum of 23°C.

Relative humidity was raised to 80% RH for adult emergence. The light/dark schedule was adjusted to 14:10 and then to 16:8 L:D with the increased temperature regimes to mimic changing day/night patterns concurrent with warming weather. The slower increase in temperatures deviated from the sawfly rearing methods outlined in Bartelt et 149

a b

Figure 8.4: Apple sawfly rearing pot (50%/50% soil/peat moss mixture). 150

Figure 8.5: Apple sawfly rearing containers in growth chamber. Elastic banding securing the upper mesh broke and was replaced repeatedly during incubation. 151

al., (1981) and Graf et al., (2001) in order to create a more graduated rise to larval metabolism instead of moving directly from a chill period to a >20°C regime.

Due to the failure of the colony to produce sawflies, further adjustments to the temperature regimes along with plans for the testing of artificial food sources and the planned usage of adults in electroantennograms were cancelled.

Year 2 – 2013

As the 2012 colonies failed to provide viable adult sawflies, a second series of sawfly colonies was established using 50% soil and 50% peat moss using the methods described above. Collection of sawfly larvae proceeded as outlined in 2012. The 3 month

2012 pre-chill period was reduced to 1 month incubation at 23°C and 80% RH.

Temperature during the 4 month chill period was raised to 4°C, humidity was maintained at 80 ± 5% RH and the previous 9:15 L:D was switched to a 10:14 L:D photoperiod. The

2013 colonies were then set on the same post-chill regime as used in 2012: after the chill period, the temperature was gradually raised. 10°C, 80 ± 5% RH and 12:12 L:D for one week, 15°C, 80 ± 5% RH and 14:10 L:D and 20°C, 80 ± 5% RH and 16:8 L:D, followed by a six-week observation period to monitor the emergence of adult apple sawflies.

8.2.2 Results

Year 1 – 2012

Pupae were found in all substrate types of the 2012 colonies except for 100% sand

(Fig. 8.6). Desiccated larvae were discovered in all colonies using 100% or 50% sand

(Fig. 8.7). Substrates with vermiculite also trended towards higher pre-pupal mortality 152

rates: 23% in 100% vermiculite and 33% in the 33% soil/sand/vermiculite mixture (Table

8.2).

Sawfly pupae were found in all other substrates, indicating that the sawfly larvae died shortly after leaving the fruitlets. Approximately 30 larvae/pupae were found per pot. Recovered numbers of pupae/larvae was in keeping with the 2-3 layers of approximately 10 infested fruitlets used per pot described in the methods. Dissection of pupae failed to reveal the presence of any parasitoids. One adult codling moth was found in the 50/50 soil/sand mixture (Fig. 8.6). No other bycatch was noted.

Year 2 – 2013

The 2013 colony failed to produce any viable adults. The mixture of peat moss/soil reduced mortality at the larval stage, as all recovered apple sawflies died during pupation.

8.2.3 Discussion

Data from the attempted colony rearing indicates several important features; temperatures in excess of 25°C will increase mortality of insects while a regime closer to

15°C will prolong their lifespan. In the rearing of sawfly pupae, substrate plays an important role in the development of the larvae; sand will lead to extremely high mortality among the larvae, keeping many of them from pupating at all. This support the observations made in Zijp and Blommers (2001) when it was determined that substrate type and soil composition affects apple sawfly mortality. Colony survival supported the observations made in Graf et al., (1996). Temperatures of 25°C or higher resulted in

153

Figure 8.6: Hoplocampa testudinea pupae and larvae collected from 50%/50% soil/sand mixture. Adult codling moth in upper right corner. 154

Figure 8.7: Desiccated Hoplocampa testudinea larvae taken from a 100% sand substrate container. 155

Table 8.2: Summary of 2012 development of Hoplocampa testudinea colonies by substrate. 100% mortality was observed in all trials. Substrate No. Pupae No. Larvae Total 100% Soil 37 1 38 100% Sand 0 21 21 100% Vermiculite 24 7 31 50/50% sand/soil 1 28 29 50/50 sand/vermiculite 2 27 29 50/50 soil/vermiculite 29 2 31 33/33/33 sand/soil/ vermiculite 16 8 24 156

increased mortality among sawfly populations. Temperatures of 15°C prolong

Hoplocampa testudinea’s life span.

The use of sand as a substrate even at 50% resulted in high mortality. In 100% sand mixtures, none of the sawfly larvae pupated before dying even at 80% RH and periodic moistening of the substrate. Consistent levels of humidity are necessary for the successful rearing of apple sawfly larvae. Results from Zijp and Blommers (2001) rearing attempts showed that pots with a volume of soil of 215 cm3 required a consistent water of content in the soil of 5-6% of total volume as well repeated additions of 8 ml of water.

Treatments lacking water replacement and/or moisture content was below 5% showed an increase in pre-emergence mortality (Zijp & Bloomers, 2001). These results support observations made in colony rearing attempts. The misting treatments that the 2012 and

2013 colonies received may have been insufficient to keep the moisture level high enough for the survival of pupae and adult emergence, leading to the observed total mortalities.

Additional colony development using the 2013 substrate mixture and the moisture regime described in Zijp and Blommers (2001) is recommended in addition to comparisons between a full pupation cycle and the accelerated growth regime tested in this study. This can be used to determine if Hoplocampa testudina can be reared in a laboratory setting faster than its normal 10-month pupation cycle. This will provide further information on the establishment of productive colonies of this insect for further study.

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8.3 Enhanced sleeve cage infestation survey

8.3.1 Materials and Methods

A no-choice bioassay using mesh sleeves was performed in 2013 to test rates of infestation in wild sawflies when only a single host plant was available. Fine-meshed bags were used to allow airflow but prevent insects moving off of the branch. Three blossom clusters on 4 trees from each of the 11 cultivars in this study were bagged (Fig.

8.8). Each limb was tapped prior to enclosure to shake off any organisms already present.

Twenty-four sleeves out of the total of 132 cages were set up with a pair of adult apple sawflies: these breeding pairs were placed in 2 sleeve cages per cultivar. Silken and

Rogers McIntosh each had 3 sleeve cages with adult pairs. The remaining one hundred and eight cages without breeding pairs were placed over unopened apple blossom clusters. All sleeve cages were set up at the midpoint of apple bloom at the height of sawfly activity. Sleeve cages without a breeding pair were assumed to be infested for the purposes of this experiment.

Sleeve cages were left in place for 5 weeks to allow for adult breeding, oviposition and larval development. After this period, the cages were removed from the trees. Number of fruitlets and infested fruitlets were counted in each sleeve and the results used to develop mean infestation per cultivar in based on all sleeves and those that had adult breeding pairs placed within. Infestation was determined based on the presence of C-scars, burrow holes and the presence of H. testudinea larvae.

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Figure 8.8 Apple tree with sleeve cage over limb 159

8.3.2 Results

Results from the sleeve cage no-choice bioassays show a significant difference in preference between apple cultivars (Table 8.3). Zestar! was a preferred cultivar when all sleeve cages were analyzed as well as those only with adults (Fig. 8.9). When the infestation rates of all sleeve data are considered, the general trend is similar to that seen in infestation surveys (Figs. 3.7 & 8.9). Zestar! and s23-06-153 are heavily infested relative to the other cultivars studied. Royal Gala and COOP 39 have lower rates of infestation (Fig. 8.9), than that observed in infestation surveys (Fig. 3.7)

When the data from those sleeve cages with adults was used, significant differences in host plant preference for oviposition were indicated (Table 8.3). The overall pattern remained close to that observed in all sleeve cage data: Pinova and Zestar! remained comparatively highly infested compared to the other cultivars studied.

Variation in cultivar preference was observed, including increase in infestation among less-preferred cultivars. Jubilee Fugi increased in rate of infestation from 6.08 ± 1.83%

(Table 3.6) in field surveys to 54.55 ± 2% (Fig. 8.9).

Data from sleeve cages without adult pairs shows evidence of H. testudinea infestation, supporting the assumption that these blossom clusters were infested prior to bagging. Infestation rates confirm the observations made during field survey preferences.

Zestar! and s23-06-153 were found to be the most heavily infested cultivars such as

Silken and Ambrosia were less-preferred for oviposition and larval infestation in this study (Fig. 8.9). These patterns of preference are similar to those observed with adult visitation and larval infestation (Figs. 3.6 & 3.7).

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Table 8.3: Summary of Kruskal-Wallis analyses of rates of infestation among sleeve cages.

Fruitlets Percentage of

Surveyed (n) Infested Fruitlets SE H Pcrit All cages 663 26.366 0.558 23.451 0.009 Adult-only cages 108 36.081 1.212 16.421 0.088 Cages without adults 554 18.355 0.577 27.842 0.002 161

100 All

90 Adults No Adults 80

SE) / % SE) /

n(+/ \ 40

30 cage study study cage 20

Mean apple sawfly infested fruitlets from enhanced sleeve sleeve enhanced from fruitlets infested applesawfly Mean

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Silken

Pinova

Zestar!

Summer Summer

McIntosh

COOP 39COOP

Ambrosia

Royal Gala Royal

s23 Jubilee Fugi Jubilee

Cultivar McIntosh Rogers

Figure 8.9: Infestation levels of Hoplocampa testudinea in enhanced sleeve cage study using data from all sleeve cages (P = 0.009), sleeve cages with adult apple sawflies (P= 0.088) and sleeve cages without adult apple sawflies (P= 0.002). Cultivars are ranked by infestation across all sleeve cages. 162

8.3.3 Discussion

The data from the sleeve cage study supported the results obtained from the field studies and behavioural bioassays. The sleeve cages containing adults showed the highest levels of infestation in the Zestar!, s23-06-153 and Pinova cultivars, confirming patterns of preference in the visitation and infestation surveys (Figs. 3.6 & 3.7). The sleeve cage study indicates that in the absence preferred cultivars, less-preferred host plants will be infested to a greater degree by H. testudinea. Lacking options for oviposition sites, the caged apple sawflies should have infested each set of fruitlets in equal measure.

However, the significant differences observed in the rate of infestation between cultivars suggests some factor or factors working against this.

A pattern of cultivar preference similar to that observed in the cages with adults was observed in the sleeve cages without adult apple sawflies. As these blossoms were infested prior to caging, the pattern of infestation should have been similar to that in the survey of uncaged fruitlets (Fig. 3.7). This appeared to be the case; cultivars determined to be preferred or non-preferred in field surveys had similar levels of infestation in the sleeve cage study (Table 6.1).

Differences among levels of infestation between the cages without adult apple sawflies and the field survey (Fig. 3.7) are likely the result of the cages preventing additional visitations by adult apple sawflies and larval infestation of the blossoms and developing fruitlets. The pattern of infestation in the cage study was similar to that in the field surveys, providing support for preferences exhibited by H. testudinea.

Variations in rate of infestation in this enhanced sleeve cage survey may have been the result of several factors; apple sawflies normally fly to their preferred 163

feeding/oviposition site (Miles, 1932) and the sleeve cages prevented this. The restriction to flight may have influenced their behaviours and made it more difficult for the insects to orient themselves towards and locate the blossoms/fruitlets within the cages.

Odours from other cultivars were present in the B137 and B138 orchards. As indicated by field surveys and bioassays in this thesis, apple sawflies will respond strongest to preferred cultivars when given a choice between these host plants and less- preferred cultivars. The odours from more-preferred cultivars may have prevented the caged insects from seeking out the less-preferred oviposition sites present in the sleeves.

These results may also be a consequence of the low numbers of paired adults used in the sleeve cage study and the variation in blossom/fruitlet productivity between cultivars. Some cultivars such as Pinova were highly productive with up to 108 blossoms and potential oviposition sites in total. Other cultivars, such as Jubilee Fugi had far fewer blossoms. This difference in the quantity and concentration of odour sources may have contributed to the observed differences in infestation.

In orchards without preferred cultivars, growers with monoculture orchards can therefore expect greater damage from apple sawfly infestations as the insects make use of available food sources and oviposition sites. Orchards with several cultivars may expect to see differences in crop loss and infestation between apple types if a preferred cultivar is present. Repeating this experiment earlier in the season with uncontaminated blossoms and with a complete set of paired adult apple sawflies per sleeve cage on more productive cultivars is recommended. The use of monoculture orchards is also recommended to determine whether proximity to the odours from other, more preferred cultivars may influence rate of infestation in a no-choice oviposition survey such as this.

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9. Appendix B: Rhagoletis pomonella

9.1 Colony Development

9.1.1 Materials and Methods

Apple maggots were raised in a manner similar to that described by Nielson

(1965). Initial supplies of maggots were collected from two sources: captured adults and pupae from field-infested apples. Pupae from this population were taken from drop trays and subjected to the pre-chill, chill and post-chill temperature regimes outlined below.

Adult apple maggot flies were collected upon emergence. In 2012, this was between July 10 and August 31. In 2013, apple maggots were collected between July 11 and September 9. Adult apple maggots were taken from several unsprayed orchard blocks using an aspirator (Fig. 3.1) and placed in an 18” x 12” x 12” fine-meshed cage. Food sources were comprised of sugar cubes and a protein/vitamin mixture recommended by

Nielson and MacAllan (1965) and described in detail in Nielson (1965): vitamins and salt-free casein acid hydrolysate, tryotophane arginine, histidine and cystinie mixed with salt mixture No. 2 USP XIII at a 4:1 ratio. Water sources were provided via cotton cloths soaked in a 500 mL beaker of water. Fresh NovaSpy and NovaMac apples were collected throughout the colony’s development in July and August. These apples were taken from an untended orchard at the AFHRC. These were provided for oviposition as a means of harvesting larvae from this population.

In 2012, apple maggots were collected for the development of a colony line.

Infested apples were taken from the colony as well as collected from a

Novaspy/Novamac orchard and placed in ‘drop cages’ over pans containing 5-7 cm of a sterilized peat moss/soil mixture. This substrate was used instead of the sifted sand, 165

vermiculite, burlap and/or felt substrates described in Nielson 1965 because of availability and to improve moisture retention and reduce desiccation. Apples were examined every 2-3 days for evidence of larval emergence (exit holes) and replaced with fresh infested apples if these exit holes were observed. Rotten apples were discarded.

The peat moss/soil mixture was 50:50 by volume and sterilized by autoclave twice over a 24-hour period. Once the maggots exited the apples and burrowed into the soil, the trays were placed in a growth chamber and subjected to a chill period of 16 weeks at 3°C and 80% humidity with a 9:15 L:D cycle. After this chill period, the temperature was raised to 23°C at 80% humidity with a 16:8 L:D cycle until adult emergence. One week after the post-chill temperature regime was started, pupae were

‘fished’ from the pans and transferred to a 500 mL beaker containing the sterilized peat moss/moss mixture. The open top of the beakers was secured with mesh to prevent emergent adults from escaping. The sealed beakers were returned to the growth chamber.

Low rates of adult emergence and the total mortality of the adults that did emerge within 3 days of emergence prevented further work with the colony.

9.1.2 Results

The 2012-2013 colony produced some adults; approximately 5-10% of harvested pupae emerged as adults, although these specimens did not survive more than three days, dying before they became sexually mature or could be tested in bioassays and/or electrophysiological tests. A fungal infection was responsible for most of the post- emergence deaths. Most of these mortalities occurred over a single weekend when adult flies emerged after the Friday check of the colony and their corpses were found in the

166

enclosures the following Monday. No bycatch or parasitoids were observed in the population.

9.1.3 Discussion

Adult apple maggot flies were produced using the rearing methods described in

Chapter 9.1, however the high mortality of emergent adults and the microbial infestation which caused it was unexpected. The substrate used in the apple maggot colonies was sterilized twice prior to use and a fresh batch of sterile substrate was used after the pupae were removed from the pans. The presence of fungus may have occurred as a consequence of ‘fishing’ the pupae out, which required briefly soaking the substrate and the pupae in room-temperature water and removing the pupae, although this method is routinely used in the rearing of Rhagoletis mendax (Ginette Pitcher, pers. comm., July,

2013) without issue. Contamination from an unidentified source is likely to have resulted in the fungal infestation that killed off the emergent adults. Further rearing attempts are recommended, increasing the ambient humidity to reduce desiccation in pupating larvae and ensuring the sterility of the growth chamber.

9.2 Wind Tunnel

9.2.1 Materials and Methods

A wind tunnel at the AFHRC was set up for the use of apple maggots in bioassays

(Fig. 9.1). Protocols from the wind tunnel bioassays described Nojima et al., (2003) and

Zhang et al., (1999) were modified for this study. All wind tunnel tests used apple

167

Figure 9.1: Flight chamber at the Agri-Food and Horticultural Research Centre

168

maggots acquired at the AFHRC site. Red bait spheres (9.5 cm diameter) were used as lures (Fig. 9.2).

An aliquot of 10 μL of concentrated volatile extract was combined with 200 μL dichloromethane in a red rubber septa. The volatile extracts were drawn from the same cultivars as used in the Y-tube behavioural bioassay. Preferred blends of Zestar!, Pinova,

COOP 39 and Royal Gala were used as well as ‘non-preferred’ blends of Ambrosia and

8S-27-43 and a pentane solvent control.

The septa were anchored 3 cm below red bait spheres in a wind tunnel for a two- choice bioassay. The spheres were 25 cm off the ground, 35 cm apart and 15 cm from each side of the wind tunnel. The flight chamber itself measured 93 x 87 x 57 cm. Five female and five male apple maggot flies were tested individually against several blend combinations. Each individual was tested three times. A pentane control and a preferred/non-preferred volatile blend were used in each test.

After removal from the colony, each fly was placed in a fine mesh-topped specimen jar in the fight chamber for 5 minutes to allow to acclimation to the conditions in the wind tunnel. After this period of acclimation was over, the container was opened and the fly was released into the flight chamber. Each test period lasted 5 minutes.

Individual flies were recovered from the flight chamber and given a rest period of 5 minutes before being tested again. Type of preference towards apple cultivars was determined on the reactions of the flies: if an apple maggot fly moved towards a particular odour source, this was considered a weak preference. If it landed on the test sphere, this would be a strong preference.

169

Figure 9.2: Set up of red bait spheres with odour sources on top of stands.

170

After an initial series of bioassays produced no responses, the septa were mounted above the test spheres, 35 cm off the ground. All other environmental factors were as indicated previously. When test insects still did not respond consistently to the odour sources, a zero-choice bioassay was conducted using intact, uninfested Zestar! apples.

The apples were secured 20-25 cm off the floor of the flight chamber and placed in the center of the airflow, 20 cm from the ‘downwind’ end. Two females and two males were tested three times each in this no-choice bioassay.

Light sources were provided by fluorescent lamps built into the wind tunnel and within the testing room as well as a red-light lamp borrowed from CABL at Acadia

University. Due to a lack of response in test subjects, several variations of light levels were used: low-light conditions within the wind tunnel and high levels outside, high light levels within the tunnel and with no outside ambient light. The red light lamp was used to observe fly behaviours in the test without other light sources. ‘Low’ light conditions were considered to be <150 Lux, measured with a Sper Scientific® Advanced Light Meter.

Maximum light provided by the wind tunnel was approximately 280 Lux at the center and 260-230 Lux at the position of each test stand. Light levels increased to 330 Lux within the center of the wind tunnel and 260-230 Lux at the downwind end with both wind tunnel and room lights on. Humidity in the testing room was between 60% and 80%

RH, higher than the 50-70% RH recorded in Zhang et al (1999). Wind tunnel bioassays were performed within apple maggot flight times and were done no earlier than 9:00 AM and no later than 4:00 PM.

171

9.2.2 Results

Three weak responses were noted in total: 1 to COOP 39, 1 to Royal Gala and 1 to Ambrosia. These responses occurred at light levels of 211-245 Lux within the flight chamber. These results are not statistically significant. In all other trials, regardless of stimulus, light levels or airflow, the apple maggot flies did not react to any other volatile blend. Trials using apples elicited no responses.

Flight was rarely observed in all wind tunnel trials and when it did occur was limited and infrequent. Specimens would fly to the top of the chamber and remain there, walk along the periphery, crawl along the walls or remain on the floor. Only insects on the ceiling of the flight chamber exhibited an attraction to the volatile extracts. These insects would not fly towards the odour source, but would walk along the ceiling towards the preferred odour source and drop onto it.

9.2.3 Discussion

The lack of positive responses to the wind tunnel bioassays is likely a result of issues with airflow and/or flight chamber structure. Light levels, specific volatile blends, position of bait spheres within the flight chamber, position of septa on bait spheres and in air column, sources of attraction, acclimation periods and time of day of trials were all tested in attempts to elicit consistent positive responses from insects. Environmental conditions including light levels, temperature and humidity speed of air flow in the flight chamber and room were within the protocols outlined by Zhang et al., (1999). Volatile extracts were prepared as described in Nojima et al., 2003. Issues during preparation of the septa may have altered the volatile profile of the extracts, making them

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unrecognizable by the test insects. However, this would not explain the lack of responses to the Zestar! apples used in subsequent trials as field surveys of adult apple maggot flies and apple maggots and behavioural bioassays indicated that this was a preferred cultivar.

As apple maggot flies use odour as a primary means of orienting themselves towards a potential food source/oviposition site (Fell et al., 1982; Rull & Prokopy, 2005) and visual cues to determine suitability of that site (Prokopy, 2011), a consistent pattern of preference to the apples should have been observed.

The consistent lack of responses to extracts from preferred and non-preferred cultivars, control extracts and mature fruit indicates that the apple maggot flies were unable to sense or orient themselves towards the odour sources. The airflow in the flight tunnel may not have carried the odours to the test insects or the internal surfaces may have disoriented the flies, preventing them reacting normally to the presence of preferred odour sources.