P>The Impacts of Climate Change and Belowground Herbivory on Aphids Via Primary Metabolites

The impacts of climate change and belowground herbivory on aphids via primary metabolites

James M. W. Ryalls

BSc (Hons), MRes

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Hawkesbury Institute for the Environment Western Sydney University

2016

Declaration of Authenticity

The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution.

Abstract

Global climate and atmospheric change (summarised as climate change for brevity) may alter patterns of crop damage by insect herbivores, but little is known about how multiple climate change factors, acting in tandem, shape such interactions. Crucially, the specific plant-mediated mechanisms underpinning these effects remain largely unknown. Moreover, research into the effects of climate change on leguminous plant species, which have the ability to fix atmospheric nitrogen (N2) via their association with root nodule-dwelling rhizobial bacteria, and their associated insect herbivores, is surprisingly scarce considering their increasing importance in terrestrial ecosystems worldwide. Using a model legume, lucerne, otherwise known as alfalfa, Medicago sativa (Fabaceae), and a model pest species, the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae), this work addresses how predicted changes in carbon dioxide

(CO2) concentrations, temperature and rainfall patterns as well as interactions with other organisms, including the root-feeding weevil Sitona discoideus (Coleoptera: Curculionidae), might shape legume-feeding aphid populations in the future.

Recent literature on the impacts of climate change on aphids and the biology and trophic interactions of lucerne aphids specifically were synthesised in chapters one and two, respectively. These chapters highlighted the importance of the interactions between multiple abiotic and biotic variables in shaping aphid population dynamics.

Empirical research chapters three to six, using up to five lucerne genotypes (i.e. cultivars) in glasshouse and field experiments, addressed how A. pisum responded to the isolated and combined effects of climate change and root herbivory. In particular, chapter three determined the effects of elevated temperatures (eT) and elevated atmospheric CO2 concentrations (eCO2) on root-feeding S. discoideus larvae and their interaction with A. pisum. Chapter four addressed whether the effects of eT, eCO2 and simulated root damage on aphids could be explained by changes in plant amino acid concentrations. Chapter five built on the mechanistic findings from chapter four to determine whether specific groups of amino acids were responsible for driving the effects of eT and eCO2 on aphid fecundity, longevity and intrinsic rate of increase (rm).

ABSTRACT

Chapter six extended this research to the field to determine the plant-mediated effects of water stress and root herbivory on aphids in a mixed grass–legume system.

Lucerne demonstrated an overcompensatory growth response to root herbivory by S. discoideus larvae by increasing net root biomass and nodulation by 31% and 45%, respectively. eT negated the positive effects of eCO2 on weevil larval development, as well as on a number of lucerne characteristics (e.g. nodulation and amino acid concentrations) and aphid performance parameters (e.g. population growth, fecundity and rm). Root herbivory by S. discoideus negatively impacted aphids in general, although effects were dependent on feeding duration and herbivore arrival sequence (i.e. whether aphids fed on the plant before or after root herbivory). While drought negatively impacted aphid abundance, potentially via reduced phloem turgor and sap viscosity, the effects of eT, eCO2 and root herbivory on aphids were often driven by concentrations of specific amino acid groups. Nitrogen (N) leached from lacerated lucerne root nodules by S. discoideus led to increased concentrations of N in a neighbouring grass, Phalaris aquatica (Poaceae), with knock-on effects on plant competition and community dynamics.

The opposing effects of eT and eCO2 on plant characteristics and both aboveground and belowground herbivores demonstrates the importance of combining trophic complexity with multiple climatic factors as a means of gaining realistic insights into how insect and plant communities will respond under future conditions. Identifying the specific amino acid changes underpinning aphid responses to climate change and root herbivory offers the potential for breeding aphid resistance traits into lucerne cultivars and informing adaptation strategies against future threats. Changes in precipitation patterns and plant-mediated indirect aboveground–belowground herbivore interactions can alter the outcome of competition between N-fixing legumes and non-N-fixing grasses, with important implications for plant community structure and productivity.

Avenues for future research are explored and other causal agents of changes in aphid performance are discussed, which may further elucidate the mechanisms underpinning climate change and belowground herbivory impacts on aphid pests.

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Preface

This thesis comprises original work conducted by myself with guidance from my supervisors; Scott Johnson (primary supervisor), Markus Riegler and Ben Moore. I conceptualised the research project and established the experimental design together with my supervisory panel. I have collected, analysed and interpreted all the data in discussion with my supervisory panel. I have written the thesis and all publications therein with guidance from my supervisory panel.

Five of the chapters in this thesis (2 to 6) have been presented in a format appropriate for peer-review journals. Some repetition was unavoidable where recurrent methodologies were used therefore.

The term ‘climate change’ within the abstract and thesis title was used to describe both climate (elevated temperature and changes in rainfall patterns) and atmospheric change (elevated carbon dioxide concentrations) for brevity. Distinctions between climate and atmospheric change are made within chapters thereafter to avoid confusion.

Acknowledgements

This work was undertaken as part of a PhD research scholarship funded by the Hawkesbury Institute for the Environment (HIE), Western Sydney University. Thank you to all the students and staff at HIE for making me feel so welcome and for all their advice and help throughout my PhD.

My PhD supervisor, Scott Johnson, deserves utmost praise for his unfaltering support, guidance and friendship over these three years. My two secondary supervisors, Ben Moore and Markus Riegler, have always provided invaluable feedback and support when needed, for which I am truly grateful. Thank you to Aidan Hall, Chris Mitchell, Goran Lopaticki, David Fidler and, in particular, to Andrew Gherlenda, for their help with field and laboratory work. All the people in my office have kept me sane and jolly with their lovable personalities and willingness to help. My beautiful fiancée, Lisa Bromfield, jumped continents with me and provided more help and support than she could ever have imagined. Thank you to my wonderful parents, Caroline and Mike Ryalls, for their encouragement and regular visits. I would like to express my sincerest thanks and appreciation to my MRes mentor, Hefin Jones, for setting me up for a great three years. Thanks also to Philip Smith for his exhaustive proofreading. Finally, I’d like to thank my examiners, Michael Rostás and Nigel Andrews, for their consideration of this thesis and the suggestions they made for its improvement. To all the people that offered help along the way, your kindness will continue to echo.

List of Tables

Table 3-1. Effect of cultivar on plant height, root nodulation, aphid colonisation and weevil emergence. Overall means (± SE) and frequencies shown. *Significance indicated by linear model outputs and ANOVA (2) test results...... 46

Table 4-1. Summary of final model statistical analyses of individual amino acid responses to plant-damage (Treatment), temperature (Temp) and CO2 treatments...... 62

Table 5-1. Results from multivariate permutational analysis (PERMANOVA) of cultivar, temperature and CO2 treatment effects on aphid performance overall (i.e. the combination of all aphid performance parameters). In general, aphid performance decreased as cultivar resistance increased and eT counteracted the increase in aphid performance observed under eCO2. *indicates significance (P < 0.05)...... 77

Table 5-2. Table of statistics from mixed models of individual amino acid responses to different lucerne genotypes, temperature and CO2 treatments...... 82

List of Figures

Figure 1-1. Main aims for each thesis chapter. Experimental chapters 3, 4 and 5 were conducted in a glasshouse environment under controlled conditions of ambient and elevated carbon dioxide and temperature, while chapter 6 was conducted in the field with three watering treatments (drought, ambient precipitation and elevated precipitation). The review identified knowledge gaps in the literature to support glasshouse experiments, which subsequently provided functional support for the field experiment...... 11

Figure 2-1. Lucerne-feeding aphids: cowpea aphids Aphis craccivora (top left), bluegreen aphids Acyrthosiphon kondoi (top right), spotted alfalfa aphids Therioaphis trifolii maculata (bottom left) and pea aphids Acyrthosiphon pisum (bottom right). Images (clockwise from top left) by Z. Ludgate, State of Queensland, D. Ironside and State of Queensland taken from the Department of Agriculture, Fisheries and Forestry...... 16

Figure 2-2. Significant parameters of Acyrthosiphon pisum reproduction (a, b), survival (c) and activity (d) between two lucerne cultivars: Radius (containing high saponin and low flavonoid concentrations) and Sapko (containing low saponin and high flavonoid concentrations). Potential drops (pds) correspond to cell wall punctures and short insertions of the aphid stylets into the cells using electrical penetration graph recordings. Data are the mean ± SE shown after Goławska et al. (2008). .... 23

Figure 2-3. Reported trophic interactions affecting lucerne-feeding aphids. Dotted arrows represent indirect interactions, solid lines represent direct interactions. Positive (+) and negative interactions (−) indicated. Roman numerals (i–viii) refer to interspecific interactions discussed in the text...... 26

Figure 2-4. Effects of two endosymbionts, Serratia symbiotica (PASS) and Rickettsia (PAR), on the life history traits (a-d) of bluegreen aphids reared on lucerne. Data are the mean ± SE shown after Chen et al. (2000)...... 28

Figure 2-5. Effect of parasitism by Aphidius eadyi on the reproductive period (a) and fecundity (progeny production) (b) of pea aphid. Aphid parasitism: P4, parasitized fourth instar; P, parasitized adult; N, unparasitized adult. Data are the mean ± SE shown after He et al. (2003)...... 32

Figure 3-1. Effects of temperature and cultivar (Hunter River, H; Sequel, S and Trifecta, T) on plant growth (height) 10 weeks after sowing. Mean values (± SE) of significant treatment factors (temperature and cultivar) in the final model shown...... 45

Figure 3-2. Height of three lucerne cultivars (Hunter River, H; Sequel, S and Trifecta, T) 14 weeks after sowing in treatments with and without aphids and weevils (plants with weevils, W+; plants without weevils, W-). Graphs (a-d) represent the four “CO2 × temperature” treatments. Mean values (± SE) of significant treatment factors (cultivar, aphid presence, CO2: temperature and temperature: weevil presence) in the final model shown...... 46

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LIST OF FIGURES

Figure 3-3. Effects of temperature on the number of root nodules per plant under aCO2 (open bars) and eCO2 (closed bars). Mean values (± SE) shown. Bars with the same letters were not significantly different (P < 0.05)...... 47

Figure 3-4. Effects of temperature on the proportion of plants containing weevils that reached adulthood and emerged from the soil at aCO2 (open bars) and eCO2 (closed bars). All columns were significantly different (P < 0.01) from one another...... 48

Figure 4-1. Hypothesised treatment effects of root cutting (before and after aphid infestation), elevated temperature and CO2 on aphids and plant characteristics, with example references given as a basis for our hypotheses. H1, H2 and H3 refer to hypotheses (i), (ii) and (iii), respectively (A). Methodological timeline of experimental events (B)...... 57

Figure 4-2. Effects of elevated temperature and CO2 on plant height (a), root biomass (b), shoot biomass (c) and root C:N (d). Mean values (± standard errors) shown. Statistically significant effects indicated *(P < 0.05). Bars with the same letters were not significantly different (P < 0.05)...... 60

Figure 4-3. Effects of elevated temperature and CO2 on total amino acids (a), essential amino acids (b) and aphid abundance (c). Mean values (± standard errors) shown. Statistically significant effects indicated *(P < 0.05). Bars with the same letters were not significantly different (P < 0.05)...... 61

Figure 4-4. Effects of plant-damage treatment on plant height (a), root biomass (b), shoot biomass (c) and root C:N (d). Mean values (± standard errors) shown. Bars with the same letters were not significantly different (P < 0.05)...... 63

Figure 4-5. Effects of plant-damage treatment on total foliar amino acid concentrations. Mean values (± standard errors) shown. Bars with the same letters were not significantly different (P < 0.05)...... 64

Figure 4-6. Effects of root cutting on aphid colonisation success (i.e. percentage of plants with aphids present upon harvest) under ambient and elevated temperatures. Statistically significant effect indicated *(P < 0.05). Bars with the same letters were not significantly different (P < 0.05)...... 65

Figure 4-7. Schematic diagram of the significant positive and negative effects of temperature, CO2 and root cutting treatments on plant characteristics and aphids. Numbers and letters correspond to figures associated with those effects...... 65

Figure 5-1. Acyrthosiphon pisum performance under elevated temperature and CO2 conditions. Means (± SE) of (a) fecundity, (b) rm and (c) longevity, and (d) percentages (± SE) of aphid colonisation shown. Terms that were significant in general linear mixed models indicated by *(P < 0.05). Bars with the same letters were not significantly different (P < 0.05) according to post hoc tests performed on interactive effects (a-c) in mixed models. Sample sizes are shown in Table 5-1 and Appendix II Table S5-1...... 78

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LIST OF FIGURES

Figure 5-2. Acyrthosiphon pisum performance on five lucerne genotypes. Coloured bars represent susceptible (white bars), low resistance (mid-grey bars) and moderate resistance cultivars (dark-grey bars). Means (± SE) of (a) fecundity, (b) rm and (c) longevity and (d) percentages (± SE) of aphid colonisation shown. Bars with the same letters were not significantly different (P < 0.05) according to post hoc tests performed on general linear mixed models. Sample sizes are shown in Table 5-1 and Appendix II Table S5-1...... 79

Figure 5-3. Principal component analysis of foliar amino acid concentration data (N = 311) with attribute loadings on the first two components PC 1 and PC 2. Amino acid abbreviations are denoted in Table 5-2. Sample scores on PC 1 and PC 2 explain, respectively, 37% and 13% of the variation in the dataset. Plots and ellipses (representing 68% of the predicted data) were coloured according to temperature and CO2 conditions with means (± SE) of total amino acid concentrations shown beside treatment combinations...... 81

Figure 5-4. Effects of eT and eCO2 on total concentrations of groups 1 and 2 amino acids (a) and the effects of cultivar on total concentrations of group 3 amino acids (b). Mean values (± SE) are shown. N = 233. Terms that were significant in PERMANOVA indicated by * (P < 0.05) and ** (P < 0.01). Correlations between aphid fecundity and grouped amino acid concentrations with 95% confidence intervals displayed (c and d)...... 83

Figure 6-1. Schematic diagram of experimental plots beneath one rain-exclusion shelter...... 96

Figure 6-2. The interactive effects of precipitation and herbivore treatment on soil water content. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05)...... 99

Figure 6-3. The impacts of precipitation and herbivore treatment on the height of Harding grass (A and C) and lucerne (B) at week six (plant transplant period), week eight (weevil inoculation period) and week twelve (harvest period). Herbivore treatment had no significant effect on lucerne height. Mean values (± SE) are shown. Statistically significant effects are indicated by *(P < 0.05) and bars with the same letters were not significantly different (P < 0.05)...... 100

Figure 6-4. The impacts of precipitation on the root and shoot biomass of Harding grass (A) and lucerne (B). Herbivore treatment had no significant effect on biomass. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05)...... 101

Figure 6-5. The impacts of herbivore treatment on the root and shoot nitrogen concentrations of Harding grass. CON were the control plants (no insects), A had aphids alone, W had weevils alone and WA had both insects. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05)...... 101

Figure 6-6. The impacts of precipitation and herbivore treatment on lucerne nodulation (A and B) and the impacts of precipitation and herbivore treatment on root N content (C) and root N concentration (D), respectively. CON were the control ix

LIST OF FIGURES

plants (no insects), A had aphids alone, W had weevils alone and WA had both insects. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05)...... 102

Figure 6-7. The impacts of precipitation on individual groups of amino acids (A), namely group 1 (glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine), group 3 (aspartate and glutamate) and group 4 (glutamine and serine) and the impacts of herbivore treatment on proline concentrations (B). CON were the control plants (no insects), A had aphids alone, W had weevils alone and WA had both insects. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05)...... 103

Figure 6-8. The impacts of precipitation (A) and weevil presence (B) on the number of aphids. A have aphids alone and WA have both weevils and aphids. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05)...... 104

Figure 6-9. Summary of key results illustrating plant–herbivore interactions under drought. Red arrows indicate negative impacts and blue arrows represent positive impacts. Numbers refer to relevant figures in Chapter 6 associated with each effect. *Amino acids represent concentrations of groups 1 and 3 amino acids, namely glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine, aspartate and glutamate...... 105

Figure 7-1. Warming frame schematic ...... 125

Table of Contents

Declaration of Authenticity ...... i Abstract...... ii Preface ...... iv Acknowledgements ...... v List of Tables ...... vi List of Figures ...... vii

Chapter 1: General introduction ...... 1 1.1 Predicted climate and atmospheric change ...... 1 1.2 Aphids as insect herbivore pests ...... 1 1.3 Direct effects of climate and atmospheric change on aphids ...... 2 1.4 Plant-mediated effects of climate and atmospheric change on aphids ... 4 1.5 Aboveground–belowground interactions ...... 10 1.6 Thesis overview ...... 10 1.7 Thesis outline ...... 11

Chapter 2: Biology and trophic interactions of lucerne aphids – review ...... 14 2.1 Abstract...... 14 2.2 Introduction ...... 15 2.3 Aphids as lucerne pests ...... 17 2.4 Cultivar resistance ...... 19 2.5 Alfalfa Mosaic Virus ...... 24 2.6 Trophic interactions ...... 25 2.7 Future climate change ...... 33 2.8 Conclusions and future perspectives ...... 35 2.9 Acknowledgements ...... 38

Chapter 3: Effects of elevated temperature and CO2 on aboveground–belowground systems: a case study with plants, their mutualistic bacteria and root / shoot herbivores ...... 39 3.1 Abstract...... 39 3.2 Introduction ...... 40 3.3 Materials and methods...... 42 3.4 Results...... 45 3.5 Discussion ...... 48 3.6 Acknowledgements ...... 51

Chapter 4: Amino acid-mediated impacts of elevated carbon dioxide and simulated root herbivory on aphids are neutralized by increased air temperatures ...... 52 4.1 Abstract...... 52 4.2 Introduction ...... 53 4.3 Materials and methods...... 55 4.4 Results...... 60 4.5 Discussion ...... 66 4.6 Acknowledgements ...... 69

TABLE OF CONTENTS

Chapter 5: Climate and atmospheric change impacts on sap-feeding herbivores: a mechanistic explanation based on functional groups of primary metabolites ...... 70 5.1 Abstract...... 70 5.2 Introduction ...... 71 5.3 Materials and methods...... 73 5.4 Results...... 77 5.5 Discussion ...... 84 5.6 Acknowledgements ...... 88

Chapter 6: Above–belowground herbivore interactions in mixed plant communities are influenced by altered precipitation patterns ...... 89 6.1 Abstract...... 89 6.2 Introduction ...... 90 6.3 Materials and methods...... 94 6.4 Results...... 98 6.5 Discussion ...... 105 6.6 Acknowledgements ...... 111

Chapter 7: General discussion ...... 112 7.1 Main findings and synthesis ...... 112 7.2 Constraints, caveats and further study ...... 120 7.3 Conclusions and future outlook ...... 123

Bibliography ...... 126

Appendices ...... 153 Appendix I – Chapter 4 supplementary material ...... 153 Appendix II – Chapter 5 supplementary material ...... 157 Appendix III – Chapter 6 supplementary material ...... 163 Appendix IV – Supplementary data in review ...... 169

List of Publications ...... 172

Experimental images ...... 173

Short story: A cultural evolution ...... 176 Part One: Pim ...... 176 Part Two: George ...... 176 Part Three: Prison ...... 176 Part Four: Parasites ...... 177 Part Five: Salvation ...... 177

Chapter 1

General introduction

1.1 Predicted climate and atmospheric change Global climate and atmospheric change encompasses multiple factors, including rising atmospheric carbon dioxide (CO2) and ozone concentrations, warming and altered precipitation patterns, which have major impacts on plants and insect herbivores. CO2 concentrations are predicted to continue to increase from current levels of c. 400 µmol mol-1 to between 500 and 1000 µmol mol-1 by 2100 (Franks et al., 2013; IPCC, 2014). Meanwhile, average global surface temperatures are projected to increase by 1 to 6.4 °C within this century (IPCC, 2007, 2014), with Australian temperatures predicted to rise by 2 to 5 °C by 2070 (CSIRO, 2007, 2012). The incidence of extreme precipitation events, including droughts and floods, is expected to increase in many regions throughout the world, with a generally drier future predicted for south-eastern Australia (Chiew et al., 2011; Dai, 2011). Moreover, prolonged drought periods are likely to be accompanied by increased temperatures and CO2 concentrations, particularly in Africa, South America and Australia (CSIRO, 2007; Meehl et al., 2007; Bates et al., 2008; IPCC, 2014). Predicting how ecosystems will respond under future conditions is becoming increasingly important, especially as the magnitude of climate and atmospheric change increases (Staudt et al., 2013).

1.2 Aphids as insect herbivore pests

Elevated CO2 concentrations (eCO2), elevated temperatures (eT) and changes in rainfall patterns may alter ecological processes in different ways, with independent or interacting direct and indirect effects on plants and insect herbivores. For example, these environmental factors can alter plant physiological processes, together with the concentration and composition of plant secondary metabolites (PSMs) and primary metabolites in the phloem (e.g. amino acids), which are especially important for aphids (Docherty et al., 1997; Douglas, 2003; Aslam et al., 2013). Aphids (Hemiptera: Aphididae), with their capacity for rapid population growth and role as a disease vector, are renowned pests of agriculture, horticulture and forestry. Generalist aphid species

CHAPTER 1 GENERAL INTRODUCTION

(e.g. Myzus persicae) can feed on multiple plant species across many families. Most of the c. 6,000 species worldwide, however, have developed intimate relationships with a narrow range of closely-related host-plants and are highly responsive to climate and atmospheric change (Huberty and Denno, 2004; Sun and Ge, 2011; Bell et al., 2015).

1.3 Direct effects of climate and atmospheric change on aphids Climate change can have direct individual- and population-level impacts on aphids. Individual-level impacts include changes in behaviour, metabolism and life-history traits, such as development time, longevity and fecundity. Population-level impacts include genotypic variation within populations or changes in population dynamics (e.g. phenology, abundance, mortality rate and distribution). While any significant effects of atmospheric change (elevated CO2) on aphids are expected to be plant-mediated (Sun and Ge, 2011), direct effects on aphids could occur through changes in gas exchange dynamics within their tracheal system, which was briefly discussed as a possibility in Ryan et al. (2015). Direct effects of wind or increased rainfall may also cause mortality by preventing aphid movement or through drowning (Menon and Christudas, 1967; Walters and Dixon, 1984; Harrington and Taylor, 1990). Temperature, however, is considered the major factor directly driving the responses of aphids to climate change.

As for most insects, the intrinsic rate of increase (rm) of aphids tends to increase with temperature up to a certain threshold, after which it decreases abruptly (van Baaren et al., 2010). The shape and magnitude of the curve is region- (i.e. determined by local adaptation), aphid species- and genotype-dependent (Awmack and Leather, 2007). The lower developmental temperature thresholds of Sitobion avenae (grain aphid) and Brevicoryne brassicae (mealy cabbage aphid), for example, have been reported as −3.6 °C and 7.1 °C, respectively (Campbell et al., 1974; Carter et al., 1982), although thresholds often depend on the analytical methods used and may vary between aphid genotypes that have adapted to different regions (Bale et al., 2007).

In warm temperate climates many aphid species reproduce parthenogenetically by producing female clones throughout the year, and this leads to rapid population growth. In cooler climates, however, aphids need overwinter to avoid cold winter temperatures in the mobile nymphal or adult stages as these are not very tolerant of low temperatures. Overwintering is achieved by producing sexual males and females in

CHAPTER 1 GENERAL INTRODUCTION autumn, which mate and produce cold-hardy eggs that hatch in spring (Dixon, 1998). Many species display both types of reproduction, with some clones genetically pre- determined to produce winter eggs (i.e. sexual clones) and some to continue parthenogenesis throughout the year. The minimum temperature threshold for survival of eggs tends to be considerably lower than the minimum temperature of their winter environments that they are adapted to, and thus overwintering success of eggs is largely independent of annual variations in winter minimum temperatures (Bale, 1999; Bale et al., 2007). As winter temperatures increase, so does survival of those aphids overwintering in the mobile stages and, over time, it is likely that selection will lead to a larger proportion overwintering in the mobile rather than the egg stage in species that have both options. This is likely to lead to increased damage in both winter- and spring-sown crops (Bale and Hayward, 2010).

Almost all aphids form an obligate association with the primary endosymbiont Buchnera aphidicola, which synthesises essential amino acids and other nutrients that aphids cannot acquire from their phloem diet (Buchner, 1965; Baumann et al., 2013; described further in section 1.3.1). Aphids may also harbour one or more secondary (or facultative) endosymbionts, which exert diverse effects on their aphid hosts, including changes in body colour, protection against parasitoids or resistance to heat stress (van Baaren et al., 2010). The effects of heat stress on Acyrthosiphon pisum (pea aphid) fecundity and the number of bacteriocytes (in which B. aphidicola resides), for example, were reduced by the presence of the pea aphid secondary symbiont “Candidatus Serratia symbiotica”, although the presence of another secondary symbiont, pea aphid Rickettsia, negated the beneficial effects of S. symbiotica to heat-stressed aphids (Montllor et al., 2002). Dunbar et al. (2007) identified a point mutation in B. aphidicola that governs the thermal tolerance of A. pisum. At low temperatures, aphid fecundity increased when they contained the mutated symbiont. When aphids were exposed to heat, however, the fecundity of those containing the mutated symbiont (present in ~20% of field populations) dramatically decreased compared with non-mutants. Thus, symbiotic associations play a fundamental role in aphid biology, with implications for predicting aphid responses to environmental change and incorporating novel biological control strategies (Goggin, 2007).

CHAPTER 1 GENERAL INTRODUCTION

While this thesis does not address predator or parasitoid impacts on aphids, it is necessary to note the direct impacts of other trophic levels on aphid populations that might obscure their responses to climate change (Barton and Ives, 2014). For example, thermal optima often vary between species and rising temperatures may release aphids from control by their natural enemies by minimising the overlap between their distributions, or vice versa. Moreover, elevated temperatures (eT) may influence other aphid population parameters, including fecundity, longevity, and predator or parasitoid attack rate. At temperatures below 11 °C, for example, A. pisum reproduces faster than the predator Coccinella septempunctata can consume it, but the reverse occurs above 11 °C (Harrington et al., 2001). Understanding how climate change impacts aphid biology is key to identifying the underlying mechanisms that influence population-level responses, including phenology, abundance, mortality, distribution and genetic variability.

1.4 Plant-mediated effects of climate and atmospheric change on aphids 1.4.1 Aphid nutritional requirements Aphids have mouthparts adapted to form stylets for probing between plant cells and penetrating the phloem tubes, into which they inject saliva and from which they withdraw sap (Pollard, 1973). Their phloem sap diet is rich in carbohydrates (predominantly sucrose) but deficient in nitrogenous compounds (Mittler, 1953; Patrick et al., 2001). In order to meet their minimum nitrogen requirements, they consume large quantities of sugar-rich sap and excrete the majority of phloem sugars as honeydew (Mattson, 1980; Febvay et al., 1995; Ashford et al., 2000). Free amino acids are the principal nitrogenous compounds in phloem sap (Douglas, 1993). Their importance for aphids has been made apparent, with clear evidence that plant amino acid composition and concentration have the strongest effects on aphid performance compared with other plant metabolites (Mittler, 1967a; Douglas, 1993; Dixon, 1998; Ponder et al., 2000). Ten of the twenty amino acids required for aphid survival, deemed “essential” amino acids, cannot be synthesised by the aphid itself (Douglas, 1993). Instead, they are supplied by primary intracellular bacterial symbionts (B. aphidicola) in exchange for other nutrients (Houk and Griffiths, 1980). For example, aphids acquire the “non-essential” amino acid glutamine from the host plant phloem and convert it to another “non-essential” amino acid (glutamic acid, otherwise known

CHAPTER 1 GENERAL INTRODUCTION as glutamate) within their bacteriocytes (where B. aphidicola resides). Glutamate is taken up by B. aphidicola and used to synthesise other “essential” amino acids for the aphid host (Sasaki and Ishikawa, 1995; Febvay et al., 1999; Haribal and Jander, 2015). This exchange allows aphids to utilise a nutritionally poor and imbalanced phloem diet which generally has a low “essential” amino acid content (Ryan, 2012).

Aphids form intricate relationships with their specific host-plants, often feeding from a single phloem sieve element cell for hours or days, while potentially evading and manipulating host-plant defences and nutritional compounds, respectively (Jiang and Miles, 1993; Will et al., 2007; Edwards et al., 2008; Gao et al., 2008b). Just as aphids can compromise host plant fitness (Dixon, 1998; Hussain et al., 2014), bottom-up effects of host plants, including nutritional quality, affect the performance of aphids (Awmack and Leather, 2002), which tend to favour plants with higher N and amino acid concentrations (Ponder et al., 2000; Nowak and Komor, 2010). Seasonal variation and environmental change can alter host plant quality, producing chemical or nutritional changes that may benefit or deleteriously affect aphids (Ryan et al., 2014b; Ryan et al., 2015). Sap-sucking aphids, as opposed to chewing insect guilds, show considerable variation in their plant-mediated responses to climate and, especially, atmospheric change, making long-term predictions challenging (Pritchard et al., 2007; Sun and Ge, 2011).

1.4.2 Effects of eT on aphid–plant interactions Direct effects of temperature on aphids and plants are prevalent, as described above, although eT may also indirectly impact aphids via changes in plant quality, morphology, phenology or defensive compounds (Holopainen and Kainulainen, 2004; Adler et al., 2007). In particular, aphid development rates and the subsequent number of generations in a given year may increase if rising temperatures increase the length of the host-plant growing season (Bale et al., 2002; Ziter et al., 2012). Additionally, leafy plant morphs can, through shading, provide protection against the negative effects of direct sunlight, high temperatures or other adverse weather conditions on aphids (Soroka and Mackay, 1991; Dunbar et al., 2007; Buchman and Cuddington, 2009). Plant genotype resistance to aphids may also alter the effects of eT on aphid success. For example, eT reduced A. pisum fecundity on both an aphid-resistant and -susceptible

CHAPTER 1 GENERAL INTRODUCTION lucerne (Medicago sativa) cultivar but to a lesser degree on the resistant cultivar (Karner and Manglitz, 1985). While short-term studies are important for identifying mechanisms linking climate change with plants and aphids, long-term studies instil realism into climate change predictions. The effects of long-term experimental warming on the aphid species Obtusicauda coweni feeding on big sagebrush (Artemisia tridentata) showed that warming tended to decrease aphid density, although no significant relationship between aphids and plant C:N ratio or size was observed (Adler et al., 2007). Empirically, disentangling the direct and plant-mediated effects of temperature on aphids proves logistically demanding. In previous glasshouse studies, for example, plants grown at different temperatures have been inoculated with insects under the same temperature conditions (e.g. Murray et al., 2013b) to distinguish between the direct and indirect effects of temperature on aphids and other insects. Artificial diet experiments (Akbar et al., 2016) can also be used to remove plant variance from the equation and complement results from glasshouse experiments.

1.4.3 Effects of water stress on aphid–plant interactions Predicted increases in temperature should be considered alongside changes in rainfall patterns since global warming effects on aphids often interact with soil moisture conditions (Romo and Tylianakis, 2013), although few studies have considered both simultaneously. Sustained water stress (e.g. drought) in plants is likely to impact aphids negatively via changes in phloem physiology (e.g. increases in osmotic pressure or sap viscosity and decreases in turgor), which may outweigh the impact of any increases in N (Johnson et al., 2011b) or amino acid concentrations (Hale et al., 2003) under drought conditions. Studies have observed positive (Wearing, 1972; Miles et al., 1982), negative (Kennedy et al., 1958; Xing et al., 2003) or no (Pons and Tatchell, 1995; Bethke et al., 1998; Aslam et al., 2013) effects of drought on aphid abundance, although the plant- mediated mechanisms are poorly understood. Additionally, some aphid species may be more resilient under water stress than others. Population growth rates of A. pisum, for example, decreased on water-stressed M. sativa plants, whereas Therioaphis maculata (spotted alfalfa aphid) was insensitive across a wide range of moisture levels (Forbes et al., 2005). Similarly, drought stress increased populations of the generalist aphid Myzus persicae (peach–potato aphid) but had no effect on the crucifer specialist Brevicoryne brassicae (mealy cabbage aphid) (Mewis et al., 2012). In that study, increases in M.

CHAPTER 1 GENERAL INTRODUCTION persicae under drought were attributed to increases in plant (Arabidopsis thaliana) sugar and specific amino acid concentrations (namely, glutamate, proline, isoleucine and lysine), while water-logged conditions decreased these amino acid concentrations. Other studies have observed changes in aphid population demography with, for example, ratios of nymphs to adults increasing on drought-stressed plants (Aslam et al., 2013), which may reflect intraspecific variation in aphid developmental rates (Sandström and Pettersson, 1994; Xing et al., 2003).

The majority of studies consider sustained water stress events, such as drought or elevated precipitation. Realistically, however, stress-related fluctuations in precipitation are discontinuous, resulting in pulsed stress events (Huberty and Denno, 2004) or changes in drought intensity (English-Loeb et al., 1997) that may periodically alter plant nutritional quality or hydraulic properties. Plants recovering from drought, for example, may regain phloem turgor and increase concentrations of plant nutritional compounds (e.g. N and amino acids), which aphids may capitalise on. However, if plants remain stressed due to other environmental pressures or if aphids remain on the plant, stress-related increases in plant quality are less likely (Huberty and Denno, 2004). Tariq et al. (2012) identified an increase in aphid performance on medium drought- stressed plants (50% of ambient water) compared with unstressed (ambient water) and highly drought-stressed plants (25% of ambient water), although no effect of pulsed drought stress was observed. Multiple theorems, including the plant stress (White, 1969; 1984), plant vigour (Price, 1991) or pulsed stress (Huberty and Denno, 2004) hypotheses, are invoked to explain these conflicting trends, which demonstrate the complexity of these plant–aphid interactions in water-stressed environments (Pritchard et al., 2007).

The field would benefit from more studies incorporating a range of predicted precipitation patterns, including pulsed stress, as well as seasonal or temporal differences that may contribute to the variation in plant quality and aphid performance. In the Western Australian wheatbelt, aphid flights into crops in winter correlate with rainfall events in the preceding summer, which support the growth of alternative host plants on which aphids proliferate before flying to crops (Thackray et al., 2004; Knight and Thackray, 2007). While controlling these neighbouring plants, especially during

CHAPTER 1 GENERAL INTRODUCTION months with higher rainfall, may serve as a way to limit aphid expansion and damage to crops, this strategy would almost certainly prove too expensive and environmentally destructive. Understanding the mechanisms underpinning crop susceptibility to aphids and building resistance into new crop genotypes may serve as the best way to mitigate the damage caused by aphids.

1.4.4 Effects of eCO2 on aphid–plant interactions Compared with other factors associated with environmental change, a wealth of knowledge has been accumulated on the effects of eCO2 on aphids, which are thought to be entirely plant-mediated, yet few general trends have emerged (Sun and Ge,

2011). Positive, negative and neutral responses of aphids to eCO2 have been observed, suggesting that aphid species respond idiosyncratically to eCO2 (Bezemer et al., 1999; Hughes and Bazzaz, 2001; Pritchard et al., 2007). The plant-mediated mechanisms underpinning responses to eCO2 remain uncertain but are most likely driven by changes in plant amino acids (Weibull, 1987; Dixon et al., 1993; Ponder et al., 2000; Karley et al., 2002). Recent studies by Ryan et al. (2014b; 2015), for example, suggest that aphid responses to eCO2 are linked to changes in individual plant amino acids. Rhopalosiphum padi (bird cherry–oat aphid) abundance on tall fescue (Festuca arundinacea) decreased under eCO2, associated with a decrease in some, mostly essential, amino acids (Ryan et al., 2014b). In contrast, R. padi abundance decreased on barley (Hordeum vulgare) under eCO2 (Ryan et al., 2015), suggesting that aphids are able to overcome the disadvantageous decrease in plant N concentrations under eCO2, potentially by reducing developmental times and increasing fecundity (Sun and Ge, 2011). Theoretical models by Newman and colleagues investigating the population dynamics of cereal aphids in Great Britain (Newman et al., 2003; Hoover and Newman, 2004; Newman,

2004; Newman, 2005) suggest that eCO2 will have positive effects on aphids when nitrogen is not limiting.

While it currently remains difficult to predict the effects of eCO2 on aphids feeding on most plants, aphid performance is likely to improve on leguminous plants, which contain rhizobial bacteria that are able to fix atmospheric nitrogen and compensate for the N-diluting effect of eCO2 experienced by other plant types (Rogers et al., 2009;

Sulieman et al., 2015). For example, eCO2 increased the nutritional quality (N and

CHAPTER 1 GENERAL INTRODUCTION amino acid content) and decreased A. pisum resistance of Medicago truncatula, associated with the down-regulation of genes in the ethylene signalling pathway (Guo et al., 2014). Furthermore, A. pisum enhanced amino acid metabolism in the host plant

(M. truncatula) to increase the aphid’s fitness (population abundance) under eCO2 (Guo et al., 2013).

Some plant–aphid interaction studies have incorporated multiple genotypes of individual plant species and identified breakdowns in aphid resistance of certain cultivars under eCO2 (Martin and Johnson, 2011; Hentley et al., 2014; Johnson et al.,

2014), whereas others have observed changes in aphid performance under eCO2 that have been consistent across host plant genotypes (Gao et al., 2008a). Contrasting differences have also been observed across studies comparing aphid genotypes under eCO2. For example, Mondor et al. (2005) identified neutral and positive effects of eCO2 in pink and green genotypes of A. pisum, respectively, whereas Ryan et al. (2014a) identified no interaction between eCO2 and R. padi genotype.

1.4.5 A multifactorial approach Studies into the effects of individual climate change factors remain important, although future increases in temperature and CO2 concentrations will occur in tandem. Only a handful of empirical studies (e.g. Himanen et al., 2008; Murray et al., 2013b) have incorporated the effects of both eT and eCO2 on plants and insects, and fewer still have examined their effects on aphids. Flynn, Sudderth and Bazzaz (2006) suggested that the combined effects of eT and eCO2 will exacerbate Macrosiphum euphorbiae (potato aphid) damage to certain plants, including the C3 perennial Solanum dulcamara.

In summary, changes in host plant quality attributable to global climate change can shape the extent to which plants are attacked by aphids, yet marked differences have been observed between both aphid and plant species and genotypes. From this standpoint, it remains difficult to extrapolate general trends from such a variety of individual responses. However, a more holistic approach combining the effects of multiple climate change factors on a multi-trophic scale, while extrapolating across plant types (e.g. leguminous and non-leguminous plants) may pave the way towards a general understanding of these responses.

CHAPTER 1 GENERAL INTRODUCTION

1.5 Aboveground–belowground interactions Aphids do not live in isolation. They share their host plant with many other herbivorous insects that live above and below the soil surface. Aboveground and belowground herbivores interact with one another indirectly through plant-mediated mechanisms (Wardle et al., 2004; van der Putten et al., 2009). Many studies report on the plant- mediated mechanisms linking aphids to spatially-separated root herbivores (Poveda et al., 2005; Johnson et al., 2009; Kutyniok and Müller, 2012; Soler et al., 2012; McKenzie et al., 2013b), yet few have considered these interactions in the context of global environmental change (Salt et al., 1996; McKenzie et al., 2013a). Belowground herbivores generally have positive effects on aphids (Johnson et al., 2012), associated with impaired root function and the stress-related accumulation of nitrogen in the foliage (Masters et al., 1993). This general trend, however, may be reversed in legumes, which have the capacity to biologically fix atmospheric nitrogen via their association with rhizobia bacteria that are housed by nodules on the roots (Wynn- Williams, 1982; Haag et al., 2013). Specifically, root-feeding herbivores have the potential to reduce N-fixation in legumes by damaging root nodules (Keane and Barlow, 2001), although other factors, including the ability of the plant to compensate for nodule loss (Quinn and Hall, 1992) or the influence of climatic or atmospheric conditions, will play a role. It is widely reported, for example, that root nodulation increases in response to eCO2 (Ryle and Powell, 1992; Lüscher et al., 2000) but decreases with eT (Munns et al., 1979; Zahran, 1999) and drought (Gil-Quintana et al., 2013). Fully factorial experiments combining trophic complexity with multiple environmental factors simultaneously are challenging to conduct and interpret but necessary to make accurate predictions of how plants and insects will interact in future environments.

1.6 Thesis overview Using a series of field-, glasshouse-, and laboratory-based approaches, this work addresses how climate and atmospheric change and interactions with belowground herbivores might shape aphid populations in the future. The main study species and basis of this PhD project (lucerne or alfalfa, M. sativa) is arguably the most important forage legume in the world. The most abundant lucerne-feeding aphid species in this region (New South Wales, Australia), the pea aphid (A. pisum), is a global pest and

CHAPTER 1 GENERAL INTRODUCTION exemplifies a well-studied model species. A major objective was to identify some of the plant-mediated indirect mechanisms driving climate, atmospheric and belowground herbivory impacts on lucerne aphids. Nitrogen dynamics and amino acid concentrations, in particular, are believed to play a major role in determining the population success of aphids and emphasis was placed on identifying specific amino acids that may be more important than others in driving aphid responses to climate and atmospheric change. The main aims of this thesis are depicted in Fig. 1-1.

1.7 Thesis outline The thesis is structured into several main objectives and chapters. This introductory chapter has covered general and contemporary theory concerning the direct and plant- mediated effects of climate and atmospheric change on aphids, as well as interactions between aphids and belowground herbivores.

CHAPTER 1 GENERAL INTRODUCTION

Chapter 2 reviews and synthesises research on the biology, management and trophic interactions of lucerne aphids, focussing on cultivar resistance, climate and atmospheric change and the interactions between lucerne aphids and other organisms, including predators, parasitoids, pathogens and bacterial endosymbionts. This chapter also demonstrates the importance of the lucerne industry and identifies a need for further research. This review entitled ‘The biology and trophic interactions of lucerne aphids’ (James M. W. Ryalls, Markus Riegler, Ben D. Moore and Scott N. Johnson) was published in Agricultural and Forest Entomology, vol. 15: 335-350, on 29 August 2013.

Chapter 3 focusses on aboveground–belowground herbivore interactions and investigates how elevated temperature (eT) and elevated atmospheric carbon dioxide concentrations (eCO2) affect larval development of the lucerne weevil (Sitona discoideus Gyllenhal) and colonisation by A. pisum, on three cultivars of a common host plant, M. sativa. This research entitled ‘Effects of elevated temperature and CO2 on aboveground–belowground systems: a case study with plants, their mutualistic bacteria and root/shoot herbivores’ (James M. W. Ryalls, Markus Riegler, Ben D. Moore, Goran Lopaticki and Scott N. Johnson) was published in Frontiers in Plant Science, vol. 4: 445, on 11 November 2013.

Chapter 4 investigates the plant-mediated mechanisms linking aphids to environmental change. Specifically, the study determines how simulated root herbivory of M. sativa before and after aphid infestation affects A. pisum under eT and eCO2. This research entitled ‘Amino acid-mediated impacts of elevated carbon dioxide and simulated root herbivory on aphids are neutralized by increased air temperatures’ (James M. W. Ryalls, Ben D. Moore, Markus Riegler, Andrew N. Gherlenda and Scott N. Johnson) was published in Journal of Experimental Botany, vol. 66: 613-623, on 16 November 2014.

Chapter 5 investigates the amino acid-mediated impacts of eT and eCO2 on the reproductive performance of A. pisum feeding on five M. sativa cultivars of varying aphid resistance. This chapter specifically determines whether the effects of eT and eCO2 on aphids are driven by groups of correlated amino acids and whether these effects are consistent across the five cultivars tested. This research entitled ‘Climate and atmospheric change impacts on sap-feeding herbivores: a mechanistic explanation

CHAPTER 1 GENERAL INTRODUCTION based on functional groups of primary metabolites’ (James M. W. Ryalls, Ben D. Moore, Markus Riegler, Lisa M. Bromfield, Aidan A. G. Hall and Scott N. Johnson) was accepted for publication in Functional Ecology (doi: 10.1111/1365-2435.12715) on 11 July 2016.

Chapter 6 investigates the effects of water stress on aboveground–belowground herbivore interactions within a grass–legume system. Specifically, the study investigates how simultaneous attack by belowground weevils (S. discoideus) and aboveground aphids (A. pisum) feeding on M. sativa affects a neighbouring grass (Phalaris aquatica) when subjected to drought and elevated precipitation in the field. This research entitled ‘Above–belowground herbivore interactions in mixed plant communities are influenced by altered precipitation patterns’ (James M. W. Ryalls, Ben D. Moore, Markus Riegler and Scott N. Johnson) was published in Frontiers in Plant Science, vol. 7: 345, on 7 March 2016.

Chapter 7 synthesises the key findings of this research within the framework of a central climate and atmospheric change theme and discusses the wider implications of these data in the context of potential future directions.

Chapter 2

Biology and trophic interactions of lucerne aphids – review

Published as Ryalls et al. 2013, Agricultural and Forest Entomology, 15, 335-350

2.1 Abstract Lucerne or alfalfa, Medicago sativa, is the most important temperate forage legume worldwide. Only one or two varieties of lucerne were grown in the U.S.A. and Australia (the two leading exporters of lucerne) before the late 1950s and late 1970s, respectively. These dates coincided with the arrival of aphid species, which devastated lucerne stands and prompted the development of aphid resistant cultivars. Lucerne- feeding aphids, including bluegreen aphids Acyrthosiphon kondoi, pea aphids Acyrthosiphon pisum, spotted alfalfa aphids Therioaphis trifolii maculata and cowpea aphids Aphis craccivora, however, still present significant risks for the lucerne industry worldwide and account for 25% of global production losses. Moreover, increased production costs, negative environmental effects and emerging aphid resistance to insecticide applications have led to a narrowing of management options against lucerne aphids. Understanding lucerne aphid biology and trophic ecology will be needed to develop future management practices, including biological control. We review and synthesize research on the four lucerne aphid species, focussing on cultivar resistance and their interactions with other organisms, including predators, parasitoids, entomopathogens and bacterial symbionts. The effects of global climate change are considered, with a particular emphasis on the potential for compromised aphid resistance in lucerne cultivars under future climates. We conclude by identifying future research questions and perspectives for the sustainable management of lucerne aphids. These include the characterization of plant secondary metabolites associated with natural enemy recruitment, an understanding of the role of endosymbionts in cultivar resistance and a better comprehension of multi-trophic interactions of lucerne aphids, both with other herbivores and higher trophic groups.

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2.2 Introduction Lucerne or alfalfa, Medicago sativa L., the principal economic species of the 87 species of Medicago and the world’s most important and widely grown temperate forage crop, is a perennial legume adapted to a wide range of climatic conditions (Yang et al., 2008; Small, 2011). Originating in south-western Asia, lucerne was first cultivated in Iran and is now distributed worldwide due to its popularity as an agricultural species (Sullivan, 1992). Its high nutritional quality (e.g. high protein content) makes it desirable as a hay, silage and pasture crop for sheep and cattle, with new uses including sprouts for salad and nutritional supplements for human diets (Bouton, 2012). Lucerne possesses many ecologically important characteristics, including the ability to fix atmospheric nitrogen via its association with rhizobial bacteria that form nodules on the roots. This has the potential to mitigate N-limitation induced by elevated atmospheric CO2 (Soussana and Lüscher, 2007) and reduces the requirement for nitrogenous fertilisers for lucerne and for subsequent crops (McDonald et al., 2003). The deep root system of lucerne gives it the ability to reduce the likelihood of soil salinity by absorbing water that would otherwise raise the watertable (Slavich et al., 2002; McDonald et al., 2003). Dryland salinity, resulting from the continual use for cropping of areas previously covered by trees, is an increasing problem worldwide with, for example, a large proportion of south-western Australia (4.4 million ha) at high risk from shallow watertables (National Land and Water Resources Audit, 2001). The high water-use of lucerne and its ability to help control this problem has therefore attracted much attention (Crawford and Macfarlane, 1995; Ridley et al., 1998; Humphries and Auricht, 2001). The lucerne industry generates US$7 billion annually and covers 32 million ha, a third of which is in the USA (Michaud et al., 1988; Şakiroğlu and Brummer, 2007). In Australia, the lucerne seed industry alone in 2007 was valued at A$95 million, with an export value of A$28 million (Carter and Heywood, 2008). These benefits and the high economic value of the lucerne industry in Australasia (Bullen et al., 2002), NorthAmerica (Small, 2011) and Europe (Veronesi et al., 2006) necessitate management strategies to maximise crop security.

Over 100 insect species, including weevils (Hypera spp. and Sitona spp.), grasshoppers (Melanophus spp.), blister beetles (Epicauta spp.) and caterpillars (e.g. Colias eurythene) are significant pests of lucerne (Frame et al., 1998; Sorensen and Manglitz,

CHAPTER 2 LUCERNE APHIDS - REVIEW

1998). Amongst the most globally-significant pests of lucerne are aphids (Hemiptera: Aphididae), which, in some regions, feed continuously and exclusively on lucerne throughout the year (Klingler et al., 2007). As invasive species, aphids can devastate lucerne and have been traditionally controlled by insecticides, although increased production costs, negative environmental effects and selection for resistance associated with insecticide applications has led to a narrowing of management options for lucerne producers (Oerke and Dehne, 2004). Greater understanding of the biology of lucerne aphids is therefore required to develop alternative control strategies, such as biological control. The purpose of this review is to synthesise existing knowledge about the four aphid pests of lucerne (spotted alfalfa aphid (SAA) Therioaphis trifolii maculata (Monell), bluegreen aphid (BGA) Acyrthosiphon kondoi (Shinji), pea aphid (PA) Acyrthosiphon pisum (Harris) and cowpea aphid (CA) Aphis craccivora (Koch)) (Fig. 2-1), including their interactions with lucerne and other organisms, the effects of climate change, and existing management options. In particular, we focus on cultivar resistance and identify future prospects for sustainable control of lucerne aphids to maximise lucerne crop security.

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2.3 Aphids as lucerne pests Aphids, with their piercing and sucking mouthparts, are specialised to feed from a single cell type, the phloem sieve element, and can feed from the same cell on lucerne for hours (Edwards et al., 2008). Like many other species, lucerne aphids can move their stylets between cells to evade many plant defences and have recently been shown to manipulate the host plant by secreting saliva into the phloem sieve elements (Will et al., 2007; Gao et al., 2008b). For example, Jiang and Miles (1993) suggested that SAA may induce redox changes in lucerne due to oxidases introduced in the aphid’s saliva. Aphid populations, including lucerne aphids, expand rapidly under optimal conditions, aided by asexual reproduction (viviparous parthenogenesis) and the telescoping of generations (i.e. females give birth to live young in which embryos of the next generation are already developing) (Moran, 1992). When a host plant can no longer support the growing population, aphids produce winged (alate) morphs that disperse with the wind (Lawrence, 2009).

In temperate climates, lucerne aphids reproduce by viviparous parthenogenesis in spring and throughout summer. In late autumn, sexual females and males are produced, which mate and produce eggs that overwinter and hatch in spring. In warm temperate climates, including some regions of Australia, the sexual phase is not necessary for producing overwintering eggs so aphids reproduce asexually throughout the year (Edwards et al., 2008). The harmful effects of aphids derive from the transmission of plant viral diseases [e.g. alfalfa mosaic virus (AMV)], the injection of toxins, secondary fungal growth (sooty mould) on honeydew (i.e. sap-feeding-induced sugary liquid secreted by aphids) and the direct ingestion of plant nutrients. They also have the ability to overcome host plant resistance and develop resistance against insecticides through the evolution of new aphid biotypes or host races (i.e. populations of a species that have been partially reproductively isolated from other conspecific populations as a direct consequence of adaptation to a specific host) (Garran and Gibbs, 1982; Diehl and Bush, 1984; Gao et al., 2007b; Lawrence, 2009).

Lucerne-feeding aphids are the most devastating insect pests of lucerne, causing an estimated production loss of 25% worldwide (He and Zhang, 2006). They present a significant problem for the lucerne industry by inhibiting seedling establishment, plant

CHAPTER 2 LUCERNE APHIDS - REVIEW growth and persistence, and reducing the quality and quantity of lucerne produced (Irwin et al., 2001). For example, during the late 1970s, the arrival of two exotic aphids, SAA and BGA, followed shortly after by PA in 1980 caused significant Australian lucerne declines (Berg and Boyd, 1984). Similarly, during the late 1950s, SAA invaded and devastated lucerne crops in western USA, leaving only a small proportion of resistant varieties standing, which were subsequently selected for breeding (Hughes and Hughes, 1987).

2.3.1 Spotted alfalfa aphid (SAA) Therioaphis trifolii maculata, originally with a Palaearctic distribution (Europe, the Mediterranean, North Africa, the Middle East, India and Pakistan), spread to south- western USA (~1953), Australia (~1977), South Africa (~1980), Japan (~1980), and New Zealand (~1982) (Blackman and Eastop, 2000). It is a yellowish-green aphid about 1.5 mm long with four to six rows of dark spots running along the length of its back. Spotted alfalfa aphids are adapted to warm, dry conditions, with peak activity during late spring, summer and autumn. They are prominent pests of lucerne and are most often found on the underside of lower leaves, which turn yellow and die due to both the removal of phloem sap (i.e. general degeneration) and the toxic nature of the aphids’ saliva (Berg and Boyd, 1984). They also cause veinal chlorosis in the growing tips and secrete large amounts of honeydew, often leading to the growth of black sooty mould (Madhusudhan and Miles, 1998). A combination of these effects greatly reduces the palatability and productivity of lucerne.

2.3.2 Bluegreen aphid (BGA) Acyrthosiphon kondoi, originating in Asia (known from Japan, Korea, India, Pakistan, Afghanistan and Iran) was accidentally introduced to California and New Zealand in 1974–1975 and subsequently spread to Australia, USA, Chile and South Africa (Blackman and Eastop, 2000). BGA adults are about 2.5 mm long and, like pea aphids, have two long cornicles protruding from their abdomens. They are blue-green in colour and feed primarily on the growing tips of lucerne stems, injecting salivary toxins as they feed (Knowles, 1998). High population densities can severely stunt the plant, causing leaves to yellow and die.

CHAPTER 2 LUCERNE APHIDS - REVIEW

2.3.3 Pea aphid (PA) Acyrthosiphon pisum is a large (~3 mm long as adults), green or pink aphid with long appendages, usually abundant from spring to early summer and autumn. PA is adapted to warmer temperatures than BGA, but cooler temperatures than SAA (Rohitha and Penman, 1983). Originally a Palaearctic species, A. pisum now occurs in most parts of the world and is a well-known pest of lucerne in Europe, America and Oceania (Julier et al., 2004). Strong genetic diversity can occur between populations with different host ranges and preferences (van Emden and Harrington, 2007). For example, populations colonising peas, lucerne and red clover in France were found to be genetically divergent from one another (Simon et al., 2003). PA, apart from being slightly larger, resembles BGA in appearance. It is often difficult to differentiate them without magnifying their antennae, which possess several dark, narrow bands in PA, but are uniformly brown in BGA. PA also infests the growing tips of lucerne stems, but tend to be less specific about where they feed on the plant than BGA (Knowles, 1998). They cause general degeneration in lucerne, but no local symptoms, so are not considered ‘toxic’ like SAA and BGA. Infested plants wilt, the top leaves become paler, and lower leaves turn yellow and drop off.

2.3.4 Cowpea aphid (CA) Aphis craccivora is a small (~2 mm long as adults), dark brown aphid with a wide range of host plants in different families (~50 crops across 19 families). Like PA, CA has an almost worldwide distribution, but probably originates from warmer areas of southern Europe (van Emden and Harrington, 2007). It is a comparatively minor pest of lucerne, with most activity occurring during early spring and late autumn (Berg and Boyd, 1984). It is most commonly associated with grain legumes in Australia but has the potential to become more destructive to pasture legumes (e.g. lucerne) with climate change (Edwards, 2001).

2.4 Cultivar resistance Because of the costly nature and negative environmental impacts of insecticide applications, the use of cultivar resistance (i.e. bottom-up control) against aphids presents a more economical and practical option for increasing lucerne yield, quality and stand persistence (Irwin et al., 2001; Edwards and Singh, 2006; He and Zhang,

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2006; Gao et al., 2007b). Aphid-resistant varieties of lucerne were selected by plant breeders soon after the late 1950s when SAA devastated lucerne stands in the USA, which at that time, were dominated by the cultivars ‘Vernal’ and ‘CUF101’ (Hughes and Hughes, 1987; Bouton, 2012). Research was undertaken to understand the nature of aphid resistance. Painter (1968) defined three mechanisms of plant resistance to insect attack: i) antibiosis (Greek for “resistance to life functions”) – the ability of a plant to reduce insect survival, extend insect development time, or reduce insect growth or fertility; ii) tolerance – the ability of a plant to show a reduced damage response (e.g. yields maintained despite aphid attack); iii) non-preference or antixenosis (Greek for “resistance to outsiders”) – refers to plant properties that reduce the likelihood of colonisation (Kogan and Ortman, 1978; van Emden, 2007). Reports of a breakdown in plant resistance to new aphid biotypes were soon prevalent due to various factors including time of year (Barnes, 1963), plant age (Howe and Pesho, 1960) and plant nutritional differences (Kindler and Staples, 1970), requiring further efforts in evaluating and investigating mechanisms for resistance.

Prior to the arrival of SAA and BGA in 1977, the Australian lucerne industry was reliant on one cultivar of lucerne, ‘Hunter River’, a locally-adapted type derived from the French variety ‘Provence’, which was highly susceptible to aphid attack (Lodge and Frecker, 1989). In response to the devastation of ‘Hunter River’, seeds of well- developed aphid-resistant cultivars were imported from the USA (Holtkamp and Clift, 1993). Australian breeding programmes have used ‘CUF101’ extensively as a progenitor of SAA and BGA resistance (Irwin et al., 2001). ‘Trifecta’, released in 1983, was the first cultivar to achieve at least moderate (>20% of plants) levels of aphid resistance (Oram, 1990), which led to significant yield increases over ‘Hunter River’ and ‘CUF101’ (Clements et al., 1984). The problem with cultivar introductions overseas, however, is that aphid responses can vary outside their country of origin (He and Zhang, 2006) so further studies are required to assess local levels of lucerne resistance, environmental adaptation and mechanisms for resistance to aphids. After more than 30 years of breeding and variety introductions, over 50 lucerne varieties are now available for Australian growers (Lattimore, 2012).

CHAPTER 2 LUCERNE APHIDS - REVIEW

In order to standardise the assessment of resistance to aphids, a rating scheme was developed (McDonald et al., 1988) using five classes ranging from susceptible to highly resistant. Multiple studies, both laboratory- and field-based, have focussed on the responses of different lucerne cultivars to aphid attack, and vice versa (Turner et al., 1981; Lloyd et al., 1983; Brownlee et al., 1984; Holtkamp and Clift, 1993; Humphries and Auricht, 2001; Gao et al., 2007b; Goławska et al., 2008). Glasshouse studies, however, may not accurately reflect the field performance of resistant lucerne cultivars (Hamilton et al., 1978; Ridland and Berg, 1981). Lodge and Greenup (1983) investigated whether the field performance of a number of cultivars in New South Wales reflected the range of SAA resistance values observed by glasshouse screening tests. In general, these tests were poor indicators of resistance in the field. Field tests showed that total aphid numbers per seedling and dry matter yield were poorly correlated with observations in glasshouse studies, whereas total numbers of SAA and numbers of large nymphs per stem generally reflected resistance levels found in glasshouse studies. Resistance differences between lucerne growth stages have also been questioned. Bishop et al. (1982) found an increase in resistance to BGA with increasing plant size. Lloyd et al. (1983), however, observed no change in the order of resistance to SAA between growth stages of mature plants and seedlings, although mature plants were more tolerant of SAA than seedlings.

Various methods, both qualitative and quantitative, are used to measure resistance to aphids. In an antibiosis test, biological traits (e.g. aphid mortality, larval development, reproduction) are scored. Girousse and Bournoville (1994), for example, evaluated lucerne cultivar resistance to PA by estimating the net reproductive rate over two weeks. Similarly, Sandmeyer et al. (1971) used longevity, number of nymphs, development and reproductive period of PA and SAA to categorize resistance of six lucerne cultivars. Antibiosis studies, although reliable, are resource-intensive, especially when large numbers of cultivars are tested. Additionally, they do not take into account differences in plant tolerance of aphids (Julier et al., 2004). Tolerance has been addressed by others (Berberet et al., 1991; Girousse et al., 1998; Bournoville et al., 2001), focussing on lucerne development (e.g. seedling biomass) under aphid infestation. He and Zhang (2006) evaluated the resistance of nine lucerne cultivars to

CHAPTER 2 LUCERNE APHIDS - REVIEW three aphid species (SAA, CA and PA) in China, focussing primarily on ‘aphid damage index’ and ‘% of resistant plants’ as parameters to evaluate cultivar resistance.

Various studies have focussed on secondary chemistry of lucerne as mechanisms for resistance. For example, lucerne contains saponins, naturally occurring glycosides that are induced by herbivore attack, which, like constitutive (constant and active) defences, can vary with cultivar (Njidda and Ikhimoya, 2011; Goławska et al., 2012). High saponin concentrations in some lucerne cultivars was correlated with resistance against PA but not SAA (Pedersen et al., 1976) and reduced reproduction and survival was seen in PA when fed on a high-saponin line of lucerne (Goławska et al., 2006). Goławska et al. (2008) compared the feeding behaviour of PA on two European cultivars and suggested that the lucerne cultivar ‘Radius’, containing a higher concentration of saponins and a lower concentration of flavonoids than cultivar ‘Sapko’, had a higher resistance to PA (Fig. 2-2). In particular, reproductive period, fecundity and survival were higher on ‘Sapko’ (Fig. 2-2 A-C) and the number of potential drops (cell punctures by aphid stylets) was higher on ‘Radius’ (Fig. 2-2 D). Goławska et al. (2012) extended this research to include four cultivars, similarly showing that PA numbers were inversely related to saponin content. They also identified specific saponins (e.g. the low saponin line did not contain zanhic acid or medicagenic acid), which are important to consider because the biological activity of saponins and other plant secondary metabolites (PSMs) is often dependent on specific components (i.e. qualitative differences) as opposed to total concentrations (Bagheri et al., 2001; Francis et al., 2002). The presence of epicuticular lipids on lucerne leaves has also been suggested as a mechanism for resistance to SAA, which were found to feed on older leaves because they contain lower lipid concentrations (Bergman et al., 1991). An alternative mechanism suggests that higher levels of an aphid alarm pheromone ((E)-β-farnesene) observed in some lucerne cultivars increases BGA resistance by acting as a chemical cue for aphid non-preference (i.e. repellent) (Mostafavi et al., 1996). This provides scope for the incorporation of increased levels of (E)-β-farnesene into new lucerne cultivars, a method already incorporated into wheat to repel cereal aphids (e.g. Sitobion avenae) (Rothamsted Research, 2013). Soluble antioxidants, such as ascorbate and glutathione, have been identified as effective enhancers of the plant’s defensive system, with a reduction in reproductive rate seen in BGA and SAA that were fed on lucerne cultivars

CHAPTER 2 LUCERNE APHIDS - REVIEW artificially infiltrated with ascorbate and glutathione (Miles and Oertli, 1993). Febvay et al. (1988) noted a few amino acids that may be important for lucerne resistance to PA with, for example, low concentrations of methionine and lysine found in the resistant cultivar ‘Lahontan’, compared to the susceptible cultivar ‘Resistador’ lacking no single amino acid. Girousse and Bournoville (1994), however, found no evidence that sugar or amino acid deficiencies in the phloem from two lucerne cultivars were responsible for resistance to PA, although variations in a few amino acids (alanine, leucine, isoleucine, arginine and ornithine) were observed, which may be linked to resistance. They identified a reduced flow of phloem in the resistant cultivar, suggesting ingestion difficulty as a potential mechanism for resistance (Klingler et al., 2005).

18 60 16 (a) (b) 50 14 12 40 10 30 8 6 20 4 10 2

Reproductiveperiod (days) 0 0

Number of nymphs produced of nymphs Number 35 40 (c) (d) 30 25 30 20 20 15 10

Number of pds Number 10

Survival(days) 5

0 0 Radius Sapko Radius Sapko Cultivar Cultivar

CHAPTER 2 LUCERNE APHIDS - REVIEW

Improvement programmes have focussed on the development and maintenance of aphid resistance as newly adapted aphid biotypes emerge (Humphries and Auricht, 2001; NAAIC, 2004). Recently, for example, a new BGA biotype was found in south- eastern Australia that causes substantial damage to previously resistant lucerne cultivars (Humphries et al., 2012). Similarly, in 1991, previously resistant lucerne varieties in California were devastated by a new BGA biotype (Zarrabi et al., 1995). Cultivar resistance may reduce aphid growth (antibiosis) or preference (antixenosis), or ameliorate the negative effect of aphid infestation on plant growth (tolerance), but the effectiveness of most plant resistance mechanisms are limited to a single aphid species, or even to single biotypes of a species (Clements et al., 1984; Gao et al., 2007b). For example, Salisbury et al. (1985) found a strong correlation between BGA and PA resistance in Australian-bred lucerne lines but no correlation in lines from the USA. Trials incorporating multiple pest-resistance are ongoing, with, for example, new cultivars outyielding ‘CUF101’ by up to 58% in California (Putnam et al., 2011). Similarly, in eastern Australia, cultivars have out-yielded ‘Hunter River’ by over 300%, suggesting that trials incorporating multiple pest resistance have dramatically increased lucerne productivity (Irwin et al., 2001), although other growth factors incorporated in breeding programmes must be considered. Continual field testing of aphid-resistant varieties, involving phenotypic selection for resistance and control of alfalfa mosaic virus, is required to maintain aphid resistance. Regular collections of aphids and laboratory aphid culture replacements are also important to ensure that virulent strains are being used (Humphries and Auricht, 2001).

2.5 Alfalfa Mosaic Virus Alfalfa mosaic virus, a common seed-borne virus that causes significant lucerne crop losses, is the only lucerne-infecting virus known to be transmitted by aphids (Ohki et al., 1986; Miczynski and Hiruki, 1987; Hajimorad and Francki, 1988). All four lucerne- feeding aphids (BGA, PA, CA and SAA) have been shown to transmit AMV in a non- persistent manner (i.e. short-term transmission) (Jaspars and Bos, 1980; Jones, 2004a). Lucerne is a perennial crop so acquisition of AMV due to aphid feeding has significant consequences for crop health and economic value over a number of cropping seasons (as opposed to annual crops). AMV incidence levels and rates of transmission through lucerne seed vary depending on virus strain, environmental conditions, plant age, aphid

CHAPTER 2 LUCERNE APHIDS - REVIEW preference and cultivar type (Bailiss and Offei, 1990). Feeding-preference tests by Garran and Gibbs (1982), for example, revealed a preference by SAA for AMV-infected lucerne cultivar ‘Siriver’ over healthy ‘Siriver’ but no preference was observed between AMV-infected and healthy ‘Hunter River’ lucerne. Additionally, younger lucerne crops in Australia were found to have a higher incidence of AMV than mature crops, although contrasting results were seen in Great Britain in the 1950s where mean AMV incidences of 5, 7 and 17% were observed in crops that were two, three and four years old, respectively (Gibbs, 1962). This contradicts the idea that seed-borne viruses are often present in younger crops (Tomlinson, 1962) but the difference may be related to the lack of AMV found in some of the commercial seed of the cultivar ‘Du Puits’ in Great Britain in 1960, therefore resulting in less seed–seed transmission compared to Australia. AMV was reported in Australia in the 1960s, most likely introduced through importation of contaminated seed stock, but was not widespread until the 1980s when lucerne-feeding aphids arrived. Similarly, introductions of these aphids to New Zealand coincided with an increase in AMV in lucerne crops (Forster et al., 1985). Controlling aphids should help to reduce the incidence of AMV in lucerne.

2.6 Trophic interactions Several trophic interactions have been reported for lucerne-feeding aphids, including interactions with bacterial endosymbionts, predators, pathogens and parasitoids (Hughes and Bryce, 1984; Leathwick and Winterbourn, 1984; Rakhshani et al., 2006; Simon et al., 2011). The first of these interactions, endosymbiosis, can represent a mutualistic interaction whereas the latter three are antagonistic interactions, which are often considered in the context of biological control (Fig. 2-3).

CHAPTER 2 LUCERNE APHIDS - REVIEW

2.6.1 Endosymbionts Most aphids examined to date harbour the primary (or obligate) bacterial symbiont, Buchnera aphidicola, which compensates for the deficiency of nutrients in their phloem diet by synthesising essential amino acids such as arginine and tryptophan, as well as other essential nutrients (Prosser and Douglas, 1991; Liadouze et al., 1995; Shigenobu et al., 2000; Wilkinson et al., 2007). Buchnera aphidicola lives in specialised host cells called bacteriocytes (or mycetocytes) in the aphid hemocoel and is transmitted vertically (maternally). Some aphids also carry one or more secondary (or facultative) symbionts that are mostly vertically, and on an ecological timescale rarely horizontally, transmitted and exert diverse effects on hosts [interaction (i) Fig. 2-3]. These effects have mostly been studied in PA. The best-characterised secondary symbionts of PA include “Candidatus Regiella insecticola” (pea aphid U-type symbiont), “Candidatus

CHAPTER 2 LUCERNE APHIDS - REVIEW

Hamiltonella defensa” (pea aphid Bermisia-type symbiont or T-type symbiont), “Candidatus Serratia symbiotica” (pea aphid secondary symbiont or R-type symbiont) and pea aphid Rickettsia symbiont (Rickettsia) (Moran et al., 2005). As the majority of lucerne-feeding PA clones carry H. defensa, compared to R. insecticola in clover feeders (Darby et al., 2003; Ferrari et al., 2004; McLean et al., 2010) it has been suggested that secondary symbionts can promote host plant specialisation (or plant choice) by lucerne aphids [interaction (ii) Fig. 2-3], however, this is not as clear as originally interpreted. In a first set of experiments, Chen et al. (2000) evaluated the effects of S. symbiotica and Rickettsia on PA and BGA (Fig. 2-4). Rickettsia had no significant effect on fecundity of PA feeding on M. sativa but significantly reduced the fecundity of PA feeding on bur clover M. hispida. Serratia symbiotica reduced fecundity and longevity of BGA on both lucerne (Fig. 2-4 B and C) and clover. Similarly, Leonardo and Muiru (2003) showed that pea aphids with R. insecticola had twice as many offspring as other aphids on clover, but populations were declining on lucerne, suggesting that aphid specialisation on host plants is influenced by symbiont identity. Although many original studies identified an association between host plant and endosymbiont presence (e.g. Tsuchida et al., 2004), it is difficult to show that endosymbionts unequivocally influence plant utilisation; the relationship may be incidental (e.g. different bacteria may simply have colonised after genetically different aphid lineages specialised on a particular host plant) or it might reflect differences in natural enemy pressures experienced by aphids on different plant species. Using antibiotic treatment to remove R. insecticola from PA, Leonardo (2004) found that R. insecticola was not responsible for causing host plant specialisation. Similarly, McLean et al. (2010) found no evidence that secondary symbionts influence plant host specialisation, although effects varied among aphid and host plant genotypes.

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25 120 (a) (b) 20 100 80 15 60 10 40

5 20

Reproductive period (days) period Reproductive 0 produced nymphs of Number 0 40 10 (c) (d) 8 30 6 20 4

Longevity (days) Longevity 10 2

0 (days) reproduction of first Age 0 None PASS PAR None PASS PAR Endosymbiont present

Furthermore, endosymbionts have been shown to influence a wide range of PA characteristics including body colour (Tsuchida et al., 2010) and resistance to heat stress (Montllor et al., 2002; Russell and Moran, 2005; Dunbar et al., 2007). Experiments have also shown that secondary symbionts can confer a physiological defence mechanism in PA against parasitoids (Oliver et al., 2009; Oliver et al., 2010) and entomopathogenic fungi (Scarborough et al., 2005) by restricting parasitoid egg development and lowering the rate of fungal transmission, respectively. Oliver et al. (2005) found one strain of H. defensa derived from CA that conferred resistance to the parasitic wasp Aphidius ervi in PA, suggesting that H. defensa may provide a protective role in other aphid species and that resistance may be transferred interspecifically. It is important to consider the aphid–symbiont association holistically as any changes in host plant nutritional composition and aphid activity (e.g. cultivar preference and reproduction) will inevitably alter the identity or frequency of symbionts present in aphid populations. Effects of symbionts on different aphid characteristics are also likely 28

CHAPTER 2 LUCERNE APHIDS - REVIEW to influence other multi-trophic interactions with antagonists (Tsuchida et al., 2010). They thus play a fundamental role in aphid biology, beyond providing essential nutrients required for aphid survival, and offer potential targets for novel biological control strategies (Goggin, 2007).

2.6.2 Predators Natural enemies of aphids include predators, pathogens and parasitoids, all of which have the potential to reduce aphid populations (Ekbom, 1994). Only a few studies have considered the importance of arthropod predators in controlling lucerne aphid numbers. Milne and Bishop (1987) identified brown lacewings (e.g. Micromus sp.), ladybirds (e.g. Coccinella repanda), predatory mites and syrphid larvae as important predator groups of BGA and SAA in lucerne. These predators significantly reduced aphid abundance in New South Wales, Australia, and were identified as potential pest management tools [interaction (iii) and (iv) Fig. 2-3]. In contrast, Ekbom (1994) found no evidence that predators have an important impact on numbers of PA in lucerne. Assessing the impact of predators on aphid populations requires careful analysis of temporal and spatial synchrony, prey choice and environmental condition (e.g. temperature) preferences (Cameron et al., 1980; Leathwick and Winterbourn, 1984; Ekbom, 1994).

2.6.3 Entomopathogens Pathogens also provide an important means of biological control. In California, after the outbreak of SAA in 1954, two pathogenic fungi, Entomophthora virufenta and Entomophthora exitialis, were widely distributed, although the occurrence of natural epizootics confounded their effectiveness (Hall and Dietrick, 1955; Hall and Dunn, 1957). After the outbreak in Australia, Milner and Soper (1981) screened five species of fungi for activity against SAA. Entomophthora sphaerosperma, an important pathogen of SAA in Israel, was highly active and presented a promising microbial control agent, which was confirmed by releases in Australia. Another entomopathogenic fungus, Zoophthora radicans, known to cause extensive epizootics in SAA populations in Israel (Kenneth and Olmert, 1975), was imported and released into Australian lucerne fields during 1979 [interaction (v) Fig. 2-3]. These fungi can be mass-produced and easily applied, are able to persist between seasons and have great potential as microbial

CHAPTER 2 LUCERNE APHIDS - REVIEW control agents, although a high relative humidity (~90%) is required for effective transmission (Shah and Pell, 2003). Significant SAA declines were partly due to Z. radicans, but also because of the introduction of the parasitic wasp Trioxys complanatus (Butt et al., 2001).

2.6.4 Parasitoids Various parasitoids (Hymenoptera: Braconidae) attack lucerne aphids, therefore providing a mechanism for protection of lucerne crops [interaction (vi) and (vii) Fig. 2- 3]. In Iran, for example, specific parasitoids of SAA include T. complanatus and Praon exsoletum. Common parasitoids of BGA and PA include Aphidius ervi, Aphidius smithi and Aphidius eadyi and CA is mainly attacked by Lysiphlebus fabarum (Rakhshani et al., 2006). In California during the period of severe infestation of SAA around 1955, three hymenopteran parasitoid species were successfully introduced and contributed significantly to the biological control of SAA (van den Bosch et al., 1964). One parasitoid in particular, T. complanatus, was generally dominant over the others. The three parasitoids were also released in Arizona in ~1956 and T. complanatus became an important control agent (Barnes, 1960). Delays in getting the parasitoid established, however, meant that other factors, including the use of insecticides against lucerne weevils and the incorporation of resistant lucerne varieties, may have influenced the aphid decline (Hughes et al., 1987). In 1958, the parasitic wasp, A. smithi, identified as an important factor in the natural control of PA in India, was imported and released in California. Aphidius smithi became established and exerted considerable control of PA (Hagen and Schlinger, 1960).

Many parasitoids were introduced into Australia in the 1980s following the outbreak of SAA, BGA and PA. Long periods of time are required to verify the effectiveness of biological control mechanisms and perhaps the first comprehensively evaluated case of biological control of an aphid species was the control of SAA in Australia by T. complanatus. In New South Wales alone, this biological control has saved ~A$2 million per year by avoiding the need to apply insecticides to lucerne fields and reducing the urgency for replacing susceptible lucerne with aphid-resistant cultivars (Hughes et al., 1987). Trioxys complanatus has also been shown to exhibit a preference for aphids feeding on lucerne rather than clover, despite the presence of aphids on both legumes

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(Milne, 1997). During 1978–1981, the Commonwealth Scientific and Industrial Research Organisation (CSIRO) imported the parasitoid A. ervi from California and Europe in an effort to control BGA in New South Wales. Surveys confirmed the successful dispersal and establishment of A. ervi despite some problems caused by drought in some areas (Milne, 1999). The impact of A. ervi on aphid abundance, however, was determined by the initial ratio of parasitoids to aphids (Wellings, 1986). The three Aphidius parasitoids of BGA and PA were also introduced into New Zealand around the same time. Aphidius smithi failed to establish but A. eadyi established quickly and spread rapidly throughout the North and South Islands due to its fast development and high fecundity. It took several attempts to establish A. ervi, which eventually replaced A. eadyi as the predominant parasitoid in the North Island (Cameron and Walker, 1989).

Successful biological control of aphids by parasitoids depends on the ability of the parasitoid to reduce aphid reproductive potential and the age structure of aphid populations can determine this success. Parasitoids that attack early instar aphids are likely to be more effective at reducing aphid populations than those that attack late instar aphids (Sequeira and Mackauer, 1988). He et al. (2003; 2005) found that parasitism of early instar PA by A. eadyi prevented any reproduction and when late instars and adults were parasitised, progeny were still produced but reproductive potential was lower than unparasitised aphids (Fig. 2-5). Specifically, parasitised fourth instar and adult aphids had a shorter reproductive period (Fig. 2-5 A) and lower fecundity (Fig. 2-5 B) than unparasitised adults. Growing aphid populations often contain large numbers of early instar individuals, which are present early in the season (Schowalter, 2006). Parasitoid attack early in the season should therefore significantly reduce aphid populations and prevent build-up later in the season. This would particularly apply in cooler conditions when aphids develop slowly, making populations vulnerable to attack by parasitoids. If similar effects on aphid populations are seen between species, this model may help to predict population dynamics regardless of species (Sequeira and Mackauer, 1988). The impact of this biological control mechanism, however, may also be dependent on the population growth of the parasitoid itself, which can be influenced by, for example, the presence of alternative aphid species (that are not feeding on lucerne) as hosts, so variation in parasitoid

CHAPTER 2 LUCERNE APHIDS - REVIEW population dynamics would make long term predictions difficult (He et al., 2005). Long- term studies are therefore required to assess changes in trophic synchrony over time.

18 (a) 16 14 12 10 8 6 4

Reproductive period 2 0 140 (b) 120

100

Number of nymphs produced 0 P4 P N

Parasitism Figure 2-5. Effect of parasitism by Aphidius eadyi on the reproductive period (a) and fecundity (progeny production) (b) of pea aphid. Aphid parasitism: P4, parasitized fourth instar; P, parasitized adult; N, unparasitized adult. Data are the mean ± SE shown after He et al. (2003).

2.6.5 Interactions between natural enemies Competition between natural enemies (e.g. predators killing other predators or parasitoids) may also make predictions difficult. Snyder and Ives (2003) investigated how different natural enemies, including parasitoids and predators, and their interactions, can affect the successful control of PA on lucerne. Parasitoids caused a delayed reduction in PA populations relating to the generation time of the parasitoid, whereas generalist predators, including coccinellid and carabid beetles, web-building

CHAPTER 2 LUCERNE APHIDS - REVIEW spiders and Nabis and Orius bugs, caused an immediate reduction in population growth. In an earlier study, carabid beetles had little influence on the population of PA due to avoidance tactics by the aphids but they fed on parasitised aphid mummies, therefore disrupting the biological control of aphids by parasitoids [interaction (viii) Fig. 2-3] (Snyder and Ives, 2001). A further level of complexity is added by the potential for endosymbionts to increase the resistance of aphids to parasitoids. The parasitoid A. ervi, for example, has been shown to increase the deposition of eggs in PA infected with the symbiont H. defensa to overcome symbiont-based defence (Oliver et al., 2012). Additionally, changing abiotic environmental conditions can alter interactions with, for example, A. ervi proving less effective at reducing aphid populations in drier, inland areas and most effective in high rainfall areas of eastern Australia (Milne, 1999). Similarly, the difficulty in establishing A. eadyi and A. ervi in New Zealand indicates that they were suited to different conditions (Cameron and Walker, 1989) and the parasitoid T. complanatus was less effective at high Australian temperatures (Hughes et al., 1987).

2.7 Future climate change Few studies to date have considered the effect of climate change on aphids feeding on lucerne. Amino acid concentrations in the phloem, which are important for aphid performance, can be altered by a variety of abiotic environmental factors, including temperature, atmospheric carbon dioxide and water availability (Docherty et al., 2003; Douglas, 2003; Aslam et al., 2013). In general, elevated temperatures (eT), anticipated to occur with climate change, are expected to increase nutrient uptake efficiency resulting in larger plants that contain lower concentrations of some PSMs (Rufty et al., 1981; Bale et al., 2002; Richardson et al., 2002). Drought is predicted to reduce nutrient uptake efficiency and eT may increase the impacts of drought by creating a higher demand for water (Gregory, 2006; Staley and Johnson, 2008), whereas plants under elevated carbon dioxide concentrations (eCO2) may perform better under drought because stomata close to minimise water loss and water use efficiency increases. Additionally, the nutrient composition of the phloem is likely to be particularly affected due to increasing demands for phloem solutes under drought

(Pritchard et al., 2007). In many plants, eCO2 is expected to increase plant productivity by increasing photosynthetic rates. This, however, generally leads to a reduction in tissue quality and, specifically, an increase in C/N ratio. This can be exacerbated when

CHAPTER 2 LUCERNE APHIDS - REVIEW increased plant growth leads to N-limitation (Bezemer and Jones, 1998; Veteli et al., 2002; Ainsworth and Long, 2004; Johnson and McNicol, 2010). Legumes such as lucerne, however, may partially overcome eCO2-induced N limitation by increasing biological nitrogen fixation, which may alter the dynamics of aphids feeding on the plant (Johnson and McNicol, 2010). Despite a wealth of studies on the effects of eCO2 on insect herbivores, however, the response of aphids and, in turn, the response of higher trophic levels, has been difficult to generalise (Pritchard et al., 2007; Sun and Ge, 2011).

Aphids have the potential to respond rapidly to climate change due to their short generation times and high reproductive capacity (Harrington et al., 1995), although changes in climate will affect aphid species which possess idiosyncratic characteristics differently. PA, for example, is prevalent in summer, whereas BGA is adapted to cooler temperatures and predominates in early spring and autumn when there are fewer natural enemies (Rohitha and Penman, 1983). Future temperature changes may increase the temporal overlap of these aphids and warmer winters may lead to the disappearance of sexual overwintering stages, causing greater devastation over extended periods, although many other factors will play a role. Chen et al. (2000) observed no effect of the endosymbionts S. symbiotica and Rickettsia on the fitness of PA at 20 °C but both increased the fitness of one parent clone reared for three generations at 25 °C on lucerne. These fitness effects, however, were not observed for two other clones at 25 °C. Montllor et al. (2002) also identified a beneficial effect of S. symbiotica and Rickettsia on PA under heat stress, although others have suggested that obligate symbionts (e.g. B. aphidicola) limit the ability of aphids to cope with rising temperatures (Harmon et al., 2009; Wernegreen, 2012).

One study (Johnson et al., 2014) has considered the effect of eCO2 on pea aphids feeding on five different lucerne cultivars of varying resistance. Compared with ambient CO2, they saw an increase in colonisation success (from 22% to 78% of plants) -1 and reproduction rates (from 1.1 to 4.3 nymphs week ) under eCO2 on the resistant cultivar ‘Sequel’. The moderately resistant cultivar ‘Genesis’, however, became more resistant at eCO2, shown by a decrease in colonisation success (from 78% to 44% of plants) and reproduction rates (from 4.9 to 1.7 nymphs week-1). Additionally, under

CHAPTER 2 LUCERNE APHIDS - REVIEW

eCO2, concentrations of the essential amino acid lysine increased in ‘Sequel’ but decreased in ‘Genesis’. This demonstrates the importance of considering different host varieties when studying the effects of climate change. Additionally, future CO2 concentrations are likely to be accompanied by elevated temperatures and while a number of studies have observed the effect of eCO2 on aphid populations, the combination of eT and eCO2 has received little focus (Newman, 2004; Himanen et al.,

2008; Auad et al., 2012). To predict the effects of eCO2 and eT on lucerne resistance to aphids it is necessary to understand how these abiotic factors influence lucerne nutrition and defence, including what nutritional and defensive properties affect resistance, of which little is known at present.

2.8 Conclusions and future perspectives While the impact of aphids on lucerne productivity may be influenced by a wide range of factors under eCO2 and eT, the effectiveness of natural enemies in controlling lucerne aphids will be a major influence (Hughes et al., 1987; He et al., 2005; Thomson et al., 2010). The interaction of abiotic and biotic variables will determine future outcomes. Any efforts to conserve or facilitate natural enemies for biological control may be negatively influenced by interactions between abiotic and biotic variables and inhibited by trophic asynchrony (Dyer et al., 2013). Although PA is best-studied at present, it is important to consider the other lucerne-feeding aphids to determine interspecific differences and provide effective management strategies. Few studies of CA, for example, have been reported, but this aphid may become more problematic in future as conditions change. Many effective resistant lucerne cultivars are already established (Lattimore, 2012) but future resistance studies should include CA to allow for effective control at the risk of an outbreak.

Longer-term studies are required to determine bacterial, predator, pathogen and parasitoid roles, as well as the effects of climate change, plant nutrition and PSM quality and quantity. Most studies focus on total concentrations of PSMs, with few considering specific components that may explain variations in plant–herbivore– symbiont–natural enemy (multi-trophic) dynamics. Factors to consider may include the effect of light availability and plant age on PSMs, such as saponins (Francis et al., 2002). PSMs, such as indole acetaldehyde in lucerne (Hatano et al., 2008) and aphid

CHAPTER 2 LUCERNE APHIDS - REVIEW pheromones, such as (E)-β-farnesene (Verheggen et al., 2012), can act as cues for natural enemies of aphids, which may differ according to cultivar type and in response to climate change. Further study may also focus on jasmonic acid signalling, a phytohormonal response that has been implicated in R gene resistance (i.e. genes in the plant genome that confer resistance) to BGA in another Medicago species, M. truncatula (Gao et al., 2007a). Incorporating resistance genes into commercial lucerne lines may provide further scope for effectively managing lucerne crops and preventing aphid infestation (Goggin, 2007; Bouton, 2012).

Secondary endosymbionts of aphids influence a wide range of lucerne aphid characteristics. Studies should also be undertaken to determine whether symbionts play a role in cultivar specialisation within the same host plant species, which may alter resistance test outcomes. It is, however, important to consider factors that may influence associations between aphid endosymbionts and host plant use, including risk of attack by natural enemies and symbiont colonisation history (Frago et al., 2012). Biological control of lucerne aphids by parasitoids, pathogens or predators presents an effective management strategy, although intraguild or predator–parasitoid predation must be considered at the same time. For example, Snyder and Ives (2003) found biological control to be most effective when both types of natural enemy were present but they suggest that long term experiments would lead to non-additive effects on aphid control as the predators would displace parasitoids. It is also important to consider the effects of climate change on host–enemy synchronisation (Hance et al., 2007) and the effectiveness and establishment of biological control agents, which are often suited to different environmental conditions (e.g. Milne, 1999).

Aphids also have to contend with indirect influences from spatially-separated root herbivores present belowground. Indeed, in a recent meta-analysis, root herbivores had the biggest effect on aphids compared with other aboveground herbivores (Johnson et al., 2012). Very few studies concerned with crop security have considered aboveground–belowground interactions (van der Putten et al., 2009) and no studies to date have considered the effect of belowground organisms on aboveground aphids feeding on lucerne. Lucerne aphids might be particularly susceptible to effects of belowground herbivores. In particular, the lucerne weevil Sitona discoideus and white

CHAPTER 2 LUCERNE APHIDS - REVIEW fringed weevil Naupactus leucoloma have the capacity to reduce nitrogen fixation in lucerne by damaging root nodules that house nitrogen fixing bacteria (Keane and Barlow, 2001; Barnes and De Barro, 2009). Given the effects of such root herbivory on nitrogen fixation it seems likely that the amino acid composition of the foliage and phloem could also vary, which in turn could affect both aphids and their primary and secondary endosymbionts (Douglas, 1993, 1998). Replicating trophic complexity in an experimental setting is challenging but necessary to provide a more holistic insight into the mechanisms underpinning multi-trophic interactions in natural terrestrial systems (Bardgett and Wardle, 2010). In general, sap-feeding aboveground herbivores, such as aphids, are thought to benefit most from plant attack by belowground herbivores. As phloem-feeders they may circumvent any induced defence compounds, which usually occur in low concentrations in phloem sap, while reaping the benefits of stress-induced increases in aboveground nutrition due to root damage belowground (Bezemer et al., 2005; Johnson et al., 2008), although many factors play a role. The opposite might be true in lucerne since the root nodules (and therefore sites of N acquisition) are often the principal site of attack by root herbivores.

There is not one lucerne variety that will persist everywhere, which is why it is important for new cultivars to be developed and targeted for different geographic ranges (Bouton, 2012). In southern Queensland, for example, aphid resistant varieties are desirable because insecticidal control is less economical on rain-grown pastures (Franzmann et al., 1979). Similarly, new cultivars are required to counteract new aphid biotypes that are able to break resistance. The capacity of lucerne aphids to develop resistance-breaking biotypes has already been demonstrated by BGA in the USA (Zarrabi et al., 1995) and Australia (Humphries et al., 2012). Lucerne breeders, however, need to focus on what producers are willing to pay. Communication failures between researchers, breeders and producers are often the reason why only a small percentage of resistance mechanisms have been incorporated into commercial lines (Edwards and Singh, 2006). Focus should be placed on incorporating multi-trophic interactions into long-term field studies, as opposed to short-term glasshouse studies, using mixed or various individual cultivars. Gutierrez and Ponti (2013) identified host plant resistance as the most important mortality factor of SAA in California. They also predicted that, alone, each mortality factor failed to control SAA, whereas successful

CHAPTER 2 LUCERNE APHIDS - REVIEW control was achieved by a combination of factors. An integrated pest management approach combining top-down (biological control) and bottom-up control (cultivar resistance) should provide a more accurate representation of potential management practices aimed at reducing lucerne aphid populations and avoiding outbreaks.

2.9 Acknowledgements Thanks to Cavan Convery of the James Hutton Institute (UK) for providing the trophic schematic diagram. Thanks also to the Department of Agriculture, Fisheries and Forestry for providing high resolution aphid images.

Chapter 3

Effects of elevated temperature and CO2 on aboveground– belowground systems: a case study with plants, their mutualistic bacteria and root / shoot herbivores 2 Published as Ryalls et al. 2013, Frontiers in Plant Science, 4, 445

3.1 Abstract Interactions between above- and belowground herbivores have been prominent in the field of aboveground–belowground ecology from the outset, although little is known about how climate change affects these organisms when they share the same plant. Additionally, the interactive effects of multiple factors associated with climate change such as elevated temperature (eT) and elevated atmospheric carbon dioxide (eCO2) are untested. We investigated how eT and eCO2 affected larval development of the lucerne weevil (Sitona discoideus) and colonisation by the pea aphid (Acyrthosiphon pisum), on three cultivars of a common host plant, lucerne (Medicago sativa). Sitona discoideus larvae feed on root nodules housing N2-fixing rhizobial bacteria, allowing us to test the effects of eT and eCO2 on three trophic levels. Moreover, we assessed the influence of these factors on plant growth. eT increased plant growth rate initially (6, 8 and 10 weeks after sowing), with cultivar ‘Sequel’ achieving the greatest height. Inoculation with aphids, however, reduced plant growth at week 14. eT severely reduced root nodulation by 43%, whereas eCO2 promoted nodulation by 56%, but only at ambient temperatures. Weevil presence increased net root biomass and nodulation, by 31 and 45%, respectively, showing an overcompensatory plant growth response. Effects of eT and eCO2 on root nodulation were mirrored by weevil larval development; eT and eCO2 reduced and increased larval development, respectively. Contrary to expectations, aphid colonisation was unaffected by eT or eCO2, but there was a near-significant 10% reduction in colonisation rates on plants with weevils present belowground. The contrasting effects of eT and eCO2 on weevils potentially occurred through changes in root nodulation patterns.

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES

3.2 Introduction Many studies report on plant-mediated interactions between spatially-separated insect herbivores that live above- and belowground, yet few studies have considered these interactions in the context of global climate change. Climate change involves multiple factors such as warming and rising atmospheric carbon dioxide (CO2) concentrations. While the effects of predicted increases in global average surface temperatures (by 1–4

°C within this century) and atmospheric CO2 concentrations (from current levels of 400 µmol mol-1 to over 550 µmol mol-1 by 2050) on insect–plant interactions have been characterised separately, only a handful of studies have considered more than one climate change variable simultaneously (Robinson et al., 2012; Stevnbak et al., 2012; Murray et al., 2013b). Moreover, the role of plant microbes, such as mutualistic rhizobial bacteria which form intimate associations with plants, have not yet been investigated. Combining trophic complexity and multiple climatic factors is challenging but necessary to provide a more holistic insight into the mechanisms underpinning insect–plant interactions (Ryalls et al., 2013a).

All legumes form symbioses with rhizobial bacteria that fix atmospheric nitrogen (N2) and are carried in root nodules (Haag et al., 2013). Root nodulation is important for the larval development of Sitona weevil species (Coleoptera: Curculionidae), including S. discoideus, which feeds on lucerne root nodules throughout its larval growth stages (Allen, 1971; Goldson et al., 1988a; Vink and Phillips, 2007). Sitona discoideus therefore has the potential to reduce N-fixation in lucerne by damaging root nodules (Keane and Barlow, 2001). The net effect of S. discoideus on nodule numbers depends on the ability of the plant to compensate for nodule loss (Quinn and Hall, 1992). The abundance and size of root nodules can also be influenced by climate change. For example, it is widely reported that root nodulation (and biological N-fixation) increases in response to elevated CO2 concentrations (eCO2) (Ryle and Powell, 1992; Lüscher et al., 2000) but decreases with elevated temperatures (eT) (Munns et al., 1979; Zahran, 1999).

Root-feeding organisms can influence the chemical composition and biomass of aboveground plant parts, which, in turn, can influence the survival of aboveground insect herbivores (Soler et al., 2012). A recent meta-analysis (Johnson et al., 2012)

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES confirmed that root herbivory by beetle larvae usually had beneficial effects on aboveground aphids. This potentially arises through impaired root function and stress- related accumulation of N in the foliage (Masters et al., 1993). Given that, unlike in most other plants, rhizobial nodules underpin N balance in legumes, there is good reason to hypothesise that the presence of belowground herbivores that specifically target root nodules (and therefore have a greater impact on N uptake than generalised root feeders) will reverse this trend for better aphid performance on plants with root herbivores.

The net effect on plant growth when subjected to above- and belowground herbivory under the influence of multiple climatic factors is unknown and untested. Here we aim to characterise this in a model legume system that incorporates multiple organisms, including Rhizobium bacteria, the nodule-feeding lucerne weevil (S. discoideus) and the sap-sucking pea aphid (Acyrthosiphon pisum) (Hemiptera: Aphididae), with both insects feeding on a common host plant, lucerne (Medicago sativa L.) (Fabales: Fabaceae). Lucerne is the most important and widely-grown temperate forage legume globally (Small, 2011; Bouton, 2012). Acyrthosiphon pisum is a widespread pest of lucerne and a concerted programme of incorporating aphid resistance into cultivars since the introduction of lucerne-feeding aphids, including A. pisum, to Australia in 1980, has helped to control aphid populations. Occasional outbreaks, however, driven by environmental factors including climatic variability and interactions with other trophic groups (e.g. release from natural enemies), still occur (Zarrabi et al., 1995; Humphries et al., 2012). Moreover, the susceptibility of different cultivars to A. pisum can be modified by such factors (Ryalls et al., 2013a), and we therefore included cultivars with moderate (‘Trifecta’), low (‘Sequel’) and no (‘Hunter River’) A. pisum resistance.

We present a novel case study that examines the interactive effects of temperature

(daytime temperature 26 and 30 °C, aT and eT, respectively) and atmospheric CO2 -1 concentration (400 and 640 µmol mol , aCO2 and eCO2, respectively) on the interactions between lucerne cultivars, rhizobial bacteria, a root herbivore and an aphid. Specifically, we set out to investigate how: (i) aphid herbivory affects plant growth (height) under eCO2 and eT; (ii) eCO2 and eT affect root nodulation and the performance of S. discoideus; (iii) the presence of nodule-feeding S. discoideus affects

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES the ability of A. pisum to successfully colonise and reproduce on three cultivars of lucerne of varying resistance to A. pisum under eCO2 and eT. We hypothesised that: (i) eCO2 and eT would promote plant growth but aphid herbivory would reduce the rate of growth; (ii) eCO2 and eT would promote and reduce nodulation, respectively, and S. discoideus would perform better (i.e. complete larval development) at lower temperatures and higher CO2 concentrations; and finally, (iii) nodule-feeding by S. discoideus would have a negative impact on aphid abundance via impaired root function and decreased plant quality. For the purpose of this case study, weevil emergence refers to the number of plants with S. discoideus that grew to adulthood and emerged from the soil.

3.3 Materials and methods 3.3.1 Growth conditions and experimental design

Four glasshouse chambers, providing two atmospheric CO2 concentrations (400 and -1 640 µmol mol , aCO2 and eCO2 respectively) and two temperature treatments (daytime temperature 26 and 30 °C, aT and eT, respectively) combined factorially, were used in this study. aT (maintained at 26/18 °C day/night on a 15L:9D cycle) represents the daily average temperature data (November to May) for Richmond, NSW (latitude −33.611098, longitude 150.742368) (Australian Bureau of Meteorology) and eT (30/22 °C day/night) was based on the predicted maximum temperature increase for this region within this century (CSIRO, Bureau of Meteorology, 2007). Humidity was controlled at 55%. Photosynthetic active radiation (PAR) was measured every hour between 10am and 3pm. PAR ranged from 210 µmol m-2 s-1 to 580 µmol m-2 s-1; no significant differences in PAR were observed between chambers overall. As an attempt to compensate for the effects of pseudo replication (though not excluding them) arising from the lack of replication of climates (climate–temperature combinations), we rotated the position of pots randomly within chambers twice weekly (Quirk et al., 2013; Sherwin et al., 2013). The environmental conditions within the chambers were logged and monitored continuously throughout the experiment to maintain temperature and

CO2 differences between chambers and temperature readings were cross-checked with transportable temperature loggers (DS1922L Thermochron temperature sensors, Thermodata, Victoria, Australia), as in Murray et al. (2013b).

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES

Lucerne seeds (sourced from Seedmark, Adelaide, South Australia) were inoculated with Rhizobium bacteria one hour prior to planting by submerging in a solution containing 250 g Nodule N lucerne seed inoculant (New Edge Microbials, Albury, NSW) and 800 mL distilled water. In each chamber, 120 lucerne seeds (40 of each of the three cultivars) were individually planted in 70 mm pots filled with sieved (2 mm) local loamy-sand soil collected from the Hawkesbury Forest Experiment in Richmond, NSW (Barton et al., 2010). Soil was kept moist by watering daily (c. 15 mL). Acyrthosiphon pisum cultures, reared from a single parthenogenetic adult female collected in Richmond, NSW were maintained at 22/14 °C day/night on the susceptible cultivar ‘Hunter River’ until required. Additionally, twenty sexually-mature S. discoideus adults, also collected by sweep-netting in July 2012 from local lucerne fields in Richmond, NSW, were reared on ‘Hunter River’ and eggs were collected every 24 hours and stored on damp filter paper at 4 °C until required. Hatching success was assessed and confirmed (>95% hatched within 5 days) by placing 200 eggs on 10 Petri dishes at 25 °C. ‘Hunter River’ was included as an experimental cultivar to provide a baseline for comparison with the resistant cultivars.

3.3.2 Experimental procedure

Plants in each of the four chambers (aCO2×aT; eCO2×aT; aCO2×eT; eCO2×eT) were distributed randomly among four treatments: weevils only (W), aphids only (A), weevils and aphids (WA) and no insects or control (C), giving 10 replicates of each treatment per cultivar (Trifecta, Sequel and Hunter River) in each chamber. When plants were six weeks old, half of the plants (treatments W and WA) were inoculated with 20 S. discoideus eggs per plant. This egg density (6027 eggs per m2) resembles S. discoideus eggs densities recorded in NSW during June (5185 eggs per m2) (Aeschlimann, 1983). Eggs were placed on top of the soil beside the stem of each plant. After a further four weeks, two teneral adult pea aphids were transferred to each plant in treatments A and WA. Plants were placed on plastic plinths within water-filled trays, which acted as moats to prevent aphid movement between plants. Aphid presence was recorded 7 and 14 days later and aphids were removed after 21 days. Six weeks after egg inoculation, plants were checked every 12 hours for emerging adult weevils (i.e. those that had completed larval development and emerged from the soil leaving an exit hole). One week after aphids were removed, roots were separated from the soil and the

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES numbers of root nodules were counted. Plant heights (from ground level to the base of the highest leaf) were measured 6 (weevil egg inoculation period), 8, 10 (aphid inoculation period) and 14 (harvest period) weeks after planting. Lucerne height is strongly correlated with biomass (Michalk and Herbert, 1977) so this represents an excellent non-destructive proxy for plant growth.

3.3.3 Statistical analyses 3.3.3.1 Plant responses

The effects of CO2 and temperature on lucerne height measurements were determined using general linear models within the R statistical interface v2.15.1. The main effect terms of ‘CO2’, ‘temperature’, ‘cultivar’ and ‘treatment’ (i.e. aphid- and weevil-treated), as well as their associated interactions, were included. The effects of temperature,

CO2, cultivar and weevil presence on the number of nodules were assessed using multifactorial ANOVA in R. The dependent variable ‘nodule number’ was log- transformed to standardise residuals. Pairs of mean estimated effects were compared using a Tukey-Kramer post hoc test. ANOVA was used to compare mean differences in root mass between weevil-treated and untreated plants. Root mass was square root transformed to normalise data.

3.3.3.2 Insect responses

The effects of temperature and CO2 treatment on the proportion of plants containing weevils that reached adulthood and emerged from the soil were assessed using a generalised linear model with a binomial error structure and logit link function within R.

The full model included main effect terms for ‘cultivar’, ‘temperature’, ‘CO2’ and ‘aphid- treatment’ (i.e. whether plants had aphids applied), as well as the interactions between these terms. The effects of CO2, temperature, cultivar and weevil presence on aphid success were determined using a generalised linear model with a binomial error structure and logit link function, using aphid success (colonisation) as the dependent variable. Models were reduced in a stepwise manner by removing the non-significant terms in order of least significance and plants that had died before harvest were not included in analyses.

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES

3.4 Results 3.4.1 Plant responses 3.4.1.1 Height

Plant height was significantly greater at higher temperatures at week 6 (F1,468 = 59.03; P

< 0.001), week 8 (F1,468 = 40.11; P < 0.001) and week 10 (F1,468 = 8.56; P = 0.004), whereas CO2 did not affect plant height in these weeks (see Fig. 3-1 for significant effects at week 10). In addition, there were significant differences in height between cultivars in these same weeks (F2,468 = 14.52, P < 0.001; F2,468 = 9.03; P < 0.001; F2,468 = 9.45; P < 0.001), with Sequel being significantly bigger than the other two cultivars overall (Table 3-1). At week 14, aphids reduced plant height (t1,195 = -2.637; P = 0.008) and there was a significant interaction between CO2 and temperature (F2,468 = 8.21; P =

0.004) whereby eCO2 increased plant growth at 26 °C (from 57.5±4.2 mm at aCO2 to

66.4±4.2 mm at eCO2) but not at 30 °C. The presence of weevils also interacted with temperature effects (F2,468 = 6.94; P = 0.009), with weevils decreasing overall plant height at 26 °C (from 66.9±4.4 mm without weevils to 58.1±3.9 mm with weevils) but not at 30 °C (see Fig. 3-2 for all significant treatment factors).

70 cv. H 60 cv. S cv. T 50

Plant heightPlant at week 10 (mm)

0 26 30

Temperature Figure 3-1. Effects of temperature and cultivar (Hunter River, H; Sequel, S and Trifecta, T) on plant growth (height) 10 weeks after sowing. Mean values (± SE) of significant treatment factors (temperature and cultivar) in the final model shown.

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES

(mm) nodules per colonised by emergence (%) plant aphids (%) Hunter River 46.3 ± 2.1 9.8 ± 0.8 27.5 6.3 Sequel 54.8 ± 2.1 12.4 ± 1.1 20.0 13.8 Trifecta 45.6 ± 1.9 7.5 ± 0.9 32.5 8.8 2 2 F2,468 = 3.96; F2,409 = 9.59;  (df = 2) 3.32;  (df = 2) 2.90; Test results: P = 0.020* P < 0.001* P = 0.190 P = 0.235

120 (b) aCO (a) aCO2 × aT cv.H 2 × eT 100 cv.S cv.T 80

0 120 (c) eCO2 × aT (d) eCO2 × eT 100

Plant height at week 14 (mm) 40

0 W- W+ W- W+ W- W+ W- W+ No aphids Aphids No aphids Aphids Treatment

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES

3.4.1.2 Nodulation and root mass eT resulted in reduced nodulation (F1,409 = 34.78; P < 0.001) but the interaction between

CO2 and temperature also affected root nodulation (F1,417 = 4.11; P = 0.042); nodulation increased under eCO2 at 26 °C (Tukey HSD; P < 0.01) but not at 30 °C (Tukey HSD; P = 0.983) (Fig. 3-3). Plants with S. discoideus had a significantly higher number of nodules and greater root mass than those without (Table 3-2).

20 a

b 10 c bc

Number of nodules Number 5

0 26 30

Temperature (°C)

Table 3-2. Mean (± SE) numbers of nodules and root mass between weevil-treated and -untreated plants. Summary statistical results of tests for differences between the variable means are provided. N Number of nodules Root mass (g) Weevil-treated 209 11.71 ± 0.94 0.093 ± 0.004 Weevil-untreated 209 8.08 ± 0.57 0.071 ± 0.003

Test results: F1,417 = 11.52; P < F1,417 = 11.55; P < 0.001 0.001

3.4.2 Insect responses 3.4.2.1 Weevils eT had a negative effect on weevil emergence (i.e. the proportion of plants with emerging weevils) (z1,234 = -3.29; P < 0.001), whereas eCO2 positively affected weevil emergence (z1,234 = 2.00; P = 0.045) (Fig. 3-4). Weevil emergence was not influenced by

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES cultivar type (Table 3-1). These responses resembled those seen for nodulation, i.e. negative and positive effects of eT and eCO2, respectively.

larval (%) development 5 No adults

Plants with weevilsPlants that completed 0 26 30

Temperature (°C) Figure 3-4. Effects of temperature on the proportion of plants containing weevils that reached adulthood and emerged from the soil at aCO2 (open bars) and eCO2 (closed bars). All columns were significantly different (P < 0.01) from one another.

3.4.2.2 Aphids

Acyrthosiphon pisum was not significantly affected by changes in temperature and CO2, cultivar type or the presence of weevils, although there was a trend for the proportion of plants that were successfully colonised by A. pisum to be lower in plants inoculated with S. discoideus eggs (2 (df = 1) 3.15; P = 0.076).

3.5 Discussion

Our study is first to incorporate the effects of both eT and eCO2 on a system combining above- and belowground insect herbivores. The results demonstrate that eT can negate the positive effects of eCO2 on lucerne nodulation and herbivory belowground, an important consideration for determining future outcomes of climate change.

Temperature promoted plant growth initially and aphid herbivory reduced plant growth at week 14. The reduction in height of plants with aphids present, however, was facilitated by root feeding by S. discoideus, indicated by an interaction between

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES temperature and weevil presence at week 14; the presence of S. discoideus decreased lucerne height at aT but not at eT. Few weevils, however, emerged at eT, suggesting that they did not account for the changes in plant growth observed at eT. 30 °C may represent an upper developmental threshold (i.e. the temperature at which development rate becomes suboptimal) for S. discoideus. Arbab et al. (2008) identified 28 °C as the optimum temperature for S. discoideus development with development time decreasing with increasing temperatures within the range of 8.5 to 28 °C (from a hatching rate of 69.01±0.92 days to 8.46±0.14 days, respectively). Additionally, egg development rates at 26 °C matched rates at 30 °C and they calculated the upper developmental temperature threshold as 30 to 32.6 °C. Temperature increases towards the thermal optimum may positively influence the abundance of S. discoideus, whereas temperatures above 30 °C may negatively affect S. discoideus numbers.

Effects of CO2 on root nodulation mirrored their effects on the emergence of S. discoideus at 26 °C, suggesting that S. discoideus performance was influenced by the number of root nodules on lucerne. eCO2 has been shown to promote nodulation and performance of another Sitona species (S. lepidus) on white clover (Trifolium repens) (Johnson and McNicol, 2010). In that study, the effects of temperature were not tested, though the authors speculated that enhanced nodulation and S. lepidus larval performance seen under eCO2 might be tempered by eT. In the present study, we observed that eT negated the positive effects of eCO2 on nodulation. Such negation of eCO2 effects by eT has also been found in aboveground insect–plant interactions (Murray et al., 2013b) and further emphasises the need to consider multiple climate change factors simultaneously when assessing the effects of global environmental change.

Modest root herbivory often produces compensatory responses by the plant which may increase root growth to offset losses due to herbivory (Andersen, 1987; Brown and Gange, 1990; Blossey and Hunt-Joshi, 2003). In the present study, we observed even larger increases in root growth and nodulation in the presence of S. discoideus above and beyond those observed in plants without S. discoideus, i.e. overcompensation. Overcompensatory nodulation has also been demonstrated in lucerne in response to Sitona hispidulus, which, after 10 days of nodule-feeding, increased the number of

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES nodule units per plant by 89±9% compared to 25±7% in control plants (Quinn and Hall, 1992). The exact mechanisms underpinning herbivory-induced increases in the formation of root nodules are unclear but it could be due to the increased exudation of metabolites (e.g. flavonoids) that attract N-fixing bacteria (Hirsch et al., 2001; Wasson et al., 2006) and/or changes in the expression of plant or bacterial cell cycle regulators (Caetano-Anollés and Bauer, 1988; Mergaert et al., 2006). Our study used egg densities (c. 6000 eggs per m2) similar to those seen in the field in NSW, Australia during June (c. 5000 eggs per m2), see Section 2.2 (Aeschlimann, 1983). Higher egg densities, such as those reported by Aeschlimann and Vitou (1988) of 9500 eggs per m2 , however, could result in root herbivory that was too severe for compensatory growth to occur.

Few changes were observed in aphids feeding aboveground, although numbers of aphids that colonised plants (i.e. remained on the plant and reproduced) were low, making aboveground–belowground and cultivar interactions difficult to interpret. Johnson et al. (2012) confirmed that, generally, aboveground aphids are positively affected by belowground root feeders. Unlike most other root herbivores, S. discoideus attacks root nodules (and therefore sites of N acquisition). Negative effects of root herbivory on aphids could therefore arise through both reduced phloem turgor via impaired root function and lower quality phloem sap from nodule damage specifically (e.g. Murray et al., 1996). This is supported by Goldson et al. (1988b), who reported that S. discoideus reduced the biomass and nitrogen concentration in lucerne as a direct result of nodule damage. Indeed, there was a trend for aphids to be less able to colonise plants with weevils present, albeit not at a 95% confidence interval (P = 0.076). The low number of aphids at the end of this experiment may have contributed to obscuring these negative impacts. Alternatively, given that weevils began to stimulate nodulation the negative impacts of initial nodule damage on aphids may have begun to be reversed, or at least alleviated. This demonstrates the importance of considering the exact nature of root herbivory, for instance which parts of the roots are targeted by root herbivores, the sequence and duration of the attack and ultimately how these effects change over time (e.g. triggering of compensatory responses).

Root and nodule feeding also have the potential to affect neighbouring plants by altering nitrogen availability in the soil (Murray and Hatch, 1994; Ayres et al., 2007).

CHAPTER 3 ABOVE- AND BELOWGROUND HERBIVORES

This arises because N leaks out of lacerated nodules and is taken up by co-occurring plants. Traditionally, studies between belowground and aboveground herbivores are conducted on the same shared host plant, but in this instance it could be envisaged that belowground herbivory affects aboveground herbivores on neighbouring plants of different species. Essentially, root herbivory by S. discoideus may reduce the quality of lucerne while indirectly improving the quality of neighbouring host plants. Where aphids can feed on both lucerne and the neighbouring plant, this may cause aphids to migrate between plants.

Results suggest that the contrasting effects of eT and eCO2 on weevils likely occurred through changes in root nodulation patterns. Further study, including repeated experimental runs to reduce problems associated with pseudo replication, should be undertaken to determine how nodule feeders belowground will affect aphids feeding aboveground, with potential pest management implications for the lucerne industry, as well as other legumes that are attacked by both insects. Incorporating such trophic complexity and multiple climatic factors represents a significant challenge for biologists, yet one that we must address to gain realistic insights into how global climate change will affect insect–plant interactions.

3.6 Acknowledgements We would like to thank Aidan Hall, Andrew Gherlenda and Lisa Bromfield for support and help with glasshouse work.

Chapter 4

Amino acid-mediated impacts of elevated carbon dioxide and simulated root herbivory on aphids are neutralized by increased air temperatures 3 Published as Ryalls et al. 2015, Journal of Experimental Botany, 66, 613-623

4.1 Abstract Changes in host plant quality, including foliar amino acid concentrations, resulting from global climate change and attack from multiple herbivores has the potential to modify the pest status of insect herbivores. This study investigated how mechanically- simulated root herbivory of lucerne (Medicago sativa) before and after aphid infestation affected the pea aphid (Acyrthosiphon pisum) under elevated temperature

(eT) and carbon dioxide concentrations (eCO2). eT increased plant height and biomass and eCO2 decreased root C:N. Foliar amino acid concentrations and aphid numbers increased in response to eCO2, but only at ambient temperatures, demonstrating the ability of eT to negate the effects of eCO2. Root damage reduced aboveground biomass, height and root %N and increased root %C and C:N, most likely via decreased biological nitrogen fixation. Total foliar amino acid concentrations and aphid colonisation success were higher in plants with roots cut early (before aphid arrival) than those with roots cut late (after aphid arrival), however this effect was counteracted by eT. These results demonstrate the importance of amino acid concentrations for aphids and identify individual amino acids as being potential factors underpinning aphid responses to eT, eCO2 and root damage in lucerne. Incorporating trophic complexity and multiple climatic factors into plant–herbivore studies enables greater insight into how plants and insects will interact in the future, with implications for sustainable pest control and future crop security.

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

4.2 Introduction Host plant quality and suitability for herbivorous insects shapes the extent to which plants are attacked (Bernays and Chapman, 1994), and sudden improvements in host plant quality can lead to pest outbreaks (Barbosa et al., 2012). Biotic and abiotic factors often induce changes in secondary metabolites that might deleteriously affect herbivores (Iason et al., 2012), but these can also change the nutritional quality of plants leading to increased susceptibility of plants to herbivores (White, 1984; Johnson et al., 2009). This is particularly true for mobile herbivores that have short generation times and respond quickly to changes in host plant nutrition, such as aphids (Dixon, 1998; Douglas, 2003). In particular, aphids respond to changes in the amino acid quality of phloem sap, which has been shown to be affected by both global climate change (Harrington et al., 1995; Newman, 2004; Pritchard et al., 2007; Sun and Ge, 2011) and root herbivory (Johnson et al., 2012; Johnson et al., 2013). To our knowledge, how these factors operate and interact remains largely unknown. In this study, we set out to examine how elevated temperature (eT) and atmospheric carbon dioxide concentrations (eCO2), as well as simulated root herbivory, acted alone and together to affect foliar amino acids and plant susceptibility to aphids.

-1 Future CO2 concentrations (from current levels of 400 µmol mol to over 550 µmol mol−1 by 2050) are likely to be accompanied by increased temperatures (1–4 °C within this century) and while a number of studies have observed effects of eCO2 on aphid populations, the combination of eT and eCO2 has received little attention (Newman,

2004; Himanen et al., 2008; Murray et al., 2013b). In general, eCO2 is expected to increase plant growth by accelerating rates of photosynthesis, which reduces tissue quality and increases the carbon to nitrogen (C:N) ratio (Robinson et al., 2012). This can be exacerbated when increased plant growth leads to N-limitation (Rogers et al., 2009). Legumes, such as lucerne (Medicago sativa L.) can, however, avoid N-limitation and maintain tissue N concentrations under eCO2 by enhancing biological nitrogen fixation (Soussana and Hartwig, 1995; Johnson and McNicol, 2010). This can alter the dynamics of foliar-feeding aphids (Douglas, 1993), which tend to perform better on plants with higher N (Nowak and Komor, 2010; Sun and Ge, 2011) and amino acid concentrations (Ponder et al., 2000; Karley et al., 2002; Guo et al., 2014). In contrast, eT may combat eCO2 effects on plant nutrient quality by decreasing biological nitrogen

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS fixation, often associated with a low tolerance of N-fixing rhizobial bacteria to increased temperatures (Zahran, 1999; Whittington et al., 2013), which ultimately decreases amino acid concentrations and aphid abundance (Ryalls et al., 2013a).

Plants, in addition to aboveground herbivores, often have to contend with root damage by herbivores, which may increase amino acid concentrations in the foliage and, in turn, make the plant more susceptible to aphid attack aboveground (Masters et al., 1993; Johnson et al., 2009). This might be different for legumes, however, since the roots are the source of N acquisition through biological nitrogen fixation. The general prediction that root herbivory usually has beneficial effects on aboveground aphids may also be altered by changes in herbivore synchronisation (Erb et al., 2011). In a meta-analysis, Johnson et al. (2012) identified the sequence of herbivore arrival (e.g. whether aphids arrive before or after roots are damaged) as the most influential factor affecting aboveground–belowground linkages. Root damage before aphids arrive may impair water uptake and lead to a stress-related accumulation of amino acids in the phloem, but crucially gives the plant a chance to recover and regain hydraulic properties (e.g. phloem turgor) which might allow aphids to capitalise on this. Root recovery is often rapid and can be over-compensatory (i.e. more root nodules are produced) in legumes (Quinn and Hall, 1992; Ryalls et al., 2013b). Conversely, if aphids are already feeding on a plant when root damage occurs, root recovery and stress-related increases in foliar nutrients may be less likely because the plant remains in stress. This mirrors the pulsed stress hypothesis (Huberty and Denno, 2004), which posits that phloem-feeding insects exhibit poor performance on continuously stressed plants, yet respond positively when plant recovery is possible.

Using the model legume, lucerne, this study combines aboveground sap-feeding by the pea aphid, Acyrthosiphon pisum (Harris), and simulated root herbivory in the form of root cutting, to characterise the effects of herbivore arrival sequence on aboveground– belowground interactions. While artificial herbivory may not mimic natural damage exactly (Blossey and Hunt-Joshi, 2003), it enables control of type, timing and intensity of damage, especially in complex systems such as this (Hjältén, 2004; Rogers and Siemann, 2004; Borowicz, 2010). Lucerne itself is the most important and widely- grown forage legume worldwide (Small, 2011). The late 1970s saw the invasion of

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

Australia by three aphid pests, including the pea aphid, which devastated lucerne stands in eastern Australia (Bouton, 2012). The development of resistant cultivars has helped to abate the aphid problem, but outbreaks driven by environmental factors and changes in the nature and intensity of trophic interactions (e.g. release from natural enemies) still occur (Zarrabi et al., 1995; Reddy and Hodges, 2000; Humphries et al., 2012; Ryalls et al., 2013a).

This study specifically aimed to determine the effects of: eT and eCO2, individually and in combination, on lucerne growth (height and biomass), chemistry (root C:N and foliar amino acid concentrations and composition) and pea aphid abundance; and simulated root herbivory (root cutting before and after aphid arrival) on lucerne growth, chemistry and pea aphids. We hypothesised that: (i) eCO2 would increase lucerne growth and promote biological nitrogen fixation (i.e. decrease root C:N), which would increase foliar amino acid concentrations and ultimately aphid abundance. In contrast, (ii) eT would increase lucerne growth but reduce biological nitrogen fixation (i.e. increase root C:N), which would reduce foliar amino acid concentrations and aphid abundance; (iii) root cutting would reduce lucerne growth and biological nitrogen fixation (i.e. increase root C:N) generally. Early root cutting would benefit aphids via increased foliar amino acid concentrations because stress and recovery of lucerne would be possible. In contrast, root cutting during aphid feeding would present above- and belowground stress, making recovery less likely, and would negatively impact aphids via decreased foliar amino acid concentrations. Hypothesised treatment effects of early- and late root cutting, elevated temperature and CO2 on aphids and plant characteristics are summarised in Fig. 4-1a.

4.3 Materials and methods 4.3.1 Glasshouse conditions Experiments were conducted in four glasshouse chambers at the University of Western Sydney Hawkesbury campus, NSW (latitude -33.611141, longitude 150.745315) in which CO2 was maintained at either ambient CO2 concentrations, aCO2 (400 µmol −1 -1 mol ), or eCO2 (640 µmol mol ) and temperature was maintained at either ambient temperature, aT (26/18 °C day/night on a 15:9 light:dark cycle), or eT (30/22 °C day/night on a 15:9 light:dark cycle). aT (26 °C) represents the average daily maximum

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS temperature for Richmond, NSW over the last 30 years and eT was consistent with the predicted maximum temperature increase of 4 °C for this region within this century (CSIRO, 2007). Humidity was controlled at 55% and no significant differences in light conditions were observed between chambers (Ryalls et al., 2013b). Temperature, CO2 and humidity within glasshouse chambers were monitored continuously throughout the experiment and plants were randomly reallocated among positions within chambers twice weekly to minimise potential within-chamber effects. The experiment itself was repeated three times, which was incorporated into statistical models to reduce the influence of pseudo-replication of CO2 and temperature treatments (Newman et al., 2011).

4.3.2 Aphid cultures Acyrthosiphon pisum adults used in the experiment were taken from four established cultures, all originating from an individual parthenogenetic adult female collected from a local lucerne field in Richmond, NSW (latitude -33.609189, longitude 150.746953) in January 2013. Cultures were maintained on propagated lucerne plants in each of the four experimental chambers for at least six generations prior to the inoculation period.

4.3.3 Experimental procedure

Lucerne plants (54 plants of cultivar ‘Sequel’) in each of the four chambers (aCO2, aT; eCO2, aT; aCO2, eT; eCO2, eT) were distributed randomly among six treatments: aphids only (A), early cut no aphids (EC), early cut and aphids (ECA), late cut no aphids (LC), late cut and aphids (LCA) and control plants with no cutting or aphids (C), giving 9 replicates of each treatment in each chamber. Five weeks after sowing, treatments EC and ECA were subjected to early root damage by severing the entire root system 35 mm below the soil surface using a sharp steel blade inserted in a narrow opening cut into the plastic pot. One week later, treatments A, ECA and LCA were inoculated with two teneral adult pea aphids. Organza bags (125 x 170 mm) were secured around each plant to confine individual aphids to the plant. One week after aphid inoculation, treatments LC and LCA were subjected to simulated late root herbivore damage using the same technique as those cut earlier. Aphids were counted and removed a week later (eight weeks after sowing). One week after aphids were removed, roots were separated from soil and shoots and all plant material was snap frozen in liquid N and

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS stored at -20 °C. Material was then freeze-dried and weighed to determine dry mass before chemical analysis. Plant heights (from ground level to the base of the highest leaf) were measured five (early cutting period), six (aphid inoculation period) and nine (harvest period) weeks after planting (Fig. 4-1b).

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

4.3.4 Chemical analyses Four samples, selected at random, from each treatment combination per run were chosen for chemical analysis (288 samples overall). Freeze-dried roots and shoots were ball-milled for 90 seconds to a fine powder. Soluble amino acids were extracted from milled shoot samples (10 mg) by shaking in 1.5 mL 80% ethanol for 20 min at 50 °C. After centrifugation at 12,000 rcf for 5 min to remove solids, 1 mL supernatant was extracted and evaporated to dryness at 30 °C in a vacuum concentrator (Eppendorf). Amino acids were analysed by reverse-phase high performance liquid chromatography (HPLC) in an Agilent 1260 Infinity HPLC system after pre-column derivatization using phenylisothiocyanate (PITC) (Crafts-Brandner, 2002; Reason, 2003). Derivatization steps were as follows: Dried extracts were redissolved in 100 µL coupling solution (acetonitrile:pyridine:triethylamine:water 10:5:2:3), vortexed and redried under vacuum at 30 °C. They were then redissolved in 100 µL coupling solution, followed by 5 µL PITC, vortexed and allowed to react for 5 min at room temperature before redrying at 30 °C. 100 µL Milli-Q water was then added, vortexed and redried at 45 °C. Samples were redissolved in 250 µL analysis solvent (water:acetonitrile 7:2) and filtered through a 0.45 µm 4 mm PVDF filter prior to HPLC analysis. HPLC was performed using an Agilent Poroshell 120 EC-C18 column (4.6 x 75 mm, 2.7 µm) at a column temperature of 40 °C. The gradient, composed of eluent A (1 L Milli-Q water, 19 g sodium acetate trihydrate, 0.5 mL triethylamine, pH 5.7, 63.8 mL acetonitrile, 1.07 mL EDTA dipotassium salt 1 g/L solution) and eluent B (acetonitrile:water 6:4), was as follows: 0– 3.75 min, 0–40% B; 3.75–4.5 min, 40–80% B; 4.5–5.75 min, 80–100% B; 5.75–7 min, 100% B; 7–9 min, 100–0% B. The flow rate was 1.4 mL min-1. Amino acid derivatives were detected by ultraviolet absorbance at 254 nm. Amino acid standards (5, 10, 15, 20 and 25 nmol) containing 17 amino acids (see below) and an internal standard (12.5 nmol L-Norleucine) were used to calibrate the analysis. Nine essential amino acids (i.e. those that cannot be synthesised by insects de novo), including arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine (Morris, 1991) and eight non-essential amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine and tyrosine) were detected using this method. Foliar amino acid composition was measured as it has been demonstrated to be a reliable indicator of phloem amino acid composition (Winter et al., 1992; Johnson et al., 2014). Root C and N concentrations of 4–6 mg milled root samples were determined using a

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

Carlo Erba CE1110 elemental analyser with thermal conductivity and mass spectrometric detection (of N2 and CO2). The percentage of N and C in the sample was calculated by comparison with known standards.

4.3.5 Statistical analyses Linear mixed effect models were produced using the nlme (v3.1-109) and lme4 (v0.999999-2) statistical packages for aphid abundance and aphid colonisation, respectively, in the R statistical interface v3.0.1. Models described the effects of simulated root herbivory, eT and eCO2 on aphids. The fixed terms included were plant- damage treatment (C, A, EC, ECA, LC and LCA), temperature (26 and 30 °C) and CO2 (ambient and elevated) as well as the interactions between these terms. The random terms included were experimental run, with chamber as a nested factor, to account for pseudoreplication and chamber effects that could confound temperature and CO2 effects. The dependent variable ‘aphid abundance’ was log+1 transformed to normalise the model-standardised residuals. Aphid colonisation (i.e. proportion of plants hosting aphids at harvest) was analysed using a binomial error structure. Models were reduced by deleting non-significant fixed terms in a stepwise manner using AIC values and associated P-values taken from ANOVA model comparisons. Post-hoc Tukey’s tests and the R package LMERConvenienceFunctions were used for pairwise comparisons of means for treatment and interaction effects. The effects of simulated root herbivory, aphid presence, eT and eCO2 on lucerne shoot biomass, height and root biomass were also analysed with mixed models using nlme in R. Dependent variables were log-transformed to normalise model residuals. The random terms included were run; chamber and fixed terms were reduced in a stepwise manner, as above.

Temperature, CO2 and plant-damage treatment effects on total, essential and individual amino acid concentrations were also analysed using linear mixed models with log- transformed dependent variables to normalise residuals. Similar models were used to determine the effects of temperature, CO2 and plant-damage treatment on root %N, C and C:N. Principal Components Analysis (PCA) and Permutational Multivariate Analysis of Variance (PERMANOVA) were used to explore the impacts of eT, eCO2 and plant- damage treatment on amino acid composition. Groupings of individual amino acids were determined using a correlation matrix and PERMANOVA results were compared with those obtained from individual mixed models.

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

4.4 Results

4.4.1 Impacts of eT and eCO2

Temperature in interaction with CO2 had a significant effect on lucerne height (F1,6 =

7.80, P = 0.032), root biomass (F1,6 = 21.76, P = 0.003) and shoot biomass (F1,6 = 6.45, P

= 0.044). At 30 °C, plants were taller under eCO2, whereas, at 26 °C, plants were shorter under eCO2 (Fig. 4-2a). Lucerne root and shoot mass increased under eCO2 at

30 °C, but no effects of eCO2 were observed at 26 °C (Fig. 4-2b and c). eCO2 decreased root C:N significantly (F1,8 = 7.90; P = 0.023) (Fig. 4-2d) but did not significantly increase root %N (F1,8 = 4.55; P = 0.065) or decrease root %C (F1,8 = 2.59; P = 0.146) individually.

80 20 aCO d (a) (b) 2 *Temp × CO2 *Temp × CO2 b

eCO2 60 c 15 a a a a b 40 10

Height (mm) 20 5

Root biomass (mg) Root biomass

0 0 26 30 26 30 40 35 c (c) (d) *Temp × CO2 *CO2 30 30 b 25 a a 20 20 15

Root C:N 10 10

Shoot biomass (mg) Shoot biomass 5

0 0 26 30 26 30 Temperature (°C) Temperature (°C)

Significant interactions of temperature and CO2 were also observed for total (F1,287 =

7.63; P = 0.006) and essential amino acid concentrations (F1,292 = 10.072; P = 0.002), as well as aphid abundance (F1,6 = 6.45, P = 0.044), whereby eCO2 significantly increased total amino acids (Fig. 4-3a), essential amino acids (Fig. 4-3b) and pea aphid numbers

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

(Fig. 4-3c) at 26 °C but not at 30 °C. Results from individual amino acid analyses (Table 4-1 and S4-1) showed that eT decreased concentrations of Arg, Asp, Glu and His and eCO2 increased concentrations of Ala, Glu, Leu, Lys, Phe, Pro and Ser. The interaction of eT and eCO2 affected Gly and Met, whereby concentrations of both amino acids increased under eCO2, but only at 26 °C.

40 aCO b 2 *Temp × CO2 (a)

eCO2 30 a a a

dry mass) dry 20

-1

(µmol g (µmol

Amino acid concentrationAmino 0 26 30 16 *Temp × CO (b) 14 b 2 12

10 a a a dry mass) dry 8

-1 6 4

(µmol g (µmol Essential acids amino 2 0 26 30 10 *Temp × CO2 (c) b 8

a 6 a a

Number of aphidsNumber plant per 0 26 30

Temperature (°C)

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

PCA revealed no clear separation of treatments, although greater variation was seen in plants grown in eCO2 at 26°C. The first two principal components (PC 1 and PC 2) accounted for 62% of the variation in the dataset (Appendix I Fig. S4-1). PERMANOVA on correlated amino acid groups, consisting of group 1 (Ala, Gly, Iso, Leu, Lys, Phe, Pro, Ser, Thr, Val), group 2 (Arg, Asp, His, Lys) and four individual amino acids (Glu, Met, Cys, Tyr), confirmed patterns observed in individual models (see Table S4-2 for full results), whereby group 1 was significantly affected by CO2 and group 2 was significantly affected by temperature (Appendix I Fig. S4-2a and c, respectively). Temperature and

CO2 interactively affected all ungrouped individuals.

Table 4-1. Summary of final model statistical analyses of individual amino acid responses to plant- damage (Treatment), temperature (Temp) and CO2 treatments.

CO2 Temp Treatment Temp × CO2 Treatment × Temp

Amino acid F1,6 P F1,6 P F5,264 P F1,6 P F5,264 P Alanine (Ala)1 28.26 0.002 - - 3.48 0.005 - - - - Arginine* (Arg)2 - - 14.41 0.009 2.41 0.037 - - - - Aspartic acid (Asp)2 - - 22.9 0.003 3.62 0.004 - - - - Cysteine (Cys)4 ------Glutamic acid (Glu)3 10.33 0.02 17.23 0.006 5.78 <0.001 - - - - Glycine (Gly)1 ------8.99 0.024 - - Histidine* (His)2 - - 11.66 0.014 4.79 <0.001 - - - - Isoleucine* (Iso)1 - - - - 2.77 0.019 - - - - Leucine* (Leu)1 9.22 0.023 ------Lysine* (Lys)1,2,3,5 23.91 0.003 ------10.88 <0.001 Methionine* (Met)4 - - - - 3.83 0.002 15.49 0.008 - - Phenylalanine* (Phe)1 10.64 0.017 ------3.74 0.003 Proline (Pro)1 7.43 0.034 - - 2.36 0.04 - - - - Serine (Ser)1 9.25 0.023 - - 2.31 0.045 - - - - Threonine* (Thr)1 ------Tyrosine (Tyr)5 ------6.99 <0.001 Valine* (Val)1 ------

Treatments that were not significant (-) and interactive effects that are not displayed were absent from the final models. All dependent variables were log transformed to standardise residuals. Superscript numbers indicate groupings used for PERMANOVA based on correlations (>0.5). *Essential amino acids as defined by Morris (1991).

4.4.2 Impacts of root damage

Plant-damage treatment had a significant effect on lucerne height (F5,504 = 10.73, P < 0.001), whereby control (uncut) plants were significantly taller than those with damaged roots (Fig. 4-4a). Similar results were found for both root (F5,496 = 22.76, P <

0.001) and shoot biomass (F5,495 = 7.44, P < 0.001). Overall, early cutting and late cutting reduced root mass by 46% and 37%, respectively (Fig. 4-4b). Similarly, shoot mass was reduced by 26% and 22% in early- and late-cut plants, respectively (Fig. 4-4c).

Significant effects of plant-damage treatment were also seen for root %N (F5,207 = 2.58;

P = 0.028), %C (F5,207 = 5.05; P < 0.001) and C:N (F5,207 = 3.33; P = 0.006), whereby control 62

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS plants had a significantly higher root %N and lower root %C and C:N than late-cut plants. Consistent general increases in C:N were seen across cutting treatments depending on the time at which the plants were cut (Fig. 4-4d).

80 25 Early cut (a) (b) b No cut b Late cut b 20 60 a a b a a 15 40 a a a a 10

Height (mm) 20 5

Root (mg) biomass

0 0 Without aphids With aphids Without aphids With aphids 40 35 (c) (d) b 30 b 30 b ab b ab 25 a a a a a a 20 20 15

Root C:N 10 10

Shoot (mg) biomass 5

0 0 Without aphids With aphids Without aphids With aphids Treatment Treatment

Plant-damage treatment also significantly affected total amino acids (F1,287 = 3.042; P = 0.011) but had no effect on essential amino acids. On plants without aphids present, total amino acid concentrations were greater in roots that were cut early compared with those cut late, although neither early-cut or late-cut plants were significantly different from uncut controls, which contained intermediate concentrations. When aphids were present, amino acid concentrations were generally higher but no significant root-damage effects were detected. However, late-cut plants with aphids contained significantly higher concentrations of amino acids than late-cut plants without aphids (Fig. 4-5). Similar to individual models (Table 4-1), Glu and group 2

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS amino acids were significantly affected by plant-damage treatment (Table S4-2). Group 2 amino acids showed dramatic increases in late-cut plants when aphids were present (Appendix I Fig. S4-2d).

40 Early cut No cut FIGURELate 5 cut a 30 a a ab a b

dry mass) 20

-1

(µmol g (µmol

Amino Amino acid concentration

0 Without aphids With aphids

Treatment Figure 4-5. Effects of plant-damage treatment on total foliar amino acid concentrations. Mean values (± standard errors) shown. Bars with the same letters were not significantly different (P < 0.05).

4.4.3 Interaction effects of environment and root damage Plant-damage treatment and temperature interactively affected Lys, Phe and Tyr (Table 4-1). Specifically, at 26 °C, concentrations of all three amino acids were consistently higher when plants were cut early compared to control plants or plants cut late. Additionally, at 30 °C, concentrations of all three amino acids were significantly higher when late-cut plants were colonised by aphids compared with other treatments. Lys was strongly correlated with group 1, group 2, Glu and Tyr (Table S4-2). Plant-damage treatment in interaction with temperature also had a significant effect on aphid colonisation success, whereby, at 26 °C, pea aphid colonisation success was significantly higher when lucerne roots were cut before aphid inoculation compared with those cut after aphid inoculation, although neither were significantly different from uncut control plants (Fig. 4-6). Aphids were significantly more successful at colonising plants with roots that were cut early at 26 °C compared with 30 °C. No significant differences between treatments were seen at 30 °C. Significant effects of different factors and their associated Figs. are summarised in Fig. 4-7.

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

70 *Treatment × Temp Early cut a No cut 60 Late cut ab 50 ab ab b 40 b

Aphid Aphid colonisation success (%) 0 26 30

Temperature (°C) Figure 4-6. Effects of root cutting on aphid colonisation success (i.e. percentage of plants with aphids present upon harvest) under ambient and elevated temperatures. Statistically significant effect indicated *(P < 0.05). Bars with the same letters were not significantly different (P < 0.05).

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

4.5 Discussion

This study is the first to incorporate the effects of both eT and eCO2 on the sequence of herbivore arrival within an aboveground–belowground framework. These combined approaches incorporating multiple factors enable greater understanding into how plants and insects will interact in the future (Ryalls et al., 2013a). The results demonstrate that eT can counter the positive effects of eCO2 and root damage on amino acid concentrations and aphid populations, with important implications for future aphid outbreaks and crop security.

4.5.1 Impacts of eT and eCO2

Expected increases in aboveground biomass and height in response to eCO2 were observed at 30 °C, although, at 26 °C, eCO2 decreased height and had no effect on biomass. This is likely due to the high numbers of aphids present on the plants at these conditions since data were analysed across all treatments. The decrease in root C:N at eCO2 suggests that lucerne increases biological nitrogen fixation to compensate for N- limitation under these conditions. The hypothesis that elevated CO2 stimulates N- fixation in legumes is broadly supported (Rogers et al., 2009).

Concentrations of amino acid group 2 significantly decreased under eT, suggesting that these amino acids were the main temperature response drivers, although no overall decrease in total amino acid concentrations were observed under eT. Total concentrations did however increase in response to eCO2 at 26 °C and the amino acids in group 1 were the major drivers of this CO2 response. Studies have reported both positive and negative effects of eCO2 on amino acid concentrations, although plant type plays an important role in determining the direction of the effect (Docherty et al., 1997;

Himanen et al., 2008; Rogers et al., 2009). While tissue dilution effects of eCO2 often lead to decreases in amino acid concentrations in plants (Sun and Ge, 2011), legumes often show increases in amino acid concentrations under eCO2 due to increases in biological nitrogen fixation (Guo et al., 2013). Responses of different cultivars, varieties or genotypes of the same species to eCO2, however, can also vary. For example, Johnson et al. (2014) found 86% and 56% increases in essential amino acid concentrations and pea aphid colonisation success, respectively, on the cultivar ‘Sequel’ under eCO2, whereas essential amino acid concentrations and aphid colonisation

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS success decreased by 53% and 33%, respectively, on the cultivar ‘Genesis’ under the same conditions. In other words, some cultivars may become more or less susceptible to aphid attack under climate change conditions, an important consideration for determining future outcomes. Further study combining both eCO2 and eT would help to clarify the effects of climate change on aphid responses to different cultivars.

This study used the widely-grown public variety ‘Sequel’, with similar results to those found by Johnson et al. (2014) using the same cultivar. As with that study, aphid numbers were lower than the reproductive potential of A. pisum when feeding on susceptible plants and cultivars (e.g. the lucerne cultivar, ‘Hunter River’). This most likely reflects aphid populations in the field since only cultivars with some aphid resistance are now grown; ‘Hunter River’ was phased out over 30 years ago (Ryalls et al., 2013a). The exact mechanisms underpinning lucerne resistance to aphids are poorly characterised (Ryalls et al., 2013a), but probably include plant traits conferring both antibiosis (e.g. low fecundity) and antixenosis (e.g. inability to colonise) resistance. We saw evidence for both in the present study. In addition to primary chemistry, secondary chemistry may play a role in lucerne resistance to aphids. In particular, saponins have also been identified as potentially important factors associated with aphid resistance (Goławska et al., 2014). Moreover, the extent to which endosymbionts (obligate and facultative) might help aphids cope with such induced changes in primary and secondary chemistry are unknown in this system, but could also play a role. Simultaneously quantifying both saponins and amino acids, while considering the role of endosymbionts, might therefore provide an important insight into lucerne susceptibility to aphids.

Effects of eT and eCO2 on aphids mirrored their effects on amino acid concentrations, suggesting that aphids rely on amino acid concentrations to effectively reproduce and colonise on lucerne. In general, when aphids were present, amino acid concentrations were higher, suggesting that pea aphids promote amino acid metabolism in lucerne to favour population growth. Similar results were found for pea aphid populations fed on N-fixing-deficient Medicago truncatula plants, which had decreased activities of N assimilation-related enzymes and amino acid concentrations under eCO2, compared with increases seen in control plants (Guo et al., 2013). Pea aphids performed better

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

on plants grown under eCO2 conditions at 26 °C but not at 30 °C. Such negation of eCO2 effects by eT has also been shown in other systems (e.g. Murray et al., 2013b), demonstrating the importance of considering multiple climate change factors. Both plant amino acid and aphid responses to eCO2 were either reduced or negated at 30 °C, suggesting that future temperature increases may have beneficial effects on lucerne by reducing aphid numbers, although this will also depend on how aphid natural enemies are affected by such changes (Ryalls et al., 2013a).

4.5.2 Impacts of root damage and interactions with climate Aboveground biomass and height of control plants were expectedly higher than those with severed roots. Overall and relative losses in root and shoot mass due to root cutting were similar to the overall reductions in root biomass (36.3%) and aboveground growth (16.3%) that were averaged over 85 experimental studies by Zvereva and Kozlov (2012). Root C:N was higher in plants with severed roots, which is likely due to the removal of nodules containing nitrogen-fixing rhizobial bacteria and the subsequent impairment of biological nitrogen fixation. Root cutting may also have caused carbohydrates to be diverted away from the foliage towards the roots (Blossey and Hunt-Joshi, 2003; Johnson et al., 2011a). Pea aphids responded positively to early-cut plants, associated with an increase in amino acid concentrations, but this effect was reversed by eT. One essential amino acid that may be important to consider includes lysine, which was strongly correlated with other amino acid groups and closely matched amino acid and aphid responses to different treatments. For example, concentrations of lysine were higher at eCO2 (also shown by Johnson et al. (2014)) and in early-cut plants maintained at 26 °C. In these conditions, aphids may respond to specific amino acids, including lysine, and even influence their composition in the phloem (e.g. Guo et al., 2013), as suggested by the increase in total amino acid concentration in late-cut plants with aphids present compared with those without aphids present. If aphids are already present on the plant when roots are severed, stress-related increases in foliar nitrogen and amino acid concentrations are less likely (Huberty and Denno, 2004) and may be the reason why aphid colonisation decreased on late-cut plants at 26 °C. Realistically, lucerne is likely to go through periods of discontinuous stress resulting from variable root damage that would allow aphids to take advantage of periodic increases in foliar nitrogen or amino acid concentrations (Huberty and Denno, 2004;

CHAPTER 4 SIMULATED ROOT HERBIVORY AND APHIDS

Johnson et al., 2012). In this case, changes in temporal patterns of herbivore arrival under future conditions would be important to consider.

Understanding how multiple climate change factors shape crop susceptibility to insect pests is clearly a priority for achieving food security (Gregory et al., 2009), with the amount damaged by insects sufficient to feed one billion people (Birch et al., 2011). The present study demonstrates how some aspects of climate change can promote aphid pests, with simulated root herbivory having similar positive effects on the same pests. This could clearly be problematic for plant production. Both these effects, mediated by increases in foliar amino acids, were, however, negated by predicted increases in temperature. Research into future patterns of crop susceptibility to insect pests is at a crossroads (Gregory et al., 2009). Logistic constraints often limit the ability to test multiple climatic factors and multi-trophic factors, but nonetheless, we will only get a realistic insight into plant–insect interactions if we meet this challenge.

4.6 Acknowledgements We would like to thank Lisa Bromfield and Aidan Hall for glasshouse assistance, Christopher Mitchell for HPLC assistance, Hilary Stuart-Williams for analysing carbon and nitrogen concentrations and Benjamin Punzalan for providing the schematic diagrams.

Chapter 5

Climate and atmospheric change impacts on sap-feeding herbivores: a mechanistic explanation based on functional groups of primary metabolites 4 Published as Ryalls et al. 2016, Functional Ecology, doi: 10.1111/1365-2435.12715

5.1 Abstract Global climate and atmospheric change are widely predicted to affect many ecosystems. Herbivorous insects account for 25% of the planet’s species so their responses to environmental change are pivotal to how future ecosystems will function. Atmospheric change affects feeding guilds differently, however, with sap-feeding herbivores consistently identified as net beneficiaries of predicted increases in atmospheric carbon dioxide concentrations (eCO2). The mechanistic basis for these effects remains largely unknown and our understanding about how multiple environmental changes, acting in tandem, shape plant–insect interactions is incomplete. This study investigated how increases in temperature (eT) and eCO2 affected the performance of the pea aphid (Acyrthosiphon pisum) via changes in amino acid concentrations in the model legume, lucerne (Medicago sativa). Aphid performance increased under eCO2 at ambient temperatures, whereby aphid fecundity, longevity, colonisation success and rm increased by 42%, 30%, 25% and 21%, respectively. eT negated the positive effects of eCO2 on both fecundity and rm, however, and performance was similar to when aphids were reared at ambient CO2. We identified discrete functional groups of amino acids that underpinned the effects of climate and atmospheric change, in addition to plant genotype, on aphid performance.

Effects of eT and eCO2 held true across five M. sativa genotypes, demonstrating the generality of their effects. Combining this knowledge with amino acid profiles of existing cultivars raises the possibility of predicting future susceptibility to aphids and preventing outbreaks of a global pest. Moreover, environmentally-induced changes in the nutritional ecology of aphids have the capacity to change life-history strategies of aphids and their direct and indirect interactions with many other organisms, including mutualists and antagonists.

CHAPTER 5 AMINO ACIDS AND APHIDS

5.2 Introduction Global climate and atmospheric change effects on plants are likely to affect many herbivore populations by altering the quantity and quality of their food resources (Kuokkanen et al., 2004; Zvereva and Kozlov, 2006; Steffen et al., 2009). These changes can threaten ecosystem function, of which insect herbivores are a fundamental component (Polis, 1998). Many insect herbivores have extremely high rates of reproduction and if host plant resources become more abundant or accessible then population outbreaks could occur (Barbosa et al., 2012). Insect herbivores currently account for crop losses equivalent to feed more than one billion people (Birch et al., 2011), so future outbreaks could be particularly threatening to food security (Gregory et al., 2009).

Functional insect and plant groups, including sap-feeders and legumes, respectively, vary in their responses to climate and atmospheric change. Sap-feeding herbivores (e.g. aphids), in particular, have been repeatedly identified in reviews and meta- analyses as being a net beneficiary of predicted increases in atmospheric carbon dioxide concentrations (eCO2) (Bezemer and Jones, 1998; Coviella and Trumble, 1999; Stiling and Cornelissen, 2007; Robinson et al., 2012), although there are several exceptions (e.g. Díaz et al., 1998; Hughes and Bazzaz, 2001). Aphids often respond to changes in amino acid composition of the phloem and perform better on plants containing higher total amino acid concentrations (Karley et al., 2002; Douglas, 2003; Sun and Ge, 2011). Indeed, aphid life-history traits are intimately linked to the changing nutritional quality (i.e. amino acid concentrations) of their host plants, with decreases triggering physiological responses such as diapause and dispersal (Dixon, 1998; Price et al., 2011). Moreover, aphids are regarded as keystone interactors with other organisms in the ecosystem (Styrsky and Eubanks, 2007), ranging from mutualists such as bacterial endosymbionts (Douglas, 1998) and tending ants (Ness et al., 2010) to antagonists (Dixon, 2000). Environmentally-induced changes in host plant quality therefore have the capacity to affect not just aphids but the much broader community.

The mechanism by which eCO2 affects aphids is thought to be entirely plant-mediated and most-likely driven by changes in amino acid concentrations and composition (Sun and Ge, 2011). Only recently, however, have these mechanisms been explored

CHAPTER 5 AMINO ACIDS AND APHIDS experimentally (Sun et al., 2009; Guo et al., 2013; Johnson et al., 2014; Ryan et al., 2014a; Ryalls et al., 2015) and few general trends have yet emerged.

The meta-analysis of Robinson et al. (2012), recent reviews (Selvaraj et al., 2013; Facey et al., 2014) and experimental studies (Murray et al., 2013b, b; Scherber et al., 2013) all illustrate the importance of considering multiple simultaneous aspects of atmospheric and climate change. In particular, air temperature is predicted to increase as concentrations of greenhouse gases, especially CO2, increase (IPCC, 2014) so the two factors will act in tandem and may interact to shape the overall effect on insect herbivores via plant-mediated mechanisms. Newman et al. (2003) highlighted the situation more than a decade ago using aphids as a model system. By modelling population dynamics, they predicted that when nitrogen is not limiting (i.e. when soil N inputs are high and aphid N requirements are low), aphids will most likely benefit from eCO2 but this effect will be reduced when air temperature increases (Newman, 2004). Empirical support for these predictions remains lacking, however. Legumes, with their ability to biologically fix atmospheric nitrogen via associations with rhizobial bacteria, are usually well provisioned with N, and therefore provide excellent candidates for testing these predictions.

Plants, in general, are expected to respond to eCO2 by increasing rates of photosynthesis and growth, which dilutes limiting nutrients such as nitrogen (in the form of free amino acids and soluble protein) (Robinson et al., 2012; Zeng et al., 2012; Dwivedi et al., 2013). Legumes tend to diverge from this general consensus because they can enhance biological nitrogen fixation under eCO2 and maintain N concentrations (Rogers et al., 2009; Johnson and McNicol, 2010). Biological nitrogen fixation and total amino acid concentrations in lucerne Medicago sativa (Linnaeus), for example, have been shown to increase under eCO2 and these changes have been responsible for increasing pea aphid Acyrthosiphon pisum (Harris) colonisation success (Johnson et al., 2014; Ryalls et al., 2015). However, nodulation and biological nitrogen fixation in legumes decrease under elevated temperatures (eT) due to the poor heat- tolerance of N-fixing rhizobial bacteria (Zahran, 1999; Whittington et al., 2013), resulting in plants with lower concentrations of amino acids (Ryalls et al., 2015). The objective of this study was to identify amino acid-based mechanisms for the

CHAPTER 5 AMINO ACIDS AND APHIDS

potentially-interactive effects of eT and eCO2 on the performance of A. pisum feeding on five lucerne genotypes (or cultivars) with varying resistance to aphid colonisation. Acyrthosiphon pisum is a model aphid species of major agronomical importance with a fully sequenced genome and widely understood life cycle (van Emden and Harrington, 2007). It feeds on a number of leguminous plant species, including lucerne, which is one of the most important and widely grown forage legumes worldwide (Small, 2011).

The specific aims of this study were to determine the individual and combined effects of eT and eCO2 on aphid performance (development time, colonisation success, fecundity, longevity and intrinsic rate of increase) in relation to lucerne growth and amino acid concentrations. We used five distinct lucerne genotypes to assess the generality of any mechanisms linking aphid performance to amino acid changes in response to eT and eCO2. It was hypothesised that: (i) eT and eCO2 would both increase plant growth but they would have contrasting effects on amino acid concentrations and aphid performance; eCO2 would increase amino acids and aphid performance, whereas eT would negate the effects of eCO2 by decreasing amino acids and aphid performance; (ii) plant growth would not vary between genotypes, which were developed as high- yielding varieties, but aphid performance would decrease on more resistant genotypes via decreased amino acid concentrations. Understanding how such environmental changes affect host plant quality has implications for crop protection from aphids but also community ecology through the keystone interactions they have with the wider community (e.g. Styrsky and Eubanks, 2007).

5.3 Materials and methods 5.3.1 Insect cultures and plant material Four A. pisum cultures were established in January 2013 from a single parthenogenetic adult female collected from a pasture containing grasses and legumes, including lucerne, at the Hawkesbury Campus of the University of Western Sydney in Richmond, NSW, Australia (latitude −33.608847, longitude 150.747016). Cultures were maintained on propagated lucerne plants in each of the four CO2 and temperature combinations (conditions below) for at least six generations (c. seven weeks) prior to the experiment. Five lucerne genotypes of varying resistance to A. pisum, including susceptible [Hunter River (HR)], low resistance [Sequel (S) and Trifecta (T)] and moderate resistance [Aurora

CHAPTER 5 AMINO ACIDS AND APHIDS

(A) and Genesis (G)], were used in this study. For full cultivar details see Johnson et al. (2014), although, since publication, cultivar S has been reclassified as low resistance (Johnson pers. comm., Seedmark, 2015).

5.3.2 Growth conditions and experimental procedure The experiment was run three times in randomised chambers to compensate for pseudo-replication of CO2 and temperature treatments and plants were repositioned at random within each of four chambers to minimise effects of chamber positions (Newman et al., 2011; Johnson et al., 2016b). Twenty five lucerne seeds (five of each genotype) provided from Seedmark, Adelaide, South Australia were individually planted in 70 mm pots filled with local field soil (Barton et al., 2010) and grown in each of four

CO2 and temperature-controlled glasshouse compartments using a 2 × 2 factorial −1 −1 design of ambient CO2 (aCO2; 400 µmol mol ) and eCO2 (640 µmol mol ) at ambient (aT) and elevated temperature (ambient + 4°C; eT). aT was set at 26/18°C day/night on a 15 h light: 9 h dark cycle and represents the average daily temperature data (November to May) over the past 30 years for Richmond, NSW (BOM, 2016). eT (30/22 °C day/night) represents the maximum predicted temperature increase for this region within this century (CSIRO, 2007; IPCC, 2014). Environmental conditions were monitored continuously throughout the experiment and temperature readings were cross-checked with transportable temperature loggers (DS1922L, Thermodata, VIC, Australia). Plants were kept moist by irrigating daily (c. 15 mL). Full operational details are given in Johnson et al. (2014).

Five weeks after sowing, one teneral A. pisum adult was placed on the underside of the youngest leaf of each plant and one organza bag (125 x 170 mm) was secured around each plant to confine each individual aphid to the plant. Following the aphid performance protocol by Mitchell et al. (2010), after an initial 12 hours, all nymphs were removed from the plants and after a further 12 hours the adult and all but one nymph were removed. The remaining individual nymphs were subsequently examined at 24-hour intervals until adult mortality occurred, and new nymphs were removed from each plant daily. The following parameters were recorded: (1) time to adulthood (development time or number of days until fourth moult), (2) colonisation success (i.e. whether aphids reproduced or not), (3) total reproduction or fecundity (total number of

CHAPTER 5 AMINO ACIDS AND APHIDS nymphs produced in a lifetime), (4) longevity (total number of days alive) and (5) the intrinsic rate of increase (rm), which was calculated using the formula in Wyatt and White (1977): rm = c(loge Md)/d where Md is the total number of offspring produced, d is the development time and c is a correcting constant (0.738). Plants were harvested after adult mortality by removing aboveground plant parts to ground level; these were then snap-frozen in liquid N and stored at −20 °C. Plant heights were measured before plants were inoculated with aphids and upon harvest. All plant material was freeze-dried and weighed. Leaves were separated from stems and ball-milled to a fine powder prior to chemical analysis.

5.3.3 Chemical analyses Soluble amino acids were extracted and analysed from milled foliar samples (10–15 mg) following the protocol set out by Ryalls et al. (2015). Amino acid standards within the AA-S-18 (Fluka, Sigma-Aldrich) reference amino acid mixture were supplemented with asparagine and glutamine (G3126 and A0884, respectively, by Sigma, Sigma-Aldrich). Nine essential amino acids (i.e. those that cannot be synthesised by insects de novo), including arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine (Morris, 1991) and 10 non-essential amino acids (alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine) were detected using this method.

Control plants (i.e. those not inoculated with aphids) grown in the first experimental run were used to compare foliar amino acids obtained from whole foliar tissue with phloem amino acid compositions. Phloem exudate and foliar samples were collected from two control plants of each genotype grown in each chamber. Phloem extraction, using the EDTA exudation technique of King and Zeevart (1974), provided information about the relative abundance (i.e. a qualitative indication) of phloem amino acids, compared with the quantitative indication provided by analysing whole tissue (Steinbauer, 2013; Ryan et al., 2015). Plant leaves were excised at the petiole base midway along the shoot axis and inserted immediately into 0.2 mL 5 mmol L−1 EDTA solution, pH 7.5. The samples were incubated for 90 min in the dark in a sealed chamber equilibrated at 25 °C, which contained a dish of saturated K2HPO4 solution to

CHAPTER 5 AMINO ACIDS AND APHIDS maintain high humidity. Excised plants and EDTA samples were frozen at −20 °C prior to amino acid analysis. EDTA samples were dried under vacuum at 30 °C before derivatization with PITC, as in Ryalls et al. (2015).

5.3.4 Statistical analyses Linear mixed models using the nlme statistical package (Pinheiro et al., 2014) within the

R statistical interface v3.1.1 described the effects of eT and eCO2 on aphids

(development time, total reproduction, longevity and rm) and plant characteristics (height and mass) across plant genotypes. The fixed effects included plant genotype

(HR, S, T, A, G), CO2 (aCO2, eCO2) and temperature (aT/26, eT/30) as well as the interactions between these terms. The random effects included run (1, 2 or 3) as a block term, with chamber as a nested factor, to account for chamber effects that could confound temperature and CO2 effects. The dependent variables were transformed to normalise the model standardised residuals where necessary (Table S5-1). The effects of eT and eCO2 on the colonisation success of aphids fed on different lucerne genotypes were assessed using a generalised linear mixed model with a binomial error structure using the lme4 statistical package (Bates et al., 2014) within R. Fixed and random effects remained the same as the general linear mixed models described above. Permutational analysis of variance (PERMANOVA), combining all aphid performance parameters in an individual model, was used to determine the effects of eT, eCO2 and plant genotype on aphid performance overall, using the function strata to account for the stratified design (i.e. by including the random effects of chamber nested within run). The effects of eT and eCO2 on total, essential and individual amino acid concentrations in different lucerne genotypes were analysed with mixed models using nlme in R with the same fixed and random effects as above. Post hoc Tukey’s tests and the R package LMERConvenienceFunctions (Tremblay and Ransijn, 2015) were used for pairwise comparisons of means for treatment and interaction effects. Principal components analysis (PCA) and PERMANOVA were used to compare relative molar contents of phloem and foliar amino acids and to determine the effects of eT and eCO2 on amino acid concentrations in different lucerne genotypes. The stratified design accounted for run and chamber, as described earlier. Groupings of covarying individual amino acids were determined using a correlation matrix, and PERMANOVA results were compared with those from individual mixed models. Linear model predictions were

CHAPTER 5 AMINO ACIDS AND APHIDS used to determine a relationship between aphid performance and grouped amino acid concentrations, which were measured from the same plants.

5.4 Results

5.4.1 Impacts of eT and eCO2 on aphid performance Aphid performance overall (over all lucerne cultivars tested) was significantly affected by the interaction between temperature and CO2 (though not when considered separately). No genotype × environment interaction was observed (Table 5-1). Mixed models of individual aphid parameters revealed that temperature, in interaction with

CO2, had a significant effect on fecundity and rm, and CO2 significantly affected longevity (Appendix II Table S5-1) and colonisation success (see Appendix II Table S5-2 for model reduction AIC comparisons). Specifically, eCO2 increased the number of nymphs produced and rm at 26 °C (Tukey P = 0.034 and 0.033, respectively) but not at 30°C (Tukey P = 0.534 and 0.718, respectively) (Fig. 5-1a and b, respectively). Additionally, eT had a positive effect on rm at aCO2 (Tukey P = 0.041) but not at eCO2 (Tukey P =

0.630). Moreover, aphids lived longer at eCO2 than aCO2 (Fig. 5-1c; t1,8 = 2.37, P =

0.045). Aphids were also more successful at colonising plants at eCO2 than aCO2 (Fig. 5- 1d; z = 2.421, P = 0.015, df = 300). No significant difference in development time (i.e. time to adulthood) was observed between temperature or CO2 treatments.

df SS MS Pseudo-F P(perm)

Temperature 1 0.274 0.274 2.861 0.066 CO2 1 0.128 0.128 1.343 0.241 Cultivar 4 1.029 0.257 2.691 0.018* Temperature × CO2 1 0.534 0.534 5.581 0.003* Temperature × Cultivar 4 0.292 0.073 0.763 0.667 CO2 × Cultivar 4 0.197 0.049 0.514 0.871 Temperature × CO2 × Cultivar 4 0.777 0.194 2.031 0.084 Residuals 218 20.841 0.096 Total 237 24.070

CHAPTER 5 AMINO ACIDS AND APHIDS

40 30 aCO 2 *Temp × CO2 (a) *CO2 (c) eCO2 30 b 20 20 bc ac 10

Longevity Fecundity a 10

0 alive) of days (number 0

(number of nymphs produced) of nymphs (number 100 *Temp × CO *CO 0.3 2 (b) 2 (d) 80

b 60 ) 0.2 b b

(%) ( a 40 0.1 20

0.0 0

Intrinsic rate of increase of Intrinsic rate 26 30 aphids by colonised Plants 26 30 Temperature (°C) Temperature (°C)

5.4.2 Aphid performance in relation to plant genotype Plant genotype had a significant effect on aphid performance overall (Appendix II Table

5-1), with differences in fecundity, rm, longevity (Appendix II Table S5-1) and colonisation success (Appendix II Table S5-2) observed between cultivars. Specifically, aphids on the susceptible cultivar HR produced more offspring than on the moderately- resistant cultivars A and G (Fig. 5-2a; Tukey P = 0.014). Aphids on cultivar HR also had a higher rm (Fig. 5-2b; Tukey P = 0.047), lived longer (Fig. 5-2c; Tukey P = 0.001) and colonised more successfully (Fig. 5-2d; z = 3.097, P = 0.002) than on cultivar A. All other cultivar comparisons were not statistically significant.

CHAPTER 5 AMINO ACIDS AND APHIDS

40 30 (a) (c) 30 a a 20 ab ab 20 ab ab ab b b b 10

Longevity Fecundity 10

0 alive) of days (number 0

(number of nymphs produced) of nymphs (number 100 0.3 (b) (d) 80 a a 60 ab ) 0.2 ab ab ab ab

m ab

r b

(%) ( b 40 0.1 20

0.0 0

Intrinsic rate of increase HR S T A G Plants aphids colonised by HR S T A G Cultivar Cultivar

5.4.3 Plant growth and amino acid responses to eT, eCO2 and plant genotype

Temperature and CO2 had a significant effect on lucerne shoot mass (Appendix II Table

S5-1), whereby mass increased at eT and eCO2 compared with ambient conditions (Appendix II Fig. S5-1a). This was consistent across all cultivars (no environment × genotype interactions were observed). Similar results were observed for plant height

(Appendix II Fig. S5-1b), which was correlated with plant mass (rPearson = 0.834, P < 0.001, df = 300). Shoot mass and height were significantly higher in the susceptible and low-resistance cultivars HR and S, respectively, compared with other cultivars (Appendix II Fig. S5-1c and d, respectively).

Phloem and foliar amino acid compositions analysed using uninfested control plants were correlated (rPearson = 0.592, P = 0.012, df = 17) and extraction technique (i.e. phloem collection using EDTA vs. tissue extraction) had no significant effect on relative

(% mol) amino acid compositions (F1,33 = 1.848, P = 0.141; Appendix II Fig. S5-2). This

CHAPTER 5 AMINO ACIDS AND APHIDS justified the use of foliar amino acids as a proxy for phloem amino acids for subsequent amino acid analyses. Temperature had a significant effect on total foliar amino acid concentrations (Table 5-2; Appendix II Table S5-3), whereby concentrations significantly decreased under eT overall. Similarly, PCA revealed a clearer separation between temperatures compared with CO2 concentrations (Fig. 5-3). Individual amino acid mixed model analyses (Table 5-2) showed that eT decreased concentrations of Arg, Asn, Asp, Glu and His, although Arg, Asn and Asp were also affected by interactions with plant genotype, with all three amino acids generally present in higher concentrations in the low-resistance cultivar T. Three-way interactions between plant genotype, CO2 and temperature were observed for Arg and Asp. Specifically, concentrations of Arg in cultivars A and HR increased in response to eCO2 at 26 °C but not at 30 °C. Similar responses of Asp were observed in cultivars A and G. In contrast, concentrations of both amino acids in cultivar S showed a decrease under eCO2 at 26 °C and an increase under eCO2 at 30 °C. Other cultivars showed a general decrease in the concentrations of both Arg and Asp under eT but no effect of eCO2 was observed. Cultivars differed significantly in concentrations of five amino acids (Phe, Pro, Ser, Thr and Val), whereby concentrations decreased in cultivars with higher pea aphid resistance. Specifically, all five amino acids followed the pattern (in descending order from highest to lowest amino acid concentrations): HR > T > S > G > A.

CHAPTER 5 AMINO ACIDS AND APHIDS

Table 5-2. Table of statistics from mixed models of individual amino acid responses to different lucerne genotypes, temperature and CO2 treatments. Cultivar × Cultivar × Cultivar × Temperature Cultivar Temperature CO2 CO2 × Amino acid Temperature CO × CO 2 2 Temperature

F4,206 P F1,6 P F1,6 P F4,206 P F4,206 P F1,6 P F4,206 P

Asparagine (Asn)1 0.794 0.530 10.003 0.020 1.674 0.243 3.146 0.016 1.747 0.141 1.355 0.289 2.398 0.052 Aspartate (Asp)1 1.973 0.100 13.347 0.011 0.734 0.424 1.838 0.123 3.001 0.020 0.017 0.901 5.039 0.001 Glutamate (Glu)1 1.987 0.098 6.183 0.047 1.000 0.356 0.548 0.701 1.660 0.161 2.797 0.146 1.215 0.306 Arginine† (Arg)2 1.659 0.161 6.761 0.041 0.109 0.753 2.173 0.073 1.145 0.336 0.616 0.463 3.012 0.019 Histidine† (His)2 1.668 0.159 6.506 0.043 0.830 0.397 2.249 0.065 1.558 0.187 0.218 0.657 1.633 0.167 Alanine (Ala)3 1.953 0.103 0.014 0.911 0.364 0.568 0.864 0.486 1.002 0.407 0.154 0.708 0.655 0.624 Glutamine (Gln)3 0.896 0.467 0.438 0.533 2.175 0.191 1.328 0.261 0.379 0.824 0.562 0.482 1.823 0.126 Glycine (Gly)3 2.211 0.069 1.972 0.210 0.396 0.552 0.952 0.435 1.558 0.187 0.396 0.552 1.367 0.247 Leucine† (Leu)3,6 0.303 0.876 0.681 0.441 0.092 0.772 0.479 0.751 2.294 0.061 0.043 0.843 0.373 0.828 Lysine† (Lys)3,5 1.021 0.397 1.867 0.221 5.764 0.053 0.631 0.641 0.724 0.577 0.688 0.439 0.499 0.737 Phenylalanine† (Phe)3 2.944 0.021 2.546 0.162 0.782 0.411 0.939 0.442 1.630 0.168 0.588 0.472 0.289 0.884 Proline (Pro)3 4.453 0.002 0.010 0.925 0.544 0.489 1.485 0.208 1.717 0.148 0.142 0.719 0.839 0.502 Serine (Ser)3 2.515 0.043 0.304 0.601 0.088 0.777 0.885 0.474 1.684 0.155 0.631 0.457 2.339 0.057 Threonine† (Thr)3 2.997 0.020 0.198 0.672 0.047 0.835 2.354 0.055 2.008 0.095 0.056 0.821 1.497 0.204 Valine† (Val)3 3.856 0.005 0.004 0.951 0.029 0.871 2.007 0.095 1.222 0.302 0.251 0.634 0.606 0.659 Isoleucine† (Iso)4 2.257 0.064 0.090 0.775 0.006 0.941 0.818 0.515 1.689 0.154 0.002 0.967 1.611 0.173 Tyrosine (Tyr)4 1.040 0.387 0.970 0.363 0.107 0.755 0.318 0.866 0.995 0.411 0.002 0.964 1.359 0.249 Cysteine (Cys)5 0.216 0.929 0.580 0.475 0.448 0.528 0.939 0.442 0.414 0.799 1.523 0.263 1.136 0.341 Methionine† (Met)6 0.256 0.906 5.440 0.058 2.051 0.202 0.976 0.422 0.390 0.816 0.882 0.384 1.437 0.223

Essential amino acids 1.529 0.195 0.045 0.839 0.012 0.915 1.205 0.310 2.279 0.062 0.122 0.739 1.115 0.351 Total amino acids 1.574 0.183 9.689 0.021 0.595 0.470 1.481 0.209 2.354 0.055 0.035 0.858 2.022 0.093

One mixed model was run for each amino acid to clarify findings from PERMANOVA tests of grouped amino acid concentrations. All dependent variables were log transformed to standardise residuals. Superscript numbers indicate groupings used for PERMANOVA based on correlations (>0.5). P-values highlighted in bold indicate significance (P < 0.05). Amino acids are listed alphabetically within correlated groups. †Essential amino acids as defined by Morris (1991).

CHAPTER 5 AMINO ACIDS AND APHIDS

5.4.4 Linking aphid and amino acid responses to eT, eCO2 and plant genotype PERMANOVA on correlated amino acid groups (i.e. those that were > 50% correlated; Table S5-4), consisting of, group 1 (Asn, Asp, Glu), group 2 (Arg, His), group 3 (Ala, Gln, Gly, Leu, Lys, Phe, Pro, Ser, Thr, Val), group 4 (Iso, Tyr) and two individual amino acids (Cys and Met) complemented results from individual models, whereby temperature had a significant effect on groups 1 and 2 amino acids (Fig. 5-4a) and plant genotype had a significant effect on group 3 amino acids (Fig. 5-4b). These grouped amino acid concentrations were positively correlated with aphid fecundity (Fig. 5-4c and d), rm

(groups 1 and 2: F1,168 = 3.95, P = 0.048; group 3: F1,168 = 8.17, P = 0.005) and longevity

(groups 1 and 2: F1,168 = 11.58, P < 0.001; group 3: F1,168 = 4.03, P = 0.046) but not correlated with development time (groups 1 and 2: F1,168 = 0.12, P = 0.726; group 3:

F1,168 = 2.68, P = 0.104).

10 5.0 aCO 2 Groups 1 and 2 (a) Groups 1 and 2 (c) eCO 8 2 4.5 **Temperature F1,168 = 6.98, P = 0.009 *Temp × CO P = 0.014 4.0 6 2

dry mass) dry 3.5 -1 4 3.0 2 2.5

(µmol g (µmol 0 fecundity) (aphid Log 2.0

Amino acid concentration acid Amino 26 30 0 5 10 15 20 25 30 Temperature (°C) Amino acid concentration (µmol g-1 dry mass) 16 5.0 Group 3 (b) Group 3 (d) 4.5 12 F1,168 = 6.93, P = 0.009 4.0

dry mass) dry 8 3.5

-1 3.0 4 2.5

(µmol g (µmol 0 fecundity) (aphid Log 2.0

Amino acid concentration acid Amino HR S T A G 0 10 20 30 40 50 Cultivar Amino acid concentration (µmol g-1 dry mass)

CHAPTER 5 AMINO ACIDS AND APHIDS

5.5 Discussion This study highlights how abiotic factors interact to drive specific changes in plant quality and, consequently, insect performance. Results show that plant genotype resistance to A. pisum was maintained under eT and eCO2 and amino acid-mediated increases in aphid performance under eCO2 can be counteracted by higher temperatures predicted in the future. This situation held true across all five plant genotypes tested, demonstrating the generality of this response.

5.5.1 Insect–plant responses to eT and eCO2

Lucerne above-ground mass showed the expected increase under eT and eCO2. Similar effects of eCO2 on the shoot weight of another legume (Vicia faba L.) was shown by

Awmack and Harrington (2000). eT negated the positive effects of eCO2 on aphid fecundity and rm, demonstrating the importance of considering multiple environmental factors at once. This neutralising effect of eT has previously been shown in aphid studies on lucerne (Ryalls et al., 2013b; Ryalls et al., 2015) and in other plant-insect systems (Murray et al., 2013a). Direct effects of eT on aphids are also possible (Bell et al., 2015), which may override the plant-mediated effects of eCO2, as prolonged exposure to high temperatures can kill or severely impair reproduction in pea aphids (Bale et al., 2002; Montllor et al., 2002). Eastern Australian populations of A. pisum are adapted to temperatures above 35 °C, although their optimum temperature for development is said to be c. 20-25 °C. Temperatures above 28 °C are likely to negatively impact populations by reducing aphid growth and development (Bieri et al., 1983; Lamb and MacKay, 1988; MacKay et al., 1993). However, only by complementing these data with artificial diet experiments (Karley et al., 2002; Akbar et al., 2016) or by growing plants at different temperatures and inoculating those plants with insects under the same temperature (Murray et al., 2013b) could direct effects be separated from the plant-mediated effects of temperature on insect herbivores. While plant- crossing experiments would serve to disentangle the indirect interactions between multiple environmental factors, artificial diet experiments would only reveal direct effects of temperature on aphids. This may be a moot point, however, since the overall impact will be the same.

CHAPTER 5 AMINO ACIDS AND APHIDS

Phloem and foliar amino acids analysed from control plants were significantly correlated and no effect of extraction technique on amino acid proportions was observed, suggesting that foliar amino acids can be used as a reliable indicator of phloem amino acid composition in lucerne, as proposed by Riens et al. (1991) and Winter et al. (1992) for spinach and barley, respectively. Amino acid concentrations declined at eT, which may explain why eCO2 caused aphids to perform better at aT but not at eT. Additionally, this may exacerbate any direct negative effects of eT on aphid physiology under eCO2 conditions. Ryalls et al. (2015) showed similar responses of aphids to eT and eCO2, although in that study we analysed amino acid concentrations from control plants on which aphids were absent. In the current study, amino acid concentrations were analysed from plants hosting a single aphid, which enabled direct correlations between aphid performance and amino acid concentrations. Aphids may compensate for lower concentrations of amino acids by increasing phloem ingestion rates (Sun et al., 2009; Sun and Ge, 2011) or promoting amino acid metabolism in the host plant (Guo et al., 2013), although similarities in the amino acid-mediated responses of aphids between Ryalls et al. (2015) and the present study suggest that this did not occur. Specific amino acids, including Arg, Asp, Glu and His, were identified as major responders to temperature in Ryalls et al. (2015) and all responded the same way in this study. In particular, group 1 and 2 amino acids (Arg, Asn, Asp, Glu and His) were responsible for the decrease in total concentrations of amino acids at eT. These amino acids showed responses similar to aphids under both eT and eCO2 and were correlated with a number of aphid performance parameters, which demonstrates the importance of characterising the responses of individual amino acids. The temperature treatment in this study simply shifted mean temperatures, although, realistically, the effect of temperature is non-linear and an increase in extreme temperatures may have an even greater impact on rhizobial bacteria (responsible for increasing biological nitrogen fixation) and, subsequently, foliar N and amino acid concentrations. Moreover, uncertainty exists as to whether rhizobia will become more tolerant under long-term warming, with implications for legume production, carbon sequestration and resistance against aphid pests. Other climate change factors, including changes in rainfall patterns (e.g. drought), have been shown to alter N dynamics, amino acid concentrations and aphid populations within the same system (Ryalls et al., 2016). It is therefore likely that water stress will interact with plant and aphid responses to eT and eCO2. While

CHAPTER 5 AMINO ACIDS AND APHIDS incorporating all three environmental factors into plant–insect studies would be challenging, it should be an important next step in determining how aphids and other species will respond under future conditions.

5.5.2 Insect-plant responses to different lucerne genotypes Lucerne shoot mass was greater in the susceptible and low-resistance cultivars HR and S, respectively, compared with the more resistant cultivars. In this case, lucerne mass may be related to resource allocation trade-offs, with resources directed towards defence strategies (i.e. resistance) and away from growth (Fritz and Simms, 1992). Aphids generally performed better on less resistant cultivars that had higher concentrations of group 3 amino acids, notably Phe, Pro, Ser, Thr and Val, compared with more resistant cultivars. This was consistent across CO2 and temperature treatments. In other words, resistance was maintained under future conditions, which promotes successful plant breeding efforts. This has been shown in other studies on aphids (Gao et al., 2008a; Ryalls et al., 2013b), in contrast to those that have identified a breakdown in resistance under eCO2 (McMenemy et al., 2009; Martin and Johnson, 2011; Hentley et al., 2014). Studies linking chemical traits to lucerne resistance remain scarce (Ryalls et al., 2013a), although saponins and amino acid concentrations have been identified as major chemical contributors. Reductions in saponin concentrations, for example, may make plants more susceptible to aphid pests (Goławska and colleagues2006; 2008; 2012; 2014). The current study suggests that lucerne resistance is driven to a large extent by functional groups of amino acids, and this may inform selection programs that target improved aphid resistance of cultivars.

5.5.3 Wider ecological impacts Aphid life history is driven by the nutritional quality (primarily the amino acid quality and quantity), of the phloem sap of their host plants (Douglas, 2003, 2013). Deterioration in host plant quality triggers diapause and dispersal (production of winged alatae), whereas increases cause population outbreaks (Dixon, 1998; Awmack and Leather, 2002; Price et al., 2011). Increases in aphid abundance arising through enhanced host plant quality often have cascading positive effects on higher trophic levels (Awmack and Leather, 2002; Johnson et al., 2013), so CO2 and temperature have the capacity to promote and diminish, respectively, populations of natural enemies.

CHAPTER 5 AMINO ACIDS AND APHIDS

Moreover, around 40% of aphids (although not A. pisum) have mutualistic relationships with ants (Way, 1963; Ness et al., 2010), whereby the aphids provision ants with sugary honeydew deposits (a by-product of having to process large quantities of relatively low- N phloem sap). Ants, in return, act as sentinels and protect aphids from their natural enemies (Stadler and Dixon, 2005). The amount and quality of honeydew produced depends on a number of factors, but is significantly affected by the amino acid quality of the phloem sap. Honeydew quality and quantity influences the degree of mutualism and tending of ants, with higher reward value increasing ant protection. Lower reward value (i.e. less or lower quality honeydew), however, decreases attendance and can even lead to predation (Cushman, 1991). Given the large effects of CO2 and temperature on amino acid groups reported here, it is intriguing to speculate how these changes will affect this keystone interaction (Styrsky and Eubanks, 2007) in other lucerne aphids that are attended (e.g. cowpea aphid). Enhanced amino acid concentrations under eCO2 may result in aphids having to process less phloem sap and therefore producing less honeydew, which would presumably reduce reward value to ants. Honeydew carbohydrate quality may also determine ant attendance, however, with sugar-rich honeydews often attracting more ants (Woodring et al., 2004). Climate change studies involving aphid behavioural ecology tend to focus on interactions with natural enemies (Hentley and Wade, 2016), but it is possible that mutualistic interactions will be affected too.

In addition to changing higher trophic-level interactions, the environmentally-driven changes in plant quality reported here (i.e. the decrease and increase in amino acid concentrations under eT and eCO2, respectively) have the capacity to change the relationship aphids have with microbial endosymbionts. Aphids possess two types of symbiotic bacteria; the obligative primary symbiont Buchnera aphidicola, found in almost all aphids, and facultative secondary symbionts, which do not occur universally. Buchnera aphidicola are housed in specialised cells in the aphids, the bacteriocytes or mycetocytes, and supplement aphid nutrition with the provision of essential amino acids (Douglas, 1998). Host plant nutritional quality can affect the balance of primary and secondary endosymbionts in aphids, with primary symbionts being promoted with increasing host plant quality (nitrogen availability), whereas secondary symbionts attain higher densities when host plant quality is low (Wilkinson et al., 2007; Chandler et al.,

CHAPTER 5 AMINO ACIDS AND APHIDS

2008; Feldhaar, 2011). Under high nitrogen concentrations, for example, the secondary symbiont Serratia symbiotica constituted 1.5% of bacterial load in A. pisum, but 8.2% under low nitrogen concentrations. Buchnera densities showed the opposite trend, in contrast (Wilkinson et al., 2007). Currently, it is uncertain whether the profiles of endosymbionts change in response to changing environmental conditions, but our findings and other studies (e.g. Wilkinson et al., 2007) suggest that they at least have the capacity to.

5.5.4 Conclusions Multiple climate change factors can clearly interact and impact aphid populations, which are amongst the most damaging pests globally (Blackman and Eastop, 2000; Bell et al., 2015). Aphids are likely to become more serious pests of lucerne and other legumes if atmospheric CO2 concentrations continue to rise (Awmack and Harrington, 2000), although eT may limit this (Newman, 2004; Ryalls et al., 2015). In addition to their pest status, aphids can have disproportionately large impacts on food webs and insect community structure simply because they can reproduce so rapidly in response to beneficial environmental conditions. Moreover, aphids engage in an extraordinary array of obligate and facultative interactions with other organisms, including microbes (Douglas, 1998), mutualists (Ness et al., 2010) and antagonists (Dixon, 2000), so climate change impacts on this group are likely to have cascading effects within some ecosystems. In the current study, eT counteracted any positive effects of eCO2 on amino acid concentrations and aphid performance. Gaining a mechanistic understanding of how herbivorous insects are affected by different aspects of environmental change, individually and in combination, is therefore essential for both preventing pest outbreaks and understanding community dynamics (Hancock et al., 2015; Simpson et al., 2015). The broader understanding into how aphids and other insects influence community dynamics, in particular, may help to unravel the complex interactions between global change and ecosystem function.

5.6 Acknowledgements Thanks go to Lisa Bromfield and Aidan Hall for their assistance in the glasshouse.

Chapter 6

Above–belowground herbivore interactions in mixed plant communities are influenced by altered precipitation patterns 5 Published as Ryalls et al. 2016, Frontiers in Plant Science, 7, 345

6.1 Abstract Root- and shoot-feeding herbivores have the capacity to influence one another by modifying the chemistry of the shared host plant. This can alter rates of nutrient mineralisation and uptake by neighbouring plants and influence plant–plant competition, particularly in mixtures combining grasses and legumes. Root herbivory- induced exudation of nitrogen (N) from legume roots, for example, may increase N acquisition by co-occurring grasses, with knock-on effects on grassland community composition. Little is known about how climate change may affect these interactions, but an important and timely question is how will grass–legume mixtures respond in a future with an increasing reliance on legume N mineralisation in terrestrial ecosystems. Using a model grass–legume mixture, this study investigated how simultaneous attack on lucerne (Medicago sativa) by belowground weevils (Sitona discoideus) and aboveground aphids (Acyrthosiphon pisum) affected a neighbouring grass (Phalaris aquatica) when subjected to drought, ambient and elevated precipitation. Feeding on rhizobial nodules by weevil larvae enhanced soil water retention under ambient and elevated precipitation, but only when aphids were absent. While drought decreased nodulation and root N content in lucerne, grass root and shoot chemistry were unaffected by changes in precipitation. However, plant communities containing weevils but not aphids showed increased grass height and N concentrations, most likely associated with the transfer of N from weevil-attacked lucerne plants containing more nodules and higher root N concentrations compared with insect-free plants. Drought decreased aphid abundance by 54% but increased total and some specific amino acid concentrations (glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine, aspartate and glutamate), suggesting that aphid declines were being driven by other facets of drought (e.g. reduced phloem hydraulics). The presence of weevil larvae belowground decreased aphid numbers by 30%, likely associated with a significant

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS reduction in proline in weevil-treated lucerne plants. This study demonstrates how predicted changes to precipitation patterns and indirect interactions between herbivores can alter the outcome of competition between N-fixing legumes and non-N- fixing grasses, with important implications for plant community structure and productivity.

6.2 Introduction 6.2.1 Species interactions and climate change Ecological communities form a network of directly and indirectly interacting species (Wootton, 1994; Polis, 1998). These networks are often categorised into tractable ecosystem components (e.g. food chains, aboveground–belowground interactions, plant–insect interactions, competitive interactions) used to elucidate the causal mechanisms that underpin species interactions and community dynamics (Holt, 1997; Rudolf, 2012). Notably, plants are exploited by multiple root and shoot herbivores, which can interact indirectly through plant-mediated mechanisms. They have the capacity to influence one another by modifying the chemistry of the shared host plant (Blossey and Hunt-Joshi, 2003; Johnson et al., 2012). Changes in host plant nutrients (e.g. amino acid concentrations) often underpin interactions between aboveground and belowground herbivores, with knock-on effects on plant community structure and productivity (Johnson et al., 2013). In particular, altered rates of root exudation in response to herbivory can influence rates of nutrient mineralisation and acquisition by neighbouring plant species (Bardgett et al., 1999; Ayres et al., 2007). However, all trophic interactions, both direct and indirect, are specific to the species combinations involved (Kos et al., 2015) and may be altered by climate change (McKenzie et al., 2013a). Such changes may propagate through communities and sway competitive advantages between species (Johnson et al., 2011b; Barton and Ives, 2014; Jing et al., 2015). The incidence of extreme precipitation events, including droughts and floods, is expected to increase in many regions throughout the world, with a generally drier future predicted for south-eastern Australia (Chiew et al., 2011; Dai, 2011). Drought is one of the most important environmental stressors for plants, often altering root to shoot biomass ratios (Gargallo-Garriga et al., 2014) and sometimes altering plant N and amino acid concentrations (Girousse et al., 1996; Garten et al., 2009; Johnson et al., 2011b). Excess water can also create anaerobic conditions around plant roots, affecting

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS growth and nutrient status, although in areas with sufficient drainage, increased water availability is likely to positively affect plant productivity. Nonetheless, effects of drought are of longer duration and are considered to have far more disruptive effects on plant–insect interactions (Pritchard et al., 2007).

6.2.2 Grass–legume ecosystems Ecosystem processes, including primary productivity and nutrient cycling, tend to increase in biologically diverse plant communities, especially in mixtures containing leguminous plant species (Hooper et al., 2005; Hatch et al., 2007). Most legumes have the ability to fix atmospheric N2 via their symbiotic relationship with rhizobial bacteria (Long, 1989). Rhizobial bacteria are accommodated by nodules on legume roots, the development of which is strongly affected by soil conditions including pH, nutrient availability, temperature and moisture (Ramos et al., 2003; Ferguson et al., 2013). Drought, in particular, can negatively impact nitrogenase activity in legume nodules (Sprent, 1972), although the mechanisms remain unclear (Streeter, 2003; Nasr Esfahani et al., 2014). Benefits of mixed plant communities (e.g. enhanced resource acquisition) arise from species complementarity (i.e. resource niche differentiation) or from selection effects (i.e. mixed communities are more likely to harbour a dominant productive species) (Loreau and Hector, 2001; Turnbull et al., 2013). Legumes, in particular, fix more atmospheric N2 when mixed with grasses than when planted in monocultures, allowing companion grass species to utilise plant-available N for production (Chmelíková et al., 2015). Hence, diverse communities can enhance primary production and increase soil fertility (Hatch et al., 2007). Compared with legume monocultures, grass–legume mixtures also show more efficient water utilisation and improved ground cover, which reduces runoff and erosion (Ta and Faris, 1987; Humphries, 2012). Moreover, mixed plant communities in general are often more productive (Tilman et al., 2001) and experience reduced herbivore damage by diluting the availability of specific host plants (Root, 1973; Haddad et al., 2009; Fabian et al., 2012). Water stress may have important consequences for grass–legume ecosystems. In particular, grasses, with their shallow root systems, tend to be more sensitive to drought than legumes (Hayes et al., 2012) but less sensitive to waterlogging (Neal et al., 2009). Hence, changes in rainfall patterns may disrupt the ability of plant species to coexist in a mixture (Tow, 1993; Tow et al., 1997).

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS

6.2.3 Belowground herbivory Root-feeding herbivores of legumes, such as Sitona weevils, have the potential to disrupt the interactions between grasses and legumes by altering the flow of N between plant species (Ta et al., 1986; Murray and Hatch, 1994). Sitona adults feed on the foliage of legumes while the larvae feed on and in the N-fixing root nodules and on roots as they develop (Aeschlimann, 1979; Arbab and McNeill, 2014). Larval feeding can enhance legume root exudation and soil N availability and result in the increased availability of N for neighbouring grasses (Murray and Clements, 1998). Previous studies, for example, have demonstrated that root pruning and herbivory by Sitona spp. enhanced the direct transfer of N from white clover (Trifolium repens L.) to a neighbouring grass species (Lolium perenne L.) (Hatch and Murray, 1994; Murray and Hatch, 1994). Sitona larvae are consistently more damaging than foliar-feeding adults and peak larval densities are often driven by prevailing moisture conditions (Cantôt, 1979; Goldson et al., 1986; Goldson et al., 1988a). While root nodule herbivory can be highly damaging to the legume, reducing nodulation and impairing N-fixation, legume root recovery can also be over-compensatory, producing higher numbers of nodules and increasing N-fixation in response to herbivory (Quinn and Hall, 1992; Ryalls et al., 2013b).

6.2.4 Aboveground herbivory Root damage by weevil larvae is likely to indirectly affect aboveground phloem-feeding herbivores by altering phloem turgor pressure and/or through changes in phloem amino acid concentrations. Aphids, in particular, tend to perform better on plants with higher N and amino acid concentrations (Ponder et al., 2000; Karley et al., 2002; Nowak and Komor, 2010; Ryalls et al., 2015). Moreover, aphid performance on plants could be affected not only by the overall amino acid concentration, but also by the proportional composition of different amino acids (Mittler, 1967b; Srivastava and Auclair, 1975; Pritchard et al., 2007). Masters et al. (1993) argued that root herbivory promotes aphid performance by impairing soil water and nutrient uptake, which reduces the relative water content of foliage and increases soluble N (e.g. amino acids) and carbohydrate concentrations in the phloem. In contrast, Ryalls et al. (2013b) suggested that negative effects of Sitona discoideus Gyllenhal on pea aphids (Acyrthosiphon pisum Harris) could arise through lower quality phloem sap from nodule damage specifically, or reduced

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS phloem turgor and increased sap viscosity (via impaired root function), which would make the phloem more difficult to access.

The majority of sap-sucking invertebrates, including aphids, respond negatively to host plant water stress, which may relate similarly to a decrease in turgor pressure and an increase in phloem sap viscosity (Raven, 1983; Huberty and Denno, 2004). Moreover, increases in plant N (Johnson et al., 2011b) and amino acids (Hale et al., 2003) under drought do not necessarily benefit aphids due to these accompanying changes in phloem physiology (Aslam et al., 2013). Effects are likely to be contingent on a range of factors including plant functional group or whether species are growing alone or in competition with others (van der Putten et al., 2004; Johnson et al., 2011b).

6.2.5 Study system, objectives and hypotheses Using a model grass–legume mixture of Harding grass (Phalaris aquatica L.) and lucerne (otherwise known as alfalfa, Medicago sativa L.), this study addressed the effects of water stress (simulated drought and elevated precipitation) and root nodule herbivory by S. discoideus on both the plant–plant interactions between lucerne and Harding grass and on foliar-feeding pea aphids (A. pisum), one of the most damaging pests of lucerne (see Ryalls et al. (2013a) for review). The long-term co-existence of lucerne and grass can be perilous, with one often failing to persist under competition with the other (Bishop and Gramshaw, 1977; Dear et al., 1999). Harding grass, one of the most persistent sown temperate perennial grasses in south-eastern Australia, is one species that complements lucerne and achieves a dynamic balance in a mixture with lucerne (Sherrell, 1984; McKenzie et al., 1990; Culvenor et al., 2007).

This study aimed to determine whether (i) water stress and insect herbivory on lucerne influenced plant growth and N dynamics in both lucerne and Harding grass and (ii) whether water stress and insect herbivory affected total and individual foliar amino acid concentrations in lucerne and, subsequently, the population growth of A. pisum. We hypothesised that: (i) drought and elevated precipitation would decrease and increase plant growth, respectively, although both would negatively impact plant N concentrations in general. Lucerne nodule herbivory by S. discoideus was predicted to cause N leakage into the soil, which would positively affect the productivity of the co-

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS occurring grass species; (ii) the negative impacts of drought and weevil herbivory on nodulation and N acquisition would decrease foliar amino acid concentrations and/or phloem turgor pressure in lucerne, which would reduce aphid populations. Moreover, specific decreases in individual amino acid concentrations would cause aphid populations to decline.

6.3 Materials and methods 6.3.1 Rain-exclusion shelters Rain-exclusion shelters (249 cm x 188 cm) located at the Hawkesbury campus of Western Sydney University (latitude -33.609396, longitude 150.737800), as described by Johnson et al. (2015), were used to exclude 100% of ambient rainfall from four mesocosms beneath each of 18 shelters. Mesocosm pots (41 cm x 41 cm x 31 cm) were arranged in a 2 × 2 formation and dug into the ground so that the rim of the pot was flush with the soil surface. Each of the 72 mesocosms was filled with the excavated soil, which was air-dried and sieved to < 4 mm. Removable mesh cages (34 cm x 34 cm x 36 cm) were fitted to each mesocosm to prevent escape of experimental insects or entry to non-experimental (free-living) insects. The cages were designed to maximise air movement and allow good light transmittance (Johnson et al., 2015).

6.3.2 Insect cultures Acyrthosiphon pisum cultures, originating from a single parthenogenetic adult female collected from a local lucerne field in Richmond, NSW in August 2014, were maintained on propagated lucerne plants at 26/18 °C day/night on a 15L:9D cycle until required. Eggs produced from thirty sexually mature S. discoideus adults collected from the same field and reared under the same conditions were collected every 24 hours and stored on damp filter paper at 4 °C until required. Hatching success of two-month old eggs was confirmed (> 97% hatched within one week) by placing 200 eggs on 10 Petri dishes at 25 °C.

6.3.3 Experimental procedure Lucerne (cv. Sequel) seeds were inoculated with Rhizobium bacteria one hour prior to sowing by submerging in a solution containing 250 g Nodule N lucerne seed inoculant (New Edge Microbials, Albury, NSW) and 800 mL distilled water. Lucerne seeds (N =

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS

144) were sown and grown in seed cells (38 mm x 57 mm), with one lucerne plant per cell, under field conditions. Six grass seeds were sown in each of 72 seed cells and grown under the same conditions. After four weeks (1 October 2014), grass from one cell was transplanted into the centre of each mesocosm and two lucerne plants were transplanted either side of the grass (see Fig. 6-1). The root systems of the individual lucerne plants were contained within porous organza bags. This was done to confine weevil larvae to the soil around each lucerne plant, prevent their movement between plants and simplify inoculation with weevil eggs, but at the same time to not restrict transfer of water and nutrients within each mesocosm. Seed numbers within each mesocosm were based on a 1:1 lucerne:grass seed weight ratio (Boschma et al., 2010; Sandral, 2013).

Shelters were assigned at random to one of three rainfall treatments giving six shelters per rainfall regime. The ambient treatment was set at the 65.4 mm average precipitation in Richmond between September–November from 1881 to 2014 (Bureau of Meteorology, Australia). The drought treatment was 50%, and elevated precipitation treatment was 150% of the ambient rainfall amounts. This translated to an application of 586 mL (ambient precipitation), 293 mL (drought) and 878 mL (elevated precipitation) of rainfall water collected at the site three times per week. Soil moisture measurements were taken weekly using a 12 cm Hydrosense II probe (Campbell Scientific, Queensland, Australia).

Two weeks after transplantation, both lucerne plants in two of the mesocosms under each shelter were inoculated with 50 S. discoideus eggs, which were placed on top of the soil beside the stem of each plant. Egg numbers (i.e. 50 eggs per plant) were based on average densities of 4000 eggs and 80 lucerne plants per m2 (Aeschlimann, 1983; Goldson and French, 1983). After a further two weeks, three teneral A. pisum adults were applied to each of the two lucerne plants in one mesocosm containing S. discoideus and one mesocosm without S. discoideus. The factorial insect treatment design under each shelter therefore comprised one mesocosm with S. discoideus alone (W), one mesocosm with A. pisum alone (A), one mesocosm with both insects (WA) and one mesocosm with neither (CON) (Fig. 1).

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS

Acyrthosiphon pisum individuals were counted and removed four weeks after being applied, whereupon the experiment was harvested. The number of plants that were colonised by aphids (i.e. aphid colonisation success) was recorded. Grass tiller numbers and plant heights of both lucerne (from ground level to the base of the highest leaf) and Harding grass were measured six (weevil inoculation period), eight (aphid inoculation period) and 12 (harvest period) weeks after sowing. Roots were separated from the soil and shoots and the number of lucerne nodules were counted. Roots and shoots were frozen at −20 °C, freeze-dried and weighed prior to chemical analyses.

Lucerne (Medicago sativa) Harding grass (Phalaris aquatica) WA A CON

W WA

A – aphids only CON – control (no aphids or weevils) W – weevils only WA – weevils and aphids

Figure 6-1. Schematic diagram of experimental plots beneath one rain-exclusion shelter.

6.3.4 C/N and amino acid analyses Freeze-dried root and leaf material was ball-milled to a fine powder and carbon (C) and nitrogen (N) concentrations of 4–6 mg of milled samples were determined using an elemental analyser (LECO TruSpec Micro, LECO Corp., St. Joseph, MI, USA). The N content of lucerne roots was calculated by multiplying root dry mass by the concentration of N in roots. Soluble amino acids were extracted and analysed from milled lucerne foliage samples (15–20 mg) following the protocol set out by Ryalls et al. (2015). Foliar amino acids were used as a reliable and quantifiable proxy for the composition of phloem amino acids, as proposed by Riens et al. (1991) and Winter et al.

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS

(1992) for spinach and barley, respectively. Amino acid standards within the AA-S-18 (Fluka, Sigma-Aldrich) reference amino acid mixture were supplemented with asparagine and glutamine (G3126 and A0884, Sigma, Sigma-Aldrich). Nine essential amino acids (i.e. those that cannot be synthesised by insects de novo), including arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine and valine (Morris, 1991) and 10 non-essential amino acids (alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine) were detected using this method.

6.3.5 Statistical analyses Precipitation and herbivore treatment effects on plant growth (height, mass, grass tiller numbers and lucerne nodulation), chemistry (carbon and nitrogen) and aphids (population numbers and colonisation success) were analysed using mixed models in the nlme (Pinheiro et al., 2014) and lme4 (Bates et al., 2014) statistical packages within the R statistical interface v3.1.1. The fixed effects included herbivore treatment (CON, A, W and WA) and precipitation (drought, ambient and elevated precipitation) as well as the interactions between these terms. The random terms included shelter (1 to 18) as a block term, with plot number (i.e. positioning under each shelter) as a nested factor to account for shelter and plot effects that could confound any treatment effects. Plant heights measured at the three time-points (i.e. weeks six, eight and 12) were combined into an individual model incorporating time as a fixed effect to account for repeated measures. Post hoc Tukey’s tests using the glht function in the R package multcomp (Hothorn et al., 2008) and the R package LMERConvenienceFunctions (Tremblay and Ransijn, 2015) were used for pairwise comparisons of means for treatment and interaction effects. The effects of precipitation and herbivore treatment on all individual amino acid concentrations were determined using principal components analysis (PCA) and groupings of covarying individual amino acids were determined using a correlation matrix and hierarchical clustering within R. Permutational analysis of variance (PERMANOVA) was used to determine the effects of precipitation and herbivore treatment on concentrations of correlated amino acid groups.

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6.4 Results 6.4.1 Soil water Soil water content was significantly affected by both precipitation and herbivore treatment, and their interaction (Table 6-1). Specifically, under ambient and elevated precipitation, soil water increased in plots containing weevils alone (Fig. 6-2), although soil water did not increase significantly when both weevils and aphids were present.

Table 6-1. Soil, plant and aphid responses to precipitation and herbivore treatments from linear mixed models. P-values highlighted in bold indicate significance (P < 0.05). Where appropriate, response variables were transformed (*Log, #Square root) before analysis. Herbivore Herbivore Response Precipitation treatment × treatment variable Precipitation Fig. F P df F P df F P df Soil characteristics Soil water (%)# 2 16.10 0.001 3,45 223.00 <0.001 2,15 2.44 0.039 6,45 Plant characteristics Phalaris aquatica Height (week 6) 3 1.91 0.141 3,45 0.35 0.708 2,15 0.58 0.741 6,45 Height (week 8) 3 2.41 0.080 3,45 28.09 <0.001 2,15 1.85 0.111 6,45 Height (week 12) 3 6.05 0.002 3,45 48.61 <0.001 2,15 1.38 0.245 6,45 Number of tillers S1 3.12 0.035 3,45 84.22 <0.001 2,15 0.53 0.786 6,45 Shoot mass# 4 0.91 0.445 3,45 91.58 <0.001 2,15 1.67 0.149 6,45 Root mass# 4 2.24 0.097 3,45 21.15 <0.001 2,15 0.22 0.968 6,45 Shoot %N* 5 2.92 0.044 3,45 2.57 0.110 2,15 0.76 0.607 6,45 Root %N* 5 3.10 0.036 3,45 2.07 0.161 2,15 2.21 0.059 6,45 Shoot %C S2 0.37 0.775 3,45 3.82 0.046 2,15 0.77 0.600 6,45 Root %C S2 1.84 0.154 3,45 1.65 0.226 2,15 0.75 0.616 6,45 Medicago sativa Height (week 6) 3 0.47 0.702 3,45 0.08 0.926 2,15 0.52 0.793 6,45 Height (week 8) 3 0.69 0.565 3,45 3.90 0.043 2,15 0.31 0.926 6,45 Height (week 12) 3 1.67 0.187 3,45 70.57 <0.001 2,15 0.85 0.538 6,45 Shoot mass* 4 0.70 0.558 3,45 19.99 <0.001 2,15 0.47 0.827 6,45 Root mass* 4 1.51 0.226 3,45 9.44 0.002 2,15 0.50 0.801 6,45 Nodulation 6 4.38 0.009 3,45 3.84 0.045 2,15 0.26 0.952 6,45 Root %N 6 3.48 0.024 3,43 1.40 0.277 2,15 0.67 0.672 6,43 Root N content* 6 2.29 0.092 3,43 18.25 <0.001 2,15 1.02 0.423 6,43 Root %C 7 0.43 0.731 3,43 5.39 0.017 2,15 2.63 0.029 6,43 Aphid responses Population# 9 4.56 0.040 1,33 5.38 0.017 2,15 2.14 0.134 2,33 Colonisation S5 3.35 0.076 1,33 3.73 0.048 2,15 3.32 0.049 2,33

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS

18 e 16 None (CON) Aphids (A) de de 14 Weevils (W) d Both insects (WA) 12 10 c b bc bc 8 6 a a Soil water (%) water Soil a 4 a 2 0 Drought Ambient Elevated

Precipitation Figure 6-2. The interactive effects of precipitation and herbivore treatment on soil water content. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05).

6.4.2 Plant growth Precipitation affected both grass and lucerne height at week eight and week 12 (Table 6-1). Specifically, grasses and lucerne plants subjected to drought at week eight were shorter than those grown under ambient and elevated precipitation, although height did not vary significantly between plants grown under ambient and those grown under elevated precipitation. By harvest (week 12), the height of both grasses and lucerne plants had increased with increasing precipitation (Fig. 6-3A and B). Additionally, grasses were significantly taller in plots where lucerne plants had been inoculated with weevils (i.e. treatments W and WA) (Fig. 6-3C). For lucerne, herbivore treatment had no significant effect on height at any point (Table 6-1). Combining plant heights into individual models (accounting for repeated measures) revealed similar results (Appendix III Table S6-1).

CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS

600 *Precipitation × Week A *Precipitation × Week B 600 Drought *Precipitation Drought *precipitation 500 Ambient precipitation Ambient precipitation 500 Elevated precipitation Elevated precipitation 400

400 *Precipitation 300 *precipitation

NS NS 300 200

Grass height (mm) height Grass

200 (mm) height Lucerne Weevil Aphid 100 Weevil Aphid inoculation inoculation inoculation inoculation 100 0 6 7 8 9 10 11 12 6 7 8 9 10 11 12 Plant age (weeks) Plant age (weeks)

*Herbivore treatment × Week C 500 None (CON) d d Aphids (A) c c Weevils (W) 400 Both insects (WA) b b b b 300 a a a a

Grass height (mm) height Grass 200

100 6 7 8 9 10 11 12 Plant age (weeks)

Grass tiller numbers increased with increasing precipitation (Appendix III Fig. S6-1A), with grasses grown under drought having, on average, 51% and 65% fewer tillers than those grown under ambient and elevated precipitation, respectively (Appendix III Fig. S6-1A). Moreover, grasses within plots inoculated with weevils alone contained 23% more tillers than those in control plots (Appendix III Fig. S6-1B). Grass and lucerne shoot mass increased with increasing precipitation (i.e. the biomass of grasses increased and decreased under elevated precipitation and drought, respectively, compared with those subjected to ambient precipitation) , although root biomass did not vary significantly between those grown under ambient and elevated precipitation (Fig. 6-4A and B). Root to shoot ratios of both plants decreased as water availability increased. Herbivore treatment had no effect on lucerne or grass biomass in general, individually or interactively (Table 6-1).

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20 c 6 Shoot mass A Shoot mass c B Root mass Root mass 5 15 b 4 b 10 3

2 a e 5 e Grass biomass (g) Grass biomass a Lucerne biomass (g) biomass Lucerne 1 e e d d 0 0 Drought Ambient Elevated Drought Ambient Elevated Precipitation Precipitation

6.4.3 Carbon, nitrogen and nodulation Precipitation had no effect on grass root or shoot N concentrations, although shoot C concentrations decreased in grasses subjected to elevated precipitation compared with drought-stressed plants (Appendix III Fig. S6-2A; Table 6-1). Herbivore treatment significantly affected grass N concentrations, whereby plots inoculated with weevils alone contained grasses with higher root and shoot N concentrations compared with grasses in control plots (Fig. 6-5).

6 Root %N Shoot %N 5 ab b a a 4

cd d 2 c cd

Grass N concentration (%) Grass N concentration

0 CON A W WA

Herbivore treatment Figure 6-5. The impacts of herbivore treatment on the root and shoot nitrogen concentrations of Harding grass. CON were the control plants (no insects), A had aphids alone, W had weevils alone and WA had both insects. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05).

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Precipitation significantly affected the number of lucerne root nodules, with plants grown under drought containing, on average, 20% and 30% fewer nodules than those grown under ambient and elevated precipitation, respectively (Fig. 6-6A). Herbivore treatment also significantly affected nodulation, whereby lucerne plants inoculated with weevils alone contained, on average, 48%, 77% and 42% more root nodules than those containing aphids alone, those containing both insects and control plants, respectively (Fig. 6-6B).

Roots of lucerne plants subjected to drought contained 52% and 60% less N than those under ambient and elevated precipitation, respectively (Fig. 6-6C). Precipitation did not significantly impact the concentration of N in lucerne roots, although concentrations significantly increased in plants inoculated with weevils alone compared with control plants (Fig. 6-6D). Precipitation interacted with herbivore treatment to significantly affect lucerne root C concentrations, with roots of plants inoculated with weevils alone containing a significantly higher concentration of C than control plants, but only under drought conditions (Appendix III Fig. S6-2B).

15 15 A b B b b a 10 10 a a a

5 5

Number of of nodules Number

0 0 Drought Ambient Elevated CON A W WA Precipitation Herbivore treatment 0.05 3.0 C D b b 0.04 2.8 ab b ab a 0.03 2.6

0.02 a 2.4

Root N content Root 0.01 2.2

Root N concentration (%) N concentration Root

0.00 2.0 Drought Ambient Elevated CON A W WA

Precipitation Herbivore treatment Figure 6-6. The impacts of precipitation and herbivore treatment on lucerne nodulation (A and B) and the impacts of precipitation and herbivore treatment on root N content (C) and root N concentration (D), respectively. CON were the control plants (no insects), A had aphids alone, W had weevils alone and WA had both insects. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05).

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6.4.4 Amino acids Precipitation and herbivore treatment had no significant effect on total amino acid concentrations (Appendix III Table S6-2) and PCA combining all amino acid concentrations revealed no separation between precipitation or herbivore treatments (Appendix III Fig. S6-3). Correlated amino acid groups (Appendix III Fig. S6-4) consisted of group 1 (glycine, lysine, methionine, tyrosine, cysteine, histidine and arginine), group 2 (isoleucine, phenylalanine, leucine, threonine and valine), group 3 (aspartate and glutamate), group 4 (glutamine and serine) and three independently varying amino acids (alanine, asparagine and proline). PERMANOVA revealed a significant effect of precipitation on amino acid groups 1, 3 and 4 (Appendix III Table S6-2). Specifically, concentrations of amino acid groups 1 and 3 increased in lucerne plants subjected to drought and concentrations of amino acid group 4 increased in those under elevated precipitation (Fig. 6-7A). Herbivore treatment only affected one amino acid, with concentrations of proline significantly decreasing in lucerne plants that were inoculated with weevils (i.e. treatments W and WA) (Fig. 6-7B).

6 Drought e A 5 Ambient precipitation Elevated precipitation d

4 cd c ab dry mass) dry 3 a ab -1 b b 2

(µmol g (µmol 1

Amino acid concentration acid Amino

0 Group 1 Group 3 Group 4 Amino acid group 2.5 B a 2.0

ab 1.5 b

dry mass) dry

-1 b 1.0

(µmol g (µmol 0.5

Proline concentration Proline

0.0 CON A W WA Herbivore treatment Figure 6-7. The impacts of precipitation on individual groups of amino acids (A), namely group 1 (glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine), group 3 (aspartate and glutamate) and group 4 (glutamine and serine) and the impacts of herbivore treatment on proline concentrations (B). CON were the control plants (no insects), A had aphids alone, W had weevils alone and WA had both insects. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05). 103

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6.4.5 Aphid responses Precipitation and herbivore treatment independently affected the number of aphids (Table 6-1). Lucerne plants subjected to drought supported 54% and 61% fewer aphids than those under ambient and elevated precipitation, respectively (Fig. 6-8A). Additionally, aphid numbers decreased by 30% on lucerne plants inoculated with weevils compared to those with aphids alone (Fig. 6-8B). Precipitation interacted with herbivore treatment to affect aphid colonisation success, with weevils reducing aphid colonisation success on plants under elevated precipitation. Drought generally reduced aphid colonisation success, especially on plants with aphids alone (Appendix III Fig. S6- 5).

14 b A 12 b 10

6 a

Number of aphids Number

0 Drought Ambient Elevated Precipitation 12 a B 10 a 8 b

6 b

Number of aphids Number 2

0 A WA Herbivore treatment Figure 6-8. The impacts of precipitation (A) and weevil presence (B) on the number of aphids. A have aphids alone and WA have both weevils and aphids. Mean values (± SE) are shown. Bars with the same letters were not significantly different (P < 0.05).

The main findings are summarised in Fig. 6-9, with numbers referring to relevant figures associated with each effect.

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6.5 Discussion To our knowledge, this study is the first to investigate the combined effects of herbivory (both above- and belowground) and climate change (i.e. drought and elevated precipitation) in a grass–legume system. The main results demonstrate that drought and weevil root herbivory have contrasting effects on plant growth and lucerne nodulation, whereas both factors reduced aphid population growth. These effects may scale-up to play important roles in governing ecosystem function.

6.5.1 Soil water availability Overall, drought reduced soil water content by 55% and elevated precipitation increased soil water content by 65% compared with ambient precipitation. Plots inoculated with weevils alone had increased soil water availability, suggesting that damage to nodules and roots by weevil larvae likely reduced water uptake by lucerne

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CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS roots. Alternatively, burrowing activity of the weevils may have increased water penetration and reduced run-off, as observed in Johnson et al. (2015) with a soil conditioning ecosystem engineer (dung beetle; Bubas bison L.). The lack of an effect when aphids were feeding on lucerne simultaneously suggests that aphids counteracted the effects of weevils on soil water availability by either promoting soil water uptake or reducing weevil damage. In fact, aphid presence appeared to nullify the effects of weevils on multiple plant characteristics throughout this study, demonstrating the strong interactions between above- and belowground stressors.

6.5.2 Plant growth under water stress and herbivory Drought clearly inhibited growth of both lucerne and Harding grass. Once water availability reaches an optimum level, more resources may be allocated to shoot growth, which likely explains why root biomass of both plant species did not vary significantly between ambient and elevated precipitation. Similarly, the ratio of roots to shoots decreased as water availability increased. This is consistent with the resource optimisation hypothesis, which posits that plants will allocate fewer resources to their roots when water and nutrient availabilities are high (Gargallo-Garriga et al., 2014). Under drought, the biomass of lucerne and grass shoots decreased by 63% and 70%, respectively, relative to those under ambient precipitation. Grasses tend to be more sensitive to drought due to their shallower roots compared with the deeper taproots of lucerne (Hayes et al., 2012). While elevated precipitation increased plant growth in general, grasses dominated lucerne in plots subjected to elevated precipitation (i.e. shoot biomass increased by 37% and 72% in lucerne and grass, respectively, under elevated precipitation relative to ambient precipitation). Increased precipitation may therefore significantly disrupt the long-term ability of these plant species to co-exist (Tow, 1993; Tow et al., 1997). By harvest, weevil presence has significantly increased grass height, most likely associated with the fertilising effect of increased N leached from lacerated lucerne nodules. Alternatively, the increase in soil water availability for grasses in plots with weevils present alone may have been responsible for increasing the height of grasses, although that would not explain the concurrent increase in grass height when both insects were present. The significant effect of herbivory on grass height and tiller numbers, however, was not reflected in grass biomass.

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6.5.3 Impacts of water stress on C and N dynamics When water is a limiting factor, metabolites involved with energy production and growth (especially carbohydrates) are often shifted from shoots to roots (Gargallo- Garriga et al., 2014), although drought may also lead to a decrease in the belowground C demand (Hasibeder et al., 2015). Drought effects on C allocation have also been found to be dependent on community composition. For example, Sanaullah et al. (2012) noted an increased allocation of C from shoots to roots under drought in monocultures of Festuca arundinacea (Schreb.), L. perenne and M. sativa. When these three species were combined into a grass–legume mixture, however, C allocation to shoots increased when exposed to drought. The current study identified an increase in grass shoot C concentrations under drought compared with elevated precipitation, suggesting that Harding grass allocates more C to anti-stress mechanisms under drought (Peñuelas and Estiarte, 1998; Jentsch et al., 2011), although concentrations in drought-stressed grasses were not significantly higher than in those under ambient precipitation.

Drought reduced root N content and nodulation in lucerne plants, likely associated with decreased root N-fixation activity. The negative impact of drought stress on nitrogenase activity in leguminous plants is well known, although the exact mechanisms remain unclear (Gil-Quintana et al., 2013). A decline in the concentration of root C suggests that the supply of reduced C to bacteria may limit nitrogenase activity and nodule development. Root N concentrations, however, did not decrease under drought, suggesting that drought-stressed lucerne plants were not N-deficient. Under drought conditions, root nodulation and, subsequently, nitrogen fixation activity may decline due to a lower demand for N to support growth (Streeter, 2003). Predicting the responses of legumes to environmental change will be particularly important to maintain the N dynamics within the many terrestrial systems that are currently dominated by inputs of fixed N by legumes (Whitehead, 2000; Lilley et al., 2001; Peoples and Baldock, 2001; Angus and Peoples, 2012).

6.5.4 Impacts of weevils on C and N dynamics Lucerne nodulation and root N concentrations increased in response to weevil herbivory, suggesting overcompensatory growth in response to nodule damage. Other

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CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS studies have also shown nodule overcompensation in lucerne after root feeding by Sitona hispidulus Fabricius (Quinn and Hall, 1992) and S. discoideus (Ryalls et al., 2013b). No significant increase, however, was observed when plants were inoculated with both insects. When both weevil larvae and aphids are feeding on the plant, root recovery and stress-related increases in plant nutrients or the diversion of resources above- or belowground may be less likely because the plant remains in a constant state of stress (Ryalls et al., 2015). Weevils mitigated the effects of drought on lucerne root C concentrations by increasing root C concentrations. This is surprising considering that drought would most likely decrease weevil damage associated with declines in larval numbers (Goldson et al., 1986; Johnson et al., 2010). The difficulty in extracting S. discoideus larvae from the soil (Wightman, 1986) made it impossible to corroborate this assumption but additional studies using a more conspicuous species (e.g. whitefringed weevil, Naupactus leucoloma Boh.) may benefit from counting and weighing larvae.

Grass root and shoot N concentrations increased in plots that were inoculated with weevils alone, suggesting that larval damage to lucerne roots and nodules caused N leakage into the soil, which subsequently increased the uptake of N by Harding grass (Bardgett et al., 1999; Murray et al., 2013a). This information may be particularly useful for ecosystems that suffer from legume-feeding pests or processes that damage legume roots, with consequences for productivity, nutrient balance and species richness within ecological communities. Murray and Clements (1998) similarly identified an increase in N in wheat (Triticum arvense L.) from weevil-infested white clover. Other studies have identified a transfer of N from white clover to perennial ryegrass in response to herbivory by root-feeding nematodes (Bardgett et al., 1999; Dromph et al., 2006). In contrast, Ayres et al. (2007) noted a 13% reduction in N transfer from white clover to perennial ryegrass when clover roots were damaged by nematodes. They also identified a significant increase in grass root N when clover was subjected to simulated defoliation (through clipping). In this case, Sitona discoideus adults are defoliating insects so simultaneous feeding of larvae and adults may lead to even greater increases in N transferred to grasses. Increasing community complexity can alter the nutrient balance of plants differently, even if one plant (in this case, Harding grass) is not directly impacted. Given the prevalence of N-limitation in

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CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS terrestrial ecosystems (Vitousek and Howarth, 1991), increased N supply has the potential to influence plant productivity, especially in grassland ecosystems (Ayres et al., 2007).

6.5.5 Amino acid and aphid responses to water stress and weevils Sustained water stress in plants tends to affect aphids negatively (Huberty and Denno, 2004). Acyrthosiphon pisum densities, in particular, have been closely linked to lucerne water content, with populations suffering lower growth rates when lucerne is drought stressed (Forbes et al., 2005). In the current study, drought negatively impacted aphids, although amino acid concentrations generally increased in plants under drought compared with those under ambient precipitation. Hale et al. (2003) also reported a reduction in aphid (Rhopalosiphum padi L.) performance alongside an increase in amino acid concentrations in plants subjected to drought, suggesting that lower sap ingestion rates on drought-stressed plants are responsible for reducing aphid performance overall and override any positive effects on amino acid concentrations. From a community perspective, drought may somewhat alleviate the impacts of sap-feeding herbivores on plants. In contrast, other studies (e.g. Mewis et al., 2012) have identified increases in specific amino acids alongside increases in aphids in response to drought. Proline, in particular, often confers tolerance to water stress and has been reported to accumulate under such conditions (Premachandra et al., 1995; Kaur and Asthir, 2015). The role of proline in stress tolerance, however, remains controversial, with other studies reporting decreased proline levels under water stress (Delauney and Verma, 1993; Lutts et al., 1996; Hien et al., 2003; Silvente et al., 2012). Fukutoku and Yamada (1984) reported that proline accumulation in soybean only occurs when protein metabolism is disturbed under severe water stress. The stage of the plant (e.g. vegetative or flowering) and relative water content of the leaves also seem to be critical determinants of proline synthesis (Silvente et al., 2012).

Many studies have demonstrated how root feeders can influence aphid populations through plant-mediated mechanisms. Generally, aboveground aphids are positively affected by belowground root feeders (Johnson et al., 2012), although species that feed on legumes should be considered separately from those that feed on non-leguminous plants since legume root herbivores feed directly on the sites of N fixation and are more

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CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS likely to negatively impact aphids by impairing root function and lowering the quality of phloem sap (Goldson et al., 1988b; Ryalls et al., 2013b; Ryalls et al., 2015). Here, proline concentrations decreased when weevils were present, which may have contributed to the reduction in aphid numbers when weevils were feeding on the plant simultaneously. Proline concentrations in plants that were inoculated with aphids alone, however, were not significantly lower than concentrations in plants that were inoculated with both insects. Aphids may have mitigated the negative effects of weevil nodule feeding on foliar amino acid concentrations by promoting the metabolism of amino acids in lucerne (Guo et al., 2013) and masking the decrease in proline caused by weevils. While weevil larvae increased nodulation in lucerne, the effects on aphids may not have been apparent by the end of the experiment. Uncertainty exists as to whether the stimulation of nodulation over a longer timeframe would actually have a positive impact on aphids considering the dependency of aphids on plant nitrogen (Butler et al., 2012). Incorporating weevil larval performance data and quantifying root damage would provide useful insights into damage intensity and thresholds in lucerne, as described by Goldson et al. (1987; 1988a) in New Zealand. Moreover, these data would allow us to examine aboveground–belowground interactions in the opposite direction (i.e. the impacts of shoot herbivory on root herbivores). No clear trend has yet emerged from studies analysing the effects of shoot herbivory on root herbivores (Johnson et al., 2016c and references therein), although it is uncertain whether patterns in lucerne or other legumes would emerge if these data were available. Understanding the complex interactions between above- and belowground communities and how they fluctuate over time is essential for characterising the fundamental mechanisms driving plant community structure (De Deyn and Van der Putten, 2005; De Deyn et al., 2007).

6.5.6 Conclusions Global climate change and herbivory can modify the transfer of N and shape the competitive interactions between legumes and non-N-fixing grasses, with important implications for plant community structure. The results demonstrate how legume nodule herbivory can increase N uptake and productivity of a companion grass species and decrease aphid populations via changes in individual amino acid concentrations. Moreover, drought decreased plant productivity in general and reduced aphid

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CHAPTER 6 INSECT–PLANT COMMUNITY INTERACTIONS populations on lucerne, potentially via reduced phloem turgor pressure. With the frequency and length of droughts projected to increase under global climate change (Trenberth et al., 2003; IPCC, 2007; CSIRO-ABM, 2014; Gargallo-Garriga et al., 2014), understanding how these factors can shape plant susceptibility to insect pests and maintain the balance between an efficient grass–legume mixture is clearly a priority for achieving food security (Ayres et al., 2007; Gregory et al., 2009; Boschma et al., 2010). This will be especially important in terrestrial ecosystems that rely more on mineralisation as a source of N for grasslands and other plant communities in the future (Newbould, 1989; Angus et al., 2006; Grace et al., 2015). Ultimately, these data inform adaptation strategies aimed at relieving the disruption caused by insect herbivore pests. With the threats of climate change on ecological communities apparent, combining multiple interacting species in environmental studies is key to maintaining ecosystem services and protecting our food resources, especially as human population levels climb towards 10 billion by the end of the century (Lal, 2006; Birch et al., 2011).

6.6 Acknowledgements We would like to thank Andrew Gherlenda and David Fidler for their help with harvesting. Thanks also to Sarah Facey, Kirk Barnett and Lisa Bromfield for their help building the insect mesh cages and Goran Lopaticki for his help preparing the field site.

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Chapter 7

General discussion 6 7.1 Main findings and synthesis The overall objective of this thesis was to determine the effects of climate and atmospheric change on aphids and their interaction with belowground root herbivores in lucerne. Initially, important research and knowledge gaps associated with aphids (chapter 1) and, more specifically, lucerne aphids (chapter 2) were identified. Empirically, this research aimed to investigate: (i) how root herbivory interacts with aboveground aphids on lucerne (chapters 3, 4 and 6); (ii) how climate and atmospheric change factors, individually and interactively, impact aphids (chapter 5) and their interaction with belowground herbivores (chapters 3, 4 and 6); (iii) how water stress and root herbivory impact plant community structure and productivity by altering the C and N dynamics between legumes and neighbouring grasses (chapter 6); and (iv) the amino acid-mediated mechanisms linking aphids to climate and atmospheric change and root herbivory (chapters 4, 5 and 6).

Lucerne remains one of the most important legumes throughout the world and is increasingly used as a source of fixed N in crop rotations or within grass mixtures to improve or maintain soil fertility (Chmelíková et al., 2015). As with other plants, predicting how lucerne will respond in the future relies on understanding the mechanisms linking climate and atmospheric change with trophic interactions, whether it be with neighbouring plants, insects and higher trophic levels (e.g. predators and parasitoids) or with the nodule-dwelling bacteria present in all legumes (chapter 2). The interactions of multiple abiotic and biotic variables will influence the effectiveness of biological control and resistance mechanisms to determine future susceptibility of lucerne to aphid pests.

Legumes have the ability to fix N2 from the atmosphere and convert it into plant- available N and often differ in their responses to climate and atmospheric change compared with other plants (Jensen et al., 2012). It would therefore be fruitful to further distinguish legumes from non-leguminous plant species, especially in studies

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CHAPTER 7 GENERAL DISCUSSION combining the effects of climate and atmospheric change on aphids. In fact, the effects of eT and eCO2 on legume-feeding aphids specifically (i.e. negative and positive, respectively) tend to follow uniform trends and form generalisations despite responses traditionally being labelled as idiosyncratic (i.e. species-dependent) when considering plants and ecosystems as a whole. Muddied generalisations often stem from collating results from both legumes and non-leguminous plants despite their often contrasting responses to climate and atmospheric change. Newman and colleagues, as far back as 2003, identified the importance of nitrogen availability in predicting the plant-mediated responses of aphids to eCO2 (Newman et al., 2003) so plants that avoid the N-diluting effect of eCO2 (i.e. legumes) should be distinguished from others and accounted for in whole-ecosystem studies (Robinson et al., 2012).

7.1.1 Impacts of belowground herbivory on aphids Root- and shoot-feeding herbivores have the capacity to influence one another by modifying the chemistry and growth of the shared host plant (Gange and Brown, 1989; Blossey and Hunt-Joshi, 2003). Changes in host plant nutrients (e.g. soluble N and amino acids) often underpin aboveground–belowground herbivore interactions, although plant responses to herbivory may be species- or family-specific. Masters et al. (1993) proposed that root herbivory impairs water uptake and induces a stress response within the host plant, leading to an accumulation of soluble N (amino acids) in the foliage, which, in turn, causes the plant to become more susceptible to aphid attack aboveground. Root herbivores of legumes, in contrast, do not necessarily induce this stress-related accumulation of N in the foliage because the roots are less damaged. Instead, they are likely to negatively impact N acquisition and uptake by targeting and damaging root nodules specifically, therefore impairing biological N fixation (Goldson et al., 1988b; Murray et al., 1996). In other words, legume nodule herbivory reduces overall nitrogen supply to the plant, which becomes deficient in soluble nitrogen (amino acids) and negatively impacts aphids (chapters 3 and 6). This highlights the importance of considering insects feeding on legumes separately from those that feed on non-leguminous plants and offers the potential for identifying generalisations within legume systems. Herbivore synchronisation may present a barrier to achieving generalisations, however, as demonstrated in chapter 4. In particular, aphid populations increased on plants with roots damaged before aphid arrival compared

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CHAPTER 7 GENERAL DISCUSSION with those with roots damaged after aphid arrival. Late cutting of roots did not allow the plant to sufficiently recover and compensate for the damage and as a result the negative interactions on aphids persisted. The ability of the plant to compensate for damage caused by herbivory (Quinn and Hall, 1992) plays an important role in determining how aphids respond to belowground herbivory. Sitona discoideus, in particular, enhanced nodulation in lucerne, demonstrating an over-compensatory growth response of lucerne to root herbivory (chapters 3 and 6). While a negative effect of root herbivory on aphids was observed within these experimental studies, it is unclear whether the over-compensatory nodulation response observed would have eventually led to an increase in N fixation and accumulation in the foliage. If so, this could have potentially had a positive effect on aphids aboveground. In fact, chapter 3 drew attention to the possibility that when S. discoideus began to stimulate nodulation toward the end of the experiment, the negative impacts of initial nodule damage on aphids may have begun to be reversed, or at least alleviated. Research into the sequence and duration of herbivory is clearly a priority for establishing links between aboveground and belowground herbivores.

7.1.2 Increased temperature and CO2 impacts on aphids and their interaction with belowground herbivores Chapters 3 and 4 within this thesis are the first to examine the combined effects of eT and eCO2 on aboveground–belowground interactions. The plant-mediated impacts of eCO2 individually (i.e. in isolation of eT) on aboveground–belowground interactions have been investigated in a few systems. One of the earliest studies examined the interaction between a root-feeding (Pemphigus populitransversus) and a shoot-feeding (Aphis fabae) aphid feeding on the brassica Cardamine pratensis (Salt et al., 1996). The baseline interaction (i.e. that occurring under aCO2) was for the shoot-feeder to suppress populations of the root-feeder. Exposing the system to eCO2 did not alter the nature of the interaction and aphid populations remained at similar levels as those reared under aCO2 (Johnson et al., 2016c).

The responses of the clover root weevil (Sitona lepidus) feeding on white clover

(Trifolium repens) grown under aCO2 and eCO2 have also been investigated (Staley and Johnson, 2008; Johnson and McNicol, 2010). As with many insect herbivores with root-

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CHAPTER 7 GENERAL DISCUSSION feeding life-stages, the larval stages feed belowground whereas adults feed on foliage (Brown and Gange, 1990; Johnson et al., 2016a). Adult weevils consumed significantly more foliage under eCO2, perhaps indicative of compensatory feeding since foliage was of lower quality (higher C:N), and laid 23% fewer eggs into the soil. Despite fewer eggs being laid, larval populations grew by 38% under eCO2, which was directly correlated with increased levels of root nodulation (housing N-fixing bacteria) under eCO2 (Staley and Johnson, 2008; Johnson and McNicol, 2010). Sitona lepidus larval performance is tightly linked to availability of root nodules (Gerard, 2001) so larvae were able to take advantage of increased resource availability. In this system, it was clear that eCO2 could alter the relationship between adults aboveground and their belowground offspring. This raises interesting questions about whether eCO2 might introduce a ‘parent-offspring’ conflict in species which have above- and belowground herbivorous life stages (Clark et al., 2011), as described by Johnson et al. (2016c).

Like white clover and S. lepidus, eCO2 promoted nodulation and survival of S. discoideus on lucerne (chapter 3). Higher temperatures, however, reduced lucerne nodulation and performance of larval S. discoideus, which most probably reduced its impact aboveground. Additionally, eT negated the positive effects of eCO2 on aphid abundance (chapter 4) and reproductive performance (chapter 5). This neutralising effect of eT has also been shown in other plant–insect systems (e.g. Murray et al., 2013b), demonstrating the importance of incorporating multiple environmental change factors simultaneously. Moreover, these effects were consistent across five lucerne genotypes of varying pea aphid resistance, demonstrating the generality of these effects. In contrast, other studies have demonstrated a breakdown in cultivar resistance to aphids under eCO2 in lucerne (Johnson et al., 2014) and in other plants (Martin and Johnson, 2011; Hentley et al., 2014). Aphid genotypes have also shown contrasting responses to eCO2, as described in chapter 1 (section 1.2). The interactions of biotic and abiotic factors identified in this thesis and the aforementioned studies showed that there was a complex interplay of mechanisms operating in addition to the interaction between the above- and belowground subsystems.

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7.1.3 Impacts of water stress and root herbivory on aphids and grass–legume dynamics A few studies have been conducted to experimentally manipulate soil water content to mimic the effects of altered precipitation patterns on aboveground–belowground herbivore interactions (e.g. Johnson et al., 2015) and competitive plant–plant dynamics (e.g. Barton and Ives, 2014). The majority of these focus on drought, which is predicted to increase in severity and frequency in many regions under a future climate. In the first study to investigate the effects of soil water content on interactions between phytophagous invertebrates feeding above- and belowground, Gange and Brown (1989) found that both a low water treatment and a root-feeding scarab beetle larvae (Phyllopertha horticola) increased the growth rate and weight of black-bean aphids (Aphis fabae) feeding aboveground on an annual Brassicaceae species (Capsella bursa- pastoris). Aphid fecundity and longevity were also increased by root herbivory, but not affected by the water treatment. Moreover, the effect of root herbivores on aphid performance was reduced under a higher soil water content treatment.

Chapter 6 is the first study to combine the effects of herbivory (both above- and belowground) and climate change (drought and elevated precipitation) on the competitive dynamics between a grass and a legume. Results demonstrated how grass N concentrations and productivity increased in response to root herbivory by weevil larvae, suggesting that N leached from lacerated lucerne root nodules was taken up by the neighbouring grass. Rates of nutrient mineralisation are particularly important in grass–legume systems, with one species often failing to persist under competition with the other if N dynamics are not maintained (Dear et al., 1999). In relation to aboveground–belowground herbivore interactions, root herbivory by S. discoideus negatively impacted aphid populations, associated with decreases in phloem proline concentrations. Moreover, drought decreased the productivity of both plant species and reduced aphid populations on lucerne, probably due to reduced phloem turgor and increased sap viscosity (Hale et al., 2003). While elevated precipitation has the potential to alter plant competitive dynamics by causing the grass to dominate lucerne, no effect on aphids or their interaction with belowground weevils was observed. However, indirect negative effects on aphids are likely to occur if their host plant is suppressed by the neighbouring grass. In situations where aphids can feed on both

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CHAPTER 7 GENERAL DISCUSSION plants, suppression of one species may cause aphids to migrate to the other. These results demonstrate how climate change and interactions between herbivores (both above- and belowground) can shape the competitive interactions between N-fixing legumes and non-N-fixing grasses, with consequences for plant community structure and productivity.

7.1.4 Amino acid-mediated mechanisms linking aphids to climate change, atmospheric change and root herbivory Difficulties associated with extracting phloem for biochemical analyses may be partly responsible for the paucity of research on amino acid-mediated mechanisms driving aphid responses to environmental change (Pritchard et al., 2007). Several quantitative and qualitative methods are available for analysing amino acids. Phloem collection can be achieved using two common methods, ethylenediaminetetraacetic acid (EDTA)- facilitated exudation (King and Zeevaart, 1974) and aphid stylectomy (Unwin, 1978), each with their advantages and disadvantages. The former is a technically simple method providing information about the relative abundance (mol %) of phloem metabolites. Excision of the plant petiole or stem allows phloem to exude into a solution of EDTA which prevents the sieve pores from sealing. This method, however, is confined to qualitative assessments of the relative proportions of phloem sap components, as opposed to absolute concentrations. Additionally, the invasive nature of this method may induce a wounding response and alter the properties of the exudate or contaminate samples through contact with other parts of the plant tissue. The stylectomy method is the most effective way of obtaining absolute concentrations of pure phloem sap and involves severing the stylet (using laser removal, scissors or microcautery) of a feeding aphid and extracting the sap directly from the severed stylet (Fisher and Frame, 1984). This method makes it easy to control for evaporation and contamination, although it is an extremely time-consuming technique, with studies reporting low exudation success rates and obtaining small volumes of exudate (Fisher and Frame, 1984; Gaupels et al., 2008; Dinant et al., 2010). The majority of studies attempting to relate phloem feeder performance to plant chemistry measure foliar or whole-tissue amino acid concentrations as a reliable proxy for phloem amino acids (Riens et al., 1991; Winter et al., 1992; Robinson et al., 2012) due to the inherent difficulty in extracting phloem samples relative to foliar or whole-tissue samples

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(Pritchard et al., 2007). Some studies suggest that greater reliance should be placed on amino acids extracted from the phloem directly, with potential variation in composition between phloem and foliar amino acids (Weiner et al., 1991; Ryan et al., 2014a; Ryan et al., 2015). Chapter 5 compared the relative compositions of phloem and foliar amino acids and demonstrated a correlation between two sampling methods (i.e. phloem extraction using EDTA and foliar extraction), with no significant effect of extraction technique on amino acid proportions. This result indicates that amino acids extracted from the foliage can be used as a reliable indicator of phloem amino acids in lucerne, as proposed by Riens et al. (1991) and Winter et al. (1992) for spinach and barley, respectively.

Primary metabolites, including amino acids, can be modified by climate and atmospheric change and mediate interactions between above- and belowground herbivores (Girousse et al., 1996; Hale et al., 2003; Bezemer and van Dam, 2005; Staley and Johnson, 2008; Oehme et al., 2013). Furthermore, climate, atmospheric and root herbivory impacts on amino acid concentrations, and subsequently aphids, are often determined by changes in the relative concentrations of individual amino acids (Mittler, 1967b; Srivastava and Auclair, 1975; Pritchard et al., 2007). Masters et al. (1993) argued that root herbivory can impair water uptake in plants, which potentially promotes shoot herbivore performance via stress-induced accumulation of soluble N compounds (e.g. amino acids) in the foliage. Leguminous plants, with their N-fixing ability, are likely to differ in their response to root nodule damage. Chapter 3 suggested that negative effects of S. discoideus on aphids could arise through lower quality phloem sap from nodule damage specifically or reduced phloem turgor and increased sap viscosity via impaired root function, which would make the phloem more difficult to access. Two chapters (4 and 6) investigated the amino acid-mediated mechanisms linking aboveground–belowground interactions and demonstrated how the negative effects of root herbivory on aphids are driven by decreases in amino acid (particularly proline) concentrations (chapters 4 and 6). However, the sequence of herbivore arrival affected amino acid accumulation (chapter 4), with amino acids increasing when roots were damaged before aphid arrival compared with those damaged after aphid arrival. Root damage also interacted with temperature, whereby root cutting early in the experiment under ambient temperature resulted in increased

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CHAPTER 7 GENERAL DISCUSSION aphid performance, associated with higher concentrations of some foliar amino acids (lysine, phenylalanine and tyrosine).

The amino acid-mediated mechanisms driving the responses of aphids to climate and atmospheric change were evident from three experimental chapters (4, 5 and 6). Foliar amino acid concentrations and aphid performance increased in response to eCO2, whereas reduced nodulation under eT led to lower concentrations of amino acids in the foliage and diminished the performance of aphids. In particular, a specific group of individual amino acids (namely, arginine, asparagine, aspartate, glutamate and histidine) decreased under eT (chapters 4 and 5). These results identified the importance of characterising the responses of individual amino acids as opposed to total concentrations alone. Furthermore, the contrasting effects of eT and eCO2 on amino acids, and subsequently aphid performance, demonstrate the importance of combining multiple environmental change factors. While it is clear that eCO2 effects on aphids are mediated by the plant, the decreases in amino acid concentrations observed under eT may have simply exacerbated the direct negative effects of eT on aphids. In other words, both indirect and plant-mediated effects of temperature on aphids are likely in play (see section 1.2). Only by growing plants at different temperatures and inoculating those plants with insects under the same temperature, however, could direct effects be separated from plant-mediated effects of temperature on insect herbivores (Murray et al., 2013b). Other climatic factors (i.e. drought and elevated precipitation) increased the concentrations of some foliar amino acids but these increases were not consistent with changes in aphid population size, suggesting that other mechanisms (i.e. changes in phloem turgor pressure) underpin aphid responses to water stress (chapter 6).

As with aphid performance, effects of eT and eCO2 on amino acid concentrations (i.e. generally negative and positive, respectively) were consistent across different lucerne cultivars (chapter 5). These are promising results for plant breeding programmes that aim to maintain resistance under future conditions, although one previous study observed inconsistent changes in amino acid concentrations across different lucerne cultivars (Johnson et al., 2014) and other plants have shown a breakdown in aphid resistance (see section 7.1.2 above). In this case, primary producers may revert back to

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CHAPTER 7 GENERAL DISCUSSION using insecticide applications to suppress disease impacts on crops, which are associated with adverse environmental effects, rapid resistance breakdown and high production costs (Jones, 2004b; Oerke and Dehne, 2004). Climate change studies incorporating multiple cultivars are key to identifying the diversity of underlying mechanisms driving plant resistance to aphids and developing effective cultivars with long-lasting resistance. Moreover, cultivar resistance to A. pisum, and potentially many other aphid species, may be increased by breeding cultivars with (i) lower concentrations of amino acids required for the synthesis of essential amino acids by aphids’ endosymbiotic bacteria and/or (ii) lower concentrations of specific amino acids identified here as more important than others (i.e. those that concurrently decreased with aphid performance). Plants also require amino acids for growth however, so pinpointing their optimal amino acid requirements under future conditions would benefit this research by identifying which amino acids could be promoted or inhibited, and to what extent without limiting plant productivity. As the chemical and molecular mechanisms underpinning aphid responses to future environmental conditions emerge, we should see improvements in large-scale management practices, and ultimately increased crop production.

7.2 Constraints, caveats and further study Aphid responses to changes in host plant quality, like in the experimental chapters of this thesis, are often inferred from correlations or associations between plants and aphids. This does not necessarily imply causation. The complex nature of biochemical pathways within plants make it feasible that any measure of a given plant metabolite may correlate with a number of other biochemical components (Ryan, 2012), which calls for stable isotopic analyses (Haribal and Jander, 2015) and/or artificial diet experiments to complement these studies (Karley et al., 2002). While studies such as those presented in this thesis are useful for identifying factors that help explain changes in aphid performance, artificial diet experiments, for example, could be used to identify the causal basis for future changes in aphid performance. Specifically, by manipulating amino acid compositions, they would be able to pinpoint whether changes in specific amino acids affect the direction and magnitude of aphid responses observed under climate and atmospheric change.

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While plant nitrogen and amino acid concentrations often determine the performance of aphids, it remains fruitful to consider other plant-mediated mechanisms that may contribute to the variety of aphid responses observed under eCO2, including other defensive or chemical mechanisms. For example, Goławska et al. (2006; 2008; 2012; 2014) noted a reduction in A. pisum activity (e.g. probing behaviour) and performance (e.g. reproduction) on lucerne plants with higher saponin concentrations and identified higher concentrations of saponins in plants infested with A. pisum, although no effects of atmospheric (or climate) change on saponin concentrations have yet been observed.

Plants may be able to compensate for aphid infestation under eCO2 by reallocating resources towards shoot tissue maintenance, as suggested by a reduction in flowering of broad beans (Vicia faba) caused by A. pisum under eCO2 (Awmack and Harrington, 2000). Additionally, as predicted by the Carbon Nutrient Balance Hypothesis (Bryant et al., 1983) and Growth Differentiation Balance Hypothesis (Herms and Mattson, 1992;

Glynn et al., 2007), eCO2 conditions may cause plants to allocate more resources to defensive compounds, such as condensed tannin concentrations, and alter enzyme composition in aphids (Hartley et al., 2000; Wu et al., 2011). Molecular mechanisms, including jasmonic, salicylic and abscisic acid signalling, have been implicated in genetic resistance to aphids under climate and atmospheric change (drought-stress and eCO2) in another Medicago species, M. truncatula (Guo et al., 2014, 2015), with potential for testing and incorporating genetic resistance into lucerne and other plants.

Incremental increases in CO2, temperature and water availability that are used in most empirical studies addressing climate and atmospheric change may not accurately reflect the non-linear changes that we are likely to observe in the future. For example, phytophagous invertebrates are predicted to respond differently to moderate and severe drought stress (Larsson, 1989; Tariq et al., 2012) so experiments applying just one level of drought or low water availability may be over-simplified. Similarly, root herbivore impacts are likely to fluctuate in the field, with plants and insects experiencing periods of discontinuous stress (discussed in chapter 4). Abiotic and biotic changes that cover a range of scenarios are vital for understanding how interactions between above- and belowground herbivores may be altered under climate and atmospheric change. Moreover, while drought has significant impacts on agricultural

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CHAPTER 7 GENERAL DISCUSSION grass–legume systems (chapter 6), responses of these systems and many other ecological communities to eT and eCO2 are untested.

Chapters 3, 5 and 6 discussed how longer-term experiments would be beneficial for identifying whether the over-compensatory nodulation response to weevil larval herbivory would eventually lead to positive effects on aphid populations, which further promotes the need to test additional scenarios. Also of concern is the realism associated with inoculating plants with root herbivores. While soil was inoculated with weevil eggs (chapters 3 and 6) that hatched successfully within a few days, the exact numbers that hatched and remained alive is uncertain without confining them to specific sections of the root and biasing their feeding regimen. Moreover, while weevil eggs were inoculated one week prior to aphid inoculation to allow larvae time to hatch and start feeding, the point at which nodule damage was significant enough to influence aphids was uncertain. Without simulating herbivory (chapter 4) and controlling for the timing and intensity of root herbivore damage (Hjältén, 2004; Borowicz, 2010), the difficulty in extracting root herbivores, especially S. discoideus, from the soil (Wightman, 1986) and the inconspicuous nature of soil-dwelling invertebrates in general makes identifying effects on belowground herbivores particularly challenging. A number of methods have been employed to non-invasively track patterns of insect movement and behaviour within the soil (Southwood and Henderson, 2000). These commonly include using artificial transparent soil (Downie et al., 2012) or slant boards (e.g. plant roots in-between a transparent medium such as glass) to visualise effects (Murray and Clements, 1992). Additionally, telemetric techniques can be used to detect insect movement in pots or, less accurately, in field conditions (Reynolds and Riley, 2002). Acoustic detection (Mankin et al., 2001; Zhang et al., 2003) or, more recently, x-ray microtomography (Jenneson et al., 2003), are likely to provide useful insights into the behaviour of belowground insects and their effects on plants and other herbivores, especially as their technology develops and detection limits improve (Johnson et al., 2004, 2007; Dawson and Byers, 2008; Mankin et al., 2008). Another approach to quantify the effects of belowground herbivory without detecting real-time movement would be to manipulate weevil larval densities under field conditions. This would provide an ecologically realistic way of comparing and contrasting multiple levels of herbivory.

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Three empirical research chapters presented in this thesis incorporate the effects of belowground herbivory on aboveground aphids. Examining the interaction in the opposite direction (i.e. impacts of shoot herbivory on root herbivores), Masters et al. (1993) proposed that shoot herbivores would detrimentally affect root herbivores because the plant would divert resources away from roots to allow for compensatory growth aboveground. This proposition is now largely discounted as many plants actually translocate photoassimilates to the roots for storage after episodes of shoot herbivory (Schultz et al., 2013). Moreover, studies subsequent to the model of Masters et al. (1993) frequently show root herbivore performance is either unchanged (Blossey and Hunt-Joshi, 2003; Johnson et al., 2012 and references therein) or even improved in the presence of shoot herbivores (Johnson et al., 2009; Kaplan et al., 2009; McKenzie et al., 2013b), as discussed by Johnson et al. (2016c). Whether different patterns in lucerne or other legumes would emerge is uncertain and would provide an interesting topic for further study.

Additional notable areas of research to consider for further study include the incorporation of higher trophic levels (e.g. predators and parasitoids) that could be considered as viable control agents, other root herbivores and additional aphid genotypes (discussed in chapter 1). Impacts of climate and atmospheric change on aphids are often genotype-specific and biological control efforts are often dependent on the responses of higher trophic levels (chapter 2). Moreover, conservation efforts may be inhibited by trophic asynchrony and/or the interactions between abiotic and biotic variables (Dyer et al., 2013).

7.3 Conclusions and future outlook The increasing dependency on lucerne as an agricultural forage crop and the growing reliance on soil N mineralisation for primary producers (Newbould, 1989; Angus et al., 2006; Grace et al., 2015) make studies into lucerne and other legumes crucial. Crop devastation events and aphid outbreaks such as those experienced with lucerne in the 1950s and 1970s in North America and Australasia, respectively, must be prevented if we are to feed another three billion people by 2050 (Cohen, 2003; Birch et al., 2011). Despite the development of aphid-resistant cultivars to thwart these invasions, aphid outbreaks driven by environmental factors, including climatic variability and

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CHAPTER 7 GENERAL DISCUSSION interactions with other trophic groups still occur (Reddy and Hodges, 2000; Humphries et al., 2012). Avoiding aphid-induced crop devastation may be achieved by pinpointing the exact plant-mediated mechanisms driving aphid responses to future environmental change. This would provide a mechanistic basis for building resistance into new cultivars. The work presented here takes a step towards this by identifying discrete groups of amino acids that underpin the effects of climate and atmospheric change, in addition to belowground herbivory and plant genotype, on aphid performance. The overall main findings of this work are summarised as follows:

 Belowground herbivory had negative effects on aphids aboveground.  Elevated temperatures (i.e. 4 °C above the average ambient temperature of -1 26 °C) negated the positive effects of elevated CO2 (640 µmol mol ) on characteristics of lucerne (nodulation and amino acid concentrations) and

aphids (population growth, fecundity and rm).  Drought and root herbivory influenced competitive grass–legume dynamics by decreasing plant growth at different rates and increasing grass N uptake, respectively.  Climate, atmospheric and root herbivory impacts on aphids were strongly driven by changes in discrete functional groups of amino acids.

By extrapolating these results it could be envisaged that root and shoot herbivory by weevils and aphids, respectively, will cause greater damage to crops under eCO2. Results suggest that eT will alleviate this damage, although the extent of climate change, the difficulty in predicting temperature fluctuations relative to CO2 increases and its potential counteractive effect in different regions is uncertain. Perhaps more importantly, the identification of specific amino acid-mediated mechanisms underpinning aphid responses to climate and atmospheric change has wider implications for predicting future crop susceptibility. In particular, this knowledge can be incorporated into plant-breeding programmes and may serve to combat any increases in aphid performance under eCO2 regardless of the effects of eT or water stress on aphids. Moreover, combining this knowledge with the emerging understanding of the molecular mechanisms underpinning aphid responses to global environmental change should provide meaningful insights into aphids acting on lucerne and other plants. 124

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Complementing glasshouse studies with long-term field studies to compare and contrast results observed within this thesis, in addition to the topics of further study mentioned above, would greatly enhance our understanding of how legumes and legume-feeding insects will respond under future conditions. Free Air Carbon dioxide Enrichment (FACE) studies, for example, could be used to elucidate the mechanisms underpinning aphid responses to eCO2 in the field. Ongoing research is currently underway (thanks to the Western Sydney University E.A. Southee Award for ecological science in agriculture) to determine how predicted changes in temperature shape the pest status of aphids. By simulating changes in temperature in the form of 12 field warming frames (Fig. 7-1), findings from glasshouse experiments (chapters 3, 4 and 5) will be extended to the field as a follow-up research project of this thesis.

Figure 7-1. Warming frame schematic

Multi-factorial approaches combining several environmental and chemical factors with multiple trophic levels would serve to clarify impacts on ecological communities and ecosystems as a whole. Plant and insect responses to climate and atmospheric change, in particular, can cascade across species networks spanning trophic levels and above- and belowground sub-systems, which may scale-up to play important roles in governing ecosystem function (McKenzie et al., 2013a). Incorporating physiological and behavioural aphid and plant responses into climate change models will further elucidate the potential impacts of environmental variability on plant–insect interactions, allowing more informed management strategies to defend our food resources against global pests such as aphids.

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Appendix I – Chapter 4 supplementary material

-1 Table S4-1: Single-factor treatment effects of CO2, temperature and herbivore damage on individual amino acids (µmol g dry mass). Mean values (± standard errors) shown. EC = early-cut no aphids, C = control (no cut) no aphids, LC = late-cut no aphids, ECA = early-cut and aphids, A = no cut and aphids, LCA = late-cut and aphids.

CO2 Temp (°C) Treatment Amino acid a e 26 30 EC C LC ECA A LCA Alanine (Ala) 2.276 ± 0.078 3.077 ± 0.137 2.548 ± 0.110 2.774 ± 0.115 2.730 ± 0.194 2.830 ± 0.238 2.220 ± 0.138 2.808 ± 0.237 3.022 ± 0.179 2.277 ± 0.141 Arginine (Arg) 1.140 ± 0.153 2.012 ± 0.390 2.182 ± 0.360 0.816 ± 0.088 2.175 ± 0.450 1.602 ± 0.804 1.252 ± 0.315 1.424 ± 0.427 1.019 ± 0.170 1.817 ± 0.560

Aspartic acid (Asp) 4.633 ± 0.244 6.433 ± 0.667 6.992 ± 0.587 3.720 ± 0.206 5.239 ± 0.488 5.969 ± 1.326 3.772 ± 0.393 5.757 ± 0.528 6.051 ± 0.541 5.982 ± 1.212 Cysteine (Cys) 0.813 ± 0.031 0.809 ± 0.030 0.817 ± 0.030 0.805 ± 0.030 0.828 ± 0.041 0.768 ± 0.041 0.771 ± 0.051 0.875 ± 0.048 0.846 ± 0.070 0.762 ± 0.055 Glutamic acid (Glu) 1.262 ± 0.039 1.493 ± 0.058 1.485 ± 0.053 1.238 ± 0.041 1.390 ± 0.096 1.252 ± 0.077 1.116 ± 0.063 1.355 ± 0.081 1.619 ± 0.093 1.467 ± 0.081 Glycine (Gly) 0.372 ± 0.014 0.451 ± 0.022 0.441 ± 0.019 0.372 ± 0.016 0.422 ± 0.033 0.400 ± 0.031 0.365 ± 0.039 0.423 ± 0.034 0.444 ± 0.025 0.398 ± 0.027 Histidine (His) 0.194 ± 0.017 0.316 ± 0.034 0.337 ± 0.031 0.152 ± 0.013 0.241 ± 0.044 0.159 ± 0.024 0.271 ± 0.041 0.281 ± 0.059 0.174 ± 0.032 0.383 ± 0.059 Isoleucine (Iso) 0.963 ± 0.036 1.117 ± 0.073 1.033 ± 0.060 1.037 ± 0.049 1.006 ± 0.089 0.929 ± 0.072 0.937 ± 0.095 1.048 ± 0.130 0.988 ± 0.061 1.297 ± 0.112 Leucine (Leu) 1.326 ± 0.065 1.645 ± 0.091 1.528 ± 0.083 1.415 ± 0.072 1.554 ± 0.147 1.507 ± 0.140 1.351 ± 0.121 1.461 ± 0.149 1.498 ± 0.121 1.477 ± 0.140 Lysine (Lys) 0.986 ± 0.083 1.523 ± 0.136 1.274 ± 0.111 1.197 ± 0.112 1.203 ± 0.148 1.147 ± 0.244 1.102 ± 0.159 0.969 ± 0.103 1.094 ± 0.153 1.901 ± 0.271 Methionine (Met) 0.350 ± 0.027 0.395 ± 0.026 0.410 ± 0.024 0.325 ± 0.030 0.423 ± 0.053 0.238 ± 0.027 0.354 ± 0.040 0.350 ± 0.044 0.416 ± 0.063 0.439 ± 0.038 Phenylalanine (Phe) 0.695 ± 0.047 0.977 ± 0.076 0.822 ± 0.059 0.834 ± 0.067 0.809 ± 0.098 0.753 ± 0.068 0.784 ± 0.100 0.787 ± 0.122 0.717 ± 0.071 1.111 ± 0.160 Proline (Pro) 2.001 ± 0.229 3.341 ± 0.405 2.742 ± 0.316 2.501 ± 0.332 2.773 ± 0.644 1.581 ± 0.215 2.012 ± 0.409 3.490 ± 0.771 2.397 ± 0.364 3.493 ± 0.696 Serine (Ser) 1.466 ± 0.057 2.040 ± 0.143 1.718 ± 0.119 1.755 ± 0.087 1.889 ± 0.249 1.498 ± 0.129 1.528 ± 0.185 1.963 ± 0.225 1.561 ± 0.103 1.964 ± 0.169 Threonine (Thr) 0.745 ± 0.032 0.926 ± 0.054 0.840 ± 0.043 0.818 ± 0.044 0.864 ± 0.080 0.767 ± 0.067 0.714 ± 0.071 0.903 ± 0.092 0.824 ± 0.053 0.900 ± 0.084 Tyrosine (Tyr) 0.578 ± 0.033 0.632 ± 0.042 0.547 ± 0.032 0.668 ± 0.043 0.543 ± 0.050 0.568 ± 0.053 0.615 ± 0.072 0.540 ± 0.061 0.600 ± 0.055 0.753 ± 0.087 Valine (Val) 0.998 ± 0.045 1.364 ± 0.100 1.225 ± 0.085 1.106 ± 0.061 1.194 ± 0.139 1.052 ± 0.106 1.023 ± 0.134 1.233 ± 0.164 1.148 ± 0.078 1.360 ± 0.150

APPENDICES APPENDIX I

Table S4-2: Results from multivariate permutational analysis (PERMANOVA) of temperature, CO2 and plant-damage (p-d) treatment effects on amino acid groupings. For the purpose of PERMANOVA, ungrouped individuals (Glu, Met, Cys and Tyr) were grouped with the highest correlated individual amino acid. *indicates significance (P<0.01) adjusted for absence of mixed effects.

df SS MS Pseudo-F P(perm)

Group 1 (Ala, Gly, Iso, Leu, Lys, Phe, Pro, Ser, Thr, Val) Temp 1 0.171 0.171 2.793 0.055 CO2 1 0.801 0.801 13.050 0.001* P-d treatment 5 0.499 0.099 1.627 0.072 Temp × CO2 1 0.301 0.301 4.911 0.011 Temp × P-d treatment 5 0.309 0.062 1.006 0.414 CO2 × P-d treatment 5 0.184 0.037 0.601 0.867 Temp × CO2 × P-d treatment 4 0.096 0.024 0.390 0.969 Residuals 255 15.647 0.061 Total 277 18.008 Group 2 (Arg, Asp, His, Lys) Temp 1 2.657 2.656 31.501 0.001* CO2 1 0.290 0.290 3.442 0.025 P-d treatment 5 1.429 0.286 3.390 0.001* Temp × CO2 1 0.256 0.256 3.039 0.030 Temp × P-d treatment 5 0.580 0.116 1.374 0.145 CO2 × P-d treatment 5 0.401 0.080 0.950 0.450 Temp × CO2 × P-d treatment 4 0.167 0.042 0.495 0.908 Residuals 255 21.504 0.084 Total 277 27.283 Group 3 (Glu, Lys) Temp 1 0.721 0.720 17.234 0.001* CO2 1 0.435 0.435 10.404 0.001* P-d treatment 5 0.612 0.122 2.930 0.004* Temp × CO2 1 0.258 0.258 6.167 0.004* Temp × P-d treatment 5 0.171 0.034 0.816 0.598 CO2 × P-d treatment 5 0.315 0.063 1.505 0.130 Temp × CO2 × P-d treatment 4 0.048 0.012 0.288 0.975 Residuals 255 10.661 0.042 Total 277 13.220 Group 4 (Met, Cys) Temp 1 0.639 0.639 12.143 0.001* CO2 1 0.103 0.103 1.953 0.124 P-d treatment 5 0.283 0.057 1.077 0.403 Temp × CO2 1 0.538 0.538 10.226 0.001* Temp × P-d treatment 5 0.161 0.032 0.612 0.810 CO2 × P-d treatment 5 0.233 0.047 0.885 0.559 Temp × CO2 × P-d treatment 4 0.187 0.047 0.889 0.554 Residuals 255 13.412 0.053 Total 277 15.555 Group 5 (Tyr, Lys) Temp 1 0.635 0.635 9.264 0.001* CO2 1 0.497 0.497 7.248 0.002* P-d treatment 5 0.433 0.087 1.264 0.229 Temp × CO2 1 0.388 0.388 5.666 0.006* Temp × P-d treatment 5 0.426 0.085 1.242 0.216 CO2 × P-d treatment 5 0.201 0.040 0.588 0.878 Temp × CO2 × P-d treatment 4 0.067 0.017 0.246 0.997 Residuals 255 17.471 0.069 Total 277 20.118

154

APPENDICES APPENDIX I

Figure S4-1. Principal component analysis of foliar amino acid data with attribute loadings on the first two components PC 1 and PC 2. Amino acid abbreviations are denoted in Table 1. Sample scores on PC 1 and PC 2 explain, respectively, 50% and 12% of the variation in the dataset. Plots were coloured according to chamber (i.e. temperature and CO2) conditions (A) and plant-damage treatment (B).

155

APPENDICES APPENDIX I

2.5 2.5 aCO2 Group 1 A Early cut Group 1 B *CO NS 2.0 eCO2 2 2.0 No cut Late cut

1.5 1.5

dry mass) dry

-1 1.0 1.0

0.5 0.5

(µmol g (µmol

Amino acid concentration acid Amino 0.0 0.0 26 30 Without aphids With aphids 4 4 Group 2 C Group 2 D *Temperature *Treatment 3 3

dry mass) dry 2 2

-1

1 1

(µmol g (µmol

Amino acid concentration acid Amino 0 0 26 30 Without aphids With aphids Temperature (°C) Treatment

Figure S4-2. Temperature, CO2 and plant-damage treatment effects on average amino acid concentrations (mean ± standard error) of group 1 (A and B) and group 2 (C and D) amino acids. Significant treatment factors according to PERMANOVA indicated * (P < 0.01).

156

Appendix II – Chapter 5 supplementary material

Table S5-1. Mixed model statistical analyses of aphid and plant responses to cultivar, temperature and CO2 treatments. Effects are illustrated in Figs. 5-1, 5-2 and S5-1.

Temperature × Cultivar × Cultivar × Temperature × Cultivar Temperature CO2 CO2 × Temperature CO2 CO2 Cultivar

Factor F P df F P df F P df F P df F P df F P df F P df

Aphid responses

Fecundity* 2.73 0.030 4,211 0.07 0.805 1,6 0.55 0.485 1,6 0.27 0.899 4,211 0.36 0.837 4,211 9.04 0.024 1,6 2.09 0.083 4,211

rm 2.97 0.021 4,189 1.68 0.243 1,6 0.45 0.527 1,6 0.55 0.700 4,189 0.08 0.987 4,189 6.43 0.044 1,6 1.74 0.143 4,189

Longevity# 4.34 0.002 4,267 0.88 0.384 1,6 6.70 0.041 1,6 2.15 0.075 4,267 0.22 0.925 4,267 1.35 0.289 1,6 1.04 0.385 4,267

Development 0.22 0.93 4,211 3.74 0.101 1,6 2.19 0.189 1,6 0.76 0.552 4,211 0.82 0.512 4,211 0.08 0.786 1,6 1.10 0.361 4,211 time Plant responses

Mass* 6.03 <0.001 4,259 50.52 <0.001 1,6 12.50 0.012 1,6 1.16 0.329 4,259 0.94 0.442 4,259 0.57 0.480 1,6 2.27 0.062 4,259

Height* 4.56 0.001 4,259 78.97 <0.001 1,6 7.21 0.036 1,6 0.08 0.988 4,259 0.20 0.936 4,259 0.15 0.786 1,6 2.17 0.073 4,259

Those highlighted in bold indicate significance (P < 0.05). Where appropriate, data were transformed (*Log, #Square root) before analysis.

APPENDICES APPENDIX II

Table S5-2. Linear mixed effect model AIC comparisons to determine the effect of cultivar, temperature and CO2 on aphid colonisation success using a binomial error structure.

Model refinement df AIC

Cult+Temp+CO 2 22 366.87 +Temp:CO2+Cult:CO2+Cult:Temp+Cult:Temp:CO2+(1|Run/Ch)

Cult+CO2+Temp +Temp:CO2+Cult:CO2+Cult:Temp+(1|Run/Ch) 18 363.38

Cult+CO2+Temp +Cult:CO2+Cult:Temp+(1|Run/Ch) 17 362.41

Cult+CO2+Temp +Cult:Temp+(1|Run/Ch) 13 357.80

Cult+CO2+Temp +(1|Run/Ch) 9 350.11

Cult+CO2 +(1|Run/Ch) 8 348.70

Cult +(1|Run/Ch) 8 353.11

CO2 +(1|Run/Ch) 4 352.42

The final model (with the lowest AIC) after stepwise deletions is highlighted in bold, with the starting model shown at the top of the table. Abbreviations are as follows: Cult (cultivar), Temp (temperature), Ch (chamber).

158

−1 Table S5-3. Single-factor effects of cultivar, temperature and CO2 treatments on individual amino acids (µmol g dry mass). Mean values (± SE) shown. HR = Hunter River, S = Sequel, T = Trifecta, A = Aurora, G = Genesis.

CO2 Temperature (°C) Cultivar Amino acid a e 26 30 HR S T A G Alanine (Ala) 1.529 ± 0.072 1.339 ± 0.060 1.444 ± 0.066 1.439 ± 0.070 1.703 ± 0.128 1.382 ± 0.090 1.541 ± 0.139 1.258 ± 0.075 1.374 ± 0.098 Arginine (Arg) 0.617 ± 0.074 0.715 ± 0.128 0.835 ± 0.130 0.474 ± 0.040 0.684 ± 0.129 0.717 ± 0.125 0.825 ± 0.202 0.639 ± 0.215 0.458 ± 0.061 Asparagine (Asn) 2.944 ± 0.398 3.432 ± 0.415 4.411 ± 0.491 1.889 ± 0.237 3.102 ± 0.627 2.810 ± 0.514 4.341 ± 0.885 2.931 ± 0.576 2.740 ± 0.577 Aspartate (Asp) 1.241 ± 0.099 1.331 ± 0.118 1.541 ± 0.114 1.001 ± 0.092 1.085 ± 0.112 1.319 ± 0.149 1.646 ± 0.235 1.237 ± 0.145 1.119 ± 0.176 Cysteine (Cys) 0.886 ± 0.086 1.077 ± 0.134 0.946 ± 0.114 1.002 ± 0.104 0.862 ± 0.131 1.100 ± 0.196 0.976 ± 0.171 1.034 ± 0.189 0.878 ± 0.157 Glutamate (Glu) 0.722 ± 0.037 0.669 ± 0.042 0.763 ± 0.037 0.625 ± 0.040 0.692 ± 0.070 0.707 ± 0.053 0.713 ± 0.073 0.754 ± 0.064 0.615 ± 0.047 Glutamine (Gln) 0.320 ± 0.023 0.287 ± 0.034 0.330 ± 0.033 0.274 ± 0.021 0.393 ± 0.076 0.309 ± 0.037 0.320 ± 0.052 0.267 ± 0.032 0.250 ± 0.020 Glycine (Gly) 0.582 ± 0.036 0.571 ± 0.031 0.603 ± 0.033 0.549 ± 0.035 0.649 ± 0.063 0.486 ± 0.040 0.689 ± 0.074 0.543 ± 0.047 0.540 ± 0.042 Histidine (His) 0.223 ± 0.017 0.253 ± 0.023 0.266 ± 0.022 0.197 ± 0.012 0.213 ± 0.040 0.222 ± 0.017 0.308 ± 0.054 0.230 ± 0.022 0.216 ± 0.022 Isoleucine (Iso) 1.008 ± 0.063 0.958 ± 0.050 0.969 ± 0.045 1.003 ± 0.071 1.019 ± 0.073 0.809 ± 0.070 1.020 ± 0.077 1.119 ± 0.127 0.954 ± 0.085 Leucine (Leu) 0.974 ± 0.056 0.905 ± 0.058 0.885 ± 0.056 1.004 ± 0.059 1.117 ± 0.118 0.886 ± 0.082 0.955 ± 0.086 0.826 ± 0.069 0.967 ± 0.098 Lysine (Lys) 0.787 ± 0.038 0.669 ± 0.035 0.779 ± 0.039 0.684 ± 0.034 0.792 ± 0.056 0.703 ± 0.059 0.777 ± 0.066 0.714 ± 0.050 0.692 ± 0.061 Methionine (Met) 0.225 ± 0.009 0.237 ± 0.009 0.244 ± 0.009 0.216 ± 0.009 0.236 ± 0.017 0.232 ± 0.016 0.242 ± 0.017 0.226 ± 0.012 0.218 ± 0.012 Phenylalanine (Phe) 0.732 ± 0.045 0.751 ± 0.067 0.690 ± 0.053 0.796 ± 0.058 0.996 ± 0.137 0.698 ± 0.091 0.741 ± 0.071 0.612 ± 0.048 0.693 ± 0.072 Proline (Pro) 1.369 ± 0.181 1.781 ± 0.316 1.410 ± 0.180 1.718 ± 0.309 2.830 ± 0.750 1.288 ± 0.215 1.758 ± 0.391 0.976 ± 0.177 1.125 ± 0.186 Serine (Ser) 1.599 ± 0.108 1.522 ± 0.090 1.488 ± 0.075 1.647 ± 0.125 2.004 ± 0.282 1.486 ± 0.121 1.662 ± 0.135 1.320 ± 0.102 1.415 ± 0.105 Threonine (Thr) 0.539 ± 0.023 0.565 ± 0.039 0.557 ± 0.032 0.544 ± 0.029 0.693 ± 0.088 0.515 ± 0.033 0.593 ± 0.040 0.477 ± 0.030 0.500 ± 0.035 Tyrosine (Tyr) 0.478 ± 0.023 0.459 ± 0.023 0.486 ± 0.022 0.451 ± 0.024 0.442 ± 0.028 0.422 ± 0.027 0.486 ± 0.035 0.541 ± 0.048 0.447 ± 0.028 Valine (Val) 0.809 ± 0.041 0.838 ± 0.048 0.811 ± 0.040 0.835 ± 0.049 1.040 ± 0.098 0.774 ± 0.058 0.879 ± 0.071 0.692 ± 0.054 0.764 ± 0.058

APPENDICES APPENDIX II

Table S5-4. Results from multivariate permutational analysis (PERMANOVA) of cultivar, temperature and CO2 treatment effects on different groups of correlated (> 0.5) amino acids. For the purpose of PERMANOVA, ungrouped individuals (Met and Cys) were grouped with the highest correlated individual amino acid. * indicates significance (P < 0.01) adjusted for absence of mixed effects.

df SS MS Pseudo-F P(perm)

All amino acids Temperature 1,214 0.657 0.657 7.776 0.001* CO2 1,214 0.087 0.087 1.029 0.366 Cultivar 4,214 0.583 0.146 1.727 0.032 Temperature × CO2 1,214 0.143 0.143 1.786 0.029 Temperature × Cultivar 4,214 0.603 0.151 1.696 0.136 CO2 × Cultivar 4,214 0.402 0.101 1.192 0.264 Temperature × CO2 × Cultivar 4,214 0.465 0.116 1.376 0.112 Group 1 (Asn, Asp, Glu) Temperature 1,214 1.948 1.948 15.152 0.001* CO2 1,214 0.190 0.190 1.474 0.201 Cultivar 4,214 0.523 0.131 1.017 0.406 Temperature × CO2 1,214 0.319 0.319 2.480 0.014 Temperature × Cultivar 4,214 1.151 0.288 2.238 0.048 CO2 × Cultivar 4,214 0.652 0.163 1.268 0.231 Temperature × CO2 × Cultivar 4,214 0.984 0.246 1.913 0.052 Group 2 (Arg, His) Temperature 1,214 0.607 0.607 5.607 0.004* CO2 1,214 0.022 0.022 0.201 0.885 Cultivar 4,214 0.484 0.121 1.117 0.334 Temperature × CO2 1,214 0.101 0.101 1.502 0.137 Temperature × Cultivar 4,214 0.650 0.163 0.932 0.438 CO2 × Cultivar 4,214 0.328 0.082 0.757 0.678 Temperature × CO2 × Cultivar 4,214 0.779 0.195 1.798 0.053 Group 3 (Ala, Gln, Gly, Leu, Lys, Phe, Pro, Ser, Thr, Val) Temperature 1,214 0.084 0.084 1.331 0.221 CO2 1,214 0.064 0.064 1.027 0.354 Cultivar 4,214 0.613 0.153 2.442 0.007* Temperature × CO2 1,214 0.061 0.061 0.967 0.359 Temperature × Cultivar 4,214 0.292 0.073 1.164 0.283 CO2 × Cultivar 4,214 0.252 0.063 1.003 0.420 Temperature × CO2 × Cultivar 4,214 0.221 0.055 0.881 0.558 Group 4 (Iso, Tyr) Temperature 1,214 0.087 0.087 1.657 0.177 CO2 1,214 0.018 0.018 0.335 0.706 Cultivar 4,214 0.410 0.103 1.962 0.071 Temperature × CO2 1,214 0.031 0.031 0.601 0.519 Temperature × Cultivar 4,214 0.163 0.041 0.779 0.609 CO2 × Cultivar 4,214 0.193 0.048 0.920 0.488 Temperature × CO2 × Cultivar 4,214 0.260 0.065 1.244 0.266 Group 5 (Cys, Lys) Temperature 1,214 0.128 0.128 1.385 0.211 CO2 1,214 0.136 0.136 1.464 0.218 Cultivar 4,214 0.190 0.048 0.513 0.857 Temperature × CO2 1,214 0.293 0.293 3.160 0.051 Temperature × Cultivar 4,214 0.271 0.068 0.732 0.654 CO2 × Cultivar 4,214 0.161 0.040 0.434 0.909 Temperature × CO2 × Cultivar 4,214 0.236 0.059 0.637 0.751 Group 6 (Leu, Met) Temperature 1,214 0.224 0.224 2.740 0.049 CO2 1,214 0.065 0.065 0.795 0.452 Cultivar 4,214 0.389 0.097 1.189 0.291 Temperature × CO2 1,214 0.104 0.104 1.271 0.246 Temperature × Cultivar 4,214 0.222 0.055 0.678 0.698 CO2 × Cultivar 4,214 0.277 0.069 0.846 0.561 Temperature × CO2 × Cultivar 4,214 0.264 0.066 0.805 0.584

160

APPENDICES APPENDIX II

0.08 aCO2 *Temperature (a) Susceptible (c) eCO *CO2 Low resistance 0.06 2 Moderate resistance a 0.04 a b b b

Mass (g) 0.02

0.00 140 *Temperature (b) (d) 120 *CO2 100

80 a a 60 b b b 40

Height (mm) 20

0 26 30 HR S T A G Temperature (°C) Cultivar

Figure S5-1. Lucerne dry mass and height between temperature and CO2 treatments (a and b) and lucerne genotypes (c and d). Mean values (± SE) are shown. Statistically significant effects are indicated by *(P < 0.05). Cultivar bars with the same letters were not significantly different (P < 0.05).

161

APPENDICES APPENDIX II

Figure S5-2. Principal component analysis of phloem and foliar amino acid data with attribute loadings on the first two components PC 1 and PC 2. Amino acid abbreviations are denoted in Table 2. Sample scores on PC 1 and PC 2 explain, respectively, 37% and 18% of the variation in the dataset. Plots and ellipses (representing 68% of the predicted data) were coloured according to the extraction technique (i.e. foliar or phloem), which did not have a significant effect on amino acids (%mol).

162

Appendix III – Chapter 6 supplementary material

Table S6-1. Mixed model results combining height data for lucerne and Harding grass over time (accounting for repeated measures by including week as a fixed effect). Grass Lucerne (Phalaris aquatica) (Medicago sativa) Factor df F P df F P Herbivore treatment 3,45 6.63 <0.001 3,45 1.13 0.347 Precipitation 2,15 70.06 <0.001 2,15 15.51 <0.001 Week 2,120 321.96 <0.001 2,264 707.16 <0.001 Herbivore treatment × Precipitation 6,45 1.65 0.156 6,45 0.42 0.865 Herbivore treatment × Week 6,120 2.23 0.045 6,264 0.54 0.776 Precipitation × Week 4,120 80.67 <0.001 4,264 100.52 <0.001 Herbivore treatment × Precipitation 12,120 0.35 0.978 12,264 0.88 0.573 × Week

163

APPENDICES APPENDIX III

Table S6-2. Results from multivariate permutational analysis (PERMANOVA) of herbivore treatment (Treatment) and precipitation effects on different groups of correlated amino acids. A statistically significant effect is indicated by *(P < 0.05).

df SS MS Pseudo-F P(perm)

All amino acids Treatment 3,60 0.107 0.036 0.737 0.716 Precipitation 2,60 0.187 0.093 1.928 0.065 Treatment × Precipitation 6,60 0.091 0.015 0.312 1.000 Group 1 (Glycine, Lysine, Methionine, Tyrosine, Cysteine, Histidine, Arginine) Treatment 3,60 0.104 0.035 0.796 0.646 Precipitation 2,60 0.192 0.096 2.205 0.024* Treatment × Precipitation 6,60 0.183 0.031 0.702 0.858 Group 2 (Isoleucine, Phenylalanine, Leucine, Threonine, Valine) Treatment 3,60 0.039 0.013 0.485 0.741 Precipitation 2,60 0.038 0.019 0.715 0.509 Treatment × Precipitation 6,60 0.034 0.006 0.213 0.994 Group 3 (Aspartate, Glutamate) Treatment 3,60 0.151 0.050 1.165 0.306 Precipitation 2,60 0.239 0.120 2.762 0.039* Treatment × Precipitation 6,60 0.134 0.022 0.516 0.871 Group 4 (Glutamine, Serine) Treatment 3,60 0.107 0.036 0.738 0.625 Precipitation 2,60 0.308 0.154 3.176 0.019* Treatment × Precipitation 6,60 0.128 0.021 0.439 0.937 Alanine Treatment 3,60 0.099 0.033 1.177 0.325 Precipitation 2,60 0.026 0.013 0.457 0.685 Treatment × Precipitation 6,60 0.178 0.030 1.058 0.392 Asparagine Treatment 3,60 0.150 0.050 0.591 0.708 Precipitation 2,60 0.385 0.192 2.277 0.087 Treatment × Precipitation 6,60 0.134 0.022 0.264 0.995 Proline Treatment 3,60 0.551 0.184 2.484 0.027* Precipitation 2,60 0.022 0.011 0.146 0.960 Treatment × Precipitation 6,60 0.345 0.057 0.777 0.621

164

APPENDICES APPENDIX III

(A) (B) 50 c 40 b ab 40 a ab b 30 30 20 20 a

Number of tillers Number Number of tillers Number 10 10

0 0 Drought Ambient Elevated CON A W WA Precipitation Herbivore treatment

Figure S6-1. The impacts of precipitation (A) and weevil presence (B) on the number of grass tillers. CON are the control plants (no insects), A have aphids alone, W have weevils alone and WA have both insects. Mean values ± standard errors are shown. Bars with the same letters were not significantly different (P < 0.05).

165

APPENDICES APPENDIX III

50 Root %C (A) Shoot %C 48 a ab 46 b

44 c c c

Grass C concentration (%) Grass C concentration

38 Drought Ambient Elevated 48 None (CON) (B) Aphids (A) Weevils (W) Both insects (WA) 46 b b b b b b b b b ab ab a

Lucerne Lucerne 44

root C concentration (%) C concentration root

42 Drought Ambient Elevated Precipitation

Figure S6-2. The impacts of precipitation on the root and shoot carbon concentrations of Harding grass (A) and the interactive effects of precipitation and herbivore treatment on the root carbon concentrations of lucerne (B). Mean values ± standard errors are shown. Bars with the same letters were not significantly different (P < 0.05).

166

APPENDICES APPENDIX III

Figure S6-3. Principal component analysis of foliar amino acid concentration data with attribute loadings on the first two components PC1 and PC2. Amino acid abbreviations are denoted in Figure S4. Plots and ellipses (representing 68% of the predicted data) were coloured according to precipitation (A) and herbivore treatments (B). CON are the control plants (no insects), A have aphids alone, W have weevils alone and WA have both insects.

167

APPENDICES APPENDIX III

Figure S6-4. Hierarchical tree of clustered amino acids. Grouped amino acids were all correlated (>0.7). Amino acid abbreviations are as follows: asparagine (asp), alanine (ala), serine (ser), glutamine (gln), glycine (gly), lysine (lys), methionine (met), tyrosine (tyr), cysteine (cys), histidine (his), arginine (arg), proline (pro), isoleucine (iso), phenylalanine (phe), leucine (leu), threonine (thr), valine (val), aspartate (asp) and glutamate (glu).

Aphids (A) c 100 Both insects (WA) bc 80 ab ab

60 a a

Plants colonised by aphids (%) aphids by Plants colonised Drought Ambient Elevated Precipitation

Figure S6-5. The interactive effects of precipitation and weevil presence on the percentage of plants colonised by aphids (± standard errors). Bars with the same letters were not significantly different (P < 0.05).

168

Appendix IV – Supplementary data in review

Dataset used for the submitted paper 'The importance of testing multiple environmental factors in legume-insect research: replications, reviewers and rebuttal' by Scott N. Johnson, Andrew N. Gherlenda, Adam Frew and James M. W. Ryalls’. Treatment % change in biomass relative to Plant No. CO2 Temperature (aphid / aphid-free) aCO2 equivalent 1 Elevated Ambient CON 52.06 2 Elevated Ambient CON 52.45 3 Elevated Ambient CON 59.00 4 Elevated Ambient CON 59.39 5 Elevated Ambient CON 61.51 6 Elevated Ambient CON 61.70 7 Elevated Ambient CON 68.84 8 Elevated Ambient CON 70.77 9 Elevated Ambient CON 78.48 10 Elevated Ambient CON 81.76 11 Elevated Ambient CON 81.76 12 Elevated Ambient CON 85.81 13 Elevated Ambient CON 98.92 14 Elevated Ambient CON 107.79 15 Elevated Ambient CON 110.10 16 Elevated Ambient CON 117.62 17 Elevated Ambient CON 128.61 18 Elevated Ambient CON 139.60 19 Elevated Ambient CON 145.58 20 Elevated Ambient CON 178.94 21 Elevated Ambient CON 201.88 22 Elevated Ambient CON 232.93 23 Elevated Ambient CON 279.01 24 Elevated Ambient APH 36.13 25 Elevated Ambient APH 43.25 26 Elevated Ambient APH 48.45 27 Elevated Ambient APH 49.55 28 Elevated Ambient APH 53.65 29 Elevated Ambient APH 54.20 30 Elevated Ambient APH 54.75 31 Elevated Ambient APH 64.33 32 Elevated Ambient APH 69.53 33 Elevated Ambient APH 71.72 34 Elevated Ambient APH 79.38 35 Elevated Ambient APH 79.38 36 Elevated Ambient APH 80.75 37 Elevated Ambient APH 82.67 38 Elevated Ambient APH 91.97 39 Elevated Ambient APH 92.52 40 Elevated Ambient APH 99.64

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APPENDICES APPENDIX IV

Treatment % change in biomass relative to Plant No. CO2 Temperature (aphid / aphid-free) aCO2 equivalent 41 Elevated Ambient APH 104.02 42 Elevated Ambient APH 112.50 43 Elevated Ambient APH 116.61 44 Elevated Ambient APH 122.63 45 Elevated Ambient APH 140.15 46 Elevated Ambient APH 148.91 47 Elevated Ambient APH 168.07 48 Elevated Ambient APH 171.08 49 Elevated Ambient APH 173.00 50 Elevated Ambient APH 176.28 51 Elevated Ambient APH 181.21 52 Elevated Ambient APH 207.49 53 Elevated Ambient APH 247.73 54 Elevated Ambient APH 294.54 55 Elevated Elevated CON 34.30 56 Elevated Elevated CON 62.97 57 Elevated Elevated CON 63.27 58 Elevated Elevated CON 64.16 59 Elevated Elevated CON 65.04 60 Elevated Elevated CON 72.73 61 Elevated Elevated CON 77.17 62 Elevated Elevated CON 84.85 63 Elevated Elevated CON 88.99 64 Elevated Elevated CON 92.24 65 Elevated Elevated CON 94.61 66 Elevated Elevated CON 95.79 67 Elevated Elevated CON 105.85 68 Elevated Elevated CON 106.73 69 Elevated Elevated CON 110.87 70 Elevated Elevated CON 111.46 71 Elevated Elevated CON 113.53 72 Elevated Elevated CON 116.78 73 Elevated Elevated CON 129.79 74 Elevated Elevated CON 136.30 75 Elevated Elevated CON 155.52 76 Elevated Elevated CON 162.02 77 Elevated Elevated CON 196.02 78 Elevated Elevated CON 210.80 79 Elevated Elevated CON 227.95 80 Elevated Elevated CON 247.76 81 Elevated Elevated CON 263.43 82 Elevated Elevated CON 312.21 83 Elevated Elevated CON 376.67 84 Elevated Elevated CON 440.82 85 Elevated Elevated CON 457.38 86 Elevated Elevated APH 35.59

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APPENDICES APPENDIX IV

Treatment % change in biomass relative to Plant No. CO2 Temperature (aphid / aphid-free) aCO2 equivalent 87 Elevated Elevated APH 49.06 88 Elevated Elevated APH 53.54 89 Elevated Elevated APH 55.79 90 Elevated Elevated APH 64.13 91 Elevated Elevated APH 71.18 92 Elevated Elevated APH 76.31 93 Elevated Elevated APH 78.23 94 Elevated Elevated APH 78.55 95 Elevated Elevated APH 89.78 96 Elevated Elevated APH 92.34 97 Elevated Elevated APH 95.87 98 Elevated Elevated APH 106.13 99 Elevated Elevated APH 106.77 100 Elevated Elevated APH 106.77 101 Elevated Elevated APH 108.69 102 Elevated Elevated APH 111.58 103 Elevated Elevated APH 112.22 104 Elevated Elevated APH 113.18 105 Elevated Elevated APH 125.36 106 Elevated Elevated APH 147.49 107 Elevated Elevated APH 147.81 108 Elevated Elevated APH 153.26 109 Elevated Elevated APH 165.12 110 Elevated Elevated APH 168.33 111 Elevated Elevated APH 178.27 112 Elevated Elevated APH 184.68 113 Elevated Elevated APH 191.73 114 Elevated Elevated APH 221.55 115 Elevated Elevated APH 239.51 116 Elevated Elevated APH 275.74 117 Elevated Elevated APH 304.92

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APPENDICES

List of Publications

Ryalls JMW, Riegler M, Moore BD, Johnson SN. 2013. Biology and trophic interactions of lucerne aphids. Agricultural and Forest Entomology 15, 335-350.

Ryalls JMW, Riegler M, Moore BD, Lopaticki G, Johnson SN. 2013. Effects of elevated temperature and CO2 on aboveground–belowground systems: a case study with plants, their mutualistic bacteria and root / shoot herbivores. Frontiers in Plant Science 4, 445.

Johnson SN, Ryalls JMW, Karley AJ. 2014. Global climate change and crop resistance to aphids: contrasting responses of lucerne genotypes to elevated atmospheric carbon dioxide. Annals of Applied Biology 165, 62-72.

Ryalls JMW, Moore BD, Riegler M, Gherlenda AN, Johnson SN. 2015. Amino acid- mediated impacts of elevated carbon dioxide and simulated root herbivory on aphids are neutralized by increased air temperatures. Journal of Experimental Botany 66, 613-623.

Ryalls JMW, Moore BD, Riegler M, Johnson SN. 2016. Above–belowground herbivore interactions in mixed plant communities are influenced by altered precipitation patterns. Frontiers in Plant Science 7, 345, doi: 10.3389/fpls.2016.00345.

Johnson SN, Gherlenda AN, Frew A, Ryalls JMW. 2016. The importance of testing multiple environmental factors in legume–insect research: replication, reviewers and rebuttal. Frontiers in Plant Science 7, 489, doi: 10.3389/fpls.2016.00489.

Johnson SN, Ryalls JMW, Staley JT. 2016. Impacts of climate and atmospheric change on aboveground–belowground invertebrate interactions. In: Johnson SN, Jones TH, eds. Invertebrates and Climate Change. Oxford, UK: Wiley, in Press.

Ryalls JMW, Harrington, R. 2016. Climate and atmospheric change impacts on aphids as vectors of plant diseases. In: Johnson SN, Jones TH, eds. Invertebrates and Climate Change. Oxford, UK: Wiley, in Press.

Ryalls JMW, Moore BD, Riegler M, Bromfield LM, Hall AAG, Johnson SN. 2016. Climate and atmospheric change impacts on sap-feeding herbivores: a mechanistic explanation based on functional groups of primary metabolites (in press). Functional Ecology doi: 10.1111/1365-2435.12715.

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APPENDICES

Experimental images

Lucerne weevil (Sitona discoideus) emerging from an egg.

Viable (black) and newly laid (white) eggs of Sitona discoideus beside a newly-hatched larva of the same species.

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EXPERIMENTAL IMAGES

Magnified pea aphids (Acyrthosiphon pisum) feeding on an experimental lucerne (Medicago sativa) leaf.

Experimental plants surrounded by water to prevent aphid movement between plants.

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EXPERIMENTAL IMAGES

Pea aphid confined to a lucerne plant using an organza bag.

Four plots covered with mesh cages underneath a rain exclusion shelter.

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APPENDICES

Short story

A cultural evolution

Part One: Pim Pim had friends, a lot of friends, but none like her. Well, in a physical sense, she was not dissimilar. Like most of the others, she was green with two banded antennae, six legs and a long stylet to feed on the phloem sap of various leguminous plants. Unlike the others, Pim had always wanted more adventure from the world, a desire she would come to regret.

Part Two: George Hours were spent in the laboratory constructing prototypes. George’s meticulous nature made it essential for him to fashion the most effective culture container possible. Having tried most of the materials available to him in the lab, George was finally satisfied with his creation: A pea aphid culture container of epic proportions. Three individual pea aphids were plucked from a nearby alfalfa field and placed within the final construct. The waiting game commenced.

Part Three: Prison Pim’s world had become dark and enclosed. She was trapped in what looked like a large plastic cage and her friends were nowhere to be seen. Two other aphids she had never seen before were looking equally bemused beside her. Before she could even think, Pim was hit by an overwhelming hunger and any reasoning behind her sudden imprisonment bore little importance in comparison. Instinct took hold as she travelled from leaf to leaf searching for an appropriate food source. She had never experienced this alfalfa variety before and could barely stomach the sap. The other aphids had migrated high above and were feeding happily on the apex of the plant but they had an advantage over Pim; they contained specialised symbiotic bacteria that enabled them to feed on this particular variety. As Pim became weak and fragile, the others grew larger and in a matter of hours they began reproducing. Without the required amino acids, Pim knew she wouldn’t last much longer. She slowly ascended the alfalfa stem

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SHORT STORY A CULTURAL EVOLUTION with what little strength she had left, probing desperately for any form of nourishment. Pim’s brittle proboscis could barely penetrate the older leaves to even confirm that sap was present. Her only hope was to make it to the apex and pray that the leaf defences of the younger foliage were more forgiving. As she reached the apex of the plant she found herself surrounded by newborn aphids. She pushed her way through the crowded crèche and plunged her proboscis into the youngest leaf. The sap tasted vulgar yet she no longer cared. Her body filled with fluid and honeydew erupted from her backside straight into the face of a newborn. Any embarrassment or concern for the little aphid was immediately trumped by Pim’s instinctive need to feed. Once she had regained some strength, however, Pim quickly became aware of her surroundings. The poor nymph that had just been almost drowned in sticky fluid was still whimpering behind her so Pim raised her head to apologise. She had barely retracted her proboscis before catching a glimpse of something alien to her. The abdomen of one of the others had exploded open and a face was emerging from within, staring directly at her with, what appeared to be, malevolent intent. At that moment, she lost her grip and fell down towards the base of the prison. This was it. This was the end. Everything went black.

Part Four: Parasites Four days had come and gone and the aphid population had octupled. “Everything was going as planned”, George confidently expressed to his supervisor. The next day, however, George checked the culture, only to find a container full of parasitic wasps. This was a disaster. One week until the aphids were finally required for experiments and the bane of the parasitoid had struck hard. In a bout of rage, he threw the failed culture out the window, immediately regretting his uncontrolled outburst. George had failed his first attempt but he didn’t give up. Keen to begin his experiment within the allotted time-frame, he constructed another culture, making a game of it in his mind, despite lingering doubts that his sanity may have been compromised.

Part Five: Salvation Pim awoke to find herself staring at the sky. A small leaf had cushioned her fall and prevented near-certain death. An eclectic mix of familiar scents and sounds filled her body with an overwhelming sense of hope and her curious antennae jolted upwards as

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SHORT STORY A CULTURAL EVOLUTION they detected fresh air and a nearby alfalfa field. She could sense the brutal odour of (E)-β-farnesene emanating from her siblings in the nearby field. This potent alarm pheromone usually made her shudder but she now welcomed the familiar scent, marching towards it with bold intent. Despite her now feeble frame she marched on with all her might. She was free from the plastic prison but far from home.

Her journey was just beginning.

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