WATER STRESS, ROOT HERBIVORY AND ABOVE-GROUND MULTITROPHIC
INTERACTIONS IN A CRUCIFER-APHID SYSTEM
MUHAMMAD TARIQ B.Sc. (Hons), M.Sc. (Hons)
A thesis submitted for the Degree of Doctor of Philosophy
of Imperial College London and the Diploma of Imperial College London
Division of Biology
Silwood Park Campus
Imperial College London
September 2010 Dedication
Dedicated To
Holy Prophet Muhammad (PBUH) The greatest Educator and Reformer
My Worthy Parents
Brothers
Sisters
&
Family Members
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Declaration
The work presented in this thesis is entirely my own and has not been submitted anywhere else.
Signed...... Muhammad Tariq
Certified...... Professor Denis J. Wright
(PhD Supervisor)
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Acknowledgements
I do not have command to praise The Almighty Allah Whose blessings are abundant and Who is our benefactor and Whose favours are unlimited. I also offer my humble gratitude from the core of my heart to The Holy Prophet Muhammad (Peace be Upon Him), who is forever a torch of guidance and source of knowledge for the whole mankind.
I wish to express my sincere gratitude to my supervisors Prof. Denis Wright and Dr.
Joanna T. Staley for their substantial guidance, continuous inspiration and invaluable assistance in bringing this dissertation to its present form. I am greatly thankful to members of my PRP, Prof. Jim Hardie and Dr. Simon Leather for their valuable suggestions, constructive criticism and encouragement throughout my research endeavour. Thanks go to
Dr. Toby J. A. Bruce at Rothamsted Research, who kindly helped with the plant volatile collection and analysis.
I wish to thank to Dr. Rosemary Collier at University of Warwick for supplying the culture of Delia radicum. I would like to thank Janet Martin at Rothamsted Research for providing the culture of Diaeretiella rapae.
I do not have words at command in acknowledging that all the credit goes to my loving father, Haji Riasat Ali and sweet mother, Hajra Begum for their amicable attitude and love, immense orison, mellifluous affections, inspiration, well wishing and keen interest which hearten me to achieve success in every sphere of life. Their prayers are the roots of my success.
Many thanks are also due to the kindness and love of my family members especially
Muhammad Afzal, Muhammad Ajmal, Mubarak Ali, Muhammad Tunveer, Muhammad Tufeq,
Muhammad Akmal, Muhammad Arif and Muhammad Sajid who always encouraged me and played a pivotal role in achieving higher goals of life.
My special love is due to the innocent prayers of my all nieces and nephews, especially, Muhammad Asad, Ali Hassan, Muhammad Ahad, Muhammad Fahad, Roshan
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Waheed, Muhammad Mudser Kabir, Muhammad Saad, Hifza Ajmal, Wajiha Waheed,
Thareem Tunveer, Rabia Waheed and Teba Ajmal.
This study was made possible by the financial support by the Higher Education
Commission of Pakistan. I am grateful for Higher Education Commission of Pakistan.
Friends are co-sharer of my struggle and my work. I also pay my special gratitude to my all friends especially Mamoon Al sarai Al alawi, Henri De Parseval, Camilo Ayra Pardo, Martyn
Powell, Faiz Ahmad, Muhammad Ahsan Khan, Jan Muhammad Mari, Asim Gulzar,
Muhammad Address, Asad Ali, Anjum Aqueel, Muhammad Ali, Sofia Khalid, Fareeda Anjum,
Khalid Zafar and Muhammad Mamoon ur Rashid who always encouraged me to reach the destination. Thanks, Silwoodians!
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Abstract
Plants, insect herbivores and their natural enemies interact in multitrophic food webs that can be influenced by various stress factors. The aim of the present study was to understand the effects of two different below-ground stress factors (drought and root herbivory) on the performance of two above-ground herbivores, a generalist (Myzus persicae) and a specialist (Brevicoryne brassicae) aphid species and two of their natural enemies, a generalist (Aphidius colemani) and a specialist (Diaeretiella rapae) parasitoid species. Preliminary experiments with various Brussels sprout (Brassica oleracea) cultivars
(cv) showed that aphid performance was influenced both by host plant on which the parent aphid had been reared and by the host plant on which it was feeding when reproducing. The
Brussels sprout cv. Oliver was selected as a consistently good aphid host for subsequent experiments. Root herbivory and drought stress were shown to significantly affect the outcome of plant-mediated interactions between insect herbivores and their parasitoids. High drought stress had a negative impact on the aphid species and their parasitoids and also reduced performance of a root herbivore, the cabbage root fly, Delia radicum. Root herbivory alone or in combination with drought significantly altered foliar herbivore performance. High drought stress combined with high D. radicum density had a negative impact on foliar herbivore performance and parasitoid preference and performance. Plant volatiles and parasitoid preference were shown to decrease with drought stress and root herbivory. Plant foliar glucosinolate, nitrogen and carbon concentrations were significantly affected by drought stress. Relative water content and plant biomass were significantly affected by both stresses. The present study on a Brassica-aphid system underlines the importance of root herbivory and drought stress in mediating interactions between plant, foliar herbivores and their parasitoids. The implications of these findings for multitrophic interactions under potential climate change are discussed with particular reference to pest management.
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TABLE OF CONTENTS
Dedication...... 2
Declaration...... 3
Acknowledgements...... 4
Abstract...... 5
Table of Contents...... 6
CHAPTER 1: General introduction
1.1 Trophic interactions...... 11
1.2 Effect of climate change on ecosystems...... 12
1.3 Aim and objectives of the present study...... 16
CHAPTER 2: Review of literature
2.1 Effect of water stress on insect-plant interactions...... 19
2.1.1 Effect of water stress on aphid-plant interaction...... 20
2.1.2 Plant Stress, Plant Vigour, Pulsed Stress hypotheses...... 22
2.2 Aphids...... 24
2.2.1 Polymorphism and maternal host effects in aphids...... 24
2.2.2 Myzus persicae and Brevicoryne brassicae...... 26
2.2.3 Host plant resistance in relation to M. persicae and B. brassicae...... 27
2.3 Cabbage root fly, aphids and water stress...... 28
2.4 Plant-herbivore-parasitoids interaction……………………………………………………….28
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CHAPTER 3: General materials and methods
3.1 Glasshouse conditions...... 33
3.2 Plant materials...... 33
3.3 Insect cultures...... 34
3.3.1 Insect rearing room conditions...... 34
3.3.2 Aphid cultures...... 34
3.3.3 Parasitoid cultures...... 34
3.3.4 Root fly culture...... 35
3.4 Aphid performance...... 35
3.5 Measurement of Plant physiology and chemistry parameters...... 36
3.5.1 Relative water content...... 36
3.5.2 Total foliar nitrogen and carbon concentration...... 36
3.5.3 Glucosinolate analysis...... 37
3.5.4 Isolation of plant volatile compounds...... 38
3.6 Data analysis...... 38
CHAPTER 4: Maternal host plant effects on aphid performance: contrasts between a generalist and a specialist species on Brussels sprout cultivars.
4.1 Introduction...... 40
4.2 Materials and methods...... 42
4.2.1 Plant production and insect cultures...... 42
4.2.2 Performance experiments...... 42
4.2.3 Data analysis...... 43
4.3 Results...... 43
4.3.1 Effective fecundity...... 43
4.3.2 Intrinsic rate of increase...... 46
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4.4 Discussion...... 49
CHAPTER 5: Aphids in a changing world: testing the plant stress, plant vigour and pulsed stress hypotheses.
5.1 Introduction...... 52
5.2 Materials and methods...... 53
5.2.1 Preliminary drought stress treatment trial...... 53
5.2.2 Experiment treatments and insect cultures...... 54
5.2.3 Aphid performance...... 54
5.2.4 Foliar relative water content and plant biomass...... 55
5.2.5 Foliar chemistry...... 55
5.2.5.1 Total leaf nitrogen and carbon concentration...... 55
5.2.5.2 Glucosinolate analysis...... 55
5.2.6 Data analysis...... 56
5.3 Results...... 56
5.3.1 Aphid performance...... 56
5.3.2 Plant biomass and relative water content...... 58
5.3.3 Foliar chemistry...... 60
5.3.3.1 Total leaf nitrogen and carbon concentration...... 60
5.3.3.2 Glucosinolate analysis...... 61
5.4 Discussion...... 63
CHAPTER 6: Drought and root herbivory modify foliar herbivore performance.
6.1 Introduction...... 66
6.2 Materials and methods...... 67
6.2.1 Plants and insect culture...... 67
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6.2.2 Experimental procedure...... 67
6.2.2.1 Preliminary experiment...... 67
6.2.2.2 Drought and root herbivore experiment...... 68
6.2.2.3 Aphid performance...... 68
6.2.3 Plant physiological measurements...... 69
6.2.4 Data analysis...... 69
6.3 Results...... 69
6.3.1 Aphid performance...... 69
6.3.1.1 Fecundity...... 69
6.3.1.2 Intrinsic rate of increase...... 72
6.3.2 Relative water content...... 74
6.3.3 Foliar biomass...... 75
6.3.4 Root biomass...... 76
6.4 Discussion...... 76
CHAPTER 7: Drought modifies trophic interactions above and below ground.
7.1 Introduction...... 79
7.2 Materials and methods...... 80
7.2.1 Study species...... 80
7.2.2 Experimental treatments...... 81
7.2.3 Parasitoid preference...... 82
7.2.4 Parasitoid performance...... 83
7.2.5 Plant volatiles...... 84
7.2.6 Root herbivore performance...... 84
7.2.7 Data analysis...... 85
7.3 Results...... 85
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7.3.1 Parasitoid preference...... 85
7.3.2 Parasitism performance...... 86
7.3.2.1 Percentage parasitism...... 86
7.3.2.2 Percentage emergence...... 87
7.3.2.3 Sex ratio...... 88
7.3.2.4 Female tibia length...... 89
7.3.2.5 Adult longevity...... 91
7.3.3 Plant volatile emissions...... 92
7.3.4 Root herbivore performance...... 93
7.4 Discussion...... 94
CHAPTER 8: Summary and General Discussion
8.1 Summary of experimental findings...... 98
8.2 General Discussion...... 100
8.2.1 Host plant quality under biotic and abiotic stress...... 100
8.2.2 The role of host plant quality in insect trophic system...... 100
8.2.3 Management implications...... 102
8.3 Future work...... 103
References...... 105
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Chapter 1
General introduction
1.1 Trophic interactions
Biotic and abiotic stresses induce physiological, morphological and chemical changes in terrestrial plants that can generate bottom-up trophic cascades from plants to higher trophic levels and thereby alter community dynamics and the composition of terrestrial communities (Ohgushi, 2005; 2008; Erb et al., 2008; Trotter et al., 2008; Utsumi et al., 2009; van der Putten, 2009). Trophic interactions play a central role in the response of ecosystems to environmental changes and they occur at all trophic levels within ecological communities (van der Putten et al., 2004; 2010). Trophic interactions may be affected by constitutive plant traits, induced responses generated by different herbivore species and different secondary consumers responding to different plant traits (Rasmann & Turlings,
2007; 2008).
Trophic networks involve above- and below-ground herbivore interactions that can be influenced by either or both interactions at the same time and frequently involve plant nutrients and complex plant defences involving secondary metabolites (Masters et al., 1993; van der Putten et al., 2001; Bezemer & van Dam, 2005; Erb et al., 2008). The allocation of primary metabolites to above- or below-ground tissues make plants necessary resources for above- or below-ground herbivores which provide resources for higher trophic levels.
Therefore, above- and below-ground trophic systems interact through primary plant products and plant defence (Wardle et al., 2004; Bezemer & van Dam, 2005).
Herbivores often cause extensive damage to their host plant, which accumulates with multiple herbivore attack. Plants are simultaneously challenged by above- and below-ground herbivores and these have the potential to influence each other through a variety of direct
12 and indirect interactions (Gange & Brown, 1989; Masters et al., 1993; van der Putten et al.,
2009; Hladun & Adler, 2009). Below-ground herbivores may strongly affect the quality and quantity of resource availability to foliar herbivores. The indirect effects of below-ground herbivores on above-ground herbivores may be positive (Gange & Brown, 1989; Masters &
Brown, 1992; Masters et al., 1993; Masters, 1995a; 1995b; Olson et al., 2008), negative
(Tindall & Stout, 2001; van Dam et al., 2005; Soler et al., 2005; Staley et al., 2007b; Wurst & van der Putten, 2007) or neutral (Moran & Whitham, 1990; Salt et al., 1996). There is limited data on the extent to which above-ground herbivores and their preference and performance are dependent on below-ground herbivores and vice versa (Masters et al., 1993; Wolters et al., 2000; Blossey & Hunt-Joshi, 2003; Sinka et al., 2007; Erb et al., 2008).
Below-ground herbivores may increase water stress in plants (Smith, 1977), and thus induce changes in plant physiology that are similar to drought (Gange & Brown, 1989).
Similarly drought may alter the plant physiology that influences insect performance, diversity and abundance (Wearing & van Emden, 1967; White, 1984; Mattson & Haack, 1987;
Srivastava, 2005; Bale et al., 2007) and ultimately have profound effects on higher trophic levels (Sumerford et al., 2000; Calatayud et al., 2002; Trotter et al., 2008). Thus, drought stress may strengthen or weaken the positive or negative effects below-ground herbivores can have on foliar herbivores depending upon the system under investigation (Staley et al.,
2008).
1.2 Effect of climate change on ecosystems
One of the most important factors that directly or indirectly influence the function of an ecosystem is climate. Changes in climate can be violent and catastrophic but even slower and more subtle change can decisively affect the structure of the ecosystem (Peters, 1992;
Lovejoy & Hannah, 2005; Montoya & Raffaelli, 2010). The frequency and intensity of extreme events are expected to increase as Earth‟s climate changes, and these changes could occur even with relatively small mean climate changes. Changes in some types of 13 extreme events have already been observed, for example, increases in the frequency and intensity of heat waves and heavy precipitation events (Bates et al., 2008). Even small-scale events of rapid change in modern times have resulted in disruption of natural and agriculture ecosystems (Alley et al., 2003; Walther, 2010).
The global climate is expected to change as a result of anthropogenic increases in atmospheric greenhouse gases (Houghton et al., 2001; Solomon et al., 2007). The average global atmospheric concentration of CO2 increased by 30% between 1750 and 2000
(Houghton et al., 2001), trapping more solar energy in the lower atmosphere and this is one of the main causes of changes in climate (Hulme et al., 2002; Hulme & Broadmeadow,
2002). According to predictions, increasing concentrations of CO2 in the atmosphere will lead to higher temperatures (Solomon et al., 2007). Similarly, global warming should increase ocean and land evaporation. In principle, evaporation increases precipitation in both summer and winter in high latitude regions, but higher temperatures reduce soil moisture (Kallis, 2008; Briffa et al., 2009). Decreased snow cover and land-ice extent are expected to follow from the trend in increasing temperature (Meehl et al., 2005). The annual maximum extent of sea ice surrounding the Antarctic continent and in the Arctic seas appears to be declining; inland glaciers throughout Europe and elsewhere have receded
(Houghton & Woodwell, 1989; Kallis, 2008; Bates et al., 2008). Such extreme, large scale climatic events will probably be important drivers of change in the organization and functional characteristics of ecosystems (Wilhite & Buchanan-Smith, 2005), with extreme events such as drought becoming more common.
Drought is relatively understudied compared to other climate change parameters such as CO2 concentration and temperature, despite having potentially large effects on community structure and composition (Bale et al., 2002). However, few studies have considered the importance of drought in multitrophic interactions in the context of climate change, as the majority of studies have focused on changes in temperature and carbon dioxide (Bezemer & Jones, 1998; Bale et al., 2002; Thomson et al., 2010). Therefore, drought has been chosen as a climate parameter to investigate its impact with other stresses
14 in multitrophic interactions. Meteorologically, drought is a term used for a scarcity of water and implies a range of different stresses with which plants and other organisms have to cope. Stress is the altered physiological condition caused by any factor of the environment that tends to alter equilibrium (Shao et al., 2009). Drought occurs in many parts of the world and affects many countries on an annual basis (Wilhite & Buchanan-Smith, 2005; McCrary &
Randall, 2010). The areas affected by severe drought evolve gradually (Figure 1.1), and regions of maximum intensity shift from season to season (Wilhite & Buchanan-Smith,
2005).
Under current climate change predictions, Europe would suffer droughts on a vastly greater scale and frequency than those that occurred in the summer of 2003 (Linden, 2006;
Bates et al., 2008; Briffa et al., 2009), which records suggest was the warmest since 1500
(Luterbacher et al., 2004). The British Isles have been experiencing a tendency for wetter winters and drier summers since the mid-1800s (Jones & Conway, 1997). In recent decades, there has also been a steepening of the northwest to southeast precipitation gradient (Jones
& Conway, 1997; Mayes, 2000). The magnitude of the impact drought has is closely related to the timing of its onset, its intensity and duration (Wilhite & Buchanan-Smith, 2005). UK climate change scenarios suggest that these temporal and spatial trends will continue into this century, with average annual run off expected to decrease in southeast England by as much as 50% by 2080 (Hulme et al., 1998).
Brabson et al. (2005) predicted that the maximum mean temperature will increase from 19.5°C to 25°C in eastern England under the high emissions (A2) scenario. The A2 scenario assumes a continuation of the rapid increase in CO2 emissions from its present level of 8 gigatonnes of C (billions of metric tons) / year to 29 gigatonnes / year while the B2 scenario represents a slower CO2 emissions increase to 13 gigatonnes / year by the end of
st the 21 century (Brabson et al., 2005). With A2 scenario, the levels of soil moisture are likely to decrease and low soil moisture is increasingly regarded as a potential contributor to heat waves and drought (Brabson et al., 2005).
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A2 scenario B2 scenario
(Where HadCM3 climate model from Met Office Hadley Centre, UK and ECHAM4 climate model from Max Planck Institut für Meteorologie (MPI) and the Deutsches Klimarechenzentrum (DKRZ), Germany)
Figure 1.1 Change in the recurrence of droughts in the next 100 years, based on comparisons between climate and water use in 1961–1990 and simulations for the 2020s and 2070s ECHAM4 and HadCM3 climate models for SRES A2 and B2 ( Bates et al., 2008).
SRES = Special Report Emissions Scenarios; The A2 scenario assumes a continuation of the rapid increase in CO2 emissions while the B2 scenario represents a slower increase in st CO2 emissions by the end of the 21 century.
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Given the number of ways in which drought can have profound consequences for ecosystems, potentially affecting the species richness (Sternberg et al., 1999; van der Putten et al., 2010) and composition (Morecroft et al., 2004) of plant communities. Consequently, drought is also likely to have a significant effect on the life history parameters, dispersal, population density and movement of phytophagous insects (Mattson & Haack, 1987; Kareiva et al., 1993; Morschel, 1999; Azerefegne et al., 2001; Shone et al., 2006; Li et al., 2007).
1.3 Aim and objectives of the present study
The aim of present study was to work towards a better understanding of the effects of soil-based factors, drought and root herbivory, on insect herbivores and their natural enemies to help develop improved integrated crop management systems. A conceptual model illustrating host plant interactions between a foliar herbivore, root herbivore, parasitoids and drought and possible mechanisms involved is shown in Figure 1.2.
The specific objectives were:
1) To assess the influence of two maternal host plant species (from different genera within the same family) on the performance of second generation (G2) alates and apterous morphs of a generalist (Myzus persicae) and a specialist (Brevicoryne brassicae) aphid species feeding on five Brussels sprout cultivars (cvs).
2) To assess the performance (fecundity and intrinsic rate of increase) of both aphid species on these cultivars to determine which cultivar was best suited for use in subsequent experiments.
3) To test the Plant Stress (White, 1984), Plant Vigour (Price, 1991) and Pulsed Stress
Hypotheses (Huberty & Denno, 2004) by exposing plants to a range of water treatments and aphid performance, leaf water contents, plant biomass, N, C and glucosinolate contents. The
Plant Stress Hypothesis predicts that insect performance increases on drought stressed
17 plants due to increased availability of nutrients and/or a decrease in defensive compounds.
The Plant Vigour Hypothesis predicts that vigorous plants are more suitable hosts for herbivores than drought-stressed plants owing to high availability of nutrients. The Pulsed
Stress Hypothesis predicts better performance of phloem feeders on pulsed water-stressed plants due to increases in availability of N and recovery of turgor compared with continuously stressed plants.
4) To assess the effects of a plant root herbivore (Cabbage root fly, Delia radicum) and drought stress on the performance of M. persicae and B. brassicae. It is hypothesized that the performance of M. persicae and B. brassicae would respond negatively to changes in the host plant induced by root herbivory and drought stress.
5) To conduct multifactorial tritrophic (parasitism success) experiments with selected combinations of drought treatments using both a generalist (Aphidius colemani) and a specialist (Diaeretiella rapae) parasitoid species. For plant-mediated herbivore and parasitoid interactions it was hypothesised that drought and root herbivory will both induce a plant stress response which will reduce parasitoid preference and performance on these plants.
6) Where significant tritrophic differences occur in multifactorial experiments, to use whole plant olfactometry to determine whether plant volatiles are involved, and where significant differences occur to entrain plant volatiles for analysis.
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Parasitoids
Generalist parasitoids Specialist parasitoids
Aphids
Generalist aphid Positive Specialist aphid or Limited food resources negative response
Negative Increase response or decrease performance
Brussels sprout Changes in plant physiology (relative water contents, foliar and root biomass)
Plant stress/plant vigour Cabbage root fly & Pulse stress hypotheses
Drought (Climate change)
Figure 1.2 Model demonstrating the possible multitrophic interactions between foliar, root herbivore, parasitoids and drought. (Original, with plates from www.mizfrogspad.com/.../brussels_sprouts.gif; www.pestspotter.co.uk/pests/ pests. htm; drought.unl.edu/.../ Arkansas/WoollyHollow1.htm; www.aphidweb.com/.../Myzuspersicae.htm).
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CHAPTER 2
Review of literature
2.1 Effect of water stress on insect-plant interactions
In relation to plants, biologists consider stress as any abiotic or biotic factor in the environment that affects plant physiology, chemistry, growth and development in such a way that plants or organisms associated with plants perform below the average (Heinrichs,
1988). Stress can also be defined as any environmental conditions that have measurable negative effects on plant performance (Hsiao, 1973; Louda & Collinge, 1992; Shao et al.,
2009). Most biotic and abiotic stresses (disease, insect, pathogen, temperature, drought, salinity, anaerobic conditions, etc.) faced by plants may directly (Bale et al., 2007; Nguyen et al., 2007) or indirectly (Porter et al., 1991; Cammell & Knight, 1992) affect herbivorous insects.
Arthropods feeding on plants face nutritional hurdles in obtaining sufficient energy, N, other nutrients and water (Scriber & Slansky, 1981). Plant water content, frequently overlooked as a nutrient, can also have a major influence on insect performance (Scriber,
1977; 1979; Reese & Beck, 1978; Paul et al., 1992; Janssen, 1994). Micro-environmental conditions of plants or plant parts that insects inhabit have significant effects on insect performance (Soo Hoo & Fraenkel, 1966; Watt, 1979; McClure, 1980; Leather & Dixon,
1981; Holtzer et al., 1988) and fitness (Reese & Beck, 1978; Nguyen et al., 2007). In addition, stress conditions such as drought have an indirect impact on insect herbivores by altering the defence metabolism of the host plant and the nutritional suitability (Bruyn et al.,
2002; Huberty & Denno, 2004; Nykanen & Koricheva, 2004).
Much of the world‟s land mass is exposed to water deficits ranging from semi-arid to extremely arid (Simpson, 1981; Loucks et al., 2006). Moisture stress affects several plant
20 processes that result in changes in the physical and chemical composition of plant tissues
(Hsiao, 1973; Hsiao et al., 1976; Barlow et al., 1980; Kozlowski, 1982; Hanson & Hitz, 1982;
Hanson & Weltzin, 2000; Adams & Kolb, 2005; McDowell et al., 2008). The severity and duration of water stress directly influence several plant physiological processes, including the nutritional quality of the plant as a food for insects (Hsiao, 1973; Begg & Turner, 1976;
Kozlowski, 1976; Mattson, 1980; Mattson & Haack, 1987; Louda & Collinge, 1992) and may make the plant more susceptible to insect attack (White, 1969; Cobb et al., 1997). Variation in plant quality can affect the performance and abundance of herbivores and their parasitoids (Mattson, 1980; Awmack & Leather, 2002; Wardle et al., 2004; White, 2008).
Drought may promote phytophagous insect outbreaks (White, 1969; 1976; 1984;
2008; Mattson & Haack, 1987; Li et al., 2007; Zhang et al., 2009) that could enhance survivorship of insects by elevating plant nutrient levels (van Emden & Bashford, 1969;
Mattson & Haack, 1987; Showler & Castro, 2010) and reducing plant defences (Mattson &
Addy, 1975; Rhoades et al., 1983; White, 1984; Rhoades, 1985; Garrett et al., 2006).
2.1.1 Effect of water stress on aphid-plant interactions
The interaction between an aphid and its host plant has two main phases; phloem location and sieve element acceptance. Both can be affected by imposition of stress on the plant (Bale et al., 2007). Aphids usually use the same host for successive generations. They adapt to the plant as it grows and develops to maturity (Nguyen et al., 2007). The effects of host plant water stress on aphids have been shown empirically to be complex. Both population increases and decreases have been attributed to water stress (Kennedy et al.,
1958; Holtzer et al., 1988). Aphids are highly specialized phloem sap consumers and have a more subtle and sustained relationship with host plants than insect defoliators (Douglas,
1998; 2003).
Aphids are particularly sensitive to the nutrient profile of their host plants (Karley et al., 2002; Douglas & van Emden, 2007). The concentration of plant nutrients is modified as a
21 result of water stress (Hale et al., 2003; Bale et al., 2007), which may affect herbivore performance either positively (Wearing, 1967; 1972a; Miles et al., 1982; White, 1984; 2008;
Mattson & Haack, 1987; Mopper & Whitham, 1992; Waring & Cobb, 1992; Oswald & Brewer,
1997), negatively (Kennedy et al., 1958; Cabrera et al., 1995; Hale et al., 2003; Huberty &
Denno, 2004), or not at all (Pons & Tatchell, 1995; Bethke et al., 1998; Williams & Cronin,
2004; King et al., 2006).
Plants must maintain a lower water potential than the environment to maintain turgor pressure, which is essential to their function. Drought can alter the composition of the phloem solutes through the process of osmotic adjustment (Morgan, 1984). This is achieved through the uptake of osmotically active organic and inorganic solutes within cells (Iannucci et al., 2002). Growing plant cells tend to accumulate organic solutes (Johnson et al., 1996), whereas mature tissues can accumulate both types of solute (Patakas et al., 2002). The degree of osmoregulation undergone by plant tissue will also depend on both level of drought stress imposed and tissue age (Pritchard et al., 1991). Thus, osmotic pressure can influence aphid performance indirectly by changing the composition of the phloem sap.
Turgor pressure does not have a significant effect on aphid performance as aphids are able to feed at pressures ranging from negative to fully turgid (Bale et al., 2007).
Plant and leaf age (Ibbotson & Kennedy, 1950; Wearing & van Emden, 1967;
Wearing, 1972b), plant species (Agele et al., 2006), aphid species (Wearing & van Emden,
1967; Wearing, 1967; 1972a; King et al., 2006), and the level and duration of water stress
(Thomas & Hodkinson, 1991; English-Loeb et al., 1997; Huberty & Denno, 2004) are also known to affect the impact of water stress on aphid performance, including survival, growth, development and reproduction (Bultman & Bell, 2003; Huberty & Denno, 2004; King et al.,
2006). For example, the fecundity of B. brassicae decreases on water-stressed Brussels sprouts (Wearing & van Emden, 1967).
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2.1.2 Plant Stress, Plant Vigour and Pulsed Stress Hypotheses
White (1974; 1984; 2008) formulated the Plant Stress Hypothesis, which asserts that drought stressed plants are a higher-quality food for insects than non stressed plants.
Drought stressed plants can, therefore, lead to insect outbreaks. Such outbreaks of herbivorous insects are probably due to changes in plant physiology, particularly available N during times of prolonged water deficit and/or changes in the ratio of nutrients to chemical defence compounds. Drought has also been reported to lead to higher concentration of sugar in sieve elements (White, 1974; Mattson & Haack, 1987), resulting in high C:N ratios and high osmotic pressure. It is this high C:N ratio that is one of the problems with sieve element as a food source (Bale et al., 2007). The changes in herbivore activity and performance are often restricted by N, not by C (Hartley et al. 2000; Awmack & Leather
2002).
The Plant Stress Hypothesis generalized that herbivores eat nutritionally inadequate food (White, 1984). Plant physiological effects can have different consequences depending upon how a particular herbivore encounters and uses the plant. Thus, insect species with different feeding habits might respond differently to stress induced changes in plant tissue
(Figure 2.1) (Larsson, 1989). Plant nutrition, allelochemistry and plant growth have different effects on different feeding guilds. In this context, phloem and cambium feeders are predicted to respond more positively to plant water stress than chewing and gall-forming insects (Larsson, 1989). The Plant Vigour Hypothesis predicts that herbivores will prefer the vigorous plants in a population and/or the most vigorous parts (newly emerging leaves, shoots, petioles) within a plant for oviposition and feeding (Price, 1991). Plant vigour has been shown to have a significant effect on herbivore abundance but not on survivorship
(Cornelissen et al., 2008).
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STRESS LEVEL
Figure 2.1 Hypothetical representation of insect performance in relation to host-tree stress level for different insect feeding guilds (Larsson, 1989).
According to a meta-analysis conducted by Huberty and Denno (2004) stressed plants exhibit elevated foliar N that adversely affects the performance of aphids but has no significant effect on leaf chewing insects, borers or leaf miners. To explain the discrepancy between the observed abundance of phytophagous insects on water-stressed plants in the field and the negative effects detected in many experimental studies where plants are continuously stressed, Huberty and Denno (2004) proposed the Pulsed Stress Hypothesis whereby bouts of stress followed by the recovery of turgor allow sap-feeders to benefit from stress-induced increases in plant N. Thus, phloem-feeding insects would respond positively on intermittently stressed plants but exhibit poor performance on continuously stressed ones. It is however, not clear how environmental changes affect N availability within the plant
(Huberty & Denno, 2004; 2006). According to this hypothesis, pulsed water stress may enhance or reduce insect herbivore performance depending on the stress intensity (Mody et al., 2009).
24
Low leaf water content as a result of drought may increase the concentration of amino acids (Girousse & Bournoville, 1994; Garg et al., 2001; Hale et al., 2003) and carbohydrates in phloem sap (Garg et al., 2001), which is critical for balanced aphid nutrition
(Mattson & Haack, 1987; Bultman & Bell, 2003; Huberty & Denno, 2004). Aphid species can have different preferred feeding sites on the same plant (Qureshi & Michaud, 2005), with consequences for both phloem localization and composition. As a result of this dietary imbalance, plant N content is thought to play a central role in mediating herbivore feeding choices and population dynamics (Mattson, 1980).
2.2 Aphids
2.2.1 Polymorphism and maternal host effects in aphids
Aphids are important pest insects throughout the world (Minks & Harrewijn, 1987;
Cole, 1996; 1997c; Hulle et al., 2010), with northern temperate regions reporting the highest pest status (Dixon, 1998; Hulle et al., 2010). Aphids damage plants directly and by transmitting plant viruses (Brault et al., 2010). Aphids can have a particularly high rates of population growth, made possible by their cyclical vivipary and parthenogensis on suitable host plants (Blackman, 1974; Dixon, 1998; Blackman & Eastop, 2000). This viviparous mode of reproduction can result in single aphids giving rise to clonal lineages infesting vast areas within a few weeks (Kennedy & Stroyan, 1959; Dixon, 1998). Most aphid species are highly host specific (Dixon, 1987; Cole, 1994).
Aphids exhibit environmentally-induced dimorphism (polyphenism) or genetically- determined dimorphism (polymorphism), whereby individual aphids are either winged or wingless (Braendle et al., 2006). The winged (alate) and wingless (apterous) phenotypes in aphids differ in a range of morphological, physiological, behavioural and life history features
(Mackay & Wellington, 1975; Braendle et al., 2006). They have evolved extensive variation in form and life cycle patterns for exploitation of resources on short-lived host plants through a complex system of polyphenism, each form having a different function (Dixon, 1998;
25
Braendle et al., 2006). This alternative polyphenism system ensures their survival (Kennedy
& Stroyan, 1959; Dixon, 1998).
In most species of aphids, the condition of the sexual forms with respect to wing development has become fixed and the males and females that occur within species are usually all either alates or apterae (Johnson & Birks, 1960). The alate morphs mostly leave the plants on which they were born to colonize new plants and therefore play a major role in host selection, whereas apterae mostly stay on the plant on which they are born (Kunert et al., 2005; Powell et al., 2006). The alates of M. persicae and B. brassicae settle preferentially on the plant apex but apterous and to some extent, alate M. persicae select the most nutritious lower leaves (Wearing, 1972b).
Environmentally-induced parental effects are considered as manifestations of intergenerational phenotypic plasticity (Lacey, 1991; Mousseau & Dingle, 1991; Schmitt et al., 1992; Agrawal et al., 1999) and intergenerational acclimation (Fox et al., 1995). Adaptive environmentally-induced parental effects include seasonal changes in the parental environment (Zehnder et al., 2007) which induce changes in parental hormones that subsequently affect offspring diapause (Mousseau & Dingle, 1991; Mousseau et al., 1991) and host plant effects on survivorship of insects (Fox et al., 1997; Olivares-Donoso et al.,
2007).
Adaptive maternal effects are common in nature, including in insects (Mousseau et al., 1991; Fox et al., 1995; Mousseau & Fox, 1998; Zehnder et al., 2007). Maternal effects in insects are as diverse and complex as the species and traits that exhibit them (Mousseau et al., 1991; Olivares-Donoso et al., 2007). Crowding, temperature and photoperiod can be predictable indicators of future environmental quality, for example, nutritional quality of a host plant, that determine their suitability for insect development. Thus maternal and even grandmaternal effects often stimulate the production of diapausing or flying progeny in insects (Zehnder et al., 2007; Yanagi & Tuda, 2010).
For herbivorous insects, host plants represent heterogeneous resources due to temporal and spatial variability in both host suitability and availability. The host plant species
26 on which a mother develops can have dramatic consequences for the phenotype of her offspring (Rossiter, 1991; Rossiter et al., 1993; Wise et al., 2008), especially affecting observed patterns of host suitability (Gould, 1988; Fox et al., 1995; 1997) and subsequent population dynamics (Rossiter, 1994), mediated via maternal or parental effects. In addition to environmental characters predictive of future host quality, the host-plant condition can affect the production of alate offspring in aphids, often mediated through maternal effects
(Sutherland, 1969; Leather, 1989).
In some aphid species, particularly those that alternate between herbaceous and woody hosts, the proportion of alates produced increases as host-plant quality decreases
(Forrest, 1970). Johnson (1966) found that aphids which have been reared on different hosts produce significantly different numbers of alate and apterous progeny. Parent aphids can be influenced both by the host on which they fed during their development, and by the host on which they are currently feeding while reproducing (Johnson & Birks, 1960; Johnson, 1966).
Variation in numbers of alate and apterous progeny may affect life history parameters such as fecundity, longevity and survival of aphids (Khan & Port, 2008).
2.2.2 Myzus persicae and Brevicoryne brassicae
The cabbage aphid Brevicoryne brassicae L. (Homoptera: Aphididae) is an oligophagous crucifer specialist (Kennedy, 1958; Hughes, 1963; Lamb, 1989; Gabrys et al.,
1997) while the peach-potato aphid Myzus persicae Sulzer (Homoptera: Aphididae) is highly polyphagous (Day & Irzykiewicz, 1953; Kennedy, 1958; Theunissen, 1989). These aphids are vectors of several viruses including cabbage black ring spot, cabbage ring necrosis, cauliflower mosaic, radish mosaic and turnip mosaic virus (Hughes, 1963; Ellis et al., 1998;
Moreno et al., 2005; Brault et al., 2010).
Both of these aphid species are important and cosmopolitan pests of horticultural brassicas. Brevicoryne brassicae is one of the most damaging and common pests on temperate brassica crops (Hughes, 1963; Singh et al., 1994; Cole, 1997b); feeding almost
27 exclusively on the phloem sap of Brassica or other closely related plant genera (Singh et al.,
1994; Cole, 1997b). Myzus persicae has a worldwide distribution, and infests hundreds of species from 40 plant families (Blackman & Eastop, 2007) and has approximately twice the feeding rate of B. brassicae (Day & Irzykiewicz, 1953). Damage to Brassica crops by both of these aphid species follows contrasting patterns; B. brassicae is relatively more successful on the young leaves and is primarily an apex feeder, whereas M. persicae normally colonise the ageing leaves of Brassica plants (Kennedy, 1958; Wearing, 1972a; Gabrys et al., 1997).
Control of these pests has largely relied on treatment with carbamate, organophosphate (OPs), pyrethroid and neonicotinoid insecticides. Crops may require several treatments to keep them free of aphids during a season (Dewar, 2007; Jeschke &
Nauen, 2008; Ahuja et al., 2010). This overreliance and overuse of insecticides has led to high levels of resistance in pest populations including aphids (Foster et al., 2007). To overcome the resistance problem, pesticide companies spend millions of dollars in developing new compounds. Insecticide resistance has led growers to increase dosage and/or frequency of application and also increases cost of production. These increases enhance the rate of resistance evolution and result in the pesticide treadmill (Pedigo & Rice,
2009). Resistance has been reported to conventional insecticides, insect growth regulators, microbial insecticides and new chemistry insecticides. Insecticide resistance may lead toward other problems such as pest resurgence and secondary pest outbreaks (Pedigo &
Rice, 2009).
2.2.3 Host plant resistance in relation to M. persicae and B. brassicae
Painter (1951) defined resistance of plants to insect attack as the relative amount of heritable qualities possessed by the plant that influence the ultimate degree of damage done by the insect. Plant resistance can be measured only by comparison with other genotypes.
Due to its variability, it can be modified by physical, chemical and biological factors (Auclair,
1989; van Emden, 2007; Pedigo & Rice, 2009). The multiplication of aphids and the
28 development of an infestation are highly dependent on one or on all of these factors (van
Emden et al., 1969; van Emden, 1991; 2007; Wellings & Ward, 1994).
For phytophagous insects, host plants represent heterogeneous resources due to temporal and spatial variability in host quality and availability (Fox et al., 1995; Olivares-
Donoso et al., 2007). Plant cultivars can vary with respect to various physiological and morphological traits, including trichome number, tissue toughness, leaf shape, leaf structures, wax contents, secondary plant substances, primary metabolite content and number of genes involved (Khan et al., 1986; Sandström & Pettersson, 1994; Panda &
Khush, 1995; Castro et al., 1996; Legrand & Barbosa, 2000; Gniwotta et al., 2005; Goggin,
2007; Gorb et al., 2008; Campitelli et al., 2008). All these factors may have direct or indirect effects on herbivore performance (van Emden, 2007; Khan & Port, 2008).
Each variety and cv of B. oleracea has its own pattern of resistance against different
Brassica insect pests (Pimentel, 1961; Radcliffe & Chapman, 1966; Ellis et al., 1996; 1998;
Cole, 1997b). Varietal differences in Brussels sprout include waxy and glossy leaves (Way &
Murdie, 1965), antixenosis and antibiosis resistance (Dunn & Kempton, 1972; Ellis et al.,
1996), and concentration of glucosinolates (Cole, 1996; 1997c).
2.3 Cabbage root fly, aphids and drought stress
Ecologists have a far better knowledge of above-ground than below-ground organisms, despite a greater proportion of global diversity existing below-ground (Hooper et al., 2000; Wolters et al., 2000). Plants are simultaneously challenged by root and shoot herbivores (Gange & Brown, 1989; Masters et al., 1993; Masters, 1995b; van der Putten et al., 2001; Wurst & van der Putten, 2007; Kaplan et al., 2008; Hladun & Adler, 2009). Root herbivores can affect plant growth (Karban, 1980; Borowicz et al., 2005; Yang & Karban,
2009), reproduction (Müller-schärer, 1991; Masters et al., 1993; 2001; Maron, 1998; Hladun
& Adler, 2009), density (Müller-schärer, 1991; Müller-schärer & Brown, 1995) and nutrient status (Bardgett et al., 1999; Ayres et al., 2007; Newingham et al., 2007) and thus may
29 strongly affect the quality and quantity of resource availability to foliar herbivores. The impact of root herbivores on the performance of foliar herbivores can be positive (Gange & Brown,
1989; Masters & Brown, 1992; Masters, 1995a; 1995b; Kaplan et al., 2008; Wurst et al.,
2008; Olson et al., 2008), negative (Tindall & Stout, 2001; Wackers & Bezemer, 2003;
Bezemer & van Dam, 2005; Soler et al., 2005; van Dam et al., 2005) or neutral (Moran &
Whitham, 1990).
The cabbage root fly, Delia radicum L. (Diptera: Anthomyiidae) is a specialized, economically important, root-feeding Brassica pest found in the Paleartic regions of the world including the UK (Ellis et al., 1999). The larvae of D. radicum cause damage to seedlings and the roots of young plants during feeding (Finch & Ackley, 1977; Dosdall et al.,
1994; Jensen et al., 2002; Felkl et al., 2005; Ahuja et al., 2010), and can cause significant loss in flowering, seed production, yield and plant biomass (McDonald & Sears, 1991;
Dosdall et al., 1994; Blossey & Hunt-Joshi, 2003). Below-ground herbivores may produce a variety of responses within plants (Section 1.1) that directly influence above-ground herbivores (Masters & Brown, 1992; Masters et al., 1993; Masters, 1995a; Tindall & Stout,
2001; Bezemer & van Dam, 2005; Olson et al., 2008; Erb et al., 2008; Johnson et al., 2008).
Thus, root herbivory could have a positive or negative or neutral effect on foliar herbivores and drought stress may strengthen or weaken this effect depending upon the system under investigation (Staley et al., 2008). Above ground herbivores may produce a variety of responses within plants that directly and indirectly influence the below ground herbivores‟ behaviour and performance (Finch & Jones, 1989; Blossey & Hunt-Joshi, 2003; Soler et al.,
2007a; Johnson et al., 2008; Kaplan et al., 2008; Sinka et al., 2009).
2.4 Plant-herbivore-parasitoids interaction
Aphids are commonly attacked by two main groups of hymenopteran parasitoids, the
Aphelinidae and the Aphidiinae. The subfamily Aphidiinae (Hymenoptera: Braconidae)
30 comprises the highest number of species of aphid parasitoids (Mackauer & Starý, 1967) including Aphidius colemani (Viereck) (Hymenoptera: Braconidae) and Diaeretiella rapae
(McIntosh) (Hymenoptera: Braconidae). These are strictly endophagous parasitoids, with three larval stages, the final one of which kills the host (O'Donnell, 1987). They pupate within the mummified aphids. After emergence the female can oviposit in aphids without mating
(only males are produced by unmated females) or feeding, however, feeding can increase longevity and fecundity of the parasitoids (Godfray, 1990; England & Evans, 1997; Lee et al.,
2004; Heimpel & de Boer, 2008; Luo et al., 2010).
Aphidius colemani is a generalist parasitoid with a broad range of hosts (Stary, 1975) and is an important biological control agent against Aphididae, targeting primarily
M. persicae (Messing & Rabasse, 1995). Diaeretiella rapae is regarded as a specialist parasitoid of Brassica-feeding aphids, such as B. brassicae (Wilson & Lambdin, 1987; Pike et al., 1999). It performs better against Brassica specialist aphids than the generalist aphid,
M. persicae (Blande et al., 2004; 2007).
As discussed, plants encounter multiple environmental stresses that affect growth and change internal reallocation of resources (Joern & Mole, 2005), which directly affect the host-plant quality and quantity. This variation in host-plant quality and quantity can have differential effects on insect herbivores and their associated natural enemies (Calatayud et al., 2002; Teder & Tammaru, 2002; Gols & Harvey, 2009). The different mechanisms by which variation in plant quality can influence the preference and performance of parasitoids are shown in Figure 2.2.
Herbivore performance may increase under stressful environmental conditions such as drought (Section 2.1). Similarly the behaviour and performance of natural enemies can be influenced by their host, host diet and environmental factors including water stress (Vinson,
1976; Kalule & Wright, 2002; Bayhan et al., 2007; White, 2008; Gols & Harvey, 2009).
Parasitoid performance and preference depends on the ecology and physiology of both insect host and host plant. They may thus be particularly sensitive to changes in the environmental conditions (Hunter, 2003; Hance et al., 2007). The effects of drought stress
31 on insects are well documented (Wearing & van Emden, 1967; Hale et al., 2003; Staley et al., 2006) but the indirect effects on parasitoids are less understood (Calatayud et al., 2002;
King et al., 2006).
Herbivore Herbivore density size Plant morphology: Herbivore foraging and access growth rate: apparency
Volatile organic compounds: Herbivore attraction or repulsion chemistry or vigour
Variation in plant quality
Figure 2.2 Mechanisms by which variation in plant quality can influence the preference and performance of parasitoids. Mechanisms can include: (1) attraction or repulsion to plant volatiles, (2) plant morphology that influences parasitoid foraging or access to herbivores, (3) effects of plant quality on herbivore density, (4) effects on the size of available herbivores, (5) effects on the growth rate and apparency of herbivores and (6) effects on the chemistry of herbivore tissues or the vigour of herbivores (Hunter, 2003).
Plants have evolved a wide spectrum of defence mechanisms to protect themselves against herbivorous arthropods, including inducible defences that directly affect the physiology or behaviour of herbivores, and indirect induced defences that enhance the effectiveness of natural enemies of herbivores (Arimura et al., 2005; Ohgushi, 2005; Felton,
2008; Dicke & Baldwin, 2010). Indirect defences can be divided into three main categories:
(a) volatile organic compounds that attract natural enemies of herbivores (Turlings et al.,
1990; Vet & Dicke, 1992; Dicke et al., 2009); (b) the provision of shelter such as ant‟s nests in hollow thorns (Bluthgen & Wesenberg, 2001; Edwards et al., 2009); (c) food supplements such as extrafloral nectar for ants and parasitic wasps (Wackers & Bezemer, 2003;
Bruinsma & Dicke, 2008). Glucosinolates, alkaloids, protein inhibitors, cyanogenic glucosides and phenolics compounds are examples of direct defences (Bennett &
Wallsgrove, 1994; Fahey et al., 2001; Wu & Baldwin, 2009), while terpenes, benzenoids, phenylpropanoids, fatty acid derivatives and amino acid derived metabolites are examples of
32 volatile organic compounds (VOCs) (Hatanaka et al., 1987; Dudareva et al., 2004; Matsui,
2006; Mumm et al., 2008).
Plants release a large number of VOCs after herbivore attack and emission of these compounds is influenced by various type of stress (Dicke & Loreto, 2010; Peñuelas &
Staudt, 2010; Heil & Karban, 2010), including temperature and drought (Fink & Volkl, 1995;
Vallat et al., 2005; Vickers et al., 2009; Loreto & Schnitzler, 2010), and pathogens and herbivores (Dicke & Hilker, 2003; Agrawal & Konno, 2009; Holopainen & Gershenzon,
2010).
33
CHAPTER 3
General materials and methods
3.1 Glasshouse conditions
In the present studies, all Brassica plants used for experiments and insect cultures were grown in a glasshouse with a minimum temperature of 20 ± 2 °C during the day (16 h) and 14 °C at night (8 h). In the glasshouse, additional light (mercury halide and sodium) was supplied to ensure a minimum light intensity of 300 Wm-2 during the day.
3.2 Plant materials
The following Brassica species were used:
Chinese cabbage (Brassica chinensis L. var pekinensis cv. Wonk Bok) (Kings
Seeds, Colchester, UK)
Cabbage (Brassica oleracea L. var. capitata L. cv. Derby Day) (Tozer Seeds
Ltd, Surrey, UK),
Brussels sprout (Brassica oleracea L. var. gemmifera (DC.) Zenker
The Brussels sprout cultivars used were:
(I) Oliver (Nickey‟s Nursery Ltd, Kent, UK)
(II) Darkmar 21 (Suffolk Herbs, Colchester, UK)
(III) Fillbasket (Sutton Seeds, Paignton, UK)
(IV) Red delicious (Sutton Seeds, Paignton, UK)
(V) Winter Harvest (Sutton Seeds, Paignton, UK)
Chinese cabbage, cabbage plants and above mentioned Brussels sprout cultivars were used to choose host plants for future experiments (Chapter 4). Brussels sprout cv
34
Oliver plants were subsequently used for drought stress (Chapter 5), root herbivory (Chapter
6) and multifactorial (Chapter 7) experiments. All plants were grown in pots (10 cm diameter) in John Innes No. 2 (Fargro Ltd, Littlehampton, UK) compost in a glasshouse. Seeds were sown at regular intervals to ensure a continuous supply of plant material for experiments and also for maintaining insect cultures.
3.3 Insect cultures
3.3.1 Insect rearing room conditions
Insects used in cultures and experiments were reared in controlled temperature (CT) rooms maintained at 20 ± 2 °C with 65-75% relative humidity and a 16 h photoperiod.
3.3.2 Aphid cultures
Myzus persicae and B. brassicae were obtained from long-term cultures at Silwood
Park. Both aphid species were reared separately on 6-week-old Chinese cabbage and cabbage for varietal trials on Brussels sprout (Chapter 4). For ditrophic and tritrophic experiments (Chapters 5-7), both aphid species were reared separately on 6-week-old
Brussels sprout cv Oliver. The aphids were sub-cultured fortnightly and transferred to fresh plants.
3.3.3 Parasitoid cultures
Aphidius colemani originating from commercial stocks from Just Green (Burnham-on-
Crouch Essex, UK) and D. rapae from IACR-Rothamsted (Harpenden, UK) were reared on separate cultures of M. persicae and B. brassicae, respectively. The parasitoids were established on aphid species for at least two generations before use in the experiments
(Douloumpaka & van Emden, 2003) to minimize maternal host plant effects.
35
3.3.4 Root fly culture
Delia radicum pupae were provided from University of Warwick (UK) and reared on swede (Brassica napus L. var. napobrassica) using the method described by Finch and
Coaker (1969). Swede was obtained from Tesco Ltd. To prevent induction of diapause, all stages of the root fly were maintained at a temperature 20 ± 2 °C with 65% relative humidity.
The adult flies were fed on 10 % honey solution absorbed on a pad of cotton wool. The females laid eggs around a small cube of swede set in a shallow dish of sand. The eggs were removed after every 2 days by flotation on water and filtration on fine nylon mesh. They were transferred with a soft brush on to the washed swede in wet sand. Maximum hatching was obtained when the eggs were kept on wet swede. The wet sand provided sufficient moisture for egg hatch and the larvae were protected from desiccation by the moisture released by root tissue. Thirty days after the inoculation, the pupae were collected and transferred into new cages. One kg of swede was enough for 600-700 larvae (Finch &
Coaker, 1969).
3.4 Aphid performance
Three clip cages were fitted to the first, second and third fully-developed leaf on each plant. Each clip cage had three aphids. After 16-18 h (overnight) aphids were removed leaving one nymph per leaf. For each individual aphid, the pre-reproductive time (time from birth to first reproduction) and the number of offspring produced in an equal number of days
(Md) were recorded and the offspring removed from each cage.
Data from both aphid species were recorded for the following parameters:
1) the effective fecundity, by allowing the aphids to reproduce during a period
equivalent to the pre-reproductive time;
2) the intrinsic rate of increase rm = 0.738 (lnMd) / d (Wyatt & White, 1977)
where Md = the effective fecundity, d = the pre-reproductive time.
36
3.5 Measurement of plant physiology and chemistry parameters
3.5.1 Relative water content
One fully-expanded intact leaf close to the centre of each plant was collected and the relative water content of that leaf was measured for each plant using the method outlined by
Barrs and Weatherley (1962) and Grace (1997).
Foliar relative water content (RWC) is a measure of fresh leaf water content, and was calculated by the following equation:
fresh weight – dry weight RWC = turgid weight – dry weight
Foliage from controlled condition pot experiment was excised using sharp scissors and assessed within 2 min. of removing the leaf from the plant. Five, 5 mm diameter leaf discs were cut from the leaf using a cork borer, avoiding the midrib. The discs were weighed fresh, and then floated on distilled water for 24 h at 2 °C in a dark cold store before being weighed again in a turgid state. Finally, they were oven dried at 80 °C for 24 h and then weighed to obtain the dry weight. The remaining plant biomass was dried at 80 °C for 24 h and weighed.
3.5.2 Total foliar nitrogen and carbon concentration
For N and C analysis, leaf samples were excised and stored at -20 °C before freeze drying and milled by using a Mixer Mill MM 400 (Retsch GmbH, Haan, Germany). Total foliar
N and C was determined by an oxidative combustion method (Wilson, 1990) in a FlashEATM
1112 analyser (ThermoScientific, TOWN, USA).
37
3.5.3 Glucosinolate analysis
One fully-expanded intact leaf close to the centre of each plant from a separate batch was collected, immediately flash-frozen in liquid nitrogen and stored at -80 °C. The samples were lyophilized, and each sample was dissolved in methanol. Desulphoglucosinolates were extracted using benzyl glucosinolate as the internal standard (Kazana et al., 2007).
Individual glucosinolates were analyzed by High-performance liquid chromatography (HPLC) according to the method of Heaney et al. (1986). 10 µl of the glucosinolate extract was run on Hewlett-Packard Lichrocart. The analysis was performed in a pre-equilibrated C18 reversed-phase fully endcapped column (synergy 4 µ Hydro-RP, 150-200 mm, 4 µm particles), in Agilent Technology 1200 binary pump series (Agilent, UK) and the separation was achieved on autosampler Hewlett-Packard Agilent 1100 series (Agilent, UK), column oven and diode array detector. High-performance liquid chromatography was performed on the samples using the software Chemstation for LC 3D Systems (Copyright© Agilent
Technologies). Individual HPLC runs were carried out using a water (Solvent A) acetonitrile
(Solvent B) gradient. The desulphoglucosinolates were eluted by the following gradient: 2%-
25% (v/v) acetonitrile for 15 min., a gradient of 25%-70% (v/v) acetonitrile for 12 min., 70%
(v/v) acetonitrile for 2 min., a gradient of 70% to 2% (v/v) acetonitrile for 2 min. and a final wash in 2% (v/v) acetonitrile. All runs were carried out at a variable wavelength detector (229 nm) with a flow rate of 0.2 µl min-1 and 35 0C. The peaks were identified on the basis of their retention times and relative to the internal standard (benzyl glucosinolate). Glucosinolate concentrations were quantified as μ mole g-1 dry weight (Cole, 1997c).
3.5.4 Isolation of plant volatile compounds
Air entrainment of plants was performed as described by Webster et al. (2008). Each plant was enclosed in a 190 x 100 mm glass vessel. This glass vessel was open at the bottom and closed at the top except with two ports (inlet and outlet). Two semicircular
38 aluminium plates with a central hole to accommodate the stem were used to seal the bottom and air was pumped in through a charcoal filter with an airflow of 400 ml/min. A Porapak Q
(Alltech Associates Inc., Carnforth, UK) adsorbent glass tube (5 mm) with 50 mg Porapak Q was inserted into the outlet port and air was drawn through this tube at airflow rate 300 mlmin-1. This difference in airflow rate was used to create positive pressure to ensure that unfiltered air was not drawn into the vessel from outside. Twelve week-old plants were used for the collection of plant volatile compounds.
GC-MS (Hewlett-Packard 6890) was performed for the identification of compounds as described by Webster et al. (2008). GC-MS was equipped with a cold on-column injector, a flame ionization detector (FID), a non-polar HP-1 bonded-phase fused silica capillary column (50 m × 0.32 mm i.d., film thickness 0.52 μ) and a polar DB-WAX column (30 m ×
0.32 mm i.d., film thickness 0.82 μ). The carrier gas was hydrogen. A 1-μl aliquot solution that contained equal quantities of both enantiomers of the compounds in redistilled hexane was injected onto the chiral GC to establish that successful separation of enantiomers took place. This was followed by co-injections of the air entrainment sample, first with an authentic standard of one enantiomer and then with the second enantiomer. Peak enhancement with either enantiomer confirmed the presence of that enantiomer in the air entrainment sample.
3.6 Data analysis
Where possible, data were analysed using parametric statistics (ANOVA) after log or square root transformation if necessary. Models were simplified by removing variables that did not improve the explanatory statistical power as described by Crawley (2005; 2007).
Posthoc Tukey HSD tests were performed for the determination of significant differences between treatment levels (Crawley, 2005; 2007). All statistical analyses were performed with
R, versions 2.6.0 and 2.10.1 (R Development Core Team, 2008; 2010). (Team, 2008;
2010)
39
Chapter 4
Maternal host plant effects on aphid performance: contrasts between a generalist and a specialist species on Brussels sprout cultivars.
4.1 Introduction
Maternal effects are common in nature and often have dramatic consequences for the growth and development of progeny. For herbivorous insects, host plants represent heterogeneous resources as a result of temporal and spatial variability in both host suitability and availability. Host plant species on which a mother develops can have dramatic consequences for the phenotype of her offspring (Rossiter, 1991; Rossiter et al., 1993), particularly affecting observed patterns of host suitability (Gould, 1988; Fox et al., 1995;
1997) and subsequent offspring performance (Rossiter, 1994) as mediated via maternal effects.
The winged and wingless phenotypes in aphids differ in a range of morphological, physiological, life history and behavioural features (Braendle et al., 2006), as well as in their pre-reproductive and reproductive periods (Mackay & Wellington, 1975). The cabbage aphid
B. brassicae, is an oligophagous crucifer specialist (Kennedy, 1958; Lamb, 1989), whereas the generalist aphid M. persicae is highly polyphagous (Day & Irzykiewicz, 1953; Kennedy,
1958). Brevicoryne brassicae feeds almost exclusively on the phloem sap of Brassica spp. or other closely-related crucifers (Singh et al., 1994; Cole, 1997a). Myzus persicae infests hundreds of plant species from 40 families (Blackman & Eastop, 2007) and normally colonises the ageing leaves of Brassica plants. Brevicoryne brassicae is more successful on the young leaves and is primarily an apex feeder (Kennedy, 1958).
40
Brassica oleracea subsp. oleracea (subsequently referred to as B. oleracea) is the native wild form of many cultivated vegetables, such as Brussels sprout (Brassica oleracea
L. var. gemmifera (DC.) Zenker) and cabbage (Brassica oleracea L. var. capitata cv. Derby
Day) (Gómez-Campo & Prakash, 1999). The sowing time of brassicas varies for spring and autumn crops in terms of early or late harvest and their life spans differ. The life spans of
Chinese cabbage and cabbage are shorter (50 - 85 days) than Brussels sprout (90 - 150 days). Because of the shorter life span of the former, the aphids may move from the parent host to another host (e.g. Brussels sprout). Furthermore, M. persicae and B. brassicae are multivoltine species and are found in the field throughout the year. In this context, it is interesting to investigate the movement of aphids between different cultivars within the same genera or between different genera.
The abundance and quality of food resources are likely to be an important determinant of performance, although reproductive success might also be influenced by variation in maternal investment in offspring quality (Wainhouse et al., 2001; Awmack &
Leather, 2002; Gols et al., 2008b; McLean et al., 2009). Parent aphids can be influenced both by the host on which they fed during their development, and by the host on which they are currently feeding when reproducing. Previous studies conducted in aphids have shown that alate and apterous morphs respond differently to experimental treatments, including the rearing host (Leather, 1989; Awmack & Leather, 2002). No work has however been carried out with respect to the performance of alate and apterous morphs of M. persicae and
B. brassicae reared on different host species when feeding on a range of cultivars of another species. In the present study, the influence of two maternal host plant species (from different genera within the same family) on the performance of second generation (G2) of alates and apterous morphs of a generalist (M. persicae) and a specialist (B. brassicae) aphid species feeding on five Brussels sprout cultivars was investigated. The second objective was to compare Brussels sprout cultivars in order to choose host plants for future experiments. In this chapter, the term “maternal” does not refer specifically to effects arising from the mother but to long-term adaptation to different host plants over several previous generations.
41
4.2 Materials and methods
4.2.1 Plant production and insect cultures
Chinese cabbage, cabbage and five cultivars of Brussels sprout (Oliver, Darkmar 21,
Fillbasket, Red delicious and Winter Harvest) were grown as described in Section 3.2. For the varietal trials with Brussels sprout, the stock cultures of M. persicae and B. brassicae were reared separately on 6-week-old Chinese cabbage and cabbage. Each stock culture rearing unit had three plants and the aphids were sub-cultured fortnightly and randomized for
12 generations on fresh 6-week-old plants to minimize the single maternal host plant effect.
Insect cultures were maintained as described in Section 3.3.2.
4.2.2 Performance experiments
Experiments were carried out on 6-week-old Brussels sprout plants (8-10 true leaves). Eight plants of each cultivar were used for both M. persicae and B. brassicae and randomised in plastic trays on benches in the insect rearing unit. An overview of the experimental design is provided in Figure 4.1. Four clip cages were fitted to the underside of the first, second, third and fourth fully-expanded leaves on each plant to give 32 cages per cultivar. Three reproductive adult apterae (G1 apterae) of each aphid species from each source were released into each of 16 clip cages on the first and third leaves, and three reproductive adult alates (G1 alates) were released into each of 16 clip cages on the second and fourth leaves. This placing was chosen because B. brassicae is primarily an apex feeder, whereas M. persicae normally colonises the ageing leaves of Brassica plants
(Kennedy, 1958). After 16-18 h (overnight), the aphids were removed except for one nymph per cage, which was not the first nymph produced as these can be atypical (Murdie, 1965).
The number of offspring produced was recorded daily and removed from each clip cage. The pre-reproductive time was calculated separately for each individual and used as an average
42 per plant. The data obtained for the G2 of alates and apterae comprised as described in
Section 3.4.
Stock culture of M. persicae and B. brassicae on Chinese cabbage
Stock culture of M. persicae and Stock culture of M. persicae and B. brassicae for three generations B. brassicae for three on Chinese cabbage generations on cabbage
Randomized fortnightly over twelve Randomized fortnightly over twelve generations on Chinese cabbage generations on cabbage
Alates (G1) Apterae (G1) Alates (G1) Apterae (G1)
G2 on Brussels G2 on Brussels G2 on Brussels G2 on Brussels sprouts cultivars sprouts cultivars sprouts cultivars sprouts cultivars
Figure 4.1 Diagrammatical illustration of experimental method. First generation (G1) of both aphid species reared on Chinese cabbage or cabbage transferred to Brussels sprout cultivars to produce the second generation (G2). Aphid performance was assessed at G2 on five Brussels sprout cultivars (Oliver, Darkmar 21, Fillbasket, Red delicious and Winter Harvest).
4.2.3 Data analysis
Data for effective fecundity and intrinsic rate of increase were analysed in R, version
2.6.0 (R Development Core Team, 2008) using three-way analysis of variance to test the effects of maternal host plant, maternal morph and Brussels sprout cultivar on aphid performance. Post-hoc Tukey honestly significant difference tests were performed as described in Section 3.6.
43
4.3 Results
4.3.1 Effective fecundity
Differences referred to in the text are always significant (P<0.05). Both aphid species reared on different sources (Chinese cabbage or cabbage) differed substantially with respect to fecundity and intrinsic rate of increase on a range of Brussels sprout cultivars. For both aphid species, the fecundity of the G2 offspring of alate morphs reared on Chinese cabbage was significantly greater on all Brussels sprout cultivars compared with the G2 offspring of apterous morphs reared on Chinese cabbage or either morph from cabbage (Figures 4.2 &
4.3). The greatest fecundity for both aphid species was on cv. Fillbasket for the combination of the G2 offspring of alate morphs reared on Chinese cabbage, whereas the lowest fecundity was on cv. Red delicious for the G2 offspring of apterae morphs reared on cabbage
(Figures 4.2 & 4.3). There was a trend for the G2 offspring of alates from Chinese cabbage to have greater fecundity compared with aphids from cabbage; these differences were significant for alates of both aphid species on cultivars Fillbasket, Red delicious and Winter
Harvest (Tables 4.1 & 4.2). The fecundity of both apterae and alates were similar on cv.
Oliver.
44
Table 4.1 Analysis of variance of effective fecundity (Md) of the second generation (G2) offspring of alate and apterous morphs of Myzus persicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars.
Source of variation d.f. Sum Sq Mean Sq F value
Rearing plants 1 24.7648 24.7648 1803.17*** Cultivars 4 20.0532 5.0133 365.03*** G2 of alates and apterae 1 2.2526 2.2526 164.02*** Rearing plants x Cultivars 4 14.6852 3.6713 267.31*** Rearing plants x G2 of alates and apterae 1 3.0376 3.0376 221.17*** Cultivars x G2 of alates and apterae 4 0.2137 0.0534 3.89** Rearing plants x Cultivars x G2 of alates and apterae 4 0.2511 0.0628 4.57** ***P< 0.001; **P< 0.01; *P< 0.05
Table 4.2 Analysis of variance of effective fecundity (Md) of the second generation (G2) offspring of alate and apterous morphs of Brevicoryne brassicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars.
Source of variation d.f. Sum Sq Mean F value Sq
Rearing plants 1 823.56 823.56 1081.34*** Cultivars 4 1248.45 312.11 409.81*** G2 of alates and apterae 1 432.31 432.31 567.62*** Rearing plants x Cultivars 4 210.83 52.71 69.21*** Rearing plants x G2 of alates and apterae 1 184.90 184.90 242.78*** Cultivars x G2 of alates and apterae 4 11.08 2.77 3.64** Rearing plants x Cultivars x G2 of alates and 4 5.02 1.26 1.65 apterae ***P< 0.001; **P< 0.01; *P< 0.05
45
Figure 4.2 Effective fecundity of the second generation (G2) offspring of alate and apterous morphs of Myzus persicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars (mean ± SEM). The different aphid forms (Apterae or Alates) and host plants (Chinese cabbage or cabbage) refer to the G1 generation.
Figure 4.3 Effective fecundity of the second generation (G2) offspring of alate and apterous morphs of Brevicoryne brassicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars (mean ± SEM). The different aphid forms (Apterae or Alates) and host plants
(Chinese cabbage or cabbage) refer to the G1 generation.
46
4.3.2 Intrinsic rate of increase
The intrinsic rate of increase of the G2 of alate and apterous morphs of M. persicae and B. brassicae (Table 4.3 & 4.4) differed significantly between the rearing plant species as well as between cultivars of Brussels sprout. The greatest intrinsic rate of increase of
M. persicae was observed on cv. Darkmar-21 for the G2 of alate morphs reared on cabbage
(Figure 4.4) but, for B. brassicae the intrinsic rate of increase was greatest on cv. Fillbasket for the G2 of alate morphs reared on Chinese cabbage (Figure 4.5). On cultivars Fillbasket,
Red delicious and Winter Harvest, there was no significant effect on the intrinsic rate of increase of M. persicae, irrespective of whether the maternal aphid was an alate or an aptera (Table 4.3 & 4.4). The intrinsic rate of increase for the G2 of alates of B. brassicae from Chinese cabbage was significantly different on all Brussels sprout cultivars. The intrinsic rate of increase differed significantly between the G2 of alates and apterae reared on either cabbage species on Brussels sprout cultivars Oliver and Darkmar-21 (M. persicae) and cultivars Red delicious and Winter Harvest (B. brassicae).
Table 4.3: Analysis of variance of intrinsic rate of increase (rm) of the second generation
(G2) offspring of alate and apterous morphs of Myzus persicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars.
Source of variation d.f. Sum Sq Mean Sq F value
Rearing plants 1 0.000166 0.000166 4.92* Cultivars 4 0.061370 0.015343 454.63*** G2 of alates and apterae 1 0.002649 0.002649 78.49*** Rearing plants x Cultivars 4 0.032434 0.008108 240.27*** Rearing plants x G2 of alates and apterae 1 0.004379 0.004379 129.75*** Cultivars x G2 of alates and apterae 4 0.000317 0.000079 2.35 Rearing plants x Cultivars x G2 of alates and 4 0.000182 0.000045 1.35 apterae ***P< 0.001; **P< 0.01; *P< 0.05
47
Table 4.4: Analysis of variance of intrinsic rate of increase (rm) of the second generation
(G2) of offspring alate and apterous morphs of Brevicoryne brassicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars.
Source of variation d.f. Sum Sq Mean Sq F value
Rearing plants 1 0.0080940 0.0080940 704.16*** Cultivars 4 0.0122315 0.0030579 266.03*** G2 of alates and apterae 1 0.0016512 0.0016512 143.65*** Rearing plants x Cultivars 4 0.0019531 0.0004883 42.48*** Rearing plants x G2 of alates and apterae 1 0.0031506 0.0031506 274.10*** Cultivars x G2 of alates and apterae 4 0.0000639 0.0000160 1.39 Rearing plants x Cultivars x G2 of alates and apterae 4 0.0001101 0.0000275 2.40 ***P< 0.001; **P< 0.01; *P< 0.05
Figure 4.4 Intrinsic rate of increase of second generation (G2) of alate and apterous morphs of Myzus persicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars (mean ± SEM). The different aphid forms (Apterae or Alates) and host plants (Chinese cabbage or cabbage) refer to the G1 generation.
48
Figure 4.5 Intrinsic rate of increase of second generation (G2) of alate and apterous morphs of Brevicoryne brassicae reared on Chinese cabbage (Brassica chinensis var. pekinensis) and cabbage (Brassica oleracea var. capitata) on five Brussels sprout cultivars (mean ± SEM). The different aphid forms (Apterae or Alates) and host plants (Chinese cabbage or cabbage) refer to the G1 generation.
4.4 Discussion
The Brussels sprout cv. as well as the differential host-plant adaptation over up to 15 previous generations influenced the performance of B. brassicae and M. persicae, although differences were not always consistent between the G2 of alates and apterae. The host plant on which an apterous mother feeds during larval development may strongly influence adult fecundity in response to the diet that she receives (Sutherland, 1969). Maternal effects could affect population dynamics by influencing offspring performance (Zehnder et al., 2007).
There can be a number of plant factors responsible for the specificity of the aphid - plant interaction (Will & Van-Bel, 2006). Aphids have telescoping generations, which means that within a single aphid, there are embryos within embryos; embryos may therefore be affected directly by the host plant species of their mother or grandmother (Spitzer, 2004).
49
The fecundity of the G2 of alate morphs of both aphid species reared on Chinese cabbage was significantly greater on all Brussels sprout cultivars than the G2 of apterae morphs reared on Chinese cabbage or either morph from cabbage. These results are similar to those obtained by Leather (1989), who found that alate aphids (Rhopalosiphum padi) produce offspring that are more fecund on good quality host plants than the offspring of apterous mothers. A good quality host plant is defined as a better nutritional host that has positive effects on insect performance and fitness as described in various studies (Amarillo-
Suarez & Fox, 2006; McLean et al., 2009). Kvenberg and Jones (1974) found similar results in relation to the progeny produced by alate parents of M. persicae compared with apterous parents, and Dunn and Kempton (1972) found that some Brussels sprout cultivars possessed antibiosis that prolonged the time for offspring to mature and decreased the fecundity of the apterae. In the present study, both aphid species performed better on cv.
Oliver compared with other Brussels sprout cultivars. Various studies (Pimentel, 1961; Singh et al., 1994; Ellis et al., 1996; Cole, 1997a) have reported similar results for cv. Oliver and describe it as a standard susceptible host for culturing generalist as well as specialist aphids.
Aphids can have a dramatic negative effect on their host plants partly as a result of their capacity for extremely rapid population growth (Goggin, 2007). Maternal effects usually influence the ability of progeny to withstand competition, starvation or other environmental stresses (Fox & Czesak, 2000). In the present study, there was a trend for aphids from
Chinese cabbage to have greater fecundity compared with aphids from cabbage, although this was less marked for B. brassicae and was most apparent for alates. Variation in fecundity of alate and apterae morphs from Chinese cabbage or cabbage on Brussels sprout cultivars suggests that there are differences in nutritional and possible secondary chemical contents. This variation in reproduction is influenced by host plant species (Vereschagina &
Shaposhnikov, 1998), host plant quality or host plant age (Sutherland, 1969; Gols et al.,
2008b). Mittler & Dadd (1966) showed that single chemical differences in food could produce changes in aphid development and that this effect is mediated both via the mother and directly on the nymphs.
50
The overall lowest mean intrinsic rate of increase of M. persicae was on cv. Red delicious and the highest rate was on cv. Darkmar-21 and Oliver. Dunn and Kempton (1971) found similar results for cv. Darkmar-21. Myzus persicae from Chinese cabbage performed similarly to aphids from cabbage, except on cv. Darkmar-21. These differences in fecundity as well as in intrinsic rate of increase between the G2 of alates and apterae might relate to maternal age and their birth sequence as a result of some inherent age-dependent variability in maternal physiology as well as the Brussels sprout cultivars possessing a variable concentration of amino acids and glucosinolates (Cole, 1997a). For M. persicae, the fluctuation in the number of alates and apterae is related to maternal age and food source
(Mittler & Daad, 1966), and this effect is likely to be sufficient to influence offspring performance (Karimzadeh et al., 2004). The present study demonstrates that fecundity as well as the intrinsic rate of increase is reduced for both aphid species on cv. Red delicious.
In summary, the present study has demonstrated that differences in food plant quality
(maternal source or Brussels sprout cv.) for both M. persicae and B. brassicae have impacts on the overall fecundity as well as the intrinsic rate of increase of the G2 of alate and apterae.
51
Chapter 5
Aphids in a changing world: testing the plant stress, plant vigour and pulsed stress hypotheses
5.1 Introduction
Climate-driven forces have a direct impact on plant quality and abundance in terrestrial communities. Variation in plant quality can affect the performance and abundance of herbivores and higher trophic levels (Mattson, 1980; Awmack & Leather, 2002; Wardle et al., 2004; White, 2008). Plants encounter multiple environmental stresses that can alter their growth and the internal allocation of resources (Barlow et al., 1980; Garg et al., 2001; Joern
& Mole, 2005). Current climate change scenarios predict an increase in the frequency and intensity of summer drought and higher temperatures in many parts of the world. Drought can have major consequences for terrestrial ecosystems (Woodhouse & Overpeck, 1998) and is a major environmental factors that directly influences plant physiological processes including nutritional quality of plants for herbivores (Hsiao, 1973; Begg & Turner, 1976;
Huberty & Denno, 2004; Bale et al., 2007; Mody et al., 2009). Three hypotheses describing the relationship between stress, plant quality and herbivore performance are the plant stress, plant vigour and pulsed stress hypotheses (White, 1969, 1984; Price, 1991; Huberty &
Denno, 2004).
The Plant Stress Hypothesis (White, 1969; 1984) asserts that herbivore performance and populations increase on drought stressed plants due to an increase in the availability of nutrients (White, 1974) and/or a decrease in the concentration of defensive compounds
(Rhoades et al., 1979). The Plant Stress Hypothesis is supported by reports of positive relationships between some herbivore populations and plant stress (Mattson & Haack,
1987; Louda & Collinge, 1992; Oswald & Brewer, 1997; Cobb et al., 1997). Other studies, however, have generated conflicting or inconsistent results (Louda & Collinge, 1992;
52
Koricheva et al., 1998; Huberty & Denno, 2004; Williams & Cronin, 2004; Joern & Mole,
2005; Rouault et al., 2006; Staley et al., 2006; Cornelissen et al., 2008; Stone et al., 2010).
The effect of stress on herbivore abundance may depend on the type of plant, type of damage, herbivore feeding guild and species, stress intensity and duration (Larsson, 1989;
Nykanen & Koricheva, 2004; Huberty & Denno, 2004; Staley et al., 2006; Cornelissen et al., 2008; Mody et al., 2009). Larsson‟s modified plant stress hypothesis (insect performance hypothesis) predicts that drought-stressed plants have a non-linear relationship with herbivore performance which is maximized at medium stress levels (Larsson, 1989).
The Plant Vigour Hypothesis (Price, 1991) predicts that vigorous plants are more suitable hosts for herbivores than drought stressed plants owing to higher availability of nutrients (Price, 1991; Cornelissen et al., 2008), lower abscission rates (Preszler & Price,
1995) or higher osmotic potential in vigorous shoots (Price, 2003). The Pulsed Stress
Hypothesis (Huberty & Denno, 2004) predicts better performance of phloem feeders on pulsed or intermittent water-stressed plants due to increases in availability of nitrogen and recovery of turgor compared with continuously stressed plants.
Insect herbivores are a key component of many terrestrial food chains, and changes in plant-insect herbivore interactions have the potential to alter terrestrial community structure and function significantly (Wolters et al., 2000). The present study used a Brassica system (B. oleracea) with a generalist (M. persicae) and a specialist (B. brassicae) aphid species, which are known to respond differently to the nutrient status and secondary metabolites of host plants (van Emden, 1991; Pritchard et al., 2007), to test the above hypotheses under a range of drought treatments.
5.2 Materials and methods
5.2.1 Preliminary drought stress treatment trial
Brussels sprout cv. Oliver plants were grown as described in Section 3.2. To assess the influence of different availability of water on leaf water content, six water regimes were
53 established on 4-week-old B. oleracea plants. The following drought stress treatments were applied per plant; 25ml/week, 50ml/week, 100ml/week, 150ml/week, 200ml/week and
200ml/2 weeks. Plants were randomised in plastic trays on benches in a controlled environment room (20 ± 2 °C; 75% RH; LD 16:8 h). Seven weeks after drought treatments began, the plants were tested for relative water content. The results of the study were used to select drought stress treatments for the main experiment (Appendix 1). All the plants were dead at 25ml/week drought regime; this treatment was eliminated from the main experiment; highly stressed at 50ml/week and 200ml/2 weeks, moderately stressed at 100ml/week and
150ml/week but no stress was observed at 200ml/week water level.
5.2.2 Experiment treatments and insect cultures
Brassica oleracea were grown in pots (diameter 10 cm) in John Innes No. 2 (Fargro
Ltd, UK) compost in a glasshouse under the same conditions as described in Section 3.2.
After four weeks, B. oleracea plants were moved to a controlled environment facility
(conditions as above) where five different drought stress levels were established. The quantity of water added per pot per week was 200 ml for control, 150 ml for low drought stress, 100 ml for medium drought stress and 50 ml for high drought stress; whereas for pulsed drought stress it was 200 ml fortnightly (equal to the medium drought treatment in total volume of water supplied). Myzus persicae and B. brassicae were reared on 6-week-old
B. oleracea plants as described in Section 3.3.2. After 4 weeks of drought stress treatments, the plants were used for aphid performance experiments. These experiments were terminated after 4 more weeks of drought stress treatments.
5.2.3 Aphid performance
Experimental treatments were applied to 8 week old B. oleracea plants in plastic trays on benches in a controlled environment room (conditions as above), replicated ten
54 times in two randomised blocks. Fifty plants of B. oleracea (five of each treatment) were used in each block for both M. persicae and B. brassicae. Three clip cages were fitted to the underside of 1st, 2nd and 3rd fully-developed leaves on each plant to give 15 clip cages per treatment per aphid species and each clip cage had three aphids. As the performance of alate and apterae can differ (Leather, 1989; Tariq et al., 2010) the same form of aphid
(apterae) was used throughout both experiments. After 16-18 h (overnight) all aphids were removed leaving only one nymph per cage. For each aphid, the pre-reproductive time (d) and the number of offspring produced in the subsequent days (Md) were recorded and removed daily from each cage. Data from both aphid species were recorded as described in
Section 3.4.
5.2.4 Foliar relative water content and plant biomass
A parallel group of plants was grown under identical treatments (replication=10) for the plant physiological assessments, which were conducted at a time equivalent to the mid- point of the bioassay (10-weeks-old). One fully-expanded intact leaf close to the centre of each plant was collected and the relative water content of that leaf was recorded as described in Section 3.5.1. Plant biomass was dried at 80 °C for 24 h and weighed.
5.2.5 Foliar chemistry
A second, parallel group of plants was grown under identical treatments
(replication=10) for the plant chemical assessments, which were conducted at a time equivalent to the mid-point of the bioassay (10-weeks-old).
5.2.5.1 Total leaf nitrogen and carbon concentration
Total foliar nitrogen and carbon was determined as described in Section 3.5.2.
55
5.2.5.2 Glucosinolate analysis
Foliar glucosinolates were determined as described in Section 3.5.3.
5.2.6 Data analysis
The effect of drought treatments on aphid performance, relative water content, plant biomass, carbon, nitrogen, individual and grouped (indole and aliphatic) glucosinolate concentrations (Hopkins et al., 2009) were analysed using ANOVA in R version 2.10.1 (R
Development Core Team, 2010) as described in Section 3.6.
5.3 Results
5.3.1 Aphid performance
Drought stress significantly altered aphid fecundity (F1, 90 = 36.038, P < 0.0001), with both aphid species responding similarly (Figure 5.1). Fecundity of B. brassicae was significantly greater at medium drought stress compared with low drought stress and sequentially lower for pulsed drought stress, control and high drought stress treatments compared with medium or low drought stress (Tukey‟s HSD, P<0.05). Fecundity of
M. persicae was significantly greater at medium and low drought stress compared with all other treatments and sequentially lower for pulsed drought stress, high drought stress and control treatments.
56
Figure 5.1 Effective fecundity of Myzus persicae and Brevicoryne brassicae (mean ± S.E.M.) feeding on Brassica oleracea under drought stress treatments. Within each aphid species, means with different letters are significantly different (P < 0.05); HDS = high drought stress, MDS = medium drought stress, LDS = low drought stress, PDS = pulsed drought stress.
Drought stress significantly altered aphid intrinsic rate of increase (F1, 90 = 27.220,
P < 0.0001; Figure 5.2). The intrinsic rate of increase of B. brassicae was significantly greater at medium drought stress compared with low drought stress which was in turn significantly greater than pulsed drought stress; high drought stress and control treatments were significantly lower than all other treatments. The intrinsic rate of increase of M. persicae was significantly greater at medium drought stress compared with low drought stress and sequentially lowers for pulsed drought stress, high drought stress and control treatments compared with medium and low drought stress.
57
Figure 5.2 Intrinsic rate of increase of Myzus persicae and Brevicoryne brassicae (mean ± S.E.M.) feeding on Brassica oleracea under drought stress treatments. Within each aphid species, means with different letters are significantly different (P < 0.05); HDS = high drought stress, MDS = medium drought stress, LDS = low drought stress, PDS = pulsed drought stress.
5.3.2 Plant biomass and relative water content
Drought stress treatments significantly affected plant biomass of B. oleracea (F4, 95 =
166.82, P < 0.0001). Plant biomass was significantly greater in the control treatment as compared with other treatments, and was significantly lower in the high drought stress and pulsed treatments compared with other treatments (Figure 5.3). Drought stress treatments significantly affected the relative water content of B. oleracea foliage (F4, 95 = 146.16,
P < 0.0001). Water content was significantly greater for the control, low and medium drought stress treatments than the high drought stress and pulsed treatments (Figure 5.4).
58
Figure 5.3 Mean plant biomass of Brassica oleracea (mean ± S.E.M.) grown under drought stress treatments. Means with different letters are significantly different (P < 0.05).
Figure 5.4 Relative water contents of Brassica oleracea (mean ± S.E.M.) grown under drought stress treatments. Means with different letters are significantly different (P < 0.05).
59
5.3.3 Foliar chemistry
5.3.3.1 Total leaf nitrogen and carbon concentration
Drought stress treatments significantly affected foliar nitrogen in B. oleracea (F4, 45 =
10.296, P < 0.0001). Foliar nitrogen concentration was significantly greater in the high drought stress and pulsed treatments compared with other treatments (Figure 5.5). The foliar carbon percentage in B. oleracea was not affected by the drought stress treatments (F4, 45 =
0.516, P > 0.05, Figure 5.6).
Figure 5.5 (a) Nitrogen % dry mass of Brassica oleracea (mean ± S.E.M.) at different drought stress treatments. Means with different letters are significantly different (P < 0.05).
60
Figure 5.6 Carbon % dry mass of Brassica oleracea (mean ± S.E.M.) at different drought stress treatments. Means with different letters are significantly different (P < 0.05).
5.3.3.2 Glucosinolate analysis
Total glucosinolate concentration was significantly greater in drought stressed plants compared with control plants (Figure 5.7). Five aliphatic (sinigrin, progoitrin, glucobrassicin, glucoraphinin, glucoiberin) and two indole (4-methoxy-glucobrassicin, 4-hydroxy- glucobrassicin) glucosinolates were identified in the foliage of B. oleracea. The concentration of three of the five aliphatic glucosinolates (glucobrassicin, progoitrin, glucoiberin) was much greater in all treatments as compared to the indole glucosinolates (Figure 5.8). The drought treatments had a significant effect on foliar glucosinolate concentrations (F6, 315 = 8.253,
P < 0.0001). The concentration of the dominant glucosinolate present, glucobrassicin was significantly greater in all the drought stress treatments compared with the control. The concentration of progoitrin and 4-methoxy-glucobrassicin was significantly greater in the medium drought stress treatment compared with other treatments. The concentrations of sinigrin and glucoiberin were significantly lower in the medium drought stress treatment
61 compared with other treatments. Drought treatments had no significant affect on the concentrations of glucoraphinin and 4-hydroxy-glucobrassicin.
Figure 5.7 Total glucosinolate concentrations of Brassica oleracea (mean ± S.E.M.) at different drought stress treatments. Means with different letters are significantly different (P < 0.05).
62
Figure 5.8 Glucosinolate concentrations of Brassica oleracea (mean ± S.E.M.) at different drought stress treatments. For each glucosinolate compound, means with different letters are significantly different (P < 0.05); PRO = progoitrin, IBE = glucoiberin, SIN = sinigrin, HBR = 4-hydroxy-glucobrassicin, BRA = glucobrassicin, RAP = glucoraphanin, MBR = 4-methoxy-glucobrassicin.
5.4 Discussion
The current study does support the plant stress hypothesis (White, 1969, 1984) since the performance of both aphid species was greater on medium drought stressed plants compared with regularly stressed plants receiving the same total water volume. These
63 results are similar with the findings of various scientists (Wearing & van Emden, 1967;
Wearing, 1972a; Mattson & Haack, 1987; Oswald & Brewer, 1997), where medium drought stress improved nutritional quality of host plants resulted in increased performance of herbivores. The response of both aphid species are also consistent with Larsson‟s (1989) modification of the plant stress hypothesis, that phloem feeders‟ responses differ with the magnitude of stress and extreme drought stress is likely to have a negative effect on aphid performance. Our results contradict the findings of most other studies of insect herbivores, which found that insect herbivore perform better on vigorous plants or pulsed stress plants
(Price, 1991; Williams & Cronin, 2004; Huberty & Denno, 2004, 2006).
Herbivore performance may be determined by many factors, including the type and concentration of glucosinolates and the nutrient status of plants (Giamoustaris & Mithen,
1995; Cole, 1997c; Hopkins et al., 1998; Newton et al., 2009; 2010; Müller et al., 2010). The mean glucosinolate concentration was relatively low in control plants compared with drought stressed plants and the elevated glucosinolate concentration under stress conditions, which is consistent with Bouchereau et al. (1996) and Schreiner et al. (2009) work on Brassica plants.
In the present study, the specialist aphid species exhibited higher performance on medium drought stressed plants which may be linked to a lower concentration of sinigrin
(Newton et al., 2009; 2010). The concentration of progoitrin and 4-methoxy-glucobrassicin were significantly greater at medium drought stress. Both these glucosinolates act as feeding attractants and stimulants for crucifer-specialist herbivores and could increase herbivore performance (Newton et al., 2009; 2010; De Vos & Jander, 2009). Indole glucosinolates and/or their breakdown products however, are mostly degraded after passage through generalist herbivores (Kim & Jander, 2007) and 4-methoxy-glucobrassicin production is not induced by generalist saliva (De Vos & Jander, 2009). Therefore,
4-methoxy-glucobrassicin has a less marked effect on generalist herbivore performance at medium drought stress. Some studies suggest that herbivore performance may be reduced by the presence of indole glucosinolates in plants (Kim & Jander, 2007; Gols et al., 2008a),
64 and that phloem feeders avoid leaf defence compounds by feeding from vascular tissues containing less of these compounds (Mattson & Haack, 1987; Rouault et al., 2006).
Furthermore, plants can adjust their glucosinolate concentration in response to herbivores while they were feeding on them (Textor & Gershenzon, 2009).
Drought stress affects several plant processes that result in changes in the physical and chemical composition of the plant tissue (Hsiao, 1973; Hsiao et al., 1976; Barlow et al.,
1980; Kozlowski, 1982; Hanson & Hitz, 1982; Hanson & Weltzin, 2000; Adams & Kolb, 2005;
Bale et al., 2007; McDowell et al., 2008). The medium drought stress improved the nutritional quality of host plants and thus may enhance herbivores‟ performance (Rhoades et al., 1979; 1983; Rhoades, 1985; Louda & Collinge, 1992). Performance of phloem feeding herbivores is usually linked with the availability of nitrogen (Miles et al., 1982; Awmack &
Leather, 2002; Khan & Port, 2008). The nitrogen concentration increased with the increase in intensity of drought stress on B. oleracea. The lowest herbivore performance was on high drought stress plants that may be associated with low turgor pressure and/or increased cell- sap viscosity (Kennedy et al., 1958; Auclair, 1963), even though these plants had significantly higher concentrations of nitrogen. The leaf water content varied among all drought treatments in this study, and therefore may directly affect phloem feeders‟ performance. The response of herbivores may depend on the level of drought stress imposed on the plant (English-Loeb et al., 1997; Hale et al., 2003; King et al., 2006) and performance can also vary among aphid species (Wearing & van Emden, 1967; Wearing,
1967; 1972a; King et al., 2006).
In the present study, foliar nitrogen contents increased with drought stress but other studies have found it decreased under elevated CO2 alone and in combination with elevated temperature, whereas elevated temperature alone had no significant effect on nitrogen contents (Bezemer & Jones, 1998; Zvereva & Kozlov, 2006). An emerging conclusion is that these three dominant variables of climate change have different effects on herbivore performance: a negative response toward high drought stress, a positive response toward high temperature (Bale et al., 2002; Zvereva & Kozlov, 2006) and medium drought stress,
65 and no general response toward elevated CO2 (Bezemer & Jones, 1998). Therefore, it is possible that herbivore performance will increase many fold under the combination of elevated temperature and medium drought stress. The present study was the first simultaneous experimental test of the Plant Stress, Plant Vigour and Pulsed Stress
Hypotheses using two aphid species, in order to develop a better understanding of herbivore performance under drought conditions.
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Chapter 6
Drought and root herbivory modify foliar herbivore performance
6.1 Introduction
Increases in the frequency, duration, and/or severity of drought associated with climate change can alter the structure and composition of terrestrial ecosystems (Walther et al., 2002; Kallis, 2008). Plant herbivore interactions dominate the terrestrial ecosystem
(Jander & Howe, 2008) and the outcomes of these interactions depend on abiotic environmental conditions (van der Putten et al., 2009). Foliar and root herbivores can interact with each other indirectly by sharing the same host plant (Masters et al., 1993). Root herbivores may induce a stress response within the host plant in a similar way to drought that can alter plant physiology (Gange & Brown, 1989) and the stress response of
D. radicum can be different from drought and can induce secondary metabolites in Brassica plants (Soler et al., 2005). Root herbivores can have a positive or negative effect on foliar herbivores (Masters & Brown, 1992; Masters, 1995a; Wackers & Bezemer, 2003; Soler et al., 2005; Wurst et al., 2008) and drought stress may strengthen or weaken this effect depending upon the system under investigation (Staley et al., 2008).
Drought stress directly influences plant physiological processes including nutritional quality for insect herbivores (Hsiao, 1973; Mattson & Haack, 1987; Louda & Collinge, 1992;
Huberty & Denno, 2004). The effects of host plant water stress on foliar insects including aphids have been shown empirically to be complex (see previous chapter for full discussion).
Both population increases and decreases have been attributed to drought stress (Kennedy et al., 1958; Holtzer et al., 1988).
The performance and abundance of the micro-lepidopteran leaf miner, Stephensia brunnichella L. (Lepidoptera: Elachistidae), on Clinopodium vulgare L. (Lamiaceae) has
67 been shown to be reduced under the combined impact of drought and root-feeding wireworm activity (Staley et al., 2007b). The present study investigates the similar interactions in a
Brassica system involving a generalist, M. persicae, and a specialist, B. brassicae, foliar aphid species, with the cabbage root fly, D. radicum, as the root herbivore. The aphid species were chosen as they are known to respond differently to the nutrient status and secondary metabolites of host plants (van Emden, 1991) and D. radicum has been shown to induce glucosinolates locally and systematically in shoots (van Dam et al., 2005; van Leur et al., 2008). It is hypothesized that the performance of M. persicae and B. brassicae would respond negatively to changes in the host plant induced by root herbivory and drought stress.
6.2 Materials and methods
6.2.1 Plants and insect culture
Brassica oleracea L. (var. gemmifera) cv Oliver plants were grown as described in
Section 3.2. Delia radicum were reared on swede as described in Section 3.3.4.
Myzus persicae and B. brassicae cultures were maintained as described in Section 3.3.2.
All experiments were conducted in a control environment room as described in Section
3.3.1.
6.2.2 Experimental procedure
6.2.2.1 Preliminary experiment
A preliminary experiment was carried out to determine leaf water content in relation to drought stress treatments under high densities (five, first instar larvae) of D. radicum. Four drought stress treatments, high, medium, low and standard water regime were established on 4-week-old plants and the quantity of water added per pot per week was 50 ml (high
68 drought stress), 100 ml (medium drought stress), 150 ml (low drought stress) and 200 ml
(standard water regime) as described previously (Chapter 5). After 4 weeks, five first instar larvae of D. radicum were introduced to each 8-week-old plant by carefully placing the larvae with a camel hair brush onto the soil surface adjacent to the stem. Larvae were observed for
30 min. to ensure that they all entered the soil. The relative leaf water content of 11 week old plants (midpoint of the experiment) was used to quantify drought stress under root herbivore attack. Preliminary results (Appendix 2) showed that all the plants with a high density of
D. radicum died at 50 ml per week (high drought stress regime) and this treatment was discarded for the main experiment. Three drought stress treatments were established on the basis of relative water content data (Appendix 2): with 100 ml per week rated as high drought stress, 150 ml per week rated as medium drought stress, and 200 ml per week as the standard water regime, and these three treatments were used in the main experiment.
6.2.2.2 Drought and root herbivore experiment
The three drought stress treatments were established on 4-week-old plants. Two separate batches of plants with five replicates per treatments were used for each aphid species. After 4 weeks (8-week-old plant), two D. radicum treatments (high density with five larvae per pot, low density with three larvae per pot) and a control without larvae were applied to all drought stress treatments. Foliar herbivore performance was measured on ten- week-old plants.
6.2.2.3 Aphid performance
Two weeks after the addition of D. radicum larvae, three clip cages were fitted to the underside of the first, second and third fully-developed leaves on each plant and each clip cage had three adult aphids. As the performance of second generation of alate and apterae can differ (Leather, 1989; Tariq et al., 2010) the same form of aphid (apterae) was used in
69 the experiment. After 16-18 h (overnight) adult aphids were removed leaving one nymph per leaf. For each aphid species, the pre-reproductive time (d) and the offspring produced during an equivalent period (Md) were recorded daily and removed from each cage. The aphid performance data was obtained as described in section 3.4.
6.2.3 Plant physiological measurements
A parallel group of plants was grown under identical treatments (replication=10) for physiological assessments, which were conducted at a time equivalent to the mid-point of the aphid experiments (11-week-old). One fully-expanded intact leaf close to the centre of each plant was collected and the relative water content of that leaf was measured as described in section 3.5.1. Foliar and root biomass was dried in an oven and weighted.
6.2.4 Data analysis
Foliar herbivore performance and plant physiological data were analysed with parametric statistics (ANOVA) as described in section 3.6. Block, aphid species, drought stress treatment and D. radicum density were used to test the significance of each variable and all the interactions except those with block. (Team, 2009 )
6.3 Results
6.3.1 Aphid performance
6.3.1.1 Fecundity
The interaction between aphid species, drought stress treatments and D. radicum density was significant for aphid fecundity (Table 6.1). Fecundity of B. brassicae was significantly greater on plants that experience intermediate drought stress without D. radicum compared with all other treatments and was lowest on the high drought stress treatment with high D. radicum density (Figure 6.1). Fecundity of M. persicae was significantly greater on
70 plants that experience intermediate drought stress with low D. radicum density compared with all other treatments and was lowest at high drought stress with high D. radicum density
(Tukey‟s HSD, P<0.05).
Table 6.1 Analysis of variance of effective fecundity of Myzus persicae and Brevicoryne brassicae on Brassica oleracea plants under different Delia radicum densities and drought stress treatments.
Source of variation d.f. Sum Sq Mean Sq F value Block 1 2.91 2.913 1.75 Aphid species (A) 1 67.54 67.540 40.56*** Drought stress treatments (B) 2 192.01 96.004 57.66*** Delia radicum densities (C) 2 407.83 203.915 122.46*** A x B 2 1.13 0.565 0.34 A x C 2 18.19 9.093 5.46** B x C 4 19.84 4.961 2.98* A x B x C 4 69.39 17.347 10.42*** Residual 161 ***P< 0.001; **P< 0.01; *P< 0.05
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Figure 6.1 Effective fecundity of Myzus persicae and Brevicoryne brassicae on Brassica oleracea plants under different Delia radicum densities and drought stress treatments (mean ± SEM). Within each aphid species, means with different letters are significantly different (P < 0.05).
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6.3.1.2 Intrinsic rate of increase
The interaction between aphid species, drought stress treatments and D. radicum density was significant for intrinsic rate of increase (Table 6.2). Drought stress and
D. radicum density significantly altered aphid intrinsic rate of increase (Figure 6.2). The intrinsic rate of increase of B. brassicae was significantly greater on plants that experience intermediate drought stress without D. radicum compared with all other treatments and was lowest at high drought stress treatments with high D. radicum density. The intrinsic rate of increase of M. persicae was significantly greater on plants that experience intermediate drought stress with low D. radicum density compared with all other treatments and was lowest at high drought stress treatment with high D. radicum density.
Table 6.2 Analysis of variance of intrinsic rate of increase of Myzus persicae and Brevicoryne brassicae on Brassica oleracea plants under different Delia radicum densities and drought stress treatments.
Source of variation d.f. Sum Sq Mean Sq F value Block 1 0.0000405 0.0000405 2.15 Aphid species (A) 1 0.0019714 0.0019714 104.85*** Drought stress treatments (B) 2 0.0040942 0.0020471 108.87*** Delia radicum densities (C) 2 0.0255927 0.0127963 680.56*** A x B 2 0.0005226 0.0002613 13.90*** A x C 2 0.0002216 0.0001108 5.89** B x C 4 0.0021459 0.0005365 28.53*** A x B x C 4 0.0020791 0.0005198 27.65*** Residual 161 ***P< 0.001; **P< 0.01; *P< 0.05
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Figure 6.2 Intrinsic rate of increase of Myzus persicae and Brevicoryne brassicae on Brassica oleracea plants under different Delia radicum densities and drought stress treatments (mean ± SEM). Within each aphid species, means with different letters are significantly different (P < 0.05).
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6.3.2 Relative water contents
Drought stress (F2, 81 = 40.88, P < 0.0001) and D. radicum treatments significantly
(F2, 81 = 4.54, P < 0.05) affected the relative water contents of B. oleracea foliage. Relative water contents were significantly greater at standard water regimes with all D. radicum densities and medium drought stress with controls and low density D. radicum compared with other treatments. Relative water contents were significantly lower at high drought stress treatment with high D. radicum density compared with other treatments (Figure 6.3).
Figure 6.3 Relative water contents of Brassica oleracea (twelve-week-old) plants under different Delia radicum densities and drought stress treatments (mean ± SEM). Means with different letters are significantly different (P < 0.05).
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6.3.3 Foliar biomass
Drought stress (F2, 81 = 214.60, P < 0.0001) and D. radicum treatments significantly
(F2, 81 = 9.16, P < 0.0001) affected foliar biomass. Foliar biomass was greatest at standard water regimes with all D. radicum treatments compared with other treatments and was lowest at high drought stress with high D. radicum density (Figure 6.4).
Figure 6.4 Foliar biomass of Brassica oleracea (twelve-week-old) plants under different Delia radicum densities and drought stress treatments (mean ± SEM). Means with different letters are significantly different (P < 0.05).
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6.3.4 Root biomass
Drought stress (F2, 81 = 111.52, P < 0.0001) and D. radicum treatments significantly
(F2, 81 = 16.87, P < 0.0001) affected the root biomass. Root biomass was greatest at standard water regime without D. radicum compared with other treatments. It decreased with drought stress and D. radicum treatments (Figure 6.5).
Figure 6.5 Root biomass of Brassica oleracea (twelve-week-old) plants under different Delia radicum densities and drought stress treatments (mean ± SEM). Means with different letters are significantly different (P < 0.05).
6.4 Discussion
The performance of both aphid species decreased with increased D. radicum density under both standard water regime and high drought stress treatments. Similar effects on the performance of M. persicae and other insects have been reported for D. radicum and several other root herbivores (Bezemer et al., 2003; van Dam et al., 2005; Soler et al., 2005; Wurst
& van der Putten, 2007). In contrast, Masters (1995b) found that the performance of
M. persicae increased under attack by a root feeder (Phyllopertha horticola).
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The performance of both aphid species was greater at medium drought stress.
Combined with a high level of drought stress, high D. radicum density had an interactive negative effect on aphid performance. This is consistent with the findings of Staley et al.
(2007b) on leaf miners, where foliar herbivore performance was negatively affected by a combined soil wireworm and drought stress treatments. In the present study, the observed effects on aphid performance may be associated with increased levels of defence compounds, which have been observed in the foliage of Brassica spp. under both drought stress (Bouchereau et al., 1996; Schreiner et al., 2009) and root herbivore attack (van Dam et al., 2005; Soler et al., 2005; van Dam & Raaijmakers, 2006; Erb et al., 2008; 2009;
Hopkins et al., 2009).
Root herbivory did not affect the relative water contents of Brassica leaves under the standard water regime, which is consistent with several other studies (Masters & Brown,
1992; Masters, 1995a; van Dam et al., 2005; Staley et al., 2007b). Root herbivory did reduce the relative water contents of leaves when combined with both medium and high drought stress. Only high water stress reduced the relative water contents of leaves in the absence of root herbivory. Root herbivory did not affect foliar biomass under the standard water regime, as also reported by Bezemer et al. (2003), but foliar biomass decreased with drought stress in combination with root herbivory. The root herbivore treatment decreased the root biomass under all water treatments, but the root herbivore effect on the root biomass was more density dependent under drought stress. Root biomass has been reported to be reduced under root herbivore damage in Brassica crops by Blossey & Hunt-
Joshi (2003). There was only a negative effect on root biomass under medium and high drought stress when the D. radicum density was high.
Performance of phloem feeding herbivores is usually linked with the availability of nitrogen (Miles et al., 1982; Awmack & Leather, 2002; Khan & Port, 2008). In the present study, foliar herbivore performance was greater at medium drought stress; which is consistent with the previous results (Chapter 5). Medium drought stressed plants have also been reported to have lower concentrations of phytoalexins compared with high drought
78 stressed plants (Plumbe & Willmer, 1985). At medium drought stress, the performance of
M. persicae was increased at low density of D. radicum, whereas at all other combinations of
D. radicum and drought stress both aphid spices reduced their performance relative to those without D. radicum. These results were similar to the findings of Staley et al. (2007b), where root herbivores reduced foliar herbivore performance only in the absence of drought stress and a positive effect of root herbivores was observed on foliar herbivores performance only under circumstances of drought, respectively. Previous studies have also shown that the effects of drought stress can vary between aphid species, including M. persicae, B. brassicae and Lipaphis erysimi (Wearing & van Emden, 1967; Wearing, 1967; 1972a; King et al., 2006). Positive effects of a low density of D. radicum at medium drought stress on aphid performance may be explained through the induction of water stress, causing an increase in the amount of soluble nitrogen in the leaves (Masters, 1995b), or by root herbivores damaging production sites of secondary metabolites in the roots (Kaplan et al.,
2008). Therefore, further studies should include nitrogen and glucosinolate analysis to better understand the potential mechanisms behind the present study.
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Chapter 7
Drought modifies trophic interactions above and below ground
7.1 Introduction
Plants, insect herbivores and the natural enemies of insects interact in multitrophic food webs that influence community dynamics (Ohgushi, 2005; Trotter et al., 2008; Utsumi et al., 2009; van der Putten, 2009). Plants are simultaneously challenged by above- and below- ground insect herbivores that can affect one another through plant-mediated interactions
(Gange & Brown, 1989; Masters, 1995b; Bezemer et al., 2003; Blossey & Hunt-Joshi, 2003).
Below-ground herbivores can increase water stress in plants (Smith, 1977) and induce changes in plant physiology that are similar to drought (Gange & Brown, 1989), and may strongly affect the quality and quantity of nutrients availability to other herbivores (Masters &
Brown, 1992; Masters, 1995a; Bezemer & van Dam, 2005; Kaplan et al., 2008; Ahuja et al.,
2010). The impact of root herbivores on the performance of foliar herbivores can be positive
(Gange & Brown, 1989; Masters & Brown, 1992; Masters, 1995a; Kaplan et al., 2008; Wurst et al., 2008), negative (Tindall & Stout, 2001; Wackers & Bezemer, 2003; Soler et al., 2005) or neutral (Moran & Whitham, 1990). Drought stress may affect herbivorous insect performance, diversity and abundance indirectly via changes in plant physiology (Wearing & van Emden, 1967; White, 1984; Mattson & Haack, 1987; White, 2008).
Trophic networks frequently involve complex plant defences (van der Putten et al.,
2001; Bezemer & van Dam, 2005; Ahuja et al., 2010), including the release of volatile organic compounds (VOCs) following herbivore attack that enhance the effectiveness of natural enemies (Vet & Dicke, 1992; Kaori et al., 2002; Dicke & Baldwin, 2010). Plant VOC emissions can vary both in quality and in quantity, depending on biotic and abiotic stress, and these changes can impact the attractiveness of the plants to natural enemies of the
80 insect herbivores (Kugimiya et al., 2010; Holopainen & Gershenzon, 2010). Plant VOC emissions have been shown to be reduced under water stress (Llusià & Peñuelas, 1998;
Ormeño et al., 2007; Sharkey et al., 2008; Loreto & Schnitzler, 2010) and such a change in plant VOC emission could affect parasitoid preference and performance (Hunter, 2003).
Some studies have examined the effects of above- or below-ground interactions in multitrophic systems (Soler et al., 2007a; 2007b; Rasmann & Turlings, 2008) but there have been few studies on the effects of abiotic factors on both above- and below-ground interactions (Staley et al., 2007b; 2008).
The present study examines how a multitrophic system with above- and below- ground components is influenced by drought stress. For plant-mediated herbivore and parasitoid interactions it is hypothesised that drought and root herbivory will both induce a plant stress response which will reduce parasitoid preference and performance on these plants. Changes in parasitoid preference are predicted to be due to a reduction in VOC emissions from stressed plants (see above). The system used comprised a Brassica oleracea variety as the host plant, generalist (M. persicae) and specialist (B. brassicae) aphids as above-ground herbivores, their respective generalist (A. colemani) and specialist
(D. rapae) aphid parasitoids, and a root fly (D. radicum) as a below-ground herbivore.
Experiments were conducted with two sub-systems, one with the generalist herbivore and generalist parasitoid, and the other with the specialist herbivore and specialist parasitoid.
7.2 Materials and methods
7.2.1 Study species
Brussels sprout cv Oilver plants were grown as described in Section 3.2. Delia radicum were reared on swede as described in Section 3.3.4. Myzus persicae and
B. brassicae cultures were maintained as described in Section 3.3.2. All experiments were conducted in a control environment room as described in Section 3.3.1.
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7.2.2 Experimental treatments
To assess the influence of drought stress on aphid performance, parasitoid performance, cabbage root fly performance, olfactory responses and VOC emissions, six parallel series of plants (five replicates per treatments with two blocks) were grown in a greenhouse. Four weeks (after germination) old B. oleracea plants were moved to a control environment facility (20 ± 2 °C; 75% RH; LD 16:8 h) where two water treatments were established. The quantity of water added per pot per week was 200 ml for the standard
(control) water regime and 100 ml for drought stress as described previously (Chapter 6).
After 4 weeks of drought stress treatments, three clip cages were fitted to the underside of
1st, 2nd and 3rd fully-developed leaves on each plant. Two separate batches of plants with five replicates per treatment were used for each aphid species. Since the performance of G2 of alate and apterae can differ (Leather, 1989; Tariq et al., 2010), the same form of aphid
(apterae) was used. Clip cages and adult aphids were removed leaving one nymph per leaf on each treatment for four weeks. After 2 weeks, D. radicum treatments (five first instar
D. radicum larvae and control without larvae) were introduced on the same plants. Five larvae were introduced to each plant by carefully placing them with a camel hair brush onto the soil surface adjacent to the stem. Each plant was carefully monitored for 30 min. by using magnifying glass, to ensure that all the root fly larvae had successfully entered the soil.
After 4 weeks (12-week-old plants) of aphid treatments, 300 aphids of each species were used to measure parasitoid preference and performance and extra aphids were removed from each plant.
7.2.3 Parasitoid preference
The behavioural responses of A. colemani and D. rapae under each treatment were determined using a four-arm olfactometer (Pettersson, 1970; Kalule & Wright, 2004; Webster et al., 2010). This olfactometer had a star-shaped arena with four regions and a central
82 chamber (2 cm2); parasitoids could move freely within each region. Each region had an area of 4 cm and the distance from one end of the olfactometer to the opposite end was 10.4 cm.
Air was drawn in through the central hole and the airflow rate for each quadrant was maintained at 100 ml/min using a vacuum pressure pump (Capex 8C, Charles Austin Pumps
Ltd, Byfleet, UK). The pump was used to draw in and vent out the air from the olfactometer chamber. Prior to the experiment, a smoke test was used to confirm an equal airflow distribution.
Mummified aphids of A. colemani and D. rape were removed from their respective treatments and kept individually in small vials (2 cm diameter and 6 cm long). The naïve females were mated within 24 h and fed on 50 % aqueous solution of honey for two days.
These 2-day-old naïve females (having no experience of aphids or plants after emergence) were used in olfactometer tests as described by Kalule & Wright (2004). Test materials consist of four treatments: control plant, drought plant, control plant with D. radicum and drought plant with D. radicum. Each choice bioassay (Table 7.1) had pair wise treatment comparisons and was repeated five times with five female parasitoids (replicates) in each repetition. The 12-week-old plants with 300 aphids of each species were used to measure parasitoid preference.
All bioassays were conducted at 20 ± 2 °C under 0.04 Wm-2 (420-680nm) light intensity (Young et al., 1987). An individual parasitoid was introduced into the central olfactometer chamber and left for 8 min. In order to control the directional bias in the chamber, the olfactometer was rotated at 90° every 2 min. (Webster et al., 2010). The olfactometer was divided into five regions corresponding to each of the four arms and central part. The time spent in each region was recorded using Olfa software (F. Nazzi, Udine, Italy) as described by Webster et al. (2010). After every 10 parasitoids tested, odour sources were removed and the olfactometer was washed with Lipsol (5% v/v; Bibby Sterilin Ltd.,
Staffordshire, UK) detergent and rinsed with 80 % ethanol solution and air dried. Time spent in the treated region was converted to percent of total time.
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Table 7.1 Details of choice bioassays for Aphidius colemani and Diaeretiella rapae using a four arm olfactometer.
Aphidius colemani Diaeretiella rapae
Control plant + M. persicae vs. Control plant + B. brassicae vs. Drought plant + M. persicae Drought plant + B. brassicae
Control plant + M. persicae vs. Control plant + B. brassicae vs. Drought plant with D. radicum + M. persicae Drought plant with D. radicum + B. brassicae
Drought plant + M. persicae vs. Drought plant + B. brassicae vs. Drought plant with D. radicum + M. persicae Drought plant with D. radicum + B. brassicae
Control plant with D. radicum + M. persicae vs. Control plant with D. radicum + B. brassicae vs. Drought plant with D. radicum + M. persicae Drought plant with D. radicum + B. brassicae
7.2.4 Parasitoid performance
Newly emerged females of each parasitoid species had been paired into a 2.5 x 8 cm glass tube and fed a single droplet of honey and a droplet of water daily, by placing the droplets inside the cover of the tube as described by Sampaio et al. (2008). As the percent parasitism is similar between aphid instars (van Emden & Kifle, 2002), the present studies were conducted on mixed instars. Mixed instars of each aphid species were exposed to each species of parasitoids on a single treated plant (five replicates per treatments with two blocks). Five paired parasitoids (one pair per sixty aphids as described by Jarošík & Lapchin
2001) were released per treatment replicate under bell cloches. Each bell cloche had five air vents to reduce humidity around the treated plants. Parasitoids were removed 24 h after their release and the remaining aphids were allowed to continue their development for 10-14 days, the time required from parasitisation to mummy formation (van Emden & Kifle, 2002).
Mummies were collected individually in gelatine capsule and the following data were collected:
i. percent parasitism
ii. percentage emergence
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iii. adult longevity (Newly emerged adults of each parasitoid species had been reared in
a 2.5 x 8 cm glass tube and fed a single droplet of honey and a droplet of water daily,
by placing the droplets inside the cover of the tube. For each individual parasitoid,
the times (days) from emergence to death were recorded).
iv. Hind tibiae length (linear dimensions) is an indicator of body size (Hohmann et al.,
1988; van Emden & Kifle, 2002; Blande et al., 2004) female length of hind tibiae was
measured under a binocular microscope with a standard size of eyepiece graticule at
4x magnification.
v. sex ratio is the ratio of males to females in a population and was calculated by
number of males divided by total individuals.
7.2.5 Plant volatiles
An air entrainment method was used for isolating volatile compounds from twelve- week-old B. oleracea plants and GC-MS was used for the identification of compounds as described in Section 3.5.4. In the present study, the preference and performance of both aphid species were similar, therefore, B. brassicae were used for plant volatile studies. Plant volatile compounds were collected from:
1) uninfested control plants,
2) aphid infested control plants,
3) aphid infested drought stressed plants,
4) aphid and D. radicum infested control plants and
5) aphid and D. radicum infested drought stressed plants.
7.2.6 Root herbivore performance
Root herbivore performance was measured using the above treatments on a separate batch of plants. Five larvae were applied per plant with five replicates per
85 treatments with two blocks. Pupal weight and percent pupation has been used to measure root herbivory performance (Hopkins et al., 1999; Bjorkman et al., 2009). Therefore, pupae were collected and counted after 5 weeks of D. radicum inoculation and weighed individually after being air dried. Percentage adult emergence and adult longevity of D. radicum were also taken into account as other measures of D. radicum performance.
7.2.7 Data analysis
Parasitoid and D. radicum performance and plant volatile emission data were analysed as described in Section 3.6. For olfactory response, time spent in the treated region was converted to percentage of total time and t-test was used to compare the mean.
7.3 Results
7.3.1 Parasitoid preference
Both parasitoid species preferred the control plants compared with other treatments
(Table 7.2). Parasitoid preference decreased significantly with drought stress. Delia radicum did not affect A. colemani behaviour on drought stressed plants compared with plants receiving the full (control) water regime. Preference of D. rapae was similar on drought stressed plants with or without D. radicum.
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Table 7.2 Percentage time (mean min. ± S.E.M.) spent by parasitoids in different treatment arms (Rf = root fly) of an olfactometer compared with control arms. Student t-Test was performed at 95 percent confidence interval for the comparison of means.
Treatments Parasitoid species Aphidius colemani Diaeretiella rapae Control (-Rf) vs Drought stress (-Rf) 64.55 ± 1.53 vs 21.92 ± 1.01*** 60.71 ± 1.44 vs 23.46 ± 0.88***
Control (-Rf) vs Drought stress (+Rf) 62.89 ± 1.31 vs 22.70 ± 1.07*** 63.11 ± 1.32 vs 22.07 ± 1.29***
Drought stress (-Rf) vs Drought stress (+Rf) 47.73 ± 1.44 vs 40.67 ± 1.5** 42.46 ± 1.47 vs 44.14 ± 1.06
Control (+Rf) vs Drought stress (+Rf) 43.45 ± 1.30 vs 40.12 ± 1.38 55.78 ± 2.49 vs 29.75 ± 2.18*** *** p < 0.001, ** p < 0.01, * p < 0.05
7.3.2 Parasitoid performance
7.3.2.1 Percent parasitism
The interaction between drought stress, D. radicum and parasitoid species was significant (Table 7.3). Drought stress and D. radicum had a negative impact on percent parasitism by both the specialist parasitoid species (D. rapae) and the generalist parasitoid species (A. colemani) (Figure 7.1). The percent parasitism by A. colemani was significantly reduced under drought stress and in the D. radicum treatment. The percent parasitism by both parasitoid species decreased significantly on aphids reared on control plants with
D. radicum compared with drought stress plants. The percent parasitism by A. colemani and
D. rapae was significantly lower on aphids reared on drought stressed plants compared with other treatments. Delia radicum reduced the percent parasitism on control plants but increased the percent parasitism by A. colemani on drought stressed plants (Figure 7.1).
87
Table 7.3 Analysis of variance of parasitism success of Aphidius colemani and Diaeretiella rapae on Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml / pot / week; “Control”) and reduced water regime (100 ml / pot / week; “Drought stressed”) with and without Delia radicum.
Source of variance d.f. F test % parasitism % emergence Sex ratio
Drought stressed treatment (A) 1 123.09*** 75.61*** 212.23***
D. radicum treatment (B) 1 10.41** 29.77*** 19.31***
Parasitoid species (C) 1 61.95*** 0.26 0.28
A x B 1 45.48*** 6.99* 0.63
A x C 1 0.79 2.12 0.67
B x C 1 11.76** 8.33** 7.22**
A x B x C 1 7.51** 1.88 0.015 Residual 72 *** p < 0.001, ** p < 0.01, * p < 0.05
88
Figure 7.1 Mean percent parasitism by Aphidius colemani and Diaeretiella rapae (mean ± S.E.M.) of Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stressed”) with and without Delia radicum. Means with different letters are significantly different (P < 0.05).
7.3.2.2 Percentage emergence
The interactions between drought stress x D. radicum treatment and parasitoid x
D. radicum treatment were significant (Table 7.3). Both parasitoid species on control plants without D. radicum had a significantly greater percentage emergence compared with parasitoids developing under drought stress and D. radicum treatments (Figure 7.2).
Percentage emergence of both parasitoid species was lowest under drought stress with
D. radicum treatment. Percentage emergence of A. colemani was not significantly different on drought stressed plants with and without D. radicum. Percentage emergence of D. rapae was similar on control plants with D. radicum and drought stressed plants without
D. radicum.
89
Figure 7.2 Mean percentage emergence of Aphidius colemani and Diaeretiella rapae (mean ± S.E.M.) that developed in Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stressed”) with and without Delia radicum. Means with different letters are significantly different (P < 0.05).
7.3.2.3 Sex ratio
The sex ratio of both parasitoid species was significantly greater on drought stressed plants compared with other treatments (Figure 7.3). The sex ratio of D. rapae was significantly greater on drought stressed plants with D. radicum compared with other treatments (Table 7.3). Delia radicum did not affect the sex ratio of A. colemani but increased proportion of the males of D. rapae.
90
Figure 7.3 Sex ratio of Aphidius colemani and Diaeretiella rapae growing on Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stressed”) with and without Delia radicum. Means with different letters are significantly different (P < 0.05); where 0 represents all females and 1 represents all males.
7.3.2.4 Female tibia length
The tibia length of both parasitoid species decreased significantly with D. radicum treatment under drought stress and the control treatment (Table 7.4; Figure 7.4). The tibia length of D. rapae was significantly greater on control plants without D. radicum compared with other treatments. The tibia length of A. colemani was not significantly different on control and drought stressed plants without D. radicum treatment.
91
Table 7.4 Analysis of variance of tibia length and adult longevity of Aphidius colemani and Diaeretiella rapae on Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stressed”) with and without Delia radicum.
Source of variance d.f. F test Tibia length Male longevity Female longevity
Drought stressed treatment (A) 1 11.51*** 21.53*** 42.58***
D. radicum treatment (B) 1 200.72*** 13.62*** 54.22***
Parasitoid species (C) 1 331.35*** 145.68*** 217.41***
A x B 1 10.34** 0.55 2.31
A x C 1 22.72*** 1.83 6.67*
B x C 1 0.0005 0.050 0.066
A x B x C 1 0.0831 0.63 1.82
Residual 472 *** p < 0.001, ** p < 0.01, * p < 0.05
92
Figure 7.4 Mean female tibia length (mm) of Aphidius colemani and Diaeretiella rapae (mean ± S.E.M.) developed in Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stressed”) with and without Delia radicum. Means with different letters are significantly different (P < 0.05).
7.3.2.5 Adult longevity
Female adult longevity of both parasitoid species was significantly greater on control plants without D. radicum treatment (Figure 7.5). Males of both parasitoid species had significantly shorter adult longevity compared with females. Female longevity of both parasitoid species decreased significantly with D. radicum treatment (Table 7.4).
93
Figure 7.5 Adult longevity (days) of Aphidius colemani and Diaeretiella rapae (mean ± S.E.M.) developed in Myzus persicae and Brevicoryne brassicae reared on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stressed”) with and without Delia radicum. Means with different letters are significantly different (P < 0.05).
7.3.2 Plant volatile emissions
Total plant volatile emission was significantly greater in the control and drought stress treatments compared with other treatments (Figure 7.6). The volatile compounds identified were: alpha-phellandrene, alpha-pinene, beta-phellandrene, beta-pinene, terpinolene, limonene, alpha-terpinene, terpineol, terpinolene, nonanal, dec-2-en-1-ol, allyl isothiocyanate, 1-terpinen-4-ol, decanal, tetradecane and verticiol (Figure 7.7). There were significant differences in the emission of allyl isothiocyanate (F4, 14 = 8.31; R = 0.62; p <
0.001), alpha-phellandrene (F4, 14 = 4.311; R = 0.42; p < 0.05) and beta-phellandrene (F4, 14 =
4.08; R = 0.3378; p < 0.05) under different treatments. The emission of allyl isothiocyanate increased significantly from B. brassicae infested plants. The emission of beta-phellandrene
94 and limonene was reduced significantly on drought stress plants with D. radicum. The emission of terpinolene and 1-terpinen-4-ol were increased on control plants with D. radicum
(Infested + Root fly) compared with control plants without D. radicum (Infested). Allyl isothiocyanate was not detected from unifested plants but was emitted from plants infested with B. brassicae with the amount released being reduced when plants were also infested with D. radicum and further still when plants were drought stressed.
Figure 7.6 Total volatile emissions of Brassica oleracea (mean ± S.E.M.) under different treatments using GC-MS; DS = Drought stress, Rf = rootfly, uninfested = without aphids, infested = with aphids.
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Figure 7.7 Individual plant volatile emissions of Brassica oleracea (mean ± S.E.M.) under different treatments using GC-MS; DS = Drought stress, Rf = rootfly, uninfested = without aphids, infested = with aphids.
7.3.3 Root herbivore performance
The performance of D. radicum was influenced by drought stress (Figure 7.8). Pupal weight (F1, 58 = 244.23; p < 0.001), percentage adult emergence (F1, 18 = 25.96; p < 0.001), and adult longevity (F1, 37 = 15.52; p < 0.001) of D. radicum were significantly reduced by drought stress (Figure 7.8). The number of pupae was not significantly different on control and drought stressed plants (F1, 18 = 0.7411; p = 0.40).
96
Figure 7.8 Delia radicum performance on Brassica oleracea plants under standard water regime (200 ml/pot/week; “Control”) and reduced water regime (100 ml/pot/week; “Drought stress”). Means with different letters are significantly different (P < 0.05): (A) pupae per treatment (B) pupal weight (C) percentage emergence (D) adult longevity.
7.4 Discussion
Biotic (D. radicum) and abiotic (drought) stress influenced parasitoid preference and performance. These results were similar to the findings of Calatayud et al. (2002) on cassava plants, where the parasitoid performance was decreased by drought stress. Root herbivory influenced parasitoid preference and performance when sharing the same host plant, which may be due to changes in the volatile blend (Soler et al., 2007b). The parasitoid species examined in both sub-systems used in the present study („generalist‟ and „specialist‟ herbivore-parasitoid combinations) exhibited a preference for plants that had not been attacked by D. radicum. This may be due to the lower emission of compounds such as allyl isothiocyanate from plants with D. radicum. Kugimiya et al. (2010) found that the qualitative and quantitative differences in the amount of VOCs were necessary for parasitoids to
97 discriminate between infested and uninfested plants. Overall, there was a clear preference- performance pattern for both parasitoid species. That is the ability of parasitoids to evaluate plant-induced defences and then select the host that will maximise the performance of their offspring (Jaenike, 1978; Gripenberg et al., 2010). These results were similar to those of
Soler et al. (2007b) on Brassica plants, where parasitoids preferred plants with undamaged roots and showed a preference-performance correlation.
Since below-ground herbivory and drought stress directly affect plant quality, parasitoid success can be influenced by both factors (Calatayud et al., 2002; Soler et al.,
2005). In the present study, a direct link between plant quality and parasitoid performance was associated with the increased production of males in both „generalist‟ and „specialist‟ sub-systems, a reduction in female tibia length and parasitoid longevity under both root herbivore and drought stress. These results are in agreement with Hunter‟s (2003) proposed plant quality mechanisms for parasitoid success, where low quality plants are associated with an increase in the production of males and a decline in the size, number and longevity of the parasitoid.
Plants often optimize their defensive investments according to abiotic growing conditions and herbivore pressure (Heil, 2008). Parasitoids can distinguish between infested and uninfested plants, and also discriminate between plants infested by different herbivore species (Heil, 2008). In the present study, no clear overall pattern was observed for plant volatile emissions under different stresses as amounts of some compounds increased while others decreased. This may be due to the specific role of individual volatile compounds under single or multiple stresses (Holopainen & Gershenzon, 2010). The emission of general plant volatiles such as ß-phellandrene and limonene was greatest from uninfested plants compared with aphid infested plants whereas emission of allyl isothiocyanate, a compound characteristic of Brassicas, was highest in aphid infested plants (Figure 7.6).
The finding that drought stress and root fly infestation reduced the emission of allyl isothiocyanate was of particular interest and may help to explain why such plants were less attractive to parasitoids. Epicuticular wax layers on leaves are known to be increased on
98 stressed plants, and this can reduce or inhibit volatile emission and affect the efficiency of
A. colemani and D. rapae (Müller & Riederer, 2005; Gentry & Barbosa, 2006; Desneux &
Ramirez-Romero, 2009; Peñuelas & Staudt, 2010). Drought stress has been shown to reduce the rate of photosynthesis and increase stomatal closure, reducing the production of volatile compounds and their emission respectively (Loreto & Schnitzler, 2010).
The quantity and quality of plant volatiles can change dramatically when plants are stressed (Soler et al., 2007b; Heil, 2008; Dicke & Baldwin, 2010), and the interactions between biotic and abiotic stress factors can have additive or opposing effects on plant volatile emissions (Holopainen & Gershenzon, 2010). In the present study, parasitoid attraction decreased under drought stress and root herbivore attack. This may have been due to opposing effects of drought and root herbivory on plant volatile compounds
(attractants) such as allyl isothiocyanate which attract parasitoids (Read et al., 1970). Other studies (Rasmann & Turlings, 2007; Soler et al., 2007b) have shown that parasitoid attraction can be significantly reduced under root herbivore attack. In the present study, higher concentrations of VOCs (terpinolene and 1-terpinen-4-ol) were produced on control plants with D. radicum compared with control plants without D. radicum that may affect parasitoid attraction. Root herbivory and drought stress decreased the preference and performance of both the generalist (A. colemani) and the specialist parasitoid species
(D. rapae) examined. These results suggest that both parasitoid species used the same or similar infochemicals during foraging, as described by Steidle & Van Loon (2003). In the present study, drought stress and root herbivory reduced the performance of both the aphid species used in the present work (Chapter 5 & 6) and such effects on their hosts may have had a negative impact on both parasitoid preference and performance.
In the present study, drought stress had a negative impact on root herbivore performance. This is in agreement with previous studies (Moran & Whitham, 1988; Brown &
Gange, 1990; Staley et al., 2007a; Staley & Johnson, 2008; Johnson et al., 2010), where drought stress influenced the performance and abundance of several root herbivores. This may provide one explanation for why there was no consistent additive effect
99 for root herbivory and drought stress on parasitoid preference and performance, since the indirect effects of D. radicum on parasitoids via the plant could have been reduced under drought stress. The pupal weight of root herbivores on Brassica plants has been shown to have a positive correlation with root biomass (Bjorkman et al., 2009).
The current study has demonstrated that drought stress and root herbivory can significantly affect the preference and performance of insects at the third trophic level
(parasitoid), which suggests that such indirect interactions between abiotic and biotic factors may play an important role in the structuring of communities.
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Chapter 8
Summary and General Discussion
8.1 Summary of experimental findings
Chapter 4: Maternal host plant effects on aphid performance: contrasts between a generalist and a specialist species on Brussels sprout cultivars
Aphid performance was influenced both by the type of host on which the parent aphid
had been reared and by the host on which it was feeding when reproducing.
The fecundity of G2 of alates of both aphid species reared on Chinese cabbage
differed significantly between cultivars of Brussels sprout. These differences were
also apparent for the intrinsic rate of increase of B. brassicae but not for M. persicae.
Brussels sprout cv. Oliver was selected as a consistently good aphid host for
subsequent experiments.
Chapter 5: Aphids in changing world: testing the plant stress, plant vigour and pulsed stress hypotheses
Aphid performance on unstressed, pulsed and severely drought-stressed plants was
significantly lower compared with aphids feeding on plants with medium drought
stress.
Plant biomass and relative water contents were significantly greater for unstressed
plants than for plants subjected to high drought stress or pulsed water treatments.
Foliar nitrogen concentration was significantly greater in the high drought stress and
pulsed treatments, and the glucobrassicin concentration (dominant glucosinolate)
101
was significantly greater in all the drought stress treatments compared with the
control.
The present study did not support the plant stress, pulsed stress or plant vigour
hypotheses but did provide support for part of Larsson‟s modified plant stress
hypothesis, where aphid performance is maximised at medium stress levels.
Chapter 6: Drought and root herbivory modify foliar herbivore performance
The fecundity and intrinsic rate of increase of both aphid species decreased at high
D. radicum density and medium or high drought stress.
Aphid performance was significantly greater at medium drought stress without
D. radicum. High drought stress with high D. radicum density had a negative impact
on aphid performance and all plant parameters studied.
Delia radicum did not influence relative water contents and foliar biomass under
normal water regimes.
Root biomass was negatively affected by both D. radicum and drought stress.
Chapter 7: Drought modifies trophic interactions above and below ground
In the absence of root herbivory, the preference and performance of both parasitoid
species was reduced significantly on drought-stressed plants compared with control
plants.
In the presence of root herbivory, both parasitoid species significantly preferred the
drought-stressed plants compared with control plants, however, drought only
significantly reduced performance in the generalist parasitoid species.
Both drought stress and root herbivory reduced plant volatile emissions, which may
explain why parasitoids preferred the unstressed control plants.
102
Drought stress significantly reduced performance (pupal weight, percentage
emergence and adult longevity) of the root herbivore.
8.2 General Discussion
8.2.1 Host plant quality under biotic and abiotic stress
In the present study, foliar nitrogen concentration increased with the increase in intensity of drought stress. These results are in line with the Plant Stress Hypothesis (White,
1984) where the nitrogen concentration increases with the increase in drought stress. The foliar relative water content and foliar and root biomass decreased with the increase in drought stress and D. radicum density (Chapter 5 & 6). These results for root herbivory are compatible with the conceptual model proposed by Masters et al. (1993), where root herbivory impairs the plant‟s ability to take up water resulting in reduction in foliar relative water content. The relationship between glucosinolates and drought stress was non-linear
(Chapter 5) and it was also true for plant VOCs emission under drought and D. radicum treatments (Chapter 7). The quantity and quality of glucosinolate and plant volatiles can change dramatically when plants are stressed (Soler et al., 2007b; Heil, 2008; Hopkins et al.,
2009; Jahangir et al., 2009; Dicke & Baldwin, 2010), and the interactions between biotic and abiotic stress factors can have additive or opposing effects on glucosinolate concentration
(Jahangir et al., 2009) and plant volatile emissions (Holopainen & Gershenzon, 2010). The present study has therefore demonstrated that as the quality of plants change in response to biotic and abiotic stress there are associated effects on herbivore-natural enemy interactions.
8.2.2 The role of host plant quality in insect trophic system
The effects of host plant quality on foliar insects including aphids have been shown to be complex. The impact of drought stress and D. radicum on aphid performance was
103 discussed in Chapters 5 and 6, including the apparent non-linear relationship between aphid performance and drought stress, which was highest at moderate drought stress and at a low density of D. radicum. The performance of both aphid species decreased with the increase in drought stress and D. radicum density. A non-linear response for herbivore performance with stress has also been demonstrated in various studies (English-Loeb, 1989; 1990;
English-Loeb et al., 1997), where herbivore performance appeared to increase and then decrease as the intensity of stress intensified.
Insect herbivore performance on crucifers can be determined by the type and concentration of glucosinolates and nutrient status of plants (Giamoustaris & Mithen, 1995;
Cole, 1997c; Hopkins et al., 1998; Newton et al., 2009). In the present study, the lowest herbivore performance was on high drought stress plants with high D. radicum density and this may be associated with increased levels of defensive compounds in the foliage of
Brassica spp. under drought (Bouchereau et al., 1996; Schreiner et al., 2009) and root herbivore attack (van Dam et al., 2005; Soler et al., 2005; van Dam & Raaijmakers, 2006;
Erb et al., 2008; 2009; Hopkins et al., 2009). Therefore, nitrogen and glucosinolate analysis of the Brussels sprout cv. Oliver plants under different treatments will be carried out to understand better the potential mechanisms behind these interactions. There are many examples of positive relationships between total foliar nitrogen content and aphid performance (Miles et al., 1982; Awmack & Leather, 2002; Khan & Port, 2008). High drought stress plants however, have low turgor pressure and higher cell-sap viscosity that can have negatively impact on aphid performance (Kennedy et al., 1958; Auclair, 1963), even though these plants had significantly higher concentrations of nitrogen.
In the present study, drought stress had a negative impact on root herbivore performance (Chapter 7). As the root biomass decreased under drought stress described above, it can be speculated that negative root herbivore performance was correlated with limited food availability under drought stress. The growth of D. radicum has been correlated with sugar (Hopkins et al., 1993) and sulphur (Marazzi & Stadler, 2005) levels of Brassicae
104 roots and there are thus other aspects of food source quality that need to be investigated to get a full picture of the factors affecting D. radicum development.
In the present study, D. radicum altered the quality of plant foliage, and this may be correlated with reduced performance of aphids and their parasitoids. A direct link between plant quality and parasitoid preference and performance was also associated with the decrease in parasitoid preference and performance under both root herbivore and drought stress (Chapter 7). These results are in agreement with Hunter‟s (2003) proposed plant quality mechanisms for parasitoid success, where low quality plants are associated with decrease in parasitoid preference and performance. Since below-ground herbivory and drought stress directly affect plant quality, parasitoid success can be influenced by root herbivore (Soler et al., 2005) and drought stress (Calatayud et al., 2002).
Numerous studies have evaluated the possible effect of global change factors such as temperature and CO2 on plant-herbivore interactions. Less work has been carried out with respect to drought impact on plant-herbivore interactions in present changing climate scenarios (Bale et al., 2002). In temperate terrestrial ecosystems, moderate to high drought stress (Beier et al., 2004), elevated CO2 concentration (Houghton et al., 2001) and increase in temperature (Solomon et al., 2007) have been projected under current climate change scenarios. These changes will directly influence the host plant quality and could alter the herbivore performance (Bale et al., 2002; White, 2008). In the present study, high drought stress reduced herbivore performance and parasitoid preference and performance thus may have indirect effects on food chains in terrestrial ecosystems (Schmitz, 2008).
8.2.3 Management implications
The present study has demonstrated the effects of plant quality on herbivore-natural enemy interactions in response to biotic and abiotic stresses. Both biotic (above- and below- ground herbivores) and abiotic (drought) stresses reduced plant quality (nitrogen, glucosinolates, plant VOCs) and were shown to be correlated with lower performance of
105 both aphid species and their parasitoids. High drought stress and high D. radicum density reduced plant quality for both aphid species and the adverse effects of high drought stress and D. radicum were transmitted to a higher trophic level. The performance of the host and its parasitoid are positively correlated, although the adverse effects of food plant quality on insect performance are usually less pronounced in the parasitoid than in the herbivore (Gols
& Harvey, 2009). In the present study, the adverse effects of food plant quality were more pronounced in parasitoids as compared to aphids which may be due to the lower quality of the host aphids (Sampaio et al., 2008).
Since the number of parasitoids was lower under biotic and abiotic stress, a refined irrigation plan could enhance the effectiveness of biological control in such an agriculture system. One of the main reasons of biological control failure is drought, by itself or in combination with other factors, and the present work has shown how drought can markedly reduce both the preference and performance of natural enemies (Chapter 7).
8.3 Future work
Only a few studies have actually quantified the association between higher trophic levels and drought stress, where it was shown that drought may reduce the diversity and abundance of herbivores that could facilitate the loss of predator and parasite, thus altering the composition of terrestrial communities (Sumerford et al., 2000; Calatayud et al., 2002;
Trotter et al., 2008). Comprehensive investigations into the interactions between higher trophic levels and drought stress are thus far lacking under the framework of global change.
Future studies should therefore focus on simultaneously testing the effects of multiple environmental factors, including drought, to determine how global climatic changes may impact on plant-herbivore-parasitoid-interactions. Plant VOCs are biochemically well characterized and many genes and enzymes involved in their synthesis are known (Heil,
2008), however experimental evidence for the biosynthetic origin of Brassica volatiles under different stresses are unknown. Increased knowledge on how the production of VOCs is
106 influenced by environmental conditions is required in order to understand how these plant
VOCs can be used in crop protection programmes (Heil, 2008).
107
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Appendix 1: Preliminary results for relative water contents of Brassica oleracea (mean ±
S.E.M.) grown under drought stress treatments. Means with different letters are significantly different (n=10; P < 0.05).
148
Appendix 2: Preliminary results for relative water contents of Brassica oleracea (mean ±
S.E.M.) grown under different water stress treatments with high Delia radicum density.
Means with different letters are significantly different (n=10; P < 0.05).
149