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ANALYSIS OF SUGARS AND AMINO ACIDS IN APHID

HONEYDEW BY HYDROPHILIC INTERACTION LIQUID

CHROMATOGRAPHY – MASS SPECTROMETRY

by

PHUONG KIEU NGUYEN

B.A., University of Colorado Colorado Springs, 2016

A thesis submitted to the Graduate Faculty of the University of Colorado Colorado Springs

in partial fulfillment of the requirements for the degree of

Master of Sciences

Department of Chemistry and Biochemistry 2019

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This thesis for Master of Sciences degree by Phuong Kieu Nguyen

has been approved for the

Department of Chemistry and Biochemistry by

Janel E. Owens, Chair

Emily Mooney

Allen Schoffstall

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Nguyen, Phuong Kieu (M.Sc., Chemistry)

Analysis of Sugars and Amino Acids in Aphid Honeydew by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry

Thesis directed by Associate Professor Janel E. Owens.

ABSTRACT

The impact of phenology to herbivore abundance was studied on the subalpine plant Ligusticum porteri and the aphid herbivore Aphis asclepiadis to assess some of the impact of climate change on this herbivore-plant system. The honeydew production of A. asclepiadis feeding on L. porteri under different phenological mismatches was examined in terms of sugar and amino acid composition. Given the difficulties of analyzing sugars and amino acids, a recent alternative separation mode for HPLC, hydrophilic interaction liquid chromatography (HILIC), was employed. As HILIC separation mechanisms are based on the strong hydrophilic interaction of polar compounds, this chromatography has been widely applied for analysis of biomolecules such as saccharides, amino acids, and proteins. In this study, composition of seven saccharides in A. asclepiadis honeydew, including xylose, fructose, glucose, sucrose, trehalose, melezitose and raffinose, and five standard amino acids, including glutamic acid, glutamine, aspartic acid, serine, and asparagine, were analyzed using HILIC on a liquid chromatography tandem mass

spectrometry (LC/MS/MS) system. The process of method development to analyze these sugars and amino acids from collected honeydew was examined.

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ACKNOWLEDGEMENTS

To Dr. Janel Owens, for all of her guidance, patience and immense knowledge through the process of researching and writing this thesis.

To Dr. Emily Mooney, for the insightful discussion about aphids and statistical analysis with R.

To my parents Han Nguyen and Chi Le, and all of my family, for all the unfailing love, support and encouragement through the difficult moments in getting this degree.

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TABLE OF CONTENTS

CHAPTER

I. REVIEW OF LITERATURE 1

1. Introduction 1

1.1. Climate Change and Phenology 2

1.2. Ligusticum porteri (Oshá) 6

1.3. Aphid Species of Interest 9

1.4. Effects of Climate Change on Aphid Communities 12

1.5. Aphid-Ant-Lygus-Plant Interaction 14

2. Compounds of Interest in Aphid Honeydew 16

3. Previous Studies 18

3.1. Previous Studies on Phenology 18

3.2. Previous Analytical Techniques for Analysis of Aphid Honeydew 19

4. Analytical Techniques 23

4.1. Hydrophilic Interaction Liquid Chromatography 23

5. Hypothesis and Objectives 28

II. EXPERIMENTAL PROCEDURES 29

1. Instrumentation and Techniques 29

1.1. Chemicals and Reagents 29

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1.4. Multivariate Analysis of Variance (MANOVA) 33

2. Study Models/Systems 34

2.1. Study 1: Experimental Design for Effects of Elevated Temperature and Lygus

Feeding Position on Aphid Honeydew Quality 34

2.2. Study 2: Experimental Design for Effects of Host Phenology and Drought

Stress on Aphid Honeydew Quality 35

2.3. Study 3: Experimental Design for Effects of Prior Lygus Feeding on Aphid

Honeydew Quality 36

2.4. Study 4: Experimental Design for Effects of Experimental Snow and Water

Addition on Aphid Honeydew Quality 36

3. Sample Preparation 37

3.1. Sample Preparation for Analysis of Sugars and Amino Acids 37

3.2. Conditioning of HILIC Columns 38

3.3. Optimization of Methods for Analysis of Sugars 39

3.4. Optimization of Methods for Analysis of Amino Acids 40

III. SUGARS ANALYSIS BY LC-MS/MS IN APHID HONEYDEW 42

1. Introduction 42

2. Materials and Methods 43

2.1. Chemicals and Reagents 43

2.2. Preparation of Standards 43

2.3. Preparation of Samples for Analysis 44

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2.5. Data Analysis 47

3. Results and Discussion 48

3.1. Optimization of LC/MS Conditions 48

3.2. Figures of Merits 48

3.3. Statistical Analysis of Sugars in Aphid Honeydew 50

4. Conclusion 55

IV. AMINO ACIDS ANALYSIS BY LC/MS/MS IN APHID HONEYDEW 57

1. Introduction 57

2. Materials and Methods 58

2.1. Chemicals and Reagents 58

2.2. Preparation of Standards 58

2.3. Preparation of Samples for Analysis 59

2.4. Instrumental Conditions 60

2.5. Data Analysis 63

3. Results and Discussion 64

3.1. Optimization of LC/MS Conditions 64

3.2. Figures of Merits 64

3.3. Statistical Analysis of Amino Acids in Aphid Honeydew 65

4. Conclusion 70

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2. Future Work 72

REFERENCES 73

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LIST OF TABLES

TABLE

1. Limits of Detection (LODs) and Quantitation (LOQs) of seven sugars and five amino acids from previous studies

21

2. Quadratic Least Square Fit Equations, Correlation Coefficients, Linearity Ranges, Limits of Detection (LODs) and Limit of Quantification (LOQs) of seven studied sugars

49

3. Precursor/Product ion pairs and parameters for MRM of five amino acids used in this study

61

4. Quadratic Least Square Fit Equations, Correlation Coefficients, Linearity Ranges, Limits of Detection (LODs) and Limit of Quantification (LOQs) of five studied amino acids

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LIST OF FIGURES

FIGURE

1. Ligusticum porteri, flowers and leaf 7

2. Aphis asclepiadis on host plant 12

3. Ant-aphid mutualism on Ligusticum porteri 15

4. Lygus bug on Ligusticum porteri 15

5. HILIC proposed retention mechanism 25

6. Schiff base formation between the amine group of the stationary phase ligand and the hydroxyl group of a reducing sugar

27

7. Chromatogram of 10.0 µg/mL sugar standard used for calibration curve (Figure 8) with structures of seven sugars of interest, xylose (X),

fructose (F), glucose (G), sucrose (S), trehalose (T), melezitose (M) and raffinose (R)

46

8. Calibration curve for sugars with concentrations ranging from 0.05 µg/mL to 10 µg/mL

47

9. Effects of elevated temperature and lygus feeding position on fructose concentration in aphid honeydew

51

10. Relationship between fructose concentration and density of honeydew droplets at different feeding position and temperature treatment

51

11. Effects of host phenology and drought stress on sucrose concentration in aphid honeydew

52

12. Relationship between sucrose concentration and density of honeydew at different elevation and moisture treatment

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13. Effects of experimental snow and water addition on sucrose concentration in aphid honeydew

54

14. Relationship between sucrose concentration and density of honeydew at different snow and water treatment

55

15. Chromatogram of 10.0 µg/mL amino acid standard used for calibration curve (Figure 16) with structures of five amino acid of interest, glutamic acid (Glu, MRM), serine (Ser, SIM), aspartic acid (Asp, SIM),

asparagine (Asn, MRM), and glutamine (Gln, MRM)

62

16. Calibration curve for amino acids with concentrations ranging from 0.1 µg/mL to 10 µg/mL

63

17. Elevated temperature and lygus feeding position on asparagine concentration in aphid honeydew

66

18. Relationship between asparagine concentration and density of honeydew at different temperature and lygus bug treatment

67

19. Effects of experimental snow and water addition on glutamic acid concentration in aphid honeydew

69

20. Relationship between glutamic acid concentration and density of honeydew at different snow and water treatment

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LIST OF ABBREVIATIONS

ANOVA Univariate analysis of variance

CI Chemical ionization

EI Electron ionization

ELSD Evaporative light scattering detector

ESI Electrospray ionization

GC Gas chromatography

GC/MS Gas chromatography coupled with mass spectrometry HILIC Hydrophilic interaction liquid chromatography

HPAEC-PAD High performance anion exchange chromatography with pulsed amperometric detector

HPLC High performance liquid chromatography

ICOF-LIF In-capillary optical fiber laser-induced fluorescence

IDE Integrated development environment

LC Liquid chromatography

LC-MS/MS Liquid chromatography tandem mass spectrometry

LOD Limit of detection

LOQ Limit of quantitation

MALDI Matrix-assisted laser desorption ionization MANOVA Multivariate analysis of variance

MRM Multiple reaction monitoring

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NP-LC Normal-phase liquid chromatography

OTC Open-top warming chamber

RID Refractive index detector

RMBL Rocky Mountain Biological Laboratory

RP-HPLC Reversed-phase high performance liquid chromatography

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CHAPTER I

REVIEW OF LITERATURE

1. Introduction

Hydrophilic interaction liquid chromatography (HILIC) is an emerging analytical technique that has been applied widely in the past few decades for the separation of polar molecules in various types of samples including but not limited to biological,

pharmaceutical and food samples (1). Not only does HILIC offer better chromatographic characteristics such as better baseline separation and peak shape, this liquid

chromatography (LC) mode also allows analyses of complex sample matrices to be accomplished with little sample preparation (2). Despite the growing applications of HILIC, its use in analysis of environmental samples from insect herbivores and plant extracts has been limited.

On the other hand, extensive studies have been done to improve understanding of the insect community’s behaviors as a response to climate change (3). In particular, aphid species have received significant attention owing to their impact on crops and agricultural output (4). Specifically, in this study, changes in aphid abundance under the impact of climate change was examined in relation to host plant phenology and its multitrophic interactions with ants and predators. This can be achieved through the analysis of aphid honeydew, a byproduct excreted as aphid feed on its host plant. This study focused on the development of simple methods for analysis of sugar and amino acid composition in aphid honeydew via HILIC on a liquid chromatography tandem mass spectrometry (LC-MS/MS) system.

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1.1. Climate Change and Phenology

Climate change has been one of the most pressing issues over the past few decades. The climate system on Earth is driven originally by radiation energy from the sun, which is distributed throughout the planet by winds, ocean currents, and many other mechanisms that ultimately affect regional climates on earth (5). Factors that can cause climate changes are known as climate forcings or radiative forcings which can be divided into four main classes that are either external forcings such as astronomical, geological and human causes, or internal forcings such as intrinsic causes (natural changes within the climate system) (6). Out of all these factors, human influence has been the principal cause of the observed climate change (7). The most concerning impact is known as the “greenhouse effect” where global warming results from heat trapped in the Earth’s atmosphere, which is caused by certain gases including mainly water vapor, methane, chlorofluorocarbons and carbon dioxide (8).

Observed changes in regional climates has had significant impacts on the

environment, including an increase in average temperature and precipitation, increase in the number of heavy downpours and droughts, shrinkage of glaciers, and shifts in plant and animal ranges, to name a few (9). Extreme weather events such as heat waves and major extreme weather events, such as hurricanes, also occur more frequently and with greater intensity. As a result, impacts of climate change on human health are significant. There are several exposure pathways by which climate change can affects human health (9). Extreme weather such as heat waves, flood and droughts or severe storms not only have immediate effects but also long-term effects on human health. Climate change results in poor air quality as it affects levels of air pollutants. Food and water quality are

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also greatly reduced due to the impact on agriculture production. Significantly, changes in the distribution of infectious agents and disease vectors can result from the increase in temperature and rainfall (10). Over time, threats from climate change can lead to long-term changes in resilience and public health. Over time, public health could suffer from many severe heat related illnesses, cardiopulmonary illness, food water and vector borne diseases, and even mental health consequences and stress.

There are many different indicators of observed climate change. Some significant indicators that have been extensively studied but not limited to are global surface

temperature, Arctic and Antarctic sea ice coverage and depth, land ice, weather pattern changes in tropics and mid-latitudes, changing in ecology of birds and mammals, insect community, ocean current changes, plant pathogens, and many other aspects (11). Global surface temperature is one of the most widely studied indicator of climate change (12). Since the mid-nineteenth century, increase in surface temperature has been observed globally. The average combined land and ocean surface temperatures were calculated computationally and show an increasing trend of approximately 0.25 degree Celsius over a ten-year period (5). Different projection models were computed to predict the future trend in global temperature, which proved to be a non-linear change in nature with a significant upward trend over the last few decades (12). Addressing several uncertainties and challenges in data availability, measurement understanding, contributions of solar variability and volcanic activity is necessary to better the modelling studies of global temperature (12).

Apart from the most common studied indicators of climate change (such as temperature), insect communities play a crucial role in reflecting the impacts of the

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changing climate (3). The ubiquity of insects and their heavily dependence on weather conditions make the insect community a perfect indicator of how an ecosystem responds to climate change. Not only do insects contribute over half of the known species on Earth, but they also exist in every trophic level of the ecosystem. Insect-human relationships are quite complex and can be either parasitism, commensalism, or

mutualism depending on the insect activities and circumstances (13). Insects can act as disease vectors (mosquitoes, ticks, etc), parasites (bed bugs, lice, etc), crop pests (aphids), or can even cause major damage to structures (termites). On the other hand, humans can also benefit from the insect communities significantly. Agriculture systems depend on the services from the local ecosystems such as pollination, pest control, and nutrient cycling (14). Insects also take part in the natural and biological control of other insect populations that cause damages to agriculture and buildings. Consequently, as the insect community is altered significantly under the impact of climate change, human beings are directly influenced by the changes in insect behaviors. Since insects have internal temperature variations and depend on the environmental temperature, global warming has a

significant effect on insect development and interaction, behavior and survivor ability (3). Furthermore, other climatic conditions such as precipitation, levels of greenhouse gases, and snow cover can affect insect community dynamics. While precipitation determines environmental moisture, which directly causes diverse physiological and behavioral insect responses, greenhouse gases such as CO2 can change plant nutrition and thus vary insect consumption of plants (3).

When assessing the different impacts of climate change, changes in phenology as an outcome of climate change is inevitable. Phenology is an important but often

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overlooked aspect of ecology, which is stated as the study of the timing of periodic biological events in animal behavior and plant growth such as hibernation, migration, or flowering (15). Changes in phenological events, based on its definition, can have a significant impact on human activity and our environment (16). Through understanding of phenology, humans can improve agricultural systems with management of invasive species and crop pests or optimization of the timing to plant, fertilize and harvest crops. Prediction of many human health-related events such as allergies and improvement in the understanding of the timing of ecosystem processes such as carbon cycling are also made possible. Hence, studies have been done extensively on the causes of changes in

phenological timing with respect to biotic and abiotic sources and how these changes correspond to the changes in season and climate (15).

The way plants and animals respond to phenology can contribute to the prediction and study of their population and behavior, making phenology an excellent indicator of climate change. Global climate change such as increasing temperature, changes in precipitation, and earlier snowmelt could significantly alter phenology of plants and animals (17, 18). Increase in temperature or seasonal shifts in precipitation could affect the development timing of plants such as increase in the photosynthetic period and alter insect active seasons (3, 17). Earlier snowmelt dates could result in an earlier beginning of the growing seasons for plants and thus affect activities of insect communities that rely on those plants (18). Furthermore, studies have shown that changes in phenology are not only different among plants and animals but also varied among animal species, leading to mismatches between interacting species (3). Phenological mismatch is defined as a continuously ongoing phenomenon when species that typically interact alter the timing of

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regularly repeated phases and are no longer active at the same time in their life cycle, leading to trophic asynchrony among their populations (19). These mismatches ultimately affect the species’ strength, duration and outcome, and potentially cause disruption of mutualisms between interacting species (20). In this study, responses from an insect species, Aphid asclepiadis, which feeds on a native host plant of the Rocky Mountains, Ligusticum porteri, were studied under changes in different factors of phenology and the effect of these phenological mismatches on the mutualism between this species of aphid and ant, and interactions between the aphid species with its predators.

1.2. Ligusticum porteri (Oshá)

The genus Ligusticum can be found across the world within the mountainous regions and consists of approximately 40 to 60 perennial members (21). Native

Ligusticum species in North America include Ligusticum porteri J. M. Coult. and Rose (Figure 1), which is also commonly known as Oshá, bear root or chuchupate and has been an important medicinal plant traditionally used by Hispanics and Native Americans (22). L. porteri in the United States distributes throughout the Rocky Mountain regions, ranging from the northern states such as Montana and Wyoming, through Utah and Colorado, and to the southern states such as New Mexico (21, 23). It can be found in the montane to subalpine meadows, within diverse soil types and among altitudes up to 10,000 feet.

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Figure 1. Ligusticum porteri, flowers and leaf. Photo by E. Mooney.

L. porteri is a slow growing member of the Parsley family and can be identified by their compound umbels with white flowers, terete-ribbed fruits and basal or irregularly dissected leaves (21). The plant is typically 50 to 100 cm tall with large roots most

favored for medicinal purposes. Differentiation between L. porteri and other species, such as poison-hemlock, is quite problematic but can be made possible through careful examination of plant height, leaf, seed and root morphology, and root scent. Historically, roots of L. porteri were used by Native Americans to treat a diversity of medical ailments related to lungs and heart (24). Recently, the roots have been extensively studied and commercially harvested to treat many illnesses such as bronchitis, influenza and aches and pain. Apart from being used as medicine, leaves and even seeds and roots of L. porteri can be used as food, in flavoring or addition to salads (23, 24).

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The majority of research done on L. porteri, or Oshá, has focused on the molecular composition of the species such as derivatives of coumarins and phthalides (21). The plant extracts are also rich in antiviral and antibacterial compounds, which make it a popular herb on the current market (25). Studies have shown certain inhibition of certain bacteria and virus activities from the extracts of L. porteri. Moreover, studies on L. porteri extracts for anti-inflammatory drugs have currently been focused on avoidance of side effects caused by pharmaceutical treatments (26). On the other hand, roots of the species contain certain toxic compounds, which still requires further study and consideration of safe dosage (21).

Owing to the demand of the species in dietary supplement industry, L. porteri, which is mainly wild-harvested, is currently being adapted to garden or greenhouse environments and has shown a successful germination rate of 70% (21). However, L. porteri has been reported as a species at risk due to over-harvesting and its population can be affected by many habitual features such as climate change and heavy grazing (23). Furthermore, level of therapeutically important secondary metabolites in this Ligusticum species are found to be driven by harvest region and light environment (27). Hence, studies and research have been done extensively in the studies of cultivating methods to improve harvest sustainability as well as successful growing rate.

As part of the Rocky Mountain region, Colorado hosts an interesting array of many rare and distinct species. Among the aspen communities, L. porteri was identified as one of the dominant species in the subalpine meadows and understories (21). It is known to host many insect herbivores, with mutualisms between insects and various ant species, and between insects and its predators (28). Sap-feeding species such as aphid

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species are commonly found feeding on this medicinal plant. To study how aspects of the environment drive the abundance and distribution of species, researchers have been investigating the mechanisms of how factors of climate change and phenology affect the feeding of these aphid species on the host plant L. porteri, thus affecting relationships between aphids and ants or aphid’s predators (29). In this study, abundance of Aphis asclepiadis species on L. porteri was studied under multiple changes in different aspects of phenology.

1.3. Aphid Species of Interest

Among the insect community, aphids are herbivorous species that are quite abundant in most terrestrial habitats. They belong to the phytophagous, sap-sucking insects and are considered pests because of their impacts: destroying crops, affecting agriculture, horticulture and forestry (30). Originally, aphids used to feed mostly on trees using their piercing/sucking mouthparts (31, 32). They then evolved and diversified into colonies feeding on herbaceous plants, mosses, and ferns, which allow them to have more diverse sources of nutrition (31). These soft-bodied insects have complicated life cycles that involve many different morphological forms and can reproduce asexually or sexually (32). Three main factors that allow aphids to multiply rapidly are: 1) parthenogenesis, where growth and development of embryos can occur without egg fertilization; 2) viviparity, where fertilized eggs develop within the mother’s body and are born capable of independent existence; 3) and telescoping of generations, a combination of

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already carries a developing embryo (31). With that being said, aphids have been considered one of the most damaging insects to agriculture and forestry in the world.

There are three principal paths in which aphids damage crops and agriculture (4). The first approach is through removing plant sap thus weakening the plants and

eventually leading to lower fruit quality and quantity or even plant death. The second way is through the production of mold fungi on the host plant thus reducing the plant photosynthesis process and eventually leading to poor fruit quality and quantity. This is all due to the fact that aphids must consume a large amount of phloem, which is low in amino acids, in order to gain enough nutrients for their reproductive activity. These insects then produce a large quantity of carbohydrate-rich product, called honeydew, which provides a perfect medium for the growth of black sooty mold fungi on the host plant. Last but not least, the third route is the spread of plant viruses and diseases by aphids as efficient vectors, causing tremendous loss and damage to agricultural system (33).

Unlike most herbivores that feed on leaves, aphids consume a greatly imbalanced food resource, the phloem sap (34). The significant carbon and nitrogen sources in phloem sap are sugars and amino acids. While leaves provide a protein/carbohydrate ratio, or amino acid/sugar ratio, of roughly 0.8 to 1.5 (w/w), phloem sap gives a protein/carbohydrate ratio of only approximately 0.1 (w/w). This ratio in phloem sap indicates that aphids actually prefer a low nitrogen source, which can be explained by the basis of evolutionary adaptation when choosing this special type of food source.

However, many essential amino acids that are important for aphid performance such as arginine, lysine, and valine cannot be synthesized by aphids themselves, thus the quantity

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and quality of nitrogen source can have a significant impact on the growth and development of aphids on their host plants (34, 35).

When feeding on the host plant, aphids excrete a by-product waste of phloem ingestion called honeydew (36). Aphid honeydew has been studied and shown to

compose of many different chemical compounds of which the majority is sugars (taking up to approximately 95% in dry weight) and amino acids (37). Studies also show that factors that influence the chemical composition of honeydew include but are not limited to the host plant species, the quality of nutrition in the host plants, the species and developmental stage of aphids, the duration and rate of aphid infestation, the presence of mutualism with ants or competitors and predators (34, 37). Even though it is found that the sugar and amino acid composition of honeydew corresponds and reflects the content of the phloem sap of host plants, aphid honeydew universally contains both standard and non-standard sugars such as glucose, sucrose, melezitose and raffinose (37, 38) and both essential and nonessential amino acids such as asparagine, glutamine, aspartic acid and proline (37, 39). Owing to the numerous breakdown products of honeydew, this aphid waste has been attractive to not only many parasitoids (36) but also ant species, which eventually benefits the aphid through its mutualism with ants (38). In this study, composition of honeydew excreted by Aphis asclepiadis (Figure 2) which feeds on L. porteri, was studied.

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Figure 2. Aphis asclepiadis on host plant. Photo by E. Mooney.

1.4. Effects of Climate Change on Aphid Communities

As climate change alters plant ecology significantly, insect communities that feed on plants, such as aphids, are also strongly affected, and so do their natural enemies and mutualistic species (40). Climate change can have both short-term and long-term effects on the insect communities (41). Short-term effects involve alteration in insect behavior, life history traits, development time, metabolic rate and sex allocation, whereas long-term impacts can lead to genetic variability in climatic adaptive populations (41). Furthermore, with the consideration of disease transmission, interactions between plant, insect, virus are interrelated forming a “disease triangle” (42). Under the impact of climate and atmospheric gases on plant, aphid, virus and their interaction, the fourth node of the “disease pyramid” is formed. As a result, through understanding of the mechanism behind climate change effects on insect community, specifically aphid population, the

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underlying causes that influence population-level responses such as phenology, abundance, distribution and genetic variability can also be identified (42).

Compared to other insect communities, aphids have a short generation time through multiple reproductive modes (31) and a relatively low developmental

temperature threshold that allows them to stay active in more extreme temperatures (43). Moreover, aphids also show a variation in a wide range of traits with respect to

environmental changes, which can be manipulated to study the overall responses of organisms to climate change. All these characteristics make aphids more likely to respond strongly to environmental changes and thus become a suitable subject for studying the impacts of global climate change. On the other hand, aphid activities on crops can be greatly affected by phenology (44). Aphid abundance is greatly altered by changes in phenology of its host plant, mutualists, predators and competitors (28, 45). Understanding phenology of aphids can help predicting the type of crops as well as the growth stage of crops at which is more prone to be invaded (44).

In this study, abundance of an aphid species, Aphis asclepiadis, which feeds on L. porteri, was studied and evaluated in relation with multiple species interactions under multiple phenological conditions. A. asclepiadis, synonymous with Aphis helianthi (46), feeds on plants in both natural and agriculture systems and has become a concerning pest due to its developed resistance or tolerance towards insecticides (47). Although its appearance greatly differs among generations, the aphid species is usually olive green with black cornicles. A. asclepiadis is found to live in Cornus stolonifera (or dogwood) in the winter and migrate to L. porteri and several other host species when spring comes (29). The aphid colonizes mostly the flowering stalks and sometimes the leaves of its host

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plants, usually is tended by several ant species forming a mutualistic relationship and also is prey to several predators such as lygus bugs, ladybird beetles and hover fly larvae (28).

1.5. Aphid-Ant-Lygus bug-Plant Interaction

Mutualism is defined as an interaction between different species that would result in beneficial outcomes for the two participants (48). In this case, as aphids feed on the host plant and excrete the sugar-rich honeydew by-product, it attracts many organisms including ants and drives the mutualism between aphids and ants where aphids will benefit from this mutualistic relationship with ants by supplying them with honeydew in exchange for their protection against predators and parasites (38). Furthermore, through protection from predators, this mutualism has resulted in an increased reproductive rate as well as feeding and excretion rates, which ultimately leads to more rapid development and colony growth (49). Previous studies have shown that A. asclepiadis feeding on L. porteri were tended by several ant species such as Formica fusca, Formica rufa, and Tapinoma sessile (Figure 3) (28, 29). Beside mutualism, aphid honeydew also attracts many antagonists such as predators and parasites. Aphid species fed on wild plants are found to live in relatively small colonies with short-time generation due to competitions with other insects and natural enemies and predators (31). The flightless nymphs of lygus bugs (Tribe Mirini: Family Miridae) such as Lygus hesperus (Figure 4) are found to also feed on phloem sap and compete with A. asclepiadis on L. porteri (28). The adults of this species, on the other hand, are predators of aphids. Together with other common

predators such as ladybird beetles and hover fly larvae, these predators control the population of aphid colonies in the system. In this study, changes in abundance of A.

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asclepiadis was studied based on the sugar and amino acids composition of aphid honeydew in respond to change in host plant phenology and interactions with other species under the impact of climate change on the host plant L. porteri.

Figure 3. Ant-aphid mutualism on Ligusticum porteri. Photo by E. Mooney.

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2. Compounds of Interest in Aphid Honeydew

Multiple environmental changes could affect plant phenology and insect

abundance. Aphid herbivory, which may be strongly influenced by the effects of climate changes, has been one of the primary means by which climate impacts have been studied (40, 41). One important indicator of these effects on aphid abundance is the honeydew byproduct, which is excreted by aphids after feeding on host plants. Studies have shown that the chemical composition of honeydews varies depending on the phloem sap composition, which in turn varies between host plant species and can be affected by changes in the host plant phenology as well as the aphid species (39, 50). Compositions of secondary metabolites (such as cardenolides), sugars, and amino acids in aphid honeydews have shown to differ distinguishably among host plant species and plant multitrophic interactions with other species (39). This study focused on sugar and amino acids composition in honeydew produced by A. asclepiadis species feeding on phloem sap of the L. porteri host plant under multitrophic interactions with several ant and lygus bug species.

Aphids’ natural diet is plant phloem sap which contains high concentrations of sugars, especially sucrose, and low concentrations of amino acids (51). Aphid honeydew generally consists of a variety of sugars including but not limited to monosaccharides (xylose, fructose, glucose), disaccharides (sucrose, maltose, trehalose), and trisaccharides (melezitose, raffinose, erlose) (38, 39, 50, 52). Within these sugars, oligosaccharides such as xylose, fructose, glucose, and sucrose can be found in various plants and living

organisms (53), whereas non-reducing saccharides such as melezitose, trehalose and raffinose are usually synthesized by insect herbivores (51, 54). The main sugar content in

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phloem sap is sucrose, which is metabolized in the aphid gut into glucose and fructose to be used as substrates in respiration or in the synthesis of other oligosaccharides

(melezitose, trehalose, and raffinose) in honeydew (51). Sugars in aphid honeydew play an important role in the mutualism between ant and aphid species (52). By providing ants with sugar-rich honeydews, aphids gain protection against predators and parasites (38). Among these sugars, melezitose has been found to be particularly abundant in various aphid species and ants seem to prefer honeydew containing high concentration of melezitose (38, 52). This oligosaccharide is synthesized by many plant sap-sucking insects such as aphids from two units of glucose and one unit of fructose to regulate osmoregulation (50, 55). Specifically, a higher concentration of melezitose has shown to not only result in increase in ant attendance and feeding efficiencies but also reduce the suitability of honeydew for predators and parasitoids (50). Hence, variation in sugar composition especially in melezitose of aphid honeydew can reflect host plant fitness as well as multitrophic interactions on aphid behavior. In this study, seven standard and nonstandard sugars including xylose, fructose, glucose, sucrose, trehalose, melezitose and raffinose in aphid honeydew collected were analyzed using HILIC in order to study how various plant treatments affect plant fitness and aphid abundance reflected by the

composition and concentrations of these sugars.

On the other hand, amino acids also play an important role in aphid abundance. Aphid honeydew has been examined to have very low concentration of amino acids (56). Aphids, like other insect herbivores, are well known to not have the capability to

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extract large amounts of amino acids from their host plants and hold on to them (38). The composition of amino acids in aphid honeydew has been shown to correspond to the phloem sap content such that asparagine and glutamine were consistently found to be the two dominant amino acids in both plant phloem sap and infested aphid honeydew (58). Furthermore, predominant amino acids found in most aphid honeydew across all species are usually include asparagine, glutamine, glutamic acid, serine, and aspartic acid with the amino acid profiles varying depending on the host plants and aphid species (37, 56). A recent finding has also discovered that aphid honeydews with high concentration of amino acids also have high concentration of sugars (56). Therefore, studies on amino acid composition and concentration in honeydews may reveal information on the host plant profile and effects of climate change on host plant phenology and aphid abundance. In this study, five amino acids that are known to be universally found in most aphid

honeydews, glutamic acid, glutamine, aspartic acid, serine, and asparagine, were studied and evaluated in honeydew samples produced by A. asclepiadis species feeding on phloem sap of L. porteri using HILIC.

3. Previous Studies

3.1. Previous Studies on Phenology

Owing to the fast growth and various reproduction pathways of aphids, these insect species have become one of the most destructive pests to crops (30). Extensive studies, therefore, have been completed in order to better the understanding of aphid living mechanisms and be able to control their outbreaks (4). Previous studies on how climate change affects aphid herbivores have been done considerably within the past

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decades. The effects of changes in temperature on aphid abundance have been well documented in the past (59, 60), whereas changes in factors such as multitrophic

interactions and snowmelt timing have very few experimental studies (28). Research on the impact of several aspects of climate changes such as elevated temperature and early snowmelt date in relationship with multitrophic interactions among host plant, insect herbivores and these insects’ natural enemies are in need for understanding these insect communities.

3.2. Previous Analytical Techniques for Analysis of Aphid Honeydew

The composition and concentration of aphid honeydew, the products excreted from feeding on plants by aphids, which plays an important role in aphid abundance, are of widespread interest of insect herbivore behaviors (39, 50). Honeydew chemical analyses were usually done to measure honeydew cardenolides, sugars, amino acids, and even proteins (39, 58). Among these compounds, recent techniques used for detection and quantification of sugars in honeydew usually include the use of reversed-phased high performance liquid chromatography (HPLC) for separation coupled with an evaporative light scattering detector (ELSD) for quantification (39), or “high performance anion exchange chromatography” (or HPLC with anion-exchange column) with pulsed

amperometric detector (HPAEC-PAD) (50), or gas chromatography coupled with a mass spectrometer (GC/MS) (61). Typically, analysis of highly polar organic compounds such as sugars requires derivatization of samples to be done prior to LC or GC injection. Derivatization is the process by which a compound is converted into a derivative that has properties more suitable to a particular analytical method (62). Sugars or carbohydrates

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have such low volatility that derivatization is required for the analysis with GC or

GC/MS (61, 63). On the other hand, analysis with LC does not require the sugar analytes to be volatile, however, without derivatization, sugar analysis usually yields poor

chromatographic performance such as very poor peak separation and low sensitivity (64). Given that derivatization can be very time-consuming, costly, and not all components of the mixture derivatize equally efficiently, alternative methods are preferred to avoid a derivatization step. With the use of an ELSD (39) or a high pH HPAEC-PAD (50), sugars can be analyzed with a LC system without derivatization. These systems, however, are not universally used nor easy to operate, thus a better alternative technique for analysis of sugars without derivatization is favored.

Besides sugars, amino acids are also often compound of interest in aphid

honeydew in the study of the abundance of these species (39, 56). Analytical techniques that have been used for detection and quantification of amino acids in aphid honeydew include the use of HPLC coupled with a photodiode array detector (39, 56), or cation-exchange HPLC on a Biochrom 20 Plus amino acids analyzer (37), or GC/MS (61). Similar to sugars, derivatization of amino acids prior to separation and detection is almost always required to improve chromatographic performance (39, 56, 61). This is due to the fact that amino acids also have low volatility, which requires derivatization to make them more volatile for GC or GC/MS analysis (63). Furthermore, owing to the reactive nature of amino acids, derivatization is necessary to convert reactive polar functional groups on amino acids such as -OH, -NH2, or -SH into a nonpolar moiety (65). Furthermore, amino acids and sugars are important indicators of changes in host plant fitness and aphid

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abundance, simpler preparation techniques that would avoid a derivatization step as well as yearning for better chromatographic characteristics are necessary.

A comparison table that summarizes limits of detection (LODs) and limits of quantitation (LOQs) from previous studies for sugars and amino acids in various types of samples using HPLC coupled with different types of detector was created as reference to this study (Table 1). In this current work, hydrophilic interaction liquid chromatography (HILIC) was selected for the detection of sugars and amino acids using LC-MS/MS.

Table 1. Limits of Detection (LODs) and Quantitation (LOQs) of seven sugars and five amino acids from previous studies.

Sample Matrices Sample Preparation Analytical Instrument Analytes LOD (µg/mL) LOQ (µg/mL) Wine and grape juices (66) Dilution and filtration HPLC-refractive index detector (RID) Glucose Fructose 21 50 103 199

Soybean (67) Extraction and purification HPLC-ELSD Fructose Glucose Sucrose Raffinose 60.0 30.0 9.3 12.1 60.0 30.0 9.3 12.1 Beverages (68) Filtration and dilution

HPLC-UV detector Glucose Fructose Sucrose 0.05 0.18 0.08 0.15 0.60 0.25

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Table 1 cont. Limits of Detection (LODs) and Quantitation (LOQs) of seven sugars and five amino acids from previous studies.

Sample Matrices Sample Preparation Analytical Instrument Analytes LOD (µg/mL) LOQ (µg/mL) Human blood (69) Derivatization and amino acid labeling HPLC-In-capillary optical fiber laser-induced fluorescence (ICOF-LIF) detector Aspartic Acid Glutamic Acid Asparagine Glutamine Serine 0.041 0.040 0.021 0.048 0.011 0.140 0.134 0.069 0.162 0.037 Tea (70) Derivatization HPLC-Fluorescence detector Aspartic Acid Glutamic Acid Asparagine Serine Glutamine 0.057 0.085 0.20 0.126 0.164 0.192 0.284 0.665 0.42 0.545 Fruits (71) Extraction and filtration HILIC-HPLC- MS/MS Glutamine Serine Glutamic Acid Asparagine Aspartic Acid 0.007 0.036 0.078 0.061 0.063 0.022 0.109 0.272 0.245 0.219

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4. Analytical Techniques

4.1. Hydrophilic Interaction Liquid Chromatography

Liquid chromatography (LC) has been a widely preferred technique for environmental and biological nonvolatile compounds. Reversed-phase liquid

chromatography (RP-HPLC) is the most common separation technique of HPLC, which separates compounds based on their hydrophobic moieties (72). Even though separation of polar compounds can be done with RP-HPLC, this technique does not have a dominant polar character. Highly polar compounds such as carbohydrates, amino acids and

peptides are poorly retained on hydrophobic stationary phases of RP-HPLC and tend to elute at the beginning of the gradient elution (73). Hence, other HPLC techniques such as normal-phase liquid chromatography (NP-LC) and HILIC are preferred for analyses of these polar compounds. Even though the NP-LC and HILIC have similar retention

principles for polar stationary phases, the differences in selection of mobile phases makes HILIC an emerging separation technique for polar molecules in a wide variety of samples in the past decades.

The acronym “hydrophilic interaction chromatography” was first suggested by Alpert in 1990 even though the principle of HILIC was first described by Samuelson et al. back in 1952 for the separation of monosaccharides (74). As in NP-LC, HILIC

employs polar stationary phases, such as bare silica or silica modified with polar moieties like amino, amide, or zwitterionic functional groups (75). The resemblance in stationary phase selections leads to the similar retention principles in NP-LC and HILIC. In addition to many types of interactions observed in NP-LC such as hydrogen bond formation, charge transfer, and ionic interaction, hydrophilic interaction plays a major role in the

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retention process in HILIC (72). In comparison to RP-LC, HILIC offers much better retention for hydrophilic compounds. On the other hand, HILIC utilizes similar mobile phase solvents as in RP-LC, which are usually mixtures containing a high content of water-miscible organic solvent, typically acetonitrile in water (74). This allows favorable interfacing to MS detectors, especially with electrospray ionization (ESI-MS) because acetonitrile can help assist spray formation while improving ionization efficiency, which thus results in an enhanced detection sensitivity (75). Furthermore, acetonitrile has proven to provide much better retention of analytes and peak shapes than methanol, making it the most popular choice as the organic solvent used in HILIC (74). Another important advantage of HILIC than NP-LC is that with the difference in choice of mobile phase solvents, it gets rid of the maintenance required for dedicated instruments used in NP methods (75).

Despite all the important advantages associated with the use of HILIC, the retention mechanism behind HILIC is not completely understood (2, 75). The most widely recognized theory currently proposes that HILIC retention is caused by the

partitioning mechanism. In this mode, separation of analytes is based on their distribution between the organic eluent, acetonitrile, and a water-enriched layer immobilized on HILIC stationary phase (Figure 5). Hence, analytes with higher hydrophilicity and

polarity will partition more into the adsorbed water layer on the stationary phase, thus are more retained (2). Furthermore, as the concentration of water in the mobile phase

decreases, the retention of polar compounds increases (75). Additional observations revealed that the retention mechanism in HILIC does not involve partitioning alone but many other interactions with the specific stationary phase. Separation of analytes can thus

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depend on interactions such as polar interactions between the analytes and the active surface of the stationary phase or ionic interactions between charged analytes and stationary phase charged moieties (75).

Figure 5. HILIC proposed retention mechanism (76).

With the increase in its popularity, HILIC has been applied in various different types of fields and sample matrices. HILIC has recently been applied in metabolomic analyses, the study of metabolic responses of living systems to modifications and changes (74). With the use of HILIC with a zwitterionic column, separation, identification and quantification of cellular metabolites from bacteria can be achieved with simple sample preparation. Another application of HILIC was in the study of antidepressant behavior (77). The chromatographic behavior for the studied antidepressant was significantly improved with the use of the bare silica column for HILIC stationary phase. HILIC has also been applied in food analysis to overcome major drawbacks with traditional RP-LC (1). Separation was usually performed on a zwitterionic stationary phase. Simpler food

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preparation processes were required as well as significant improvement in sensitivity in the detection of antibiotics in food was achieved when using HILIC.

Despite the extensive application of HILIC in multiple fields and sample types, very little studies have applied HILIC in environmental samples from insect herbivore and plant extracts. Specifically, in aphid honeydew, the byproduct excreted from this insect species as they feed on phloem saps of host plant, the composition of sugars and amino acids dictates the plant fitness and aphid abundance. Accurate measurement of these analytes plays an important role in the study of this species. HILIC offers an

excellent choice for the improvement of chromatographic separation of sugars and amino acids as well as very little and simple preparation steps for honeydew samples. Especially with amino acids, derivatization has always been an irreplaceable step in sample

preparation prior to analysis. In this study, composition of sugars and amino acids were determined without derivatization using HILIC with mass spectrometric detection.

Depending on the analytes of interest, suitable column materials for HILIC were selected. Reducing sugars such as fructose and glucose usually mutarotate in solution and create an equilibrium between their a and b anomers. This has caused a major challenge for chromatographic separation of sugars (78). Previously to produce single quantifiable peaks for sugars, silica-based alkylamine columns have been chosen for the separation of saccharides (79). The amine group creates a high pH environment beneficial for

collapsing the sugar anomers. However, this environment also significantly reduces the column lifetime as the stationary phase self-deteriorates. A major disadvantage of this column use is the formation of the Schiff base as the consequence of the chemical

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6). Not only this reaction reduced the column lifetime, but it can lead to loss of sugar peaks over time as these sugar analytes are tightly bonded to the column. Similar outcomes apply to the analysis of amino acids.

Figure 6. Schiff base formation between the amine group of the stationary phase ligand and the hydroxyl group of a reducing sugar (79).

To overcome these limitations, amide bonded stationary phases have been applied in recent studies of carbohydrates and amino acids (71, 80). By eliminating the formation of imine group, it allows the full recovery of the analytes as well as improvement in separation efficiency (79). On the other hand, environmental samples such as aphid honeydews contain other chemical components that can cause matrix interferences to the analysis of sugars and amino acids in honeydew. Extensive sample preparation steps, especially derivatization, were involved in previous studies without HILIC (56, 61). To simplify these extensive steps, in this study, analyses of sugars and amino acids in aphid honeydew was completed using HILIC performed on Xbridge Amide columns (3.0 mm

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× 100 mm, 3.5 µm, Waters Corp., Milford, MA). With the developed methods, direct analyses of sugar and amino acid compositions in aphid honeydew were achieved.

5. Hypothesis and Objectives

The ultimate goal of this research was to study the impacts of climate change on insect abundance through host plant fitness and multitrophic interactions. As aphid honeydew can reveal important information on this species abundance and its host, appropriate methods are needed for the analysis of this type of environmental samples. This work developed simple methods for the analysis of sugars and amino acids (standard and non-standard) in aphid honeydew via a new emerging separation technique, HILIC on a Shimadzu LC-MS/MS. Furthermore, detection and quantification of seven sugars and five amino acids of interest were also examined. The current hypothesis is that there will be an improvement in chromatographic characteristics in comparison to previous analytical methods such as better peak resolution and peak shape. Also, it is hypothesized that limit of detection as well as limit of quantification of the developed methods with HILIC will be lower to level of part-per-million.

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CHAPTER II

EXPERIMENTAL PROCEDURES

1. Instrumentation and Techniques

Composition and concentration of both sugars and amino acids present in aphid honeydew is crucial in the understanding of how climate changes affect plant phenology and interspecies interactions affect aphid herbivores. With the use of HILIC, the primary and only technique utilized to analyze honeydew samples is liquid chromatography coupled with a tandem mass spectrometry detector.

1.1. Chemicals and Reagents

Stock sugar and amino acid solutions were prepared from standards purchased from various vendors including Fisher Scientific (Fair Lawn, NJ), Sigma-Aldrich (St. Louis, MO), and Macron Chemicals (Phillipsburg, NJ). LC-grade 18 MW DI water filtered by a Barnstead filtration system from Fisher Scientific was used for all standard preparation procedures and for LC chromatographic mobile phase preparation. LC/MS-grade solvents ≥99.9% pure methanol and ≥99.9% pure acetonitrile were purchased from Fisher Scientific, ammonium hydroxide (≥25% assay) was purchased from Sigma-Aldrich,formic acid (≥98% purity) was purchased from Honeywell FlukaTM (Muskegon, MI), and 1.00 M ammonium formate solution was made from ammonium formate

(≥99.0% purity) from Sigma-Aldrich. All other chemicals were from Fisher Scientific unless otherwise specified.

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1.2. Liquid Chromatography – Mass Spectrometry

Liquid chromatography (LC) has been employed since the early 1900s and has become the most widely used analytical technique for the separation and analysis of multiple types of chemical mixtures (81). Essentially, a sample can be introduced into the column (stationary phase) of an LC through an autosampler. As the mobile phase carries the sample through the column, components of the sample mixture can be separated based on their interactions with the column packing material as well as the column dimensions. These analytes elute from the column into the detector at a certain retention time, which allows identification and analysis of these compounds. The detector used in this study was a mass spectrometer (82).

Mass spectrometry (MS) is a powerful analytical technique that has been used in identifying and quantifying a wide range of molecules from isotopes to biomolecules such as proteins. As a sample enters the mass spectrometer, it gets ionized into multiple ions by the ion source that are separated based on their specific m/z ratio by the mass analyzer. The detector then identifies and records the relative abundance of each

separated ion type (83). Ionization sources of a mass spectrometer include hard ionization methods such as electron ionization (EI) and chemical ionization (CI), and “soft”

ionization methods such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). With EI and CI, information on the analytes of interest can be obtained through complete or partial fragmentation of ions. However, molecular mass of target ions cannot be observed with too many fragment ions. On the other hand, “soft” ionization techniques such as ESI enable molecular mass of a molecule to be obtained from much cleaner spectra. Therefore, ESI has been applied for analysis of a

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wide range of compounds with relatively high sensitivity. With ESI, the analyte molecules were first generated into highly charged droplets which is followed with charged droplet evaporation (84). Once the droplet reaches Rayleigh limit, Coulombic repulsion occurs resulting in multiple charged ions (84). In this study, sugar and amino acid compositions in aphid honeydew were analyzed by liquid chromatography coupled with a tandem mass spectrometry system (LC-MS/MS) fitted with ESI.

Quantitation with mass spectrometry can be acquired in many different scanning modes depending on the specific purpose of the study. One common mode of acquiring data with LC/MS system is with selected ion monitoring (SIM). With SIM, the mass spectrometer was set to look for a single mass unit over a very small mass range,

allowing only compounds with the selected mass to be detected (85). By monitoring just one specific mass instead of the whole mass spectrum, sensitivity of the mass

spectrometer is significantly increased. Another common mode for quantitation in LC-MS/MS is multiple reaction monitoring (MRM). MRM spectra deliver a very highly sensitive and specific quantitation of target compounds (86). MRM scanning mode was set to select a specific mass of the precursor ion (intact analyte) and one or multiple specific product ions (fragment ions from the precursor ion) after fragmentation. In this study of aphid honeydew, detection of sugars was performed in SIM mode whereas detection of amino acids was performed in SIM and MRM mode.

A Shimadzu LCMS-8030 system was used for chromatographic separation of sugars and amino acids in this study. The LC system consisted of a binary solvent delivery system equipped with triple quadrupole mass spectrometer and an autosampler. HILIC for sugar and amino acid analyses was performed on Xbridge Amide columns (3.0

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mm × 100 mm, 3.5 µm, Waters Corp., Milford, MA). Quantitation was by quadratic least squares fit with 1/x weighting from 0.05 to 5 µg/mL for sugars and from 0.1 to 10 µg/mL for amino acids. The specific parameters and conditions of LC-MS/MS system for sugar and amino acid analyses are discussed in chapter 3 and chapter 4 respectively.

1.3. RStudio Programming

Statistical analyses of collected data for sugar and amino acid concentrations in aphid honeydew were completed using RStudio version 3.6.0. RStudio is an integrated development environment (IDE) for R which is a language and environment for statistical computing and graphics (87). With the use of R, various statistical and graphical

techniques can be achieved. Specifically, R effectively handles and stores a significantly large amount of data. It also provides a wide range of intermediate tools, calculations and graphical facilities for data analysis with the use of a well-developed but simple and effective programming language (88). Being an IDE just for R, RStudio includes powerful coding tools that eases the process of data manipulation.

In this study, the statistical software RStudio version 3.6.0 was used to create graphical display of each sugar and amino acid concentration corresponding to each treatment condition in correlation with density of honeydew droplets. Besides the regular line graph for linear relationship between variables, box plots were also generated

through the help of R to describe the discrepancy between data sets. Box plots, also known as box and whisker diagram, is a visual representation of the distribution of a group of data based on what called a five-number summary: minimum, first quartile, median (second quartile), third quartile, and maximum (89). Despite displaying less

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information than histogram (bar graphs), box plots are very efficient in identifying outliers and comparing distribution between multiple groups of data. Furthermore, not only do box plots take up less space for large data groups but this type of diagram also focuses more on information about the data set rather than the mean and standard deviation for each group of data (89).

1.4. Multivariate Analysis of Variance (MANOVA)

For data analysis of sugars and amino acids in aphid honeydew, multivariate analysis of variance (MANOVA) was performed using RStudio. MANOVA is an

extension of the univariate analysis of variance (ANOVA) in which statistical differences from the effect of an independent grouping variable onto a continuous dependent variable were investigated (90). While ANOVA only deals with one dependent variable,

MANOVA examines the effects of an independent variable onto multiple dependent variables. However, performing multiple ANOVAs may not yield the same result as MANOVA owing to additional experimental errors generated from multiple ANOVA tests comparing to one MANOVA test. Furthermore, ANOVA does not take into account the correlations between multiple dependent variables, thus may not show a significant difference between the means while MANOVA does (90). In this study, MANOVA was used to simultaneously analyze variation in each of the sugars and amino acids between treatments. Density of honeydew droplets was used as a covariate in these analyses to account for variation in the amount of honeydew in each extract.

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2. Study Models/Systems

Different study models were established to analyze different changes in phenology and relationships between aphids and other species under the impact of climate change. Field experiments were conducted at the Rocky Mountain Biological Laboratory (RMBL) near Crested Butte, CO. Analysis of sugar and amino acid composition in aphid honeydew were conducted in the Department of Chemistry and Biochemistry, University of Colorado – Colorado Springs.

2.1. Study 1: Experimental Design for Effects of Elevated Temperature and Lygus Feeding Position on Aphid Honeydew Quality

The first model objective is to examine the fitness of L. porteri under heat induced plant stress and exposure to Lygus hesperus species (91). To evaluate the effect of stress levels on the Ligusticum host, one group of host plants was treated at elevated temperature using open-top warming chambers (OTC) whereas another group was treated at ambient temperature. The OTCs surrounded the tops of flowering stalks with a

cylinder of clear polyethylene film allowing flying insects to approach at the top while leaving the bottom open to ground and vegetation dwelling arthropods (28)(Appendix Figure S1). Within each temperature treatment group, lygus bug colonies were introduced at different positions on plant, either at the umbel or stem, allowing evaluation of change in plant phenology in the presence of this herbivore. Experimental aphid colonies were applied to each plant using field-collected non-alate aphids. These colonies were allowed to grow for ten days prior to honeydew collection. Composition of sugars and amino

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acids were determined in aphid honeydew collected from host plants treated in the two study groups.

2.2. Study 2: Experimental Design for Effects of Host Phenology and Drought Stress on Aphid Honeydew Quality

The second study model focused on the effects of snowmelt date on the

abundance of insect herbivore through plant phenology versus soil moisture (92). This model consisted of three main study groups: one studied plant fitness at high-elevation (3000 m) with no added water, one studied plant fitness at low elevation (2700 m) with no added water, and one studied plant fitness at low-elevation (2700 m) with added water. Snowmelt date is described as the first date of bare ground seen by Billy Barr at a weather station located at RMBL (93). High elevation sites experience a later snowmelt date than low elevation sites (94), which would eventually delay host plant phenology and shorten their growing period during dry season. Low elevation sites without added water would, as expected, experience the opposite effects, an advanced plant phenology with a longer growing period during dry weather. Low elevation sites with added water, on the other hand, will still experience an advanced phenology owing to earlier snowmelt date but a shorter growing period during dry seasons because of the added water to match soil moisture of the high elevation sites. Experimental aphid colonies were applied to colonies in each treatment group in July of 2018. These colonies were allowed to grow for ten days prior to the collection of honeydew. Composition of sugars and amino acids were then determined for aphid honeydew collected from host plants treated in the three study groups.

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2.3. Study 3: Experimental Design for Effects of Prior Lygus Feeding on Aphid Honeydew Quality

The third study model examined the effects of L. hesperus colonization on host plant quality and aphid abundance (95). Specifically, changes in L. porteri quality induced by L. hesperus were reflected through change in composition of honeydew produced by A. asclepiadis feeding on the same host. Two study groups in this model included one group with treatment of L. hesperus on L. porteri prior to A. asclepiadis introduction to plant and the other group without treatment of lygus herbivore on host plant prior to aphid introduction in August of 2018. These colonies were allowed to grow for thirteen days prior to honeydew collection. Composition of sugars and amino acids in aphid honeydew collected from lygus addition plants and lygus free controls were

analyzed to examine the effect of L. hesperus on the host plant quality.

2.4. Study 4: Experimental Design for Effects of Experimental Snow and Water Addition on Aphid Honeydew Quality

The fourth study model evaluated the multitrophic effects of snow cover and soil moisture on aphid abundance in nine replicate plots. The first set of three replicate plots examined the effects of snow cover by experimentally increasing snow depth by 3 cm relative to the untreated plots. This was done by adding snow from drifts at least 15 m away from plots and maintaining a 1 m buffer zone around plots to avoid disturbing existing snow cover. The second and third set of three replicate plots demonstrated ambient or water addition treatment. In the plots where water was supplemented, 3.5 liters of water were added every three days prior to onset of monsoonal rainfall (June 9-

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July 6) and every week for the remainder of July (July 9 – July 30) to the host plants in water addition plots. Host plants in ambient plots received no additional treatment. Experimental aphid colonies were applied to each plant at the end of July 2018; colonies began as five field-collected instars and one alate per plant. These colonies were allowed to grow for fifteen days prior to honeydew collection. Composition of sugar and amino acid in aphid honeydew were analyzed to determine the effect of snow and water addition on plants.

3. Sample Preparation

3.1. Sample Preparation for Analysis of Sugars and Amino Acids

Based on the previous methods from aphid honeydew studies (39, 50), foil squares surrounding stems or umbels below colonies were collected and contained in 50 mL centrifuge tubes. Two mL of LC-grade 18 MW DI water, heated to approximately 80 °C, was added to each centrifuge tube using an air displacement micropipette

(Finnpipette, Thermo Scientific) to completely immerse foils and remove honeydews from foils. The centrifuge tubes were then sonicated (FS20D sonicator, Fisher Scientific) for 15 min at 60 °C in degas mode. One mL of each sample was transferred into a Waters LC autosampler vial using an air displacement micropipette for direct analysis with LC-MS/MS using a method developed in-house using a Waters Application Note as a springboard (96).

For samples with high concentration of sugars and amino acids outside the range of the calibration curve, dilution of 1:20 and 1:400 of honeydew samples were prepared. From the original extract of each honeydew sample in LC-grade 18 MW DI water, 50 µL

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was transferred into a new Waters LC autosampler vial using an air displacement

micropipette. To this vial, 950 µL of a mixture of methanol and water (10/90 in v/v) was added using a Hamilton microliter gastight syringe (Bonaduz, Schweiz) to make 1:20 diluted samples. The samples were vortexed (Vortex Mai Mix II, Thermo Scientific) for 10 seconds before analysis with LC-MS/MS. The same dilution method was used to make 1:400 diluted samples with 50 µL of 1:20 diluted samples as the starting materials.

For samples with low concentration of amino acids outside the range of the prepared calibration curves, honeydew samples were concentrated ten-fold. Five hundred µL of the original extract of each honeydew sample were transferred into a new Waters LC autosampler vial using an air displacement micropipette. The samples were left in the fume hood for a week at ambient temperature to evaporate all of the water. Once all of the water had evaporated, the amino acid analytes were resuspended in 50 µL of LC-grade 18 MW DI water using an air displacement micropipette. The samples were sonicated for 15 min in degas mode at ambient temperature, and then transferred into Waters autosampler vial inserts for analysis with LC-MS/MS.

3.2. Conditioning of HILIC Columns

Before column installation, LC pumps were purged at a flow rate of 1.000 mL/min with 100% LC-grade 18 MW water for 10 mins, and 100% LC/MS-grade acetonitrile for another 10 mins. The LC system was then flushed at the same flow rate with a mixture of acetonitrile/water (50/50 in v/v) for 10 mins before installation of the newly purchased Xbridge Amide columns (3.0 mm × 100 mm, 3.5 µm, Waters Corp., Milford, MA) for sugar and amino acid analysis. The columns were then flushed with

Figure

Figure 3. Ant-aphid mutualism on Ligusticum porteri. Photo by E. Mooney.
Table 1 cont. Limits of Detection (LODs) and Quantitation (LOQs) of seven sugars and  five amino acids from previous studies
Figure 6. Schiff base formation between the amine group of the stationary phase ligand  and the hydroxyl group of a reducing sugar (79)
Figure 7. Chromatogram of 10.0 µg/mL sugar standard used for calibration curve (Figure  8) with structures of seven sugars of interest, xylose (X), fructose (F), glucose (G),  sucrose (S), trehalose (T), melezitose (M) and raffinose (R) (resolving power of
+7

References

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