The Relative Importance of Larval and Adult Nutrition in the Isotopic Profile

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8-28-2019 9:30 AM

The relative importance of larval and adult nutrition in the isotopic profile of the spermatophore produced by the male true armyworm moth (Mythimna unipuncta) (Haworth).

Aida Parvizi The University of Western Ontario

Supervisor McNeil, Jeremy N. The University of Western Ontario

Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Aida Parvizi 2019

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Recommended Citation Parvizi, Aida, "The relative importance of larval and adult nutrition in the isotopic profile of the spermatophore produced by the male true armyworm moth (Mythimna unipuncta) (Haworth)." (2019). Electronic Thesis and Dissertation Repository. 6456. https://ir.lib.uwo.ca/etd/6456

This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The wings of true armyworm (Mythimna unipuncta) are metabolically inactive tissue and as their isotopic profiles are determined by resources obtained during larval development, they provide information on natal origin of immigrants. The majority of female immigrants are mated so could the isotopic profiles of spermatophores dissected from her reproductive tract provide information about the natal origin of her mates? However, as spermatophores are produced by metabolically active male accessory glands and could use resources obtained from larval and adult feeding I conducted experiments to determine the relative importance of these two dietary sources on the isotopic profiles of spermatophores. Laboratory results showed H from both dietary sources affect the isotopic profiles of spermatophores so they provide no reliable information about the origin of the female’s mates. However, this could be a model system to examine turnover rates of resources obtained during larval and adult stages in a migratory species.

Keywords

Mythimna unipuncta, spermatophore, stable isotope, nutrient allocation, migration, metabolically active tissue, accessory gland

Summary for Lay Audience

When habitat quality declines, insect species either enter a dormant state until conditions improve or migrate to more favorable sites. The true armyworm moth, Mythimna unipuncta, is a seasonal migrant, passing the winter in southern United States and moving northward in summer to avoid high temperatures at overwintering sites. Populations of this species occur annually in eastern Canada and may in some years cause considerable crop losses. However, we know little about the natal origin of these immigrants or if they come from the same site every year. These questions are now being addressed using naturally occurring chemical signatures that are based on stable isotopes of H and C (depicted as 훿2H and 13C) in wings.

The majority of spring immigrant females captured in London, ON, are mated, as evidenced by the presence of one or more spermatophores in her reproductive tract. The spermatophore, a structure containing sperm and nutrients, is transferred from the male to the female at the time of mating. While wing tissue composition does not change after emergence and is a reflection of larval diet, the spermatophore is formed at least several days after adult emergence so its isotopic profiles could be influenced by both larval and adult dietary sources.

Controlled laboratory experiments using individuals reared on different combinations of control and 훿2H / 13C spiked larval and adult diets, found that (i) both larval and adult diets affect the isotope composition of spermatophores, and (ii) while the spermatophores are formed within the female’s reproductive tract her contribution to the spermatophore’s isotope profile was small. Overall, my findings suggest that the isotopic profiles of spermatophores do not provide any reliable information about male natal origin due to the effects of adult diet. However, this system could be used to examine the turnover of larval and adult dietary foods in metabolically active tissues, and future studies should include factors such as sustained flight, and male mating history on spermatophore isotope composition.

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Co-Authorship Statement

I prepared the different diets and reared the insects in the laboratory, as well as collecting the immigrant adults from the field. I set up the different combinations of mating pairs, dissected out and prepared the spermatophores for isotopic analyses. Professor Hobson ran all the H analyses while I helped run the C ones. I worked with Professors Hobson and McNeil to develop the experimental protocols and we all participated in the analyses and interpretation of the data. I will work with Professors Hobson and McNeil to prepare the manuscript that will be submitted for publication in the primary literature.

Acknowledgments Thank you, Dr. Jeremy McNeil, for sharing his knowledge and expertise, and for his consistent support throughout this journey. Under his leadership, not only did I succeed to complete my Masters, but I grew as a person as well.

I also extend my gratitude to Dr. Keith Hobson for his invaluable help in designing the experiments and for his generosity in granting me access to his lab to carry out the experiments.

To Dr. Nusha Keyghobadi, who served on my advisory committee, I extend my most sincere appreciation for her feedback and suggestions on how to better my experiments.

To Dr. Marc-André Lachance, I sincerely appreciate your invaluable feedback, and the time, and attention you granted me.

To all the work-study students and volunteers who helped maintain the lab and the insect colony, this would have never been possible without you.

And last, but certainly not least, I extend my most heartfelt thank you to my family for their unconditional love and support.

Table of Contents

Abstract ...... ii

Summary for Lay Audience ...... iii

Co-Authorship Statement...... iv

Acknowledgments...... v

Table of Contents ...... vi

List of Tables ...... viii

List of Figures ...... ix

List of Appendices ...... x

Chapter 1 ...... 1

1 Introduction ...... 1

1.1 Habitat deterioration ...... 1

1.2 Dormancy- “here, later” ...... 1

1.3 Migration- “there, now” ...... 3

1.4 Techniques used to study migration ...... 4

1.4.1 Stable isotope ...... 6

1.5 Stable isotope work on migratory species ...... 9

1.6 Stable isotope work on migratory insects ...... 10

1.7 True armyworm moth (Mythimna unipuncta (Haworth), Lepidoptera: Noctuidae) ...... 12

1.8 Why examine the isotopic profiles of spermatophores? ...... 14

1.9 Research objectives ...... 16

Chapter 2 ...... 17

2 Methods and Materials ...... 17

2.1 Study animal and laboratory rearing ...... 17

2.2 Laboratory experiments ...... 17

2.3 Preparation of spermatophores for isotope analyses ...... 21

2.4 Measurements of spermatophore and wing samples for δ2H analysis ...... 21

2.5 Measurements of spermatophore samples for δ13C analysis ...... 21

2.6 Field-collected spring immigrant moths ...... 22

2.7 Statistical analysis ...... 22

2.7.1 Laboratory experiments ...... 22

2.7.2 Field-collected individuals ...... 23

Chapter 3 ...... 24

3 Results ...... 24

3.1 Laboratory experiments ...... 24

3.1.1 Effect of diet and secretions from the female’s reproductive tract on spermatophore δ2H ...... 24

3.1.2 Relative importance of larval and adult resources in determining the isotope profile of the spermatophore ...... 25

3.2 Field-collected spring immigrant moths ...... 28

Chapter 4 ...... 31

4 Discussion ...... 31

4.1 Future direction ...... 34

References ...... 35

Appendices ...... 48

Curriculum Vitae ...... 53

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List of Tables

Table A- I Results from the fligner-killeen test...... 48

Table A- II Median and range of different mating pair groups used in the experiment- Effect of diet and secretions from the female’s reproductive tract on spermatophore δ2H)...... 48

Table A- III Median, range and the results of Mann-Whitney Wilcoxon test for groups in the experiment- Relative importance of larval and adult resources in determining the isotope profile of the spermatophore...... 49

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List of Figures

Figure 1 Pattern of seasonal migration pattern of the true armyworm moth, (Mythimna unipuncta) ...... 13

Figure 2 Predicted wing deuterium isoscape for the eastern distribution of the true armyworm moth (Mythimna unipuncta)...... 14

Figure 4. Experimental design used to determine the relative importance of larval and adult diet on the istope profile of true armyworm moth (Mythimna unipuncta) using both δ13C and δ2H...... 20

Figure 5. Effect of diet and secretions from the female’s reproductive tract on the spermatophore’s δ2H isotope value produced by male true armyworm moths (Mythimna unipuncta)...... 25

Figure 6. Relative importance of larval and adult resources in determining the δ2H and δ13C isotope profile of the true armyworm (Mythimna unipuncta) male moth spermatophore...... 27

Figure 7. Joint distribution of δ2H (a) and δ13C (b) values of wings from field-collected true armyworm moth (Mythimna unipuncta) male spring immigrants...... 29

Figure 8. The δ2H values of wings from field collected true armyworm (Mythimna unipuncta) spring immigrant female moths...... 30

List of Appendices

Appendix I ...... 48

Appendix II ...... 48

Appendix III ...... 49

Appendix IV...... 49

Appendix V ...... 50

Appendix VI...... 52

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Chapter 1

1 Introduction

1.1 Habitat deterioration

All organisms are faced with both unpredictable and predictable changes in habitat quality which may negatively affect normal activities such as growth and reproduction if the conditions deteriorate. As the timing of events such as fire, drought, sudden temperature changes (i.e. heatwave) or the availability of mates are unpredictable organisms have little time to react and must rely on constitutive (always present) adaptations to cope with such changes. In contrast, when facing predictable seasonal changes in habitat quality, such as the onset of winter in temperate regions or the dry season in the tropics, organisms can use environmental cues, such as decreasing temperature and daylength (token stimuli), to prepare physiologically and behaviorally for the oncoming unfavorable conditions (Tauber et al., 1986).

When considering responses to predictable habitat change, Southwood (1977) and Solbreck (1978) proposed two general strategies that insects use to cope with deteriorating conditions: i) they remain in their habitat (“here”) in some form of dormant state (diapause or quiescent) and reproduce in the same habitat when conditions improve (“later”), or ii) emigrate to other habitats (“there”) suitable for immediate reproduction (“now”).

1.2 Dormancy- “here, later”

Dormancy, a genetically programmed response to token stimuli, is an adaptive strategy where the organism suspends reproduction, growth, and development during the period of inhospitable conditions within the habitat. In insects, this process is referred to as diapause (Beck,1968; Saunders, 2002; Tauber et al., 1986). Diapause can be obligatory, as seen in many univoltine (one generation a year) species such as the western bean cutworm (Striacosta albicosta) (Douglass et al., 1957), although the duration of diapause

2 may vary depending on prevailing environmental conditions. In most multivoltine species diapause is a facultative state, induced by token stimuli, such as daylength, temperature and food quality that result in significant physiological changes (Tauber et al., 1986). Depending on the species and the environment in which they live, insects may enter diapause in the summer (estivation), fall (autumnal dormancy), spring (vernal dormancy), or winter (hibernation). Furthermore, diapause generally occurs during a specific stage in the insect’s life cycle and will vary depending on the species. For example, the gypsy moth (Lymantria dispar) overwinters as an undifferentiated egg (Leonard, 1968), the European skipper (Thymelicus lineola) as a first instar larva within the egg (McNeil and Fields, 1985), the spruce budworm (Choristoneura fumiferana) as a second instar larva (Han and Bauce, 2002), the western bean cutworm as a prepupa (Douglass et al., 1957), and the mourning cloak butterfly (Nymphalis antiopa) as an sexually immature adult (Herman and Bennett, 1975). There are also species which may have arrested development in different life stages at different times of the year. For example, the western tree-hole mosquito, Aedes sierrensis, estivates in the egg stage and hibernates as a larva (Jordan, 1980).

As outlined by Tauber et al. (1986), there are a range of behavioral and physiological changes in response to the diapause-inducing token stimuli that prepare the insect for the advent of adverse environmental conditions. Behaviorally these can include increased feeding to accumulate reserves that will sustain individuals during diapause and the selection of a specific overwintering site. At the physiological level there are changes that allow the organism to withstand greater extremes of cold, heat or desiccation, as well as accumulation of metabolic reserves through i) hypertrophy of storage organs such as fat bodies storing lipids and proteins (El-Hariri, 1966; Lees, 1955), ii) lowered metabolic activity (Lees, 1955; Wigglesworth 1972) and in some cases, iii) the degeneration and absorption of certain tissues such as wing muscles, ovaries, and testes for build-up of metabolic reserves (Lees, 1955). Once initiated, diapause development must be completed and its duration will be determined by both genetic and environmental factors before the individual resumes normal development, growth and reproduction (Tauber et al., 1986). While diapause allows individuals a temporal escape from unfavorable

3 conditions, the other option is a spatial escape by emigrating to habitats immediately suitable for reproduction (Dingle, 2014).

1.3 Migration- “there, now”

Kennedy (1985) stated “Migratory behavior is persistent and straightened out movement effected by the animal’s own locomotory exertions or by its active embarkation upon a vehicle. It depends on some temporary inhibition of station keeping responses but promotes their eventual disinhibition and recurrence”. To ensure that migrants successfully leave their deteriorating habitat normal behaviors, such as foraging, are temporarily inhibited in the early stages of migration, although subsequently migrants may feed to obtain additional resources for flight (Kennedy 1985; Dingle, 2014).

Long-lived species, such as birds and mammals, generally undertake round-trip or loop migration, where the same individuals move between overwintering and breeding habitats (Dingle, 2014). Certain insect species undertake a similar form of migration. Sexually immature adults of a lady beetle (Hippodamia convergens) migrate to high altitude overwintering sites in fall and return to breed at lower altitudes in spring (Roach & Thomas, 1991). A similar pattern is seen with the bogong moth (Agrotis infusa), where sexually immature adults migrate to aestivation sites at high altitudes in the Australian Alps and return to lower altitudes to breed in the fall (Common, 1954). However, in insects, most return migrations generally involve several generations, where individuals leaving a deteriorating habitat undertake a one-way migration to a suitable breeding site, and their descendants complete the one-way return journey. Adults of the monarch butterfly (Danaus plexippus) leaving Canada in the fall migrate directly to the overwintering sites in Mexico, but the following spring there is a stepwise return migration that involves several generations. However, within the population there are a small number of individuals that emigrate from Canada in the fall that successfully complete a round trip migration (Malcolm, 1987; Flockhart et al., 2013).

Most insect species initiate migration as sexually immature adults, which Johnson (1969) referred to as the “oogenesis-flight syndrome” as complete development of the

4 reproductive organs occurs post migration. As with species that undergo dormancy locally, migratory species respond to similar environmental cues indicative of pending habitat deterioration, resulting in major physiological and behavioral changes that facilitate long distance movement (Rankin & Riddiford, 1978; Rankin & Burchsted, 1992). For example, increased feeding leads to fat body hypertrophy, which provides energy to fuel the long-distance flight, while low titers of juvenile hormone ensure that the adults remain sexually immature during migration. However, there are a few species in which migration is undertaken by sexually mature individuals. For example, under outbreak conditions when host trees are heavily defoliated, female spruce budworm mate and lay part of their egg complement before emigrating in search of better-quality oviposition sites (Sanders, 1991).

1.4 Techniques used to study migration

To develop the most effective conservation or pest management strategies for migratory species, it is essential to have an in-depth understanding of the entire migratory process (Hobson et al., 2019). However, the ability to collect data on migrating species depends on many parameters, including the size of the organism, the manner of locomotion (e.g. walking versus flight), as well as the duration, timing and distance of the migration (Hobson et al., 2019). For example, collecting information on large terrestrial mammals can be done by direct observation, something that would not be possible for a moth that flies at night in upper air currents (Taylor, 1974; Gatehouse, 1997).

Many techniques have been used to gain information on migratory species, but each has benefits and limitations. One approach that provides insight into the distance moved is “mark and recapture”, where individuals are marked at one site and subsequently captured at another. This has resulted in data sets that have significantly improved our understanding of the fall migration of the monarch butterfly from Canada/USA to the overwintering sites in Mexico (Taylor, 1997; 1998). This is a rather special case as all of the migrants aggregate at a few specific overwintering sites, and thus the probability of recapture is high. However, this is not the case for most species as migrants spread over a broad area, so the cost associated with capturing and marking

5 individuals and the low probability of recapturing them represent serious disadvantages of such tagging systems. This is underscored by the work of Li et al. (1964), who used mark-recapture to determine if the oriental moth (Mythimna separata) undertook a northward spring migration in China. In their abstract (the article is in Chinese with an English abstract) they noted that only five of over 1,000,000 marked moths were recaptured. Thus, while the results provided meager support for the hypothesis that the species does migrate northward, little data were obtained for the actual costs incurred.

Several different trapping methods have been used to gain information on the arrival time and the relative abundance of immigrating insect species. For example, the combined use of light and sex pheromone traps has been used to provide information on spatial and temporal distributions of different insect migratory species (Cantelo, 1974; Barclay, 1983), and in the case of pest species the data obtained can be used to determine if, when, and where insecticides should be used to prevent an outbreak (Allison & Cardé, 2016). Other methods which may be used alone or in combination to provide information on migratory species include capture by an airplane equipped with nets (Glick, 1960), utilizing food bait lures (Yela & Holyoak, 1997), drop nets, malaise traps, and air flow system suction traps (Johnson, 1950; Taylor, 1962). However, these techniques provide information only at the site of capture, thus a snapshot in time, but provide little or no information on either the origin or destination of the individuals caught. Furthermore, several of these techniques, like suction and light traps, are not species-specific and there is a considerable cost associated with sorting through the total catch to separate out the species of interest (Chapman et al., 2002).

With the advent of remote sensing technologies, VHF radio transmitters and satellite tags have provided more precise information on the movement of migrants, when they are within the range of detection (Hobson et al., 2019). However, at this time, the size of the devices limits their use on small-bodied organisms and therefore are not viable for use on most insect migrants (Chapman et al., 2002; Rubenstein & Hobson, 2004; Dingle, 2014). In addition, such equipment is expensive and like mark/recapture they are labor-intensive. Furthermore, their presence could modify the behaviors of tagged individuals in ways that negatively affected survival and reproductive success.

Scanning radar has proven a reliable method in studying airborne high-altitude insects, where the orientation of migrants can be determined without interfering with their behavior, but is not species-specific (Chapman et al., 2003; Feng et al., 2014). More detailed information can be obtained with a combination of radar and ground-based light traps, as in the case of the migration of the oriental moth, where information on abundance, orientation, altitude, and speed at which migrant insects traveled was obtained (Zhao et al., 2009). However, the geographical distance travelled by migrants cannot be determined from these techniques, especially in the case of airborne migrants which are carried on prevailing, and highly variable, wind currents (McNeil et al., 1993; Gatehouse 1997; Stefanescu et al., 2007).

As noted, the data collected with many of these techniques provide information only for that specific trapping location, and thus national or international detection networks are needed to obtain data over the entire migratory pathway. For example, a network of light and suction traps extending from the north of France through England was set up and provided information on the patterns of seasonal migration of aphid pest species (Taylor, 1979). However, even with a wide network of traps, as these techniques are ground-based and stationary, airborne insects carried on upper wind currents may not be included in the catches (Chapman et al., 2003).

1.4.1 Stable isotope

Another approach for the study of migration is the use of intrinsic markers present within the individual. In particular, analysis of the naturally occurring abundance of stable isotopes of several elements in metabolically active (e.g., muscle) or inactive (henceforth fixed) tissues (e.g., insect wings/ bird feathers), have opened up ways of tracking animal movement and can be effectively used on small bodied organisms (Hobson, 1999; Hobson & Wassenaar, 2019). The underlying principle is that stable isotope ratios of various elements in nature are affected by biogeochemical processes, which results in predictable spatial isotopic patterns (forming so-called isoscapes, West et al., 2010), or are associated with primary productivity (e.g., related to photosynthetic pathway). These

7 patterns are transferred up food webs to animal consumers whose tissue stable isotope values can ultimately be related to source isotopic patterns (Wassenaar, 2019).

The type of tissues chosen for studies using stable isotopes is important. In the case of non-metabolic tissues stable isotope values do not change following formation and so they can potentially provide information on the site where the tissue was grown (Bearhop et al., 2003; Hobson, 2005). In contrast, the elemental composition of metabolically active tissues changes over time, such that their stable isotope composition is a time-integrated signal directly linked to the metabolic turnover of the tissue in question (Martinez del Rio & Carleton, 2012). Fast turnover tissues (e.g., plasma, liver) provide isotopic information on the organism’s more recent diet or location whereas slow turnover tissues (e.g., muscle, bone collagen) will represent integrations over much longer periods (Hobson, 2005). Thus, a combination of isotopic information from both fixed and active tissues from the same animal may provide greater insight about its movement and ecology, which subsequently contribute to better conservation and pest management strategies (Alisauskas & Hobson, 1993; Bond & Hobson, 2012).

Stable isotopes, which occur naturally in the environment, are different forms of an element with the same number of protons but different number of neutrons. They behave identically chemically but not kinetically. Stable isotope abundance in a tissue is measured as a ratio of the heavy to light isotope species (Hoefs, 2018), using the following equation:

δX = (Rsample/Rstandard – 1) * 1000 where X is the heavy isotope of an element and R is the ratio of the heavy to light isotope. The stable isotope ratio expressed in δ notation is the difference in relative abundance of the heavier to lighter isotope compared to international standards in parts per thousand (‰) (Criss, 1999).

The primary light elements used in studying animal migration and ecology are C, H, N, O and S (Wassenaar, 2019). For example, deuterium (2H) is the heavier rare stable isotope of hydrogen, and protium (1H) is the lighter common stable isotope; the

8 abundance of deuterium in a matter is measured as a ratio of 2H/1H and compared to the international standard VSMOW (Vienna Standard Mean Ocean Water) (Hobson & Wassenaar, 2019). International standards are essentially arbitrary and chosen by the International Atomic Energy Agency in Vienna. The abundances of both deuterium (δ2H) and 18O (δ18O, heavier isotope of oxygen) are calibrated to VSMOW which is relatively enriched in both 2H and 18O in comparison to most biological materials. Animal tissues typically have negative 2H values (i.e., they have less 2H compared to 1H relative to the VSMOW standard) (Wassenaar, 2019).

Hydrological and meteorological processes contribute to regional-, continental-, and global-scale patterns of the stable isotopes of oxygen (O), and hydrogen (H) in environmental waters ultimately incorporated in food webs (Bowen & West, 2019). To date, δ2H measurements have proven to be more useful than δ18O measurements in this regard (Hobson & Koehler 2015) as the 2H patterns seen in the amount-weighted mean annual precipitation of different regions is reflected in their respective plant communities and consequently in food webs (Bowen & West, 2019). Therefore, in many cases, 2H analysis is used to trace organisms to their origin, especially those who undertake long- distance migration (Hobson, 1999; Rubenstein & Hobson, 2004; Hobson, 2005).

Stable isotopes of carbon (δ13C), nitrogen (δ15N), and sulfur (δ34S) can be used to gain additional biological information associated with diet or local habitat (Hobson, 2019). These typically do not display predictable continental-wide patterns like δ2H (but see Hobson et al., 2012), but they can still be useful in refining spatial assignments associated with climate, land-use practices or altitude (Bowen & West, 2019). Therefore, C,N,S stable isotope measurements can be paired with H stable isotope to provide additional habitat and life history information, as well as in some cases a better resolution in inferring origins (Hobson et al., 2012; Larson et al., 2013; Haché et al., 2014; Hobson & Kardynal 2016; Bowen &West, 2019).

Carbon is a fundamental element found in all macromolecules (proteins, lipids, and carbohydrates). Consequently, 13C values in animal tissues can be used to gain insight into the food source migrants previously ate. In addition, they may provide

9 information on movement between habitats that differ in plant species composition. For example, due to different photosynthetic pathways, C3, C4 and CAM plants differ in tissue 13C values with C4 and CAM plants showing more positive values than C3 plants (mean of -12 ‰ compared to -25‰; O’Leary, 1988; Peters & Vogel, 2005). In North America, C3 plants predominate but grasses and some important agricultural crops, such as corn, millet and sorghum, are C4 plants.

Nitrogen is found largely in amino acids and values of 15N in some proteins closely reflect those of their source, whereas those in other proteins reflect the trophic position of the consumer (Layman et al., 2012; McMahon & Newsome, 2019). While the use of 15N values to infer animal origins is more complicated due to natural and anthropogenic N inputs such as fertilizer use, animal husbandry and sewage disposal (Pardo & Nadelhoffer, 2010), 15N values can serve as an indication of whether migratory individuals originated from or encountered agricultural lands along their migratory path (Westerman & Kurtz, 1974; Hobson, 1999; Yerkes et al., 2008; Hobson et al., 2018).

Within food webs, S is mainly found in a few amino acids (e.g., cysteine) and directly linked to dietary protein pathways, and is therefore a good indicator of an individual’s diet (McCutchan et al., 2003). Additionally, marine-derived sediments, volcanic rocks, sea spray and anaerobic wetland systems all typically have high 34S values compared to other terrestrial systems (Haché et al., 2014). Therefore, 34S can be used as a tracer to gain information about food webs used by migratory animals, and to distinguish among certain terrestrial and coastal ecosystems in migration studies (Fry, 2002; Weber et al., 2002).

1.5 Stable isotope work on migratory species

Collectively, the profiles of different stable isotopes from metabolically active vs. inactive tissues provide information on different aspects of an organism’s life history and ecology (Hobson & Wassenaar, 2019). For example, studies on migratory birds have used 2H from feathers, alone or in combination with other stable isotopes, to assign

10 origins of migrants or to make the link between breeding and wintering grounds (Hobson & Wassenaar, 1996; Hobson, 1999; Rubenstein et al., 2002; Hobson et al., 2012; Hobson & Kardynal, 2016; Hobson & Wassenaar, 2019). Movements between different habitats can be delineated based on distinct isotopic values present in food webs of the respective habitats (Alisauskas & Hobson, 1993). For example, movement of lesser snow geese (Chen caerulescens) between estuarine and agricultural habitats along their spring northward migration has been determined by comparing the isotopic composition of the geese’s pectoral muscle tissue to that of each of the habitats (Alisauskas & Hobson, 1993). Using the same approach, migratory and non-migratory species may be distinguished, as shown in the study of the nectarivorous southern long-nosed bat (Leptonycteris curasoae), where residents fed exclusively on CAM-based food webs, while migrants switched between C3 and CAM flowering plants during their spring and fall migration (Fleming et al., 1993).

Resource availability is an essential factor affecting species fitness and is especially important for long-distance migratory species as they require sufficient fuel to reach their destination. Exogenous (i.e. based on current dietary intake), and endogenous (i.e. based on stored body) nutrients are allocated towards different life history traits including survival, growth, and reproduction (Boggs & Ross, 1993). In the case of the southern long-nosed bat, isotope analyses led to the finding that seasonal migrations in spring and fall are associated with the blooming periods of certain columnar cacti and Agave species, indicating these plants provide a migratory corridor that provide bats the fuel necessary for migration (Fleming et al., 1993).

1.6 Stable isotope work on migratory insects

In holometabolous insects, where different stages in the life cycle often exploit different food sources (e.g., caterpillars eating leaves while adults feed on nectar), may partition the resources differently and provides a great opportunity to look at nutrient allocation and the relative importance of larval (endogenous) vs. adult (exogenous) diet (Boggs & Ross, 1993; O’Brien et al., 2004; Boggs & Freeman, 2005). Wing formation occurs during pupal development, using nutrients acquired during larval stage (Boggs &

Freeman, 2005). Consequently, as wing tissue is metabolically inactive its isotope signature reflects that of larval diet (Hobson at al., 1999; Hobson et al., 2018) and can be used to gain information on natal origins of migrants (Hobson et al., 1999; Brattström et al., 2010; Hobson et al., 2012; Yang et al., 2015; Hobson et al., 2018).

A substantial amount of stable isotope work has been done on the monarch butterfly. The stable isotopes 2H (in local isotope hydrology) and 13C (in milkweed plants) in monarch wings were used to assign the probability of natal origin in eastern (Hobson et al., 1999) and western North America (Yang et al., 2015), contributing to the development of better conservation practices (Flockhart et al., 2017). This approach has also been used to investigate correlations between distance moved (from their natal origin to capture site) and different aspects of wing morphology (Yang et al., 2015).

The same method was used in studying the highly variable and plastic migration pattern of the European red admirals (Vanessa atalanta). Wing 2H analyses were used to determine the natal origins of summer and autumn seasonal migrants and the results showed this species to have a broader range of natal origins than previously thought (Brattström et al., 2010). Similarly, the globe skimmer or wandering glider dragonfly (Pantala flavenscens), the highest flying odonate (Kumar, 1984), was thought to complete a multi-generational annual migration circuit of about 14,000 km between India and East Africa (Anderson, 2009). However, wing 2H analysis of P. flavenscens caught in the Maldives showed that individuals actually originating from much further north in India than previously assumed, indicating that the migratory distance covered is closer to 18,000 km.

The majority of isotopic studies on migratory insects have been on non-pest species but recent work on the true armyworm (Mythimna unipuncta ) has shown that this approach is useful for studying migratory seasonal pests (i.e. determining their natal origins) and has the potential to generate data necessary for creating better pest management programs (Hobson et al., 2018).

1.7 True armyworm moth (Mythimna unipuncta (Haworth), Lepidoptera: Noctuidae)

The true armyworm is an agricultural pest in North America (Breeland, 1958; Guppy, 1961; McNeil 1987) with sporadic outbreaks reported throughout central United States and eastern Canada (Guppy, 1961). This insect is present every summer in Canada where it may complete one or two generations but none of the developmental stages can survive the winter conditions (Fields & McNeil, 1984; Doward, 2018). During the fall and winter there may be several generations in the overwintering areas before the northward spring migration. Consequently, McNeil (1987) postulated that it is a seasonal migrant with adults migrating north in spring from overwintering sites as temperatures in the south are too hot in the summer, and their descendants subsequently migrating back south in the fall to avoid the lethal cold temperatures (see Figure 1). This scenario was based on the following observations: (i) sexual maturation is delayed when adults are reared under short day (10L:14D), cool temperature (10°C) conditions that match spring conditions in the south and fall conditions in the north (Turgeon & McNeil, 1982; Delisle & McNeil 1987), (ii) these delays are related to physiological changes in response to environmental conditions (Cusson &McNeil, 1989; McNeil et al., 1993), and (iii) adults captured in Canada during the fall are sexually immature (McNeil, 1987), as predicted by the “oogenesis-flight syndrome” model (Johnson 1969).

Hobson et al. (2018) demonstrated, using a spatially explicit isotope landscape (West et. al., 2010) based on the eastern distribution of the true armyworm (see Figure 2), that 2H of wing chitin could be used to assign natal origins. Furthermore, 13C profiles provided insight into the type of larval host plant used by moths captured at different times of the year in Ontario and Texas. This study provided the first experimental data supporting the hypothesis of a two-way migration proposed by McNeil (1987): spring moths captured in London clearly originating from habitats much further south while those captured in Texas during the fall originated from much further north.

Figure 1. Pattern of seasonal migration pattern of the true armyworm moth, (Mythimna unipuncta).

The lifecycle of the true armyworm is depicted on the map. Adult moths undertake a northward spring migration (indicated by the red arrows) to avoid the deleterious effects of summer temperatures. In the fall their decedents undertake a southward migration (indicated by the blue arrows) in order to avoid severe winter

14 conditions. Their migration is facilitated by dominant winds, 70% from the south in spring and 60% from the north in fall. (The figure is based on data from McNeil, 1987).

Figure 2 Predicted wing deuterium isoscape for the eastern distribution of the true armyworm moth (Mythimna unipuncta).

The isoscape shows an enrichment of deuterium in lower latitudes (less negative 2H values) and a depletion towards higher latitudes (more negative 2H values) (Figure was taken from Hobson et al., 2018, see copyrights in Appendix VI).

1.8 Why examine the isotopic profiles of spermatophores?

In Lepidoptera, secretions from the male’s accessory glands (i.e. nutrients and substances that form the spermatophore), together with the spermatozoa, are transferred to the bursa copulatrix (part of the female’s reproductive tract) (Mann, 2012; Marcotte et al., 2005). Several hours post mating, the spermatozoa are transferred to the spermatheca, leaving

15 just the chitinous part of the spermatophore in the bursa copulatrix (Marcotte et al., 2003).

The 13C profiles of spermatophores in the European corn borer (Ostrinia nubilalis) were successfully used to determine larval host plant use (Ponsard et al., 2004) and subsequent assortative mating (Malausa et al., 2005) of the two distinct host races feeding exclusively on C4 or C3 plants. Given that the majority of immigrant spring armyworm females captured in Canada are mated (McNeil, 1987; Marshall & McNeil, 1989), presumably post migration, I undertook studies to determine if the isotopic profiles of the spermatophores she contained might provide useful information about the origin of her mates.

Male accessory glands produce some secretions during metamorphosis that would originate from resources gained during larval development but there is an increase in gland activity following adult emergence (Chen, 1984). Thus, given that the male accessory glands are metabolically active tissues, the secretions produced could contain resources acquired from both larval and adult food sources. The corn borer is a non- migratory species that mates very soon after emergence (Royer and McNeil, 1993) while the true armyworm has a significant pre-reproductive period during which it constantly feeds on nectar. This period can be around 4-10 days post emergence under summer conditions and may be longer under conditions that induce migratory behavior (Delisle & McNeil, 1986,1987). Thus, the relative importance of larval and adult nutritional sources allocated towards spermatophore production could differ considerably between the two species. Therefore, to determine if the isotopic profile of spermatophores dissected from field collected females would be useful in identifying the natal sites of her mates, it is first necessary to determine the relative contribution of larval and adult resources to spermatophores in the true armyworm. Furthermore, given that the spermatophore is formed within the female’s reproductive tract, it is possible that some of her secretions are incorporated into the spermatophore and could consequently affect its isotopic profile.

1.9 Research objectives

For my thesis I undertook studies to determine if spermatophores, based on their isotope composition, may be used as a tool to gain additional information on the origin of the males true armyworm females had mated with. Thus, I ran laboratory-controlled experiments to determine 1. the effect of diet and secretions in the female’s reproductive tract on the spermatophore’s isotope profile. 2. the relative importance of larval and adult resources in determining the isotope profile of the spermatophore.

I also ran experiments using field-collected spring immigrants 1. to determine the relationship between the isotopic profiles of a male’s wing to that of the spermatophore he produces when mated with a control laboratory-reared female. 2. to compare the isotopic profile of the wings from field collected females wing and the spermatophore(s) that she contains.

Chapter 2

2 Methods and Materials

2.1 Study animal and laboratory rearing

Mythimna unipuncta eggs were obtained from a colony maintained at 25 ± 0.5°C, 70 ± 5% RH, under a 16L:8D photoperiod, established using moths captured in light traps at the Environmental Science Western Field Station (ESW; 43.07°N, 81.34°W) in 2017 and 2018. All larvae were reared individually on a pinto bean diet (Shorey & Hale, 1965) and separated based on sex upon pupation (Breeland, 1985). Male and female moths were held in separate cages and fed 8% artificial nectar (henceforth referred to as nectar). Seven days post-eclosion, a virgin male and a virgin female were put in a mating cage and observed throughout the scotophase using a flashlight covered with a Kodak red Wratten #29 filter (so that mating pairs were not disturbed by the flashlight) (Turgeon & McNeil, 1982). Once mated, females were allowed to lay eggs for 48h to ensure that the spermatophore was empty before being dissected out for analysis.

2.2 Laboratory experiments

The first set of experiments were designed to determine how overall diet affects 2H values of spermatophores, and whether secretions in the female’s reproductive tract actually contribute to the 2H composition of the spermatophore. Larvae were either reared on a standard pinto bean diet made with snow melt water having a δ2H value of - 116.8 ‰ or with tap water spiked with 99% deuterated water (Sigma Aldrich) to give a δ2H value of +600.4‰ (Fig 3a). The resulting adults were fed sugar solutions prepared with the same two water sources as spiked or unspiked nectar (Fig 3a,b). This allowed me to establish four different groups of mating pairs: (i) both adults from spiked diet (SS), (ii) both adults from unspiked diet (UU), (iii) spiked males with unspiked females (SU), (iv) unspiked males with spiked females (US) (Figure 3c). A comparison of the isotopic profiles from the spermatophores produced by UU and SS pairs allowed me to verify that

18 deuterium is incorporated into the spermatophore, while a comparison of spermatophores from UU versus US and SS versus SU allowed me to determine whether fluids from the female’s reproductive tract affect the spermatophore’s isotope composition. The following equations were used to determine the percent female’s hydrogen contribution to the spermatophore (See Appendix I for full calculations): A) (|UU-US|) / (|UU|+|SS|)

B) (|SS-S U|) / (|UU|+|SS|)

Figure 3 Experimental design used to determine if deuterium from diet is incorporated into the spermatophore, as well as the relative contribution of males and females to the overall spermatophore isotopic composition in the true armyworm moth, (Mythimna unipuncta). a) Larvae reared on spiked diet and fed spiked nectar post eclosion. b) Larvae reared on unspiked diet and fed unspiked nectar post eclosion. c) Table showing four different mating pair combinations of 7-day-old virgin male and female moths. S (blue square) and U (green squares) indicate whether the individual was reared on a spiked or an unspiked larval diet and nectar. Spermatophore δ2H values of UU pairs in comparison to those from SS pairs answered the question of whether deuterium from the diet is incorporated into the spermatophore. Comparison of spermatophore δ2H values from mating pairs reared on the same diet (UU and SS) to those reared on different diets (US

19 and SU) determined if fluid from the female’s reproductive tract is incorporated into the spermatophore, thus affecting its δ2H value.

A second series of experiments was carried out to investigate the relative importance of larval and adult diet in spermatophore composition. In these experiments all larvae were reared on unspiked pinto bean (C3) diet, while adults were fed nectar made with either maple (C3) syrup or cane sugar (C4) dissolved in spiked or unspiked water. Maple syrup and cane sugar were chosen to reflect the possible use of different nectar sources in nature, as C3 plants have a more negative δ13C value (lower amount of C13) than C4 plants. Thus, four different types of nectar treatments and mating groups were made (Figure 4): (i) unspiked C3 (UC3), (ii) spiked C3 (SC3), (iii) unspiked C4 (UC4), or (iv) spiked C4 (SC4). Therefore, by comparing spermatophore isotope values from mating pairs that received nectar with a similar isotope composition as their larval diet to those who were switched onto nectar that isotopically differed from their larval diet, it was possible to determine if adult diet affects the δ2H and δ13C composition of a spermatophore.

The following equation was used to determine to what extent (in percentage) nectar contributes to the spermatophore isotope composition (δ2H from water and δ13C from sugar) (refer to Appendix II for full calculations):

Adult nectar contribution

= Isotope (δ2H or δ13C) change in Spermatophore / Isotope (δ2H or δ13C) change in diet

Figure 4. Experimental design used to determine the relative importance of larval and adult diet on the istope profile of true armyworm moth (Mythimna unipuncta) using both δ13C and δ2H. Larvae were reared on unspiked pinto bean (C3) diet and separated based on sex upon pupation. 7-days post eclosion, virgin male and female moths were put into mating pairs within each of the treatment groups. 2.3 Preparation of spermatophores for isotope analyses

Each spermatophore was held in an individual Eppendorf tube, air dried in a fume hood for 24h, and then analyzed for stable H or C isotope composition (2H,13C). In experiments where wing isotopic profiles were also determined, one forewing of each individual was soaked in 2:1 chloroform:methanol for approximately 6h; the solvent was decanted and the wing was air dried in the fume hood for 24h before analysis.

2.4 Preparation of spermatophores for isotope analyses

Each spermatophore was held in an individual Eppendorf tube, air dried in a fume hood for 24h, and then analyzed for stable H or C isotope composition (2H, 13C). In experiments where wing isotopic profiles were also determined, one forewing of each individual was soaked in 2:1 chloroform:methanol for approximately 6h; the solvent was decanted and the wing was air dried in the fume hood for 24h before analysis.

2.5 Measurements of spermatophore and wing samples for δ2H analysis

Individual δ2H measurements were done using 0.2 ± 0.02 and 0.35 ± 0.02 mg of spermatophore and forewing, respectively. Samples were weighed into pressed silver 3.5 × 5 mm capsules and then crushed and placed in a Eurovector Uni Prep (Milan, Italy) heated (60oC) carousel interfaced with a Eurovector 3000 Elemental Analyzer. Following pyrolytic combustion on glassy carbon at 1350oC, gases were separated and introduced into a Thermo Delta V Plus isotope ratio mass spectrometer (Bremen, Germany) using helium as the carrier gas in continuous flow mode. Stable H isotope data are reported in standard delta (δ) notation relative to the Vienna Standard Mean Ocean Water (VSMOW). All δ2H values are reported for the non-exchangeable H fraction using the comparative equilibration approach of Wassenaar and Hobson (2003). Two USGS keratin standards, EC-01 (formerly CBS: Caribou Hoof Standard, -197 ‰) and EC-02 (KHS: Kudu Horn Standard, -54.1 ‰) were included with every 10 samples. Based on replicate (n=5) within-run measurements of these keratin standards, we estimate measurement error to be < 2.0‰.

2.6 Measurements of spermatophore samples for δ13C analysis

When measuring δ 13C, we used 0.2 ± 0.02 mg of spermatophores and 0.35± 0.02 mg of forewings, cane sugar and dried maple syrup. Samples were placed in 4 × 3.2 mm tin pressed capsules and then analysed using a Costech Elemental Analyzer coupled to a Thermo Delta Plus XL isotope ratio mass spectrometer (Bremmen, Germany) operated in

22 continuous flow mode with helium carrier gas. Two standards, USGS-40 and USGS-41 were included for every 10 samples. Values of δ 13C were calibrated to Vienna Pee Dee Belemnite (VPDB) using USGS-40 (δ 13C = ─26.4 ‰) and USGS-41 (δ 13C = +37.6 ‰). Measurement error for δ 13C, based on replicate within-run (n=5) analyses of standards is ± 0.1 ‰.

2.7 Field-collected spring immigrant moths

Live spring immigrant male and female moths were collected from pheromone and light traps, respectively, set up in Environmental Sciences Western (ESW) field station 2018. Each male was paired with a mature virgin female from the laboratory colony, and after mating the 2H and 13C profiles of the male’s forewing was compared with those of the spermatophore he had produced.

In the case of field-collected female spring immigrants, they were held in individual cages and allowed to lay eggs for 48h to ensure that the spermatophore(s) they carried were emptied. Then the 2H value of each spermatophore dissected out of the female was compared to that of her forewing.

2.8 Statistical analysis

2.8.1 Laboratory experiments

Data were analyzed in R version 1.1.456. Data were not normally distributed based on quantile-quantile plots (Becker et al., 1988), and normality was not achieved by log, square root, or power transformations of the absolute value (due to the presence of negative numbers) of dependent variables. Equal variance was tested using the Fligner- Killeen test of homogeneity of variances (Conover et al., 1981), variances differed significantly in all data sets except for that of the second series of experiments investigating the relative importance of larval and adult diet on spermatophore δ13C composition (Table A-I, Appendix I). Furthermore, due to small sample sizes (10-13 per group) and high spermatophore isotope variability between individuals within groups, outliers were not excluded from analyses. Therefore, because the data did not meet the

23 assumptions of a parametric test, a pairwise two-tailed Mann-Whitney-Wilcoxon (with W statistic to detect differences between groups) was used determine whether differences between groups were significant.

2.8.2 Field-collected individuals

Due to the non-normal distribution, unequal variance, and outliers, the non-parametric

Spearman’s ran correlation (with the rs statistic to determine if there is a relationship between the two tissues) (Zar, 2005) was carried out on field collected males to determine if there was a relationship between spermatophore and wing isotope values of spring male immigrants.

In the case of female spring immigrants, I did not perform a correlation test between wing and spermatophore(s), as the two variables are not related (i.e., unlike wings, spermatophore(s) originate from male individuals). Therefore, the values were graphed and analyzed on an individual basis, looking at the different δ2H values of the female’s wing in comparison to the spermatophore(s) that she carried.

Chapter 3

3 Results

3.1 Laboratory experiments

3.1.1 Effect of diet and secretions from the female’s reproductive tract on spermatophore δ2H

There was a significant effect of diet on the δ2H values as the spermatophores from mating pairs reared on spiked larval and adult (SS) diet had significantly more positive values than those reared on unspiked larval and adult (UU) diet (Mann-Whitney-

Wilcoxon W= 100, n1=n2=10, p<0.05, two-tailed; Figure. 5; Table A-II (Appendix II)).

There was a detectable evidence of a female contribution, with the spermatophores from control males mated with spiked females being significantly less negative that those when both sees were unspiked (Mann-Whitney-Wilcoxon W=0, n1=n2=10, p<0.05, two- tailed). The spermatophores for pairs where both sexes were reared on spiked diet were less negative that those from a spiked male mated with an unspiked female the difference was not significant (Mann-Whitney-Wilcoxon W= 63, n1=n2=10 p=0.352, two-tailed; Figure 5; Table A-II (Appendix II)) but the calculated power analysis indicates the probability of non-significance was low (<0.8). Using the data from all four groups (UU, US, SU, and SS), the female contribution varied from 7.8% to 13 % (see calculations in Appendix III).

Figure 5. Effect of diet and secretions from the female’s reproductive tract on the spermatophore’s δ2H isotope value produced by male true armyworm moths (Mythimna unipuncta).

Spermatophore δ2H isotope value of mating pairs when both sexes were reared on a spiked (SS) or an unspiked (UU) diet and mating pairs where one sex was reared on an unspiked and the other on a spiked diet (SU, and US). Medians with different letters differ significantly (using Mann-Whitney-Wilcoxon test, p<0.05. Boxes show 1st and 3rd interquartile range with line denoting median. Outlier is represented by a dot.

3.1.2 Relative importance of larval and adult resources in determining the isotope profile of the spermatophore

As each individual used in the previous experiments were maintained on similar isotopic diets during both larval and adult life stages, the results obtained did not provide information on the relative importance of larval and adult nutrition in determining the δ2H values of spermatophores.

However, when I manipulated only the deuterium levels in the nectar, the δ2H values of the spermatophores from unspiked C3 and C4 mating pairs (UC3, UC4) were significantly more negative than those obtained from spiked mating pairs (SC3, SC4) (pairwise Mann-Whitney-Wilcoxon, p<0.05; Figure 6a; Table A-III (Appendix IV)), and were independent of the source of carbon source used (i.e. maple syrup or cane sugar). The amount of H in the spermatophore originating from water in the nectar was 16-17% (see Appendix V for calculations).

The results obtained when using different carbon sources also support the idea that adult resources are important as the spermatophores from mating pairs given a C3 nectar (UC3, SC3), had significantly more negative δ13C values than those given a C4 nectar (UC4, SC4) (pairwise Mann-Whitney-Wilcoxon, p<0.05;Figure 6b; Table A-III (Appendix IV)), regardless of deuterium levels in the water. The amount of C derived from sugar in the nectar incorporated in the spermatophore produced by adult males was 31-34% (see Appendix IV for calculations).

Figure 6. Relative importance of larval and adult resources in determining the δ2H and δ13C isotope profile of the true armyworm (Mythimna unipuncta) male moth spermatophore.

Mating pairs were reared on spiked (S) or unspiked (U) water in nectar made from C3 or C4 sugar sources. (a) The δ2H isotope values of spermatophore (n=11/treatment) from mating pairs given spiked nectar SC3, SC4 and those given unspiked nectar UC3, UC4. (b) The δ13C isotope values of spermatophores (n=13/treatment) from mating pairs given C4 nectar SC4, UC4, and those given C3 nectar SC3, UC3. Outliers were included

28 in the range within each group that contained an outlier. Medians with different letters differ significantly (using Mann-Whitney-Wilcoxon test, p<0.05). Boxes show 1st and 3rd interquartile range with line denoting median. Outliers are represented by dots.

3.2 Field-collected spring immigrant moths

2 13 There was no correlation between  H (rs(13)=0.2, p=0.49; Figure 7a) or δ C (rs(13)= -0.46, p=0.1; Figure 7b) values of wings from field-collected spring immigrant true armyworm males and the spermatophores they produced when mated with laboratory females. However, δ13C values of the spermatophores did show that all field caught males, with the exception of one individual (δ13C = -18 ‰;), fed on C3 plant nectar, and their wing δ13C value showed that they fed on C3 plant during larval development (Figure 7b).

Consistent with the above findings for males, there was quantifiable variability between the 2H wing values of female spring immigrants and the spermatophore(s) that they contained (Figure 8). No statistical analyses were attempted due to the small sample size.

Figure 7. Joint distribution of δ2H (a) and δ13C (b) values of wings from field-collected true armyworm moth (Mythimna unipuncta) male spring immigrants captured near London, ON in the spring of 2018 and the spermatophore he produced when mated with a colony female. Spearman’s correlation showed no significant relationship between the wing and the spermatophore isotope values in either case of δ2H and δ13C (p>0.05).

Figure 8. The δ2H values of wings from field collected true armyworm (Mythimna unipuncta) spring immigrant female moths captured near London, ON, in the spring of 2018 and the spermatophore(s) that they contained at the time of capture. The numbers indicate female identification labels (ID). In cases where the female contained multiple spermatophores, a letter was assigned to each of her spermatophores.

Chapter 4

4 Discussion

Isotopic profiles of insect wings have been used to determine (i) the type of larval host plant used (i.e. C3 vs. C4 plant) based on δ13C measurements (Hobson et al., 1999; Hobson et al., 2018), (ii) if insects originated from natural or agroecosystems based on δ15N values (Pardo & Naddlehoffer, 2010; Hobson et al., 2018), and (iii) the natal origin of migratory species based on δ2H values (Hobson et al., 1999; Hobson et al., 2012; Hobson et al., 2018). The δ13C values of spermatophores dissected from field-collected European corn borer females provided information on whether the males had fed on C3 (mugwort or hop) or C4 (corn) plants during larval development (Ponsard et al., 2004). Furthermore, as in more than 95% of cases the larval host plant used by females (based on her wing δ13C value) was the same as her mates (based on the δ13C values of the spermatophores present in their reproductive tracts), Malausa, et al. 2005 concluded there was assortative mating. The question underlying my research was ‘Would the isotopic profile of the spermatophores dissected from immigrant armyworm females provide information on the natal origin of her mate(s)?’ This is important as this species is a sporadic pest and having a good understanding of the natal origin of immigrants would not only provide insight about gene flow but the data could also be useful in developing better pest management strategies. If immigrants come from the same general area each year it would be easier to develop models for the population dynamics at the overwinter sites than if they originated from different areas each year.

The isotopic values obtained for different spermatophores dissected from the same true armyworm spring immigrant female were often quite different (Figure 8). If one assumed that the δ2H values of spermatophores are a reliable indicator of male origin, one could conclude that these females had mated with males with different natal origins than their own, suggesting a high level of gene flow within the North American population. However, my results show that the data should be interpreted with caution, as

32 spermatophore isotope values can be affected by both larval and adult food resources exploited by the male, as well as by secretions from the female’s reproductive tract.

Although the female secretions are incorporated into the spermatophore (Figure 5, Appendix III), it only has a significant effect when an unspiked male is mated with a spiked female that had been fed a spiked diet during both larval and adult development. It should be noted however, that experimental conditions where individuals are always reared on unspiked diet most realistically reflect those armyworms one would encounter in nature; thus when working with field-collected material one could consider the female contribution is minor and the spermatophore δ2H values observed primarily reflecting those of the male’s body signature. Varying the abundance of either 2H or 13C in nectar confirmed that nutrients obtained by the adult male affects the spermatophore’s isotope profile (Figure 6), which could help explain why no correlation was observed between the isotope profiles of wings and those of spermatophores produced by field-collected spring immigrant males (Figure 7).

The δ13C values obtained from the wings and spermatophores of field-collected immigrant males show that, with one exception, they fed on C3 plants as larvae and as adults. There was no correlation between the δ13C values of the two structures (Figure 7b) but as wing values only reflect larval diet while those of the spermatophore could be influenced by both larval and adult diet, this could be due to the different processes involved in photosynthesis and nectar production. For example, Lüttge (1985) reported that the δ13C values of nectar produced by Abutilon striatum (a C3 species) and Sansevieria sp. (a CAM species) were less negative than those of their leaves (-26.70 ± 0.76 ‰ vs -31.00 ± 1.41‰ and -14.39 ± 0.38 ‰ vs -15.82 ± 0.49 ‰). Therefore, while the δ13C wing value of a migrant male will reflect that of the C3 host plant used during larval development, the δ13C value of his spermatophore could be differ depending on the nectar sources used during his northward migration.

There was considerable inter-individual variability in the differences between the 2H wing value and that of the spermatophore produced by spring immigrant males (Figure 7a); in some cases, they were quite similar while in others they were markedly

33 different. A number of interrelated factors may account for such variability. The size and content of the first spermatophore produced may vary as a result of the host plants used during larval development (Delisle and Bouchard, 1995; Delisle and Hardy, 1997). Furthermore, in species where males mate several times the spermatophore size and content decreases with each successive mating (Marshall and McNeil, 1989; Royer and McNeil, 1993; Marcotte et al., 2005). As accessory glands are metabolically active the relative contribution of larval and adult resources could change with successive matings, resulting in spermatophores with different isotopic profiles. This was the case for the non-migratory tobacco hornworm (Manduca sexta), when males were given nectar enriched with 13C before and after each mating (Levin et al. 2016, 2017).

The duration and intensity of flight activity, especially in migratory species can influence elemental turnover rates in metabolically active tissues (Hobson, 2019), which in turn could affect the isotopic profiles of spermatophores. In all of my experiments, males were held in cages with limited capacity to fly whereas the spring immigrants would have flown considerable distances from their natal habitats (Hobson et al. 2018) and as a consequence have expended considerably energy prior to mating. This increased expenditure could result in fewer larval resources being available for spermatophore production and consequently would influence the isotopic profile. In addition, prevailing weather conditions will affect the number of stopovers the insects make when migrating between sites (Gatehouse, 1997) and, as a result, the source(s) of adult resources that could be invested in the spermatophores. For example, the isotopic profile of the spermatophore produced by an immigrant male feeding at several sites during the northward migration may differ significantly from one produced by a male from the same natal site who completed the journey without stopovers. This could explain, at least in part, why the spermatophore and wing isotope values obtained for some immigrant males caught in London, Ontario, were quite similar, while others were not (Figure 7).

My results indicate that the isotopic profiles of spermatophores provide insight into the host plants exploited during the adult stage but, due to their contemporary nutrient allotment during mating, do not provide information on the natal origin of the males (whether they are dissected out of immigrant females or produced by field-

34 collected male immigrants). As noted above, this may be related to a number of different factors and additional controlled experiments should be carried out to clarify their relative importance in the isotopic profiles of spermatophores. In fact, the armyworm could be a useful insect model system to examine fundamental questions relating to the turnover of adult and larval resources in metabolically active tissues in migratory species of Lepidoptera. The few previous studies examining isotopic profiles of spermatophore were conducted on non-migratory species that mate very soon after emergence, so there is a short time over which adult resources are acquired prior to spermatophore production. In contrast, even under conditions ideal for reproduction, migrant lepidopterans do not become sexually mature for several days following emergence. Under conditions indicative of impending habitat deterioration, the pre-reproductive period may be extended for several weeks (McNeil, 1987; McNeil et al., 1995) so that the time over which adults nectar feed prior to the onset of reproduction is considerably longer.

4.1 Future direction

As a follow up to my thesis I would conduct studies, to determine if there are significant differences in isotopic profiles between spermatophores produced by males sequentially fed a series of nectars with increasingly negative 2H values and those from males fed only the nectars with the lowest or highest 2H levels. The first group would represent males that fed at different stopover sites during spring migration and the latter males that flew directly from the natal site to the final destination. This could be done using the same protocol I employed where males were held in small cages, but also repeated with individuals that were allowed to fly, using flight mills that provide information not only on time spent flying but also on flight speed (Rowley at al., 1968; Riley, 1997; Lu et al., 2007; Chapman et al., 2015).These experiments would certainly provide greater insight into fundamental questions relating to differential resource utilization in migrant species of Lepidoptera.

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Appendices

Appendix I

Table A- I Results from the fligner-killeen test.

Appendix II

Additional information provided for the data collected in the first and second experiments.

Table A- II Median and range of different mating pair groups used in the experiment- Effect of diet and secretions from the female’s reproductive tract on spermatophore δ2H).

Appendix III

Spermatophore δ2H values of the mating pair combinations based on larval and adult diet (UU, SS, SU, US) were used to calculate the female H contribution towards the spermatophore composition. A) ((|UU-US|) / (|UU|+| SS|) = (|128.6-100.1|) / (|128.6+75.4|)) * 100 = 13.97% B) ((|SS-SU|) / (|UU|+| SS|) = (|75.4-59.4|) / (|128.6+75.4|)) * 100 = 7.8%

Appendix IV

Appendix V

Larval vs. adult contribution to the spermatophore

Larvae were reared on unspiked pinto bean (C3) diet. Once pupated they were separated based on sex and given either maple syrup (C3) or cane sugar(C4) dissolved in spiked or unspiked artificial nectar, resulting in the following 4 artificial nectar combinations as well as mating pair groups based on their adult diet:

UC3- Unspiked C3 (maple syrup)

SC3- Spiked C3 (maple syrup)

UC4- Unspiked C4 (sugarcane)

SC4- Spiked C4 (sugarcane)

Using Hydrogen to decipher larval vs. adult contribution to the spermatophore composition

UC3-SC3:

Hydrogen change in spermatophore:

(UC3 spermatophore) + (SC3 spermatophore): 109.18 + 15.09 = 124.27

Diet difference:

(unspiked larval diet) + (Spiked artificial nectar): 113.92+ 599.51 = 713.43

Adult diet contribution:

(Hydrogen change in spermatophore) / (Diet difference): 124.27/713.43 = 17.42 %  1.03

UC4-SC4:

Hydrogen change in spermatophore:

(UC3 spermatophore) + (SC4 spermatophore): 103.05 + 7.93 = 110.98

Diet difference:

(Unspiked larval diet) + (Spiked artificial nectar): 113.92 + 599.51 = 713.43

Adult diet contribution:

(Hydrogen change in spermatophore) / (Diet difference): 110.98/713.43 = 15.56% 1.04

Using carbon to determine larval vs. adult contribution to the spermatophore composition

UC3-UC4:

Diet difference:

(Adult C3 maple syrup artificial nectar) – (Adult C4 sugarcane artificial nectar): 24.64 – 12.33= 12.31‰

Carbon change in spermatophore:

(UC3 spermatophore) – (UC4 spermatophore): 24.64– 19.92‰ = 4.16‰

Adult diet contribution:

(Carbon change in spermatophore) / (Diet difference): 4.16/12.31 = 33.79%  1.40

SC3-SC4:

Carbon change in spermatophore:

(SC3 spermatophore) – (SC4 spermatophore): 23.95 – 19.8‰ = 4.15‰

Diet difference:

(Adult C3 maple syrup artificial nectar) – (Adult C4 sugarcane artificial nectar): 24.64 – 12.33 = 12.31‰

Adult diet contribution is:

(Carbon change in spermatophore) / (Diet difference): 4.15/13.93 = 30.99%  1.40

Appendix VI

Curriculum Vitae Name: Aida Parvizi

Post-secondary University of Western Ontario Education and London, Ontario, Canada Degrees: B.S.c (Honor Spec. Biology, Major Medical Science) 2012-2017

Honours and Deans honor list Awards: 2015-2017

Innovative award Western University Student Success 2016

Related Work Summer student research Experience The University of Western Ontario 2017/05-2018/09

Teaching Assistant The University of Western Ontario 2017/09-2019/04

Outreach Entomological Society of London, Bug day 2018, 2019

Presentation of Poster presentation my research 2018 annual meeting Entomological Society of Canada/America

Oral presentation (presented by Dr.McNeil) 2018 annual meeting Entomological Society of Canada/America

Oral presentation 2019, BGRF The University of Western Ontario