Pollinator-Mediated Gene Flow in and Among Fields of Alfalfa

POLLINATOR-MEDIATED GENE FLOW IN AND AMONG FIELDS OF ALFALFA

PRODUCED FOR SEED

NATALIE KIRA BOYLE

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Entomology

MAY 2015

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of NATALIE

KIRA BOYLE find it satisfactory and recommend that it be accepted.

______Douglas B. Walsh, Ph.D., Chair

______Walter S. Sheppard, Ph.D.

______Laura C. Lavine, Ph.D.

______James D. Barbour, Ph.D.

ACKNOWLEDGEMENT

I wish to thank many individuals without whom this research would not be possible. I would like to express my deepest appreciation to Dr. Douglas B. Walsh for providing me with invaluable guidance and support while working towards this degree. Additionally, thank you to my committee, Dr. Laura Lavine, Dr. James Barbour, and Dr. Walter S. Sheppard for all of the advice and recommendations that have been provided in designing these experiments and the preparation of this manuscript. Dr. Ruth Martin, Dr. Stephanie Greene and Dr. Sandya Kesoju played an integral role in the development of some of the methods described in this work. I would also like to thank Dan Groenendale, Tora Brooks and other members of the Walsh lab for their support and involvement in providing field assistance as needed throughout the duration of this degree. I wish to thank Estela Cervantes for her assistance in preparation of the seedling germination assays and Dr. Marc Evans for statistical advice. Dr. Fang Zhu provided instruction and oversight of molecular techniques for pollen DNA extraction and qPCR preparation. I am also grateful to the alfalfa seed producers of Touchet, WA, for allowing me to conduct research in their fields year after year. Finally, I want to thank my friends and family for the enduring love and support they have so graciously provided over the past several years. A big thanks to

Dan Plotnick for staying supportive of my endeavors despite all of the stress and frustration that comes along with the graduate school experience – I couldn’t have done it without you!

iii

POLLINATOR-MEDIATED GENE FLOW IN AND AMONG FIELDS OF ALFALFA

PRODUCED FOR SEED

Abstract

by Natalie Kira Boyle, Ph.D. Washington State University May 2015

Chair: Douglas B. Walsh

Cross-pollination by bees is necessary for commercial alfalfa seed production. To maintain varietal purity in alfalfa, seed producers adhere to spatial isolation standards to minimize or prevent bee flight and subsequent pollen flow between fields. The increased use of genetically-engineered (GE) crops in agriculture has raised concerns over pollinator-mediated gene flow between transgenic and conventional agricultural varieties. The 2011 deregulation of genetically engineered glyphosate-resistant alfalfa by the USDA has generated public concern and scientific debate over current recommended bee management practices and their ability to maintain varietal purity of alfalfa grown for seed production. The primary objective of this research is to determine the roles that pollinators play in contributing to undesired gene flow between alfalfa fields.

We evaluated the impact of migratory beekeeping practices on transgenic pollen flow between spatially isolated alfalfa fields by permitting honey bees, Apis mellifera, to openly forage upon transgenic alfalfa blossoms, and transporting them 112 km to forage on caged conventional alfalfa following either 8 or 32 hours of isolation from the transgenic source.

Cross-pollination between transgenic and conventional alfalfa was nearly eliminated (0.00008%) following eight hours of isolation from the transgenic source.

The alfalfa leafcutting bee, Megachile rotundata (ALCB), is another commercially managed pollinator used extensively in alfalfa pollination. We evaluated the influence of the

ALCB on gene flow between GE and conventional alfalfa seed fields by testing for the presence of the GE trait in pollen provisions collected from domiciles located in conventional alfalfa seed fields planted directly adjacent to GE alfalfa fields. Pollen samples collected from domiciles in conventional seed fields were at variable distances from the adjacent GE fields. Alfalfa seed in the vicinity of each domicile was harvested and tested for the transgene. We found that the

ALCB frequently forages at distances which exceed current estimates for ALCB foraging range.

Additionally, GE trait expression in harvested conventional seed was detected at rates that surpass established thresholds for varietal purity. Measurable impacts of ALCB-mediated pollen flow were confirmed and can be used to inform science policy regarding the development of best management practices mitigating undesired gene flow between genetically distinct alfalfa varieties.

TABLE OF CONTENTS

ACKNOWLEDGEMENT ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

DEDICATION ...... vii

CHAPTER

1. INTRODUCTION ...... 1

Glyphosate resistant alfalfa seed production in the U.S...... 1

The agricultural significance of alfalfa seed production ...... 2

Pollinators relied upon for alfalfa seed production ...... 3

Measuring the foraging range of bee species ...... 8

References ...... 13

2. EVALUATING MIGRATORY BEEKEEPING INFLUENCES ON POLLEN FLOW IN

TRANSGENIC ALFALFA SEED FIELDS ...... 21

Introduction ...... 21

Materials and Methods ...... 23

Field experiment ...... 23

Trait confirmation of seedlings using the seedling germination assay ...... 25

1. Measuring the baseline germination rate of harvested seed ...... 25

2. Trait detection in replicates with a fixed percentage of GE seed ...... 26

3. Determining the background AP level in the original seed lot ...... 27

Seedling germination assay of experimental samples ...... 27

Results ...... 27

Field experiment ...... 28

Trait confirmation of seedlings using the seedling germination assay ...... 28

Discussion ...... 29

References ...... 31

3. MEASURING ADVENTITIOUS PRESENCE IN LARVAL PROVISIONS OF THE

ALFALFA LEAFCUTTING BEE, Megachile rotundata, TO INDICATE FORAGING RANGE

Introduction ...... 40

Materials and Methods ...... 43

Field Identification and pollen collection from ALCB domiciles ...... 43

Isolation of pollen provisions from ALCB nests ...... 45

DNA extraction from ALCB pollen provisions ...... 46

PCR REACTIONS of extracted pollen DNA ...... 47

Results ...... 48

Discussion ...... 49

References ...... 51

4. THE INFLUENCE OF THE ALFALFA LEAFCUTTING BEE IN CROSS POLLINATION

BETWEEN TRANSGENIC AND CONVENTIONAL ALFALFA SEED FIELDS ...... 59

Introduction ...... 59

Materials and Methods ...... 62

Field identifications and seed collection…………………………………………62

Seedling germination assay for harvested seed………………………………….63

vii

Results ...... 64

Discussion ...... 65

References ...... 70

viii

LIST OF TABLES

CHAPTER 2

2-1. Average seed yield and percent AP detection by field cage treatment in alfalfa seed

tested using seedling germination assay screening and test strip confirmation ...... 34

2-2. Average %AP detection in seed samples with a fixed percentage of GE:conventional

alfalfa seed, ± SE ...... 35

CHAPTER 3

3-1. GE trait detection in pollen using qPCR, in relation to distance from a GE source. Field number correlates with the fields identified in Figure 4-1. ‘n’ indicates the number of individual nests tested for the transgene at each domicile included in the study ...... 56

CHAPTER 4

4-1. Baseline germination rates for conventional seed samples collected in the field in 2013 and

2014...... 73

LIST OF FIGURES

CHAPTER 2

2-1. Organization of the experiment set-up for seed treatments containing known percentage

of GE:conventional seed ...... 36

2-2. One replicate of alfalfa seedlings following removal from the water curtain germinator.

Seedlings were evaluated individually for symptoms of glyphosate resistance ...... 37

2-3. The alfalfa seedling on the left exhibits characteristics of glyphosate resistance, while the

alfalfa seedling on the right does not. Glyphosate resistance is characterized by a

seedling’s large size, elongated root, the presence of root hairs on the root, and cotyledon

growth ...... 38

2-4. Average alfalfa seed yield in bee-pollinated and control field cages, ± SE; P = 0.001….39

CHAPTER 3

3-1. X-rayed ALCB nests, demonstrating the variable number of pollen provisions available

in each individual nest. Only nests exhibiting at least 3 individual pollen provisions were

tested for the absence of presence of the transgene ...... 57

3-2. A step-by step progression of isolating a pollen provision from an individual ALCB cell

under a dissecting microscope ...... 58

CHAPTER 4

4-1. Map of Walla Walla county alfalfa seed fields. Fields marked in red indicate a GE alfalfa

field, used as a source field to measure gene flow into conventional fields (marked in

yellow). Conventional fields are labelled to correspond with the model legend presented

in Figure 7. F3 and F5 could not be used in 2014, having been reseeded with wheat and

transgenic alfalfa that year, respectively. F6 was incorporated into the study in 2014 to

make up for the loss of F3...... 74

4-2. 2013 GE trait occurrence in harvested conventional seed for fields 1 (left) and 2 (right),

with %AP reported at each location from which seed was sampled. The transgenic field

is marked in orange ...... 75

4-3. 2013 GE trait occurrence in harvested conventional seed for field 3, with %AP reported

at each location from which seed was sampled. The transgenic field is marked in orange

...... 76

4-4. 2013 GE trait occurrence in harvested conventional seed for fields 4 (South) and 5

(North) with %AP reported at each location from which seed was sampled. The location

labelled N.D. had no detection of the GE trait. The transgenic field is marked in orange

...... 77

4-5. 2014 GE trait occurrence in harvested conventional seed for fields 1 (left) and 2 (right),

with %AP reported at each location from which seed was sampled. The transgenic field

is marked in orange ...... 78

4-6. 2014 GE trait occurrence in harvested conventional seed for field 3, with %AP reported

at each location from which seed was sampled. The transgenic field is marked in orange

...... 79

4-7. 2014 GE trait occurrence in harvested conventional seed for fields 4 (South) and 5

(North) with %AP reported at each location from which seed was sampled. Locations

labelled N.D. had no detection of the GE trait. The transgenic field is marked in orange

...... 80

4-8. 2013 data fit to an exponential decay model. Each point reflects average percent AP

expression at every domicile incorporated into the study. Domiciles were located at

variable distances from a known transgenic field, and the shape of each plotted point

corresponds to the field from which the seed was sampled (Figure 1) ...... 81

4-9. Pooled 2013 and 2014 data fit to an exponential decay model. Each point reflects

average percent AP expression at every domicile incorporated into the study. Domiciles

were located at variable distances from a known transgenic field, and the shape of each

plotted point corresponds to the field from which the seed was sampled (Figure 1) ...... 82

4-10. 2014 data fit to an exponential decay model. Each point reflects average percent AP

expression at every domicile incorporated into the study. Domiciles were located at

variable distances from a known transgenic field, and the shape of each plotted point

corresponds to the field from which the seed was sampled (Figure 1) ...... 83

xii

Dedication

To Herma Amalia, for the unwavering support and encouragement you provided me while

working towards this achievement.

xiii

CHAPTER ONE

INTRODUCTION

Glyphosate resistant alfalfa seed production in the U.S

Global reliance upon genetically engineered (GE) crops is undeniable in present day agriculture. As GE-plants continue to be developed, deregulated, and planted on a commercial scale, it is of paramount importance that we are aware of significant environmental impacts that may be associated with their release. Understanding the influence that pollinators play in pollen dispersal of commercialized GE pollen has consequently become a critical area of research. While most GE crops are not exclusively pollinator dependent at present, GE crops that benefit from or require pollination will likely be planted on a greater scale over the next several years (APHIS 2011). This suggests a need to characterize the effects of transgenic pollen movement between GE and conventional crops, for example, by examining foraging rates and distances among a suite of different pollinators.

The United States Department of Agriculture (USDA) is responsible for determining the relative risk posed by deregulating GE crops, which historically have been non-pollinator dependent annuals. In 2005, glyphosate-resistant, or Roundup Ready® alfalfa (Monsanto Co.,

St. Louis, MO, USA), became the first open pollinated perennial to undergo deregulation, resulting in planting on a massive scale exceeding 8.5 million ha domestically. However, shortly following deregulation, many seed producers became concerned that the production standards proposed in the environmental assessment conducted by Monsanto were not sufficient to prevent migration of the Roundup Ready gene into conventional alfalfa via pollinator-mediated gene

1 flow. An injunction was filed in 2007 to halt the deregulation of GE alfalfa to determine the extent of pollinator-mediated pollen flow of the glyphosate resistant trait into conventional fields

(APHIS 2011) until the Supreme Court reversed the ruling in 2011. The contribution made by pollinators to gene flow in alfalfa is relatively unknown but critical for characterizing the risk they may present to undesired cross-pollination events. Because this is the first pollinator dependent crop to be fully deregulated, data obtained from pollinators within this system will be instrumental in shaping future policies and guidelines developed to minimize cross pollination risk in other systems. This dissertation is intended to elucidate the mechanisms by which GE pollen can be dispersed by insect pollinators into GE-sensitive areas.

The agricultural significance of alfalfa seed production

Alfalfa is an economically important crop in US agriculture, planted on 8.5 million hectares nationwide (USDA-NASS 2015). With an annual value exceeding $7 billion annually, alfalfa provides a foundation for livestock and dairy industries. Alfalfa seed production is a necessary component of alfalfa production. Walla Walla County in Washington State is a major production area for alfalfa seed. In fact, 69% of state alfalfa seed production, and 12% of national alfalfa seed production in 2007 occurred within this county. Valued at $20 million within Washington State, alfalfa seed plays a pivotal role in the Western U.S. agricultural economy.

Adequate pollination services are required to ensure a profitable seed yield for alfalfa seed growers. Profitable alfalfa seed yields hinge on bee-visitation of blossoms. This is because

1) alfalfa plants are largely self-incompatible, and 2) due to the unique morphology of the alfalfa blossom, which must be ‘tripped’ in order to be pollinated (detailed in Bohart 1957). Per acre

2 seed yields in the Walla Walla valley are twice that of other alfalfa seed producers in the Pacific

Northwest. This is due in large part to the combined pollination efforts of both the alkali bee and the alfalfa leafcutting bee (Wichelns et al. 1992).

Pollinators relied upon for alfalfa seed production

The activity of pollinating insects provides critical ecological and economic services to flowering plants in natural ecosystems and modern agro-ecosystems (Klein et al. 2007). While the significance of these services is widely recognized, the specific contribution of various pollinating species has been substantially less explored. Plant-pollinator interactions are complex and variable by species (Batra 1976, Klein et al. 2004). Many different factors merit consideration when evaluating the nature of a particular plant-pollinator relationship, and can influence the relative importance of particular pollinators in certain environments (Harder and

Thomson 1989, Thomson & Goodell 2001). Some of these factors include the rate of pollen removal and deposition between blossoms, the pattern of pollinator-mediated pollen transfer in the landscape, and the foraging range of the pollinator of interest (Wilcock & Neiland 2002,

Brunet & Stewart 2010, Wright et al. 2015).

Three different bee species are relied upon for commercial pollination of alfalfa seed, each one exhibiting a unique interaction with the alfalfa blossom. Alfalfa requires that the blossom be tripped for pollination to occur, which relies on the release of the sexual column from the keel, an action dependent upon insect visitation (Bohart 1957, Tysdal 1940). Therefore, in addition to pollen removal and deposition by bees, pollen transfer in the landscape and foraging range of the pollinator, the tripping rates also warrant consideration when evaluating

3 pollination services in alfalfa seed fields. We will consider the relationship of each species with alfalfa below.

The honey bee (Apis mellifera) is the most widely studied of the three pollinators used in alfalfa seed production. Because of their wide application to dozens of different cropping systems, and the ease by which they can be intensively managed and moved to meet the pollination demands of various crops as they come into bloom, honey bees historically have been heavily relied upon for alfalfa seed production (Bohart 1957). However, it has long been recognized that honey bees are not terribly efficient pollinators of alfalfa (Henslow 1867, Bohart

1957). With few exceptions, honey bees typically exhibit a low tripping rate of the alfalfa blossom, ranging between 0.8% and 22% of blossoms visited (Cane 2002, Stephen 1955, respectively). The tripping mechanism of the blossom causes the bee’s head being struck by the staminal column. Honey bees quickly learn to obtain nectar without tripping the flowers, and thus avoid being struck by the staminal column, preventing pollination from occurring.

Furthermore, among the flowers that were successfully tripped by honey bees, fewer seeds per pod develop in comparison to those mechanically pollinated, or pollinated by bumble bees or leafcutting bees (Pharis and Unrau 1953). This finding suggests that due to the low rates of pollen removal and deposition the honey bee may not be well-suited for alfalfa seed production when compared to alkali or alfalfa leafcutting bees. Honey bees’ contribution to pollen flow and foraging range in alfalfa seed fields has been well characterized (Hagler et al. 2011a, Bradner et al. 1965), and suggest low rates of cross-pollination beyond 50 meters (165 ft) from a source plot, and a foraging range of just under 6,000 m.

The alkali bee, Nomia melanderi, is a solitary, ground-nesting bee species that features prominently in alfalfa seed pollination within the Walla Walla valley of Washington State

(reviewed in Bohart 1950, Bohart 1972). The alkali bee is native to the Western United States, although their contribution to alfalfa seed production was not fully realized until the 1940s

(Mayer & Miliczky 1998). Alkali bees commercially managed by seed producers are artificially maintained in sub-irrigated alkaline soils. These parcels of land are referred to as ‘bee beds,’ and they are designed to attract and maintain nesting females and their brood as they forage upon alfalfa for nectar and pollen for provisions. Alkali bees are univoltine and overwinter underground as prepupae. When the prepupae receive the appropriate daylight and temperature cues, they will pupate and alkali bees will subsequently emerge from the soil to begin foraging for the next 6-8 weeks (alkali bee foraging typically occurs from late May to early August in

Washington state). This life cycle synchronizes closely with peak alfalfa bloom, and partly explains why alkali bees are so attractive to seed producers. Alkali bees exhibit a penchant for alfalfa blossoms and females have been documented to trip over 80% of visited blossoms (Cane

2002). Because the alkali bee is a locally abundant endemic species, it is an attractive and affordable option for growers in the area that rely on pollination services. A productive bee bed can remain active for decades following its initial establishment, and their pollination services have been shown to significantly increase seed yield (Cane 2008) over yields achieved relying on honey bee pollination alone. In fact, seed producers reported yields ten times the national average following the implementation of artificial bee beds throughout the northwest during the

1950s (Bohart 1970). These documented yield increases provide good evidence for improved rates of pollen removal and deposition by the alkali bee when compared to the honey bee.

Estimates of the alkali bee’s foraging range extend to 1600 m from their nest, although alkali bees have been found up to 11 km from their nesting site (Delaplane and Mayer 2001).

Unfortunately, the establishment of bee beds is difficult in areas that do not already possess abundant local populations of alkali bees, thus limiting the potential for bee bed installments outside of the Walla Walla, WA region. The bees are also highly susceptible to parasites and pesticide exposure. Additionally, alkali bees forage as adults for only a few weeks out of the year, and their phenology may not synchronize with peak bloom in other regions within the U.S. (all of these limitations are discussed in Bohart 1950).

The third species and universally most important pollinator of alfalfa seed fields is the alfalfa leafcutting bee (Megachile rotundata), or ALCB (reviewed in Bohart 1972, Pitts-Singer and Cane 2011). Like the alkali bee, the alfalfa leafcutting bee is a commercially managed, solitary bee, with a tripping rate of alfalfa blossoms close to 80% (Cane 2002, Brunet and

Stewart 2010). Native to Eurasia, the ALCB was unintentionally introduced to the United States during the 1940s (Stephen 2003) and wasn’t recognized for its potential in alfalfa seed until 1961

(Stephen 1961). Since then, use of the ALCB for pollination has become standard practice in most alfalfa seed production areas. While solitary, the gregarious nature of the ALCB allows for seed producers to manage large populations of bees in relatively small, sheltered structures referred to as ‘domiciles’. Their success in alfalfa can be attributed to many factors. They nest readily in artificial nesting cavities, which makes them easy to propagate and distribute commercially. They overwinter as prepupae in cold, temperature controlled rooms and their emergence can be manipulated to synchronize with peak alfalfa bloom. Due to poor rates of high ALCB retention domestically, bees are typically imported from Canadian suppliers annually to meet the pollination demands of U.S. alfalfa seed producers. High rates and parasitism and chalkbrood in U.S. climates limit successful propagation of bees within its borders. Bees, sold as prepupae, are distributed in stocked Styrofoam nesting boards, called ‘bee boards,’ or as loose

6 cells, or cocoons, which have been physically removed from provided nesting cavities (Pitts-

Singer & Cane 2011). The ALCB is a very efficient pollinator of alfalfa and can produce seed yields up to 2200 kg per hectare (Johansen 1991), versus 392 kg per hectare using honey bee pollination alone (Pederson et al. 1955). Since the leafcutting bees are smaller in size than honey bees or alkali bees, it should not come as surprising that their foraging range also is shorter.

Therefore, to ensure uniform pollination and yield, seed producers place their bee boards in sheltered bee domiciles which are spaced at regular intervals throughout the field. Reports of

ALCB foraging range are variable, ranging from 100 – 500 m (Tasei & Delaude, 1984, Tepedino

1983) and up to 1600 m in extreme cases (Packer 1970). The generally-accepted foraging range for alfalfa leafcutting bees is 275m, based largely on ‘source and sink’ seed trials (St. Amand et al. 1999, Fitzpatrick et al. 2002, Van Deynze et al. 2008, McCaslin et al. 2000, Tasei & Delaude,

1984) – discussed further below.

Understanding the foraging range of all three managed pollinators is critical for describing the movement of transgene pollen into nontarget areas. While literature for honey bee pollination of alfalfa seed is relatively well developed (Hagler et al. 2011a), less is known about the foraging behavior of the more efficient ALCB and alkali bee. It has been estimated that alfalfa leafcutting bees often forage within a 30 m range from their domicile, with numbers dropping off precipitously at further distances (Bosch & Kemp 2005). Other literature has confirmed that alfalfa leafcutting bees are capable of flying over 1.6 km from their domicile

(Packer 1970). The foraging range of the alkali bee is also relatively unknown (Packer 1970).

Current literature suggests that alkali bees typically do not forage more than 1600 m away from their nesting site (Johansen et al. 1982). However, recent research has revealed that the alkali bee is capable of regularly foraging at least 6.4 km from her nest (Vinchesi 2014). This

7 discovery suggests a gross underestimation of the foraging range of an important pollinator of alfalfa seed. The discrepancy may be largely due to the different methods utilized to evaluate alkali bee foraging range (as discussed in the next section). We suspect that current foraging range estimations for the alfalfa leafcutting bee are similarly underrepresented. One primary aim of my research with alfalfa leafcutting bees is to determine the probability of transgene movement in an agricultural landscape. These findings would be more widely applicable to alfalfa seed producers on a national scale.

Measuring the foraging range of bee species

Considering the importance of insect pollination in economic and ecological systems, many studies have been conducted to determine the foraging range of bees. Knowing the foraging range of a bee species is important for many reasons. Foraging range can allow us to estimate the impacts of habitat degradation and landscape fragmentation on specialist plant and/or pollinator species, and it can help inform us of how to direct conservation efforts for declining bee populations. In agricultural systems, understanding the foraging range of pollinators help determine best management practices for bees providing pollination services and to characterize gene flow between fields. In particular, having precise estimates of pollinator flight range has become necessary to predict the spread of engineered genes via pollinator- mediated pollen flow from plants in agricultural fields planted to GE varieties into agricultural fields planted to related varieties and to wild or feral relatives.

Many techniques have been employed to determine how far bees forage, such as translocation, mark-recapture, harmonic radar, movable feeding stations, and seed or pollen analysis (Zurbuchen et al. 2007). Each technique is accompanied by their own pros and cons

8 which limit or restrict data interpretation in different ways. In translocation studies, marked bees are removed from their nesting site and released at variable distances from the nest. Then, the nest is observed for a period of time as the marked bees navigate back to their nest (Abrol

1988,Gathmann & Tscharntke 2002). The idea behind this technique is that only the bees familiar with the environment in which they have been released will be able to find their way back (some publications refer to this as ‘homing range’). Many argue that this method is disruptive and limits foraging range estimates, as it does not measure behavior in natural foraging conditions. Specifically, this method does not reveal the typical foraging range of bees, instead showing the maximum range that they are capable of flying. For example, Vincens and

Bosch (2000) measured a homing range for Osmia cornifrons of 1,800m. However, in the presence of locally abundant floral resources, most females foraged within 100-200 m from their nest site. Clearly, in this case foraging range and homing range are not biologically meaningful definitions.

Mark recapture studies provide an alternative method for determining foraging range in bees. This type of study is usually conducted in one of two ways. The first technique involves treating bees at their nest site with a traceable dye or powder, recapturing the bees in the field and checking them for the presence of the dye/powder (Hagler et al. 2011a). This method was employed by Hagler et al. (2011b) to determine honey bee foraging distance in alfalfa fields.

Bees were dusted with a fluorescent powder in combination with a protein the presence of which could be confirmed with a simple molecular test. Marked honey bees were then recaptured in the field along predetermined transects, and their hive of origin could be ascertained. An alternative approach to this method can be used on smaller bees that may be more difficult to recapture. In this instance, the bees are treated with a specific protein at their nest, and the

9 blossoms that they are suspected to be visiting is tested for the presence of the protein. In this case, rather than recapturing the bee, the protein is being recaptured on visited floral resources.

Biddinger et al. (2013) had success using this method by treating O. cornifrons with a chicken egg white marker in a cherry orchard, and testing randomly selected cherries blossom for the presence of the marker. Mark-recapture techniques are not available to bee species whose nests are inaccessible. Additionally, there may be occasions (for example, with the alfalfa leafcutting bee, M. rotundata in alfalfa seed fields) where the stocking densities of bees at their nests sites are so high that it is not possible to mark each bee nesting at a particular site. Additionally, recovery of marked bees can be difficult in areas of high local bee abundance (Boyle & Walsh, unpub. data).

Radiotracking and harmonic radar are newer methods developed to determine the foraging range of bees, and can provide in depth, detailed information about individual foraging routes. However, these techniques require that a transmitter be affixed to individual bees to track their movements. Researcher have successfully used harmonic radar to track bumblebee foraging activity (Osborne et al. 1997), and radiotracking has been employed by Pasquet et al.

(2008) to determine the typical foraging range of X. flavorufa. However, this technology is limited in application to larger bee species, such as bumblebees and honey bees, due to the size and weight of the transmitters currently available.

Other studies have designed movable feeding stations to determine the maximum foraging range of a particular bee species (van Nieustadt & Ruana Iraheta 1996, Greenleaf et al.

2007). In this case, bees are trained to forage at a mobile feeding station moved at away from the nest site over time. Once again, this technique does not provide any information regarding the typical foraging range of the bee, and may involve a lot of manipulation of the bee and the

10 environment around it for this type of analysis to work. Typically, movable feeding station experiments work best with specialist species, such as orchid bees, so that local floral resources do not reduce or limit the attractiveness of the feeder.

In seed production operations, we have the option of testing progeny for a known immigrant gene to determine pollinator foraging range and contribution to gene flow. Studies that examine gene flow in this way are commonplace, especially since it can address concerns over the movement of genetically engineered genes in agricultural landscapes (St. Amand et al.

1999, Fitzpatrick et al. 2002, Van Deynze et al. 2008, McCaslin et al. 2000, Tasei & Delaude,

1984). However, these ‘source and sink’ trials do not accommodate long range dispersal of bees, as they often fail to detect gene flow beyond a few meters from the bee’s nest (Fenster

1991, Ferreira et al. 2007). Most studies that endeavor to characterize foraging range of bees in alfalfa seed fields use this method. However, rather than capturing foraging range per se, this is merely an estimation of the rates of cross pollination events that occur in the field and do not provide a realistic estimation of how far bees are regularly flying.

To address some of the shortcomings of the seed testing method, some studies have endeavored to look directly at the pollen the bees are foraging upon to determine how far they fly

(O’Neill et al. 2004). Pollen is collected directly from bees as they return to the nest (Beil et al.

2008) or by examining pollen provisions from the nest itself (Tepedino 1983). Then the pollen is examined under a microscope and identified to species using morphological characters.

Tepedino (1983) used this technique to determine the rate of ALCB foraging in hybrid carrot fields. Pollen analysis in this way may not allow for foraging range estimates in mixed heterogeneous landscapes or pollination estimates between genetically distinct crop varieties.

All of the above-described techniques were considered when determining the most accurate way to measure ALCB foraging range in alfalfa. However, we opted to develop a novel strategy for assessing foraging distance in this system by examining ALCB pollen provisions for the glyphosate resistant trait using qPCR. This technique is easily incorporated into existing alfalfa seed production field conditions, and does not introduce unnatural circumstances or modify the landscape in any way for the foraging bees (discussed further in Chapter Three).

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CHAPTER TWO

EVALUATING MIGRATORY BEEKEEPING INFLUENCES ON POLLEN FLOW IN

TRANSGENIC ALFALFA SEED FIELDS

Introduction

Migratory beekeeping provides essential services for agricultural production worldwide.

Within the United States alone, honey bees’ estimated contribution to agriculture amounts to over 14 billion U.S. dollars (Morse & Calderone 2000). Globally, this figure exceeds $215 billion (Gallai et al. 2009). The agricultural success of the honey bee comes from the ease by which they can be intensively managed and moved from one region to the next. Commercial beekeeping operations rely upon frequent transport of hives to meet the pollination demands of various crops as they come into bloom. Alfalfa (Medicago sativa subsp sativa L.) is an insect- pollinated, perennial crop that relies upon managed bees for seed set. The United States is a leading exporter of alfalfa seed and hay (Tyng 2012). Since the initial 2005 deregulation of genetically engineered (GE) glyphosate-resistant (Roundup Ready®, Monsanto, St. Louis, MO,

USA) alfalfa, U.S. seed producers have expressed concern over the potential for honey bees

(Apis mellifera L.) to mediate gene flow of transgenic alfalfa pollen from GE fields into conventional alfalfa hay and seed fields (VanDeynze et al. 2008, Cornish et al. 2014).

Migratory beekeeping operations may allow for the movement of transgenic pollen beyond the natural foraging range of honey bees. Thus, migratory bee hives have the potential to contribute to transgenic cross-pollination in conventional seed stocks. Adventitious presence (AP) refers to the unintentional incidence of a particular variety or trait in harvested seed stock. In this case,

AP refers to the presence of the glyphosate resistant trait in conventional alfalfa seed stocks.

Adventitious presence is a concern for the export market. Many buyers of alfalfa seed and forage maintain strict standards to limit AP to near zero in seed or forage exports. If alfalfa seed or forage destined for such markets exceed established thresholds, the entire lot can be rejected

(Cornish et al. 2014). Therefore, it is critical to assess whether transgenic pollen latent in commercial hives contaminate conventional alfalfa seed fields during the routine movement of hives that facilitate commercial pollination.

Honey bee hairs trap pollen grains that are exchanged between bees in the hive (Free and

Durrant 1966, Free and Williams 1972). Pollen that is circulating between bees in a hive can and occasionally will lead to inadvertent cross pollination of different plant varieties during foraging

(DeGrandi-Hoffman et al., 1986). Additionally, the environment within the hive preserves viable pollen for extended periods of time. Pankiw and Bolton (1965) demonstrated that alfalfa pollen in the hive can remain viable for up to nine days.

Many studies reveal the length of time required to eliminate trace amounts of viable pollen from the hairs of worker bees. These studies are often conducted when honey bees are used to pollinate caged agricultural accessions, where the need to maintain varietal purity is high.

Sufficient honey bee isolation periods tend to center around 12 hours of confinement, although there is some variability depending on the specific crop in question. Kraii (1961) determined that 12 hours of isolation was enough time to eliminate cross pollination in cabbage, radish, wallflower and Begonia plots, while two days are required in sweetclover (Pankiw and Goplen,

1967). Honey bee colonies confined for 12 hours prior to introduction to field cages reduced seed contamination to 0.13% in sunflower plots (Wilson 1989). In alfalfa, Pankiw and Bolton

(1965) indicate that two days may be long enough to avoid undesired cross pollination between varieties in a greenhouse experiment, although this has yet to be rigorously tested. Currently, no

22 evaluation has been conducted to measure the potential for movement of viable transgenic pollen within migratory bee hives. The need for such studies in alfalfa has been recognized due to AP concerns and successful coexistence of GE and non-GE alfalfa production (Mueller 2004). Such studies are also relevant for understanding risks to varietal seed purity in conventional alfalfa seed production, and may be applicable to other honey bee-pollinated crops such as apple and plum, in which transgenic varieties are currently under development (Carter 2012, Scorza et al.

2013). This study uses the Roundup Ready® trait as a marker to assess pollination of non-GE alfalfa blossoms with transgenic pollen latent in hives isolated for an 8- or 32-hour period. We assayed for the transgene using a novel method of germinating glyphosate-treated alfalfa seedlings in a water curtain germinator, and scoring them based on seedling phenotype and root hair emergence to determine rates of AP from harvested seed.

Materials and Methods

Field experiment. Sixteen (2.7 m wide x 3.3 m long x 1.8 m high) PAK 25 mesh anti- insect screen cages (Pak Unlimited, Inc., Cornelia, GA, USA) were placed over Pioneer alfalfa variety 54V09 on the Washington State University Roza experimental farm in Prosser, WA, prior to bloom. On June 16, 2014, twelve queen-right honey bee colonies housed in five-frame nucleus hives were placed in a blooming GE alfalfa seed field in Touchet, WA (located 112 km from the Roza experimental farm in Prosser, WA), and allowed to forage openly for one week.

After one week, the hive entrances were closed at dusk (after bee activity had ceased for the day), and they were driven overnight to the caged alfalfa plots in Prosser, WA. Hive entrances were opened upon arrival. Six of the nucleus hives were immediately placed into individual field cages, while the remaining six nucleus hives were held for 24 hours in a GE-free holding yard

23 prior to placement. This comprised two treatment groups; (1) eight hours of isolation and (2) 32 hours of isolation from a GE alfalfa source. ‘Isolation’ is defined as the minimum amount of time measured between honey bees foraging on GE alfalfa and foraging on conventional alfalfa.

Although the honey bees were introduced to the cages immediately upon their arrival in Prosser,

WA, they do not forage at night, and would not commence foraging until the colony receives the correct daylight and temperature cues to begin the next morning. Therefore, we feel confident that eight hours of isolation is an accurate approximation. This practice also mirrors the standard migratory beekeeping routine of loading hives onto trucks at night to minimize the loss of foraging bees during the day.

Bees were permitted to forage on the caged alfalfa blossoms for forty-eight hours. After forty-eight hours, the bees were removed from the cages and returned to the same GE field in

Touchet, WA. We then allowed the bees to forage freely on the GE alfalfa for another week before returning them to the cages in Prosser, WA. In total, honey bee hives were introduced to the same cages on three separate occasions to ensure adequate pollination and seed yield for subsequent analysis. Four cages in Prosser, WA, received no honey bee pollination as a control.

Control cages were used to confirm that any pollination in the cages was directly attributable to the introduced pollinators.

On August 13, 2014, the caged conventional alfalfa plots were harvested. The alfalfa seed pods were dried, cleaned, scarified and weighed prior to set-up of the seedling germination assay. Adventitious presence was confirmed using AgraStrip® RUR Seed & Leaf test strips

(Romer Labs Inc., Union, MO, USA).

Trait confirmation of seedlings using the seedling germination assay. To assess AP in harvested alfalfa seed samples, we developed a novel seedling germination assay in which seedlings germinated for 14 days following exposure to a low concentration of glyphosate solution. Seedlings were then evaluated individually for traits associated with glyphosate resistance. In order to perform a reliable assay, three types of control experiments were conducted concurrently with the experimental samples. These control experiments included 1) measuring the baseline germination rate of harvested seed in distilled water, 2) confirmation of

GE trait detection in replicates with a known percentage of GE seed, and 3) determining the background presence of AP in the original seed lot of our conventional alfalfa plots.

1. Measuring the baseline germination rate of harvested seed. To establish the baseline germination rate, harvested seed samples were germinated in a distilled water treatment for 14 days. Determining the germination rate allowed us to adjust the detection of AP according to the proportion of viable seed harvested from the cages. Since all seeds harvested from the cages were from the same alfalfa variety grown under the same environmental conditions, we tested three individual replicates of 100 pooled seeds to establish the germination rate.

Seeds were counted using a vacuum aspirator with a 100-count vacuum head (Hoffman

Manufacturing, Inc., Jefferson, OR, USA). Each replicate of 100 seeds was sandwiched between labeled 23cm x 24cm seed germination papers (Anchor Paper Co., St. Paul, MN, USA) that had been saturated with distilled water. Individual replicates were placed directly on a perforated germination tray fitted for a water curtain germinator. The germination tray was placed in a perforated plastic greenhouse bag before introduction to the water curtain germinator. The germinator was set at 20 degrees Celsius with an 16:8 hour light:dark cycle. We allowed the

25 seeds to germinate for 14 days, watering the trays with an antimicrobial Plant Preservative

Mixture (PPM™) (Plant Cell Technology, Washington DC, USA) solution (1mL PPM: 1L distilled water) as needed. Upon removal from the germinator, all sprouted seedlings were counted, and the average percent germination rate was calculated.

2. Confirmation of trait detection in replicates with a known percentage of GE seed.

This trial was designed to test that the protocol we developed was robust enough to ensure high accuracy in the detection of positive seedlings. Prior to initiation of the assay, a fresh 80 ppm working solution of glyphosate (Gly Star® Original, Albaugh, Inc., Ankeny, IA, USA) was prepared. We set up nine replicates of 600 individual seeds per replicate that each contained a fixed percentage of GE seeds mixed in with conventional seeds. Three different fixed percentages, 10%, 1% and 0.16%, were tested; see Figure 1. As described above, seedlings were prepared on saturated germination towels using a vacuum aspirator with a 100-ct vacuum head.

However, instead of saturating the germination paper with a distilled water solution, the germination towels were saturated with the 80 ppm glyphosate solution. Replicates were loaded directly on to the germinator trays, and subjected to the same 14 day experimental conditions as described for the germination control. Four days following initial set-up, the seedlings were watered with a second application of the glyphosate solution.

At the end of the 14-day germination period, the seedlings were scored blindly. No information was provided to our seedling scorer regarding percent of GE seed in each treatment, or the number of replicates per treatment. Each seedling was assessed individually for symptoms of glyphosate resistance (Figure 2), such as root hair development, overall root length, thickness, and growth of the cotyledons (Figure 3). Positively scored seedlings were confirmed for the

26 presence of the GE trait using lateral flow AgraStrip test strips according to labeled directions.

The percentage of GE seed was then calculated for each replicate.

3. Determining the background AP level of the GE trait in the original seed lot. To assess AP in the experimental samples, we first needed to know what the background level of AP in the original seed lot was. Although the original seed lot was labelled as conventional, some varieties of alfalfa grown for seed may still exhibit a low level presence of the glyphosate resistant trait. If AP is detected in the original seed lot, the calculated rate of detection in the germinated seedlings would need to accommodate preexisting levels of AP from the field. Three individual replicates of 1800 seeds were tested for the presence of the transgene from the original seed lot, and the percent AP was calculated by averaging that percentage over the three replicates. Seeds were prepared for the germination assay and scored using the procedures outlined above.

Seedling germination assay of experimental samples. To assess adventitious presence within the caged plots, three replicates of 1800 individual seeds per field cage were placed in a water curtain germinator for 14 days. Due to low seed yield in some of the bee-pollinated cages we were unable to test seed from one caged plot in each of the treatment groups. Seed was also assayed from the original seed lot to determine the background presence of AP in the cages.

Prior to initiation of the assay, an 80 ppm solution of glyphosate (Gly Star® Original,

Albaugh, Inc., Ankeny, IA, USA) was prepared. Seeds were counted using a vacuum aspirator with a 300-count vacuum head (Hoffman Manufacturing, Inc., Jefferson, OR, USA) and sandwiched between labeled 46cm x 48cm seed germination papers (Anchor Paper Co., St. Paul,

MN, USA) that had been saturated with the working solution. Individual replicates were stacked six high on perforated germination trays fitted for the water curtain germinator. The germination

27 trays were placed in perforated plastic greenhouse bags before introduction to the germinator.

The germinator was set at 20 degrees Celsius with an 16:8 hour light:dark cycle. We allowed the seeds to germinate for 14 days, watering the trays with an antimicrobial Plant Preservative

Mixture (PPM™) (Plant Cell Technology, Washington DC, USA) solution (1mL PPM/1L distilled water) as needed, and applied a second 80 ppm glyphosate treatment to the seedlings three days following initial setup. Statistical analysis was conducted using Minitab® 17

(Minitab® Statistical Software 2010).

Results

Field experiment

The seed yield differed dramatically between control and bee-pollinated cages (t = -0.483, P =

0.001; Figure 4, Table 1). However, no difference in seed yield was detected between one- and two-night isolation groups (t = -1.29, P = 0.229; Table 1).

Trait confirmation of seedlings using the seedling germination assay

1. Measuring the baseline germination rate of harvested seed

We found that the baseline germination rate of seed harvested from the cages averaged 54 ±5%

(SE), suggesting that the %AP in the experimental samples can be calculated as follows:

%AP = (the number of positive seedlings)/(1800 total seeds * 0.54)

2. Confirmation of trait detection in replicates with a known percentage of GE seed

Data from this control study suggests highly accurate detection of the GE trait in samples containing a fixed percentage of transgenic seed (Table 1).

3. Determining the background AP level of the GE trait in the original seed lot

Seedling germination assay of experimental samples

No seedlings tested from the original seed lot were positive for the GE trait; therefore the original AP level in this field was considered insignificant.

Seedling germination assay of experimental samples

Results obtained from the seedling germination assay indicated that risk of cross- pollination from viable transgenic alfalfa pollen in the hive was almost completely eliminated following one night of isolation from the GE source (Table 1), even though trace levels of the transgene were detected. No significant differences in AP were found between one- and two- night isolation groups (t = 2.12, P = 0.067).

Discussion

The use of GE traits, such as glyphosate resistance, can allow us to detect rates of cross pollination at high levels of sensitivity. The seedling germination assay can serve as a valuable tool in measuring gene flow and foraging behavior in landscapes featuring transgenic alfalfa, as indicated in this study.

Caged seed yield was found to be substantially lower than would be expected in an open field environment (Bohart 1957). Although honey bees are acknowledged as inefficient pollinators of alfalfa seed (Henslow 1867, Bohart 1972), low seed yields in the cages most likely

29 resulted from a cage effect, as has been previously demonstrated (Hughes 1966, Rubis et al.

1966).

While the bees were held in the cages for a total of six days throughout the season,

Hanson (1961) showed that alfalfa pollen remains viable on untripped blossoms for 8 – 15 days, suggesting that all blossoms in the cages were capable of being pollinated despite intermittent introductions of the bees to the cages over the course of the study.

In this study, we determine the potential for transgenic pollen flow following honey bee hive transport between GE and conventional alfalfa seed fields. Typically, honey bees require no more than two days of isolation to remove germinable traces of pollen from foraging bees. Our results agree with Pankiw and Bolton (1964), indicating that any risk of cross pollination between alfalfa varieties is virtually eliminated overnight. While trace amounts of the transgene in the cage studies were detected, they were at levels that would not be of economic significance to the alfalfa seed industry (AOSCA 2012). However, organic or sensitive alfalfa seed markets that uphold a zero percent rate of detection may require more than two days of bee hive isolation from genetically engineered alfalfa to ensure 100% eradication of viable transgenic pollen.

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Table I. Average seed yield and percent AP detection by field cage treatment in alfalfa seed tested using seedling germination assay screening and test strip confirmation

Treatment Seed Yield, g (± SE) %AP (± SE)

Overnight isolation 15.9 ± 4.70 8x10-5 ± 2x10-5

Two-night isolation 26.2 ± 6.47 2x10-5 ± 2x10-5

Control 1.02 ± 0.44 0a

Original Seed Lot N.A. 0 ± 0 aControl %AP is based on a pooled sample of 100 seeds per replicate over three replicates due to low seed yield for this group (see figure 4).

Table 2. Average %AP detection in seed samples with a fixed percentage of GE:conventional alfalfa seed, ± SE.

Fixed %AP Average %AP detected in

seed experimental seedlings (± SE)

10% 11.6 ± 0.48

1% 1.33 ± 0.08

0.16% 0.16 ± 0.08

Figure 1. Organization of the experiment set-up for seed treatments containing a known percentage of GE:conventional seed. Each replicate consisted of 600 seeds total. Each treatment group (10%, 1% and 0.16%) comprised three of the nine replicates. Seedlings were scored blindly for the %AP in each replicate.

Figure 2. One replicate of alfalfa seedlings following removal from the water curtain germinator. Seedlings were evaluated individually for symptoms of glyphosate resistance.

Figure 3. The alfalfa seedling on the left exhibits characteristics of glyphosate resistance, while the alfalfa seedling on the right does not. Glyphosate resistance is characterized by a seedling’s large size, elongated root, the presence of root hairs on the root, and cotyledon growth.

30 *

Alfalfa Alfalfa Seed Yield (g) 5

0 Bee-Pollinated Control

Figure 4. Average alfalfa seed yield in bee-pollinated and control field cages, ± SE; P = 0.001

CHAPTER THREE

GENETIC DETECTION OF TRANSGENIC POLLEN FROM ALFALFA

LEAFCUTTING BEE (Megachile rotundata) LARVAL POLLEN PROVISIONS

Introduction

The alfalfa leafcutting bee is a commercially managed, solitary bee that plays a pivotal role in providing pollination services to alfalfa seed production in North America (Pitts-Singer and Cane 2011). Its value to the alfalfa seed and resultant hay industry is unquestionable, pollinating one-half of all alfalfa seed produced in the northwestern United States (Van Deynze et al. 2008). Alfalfa produced for the seed and forage markets collectively are the 4th largest crop in the United States, by both acreage and farmgate value (APHIS 2011). Over the last few years, international hay exports from the U.S. have risen to an all-time high (Tyng 2012). Most U.S. alfalfa exports are destined for Asian markets that tend to be sensitive towards genetically engineered products.

Since the 2011 deregulation of genetically engineered (GE), glyphosate-resistant alfalfa by the USDA, seed producers and buyers of alfalfa have expressed concern over the potential for pollinator-mediated gene flow in alfalfa seed fields. Many foreign markets that rely on U.S. alfalfa importation maintain strict standards limiting GE-trait detection to a fraction of a percent in conventionally produced varieties (Cornish et al. 2014). Because acreage devoted to GE alfalfa increases each year, and additional transgenic alfalfa varieties are about to enter the market, it is critical that reliable and robust best management practices are established to ensure the genetic integrity of alfalfa varieties sold in today’s agricultural market (Cornish et al. 2014).

Ensuring varietal purity is not a new concept in alfalfa seed production. Many types of alfalfa varieties are produced for different end markets. This allows alfalfa seed and forage producers to select the variety that best suits the environment they are growing in. Traditionally, seed producers assess varietal purity by growing harvested seed in a greenhouse and measuring phenotypic traits, such as growth rate, stem length, leaf serration, and other morphological characters (Nittler et al. 1964) and comparing them to an expected phenotype. The technical term for the presence of an undesired genetic trait in harvested seed is adventitious presence, or

AP. Until recently, the above-described methods of AP testing have been sufficient in meeting the purity demands in place by the alfalfa seed market. However, the advent of GE alfalfa has permitted molecular screening for the glyphosate resistant trait in seed at an unprecedented level of sensitivity (APHIS 2011). Due to the politically charged and controversial nature of genetically engineered crops, many importers of alfalfa seed and forage uphold low- or zero- tolerance for the presence of the transgenic trait in conventionally produced seed. When seed destined for export exceed the established thresholds, the entire seed lot can be rejected for sale to AP-sensitive markets. As alfalfa is the first open pollinated, perennial crop to be deregulated by the USDA, it is critical that we understand the role that pollinators may play in contributing to

GE gene flow between seed varieties (Cornish et al. 2014).

The Association of Official Seed Certifying Agencies (AOSCA) is an international non- profit organization that works with seed producers across various cropping systems. They are charged with establishing standards for seed management practices and certification. Among

42 standards for the alfalfa leafcutting bee (ALCB) recommend at least 274m of isolation between alfalfa varieties to meet an AP tolerance of 0.1% in harvested seed (AOSCA 2012). The 0.1% threshold at this distance is based upon current estimates of the ALCB foraging range, although many are concerned over the robustness of these current standards due to recent unexpected AP detection in some alfalfa varieties destined for export (Gillam 2013, Gillam 2015).

Current ALCB foraging range estimates are based on studies which test trait expression of harvested seed at known distances from a genetically distinct alfalfa variety, and are therefore indirect approximations for their true foraging range. Reports of the ALCB’s foraging range has been variable over the past several decades, ranging from 100 – 500m (Tepedino 1984, Tasei &

Delaude 1983) and up to 1600m in some cases (Packer 1970). Alfalfa seed producers manage their pollinators by spacing sheltered nesting sites (or domiciles) regularly throughout their production fields. Within the Walla Walla valley of Washington State, ALCB domiciles may be spaced anywhere between 75 and 230m apart from one another depending on the personal preferences of the grower combined with the shape and size of the field they are working with

(unpub. data). If we can more accurately predict the foraging range of the alfalfa leafcutting bee, we can make more informed recommendations for domicile spacing and management, thus mitigating the risk of undesired gene flow between adjacent fields. In this study, we aimed to assess foraging range by measuring the rate of GE pollen detection in ALCB pollen provisions collected from conventional domiciles. ALCB foraging range can then be inferred from the distance to the nearest GE field in the area at minimum. This will provide us with a ‘worst case scenario’ prediction of ALCB mediated gene flow between GE and conventional alfalfa seed fields.

As a solitary bee, the female ALCB mates once, then lays and provisions eggs over the duration of her lifespan (Richards 1994), from Mid-June to late July in Washington state. When resources are abundant, the ALCB will lay and provision on average 57 eggs in her lifetime

(Maeta 2005). When pollinating commercial alfalfa produced for seed, ALCBs lay eggs in artificial nesting cavities, 5-7mm in diameter and 95- 150 mm in length (Gerber & Klostermeyer

1970). The ALCB name is derived from the bee’s behavior of enclosing individual cells with disc-shaped pieces of alfalfa leaves and petals they collect from the field using the beveled edges of their mandibles (Pitts-Singer & Cane 2011). In a typical alfalfa seed field, it takes on average

2.5 hours for a female to construct one cell from the leaf discs, and 5 hours (17 individual trips) to provision it with a combination of pollen and nectar. This means that each ALCB cell requires about 7.5 hours to complete, limiting a female ALCB to laying 1-2 eggs per day (Klostermeyer and Gerber 1969). A full alfalfa pollen provision typically weighs about 90 mg and contains 33-

36% pollen and 64-66% nectar by weight (Klostermeyer et al. 1973, Cane et al. 2011, Rincker et al. 1987). This equates to about 1.3 million pollen grains per provision (Cane et al. 2011).

In this study we examined individual pollen provisions for the presence of the transgene, in order to better understand the typical ALCB foraging range in an agricultural landscape. This information can be used to inform science policy related to pollinator management and domicile spacing in regions of alfalfa production.

Methods

Field Identification and pollen collection from ALCB domiciles. Five sites in the

Touchet valley of central Washington State were identified in which conventional seed fields were located adjacent to known transgenic fields. To ensure adequate pollination of the seed

44 fields, growers place ALCB domiciles at regular intervals throughout their fields. This practice enabled us to place experimental nesting boards at known and variable distances from a transgenic pollen source. We used 39-hole Binderboards® (Pollinator Paradise, ID, USA) outfitted with customized cardboard liners (Spiral Paper Company, CA, USA) to attract nesting bees from the field. Binderboards were placed in a total of 7 domiciles across the five fields used in this experiment. One Binderboard was placed in each domicile on June 11, 2013, as the bees were beginning to emerge for the pollination season. In an attempt to characterize early- and late-season foraging habits, all Binderboards were removed from the field half-way through the season and replaced with new ones on July 3, 2013. The second deployment of Binderboards were removed from the fields at the end of the pollination season in late July. The end of the pollination season is characterized by seed pod maturation in alfalfa plants and a substantial reduction in both alkali and leafcutting bee populations for the year. To serve as a positive control, Binderboards were also placed in transgenic fields over the course of the season.

Similarly, Binderboards were placed in conventional fields that were isolated from transgenic alfalfa by at least 1600 m. The isolated conventional pollen samples served as a negative control for the experiment, as confirmed by PCR (see methods, below).

The foraging range of ALCBs were assessed by examining the incidence of transgenic pollen in larval bee provisions from conventional domiciles that were located a known distance from transgenic seed fields. A female ALCB provisions one nest at a time, relying on olfactory and chemical cues to navigate back to her nest from each foraging bout. For the purposes of this study, we considered each nest to serve as an independent replicate. While we acknowledge the possibility that this technique may incorporate multiple nests provisioned by the same individual over time, the probability of this is unlikely considering the stocking density of ALCBs at the

45 domiciles and the resultant high abundance of bees residing at each one. Additionally, we had to standardize testing for the variable number of cells contained within each nest. The ALCB nests collected contained a variable number of pollen provisions (exhibited in Figure 1), ranging between zero and five in each nest during 2013. In part, the low number of provisions is likely due to the disruption of collecting and replacing the Binderboards in the middle of the season. In order to standardize testing for these variable numbers of pollen provisions, I ran analysis only on nests from which 1-3 pollen provisions were provided. Sampling in this way allowed me to standardize foraging effort across nests. A nest with at least one provision positive for the gene was counted as positive for AP. Eight ALCB nests were analyzed from each domicile included in the study, although in some cases, less than eight nests were available to sample from in which case all available provisions were tested for AP. Due to the low observed nesting rate, early-and late season samples were pooled to facilitate statistical analysis.

After the Binderboards were collected from the field, they were immediately placed into a -20 C freezer overnight. The next day, I removed and labelled all of the provisioned liners for dissection and further processing. Cardboard liners were stored in 50 mL falcon tubes (BD

Biosciences, San Jose, CA) until dissection.

Isolation of pollen provisions from ALCB nests. It has been estimated that the ALCB foraging range for alfalfa leaf discs is shorter than it is for pollen and nectar. Evidence for shorter leaf disc foraging ranges are provided by the significantly shorter amount of time required for completion of individual leaf collecting trips. Jensen (2001) reported an average of

672s per ALCB foraging trip versus 101 average seconds to complete a foraging trip for an individual leaf disc. Similarly. Klostermeyer and Gerber (1969) found average pollen and leaf disc foraging periods of 894 s and 318 s, respectively. The cause of the shortened leaf disc trips

46 are likely due to a high local abundance of alfalfa leaves (Jensen 2001, Klostermeyer and Gerber

1969), thus limiting the amount of time or distance to cover in order to complete a full bout of foraging. Therefore, when preparing the pollen provisions for analysis, I carefully removed each leaf disc from the mass to ensure that the DNA extracted for this experiment was from pollen alone (Figure 2). This action was performed using a dissecting microscope and a microslide tool kit (Bioquip, USA). After each pollen ball was isolated from its cell, the provision was placed in a labelled 1.5 mL Eppendorf tube and stored in at -20°C for DNA extraction.

DNA extraction from ALCB pollen provisions. Individual pollen provisions were evaluated for the presence of the transgene using a modified CTAB buffer-based extraction protocol based on Xin and Chen (2012). Current techniques for assessing the presence of genetic traits in pollen are extremely limited. Most studies developed to screen for specific traits in pollen are performed using cross pollination assays in which germinated plant tissue can be tested for the presence of the gene in question. To our knowledge, this is the first protocol developed using qPCR to detect the incidence of transgenic pollen provisioned by bees.

DNA extraction from pollen is widely recognized as a molecular challenge. As mentioned above, ALCB pollen provisions meet a 2:1 nectar to pollen ratio. Nectar is devoid of

DNA, and the large amount of polysaccharides in the provisions can interfere with PCR amplification of DNA. Similarly, pollen itself is composed of a number of PCR inhibitors such as starch and lecithin. Therefore, DNA extraction of pollen provisions requires many clean-up stages to eliminate any interference resulting from inhibitors.

To prepare pollen for DNA extraction, individual pollen balls were removed from 1.5 mL centrifuge tubes and weighed prior to extraction. Single pollen balls were put into 2 mL Lysing

Matrix A grinding tubes (MP Biomedicals, Santa Ana, CA, USA), containing 1.2mL of CTAB

47 extraction buffer (100mM Tris pH 8.0, 20mM EDTA, 2% CTAB, 1.2M NaCl; adjusted to pH

8.0). The grinding tubes were loaded into a Geno/Grinder 2010 and ground at 15,000 rpm for two minutes. The ground samples were incubated in a water bath at 60°C for 30 minutes, then spun for 10 minutes at 10,000 rpm, and the supernatant transferred to a new 1.5 mL tube. An equal volume of 24:1 chloroform/isoamyl alcohol was added to the supernatant, and the centrifuge tubes were vortexed gently to mix. Samples were spun again at 10,000 rpm for 10 minutes. Then we transferred 375uL of the viscous aqueous phase into new 1.5mL centrifuge tubes, vortexed the samples gently and incubated them in the water bath at 60°C for 30 minutes to precipitate the DNA. The samples were spun at 10,000 rpm for 10 minutes and the supernatant was poured off, leaving a white pellet with DNA at the bottom of the tube. After adding 500 uL of wash buffer (30% TE (10mM Tris pH 8.0, 1mM EDTA), 70% EtOH), we let the samples sit at room temperature for 30 minutes then spun it for 10 minutes at 10,000 rpm.

We poured out the supernatant from the tubes and spun the samples again at 10,000 rpm for 10 minutes, and pipetted off any excess liquid at the bottom of the centrifuge tubes. The samples were allowed to dry completely for at least 15 minutes, before adding 30uL of TE buffer to resuspend the pellet. Finally, they were put in a 60 C water bath for 10 minutes and stored in the refrigerator overnight prior to PCR analysis.

PCR reactions of pollen DNA extract. Roundup Ready alfalfa contains two events referred to as J101 and J163 that can be assessed using confidential Monsanto probes in combination with forward and reverse primers for both events. qPCR was used to validate the presence of the transgene. Only samples in which both events were detected were considered positive for the GE trait.

Samples were prepared for analysis using a 20 µl reaction volume. To each well, we added 13.86 µl distilled water, 2.0 µl 10X PCR Buffer (Bioline), 1 µl 50 mM MgCl2, 0.3 µl 25 mM dNTPs, 0.26 µl J101 primer/probe mix, 0.26 µl J163 primer/probe mix, 0.32 µl Biolase Taq, and 2.0 µl of DNA template. Additionally, a water control and a positive control were implemented each time qPCR was run.

A BioRad CFX96 Real-Time PCR Detection System was used to perform qPCR. The

PCR protocol, developed in collaboration with Ruth Martin at USDA-ARS in Corvallis, OR, included a 3-minute incubation at 95.0°C followed by 50 cycles of PCR, consisting of 15 seconds at 95.0°C and 1 minute at 60.0°C.

Results

In a 100mm nesting cavity, the ALCB will lay up to 12 individually provisioned cells

(Boyle & Walsh 2014; unpub. data). However, in 2013, the number of provisions in each occupied nest was substantially lower, averaging 1.72 ± 0.069 (SE) and ranging between 1 and 5 provisions per nest. It is probable that the observed low fill in the supplied nesting liners were a consequence of the mid-season disruption of replacing the Binderboards in an attempt to capture early- and late- season differences in foraging behavior. Out of statistical necessity, 2013 early- and late-season nests were pooled together to provide the replication necessary to interpret our results.

Attempts to characterize the transgene in bee-provisioned pollen using this new technique for DNA extraction was successful, allowing for sensitive detection of transgenic pollen at the furthest distances from GE alfalfa seed fields tested. While we were able to evaluate only a limited number of pollen provisions using this method, rates of AP detection in pollen are

49 consistently higher than would be expected based on current foraging range estimates (Table 1).

Due to the small sample size, it was not possible to perform statistical analysis to determine differences in AP detection over distance. In part, this is due to the consistently high level of GE pollen detection at all sites that were tested for the transgene, ranging between 85 and 320 meters from a transgenic alfalfa pollen source. Such findings suggest a gross underestimation of the typical alfalfa leafcutting bee’s foraging range, as bees were found to frequently return to their nest with transgenic pollen from 320 m away during nest construction.

Discussion

The results of this study suggest that the potential for alfalfa cross pollination extends well beyond the current isolation distances recommended by AOSCA. While this data confirms that the ALCB is frequently flying beyond current estimations of its typical foraging range, we acknowledge that our data is limited in interpretation to pooled activities of the individual

ALCB. It has been estimated that a single pollen provision is the culmination of 17 independent foraging bouts on average, or one full day of foraging (Klostermeyer and Gerber 1969). Our protocol for detecting the transgene cannot accurately quantify the ratio of conventional to transgenic pollen in each provision. Therefore, although we saw high rates of AP in pollen samples collected from conventional domiciles, it cannot be known how many individual foraging trips were made to the transgenic field in each sample analyzed. We can only conclude what the maximum foraging distance was during the compilation of each individual provision.

Additionally, this knowledge does not reveal any information directly relatable to GE trait expression in harvested seed.

It is important to note that just because we are detecting the transgene in the pollen, this does not necessarily pose a risk for successful cross pollination at these ranges. The chance of a cross pollination event occurring between transgenic and conventional varieties is dependent upon more factors than simply observing a bee move between the two alfalfa blossoms. Other variables that merit consideration include rates of pollen removal and deposition by ALCBs, the probability of cross pollination following successive visits to intermediate alfalfa blossoms, and the probability that a pollinated alfalfa blossom yields seed at all (Detailed further in Chapter 4).

For these reasons, knowing the extent of ALCB foraging may not be enough to warrant revision of current isolation buffers. We would also need to know the rate of cross-pollination between transgenic and conventional fields resulting from ALCB activities.

Data obtained from this study demonstrate the effective application of a novel method for measuring bee-mediated pollen flow (and foraging range) in an agricultural habitat. This is the first study to measure AP in bee-provisioned pollen samples, as traditional methods for AP screening have been restricted to ‘source and sink’ seed trials (Hammon et al. 2006, Fitzpatrick et al. 2002, McCaslin et al. 2000, St. Amand et al. 2000), which relies on trait expression in harvested seed as a proxy for bee foraging between alfalfa varieties. We believe that this technique can inform us of foraging range of not only pollinators within alfalfa fields, but also within other agricultural systems in which transgenic plant varieties are currently being considered for deregulation such as apple and plum (Scorza et al. 2013, Carter 2012). This method could become a particularly useful tool as honey bee populations are in decline and farmers across many agricultural systems are increasingly turning to alternative pollinators to meet pollination demands. Testing foraging range in this way provides a natural and realistic

51 estimate of a bee’s foraging range and can be used inform bee management in many additional applications.

References

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Packer, J.S. 1970. The flight and foraging behavior of the alkali bee (Nomia melanderi) and the alfalfa leafcutting bee (Megachile rotundata). Ph.D. thesis, Utah State Univ., Logan, Utah. 119 pp.

Pitts-Singer T.L., Cane J.H. 2011. The alfalfa leafcutting bee, Megachile rotundata: The world’s most intensively managed solitary bee. Annu. Rev. Entomol. 56: 221-237

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Table 3-1. GE trait detection in pollen using qPCR, in relation to distance from a GE source.

Field number correlates with the fields identified in Figure 4-1. ‘n’ indicates the number of individual nests tested for the transgene at each domicile included in the study.

Distance Field to GE (m) n % AP 2 85 7 100 1 88 8 87.5 5 152 8 62.5 3 207 7 42.9 3 207 8 87.5 2 305 8 87.5 1 320 8 75

Figure 3-1. X-rayed ALCB nests, demonstrating the variable number of pollen provisions available in each individual nest. Only nests exhibiting at least 3 individual pollen provisions were tested for the absence of presence of the transgene.

Figure 3-2. A step-by step progression of isolating a pollen provision from an individual ALCB cell under a dissecting microscope.

CHAPTER FOUR

THE INFLUENCE OF THE ALFALFA LEAFCUTTING BEE IN CROSS

POLLINATION BETWEEN TRANSGENIC AND CONVENTIONAL ALFALFA SEED

FIELDS

Introduction

Commercial alfalfa seed production is major cash crop of the Walla Walla valley in south-central Washington State. Over 2,400 acres of alfalfa seed is grown in this region, accounting for 69% of Washington state seed production and 12% of total U.S. production in

2007 (USDA-NASS 2015). The success of alfalfa seed in Washington State comes from the combined pollination efforts of the alfalfa leafcutting bee, Megachile rotundata (ALCB), and the alkali bee, Nomia melanderi (Bohart1957, Bohart 1972). Areas which are pollinated by one or both of these bee species can provide seed yields which are five- to ten-fold the yield of alfalfa when exclusively pollinated by honey bees.

Since the introduction of alfalfa to the new world and its increased use as a forage crop for animal feed, breeders have been developing genetically distinct seed varieties for different end users. Each alfalfa variety exhibits a unique combination of characteristics that growers in different parts of the world may find desirable. Some alfalfa varieties, for instance, are bred for increased pest or disease resistance, and others for different climate regimes or different soil types (Nittler 1964). Because alfalfa is a perennial crop that is dependent on bee pollination for seed development, seed producers must manage their pollinators to maximize yield while also ensuring the genetic integrity of each alfalfa variety grown. Adventitious presence (AP) refers to

61 the presence of an undesired genetic trait in harvested seed. Pollinators of alfalfa have the potential to contribute to undesired cross-pollination between varieties, resulting in the occurrence of AP in harvested seed (Mueller 2004, Cornish et al. 2014). To mitigate this risk, seed producers often introduce isolation buffers between different alfalfa fields. Increasing the distance that bees must fly to access and cross-pollinate another field limits the occurrence of AP between varieties. When genetically engineered (GE), glyphosate-resistant alfalfa entered the market in 2011, many seed producers questioned the efficacy of current management practices and isolation buffers in place to curb undesired gene flow between fields. Genetic screening of alfalfa seed and forage for the GE trait allows for sensitive detection of AP in conventional seeds

(APHIS 2011). Many economic markets will not buy conventional alfalfa forage or seed containing even a fraction of a percent of the GE trait. Recent detection of AP in conventional alfalfa requires that we revisit current isolation recommendations for maintaining varietal purity between fields.

As detailed in chapter 3, the Association of Official Seed Certifying Agencies (AOSCA) is an international organization that works directly with seed producers in many different crops.

AOSCA establishes standards for seed management practices and certification. Among

AOSCA’s responsibilities are recommending isolation standards that mitigate bee-mediated pollen flow between adjacent crop varieties, including alfalfa. The isolation standards vary depending on the species of bee being used to provide pollination and are based upon current knowledge of their foraging ranges. Current AOSCA isolation standards for the alfalfa leafcutting bee recommend at least 274m of isolation between alfalfa varieties to meet an AP tolerance of 0.1% in harvested seed (AOSCA 2012). The 0.1% threshold at this distance is based upon the supposed foraging range of the ALCB, however, there is concern over the

62 robustness of these current standards due to recent and unexpected AP detection in Washington alfalfa fields. The 0.1% threshold is a common standard used in the alfalfa industry for ‘AP- sensitive’ seed, which is the seed class most commonly destined for international exports.

The National Alfalfa and Forage Alliance (NAFA) is a national organization that works closely with local alfalfa seed and hay associations across the U.S. They work directly with the seed producers to, among other issues, advise them on management practices to address concerns over transgenic pollen flow. In 2011, NAFA proposed that coexistence of genetically engineered and conventional alfalfa seed markets can be achieved via the establishment of ‘Grower

Opportunity Zones,’ or GOZs. A GOZ is a spatially isolated alfalfa seed production region in which a majority of the growers elect to focus on growing either GE or conventional alfalfa exclusively (Cornish et al. 2014). By segregating the production of seed to individual geographic regions, alfalfa seed growers need not concern themselves over pollinator-mediated gene flow. As of March 2015, 18 GOZs have been approved by NAFA, 16 of which have committed to GE GOZs (including Walla Walla County, WA). These actions will be extremely practical for alfalfa seed producers everywhere. However, the data presented in this chapter provides valuable insight for the alfalfa industry. Because of new GE alfalfa varieties (lignin reductase and a high tannin variety) undergoing deregulation, it is important that the genetic integrity of all these GE varieties are maintained within GE GOZs. The same applies to conventional GOZs, in which many neighboring alfalfa varieties may present the possibility for cross-pollination among fields.

The objective of this study was to address concerns over current recommended varietal isolation standards for the alfalfa leafcutting bee by assessing seed at variable distance from a transgenic field for the presence of the GE trait.

Methods

This study focuses on the role of the alfalfa leafcutting bee (ALCB) on cross-pollination between GE and conventional alfalfa seed varieties. Seed samples were collected in front of conventional alfalfa leafcutting bee domiciles at variable distances from a GE field, and tested for the presence of the transgene. Occasionally referred to as a ‘source and sink’ gene flow trial, this method is a commonly employed to measure gene or pollen flow across a landscape. All of the 2013 and 2014 was pooled and incorporated into a predictive model that can be referenced by seed producers to mitigate the risk of cross-pollination between adjacent seed fields. Ultimately, the objective of this study was to determine how robust current parameters are for mitigating

ALCB-mediated gene flow, and whether current AOSCA buffer recommendations are due for revision considering the increased sensitivity for AP testing.

Field site identification and seed collection. We identified five conventional alfalfa seed fields within Walla Walla County, WA, that were directly adjacent to a transgenic seed field

(Figure 1). In each field, we sampled mature seed from the immediate vicinity (10m or less from the domicile entrance) of regularly spaced domiciles spread throughout the conventional fields

(24 domiciles in total in 2013 and 33 domiciles in 2014; Figures 2 - 7). Mature seed was hand harvested from 8 individual plants at each domicile during mid-August of both years. A leaf tissue sample was collected from each plant in the study and the absence of the GE trait within the parent plant was confirmed using Romer Agrastrips. We sampled seed in front of the bee domiciles to maximize the likelihood that the specific plants we harvested from were pollinated by the alfalfa leafcutting bee, rather than other alternative pollinators such as honey bees or wild unmanaged bee species in the area.

Seedling germination assay for harvested seed. Seed samples were cleaned by hand, then blown and scarified in preparation for a greenhouse germination assay. Methods addressing the specifics of this assay are detailed in chapter 2. Harvested seed samples were pooled by location prior to plating on greenhouse trays. We tested 3 replicates of 2400 seeds for each sample taken over both years. Each 2400-seed replicate was sandwiched between two labelled germination papers saturated with an 80 ppm solution of glyphosate. Prepared replicates were stacked onto 19” x 21” greenhouse trays and fitted with perforated plastic greenhouse bags prior to placement in a water curtain germinator. All seed samples were maintained at a 16:8 light cycle at 20°C for 14 days and received a second 80 ppm glyphosate treatment on day four in the incubator. The seedlings were watered as needed using an antimicrobial distilled water/PPM solution.

In addition to the preparation of experimental seed samples, some seed samples were prepared in which germination papers were saturated with distilled water instead of the glyphosate solution. This treatment allows us to estimate the baseline germination rate of the harvested seed, and adjust the detection of AP according to the proportion of viable seeds from the field. The baseline germination rate was determined for every conventional field that we sampled from over 2013 and 2014. To establish the germination rate, three 100-seed replicates were prepared and germinated concurrently with their respective experimental samples. The 100 seeds selected for each replicate came from a pooled sample of all harvested seed collected within that field and year. At the end of the 14 day germination period, the number of successfully germinated seedlings are calculated as a percentage and averaged over the three replicates.

Experimental seed samples were scored for the presence of the GE trait after 14 days in the water curtain germinator. Each seedling was individually examined for symptoms indicating glyphosate resistance, such as being large in size, possessing an elongated root with visible root hairs, and/or exhibiting relatively large cotyledons. Seedlings that were suspected to be glyphosate resistant were confirmed for presence of the GE trait using Romer Agrastrips.

To determine the role of the ALCB across an agricultural landscape, we fitted the seed data obtained for both years to a negative exponential model in Matlab to determine the relationship between GE trait occurrence in seed and distance from a GE source field. The model can be used to predict the movement of the GE trait over a known distance.

Results

None of the conventional leaf tissue sampled concurrently with seed tested positive for the GE trait. Therefore, all of the seed sampled in the field was incorporated into the seedling germination assay. The baseline germination rates were calculated (Table 1) and were used to calculate the percent AP detected in each experimental domicile.

The percent AP detected in each domicile for 2013 and 2014 can be examined in Figures

2 – 7. These figures clearly demonstrate an inverse relationship between percent AP and distance from a transgenic field. It is also evident that there is a large amount of variation in GE trait detection between years for the conventionally harvested seed. Percent AP detection in

2014 was typically lower than in 2013 (even when the same domiciles were tested between years, as can be seen when comparing the rates of GE trait expression between years in figure 2 versus figure 5 and in figure 4 versus figure 7). These differences become more apparent when the results are fitted to an exponential decay (Figures 8 – 10). While the shape of the curve

66 remains the same, it appears that in the 2014 model, gene flow is more limited as AP detection decreases more quickly than for the 2013 data set. However, it should be noted that while more domiciles were sampled in 2014 than in 2013, they are at less variable distances from a transgenic source. Secondly, the reported R2 value for the 2014 model (R2 = 0.269) indicates this model does not describe the relationship of gene flow over distance as well as the 2013 model (R2 = 0.5433). Even when both 2013 and 2014 data sets are pooled into a single model

(Figure 9), R2 is still too low to confidently predict gene flow from these findings.

Discussion

It is difficult to explain the degree of year-to-year variability in ALCB-mediated gene flow between fields. From our results, it is clear that the percent AP is typically lower for 2014, and the model fit is poor (both alone and when pooled with 2013). Plans for 2015 include revisiting archived seed samples used in the 2014 germination assay to confirm the accuracy of the data collected. No changes were made to the seed sampling procedure, and sampling occurred in the same fields (and many of the same locations) for both years. There are a number of factors that may underlie the variation we saw, such as year-to-year variation in wind speed, temperature, or pesticide use, although we have not yet made any of those comparisons.

Similarly, differences in individual seed producers stocking densities or seeding practices may have influenced ALCB foraging behavior. Additionally, it is possible that an error was made in the preparation of the germination assay, as AP screening of the 2014 seedlings was unusually difficult. There are plans to determine the cause of the discrepancy over the summer of 2015.

Despite these differences, in both years that AOSCA’s determination of 0.1% AP at 274 m is not upheld when applying any of the three fitted models (at 274m, 2013: 0.47% AP; 2014:

0.14% AP, pooled: 0.39% AP). These findings suggest that current isolation standards may need to be increased to ensure that harvested seed will fall under the 0.1% AP threshold in sensitive, conventional alfalfa seed fields.

Unfortunately, we had a difficult time fitting the 2014 seed data to a model. Walla Walla

County, WA, is a NAFA-approved GE GOZ, and has been since July 2011. A typical alfalfa seed stand stays in production for two to four years and occasionally up to six years. Therefore, every year it will become more difficult to locate a transgenic alfalfa seed field surrounded by conventional varieties, because the conventional fields are taken out of production and reseeded with wheat or GE alfalfa produced for seed or forage. The protocol we developed to measure transgene flow hinges on a specific ‘source and sink’ experimental layout. Walla Walla County has been a GE GOZ for four years and our transgenic ‘source’ fields are rapidly outnumbering the conventional ‘sink’ fields. During 2014, we didn’t have the ability to place Binderboards at the variable distances that were tested from in 2013 due to these changes. Chapter 3 concluded that the incidence of AP in bee-provisioned pollen is much higher than expected considering previous estimates of ALCB foraging range. The domiciles from which the pollen provisions were sampled are the same domiciles from which the seed samples were collected in 2013 (Table

1). Part of the reason we have seen high rates of GE trait detection in pollen and not in seed may be due to the rate of pollen deposition between individual alfalfa blossoms. The amount of pollen that is picked up by a pollinator and deposited on a subsequent blossom varies depending upon the specific pollinator species and crop in question. This serves as a partial explanation for why some bees are more efficient than others in providing specific pollination services within agriculture. This concept has been coined as ‘pollinator presentation theory’ and is well established within the literature. For example, Thomson & Goodell 2001 detected differential

68 removal and deposition of pollen between honey bees and Bombus terrestris in almonds and apples. In the case of alfalfa pollination, this type of information is not currently available. It is well established that leafcutting bees are excellent pollinators of alfalfa for seed production.

However, specific data related to the movement of individual pollen grains per visit has not been characterized in this system. If we could better understand the nature of ALCB-facilitated pollen deposition and removal, we might find some limitation in the pollen hoarding or depositing capabilities of this bee. The pattern of pollen transfer in bees is characteristically leptokurtic

(Wilcock and Neiland 2002). This pattern of pollen flow has been characterized many times in the literature (Krauss 2000, Groom 1998, Kunin et al. 1997). When an alfalfa leafcutting bee visits an alfalfa blossom, the pollen collected from that blossom has a higher probability of cross- pollinating the next closest blossom than one that is farther away. Additionally, bees tend to exhibit a ‘patch foraging’ behavior as they move throughout the landscape. The concept of patch foraging suggests that bees forage in individual ‘patches’ of locally abundant floral resources. A bee will continue to forage upon floral resources within a patch until those local resources are depleted. Because flight is energetically costly for bees, foragers tend not to fly further than is necessary to access resources (Pitts-Singer & Bosch 2010). These assumptions imply that the

ALCB would preferentially work in patches that are closest to the nest, reducing the movement of pollen between patches and along greater distances during individual foraging flights.

It is also possible that pollen desiccates during the flight of the leafcutting bee in such a way that the pollen would no longer be viable after exposure to prolonged foraging conditions

(Wilcock and Neiland 2002). This would limit the probability of cross pollination of alfalfa varieties over spatially isolated distances. At present, there is little information available which discusses the viability of pollen grains collected by foragers, although this work is in currently in

69 progress (Brunet 2015 – personal comm.). Some of the things we do know about alfalfa viability is that pollen on the alfalfa blossom can remain viable for about ten days after anthesis (Hanson

1961). Secondly, pollen collected by honey bees and stored within the hive environment can remain viable for up to 9 days (Pankiw and Bolton 1964), suggesting that perhaps pollen would remain viable during a foraging bout, though this hasn’t been tested.

Finally, work done by Cane (2002) and Strickler (1999) suggest that there are physiological limitations in mature pod and seed formation. Not every blossom that gets pollinated matures and develops seed. Seed production by plants require an immense investment of resources, and thus there is a limitation to the number of seeds a single plant can produce in a season. In fact, Cane (2002) and Strickler (1999) both report that on average, only about half of tripped alfalfa blossoms go on to develop seed pods and mature seeds. This suggests that on the occasion that a bee successfully cross-pollinates a conventional blossom with transgenic pollen, there is only a 50% chance that the pollinated blossom will produce seed.

Some publications examining bee-mediated gene flow in alfalfa have identified a border effect, in which the highest detected rates of cross pollination in seed occur along field margins and are at the most risk for the detection of AP in harvested seed (Greene et al. unpub.).

Growers who may be concerned about pollen flow into conventional fields should be able to mitigate the occurrence of AP by harvesting field edges separately from the field interior. Seed from the field margins would then be sold to a separate market with a higher tolerance for GE in the seed. Based upon the decreasing exponential relationship observed between transgenic pollen and distance, this action likely would substantially decrease the percent AP detected in harvested seed.

As can be inferred from figures 2 – 7, seed producers in Touchet do not currently adhere to the recommended isolation buffers in place by AOSCA. While these seed producers acknowledge the possibility of cross-pollination between conventional and transgenic varieties, most of the seed harvested in this region is destined for the domestic market, in which AP tolerances are much higher than they are abroad.

Side-by-side analyses of trait expression in seed and bee –collected pollen are not frequently considered when characterizing gene flow in agricultural systems. While these results indicate that AOSCA isolation buffers may require some revision, they also suggest that that many factors related to plant-pollinator interactions be considered when characterizing gene flow. The combined knowledge of ALCB foraging range and their contribution to cross pollination in seed can be taken together to inform future public policy related to transgenic pollen flow. Other factors that may benefit our understanding of gene flow include the rates of pollen removal and deposition in alfalfa, the rate of pollen desiccation on active foragers, and the patch foraging behavior of the leafcutting bees while moving through an alfalfa field. Working within this system provided us with the opportunity to develop novel procedures for both determining seed and pollen gene flow between alfalfa varieties. The data obtained from this work can be applied to isolation recommendations for all different types of alfalfa varieties.

Perhaps most importantly, these results will be informative for seed producers who wish to grow glyphosate resistant GE seed, or other GE alfalfa varieties which are now entering the market.

References

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Table 4-1. Baseline germination rates for conventional seed samples collected in the field in

2013 and 2014. Field labels correspond to the labels presented in Figures 4-1 – 7.

Year Field Germination rate (%) 2013 F1 91.7% 2013 F2 85.3% 2013 F3 83.3% 2013 F4 83.7% 2013 F5 91.0% 2014 F1 53.7% 2014 F2 66.3% 2014 F4 73.7% 2014 F6 71.3%

Figure 4-1. Map of selected Walla Walla County, WA, alfalfa seed fields in 2013 and 2014.

Fields marked in red indicate a GE alfalfa field, used as a source field to measure gene flow into conventional fields (marked in yellow). Conventional fields are labelled to correspond with the model legend presented in Figure 7. F3 and F5 could not be used in 2014, having been reseeded with wheat and transgenic alfalfa that year, respectively. F6 was incorporated into the study in

2014 to make up for the loss of F3.

Figure 4-2. 2013 GE trait occurrence in harvested conventional seed for fields 1 (left) and 2

(right), with %AP reported at each location from which seed was sampled. The transgenic field is marked in orange.

Figure 4-3. 2013 GE trait occurrence in harvested conventional seed for field 3, with %AP reported at each location from which seed was sampled. The transgenic field is marked in orange.

Figure 4-4. 2013 GE trait occurrence in harvested conventional seed for fields 4 (South) and 5

(North) with %AP reported at each location from which seed was sampled. The location labelled

N.D. had no detection of the GE trait. The transgenic field is marked in orange.

Figure 4-5. 2014 GE trait occurrence in harvested conventional seed for fields 1 (left) and 2

(right), with %AP reported at each location from which seed was sampled. The transgenic field is marked in orange.

Figure 4-6. 2014 GE trait occurrence in harvested conventional seed for field 3, with %AP reported at each location from which seed was sampled. The transgenic field is marked in orange.

Figure 4-7. 2014 GE trait occurrence in harvested conventional seed for fields 4 (South) and 5

(North) with %AP reported at each location from which seed was sampled. The location labelled

N.D. had no detection of the GE trait. The transgenic field is marked in orange.

Figure 4-8. 2013 data fit to an exponential decay model. Each point reflects average percent AP expression at every domicile incorporated into the study. Domiciles were located at variable distances from a known transgenic field, and the shape of each plotted point corresponds to the field from which the seed was sampled (Figure 1).

Figure 4-9. Pooled 2013 and 2014 data fit to an exponential decay model. Each point reflects average percent AP expression at every domicile incorporated into the study. Domiciles were located at variable distances from a known transgenic field, and the shape of each plotted point corresponds to the field from which the seed was sampled (Figure 1).

Figure 4-10. 2014 data fit to an exponential decay model. Each point reflects average percent

AP expression at every domicile incorporated into the study. Domiciles were located at variable distances from a known transgenic field, and the shape of each plotted point corresponds to the field from which the seed was sampled (Figure 1).