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This is to certify that the

thesis entitled
EFFECT OF TRANSGENIC Bt CORN POLLEN AND GLYPHOSATE
RESISTANT CANOLA POLLEN ON THE SURVIVAL OF THE HONEY BEE
(Apis mellifera L.) WORKER LARVAE AND THE USE OF

Bt CORN POLLEN AS A CONTROLLING METHOD FOR THE
GREATER WAX MOTH (Galleria mellonella L.)

presented by

 

 

Anne Valdes Hanley

has been accepted towards fulfillment
of the requirements for

M. S. degree in Entomology

 

 

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6/01 c-JCIRC/DatoDuopes-pJ 5

 

EFFECT OF TRAN SGENIC Bt CORN POLLEN AND GLYPHOSATE RESISTANT
CAN OLA POLLEN ON THE SURVIVAL OF THE HONEY BEE (Apis mellifera L.)
WORKER LARVAE AND TIDE USE OF Bt CORN POLLEN AS A CONTROLLING

METHOD FOR THE GREATER WAX MOTH (Galleria mellonella L.)

By

Anne Valdes Hanley

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements

For the degree of

MASTER OF SCIENCE
Department of Entomology

2002

ABSTRACT

EFFECT OF TRANSGENIC Bt CORN POLLEN AND GLYPHOSATE RESISTANT

CANOLA POLLEN ON THE SURVIVAL OF THE HONEY BEE (Apis mellifera L.)

WORKER LARVAE AND THE USE OF Bt CORN POLLEN AS A CONTROLLING

METHOD FOR THE GREATER WAX MOTH (Galleria mellonella L.)
By
Anne Valdes Hanley

The honey bee, Apis mellifera L. (Hymenoptera: Apidae), a valuable pollinator, is one of
the most important insects in the world’s agriculture and food industry. With the recent
concern about the effects of transgenic crops on non-target insects, the effects of
transgenic crops on the honey bee must be evaluated. The effect of glyphosate resistant
canola pollen and Bt transgenic corn pollen (CrylF and CrylAb) on 4-5 (1 old honey bee
worker larvae was tested in the field. Percent larval mortality, mean pupal weight, blood
protein level of newly emerged adults, and percent adult emergence were determined for
bees fed transgenic or non-transgenic pollen. There were no significant differences in the
above parameters between larvae fed glyphosate resistant canola pollen and non-
transformed canola pollen (P>0.05). There were no differences in the parameters
between larvae fed transgenic corn pollen and non-transformed corn pollen. The use of
Bt transgenic corn pollen as a control of the greater wax moth was tested in the
laboratory. Mortality of larvae fed CrylF corn pollen was significantly different from
larvae fed CrylAb com pollen and non-transformed corn pollen (P<0.05). CrylF corn
pollen appears to be a good control of wax moth larvae in the laboratory. These results

suggest that there is a possible benefit of transgenic corn and canola pollen for bees.

Copyrighted by
ANNE VALDES HANLEY
2002

To my grandfather, Clifford Burgess Godwin, Jr.

ACKNOWLEDGEMENTS

Many people have been instrumental in my success during the past two years at
Michigan State University. I would like to acknowledge those who have supported me as
I earned my Master’s degree. To each of the following people, I extend my thanks.
Thank you to major professor, Dr. Zachary Huang, and to my guidance committee
members, Dr. Walter Pett and Dr. James Hancock, for their encouragement and support
during my time of study at Michigan State. I also thank Dr. Roger Hoopingamer and Dr.
George Ayers for their valuable advice to me in times of need. A warm thanks to Dr.
Edward Grafius, Dr. Fred Stehr, Dr. Deborah McCullough, Dr. Christine Difonzo, Dr.
James Miller, and Ron Priest for their advice and support.

Thank you to my family, especially my parents, David and Mary Hanley and my
siblings, Joe, Jeanne, and Maureen Hanley for their unwavering love and support.
Without them this degree would not have been made possible.

Many thanks to Dr. John Wise, Janice Howard, and Ryan Vanderpoppen for
introducing me to entomology at the Trevor Nichols Research Complex. Danka to Dr.

J oerg Schmidt-Bailey, Dr. Ron Lin, Sandie Michalek, Lauren Clauda, Erik Foster, Emily
Jackson, Priya Kishnamurti, and Ruiwu Wang, all of whom were instrumental in helping
me to achieve my Master’s degree.

I extend my thanks to the Michigan Beekeepers Association for their continued
interest in my research and especially to Ten'y Klein, David and Blanche Barber, and Jim
Conor for their generous help and support.

Thanks to Dan Winslow and Edward Kubiak for allowing me to collect pollen in

their fields of corn. Their kindness and hospitality is most appreciated.

I would like to thank Michael Langenberger and his supervisor, Albert Robertson,
for collecting canola pollen in Saskatoon, Saskatchewan, Canada.

I would like to extend my warm thanks to Christine Vandervoort and Tom
Garavaglia for the assistance they gave me in testing my pollen samples for the pollen
pesticide dose concentration.

A big thank you to the kind support staff in the entomology office:

Heather Lenartson, Angela J emstadt, Ballory J arriel, Linda Gallagher, and Jill Kolp for
their willingness to help graduate students. Also, thanks to Jan Eschbach for her
computer savvy and help in poster and slide making.

My kind thanks goes to Nathan Cottrell for his tips on wax moth rearing
and making graphs, to Breana Simmons for her rousing pep talks and guidance, to Brian
McComack for the lesson in webpage design, to Natalia Botero-Garces and Young Eric
Hoffmann for their lessons in Powerpoint presentations and BSA talks. Thank you to
Terry Davis for the use of the growth chamber on the first floor of the Natural Science
Building. Thanks to Barb Stinnett and Gary Parsons for their help and support in help'ng
me to become a bughouse tour guide and a museum volunteer. A warm thank you to
Moe Nielsen for teaching me to spread moths for my voucher specimens.

To all the rest of the Entomology Department I thank you for your kind support to
me in the past two years. Lastly, I would like to thank the GREEEN program at MSU

whose grant made this research possible.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................. x
INTRODUCTION ............................................................................................................... 1
Research objectives ............................................................................................................. 2
LITERATURE REVIEW .................................................................................................... 3
History of the honey bee ......................................................................................... 3

Honey bee biology .................................................................................................. 4

Honey bees as valuable pollinators ......................................................................... 4

A pest of the honey bee ........................................................................................... 5
Bacillus thuringiensis .............................................................................................. 7
History of transgenic crops ..................................................................................... 8
Canola as a forage crop ........................................................................................... 8

Effect of Bt transgenic pollen on non-target Lepidoptera ....................................... 9
Possible effects of transgenic pollen on honey bee ............................................... 10
Laboratory studies ..................................................................................... 11

Field studreslB

Research objectives ........................................................................................................... 14

CHAPTER ONE: EFFECT OF TRAN SGENIC Bt CORN POLLEN AND
GLYPHOSATE RESISTANT CANOLA POLLEN ON THE SURVIVAL OF HONEY

BEE (Apis mellifera L.) WORKER LARVAE ................................................................. 16
Abstract ............................................................................................................................. 17
Introduction ....................................................................................................................... 18
Materials and methods ...................................................................................................... 21
Pollen collection .................................................................................................... 21
Canola pollen collection ............................................................................ 21

Corn pollen collection ............................................................................... 22

Larvae mapping ..................................................................................................... 22
Experiment one — summer 2000 ........................................................................................ 22
Field study setup .................................................................................................... 22

Pollen treatments ................................................................................................... 23

Larval feeding ....................................................................................................... 23
Experiment two — summer 2001 ....................................................................................... 24
Field study setup .................................................................................................... 24

Pollen preparation ................................................................................................. 24
Pesticide test doses ................................................................................................ 24
Diazinon dose trial one .......................................................................................... 25
Diazinon and chlorfluazuron (IGR) dose trial ....................................................... 25
Experiment three — summer 2002 ..................................................................................... 25
Field study setup .................................................................................................... 25
Treatment preparation ........................................................................................... 26
Larvae mapping and feeding ................................................................................. 26

Larval mortality , pupal weight, blood protein level, and adult emergence .......... 27
Midgut dissection .................................................................................................. 27

vii

Statistical analysis ............................................................................................................. 27

Experiment one — summer 2000 ........................................................................................ 27
Experiment two - summer 2001 ....................................................................................... 28
Experiment three — summer 2002 ..................................................................................... 28
Results ............................................................................................................................... 28
Experiment one — summer 2000 ........................................................................................ 28
Experiment two — summer 2001 ....................................................................................... 29
Experiment three — summer 2002 ..................................................................................... 29
Discussion ......................................................................................................................... 29
Experiment one — summer 2000 ........................................................................................ 31
Experiment two — summer 2001 ....................................................................................... 31
Experiment three — summer 2002 ..................................................................................... 32

CHAPTER TWO: EVALUATION OF Bt CORN POLLEN AS A CONTROLLING
METHOD FOR THE GREATER WAX MOTH (Galleria mellonella L.)... ..4.............6

Abstract ............................................................................................................................. 47
Introduction ....................................................................................................................... 48
Wax moth biology ............................................................................................................. 49
Control methods ................................................................................................................ 49
Materials and methods ...................................................................................................... 49
Statistical analysis ............................................................................................................. 50
Results .............................................................................................................................. 50
Discussion .............................................................................................. 50
LITERATURE CITED ............................................................................... 53

APPENDIX 1: Record of deposition of voucher specimens...................... ................59

viii

LIST OF TABLES

Table 1: Voucher specimen data ...................................................................................... 61

ix

LIST OF FIGURES

Figure 1. Mean pupal weights of bees fed pollen ..........................................................
Figure 2. Percent larval mortality of bees fed canola pollen ..........................................
Figure 3. Percent larval mortality of bees in Diazinon and chlorfluazuron dose trial
Figure 4. Percent larval mortality of Diazinon dose response trial .................................
Figure 5. Percent larval mortality of bees fed canola pollen in trial one .......................
Figure 6. Percent larval mortality of bees fed canola pollen in trial two .......................
Figure 7. Percent larval mortality of bees fed corn pollen in trial one ..........................
Figure 8. Percent larval mortality of bees fed corn pollen in trial two ..........................
Figure 9. Mean pupal weight of bees fed canola pollen
Figure 10. Mean pupal weights of bees fed corn pollen
Figure 11. Mean blood protein level of bees fed canola pollen
Figure 12. Mean blood protein level of bees fed corn pollen ..
Figure 13. Adult emergence of bees fed canola pollen in trial one and two ..................

Figure 14. Adult emergence of bees fed corn pollen in trial one and two ......................

Figure 15. Wax moth larvae mortality measurements from four trials of a laboratory

study ................................................................................................

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.36

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.42

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..53

INTRODUCTION

The honey bee, Apis Mellifera L. (Hymenoptera: Apidae), is one of the most
important insects in the world’s agriculture and food industry. Many of the world’s crops
including tree fruits, blueberries, and cucurbit vegetables require pollination for crop
production. For nearly all of these crops, successful pollination depends either wholly or
predominantly on the honey bee (Hoopingamer et al. 1992). It is clear that the world’s
agricultural industry depends on the honey bee for its very survival. It is essential to
maintain an adequate pollinating force of honey bees for this important industry.

The use of transgenic crops in the United States is sizeable, making up 68% of
soybean acreage, 26% of the corn acreage, and 69% of the cotton acreage (Commoner
2002). Published experiments have mainly tested the effects of purified transgene
products on honey bees. There is a potential for pleiotropic effects due to the insertion of
a foreign gene into the plants, which could produce other gene products besides the
inserted gene. These products could also be toxic to bees or they could affect the
longevity of foraging bees or decrease brood survival. Because of these reasons, it is
important to determine the impacts of transgene plants by testing the effects of actual
products like pollen and nectar from transgenic plants on the honey bee and other
beneficial insects.

The greater wax moth, Galleria mellonella L., is a pest of weak honey bee
colonies and stored honey combs. Current controls of the wax moth are chemical
fumigants that can only be used on stored combs. Because CrylAb and CrylF corns are

designed for controlling Lepidopteran larvae, it is possible that they can be used as a

controlling method for wax moths in the field. It is with these two areas in mind that we
formed the objectives of our study.
Research Objectives

The main research objectives of this study were 1) To determine the effect of
transgenic glyphosate resistant canola pollen and transgenic Bt corn pollen on the
survival of honey bee, Apis mellifera L., (Hymenoptera: Apidae) worker larvae and 2) To
evaluate transgenic Bt corn pollen as a potential controlling method for the greater wax

worm, Galleria mellonella L. (Lepidoptera: Pyralidae), a pest of the honey bee.

LITERATURE REVIEW
History of the honey bee

Mankind has valued honey since times of old, as evidenced in the book of Isaiah,
7:15: “He shall be living on curds and honey by the time he learns to reject the bad and
choose the good” (New American Bible 1970). Also in Genesis 43:11, it states: “Their
father Israel told them: ‘If it must be so, then do this: Put some of the land’s best products
in your baggage and take them down to the man as gifts: some balm and honey, gum and
resin, and pistachios and almonds’” (New American Bible 1970). In the Old Testament
milk and honey usually mean excellent and abundant food (Orchard 1951).

Social bees have produced and stored honey for the last 10 to 20 million years.
The earliest records of man’s exploitation of honey are evidenced by rock paintings and
by the findings of anthropological studies of both contemporary and pre-historic tribes,
which hunted for honey in the wild (Crane 1983).
Honey bee biology

Honey bees are eusocial insects, meaning that they have a reproductive division
of labor; multiple generations exist at the same time; and they care for their young
cooperatively (Winston 1987). Three castes exist in the hive, a queen, workers and
drones. All castes have four developmental stages: egg, larva, pupa, and adult (Winston
1987). The queen lays two types of eggs, fertilized and unfertilized. The unfertilized
eggs develop into drones, while the fertilized eggs can develop into workers or queens,
depending on what they are fed during development. The queen is fed royal jelly

throughout her immature stage and for her entire life after emergence. Worker larvae are

fed royal jelly for the first three days of development and then are fed a mixture of
worker jelly and pollen.

Each caste has different tasks to perform within the colony. The drones’ only role
in the hive is to mate with the queen in the spring or summer of the year. Those that do
succeed in mating with the queen die soon after. The remaining drones are fed nectar and
pollen by worker bees. In the fall, the drones are banished from the hive by workers, so
that they will not use up the limited winter food reserves. The sole task of the honey bee
queen is to lay eggs to populate the hive. In the first two weeks after she emerges in the
summer, she goes on her first mating flight during which she usually mates with more
than one drone. There is a record of a queen mating with 17 different drones on one
mating flight (Winston 1987). After she is mated, she returns to the hive and begins to
lay eggs. Queens have been known to lay up to 2,000 eggs a day in the summer. She is
the only fertile female in the hive and usually lives for three to five years (Winston 1987).

Worker bees, which make up the majority of the hive’s population, are nearly
sterile females. Young workers have many tasks to perform in their lifetimes, which are
completed in an order that is somewhat dependent on their age. Upon emergence, they
begin cell cleaning, then they perform the tasks of capping brood, tending brood,
attending the queen, receiving nectar, cleaning debris, packing pollen, comb building,
ventilating the hive, guarding the hive, and finally the foraging flight (Winston 1987).
There is some overlap in activities.

Honey bees as valuable pollinators
Apis mellifera L. is the most popular species of honey bees. It is highly valued

today for its products such as honey, wax, pollen, and royal jelly, but more importantly

for its pollination of important fruits and vegetables. Honey bees have coevolved with
angiosperms over millions of years (Crane 1989). Proof of this intricate relationship are
the pollen baskets on their tibiae called corbiculae and the plumose hairs that cover bees’
bodies, both of which help the bee collect pollen from angiosperms. On the plants’ side,
angiosperms have evolved colors, scents, and excess nectar and pollen to attract the bee
to pollinate them (Caron 1999). Pollination is the transfer of pollen from the anther to
the stigma. For most plants, pollination must be accomplished before fertilization and the
eventual seed production can take place (Vansell and Griggs 1952). It has been estimated
that approximately one third of people’s diets in the United States comes directly or
indirectly from bee pollinated crops (Hoopingamer 1992). In 1998, it was estimated that
there were 2,500,000 colonies rented for pollination purposes in the United States (Morse
and Calderone 2000). In 2000, the value of increased crop yield and quality gained
through pollination by honey bees was $14.6 billion (Morse and Calderone 2000).

Pollen is the natural source of protein for bees, whereas nectar collected by bees
contains rather low concentrations of amino acids (Baker and Baker 1977). The pollen
consumption of newly emerged workers increases for the first week. However, when
workers reach the nursing stage, their pollen consumption decreases (Crailsheim 1990).
Large amounts of protein are required during larval development. One larva needs about
50mg royal jelly to reach a weight of 35mg (Crailsheim 1990). The protein content of
the honey bee’s hemolymph at emergence depends on the availability of food outside the
hive during the nursing season (Kunert and Crailsheim 1987).

The nutritional value of pollen for bees is defined by the content of ten essential

amino acids: arginine, histidine, lysine, tryptophan, phenylalanine, methionine, threonine,

leucine, isoleucine, and valine (Groot 1953). Honey bees are able to distinguish between
different kinds of pollen. Corn pollen, for example, is not as attractive to bees but they
will forage for it because of its great abundance and protein content (Crane 1984).
Canola pollen is an excellent bee forage source that has a higher protein content than corn
poHen.

A pest of the honey bee

Like other animals, honey bees are plagued by many diseases and pests. One of
these pests is the greater wax moth, Galleria mellonella L., a pest of stored combs and
weak colonies. Larvae of the greater wax moth cause considerable damage to beeswax
combs left unattended by bees. Beeswax combs in weak or dead colonies and those
placed in storage are subject to attack.

Treating infested comb with extreme temperatures is one of the current methods
of wax moth control (Williams 1978). Other methods of controlling the wax moth used
in the past are chemicals such as paradichlorobenzene, ethylene dibromide, methyl
bromide, and ethylene oxide to fumi gate stored equipment (Williams 1978). These
chemical fumigants have several disadvantages. First, they may be toxic to humans and
animals. Several applications are often required during an average four month storage
period. After fumigation, a closed system in the storage area is needed for fumigation
and this system is difficult and expensive to maintain (Cantwell 1981). These chemicals
are no longer approved for use in the hive.

An alternative method to chemical control of wax moth, Thuricide 90TS®, a
Bacillus thuringiensis preparation that was incorporated into beeswax foundation, was

studied in the early 1960’s (Cantwell 1981). However, it did not prove to be very

successful. In the 1980’s, a Bacillus tlzuringiensis bio-pesticide, Certan®, was developed
to treat honey bee colonies in the field (Cantwell 1981). This new bio-pesticide was
available for sale by bee supply companies but was not profitable so it is no longer
manufactured (Caron 1999). At present the only available methods for wax moth control
are extreme temperatures. This method is both time consuming and costly as it requires a
large freezer or wax melter to be used efficiently. New natural controls that could be
used in the field would be very beneficial for the beekeeping industry.
Bacillus thuringiensis

Since the first use of protection of plants against pests by transfer and expression
of foreign genes coding for entomopathogenic proteins, transgenic crops have been the
object of much concern and study (J ouanin et al. 1998). Genetically modified plants are
plants that have genes inserted into them for several reasons including: herbicide
resistance, insect resistance, and growth enhancement. One of the most popular genes
that are currently being inserted into a wide variety of plants is a gene that expresses the
Bacillus thuringiensis toxin. Bacillus thuringiensis (Bt) is a gram positive, facultative,
anaerobic bacterium that forms characteristic crystals that are made of crystal (Cry)
proteins, which are toxic to a wide variety of insects (Sonenshein 1993). Bt has been
used in agriculture since the 1920’s to control insect pests in the orders Lepidoptera,
Coleoptera, and Diptera (Beegle and Yamamoto 1992). It is very specific in its toxicity,
which makes it different from synthetic, broad-spectrum insecticides such as carbamates
and organophosphates. Bt is harvested from soil, grain bins, and also from insects which

have died from Bt infection (Harwood 1994).

Bt strains produce two types of toxins. The main types are Cry (crystal) toxins,
encoded by different Cry genes, which is how different types of Bt are classified. The
second types are the Cyt (cytolytic) toxins, which can augment the toxicity of the Cry
toxins, adding to their effectiveness in insect control (Li 1991). These toxins can be
expressed in plants’ leaves and pollen.

History of transgenic crops

There were over 28 million ha of transgenic commercial crops grown worldwide
in 1998 according to James (1998). These crops include com, cotton, soybeans, rice,
canola, tomatoes, and potatoes (James 1998). The categories of transgenic crops are as
follows: glyphosate resistant, insect resistant, virus resistant, both insect and glyphosate
resistant, and quality traits (James 1998). The use of worldwide (excluding China)
glyphosate resistant crops increased by 6,232,159 ha from 1996—1997 (James 1998). The
use of insect resistant crops worldwide (excluding China) also saw a significant increase
of 2,954,205 ha from 1996—1997 (James 1998). One of the most used genes for insect
resistance is from Bt. The major pest controlled by Bt corn is the European corn borer,
Ostrinia nubilalis L (Anon. 2000). Grower satisfaction inl996 with Bt corn yield caused
an increase from 1 percent of the national acreage to 9 percent of the 32 million ha of
corn in 1997 in the United States (James 1998).

Canola as a forage crop

Canola (Brassica napus) is a plant that originated from rapeseed. Rapseed is not

considered healthy for human consumption because of high levels of licosenic and erucic

acids which are not essential to human growth. Canadian plant breeders produced canola

by genetically altering rapeseed by reducing glucosinolates and licosenic and erucic

acids. Overtime canola has become a worldwide term not just a Canadian term. Today
annual worldwide production of canola is 7.5 million tons on 1,618,743 ha (Oplinger
1989). Canola is an excellent bee forage crop and it is grown mainly in northern
climates. Canada accounts for 15% of the world canola production, while the United
States accounts for only 1% of canola production. The major states for canola production
in the United States are North Dakota and Minnesota (Oplinger 1989). Recently farmers
have begun to use glyphosate resistant or Roundup Ready Canola seeds instead of non-
transformed canola seeds.

A study was done to determine if honey bees show a preference for genetically
modified transgenic oilseed rape or non-transgenic oilseed rape. They found that if
colonies are closer to a non-transgenic oilseed rape field than to a transgenic oilseed rape
field, the bees will bring in a minimal amount of transgenic pollen as compared to non-
transgenic pollen (Osborne et al. 2001).

In 1996, more than 121,410 ha of glyphosate resistant or Roundup Ready canola
was grown in Canada (James 1998). The glyphosate resistant genes had no effect on
yield but improved weed control which increased yield indirectly. The average yield of
glyphosate resistant canola in Canada in 1996 was 9% higher than the yield of non-
herbicide tolerant canola treated with herbicides (James 1998).

Effect of Bt-transgenic pollen on non-target Lepidoptera

A laboratory assay found that Monarch butterfly larvae fed CrylAb (Bt 11)
transgenic pollen had a decreased survival rate compared to those larvae fed non-
transformed corn pollen (Losey et al. 1999). This research caused concern about the

impact of transgenic plants as well as criticism of the methods used in the study. In

response to the Losey study, five field studies were conducted in Iowa, Maryland, New
York, and Ontario to determine the effect of exposure of monarch larvae in field
conditions. The studies found that larvae did not suffer any acute effects under natural
levels of Btll and Mon810 corn pollen (Sears et a1 2001). In another study, two
varieties, Pioneer variety 34RO7, Monsanto event 810, both of which express the
CrylAb gene in their pollen were tested on black swallowtail larvae. Both field and
laboratory studies in Illinois found no negative effects on the larvae by the corn pollen
(Wraight et al 2000).
Possible effects of transgenic crops on the honey bee

With the recent concern about the effects of transgenic crops on non-target
insects, the effects of transgenic crops on the honey bee must be evaluated in order to
safeguard the future of the honey bee. There are two ways that transgenic plants can
affect the honey bee: pollen collected by bees might prove to be toxic to them or
pleiotropic effects could make the transgenic plant less attractive to the honey bee by
producing less nectar (Burgess et al. 1996). Recently an article published in Harper’s
Magazine vigorously challenged current DNA theories. The results of the human
genome project were not as expected, finding the number of human genes amounting to a
mere 30,000, not the 100,000 genes the project scientists had predicted (Commoner
2002). A mustard-like weed has 26,000 genes, very close to the number found in
humans. The central dogma of the project was that the molecular structure or DNA is the
exclusive agent of inheritance in all living things (Commoner 2002). The results of the
genome project cannot explain the difference between humans and other organisms

having comparable numbers of genes. A possible reason for the differences is alternative

10

splicing. This is where the gene’s original nucleotide sequence is split into fragments
that are recombined to encode a multiplicity of proteins. These proteins are different in
their amino acid sequence from one another and from what the gene encoded for
originally. Thus, alternative splicing can be said to generate new genetic information
(Commoner 2002).

The genetic engineering industry in the United States is guided under the same
theory as the human genome project. They state that the transfer of a bacterial gene into
a corn plant is expected to be very predictable. In a genetically engineered transgenic
plant, the transplanted bacterial gene must interact with the plant whose system in most
cases is entirely different from its own. This difference often leads to experimental
failures that occur before a transgenic organism is produced and also to unexpected
genetic changes that occur after the gene has been transferred (Commoner 2002). The
possibility of these changes occurring in transgenic crops must be studied to ensure the
safety of the honey bee and other pollinators that may be affected by the pleiotropic
effects.

Laboratory Studies

Several studies examined the effects of transgenic products (proteins) on honey
bees. Belzunces et al. (1994) fed honey bees doses of Soybean trypsin inhibitor (SBTI),
Bowman-Birk soybean trypsin inhibitor (BBI), and orzacystatin mixed with sugar syrup.
they found that trypsin activity was reduced after 3.5 days of feeding the 0.1 and 1 mg/g
dose of BBI (Belzunces 1994). Honey bee larvae, ages 1-3 days old, were fed a purified

CrylAc toxin as a rate of 20 rig/ml. Feeding on the toxin showed no significant effect on

11

the survival of these larvae. The rate of protein that they were fed was much higher than
that actually found in the field (Sims 1995).

Picard-Nizou et a1 (1995) tested bee foraging activity on transgenic oilseed rape
plants that had a bean chitinase gene inserted which was under the control of CaMV 35S
promoter. These studies found that the transformation event did not appear to have an
effect on foragers’ choices .

The toxicity of cowpea trypsin inhibitor (CpTI), chitinase and B-1,3 proteins to
honey bees were tested by feeding or injecting the proteins to adult worker bees. No
toxic or behavioral effects were found between treatment and control bees except for
CpTI which had a significant effect on adult bee learning behavior (Picard-Nizou et al.
1997).

The toxicity of two endopeptidase inhibitors, bovine pancreatic trypsin inhibitor,
and soybean trypsin inhibitor was tested on honey bees. The study found that the two
inhibitors fed in 15% (weightzvolume) sugar solution killed honey bees within 8-16
days (Malone et al. 1995). The next step after this study was to determine whether or not
the endopeptidase inhibitors were present in the pollen and nectar of the transgenic plants
in which they were inserted.

Potato proteinase inhibitors, POT-1 and POT—2 were fed to newly emerged adult
bees in either sugar syrup or pollen food. Honey bees fed POT-1 or POT-2 in pollen or
syrup had significantly reduced lifespan. This adverse effect was stronger when applied
through pollen than when applied though the sugar syrup treatment (Malone et al. 1998).

. Newly emerged bees were fed Crlea delta-endotoxin, Bt biopesticide

preparations, Dipel 2A® and Foray 48B®, and Kunitz soybean trypsin inhibitor

12

(SBTI) in a pollen based food. The study showed that adults are unlikely to be harmed
by transgenic plants expressing Crlea or SBTI or by Bt biopesticides that are used as
recommended (Malone et al. 1999). In a similar study, newly emerged bees were fed
either Crlea toxin or aprotinin proteinase inhibitor in pollen. Bees fed Crlea did not
differ significantly from control bees in their timing of first flight. However, bees fed
aprotinin began to fly and also died three days sooner than bees fed Crlea or control
pollen (Malone et al. 2000).

Field Studies

Experiments using purified gene products mixed into bee food have the potential
to provide useful information on the impacts of transgenic plants prior to release.
However, because of possible alternative splicing, proteins other than the transgene
product might be changed. Because of this, research must continue on testing actual
plant parts/products (e.g. nectar, pollen) from transgenic plants that are already on the
market to determine their possible effects on the honey bee.

Only a few field studies using transgenic pollen have been conducted. In
Germany where honey bee colonies were placed inside gauze tents with CrylAb
expressing Bt corn plants showed no effects of Bt pollen on bee survival, foraging
frequency, behavior, or brood development during pollen tassling. Brood development
was monitored for 30 days after pollen shed and no significant effects were shown (Schur
et al. 1999 cited by Malone et al 2000).

In another field conducted study, colonies were fed a sugar syrup solution of two

concentrations of the coleopteran toxin, CryIHB at a rate of .066% or .332% over a two-

13

month period. No effect on larvae survival or pupal dry weight was shown (Arpaia
1996).

A field trial conducted in Saskatoon, Saskatchewan used a method of allowing
bees to forage freely in a Roundup Ready® canola field and in a regular canola field.
Bees foraging for two pollen types did not differ significantly in larval survival, pupal
weight, and adult survival (Huang unpublished). Further studies of this nature should be
performed in order to test the effect of transgenic pollen on honey bees.

Honey bees play an important role in agriculture as valuable pollinators of fruits
and vegetables. The latest estimate of increased yield and quality due to honey bee
pollination is valued at $14.6 billion a year (Morse and Calderone 2001). Due to the
importance of the honey bee in agriculture, toxicity of new pesticides on bees is assessed
before registration. The genes inserted into transgenic crops that are designed to produce
proteins to kill insect pests, must be tested for possible toxic effect to honey bees. The
majority of published studies have dealt with feeding trans-gene protein directly to honey
bees. Due to possible alternative splicing of the genes once they are inserted into the
corn or canola plants, more realistic tests must be performed using actual pollen from
transgenic plants containing the protein. This field study used pollen collected from corn
and canola plants to feed larvae directly. The method is a better test of whether
transgenic canola and Bt corn pollen have any negative effects on honey bee larvae.

Another aspect of this study tested pollen produced from transgenic Bt corn plants
as a possible control for the greater wax moth, Galleria mellonella L. Current controls of

the wax moth are chemical fumigants that can only be applied to stored combs.

14

Transgenic Bt corn pollen is a possible control method that could be applied in the field
to existing colonies.

Research Objectives

The main research objectives of this study were 1) To determine the effect of transgenic
glyphosate resistant canola pollen and transgenic Bt corn pollen on the survival of honey
bee, Apis mellifera L., (Hymenoptera: Apidae) worker larvae and 2) To evaluate
transgenic Bt corn pollen as a potential controlling method for the greater wax worm,

Galleria mellonella L. (Lepidoptera: Pyralidae), a pest of the honey bee.

15

CHAPTER ONE

EFFECT OF TRANSGENIC GLYPHOSATE RESISTANT CANOLA POLLEN

AND Bt CORN POLLEN ON THE SURVIVAL OF WORKER HONEY BEE

LARVAE

l6

ABSTRACT

The honey bee, Apis mellifera L. (Hymenoptera: Apidae), a valuable pollinator, is one of
the most important insects in the world’s agriculture and food industry. There were close
to 28,329,000 ha of transgenic commercial crops grown worldwide in 1998, these crops
include com, cotton, soybeans, rice, canola, tomato, and potato (James 1998). With the
recent concern about the effects of transgenic crops on non-target insects, the effects of
transgenic cr0ps on the honey bee must be evaluated to safeguard the future of the honey
bee and crops dependent on bees. Transgenic crops could produce pollen toxic to

adult bees or larvae or the expression of the foreign gene could result in pleiotropic
effects in transgenic plants that would make them less attractive or nutritious to bees.
The of feeding glyphosate resistant canola pollen and Bt transgenic corn pollen (CrylF
and CrylAb) to 5-6 d old honey bee worker larvae was determined. A positive control
of a diazinon pesticide dose of 888ug/larvae gave a ~50% mortality rate. Percent larval
mortality, mean pupal weight, blood protein level of newly emerged adults, and percent
adult emergence were determined. There were no significant differences in the above
parameters between larvae fed glyphosate resistant canola pollen and non-transformed
canola pollen (P>0.05). There were no differences in the parameters between larvae fed
transgenic corn pollen and non-transformed corn pollen. There were no significant
differences between the parameters tested on larvae fed glyphosate resistant canola pollen
and non-transformed canola pollen (P>0.05). In conclusion, glyphosate resistant canola
pollen and Bt transgenic corn pollen (CrylF and CrylAb) do not appear to have any
significant effects on percent larval mortality, mean pupal weight, blood protein level of

newly emerged adults, and percent adult emergence of larvae fed at age 4-5 (1 old.

17

INTRODUCTION

The honey bee, Apis mellifera L. (Hymenoptera: Apidae), is one of the most
important insects in the world’s agriculture and food industry. Many of the world’s crops
including tree fruits, blueberries, and cucurbit vegetables require pollination for crop
production. For nearly all of these crops, successful pollination depends either wholly or
predominantly on the honey bee (Hoopingamer 1992). It is clear that the world’s
agricultural industry depends on the honey bee for its very survival. It is essential to
maintain an adequate pollinating force of honey bees for this important industry.

There were over 28 million ha of transgenic commercial crops grown worldwide
in 1998 (James 1998). These crops include com, cotton, soybeans, rice, canola,
tomatoes, and potatoes. The categories of transgenic crops are as follows: glyphosate
resistant, insect resistant, virus resistant, both insect and glyphosate resistant, and quality
traits (James 1998). One of the most used genes for insect resistance is from Bacillus
thuringiensis. The major pest controlled by Bt corn is the European corn borer, Ostrinia
nubilalis L (Anon. 2000). Grower satisfaction inl996 with Bt corn yield caused an
increase from 1 percent of the national acreage to 9 percent of the 32 million ha of corn in
1997 in the United States (James 1998).

Canola (Brassica napus) is a plant that originated from rapeseed. Canadian plant
breeders produced canola by reducing glucosinolates and licosenic and erucic acids in
rapeseed (which are not essential to human growth). Today annual worldwide production
of canola is 7.5 million tons on 1,618,743 ha. Canola is an excellent bee forage crop and

it is grown mainly in northern climates. Canada accounts for 15% of the world canola

l8

production, while the United States accounts for only 1% of canola production. The
major states for canola production in the United States are North Dakota and Minnesota
(Oplinger 1989). Recently farmers have begun to use glyphosate resistant or Roundup
Ready® canola seeds instead of non-transformed canola seeds.

With the recent increase of concern about the effects of transgenic crops on non-
target insects, the effects of transgenic crops on the honey bee must be evaluated in order
to safeguard the future of the honey bee. Transgenic crops could produce pollen toxic to
adult bees or larvae or the expression of the foreign gene could result in pleiotropic
effects in transgenic plants that would make them less attractive or nutritious to bees.
(Malone et al. 1995, Burgess et al.1996).

Several studies examined the effects of transgenic products (proteins) on honey
bees. Belzunces et al. (1994) conducted a study where bees were fed doses of Soybean
trypsin inhibitor (SBTI), Bowman-Birk soybean trypsin in hibitor, and orzacystatin
mixed with sugar syrup and showed no negative effects. Honey bee larvae, ages 1-3 days
old, were fed a purified CrylAc toxin as a rate of 20 ug/ml. Feeding on the toxin showed
no significant effect on the survival of these larvae. The rate of protein that they were fed
was much higher than that actually found in the field (Sims 1995).

Bee foraging activity were observed on transgenic oilseed rape plants that had a
bean chitinase gene inserted which was under the control of CaMV 358 promoter. These
studies found that the transformation event did not appear to have an effect on foragers’
choices (Picard-Nizou et al. 1995).

Toxicity of cowpea trypsin inhibitor (CpTI), chitinase and B—1,3 proteins on

honrey bees was tested by feeding or injecting the proteins to adult worker bees. No

19

toxic or behavioral effects were found between treatment and control bees except for
CpTI which had a significant effect on adult bee learning behavior (Picard-Nizou et al.
1997). The toxicity of two endopeptidase inhibitors, bovine pancreatic trypsin inhibitor,
and soybean trypsin inhibitor was tested on honey bees. The study found that he two
inhibitors fed in 15% (weightzvolume) sugar solution killed honey bees within 8-16
days (Malone et al. 1995). Potato proteinase inhibitors, POT-1 and POT-2 were fed
to newly emerged adult bees in either sugar syrup or pollen food. Honey bees fed POT-1
or POT-2 in pollen or syrup had significantly reduced lifespan. This adverse effect was
stronger applied through pollen than if applied though the sugar syrup treatment (Malone
et al. 1998).

Newly emerged bees were fed Crlea delta-endotoxin, Bt biopesticide
preparations, Dipel 2A® and Foray 4SB®, and Kunitz soybean trypsin inhibitor
(SBTI) in a pollen based food. The study showed that adults are unlikely to be harmed
by transgenic plants expressing Crlea or SBTI or by Bt biopesticides that are used as
recommended (Malone et al. 1999). In a similar study, newly emerged bees were fed
either Crlea toxin or aprotinin proteinase inhibitor in pollen. Bees fed Crlea did not
differ significantly from control bees in their timing of first flight. However, bees fed
aprotinin began to fly and also died three days sooner than bees fed Crlea or control
pollen (Malone et al. 2000).

Colonies were fed a sugar syrup solution of two concentrations of the coleopteran
toxin, CryIHB at a rate of .066% or 332% over a two-month period. No effect on larvae
survival or pupal dry weight was shown (Arpaia, 1996). Effects of transgenic herbicide

resistant oilseed rape on honey bees were tested in a greenhouse setting. No significant

20

differences were found in measurements of worker bee mortality, foraging activity and
preferences, colony population, brood area, food stores, or diseases (Chaline et al. 1999).

In Germany, honey bee colonies were placed inside gauze tents with CrylAb
expressing Bt corn plants showed no effects of Bt pollen on bee survival, foraging
frequency, behavior, or brood development during pollen tasslin g. Brood development
was monitored for 30 days after pollen shed and no significant effects were shown (Schur
et al. cited by Malone et al.2000).

Honey bees play an important role in agriculture as valuable pollinators of fruits
and vegetables. The latest estimate of increased yield and quality due to honey bee
pollination is $14.6 billion a year (Morse and Calderone 2001). Due to the importance of
the honey bee in agriculture, toxicity of new pesticides on bees is assessed before
registration. The genes inserted into transgenic crops that are designed to produce
proteins to kill insect pests, must be tested for possible toxic effect to honey bees. The
majority of experiments have dealt with feeding transgene products (proteins) directly to
honey bees. Due to possible splicing of the genes once they are inserted into the corn or
canola plants, more realistic tests must be performed using actual pollen from transgenic
plants containing the protein.

Considering the importance of the honey bee in agriculture and the possible
negative effects of transgenic plants on the honey bee, our objectives were to determine
the effect of glyphosate resistant canola pollen and transgenic Bt corn pollen on the
survival of honey bee worker larvae.

MATERIALS AND METHODS

Canola pollen collection

21

The canola pollen was collected in Saskatchewan, Alberta, Canada. Four hives
were placed directly in Roundup ready and non-transgenic canola fields with pollen traps
on them to collect the pollen. The traps were checked daily for a two week period during
the canola bloom. In total, 600 grams of regular canola pollen and 450 grams of
Roundup Ready® canola pollen were collected.

Corn pollen collection

Corn pollen was collected during the week of corn tassling in late July or early
August, depending on the variety of corn in 2000 and 2001. Lawson® waterproof bags
were placed over the tassles and folded and then paper-clipped in place to ensure that the
pollen would not leak out. Pollen bags were left in place for 24 hours and then collected.
After the bags were collected the pollen was sifted using size 1, 2, and 5 Sifters to remove
any insects, tassles, and other debris. Once sorted the pollen was placed in a plastic
container, labeled and stored in the freezer at —20° C. One corn plant yielded a very small
amount of pollen so ~3000 plants were bagged over the course of the tassling season.
Larvae identification mapping

The larvae were mapped on day 4-6 of the larval stage with a transparency that
was pinned to the frame and a permanent marker was used to circle the larvae that were
to be used in the experiment. After the larvae were capped, on day 9, the tracing was
placed over them again and percent mortality was measured. Former occupants of empty
cells were assumed to be dead since workers remove dead larvae.

Experiment one - summer 2000

Field study setup

22

Miniature bee hives were established at the Michigan State University Apicultural
Building on College Road in East Lansing, MI. The hives were a fourth of the size of a
medium bee super (25 x 22.5 x 22 cm). The four frame Pettite hives were filled with ~
3,000 bees using a cup measuring scale system in the July of 2000. Newly mated
queens were placed in the hives. A pollen trap was put on the front of each hive to
prevent returning foragers from bringing outside pollen into the hive. The queen was
placed under a wire cage and allowed to lay eggs and then the cage and queen were
moved to a new part of comb.

Pollen treatments

The pollen treatments for this experiment were as follows: Untreated corn pollen,
corn pollen containing CrylAb (Event Br 11), com pollen containing CrylF (Event
TC1507 purchased from Mycogen- Dow Agrisciences Co.), and diazinon treated corn
pollen. The Diazinon- SOOAG dose used was 1.5 q to the acre, which is the dose a
farmer would spray on his corn field. This application served as a positive control to
ensure that the methodology of the experiment was valid.

Larval feeding

Approximately 2 g of pollen was placed in each hive directly on the comb around
the newly laid brood. It was then sprayed with a 50% (weight: volume ) sugar syrup
solution to make it stick to the frame. A measurement was taken of pupal weight.
Experiment two - summer 2001
Field study setup

Eight four frame beehives (nuclei) were standardized using a package bee funnel

method by which each package received approximately (6,000 bees) and a queen. Bees

23

were weighed using a cup measuring scale system as in 2000. The bees and queen were
placed in each hive after the packages had been in a dark quiet garage for one evening so
that they could settle’down. A pollen trap was placed on the front of each hive. The trap
had three layers of hardware wire cloth (5 sq. per 2.54 cm.) that the foragers had to pass
through to enter and exit the hive. The goal of this trap was to prevent external pollen
from being brought into the hive. A drawer was placed under the trap to catch any pollen
that fell off the bees’ hind legs. Queens were hived and kept in a plastic cage for three
days in order for the hive to become accustomed to their pheromone. After three days
she was released into a wire cage that was approximately 7.6 cm by 12.7 cm in size. The
wire cage restricted the queen’s egg laying to specific areas of the frame unit and the
amount of brood in the hive. After approximately one week the queens would begin to
lay eggs in the cages. After she had laid the entire area of the cage, the queen was moved
to a second area in the hive and after laying there was moved to a third cage, until the
available comb was all in use.
Pollen preparation

Canola pollen was mixed with a 75% (w/v) sugar syrup solution in a ration of 3:1.
Pollen patties were placed in petri dishes, labeled, weighed, and then placed in the hives.
Pesticide test doses

A positive control was needed to prove that the method of the experiment was
sound. Several pilot trials were run to try to determine which dose should be used in the
final experiment.

Diazinon dose trial one

24

The doses used in this trial were fed to each hive mixed with 100 g of bee
collected pollen. Two hives were fed each dose, with ten hives in total for the entire
experiement. The experiement was replicated once. The diazinon doses were as
follows: 0 dose, 0.39x10’6g, 1.95x10'6 g, 9.6x 10'6 g, and 48.25x10'6 g per 100 g bee
collected pollen.

Diazinon and chlorfluazuron (IGR) dose trial

Increased Diazinon doses and a dose of a new chemical called chlorfluazuron
were tested. The treatment doses of diazinon which were fed to three hives each were as
follows: 0 dose, 6 mg diazinon, 6.5 mg diazinon, 7mg diazinon per 100 g bee collected
pollen. The chlorfluazuron dose was 1.56 mg per 100 g bee collected pollen. This gave a
total of fifteen hives for the entire experiment.

Experiment three - summer 2002
Field study setup
Honey bees

In April of 2002, five three-pound packages were established at the Michigan
State University Apicultural Building on College Road in East Lansing, Michigan. Each
colony was medicated with Fumidil, Terramyacin, Apistan, and Cumophos strips to
protect it against Nosema, American Foul Brood, and Varroa mites respectively. They
were fed pollen patties, which were composed of a 3:1 mixture of mixed bee collected
pollen and 50% sugar syrup solution. The pollen mixture was wrapped in wax paper and
then given to the bees.

Treatment preparation

25

A positive control that is toxic to larvae but not toxic to adults was needed for the
experiment to prove the method was sound. The chemical chosen was diazinon, a
common agricultural pesticide. The diazinon dose was determined by performing a pilot
trial that resulted in 50% larvae mortality. This dose was calculated to be 888 pg / g
pollen. Untreated pollen was also treated with acetone to control for a possible effect of
solvent. The acetone was allowed to evaporate and then 5 ml of 50% sugar syrup was
added to the mixture to make it into a liquid form. There was 7.5 ug pollen per 6 ul sugar
syrup solution. The mixture was then mixed using a vortexer and stored in a freezer ( -
20° C) until use. Before each trial it was thawed and then vortexed to remix it.

The treatments were as follows: untreated (larvae not fed), bee collected pollen,
diazinon treated treated bee collected pollen (8 8 8 rig/g pollen), nontransformed corn
pollen, corn pollen from Cry1A(b) (Event Bt11)corn, corn pollen from CrylF (Event
TC1507 purchased from Mycogen 1 Dow Agrisciences) corn, non-transformed canola
pollen, and glyphosate resistant (Roundup ready) canola pollen.

Larvae identification and feeding

In five hives, groups of 4-5 (1 old larvae were selected and divided into eight
sections on a transparency. The transparency was pinned to the frame so that it would
stay in place. A tack was placed at each corner of the entire brood patch to delineate the
treated area. With a permanent marker, the 4-5 d old larvae that were to be treated were
circled on the transparency. They were then divided into the eight sections and randomly
assigned a number that corresponded to one of the treatments. After mapping, the larvae
were fed 6 ul of the designated treatment solution(7.5 ug pollen/ 6 ul sugar syrup) with a

20 111 Eppendorf repeat pipettor.

26

Larvae mortality, pupal weight, blood protein level, and adult emergence

When the larvae were capped in each of the five hives, we placed the
transparency over the brood patch once again and checked for larvae mortality. Empty
cells, indicating that the larvae had died and been carried out by a nurse bee, were
counted as dead larvae. Larvae were considered alive, if the cell had been capped with
wax by worker bees. In trial two, five pupae were collected at the blackeye stage from
three hives to compare weights between treatments. The frame of pupae was left in the
hive, until 3-4 days before emergence; then the worker bees were brushed off and the
comb was placed in an incubator to monitor adult emergence. Blood protein levels of
newly emerged bees from three hives were measured for each treatment. Adult
emergence was measured in three hives for each treatment. This was done by placing the
map over the previously capped cells and counting them as emerged if they were empty.
Midgut dissections

In order to ensure that there was no spread of pollen by nurse bees to other larvae,
midgut dissections were made 24 hours after feeding on a subpopulation of larvae that
was separate from the experiment. The pollen from the midguts was compared to slides
made of each pollen type to make sure that it matched the original pollen that was fed to
the larvae. The number of pollen grains in the non-transformed canola treatment was
measured prior to feeding with a hemocytometer. After the midguts were dissected, the
number of pollen grains in guts of larvae fed non-transformed canola were recorded,

again using a hemocytometer.

27

STATISTICAL ANALYSIS
Experiment one - summer 2000

Pupal weights were analyzed using SAS general linear model (GLM) and the
means were separated with Tukey’s HSD test (SAS Institute 1999).
Experiment two - summer 2001

Percent honey bee larval mortality in the two canola pollen treatments was
analyzed using SAS t test (SAS Institute 1999). The diazinon dose trial one and the
diazinon and chlorfluazuron dose trial data was transformed using sqrt (x+.5) to stabilize
the variance. Data was then analyzed with SAS general linear model. Means were
separated using Tukey’s HSD test (SAS Institute 1999).
Experiment three - summer 2002

All data was analyzed as a randomized complete block design, where each hive
served as a block. Percent larval mortality, pupal weight, blood protein level, and percent
adult emergence were analyzed using SAS general linear model procedure (GLM).
Means were separated with Tukey’s HSD test (SAS Institute 1999). Adult emergence
data could not be analyzed because there was no variation in seven of the eight treatments
(all but diazinon).
RESULTS
Experiment one — summer 2000

Results show a significant difference in the pupal weights of the CrylF treatment

and the diazionon treated corn pollen (P<0.05) (Figure 1).

28

Experiment two - summer 2001

Results show no significant difference between treatments in the diazinon dose
response trial (F=.47, df=4, 10, P=.7591) (Figure 3). There was no significant difference
in the diazinon and chlorfluazuron dose response trial treatments (F=.72, df=4, 10,
P>0.05) (Figure 4). These dose pilot trials proved to show puzzling results with the
control treatments having the as high of mortality as the pesticide doses being tested.
Results (Figure 5) show a significant difference between the percent mortality of the
larvae fed regular canola pollen vs. larvae fed Roundup Ready® Canola pollen (t=2.57,
df=1, P=0.0498). However, there is a large inter-hive variation in the Roundup Ready®
canola pollen treated hives, yielding a treatment mean standard error of 9.29.
Experiment three — summer 2002

The results show a significant difference in the percent mortality between larvae
fed diazinon treated pollen and the canola pollen fed larvae. However, there was not a
significant difference between larvae fed transgenic glyphosate resistant canola pollen vs.
larvae fed non-transgenic canola pollen (F: 35.94, df=4, 45, P=.3142) (Figure 6). There
is a significant difference between the diazinon treated pollen and the corn pollen
treatments. However, there is no significant difference in percent mortality between
larvae fed non-transgenic corn pollen vs. those fed transgenic CrylAb and CrylF pollen
treatments (F: 45.45, df=5, 50, P=.0556) (Figure 7).

There is a significant difference in mean pupal weight between bees fed diazinon
treated pollen and the canola pollen fed larvae. However, there was not a significant
difference between larvae fed transgenic glyphosate resistant canola pollen vs. larvae fed

non-transgenic canola pollen (F=23.99, df=4, 68, P<0.05)(Figure 8). Significant

29

differences in the mean pupal weight between larvae fed diazinon treated polbn and
those fed the three corn pollen treatment were found. However, there is no significant
difference between mean pupal weight of non-transgenic corn pollen fed larvae vs. those
fed transgenic CrylAb and CrylF pollen treatments (F=26.98, df=5, 82, P<0.05) (Figure
9).

There were no diazinon fed larvae that survived to adult emergence in the hives
tested for blood protein concentration. Thus, only the other seven treatments were
measured for blood protein concentration. There were no significant differences in blood
protein concentrations between larvae fed glyphosate resistant canola pollen and those
fed non-transformed canola pollen (F2123, df=3, 56, P>0.05)(Figure10). No significant
differences were found in blood concentration (ug/ml) of larvae fed non-transformed corn
pollen and those fed CrylF corn pollen and CrylAb corn pollen (F=1.78, df=4, 70,
P>0.05) (Figure 11). All treatments except for diazinon had a 100% emergence rate.
Thus, the data could not be analyzed in SAS since there is no variation. Diazinon had a
28.25 i 2.4 percent emergence rate.

Larvae dissection

Honey bee larvae midgut dissections were conducted a sub-sample of larvae not
used in the experiment to monitor the type of pollen that were in the larvae’s midgut.
There was no spread of the pollen by worker bees; each larvae had the correct type of
pollen in their midgut that had been given by pipette. Six ul of non-transformed canola
pollen was mixed with 394 ul of 50% (weight/volume) sugar syrup. Pollen grains were
counted on a hemocytometer. The average count of pollen grains in .lul of this solution

was 27.8 i 1.01. After dissection the larvae midguts of larvae fed non-transformed

30

canola pollen were ground with a pestle and then diluted with 394 ul of 50% sugar syrup
(weight/volume). Counts were taken from three larvae midguts. They had an average
recovery count of 22.3 i .68 pollen grains in .1ul of the solution. Only pollen of a size
50% or greater of the original was counted. Some of the pollen had been digested and
was broken into small pieces and sowas not counted. Thus, an average of 80% of the
pollen fed to the larvae was found in the midguts.
DISCUSSION
Experiment one

Further trials must be done in order to test for the effect of transgenic Bt corn
pollen on honey bee worker larvae and adults. Future studies should include larvae
mortality as well as pupal weight and blood protein measurements.
Experiment two

The methodology in these experiments needs to be improved in many ways to
avoid the inter-hive variation that was found in the pilot study. Normal hive larvae
mortality is about 10% on average due to genetic abnormalities, weather, disturbances to
the hive, or lack of food to feed the brood. This experiment placed many unnatural
controls on the hives that were used in each treatment. The first of which is that the hives
were made up of only 6,000 bees, a number that places them at a disadvantage for
warding off diseases, predators, and pests. Secondly, the queen was caged and only
allowed to lay a small amount of eggs and was moved every few days. Thus, the hive
populations were not replenished. The pollen trap might have also been a factor in
disrupting the colony and causing variation. I do not believe that the significant results of

the canola trial can be taken at face value without considering the great variation. This

31

protocol must be modified to decrease control mortality before this experiment can be
performed again.
Experiment three

The results suggest that transgenic Bt corn and glyphosate resistant canola pollen
_do not have a negative effect on worker larvae survival, pupal weight, blood protein level
or adult emergence when fed on the 4th or 5th day of development. Further studies
should be done in order to compare the foraging behaviors and longevity of workers fed
transgenic Bt corn pollen and canola pollen with those fed regular non-transformed
pollen. While the benefits of genetically engineered crops may be significant, including
reduced pesticide use and increased yield, potential negative impacts must be carefully

assessed before introducing them to the environment.

32

Mean i SE

Pupal Weight,

160 -
140 -
120 .
100 ~
80 ~
60 -
40 ~
20 -

 

 

Unt Pest Cry1 F Cry1 Ab
Pollen Treatment

Figure 1. Mean pupal weight of bees fed pollen treatments.

33

C)
O

 

..b
O

 

 

 

N
O

 

..L
O

 

 

 

Percent Larval Mortality, Mean 1 SE
on
o

0 mg 1.56 mg 6.0 mg 6.5 mg 7.0 mg
IGR Diazinon Diazinon Diazinon

Treatment

Figure 2. Percent larval mortality for diazinon and chlorfluazuron (IGR) dose

response trial.

34

 

 

 

 

3‘:
1001

+|

c -_

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g 80-

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a 60~

'1'.’

O

'5'

E

CB

.1 201

H

C

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O

h

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0 .39 1.95 9.6 48.25
Diazinon Treatment (mg/1009 pollen)

Figure 3. Percent larval mortality of larvae in diazinon dose trial.

35

 

 

 

 

 

Lu

0)

+. 35 - t=2.57, p<0.05
g a

a, 30- --

E 25

E

,g 20-

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2 15~

a

2 10-

m

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Canola Pollen Treatment

Figure 4. Honey bee larvae mortality measurement after being fed

canola pollen treatment.

36

Percent Larval Mortality
-L N 00 #- 01 O) \l
O O O O O O O

 

 

 

Diaz Mix Reg RR Unt
Pollen Treatment

Figure 5. Percent larval mortality of bees fed canola pollen in trial one.

37

Percent Larval Mortality
.1 N 00 -i> 01 O) \I
O O O O O O O

 

 

Diaz Mix Reg RR Unt
Pollen Treatment

Figure 6. Percent larval mortality of bees fed canola pollen in trial
two.

38

a

-‘ N (.0 k 01 C) \l
O O O O O O O
I r r r r r l

b b b b

Percent Larval Mortality

b

 

 

Diaz Mix Cont Cry1AbCry1F Unt
Pollen Treatment

Figure 7. Percent larval mortality of bees fed corn pollen in trial one.

39

Percent Larval Mortality
-* N 00 -I>~ 01 CD \I
O O O O O O O
l 1 1 l l 1 J

 

 

Diaz Mix Cont Cry1AbCry1F Unt

Pollen Treatment

Figure 8. Percent larval mortality of bees fed corn pollen in trial two.

40

160 -

LLI

a:
+|140-
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3120- a
E
A100“
U)

5.. 80-
%, 60-
'6

3 40~
3 2o-
3

CL

 

 

Diazinon Mix RR Reg Unt
Pollen Treatment

Figure 9. Pupal weight of bees fed canola pollen in trial one.

41

Pupal Weight (mg) Mean 1 SE

160 -
140 ~
120 ~
100 -
8O -
60 -
4O —
20 -

 

 

Diazinon Mix Cont Cry1FCry1Ab Unt
Pollen Treatment

Figure 10. Pupal weight of bees fed corn pollen in trial one.

42

+SE

 

 

| 4000-
r:
a:
(D
E
’—‘ 3000 -
E
\
m
3
7, 2000 -
>
o
..l
c
E’ 1000 -
o
h
a
‘3
2 Mix RR Reg Can Unt
m

Pollen Treatment

Figure 11. Blood protein concentration of bees fed canola pollen (ug/ml

hemolymph) in trial one.

43

Blood Protein Level (ug/ml) Mean 1 SE

5000 -

4000 -

3000 -

2000 -

1000 -

 

 

Mix Cont Cry1Ab Cry1F Unt
Pollen Treatment

Figure 12. Blood protein concentration of bees fed corn pollen (ug/ml

hemolymph) in trial one.

Trial One Trial Two

 

 

100 1 100 1
8 8
g 80 - g 80 _
E’ E’
0 0
E 60 — IE 60 —
E a
< 40 ‘ < 40 —
‘5 E
g 20 - g 20 ~
0 0
n. n.
o -
Diaz Mix RR Reg Unt Diaz Mix RR Reg Unt
Pollen Treatment Pollen Treatment

Figure 13. Percent adult emergence of bees fed canola pollen in trial one and two

45

Trial One Trial Two

 

 

100 - 100 _
a a
S 80 - 5 80 -
e 9
CD (D
E 60 1 E 60 -
E a
< 40 ‘ < 40 -
E i=3
9 20 — g 20 -
d) d)
n. r;
0 _.
Diaz Mix Cont Cry1AbCry1F Unt Diaz Mix Cont Cry1AbCry1F UnI
Pollen Treatment Pollen Treatment

Figure 14. Percent adult emergence of bees fed corn pollen in trial one and two.

46

CHAPTER TWO
EVALUATION OF THE USE OF Bt CORN POLLEN AS CONTROLLING

METHOD FOR THE GREATER WAX MOTH, (Galleria mellonella L.)

47

ABSTRACT

The greater wax moth, Galleria mellonella L. is a pest of the honey bee. The
larvae feed on the pollen of a hive and in doing so make messy burrows and tunnels of
webbing. This activity can destroy a weak hive or stored honeycomb in a short time.
New natural controls that could be used in the field would be very beneficial for the
beekeeping industry. Bt corn pollen is a potential method for controlling for the greater
wax moth for several reasons. In a laboratory study, we fed first instar wax moth larvae
three types of corn pollen: non-transformed corn pollen, Cry1Ab corn pollen, and Cry1F
corn pollen. We found that the mortality of larvae fed Cry1F corn pollen was
significantly greater than the mortality of larvae fed Cry1Ab or non-transformed corn

pollen (P< 0.05). In each trial Cry1F fed larvae had a 100% mortality.

48

INTRODUCTION
Wax moth biology

Like other animals, honey bees are plagued by many diseases and pests. One of
those pests is the greater wax moth, Galleria mellonella L., a pest of stored comb and
weak colonies. Larvae of the greater wax moth cause considerable damage to beeswax
combs left unattended by bees. The larvae feed on the pollen of a hive and in doing so
make messy burrows and tunnels of webbing. This activity can destroy a weak hive or
stored honeycomb in a short time (Williams 1978). The wax moth is a holometabolous
insect having four stages in its life cycle: egg, larvae, pupae and adult. The moth thrives
at a temperature of 30 degrees Celsius and at a humidity of 70% with total darkness
(Williams 1978).
Control methods

Methods of controlling the wax moth in the past have used chemicals such as
paradichlorobenzene, ethylene dibromide, methyl bromide, and ethylene oxide to
fumi gate stored equipment. These chemical fumigants have several disadvantages. They
may be toxic to humans and animals. During an average four month storage period,
several applications are often required. After fumigation, a closed system in the storage
area is needed and this system is difficult and expensive to maintain (Cantwell 1981).
Many of the fumigants are not legal for use in controlling wax moth.

An alternative method to chemical control of wax moth, Thuricide 90TS®, a
Bacillus tlzuringiensis preparation that was incorporated into beeswax foundation was
studied in the early 1960’s. However, it did not prove to be very successful. In the

1980’s, a Bacillus thuringiensis bio-pesticide, Certan®, was developed to treat honey bee

49

colonies in the field (Cantwell 1981). This new bio-pesticide was available for sale by
bee supply companies but was not profitable to make so it is no longer manufactured
(Caron 1999). Current control methods that are approved are extreme temperatures,
which are very time consuming for the beekeeper and requires a large freezer or heating
device. New natural controls that could be used in the field would be very beneficial for
the beekeeping industry. Bt com pollen is a potential control method for the greater wax
moth for several reasons. First of all, Bt CrylAb and Cry1F toxins are specific for
Lepidoptera, so it might be toxic to the wax moth. Secondly, Bt corn is already grown so
the pollen is available to bees. Thirdly, this method of control, should it prove
successful, is economical, non-chemical, and does not cause honey contamination.
MATERIALS AND METHODS

Laboratory biological assays were conducted in the winter of 2000 at Michigan
State University. Later instar wax worm larvae were purchased at a local shop (Grand
River Bait and Tackle Shop, Lansing, Michigan). They were then raised to adulthood on
an artificial diet. The wax moth diet consisted of the following ingredients: 8 parts
wheat bran, 6 parts corn meal, 6 parts whole wheat flour, 2 parts brewer’s yeast, 10 parts
glycerin, and 5 parts distilled water (Hoopingamer 1990).

After the larvae pupated, they were placed in 2.2 liter plastic containers (12x
30cm), at a density of about 25 pupae per container. Strips of wax paper were cut and
folded accordion style and then paper-clipped and placed into the containers. The wax
paper strips served as oviposition sites for the adult gravid female moths.

Once the females had laid eggs on the strips, they were removed and the eggs

were placed in a plastic container to hatch. After five days, newly hatched larvae were

50

transferred into small plastic containers holding the pollen being tested. The larvae were
transferred using small pieces of wax paper to pick them up being careful not to damage
them in the process.

This experiment was performed using a completely randomized design. There
were three treatments: non-transformed corn pollen, corn pollen containing the toxin
CrylAb, and corn pollen containing the toxin Cry F. There were three replicates for each
experimental unit, which consisted of small plastic Solo cups that had 2.5 g of the pollen
in each. Five larvae were placed in each cup and then placed in the growth chamber at
70% relative humidity, 30 ° C in complete darkness. After nine days, each cup was
inspected for larvae mortality. A microscope was used to locate the larvae due to their
small size. This study was repeated four times.

STATISTICAL ANALYSIS

Wax moth percent mortality in the pollen treatments CrylAb and untreated was
analyzed using logistic regression to determine the least square means assuming a
binomial distribution. Data were analyzed with SAS GLM (general linear model)
procedure. Means were separated using Tukey’s HSD test (SAS Institute 1999).
RESULTS

Wax moth larvae fed Cry1Ab corn pollen and untreated pollen do not show a
significantly different mortality from one another. Cry1F was statistically significant
from both the untreated pollen and Cry1Ab corn pollen (p< 0.05). With an unfailing
100% mortality rate, Cry1F provides excellent control for the greater wax moth in a
laboratory setting.

DISCUSSION

51

Cry1Ab does not appear to be an effective control of wax moth, as it shows no
significant difference from the untreated pollen. Although Cry1Ab is specific for
Lepidoptera, it did not have an effect on this specific moth.

Cry1F corn pollen is an effective control for wax moth in a laboratory. We did
not collect enough Cry1F pollen to perform any further tests. I would recommend that a
laboratory choice test and a field trial be done to solidify our conclusions. The field test
could be done by placing honey bee hives in the middle of a Cry1F corn field during

tassling and submitting them to wax moth infestation.

52

 

     

5”?

+l120- a

C

£100-

a? 80- E
(U .
’5 -.
2 60- b
d)

(U

E 40‘ b

_l

E. 20-

O

E

31‘:

3

Cry1 F Cry1 Ab Untreated
Pollen Pollen Pollen

Treatment
Figure 15. Wax moth larvae mortality measurements from four trials of a laboratory

study.

 

53

LITERATURE CITED

54

LITERATURE CITED

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55

 

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Malone, L., Burgess, E., Gatehouse, H., Voisey, C., Tregeigda, E. and Philip, B. 2001.
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populations: A risk assessment. PNAS Early edition 134: 1-6.

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toxicity of Bacillus thuringiensis pollen to black swallowtails under field
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58

APPENDIX

59

Appendix 1
Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in the named
museum(s) as samples of those species or other taxa, which were used in this research.
Voucher recognition labels bearing the Voucher No. have been attached or included in
fluid-preserved specimens.

Voucher No.: 2002-06

Title of thesis or dissertation (or other research projects):

Effect of transgenic Bt Corn pollen and Roundup Ready canola pollen on honey bee

(Apis mellifera L.) worker larvae and the use of transgenic Bt corn pollen as a controlling
method for the greater wax moth (Galleria mellonella (L))

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator’s Narne(s) (typed)
Anne Valdes Hanley

 

 

Date June 27, 2002

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North
America.
Bull. Entomol. Soc. Amer. 24: 141-42.

Deposit as follows:
Original: Include as Appendix 1 in ribbon copy of thesis or dissertation.

Copies: Include as Appendix 1 in copies of thesis or dissertation.
Museum(s) files.

Research project files.

This form is available from and the Voucher number is assigned by the Curator,
Michigan State University Entomology Museum.

60

Appendix 1.1

Table 1: Voucher Specimen Data

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