The Use of Creosote Bush and Grapefruit Leaves Smoke in The

Home , Creosote

THE USE OF CREOSOTE BUSH AND GRAPEFRUIT LEAVES SMOKE IN THE

CONTROL OF VARROA MITE INFESTATION IN HONEYBEE COLONIES

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In Agriculture

Option in:

Animal Science

Mary Nguyen

2021

SIGNATURE PAGE

THESIS: THE USE OF CREOSOTE BUSH AND GRAPEFRUIT LEAVES SMOKE IN THE CONTROL OF VARROA MITE INFESTATION IN HONEYBEE COLONIES

AUTHOR: Mary Nguyen

DATE SUBMITTED: Spring 2021

Department of Animal & Veterinary Sciences

Dr. Cord M. Brundage ______Thesis Committee Chair Animal & Veterinary Sciences

Dr. Hyungchul Han ______Animal & Veterinary Sciences

Dr. Melody Wallace ______Animal & Veterinary Sciences

Benjamin Lehan, M.S. ______Agricultural Sciences

ACKNOWLEDGEMENTS

I would like to express the utmost gratitude to my committee members for assisting me through this journey. To Dr Cord Brundage, thank you for offering to be my mentor and allowing me to pursue a master’s degree. To Dr Hyungchul Han, thank you for guiding me along and assisting me in completing my thesis. To Dr Melody Wallace, thank you for teaching me about bees. To Benjamin Lehan, thank you for your love of entomology and agreeing to be the agricultural insect expert in the committee.

I would like to thank Professor Mark Haag for teaching me about beekeeping as well. To Jimmy Risk from the Math and Statistics Department at Cal Poly Pomona, thank you for assisting me with my data analysis. Even though Dr Kathleen Earle is not part of my committee for my thesis, I would like to thank her for helping me with my college degree these past few years. She never stopped supporting me and encouraging me in academics, career, and life.

I would also like to thank Bill’s Bees of Southern California for allowing me to come to his apiary and performing my research on his bees. Without his generosity and assistance during the pandemic, no data could be collected. 23628 bees were used for this experiment, and more died in the process of collecting samples.

Lastly, I would like to thank my friends and my dog for making quarantine bare- able enough to allow me to be functional and capable of working through my classes, homework assignment, and other aspects of my thesis.

iii

ABSTRACT

One of the leading causes of the honeybee population decline is the ectoparasitic

Varroa mites, which infect brood larva and adult bees, leading to weakened immune systems and dead individuals. One experimental method of natural mite control includes burning creosote bush or grapefruit leaves, as this causes the mites to become disorientated and crawl off the bees. As smoking is already used in apiary management, this may be a less invasive method of mite control to ensure healthier bees around to pollinate. Prolonged exposure of this smoke kills the bees. The objective of this research is to determine what ratio of creosote bush and grapefruit leaves would cause the mites to dislodge off the honey bee without negatively affecting the honey bee’s behavior. 350

±124 drone and nurse honeybees were placed in wide mouth 2-pint canning jars with mesh lids, which allows the mites to fall through the mesh and onto adhesive paper.

Smoke application treatments were performed on 3 different days, with two weeks quarantining in between each treatment day. Treatments consisted of 100% of creosote bush (CB) with 0% grapefruit leaves (GL), 75% CB with 25% GL, 67% CB with 33%

GL, 33% CB with 67% GL, 25% CB with 75% GL, and 0% GB with 100% GL. This was compared to a control group with no smoke applied. 12 total puffs of smoke were used for these treatments. Additional treatment groups of 24 puffs of smoke of CB and GL was tested for. Smoke was applied for 1.5 minutes, then the lid removed for 5 minutes, and then the mites counted on the adhesive paper. The bees were then drowned in windshield wiper fluid, and a Varroa EasyCheck was used to account for mites unaffected by the smoke. Although the smoke (p = 0.272 for CB, p = 0.758 for GL) had a higher mite knock off rate than the control with no smoke (p = 1), we did not notice any statistically

iv significant difference or consistent trends between various concentration of smoke exposure regarding mite knock off. Creosote bush nor grapefruit leaves cannot be recommended for use in smokers as a method of mite control. Future studies are needed to determine what other smoked plant materials may be effective against Varroa mites, yet safe on the bees.

TABLE OF CONTENTS

SIGNATURE PAGE ii

ACKNOWLEDGEMENT iii

ABSTRACT iv

LIST OF TABLES viii

LIST OF FIGURES ix

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 3

2.1 Beekeeping Practices 3

2.2 Colony Collapse Disorder 3

2.3 Varroa Mites 4

2.4 Natural Mite Control Tactics 7

2.5 Chemical Miticide 9

2.6 Alternative Mite Control with Chemicals 12

2.7 Creosote Bush 15

2.8 Grapefruit 15

2.9 Smoking Habits in Other Pest Management 16

2.10 Smoke 17

2.11 Concerns for Pesticide in Beekeeping 18

2.12 Importance of Varroa Control 19

CHAPTER 3 METHODS AND MATERIAL 20

3.1 Plant Material 20

3.2 Smoking Chamber Set Up 20

3.3 Bee Sample Collection 20

3.4 Smoker Set Up 21

3.5 Smoke Application 21

3.6 Alternative Treatments Tested 22

3.7 Remaining Mite Count 22

3.8 Statistical Analysis 23

CHAPTER 4 RESULTS 24

CHAPTER 5 DISCUSSION 26

5.1 Limitations 26

5.2 Experimental Adjustments 31

5.3 Future Directions 32

CHAPTER 6 CONCLUSION 34

REFERENCES 47

vii

LIST OF TABLES

Table 1: Ratios Tested with Corresponding Number of Puffs………………………..35

Table 2: Effect of 0% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites; Control

…………………………………………………………………………………………35

Table 3: Effect of 100% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites……35

Table 4: Effect of 75% Creosote Bush, 25% Grapefruit Leaves on Varroa Mites……36

Table 5: Effect of 67% Creosote Bush, 33% Grapefruit Leaves on Varroa Mites……36

Table 6: Effect of 67% Creosote Bush, 33% Grapefruit Leaves on Varroa Mites Retested

………………………………………………………………………………………….36

Table 7: Effect of 33% Creosote Bush, % Grapefruit Leaves on Varroa Mites……….37

Table 8: Effect of 33% Creosote Bush, 67% Grapefruit Leaves on Varroa Mites Retest

………………………………………………………………………………………….37

Table 9: Effect of 25% Creosote Bush, 75% Grapefruit Leaves on Varroa Mites…….37

Table 10: Effect of 0% Creosote Bush, 100% Grapefruit Leaves on Varroa Mites……38

Table 11: Effect of 200% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites……38

Table 12: Effect of 0% Creosote Bush, 200% Grapefruit Leaves on Varroa Mites……39

Table 13: Effect of 100% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites with

2.5 minutes exposure ……………………………………………………………………39

Table 14: Effect of 0% Creosote Bush, 100% Grapefruit Leaves on Varroa Mites with

2.5 minute exposure …………………………………………………………………….40

Table 15: Percentage of Mites knocked off based on total mites in a treatment and p- values…………………………………………………………………………………….40

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LIST OF FIGURES

Figure 1: % of mites knocked off based on total number of mites in a treatment versus the % of creosote bush smoke used in the standard 12 puffs of smoke treatments……………………………………………………………………………….. 41

Figure 2: % of mites knocked off based on total number of mites in a treatment versus the number of puffs of smoke of creosote bush only applied……………………………42

Figure 3: % of mites knocked off based on total number of mites in a treatment versus the number of puffs of smoke of grapefruit leaves only applied………………………..42

Figure 4: % of mites knocked off based on total number of mites in a treatment versus the time of exposure to 12 puffs of creosote bush smoke……………………………….43

Figure 5: % of mites knocked off based on total number of mites in a treatment versus the time of exposure to 12 puffs of grapefruit leaves smoke……………………..……..43

Figure 6: % of mites knocked off vs Number of bees in 100% CB with 0% GL...…….44

Figure 7: % of mites knocked off vs Number of bees in 67% CB with 33% GL…...….44

Figure 8: % of mites knocked off vs Number of bees in 33% CB with 67% GL………45

Figure 9: % of mites knocked off vs Number of bees in 0% CB with 100% GL………45

Figure 10: % of mites knocked off vs Number of bees in 200% CB with 0% GL……...46

Figure 11: % of mites knocked off vs Number of Mites in a Trial……………………...46

CHAPTER 1 INTRODUCTION

Honey bees, or Apis mellifera, are known as the pollinators of the world. It is estimated that one third of all bites of food is the direct result of a bee’s pollination

(Glenny, et al, 2017). However, in the past few decades, the bee population has been on a decline at an alarming rate. Scientists named the issue “Colony Collapse Disorder”, or

CCD. CCD is identified by an abandoned hive – beekeepers commonly find their hives void of honey bees, with only the queen and a small number of nurse bees. As bees tend to protect the hive and stay near their queen, CCD is a significant cause of alarm for producers. One of the main causes of this bee decline is the ecto-parasitic Varroa mite and it is important to find different methods of mite control to assist the bees

(Rosenkranz, Aumeier, & Zigelmann, 2010).

The use of certain plant pesticide in the smoke is a unique way of mite control. By using these volatile smoke chemicals, beekeepers will have another method of mite control to further reduce the number of destructive mites in their colonies (Eischen &

Vergara, 2004). Additionally, by applying the chemicals in the smoke, this would treat the bees in a less invasive or destructive method compared to adding stripes of chemicals into the hive or destroying a frame of brood.

Entomologist Frank A. Eischen tested the efficiency of the smoke from various plant materials, including gobernadora, eucalyptus, coffee beans, corncobs, pine needles, and tobacco (Adams, 1997). Many of these plants are known to have some established and natural pesticidal effect to prevent pests from destroying its leaves or branches.

Eischen confined the bees into a cage and placed that in a container. He applied smoke into the container for a minute, then observed the number of mites that fell off the bees,

1 as well as the bees’ ability to recover from the smoke. The two most effective was from creosote bush and grapefruit leaves. The creosote bush was able to kill many mites but resulted in comatose bees, with some dying soon after. The grapefruit leaves merely disorientated the mites into falling off the bees but did not kill the mites. However, the bees were not harmed (Adams, 1997).

The objective of this research experiment is to determine what ratio of creosote bush and grapefruit leaves, when burnt and the smoke applied to bees, would be an effective method of mite control. This experiment is designed to determine a balance in the application of smoke as a possible miticide by comparing the number of mites to the ratio of the creosote or grapefruit plant material used. Once a proper ratio is determined for the most mite knock off, it would be beneficial to test varying time duration of smoke application to ensure the lowest exposure to these bees.

Instead of applying the smoke to the entire beehive, sample jars are used in this preliminary study to give us an idea of how the varroa mites and bees react to being exposed to smoke from creosote bush and grapefruit leaves. We will study how creosote bush smoke and grapefruit leaves smoke affects varroa mites and bees. Background information on beekeeping tactics and varroa mite control will be explored in Chapter 2.

The methods used will be detailed in Chapter 3. Results of the study is mentioned in

Chapter 4, with an evaluation of these results and future directions discussed in Chapter

5. The thesis will be concluded with Chapter 6.

CHAPTER 2 LITERATURE REVIEW

2.1 Beekeeping Practices

Basic beekeeping includes a Langstroth hive, which is formatted as a wooden box with 8-10 frames inside for the bees to create comb and honey on. Multiple boxes are stacked on each other and closed off with a top cover. This allows beekeepers to observe their bees and collect honey without destroying the hive, as is standard with wild hives.

Smoke applied to the hive calms the bees and allow safer handling. Sugary water and synthetic pollen substitute sustain the hive over the winter, when natural food sources are scarce. Additionally, beekeepers stack more boxes so the hive can expand. When there are a considerable number of frames populated with bees, the beekeepers separate the frames into two separate Langstroth hives. Empty frames are added in the boxes so the bees have space to grow and expand into. The bees will create a new queen to take care of the new colony. In the beekeeping world, the entire hive is considered the biologically significant unit when determining bee population, as bees are not capable of surviving as an individual singular bee (Fels, et al, 2019). However, due to Varroa mites, many hives do not expand in numbers and often die out, leaving less colonies to pollinate

(Rosenkranz, Aumeier, & Ziegelmann, 2010).

2.2 Colony Collapse Disorder

The phenomenon of colony collapse disorder is still not clearly explained. It is defined as the abandonment of hives by the worker bees. One third of beehives have been showing up abandoned annually and are considered as cases of CCD (Glenny, et al,

2017). There is no identified cause, but researchers theorized CCD is due to multifactorial poor conditions in the hive and stress that causes the worker bees to leave

3 the hive (vanEngelsdorp, et al, 2009). Monocropping, pesticides, and varroa mites are thought to be the three main causes of CCD (Goulsen, et al, 2015).

Monocropping may be the biggest reason for CCD. American farmers have been pushing to plant one crop for acres around. Honeybees in these fields will only have one source of food, and with this restricted diet of pollen and nectar from one type of plant, would reducing the availability of other vitamins and minerals (vanEngelsdorp, et al,

2008).

The use of pesticides, such as imidacloprid, in agriculture is another factor contributing to CCD. Imidacloprid was detected in 91% of soil samples, with 65% of samples at concentrations higher than 1 g kg-1, but only 15% of samples were collected from areas where seeds were treated in the past year, indicating the longevity of residual imidacloprid retention in fields. It was detected in 83% of sunflower pollen despite the sunflowers never being treated for it (Bonmatin, et al, 2005). Even at sublethal doses between 1-20 g kg-1, long term ingestion of residual imidacloprid causes bee mortality

(Zhu, et al, 2017).

Varroa mites, or Varroa destructor, is the third cause of CCD as these mites infect bees and cause poor health and living conditions for the bees. Elevated levels of these parasites have been correlated with a higher rate of CCD colonies (vanEngelsdorp, et al, 2009).

2.3 Varroa Mites

Varroa mites, or Varroa destructor or Varroa jacobsoni, have been a prevalent issue in the beekeeping world. Heavy infestations of Varroa mites is called varroosis.

Originally infecting only the eastern honey bee, or Apis cerena, the Varroa mite jumped

4 hosts to the western honey bee, or Apis mellifera (Ritter, 1981). These mites were originally thought to feed off the hemolymph of bees, but recent studies have proven they more readily feed off the fat bodies (Ramsey, et al, 2019). Fat bodies are vital to keeping a healthy immune system by playing a role in vitamin and hormone control, and functioning as a rudimentary liver in bees (Nilsen, et al, 2011). Although they can be spotted on the bees, mites prefer the abdominal ventral side, where the bees cannot reach easily. The bees would be unable to groom off the mites from their undersides. However, the mites are viewed occasionally on the bee’s thorax, on the dorsal side. When Varroa mites are on the dorsal side of the thorax, it is very likely they are attempting to crawl off the bee to another host (Ramsey, et al, 2019). The western honey bee is the most common bee in the United States, in both beekeeping and in the wild. If hives are left untreated, the mites cause the hive to collapse within 2-3 years (Rosenkranz, Aumeier, &

Ziegelmann, 2010).

The female Varroa mite infests hives by crawling onto foraging worker bees.

When the foraging bees come into contact with nurse worker bees, the mites will crawl over to the nurse bees. The nurse bees will then crawl back to the brood area of the hive to assist with making honey or feeding brood. When the nurse bee crawls over a cell with a larva and deposits food, the Varroa mite will land on top of the food with the larva. If there is sufficient food with the larva, the Varroa mite will become trapped in the sticky food and be unable to leave. This is ideal for the mite since she obscures herself under the larva and avoid being detected by the nurse bees. She would still be able to feed off the hemolymph of the larva as it develops, becoming sealed in with it when it caps over, and lay her eggs on it such that when the pupae hatches and emerges, it will take the Varroa

5 mite offspring with it (Donze & Guerin, 1994). If there is not enough brood food to trap her, she may feed off the larva but then crawl out of the cell to feed from a different larva. It was noticed that if there was not sufficient amount of brood food to trap the mite, she was less likely to lay eggs and thus the spread of varroosis would be lowered in the hive (Infantidis, 1988). Additionally, mites preferred the drone cells over the worker cells, with 11.6 times more mites noticed in the drone cells (Boot, et al, 1995). Since drones take approximately 3 days longer to pupate, the mites could feed off the drone for a longer period and more of the offspring will develop. Female Varroa mites will lay 5-6 eggs, and usually start laying eggs about 60 hours after the cell capped over for the bee pupae to develop in. Eggs are laid in 26-32 hours intervals, allowing the eggs to develop at different times. Usually, 4 of the mite eggs hatches and reach maturity after a few days, with 3 females and one male. The other two eggs would not reach maturity before the bee leaves the cell, so there is a mortality rate of 1.43 protonymph mites per infected cell. The males take up to 154 hours to reach maturity while the females usually take about 134 hours. However, the male is usually the first egg to be laid, and thus would be the first to hatch. The male will then mate with his sisters (Martin, 1994). Although the mother mite lays up to 6 eggs, usually only 1.3 female daughter mites will successfully move on if they were feeding off a worker pupa. On a drone pupa, 2.6 daughter mites will live

(Schulz, 1984). Sometimes, two female mites would inhabit the same cell. As two female mites and their 10 offspring take up a lot of space, it has been noted that they may alternate when they lay eggs to maximize the time the protonymph are allowed to feed and develop (Donze & Guerin, 1994).

2.4 Natural Mite Control Tactics

The bees do have their own system to control mites. 98% of Apis mellifera mellifera and 86% of Apis mellifera caucasia will attempt to groom the mites off their bodies (Bak & Wilde, 2016). Honey bees will occasionally remove pupae infested with

Varroa mites, if they detect the mites. Anywhere from 6.0-42.3% of mites will be removed due to the grooming behavior of hygienic colonies, lowering the mites’ reproductive abilities (Spivak, 1996). In another study, 44-63% of mites were damaged or removed by the bees (Rosenkranz, et al, 1997). Apis cerana, or the eastern honey bee, is more likely to remove phoretic mites than Apis mellifera, or the western honey bee, but both species still regularly groom mites off their bodies. 12.5% of mites had injuries from the western honey bee’s attempt to groom them off (Fries, et al, 1996).

Natural methods of mite control include heating the hive. Bees maintain their hives at a temperature between 33-36 degrees Celsius (Seely, 1985). At 40 degrees

Celsius, the mites begin to drop off the bees. By heating the hive to this temp for a few hours straight, this reduces the number of mites. However, 40 degrees Celsius is close to the maximum temperature that bees tolerate before they start dying too (Harbo, 2000).

Adding a perforated board to the bottom of the Langstroth hive with a glue trap prevents mites from crawling back onto the bees, especially paired with keeping the hive in a dark room for three days (Allam, et al, 2021).

Another more serious method of mite control is killing off infected brood.

Beekeepers allow the queen to lay her eggs on a frame and move her to a new frame after nine days. The original frame, with Varroa mite infestation and the larva, or brood, is naturally raised by nurse bees. When it caps over, the beekeepers destroy the brood

7 frame, before the bees emerge with the mites. Since the Varroa mites have a life cycle of

10 days, this greatly diminish the number of mites before they have the chance to reproduce (Boots, Calis, & Beetsma, 1992). However, this reduces the number of useful worker bees dramatically.

Beekeepers will discard drone frames. Mites prefer the drone brood as it gives them a longer time for the mite eggs to develop into maturity. Varroa mite only reproduce on drone brood if they are infesting an Apis cerana hive (Boot, et al, 1999).

Drones are larger than the workers, so the cells that house drones will naturally be larger than the regular combs. By adding a drone frame, or a frame with larger cells, the queen will lay drone eggs into these cells. Mites will flock to this frame to be capped in with these drones. Beekeepers remove the drone frame and freeze the frame, killing both drone pupae and mites (Calis, et al, 1997). Drones do not contribute much to the hive and are only purposed to mate with any new emerging queens. Therefore, it is not considered harmful to the hive to discard of them, aside from the wasted efforts of the worker bees to build comb and raise the drones.

Beekeepers use electricity to create heat in the foundation of the frame for 2-3 minutes and kill the mites. As the drone and mites are in close contact to the foundation, this will kill both. Any worker bees on the top of the cells are insulated against this heat via the wax of the combs (Huang, 2001). It is preset to shut off at 44 degrees Celsius, which is a temp that will kill mites but most brood will survive from (Br∅dsgaard &

Hansen, 1994). Heating the hive at 40-45 degrees for 2.5 hours had a mortality rate of 99-

100% in mites (Bičík, Vagera & Sádovská, 2016). By leaving the frames in the hive, the worker bees will notice the drone larva/pupae aren’t viable and will begin the process to

8 uncap and remove the drones and the mites (Boecking, Rath & Drescher, 1993). This means the whole frame does not have to be destroyed, nor do the bees have to start creating comb again. However, it does take time for the bees to identify and remove the deceased pupae.

2.5 Chemical Miticide

There are other methods of mite control, including chemical miticides, aimed at reducing the mite load but leaving the bees unharmed.

Apivar, or Amitraz, and Thymovar, or thymol, are both marketed treatments for

Varroa mites. Both come in plastic strips that are placed in between frames of the

Langstroth hive. In one Canadian study by Yahya Al Naggar in 2016, 45 colonies were tested for the effectiveness of the two products in assisting with overwintering survival.

Two strips of each product were placed in their respective hive in October. It was noted that Apivar was 76.5% effective in reducing mite numbers, and Thymovar was 26.7% effective. When checked seven months later, in April, 93% of the hives treated with

Apivar survived over winter and 67% of Thymovar hives survived.

Another study at the University of the Balearic Island in Spain by Dr Mar Leza in

2015 compared Apivar with Apiguard, another thymol based miticide. In spring,

Apiguard was 89.8% effective at reducing the daily mite count, compared to Apivar at

64.3%. However, in fall, Apiguard was only 30.8% effective and Apivar was 17.9%.

Apivar’s reduced effectiveness in this study was theorized to be due to the mites’ resistance since Apivar has been used for the past three decades.

Formic acid is another miticide, but with only 70% effectiveness at mite mortality

(Eischen, 1998). However, other studies have shown formic acid, in gel packets or

9 ingrained in paper, had 93.6-100% effectiveness. The gel form lasted 2 weeks due to its gradual release whereas the paper only lasted 3 days. Additionally, the gel had a lower rate of bee mortality compared to paper, but the gel form affected the brood reared (Satta, et al, 2005).

Another chemical known for its effects on mites is fluvalinate. When Dr Jeff

Pettis treated worker with Apistan, or fluvalinate strips, for 5 days instead of the recommended 3 days, then examine two months later, there was no significant mortality of worker bees. When applied to queen bees, the queens died on the fourth and fifth day of treatment. However, since death occurred past the recommended three-day treatment period, Apistan’s marketed treatment was still considered a decent treatment. When the worker bees and queen bee were treated with Apistan and tested after overwintering, or after 6 months, there was a higher rate of morality in both worker bees and queen bees

(Pettis, et al, 1991).

Dr Timothy Haarmann treated queen bees with fluvalinate for a shorter period of time than recommended, the queens with a higher dosage of fluvalinate weighed less than those with a lower dosage. However, they did not have any significant developmental abnormalities when compared to the control. Coumaphos is another chemical for Varroa mites. In the same study, queen bees exposed to coumaphos suffered from developmental abnormalities, smaller ovaries weight, and, if exposed for more than 24 hours, even death

(Haarmann, et al, 2002).

Some mites seemed resistant to amitraz and fluvalinate. The first reported case of amitraz resistance in mites was in Minnesota, in 1999. Researchers discovered the mite morality rates when exposed to these two chemicals were diminished compared to the

10 standard rates. However, coumaphos was still effective in controlling the mites, at that time (Elzen, et al, 2000). Similar mite resistance to fluvalinate was noted in Texas,

Florida, and California (Elzen et al, 1999).

One reason why beekeepers are reluctant to use Apivar is its toxicity on honey.

Beekeepers are advised not to consume or sell honey for up to 42 days after apivar was applied to a hive. However, additional research shows that there was no residue in the honey nor wax after 30 days, which indicates the honey is safe for human consumption earlier (Martel, et al, 2007). The efficiency of each type of prevention seems to vary in each study done. In one comparison, apistan was 99% effective, thymol blend was 70% effective, and formic acid was 51% effective at removing Varroa jacobsoni (Calderone,

1999). According to bee science lecturer Mark Haag, Apivar is the preferred choice of mite treatment on Cal Poly Pomona campus. There has been a push for research that explores more natural methods of mite prevention, such as testing different plant materials that contain miticide chemicals.

Dr Elzen of Rhode University compared experimental and commercial miticide of

2,6-dimethoxyphenol. The experimental mixtures of volatile chemicals had a noticeably better result of dislodging mites off the honey bees. Direct contact of the phenol drops caused mites to fall off, but without any significant differences between experimental and commercial. This may be due to the mites’ resistance to commercial chemicals from years, if not decades, of repeated use of the miticide (Elzen, Stipanovic, & Rivera, 2001).

Thymol is considered an effective miticide, and there has been research done with essential oils containing thymol. One study placed essential oil droplets onto pieces of honeycomb wax, and then introduced Varroa jacobsoni mites to the combs. Most

11 essential oils had little to no effects on the mites. However, the thymol infused oils had an effectiveness rate of 90-100% mite mortality. Additionally, the residue of thymol infused oil on the honey was low, indicating it was safe to eat sooner than conventional

Thymovar or Apiguard (Imdorf, et al, 1999). However, this study did not test the effects of the thymol or essential oils on honey bees.

Since new worker bees hatch every 21 days, the hive must be regularly treated to keep the number of mites low. With repeated use of miticides, Varroa mites may develop a resistance to the miticides. Beekeepers commonly rotate the type of miticides to prevent resistance. The miticides may leak into the honey and poison it for humans. Depending on the miticides, bee keepers discard the honey created shortly after a miticide application as it is unfit for human consumption (Rinkevich, Danka, & Healy, 2017).

2.6 Alternative Mite Control with Chemicals

Pollen traps are a useful method of mite control. When bees land in front of their hive, the traps gently scrape the pollen off the bees as the bee move to enter the hive. The trap would help dislodge any Varroa mites sticking to the bee’s underside. Brahim

Cakmak of Uludag University (2002) found that the hives with pollen traps had 50% less infected capped brood cells compared to the control of no pollen traps. He applied walnut leave smoke into the hive entrance for one minute. The hives without traps only noticed a

21% increase in mite capture. When the walnut leaves smoke was applied to the hives with a pollen trap, there was a 66% boost in mite capture compared to the control. He noted that the hives with pollen traps had a higher yield of honey, which meant the bees could be focusing more on honey production rather than brood production. Without

12 brood, there wasn’t as many opportunities for the mites to reproduce, which may account for the reduced number of mites on the pollen trap hives.

Another useful organic mite deterrent is tobacco. Tobacco smoke and extract, when applied to the beehive, results in reduced numbers of Varroa mites without affecting bee mortality. They sprayed the extract directly on the bees, but smoke wise, three grams of each test material was lit on fire and the smoke was allowed to disperse in the hive. In the same study, harmel extract was used. It had little to no effect on the

Varroa mites but caused a few bee mortalities, indicating harmel should not be used on beehives (Abdol-Ahad, et al, 2008). A combination mixture 5% of clove oil and tobacco extract was 96.483% effective with mite mortality. The brood was uncapped and placed in petri dishes, where they were exposed to the fumes from the extracts (Mahmood,

2014). In another study, an empty super, or box without frames, was added to the bottom of Langstroth hive, with a bee excluded added in between the bee-filled boxes and the empty box. A white sheet of paper or cardboard was added to the bottom of the hives to catch any debris that fell. Tobacco smoke was added into the entrance and the paper examined the following day. Varroa mites were counted on the paper (Ruijter & Eijnde,

1984). However, it is possible some of the mites on the paper was from manual grooming of the bees or the mites simply crawled off, without effects from the tobacco smoke.

Entomologist Frank A Eischen discovered two promising plants for mite control in 1997. He tested forty various natural plants’ abilities when burnt in a smoker. Bees were placed in a holding chamber and the smoke was applied for a minute. The number of mites that fell off the bees were caught by an adhesive white paper placed at the bottom of the holding chamber. Then the bees were placed in an alcohol wash to dislodge

13 remaining mites. Eischen found out that when creosote bush was applied, the mite mortality rate was 60-100%. However, some bees suffered from the smoke and were noted to be sluggish or deceased. When he burnt grapefruit leaves in the smoker and applied it, 90-95% of mites had fallen off the bees within 30 seconds. Additionally, the bees were not significantly more affected by the smoke compared to regular beekeeping smoking habits. However, the mites in the grapefruit leaves test did not die, which meant if given time, they could have crawled back up to re-infest the bees. However, due to the possible side effects of contaminating the honey, Eischen does not recommend using creosote bush nor grapefruit leaves as a method of mite control and did not write an official paper on his crude experiment (Adams, 1997).

Frank A Eischen also tested various plant material smoke on honey bee, but to test its effectiveness on the honey bee tracheal mite, or Acarapis woodi. He collected 12 bees in small jars and applied smoke into the jars. He kept the bees for 18 days to observe mortality and culled five of the bees on day 4 to count tracheal mites’ mortality. He counted the mortality of the mite larva and eggs. The study found that creosote bush was the most effective at killing tracheal mites. However, it was noted that mite mortality was negatively correlated with bee mortality. This indicated that when the bees struggled to breathe, they may have inhaled less of the smoke and thus the mites received a lowered dosage of chemical (Eischen & Vergara, 2004). As Varroa mites are external parasites, they do not depend on the bee’s ability to breathe, and thus should not be affected by the bee’s mortality.

2.7 Creosote Bush

Creosote bush, or Larrea tridentata, is known for its antifungal uses. A lower polarity of the molecules extracted from the creosote bush resins had higher antifungal properties. Resin extracted from the creosote bush were antifungal against black bread mold and various other plant harming fungi (Fernandez, Hurtado & Hernandez, 1978).

Creosote bush has two polyphenol lignans, nordihydroguaiaretic acid and methyl- nordihydroguaiaretic acid, that are very effective at stopping the fungal enzyme β-1,3- glucanase, which is responsible for pathogen-plant attacks (Vargas-Arispuro, Contreras-

Valenzuela, & Martínez-Téllez, 2009). Although creosote bush may be a considerable fungicide, it doesn’t seem to be much of a pesticide. Several different species of midge, all under the genus of Asphondylia, have been noted to reside on creosote bush (Gagne &

Waring, 1990).

2.8 Grapefruit

Grapefruit is not known as a pesticide either. Instead, it is known for its drug inhibiting interactions. It reduces or enhances the effectiveness of over 85 drugs, with a considerable number of the medication used to treat common ailments (Bailey, Dresser &

Arnold, 2013). In humans, it affects drugs taken three days ago (Greenblatt, et al, 2003).

The furanocoumarins in grapefruit has been attributed with this effect, most noticeably the compounds of “bergamottin” and “6’,7’-dihydroxybergamottin”. It inhibits the liver enzyme CYP3A4, which is responsible for metabolizing drugs and causes a higher or lower amount of the drug in the bloodstream (Kaker, et al, 2004).

Furanocoumarins may have a pest deterrent element to them. The bee-bee tree produces seven linear furanocoumarins, including 5-(6-hydroxy-3,7-dimethylocta-2,7-

15 dienyloxy)psoralen. Four of these compounds were noted as a feeding deterrent to the larva of the Egyptian cotton leafworm and the tobacco budworm, which is a moth

(Stevenson, et al, 2003). Other furanocoumarins, like benzofuran and coumarin, deters tobacco hornworms (Neal & Wu, 1994). Similarly, furanocoumarin found in plants of the family Rutaceae and Umbelliferae had a toxic effect on southern armyworm moth larvae

(Berenbaum, 1978).

2.9 Smoking Habits in Other Pest Management

Although it may not necessarily be smoke from a fire, different pest control tactics include aerosolizing pesticidal chemicals, which allows the compounds to disperse further. Foggers, also known as bug bombs, contain pyrethroid and pyrethrin and an aerosolizing component to disperse these compounds into the surrounding area rather than relying on the insects to walk into the chemicals. They are commonly used in households and gardens to control common insect pests (Hudson, et al, 2014). They work well against mosquitos, such that adult and larval Aedes aegypti numbers reduced when exposed to smoke generating pyriproxyfen and permethrin tablets (Harburguer, et al,

2009).

Pyrethrins were derived from the Chrysanthemum cinerariaefolium flower

(Bradberry, et al, 2005) but since they affected bees (Hudson, et al, 2014), they were not considered for Varroa mite control. Other aerosolized plant materials are useful as pest control. Gigochocha atroviolace bamboo smoke has a 100% mortality rate of

Coptotermes formosanus and Reticulitermes speratus termites within 14 days (Subekti &

Yoshimura, 2020).

2.10 Smoke

Smoke is already used ubiquitously in apiaries as part of the process to work with bees. When smoke is applied, the bee’s instinct is to engorge itself with honey, or fuel for energy, and then flee the hive (Newton, 1968). Smoke has been known to affect the bee’s ability to detect pheromones. The queen pheromone attracts her workers to her and to protect her and the hive, but with the pheromone covered, the beekeepers may handle the queen away from the hive. This will prevent the worker bees from swarming to protect their queen (Winston & Slessor, 1998). The effects of the smoke only lasts 10-20 minutes so using smoke is not considered harmful to the hive (Visscher, Vetter & Robinson,

1995). Other research indicates smoke does not affect the likelihood of being stung when a hive is agitated. Rather, it reduces the amount of venom released in a sting. With less venom released, the alarm pheromone is not as prevalent and thus the bees are less likely to sting again (Gage, et al, 2018). The smoke masks the scent of the alarm pheromone, further reducing the chances of other bees stinging. Additionally, the bees that engorge themselves with honey after being exposed to smoke were less likely to sting than bees with less honey in their honey sacs (Free, 1968).

Beekeepers prefer to use natural fuel based on its availability, cost, and safety.

Twine, burlap, pine needles, cedar shavings, cardboard, coconut fibers, newspaper, or leaf litter are commonly used in smokers (Robinson & Visscher, 1984). These materials are specifically used to minimize harmful chemicals in the smoke that may affect the bees or contaminate the honey. Depending on the source of plant material for fumigation, there may be up to 12 compounds found on honey. Sealed honey has more concentration of chemicals, likely due to the wax’s absorptive abilities. Open honey had less residue as

17 it allowed the compounds to dissipate from the honey (Tananaki, Gounari &

Thrasyvoulou, 2009).

2.11 Concerns for Pesticide in Beekeeping

There are three main reasons for colony collapse disorder – monocropping restricting a hive’s access to a variety of flowers, pesticide use affecting bees, and the

Varroa mites (Goulsen, et al, 2015). In 2008, surveyed beekeepers reported that 22% of their hives were lost in the winter. They stated this was a normal and expected loss

(vanEngelsdorp, et al, 2008). Since beehives die at an alarming rate, many beekeepers have abandoned their beekeeping hobbies and turned to other interests, which reduces the number of hives even further (Potts, et al, 2010). This percentage of bee loss has slowly but steadily increased in the following years.

Many pesticides are credited to the death of many honey bee colonies, which may be why many beekeepers are reluctant to use pesticides in their hives. Pesticides, such as imidacloprid, are cited as the reason for some unexplained bee death (Decourtye, et al.

2004). Even if the pesticide doesn’t kill the bee right away, they affect the bee’s abilities to forage. For example, thiamethoxam affects the bee’s abilities to return to the hive, thus causing the bee to die (Henry, et al, 2012). However, even if the beekeeper or the immediate farmer doesn’t use pesticides in their crops, pesticides, such as neonicotinoids, have been found in the soil of unplanted fields, and in dandelion weeds, indicating that neonicotinoids are absorbed via the roots (Krupke, 2012). When bees are exposed to pesticides, the number of parasitic Nosema in their gut flora increases (Pettis, et al, 2012).

It’s important to reduce the amount or potency of pesticides around the bees to prevent the bees from dying.

2.12 Importance of Varroa Control

Varroa mites are considered detrimental to bees. Not only do they feed off the fat bodies of bees, which weakens the bee’s immune system, they act as vectors for five different viruses, including deformed wing virus. Deformed wing virus originated in

Asian honeybees and were transmitted to European honeybees (Wilfert, et al, 2016).

Deformed wing virus causes the bee’s wings to be underdeveloped, shriveled and small.

The bee is unable to fly, cannot forage for food, cannot evaporate nectar into honey, and cannot help regulate hive temperature, which is detrimental over winter. Bees with deformities had a higher titer results than those without physical deformities, despite both being tested positive (Bowen-Walker, Martin, & Gunn, 1999).

Despite how serious the Varroa mite infestation could be, many smaller bee keepers refuse to mite test their colonies. Mite check could be as easy as collecting approximately 300 bees, or ½ cup of bees, and swirling them in an alcohol wash to kill and dislodge the mites. Then the number of mites dislodged is compared to 300 bees to estimate a percentage of infestation in the colony. It is recommended for larger apiaries, to test one colony for each eight colonies (Lee, et al, 2010).

It is important to control the infestation of varroa mites in a hive. There are many different ways to treat them and most beekeepers utilize a combination of various methods, both physical and chemical. It is vital to determine more way, and safer ways, of treatment. We will be testing to see what ratio of creosote bush and grapefruit leaves smoke would be most effective at knocking off mites without altering bee behavior. We hypothesize that some combination of the two would be most effective at knocking off mites without altering bee behavior.

CHAPTER 3 MATERIALS AND METHODS

3.1 Plant Material

Creosote bush (Larrea tridentata) plant material was collected in Barstow,

California in March 2020. Creosote bush was collected in off road, rural regions to limit possible roadside pollution. Grapefruit (Citrus paradisi) leaves were collected on Cal

Poly Pomona’s Fruit and Crop Unit in March 2020. Fallen grapefruit leaves were collected off the ground rather than leaves from the tree. They were stored in separate plastic storage bin and allowed to air out in the sun in ambient spring weather to dry the material to make it easier to burn.

3.2 Smoking Chamber Set Up

A 15 L storage bin, with the dimensions of 16 ¾” L x 12” W x 7” H, had a small hole drilled in to the side to apply the smoker, approximately the size of 1.5-2.0” in diameter. 8.5”x 11” shipping labels lined the bottom, with the adhesive side up. Wide mouth 2-pints mason jars had their lids replaced with wires. These wires have enough space for the small mites to fall through. Wooden platforms were created to elevate the jars above the adhesive paper. A humidity and temperature monitor was placed in the corner of the storage bin.

3.3 Bee Sample Collection

A hive was opened, and the queen bee located by inspecting various brood frames until she was identified. This frame was set aside so the queen would not accidentally be harmed. Half a cup of live bees, approximately 350 ±124 bees, were collected off a brood frame by shaking the frame over a bin, then scooping the bees out and placing it into the jars. The measuring cup was then upturned inside the mason jars then closed.

3.4 Smoker Set Up

The smoker is comprised of metal fire chamber, with a bellow behind it to pump air through the fire chamber and blow the smoke out of the nozzle. The bellow is approximately 1.35L in volume. Since creosote bush and grapefruit leaves are not ideal tinder, newspaper was used as a starter at the bottom of the fire chamber in two separate smokers before creosote bush and grapefruit leaves were heaped on top. Both smokers were set aside for 5 minutes to ensure the fire could burn though the plant material and generate enough smoke to apply on the bees.

3.5 Smoke Application

For the purpose of this experiment, a puff of smoke is defined as a 1.35L volume of air and smoke expelled out of the smoker’s nozzle when the bellow is fully compressed. 12 puffs of smoke were applied via the drilled hole for each treatment, with varying numbers of puffs from each smoker. 100% CB with 0% GL, 75% CB with 25%

GL, 67% CB with 33% GL, 50% CB with 50% GL, 33% CB with 67% GL, 25% CB with 75% GL, and 0% CB with 100% GL were tested to determine the idea concentration of both plant materials in terms of its highest effectiveness on mites but lowest effect on bees. For the 100% with 0% treatments, 12 puffs from one smoker was used. For 50% with 50%, 6 puffs of each was applied. For 67% with 33%, 4 and 8 puffs of smoke were applied. For 75% with 25%, 3 and 9 puffs of smoke were applied. The number of puffs of smoke used in each treatment is found in Table 1.

Once the smoke was applied, the hole was sealed with masking tape and the container allowed to sit for 1.5 minutes. After 1.5 minutes, the lid was opened, and the container allowed to air out for 5 minutes. Temperature and humidity were noted. Bee

21 behavior was noted. After 5 minutes, the jars were removed and upturned. Mites were accounted for at the bottom of the paper. Bee activity level was classified as ‘active’,

‘semi-active’, and ‘sedated’. Active indicates the bees collectively were crawling around with the same intensity and frenzy as prior to the smoke exposure. Sedated indicates little to no activity, with the bees huddling. Semi-active refers to an activity level in between the two. If a mite was not observably moving, it was considered dead. The total number of fallen mites were accounted for under the ‘fallen’ category, which includes the dead mites.

3.6 Alternative Treatments Tested

Two alternative treatments of smoke were tested for. The first adjustment was increasing the time of smoke exposure from 1.5 minutes to 2.5 minutes. This was performed using 100% creosote bush, then 100% grapefruit leaves on two separate treatment groups. The second experiment tested concentration by puffing 24 puffs of smokes from creosote bush rather than 12 puffs but using the same 1.5-minute parameter.

24 puffs of smoke from grapefruit leaves was tested for.

3.7 Remaining Mite Count

A Varroa easycheck cup from Veto-pharma was used to account for any mites not affected by the smoke after the 5-minute resting period. This cup has a strainer inside the cup. Windshield fluid was added in the easycheck cup strainer to cull the mites and the bees. The cup was shaken, and dislodged mites will fall through the strainer in the cup, then counted.

3.8 Statistical Analysis

Data was analyzed via R studio program to determine P values. A logistic regression model was used to determine the probability of a mite falling off depending on the treatment, and Fisher test was used to account for contingency. Any P values less than

0.05 were considered significant.

CHAPTER 4 RESULTS

Data was collected on three separate days. The 100-0% treatments, the 75%-25% treatments and the control was tested on July 7, 2020. The 67-33% treatment was tested on August 5th, 2020. Results are found on table 2 through table 14.

If we considered all the mites per treatment, the 100% CB, 0% GL (Table 3) had a

11.32% knock off rate (p = 0.272). 75% CB and 25% GL (Table 4) had 7.89% knock off rate (p = 0.304). 67% CB and 33% GL (Table 6) had 21.15% knock off rate (p = 0.007).

33% CB and 67% GL (Table 8) had 45.24% knock off rate (p = 0.467). 25% CB and

75% GL (Table 9) had 0% knock off rate (p = 1). 0% CB and 100% GL (Table 10) had

11.36% knock off rate (p = 0.758). The control, with no smoke applied (Table 2), had

3.57% knock off rate (p = 1).

Two additional treatments were tested to see if double the amount of puffs of smoke would be more effective against the mites. Considering 12 puffs of one plant material was considered an 100% treatment, “200%” was used to refer to doubling the number of puffs of one plant material. With 24 puffs of creosote bush smoke, or 200% creosote bush and 0% grapefruit leaves (Table 11), 25% of mites were knocked off (p =

0.014). With 24 puffs of grapefruit leaves smoke, or 0% creosote bush and 200% grapefruit leaves (Table 12), 4.167% of mites were knocked off (p = 0.138).

Most of the bees were active throughout the experiment except for a few cases. In the 100% creosote bush and 0% grapefruit leaves, two of the jars had an overall sedated bee activity during the smoking process but recovered and were active before the lid was removed. All the samples in 0% creosote bush and 100% grapefruit leaves had a similar

24 activity noticed of initially sedated but then later active. The 33% creosote bush and 67% grapefruit leaves had three samples of semi-active bees.

There were only two trials that had p-values below 0.05. The 67% creosote bush and 33% grapefruit leaves had a p-value of 0.007 and the 200% creosote bush and 0% grapefruit leaves had a p-value of 0.014, and thus the only results that was considered statistically significant.

The percentage of mite knock off was compared to the number of bees in a jar.

Rather than adding up the total numbers of mites in a trial, we considered each jar as individual samples. Only treatments that had at least three jars with mites knocked off from the smoke were considered, such that a trend can be observed between the percentage of mites knocked off to the number of bees in the sample. 100% CB with 0%

GL (Figure 6), 67% CB with 33% GL (Figure 7), 33% CB with 67% GL (Figure 8), 0%

CB with 100% GL (Figure 9), and 200% CB with 0% GL (Figure 10) were considered.

None had a linear trend between the number of bees in the sample to the percentage of mites that were knocked off.

CHAPTER 5 DISCUSSION

There were many errors with the experimental design and various aspects of the experiment that lead to indeterminate conclusion. As the bees all recovered from the smoking process, it is undeterminable on whether the creosote bush or grapefruit leaves had a detrimental effect on the bees. Therefore, we cannot determine which plant material, when smoked, would be effective at knocking off the mites without affecting the bees as the bees were unaffected by both, regardless of concentration.

5.1 Limitations

Due to the COVID-19 pandemic, accessibility to apiary facilities were limited.

Bill’s Bees graciously allowed the research to be performed on their bees, but the hives were still subjected to commercial beekeeping practices, including miticide treatments.

Tests were done in the field, during late summer 2020, on three different days. Due to the

2 weeks self-quarantine recommended by the Center for Disease Control and Prevention, samples could only be collected every 2 weeks, so experimentation was limited. There should have been a 50%-50% treatment done.

Parameters changed on the different days of testing. The first day of testing, the temperature and humidity ranged between 31.1-32.5 ᴼ C and 32-40%. On the second day of testing, the temperature dropped to approximately 27.2-27.3 ᴼ C and humidity rose to

46-48%. On the final day, temperature dropped even further to 24.5-25.7 ᴼ C and humidity rose to 49-54%. These changes in temperature and humidity may have an effect on the bee behavior and mite health since even subtle changes in temperature between

33-36 ᴼ C versus 40ᴼ C can affect mite drop (Seely, 1985; Harbo, 2000).

Additionally, due to the two weeks in between, the season changed slightly over the month of testing time period. July is still considered a good time for foraging, honey making and brood rearing, but August is closer to fall. Mite levels may change over the months, as well as food availability for the bees (Sajid, et al, 2020).

The bees sampled on belonged to a private beekeeper, who sells the honey. The concerningly high numbers of mites after the first day of testing led the beekeeper to apply miticide to the hives. This may explain the low level of mites in the subsequent samples. The resulting reduced mite load during the second day of testing may have caused the beekeeper to relax his miticide control as he did not reapply before the third day of testing, hence why the number of available mites spiked. Due to the miticide application before the second day of testing as well as the limited availability of mites, the data from the second day was not compared to the data collected from the other days.

The 33%-67% treatments, displayed in Table 6 and Table 8, were redone on day 3 to make up for day 2’s results. The results from day 2 is still displayed in Table 5 and Table

7 but were not analyzed.

Even with puffs of smoke setting a volume-based standard of each plant material applied, it is difficult to determine an actual concentration applied. It is possible the smokers were unevenly filled or unevenly lit on fire. The volatile abilities of each plant material may affect the results. Grapefruit leaves caught fire faster than the creosote bush and burned through faster. Creosote bush ignites slowly but burn longer and produce hotter fires (Fuentes-Ramirez, 2016). Each puff from the smoker had a 1.35 L volume of release of smoke, but the billow of smoke may vary in composition. It would be hard to accurately quantify how much smoke is applied to the bins during each treatment.

Most of the bees were active and unaffected by the smoke, aside from a few samples. With regards to this experiment, it is indeterminable if their activity is innate and a reflection of the hive they came from, or if it was due to the smoke. However, none of the samples displayed a noticeable negative effect on the bees as all were able to recover to their original activity level. The three-point scale of activity of active, semi- active and sedated had a broad range of classification, and did not note any subtle variation in activity level. Defining activity levels, such as testing individual bees for the distance they crawl or how long it takes for them to take flight, can refine how we analyze the effect of the smoke on the bees.

Therefore, the initial goal of the experiment to determine a decent ratio of creosote bush to grapefruit leaves that wouldn’t harm the bees is nullified as regardless of ratio, none of the bees were harmed. The smoke exposure, especially if repeated, may have a long-term effect of the bees. This is tested by housing a smaller number of bees in a larger container, providing honey and water to sustain them for a few weeks, exposing them to smoke on a daily basis, and checking them regularly for mortality. Frank A.

Eischen performed a similar study by exposing bees to smoke as a possible tracheal mite treatment and checking for mortality over an 18 day period (Eischen & Vergara, 2004).

Bee activity may account for the removal of mites rather than the irritating effects from the smoke on the mites. Apis cerana’s excessive grooming behavior is a reason why varroosis in the eastern honey bee is not as severe as in the western honey bee’s population (Boot, et al, 1999). More bees in a jar may equate to more surface area movement in between the bees, allowing for more mites to be dislodged. Although bees were measured in ½ cup volume, the exact number of bees varied. 350 ±124 individual

28 bees were counted in each jar after the experiment. However, as displayed in Figures 6 through 10, there were no linear trends between the number of bees in a jar compared to the percentage of mites knocked off.

However, if we considered the total number of mites in a trial to the percentage of mites that were knocked off, we can see a trend where the percentage of mites that dropped increased if the number of individuals mites presented in a trial increased

(Figure 11). The reasoning for this is unclear and needs future studies to test if and why increased number of mites may also correlated with a higher percentage of mites dropped.

Another variation was the types of bees in each sample. As each scoop was collected from various frames shook into a bin, there may be a mixture of worker bees and drone bees. Mites prefer drone brood but drones aren’t as hygienic as worker bees and less likely to groom mites off themselves (Boot, et al, 1995). The various hives may contain bees of various age, genetic, and health status, which may affect their mite resistance.

Another variation is the uneven exposure of smoke on the jars in a trial. The position of the jars may affect how much smoke the bees were exposed to, even if they were in the same chamber at the same time. Since the smoke application hole was drilled in the upper middle of one side of the smoking chamber, the jars closest to the smoking chamber would be exposed to the smoke first, and in a bigger concentration. As the smoke dispersed to the back end of the chamber, it would not be in as strong of a concentration, especially since the smoke would have to disperse to the bottom of the chamber and travel through the mesh lids to affect the bees and mites (Barclay & Aston,

1990). The lids were placed on before smoke was applied but the storage bins did not have an airtight lock. Volatile chemicals may escape from any miniscule gaps in the top of the lid.

The temperature was noted after the smoke was applied but it did not note the heat from the smoke. The temperature was not recorded before smoke application so there is no reference to compare the temperature to after the smoke application. It would be useful to have a temperature monitor that can record fluctuations in temperature as the smoke is applied. If the smoke is hot enough to cause a spike in temperature, even if momentarily, this could affect the bees and mites, even without the volatile chemicals in the smoke (Harbo, 2000).

All of these variables may contribute to why the hypothesis could not be proven with this research. We hypothesized that a combination of creosote bush and grapefruit leaves would be more effective at knocking off the mites without affecting the bee’s behavior. To reduce some of the aforementioned variables, we could select a smaller sample of bees, for example 12, such that each individual bee could be monitored separately over a few days time period (Eischen & Vergara, 2004). We could intentionally infect these bees with mites so we have an equal number of mites available for each trial. Although we would have a smaller sample size of bees, we could observe the bee behavior individually in greater detail rather than estimating the behavior of the whole jar. If we raise the bees specifically for this experiment, we could even select for similar aged worker bees or drone bees to test on, reducing the variation in age, sex, and health. If the bee was housed in a small chamber and had the smoke applied individually, this would reduce the variation in smoke as it traversed throughout the chamber.

Even if future experiments show promising miticidal effects with using creosote bush or grapefruit leaves, beekeepers should take caution when working with these plant materials. Although the compound that causes eye irritation has yet to be identified in citrus, the smoke from the grapefruit leaves, when accidentally blown into human faces, had an eye irritating effect, such that epiphora was elicited. It is recommended to wear eye protection when working with citrus (Monaghan, et al, 2011). Creosote bush is not recommended as burning material as it left a thick, tarry resin. This resin is undesirable, such that woodrats that are used to eating creosote bush, would have decrease food intake when there was an increase in creosote bush resin (Mangione, Dearing, & Karasov,

2000).

5.2 Experiment Adjustments

Due to the low percentage of mites being dropped in each sample from the smoke, we ran two treatments to see if increased exposure time of solely creosote and solely grapefruit may increase the number of mites dropped during the second day of testing.

There did not seem to be any discernable differences after 2.5 minutes of exposure, so the original plan of 1.5 minutes of smoke exposure was resumed to have a consistent comparison between the 33% with 67% trials performed on day 3 and the trials performed on day 1.

Another treatment criterion was changed for the last two treatments. Instead of 12 puffs of smoke, we increased it to 24 puffs of smoke to see if increased concentration would affect the mites. Although we only tested one type of smoke in each trial, this would still be able to test if a different concentration of smoke would be more effective at knocking off the mites. The 24 puffs of creosote bush had a higher mite drop compared to

12 puffs or no puffs control (Figure 2). Had the p values been below 0.05 for all three criteria, then more puffs of smoke would result in more mites knocked off. Grapefruit leaves did not have the same result since the 24 puffs of smoke had a smaller percentage of mites knocked off than 12 puffs of smoke (Figure 3).

The beekeeper filled the canning jars brought to the testing site. However, 23 jars were brought on the first day in case a jar broke or was misplaced. There were 3 extra jars of bees. The control and 25% CB with 75% GL treatments were tested on 4 sample each instead of the usual 5.

With the 100% grapefruit treatment, the lid was accidentally left on for an additional 30 seconds. For data analysis purposes, we assumed 30-60 seconds additional smoke exposure had a negligible effect on the mites dropped. However, it is still a factor that could affect the data for the 100% grapefruit criteria. We would need to retest the

100% grapefruit leaves treatment at 1.5 minutes of exposure to determine a more accurate result.

5.3 Future Directions

There are many ways to streamline the experiment. Had there been more time and beehives available, it would be ideal to test out various times of exposure and concentration of smoke. Additional researchers partaking in the data collection could allow data to be collected on the same day and reduce the discrepancies between the various days’ weather. A 50% CB with 50% GL treatment should be performed.

The use of newspaper as a fire starter may affect the results. Ink and paper type may leak chemicals into the smoke. Newspaper ink have agonists against aryl hydrocarbon receptors (Bohonowych, et al, 2008). Ah receptors are important for gene

32 expression roles involved with metabolism and immunity. If newspaper is used in future studies, it is important to determine if it affects the bees and mites.

Future research should investigate other plant materials. Frank Eischen’s experiment was performed two decades ago, and we would need to look into if the mites have developed a resistance to the creosote bush and grapefruit leaves chemicals.

CHAPTER 6 CONCLUSION

Based off of our research, creosote bush nor grapefruit leaves in smokers would be recommended for miticide control. This study did not show any difference between the ratios of creosote bush and grapefruit leaves exposure to mite knock off rate. There were no significant differences in the bee’s behavior and recovery that would indicate the creosote bush or grapefruit leaves would be harmful on them, but this claim would need to be further investigated. The hypothesis that a combination of the two would be most effective was not proven with this research. The findings in this study was affected by a lot of variables that may skew the results and future studies would have to address those variables to allow for a more uniform study. Further research should look into why the results were not significant compared to Frank A. Eischen’s original work.

Table 1: Ratios Tested with Corresponding Number of Puffs

Creosote bush 100% 75% 67% 50% 33% 25% 0% 200% 0% # of puffs 12 9 8 6 4 3 0 24 0 Grapefruit 0% 25% 33% 50% 67% 75% 100% 0% 200% leaves # of puffs 0 3 4 6 8 9 12 0 24

Table 2: Effect of 0% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites; Control

Date: July 7 Temp: 32.5ᴼC Humidity: 32% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 8 8 0% 379 B 1 0 Active 11 12 8.33% 327 C 0 0 Active 3 3 0% 390 D 0 0 Active 5 5 0% 331 Total 1 0 27 28 3.571% 1427

Table 3: Effect of 100% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites

Date: July 7 Temp: 31.1ᴼC Humidity: 39% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 1 0 Active 3 4 25% 286 B 2 0 Sedated at first; 5 7 28.57% 401 became active C 3 1 Sedated at first; 26 29 10.34% 329 became active D 0 0 Active 6 6 0% 317 E 0 0 Active 7 7 0% 301 Total 6 1 47 53 11.32% 1634

Table 4: Effect of 75% Creosote Bush, 25% Grapefruit Leaves on Varroa Mites

Date: July 7 Temp: 31.4ᴼC Humidity: 36% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 1 0 Active 1 2 50% 422 B 0 0 Active 4 4 0% 340 C 2 0 Active 16 18 11.11% 402 D 0 0 Active 4 4 0% 343 E 0 0 Active 10 10 0% 318 Total 3 36 38 7.895% 1825

Table 5: Effect of 67% Creosote Bush, 33% Grapefruit Leaves on Varroa Mites

Date: July 22 Temp: 27.3ᴼC Humidity: 46% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 1 1 0% 397 B 0 0 Active 5 5 0% 424 C 0 0 Active 3 3 0% 400 D 0 0 Active 0 0 0% 373 E 0 0 Active 2 2 0% 350 Total 0 0 11 11 0% 1944

Table 6: Effect of 67% Creosote Bush, 33% Grapefruit Leaves on Varroa Mites Retested

Date: August 8 Temp: 25.7ᴼC Humidity: 49% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 7 7 0% 432 B 0 0 Active 9 9 0% 362 C 2 0 Active 9 11 18.18% 473 D 5 0 Active 2 7 71% 377 E 4 0 Active 14 18 22.22% 363 Total 11 0 41 52 21.15% 2007

Table 7: Effect of 33% Creosote Bush, 67% Grapefruit Leaves on Varroa Mites

Date: July 22 Temp: 27.2ᴼC Humidity: 47% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 4 4 0% 323 B 0 0 Active 3 3 0% 446 C 0 0 Active 3 3 0% 416 D 0 0 Active 2 2 0% 404 E 0 0 Active 1 1 0% 425 Total 0 0 13 13 0% 2014

Table 8: Effect of 33% Creosote Bush, 67% Grapefruit Leaves on Varroa Mites Retest

Date: August 8 Temp: 24.8ᴼC Humidity: 51% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 1 0 Active 2 3 33.33% 382 B 13 1 Active 17 30 43.33% 403 C 2 0 Semi-active 4 6 66.67% 383 D 2 0 Semi-active 0 2 100% 419 E 1 0 Semi-active 0 1 100% 385 Total 19 1 23 42 45.24% 1972

Table 9: Effect of 25% Creosote Bush, 75% Grapefruit Leaves on Varroa Mites

Date: July 7 Temp: 31.1ᴼC Humidity: 36% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 14 14 0% 369 B 0 0 Active 9 9 0% 339 C 0 0 Active 12 12 0% 418 D 0 0 Active 3 3 0% 226 Total 0 0 38 38 0% 1352

Table 10: Effect of 0% Creosote Bush, 100% Grapefruit Leaves on Varroa Mites

Date: July 7 Temp: 32.1ᴼC Humidity: 40% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Sedated at first; 10 10 0% 389 became active B 1 0 Sedated at first; 6 7 14.29% 425 became active C 1 0 Sedated at first; 6 7 14.29% 307 became active D 2 1 Sedated at first; 9 11 18.18% 377 became active E 1 0 Sedated at first; 8 9 11.11% 382 became active Total 5 1 39 44 11.36% 1880

Table 11: Effect of 200% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites

Date: August 5 Temp: 24.9ᴼC Humidity: 52% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 5 0 Active 1 6 23.33% 321 B 1 0 Active 9 10 10% 461 C 0 0 Active 1 1 0 461 D 2 0 Active 10 12 16.67% 358 E 2 0 Active 9 11 18.18% 448 Total 10 0 30 40 25% 2049

Table 12: Effect of 0% Creosote Bush, 200% Grapefruit Leaves on Varroa Mites

Date: August 5 Temp: 24.5ᴼC Humidity: 52% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 3 3 0% 383 B 1 0 Active 53 54 0% 359 C 0 0 Active 16 16 0% 276 D 0 0 Active 3 3 0% 388 E 3 0 Active 17 20 15% 393 Total 4 0 92 96 4.167% 1799

Table 13: Effect of 100% Creosote Bush, 0% Grapefruit Leaves on Varroa Mites with 2.5 minutes exposure

Date: July 22 Temp: 27.3ᴼC Humidity: 48% Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 0 0 Active 1 1 0% 348 B 0 0 Active 4 4 0% 411 C 0 0 Active 0 0 0% 387 D 0 0 Active 1 1 0% 339 E 1 0 Active 0 1 100% 369 Total 1 0 6 7 14.29% 1854

Table 14: Effect of 0% Creosote Bush, 100% Grapefruit Leaves on Varroa Mites with 2.5 minute exposure

Date: July 22 Temp: 27.2 ᴼ Humidity: 46% C Sample Mite Mite Bee Activity Mite Total Percent Total # Fallen Dead Missed Mites Knocked of bees Off A 1 0 Active 0 1 100% 389 B 0 0 Active 2 2 0% 300 C 0 0 Active 0 0 0% 417 D 0 0 Semi-active 2 2 0% 356 E 0 0 Active 0 0 0% 409 Total 1 0 4 5 20% 1871

Table 15: Percentage of Mites knocked off based on total mites in a treatment and p- values

Treatment Criteria Total Total Percentage of P-values Number of Number mites knocked off Mites of Mites based on total knocked off in mites in a Treatment treatment Control 1 28 3.571% P = 1

100% Creosote bush, 6 53 11.32% P = 0.272 0% Grapefruit leaves 75% Creosote bush, 3 38 7.895% p = 0.304 25% Grapefruit leaves, 67% Creosote bush, 11 52 21.15% P = 0.006651 33% Grapefruit leaves 50% Creosote bush, N/A N/A N/A N/A 50% Grapefruit leaves 33% Creosote bush, 19 42 45.24% P = 0.467 67% Grapefruit leaves 25% Creosote bush, 0 38 0% P = 1 75% Grapefruit leaves 0% Creosote bush, 5 44 11.36% P = 0.758 100% Grapefruit leaves

200% Creosote Bush, 10 40 25% P = 0.014 0% Grapefruit leaves 0% Creosote Bush, 4 96 4.167% P = 0.138 200% Grapefruit leaves

% Mites Knock Off vs % Creosote Bush Smoke 50 45 40 35 30 25

in a rrial in 20 15

based on total mites total on based 10 % of Mites Knocked off Knocked Mites of % 5 0 0 20 40 60 80 100 120 % Creosote Bush Smoke Used in the 12 puff of smoke treatments

Figure 1: % of mites knocked off based on total number of mites in a treatment versus the % of creosote bush smoke used in the standard 12 puffs of smoke treatments. The % of mites knocked off versus % of grapefruit leaves can be viewed in a right to left manner as the grapefruit leaves concentration is inversely proportional to creosote bush concentration. The 24 puffs treatments were not used.

y = 0.8929x + 2.5825 % Mites Knock Off vs Numbers of CB Puffs R² = 0.9751 30

% Mites Knocked Off 5

0 0 5 10 15 20 25 30 Puffs of Creosote Bush Smoke Applied

Figure 2: % of mites knocked off based on total number of mites in a treatment versus the number of puffs of smoke of creosote bush only applied. No grapefruit leaves smoke was applied in any of these treatments.

y = 0.0248x + 6.068 % Mites Knock Off vs Number of GL Puffs R² = 0.0047 12

% Mites Knocked Off 2

0 0 5 10 15 20 25 30 Puffs of Grapefruit Leaves Puffs Applied

Figure 3: % of mites knocked off based on total number of mites in a treatment versus the number of puffs of smoke of grapefruit leaves only applied. No creosote bush smoke was applied in any of these treatments.

% Mites Knock Off vs Time Exposure of CB 16 14 12 10 8 6 4 % Mites Knocked Off 2 0 0 0.5 1 1.5 2 2.5 3 Time of Exposure of Creosote Bush Smoke (minutes)

Figure 4: % of mites knocked off based on total number of mites in a treatment versus the time of exposure to 12 puffs of creosote bush smoke. No grapefruit leaves smoke was applied in any of these treatments.

% Mites Knock Off vs Time Exposure of GL 12

% Mites Knocked Off 2

0 0 0.5 1 1.5 2 2.5 3 Time of Exposure of Grapefruit Leaves Smoke (minutes)

Figure 5: % of mites knocked off based on total number of mites in a treatment versus the time of exposure to 12 puffs of grapefruit leaves smoke. No creosote bush smoke was applied in any of these treatments.

% Mite Knocked off vs Number of Bees in 100% CB with 0% GL 30

% Mites Knocked Off 5

0 0 50 100 150 200 250 300 350 400 450 Number of Individual Bees in a Jar

Figure 6: % of mites knocked off vs Number of bees in 100% CB with 0% GL. This is based off the percentage of mites knocked off in a jar, excluding 0% knock off rates, rather than the whole trial. There is no linear trend between increasing number of bees to percentage of mite knocked off.

% Mite Knocked off vs Number of Bees in 67% CB with 33% GL 80 70 60 50 40 30 20

% Mites Knocked Off 10 0 0 100 200 300 400 500 Number of Individual Bees in a Jar

Figure 7: % of mites knocked off vs Number of bees in 67% CB with 33% GL. This is based off the percentage of mites knocked off in a jar, excluding 0% knock off rates, rather than the whole trial. There is no linear trend between increasing number of bees to percentage of mite knocked off.

% Mite Knocked off vs Number of Bees in 33% CB with 67% GL 120

100

% Mites Knocked Off 20

0 380 385 390 395 400 405 410 415 420 425 Number of Individual Bees in a Jar

Figure 8: % of mites knocked off vs Number of bees in 33% CB with 67% GL. This is based off the percentage of mites knocked off in a jar, rather than the whole trial. There is no linear trend between increasing number of bees to percentage of mite knocked off when comparing the individual jars exposed to this treatment.

% Mite Knocked off vs Number of Bees in 0% CB with 100% GL 20 18 16 14 12 10 8 6 4 % Mites Knocked Off 2 0 0 50 100 150 200 250 300 350 400 450 Number of Individual Bees in a Jar

Figure 9: % of mites knocked off vs Number of bees in 0% CB with 100% GL. This is based off the percentage of mites knocked off in a jar, excluding 0% knock off rates, rather than the whole trial. There is no linear trend between increasing number of bees to percentage of mite knocked off.

% Mite Knocked off vs Number of Bees in 200% CB with 0% GL 25

5 % Mites Knocked Off

0 0 100 200 300 400 500 Number of Individual Bees in a Jar

Figure 10: % of mites knocked off vs Number of bees in 200% CB with 0% GL. This is based off the percentage of mites knocked off in a jar, excluding 0% knock off rates, rather than the whole trial. There is no linear trend between increasing number of bees to percentage of mite knocked off.

y = 0.5788x - 10.048 % Mite Knocked off vs R² = 0.11 Number of Mites in a Trial 50 45 40 35 30 25 20 15 10

% of % Mites Knocked Off 5 0 25 30 35 40 45 50 55 Number of Mites in a Trial

Figure 11: % of mites knocked off vs Number of Mites in a Trial. This considered the numbers listed in Table 15.

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