the effect of imidacloprid combined with oxalic acid...

The Effect of imidacloprid combined with oxalic acid mite treatment on Apis mellifera mortality

Courtney Wadley

MAY 19, 2016Advanced Scientific Research 2a

Nena Tippens

The Effect of Imidacloprid Pesticide Combined With Oxalic Acid Mite Treatment onApis Mellifera Mortality Wadley1

Abstract:

This study is a comprehensive collection of information on global bee colony decline and

the factors that contribute to it. Imidacloprid and oxalic acid have each individually been known

to contribute to Apis mellifera mortality and bee colony decline. While these factors have been

studied individually, little to no research has been conducted on how the combinations of these

and other factors affect mortality rates of Apis mellifera. Results suggested that the combination

of feeding Western honey bees imidacloprid and treating them with oxalic acid leads to

extremely significant and high mortality rates compared to other individual treatment groups.


Research Question

How does exposure to Varroa mite treatment and plant pesticide affect Apis mellifera

mortality?

Background Research

Introduction:

Agricultural Research in the United States has revealed that the global population of bees

(domestic and wild) is rapidly declining. This is troubling news, as bees serve as one of

agriculture’s most important pollinators. Honey bees for many years have been successful in

supporting human agriculture through pollination (Santos, et al., 2009). Some scientists believe

that the phenomenon is occurring because bees are being exposed to sub-lethal amounts of

pesticides, while others claim miticide (chemical treatment used to control pests in hives)

indirectly affect bee mortality. Many factors such as bee diets, pathogens, parasites, diseases,

global warming, and pesticide use were each thought to be the primary cause of this population

calamity, but recent studies suggest that bees’ interaction with multiple factors may be the

problem’s root (Pettis, et al., 2013).

Rapid decline in bee population negatively impacts the global economy. Because bees

provide such an essential ecological service, humans depend on food produced as a result of

pollination to make profit in the primary sector. There has been a six percent decline in vegetable

availability, and with many crop yields producing similar patterns, profits are also on a major

decline. Humans utilize many resources, including money, to harvest the pollinated crops. When

there is too high a demand and not enough supply, global food prices increase, making synthetic

and less healthy options objectively cheaper. This evidence proves the recent increase in bee

mortality is not beneficial to the primary sector of the global market (Potts, et al., 2010).


The recent decline in bees can be attributed to many factors. But while the factors have

been thought, in many cases, to be the cause of such a population and global crisis, these factors

have never been considered together as cause for global bee colony decline. This is a pressing

issue which needs to be studied with more intensity and focus on the relationships between

factors as opposed to individual effects. The two most commonly individually studied factors

seem to be the effect of plant pesticides on Apis mellifera and the effect of mite treatments on

Apis mellifera (sources). This is why experimenting to see the effect of mite pesticides and plant

pesticide treatments combined on bee mortality is so important to the future of global

environment health.

Apis mellifera:

The subject of this experiment is the Apis mellifera (or Western honey bee). Western

honey bees have existed for millions of years serving as pollinators (Hester, 2015). Apis

mellifera were brought to North America during the seventeenth century with the American

colonists. This is why Western honey bees are also known as European honey bees (Sammataro

and Avitabile, 1998). Western honey bees are classified as eusocial insects. Eusocial insects are

organisms which cooperate in a cohesive pattern and collectively share attributes of one

organism. Therefore, each colony functions as one cohesive organ system and performs tasks as

a superorganism. Eusocial insects, such as bees, work specifically as nest-bound superorganisms,

meaning the hive members depend on each other to maintain a hive’s (equilibrium) homeostasis.

Honey bees cannot survive individually without the support of a hive or colony (Bonoan, et al,

2014).

Apis mellifera hives maintain homeostasis and functions as superorganisms through a

caste system. The hive caste system dictates which occupants of the hives perform certain tasks.

According to the Ontario Beekeeper’s Association, the queen, workers, and drones make up the


three hive castes. The queen and worker bees are all female while the drone bees are male. The

queen bee decides whether a bee will be born a male drone or female worker through

fertilization. Fertilized eggs are born female workers while unfertilized eggs are born as drones

(2015).

The queen is at the top of the caste system function, and is essential to the survival of a

colony or hive. A queen can be recognized as being slightly larger in size compared to the

average worker bee which has short wings and a narrow abdomen. Queens live mainly to

produce eggs, therefore creating more bees, for the entire hive. Queens have an average life span

of two to four years and during that time laid an average of 365,000 to 547,500 eggs per year.

The queen is vital to hive survival, and because of that, the workers keep a queen well fed and

protected. Young worker attendants care for the queen, and constantly lick and groom her body;

thereby, distributing pheromones which are essential to hive survival. Queen bees become fertile

through a behavior known as the mating flights, in which the queen leaves the hive or colony as

a virgin queen, shortly thereafter, inseminated with thousands of drone sperm. If promiscuous

mating is not successful, the queen would only mate with a low number of drones, leaving the

health of the hive at risk due to the lack of brood (bee pupae) genetic diversity (Hester, 2015).

Worker bees are second in the caste hive system and also vital to hive health, with their

population making up the majority of a hive. Worker bees are infertile females and are a hive’s

smallest bees. These bees serve primarily to keep the cohesive hive organ system running. A

worker bee has several hive responsibilities which change during her life-span. During the first

few days the newly hatched worker bee keeps cells clean and warms brood cells. The next few

days she would proceed in the days after to feed older and then younger larvae. Next, she would

transport food and repair damages to the hive. Some worker bees then go on to attend to the

queen. The penultimate job for some workers is to become a guard bee, which is a bee that


protects/surrounds the queen and hive. The final job of a female worker bee is to forage for

resources. The collection of resources is important for hive survival, and is essential to colony

homeostasis (Hester, 2015).

Drones are at the bottom of the caste system. This type of bee possesses short legs, large

compound eyes, and large flight muscles. They do not perform any tasks throughout the hive and

nor do they feed themselves, because drones either barely or do not possess the physical

characteristics required to forage in the environment. These bees only serve the purpose of

fertilizing virgin queens during mating season. Drones mate by participating in the spring mating

flight, in which bees will fly near a hive and try to fertilize a virgin queen during flight. After

mating with a virgin queen, drones fall to the ground and die. These bees live towards the bottom

of the hive and are usually kicked out of the hive by female worker bees or are dead by winter

time each year (Hester, 2015).

During the winter time bees often face varying temperatures, which signals danger for the

incoming brood and the health of the hive. It is important to note the natural maintenance

practices of the hive. Broods in the hive need to maintain a steady temperature of 35-36o Celsius

at all times for positive growth and development. To keep this temperature constant, the worker

bees of the hive cluster around the brood cells and flex their thoraces (which are pressed against

the pupae) rapidly (Bujok, 2002). Honey bees must constantly regulate temperature because their

hive is immobile. This specific temperature range must be maintained because if temperatures

are not favorable brood mortality and a hindrance in development can occur. Unfavorable

temperatures can also lead to weak immune systems, causing increases in bee risk and

susceptibility to pathogens, pesticides, and disease (Bonoan, et al., 2014).


Pesticides:

Many factors must come together in a hive so a colony can survive, but detrimental

agricultural practices such as intensive pesticide use has proven to hinder hive homeostasis. In

fact, scientists have pinpointed intensive pesticide use as the reason for such global bee

population decline. Researchers Chakrabarti, Rana, Bandopadhyay, Naik, Sarkar, and Basur

recently conducted a study investigating the effects of sub-lethal exposure to pesticide in an

agricultural landscape on the olfaction and overall health of the Indian (Western) honey bee. The

scientists asked the question, if native Apis ceranae are exposed to sub-lethal amounts (1.81

+ .04) of pesticide for a significant amount of time, then how does this affect the bees’ olfactory

system? The results found were that all honey bee samples produced similar results, there was a

decrease in Proboscis Extension Reflex (PER) in High Intensity Crop (HIC) honey bees; there

was a higher intensity of free (Calcium) Ca2+ in the brain of LIC (Low Intensity Crop) honey

bees, and multiple pesticides impact bee olfaction capacity. The results were presented in a

simple and easy to read format with an informative section which described how the results were

found. The authors came to the conclusion that bees being exposed to sub-lethal amounts of

pesticides in agricultural setting impairs the olfactory senses of bees, causing them to lose their

way back to the hive. Bees cannot survive without the support of a colony just as a colony cannot

function without workers (2015).

Research reveals the pesticides which most affects honey bees negatively are

neonicotinoids. Neonicotinoids are commercial insecticides which are used to kill or remove

unwanted pests from plants. Neonicotinoids were introduced into commercial agriculture

production in the 1990’s, and are now the most used pesticides/insecticides in the world. These

insecticides are utilized for agriculture in over one hundred and twenty different countries to

control over one hundred and forty different pests such as moths, caterpillars and other insects


harmful to crop health. Neonicotinoids are so widely used that they were valued at one point five

billion dollars in the 2008 stock market. The pesticide works by remaining present in plant

tissue, killing insects that consume the plants, but are thought to have no toxic effect on

mammals. Numerous studies, including conclusions found in the study conducted by

Chakrabarti, Rana, Bandopadhyay, Naik, Sarkar, and Basur, proves even sub-lethal exposure to

these pesticides are toxic to the honey bee. Many countries and organizations such as the

European Union have taken this information into account and have banned use of the three most

controversial pesticides. These pesticides include Imidacloprid, Clothianidin, and Thiamethoxam

(Lundin, et al., 2015). Honey bee risk is when Apis mellifera have higher chances of being killed

or hurt, and hindering bee population growth and sustainability. Neonicotinoids can hinder

cognition of honey bees, affecting how they forage and increasing Apis Mellifera mortality risk

(Tan, et al., 2014).

One research study confirmed claims that pesticides can even increase chance of

infection due to pathogens and parasites. In Crop Pollination Exposes Bees to Pesticides Which

Alters Their Susceptibility to the Gut Pathogen Nosema ceranae, scientists tested the hypothesis

that if Apis melifera experience consumption of honey bee diets, parasites, diseases and

pesticides in an agricultural setting, then the interaction the bees have with parasites will have

stronger negative effects on managed honey bee colonies. The background provided to support

the hypothesis includes information on research concerning the sub-lethal effects of pesticides on

bees, surveys on the different colony reserves and building material used, and background on

Nosema (the investigated pathogen thought to impair olfactory senses within Apis mellifera).

The results showed that all pollen collected contained pesticides, insecticides and fungicides. The

amount of pesticides found was discovered to be similarly large across all hives. In addition to

the pesticides present, researchers also found 147 out of all 630 bees were infected with Nosema.


Nosema impairs the olfactory senses of honey bees and causes them to become lost on their way

back to the hive from foraging. The researchers came to the conclusion that pesticides used by

farmers have significant effects on a bee’s susceptibility to parasite and Nosema infection (Pettis,

et. al, 2013).

Imidacloprid:

As stated previously, Imidacloprid has been banned from commercial use in certain

countries and organization membership provinces. This is because Imidacloprid is a type of

neonicotinoid pesticide. The metabolites of Imidacloprid aggravate nicotinic acetylcholine

receptors and negatively impact the pollinating behavior of Apis mellifera. Use of Imidacloprid

on crops statistically decreases pollination service quality by 6%-20%. This means that 6%-20%

of crops do not get completely pollinated by the bees. This decrease in performance also

negatively affects bees’ abilities to perform their duties such as enter the hive, forage, and even

return to the hive. A bees’ ability to return to the hive is crucial for bee and hive survival (Tan, et

al., 2014).

Imidacloprid is the most commonly used and researched pesticide (Lundin, et al., 2015).

In one research study, Imidacloprid Alters Foraging and Decreases Bee Avoidance of Predators,

Ken Tan and his fellow researchers hypothesize that if neonicotinoids (harmful insecticides) alter

honey bee foraging behavior, then neonicotinoids would also impair a bee’s ability to sense

danger and avoid predators. To support their hypothesis, Tan provided an extensive background

on how Imidacloprid (a neonicotinoid) decreases foraging activity of bees and how

neonicotinoids affect bee behavior around the world. Neonicotinoids have varying effects on

Apis mellifera around the world depending on region and indigenous species. The results of this

experiment showed there is a huge effect of pesticide concentration on the number of bees which

returned to feeders after foraging, and that increasing pesticide concentration reduces the average


amount of nectar collected, and that exposing bees to heavy concentrations of Imidacloprid

causes bees to not avoid dangerous predators. After gathering all of the results, it was concluded

that neonicotinoid pesticides impair honey bee cognitive responses and also concluded that

significant concentrations of neonicotinoids can impair a bee’s ability to sense and avoid danger.

Neonicotinoids are proving to be a key factor in global bee decline (Tan, et. al, 2014).

Miticide:

Another variable considered in the study is miticide. Miticide is man-made pest control

used heavily around the 1980s and still used today to remove mites from bee hives. In 1984,

Tracheal mites and Varroa mite infestations massively decreased United States bee population,

prompting the invention of miticide. These chemicals served as anthropogenic (man-made) hive

maintenance for years, and are supposed to not harm bees while killing off the mites. However,

evidence has shown that it is difficult to create miticide completely safe for bees yet still be

effective. Even with the advanced technology of miticide, Apis mellifera continue to die off in

colonies at alarming rates (Burley, 2007).

Bee mortality has shown a rapid increase in recent years, and rates have increased in

many regions of the world. Bee mortality rates are the average numbers of bee or colony deaths

per year. The United States has lost fifty nine percent of its bees from 1947 to 2005 and

meanwhile, Europe has lost twenty five percent of its colonies from 1985 to 2005. This

troublesome news proves that mortality rates are rapidly increasing year by year. There are

various consequences associated with this massive bee genocide: significant decrease in crop

yield (amount of food produced), decrease in obtainable profit from crops, and drastic negative

environmental impacts. When speaking specifically of harsh environmental impacts, a few

effects concerning bees are very important to understand. Western honey bees are a primary

agricultural pollinator. The crops they pollinate account for one third of all of the food humans


consume. Without bees, there would be a significant increase in demand for pollinator intensive

crops such as almonds, oil seed rape, watermelon, apples, broccoli, and many others. This

decline causes a decrease not only in biodiversity but also food supply. Pollination is the major

ecological service they contribute to human beings (Burley, 2007).

Nest Homeostasis:

Nest homeostasis is largely affected by the environmental conditions and factors of a

region/area. Homeostasis of a nest is defined as the monitoring and maintenance of internal

environmental conditions and temperatures. Hive homeostasis contributes to large brood rearing,

stable rearing conditions, forager warming, and the seasonal population of the hive. To maintain

hive homeostasis, proper worker bee characteristic (strong thorax muscles, strong wings)

development is of the upmost importance because they will often have to respond to extreme

differences in temperature throughout the year. Compounding problems limits homeostasis of the

hive. It does this by placing physical strain on the growth and development of worker bees, but

regardless of bee architecture, worker bees are still able to properly maintain a hive because of

physiological flexibility. (Winston, 1991).

Bees maintain homeostasis of the hive through temperature control. Honey bees try to

improve internal homeostasis by covering any openings with propolis. Propolis is a sticky sap

which acts as a sealant and provides proper insulation for the hive. During the winter, some

workers will even protect the hive with a curtain or cover of propolis. Comb design and structure

also contributes to hive homeostasis. This is because the hive’s brood chambers are covered with

protective temperature buffering wax comb layers. These layers are also protected by other

worker bees when they flex and contract thorax muscles in clusters around the hive between

combs. This action is often most necessary during winter due to extreme cold temperatures.

Honey bees store their energy during winter and use it through ‘hibernation’ periods to warm the


hive and maintain cell brood temperature. Workers will even consume stored honey to generate

the massive amounts of heat needed to maintain the hive. Extreme heat is not usually a problem,

with most tropical hives being located under shady areas. To produce heat during the winter

clusters, bees gathered together during the heating process, will often break apart to allow these

bees to feed for continuation of proper hive and brood warming. Even with significant honey

reserves, a hive can die during winter because bees were not allowed to occasionally leave the

cluster to feed. There is almost no brood rearing during this time period because of the constant

temperature fluctuations in the hive (Winston, 1991).

In conclusion, there are various factors at play when it comes to bee colony decline. Most

scientists in the past have believed it to be attributed to an individual factor when in truth, it may

have been a combination of factors all along. Each factor discussed in detail has proven to have

significant impacts on the behavior, but especially the mortality of Apis mellifera. The wide

disparity between studies involving factor combinations versus individual factor study without

evidence for a pinpoint source of bee colony decline suggests there might be some knowledge

left to gain. While individually the factors of Imidacloprid and miticide use have been thought to

negatively impact the mortality rate of Apis mellifera, the notion of combining these factors to

see if they have somewhat of a “synergistic” effect on Apis mellifera mortality seems to be a

concept worth exploring. Global hive homeostasis trends may truly be dependent on more factors

than just the two being studied, but in terms of research progression, the next step clearly is to

take combinations of factors into account. If the study conducted shows any amount of statistical

significance, it may be high time to start looking into more and more factor combinations. This

study focuses on looking outside the box in research methodology to find additional causes of

bee colony decline.


Hypothesis

If Apis mellifera are exposed to combinations of miticide to treat Varroa mites and

neonicotinoids, then hive mortality rates will be higher than mortality rates of those treated

individually with only miticide or only pesticide.

Justification

The purpose of this study is to expand understanding on the impact of how inadvertent

chemical combinations affect bee mortality and colony health. The combination of

neonicotinoids, a systematic pesticide widely used on food crops, with commonly used Varroa

mite treatments have individually proven in the past to be indicators of bee colony decline. Bee

colony decline is a plague which has spread throughout the globe, killing off entire populations

of bees. There have been several indicators hypothesized to be the cause of this massive bee

colony decline, but so far research has not produced conclusive results. Little to no research

however, has been conducted on how these indicators might work in tandem to cause bee colony

decline or increase bee mortality. The mite treatment and plant pesticide used in the experiment

are used most frequently on crops and hives around the world. Therefore, this study is vital to

expanding the understanding of how these indicators in combination affect bee death not just in

Western honeybees, but also bee species around the globe which are most exposed to these

commonly used plant pesticides and mite treatments.

Materials:

1 hive nucleus 1 Thermo scientific incubator (in lab)

1 Corning Stirrer/Hot Plate (In lab) 1 Teflon magnet coated stir bar (in lab)

1 Micro pipette 1 scientific balance (in lab)

Mason jars (for solutions) 140 cotton balls

Ziploc containers (cages) 2lbs Oxalic acid


2lbs Imidacloprid Screen Wire

Estimated Budget

1 hive nucleus - $150.00

16 16oz. Mason Jars (for solutions) - $57.60

20 Ziploc containers (cages) - $10.99

250g Oxalic acid - $80.70

100 mg Imidacloprid - $60.50

Cotton ball jumbo bag - $1.50

Phifer 48in by 25ft charcoal fiberglass screen - $34.86

Estimated total: $396.15

Methods: Step by Step

1. An insulated location to conduct the experiment was secured. Conducted the experiment

at the Georgia Tech ecology lab in Atlanta, Georgia.

2. In the ecology lab, there were three different solutions made for experimental use. First,

two liters of 1:1 sucrose solution were made by measuring out on a scientific balance

1000 grams of sugar and mixing it with 800 ml of water in a two liter glass bottle. The

solution was then placed on a hot plate and mixed together by a Teflon magnet coated stir

bar. Next, the Imidacloprid solution was made by placing .0125grams in a 500milliliter

bottle and mixing it with the previously prepared sucrose solution poured into the 500

milliliter bottle. .0125 g is the amount of imidacloprid bees are on average exposed to in

the field. Finally, the Oxalic acid solution was made by mixing 35g of Oxalic acid

powder in a 500ml glass bottle with sucrose solution.


3. After the solutions were made, frames were collected with a majority of capped brood

and placed in a Thermo-incubator. The incubator temperature was set to thirty-five

degrees Celsius and the brood were left to emerge from their caps overnight.

4. Twenty bee cages were then constructed to hold Apis mellifera for experimentation.

a. Twenty Ziploc containers were purchased. Then, a square three inch by three inch

hole was cut in each container lid and pre-cut screen wire was duck taped to the

lid top.

b. Twenty cages were created and each cage was labeled by group. The four groups

were Control (1:1 Sucrose Solution), Imidacloprid w/Oxalic acid, Imidacloprid

Only, and Oxalic acid w/ 1:1 Sucrose Solution. There were five cages designated

to and labeled for each experimental group.

5. The next day, the newly emerged bees were taken from their frames and placed in cages.

Ten bees were placed in each cage, meaning there were fifty bees designated to each

experiment group.

6. Each cage was then treated with its designated group treatment

a. Control Group – Cotton balls were drenched with 1:1 Sucrose solution, put on a

microgram (mg) weigh boat, and placed in each control group cage to orally feed

the Apis mellifera.

b. Imidacloprid w/Oxalic acid Group – Cotton balls were drenched in Imidacloprid

solution and placed in each Imidacloprid/Oxalic acid cage on an mg weigh boat.

Then oxalic acid was applied by using a micro-pipette to place one twenty micro-

liter drop on each bee in the cages.

c. Imidacloprid Only - Cotton balls were drenched in Imidacloprid solution and

placed in each Imidacloprid only cage on an mg weigh boat.


d. Oxalic acid w/ 1:1 Sucrose Solution - Cotton balls were drenched with 1:1

Sucrose solution, put on a microgram (mg) weigh boat, and placed in each Oxalic

acid w/ 1:1 Sucrose Solution group cage to orally feed the Apis mellifera. Then

oxalic acid was applied by freezing the cages for ninety seconds and afterwards

using a micro-pipette to place one twenty micro-liter drop on each bee in the

cages.

7. After administering the different treatments, the bee cages were placed back in the thirty-

five degree Celsius incubator and left over night.

8. After twenty-four hours, each cage was removed out of the incubator and a death count

was taken. Results and observations were then recorded and analyzed.

Results:

The project measured the effect of imidacloprid pesticide combined with oxalic acid mite

treatment on Apis mellifera mortality through an unpaired t-test and averages. By looking at

figure one, it is obvious to see the differences in mortality using oxalic acid vs. not using oxalic

acid. The control group were bees which had no oxalic acid administered. The graph shows that

no apparent deaths were recorded after the twenty-four hour experiment period. Results were

also recorded for Apis mellifera which were fed imidacloprid only. The graph shows that feeding

Apis mellifera imidacloprid only produces small amounts of mortality. On average those bees

which were fed imidacloprid only had low mortality rates. Results were also recorded for Apis

mellifera which were fed imidacloprid solution and treated with oxalic acid. The graph shows

very high mortality rates for this category, and has the highest mortality rates out of all the

experimental groups. The last group results were recorded for was oxalic acid treatment with

bees fed imidacloprid solution. This group also showed high mortality rates after a twenty-four

hour period, but proved to have an average mortality rate ten percent less than that of the


imidacloprid w/oxalic acid group. A Chi Square test was used to measure the significance of

these results combined. A Chi Square test was used because it compares the expected to

observed results of two or more variable groups. This test had more than two variable groups and

means, therefore requiring the use of a Chi Square test to measure significance. The Chi Square

test found a P-value of 5.9 x 10−13. The P-value rejects the null hypothesis the number of bees

that survived are equally distributed. This result is strongly significant, and suggests that the

combination of imidacloprid pesticide and oxalic acid mite treatment greatly increases average

Apis mellifera mortality.

Figure one displays the collective results of Apis mellifera death after twenty-four hours.

By looking at figure one and Apis mellifera death raw data one can infer that control had no bees

die during after each trial. It is also safe to infer that any combination using oxalic acid, whether

it be with sugar water or imidacloprid, results in high amounts of bee deaths after each trial.

From these results it is clear to see that the combination of imidacloprid with oxalic acid and the

administration of oxalic acid and sugar water (Appendix C).

Figure 1: Apis mellifera Death after 24hrs


From figure two, it is clear to see that Apis mellifera not treated and only fed sucrose

solution have on average no mortality and an extremely high percent survival rate after a twenty

four hour period.

Figure 2: Control Group Average Death & Survival


From figure three, it is clear to see that Apis mellifera treated with imidacloprid only have a

relatively low average mortality rate and moderate average survival rate after a twenty four hour

period. There exists in the graph on average a twenty percent survival rate and eighty percent

death rate.

Figure 3: Imidacloprid Only Group Death Average Death & Survival


From figure four, it is clear to see that Apis mellifera treated with oxalic acid and fed 1:1

Sucrose solution have a relatively high average mortality rate and moderately low average

survival rate after a twenty four hour period.

Figure 4: Oxalic Acid w/ 1:1 Sucrose solution Group Average Death & Survival

From figure five, it is clear to see that Apis mellifera fed Imidacloprid and treated with

oxalic acid have an extremely high average mortality rate and an extremely low average survival

rate after a twenty four hour period.

Figure 5: Oxalic Acid w/Imidacloprid Average Death & Survival


From these various averages, it can be determined that on average the combination of

imidacloprid and oxalic acid is ten percent more potent than the mortality rates of Apis mellifera

just being treated with oxalic acid and fed the 1:1 sucrose solution. It can also be determined that

the mortality rate average of bees treated with oxalic acid and fed with imidacloprid is seventy-

two percent higher than the average mortality rate of bees just fed imidacloprid only.

International data referenced in the lit review suggests that imidacloprid on average resulted in

the most bee deaths, due to imidacloprid’s ability to interfere with Apis mellifera olfactory

senses (Chakrabarti, et. al, 2015). That study focuses solely on one factor (or perceived cause) of

the recent trend in bee mortality increase, and its results contradict the findings of this study.

Thus, suggesting that research with combinations of factors needs to be more thoroughly

researched when it comes to finding the ultimate cause of global Apis mellifera mortality

increase.

Discussion:

The purpose of this study is to expand understanding on the impact of how inadvertent

chemical combinations affect bee mortality and colony health. The combination of

neonicotinoids, a systematic pesticide widely used on food crops, with commonly used Varroa

mite treatments have individually proven in the past to be indicators of bee colony decline.

Researchers have studied these factors individually multiple times, often resulting in numerous

single-cause studies, but currently there is little to no research on how combinations of these

factors affect Apis mellifera mortality. Bees come into contact with many chemicals, pesticides,

and particulates in the field, making it easy for them to “pick-up” two or more possibly

synergistic factors. More research on combinations of strong-correlation factors regarding Apis

mellifera should be conducted, because there are many individual factors that could possibly

have a negative synergistic effect on bees.


From the results of this project, it is clear that the combination of feeding Apis mellifera

imidacloprid pesticide solution produces significantly high mortality rates for bees compared to

other experimental groups. According to the Graph Pad Software Inc. unpaired T-test, the results

of this group compared to that of other experimental group mortality rate showed a statistical

significance of .9286. This is extremely significant, and indicates that the combination of

neonicotinoid pesticides such as imidacloprid with the mite treatment of oxalic acid is very

potent to bees. The high mortality rates of even just that of bees treated with oxalic acid and fed

1:1 Sucrose solution (average of 82% mortality) indicates also that the use of oxalic acid to treat

mites might be hazardous to the health of bees. This is important to consider for any commercial

beekeepers or even hobby beekeepers in the United States especially, because oxalic acid

globally is presumed to be safe for usage and is slowly starting to gain tentative approval for use

by beekeepers. The results presented suggest overall, it is not safe to use oxalic acid dribble to

treat mites and treating crops with imidacloprid most likely will enhance the negative effects of

utilizing it.

This project is the first project to try to find a correlation between combinations of

pesticide and miticide honey bee exposure and globally decreasing Apis mellifera population.

Because of this, there is little to no information to compare it to. However, there is other data

about the effects of these factors individually correlating to global bee decline. In China, the data

for imidacloprid showed that testing at the experiment’s highest dose the result significance was

P = .012, respectively . This shows low significance in terms of p-value testing. Data in India

studying only the effects of intensive pesticides found olfactory senses were impaired

significantly by p<.01 using the 1 tail Mann Whitney U Test (Chakrabarti, et al., 2015). Those

results are of generally low significance. The results of the experiments in China and India

indicate why it is becoming more and more relevant to study factor combinations. Studying


factors individually does not provide enough statistically significant evidence to pinpoint one

factor as the main cause of the recent trends in global Apis mellifera decline.

There are three major improvements which should be made to this project. One major

improvement suggestion is to possibly wait a longer period of time before applying treatment to

the Apis mellifera. It would possibly be best to wait a day or so before administration of these

chemicals and pesticides because new-born bees have soft cuticles, which absorb liquids and

other chemicals relatively quickly compared to those bees of 2-3 days old with hardened cuticles.

Another improvement suggestion is to possibly lower the dose of oxalic acid administered to the

Apis mellifera. This might improve the experiment because bees are exposed to different dosages

in different countries, including India, often with lower dosage standards than the United States

gives for commercial usage of the product. A final improvement on experiment suggestion is to

handle the bees more carefully. While putting the new bees in their cages some exhibited

symptoms of diarrhea and vomit. This was probably due to the fact that many bees had to

quickly be placed in the cages, possibly causing discomfort and panic. This may affect results

being that they do not experience that process in their natural environment.

There are multiple ways to expand and extend this project. One future experiment to

conduct would be the effect of cuticle formation on Apis mellifera toxin susceptibility. That

experiment could be used to either support or deny the notion that soft cuticles make bees more

susceptible to absorption of harmful substances. The project could also indicate what time of

year is best to treat Apis mellifera as a commercial or hobby beekeeper. Another future

experiment would possibly explore honey bee diet, and look to see the effect of honey type on

the overall health of bees. This project could explore the various contents of multiple honey

types around the world, and even examine what content is in the honey of the country with the

largest population of bees. A final future experiment suggestion is a honey bee psychological


study which focuses on how honey bee foragers choose the flowers they pollinate. This will help

scientists and the entire beekeeping community learn which flowers honey bees are most

attracted to and possibly need to plant more of. There are various future experiments which can

extend further upon the effect of imidacloprid pesticide combined with oxalic acid mite

treatment on Apis mellifera mortality.


Bibliography

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dissipation in the honey bee (Apis mellifera) hive. Naturwissenschaften, 459- 465.

Doi: 10.1007/s00114-014-1174-2

Bujok, B., Kleinhenz, M., Fuchs, S., & Tautz, J. (2002). Hot spots in the bee hive.

Naturwissenschaften, 89 (1), 299-301. Doi: 10.1007/s00114-002-0338-7.

Burley, L. (2007). The Effects of miticides on the reproductive physiology of honey bee (Apis

Mellifera L.) queens and drones, 1(1), 1-91.

Chakrabarti, P., Rana, S., Bandopadhyay, S., Naik, D., Sarkar, S., & Basu, P. (2015).

Field populations of native Indian honey bees from pesticide intensive agriculture

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Retrieved From http://www.nature.com/articles/srep12504.

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and their impacts on bees: a systematic review of research approaches and identification

of knowledge gaps. PLoS ONE, 10(8): e0136928. doi:10.1371/journal.pone.0136928

Pettis, J., Lichtenberg, E., Andre, M., Stitzinger, J., Rose, R., & VanEngelsdorp, D. (2013).

Crop pollination exposes bees to pesticides which alters their susceptibility to the gut

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345-353. doi:10.1016/j.tree.2010.01.007

http://www.nature.com/articles/srep12504


Tan K, Chen W, Dong S, Liu X, Wag Y. (2014). Imidacloprid alters foraging and

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Sammataro, D., A. Avitabile. 1998. The beekeeper's handbook, 3rd edition. Ithaca, New York,

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Appendix

Appendix: Page 24 Table of Contents

Appendix A: Page 25 Pictures of Materials

Appendix B: Page 26-27 Pictures of Experiment

Appendix C: Page 28 Apis mellifera Death after 24 Hours Raw Data

Appendix D: Page 29 Apis mellifera Death after 24 Hours Graph

Appendix E: Page 30 Apis mellifera Average Death after 24 Hours Raw Data

Appendix F: Page 31-32 Apis mellifera Average Death after 24 Hours Graphs

Appendix G: Page 33 Acknowledgements


Appendix A

a. Bee Cages b. Honey Bee Brood Comb

c. Sucrose Solution d. Oxalic Acid

e. Imidacloprid f. Incubator Chamber


Appendix B

A. Bee Cages with Hive Nucleus B. Capped Brood Cells

C. Bee Cage with Cotton Ball D. Glass Solution Jars


E. Making Sucrose Solution F. Dissolving Sucrose into water

G. Measuring Out Imidacloprid H. Georgia Tech Urban Beekeeping Rooftop


Appendix C


Appendix D


Appendix E

Apis mellifera Death after 24hrs

Treatment Type Percent Survival Percent DeathControl (1:1 Sucrose Solution) 100% 0%Imidacloprid w/Oxalic Acid 8% 92%Imidacloprid Only 80% 20%Oxalic Acid w/Sucrose Solution 18% 82%


Appendix F


Appendix F:

Acknowledgements

First and foremost, I would like to thank my wonderful, amazing, talented mentor for

helping me through this process. I certainly could not have asked for a better person to not only

advocate for my project, but also be an advocate for me. When I started this project I did not

think I would ever apply for a grant, acquire membership into the Metro Atlanta Beekeeper’s

Association or even the Georgia Beekeeper’s Association, attend an amazing seminar on

beekeeping, use a smoker, and even have the ability to conduct my experiment in a professional

ecology lab. To me you truly are the world’s greatest mentors and one of the world’s greatest bee

lovers, and I could not have asked for a better advocate or a better person to help me on this

journey.

Second, I would like to thank the Georgia Beekeeper’s Association. Without your help, support,

and grant for my research, this project may not have been possible. It is truly inspiring to me to

have a group of people who believe in me and support my ambitious endeavors. I will be forever

grateful

Finally, I would love to thank Dr. Jennifer Leavey and the Georgia Tech Ecology lab for

allowing me to conduct my experiment in their facilities. It is a young researcher’s dream come

true. I can only imagine what would have happened if I had gone another direction without a

proper lab, so thank you for supporting such an ambitious project and an even more ambitious

young lady.

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