AN ABSTRACT OF THE THESIS OF
DEBORAH ANNE DELANEY for the degree MASTER OF SCIENCE
in ENVIRONMENTAL SCIENCES presented on December 10,
2003.
Title: CONSEQUENCES OF COUMAPHOS AND VARROA
DESTRUCTOR ON DRONE HONEY BEE SPERM QUANTITY.
Abstract approved
Redacted for privacy
The number of drones and genetic diversity among drones are
essential components to a well mated queen. Varroa destructor
preferentially parasitizes drone brood, and is thought to be
responsible for the loss of feral populations that once provided
additional drones for honey bee mating areas. It is necessary to
use miticides (e.g. coumaphos) in managed colonies to control V.
destructor. Little is known about the sublethal effects of these
compounds, which are directly introduced into the hive. In
response to growing concerns about the successful mating of honey
bee queens, drone honey bees were exposed to coumaphos, during
drone development. Sperm and seminal vesicles were sampled
among drones that were exposed to coumaphos and drones that
were not exposed to coumaphos, but were parasitized by Varroa
destructor. There were no significant differences found between the
two treatments in terms of seminal vesicle size and sperm
numbers. These results indicate that drones parasitized by V.
destructor have similar sperm quantities as drones exposed to
coumaphos.
Consequences of Coumaphos and Varroa destructor on Drone
Honey Bee Sperm Quantity
by
Deborah Anne Delaney
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented December 10, 2003
Commencement June 2004
Master of Science thesis of Deborah Anne Delaney presented on
December 10, 2003.
APPROVED:
Redacted for privacy
'
Professor,
senting Environmental Sciences
Redacted for privacy
Co-Major Pr4'fessói reØi-eentirg Enironmental Sciences
Redacted for privacy
Director of tltéEnvironmental Sciences
Redacted for privacy
Dean of the Grflafe School
I understand that my thesis will become part of the permanent
collection of Oregon State University libraries. My signature below
authorizes release of my thesis to any reader upon request.
Redacted for privacy
Deborah Anne De1hey, Author
ACKNOWLEDGEMENTS
I would like to thank Dr. Lynn Royce. She has been a
wonderful mentor and a true friend. Without her instruction and
guidance I would not have been able to complete this degree. I
would also like to thank Dr. Dennis Michael Burgett for believing in
me and introducing me to the wonderful world of bees. His stories,
humor and candle making are wonderful memories that I will have
forever. Sincere appreciation is also extended to Dr. David Williams
for serving on my graduate committee.
I am grateful to Dr. William Winner, former head of the
Environmental Science program, for advising me through the
program. I also want to thank Dr. Lynda Ciuffetti and her lab for
sharing their camera and microscope.
I also want to acknowledge the help of Dr. Nancy Baumeister,
Leigh Ann Harrod and Marina Meixner. Their statistical expertise
was greatly appreciated. I want to give a special thanks to Dr.
Walter S. Sheppard for believing in me. I could not have completed
this task without your help and support. To my friends and family
in Corvallis, thank you for your friendship and community.
Most of all I wish to thank my husband and my son,
Michael and Levi MorreU, who are a constant source of strength
and inspiration.
TABLE OF CONTENTS
Page
INTRODUCTION
1
MATERIALS AND METHODS
9
Drone Rearing
9
Parasitism Assessment
13
Seminal Vesicle Dissection
13
Semen Extraction
14
Photographing Sperm and Sperm Counts
15
Data Analysis
16
RESULTS AND DISCUSSION
16
CONCLUSION
23
BIBLIOGRAPHY
25
LIST OF FIGURES
Figure
Page
Drone honey bee semen stained with DAPI.
15
The effects of treatment on sperm counts.
19
LIST OF TABLES
Table
Average number of sperm per drone
from each colony.
17
Average sperm count per drone
sample for each
treatment.
18
Descriptive statistics of sperm
count means for coumaphos
and control honey bee colonies.
18
Analysis of Variance for sperm
Counts of drone honey bees.
19
Analysis of Variance for seminal
vesicle measurements of drone
honey bees.
20
6
Average seminal vesicle measurements
for drone honey bees for each colony.
20
7
Average seminal vesicle measurements
for drone honey bees for each
treatment.
21
1
2
3
4
5
Consequences of Coumaphos and Varroa
destructor on Drone Honey Bee Sperm
Quantity
INTRODUCTION
Drones are an important biological component of honey bee
populations. Although they do not forage or perform work inside
the honey bee colony, they carry important genetic information
which they contribute to the next generation of workers. The loss
of feral populations of the honey bee to the parasitic mite, Varroa
destructor Anderson and Truman, has resulted in a reduction of
genetic variability which was once available for breeding with
managed colonies (Collins & Pettis, 2001).
Drones are fed and cared for by the workers, and their large
body size requires significant resources. The queen produces
drones in early spring (Winston, 1987). The colony expands quickly
in late spring and mature drones and virgin queens are required at
this time to re-colonize the hive when the colony swarms.
V.
destructor matches their peak in reproduction with drone
production in their honey bee host (Ritter, 1988). Beekeepers often
treat honey bee colonies with mite control products, such as
coumaphos, during the early spring to reduce levels of this mite.
Thus coumaphos can be present in the hive during drone rearing.
A drone's main function in life is to mate. Mating in honey
bees occurs at specific sites called drone congregation areas
(Winston, 1987). Drones gather at these aerial sites awaiting the
arrival of virgin queens. These congregation sites vary in size, from
30-200 m in diameter. The drones typically fly at a height 10-40 m
above the ground (Ruttner and Ruttner, 1966). Depending on the
weather and the number of colonies present these sites contain
from a few hundred to thousands of drones. Once a queen arrives,
drones pursue the queen and often form a comet-shaped cloud
following her (Winston, 1987).
Due to the genetic. contribution drones provide to future
generations, it is important to have large numbers of drone brood in
the colony during mating season. Seeley (2002) suggested that
more drone brood present in a colony fosters a more favorable
environment for mite production. Seeley's study found that the
natural amount of drone comb in colonies was 20%. Given the
resources required to rear drones and the fact that V. destructor
reproduces quite well on drone brood, some beekeepers attempt to
remove and kill drones. However, large numbers of drones are very
important to the successful mating of queens. Using micro-
satellites, Estoup et al. (1994), estimated that an Apis
meltfera L.
queen mates with 20-27 drones. Micro-satellites are areas in the
3
DNA that are non-coding and quite variable due to errors in
replication. This type of genetic marker can be used to determine
differences among populations. Rinderer et al. (1998)
recommended that 60 drones should be supplied for each queen,
and that twenty drones that successfully mate are crucial. A poorly
mated queen can result in an early supersedure (Camargo &
Goncalves, 1971). Clearly, good drone populations are critical to
the diversity of the gene pool of A. meUfera.
Since 1987, beekeepers in the United States have had to
control populations of V. destructor, an introduced honey bee mite
from Asia. It is an ectoparasite of Apis cerarta that host-shifted to
A. met itfera within historical times. Varroajacobsoni was initially
described in 1904 as a parasite of A. cerana in Sumatra by A.C.
Oudemans (Dc Jong et al. 1982b). In the 1960's it was
rediscovered in the Philippines and reported to be a parasite of A.
melt fera (De Jong et al. 1982b). Although, A. mellifera and A.
cerana were originally allopatric species, A. mellfera has been
extensively imported into much of the range of A. cerana in
southeast and eastern Asia. More recent taxonomic analysis
classified the mite that colonized A. mettfera as V. destructor
(Anderson & Truman, 2000).
V. destructor is a parasite restricted to cavity nesting Apis
species. The constant microclimate the bees maintain in their nest
4
allows the mites to flourish regardless of the ambient temperature.
V. destructor has co-evolved with A. cerana, and has developed
mechanisms that allow it to co-exist with this mite. A. cerarta will
groom the mite off the bodies of their sisters and remove them from
worker brood (Peng et al. 1987). Thus, V. destructor only infests
drone brood in A. cerana colonies, and infestations do not lead to
colony death (Harbo & Hoopingarner, 1997). In A. nie1Lfera
colonies, V. destructor parasitizes both worker arid drone brood, but
prefers drone brood. The number of mites in an infested colony can
range from 3,000 to 11,000 mites (De Jong et al. 1982b). An adult
worker may cany up to 5 mites and a drone may carry up to 12
mites. Twenty mites have been found in a single drone cell (De
Jong et al. 1 982b). This preference for drone brood has been
shown to decrease the number of mature drones available for
mating (Donze & Guerin, 1997).
When populations of temperate zone A. meLIfera are infested
with V. destructor, colonies typically die within one to two years. It
is thought that feral honey bee populations in the United States
were decimated with the arrival of this parasite (Collins & Pettis,
2001). The reduction of feral colonies could then be responsible for
a decrease in the number of viable drones that surround queen
mating yards in many areas.
In response to increasing population levels of V. clestructor, a
cascade of symptoms occurs in honey bee colonies, called parasitic
mite syndrome. (Shimanuki et al. 1994). The symptoms include
reduced weight of workers and drones (Choi, & Woo, 1974), a
reduced level of glycoprotein expression in drone sperm (Del Cacho
et al. 1996) and overall colony population reduction. Wing and
limb deformities are also associated with V. destructor parasitism
(De Jong et al. 1982a). Other work suggests that Varroa increases
the susceptibility of honey bees to viruses and diseases leading to
colony mortality, such as Acute Paralysis Virus (APV) (Ball & Allen,
Rinderer et al. (1998) showed that V. destructor adversely
affects drones and leads to a decrease in the number of drones that
reach sexual maturity. Schneider (1986) found a correlation
between the intensity of infestation of the drone brood by V.
destructor and a decrease in drone emergence and, weight and size
of seminal vesicles and mucus glands. The number of spermatozoa
was also shown to decrease by 50% when three or more mites were
found parasitizing drone pupa. This is understandable, considering
that maturation of the sperm takes place during the pupal phase
when V. destructor is feeding on the hemolymph (Collins & Pettis,
2001). Ritter (1988) also found that V. destructor present in drone
cells caused a reduction in body weight, seminal vesicles and the
mucus glands of parasitized drones. Del Cacho et al. (1996)
studied a group of proteins called lectins. These proteins aid sperm
in binding to the egg prior to fertilization. V. destructor reduced the
amount of cells that provided good lectin binding sites.
Because the loss of untreated colonies to V. destructor is
almost a certainty in temperate climates, beekeepers routinely use
chemical intervention in their hives. Presently, the two most widely
used chemicals registered for use for V. destructor control in the
United States are Apistan® and CheckMite+®. The active
ingredient in Apistan® is fluvalinate, a synthetic pyrethrin. It is
applied to colonies in a plastic strip that is left in the colony for
about eight weeks. Although originally quite effective, the efficacy
of fluvalinate is dwindling due to an increased mite resistance
worldwide (Milani, 1995; Elzen et al. 2000).
Checkmite-i-® is a formulation containing coumaphos, an
organophosphate, as the active ingredient. This prOduct was given
a section 18 emergency use approval by the Environmental
Protection Agency in 1998 for use in colonies in response to the
growing resistance of V. destrcutor to Apistan® and the arrival of
another introduced honey bee pest, the small hive beetle. This
chemical is applied to the colony in a similar formulation to
Apistan®, as a plastic strip impregnated with 10% coumaphos by
weight (Weick & Thorn, 2002).
7
Coumaphos is lipid soluble, and is retained in the wax comb
in the hive. This propensity to migrate into wax has led to specific
use restrictions and label changes to reduce contamination of
honey. Resistance to Checkmite® by V. destructor has already
been documented in the United States (Elzen & Westervelt, 2002).
Few studies have shown effects of these chemicals,
coumaphos and fluvalinate, on the different honey bee castes.
Haarmann et al. (2002) looked at the effects of fluvailnate and
coumaphos on developing queen honey bees. The presence of
coumaphos in colonies was negatively correlated with queen and
ovary weights. Rinderer et al. (1998) examined the effects of
Apistan® on drones. They reported that Apistan® reduced the
number of drones that reach sexual maturity in a colony. Drones
in colonies treated with Apistan® had a higher mortality than
drones in colonies infested with V. destructor. This study also
showed that Apistan® reduced the weight of drones by 5%, and
also significantly reduced the weight of seminal vesicles.
Organophosphates are paralysis-inducing insecticides, which
act by inhibiting Acetylcholine esterase. Acetylcholine plays an
important role in the CNS of invertebrates (Weick and Thorn, 2002).
Organophosphates have also been shown to affect male
reproductive function. Sperm motility and quantity were reduced
when organophosphates were applied to the skin of bovines
8
(Gagnon, 1990). Phorate, another organophosphate, was shown to
lower the amount of spermatids and spermatocytes in the testis of
gerbils (Davies, 1980). A recent study by Weick and Thom (2002)
showed that the organophosphate diazinon, had effects on odor
learning in A. me11fera when small doses were applied dermally or
injected intracranially. Coumaphos did not show inhibitory effects
on the odor learning behavior of honey bees when applied in these
fashions.
There is no doubt that drone fitness is crucial to the health of
honey bee colonies. The number of drones and genetic diversity
among drones are essential components to a well-mated queen.
Therefore, any reduction in drone numbers or drone fitness is
detrimental to the beekeeping community.
If V. destructor has reduced the feral population that provided
drones for honey bee mating areas, managed colonies must provide
the source of drones for the future of US beekeeping. Unfortunately
it is necessary to use miticides in managed colonies to control V.
destructor. It is important to investigate the subtle effects of
compounds that are directly introduced into the hive. The effect of
coumaphos on drone survival and fitness is not well understood.
To date, there are no studies showing the effects of coumaphos on
drone performance and fitness. The research presented here
examines the effects of coumaphos on sperm quantity in drone
honey bees in Apis meUfera.
MATERIALS AND METHODS
Drone Rearing
During the first field season, three treatment regimens were
followed. The colonies were started in April of 2000 from fifteen
three pound packages. The packages were initially treated with
Apistan ®, to reduce mite levels in all the colonies. The colonies
were randomly assigned to one of three different treatments: high
mite, low mite/no mite and with coumaphos. Once the first drone
eggs were laid, coumaphos was put into the coumaphos treatment.
The colonies started with low to zero mites. Therefore, one frame of
brood was taken from colonies with mite infestations and was
inoculated into each colony in the high mite treatment.
Drone brood was removed just prior to emergence and
allowed to emerge in an incubator. Once the drones emerged in the
incubator they were marked and put back into their respective
colony until they reached maturity. Drones reach maturity about
12 days after emergence when their reproductive organs are
finished developing and their semen has matured. During this field
season two problems arose. The first problem was drone
10
emergence. The drones in the incubator did not emerge in
adequate numbers. Over 50% of the drones died prior to
emergence. The second problem arose when the drones were
recaptured from the hive after they reached maturity (12-14 days).
Less than 30% of drones were recaptured and survived to maturity.
Due to the small sample number the experiment underwent
changes in the design.
The following season (2001), cages were designed that
allowed the drones to be incubated directly in each colony.
However, only ten colonies remained that did not contain
coumaphos residues. The five colonies that contained coumaphos
the previous year were abandoned, because of any possible effects
coumaphos exposure might be having on the queens. The ten
remaining colonies were randomly assigned to either coumaphos or
a non-coumaphos mite treatment.
Ten colonies, housed in standard Langstroth hives were used for
drone rearing. These ten hives were treated with Apistan in the fall
of 2000 to equalize the mite loads in the hives. In March of 2001
brood and honey stores were equalized in all the hives. Hives were
randomly assigned treatment regimes, and five hives were treated
according to label instructions with coumaphos (Checkmite ®+) in
April of 2001. The other five hives were untreated. Natural mite
drop was measured to assess the initial mite load of the ten
11
colonies. Sticky boards were placed in the bottom of the hive body
under screens for 72 hours. After 72 hours the boards were
removed and all mites were counted.
Cages were assembled from drawn out deep frames, 23 x 42
cm, containing drone comb, and 1/8-inch mesh and queen
excluder material. The mesh was fitted around the frame to provide
an enclosed cage. A square of queen excluder material was fitted in
the middle of the cage and served as a door to the cage. This
design allowed the queen to lay in the enclosed drone comb at a
specified time.
Drone cells are larger than worker cells.
The
worker cells are generally 5.2-5.4 mm in diameter, and drone cells
are typically 6.2-6.4
nmTI
in diameter.
It was important for the
combs to have as much drone comb as possible to ensure that a
large number of drone eggs were laid. This cage design also allowed
the workers to feed the drones once they emerged. Each of the ten
hives had one cage. Due to the large size of the cage, each brood
chamber that held a cage contained a total of eight frames. The
queen from each colony was put inside the cage and allowed to lay
eggs for 1-3 days, ensuring that all the drones that emerged in the
cage were similar in age. Drones remain in the egg stage for three
days. Once the queen laid a sufficient number of drone eggs she
was removed and put back into the main hive body. From the time
the egg is laid, drones develop for ten days before they are capped
12
with wax by workers. This capping period and the formation of a
silken cocoon within the cell marks the beginning of their pupal
stage. It takes about 24 days from the time the egg is laid for a
fully formed drone to finish developing and chew out of the capped
cell.
The drones were observed every three days for twenty-four
days or until emergence.
Once the drones emerged, 30 to 50 drones per hive were
marked with acrylic paint and placed back into the cage where they
could be fed by workers. The drones were marked each day over a
three day period, ensuring that all the drones would only be about
three days apart in age. The drones were marked to track their age
from the time of emergence. Drones do not reach sexual maturity
until around twelve days after emergence. At this point they can
yield semen. The colors were specific to the hive and the treatment,
providing a color key to the parent home for each sample of drones.
At twelve days, marked drones were collected and their seminal
vesicles were measured. A sperm sample was taken from each of
the two seminal vesicles for each drone.
13
Parasitism Assessment
From four out of the five surviving non-coumaphos colonies
drone brood was pulled while emerging, and the number of adult
Varroa mites and deutonymphs were counted on the drone and in
the brood cell it emerged from. This procedure was done for each of
the surviving non-coumaphos colonies in order to assess parasitism
levels during drone development.
Seminal Vesicle Dissection
Due to the variability in the amount of sperm produced from
mature drones, the seminal vesicles were dissected. This allowed
for a consistent volume of sperm to be extracted from each vesicle.
This same procedure is used in other insect studies attempting to
quantiQr sperm production (Beaver et al. 2002). Before sperm
extraction, each seminal vesicle was measured (length and width).
Drone cages from both a coumaphos colony and an untreated
colony were collected and put into separate nucleus hives with 3-5
frames of workers and honey. The nucleus hives with drone cages
were transported to the laboratory for analysis. A total of 91 drones
were sampled between May and July of the 2001 field season. Ten
mature marked drones from each treatment were collected from
cages and put into the freezer until immobilized. Dissections were
as follows: the head and thorax were removed. The abdomen was
14
pinned into a Petri dish lined with beeswax and incised down the
middle with micro-scissors. The seminal vesicles were dissected
out into 0.85% saline solution. The length and width were
measured under 8X magnification using an ocular micrometer. The
length and width of both seminal vesicles were recorded.
Semen Extraction
A semen sample of 0.5 ul was drawn into a capillary tube with a
syringe from each seminal vesicle. The semen was put onto a slide,
and diluted with 50u1 of 0.85% saline solution, and then mixed to
separate any sperm bundles or clusters. A sub-sample was
collected and streaked across a slide for each vesicle. A total of 10
drones were sampled per colony except in hive 7a , which had 9
drones sampled, 4a which had 8 drones sampled and Gc. In colony
6c only four surviving drones were sampled. Three sperm samples
were counted per seminal vesicle, giving a total of six sperm
samples per drone.
The semen smears were allowed to dry to facilitate all the sperm
settling onto one viewing plane. The sperm was stained with vectashield DAPI, a fluorescent nuclear stain, which allowed the nuclei
of each sperm to be readily visible (Figure 1).
II'
photograph on the semen smears. Pictures of semen were recorded
and saved as a j .pg file for later counting. Image Pro© software was
used to count the sperm.
Data Analysis
Sperm totals and the averages of sperm per drone for each
colony and treatment were calculated. These calculations were
double checked by entering all the raw data into an excel
spreadsheet, and making pivot tables. Statistical tests, ANOVA and
MANOVA, were used to compare means. This analysis was
performed with the software package STATISTICA©.
RESULTS AND DISCUSSION
The average numbers of sperm per drone sample from each of the
experimental colonies are shown in Table 1
17
Table 1. Average number of sperm per drone sample
from each colony.
Colony/Treatment
8c/coumaphos
9c/ coumaphos
5c/coumaphos
6c/coumaphos
lOc/coumaphos
la/control
7a/control
3a/control
Sperm
Counts
3,830
2,104
2,568
670
1,474
1,977
1,461
7a*/control
4a/control
927
764
2,081
Table two shows the average number of sperm per
drone for each colony arid per treatment. The average number of
sperm per drone for the coumaphos colonies was 2,334. The
average number of sperm per drone for the non-coumaphos
colonies was 1,585.
18
Table 2. Average sperm count per drone
sample for each treatment.
Average Sperm! St
St
Dev
Drone Sample
Error
8c/coumaphos
3,830
192
609
9c/coumaphos
2104
201
63
5c/coumaphos
2568
110
350
6c/coumaphos
670
47
150
lOc/coumaphos
1474
53
169
Hive#
Avg.
Sperm! Treatmt.
Hive#
la/control
7a/control
3a/control
7a*/control
4a/control
2334
394
Average Sperm! St
St
Dev
Drone Sample
Error
1977
283
89
1461
73
231
927
45
145
764
61
195
2081
89
281
Avg.
Sperm/Treatmt.
244
1585
The data were analyzed using ANOVA, and the summary
data is shown in Tables 3 and 4. There was no significant difference
in sperm numbers between the two treatment groups (P= .26).
Table 3. Descriptive statistics of sperm count means
for coumaphos and control honey bee
colonies.
treatment
sperm count
means
control
274
coUmaphos
377
All Grps
326
sperm
count N
5
5
10
sperm count
Std.Dev.
88.6
167.6
137.6
19
Table 4. Analysis of Variance for sperm counts of drone honey bees.
Variable
sperm
count
COUM
26662
df
1
MS
CONTROL
df
MS
143795
8
17974
26662
F
p
1.48
0.26
Figure 2 shows the overlap between the two treatments Hive
8c in the coumaphos group yielded exceptionally high amounts of
sperm. When treated as an outlier and removed from the data set,
the statistical comparison between the two treatments did not
change.
600
550
500
450
H
400
E
a
350
300
250
200
Mean
150
Control
COUnphOS
treatment
Figure 2. The effects of treatment on sperm counts.
fl
I ±SD
±SE
20
The seminal vesicle data was analyzed using ANOVA. There
was no significant difference in the length and width(mm) of
seminal vesicles in the coumaphos colonies versus the noncoumaphos colonies (P=.49) (Table 5).
Table 5. Analysis of Variance for seminal vesicle measurements of
drone honey bees.
treatment; LS Means (sexninal_vesicle.sta)
Wilks lambda.81795, F(2, 7).77899,
p.49= NO DIFFERENCE
Effective hypothesis decomposition
treatment
coumaphos
control
L
L
L
L
W
W
W
W
N
.30
.014
.27
.33
.072
.09
.07
.075
5
.29
.014
.25
.32
.071
.09
.069
.074
5
The length and widths(mm) of seminal vesicles for the two
treatments are presented in tables 6 and 7.
Table 6. Average seminal vesicle measurements for
drone honey bees for each colony.
Colony/Treatment Length (mm)
Width (mm)
8c/coumaphos
.31
9c/coumaphos
.29
5c/coumaphos
.30
6c/coumaphos
.31
lOc/coumaphos
.30
la/control
7a/control
3a/control
7a*/control
4a/control
.29
.28
.24
.30
.35
.07
.07
.072
.075
.076
.069
.071
.075
.072
.07
21
Table 7. Average seminal vesicle measurements for
drone honey bees for each treatment.
Treatment
coumaphos
control
Length (mm)
Width (mm)
.30
.29
.072
.071
Sperm quantity as measured by Image Pro© and Excel© was
similar between the two treatments. No significant differences were
detected using an ANOVA. This suggests that coumaphos had no
effect on sperm quantity. However, the colonies without
coumaphos had high mite levels entering the 2001 sampling
season, which I believe, affected the sperm quantity in the noncoumaphos colonies. Seventy drones, from the four out of five
surviving colonies, had an average of 3.24 mites per drone (SE .48).
Mites were counted from emerging drones and the inside of the
brood cells. Wing deformities were seen among many of the
emerging drones. Drones that had more than three mites had
abdominal and limb deformities and discoloration of the abdomen
and thorax was common. De Jong et al. (1982a) reported that wing
and limb deformities were a result of parasitism by V. destructor in
workers. Therefore, the sperm counts attributed to the noncoumaphos treatment colonies were sperm counts from drones
reared under high levels of parasitism by V. destructor. It is known
22
that drone pupae with three or more mites experience a 50%
reduction in sperm produced (Ritter, 1988). Therefore, the fact that
colonies with low mite levels and coumaphos had sperm counts
that were not significantly different from colonies that had no
coumaphos and high mite levels implies that coumaphos may
possibly be causing a reduction in sperm quantity.
Future research will help clarify the consequences
coumaphos has on drone fitness. The use of a third control with no
coumaphos and low mite levels will help to reveal any differences
among the treatments. Without a third control group with low mites
it is difficult to correlate the quantity of sperm to the presence of
coumaphos. However, other research provides some possible
insight on the effects of Varroa on sperm quantity, which allows
some hypotheses to be made on why the mite levels were similar
between the two treatments.
The exposure of developing drones to mites leads to a
reduction in body weight, seminal vesicle and mucus gland size
(Ritter, 1988). As mentioned earlier, a reduction in the amount of
sperm produced is known to occur in parasitized drones (Ritter,
1988). Collins and Pettis (2001) observed morphological
deformities in drones that were infested with Varroa during drone
development. Varroa infestation also causes early mortality in
drones (Sylvester et al. 1998). It is clear that Varroa parasitism has
23
profound effects on drone development, morphology and
physiology.
CONCLUSION
Due to the high level of mites in the control colonies in my
study, the normal sperm load sampled was compromised. The
sperm levels between the control colonies and the coumaphos
colonies had similar numbers of sperm. Since mites are known to
decrease sperm levels in drones, it is possible that coumaphos also
has a negative effect on sperm numbers.
Coumaphos effects could be better tested by designing an
experiment with different treatments each having varying levels of
coumaphos. A toxic threshold could be established, and the
developmental, physiological and morphological consequences
could be identified. This type of study could establish the toxic
effect of coumaphos on sperm and other aspects of drone fitness.
A future study looking at the effects of coumaphos on
honey bees must include a third control treatment. Without a
normal population present it is hard to assess whether the treated
population has deviated from the norm.
This is the first study that looked at the affects of coumaphos
on sperm quantity, and the results are still unclear. The mode of
action for coumaphos, like other organophosphate insecticides, is
24
the inhibition of acetyicholinesterase. This leads to repetitive nerve
firing. Organophosphates, therefore affect proper nerve
functioning. Due to the mode of action of this pesticide it would be
useful to examine flight performance in drones that were reared in
the presence of coumaphos.
It is important for scientists and beekeepers to understand
the consequences of the chemicals to which we expose honeybees.
The recent problems with queen mating and acceptance suggest
that something has changed within the mating arena in the recent
years. Be it loss of genetic diversity due to the reduction in feral
drones contributing to the gene pool, or the sublethal affects of the
miticides that are now used in honeybee colonies, the introduction
of V.
destructor to
US beekeeping has profoundly changed the
management of honey bees.
25
BIBLIOGRAPHY
Anderson, D. L. and J. W. H. Trueman. 2002. Varroajacobsoni
(Acari: Varroidae) is more than one species. Experimental and
Applied Acarology 24: 165-189.
Ball, B. V. and M. F. Allen. 1988. The prevalence of pathogens in
honey bee (Apis mellfera) colonies infested with the parasitic mite
Varroajacobsoni. Annals of applied Biology. 113: 237-244.
Beaver, L. M., B. 0. Gvakharia, B. Rush, T. S. Vollintine, D. M.
Hege, R. Stanewsky, J. M. Giebutowicz. 2002. Loss of circadian
clock function decreases reproductive fitness in males of Drosophila
melanogastor. PNAS 99: 2134-2139.
Camargo, J. M. F. and L. S. Goncalves. 1971. Manipulation
procedures in the technique of instrumental insemination of the
queen honey bee Apis rnellifera L. (Hymenoptera: Apidae).
Apidologie 2: 239-246.
Choi, S. Y. and K. S. Woo. 1974. Studies on the bionomics of bee
mite, Varroajacobsoni Oudemans, and its chemical control (II).
Research Review ORD 16(L): 69-76.
Collins, A. M. arid J. S. Pettis. 2001. Effect of Varroa Infestation
on Semen Quality. American Bee Journal 141: 590-593.
Davies, A. G. 1980. Annual Research Reviews: Effects of
Hormones, Drugs and Chemicals on Testicular Function. Vol. 2
Westmount, Quebec: Eden Press
De Jong, D., P. H. Dc Jong, L. S. Goncalves. 1982a. Weight Loss
and Other Damage to Developing Worker Honeybees from
Infestation with Varroajacobsoni. Journal of Apicultural Research
21: 165-167.
De Jong, D., R. A. Morse, GC. Eickwort. 1982b. Mite pests of
honey bees. Annual Review of Entomology 27: 229-252.
Del Cacho, E., JJ. Marti, A. Josa, J. Quilez, C. Sanchez-Acedo.
1996. Effect of Varroajacobsoni parasitization in the glycoprotein
expression on Apis mellfera spermatOzoa. Apidologie 27: 87-92.
26
Donze, G. and P. M. Guerin. 1997. Time-Activity Budgets and
Space Structuring by the Different Life Stages of Varroajacobsoni in
Capped Brood of the Honey Bee, Apis mellifera. Journal of Insect
Behavior 10: 371-393.
ELzen, P. J., J. R. Baxter, M. Spivak, W.T. Wilson. 2000. Control of
Varroajacobsont Oud. resistant to fluvalinate and amitraz using
coumaphos. Apidologie 31: 437-441.
Elzen, P. J. and P. Westervelt. 2002. Detection of coumaphos
resistance in Varroa destructor in Florida. American Bee Journal
142: 29 1-292.
Estoup, A., M. Solignac, J-M. Cornuet. 1994. Precise assessment
of the number of patriline and of genetic relatedness in honey bee
colonies. Proceedings of the Royal Society of London, Series B 258:
1-8.
Gagnon, C. 1990. Controls of sperm mobility: Biological and
Clinical Aspects. Boca Raton, Florida: CRC Press
Haarmann, T., M. Spivak, D. Weaver, B. Weaver, T. Glenn. 2002.
Effects of Fluvalinate and Coumaphos on Queen Honey Bees
(Hymenoptera: Apidae) in Two Commercial Queen Rearing
Operations. Journal of Economic Entomology 95: 28-35.
Harbo, J. R., and R. A. Hoopingarner. 1997. Honey Bees
(Hymenoptera: Apidae) in the United States That Express
Resistance to Varroajacobsoni (Mesostigmata: Varroidae). Journal
of Economic Entomology 90(4): 893-898.
Milani, N. 1995. The resistance of Varroajacobsoni to pyrethroids:
a laboratory assay. Apidologie 26:415-429.
Peng, Y. S., Y. Fang, S. Xu, L. Ge. 1987. The resistance
mechanism of the Asian honey bee, Apis cerana Fabr., to an
ectoparasitic mite, Varroajacobsoni Oudemans. Journal of
Invertebrate Pathology 49: 54-60.
Rinderer, T., L. I. De Guzman, V. A. Lancaster, 0. T. Delatte, J. A.
Steizer. 1998. Varroa in the Mating Yard: I. The Effects of Varroa
jacobsoni and Apistan® on Drone Honey Bees. American Bee
Journal 139: 134-139.
27
Ritter, W. 1988. Varroajacobsoni in Europe, the tropics, and
subtropics. In: Needham, 0. R., Page, R. E. Jr., Delfinado-Baker,
M., and Bowman, C. E. Africanized Honey Bees and Bee Mites. Ellis
Horwood, Ltd., 349-359.
Ruttner, F. and H. Ruttner. 1966. Untersuchungen uber die
flugaktivitat und das Paarungsverhalten der Drohnen, III. Z.
Bienenforsch. 8: 332-354
Schneider, P. 1986. Varroa workshop, Feldafing, 20. (as cited on
p. 356 of Ritter, W. 1988. Varroajacobsoni in Europe, the tropics,
and subtropics. In Needham, 0. R., Page, R. E., Jr., DelfinadoBaker, M., and Bowman, C. E. Eds. Africanized Honey Bees and
Bee Mites. Chichester, Ellis Horwood Ltd. 1988, pp. 349-359.
Seeley, T. D. 2002. The effect of drone comb on a honey bee
colony's production of honey. Apidologie 33: 75-86.
Shimanuld, H., N. W. Calderone, D. A. Knox. 1994. Parasitic mite
syndrome: The symptoms. American Bee Journal 134: 827-828.
Sylvester, H. A., R. P. Watts, L. I. Guzman, J. A. Stelzer, T. E.
Rinderer. 1999. Varroa in the mating yard: II. The effects of Varroa
and fluvalinate on drone mating competitiveness. American Bee
Journal 139: 225-307.
Weick, J. and Thorn, R. S. 2002. Effects of Acute Sublethal
Exposure to Coumaphos or Diazinon on Acquisition and
Discrimination of Odor Stimuli in the Honey Bee (Hymenoptera:
Apidae). Journal of Economic Entomology 95: 227-236.
Winston, M. L. 1987. The Biology of the Honey Bee. Cambridge,
Massachusetts: Harvard University Press.