ESTONIAN UNIVERSITY OF LIFE SCIENCES
Institute of Forestry and Rural Engineering
Department of Forest Management
Kristiina Maalaps
Sperm management in honey bees: Counting sperm on honey
bee eggs to measure queen fertility
Master thesis in natural resources management
Supervisors:
Dr. Susanne den Boer
Prof. Marika Mänd
Tartu 2014
INTRODUCTION ................................................................................................................. 5
1. Social insects and honey bee reproduction ........................................................................ 9
1.1. Social insects ............................................................................................................... 9
1.2. Honeybees ................................................................................................................. 10
1.3. Honeybee reproduction ............................................................................................. 12
1.3.1. The mating flight ................................................................................................ 12
1.3.2. The male’s role in mating ................................................................................... 14
1.3.3. The female’s role in mating................................................................................ 15
1.3.4. Egg fertilization.................................................................................................. 17
2. Material and Methods ...................................................................................................... 18
2.1. Experiment 1 ............................................................................................................. 18
2.1.1. Collecting freshly laid eggs ................................................................................ 18
2.1.2. Sperm count on freshly laid eggs ....................................................................... 20
2.1.3. Assessing the effect of sperm number and sperm viability ................................ 23
2.2. Experiment 2 ............................................................................................................. 25
2.2.1. Queen rearing ..................................................................................................... 26
2.2.2. Drone and semen collection ............................................................................... 27
2.2.3. Artificial insemination........................................................................................ 29
2.2.4. Measuring sperm use, sperm number and sperm viability in the inseminated
queens ........................................................................................................................... 31
2.3. Statistics .................................................................................................................... 31
3. Results .............................................................................................................................. 33
3.1. Sperm use during fertilization in naturally inseminated queens ............................... 33
3.2. Effects on sperm use ................................................................................................. 34
3.2.1. Queen age ........................................................................................................... 34
3.2.2. Sperm viability ................................................................................................... 36
3.2.3. Sperm number and volume ................................................................................ 38
3.3. Effects on sperm viability ......................................................................................... 40
3.3.1. Queen age ........................................................................................................... 40
3.3.1. Effect of volume ................................................................................................. 41
3.4. Queen age effects on sperm number in spermatheca ................................................ 42
3.5. Summary and Principle Component Analysis........................................................... 44
DISCUSSION ...................................................................................................................... 46
CONCLUSION .................................................................................................................... 52
ACKNOWLEDGEMENTS ................................................................................................. 54
LITERATURE ..................................................................................................................... 55
APPENDIXES ..................................................................................................................... 60
Appendix 1. Detailed summary of the thesis in Estonian ................................................ 60
Eesti Maaülikool
Kreutzwaldi 1, Tartu 51014
Magistritöö lühikokkuvõte
Autor: Kristiina Maalaps
Õppekava: Loodusvarade kasutamine ja kaitse
Pealkiri: Sperma kasutus meemesilastel: Spermatosoidide loendus viljastatud
munarakkudel ema viljakuse mõõtmiseks
Lk.: 64
Jooniseid: 25
Tabeleid: 3
Lisasid: 1
Osakond:
Metsandus- ja maaehitus
Uurimisvaldkond:
Sotsiaalsed putukad
Juhendajad:
Marika Mänd, Susanne den Boer
Kaitsmiskoht ja aasta:
Tartu 2014
Meemesilastel (Apis mellifera) on ühe pere kohta vaid üks viljastumisvõimeline emane,
kes paaritub reeglina vaid kord keskmiselt kuni 12 isasega. Kogutud sperma hoiustab ema
elu jooksul spermapauna, kasutades seal olevat spermat aja jooksul munarakkude
viljastamiseks.
Käesoleva töö keskseks uurimisalaks on uurida sperma kasutust muna viljastamise
protsessis ning kuidas sperma kasutus on mõjutatud ema vanusest, sperma elujõulisusest
spermapaunas pärast ema lahkamist ning erinevatest sperma kogustest spermapaunas.
Selleks, et eelmainitud faktoreid ning nende mõju sperma kasutusele uurida, koostati kaks
uurimistööd. Peamine meetod, mille kasutus läbib mõlemat uuringut, on spermatosoidide
loendusmeetod värskelt unetud munadelt, kasutades fluoretsents värvi DAPI. Esimeses
projektis on vaatluse all 22 loomulikul teel paaritunud emamesilased. Teises pojektis
kasutatkse kahte gruppi paaritumata measeid, keda viljastatakse eri seemne hulkadega.
Lisaks rakendatakse ka järgmisi tehnikaid: emade lahkamine ning elujõulise sperma
koguse uurimine spermapaunas.
Tulemused näitavad, et noored emad (1-4 kuud vanad) kasutavad keskmiselt 7-9
spermatosoidi muna kohta, vanemad (7-28 kuud vanad) vaid 1-2 spermatosoidi.
Noorematel emadel on sperma elujõulisus suurem kui vanadel. Sperma kasutus munaraku
viljastamisel on oluliselt mõjutatud sperma kogusest spermapaunas ning sperma
elujõulisusest, sellest hoolimata leidsin, et peamiseks faktoriks, mis mõjutab sperma
kasutust on siiski ema vanus.
Antud tulemused ei ole kasulikud mitte ainult mesilasbioloogia paremaks arusaamiseks
vaid ka mesinikele, kellel üheks olulisemaks faktoriks mesilate tugevuse tagamisel on
efektiivne, viljakas emamesilane.
Märksõnad: Apis mellifera, meemesilane, sperma kasutus, spermatosoidide loendus,
spermapaun, sperma elujõulisus, kunstlik viljastamine, DAPI
Estonian University of Life
Abstract of Master’s Thesis
Sciences
Kreutzwaldi 1, Tartu 51014
Author: Kristiina Maalaps
Curriculum: Natural resources management
Title: Sperm management in honey bees: Counting sperm on honey bee eggs to measure
queen fertility
64 pages
25 figures
3 tables
1 appendix
Department:
Institute of forestry and rural engineering
Field of research:
Social insects
Supervisors:
Susanne den Boer, Marika Mänd
Place and year of defence:
Tartu, 2014
Honey bee (Apis mellifera) colonies are headed by a single reproductive female. This
queen mates just once in her life with 12 males on average. The sperm is then stored in
her spermatheca and used for egg fertilization throughout a queen’s life.
The central aim of the current thesis is to investigate sperm use patterns in honey bee
queens and the possible factors influencing it: queen’s age, sperm viability and sperm
number in the spermatheca.
To compare the effects of aforementioned factors two projects were conducted. The main
technique used in both projects, was a method developed to directly visualize sperm
present on freshly laid eggs using a fluorescent dye and microscope. In the first project
22 naturally mated queens of different ages were examined. In the second project two
groups of virgin queens were artificially inseminated with different volumes of semen.
The results showed that young queens use more sperm when fertilizing an egg than older
queens (amounts being 7-9 sperm in 1-4 months old and 1-2 in 7-28 months old queens).
The results also showed that in general younger queens have higher sperm viability in
their spermatheca and also the total number of sperm is higher. All of these factors seem
to affect sperm use, but queen age seems to be the most important determinant.
The results of this thesis benefit the overall basic understanding of the honeybee biology
and therefore social insects in general. Furthermore, with actual numbers on sperm use it
gives the beekeepers a better insight to the factors that influence queen fertility and
which might be of importance for managing hives.
Keywords: Apis mellifera, sperm use, sperm count, spermatheca, sperm viability,
artificial insemination, DAPI, fertilization
INTRODUCTION
Queens in honey bee (Apis mellifera) species only mate once early in life with multiple
males and store the sperm they receive inside their sperm storage organ, the spermatheca.
They have to use this one sperm supply that will never be renewed, to internally fertilize
eggs and produce all the workers and new virgin queens during their lifetime. With the
amount of sperm in their spermatheca decreasing over time, the end of a queen’s
reproductive life is determined by the exhaustion of her sperm supply. In case of excessive
sperm use, queens can prematurely run out of sperm and start laying unfertilized eggs that
will develop into drones (males). This is a point of danger for the queens, as once they stop
laying fertilized eggs that develop into worker bees (or queens), they simultaneously stop
excreting specific pheromones that inform the workers about the their productivity. This
sort of behaviour induces supersedure of the queen, where she is replaced with a new queen
by workers to ensure the continuation of the colony (Winston 1991, Kraus, Neumann et al.
2004, Baer 2005). Supersedure is the nature’s way of re-queening the colony when the bees
are not satisfied with the performance of the existing queen. Once worker bees have an
indication that the queen isn’t able to maintain the hive’s strength they need to replace the
dysfunctional queen by building a queen cell on top of a normal worker cell where the old
queen has previously already laid a fertilized egg. The workers continue feeding the
developing larvae with royal jelly which determine larvae’s growth into new queens (also
used in queen rearing see below).
With the pressure on performance, it is critical for the queen to have a balanced sperm use she should use sufficient numbers of sperm per egg to ensure success of fertilization, but at
the same time she should limit the sperm use per egg to prevent wasting sperm cells and
5
depleting her sperm store faster than needed, all to ensure longevity of her reproductive
lifespan. Over evolutionary time, queens should therefore have been selected to use as few
sperm as possible without leaving eggs unfertilized. Even if some eggs occasionally would
be left unfertilized it would not be too harmful to a queen’s fitness, as eggs can be replaced
while sperm cannot (queens never re-mate later in life).
The sperm use in some other social species has been shown to be relatively low. For
example, in Solenopsis invicta, the fire ant the queen uses under 3.5 sperm to fertilize an
egg. In Atta colombica, the leaf-cutter ants use on average 2 sperm per egg (Foster and
Ratnieks 2001, den Boer, Baer et al. 2009). The significance of sperm use during
fertilization and sperm use patterns in Apis mellifera haven’t been studied in great detail.
With the exception of the current study, no in vivo study has been performed that would
allow the visualisation of sperm use on eggs. To date, the average sperm use in honeybees
has been mainly calculated by dividing the total number of sperm stored by the total
number of estimated offspring produced, giving just a rough insight into the expenditure of
the stored sperm with estimates of 4 - 100 sperm/egg (Bresslau 1905, Adam 1912, Harbo
1979). At this point an in-depth study is needed to understand more specifically the
management of a queen’s sperm supply during egg fertilization and the factors affecting it.
Understanding the sperm use patterns during the process of inseminating the egg in honey
bee queens would enhance our ability to comprehend the mechanisms of the fertilization
process better. Furthermore it would allow the beekeepers to reconsider the need to requeen their hives at an early stage of the queen’s life. In industrial beekeeping as a
management of natural resources (honey, bees) queens are killed at a very early stage of
their life (1-2 years of performance in Mediterranean areas) as a precaution measure - this
is based on the observation that in general older queens produce fewer fertilized eggs (and
therefore worker bees) and are less able to maintain a strong hive.
6
Aims and predictions
The central aim of the study is to investigate sperm use patterns in the queens of Apis
mellifera. How does a queen distribute sperm during fertilization of the eggs to ensure
optimal sperm use throughout her life? The study will firstly provide information on the
actual numbers of sperm used for fertilizing eggs and secondly shows how various factors
influence the characteristics of sperm use during fertilization, such as queen senescence,
sperm viability and number of sperm stored in spermatheca.
To answer the aforementioned questions, two subsequent projects were conducted.
1. In the first project the effect of queen age, sperm viability and sperm number in the
spermatheca after dissection were examined, using queens that had mated naturally. It is
hypothesized that sperm use is mainly influenced by queen age and I predict that the
number of sperm used per egg is higher in younger queens and lower in the older queens. In
the beginning of their reproductive lives queens need to show their efficiency in
productivity and therefore possibly need to use more sperm, whereas mature queens need to
maintain an optimal sperm use level to avoid running out of sperm too early in their
reproductive life.
2. In the second project I examined artificially inseminated queens of the same age (thereby
controlling for factor queen age) with two different volumes of sperm to see the effect of
different insemination volumes and the subsequent difference of numbers of sperm stored
in the spermatheca. It was hypothesised that the amount of sperm stored in the spermatheca
affects sperm use during fertilization and I predict that queens that have fewer sperm in
storage, use fewer sperm to fertilize eggs. It is theorized that the level of liquid in
spermatheca is constant at all time, as spermathecal glands will secrete spermathecal fluid
into the spermatheca so it is never empty (Harbo 1979). If the volume of the spermatheca is
fixed (the spermatheca morphology seems to suggest it cannot expand), it means that if
there are many sperm in the spermatheca, there is less fluid and vice versa; if there are few
sperm in the spermatheca there is more fluid. Assuming that the volume of liquid that is
released from the spermatheca during egg fertilization is constant, it could be possible that
with a higher concentration of sperm in spermatheca (in younger queens for example), the
7
number of sperm used to fertilize eggs is higher than with a lower concentration (as in older
queens or queens inseminated with lower levels of sperm as seen in project 2).
Besides looking at queen age and sperm volume, I also measured the viability of stored
sperm in both experiments. To support my hypothesis of sperm use going down with age I
also see how sperm viability might affect the sperm use and how it is correlated with the
amount of sperm that the queens were inseminated with and with the amount of sperm that
was still stored in queens (who had previously already been reproductive) of different age.
Approach
The main technique used for these projects was originally developed to directly visualize
sperm present on freshly laid eggs in leaf-cutter ant species Atta colombica (den Boer, Baer
et al. 2009). I adapted this technique for use in the honeybees, and in that way it was the
first time that sperm use data could be collected by direct observation, instead of indirect
calculations. I furthermore used a combination of field based and laboratory techniques,
such as beekeeping, fluorescent microscopy, semen collection, artificial insemination of
virgin queens and dissection of queens to measure the effects of queen senescence and
sperm volume stored in the spermatheca on sperm use patterns and viability.
The study was conducted during 14 months in 2012-2013 within the Centre of Integrative
Bee Research (CIBER) on the grounds of the University of Western Australia, Perth, WA,
Australia. CIBER is a research group created to study social insects, with research areas
including the evolutionary biology (sociobiology, sexual selection theory) of social insects
(bees, ants, termites), proteomics and artistic expressions. The beekeeping season in
Western Australia is longer than in Europe, allowing for annual research. In warmer
climates bees do not hibernate according to the in European standards, though their activity
still ceases noticeably compared to summer. The technology necessary for the current study
was provided by CIBER.
8
1. Social insects and honey bee reproduction
1.1. Social insects
Social insect societies are characterised by obligate group living (with multiple generations
living in the same nest), cooperative brood care, and a reproductive “royal” caste and sterile
or semi sterile “worker” caste division of members of the colonies. The workers are
furthermore differentiated into groups of specialised labour (Wilson 1979). Social insects,
also referred to as eusocial (meaning “truly” social), are made up mainly of four familiar
groups: all of the ants (family Formicidae of the order Hymenoptera. 9500 species known),
some of the bees (from family Halictidae and Apidae, around 10 000 species within
abovementioned bee families are social), some of the wasps (around 800 species from
families Vespidae and Sphecidae) and all termites (Isoptera with about 2000 species)
(Wilson 1979, Hölldobler 1994).
In contrast to most non-social insect species, in social insects an individual’s sex is
determined not by the presence or absence of a sex chromosome, but instead by the number
of copies of the genome in an individual’s cells (called 'haplodiploidy') (Trivers and Hare
1976, Mueller 1991). The males in social insect species develop from unfertilized eggs,
meaning, that the eggs are haploid and contain a single copy of the genome derived from
the mother. Males thus have no father. Diploid fertilized eggs containing two copies (one
from each parent) and become females. Females can be either queens or sterile workers.
9
The reproductive individuals in eusocial insects are the queens and the males (kings in
termite species). Forming the largest colonies in the world known, most eusocial species
are headed by one reproductive female that has mated with one male (monoandry).
However, in some species there more than one queen per colony (polygyny) and in some
other species the queens mate with multiple males (polyandry) (Hughes, Ratnieks et al.
2008, Jaffé, Pioker-Hara et al. 2014). For example colonies of certain army ants (Dorylus
spp.) and leaf-cutter ants (Atta spp.), that have gigantic societies reaching up to millions of
individuals are headed by a single, multiply mated queen. Polyandry also occurs in honey
bees (Apis spp.). Among the bee species with the largest colonies, reaching up to tens of
thousands of individuals, the colonies are headed by one extremely polyandrous queen,
who is able to mate with more than 50 males (Hughes, Ratnieks et al. 2008, Jaffé, PiokerHara et al. 2014). It has been established that multiple queen mating is beneficial as it gives
rise to more genetically diverse offspring. This in turn is thought to facilitate division of
labour between those offspring and will make a colony more resistant against diseases and
parasites. It is therefore not a coincidence that multiple mating is seen in species where
queens produce the biggest colonies. (Hughes, Ratnieks et al. 2008). Although worker
honeybees are unable to mate and produce diploid offspring, they are capable of laying
unfertilized eggs that become males. This rarely occurs when the queen is alive because
workers police other workers, destroying any eggs laid (Ratnieks 1988, Foster and Ratnieks
2001) Therefore, the reproduction of the colony lies solemnly on the queen and the males
(drones).
1.2. Honeybees
By classification, honey bees (Apis mellifera) belong to Animalia kingdom, Insecta class,
Hymenoptera order, family Apidae, genus Apis. Honeybees are known to originate from
tropical Africa and spread from South Africa to Northern Europe, India and China. The
natural habitat of the honey bee is broad, reaching from the southern areas of Africa
10
through savannahs, rain forests, deserts and Mediterranean and reaching up to Northern
Europe and Scandinavia (Winston 1991). The adaptions to the different climate zones has
resulted in numerous sub-species of honey bees (Louveaux 1966).
Members of a typical honey bee colony are hierarchically divided into three groups: a
queen, drones and the worker bees. The queen and the drones are the only sexuals in the
complex of a colony. Worker bees are all female, ordinarily unable to perform in any
reproductive processes and, as their name implies, they are responsible for most of the
labour in the hive. Their functions include secreting wax from glands on their abdomen to
create the honeycomb and brood comb from it. The comb is structured of hexagonal cells
that are large enough to withhold a developing drone, worker, honey or pollen. The workers
are also the care takers of the queen, young drones and brood. Workers feed young larvae,
seal the pupa into cell and feed the emerging young adults until they are old enough to feed
themselves. Worker bees also participate in gathering operations, collecting nectar to make
honey, pollen from the flowers in season and resin for making propolis. The worker is also
responsible for defending the hive with her barbed stinger and a muscular venom pouch
that rhythmically pumps venom into the intruder when stinging. The worker bee dies after
the act due to the abdominal rupture caused by leaving her sting into the intruder (Winston
1991).
The importance of the honey bee in the biosphere is extensive. In biodiversity bees provide
a key ecosystem service through pollination. Bees are of inestimable value as agents of
cross-pollination, and many plants are entirely dependent on particular kinds of bees for
their reproduction.
Economically, bees are important as industrial pollinators, the providers of honey which we
harvest from honey bees as well as pollen and propolis for their nutritional value and
medical applications. Other raw materials like wax are used for craft and manufacturing. In
the human food section, according to the U.S. Department of Agriculture, bees pollinate 80
11
percent of flowering crops which constitute 1/3 of everything we eat, including apples,
broccoli, strawberries, nuts, asparagus, blueberries and cucumbers (USDA, 2014). The
economic value of honey bees and bumble bees as pollinators of commercially grown
insect pollinated crops in the UK has been estimated at over £200 million per year. The
gross product value of natural honey in the world in 2011 was 6,181 million US dollars, in
Estonia 6,91 million US dollars (Faostat, 2014) (Figure 1).
Honey gross production value of 2011 by country
(millions; US dollars)
1050
900
750
600
450
300
150
0
Figure 1. Economic value of honey in honey gross production value (GPV) by country in
2011 is brought in current millions US dollars. The figure is showing that China has the
highest GPV with 874.01 million US dollars, Estonia 6,91 million US dollars. Data from
Faostat (2014).
1.3. Honeybee reproduction
1.3.1. The mating flight
Mating in honey bee species takes place in mid-air, in a specific area called the drone
congregation area. As the only reproductive female in colony, honey bee queen mates once
12
or very few times at the beginning of her life (Ruttner 1956). Once sexually mature, the
virgin queen flies to a congregation area where hundreds or thousands of unrelated drones
await to mate. The duration of one successful mating flight can take up to 15-30 minutes
(Woyke 1975, Koeniger, Koeniger et al. 2005). At a height of 10- 40 m above the ground,
drones form temporary clusters, the “drone comets” behind the queen of more than 100
males chasing a female sexual at the time (Gries and Koeniger 1996), resulting in extreme
degree of polyandry in Apis mellifera queens. Even though the ejaculate of a single drone
would be able to fill the spermatheca (Woyke 1962), the number of drones mated with
queen can reach up to 90, with an average of 12 drones (Koeniger and Koeniger 2000,
Tarpy, Nielsen et al. 2004). It has been estimated that in Apis mellifera the average number
of drones visiting a drone congregation area in total is around 12 000-25 000, though only
limited number of males have access to the queen and perform at nuptial flight (Page 1986,
Koeniger and Koeniger 1991, Schluns, Koeniger et al. 2004, Koeniger, Koeniger et al.
2005). Queen mates only on one day, very rarely at more times in her life. Once mated, it
takes a few days until the queen starts laying eggs with the frequency of 200 000 eggs per
annum (Snodgrass 1984, Winston 1991).
Usually in spring, given that the affirmed queen is reproductive and hive strong enough, the
honey bee colony undertakes a process (swarming) to establish a new colony. Swarming
happens when the old queen leaves the hive (nest) with approximately half of the worker
bees to start a new one. In order to prepare for swarming, scouting bees (foraging bees with
most experience within the colony) start exploring nearby areas suitable for the swarm to
temporarily cluster. The hive is ready to swarm as soon as after the old queen has laid eggs
to queen cells they have reached the stage in their development where they are ready to be
capped. Virgin queens emerge about two days after the swarm has departed. The swarm
(old queen and approximately half of the colony) relocates itself at first to a suitable area
close to the hive, usually clustering around a tree or a branch. From there, 20-50 bees are
sent out to find the best possible place to facilitate the colony for nesting. Once the new
location is decided (by the characteristics of the returning scout’s descriptive dance of her
find), the cluster is ready to swarm. A honey bee swarm contains about 5000 - 50 000
13
workers, zero to few thousand drones and one queen, occupying approximately 15 – 30 m
in diameter and 1.5 – 6 m high area of air space. Their speed through air has been recorded
to be up to 24 km/h (Ribbands 1953, Morse 1963). The cluster finds the spot by the
directions given and the pheromones previously secreted to the new location by the scouts
(also referred to as “leader bees”) (Avitabile, Morse et al. 1975).
1.3.2. The male’s role in mating
During mating flight, the drones pursue queen and several mate with her in flight. After
mounting the queen, drones insert their copulatory apparatus called endophallus, and
ejaculate the semen into the queen. The ejaculation happens when the drone everts the
endophallus fully. When the drones are excited and their thorax is squeezed during mating,
their abdominal muscles contract and the pressure of the hemolymph inside the abdomen
increases. As a result, the endophallus is pushed out of the abdomen and is everted. When
the eversion is only partial, the dorsal walls of the drones cervical duct do not open and no
semen is ejaculated (Woyke 2008). The queen initially receives around 2,2 µl of sperm
from each mated drone (approximately 12.7 million sperm cells) (Woyke 1955, Haberl and
Tautz 1998, Koeniger and Koeniger 2000). Along with the ejaculation, drones also leave
within the queen a mating sign consisting of several gland secretions and a chitinous plate
as an indication of copulation when she returned to the hive (Baer 2005, Schlüns, Moritz et
al. 2005).
The copulation is lethal to drones of all Apis species, as the eversion of the males
endophallus is irreversible (Ruttner, 1954). When the male separates from the queen after
the ejaculation (with the endophallus still attached to the queen), he falls on the ground to
die, paralysed. The emasculated drones die very quickly (Ruttner 1956, Koeniger and
Koeniger 1991). As a consequence, males can copulate only once in their life, committing
their entire reproductive effort to a single queen.
14
1.3.3. The female’s role in mating
After receiving sperm from several drones during nuptial flight, semen is temporarily
stored in lateral oviducts of the queen (Figure 1) with the amounts initially stored reaching
80-90 million sperm (Koeniger and Ruttner 1989, Kraus, Neumann et al. 2004, Lodesani,
Balduzzi et al. 2004).
Figure 2. Reproductive system of the honey bee queen. Spermatheca (spherical ball in the
middle of the reproductive organs on the figure) stores sperm until releasing a certain
amount for fertilizing an egg. Eggs are produced in the ovaries and they move down
through oviducts; at the same time sperm is released from the spermatheca and is
transported through spermathecal duct for insemination of the egg. Spermathecal glands on
each side of the spermatheca are known to contribute to sperm survival in spermatheca
during storage. Figure also illustrates the positions of the sting in queens and the venom sac
(Bujalska 2013).
15
Sperm is moved from the lateral oviducts to the spermatheca (vesicula seminalis) (Figure 2)
through the spermathecal duct, probably caused by of a combination of active sperm
swimming, contractions of the lateral oviduct and the effectivity of a so–called ‘sperm
pump’ in the spermathecal duct (Bresslau, 1905). This sperm migration happens within 1020 hours after mating (Woyke 1955), and only 5% of all sperm is stored while the rest is
discarded from the lateral oviducts (Baer, 2005).
The spermatheca in Apis mellifera queens is a spherical organ for sperm storage (Martins
and Serrao 2002). The 5% of sperm that reaches the spermatheca is kept there until used for
egg fertilization for as long as the queen will live. This means that sperm might need to be
maintained viable up to several years (Life expectancy of the queen is up to 7 years,
depending on the area in the world) (Gessner 1976, Eberhard 1996, Simmons 2001). There
are probably many mechanisms as to how queens ensure sperm survival for such long
periods of time, some of them mentioned in this paper.
Lying in intimate contact with the cells of the spermathecal wall is a dense network of
tracheae (Figure 3) that is probably there to facilitate gas exchange with the stored sperm.
On the upper half of each side of the spermatheca lies a long spermathecal gland that is
linked to spermathecal duct (Figure 2). Obtaining sperm transport function, spermathecal
duct possesses a robust muscle layer (an S-shaped bend) just below the duct towards
spermatheca (Laidlaw 1944, Dallai 1975). The bend has been described to act as a valve to
prevent the spermathecal fluid escaping, as well as a muscular arrangement of the sperm
duct to control sperm transfer during fertilization (Bresslau’s sperm pump) (Cheshire 1884,
Bresslau 1905). The paired spermathecal glands (Figure 2) produce secretions into the
spermatheca that likely provide an adequate physiochemical environment for sperm
survival (Dallai 1975, Gessner 1976, Weirich, Collins et al. 2002). It is also shown that
spermathecal fluid has a positive effect on sperm survival (den Boer, Boomsma et al. 2009).
16
1 mm
Figure 3. The image shows a close-up of a spermatheca (approximately 1.1 mm),
intimately covered with a tracheal network. On either side of the spermatheca can be seen
two glands. Where the arrow is pointing, the glands merge together with the spermathecal
duct and the site of entrance to the spermatheca. The pinkish colour of the spermatheca is
caused by stored sperm, and is a good indicator as to whether a queen has mated or not.
Virgin queens have a clear spermatheca. Photo: Susanne den Boer
1.3.4. Egg fertilization
In honey bees it is unknown how the mechanisms of egg fertilizations precisely work,
although it is believed to be regulated by valve and a pump on the spermathecal duct and
suggested that the queen manipulates the sperm release accordingly to the environmental
cues (Bresslau 1905). In “The insects. Structure and Function”, Chapman (1998) has
described the process of fertilization in the species of Schistoceca (commonly called bird
grasshoppers). Simultaneously to the egg passing into the genital chamber, a number of
sperm is released and the fertilization of the egg takes place. Sperm release is a mechanical
response to the egg entering the genital chamber. When the egg is on the movement
towards the chamber, the activity of the oviducal muscles are induced so that the egg stops
moving and allows through the activation of muscles in spermathecal duct to squeeze the
sperm towards the egg.
17
2. Material and Methods
2.1. Experiment 1
In experiment 1 I examined sperm use patterns of 22 naturally inseminated queens of
different ages (1, 4, 10, 18 and 25 months after enclosure) that were provided by local
beekeepers in Western Australia. I chose an age range that represented very young queens
on one end (1 month: queens that have mated and started laying eggs only recently) to very
old queens (25 months: queens that are normally replaced by beekeepers as they are more
likely to show inconsistent egg laying patterns). All queens were kept in 6-framed deep cut
Langstroth hives and maintained daily at the university campus. This experiment was
conducted within a timeframe of 14 months (1st of March 2012 – 20th of April 2013), and
some queens were tested multiple times during this period. Queens were marked on the
thorax with a small paint dot, which made it easier to find them in the colony. About twice a
week, queens were taken out of their hive to collect their freshly laid eggs as described
below. All other days of the week, queens were left undisturbed in the hive to reduce stress.
2.1.1. Collecting freshly laid eggs
In order to collect freshly laid eggs, a queen was located in the hive, placed onto an empty
frame and covered with a queen excluder box (Figure 4). The excluder box was built to
keep the queen on a certain area of the frame (in contrast to smaller worker bees, queens are
too big to fit through the bars of the excluder box). The box ensured that she only laid eggs
on a small area of the frame. Placing queens on the empty frame was either done by gently
guiding them to walk from one frame to another or carefully lifting them onto the frame
18
provided by holding the individual in-between the thumb and the index finger. Once the
excluder box was pressed into the empty frame, the queen was locked in. The queen was
accompanied by at least 5 workers at lock-in to reduce the stress. The frame was then
placed into the hive in-between brood frames, so that other workers could also freely move
to and from the excluder box. The queen remained in the excluder box for 1.5-3 hours to
ensure enough time for egg laying. The empty frames used were not treated, nor did they
contain any pollen, nectar or eggs. All eggs were collected in-between 7am – 5 pm.
Figure 4. The photograph shows a hive from where the egg collection was conducted, an
empty frame and a queen who is caged in the yellow excluder box. Photo by the author.
After 1.5-3 hours, queens were released back into the hive and the empty frame was taken
to the lab for egg collection. Eggs can be seen sitting in the bottom of cells (Figure 5). As it
was made sure that the frame was egg-free at the start of the experiment, any egg that could
be seen within the ‘queen excluder area’ should have been less than 3 hours old. Eggs were
removed with a specific needle, and placed on a microscope slide in a box with high
19
humidity to (closed box with a damp tissue), to prevent the eggs from drying out. All of the
freshly laid eggs were removed from the frame and counted. Only 15-25 randomly picked
eggs per queen were used at a time for sperm count
Figure 5. Freshly laid eggs (approximately 1.5 hours old) are visible in the cells of the
empty frame and on a microscope slide. Photo by the author.
2.1.2. Sperm count on freshly laid eggs
The total number of sperm used to fertilize each egg was established by 1) counting the
sperm visible on the fertilized egg - it was assumed that all sperm released by queens
during fertilisation would fixate on the egg and would be visible when examined with
DAPI (Den Boer et al 2009); 2) +1 sperm was added to each sperm count representing the
sperm already integrated with the nucleus of the egg during fertilization and therefore could
20
not be visible under the microscope (den Boer, Baer et al. 2009). It is realistic to add +1 to
these counts, as it has been shown that sperm penetrates the egg 15 minutes after release
onto the egg, and the paternal and maternal nuclei fuse at 93 mins (well before I removed
the eggs from the frame and prepared the egg for sperm counts), which would change the
appearance of sperm so that there are no longer recognizable as such (Yu and Omholt
1999).
Sperm counts were conducted by staining the eggs with the blue-fluorescent DAPI Nucleic
Acid Stain. DAPI binds DNA and RNA for easy visualization of the sperm heads, allowing
the counting of sperm nuclei (Figure 6). Eggs were individually placed on a microscope
slide and stained with 5 ml of a DAPI working solution, i.e. 2 ml of a DAPI stock solution
(2 mg DAPI in 1 ml dimethylsulfoxide) in 1 ml 0.1M NaPO4 buffer, pH 7.0. Because of
DAPI’s light sensitive characteristics, all stained eggs had to be kept in the dark to prevent
the stain fading.
In order to ensure equal distribution of DAPI solution in and out of the egg, cover slides
were placed over the eggs with the intention to burst them. With the cytoplasm flowing out,
DAPI was allowed to stain sperm cells present both on the egg’s chorion and those
potentially in the cytoplasm (Figure 7, 8). Eggs were examined using a fluorescence
microscope (ZEIZZ Imager.A1, AXIO) with a filter for DAPI (EXFO X-Cite 120
illuminator) so that sperm cells would fluoresce blue when viewed. The number of sperm
cells associated with each egg was counted over time for a total of 27–60 eggs per queen.
21
Figure 6. Image of sperm heads (bright light-blue cylindrical shapes) visible on
fertilized honey bee egg (hazy-blue shape in the background). Photo: author.
Figure 7. A close-up of sperm cells present on eggs laid by
A. mellifera queens. Photo by the author.
22
Figure 8. A DAPI treated burst egg, allowing visualization of sperm
heads both on the egg’s chorion and in the cytoplasm. Photo by the author.
2.1.3. Assessing the effect of sperm number and sperm viability
To assess the effect of the viability of stored sperm and its volume, the queens were
dissected under a stereomicroscope after sufficient eggs had been analysed for sperm use.
The dissection was done immediately after the last sperm count on eggs. Prior to dissection,
a queen was sedated with CO₂ and pinned down to the dissection plate under the
microscope. After killing the queen quickly by the removal of the head with scalpel, 1 st, 2nd
and 3rd abdominal segments were opened in tearing motion to access the spermatheca. The
spermatheca was located under the first segment near the stinger in front of the ovaries. The
spermatheca could be easily dissected and the tracheal network surrounding the
spermatheca removed. The clean spermatheca was transferred into the sterilized lid of an
Eppendorf tube containing 10 µl of Hayes saline solution (9 g NaCl, 0.2 g CaCl2, 0.2 g
KCl, and 0.1 g NaHCO3 in 1,000 ml H2O). The spermatheca was then gently ruptured until
the sperm was equally distributed in the liquid. The lid with its contents were placed on to
23
the eppendorf containing 190 µl of Hayes and shaken gently 10 times to allow the equal
distribution of the sperm in Hayes. The mixture – stock solution- became the base for the
following examinations.
SPERM NUMBER
5 µl of the stock solution was placed into 295 µl of distilled water and shaken well (32
times) manually. 4 droplets of 1 µl were placed on to a microscope slide, a full circle was
drawn around it (avoiding touching the droplets) to distinguish the areas needed at a later
stage of the experiment. The droplets were left to dry. 2 µl of DAPI (prepared as described
above) was added to each droplet when dry, covered with coverslips and examined under
the fluorescent microscope. Sperm number was found by counting the number of sperm in
each droplet in the range of the circles previously drawn.
The number of sperm counted on the subsample was multiplied by 12 000 (the dilution
factor) to calculate the number of sperm in the spermatheca. The final number of sperm
cells in the spermatheca was calculated by taking the average from the results of four
subsamples per queen examined.
SPERM VIABILITY
Two fluorescent dyes, SYBR-14 and propidium iodide (PI) were used for sperm viability
staining. SYBR-14 contains a membrane-permeate nucleic acid stain, colouring viable
sperm bright green. Propidium iodide is absorbed by sperm that has damaged plasma cells
(dead sperm), staining nuclei bright red (figure 9 & 10) (Damiens, Bressac et al. 2002). PI
and SYBR-14 are both fluorescent dyes and are extremely light sensitive, fading in
exposure to light. Therefore, to ensure clear images under the microscope, it was necessary
to ensure that samples were kept in the dark.
24
Figures 9 & 10. Photographs visualizing sperm viability where green sperm stained with
SYBR-14 represent the viable and red stained with PI represent the unviable sperm.
Photos by the author.
50 µl of stock solution was added to 150 µl of Hayes into an Eppendorf tube, the liquids
were mixed gently by slowly turning the tube 10 times manually. As any rapid mechanical
handling of sperm could possibly harm it and alter the viability of it, the sperm was handled
with caution and equally for every queen dissected.
Three drops of 5 µl of the mixture were placed on a microscope slide and 5 µl of SYBR-14
was added to each drop. The slides were then placed into a light repellent box for
incubation in the dark. After 10 minutes of incubation, 2 µl of propidium iodide was added
to each drop and left to incubate for another 7 minutes. A round cover slide was positioned
on the samples and 400 sperm were counted on each drop. To count the samples a
fluorescence microscope was used with filter set to a visible wavelength (as when bound to
DNA, the fluorescence emission maxima of SYBR14 and PI dyes are 516 nm and 617 nm)
with a grid focus calibration.
2.2. Experiment 2
In experiment 2 I examined sperm use patterns of 15 queens that were reared by an apiarist
and artificially inseminated with two different amounts of semen. Semen was all freshly
25
collected from drones. About 1200 mature drones were used for this project. The volumes
chosen for insemination were 3µl and 12µl of semen to magnify possible changes occurring
in sperm use, whereas the average amount of sperm used for 'normal' artificial insemination
in beekeeping is 6-8µl semen per queen. As in the previous experiment, all queens were
kept in 6-framed deep cut Langstroth hives and maintained daily at the university campus.
This experiment was conducted within a timeframe of 2.5 months (February 2013 – April
2013).
2.2.1. Queen rearing
Virgin queens were produced from the hives present in the bee-yard of the University of
Western Australia, using the grafting method. 40 larvae, less than 36 hours of age were
collected from unsealed cells from a breeder queen and placed into artificial (plastic) queen
cell cups (figure 11). The cups mimic the shape and size of natural queen cells that is
different from worker and drone cells, indicating to workers what to feed the developing
larvae. For queen development the larva has to be fed with nutrient-dense glandular food
(secreted by nurse bees) called royal jelly. The feeding has to take place throughout the
queens larval stage of life, therefore the frame with grafting cells was returned to a nucleus
hive where the worker bees could tend to it, and placed between the honey and pollen
frames. The nucleus box was left overnight in a dark, cool place. The frame with the queen
cells was transferred to a full size (feeding) colony about 20 hours after grafting, in a way
that the queen who already existed in the feeding hive couldn’t have any access to the
developing queen cells. In the feeding hive the queen cells were reared to mature, utilizing
the supersedure behaviour of the colony. The frame with queen cells was removed from the
feeding colony 10 days after being grafted. The queen cells were distributed into 3-framed
queen-less nucleus hives with one queen cell per hive. Queens hatched about 2-3 days after
being placed into the nucleus hives. As the queens hatched in queen-less hives, the worker
bees accepted them as one of their own. All queens were reared at the same time to ensure
equality in age when hatching. With a high mortality rate, about 20 queens were expected
26
to successfully hatch and be introduced to the hives from the 40 reared. The surviving
queens were left in their new hives to mature. The time for queen development was 2.5
weeks with an additional week of maturation before artificial insemination. A success rate
of 75% of the inseminated queens appeared, resulting in a total of 15 queens examined in
the study.
Figure 11. Twenty artificial queen cell cups of successfully reared queens
with a close-up (top right corner) of worker-built cell on top of a cell cup.
Photo: author.
2.2.2. Drone and semen collection
Drones were collected 1-3 days before their semen was harvested. The collection was
spread out to 3 days to maximize the possibility of collecting enough drones needed for the
experiment. The collection was done using a simple technique described below. The semen
27
could only be collected from mature drones therefore not every drone in the hive was
suitable for collection. Mature drones perform a nuptial flight at about 1.30 – 4.30 pm,
allowing catching them from the hive door by simply picking them up before or after
flying, using soft-tipped tweezers. Males that are not mature do not yet engage in flights.
The drones were collected into 4 drone cages on 3 different days as only a certain amount
of drones perform the nuptial flight, keeping approximately 300 drones in each cage. The
cages were stored in hives, allowing the workers to naturally care for the drones and reduce
their stress levels before using them for semen collection.
Semen collection was performed immediately prior to the insemination process. Capillaries
were calibrated to allow the collection of either 3µl or 12µl semen, and 10 virgin queens
were used for each of these treatment groups. These quantities were chosen as they
represent the low end and high end of what is used in artificial insemination in the
beekeeping industry (there average volume is 6-8ul). The males were stimulated to evert
their endophallus by placing them in a container on a paper towel with chloroform.
Chloroform forces the abdomen of the drone to contract and a pair of yellow-orange
cornula to be exposed (Figure 12). Only the drones everted were used as they were fully
sexually mature. For complete eversion the treated drones were squeezed between the
thumb and forefinger, applying pressure along the sides of the anterior abdomen towards
the tip.
28
Figure 12. Left: partially everted drone with cornula exposed. Right: fully everted drone
after squeezing in a rolling motion between fingers to achieve the semen to expose. The
white substance in the tip of the capillary is collected semen. Photos by the author.
With the endophallus fully out, pearl-coloured semen would appear on a bed of white
mucus. The semen was collected under a microscope into a prepared capillary avoiding the
collection of the mucus layer. Saline solution was used to prevent the blockage of the tip of
the capillary. Semen from drones who defecated whilst squeezing was not used as the semen
might have been contaminated with faeces and consequently result in failing the artificial
insemination. In order to fill one capillary with the requested amount, semen from several
drones was collected using the same method.
2.2.3. Artificial insemination
ARTIFICIAL INSEMINATION (AI) has been described in great detail in many published
articles and books (Laidlaw 1944, Woyke 1975, Buys 1993, Baer and Schmid-Hempel
2000), therefore it will only be briefly described in this thesis. AI was conducted with virgin
queens 7 days of age, being sexually mature. The queens were placed into a queen holder
with at least 3 segments of the abdomen exposed and sedated with CO₂. The queen’s sting
chamber was opened with ventral and dorsal hooks where the ventral hook was positioned
by securing it on the tip of the queen’s abdominal segment whereas the dorsal hook clasped
the sting (Figure 13).
29
Figure 13. Insemination of the honey bee queen. Image on the left shows the placement of
the capillary to vaginal entrance of the queen and positions of the hook for optimal
performance. Image on the right describes the insemination process with semen entering to
the queens’ oviducts (Cobey 2013).
In order to allow the vaginal entrance to be properly exposed, the sting was pulled to the
outside. These motions allowed the tissue to stretch to a V-like shape, being the entrance
point for the syringe containing semen. For the injection of the semen 2µl of saline would
be used to act as lubricant on the capillary tip. The tip was positioned north-east of the
tissue and inserted approximately 0.5 mm, moved slightly to the left to overcome the bifold valve and continued inserting for another 0.5 mm to allow the passage of the semen.
Figure 14. Left: Group working on queen management during the insemination, semen
After
the syringe
tip was
slipped intoRight:
the median
oviduct,
the semen
was injected
collection
and artificial
insemination.
Insertion
of semen
into a sedated
honey(Figure
bee
queen. Two hooks are used to open the queen’s vaginal entrance, allowing the insertion of
the syringe into the median oviduct. Photos by the author.
30
14). This procedure was repeated on 20 queens, 10 of which were inseminated with 3µl of
semen and the other 10 queens with 12µl of semen. The queens were instantly transported
in queen cages each to a separate, queenless colony ,and left into the cages for
approximately 3-5 days, the time it takes them to eat themselves out of the queen cage and
for workers to be sufficiently exposed to the queen’s scent to accept the her. The success
rate of the insemination process was 75% with the survival of 15 queens after insemination
and maturation within the hive.
2.2.4. Measuring sperm use, sperm number and sperm
viability in the inseminated queens
The techniques used for collecting eggs, measuring sperm use, sperm number and viability
were conducted the same way as described in project 1 (see above).
2.3. Statistics
All data were analysed using statistics programs IBM SPSS Statistics 20 and R. Nonparametric statistics were used to examine differences in sperm use between queens, as the
distribution of sperm use was highly skewed (towards the lower numbers) and was not
normally distributed in queens of different ages (Figure 15). For the subsequent (parametric
or non-parametric) analyses I therefore used the median number of sperm per fertilized egg
per queen instead of the mean. In cases where queens were measured multiple times,
median sperm use per age class (rounded up/down to the nearest whole month after
hatching) was calculated, only including age classes in the statistical analysis if more than 5
eggs in that age class could be examined. As a result, multiple sperm use medians for 6 out
of the 22 queens could be calculated, with each of these 6 queens being examined 2 or 3
times, with 3.6 months between measurements on average.
31
When examining the effect of queen age,
Queen 11
sperm use and sperm viability on sperm use,
I used the median sperm use value of the
latest a month before the queen was
dissected if she was measured over a longer
period of time, instead of using the median
over the entire time she was measured.
Queen 12
Effects of single factors on sperm use were
examined using correlations when the
independent variable was a continuous
variable
(sperm
viability
and
sperm
number), with a Mann Whitney U test if
there
were
two
groups
within
the
independent variable (insemination volume)
Queen 13
or with a Kruskall Wallis test if there were
multiple groups (queen ID).
After examining single factor effects on
sperm use I tried to examine all effects in
the
same
model
using
a
Principle
Component Analysis. The procedure for this
is described at the end of the results section.
Figure 15. Histograms showing examples of
frequency distributions of sperm use. Three
queens were randomly chosen for this graph,
and the graphs represent their sperm use when
they were all one month old. It shows that
sperm use is not normally distributed (skewed)
and that there are differences between queens
in the number of sperm they use to fertilize
eggs. Note that the scale of the x-axis in the
third graph is different.
32
3. Results
3.1. Sperm use during fertilization in naturally
inseminated queens
I found that the distribution of sperm use during egg fertilization of 22 individual queens
was highly skewed (figure 15 & 16). Overall median sperm use was relatively low, with
only 2 sperm per egg. However, queens differed significantly in the number of sperm they
used per egg (Figure 15 & 16, Kruskall-Wallis, H= 517.496, df=21, p<0.001). The majority
of queens (17 out of the 22 queens tested) used between 1 and 3 sperm. The remaining 5
queens used a median of 6, 9, 12, 14.5 and 15.5 sperm per egg. This last queen (queen 13,
see figure 15 & 16) used a particularly large amount of sperm for some of her eggs; out of
the 44 eggs that I analysed for her in total 4 (9%) had 100 or more sperm cells on them and
11 (25%) had 50 or more sperm on them.
33
Figure 16. Boxplots illustrate sperm use in all 22 queens, showing the difference in
the median sperm use in individual queens (indicated by the thick line). Data points,
where queens were measured at more than one age, were pooled. Whiskers of the
boxplot illustrate minimum/maximum value of the data, excluding outliers which are
marked as circles and rectangles.
3.2. Effects on sperm use
3.2.1. Queen age
The effect of age on the number of sperm used per egg was examined. It was found that
median sperm use decreased with queen age (Figure 17; r = -0.529, n = 28, p = 0.004), with
queen age explaining 28 per cent of the variance in sperm use. Medians of sperm use are
34
shown in table 1. The sperm use in one and three month old queens was found to be high
(median of 7 and 9 sperm per egg) and then drop around 4+ months of age to a steady use
of 1 -4 sperm per egg. When separately looking at the queens that were measured multiple
times (Queen 1; 3; 5; 13; 14 and 16), I found a decrease in sperm use with age in 4 of the 6
queens. In queen 14 sperm use stayed the same (over a 2 month period) and in queen 3
sperm use even went up with one sperm per egg over a 3 month period.
Queen ID
35
Sperm use per egg during fertilization (median)
30
25
20
15
10
5
0
0
5
10
15
20
25
30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Age of the queen (months)
Figure 17. Number of median sperm used per queen shows a decrease with age. Individual
queens are marked with different colours. All queens measured multiple on multiple ages
are connected with line.
35
Table 1. Sperm use medians in queens of different ages show higher use of sperm in the
first months of the queens life (median of 7 – 9 sperm), then lowering to 4 sperm per egg in
4 months old and down to 1-3 sperm in queens aged 7-28 months. When there were
multiple queens of the same age, their sperm uses at that age were pooled. N is the number
of eggs analysed in a particular age group in total. All medians are reported with standard
error.
Age
(months)
1
3
4
7
10
18
19
20
25
27
28
Total
Median
7.00
9.00
4.00
1.00
2.00
1.00
1.00
2.00
3.00
1.00
1.00
2.00
N
163
15
211
29
154
211
61
19
62
25
19
969
St. Error
3.55
5.95
0.51
0.07
0.34
0.13
0.14
0.44
0.77
0.04
0
3.2.2. Sperm viability
I examined the relationship between the quality of sperm stored in the spermatheca
(examined as sperm viability) and the number of sperm used by that queen to fertilize eggs.
Sperm viability and sperm use were measured both in naturally inseminated queens
(experiment 1) as well as artificially inseminated queens (experiment 2). When looking at
the naturally inseminated queens, sperm viability on its own had a significant effect on
sperm use; the higher the viability of sperm the more sperm the queen used for the
fertilization of a single egg (r=0.773, n=14, p=0.001) (Figure 18). Only 14 queens were
measured out of 22 as during the project some queens died (a natural death) , leaving only
14 to be used for dissecting and further studies.
36
Median sperm use per egg during
fertilization in naturally inseminated
queens
16
14
12
10
8
6
4
2
0
0
20
40
60
80
100
Sperm viability (%)
Figure 18. Sperm viability was measured instantly after dissection of 14 queens of different
age (naturally inseminated) and was plotted against sperm use (measures of the last age of
the queens were used in queens that were measured multiple times). The figure suggests
that sperm viability and sperm use are positively correlated.
This significant correlation remained when the two queens with the highest sperm use at the
end of their lives (queen 12 with 12 sperm per egg, and queen 18 with 15.5 sperm per egg)
were removed from the dataset, leaving only queens with a final sperm use of 4 sperm per
egg or less (r=0.754, n=12, p=0.002).
When looking at artificially inseminated queens with different volumes, sperm viability
didn’t have a significant effect on sperm use during fertilization (r=0.296, n=15, p=0.284)
(Figure 19). When removing the one queen with a high sperm use from the dataset (queen
number 7 with median of 26 sperm per egg), no changes could be tracked, sperm viability
was still not significantly correlated with sperm use (r=0.428, n=14, p=0.126).
37
However, when the dataset was split up into the two treatment groups, I found that within
the low AI volume group (inseminated with 3 µl) sperm viability was not affecting sperm
use (r=-0.119, n=7, p=0.799), but it was in the high volume treatment group (inseminated
with 12 µl , r=0.782, n=7, p=0.038).
Median sperm use during fertilization in AI
queens
30
Insemination
volume
25
3 ul
20
12 ul
15
10
5
0
0
20
40
60
80
Live sperm in spermatheca after dissection (%)
Figure 19. No overall significant increase in the number of sperm used per egg during
fertilization can be seen in relation to an increase in the percentage of viable sperm in
the spermatheca (measured instantly after dissecting the queens), measured in 15
artificially inseminated queens. When examined separately, an there is a significant
postive relationship in the queens inseminated with 12 µl of semen (open circles on
figure).
3.2.3. Sperm number and volume
In experiment 1 I examined sperm number in naturally mated queens to see if sperm
number in spermatheca (counted after dissecting the queens) had an effect on median
38
number of sperm used during fertilization. I found a significant effect (r=0.609, n=14,
p=0.021), with sperm number explaining 37 % of the variation in sperm use. This
indicatesthat the higher the amount of sperm in spermatheca, the more sperm the naturally
Median seprm use during
fertilization
mated queen uses for fertilizing eggs (Figure 20).
18
16
14
12
10
8
6
4
2
0
0
1000000 2000000 3000000 4000000 5000000 6000000
Sperm number in spermatheca after dissection in
naturally inseminated queens
Figure 20. Illustration of the amount of sperm stored in the spermatheca after dissection on
sperm use shows, that the more sperm is stored in naturally inseminated queens, the more
sperm they would use to internally fertilize an egg.
In the second experiment I more directly examined sperm number by artificially
inseminating queens with either 3 µl or 12 µl and studying their sperm use afterwards. To
check whether queens that where inseminated with 3 µl of sperm actually had less sperm in
storage than queens that were inseminated with 12 µl sperm, a sperm volume count was
done. I saw that smaller insemination volume in fact resulted in smaller amount of sperm
stored in spermatheca (mean of stored sperm in the spermatheca of 3 µl inseminated queens
1,076,907.14; SE=491,531.99), and a larger volume inseminated lead to a larger number of
sperm in storage (mean number of sperm in the spermatheca of 12 µl inseminated queens
2,747,187.50; SE=223,312.51; volume inseminated with was)
39
I tested the effect of the amount of sperm stored in the spermatheca on sperm use. As was
found in the naturally inseminated queens, the number of stored sperm was shown to
significantly affect sperm use (r=0.611, n=14, p=0.020, one outlier removed from the
dataset, figure 21).
Median sperm use during
fertilization in AI queens
30
25
20
15
3 ul
12 ul
10
5
0
0
2000000
4000000
6000000
Sperm number in spermatheca after dissection
Figure 21. Sperm stored in the spermatheca (measured after dissection of the
queens) has a significant effect on the median of sperm use in artificially
inseminated queens (insemination volumes of 3 µl and 12 µl).
3.3. Effects on sperm viability
Besides examining effects on sperm use, with my dataset I could also analyse whether
queen age and sperm number affects sperm viability.
3.3.1. Queen age
The results of experiment 1 revealed a significant negative effect of queen age on the
viability of sperm stored in spermatheca (r=-0.767, n=14, p < 0.001) (figure 22). The older
the queen, the less viable their sperm is after dissection from the spermatheca.
40
Figure 22. A negative correlation between viable sperm in spermatheca (percentage of total
amount of sperm counted in spermatheca) and the age of the queens shows decline of
viability with the queen age.
3.3.1. Effect of volume
In the second experiment I artificially inseminated queens of the same age with either 3µl
or 12 µl of semen. It was found that the percentage of viable sperm in the spermatheca after
dissection does not differ significantly between treatment groups (Mann-Whitney U=14.00;
Z=-1.342, p=0.209), as can be seen in figure 23 (error bars are overlapping, indicating no
significant difference between treatment groups).
41
Sperm viability after dissection (%)
65
60
55
50
45
40
1
3 µl
12 µl
Volume of sperm used for artificial insemination (µl)
Figure 23. The graph illustrates the difference of mean values of viable sperm present in
spermatheca after dissection with standard errors (brought out as whiskers on the graph).
Queens inseminated with 3 µl of sperm have slightly lower percentage of viable sperm in
their spermatheca (52.57 with standard error of 4.41) than queens inseminated with 12 µl.
(55.85 with standard error of 5.29).
3.4. Queen age effects on sperm number in spermatheca
Lastly, in the first experiment I could also examine whether the number of stored sperm
was affected by queen age. The results reveal a highly significant negative correlation
between the two factors (N =505, Pearson correlation=-0.71, p<0,01) (Figure 24); the older
queens are, the less sperm is stored in the spermatheca. The mean of the sperm number in
the spermatheca of 1-2 month old queens was 3,549,750, and 511,200 in queens that were
18-27 months old. Means of sperm stored in the spermatheca for all the different age
classes is shown in Table 2.
42
Table 2. Number of sperm in spermatheca (mean) in queens of different
ages show that the younger the queen, the more sperm is in the spermatheca.
Age (months)
1
2
3
7
10
18
25
28
Mean
3,740,534
1,112,150
1,112,150
1,182,950
890,095
357,739
799,450
525,100
Std. Deviation
1401101.56
0.00
0.00
0.00
139151.54
236668.11
0.00
0.00
Number of
queens
3
1
1
1
4
3
1
1
Figure 24. The graph illustrates the correlation between age and the number of sperm in
spermatheca (µl) after dissection of the queens showing, that the younger the queen, the
more sperm is in the spermatheca.
43
3.5. Summary and Principle Component Analysis
Table 3. Summary of the results of project 1 and project 2.
Dependent variable
Sperm use
Sperm viability
Sperm number
Independent variable
Effect Direction of effect
Queen Age
Sperm Viability
1.in naturally mated queens
2. in queens AIed with 3 µl
3. in queens AIed with 12 µl
Stored sperm number
Queen Age
Insemination volume (in AI)
Yes
Negative
Yes
No
Yes
Yes
Yes
No
Positive
N/A
Positive
Positive
Negative
N/A
Queen Age
Yes
Negative
A summary of all effects is presented in table 3. It seems that there are various factors that
could influence sperm use. However, it is unlikely that these factors operate independently.
On the contrary, it is clear that the factors sperm number in the spermatheca, sperm
viability of sperm in the spermatheca, and queen age are correlated. The question now is
whether these separate effects still influence sperm use when they are entered into the same
multiple regression model. However because these factors are correlated, you violate the
assumption of no (multi)collinearity between predictor variables for this type of analysis. A
Principle Component Analysis (PCA) was therefore conducted to convert the correlated
predictor variables into linearly uncorrelated variables, called principle components.
In experiment 1, the effect of the independent variables queen age, sperm number and
sperm viability on sperm use were examined. Three principle components (PCs) were
calculated. As PC1 and PC2 together already explained 93.31% of variance, PC3 was left
out of the subsequent analysis. PC 1 mostly represents queen age (loading onto the
component -0.926), but also correlates with sperm number (loading 0.859) and sperm
viability (loading 0.906), and explained 80.56% of variance. This opposed signs between
queen age on the one hand and sperm viability and sperm number on the other hand suggest
44
that sperm viability increases whenever sperm number increases, but both decrease with
queen age. PC2 was correlated with sperm number (loading 0.508) and explained 12.75%
of the variance. PC1 and PC2 and their interaction term were entered as covariates in a
multiple regression analysis examining the effect on the dependent variable sperm use. The
analysis showed that PC1 had a significant effect on sperm use (F1,14=13.429, p=0.004),
PC2 and the interaction term had no effect, even though the latter two were very close to
significance (F1,14=3.678, p=0.084 and F1,14=4.506, p=0.060 respectively). The fact that
PC1 has a significant effect can be interpreted as queen age has an effect on sperm use,
either directly, or through its effect on sperm viability and sperm number in the
spermatheca. That PC2 has no significant effect suggests that sperm number on its own
does not influence sperm use. However, as stated above, this effect was nearly significant,
so it is very likely that a significant effect is found when more queens are tested. In
conclusion, when running all predictor variables in the same model to examine their effect
on sperm use, it was found that all have an effect (significant or near-significant), but that
queen age is the main predictor for sperm use.
45
DISCUSSION
In this study a new method of counting sperm on freshly laid eggs was successfully applied
to honey bees (Apis mellifera). After its previous application in the ant species Atta
colombica, I have shown with these two projects, that the method of using the fluorescence
stain (DAPI) is also suitable to stain and visualize sperm cells on freshly laid eggs in honey
bees. This study therefore represents the first study to directly determine sperm use per egg
in honey bees, while previous studies calculated sperm use using data on stored sperm
number and lifetime egg production.
The analysis of the first experiment using naturally inseminated queens reveal that overall,
queens of Apis mellifera use a median of 2 sperm per egg during fertilization. This result is
smaller than the estimated sperm use calculated so far (4 - 100 sperm/egg) (Bresslau 1905,
Adam 1912, Harbo 1979). In the artificial insemination experiments, sperm use medians for
queen inseminated with 3µl and 12µl of semen used were 3 and 5 sperm per egg
respectively. These numbers are higher than those found for most of the queens in the first
experiment. One explanation for that fact could be that the queens used for AI (second
experiment) were much younger than most of the queens used in experiment 1. The median
sperm use in very young queens in first experiment (1-3 months old) was 7-9 sperm per
fertilized egg. As this example also suggests, I found that age has a significant effect on
sperm use. With age measured in 22 queens in experiment 1, the analysis shows that sperm
use goes down during a queen’s lifetime. Sperm use starts off high, with a median of 7 in 1
month old queens. A relatively sudden drop in sperm number occurs at about 4 months of
age (table 1), continuing to decrease with age.
46
Even though a sperm use decrease with age seems to be a general pattern, it is not true for
all of the 6 queens that I measured over a longer time span. In 4 out of six queens sperm use
went down over time. However, sperm use in queen 3 went up in 3 months’ time and in
queen 14 sperm use remained at one sperm per egg irrespective of her age (measured over a
2 months period) (Figure 19). This suggests that even though sperm use decreases with age
at a population wide level, there is variation between queens within such a population.
However, these two queens were only measured over a time span of 2-3 months, which
might be too short to conclude about a general pattern for these queens. It might also be that
queens fluctuate their sperm use from month to month and season to season, possibly due
to some individual or intracolonial factors (like queen stress, hive strength) or external
factors (like outdoor temperature, humidity, seasonal changes). It is known for example that
at the end of the season, when queens go into hibernation less workers are produced
(Winston 1991). Queens don't lay that many eggs, and this decrease in egg laying rate
might also have influence on a queen's sperm use. Even though honeybee colonies in
Australia do not hibernate as they do in more temperate climates, it is still very possible
that egg laying rates fluctuate during the season. Future work is needed to examine whether
egg laying rate indeed affects sperm use.
It is relevant for the queen to optimize her sperm use already at an early stage of her
reproductive life. In order to be fully accepted in the hive as a freshly sexually matured
female, the young queen needs to prove her reproductive efficiency. Using a higher number
of sperm for fertilizing each egg at the earliest period of her reproductive life, a queen
likely makes sure that all the eggs she lays are fertilized. This signals to the workers her
quality - her ability to maintain the strength of the hive and efficiently produce offspring.
As the total volume of sperm stored in her spermatheca decreases with every fertilized egg
that is laid, a queen quickly needs to minimize the number of sperm used per egg as much
as possible, in order to prolong her reproductive life and thus her position in the hive. The
noticeable drop in sperm use that I find already in first few months could thus be caused by
the need to stabilize the sperm use to ensure long-term fertility.
47
How sperm release is regulated on a physiological level remains unclear. As written in the
introduction, Harbo (1979) hypothesised that the volume of fluid in the spermatheca (which
is a combination of sperm and secretion produced by the spermathecal glands) probably
remains the same over a queen's lifetime as spermathecae don't seem to change in size. As
queens produce on average 200 000 fertilized eggs yearly (Snodgrass 1984, Winston 1991)
the concentration of sperm in spermathecal fluid decreases every time sperm is released. I
hypothesise that each time a queen lays an egg, she releases a specific volume of sperm-
Number of sperm remaining in
the spermatheca
containing fluid from the spermatheca.
120
The sperm plus fluid are then replaced
100
80
by fluid alone, which gradually reduces
60
the concentration of the sperm in the
40
spermatheca. This would mean that the
20
number of sperm used per egg is
0
0
20
40
60
Number of eggs released after the
mating flight
reduced when a queen ages due to a
lowered number of sperm in the
spermatheca. Indeed, I find exactly such
a pattern in my data: sperm use
Figure 25. Predicted sperm release pattern
according to Harbo's theory
decreases with queen age and sperm
number.
Harbo's
hypothesis
even
explains why there is a sudden drop in sperm use in the first few months, after which sperm
use levels out. Imagine a hypothetical scenario where a queen stores 100 sperm cells in her
spermatheca and the total volume of fluid in that spermatheca is 100μl. Imagine that every
time fluid is released from the spermatheca she releases 10μl, which is 1/10 of what is
stored in the spermatheca at that time. And every time some fluid is released it is replaced
with 10μl of spermathecal gland secretion. Now when the queen lays her very first eggs
1/10 of the sperm cells (1/10*100=10) are released in 10μl fluid, leaving the queen with
100-10=90 sperm cells. 10μl of fluid is added so now we have 90 sperm cells in 100μl. For
her second egg, the queen again releases 10μl fluid (1/10 of her total volume), which
corresponds to 1/10*90=9 sperm cells. She now has 90-9=81 sperm cells in storage. For the
third egg, again 10μl is released, which contain 1/10*81=8,1 sperm cells, leaving her with
48
81-8,1=72,9 sperm cells and so on and so on. If we plot this we get the graph in figure 25.
Figure 25 looks identical to figure 17 where the actual data are plotted. Harbo's theory
therefore can explain our data and I can therefore conclude that the mechanism of sperm
release from the spermatheca is regulated via the release of a fixed volume of fluid that is
replaced with sperm free fluid after every egg.
However there is one big difference between graph 26 with the hypothesised data and graph
17 with my actual data. In graph 25, sperm use will eventually come close to zero. In my
actual dataset median sperm use never gets below two. This seems to suggest that over
evolutionary time queens have been selected to use as few sperm as possible, but that the
lowest number of sperm that could actually be used for queens to have reproductive success
is two, two is the absolute limit for minimum sperm use. Queens that used less than two
sperm per egg probably laid too many unfertilized eggs and were replaced by workers.
Such queens had very low reproductive success, so that their genes were less likely to be
passed on to a next generation. The genotypes coding for extreme low (<2) sperm use
disappeared over evolutionary time (as well as the genotypes for extreme high sperm use)
leading to a 'fine tuned' genotype for low, but not too low sperm use.
The results do not only show effects of queen age on sperm use, but also effects of queen
age on sperm number and sperm viability. Both sperm number and sperm viability go down
when queens get older. In naturally mated queens sperm viability and sperm number will
always be tied to queen age; even though there is variation between queens, younger
queens will on average always have more sperm, and sperm of higher quality, in storage
then older queens. In the artificial insemination experiment, queens were all the same age,
thereby 'removing factor age from the equation' so that the effects of sperm number and
sperm viability on sperm use could be examined independent of queen age. These analyses
show that there is a relationship between sperm number and sperm use, which can be
explained with the Harbo interpretation above. The effect of sperm viability was not so
clear cut. Sperm viability was only correlated with sperm use in the queens that had been
49
inseminated with a larger volume of sperm and not in queens that were inseminated with a
smaller volume of sperm. The average number of sperm that was stored in the 3μl group
was about 1 million, and 2,75 million in the 12μl group. These numbers lie far below the
number found in naturally mated queens of the same age; 3,75 million in 1 month old
queens (table 2). Whether this has anything to do with the finding that viability only had an
effect in the queens that had more sperm in storage is unclear, at present I cannot find any
explanation for this. It is important to note that the percentages of viable sperm that I found
in the saline solution do not necessarily reflect the percentage of live sperm as it is in the
spermatheca. It is known that sperm is killed due to the treatment as well. It might therefore
be that all sperm were alive when they were in the spermatheca, but are differently affected
by pipetting and dilution between queens & treatments. For example, sperm in older queens
might be more fragile, and therefore the effect of treatment on sperm in older queens is
greater, resulting in lower sperm viability overall. Propidium iodide stains sperm which has
damaged plasma membranes red. But these sperm cells might be in the act of dying (=not
yet dead in the spermatheca) and this is not possible to distinguish between these two
possibilities. The idea that sperm is actually not dead while still in the spermatheca is
supported by fact that even though 75% (see Figure 23) of unviable sperm was detected in
queens that were 28 months of age, the colonies they headed were strong and healthy, even
though the median sperm use on the same queen was one sperm per egg (see table 1)
Furthermore, it was found in mated stingless bee queens that the epithelial cells of
spermathecal walls contained sperm pieces (Cruz-Landim, Yabuki et al. 2003), suggesting
that dead sperm cells were absorbed by the spermathecal wall. This in turn leads to the
prediction that sperm is not just kept alive in the spermatheca, but also that only live sperm
would be accommodated in the spermatheca and dead sperm removed. If this was the case
in honey bees as well, it would be possible that sperm which has so far been counted as
dead would actually not have been dead in the spermatheca and would be perfectly able to
fertilize eggs. That I find a correlation between sperm viability and sperm use therefore
does not necessarily indicate a causal relationship between the two. It might simply be
because sperm viability is so closely linked to queen age and queen age has an effect on
sperm use. This might also explain why I only got a clear cut result for sperm viability
50
effects in the first experiment where queens of different ages were used, but only partly in
the second experiment, where queens of one age class were used.
The results obtained could provide some guidelines for bee keepers for the process of queen
replacement. Beekeepers in Western Australia standardly replace 1 year old queens because
they might not perform optimally in the near future. This prematurely killing and
replacement of honey bee queens ensures the beekeepers that their hives remain strong.
However, queen replacement is a lengthy and therefore costly procedure. More insights into
sperm use pattern to better predict the end of a queen's reproductive life could therefore be
beneficial. Using the data in this study I can make some calculations. With an annual
insemination rate of 200 000 eggs (Snodgrass 1984, Winston 1991), median sperm use of 8
sperm per egg in the first 3 months of the queen's life and overall median sperm use of 2
sperm per egg for the rest of her life, it can be calculated that within 2 years queens have
used up roughly 1 100 000 sperm (400 000 sperm within the first 3 months and 700 000
sperm in the following 21 months). In this study we saw that the mean number of sperm in
the spermatheca in queens of 1 month old was approximately 3 740 000 sperm and queens
of 28 months of age still about 525 100 sperm in their spermatheca. Theoretically the old
queens of 28 months could still fertilize at least 250 000 eggs (meaning equal amount of
workers), which is more than a year worth of eggs.
51
CONCLUSION
The aim of the current study was to investigate sperm use patterns in honey bee (Apis
mellifera) queens during egg fertilization, and the factors influencing it. For this purpose I
examined the actual number of sperm used by the queen to fertilize an egg. The influencing
factors that were measured in this study are queen age, sperm number in spermatheca and
sperm viability.
Firstly the study showed that it is possible to successfully apply a new method to allow
quantification of the sperm number on freshly laid eggs using DAPI fluorescence stain to
honey bees (Apis mellifera). DAPI effectively stained the nuclei that situates in the sperm
heads, allowing an easy method to count and quantify the sperm on fertilized eggs. This
kind of method had previously been used on Atta colombica, the leaf cutter ants, but not yet
been practiced on honey bees.
The main results of the study showed that young queens indeed use more sperm to fertilize
an egg compared to the old queens and therefore proving the first hypothesis. The medians
of sperm use in queens of 1-3 months old are 7-9 sperm per egg (n=178 eggs). In 4 months
old queens the sperm use has dropped down to 4 sperm per egg in median (n=211 eggs),
whereas 7-28 months old queens have developed a quite stable sperm use with just 1-2
sperm per egg (n=580 eggs measured for sperm use). Sperm number in spermatheca also
had a significant effect on sperm use, showing that with higher number of sperm present in
spermatheca queens use more sperm per egg than with lower numbers. The experiments
also showed that sperm viability only had a significant effect on sperm use in certain
52
conditions: 1) In naturally mated queens sperm viability was significantly higher in
younger queens than older (this could be explained through the aging of the queens); 2) In
queens inseminated with large volume (12 µl) of sperm, the significance was seen (in
contrast to the queens inseminated with 3µl, where no such effect occurred).
In the end all three main factors influencing sperm use (sperm viability, sperm number in
spermatheca after dissection and queen age) was put into one test to distinguish the main
affecter on sperm use during fertilization. It was found that queen age influences sperm use
the most, with sperm use being higher in queens of young age (1-3 months) compared to
old age (up to 28 months as measured in this study).
A possible explanation to the results obtained about the significant effect of queen’s age and
sperm number in spermatheca on sperm use could be through the hypothesis made in late
1970’s that the concentration of sperm in spermathecal fluid decreases every time sperm is
released. It is possible that each time a queen lays an egg she releases a specific volume of
sperm-containing fluid from the spermatheca. The sperm plus fluid are then replaced by
fluid alone, which gradually reduces the concentration of the sperm in the spermatheca.
This would mean that the number of sperm used per egg is reduced when a queen ages due
to a lowered number of sperm in the spermatheca. This sort of pattern is indeed found in
my data, where sperm use decreases with queen age and sperm number.
The results obtained could possibly provide some guidelines for bee keepers for the process
of queen replacement. Approximate suggestive calculations based on the results obtained
from this study show, that the queen would still be fertile after 2 years of constant
fertilization of the eggs, being hypothetically able to produce at least another 250 000
worker bees (with the insemination rate of 200 000 eggs per year and fertilization rate with
a median of 2 sperm per egg).
53
ACKNOWLEDGEMENTS
The author would most gratefully like to thank Susanne den Boer and Marika Mänd for
their excellent supervision and Boris Baer for his helpful advice, inspiring and teaching the
author all techniques necessary, as well as for offering moral support throughout the
process. In addition the author would like to thank Tiffane Bates and Ben Liebenberg for
creating the space with bees and offering practical and theoretical knowledge about
beekeeping. The author is also grateful to David Nash (from the University of Copenhagen)
and Allan Sims (from Estonian University of Life Sciences) for providing with helpful
information on statistics, CIBER group for its support and generosity in sharing their
knowledge in many aspects, as well as University of Western Australia for hosting the
author of the thesis in their university.
54
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58
Insemination of the honey bee queen. Susan Cobey.
[http://www.extension.org/pages/28329/insemination-of-the-honey-bee-queen]
(28.04.2013)
Queen bee anatomical drawings. Katherine Bujalska.
[http://www.katherinebujalska.co.uk] (23.05.2013)
Honey: World Production, Top Exporters, Top Importers, and United States Imports
by Country.
http://worldtradedaily.com/2012/07/28/honey-world-production-top-exporters-topimporters-and-untied-states-imports-by-country/ 28/07/2012 by Isaac Thompson
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http://marketnews.usda.gov/portal/fv/honey
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APPENDIXES
Appendix 1. Detailed summary of the thesis in Estonian
Sperma kasutus meemesilastes: spermatosoidide loendus
viljastatud munarakkudel mõõtmaks ema viljakust
Meemesilastel (Apis mellifera) on ühe pere kohta vaid üks viljastumisvõimeline emane
ning mitu isast, kelle ainsaks ülesandeks elus on tagada oma päriliku materjali edasi
andmine ehk ema viljastamine. Ema paaritub reeglina vaid kord elus paaritumislennu ajal,
kus teda viljastab keskmiselt 12 isast. Isased surevad peale suguühet, kuna paaritumise
tagajärjel rebeneb nende suguelund nende küljest, jäädes ajutiselt emamesilasse. Isased
kukuvad paralüseeritult maha surema. Ema kogub esialgse isastelt saadud sperma mahu
esmastesse suguteedesse, millest jõuab lõppkokkuvõttes spermapauna kokku vaid väike
osa.
Spermapaun on organ, kus hoiustatakse elujõulist spermat ema paaritumise ning muna
viljastamise vaehpealsel ajaetapil. Munarakkude viljastamine toimub ema siseselt, munedes
aastas ligi 200 000 viljastatud muna. Kuna ema paaritub vaid korra oma elu alguses, tuleb
tal tagada spermapaunas hoiustatava sperma elujõulisus kuni aastateks. Sperma jaotus
munarakkudele kogu ema elu vältel on mõjutatud kahest limiteerivast tegurist, millest
sõltuvalt optimeerib ema spermatosoidide arvu, mida ta kasutab muna raku viljastamiseks.
Esiteks, kuna pere üheks oluliseks tugevuse näitajaks on toodetud tööliste arv, peab ema
optimeerima kasutatud spermatosoidide arvu nii, et tööliste toodag oleks tagatud ka ema
kõrgemas eas. See eeldab spermatosoidide võimalikult madalarvulist kasutamist. Samas on
täheldatud sperma kvaliteedi langust paralleelselt ema vanuse suurenemisega, mistõttu peab
ema viima spermatosoidide arvu piisavalt kõrgele, et tagada viljastumise efektiivsus. Kui
ema ei suuda enam läbi viljastatud munade munemise toota piisavalt töölisi, asendatakse ta
60
pere poolt uue, tõenäoliselt efektiivsema emaga. Seetõttu on oluline, et ema sperma kasutus
oleks optimaalne, et tagada ühelt poolt pikk vijastumisiga ning teiselt poolt kasutada
piisavalt palju spermatosoide, et tagada munarakkude efektiivne viljastamine igas
ajahetkes.
Seniajani ei oldud viidud läbi uuringut, mis võimaldaks täpse, värskelt viljastatud
munarakul oleva spermatosoidide arvu kindlaks tegemist. Kogutud andmed sperma
kasutuse kohta olid saadud vaid läbi kaudsete arvutuste, jagades kogu spermapaunas oleva
sperma arvu toodetud tööliste arvuga. Sellisel meetodil saadud spermatosoidide arv
varieerus 4-100 spermatosoidini, olles äärmiselt umbkaudne hinnang.
Sperma kasutuse uurimise olulisus lasub peamiselt kahel eri suunitlusega alal. Esiteks
omavad
tulemused
suurt
rolli
mesilaste
reproduktiivsussüsteemide
mõistmisel
arengubioloogia vallas, aidates vastata küsimustele eri organite funktsioonide koha pealt
seoses sperma kasutusega emas. Teisalt saavad tulemustest kasu ka need mesinikud, kes
tegelevad intensiivsema mesindusega, kus emasid peredes vahetatakse enneaegselt emade
loomulikku surma. Mainitud käitumise üheks põhjuseks on uskumus, et vanemad emad ei
suuda tagada pere tugevust läbi piisava arvu tööliste tootmise, kuna nende spermapaun võib
elujõulisest seemnest tühi olla.
Käesoleva töö esmaseks eesmärgiks oli kohandada ning praktikasse panna uuringu
põhimeetod spermatosoidide visualiseerimiseks värskelt munetud munadel meemesilastes.
Meetod on välja töötatud CIBER (Centre of Integrative Bee Research) grupi liikmete
Susanne den Boer ning Boris Baer poolt, kes kasutasid seda efektiivselt esialgselt
sipelgaliigi Atta colombica peal. Uuringu uudsus seisneb meetodi esmakordses kasutamises
meemesilases. Meetodi unikaalsus ning lihtsus peitub fluoretsents värvi kasutuses, kus
terve muna katmisel kõnealuse värviga on võimalik esile tuua vaid DNA pärilikku materjali
sisaldavad elemendid ehk spermatosoidide pead. Olemasolul värvub ka juba arenguteed
alustav sügoot.
61
Käesoleva töö keskne uurimisala on sperma kasutuse muutused, mida vaadeldakse
mõjutatuna eri teguritest. Antud juhul on peamiseks mõjuatatvaks faktoriks ema vanus
(seprma kasutuse muutused vastavalt eri vanuselistele emadele), kuid vaadeldakse ka
sperma kasutuse (viljastamisel munarakuga) seost sperma elujõulisusega spermapaunas
peale ema lahkamist ning spermatosoidide koguarvu mõju spermapaunas (leitud peale
emade lahkamist) sperma kasutusele. Peamise hüpoteesina väidetakse antud uurimistöös, et
sperma kasutus on erivanuselistes emades erinev, kus spermatosoidide arv munaraku
viljastamiseks väheneb ema vanuse suurenedes. Teiseks, väidetakse antud uurimistöös, et
sperma kasutus on erinev erinevate sperma koguste juures spermapaunas, kus
spermatosoidide
arv
munaraku
viljastamiseks
suureneb
spermatosoidide
arvu
kõrgenemisega spermapaunas.
Töö läbivateks etappideks on: 1) tarude ettevalmistus; 2) värskelt munetud munade korje
läbi kahe projekti, kokku ligikaudu 13 kuud; 3) spermatosoidide loendus värskelt munatud
munadel; 4) emade lahkamine ning spermapaunade laboratoorne analüüs. Lisaks
eelmainitule on teises projektis juures emade kunstlikult viljastamise etapp, mis hõlmab
endas muuhulgas emade aretamist, isaste korjet, seemne korjet ning emade viljastamist
laboratoorsetes tingimustes. Saadud andmed on analüüsitud statistilisi meetmeid kasutades.
Esimeses uuringus vaadeldi ema vanuse mõju sperma kasutusele. Uurimisobjektideks olid
22 erivanuselist ema, kelle sperma kasutust varieeruvatel aegadel aasta jooksul mõõdeti.
Teises uuringus kasutati lõpptulemusena 15 kunstlikult viljastatud ema, kus kaheksa ema
viljastati kunstlikult 12 µl vastselt enne viljastamist kogutud sperma kogusega ning seitse 3
µl sperma kogusega. Nii esimeses kui teises uuringus oli vaatluse all sperma kasutuse
mõjutatus
munaraku
viljastamisel
sperma
kogusest
spermapaunas
ning
sperma
elujõulisusest spermapaunas, mõõdetuna peale emade lahkamist.
Tulemustena saadi positiivne kinnitus püstitatud hüpoteesile, kus sperma kasutus langes
ema vanuse tõusmisel (r = -0.529, n = 28, p = 0.004). Kasutatud spermatosoidide
mediaaniks 1-3 kuu vanuselistes emades on 7-9 spermatosoidi muna kohta, langedes järsult
4 spermatosoidini nelja kuulistes emades ning taandudes juba 7-kuulistes emades 1-2
62
spermatosoidini muna kohta, püsides sellisel tasemel ka vanimates emades, kelle sperma
kasutust antud uuringus mõõdeti (28 kuud). Üldine mediaan on 2 spermatosoidi muna
kohta vaadelduna üle kõigi emade.
Erivanuselistes emades leiti spermapaunas oleva sperma elujõulisuse oluline mõju sperma
kasutusele (r=0.773, n=14, p=0.001) näidates, et mida suurem on sperma elujõulisus seda
kõrgem on omakorda sperma kasutus munaraku viljastamisel. Kunstlikult viljastatud
emades mõjutas sperma elujõulisus oluliselt vaid nende emade sperma viljastamiskasutust,
keda oli eelnevalt seemendatud suurema spermakogusega (12 µl).
Saadud spermatosoidide arvukuse mõju spermapaunas sperma kasutusele munaraku
viljastamisel kinnitas samuti püstitatud hüpoteesi, kus on näha, et mida kõrgem on sperma
arvukus spermapaunas (peale emade lahkamist), seda rohkem spermatosoide kasutavad
emad munaraku viljastamiseks. Antud tugevat korrelatsiooni oli näha nii loomulikul teel
paaritunud emades (r=0.609, n=14, p=0.021) kui ka kunstlikul teel viljastatud emades
(r=0.611, n=14, p=0.020). Keskmised spermatosoidide arvukused spermapaunades olid: 1-2
kuu vanuselistes emades 3 549 750 spermatosoidi; 18-28 kuu vanuselistes emades 511 200
spermatosoini keskmiselt. Kunstlikult viljastatud emades olid keskmised spermatosoidide
arvukused spermapaunades järgnevad: emades, kes olid viljastatud 3 µl seemne kogusega
oli spermatosoidide arv spermapaunas 1 076 907 ning 12 µl seemendamiskogusega emades
vastavalt 2 747 187 spermatosoidi.
Ema vanuse mõju spermatosoidide elujõulisusele oli oluline (r=-0.767, n=14, p < 0.001)
näidates, et mida vanem on ema, seda madalam elujõulisus spermal on.
Vanuse mõju spermatosoidide arvule sperma paunas oli langeva iseloomuga (N =505,
Pearson correlation=-0.71, p<0,01) – mida vanem ema, seda vähem spermat paunas, kus
keskmine sperma kogus oli suurim1 kuulistes emades 5.56 miljonist isendiga ning
madalaim 10-28 kuulistes emades, kus spermatosoidide arvuks oli ligikaudu 56 050– 890
000 isendit.
63
Viimaks testiti kolme põhifaktori mõju sperma kasutusele munaraku viljastamisel (ema
vanus, spermatosoidide arv ning elujõulisus spermapaunas) küsides, milline mõõdetud
faktoritest omab suurimat mõju sperma kasutusele. Selgus, et suurimaks sperma kasutuse
mõjuteguriks on emade vanus.
Saadud tulemused toetavad 1970. aastate lõpus tõstatatud teooriat, kus väidetakse, et
spermatosoidide konsentratsioon spermapauna vedelikus kahaneb aja jooksul ning
tulemuslikult mõjutab seega spermatosoidide arvu munaraku viljastamisel aja jooksul.
Kuigi seniajani ei ole tõestatud, kas spermapauna vedelik on tegelikult konstantne läbi aja,
on antud uurimuse tulemustest näha, et sperma kasutus kahaneb ema vanuse kahanedes.
1970ndatel tõstatatud teooriat toetab ka antud uuringus saadud sperma kasutamise languse
järsk muutus peale 3-4ndat elukuud. Vaadates näiteks antud uuringu tulemusi näeme, et
emad kasutasid kuni 3-4 elukuuni suhteliselt suurt kogust spermatosoide munaraku
viljastamiseks ning siis optimeerisid sperma kasutuse järsult 1-2 spermatosoidi peale.
Antud tulemused ei ole kasulikud mitte ainult mesilasbioloogia paremaks arusaamiseks
vaid ka mesinikele, kellel üheks olulisemaks faktoriks mesilate tugevuse tagamisel on
efektiivne, viljakas emamesilane. Teades, et 28-kuudes emades (antud emad on aastaringselt aktiivsed ning nende metabolism on liirem kui Eestis kasutatavad emad) on sperma
paunas hoitava sperma kogus ligikaudu 500 000 spermatosoidi ning teades, et emad
kasutavad keskmiselt 2 spermatosoidi munaraku kohta, saame umbkaudselt arvutada, et 28
kuused emad suudaksid veel viljastada ligi 250 000 munarakku eeldades, et spermapaunas
hoisustatud sperma on 100% elujõuline. Kuna antud uurimuses saime teada, et 28 kuustes
emades on sperma elujõulisus ligi 25%, siis saame arvutustest teada, et emad on
tõenäoliselt võimelised viljastama ligikaudu 62 500 munarakku.
64