Apidologie (2011) 42:29–38
c INRA/DIB-AGIB/EDP Sciences, 2010
DOI: 10.1051/apido/2010026
Original article
Effects of age, season and genetics on semen and sperm
production in Apis mellifera drones*
John W. R1 , Steven H1 , Robert S-H2 ,
Denis L. A3 , Gretchen W4
1
NSW Department of Primary Industries (DPI), Tamworth Agricultural Institute, 4 Marsden Park Road, Calala,
NSW 2340, Australia
2
University of Western Sydney, Centre for Plant and Food Science, Hawkesbury Campus, Locked Bag 1797,
Penrith South DC, NSW 1797, Australia
3
CSIRO Entomology, PO Box 1700, Canberra, ACT 2601, Australia
4
PO Box 223, Richmond, NSW 2753, Australia
Received 6 October 2009 – Revised 11 January 2010 – Accepted 20 January 2010
Abstract – Adult drone honey bees from 4 Australian breeding lines were reared under similar conditions
and examined for semen and sperm production when 14, 21 and 35 days old, during spring, summer and
autumn. Almost half (40.5%) of all drones examined did not release any semen when manually everted. For
those that released semen, the average volume released per drone was 1.09 µL (range 0.72 (±0.04)–1.12
(±0.04) µL) and the average number of sperms in the semen per drone was 3.63 × 106 (range 1.88 (±0.14)–
4.11 (±0.17) × 106 ). The release of semen was dependent on breeding line and age (P < 0.05), but not on
the rearing season. The volume of semen released per drone was dependent on season, age, and breeding
line (P < 0.05), while the concentration of sperm in the semen was dependent on season and breeding line
(P < 0.05). Hence our data indicate that genetics underpins the maturation of drone honey bees as well as
the volume of semen they release and the concentration of sperm in that semen.
Apis mellifera / drones / semen production / sperm
1. INTRODUCTION
Mating is the most significant function of an
adult drone honey bee (Apis mellifera). During mating, semen is transferred from a drone
into the genital orifice of a virgin queen on
the wing, via the drone’s endophallus, which
is everted during copulation (Bishop, 1920a;
Woyke, 1964). After mating, secretions and
parts of the drone reproductive organs remain
in the genital tract of the queen and signify
a ‘mating sign’ (Woyke and Ruttner, 1958),
which may function to prevent semen from
Corresponding author: D.L. Anderson,
denis.anderson@csiro.au
* Manuscript editor: Klaus Hartfelder
flowing out of the vagina (Bishop, 1920b).
The semen consists of sperm, produced in the
testes, and mucus, produced by large mucus
glands. Sperm production is largely considered completed by the time drones reach sexual maturity, or when they are about 9–12 days
old (Bishop, 1920a). Sperm is stored in the
seminal vesicles of drones until they mate with
virgin queens, at which stage it is ejaculated
with mucus (semen) when the endophallus is
everted. Normally, several drones will mate
with a single virgin queen and copulations
during flight follow one another about every two seconds (Koeniger et al., 1979). Each
drone ejaculates from 6–12 million sperms
and sperm from each mating accumulates in
the queen’s lateral oviducts (Woyke, 1960;
30
J.W. Rhodes et al.
Kerr et al., 1962). Most of this sperm is
subsequently expelled from the queen (Page,
1986), but about 5–6 million migrate into the
spermatheca, where they are stored until released in small quantities when the queen
begins laying eggs (Zander, 1916; Bishop,
1920a, b; Page, 1986).
Previous studies in south east Australia
found low numbers of sperm in the spermathecae of a high proportion of ovipositing commercially reared queen bees less than 35 days
old (Rhodes and Somerville, 2003). This has
serious implications for the productivity of
colonies headed by such queens, as queen bees
are generally superseded and killed by their
colonies when they run out of sperm (Winston,
1987). Possible explanations could be that
there were low numbers of drones of the right
age available to mate with these queens, or
sufficient numbers of drones of the right age
but problems with their semen production, or
a combination of both.
Limited data are available on the proportion of drones that release semen at the tip
of the endophallus following manual eversion. Collins and Pettis (2001) reported 60%
of 12-day-old and Anderson (2004) 90% of
20-day-old A. mellifera drones did not release
semen after manual eversion. Several studies
have been reported on the volume of semen
produced by drones and the concentration of
sperm in that semen. Woyke (1960; quoted
in Rinderer, 1986) stated that an A. mellifera
drone produces about 10 × 106 sperm, and
1 µL of semen contains about 7.5 × 106 sperm.
Koeniger et al. (2005) compared sperm numbers between A. dorsata and A. mellifera
drones and provided a comparison of sperm
counts from A. mellifera drones from various
authors. Sperm counts ranged from a mean
of 4 (2 ± 0.1) × 106 sperms per seminal vesicle for European A. mellifera (Rinderer et al.,
1999), to 11.9±1.0×106 sperms per individual
drone from an A. m. carnica colony (Schlüns
et al., 2003). Collins and Pettis (2001) considered that the large variation reported in sperm
numbers in semen might be due to using viscous and sticky semen to obtain counts. This
may cause sperm cells to clump after being experimentally diluted, resulting in measurement
errors. However, Bishop (1920a) reported that
sperm release themselves more easily in the
ejaculatory duct when mucus is more viscous.
Koeniger et al. (2005) also stated that, in general, sperm numbers of individual drones show
high variance and also suggested this could be
due to errors in the sperm counting method.
Nevertheless, they concluded that the cause
of differences in sperm concentration reported
from individual drones was yet to be determined.
The current study was carried out on A. mellifera drones in south east Australia, where
studies had previously shown that commercially reared A. mellifera queens contained low
concentrations of sperm in their spermathecae (Rhodes and Somerville, 2003). The aim
was to determine whether particular characteristics of drones in that region might have
been a contributing factor. We assessed the effects of age, genetics and season on semen
and sperm production by examining 14, 21
and 35-day-old drones reared from four different breeding lines during spring, summer and
autumn.
2. MATERIALS AND METHODS
2.1. Obtaining drones and measuring
semen and sperm production
Three to five open-mated queen bees were produced by standard queen-rearing methods from
each of four instrumentally inseminated breeder
queen bees that were similar to A. mellifera ligustica and maintained by different commercial queenrearing operations. The daughter queens were used
to provide drones for the current study and each represented dissimilar genetic backgrounds and identified as representing lines 1, 2, 3 and 4. Each of the
daughter queens was marked and kept in a doublestory hive in a single apiary at Richmond, New
South Wales, Australia. To reduce nutritional effects on the drone characteristics being examined,
the hives were continuously replenished with pollen
and sugar syrup throughout the study. All hives
were also free of clinical signs of disease.
Drones were reared from each line during spring
2003 (Oct.–Nov.), summer 2004 (Feb.–Mar.) and
autumn 2004 (April). Drone rearing and maintenance involved placing a queen bee in a queen excluder cage on a full frame of drone comb for 24 h.
31
Semen and sperm production in drones
After the queen had laid eggs in the comb, the
comb was removed from the cage and placed in
the middle of the brood combs in the brood chamber. Seventy-two hours before the drones were due
to emerge the drone comb was placed back in the
cage and the cage placed back in the brood chamber.
Adult drones that emerged in the cage were marked
with a coloured Posca marking pen and released
into the hive in which they were reared. All rearing
and maintenance of adult drones was carried out in
the hive headed by their respective queen mothers.
Marked drones were confined to their hive, to prevent loss of drones, by a queen excluder placed between the brood chamber and bottom board and a
second queen excluder placed on top of the brood
chamber, beneath the super box.
Adult drones were examined at three ages, when
14, 21 and 35 days old. Within each age group,
the oldest drones may have been up to 24 h older
than the youngest drones, due to the time allowed
for the queens to lay sufficient numbers of eggs in
each comb. For each examination a sample of 30
drones, or the number of drones surviving to the
age being sampled, was collected in the morning
before drone flight commenced. Collected drones
were held in cages, 16 × 10 × 3 cm, with one side
made of queen excluder and placed between frames
of unsealed brood in a strong colony until required
for laboratory analysis.
Drones were examined for the proportion that
released semen at the tip of the endophallus after manual eversion and for the volumes of semen
and numbers of sperm produced. Drones were everted manually following the methods described by
Collins and Donoghue (1999). In brief, drones of a
known age were stimulated to ejaculate by pressing
on the thorax, which usually resulted in partial eversion of the endophallus. Further pressure on the abdomen forced the haemolymph into the endophallus and completed the process. Semen was collected
from the tip of the endophallus, with particular care
taken to avoid the collection of mucus, which may
have caused semen to become viscous and sticky.
Semen was collected using a Schley II apparatus
and the semen volume, µL, read directly from a
Gilmont micrometer syringe (model GS-1100) attached to the collection tip, to an accuracy of 0.1 µL.
Drones that did not ejaculate semen or only a small
amount of semen not sufficient for measurement
were recorded as not releasing semen. The number
of sperm in the semen was determined by placing
0.6 µL of the semen (used to measure volume) in
1.5 mL water, mixing with a vortex, and counting
the number of sperm present in 25 squares of an improved Neubauer haemocytometer, depth 0.1 mm,
1/400 mm2 with the aid of a light microscope. The
number of sperm produced by each drone was determined from the formula:
Number of sperm/drone(×106 ) = Volume
of semen (µ L) × total number
of sperm counted in 25 squares
of the haemocytometer × 25 000.
2.2. Statistical analysis
Counts of drones with measurable amounts of
semen are binomial variates. These were analysed
using a logistic regression model, which is a generalised linear model with a logit link and binomial
variance function. Season, age, line and all interactions were terms in the model and the significances
of these terms were assessed by sequentially adding
each term to the model and the changes in deviance
between the nested models were compared to a Chi
squared distribution with degrees of freedom being the degrees of freedom of the added term. Semen volume and number of sperm per drone for
those drones with semen were analysed as a linear
model with season, drone age, breeding line and all
interactions included in the model. A square root
transformation for both variables was necessary
to meet the assumptions of normally distributed
residuals. Predictions on the back-transformed scale
were shown for all levels of the main effect terms
(season, drone age and breeding line) and averaged
over all other terms in the model. The logistic regression model was fitted using the statistical software R (R Development Core Team, 2007), utilising the general linear model (GLM) function and
the linear model was fitted using ASReml (Gilmour
et al., 2006). Significance of terms was assessed at
a probability of 5 percent (P ≤ 0.05). Note that
this study was conducted over a one-year period
and hence any seasonal effects found may or may
not be replicated again had the study been repeated
in another year. Each season had only one replication and hence pseudo-replication is involved in any
analysis with season as a factor.
3. RESULTS
3.1. Semen release and semen volumes
Only 59.4% of drones examined from the
4 breeding lines released measurable amounts
J.W. Rhodes et al.
32
Table I. The number of drones marked at emergence and the proportion of drones releasing a measurable
amount of semen after manual eversion from the number examined for three seasons, for four breeding lines
at three ages.
Season
2003–04
Spring
Summer
Autumn
TOTALS
Line
1
2
3
4
1
2
3
4
1
2
3
4
No. of
drones
marked
*
601
427
190
284
219
640
750
710
129
700
460
820
5,930
Number of drones releasing semen/
number of drones examined (%)
Age (days)
14
21
35
6/30 (20.0)
9/30 (30.0)
9/13 (69.2)
28/30 (93.3)
26/30 (86.7)
14/15 (93.3)
20/30 (66.7)
6/30 (20.0)
4/6 (66.7)
15/30 (50.0)
19/30 (63.3)
21/29 (72.4)
12/30 (40.0)
14/30 (46.7)
1/3 (33.3)
28/30 (93.3)
28/30 (93.3)
27/30 (90.0)
11/30 (36.7)
8/30 (26.7)
17/30 (56.7)
18/30 (60.0)
17/30 (56.7)
23/27 (85.2)
13/30 (43.3)
15/30 (50.0)
na
28/30 (93.3)
20/22 (90.9)
na
9/30 (30.0)
9/30 (30.0)
na
23/30 (76.7)
6/13 (46.2)
na
211/360 (58.6) 177/335 (52.8) 116/153 (75.8)
TOTALS
24/73 (32.8)
68/75 (90.6)
30/66 (45.4)
55/89 (61.7)
27/63 (42.8)
83/90 (92.2)
36/90 (40.0)
58/87 (66.6)
28/60 (46.6)
48/52 (92.3)
18/60 (30.0)
29/43 (67.4)
504/848 (59.4)
na: No drones survived to provide data. * Number of drones marked at emergence.
of semen at the endophallus after manual eversion (Tab. I). The percentage of drones that
released semen was quite variable among the
different genetic lines, with 91.7% from line
2 releasing semen, 40.3% from line 1, 38.9%
from line 3 and 64.8% from line 4 (Tab. II).
The percentage of drones that released semen
when aged 14, 21 and 35 days-old was 58.6%,
52.8% and 75.8%, respectively, while the percentage that released semen in spring, summer
and autumn was 58.4%, 61.8% and 57.2%, respectively (Tab. II).
Statistical analyses showed that semen release in the drones was dependent on their genetics (the breeding line they came from) and,
to a lesser extent, on their age and the season
in which they were produced. The deviance for
each term and their associated degrees of freedom and P values are shown in Table V.
For drones that released a semen sample after manual eversion, the mean semen volume
was 1.09 (range 0.1–3.6) µL per drone, with
a predicted mean of 1.03 µl when adjusted
for imbalance in drone numbers for age, season and breeding line. Predicted semen volumes and ranges of volumes for drones that
Table II. The proportion of drones releasing a measurable amount of semen after manual eversion
from the total number examined for three seasons,
three age groups and four breeding lines.
Variable
Season
Spring
Summer
Autumn
Age (days)
14
21
35
Line
1
2
3
4
Overall mean
Proportion of
drones releasing
semen (%)
Stat. Sig.*
NS
177/303 (58.4)
204/330 (61.8)
123/215 (57.2)
211/360 (58.6)
177/335 (52.8)
116/153 (75.8)
79/196 (40.3)
199/217 (91.7)
84/216 (38.9)
142/219 (64.8)
504/848 (59.4)
S
b
b
a
S
c
a
c
b
* Stat. Sig. = statistical significance. NS = not significant; S = significant. See text for detail. Means
with letters in common are not significantly different, P ≤ 0.05.
33
Semen and sperm production in drones
Table III. Semen volume and range of volumes produced by drones for season, age and breeding line
effects for drones that released a measurable (equal to or more than 0.1 µL) amount of semen after manual
eversion.
Variable
Season
Spring
Summer
Autumn
Total
Age(days)
14
21
35
Total
Line
1
2
3
4
Total
Overall Mean
Stat. Sig.+
Semen volume/drone µL
Predicted mean ± s.e.*
Range
177
204
123
504
1.03 ± 0.04
0.92 ± 0.05
0.82 ± 0.04
0.2–3.6
0.1–3.0
0.1–1.6
S
a
ab
b
211
177
116
504
0.98 ± 0.03
1.07 ±0.04
0.73 ± 0.04
0.1–2.2
0.2–3.6
0.2–3.0
S
a
a
b
79
199
84
142
504
0.72 ± 0.04
1.12 ± 0.04
0.85 ± 0.05
1.01 ± 0.04
0.2–1.8
0.1–3.0
0.1–2.6
0.2–3.6
S
c
a
b
a
N∼
1.03 ± 0.02
+
Stat. Sig. = statistical significance. S = significant. See text for detail. Means with letters in common are
not significantly different, P ≤ 0.05.
released a measurable volume of semen are
shown in Table III for season, age and breeding
line. Semen volumes were significantly higher
for spring than summer and autumn reared
drones, which produced the smallest volumes.
Volumes were also significantly higher in the
14- and 21-day-old drones than in 35-day-old
drones. A significant line effect was also identified, with lines 2 and 4 producing greater volumes than line 3, with line 1 producing the
smallest volumes. Significance for all terms in
the model are shown in Table V. The relative
sizes of the F values and a perusal of the predicted values showed that genetics (breeding
line) had the largest effect on semen volume,
followed by the age of drones with all other
terms having much smaller effects.
3.2. Sperm numbers
The mean number of sperm produced per
drone was 3.63 (range 0–19.1) × 106 , with a
predicted mean of 3.17 × 106 when adjusted
for imbalance in drone numbers for age, season and breeding line. Predicted means of
the number of sperm produced per drone and
range of sperm numbers for drones releasing
a measurable volume of semen are shown in
Table IV for season, age, and breeding line.
A season effect was identified, with autumn
reared drones producing significantly more
sperm than summer and spring reared drones,
which produced the least sperm. An age effect
was also identified, with 21-day-old drones
producing more sperm than 14- and 35-dayold drones. Genetics also affected sperm production (P < 0.05), with drones from line 2
producing more sperm than drones from lines
3 and 4 and line 1, which produced the least
sperm. Significance for all terms in the model
are shown in Table V. The relative sizes of the
F values and a perusal of the predicted values
showed that the season in which drones were
reared had the largest effect on sperm numbers,
followed by breeding line (genetics) and, to a
lesser extent, age.
J.W. Rhodes et al.
34
Table IV. Number of sperm/drone × 106 for season, age and line effects for drones, which released a
measurable (equal to or more than 0.1 µL) amount of semen after manual eversion.
Variable
N∼
Season
Spring
Summer
Autumn
Total
Age (days)
14
21
35
Total
Line
1
2
3
4
Total
Overall Mean
No. sperm/drone 106
Predicted means ± s.e.*
Range
Stat. Sig.+
177
204
123
504
1.88 ± 0.14
3.12 ± 0.21
4.24 ± 0.25
0.12–9.43
0.12–19.13
0.04–11.86
S
c
b
a
211
177
116
504
2.83 ± 0.15
3.36 ± 0.18
2.83 ± 0.21
0.12–11.52
0.18–13.52
0.12–19.13
S
a
b
a
79
199
84
142
504
2.12 ± 0.19
4.11 ± 0.17
2.84 ± 0.22
3.11 ± 0.17
0.13–7.78
0.12–19.13
0.04–7.16
0.02–13.52
S
c
a
b
b
3.17 ± 0.11
+ Stat. Sig. = statistical significance; S = significant. See text for detail. Means with letters in common are
not significantly different, P ≤ 0.05.
Table V. Significance of season, age, line and all interactions. Deviance statistics from logistic regression
model for semen release and F statistics from linear model for semen volume and sperm numbers together
with their respective P values.
Term
Season
Age
Line
Season.Age
Season.Line
Age.Line
Season.Age.Line
DF
2
2
3
3
6
6
9
Semen release
Deviance
P
χ2DF
value
1.4
23.4
177.1
0.7
8.3
10.2
16.4
0.51
<.001
<.001
0.88
0.22
0.12
0.06
Semen volume
F
P
(ndf = DF, value
ddf = 461)
3.2
0.04
11.7
<.001
12.5
<.001
2.3
0.08
2.2
0.051
3.0
0.01
2.5
0.01
Sperm numbers
F
P
(ndf = DF, value
ddf = 460)
69.6
<.001
7.9
<.001
20.1
<.001
3.1
0.03
3.3
0.004
1.3
0.27
2.6
0.01
Semen and sperm production in drones
4. DISCUSSION
The total numbers of drones that survived
to ages 14, 21 and 35 days in each of our experiments are not known, but from the number
of drones marked at emergence, it was clear
that most did not survive longer than 35 days
(Tab. I). Only about 4% of the 3821 marked
spring- and summer-reared drones survived to
35 days, whereas none of the 2109 marked
autumn-reared drones survived to 35 days
(Tab. I). Hence, the life span of drones in our
study was similar to that reported in other
studies (Witherell, 1972; Fukuda and Ohtani,
1977).
The volumes of semen in drones of the four
breeding lines (Tab. II) were also mostly comparable with those reported in other studies.
The mean volume of semen per drone in our
studies of 1.09 µL (range 0.72 ± 0.04−1.12 ±
0.04 µL) (Tab. III) was within the range reported by Woyke (1960; quoted in Rinderer,
1986) of 1.3 µL per drone, and by Collins and
Pettis (2001) of 0.95 (range 0.48–1.67) µL per
drone. However, the mean number of sperm
produced per drone in our study, 3.63 (range
1.88 (±0.11)–4.11 (±0.17)) × 106 (Tab. IV)
was at the lower end of sperm numbers reported in studies by Schlüns et al. (2003) of
11.9 (±1.0) × 106 , Woyke (1960; quoted in
Koeniger et al., 2005) of 11–12 × 106 , Collins
and Pettis (2001) of 8.66 × 106 , Köhler (1955;
quoted in Ruttner, 1956) of 4.5 × 106 and
Anderson (2004), 3.19 (±2.37) × 106 . Interestingly, the sperm count obtained by Anderson
(2004) was similar to that obtained here, was
obtained using a similar counting method to
ours, and was also obtained using drones in a
commercial queen-rearing apiary in the same
region as our study (south east Australia).
Hence, while the volumes of semen produced
by drones in our study were comparable with
those from drones observed in other studies,
the concentration of sperm in their semen was
generally less. As the drone mother colonies
in our studies were fed pollen and sugar syrup
on a continuous basis, it is unlikely that the
reduced sperm concentrations were due to nutritional effects. Perhaps the large differences
in sperm numbers reported to date by different authors may be due to differences in the
35
counting method used. In our study sperm was
counted in semen released at the tip of the endophallus following manual eversion and dilution in water, whereas, in some other studies,
sperm numbers have been determined in seminal vesicles.
In our study, increasing semen volume did
not always correspond with increased sperm
numbers. The predicted means for semen volume (Tab. III) showed a seasonal effect, with
spring drones producing significantly higher
volumes of semen than summer and autumn
drones. Yet, autumn drones produced significantly more sperm than summer drones,
which, in turn, produced more sperm than
spring drones. The higher volume of semen
produced by spring drones but lower numbers
of sperm (Tab. IV) indicates a seasonal effect
on sperm production.
The drones examined in our studies released semen at a much older age than has
been previously reported. Earlier studies reported that sperm production in drones is
largely completed by the time adult drones
reach sexual maturity and can be released in
the seminal fluids of drones at that stage, or
when they are about 9–12 days old (Bishop,
1920a). Yet, in our study, a far higher proportion of drones released semen at the endophallus after manual eversion when aged 35 days
(75.8%) than when aged 14 days (58.6%) or
21 days (52.6%) (Tab. II). Anderson (2004)
also reported that adult drones older than
20 days produced more semen than younger
drones and, as mentioned, that study was carried out in a similar geographical region to that
of the current study. Thus, there appears to be
greater variation in the time that drones reach
sexual maturity, which is the stage at which
they release seminal fluid and sperm from the
endophallus (Bishop, 1920a), than is currently
recognized. Our results also indicated that semen release (and thus the time taken for drones
to reach sexual maturity) is dependent on the
genetics of the drones. For instance, significantly more drones from line 2 released semen
at day 14 than drones of the other lines (Tab. I).
Line 2 queen mothers were sourced from a
honey bee breeding program which has been in
operation in excess of 20 years with selection
based on field evaluation combined with the
36
J.W. Rhodes et al.
use of instrumental insemination to produce
the following season’s breeding stock. While
not specifically selecting drones producing a
large volume of semen, these drones may have
inadvertently been selected over drones producing a smaller volume of semen.
Sperm numbers in drone honey bees have
been found to depend on body size (Schlüns
et al., 2003). Even though we did not measure
body size here, our results clearly showed that
sperm numbers in drones also depend on the
genetics of the drones. Drones from line 2 produced significantly more sperm after manual
eversion than drones derived from the other
3 lines (Tab. IV). Indeed, our data indicates
that the genetics of the drones underpins all aspects of seminal fluid and sperm production in
drones, including the proportion of drones that
release semen, the volume of semen produced
per drone, and the number of sperms produced
per drone.
A relatively large proportion (40.6%) of all
drones examined here when 14 to 35 days
old did not release semen (and, hence, sperm).
This was due to the drones either releasing no
semen or insufficient amounts to be collected
(Tab. I). High percentages of drones producing
no or small amounts of semen after manual eversion were also reported by Collins and Pettis
(2001) and Anderson (2004) for A. mellifera
drones. Should queen bee mating involve one
or more flights over one or more days mating
with a sufficient number of drones until a sufficient, but unknown volume of semen containing an unknown number of sperm has transferred to the spermatheca, which then acts as
the trigger for halting mating flights, then semen quality, semen volume and sperm numbers, of individual drones is reduced in importance. Data showing a high proportion of
young, recently mated, ovipositing queen bees
with low numbers of sperm in their spermathecae (Rhodes and Somerville, 2003) suggest
the presence of a mechanism in the queen
bee mating procedure which results in queen
bees halting their mating flights and commence ovipositing irrespective of the number
of sperm present in their spermatheca. Should
such a mechanism be present, then the importance of sufficient numbers of drones of high
quality at queen bee mating areas at the time
of queen mating becomes greatly enhanced,
from the perspective of the production of commercially reared queen bees. The issue of large
numbers of apparently mature drones (drones
aged 9–18 days) releasing little or no seminal
fluid and sperm after manual eversion clearly
warrants further investigation, particularly to
determine if those drones are equally able to
mate with queens as effectively as drones that
produce large amounts of semen and sperm,
and if queens that receive fewer sperms during mating store less sperm in their spermathecae than queens that receive more sperms during mating. This is because queen bees with
fewer sperms in their spermathecae after mating tend to be superseded or replaced sooner
by colonies than queens with higher numbers
of sperms in their spermathecae.
Finally, our studies have shown that particular characteristics of adult drones in south
east Australia, such as their slower maturation period and their production of relatively
low numbers of sperms, may be contributing to a problem of low numbers of sperm in
the spermathecae of commercially-produced
queen bees in this particular region (Rhodes
and Somerville, 2003). As those drone characteristics are also strongly influenced by genetics (see results), it would be advisable for
queen breeders and producers in south east
Australia (and, indeed, in all other regions)
to routinely test drones of breeding lines for
semen release and sperm production, ideally
when drones are 14 days old.
ACKNOWLEDGEMENTS
The Honey Bee Program of the Rural Industries
Research and Development Corporation, Canberra,
Australia, funded this project. The NSW Department of Primary Industries, and the Centre for Plant
and Food Sciences, University of Western Sydney
provided staff and facilities. We would like to thank
Mr. Michael Duncan, University of Western Sydney, for the provision of bees, apiary materials, and
assistance with apiary management. We gratefully
acknowledge the assistance of Ms Sharon Nielsen,
Biometrician, NSW Department of Primary Industries, who assisted with the statistical analysis and
provided comments on an earlier version of this
paper.
Semen and sperm production in drones
Effets de l’âge, de la saison et de la génétique sur
la production de sperme et de spermatozoïdes
chez les mâles d’Apis mellifera.
Apis mellifera / mâle / production de sperme /
variabilité
Zusammenfassung – Die Auswirkungen von
Alter, Jahreszeit und Genetik auf die Samenund Spermaproduktion von Apis mellifera
Drohnen. Die vorliegende, in Südost-Australien
durchgeführte Studie sollte überprüfen, ob und
welche Rolle die in dieser Region vorkommenden
Drohnen spielen könnten in der Frage der geringen
Spermienzahlen, die in Spermatheken von Königinnen gefunden worden waren. Diese Befunde einer
früheren Studie hatten gezeigt, dass kommerziell
produzierte Königinnen in dieser Region nur
geringe Spermienzahlen in ihren Spermatheken
aufwiesen (Rhodes und Somerville, 2003).
Dazu untersuchten wir natürlich begattete Tochterköniginnen von künstlich besamten Apis mellifera
ligustica-ähnlichen Mutterköniginnen, die wir
von vier kommerziellen Züchtern erhalten hatten.
Drohnen, die von Völkern dieser Mutterköniginnen im Frühjahr (Okt.–Nov. 2003), Sommer
(Febr.–März 2004) und Herbst (April 2004)
aufgezogen wurden waren, wurden im Alter von
14, 21 und 35 Tagen untersucht. Zur Gewinnung
von Spermaproben wurden sie manuell evertiert.
Als Parameter wurden der Anteil an Drohnen mit
messbarem Samenvolumen (0,1 µL oder mehr
auf dem evertierten Endophallus), das Volumen
an produziertem Samen sowie die Spermienzahl
pro Drohn bestimmt. Die Daten wurden anhand
eines logistischen Regressionsmodells untersucht,
das ein generalisiertes lineares Modell mit LogitVerknüpfung und binomialer Varianzfunktion
darstellte.
Nur bei 59,4 % der untersuchten Zuchtlinien konnten wir nach Eversion des Endophallus messbare
Samenmengen finden. Die statistischen Analysen
zeigten, dass vor allem die Genetik (d.h. die
jeweilige Zuchtlinie) die produzierte Samemenge
bestimmte. Das Alter war hingegen nur von geringer Bedeutung und die Jahreszeit spielte überhaupt
keine Rolle.
Bei Drohnen, die ein messbares Samenvolumen
freigesetzt hatten, betrug das mittlere Samenvolumen 1,0 µL, mit einem Minimum von 0,72 ±
0,04 und einem Maximum von 1,12 ± 0, 04 µL.
Das Samenvolumen bei Frühjahrsdrohnen war
signifikant größer (P < 0,05) als das von Sommeroder Herbstdrohnen, wobei letztere die gerinsten
Volumina produzierten (P < 0,05). Bei den Spermienzahlen verhielten es sich jedoch genau
umgekehrt, mit den höchsten Spermienzahlen
bei Herbstdrohnen im Vergleich mit Frühjahrsoder Sommerdrohnen (P < 0,05). Die mittlere
Spermienzahl pro Drohn lag bei 3,63 Millionen,
mit einem Minimum von 1,88 ± 0,14 und einem
37
Maximum von 4,11 ± 0,17 × 106 , unabhängig
von Alter, Zuchtlinie oder Jahreszeit. Diese Werte
liegen damit am unteren Ende bereits publizierter
Werte zu Spermienzahlen. 21 Tage alte Drohnen
produzierten signifikant höhere Samenvolumina
und Spermienzahlen als 14 und 45 Tage alte
Drohnen (P < 0,05), während Drohnen bestimmter
Zuchtlinien ein höheres Samenvolumen und mehr
Spermien aufwiesen, was darauf hinweist, dass
die Genetik eine wichtige Rolle hinsichtlich dieser
Parameter spielt.
Die Daten zeigten außerdem eine breitere Variation
hinsichtlich des Zeitpunkts der sexuellen Reife als
früher berichtet, was von Bedeutung sein sollte
für Züchter, wenn sie routinemässig Drohnen von
Zuchtlinien hinsichtlich freigesetzter Samenmenge
und Spermaproduktion untersuchen.
Apis mellifera / Drohnen / Samenproduktion /
Sperma
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