Published in Journal of Insect Physiology 48, issue 2, 197-203, 2002
which should be used for any reference to this work
1
Sperm survival in the female reproductive tract in the fly Scathophaga
stercoraria (L.)
G. Bernasconi
a
b
a,*
, B. Hellriegel b, A. Heyland
1,b
, P.I. Ward
b
Institute of Environmental Sciences, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland
Zoological Museum, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland
Abstract
While sperm competition risk favours males transferring many sperm to secure fertilizations, females of a variety of species
actively reduce sperm numbers reaching their reproductive tract, e.g. by extrusion or killing. Potential benefits of spermicide to
females include nutritional gains, influence over sperm storage and paternity, and the elimination of sperm bearing somatic mutations
that would lower zygote fitness.
We investigated changes in sperm viability after in vivo and in vitro exposure to the female tract in the polyandrous fly, Scathophaga stercoraria. Sperm viability was significantly lower in the females’ spermathecae immediately after mating than in the
experimental males’ testes. Males also varied significantly in the proportion of live sperm found in storage in vivo. However, the
exact mechanism of sperm degradation remains to be clarified. In vitro exposure to extracts of the female reproductive tract,
including female accessory glands, failed to significantly lower sperm viability compared to controls. These results are consistent
either with postcopulatory sperm mortality in vivo depending entirely on the male (with individual differences in sperm viability,
motility or longevity) or with postcopulatory sperm mortality being subtly affected by female effects which were not detected by
the in vitro experimental conditions. Importantly, we found no evidence in support of the hypothesis that female accessory glands
contribute to sexual conflict via spermicide. Therefore, female muscular control remains to date the only ascertained mechanism
of female influence on sperm storage in this species.
Keywords: Female reproductive tract; Sperm storage; Sperm viability; Spermicide; Sexual conflict; Diptera; Female accessory gland; Scatophaga
1. Introduction
In many taxa the number of sperm stored by females
is much lower than that transferred by males at mating.
For instance, in the beetles Tribolium castaneum (Bloch
Qazi et al., 1996) and Callosobruchus maculatus (Eady,
1994), only 4% and 15%, respectively, of the sperm
transferred reach the spermatheca. Females of some
species directly extrude sperm after mating (e.g. carrion
flies, Otronen and Siva-Jothy, 1991; millipedes, Barnett
et al., 1995; feral fowl, Pizzari and Birkhead, 2000).
* Corresponding author. Tel.: +41-1-635-4807; fax: +41-1-635-5711.
E-mail addresses: bernasco@uwinst.unizh.ch (G. Bernasconi),
barhell@zool.unizh.ch (B. Hellriegel), aheyland@zoo.ufl.edu
(A. Heyland), pward@zooolmus.unizh.ch (P.I. Ward).
1
Present address: Department of Zoology, University of Florida,
223 Bartram Hall, POB 118525, Gainesville, FL 32611-8525, USA.
Moreover, the female reproductive tract can be hostile
to sperm (Birkhead et al., 1993), reducing the viability
and/or fertilization ability of sperm reaching storage, as
suggested by evidence of sperm digestion starting
immediately after sperm transfer at copulation
(flatworms, Michiels and Bakovski, 2000), sperm degradation in the bursa copulatrix (bruchid beetles, Eady,
1994), and that female accessory gland secretions debilitate spermatozoa (house fly, Degrugillier, 1985).
Numerical and functional elimination of sperm prior to
fertilization counters male interests because males are
selected to respond to increased sperm competition risk
by maximizing their representation in the fertilization set
(Parker, 1990; Hellriegel and Ward, 1998).
Providing a hostile environment for sperm may allow
females to (i) promote male competition (Birkhead et al.,
1993; Keller and Reeve, 1995; Bernasconi and Keller,
2001), (ii) obtain nutritional benefits (Wickler, 1985;
Arnqvist and Nilsson, 2000), (iii) eliminate sperm bear-
2
ing somatic mutations (Jones et al., 2000; Siva-Jothy,
2000), (iv) counter antagonistic male adaptations, e.g.
to neutralize male-derived substances with detrimental
effects on female fitness (Chapman et al., 1995; Rice,
1996; Johnstone and Keller, 2000; Andrès and Arnqvist,
2000), or (v) influence paternity (Birkhead et al., 1993;
Birkhead, 1998; Hellriegel and Ward, 1998; Greeff and
Parker, 2000).
In the fly Scathophaga stercoraria, females have
influence over paternity (Ward, 2000a). Theory suggests
two mechanisms by means of which females can bias
paternity: control of storage rates to multiple spermathecae, and spermicide (Hellriegel and Ward, 1998;
Greeff and Parker, 2000). Evidence indicates that sperm
storage rates depend on female muscular activity
(Simmons et al., 1999), and this results in different proportions of sperm from a female’s mates being stored
across her multiple spermathecae (Hellriegel and Bernasconi, 2000). In contrast, it is not known whether spermicide occurs, nor are potential mechanisms and their
quantitative effects identified. Female tract physiology
and products have not been as extensively investigated
in Diptera as male-derived products (e.g. Drosophila
males, Chapman et al., 1995; Rice, 1996). In S. stercoraria, female reproductive accessory gland secretions are
known to be released during copula (Hosken and Ward,
1999). The glands were found to be larger when females
were mated to several males vs. one male after 10 generations (Hosken et al., 2001). Moreover, if sperm are
exposed to a hostile environment prior to storage, males
may differ with respect to their ability to resist sperm
degradation in the female tract, providing a new predictor of variation in paternity success among males. Here
we investigate sperm survival after exposure (i) to the
female reproductive tract in vivo using virgin males of
different age at first mating and (ii) to extracts of different regions of the female tract, including female accessory glands, in vitro.
nm). Both solutions stain sperm heads only (Fig. 1). We
used Schneider’s Medium (GIBCO BRL 21 720-024)
with heat-inactivated (30 min/56 °C) 10% fetal calf
serum (EUROBIO 01 0056) as buffer. This protocol is
an optimization of the protocol of Molecular Probes for
our study organism. Samples were analyzed for epifluorescence (Reichert-Jung Polyvar microscope, Hamamatsu C5405, Argus-20). To ensure accuracy, we examined 46 images and recorded them under both light and
fluorescence microscopy. Ninety-six percent of the cells
seen in light microscopy could be found in the fluorescence image. Overall, 5% (678 out of 13 654 sperm
cells) of the cells were doubly-stained (green in centre,
red at ends), and were included as dead cells. In a preliminary experiment, we ensured that no dead sperm
stain green by heating the testes (10 min/60 °C), which
resulted in all cells staining red. We counted live sperm
(green, 485/10, BP 540/20) automatically (NIH Image),
after calibration to sperm head size in a preliminary
experiment (320 images, 4 males). Dead sperm (red,
557.5/27.5) were counted manually, to avoid counting
dead tissue of a size similar to sperm heads.
2. Material and methods
We collected S. stercoraria mating pairs in the field
(Fehraltdorf, Switzerland, November 1999) and raised
their progeny in the laboratory (larvae: 14 °C; adults: 19
°C, light/dark cycle 13/11 hours, Ward and Simmons,
1991). After emergence, virgin flies were kept singly and
supplied with water, sugar and Drosophila as prey.
2.1. Sperm viability assessment
We assessed sperm viability with the live/dead
Sperm Viability Kit (L-7011, Molecular Probes), which
consists of a membrane-permeant nucleic acid stain
(SYBR14; diluted 1:50; emission max. 516 nm) and a
dead-cell stain (probidium iodide; emission max. 617
Fig. 1. Live sperm stain green (485/10, BP 540/20), dead sperm red
(557.5/27.5; live/dead Sperm Viability Kit L-7011, Molecular
Probes).
3
2.2. Sperm viability after in vivo exposure to the
female tract
We compared the proportion of live sperm in the spermathecae (i.e. after exposure to the female tract) to the
proportion of live sperm in the males’ testes for 20 mating pairs. Flies were dissected at the end of copulation
(see Hellriegel and Bernasconi, 2000). Because male age
could either affect sperm viability in the male or its
ability to resist degradation within the female tract, in
10 mating pairs we used young males (18±2 days posteclosion) and in 10 pairs older males (50±1 days). All
males were virgin, thus male age corresponds to age at
first mating. The females’ spermathecae were placed in
60 µl buffer with 5 µl of each stain, vortexed and transferred to a microslide in 40 µl. Sperm were released by
exerting pressure on the cover glass (see control
experiment). For the male, we opened each testis separately in buffer on a microslide and released the sperm
from the third proximal to the ejaculatory duct. For each
testis we added 5 µl of each stain to 60 µl of suspension,
vortexed, and transferred 40 µl to a microslide. We
counted live/dead sperm on each of 20 (19–21) images
for each testis and for the spermathecae. Images were
recorded 20±6 min after dissection and 9±4 min after
adding stains. Images had 35±23 live and 7±11 dead
sperm; 57 out of 1197 (5%) with tissue debris were
excluded from analysis. Females were 36±6 days old.
In addition, we carried out a control experiment to
ensure that our procedure for preparing sperm from the
testis vs. spermathecae did not affect sperm viability. We
dissected both testes of each of six males and prepared
two separate sperm suspensions which we stained as
described for testis (see above). For one of the male’s
testes we transferred 40 µl of stained suspension to a
microslide and examined it directly. For the other testis,
we transferred 40 µl of stained suspension to a
microslide on which we placed the spermathecae of a
virgin female (i.e. containing no sperm) and exerted
pressure on the cover glass until the spermathecal walls
opened, ensuring comparable pressure as for preparing
spermathecal sperm in the in vivo experiment. We examined 320±123 sperm/male (72 images, 6 males).
2.3. Sperm viability after in vitro after exposure to
female reproductive tissue
To identify whether, and which part of, the female
tract might be responsible for sperm mortality, we
exposed samples of sperm (from 24 males, 12 in each
of two experimental blocks) to a set of suspensions containing buffer and isolated female reproductive tissues
(female accessory glands, spermathecae, bursa copulatrix, bursa copulatrix with female accessory glands)
or to a control (male ejaculatory bulb, male flight muscle, female flight muscle, buffer only). All suspensions
were kept frozen (⫺20 °C, 6 days), and thawed and centrifuged (13 000g/2 min/4 °C; Haereus Biofuge Fresco)
before use. Sperm obtained from virgin males (n=24)
were released in 100 µl buffer. We incubated (x±SD:
11±2 min, room temperature) 15 µl sperm suspension
with 30 µl buffer and 15 µl tissue suspension. After incubation, we added 5 µl of each stain, vortexed and
immediately examined 30 µl of the mixture. We
recorded 108 images/male, 5±1 min after staining. Each
image had 17±10 live and 4±3 dead sperm in the first
and 13±8 live sperm and 3±3 dead sperm in the second
block. Results of ANOVA adjusted for covariates (time
from testis dissection, p=0.64; duration of incubation,
p=0.11) were consistent with the analysis without
covariates.
The temporal sequence of all procedures was randomized, sperm counts and incubation sequence were
blind. All dissections were under CO2. Unless specified,
data are given as mean±SD. Angularly transformed
viabilities were analyzed using GENSTAT 5.3.2 (1995,
Lawes Agricultural Trust, Rothamsted Experimental
Station) with appropriate F-tests.
3. Results
The proportion of live sperm in the spermathecae
immediately after mating and exposure to the female
tract was significantly lower than in the male’s testes
(Table 1, F1,18=12.98, p=0.002, Figs. 2 and 3). In the
spermathecae, viability was 79.2±10.3% (range 62.2–
95.3%), in the testis dissected first it was 88.6±4.7%
(80.2–97.1%) and in the testis dissected last 82.7±3.7%
(82.7–94.2%). The difference between sperm viability in
the spermathecae and the average sperm viability of the
two testes was 9.8±11.4%, with the most extreme difference for a given male being 30.1% lower viability in the
spermathecae. These results indicate that sperm can die
very soon after insemination.
Sperm viability did not differ significantly between a
male’s testes (Table 1, F1,18=0.39, p=0.54), indicating
that there was no significant overall difference in the
proportion of live sperm between the testis dissected first
and second after a single mating (when averaged, the
slope of the lines connecting individual values of sperm
viability in testis 1 and testis 2 in Fig. 3 is flat). That
there was no significant difference between the testis dissected first and second ensures that the procedure to
assess sperm viability was rapid enough to avoid artifacts. Sperm survival through storage processes, i.e. the
difference between sperm viability in the testes and in
the spermathecae, varied widely and significantly among
males (F18,1080=15.56, p⬍0.001, Fig. 3). However, this
variation was not significantly explained by the age at
which males were allowed their first mating (young:
18±2 days post-emergence, n=10; old: 50±1 days, n=10,
4
Table 1
Accumulated analysis of variance for male age at first mating and sperm source (testes or spermathecae) on angularly-transformed sperm viability
in vivo
Source of variation
Male age
Latency to copulation
Copulation duration
Residual among males
Source of sperm
Planned contrasts
spermathecae vs. testesa
testis 1 vs. testis 2b
Male age×source of sperm
Individual male×source of sperm
individual male×(spermatheca vs. testes)
individual male×(testis 1 vs. testis 2)
Residual among images
Total
a
df
1
1
1
16
2
1
1
2
36
18
18
1080
1139
ss
ms
0.04
2.12
0.03
14.7
22.4
0.04
2.12
0.03
0.92
22.4
22.3
0.11
1.99
36.0
30.9
5.1
159.1
236.4
22.3
0.11
1.99
2.0
1.7
0.28
0.15
0.21
Fobs
p(F⬎|Fobs|)
0.05
2.30
0.03
0.83
0.15
0.86
11.17
⬍0.001
12.98
0.39
1.00
1.36
15.56
1.93
0.002
0.54
0.38
0.08
⬍0.001
0.01
Effect of exposure to female tract. bVariation between a male’s testes.
Fig. 3. Effect plot for sperm viability in the testes (1 and 2) of individual males and in the spermathecae of their mate. Lines connect
values for each individual male. Sperm viability is angularly transformed [(180/π)×arcsin sqrt(proportion)].
Fig. 2. Viability (mean±SE, n=20 mating pairs) of sperm from the
spermathecae of singly-mated females and their mate’s testes.
F1,18=1.15, p=0.30), nor did male age, latency to (4±4
min) or duration of copulation (36±10 min, n=20) predict
viability (all pⱖ0.15).
In the control experiments, viability in samples from
the testes examined directly after staining (92.7±5.3%)
did not differ significantly from viability in samples from
the testes on which we applied pressure sufficient to
break open a spermatheca (92.0±7.9%; paired t-test:
t=⫺0.23, df=5, p=0.83). This indicates that the handling
procedures to obtain sperm from the spermathecae vs.
from testes did not affect sperm viability differently.
To identify whether parts of the female tract release
substances which are toxic to sperm, we exposed sperm
in vitro to isolated female reproductive tissues (female
accessory glands, spermathecae, bursa copulatrix, and
bursa copulatrix with female accessory glands), and to
control tissues (male ejaculatory bulb, male flight muscle, female flight muscle, buffer only). Exposure to
female reproductive vs. control tissues failed to reveal a
spermicidal function of any of these extracts from parts
of the female tract (F7,154=1.01, p=0.43, 24 males, Tables
2 and 3). None of the linear contrasts comparing each
5
Table 2
Analysis of variance for angularly-transformed in vitro sperm viability after exposure to female reproductive tissue (female accessory gland, bursa,
spermathecae, bursa copulatrix with female accessory glands) vs. controls (male ejaculatory bulb, flight muscles of male and female, buffer only)
in two experimental blocks
Source of variation
df
ss
Block
1
Residual among males
22
Treatment
7
Treatment×block
7
Residual among
154
treatments within
males
Residual among
1529(7)
images
Total
1720(7)
ms
Fobs
p(F⬎|Fobs|)
619.2
32224.2
1683.3
2549.2
36683.0
619.2
1464.7
240.5
364.2
238.2
0.42
6.15
1.01
1.53
1.85
0.52
196734.3
128.7
0.43
0.16
270355.5
Table 3
Sperm viability (mean±SD; %) after in vitro exposure to (a) female reproductive tract extracts and (b) controls in two experimental blocks
(a)
Accessory gland
Bursa copulatrix
Spermathecae
Accessory gland and
bursa copulatrix
Block 1
Block 2
78.7±14.8
82.9±15.1
83.0±11.5
80.9±13.2
79.3±14.2
82.3±14.7
80.5±14.4
84.0±13.2
(b)
Male muscle
Buffer only
Ejaculatory bulb
Female muscle
Block 1
Block 2
83.0±12.1
85.7±13.4
82.0±13.1
82.3±12.6
81.4±11.4
83.0±14.9
82.9±12.6
77.1±21.0
female reproductive tissue to the controls was significant
(all p⬎0.30). Control tissues did not differ among each
other (p=0.16, Table 2).
4. Discussion
The proportion of live sperm of a given male was significantly lower in the spermathecae (i.e. after mating
and sperm storage) than it was in his testes after copulation. This reduction in sperm viability among stored
sperm immediately after a single copulation indicates
that sperm degradation already occurs during mating and
sperm storage. In extreme cases sperm viability in the
spermathecae can be up to 30% less than in the same
male’s testes, thus potentially putting a male at disadvantage compared to rival males competing for sperm storage and fertilization of the female’s ova. By contrast,
reduced sperm viability does not necessarily impact
female fertility because after one mating females store
over 1000 sperm cells (Otronen et al., 1997), which is
sufficient to produce at least four fully fertile clutches
(Parker, 1970), each of which consists of 55–65 eggs
(Blanckenhorn, 2000). The control experiment confirmed that the lower sperm viability observed in vivo
in the spermathecae cannot be ascribed to differences in
the procedure to prepare sperm for examination. Also,
sperm viability in the testes of virgin males (control
experiment) was not lower than in the testes of singlymated males (in vivo experiment). This suggests that low
sperm viability in the spermathecae cannot be due to
“old” sperm having been transferred at mating. It is also
unlikely that the sperm viability difference between
testes and spermathecae could arise through preferential
storage of dead sperm. Although sperm may be transported up the female tract independently of their motility
(Simmons et al., 1999), under female musculature control (Hellriegel and Bernasconi, 2000), it would be counter-adaptive if storage were more efficient for dead
sperm. Mechanical damage (sperm are tightly packed in
the ducts during transfer and the duct surface is rough,
Hosken et al., 1999) or failure in sperm and/or seminal
fluid (loss of sperm function, failure in sperm motility,
lack of sperm nutrients or necessary cofactors) may also
provide an explanation, although selection to avoid such
malfunctions clearly should be strong. In sum, the lower
sperm viability in the spermathecae than in the testes
immediately after mating is an intrinsic property of
males (varying in sperm quality, motility, longevity)
and/or is mediated by the female tract.
Importantly, we found significant and substantial variation among males in the proportion of live sperm reach-
6
ing storage. This implies that a male’s proportional contribution to the set of stored sperm when females mate
multiply (e.g. 2–79% for the second of two males,
Hellriegel and Bernasconi, 2000) need not equal his
chance of fertilization, because males can also differ in
sperm viability and survival to storage. Recent work has
focused on understanding sources of variance in male
fertilization success (Lewis and Austad, 1990; Arnqvist
and Danielsson, 1999) and sexual selection on sperm
traits associated with fertilization ability (Keller and
Reeve, 1995, Bernasconi and Keller, 2001). Future studies need to establish whether males vary heritably in
sperm degradation resistance, and how this correlates
with fertilization ability in competitive situations. Interestingly, in the present study variation among males in
the proportion of live sperm reaching storage was independent of when males mated for the first time. That is,
male age at first mating, and therefore possibly sperm
age (Stockley, 1999), did not significantly affect either
sperm viability in the male or sperm ability to resist
degradation during transfer within the female tract. In S.
stercoraria, males vary heritably in sperm length,
although the causes of this variation are still unidentified
(Ward, 2000b). It would thus be interesting to investigate
whether variation among males in the sperm ability to
resist degradation in the female tract is at least partly
explained by variation in sperm morphology.
The mechanism of sperm death remains to be established, as none of the isolated female reproductive
tissues (female accessory glands, spermathecae, bursa
copulatrix, and bursa copulatrix with female accessory
glands) to which we exposed sperm in vitro more
strongly affected sperm mortality than control tissues
(male ejaculatory bulb, male flight muscle, female flight
muscle, buffer only, Table 3). In particular, in vitro the
female accessory glands did not have the spermicidal
function reported based on preliminary data in a previous
study (Hosken et al., 2001). This lack of significance of
the in vitro experiment does not allow us to determine
whether reduced sperm viability in vivo after storage
results from intrinsic male traits or from female influence
(whereas a significant spermicidal effect of one or more
of the female tract extracts would have strongly supported female influence). In most studies, lack of evidence does not imply lack of effect (Cohen, 1988). However, the statistical power of the in vitro experiment is
very high, suggesting that one could conclude that none
of the extracts of the female tract we investigated has a
spermicidal function. Alternatively, lack of significance
can be due to in vitro conditions that did not recreate
the relevant physiological conditions, thus masking subtle treatment effects. Indeed, this may have been the case
because viability values were generally low in vitro
(Table 3).
Recently, female accessory gland were found to be
larger in females mated to several rather than one male
after 10 generations, suggesting a spermicidal function
(Hosken et al., 2001). The present study, however, found
no evidence in support of a spermicidal function, despite
the high statistical power. Thus, it is possible that these
glands, whose secretions are released during mating
(Hosken and Ward, 1999), have a different function
which is also related to the experimental regime of
polyandry/monandry. For instance, lubrication to lower
mating costs would be a function that is consistent with
the design used by Hosken et al. (2001), which did not
separate the two components of polyandry, i.e. mating
with genetically different males and mating with more
numerous males (see Bernasconi and Keller, 2001, for
a design that separates these components). In sum,
further work is needed to clarify the function of these
glands.
In conclusion, we found significantly lower sperm
viability in a female’s spermathecae than in her mate’s
testes. Also, we found significant differences among
males in the proportion of viable sperm that their mates
store. Sperm mortality during mating and storage can
result from intrinsic male properties (such as variation
in sperm longevity) and/or from a spermicidal environment in the female tract. However, the mechanism
remains to be identified. In vitro exposure to female
reproductive accessory glands and other parts of the
female tract failed to reveal a female spermicidal effect.
Thus, to date, female muscular control of storage rates
remains the only ascertained mechanism of differential
sperm storage (Hellriegel and Bernasconi, 2000) in this
species, confirming at least one of the mechanisms of
paternity bias suggested by theory (Hellriegel and Ward,
1998). Indeed, differential storage across multiple spermathecae may be the most flexible mechanism of cryptic
female choice at the time of egg-laying in a species
where the relevant ecological conditions for optimal
choice vary at a microgeographical scale (Ward, 2000a).
Acknowledgements
We thank Wolf Blanckenhorn, David Hosken, Eric
Kubli, Bernhard Schmid and the referees for comments
and discussion, Ruth Böhni, Dieter Burkhard, Jeannette
Fanti, Gilbert Gradinger, Stefan Keller, Marc Kéry,
Michel Nakano, Stefan Sommer and Anna Willimann
for help, and Urs Greber for facilities. This work was
supported by the Swiss Federal Program for Academic
Recruitment (No. 409 GB, No. 167 BH) and Swiss NSF
(No. 31-46861.96, PIW).
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