Effects of multiple mating and male eye span on female reproductive output in the stalk-eyed fly, Cyrtodiopsis dalmanni
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Behavioral Ecology Vol. 12 No. 6: 732–739
Effects of multiple mating and male eye span
on female reproductive output in the
stalk-eyed fly, Cyrtodiopsis dalmanni
Richard H. Baker, Robert I. S. Ashwell, Thomas A. Richards, Kevin Fowler, Tracey Chapman, and
Andrew Pomiankowski
The Galton Laboratory, Department of Biology, 4 Stephenson Way, University College London,
London NW1 2HE, UK
Females of the stalk-eyed fly, Cyrtodiopsis dalmanni, mate repeatedly during their lifetime and exhibit mating preference for
males with large eye span. How these mating decisions affect female fitness is not fully understood. In this study, we examined
the effects of multiple mating and male eye span on short-term reproductive output in this species. Experiments that manip-
ulated the number of copulations and partners a female received suggested that obtaining a sufficient sperm supply is an
important benefit associated with multiple mating. The average percentage of fertile eggs laid by females increased as a function
of mating frequency and ranged from 40% for females mated once, to 80% for females mated continuously. In addition, a high
proportion of copulations in this species appeared to be unsuccessful. One-third of all females mated once laid less than 10%
fertile eggs. There was no significant difference in reproductive performance between females mated to multiple partners and
females mated to a single partner. There was also no indication that females received any short-term reproductive benefits from
mating with males with large eye span. In fact, females mated to males with short eye span laid a higher percentage of fertile
eggs than females mated to large eye span males. Key words: diopsid, exaggerated morphology, eye span, multiple mating, sexual
selection. [Behav Ecol 12:732–739 (2001)]
M ultiple mating by females (polyandry) occurs in many
species. We expect that males are commonly under
strong selection to mate multiply. But the reasons for female
Regardless of the number of matings a female receives,
choice of a specific mate will also directly impact on female
reproductive performance (Iwasa and Pomiankowski, 1999;
multiple mating are poorly understood, especially as several McLain, 1998; Savalli and Fox, 1998; Thornhill, 1976). Fe-
male mating characteristics have been shown to be harmful males of numerous species exhibit mate preferences for males
to females (Chapman et al., 1995; Fowler and Partridge, 1989; with exaggerated ornamental characters (see Andersson, 1994
Rowe et al., 1994). Numerous hypotheses have been present- for review). Many studies examining the adaptive significance
ed to explain the adaptive significance of female multiple mat- of mate choice have focused on genetic benefits associated
ing and have generally been placed into two categories—di- with Fisher’s runaway process or good genes (Pomiankowski,
rect benefits and genetic benefits (see Arnqvist and Nilsson, 1987; Wilkinson et al., 1998b). But mate choice may also be
2000; Jennions and Petrie, 2000 for recent reviews). Common related to more immediate changes in female reproductive
direct benefits associated with multiple mating are nutritional success. The expression of ornamental characters is often de-
gains derived from male feeding of females, for instance pendent on male condition (David et al., 1999; Rowe and
through nutrient containing spermatophores (Butlin et al., Locke, 1996; Wilkinson and Taper, 1999). If ejaculate quality
1987; Gwynne, 1984; Lamunyon, 1997; Pardo et al., 1995), also depends on condition, then female preference for males
and sperm replenishment (Ridley, 1988; Walker, 1980) espe- with exaggerated characters may be associated with improved
cially in species with relatively small ejaculates (Pitnick, 1993; fertility and fecundity. A few studies have established a link
Pitnick and Markow, 1994). Several genetic benefits of mul- between female preference and increased fecundity and fer-
tiple mating have been proposed but these have been more tility. In the seed beetle, Stator limbatus, females prefer to
difficult to demonstrate experimentally. A few recent studies mate with large males and this mating results in increased
have provided experimental evidence that females benefit fecundity (Savalli and Fox, 1998). In the stink bug, Nezara
from multiple mating by reducing genetic incompatibility be- viridula, females allowed to choose their mates laid more fer-
tween mates (Newcomer et al., 1999; Tregenza and Wedell, tile eggs during their lifetime than those assigned males ran-
1998) or creating sperm competition that promotes fertiliza- domly (McLain, 1998). However, neither of these studies ex-
tion by genetically superior males (Madsen et al., 1992; Olsson amined female choice with respect to a highly exaggerated
et al., 1996). Alternatively, multiple matings may have no male trait.
adaptive value for females resulting instead from incomplete In this study, we examined the effect of variation in mating
female control over matings (Ridley, 1990, Rowe et al., 1994) frequency and male eye span on short-term female fitness in
or as a correlated response to multiple mating in males (Hal- the stalk eyed fly, Cyrtodiopsis dalmanni. This species is a use-
liday and Arnold, 1987). ful model system as females show high rates of multiple mat-
ing, typically mating several times each morning (Wilkinson
et al., 1998a). In addition, male eye span is highly exaggerated
Address correspondence to A. Pomiankowski. E-mail: ucbhpom@ and females prefer to roost and mate with males with large
ucl.ac.uk. eye span (Burkhardt and de la Motte, 1988; Hingle et al.,
Received 1 August 2000; revised 16 February 2001; accepted 26 2001; Wilkinson et al., 1998a). It has also recently been estab-
February 2001. lished that male eye span is strongly condition dependent,
2001 International Society for Behavioral Ecology more so than other non-sexual traits, and there is a geneticBaker et al. • Male mating effects in a stalk-eyed fly 733
component underlying this condition dependence (David et in the experiment was 8.861 ⫾ 0.046 (SE) mm. The experi-
al., 1998, 2000). Overall, little is presently known about the ment was divided into three blocks spaced a week apart with
fitness consequences of multiple mating or mate choice in 15 females from each group used in each block. Therefore, a
diopsids. Our study examines two aspects of female mating total of 45 females were assigned to each group.
behavior, one related to mating frequency and one related to Females were individually housed in circular 400 ml plastic
mating preference. In the first part of the study, we manipu- containers (height ⫽ 95 mm, diameter ⫽ 75 mm) with a
lated the number of matings a female received and examined roosting string hanging from the top and moist tissue paper
the effects on female fecundity and fertility. Our experimental and a food tray at the base. Females were placed in their con-
design differentiates between the effects of multiple partners tainers a week prior to the beginning of the experiment in
and multiple copulations, similar to the studies of Tregenza order to acclimatize them to the experimental conditions. Fe-
and Wedell (1998) and Newcomer et al. (1999). This provides males laid eggs primarily on the tissue paper but occasionally
a means for distinguishing direct benefits from genetic ben- on the food. The tissue and food were removed from the
efits. In the second part of the study, we mated females to containers three times a week and all the eggs on both sub-
males of differing eye span and examined the effects on fe- strates were counted. Fecundity was recorded for all groups
cundity and fertility. from the day following the final mating of the second treat-
ment and for the next 14 days. In order to determine the
fertility of the eggs, the tissue paper was transferred to a petri
METHODS dish containing a moist cotton pad and stored at 25⬚C for 5
Study animal days. Eggs of C. dalmanni take 2–3 days to hatch and, follow-
ing this period, hatching success can be assessed by visual in-
Cyrtodiopsis dalmanni is distributed throughout Southeast spection of the eggs at 10⫻ magnification. For fertile eggs that
Asia and is found predominantly at the edge of forest streams. have hatched, only the outer shell of the chorion remains,
This species forms aggregations at night on roothairs, and while unhatched eggs appear full with the embryo still inside
males compete with each other for control of these roothairs the chorion. A few eggs showed clear signs of fertilization and
where the majority of matings occur at dawn and dusk (Wil- development (i.e., segmental striations and early mouthpart
kinson and Reillo, 1994). Individuals of C. whitei, a closely formation) but had not yet hatched. These were recorded as
related diopsid species, have been observed to mate over 10 fertile. Eggs collected from females in the stock population
times a day in the field (Burkhardt et al., 1994; Lorch et al., cages and kept under identical conditions have a hatching
1993) and similarly high mating rates have been found in C. success rate greater than 90%, suggesting that nearly all fer-
dalmanni laboratory populations (Baker et al., unpublished tilized eggs hatch and that hatching success provides a close
data; Wilkinson and Reillo, 1994). In both of these species, approximation of fertility. Eggs laid on the corn could not be
copulation duration is relatively short, usually lasting less than assessed for fertility and were not counted. These eggs, how-
60 s (Wilkinson and Reillo, 1994) and sperm transfer is ac- ever, represent only 12.6% of the total number laid. A few
complished using a spermatophore (Kotrba, 1996). females died during the experiment. Those that survived at
The C. dalmanni flies used in the experiments were from least 7 days were included in the final analysis and the fecun-
a large laboratory colony collected from Gombak, Malaysia in dity per day was calculated by dividing the total number of
1993. These flies have been maintained in several population eggs laid by the female’s lifespan. No males died during the
cages of at least 200 individuals per cage with a 1:1 sex ratio. experiment.
Flies are fed ground corn medium and kept at 25⬚C, with a
12-h day/night light cycle. Experimental flies were collected Experiment 2
as eggs. Emerging adults were segregated by sex and allowed Three experimental groups were set up to differentiate the
to reach sexual maturity (approximately 3–4 weeks after pu- effects of multiple partners from multiple copulations on fe-
pation). All individuals used in this experiment were 6–8 male reproductive output. Females were mated once (single
weeks old at the start of each experiment. mated), mated three times to the same male (triple mated-1
The lifespan of C. dalmanni females in the laboratory av- male) or mated once to three different males (triple mated-
erages 4 months and can easily reach over 6 months (Reguera 3 males). Every male in the experiment was mated to a female
P, unpublished data), making it difficult to monitor lifetime from each group over a 3-week period. So, for example, in
fecundity and fertility. Therefore, we chose to examine these week one a given male was mated on the third day to a single
variables for a limited period of time, usually 14 days. In ad- mated female, in week two he was mated once on the first,
dition, because C. dalmanni females have high remating rates, second, and third days to a female from the triple mated-1
the effect of a single or a few matings is probably most pro- male group, and, finally, in week three he was mated once on
nounced in the short term. the first, second and third days to three different females from
the triple mated-3 males group. Groups were set up in a block
Effects of multiple mating design over a 3-week period, with 15 virgin females in each
group being mated in each week. This meant that a total of
Experiment 1 45 females were assigned to each experimental group. Using
Three experimental groups were set up to investigate how each male in all three groups has the advantage of controlling
mating alters female fecundity and fertility. Females were un- for variation in male effects other than those caused by the
mated, mated three times, or mated repeatedly. In the un- different treatments (Tregenza and Wedell, 1998).
mated group, virgin females were housed by themselves for Females were individually housed in 400 ml containers. Tis-
the duration of the experiment. In the second group, virgin sue paper and food was collected from the containers three
females were allowed to mate once on three consecutive days times a week and the number of eggs laid on both substrates
with the same male. Each morning, females were transferred was recorded. The hatching success of these eggs was deter-
to containers with a single male, observed until a copulation mined as described in the previous experiment. The males
occurred and then moved back to their original containers. used in the experiment were not virgins and had an average
In the third group, a single virgin female was placed with a eye span of 8.880 ⫾ 0.047 mm. Prior to the start of the ex-
virgin male and the pair were kept together for the remainder periment, males were housed in large cages (20 ⫻ 23 ⫻ 33
of the experiment. The average eye span of the males used cm) with females. At the start of the experiment males were734 Behavioral Ecology Vol. 12 No. 6
moved into individual 400 ml containers. They were kept in
these throughout the remainder of the experiment except
during the time for their assigned matings.
In both experiment 1 and experiment 2, a large number of
females had low hatching success. As the experiment was de-
signed to assess the effect of mating on female egg fertility, it
was important to remove sterile females from the data set. To
establish whether low hatching success was due to female ste-
rility, females with less than 10% hatching success were further
investigated after the main experiment had finished. The 10%
hatching success cut-off is an arbitrary designation. As there
are occasional errors in scoring hatching, 10% was chosen as
a conservative limit to ensure that all sterile individuals were
identified. Individual females were housed continuously with
two new males in a 400 ml container. Eggs were collected
three times over the following 7 days and scored for egg fer- Figure 1
tility. These females were taken to be sterile if their hatching The mean number of eggs laid per day (shaded bars) and the
success remained below 10% under these new conditions of mean percentage of total eggs that hatched (open bars) for female
constant access to males. Sterile females were excluded from groups that received different numbers of matings. Egg laying was
further analysis. monitored for 14 days. The intermediate treatment involved one
mating per day for three consecutive days. Bars represent 95%
Finally, the contribution of male sterility to variation in fe- confidence intervals.
male hatching success was investigated. To identify sterile
males, any male with less than 10% hatching success in both
the single mated and triple mated-1 male groups were further Females that died during the experiment were not replaced,
investigated after the main experiment had finished. These and the egg laying variable was standardized by dividing the
males were housed continuously with two virgin females in a total number of eggs laid by the four females by the sum of
400 ml container. Eggs were collected three times over the their life spans.
following 7 days and scored for hatching success. These males
were taken to be sterile if their hatching success remained
below 10% under these new conditions of constant access to Statistical analysis
females. For the multiple mating experiments, fecundity, measured as
the number of eggs laid per day, was compared among groups
Effects of male eye span using a square root transformation of the data. For the male
eye span experiment, fecundity had a normal distribution and
Two experimental groups were set up to investigate whether was analyzed without transformation. However, due to a large
mating with males of a particular eye span altered female egg number of very low hatching success scores in all experiments,
laying rate or egg fertility. Females were either mated repeat- this variable could not be normalized using standard trans-
edly to small or large eye span males. Small eye span males formations. Therefore, nonparametric tests were used to ex-
included flies with an eye span less than 7.5 mm while large amine differences in percent hatching success among groups.
eye span males comprised flies with an eye span greater than All tests were calculated using the JMP statistical package (ver-
8.5 mm. The means of the two groups were 6.642 ⫾ 0.101 sion 3.2.2, SAS Institute Inc.). Identification of significant
mm and 9.023 ⫾ 0.035 mm. Female choice experiments have pairwise differences between groups was made using either
demonstrated that a difference in eye span of this order the Tukey-Kramer HSD test or Dunn’s nonparametric test for
(2.381 mm) is sufficient to cause strong female preference for multiple comparisons (Zar, 1996). As percentages are not re-
the large eye span male in binary choice experiments (Hingle liable indicators when the sample size is very small, any female
et al., 2001; Wilkinson et al., 1998a). The regression coeffi- that did not lay at least ten eggs over the course of the ex-
cient for eye span on body length is 0.969. Therefore, these periment was excluded from the analysis. Data are presented
two variables represent equivalent effects in this experiment. as mean ⫾ SE unless otherwise specified.
Eye span was measured from the outer edge of each eye and
body length from the front of the face to the tip of the wing.
Measurements were made using a monocular microscope con- RESULTS
nected to a computer with the NIH Image software package
Effects of multiple mating
(version 1.55).
Four virgin females were housed with each male in a 1500 Experiment 1
ml (height ⫽ 164 mm, diameter ⫽ 120 mm) container that In the first experiment, females were unmated, mated three
included a central roosting string hanging from the top and times, or mated repeatedly. There was a significant difference
moist tissue paper and a food tray at the base. As in the pre- in fecundity among blocks (F2,122 ⫽ 12.291, p ⬍ .001), so a
vious experiments, tissue paper and food were collected three block variable was added as an effect in the ANOVAs, but
times a week for 2 weeks. All eggs on both paper and food there was no block effect on hatching success (Kruskal-Wallis
were recorded and hatching success was calculated as de- Test, H corrected for ties ⫽ 3.702, p ⫽ .157).
scribed in experiment 1. The sample size was 40 containers The three groups showed heterogeneity in the mean num-
for each male type and the experiment was divided into two ber of eggs laid per day (ANOVA, F2,120 ⫽ 32.021, p ⬍ .001).
blocks with 40 containers in each block. Unmated females had the lowest fecundity and females con-
Males and females were placed together a week prior to the stantly exposed to a male had the highest fecundity (Figure
collection of eggs and kept together for the duration of the 1; unmated, 1.507 ⫾ 0.228 eggs, n ⫽ 39; mated three times,
experiment. Males that died during the experiment were ex- 3.007 ⫾ 0.288 eggs, n ⫽ 45; mated repeatedly, 5.590 ⫾ 0.565
cluded from the final analysis and a small male that was un- eggs, n ⫽ 41). Paired comparisons indicated that all treat-
able to engage his genitalia with females was also excluded. ments were significantly different from each other (unmatedBaker et al. • Male mating effects in a stalk-eyed fly 735
.02; single mated versus triple mated-3 males, Q ⫽ 4.169, p ⬍
.001; mated triple mated-1 male versus triple mated-3 males,
Q ⫽ 1.479, p ⬎ .5). While not significant, the difference in
hatching success between the two multiply mated groups
(61.9% versus 73.4%) is large enough to suggest a significant
effect might be detected if sample sizes were increased. This
11.5% difference, however, is probably higher than the real
difference for the flies in our population. The difference be-
tween the two groups is only 4% if the number of eggs and
number of hatched eggs for each female in a group are
summed together prior to calculating hatching success
(67.6% versus 72.0%) or if the females laying less than 10 eggs
are included in the analysis (58.5% versus 62.4%).
Overall, the pattern of hatching success scores suggested
that individual copulations did not provide an adequate sup-
ply of sperm for females. The mean hatching success for sin-
Figure 2 gle mated females was only 40% and the distribution of hatch-
The mean number of eggs laid per day (shaded bars) and the ing success for each of the groups (Figure 3) indicated that
mean percentage of total eggs that hatched (open bars) for female over one-third (34.2%) of single matings were unsuccessful
groups that received different numbers of matings and numbers of
partners. Egg laying was monitored for 14 days. The multiply mated
(i.e., hatching success scores less than 10%). When the num-
treatments received a total of three matings, one per day for three ber of matings increased to three the number of infertile
consecutive days. Bars represent 95% confidence intervals. scores dropped dramatically (12.5% for triple mated-1 male
and 3% for triple mated-3 males).
To examine whether the low female hatching success scores
versus mated three times, q ⫽ 5.209, p ⬍ .005; unmated versus were caused by a large number of sterile males, experimental
mated repeatedly, q ⫽ 13.597, p ⬍ .001; mated three times males with low hatching success (less than 10% in both the
versus mated repeatedly, q ⫽ 5.462, p ⬍ .005). single mated and triple mated-1 male groups) were given con-
There was also a significant difference between the two mat- tinuous access to two virgin females and scored for hatching
ed groups in hatching success (Mann-Whitney U test, U ⫽ success. Despite the high occurrence of low hatching success
423, p ⬍ .001), with the group mated repeatedly laying a high- among the experimental females, only one male was shown
er percentage of fertile eggs than the group mated three times to be sterile (i.e., continued to exhibit hatching success less
(Figure 1; mated three times, 61.9 ⫾ 5.0%, n ⫽ 40; mated than 10%). This suggests that the lack of fertility among ex-
repeatedly, 81.3 ⫾ 3.2%, n ⫽ 39). perimental females arose from unsuccessful copulations rath-
er than male sterility.
Experiment 2
In this experiment the three experimental groups differed
with respect to the number of partners as well as the number Effects of male eye span
of copulations. Females were mated once (single mated), mat- In the final experiment, females were mated repeatedly to
ed three times to the same male (triple mated-1 male) or males with either small or large eye span. Similar to the pre-
mated three times to three different males (triple mated-3 vious experiments, there was a block effect on fecundity (t
males). There was a block effect on fecundity (F2,128 ⫽ 11.737, test: t ⫽ 5.579, p ⬍ .001), but not hatching success (Mann-
p ⬍ .001), so we included block as an effect in the ANOVAs, Whitney U, U ⫽ 679.5, p ⫽ .9612), so we included block as
but there was no block effect on hatching success (Kruskal- an effect in the fecundity ANOVA. Male eye span did not
Wallis Test, H corrected for ties ⫽ 5.530, p ⫽ .063). affect female fecundity (Figure 4; ANOVA, F1,72 ⫽ 0.315, p ⫽
Unlike the previous experiment, there was no difference .577). There was no significant difference between females
among the three groups in fecundity (Figure 2; ANOVA, F2,126 mated to small and large eye span males in the number of
⫽ 0.938, p ⫽ .394; single mated, 3.121 ⫾ 0.329 eggs, n ⫽ 44, eggs laid per day (small, 4.999 ⫾ 0.330 eggs, n ⫽ 34; large,
triple mated-1 male, 3.007 ⫾ 0.228 eggs, n ⫽ 45, triple mated- 4.867 ⫾ 0.334 eggs, n ⫽ 41). However, male eye span did
3 males, 2.594 ⫾ 0.259 eggs, n ⫽ 42). affect female hatching success (Figure 4; Mann-Whitney U, U
The hatching success scores for the experiment were char- ⫽ 474.5, p ⫽ .0258). Females mated to small males had a
acterized by a substantial number of infertile matings. Of the higher percent hatching success than females mated to large
110 females that laid at least 10 eggs, 19 had hatching success males (small, 83.3 ⫾ 4.0 %, n ⫽ 34; large, 69.1 ⫾ 5.4 %, n ⫽
scores less than 10%. To determine if any of these scores were 40).
due to female sterility, we placed each of these females with
three new males after the experiment had finished. The
hatching success of their eggs was then scored for 7 days. By DISCUSSION
this method, only two out of the 19 females were considered Effects of multiple mating
sterile (i.e., continued to produce less than 10% fertile eggs)
and were excluded from further analysis. In this study we showed that additional matings increased the
The experiment revealed that the number of matings a fe- egg laying rate of C. dalmanni. This effect was only evident
male received affected hatching success (Kruskal-Wallis Test, when there was a substantial difference in the number of mat-
H corrected for ties ⫽ 18.318, p ⬍ .001; single mated, 40.0 ⫾ ings females received. Females that were allowed unlimited
5.5%, n ⫽ 38, triple mated-1 male, 61.9 ⫾ 5.0%, n ⫽ 40, triple matings produced more eggs than females mated three times
mated-3 males, 73.4 ⫾ 4.0 %, n ⫽ 32). Females that mated and both of these groups laid more eggs than did virgins (Fig-
only once had a significantly lower percentage of fertile eggs ure 1). However, females mated once had the same fecundity
than the two groups of females mated three times, but these as females mated three times (Figure 2).
multiply mated groups did not differ from each other (Figure While numerous studies have demonstrated that fecundity
2; single mated versus triple mated-1 male, Q ⫽ 2.867, p ⬍ rates are increased by male-derived nutrients transferred dur-736 Behavioral Ecology Vol. 12 No. 6
Figure 4
The mean number of eggs laid per day (shaded bars) and the
mean percentage of total eggs that hatched (open bars) for female
groups mated to small or large eye span males. The small category
includes males with an eye span less than 7.5 mm and the large
category includes males with an eye span greater than 8.5 mm. Egg
laying was monitored for 14 days. Bars represent 95% confidence
intervals.
but a significant increase in fecundity between females mated
three times and those mated repeatedly (Figure 2).
An alternative interpretation of our fecundity results is that
the egg laying rate increased as a response to chemical or
physical stimuli received during mating. In Drosophila melan-
ogaster, as well as some other Diptera (see Chapman et al.,
1998 for review), accessory proteins, such as the ‘‘sex pep-
tide,’’ stimulate egg production and increase laying rate
(Chen et al., 1988). Whether this is happening in C. dalmanni
will require further experiments manipulating the levels of
accessory gland products given to female.
The fertility results from our study provided no evidence
for genetic benefits that have been reported in other species
(Newcomer et al., 1999; Tregenza and Wedell, 1998). The sec-
ond experiment was designed to separate the effects of mul-
tiple partners from multiple copulations on female reproduc-
tion. Of the three treatments, one pair differed with respect
to the number of copulations (one versus three) but not the
number of partners, while another pair differed with respect
Figure 3 to the number of partners (one versus three) but not the
A histogram of egg-hatching success scores for female groups that number of copulations. Genetic effects are expected to occur
received different numbers of matings and numbers of partners. as a result of an increased number of partners. Contrary to
The triple mated treatments received one mating per day for three this prediction, hatching success did not differ between fe-
consecutive days. males mated three times to a single male and females mated
three times to three different males. The significant differ-
ence in this experiment was between the single mated females
ing copulation (Butlin et al., 1987; Gwynne, 1984; Lamunyon, and the multiply mated females (both those mated to one
1997; Pardo et al., 1995), the mating conditions in C. dal- male and those mated to three males). It is important to note,
manni are not generally similar to these other species. The however, that the hatching success measure used in this study
majority of insects that provide nutritional gifts produce large provided only a partial indication of offspring fitness. It is
spermatophores that comprise a substantial proportion of a possible that genetic effects will be evident when larval mor-
male’s body weight (Gwynne, 1984; Savalli and Fox, 1998). tality and pupation success are also examined.
Copulation durations for these insects are usually greater than Our results suggest obtaining a sufficient sperm supply is
30 min and often take hours (Butlin et al., 1987; Rutowski et the primary reason for the high frequency of female mating
al., 1987; Taylor et al., 1998; Taylor and Yuval, 1999). In con- in C. dalmanni. Females in constant contact with a male had
trast, C. dalmanni produces a spermatophore that is smaller significantly higher hatching success than females mated
than most other diopsids (Kotrba, 1996) and has copulations three times, and in turn females mated three times had sig-
that generally last less than 60 s, suggesting little nutritional nificantly higher hatching success than females mated only
substance is transferred. If C. dalmanni males do provide once. Following the work of Bateman (1948), evolutionary
small scale nutrient transfers during mating, they could still biologists have generally focused on identifying factors other
be important as females mate many times per day. Such an than sperm-limitation to explain the evolution of multiple
interpretation is consistent with the observation of no fecun- mating in females (Butlin et al., 1987; Gwynne, 1984; Lamun-
dity difference between females mated once and three times, yon, 1997; Madsen et al., 1992; Newcomer et al., 1999; OlssonBaker et al. • Male mating effects in a stalk-eyed fly 737
et al., 1996; Pardo et al., 1995; Tregenza and Wedell, 1998). other than sperm limitation become important in determin-
However, Eberhard (1996) lists over 25 insect species in which ing female mating rate.
male ejaculate size is smaller than the storage capacity of fe- Finally, another factor that may be important in the evolu-
males and comprehensive surveys of insects have found that tion of multiple mating in C. dalmanni is meiotic drive. An
a single copulation is rarely sufficient to maximize female fe- X-linked meiotic drive system that produces a female-biased
cundity (Ridley, 1988; Arnqvist and Nilsson, 2000). C. dalman- sex ratio has been found in C. dalmanni (Presgraves et al.,
ni appears to be another example. The exact number of 1997). Wilkinson et al. (1998a) point out that, among three
sperm transferred by males of C. dalmanni is unknown but it Cyrtodiopsis species, mating rate correlates with the frequency
is likely to be small. Its sister taxa, C. whitei, has been esti- of the driving X chromosomes. Meiotic drive has been sug-
mated to transfer an average of 90 sperm per mating (Lorch gested as a factor promoting the evolution of polyandry, as
et al., 1993) and the spermatophores of these two species are females can reduce the proportion of offspring fertilized by
of similar size (Kotrba, 1996). In any case, it is clear that a sperm carrying the drive locus by mating with several males
single mating rarely results in maximum fertility. Only 18% of (Haig and Bergstrom, 1995). The laboratory population used
the females mated once had hatching success scores above in our experiment, however, shows no evidence of biased sex
80% (Figure 3), a value which is the average hatching success ratios and probably lacks meiotic drive. So the suggested in-
for females mated repeatedly to a single male (Figure 1). volvement of meiotic drive as a major component favoring
The mating system of C. dalmanni is similar to that found multiple mating remains to be investigated.
in some Drosophila species that are distinguished by frequent
matings and small ejaculates (Markow, 1985; Pitnick, 1993;
Effects of male eye span
Pitnick and Markow, 1994). One prediction of sperm com-
petition theory (Parker, 1990) is that ejaculate size should in- In most insect species there is a positive correlation between
crease as a function of mating frequency because larger ejac- body size and sexually selected fitness components (Partridge,
ulates ensure greater fertilization success when fertilization is 1988), and the mating advantage of males with large eye span
a raffle process among sperm from several males. This model, in sexually dimorphic diopsid species has been well docu-
however, assumes female mating frequency is driving the evo- mented (Burkhardt and de la Motte, 1988, 1994; Hingle et
lution of ejaculate size and not vice versa. Mating systems that al., 2001; Lorch et al., 1993; Panhuis and Wilkinson, 1999;
provide the opportunities for numerous copulations by a sin- Wilkinson and Reillo, 1994; Wilkinson et al., 1998a). In C.
gle male may select for males that partition their ejaculate dalmanni, females show strong preference for large eye span
among many females (Pitnick and Markow, 1984). In C. dal- males (Hingle et al., 2001; Wilkinson et al., 1998a). Recent
manni, the aggregation behavior exhibited by females may experiments have shown that male eye span is highly condi-
have promoted an evolutionary transition in males toward tion-dependent, and that there is genetic variation underlying
higher mating frequency with reduced sperm transfer. Mono- this response as predicted by handicap models of sexual se-
morphic species that are phylogenetically ancestral to C. dal- lection (David et al., 1998, 2000). Selection experiments in-
manni and which do not form mating aggregations, such as dicate that large eye span also provides a signal of male quality
C. quinqueguttata and Teleopsis quadriguttata (Presgraves et by indicating resistance to meiotic drive (Wilkinson et al.,
al., 1999), produce a larger spermatophore (Kotrba, 1996), 1998b). The possibility of more immediate female fecundity
mate less often (Wilkinson et al., 1998) and for a longer pe- and fertility benefits from mating with large eye span males
riod of time (Kotrba, 1996) than C. dalmanni. has not been investigated before. Results from this study pro-
Not only do males transfer small amounts of sperm in a vide no indication of such benefits. In fact, the small males
given mating, but a significant proportion of copulations in used in this study outperformed large males with respect to
this species are unsuccessful. One-third of the females mated their ability to enhance female fertility.
once had hatching success scores less than 10%. Mating sys- Other studies have found that small males outperform large
tems with a substantial number of unsuccessful copulations males in some situations. In both Drosophila melanogaster (Pit-
have been found in several other species (Hoogland, 1998; nick, 1991) and the water strider, Gerris incognitus, (Arnqvist
Lamunyon, 2000; Petersson, 1990; Whittier and Shelly, 1993). et al., 1997) small males had a higher hatching success than
In C. dalmanni, this may occur for several reasons including large males and it was suggested that this results from large
improper spermatophore construction or orientation, female males, who have more opportunities for matings, allocating
spermatophore rejection, or extremely low levels of sperm less sperm per ejaculate. This is unlikely to be a factor in our
transfer. A study on C. whitei indicated male mating duration study because males were allowed constant contact with fe-
had a bimodal distribution with only matings longer than 35 males. All of the males in our experiment were observed to
s resulting in sperm transfer (Lorch et al., 1993). All of the mate but the exact number of matings during the experiment
matings in our experiment, however, were longer than 35 s. was not recorded. Under similar experimental conditions
In the future, it will be important to determine the proportion (i.e., one male housed with four females), males have been
of long copulations that result in proper spermatophore con- observed to mate, on average, nearly four times in a 1.5-h
struction and whether certain males are generally more suc- period each morning (unpublished data). Therefore, over the
cessful at constructing spermatophores. course of the experiment (including the week long acclima-
The results from this study clearly indicate that, as a result tion period) each female would have received, on average, at
of the combination of small ejaculates and unsuccessful cop- least 20 matings. With such a high number of matings, effects
ulations, numerous matings are required by females to maxi- due to variation in ejaculate size are likely to be small relative
mize fertility. Unfortunately, the mating frequency of females to the effects of overall sperm quality or proper spermato-
in the field has not been determined so it is unclear if females phore construction. Studies that control for the number of
mate more or less often than is necessary to ensure high fer- matings and directly measure the ejaculate size produced by
tility rates. When provided with continual access to several small and large males could help identify whether fertility or
males in the lab, females will, on average, mate over four times fecundity differences exist between these types of males.
in a 90 min dawn period (unpublished data). This high mat- It has been suggested that selection for increased compet-
ing rate suggests females may copulate beyond the level need- itive ability of sperm may create characteristics that cause
ed for fertility assurances and additional research, in both the weaker fertilization ability in the absence of competition
field and the lab, is needed to determine if and when factors (Eberhard, 1996). Burkhardt and de la Motte (1994) found738 Behavioral Ecology Vol. 12 No. 6
that large males of C. whitei had higher fertilization success Jennions, MD, Petrie M, 2000. Why do females mate multiply? A re-
than small males when they both mated the same female. view of the genetic benefits. Biol Rev 75:21–64.
Therefore, the sperm of large males may have superior com- Kotrba M, 1996. Sperm transfer by spermatophore in Diptera: new
petitive ability, but not superior fertilization ability, than that results from the Diopsidae. Zool J Linn Soc 117:305–323.
Lamunyon CW, 1997. Increased fecundity, as a function of multiple
of small males. The advantage found for small males in this mating, in an arctiid moth, Utetheisa ornatrix. Ecol Entomol 22:69–
experiment occurred in isolation from other males and may 73.
not persist when more interaction among males is allowed. It Lamunyon CW, 2000. Sperm storage by females of the polyandrous
will be important for future studies to examine the perfor- noctuid moth Heliothis virescens. Anim Behav 59:395–402.
mance of sperm from large and small males of C. dalmanni Lorch P, Wilkinson GS, Reillo PR, 1993. Copulation duration and
in various competitive and non-competitive situations. sperm precedence in the Malaysian stalk-eyed fly, Cyrtodiopsis whitei
(Diptera: Diopsidae). Behav Ecol Sociobiol 32:303–311.
Madsen T, Shine R, Loman J, Håkansson T, 1992. Why do female
We thank Matthew Denniff for invaluable assistance maintaining cul-
adders copulate so frequently? Nature 355:440–441.
tures, measuring flies, and counting eggs. Jeremy Field kindly provid-
Markow TA, 1985. A comparative investigation of the mating system
ed helpful advice on the data analysis. Funding for this project was
of Drosophila hydei. Anim Behav 33:775–781.
provided by NERC (grant to T.C., K.F., and A.P. for support of R.B.),
McLain DK, 1998. Non-genetic benefits of mate choice: fecundity en-
and the Royal Society (summer research studentship to T.R.).
hancement and sexy sons. Anim Behav 55:1191–1201.
Newcomer SD, Zeh JA, Zeh DW, 1999. Genetic benefits enhance the
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