Behav Ecol Sociobiol (1989) 25: 237-246
Behavioral Ecology
and Sociobiology
9 Springer-Verlag 1989
Effects of spermatophores
on male and female monarch butterfly reproductive success
Karen S. Oberhauser
University of Minnesota, Department of Ecology and Behavioral Biology, J09 Zoology, Minneapolis, MN 55455, USA
Received February 14, 1989 / Accepted May 9, 1989
Summary. I present the results of experiments designed to measure the effects of spermatophores
produced by male monarch butterflies on male and
female reproductive success. There was wide variation in the number of matings by captive males,
suggesting the potential for strong sexual selection
on males. Male lifespan was not affected by total
number of matings, nor did it differ between males
that were allowed to mate and those not exposed
to females. Two effects of spermatophores on female behavior or fecundity are reported: (1) Females that received large spermatophores delayed
remating longer than those receiving small ones.
(2) Females allowed to mate several times laid
more eggs than singly-mated females. The relative
importance of these effects is discussed in relation
to monarch mating patterns.
Introduction
Like other Lepidoptera, male monarch butterflies
(Danaus plexippus) produce a spermatophore during mating that often represents a large material
investment, comprising up to 10% of their body
mass (Oberhauser 1988). Accessory gland products
contained in spermatophores have been shown to
function in sperm activation and stimulation of
oogenesis and oviposition (Leopold 1976). However, lepidopteran spermatophores often contain
more material than is necessary for these functions; even the very small spermatophores transferred by recently-mated males result in egg fertilization and oviposition (Rutowski et al. 1987, present study, personal observations). Spermatophores
contain significant quantities of nitrogen (Boggs
1979, personal observations, largely in the form
of protein (Marshall 1982). Because the adult food
of monarch butterflies, nectar, is a poor nitrogen
source, most spermatophore nitrogen must come
from larval resources and could represent an important limiting resource for males.
Spermatophore production provides an opportunity to study costs and benefits of reproductive
investment, and possible conflicts between the
sexes. There are several potential costs that males
could incur during spermatophore production:
time costs of mating itself, the time required to
replenish accessory gland products used to form
spermatophores, and material costs, which might
decrease the resources available for somatic maintenance.
Two potential benefits of producing large spermatophores have been suggested. First, spermatophore constituents may be used by females to increase offspring quantity or quality, thereby increasing male reproductive success as well. Several
studies have shown that the contents of lepidopteran spermatophores are incorporated into both
eggs and female somatic tissue (Boggs and Gilbert
1979; Marshall 1980; Boggs 1981 ; Boggs and Watt
1981; Greenfield 1982), and two demonstrated an
increase in female fecundity when more spermatophore material was received (Rutowski et al. 1987;
Watanabe 1988). Second, large spermatophores
could delay female remating (Sugawara 1979;
Boggs 1979; Boggs 1981; Rutowski 1980; Rutowski et al. 1981). Many female Lepidoptera exhibit polyandry (Drummond 1984) and there is a
pattern of sperm precedence by the last male to
mate (Gwynne 1984). Thus sperm from males that
produce large spermatophores could fertilize a
higher proportion of a female's eggs without necessarily increasing her lifetime reproductive success.
These two effects are not mutually exclusive; males
may benefit both by increasing female reproductive
output and by decreasing female receptivity to sub-
238
sequent mating. However, since a spermatophore
that has been digested will not be as effective at
delaying remating, there must be some tradeoff between the two effects. This could lead to conflict
between male and female interests in the rate of
sperrnatophore digestion.
Methods
General rearing and maintenance
All experimental butterflies were reared from eggs laid by wildcaught females (Oberhauser 1988). Experiments were done outside during the summers of 1986-1988. Most matings took
place in large screen cages (mating cages) that were 4 m x 2 m x
2 m or 2 m x 2 m x 2 m. Females whose fecundity was being
measured were kept singly in oviposition cages (0.7 m x 0.7 m x
0.7 m). Butterflies being used in experiments were removed
from their cages and fed a 30% honey solution to satiation
every morning (except as noted below); otherwise they were
in outdoor cages at all times. Those not in current use were
kept in glassine envelopes at room (1986-1987) or ambient
(1988) temperatures and fed every other day. All butterflies
were exposed to natural light/dark periods.
Male mating experiment
Sixty-nine males that emerged 8-12 July 1988 were randomly
assigned to virigin (n = 12) and mating (n = 57) treatments. Virgin males were put into a mating cage every day without females. Mating males were p u t into a cage daily with an approximately equal n u m b e r of females (female n u m b e r varied with
female availability: actual F : M ratios ranged from I to 0.7).
Most females were virgins, although after the first week some
previously-mated females were used. Since this experiment was
designed to assess effects of mating frequency on male lifespan,
the numbers a n d types of females were not crucial. The experiment ended when all males h a d died. All matings and the age
at death for each male were recorded.
Previous dissections of recently-mated females (Oberhauser
1988) allowed an estimate of the spermatophore mass transferred during each mating. Spermatophore mass transferred by
virgin males was estimated using the following equation:
Mass = - 5.469 + 1.855" (male age at mating) + 0.0480" (male
mass at emergence). Spermatophore mass from previouslymated males was estimated as: M a s s = - - 4 . 8 7 1 + 2 6 . 7 9 * ( l o g
time since last mating by male) - 1.478" (number of previous
matings by male) + 0.0321* (male mass at emergence). These
models provide a fairly accurate estimate of spermatophore
mass transferred (R z for first mating = 0.722; R 2 for subsequent
matings = 0.858).
Female remating and initial spermatophore size
I examined the effects of spermatophore size on female intermating interval by allowing females to mate with males that
transferred either large or small spermatophores. The experiment was done in three blocks because of space and sample
size limitations. Initial matings in block 1 occurred on 29 July
1987; block 2, 28 July 1988; and block 3, 13 August 1988. In
each block 15 females were randomly assigned to each of two
treatments, mating with six- to ten-day old virgin males or
males that had mated one to two days previously (spermatophore masses of approximately 30-37 m g and 7-15 rag, respectively, Oberhauser 1988). Actual sample sizes were less than
15 because some females did not mate on the first day they
were exposed to males. All females were six or seven days old
for the first mating. Previous work h a d shown that this was
the age at which captive females were most likely to mate for
the first time (Oberhanser unpublished), although I am not
sure how it corresponds to the age at which wild females first
mate.
Each day after the first mating, all females were put into
a mating cage with half as many virgin males as females present.
When an odd number of females was present, male fractions
were rounded up. Fresh Asclepias syriaca was provided as an
oviposition substrate. W h e n a female remated, she was dissected to make sure that she had actually received two spermatophores. Females that died or escaped before remating and
any that had not remated 18 days after their initial mating were
not included in the analysis.
Female matingfrequencies
In 1986, 38 females were used to determine female mating fiequencies. Females mated for the first time at age 6-9 days and
were put into mating cages with males every day thereafter
(unless i t was raining or below 16 ~ C, when matings do not
occur, personal observations). There were approximately equal
numbers of males and females in the cages, and butterfly density
was not over 40 per cage. All matings were recorded. Males
were either virgins or had not mated within four days, so spermatophores transferred h a d masses of approximately 25 mg or
more (Oberhauser 1988). Fresh Aselepias syriaea was provided
for oviposition. The experiment ended on 31 August, even
though some females were still alive. A t this point, all females
were over five weeks old, after which few matings occur (personal observations), and no matings had occurred for 6 days.
Therefore mating frequencies observed should provide a fairly
accurate representation of what they would have been if the
experiment had run until all females died.
Fecundity experiments
I performed two experiments to test the hypothesis that receiving m o r e spermatophore material affects female lifespan, fecundity, or egg fertility (here I use fertility to represent the proportion of eggs that were fertile, not total gamete production).
In 1987, females were assigned to large or small spermatophore
treatments, mating with either eight day-old virgin males or
males that had mated two days previously. These two male
groups transferred spermatophores of approximately 35 and
17 rag, respectively (Oberhauser 1988). All females mated the
same day. They were kept in individual oviposition cages and
given fresh Asclepias syriaca daily until they died naturally.
Eggs were counted daily, and kept on the plant on which they
were laid until hatching, when larvae were counted. Initial sample sizes for small and large spermatophore recipients were 11
and 14 respectively.
This experiment was modified in 1988 by increasing the
difference between spermatophore treatments. This was done
to increase the chance of finding an effect of the amount of
spermatophore material received on female fecundity if one
existed. I also added two feeding treatments to determine
whether the importance of spermatophore nutrients was affected by other nutrition the female received. All females mated
for the first time at age 5 to 7 days to 5 to 8 day-old virgin
males. After the initial mating, they were randomly assigned
to four treatments in a two by two factorial design, with singleand multiple-mating levels, and low and high food concentrations. The initial sample size for each treatment was nine indi-
239
viduals. Females in the single-mating treatment were not exposed to males after their first mating. When a female in the
multiple-mating treatment had not mated for three days, a virgin male was put into her cage after 1400 h to allow time for
her to lay eggs without distraction. If she mated, no male was
put into her cage for three days. If she did not mate, a different
male was added the following day, until she mated. No males
were added after 18 days of egg laying for two reasons. First,
fecundity is low after this time, so additional matings were
unlikely to affect lifetime fecundity. Second, the mating fiequency experiment showed that few females mate after age
24 days. Females in the low feed treatment received a 15%
honey solution daily, while those in the high feed treatment
were fed the 30% solution used for all other butterflies. It was
not feasible to monitor the volume of honey solution ingested
by all females, but data obtained by weighing butterflies before
and after feeding showed that both groups of females ingested
approximately equal volumes of solution. Therefore females
receiving the 15% honey solution received fewer carbohydrates.
Females were weighed every three days to determine if the treatments affected their masses.
Data on egg nmnbers and egg fertility were collected as
in 1987, with two exceptions. First, after eggs were counted
a sample of twenty for each female (or all eggs if 20 or fewer
were laid) was collected. Portions of leaves containing these
eggs were cut from the plant and kept in individual petri dishes
until hatching. Daily egg fertility was estimated by assuming
that this sample was representative of all eggs laid. This method
of measuring fertility was more accurate than the one used
previously because it was impossible for larvae to escape. Second, i counted all eggs that were fertilized instead of only those
that hatched. Thus eggs that were visibly fertile (had turned
black) when other eggs had hatched were included. This was
done because I was interested in sperm viability, and there was
no reason to think that the problem with fertilized eggs that
did not hatch lay in sperm, and not ova quality.
Results
Male mating frequencies and costs
The number of matings for males permitted to
mate ranged from 0 to 11 (mean = 3.0, median = 2,
Fig. I a). Figure I b illustrates the estimated total
spermatophore mass transferred for males with different numbers of matings. It shows that males
can transfer significant quantities of spermatophore material during their lives (compare to male
masses of approximately 500 mg), and also that
there is wide variation in the total amount of material transferred. The scatter in estimated mass for
males that mated the same number of times is
caused by differences in the time between matings;
if a male mates a given number of times in quick
succession, he will transfer less material than a
male that waits longer between matings (Oberhauser 1988).
Lifespan data were analyzed by comparing the
virgin and mating treatment groups, and by comparing males within the mating treatment (Fig. 2).
One male lived twice as long as any other males;
residual analysis showed that this male was an out-
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Fig. 1 a. Frequency distribution of the total number of matings
by males exposed to females every day after age six days. b
Estimates of the total amount of spermatophore material transferred by the same males. Values were obtained by estimating
masses of individual spermatophores and summing over each
male's lifetime. See text for further explanation
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Treatment
2
4
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5
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of Matings
9
11
Fig. 2. Effect of mating on male lifespan. The first two bars
represent the mean age (in days) obtained by males in the virgin
(V) or mating (M) treatments. Subsequent bars represent mean
ages obtained by males within the mating treatment that mated
different numbers of times. The male that mated 11 times was
an outlier (see text); without him there are no significant treatment effects, nor did the number of times a male mated affect
his lifespan. Bars represent one standard error
240
1
Table 1. Linear regression of male lifespan
Predictor
Coefficient
Std. error
Constant
Mass
Times mated
18.011
6.076
0,137
3.618
7.122
0.130
P
0.8-
<0.001
0.398
0.296
O
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0.8-
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Adj. R 2 = - 0 . 0 1 6 , N = 4 6 , Overall P = 0 . 5 8 5
0.4
Predictor
Coefficient
Std. error
Constant
Mass
Total sp.
i 8.991
3.854
0.0082
3.282
6.524
0.0086
9
P
< 0.001
0.558
0.350
Adj. R z = - 0 . 0 2 1 , N = 46, Overall P = 0.656
Neither the model which included male mass and total times
mated nor that which included male mass and total spermatophore mass (total sp.) showed any significant effects of the
predictors on male lifespan
Large SP ( n = 3 3 )
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Days after First Mating
Fig. 3. The cumulative proportion of females receiving small
and large spermatophores that had remated each day after theirinitial mating. Females that received small spermatophores remated significantly sooner (rank sum test, 1-tailed P = 0.018)
15
lier ( t = 13.59, 45 df, P<0.001), so he was excluded
from the analyses.
There was no difference between the two treatment groups in lifespan ( t = - 2 . 0 1 , 10.3 df [unequal variances], P=0.072). In fact, the trend is
not in the direction predicted by the hypothesis
that mating is costly; males in the virgin treatment
had shorter average lifespans than mating males.
Because males within the mating group did not
mate an equal number of times, I examined the
effect of mating on lifespan within this group using
multiple linear regression. Independent variables
were total number ofmatings, total spermatophore
mass transferred and mass at emergence. Because
number of matings and spermatophore mass were
highly correlated, only one of them was included
in any given model. None of the independent variables had significant effects on lifespan (Table 1).
13
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9
7
5
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2
3
4
5
6
Number
of Matings
Fig. 4. Frequency distribution of the total number of matings
by females exposed to males every day after age six to nine
days. All females mated at least once
Female mating frequencies and costs
Effect of spermatophore size on female intermating
interval
Figure 3 shows the cumulative fraction of females
that had remated as a function of the number of
days elapsed since the initial mating. Females
which received large spermatophores waited significantly longer to remate (rank sum 1-tailed P =
0.018) than those which received small ones. Three
females died or escaped before remating and three
did not remate. Of those that did not remate, two
received large spermatophores and one received a
small spermatophore. The time at which females
mated initially did not affect the time to female
remating (Kruskal-Wallis test statistic = 0.497, P =
0.780), so data from the three time blocks were
combined for analysis.
Females exposed to males every day mated from
one to six times (mean = 3.5, Fig. 4). Figure 4 includes both females that died a natural death (n =
13) and those that were still alive on 31 August
(n=25).
This experiment identified a possible cost of
mating for females. Both of the females that mated
six times and two that mated four times (Fig. 4)
were either killed or severely debilitated when their
bursae became so full that they ruptured. In all
of these cases, females had mated at least three
times within seven days (one female mated four
times in five days). In each case, the last spermatophores were not in the bursa proper, and the sperm
in them had not been transferred to the spermatheca. Forced copulations by males were observed
241
Table 2. Effects of spermatophore mass on female fitness
Year
Treatment
N
Total eggs
Lifespan
Egg fertility
1987
Large sp.
Small sp.
10
13
884.2 (42.13)
881.5 (45.28)
33.7 (2.75)
41.2 (0.89)
79.1 (0.9)
76.0 (1.3)
1988
MM, low feed
MM, high feed
SM, low feed
SM, high feed
620.1
526.0
477.7
285.0
28.1
29.1
35.6
26.6
81.6
84.6
79.3
81.7
6
7
7
5
(79.56)
(68.60)
(57.11)
(112.0)
(3.78)
(2.91)
(2.73)
(7.11)
(3.0)
(4.8)
(5.2)
(4.4)
Females in 1987 all mated once, with one group receiving small and one large spermatophores. In 1988, there were two levels
of mating (MM = multiple mating, SM = single mating), and two concentrations of feed. Total Eggs, Lifespan, and Egg Fertility
given as: mean (std. error). Egg Fertility (given in percentages) was measured in 1987 as total larvae emerged/total eggs, and
in 1988 as total fertilized eggs/total eggs
Table 3. Effects of female mass at emergence and spermato-
phore size on lifespan and fecundity, 1987
a Lifetime fecundity
Predictor
Coefficient
Std. error
P
Constant
Mass
Sp. size
460.33
0.9857
-53.010
512.21
1.0028
59.709
0.380
0.338
0.386
N = 2 2 , R2=0.068, P=0.570.
b Lifespan
Predictor
Coefficient
Std. error
P
Constant
Mass
Sp. size
81.096
- 0.1018
7.2483
35.628
0.0704
4.3255
0.035
0.166
0.111
N = 2 2 , RZ =0.080, P=0.171
Results of multiple regression analyses of two measures of female fitness on female mass at emergence and spermatophore
size (a dummy variable was used to indicate small or large
spermatophore). Neither predictor had a significant effect on
lifetime fecundity or female lifespan
both in this study and by Pliske (1975). It is likely
that these females were forced to mate, although
initiations of all relevant matings were not observed. Thus, the cost is actually one of over-mating, and not of mating per se.
Effects of spermatophore material
on female fecundity
1987 data on fecundity and lifespan (Table 2) were
analyzed using linear regression models, with treatment and mass at emergence as independent variables, and lifetime fecundity and lifespan as dependent variables. Mass was included in the models
because of the common finding that fecundity in
insects is correlated with mass (Suzuki 1978; Le-
derhouse 1981 give data on Lepidoptera). Spermatophore size treatments (small or large) were represented with d u m m y variables. There were no significant effects in either regression (Table 3).
Females could benefit by mating with a virgin
male if they do not receive sufficient sperm from
a previously-mated male to fertilize all eggs they
could lay over their life. There is evidence that
recently-mated male Lepidoptera transfer fewer
sperm (Sims 1979), and visual comparisons of the
amount of sperm in females' spermathecae indicate
that this is true in monarchs (personal observations). However, mean egg fertilities in the two
treatment groups were not significantly different
(Table2, t=1.79, 21 df, P=0.088), and both
groups of females were laying fertile eggs at the
end of their lives.
Lifetime fecundity and lifespan data from 1988
(Table 2) were first tested with two-way analyses
of variance to determine the effects of mating and
feed treatments (Table 4a and b). There were no
significant effects due to feed treatment or the interaction between feed and mating treatments in
either ANOVA, but mating treatment did affect
fecundity, with multiply-mating females laying
more eggs. Sample sizes are less than the original
nine per treatment because four females died fewer
than four days after their first mating, when mating treatment could have had no effect, three laid
no eggs during the entire experiment, and one in
the multiple-mating treatment only mated once,
despite having additional males put into her cage.
These females were not included in any analyses.
Two females escaped and one drowned during the
experiment; these were included in daily analyses
before they were gone (see below), but not in analyses of lifetime fecundity or lifespan.
Three females in the single-mating treatment
in 1988 died before age 18 days, and as a result
had low lifetime fecundity. To remove any undue
influence of their early mortality, I used a repeated
242
Table 4. Analyses of variance for fecundity and lifespan, 1988
60
a Lifetime fecundity
50
Source
Feed
Mate
Feed* mate
Feed* mate* rep
(error)
DF
1
1
1
21
SS
164600
294070
19423
775480
F
4.46
7.96
0.53
P
Z _
I
40
0.057
0.010
0.476
L
30
20
o~
r
b Lifespan
Source
DF
SS
F
P
0
,
1
Feed
Mate
Feed* mate
Feed* mate* rep
(error)
1
1
1
21
206.53
14.529
121.09
2075.1
2.09
0.15
1.23
0.163
0.705
0.281
J
3
4
5
6
7
8
9
Three Day Period
Fig. 5. The mean number of eggs laid per day averaged over
three day time periods for females that mated once (cross-hatching) or were allowed to mate multiply (open bars). Bars represent one standard error
c Repeated measures A N O V A for daily fecundity
Source
Mate
Time
Mate* time
M a t e * time* rep
(error)
DF
1
4
4
110
SS
F
3048.7
9386.4
783.76
32175
10.42
8.02
0.67
P
0.002
<0.001
0.617
Two way A N O V A ' s of female fecundity and lifespan, a W h e n
females were allowed to mate multiply, they had higher lifetime
fecundity t h a n females that mated once. Neither feed treatment
nor the interaction between feed and mating treatments affected
lifetime fecundity significantly, b There were no significant effects in the A N O V A of lifespan, c Females allowed to mate
multiply had higher daily fecundities than those that mated
once. The significant effect of time is a reflection of decreasing
fecundity over female lifespan (see Fig. 5)
sampling A N O V A model to test the effect of mating treatment on daily instead of lifetime egg totals
(Table 4c). There was a significant mating effect
even when the influence of lifespan was removed
from the analysis. In this model, single- and multiple-mating were whole-plot treatments. Subplot
treatments were three-day time periods in which
each observation consisted of the mean number
of eggs per day laid by a female during that time
period (Fig. 5 shows mean values during each time
period for females in both treatments). I compared
egg totals over three-day periods to even out daily
weather effects, but the results of the analysis were
the same when one- or two-day time periods were
used. During the first time period no difference
is expected, because females in the multiple-mating
treatment were only allowed to remate three days
after their initial mating. After the sixth time period sample sizes in each mating treatment group
were so small due to female mortality (n < 9 in
all cases) that the A N O V A became too unbalanced
to analyze meaningfully. Because egg totals were
low during the last three time periods, this should
not affect the results. Thus the analysis only included time periods 2 through 6 shown in Fig. 5.
Females within the multiple-mating treatment
mated different numbers of times; four mated
twice, seven mated three times and two mated five
times. The number of matings within this treatment did not affect female fecundity or lifespan
(Kruskall-Wallis analysis of variance; fecundity
test statistic = 4.208, P = 0.240; lifespan test staffstic = 4.210, P = 0.239).
To determine whether feed treatments affected
females, I compared female masses in the two feed
treatment groups over the course of the experiment
(Fig. 6). On the first weighing after the feeding regimes were initiated, masses were no different, but
in all-subsequent weighings females receiving the
lower honey concentration had lower mean
masses. The differences from 31 July-12 August
are all significant at the 0.05 level (t-tests).
Finally, I compared fertility in single- and multiply-mated females (Table 2). As in the case of
females that had received small and large spermatophores, there were no significant differences (t =
- 0 . 6 5 , df=23, P=0.261). Both groups were laying fertile eggs at the end of the experiment, indicating that singly-mated females did not run out
of sperm.
243
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Fig. 6. Mean masses of females (in grams) receiving 15% (open
bars) and 30% (cross-hatching) honey solutions. Feeding regimes were initiated the day after females' first matings,
25-27 July 1988. Bars represent one standard error. There were
no differences in the two groups at eclosion (eel.), but on eight
out of nine weighings after initiation of feed treatments, females
receiving i5% honey had lower masses. The differences from
31 July-12 August are individually significant (t tests, P < 0.05)
Discussion
Cost of spermatophore production to males
and effects on strength of sexual selection
Because lepidopteran spermatophores have the potential to increase offspring quantity or quality and
to decrease future male success, they are classified
as either parental investment (Trivets 1972; Low
1978) or nonpromiscuous mating effort (Gwynne
1984). This type of male reproductive effort decreases the difference between the sexes in investment that benefits the offspring, and could therefore decrease the strength of sexual selection on
males (Trivets 1972). Although I did not measure
variance in male reproductive success per se and
more direct measures of the strength of sexual selection are needed, the distribution of male mating
success (Fig. I a) suggests that there is at least the
potential for strong sexual selection on male monarchs. This apparent contradiction of theory indicates that it is important to learn the magnitude
of the cost of nonpromiscuous mating effort.
There are several possible costs incurred by lepidopteran males in transmitting spermatophores.
First, mating costs time. Monarchs spend several
hours in copula (commonly 16-18, Svfird and Wiklund 1988b, personal observations). However,
most of this time is during the night, when they
would not be foraging or looking for other mates.
In addition, many Lepidoptera spend much less
time in copula (e.g. Sims 1979; Rutowski 1984;
Svfird 1985; Rutowski and Gilchrist 1986; Svfird
and Wiklund 1986), and during much of the time
that monarchs are in copula males are not actually
transferring accessory gland material (Sv/ird and
Wiklund 1988b, personal observations). This suggests that prolonged mating has a function other
than just spermatophore transfer (Svgrd and Wiklund 1988b). A more important time cost is probably that required to replenish the accessory gland
materials that compose spermatophores (Johnson
1979; Svfird 1985; Svfird and Wiklund 1986; Oberhauser 1988). Although male monarchs do not delay remating until they can produce a large spermatophore (Oberhauser 1988), the success of matings
in which a small spermatophore is transferred is
likely to be low, given the effect of large spermatophores on delayed female remating. In at least one
other species, Pieris protodice, males are less persistent in courting females just after a mating (Rutowski 1979). Finally, spermatophores must have
a material cost, which could be manifested in
shorter lifespans for males that mate more often.
Three studies have looked for this effect. Shapiro
(1982) found that refrigerated male Tatochila sterodice that mated had shorter lifespans than refrigerated virgin males. Svfird (1985) found that multiply-mated male Pararge aegeria did not have
shorter lifespans than those that mated once. In
the present study, males that were exposed to females and allowed to mate several times lived as
long as those never exposed to females.
Given that there must be some material cost
to producing spermatophores, there are two likely
reasons for the negative results in Sv~ird (1985)
and the present study. Both are expected to lessen
the difference between mating and non-mating
males in reproductive expenditure. First, the costs
of nonpromiscuous mating effort could be small
relative to other metabolic costs incurred by males.
Monarch males are very active, both in mating
cages and in the wild, as they attempt to find females and secure copulations (personal observations). Although this is mating effort, it is promiscuous, and thus not expected to decrease the
strength of sexual selection on males (Trivers
1972). Because the males used by Shapiro (1982)
were refrigerated, the cost of nonpromiscuous mating effort relative to other metabolic costs was
probably much higher in his study. A second possibility that might decrease the difference in expenditure between mating and non-mating males is that
even non-mating males pay high costs of spermatophore production. All males accumulate spermato-
244
phore material in their accessory glands for at least
40 days after eclosion (Johnson 1979), and this material could be unavailable for other expenditures
even if males do not mate. Although accumulation
is faster just after a mating (Oberhauser 1988), the
material produced during this time might be
cheaper to males, because it consists of a higher
proportion of water and less protein, personal observations. If either of the above explanations is
important, it is likely that any increase in cost incurred by mating males relative to non-mating
males is small. Assuming that the variance in male
lifespan observed in this study is a good estimate
of population variance, I would have needed sample sizes of 185 males in each treatment to detect
a difference in lifespan of one day (Type 2 error
rate = 0.1).
Both of the above factors could have been responsible for the lack of lifespan differences between mating and non-mating males. However, the
potential for strong sexual selection on male monarchs suggests that the first is at least partly responsible; relative to other metabolic costs, nonpromiscuous mating effort is not very costly to
male monarchs. The second possibility, that mating and non-mating males pay similar costs of spermatophore production, should not by itself affect
the relative expenditure by males and females on
offspring, and is thus not expected to affect the
strength of sexual selection on males.
Benefits of spermatophores to males and females
Since most Lepidoptera show a pattern of sperm
precedence of the last male to mate (Drummond
1984), males gain fertilizations by delaying subsequent matings as long as possible. Other researchers have reported evidence that large spermatophores delay female remating in the Lepidoptera
(Sugarawa 1979; Boggs 1979; Rutowski 1980; Rutowski et al. 1981). An alternative hypothesis for
the results of Boggs (1979), Rutowski (1980), and
Rutowski et al. (1981) is that previously-mated females are receptive only when they run out of viable sperm, since both sperm supply and spermatophore size decrease with time since mating, and
previously-mated males transfer fewer sperm (Sims
1979). However, female monarchs mated once to
previously-mated males laid as many fertile eggs
throughout their lives as females mated once to
virgin males (Table 2). This implies that they did
not run out of sperm.
Delayed female remating is only beneficial to
males if there is a good chance that females will
remate, but accurate estimates of female mating
frequencies are difficult to obtain. It is easy to
count all matings by captive butterflies, but the
unnatural conditions of captivity might affect mating frequency. Studies of female mating frequencies in the wild are done by collecting living butterflies and counting their spermatophores; it is impossible to tell if an individual would have mated
again had she not been collected. The mean mating
frequency for female monarchs in this study (3.5,
Fig. 4) is slightly lower than that found by Pliske
(1973) in wild females in the most worn category
(4.3), and much lower than that reported by Suzuki
and Zalucki (1986) in their most worn category
(7.3). Lower mating frequencies in my study could
be explained by the fact that females received
larger spermatophores in the female remating experiment than they would in the wild, since I used
no recently-mated males. This should increase the
time between female matings, and thus decrease
the total number of matings. All of these studies
suggest that males should "expect" that females
will remate, and would benefit by delaying this
remating as long as possible.
While there is little disagreement on the effect
of spermatophores on delaying female remating,
their effect on female fecundity is controversial.
In a study by Rutowski et al. (1987), Colias eurytheme females that received small spermatophores
laid fewer eggs than those that received large ones.
Watanabe (1988) showed that singly-mated female
Papilio xuthus laid fewer eggs than multiply-mated
females. However, other studies (Greenfield 1982;
Jones etal. 1986), including one on monarchs
(Sv/ird and Wiklund 1988a) have not shown this
effect. Sv~rd and Wiklund (1988a) also compared
fecundity in multiply- and singly-mated females.
The difference in our results could be explained
by the fact that the present experiment was done
under ambient conditions, whereas their study was
done under controlled laboratory conditions, as
were those of Greenfield (1982) and Jones et al.
(1986). 1988 was an unusually hot and dry summer, and heat stress probably contributed to the
early deaths and lack of egg-laying of some of my
females. The weather could also have contributed
to the low average fecundities observed in 1988
(Table2). As Sv/ird and Wiklund (1988a) suggested, harsh environments might cause spermatophore-derived nutrients to assume more importance to females. Since I found no effect of low
quality diet on female fecundity, harsh weather
conditions may be more stressful than low adult
food quality. The lack of correlation between mate
number and fecundity within the multiply-mating
group of females might indicate that my sample
245
size was too small to detect this effect, or that
females are finely-tuned to accept the amount of
spermatophore nutrients that will maximize their
individual fecundity.
Female multiple-mating
It is likely that female Lepidoptera in at least some
species benefit from spermatophore-derived nutrients, and that this benefit helps to explain the
frequency of multiple-mating among females in the
order. All published studies that have followed
spermatophore nutrients after mating have shown
that they are digested and used in female tissue
(Boggs and Gilbert 1979; Marshall 1980; Boggs
1982; Boggs and Watt 1981; Greenfield 1982). It
is possible that females in benign conditions do
not benefit from the nutrients, but it is more likely
that the benefit under such conditions is too small
to be measurable with feasible sample sizes. If spermatophore-derived nutrients are ever useful to female Lepidoptera, they may have evolved to use
the amount of spermatophore material in their
bursa copulatrix as a cue to tell them when to
remate, avoiding copulations when they would be
detrimental, but mating if the material is depleted.
Male-female conflict?
Rutowski et al. suggested that the two effects of
spermatophores (increased female fecundity and
delayed remating) have "coevolved so that the effect of the secretions on a female's behavior maximizes the male's genetic return on nutrients passed
to the female" (1987, p. 321). I think it is more
likely that these two effects are conflicting (see also
Boggs 1981; Drummond 1984); a spermatophore
that has been broken down will not be as effective
at delaying remating. Evidence from these and
other experiments suggests that the benefit of delaying female remating in those species in which
it exists is more important to males. If a large spermatophore delays remating by even a single extra
day, the male gains paternity of all of the eggs
the female lays in that day. Even though multiplemating increased lifetime fecundity in this study
(Tables 2 and 4), the benefit to an individual male
is only the difference in female fecundity caused
by his spermatophore in the time between his mating and her next mating. The fact that I could
not detect a difference in fecundity when females
received either one large or one small spermatophore (Tables 2 and 3), suggests that the benefit
to males of increased female fecundity is small relative to the benefit of delayed remating. I think
that it is more likely that the primary function
(sensu Williams 1966) of large spermatophores is
to delay female remating, but that females have
evolved to take advantage of the male investment
by digesting and using its constituents. Conflict
between the sexes on how spermatophores should
be used is likely, with females benefiting most by
digesting their contents as rapidly as possible, and
males by delaying their breakdown.
Acknowledgements. I thank T. Carlson, W. Herman, P. Oberhauser, S. Oberhauser, B. Sharp, P. Van Meter, and especially
Don Alstad for help and advice during various stages of this
work. P. Abrams, D. Alstad, C. Boggs, D.G. Brown, J. Curtsinger, and C. Wiklund suggested useful changes in earlier versions of the manuscript. Research was supported by an NSF
doctoral dissertation improvement grant (BSR 8805884), a University of Minnesota Doctoral Dissertation Research Grant,
and the Dayton and Wilkie Funds for the Study of Natural
History, administered by the Bell Museum of Natural History
at the University of Minnesota.
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