Anita. Behav., 1988, 36, 1384-1388
Male monarch butterfly spermatophore mass and mating strategies
K A R E N S. O B E R H A U S E R
Department of Ecology and Behavioral Biology, 109 Zoology, University of Minnesota, Minneapolis,
Minnesota 55455, U.S.A.
Abstract. Male monarch butterflies, Danaus plexippus, produce a spermatophore which can represent
approximately 10% of their body mass. Spermatophore mass increased with age in virgin males, and with
the time since last mating in non-virginmales. Male monarchs did not delay re-mating until they were able
to produce a large spermatophore. Recently mated males were as likely as virgins to copulate with both
virgin and non-virgin females. Monarchs provide an example of Bateman's principle, mating whenever
possible, despite the non-trivial cost involved.
Bateman's (1948) study of reproductive success in
Drosophila melanogaster showed that male reproductive success was correlated positively with the
number of copulations they obtained. His study
provided a quantitative example of Darwin's
(1871) suggestion that males maximize their fitness
by copulating as often as possible. However, there
are exceptions to Bateman's principle. In species
whose females mate synchronously, it may be
impossible for males to show a high degree of
polygyny (Knowlton 1979). In other species, it may
be in the male's best interest to be monogamous if
he can provide parental care that substantially
increases offspring survival, or if guarding a mate
prevents her from copulating with another male
(Emlen & Oring 1977). A third category of exceptions may occur in polygynous species in which
males delay re-mating to increase the expected
number of offspring they gain from each mating.
Bateman's principle is based on the assumption
that males incur little or no cost in ejaculate
production, but mating success in some species is
affected by the transfer of substances that must be
replenished after mating. Therefore, selection
might favour males that wait between rantings until
the amount of material available for transfer to the
female ensures paternity of more of her offspring
(Dewsbury 1982).
In this paper I report a study of re-mating
strategies in captive monarch butterflies, Danaus
plexippus. Male monarchs, like all Lepidoptera,
produce spermatophores consisting of a sperm sac
embedded in a gelatinous body formed from
accessory gland secretions. Several aspects of
monarch biology suggest that males may increase
their reproductive success by delaying re-mating:
spermatophore size increases with the time elapsed
since the last mating by a male (see below),
monarchs are highly polyandrous (Pliske 1973;
personal observation), and large spermatophores
delay female re-mating (Oberhauser, unpublished
data). Thus by waiting to re-mate, males might
increase the number of ova from each mate that are
fertilized by their sperm, thereby violating Bateman's principle.
METHODS
The study animals were offspring of five to seven
wild females captured in Wisconsin and Minnesota
in late May and June 1985-1987. These females
were kept in oviposition cages (65 x 65 x 65 em)
with host plants, Asclepias syriaea, and their eggs
were collected daily. Larvae were reared in outdoor
cages and fed on host plants daily until pupation.
On the morning following adult eclosion (when
they had dried and were able to fly), the butterflies
were individually numbered, weighed, fed and
stored in glassine envelopes measuring 10 x 12 cm.
Butterflies kept in envelopes were fed to satiation
on a 30% honey solution every other morning, or
daily during experiments when they flew in outdoor
cages. In 1985, butterflies were kept at 25 ° on an
LD 16:8 photoperiod. In 1986 and 1987 they were
kept at 20-25 ° near a window, where they experienced natural daylength. There was no evidence
that these two regimes affected the physiological or
behavioural characteristics under study.
Mating experiments were carried out in large
outdoor screen cages, measuring 2 x 2 x 2 m and
2 x 4 × 2 m (width x length x height). From 30 to 45
individuals at a time were introduced into these
1384
Oberhauser. Monarch butterfly mating
cages, in sex ratios appropriate to particular experiments. The butterflies were released into the cages
between 0900 and 1100 hours and were removed at
dawn on the following morning. The cages were
monitored for mating pairs every half hour until
dark. Since monarchs remain in copula for several
hours, and do not initiate matings at night (personal observation), it is unlikely that matings were
missed.
To determine spermatophore mass, mated
females were dissected in insect saline soon after
removal from the mating cage. Spermatophores
were dissected from the bursa copulatrix, blotted to
uniform dryness, and weighed to the nearest 0"01
mg on a Mettler semi-micro analytical balance.
Most copulations end early in the morning (usually
between 0200 and 0600 hours, personal observation), so females were dissected from 3 to 8 h after
copulation ended.
Two experiments were performed to determine
whether males delay re-mating until they can
produce larger spermatopores. First, mating propensity was compared in males with different
mating histories. Virgin males and males that had
mated from 1 to 8 days previously were exposed to
virgin females, and all matings were recorded. In
each cage 18-20 females and 22-25 males were
used. Since weather affects mating probability
(personal observation), I used males with many
different mating histories simultaneously to even
out weather effects. These experiments were carried
out in 1985 (on 36 different days) and 1986 (23
days).
In 1987, I did a second mating experiment to
determine whether recently mated males were less
likely than virgins to mate with recently mated and
presumably reluctant females, as suggested by
Rutowski (1979) in his study of Pierisprotodice. On
day 1, 40 virgin males and females were released
into mating cages. Mated individuals from day I
(30 pairs) and 30 virgin males and 30 virgin females
were assigned to four pairwise treatments 2 days
later (day 3): 15 non-virgin males confined with 15
non-virgin females, 15 non-virgin males with 15
virgin females, 15 virgin males with 15 non-virgin.
females, and 15 virgin males with ! 5 virgin females.
All matings were recorded. Butterflies in each
treatment were confined in separate 2 x 2 m cages
in an open field, such that none was shaded at any
time during the experiment. Butterflies were
assigned to treatments randomly, and all were 6 + 1
days old on day 1. Those that had been in cages on
1385
day 1 and had not mated were not used again on
day 3. Data were examined using a loglinear
analysis (Fienberg 1985), in which the dimensions
of a three-dimensional contingency table were male
(virgin or non-virgin), female (virgin or non-virgin)
and mated (yes or no).
RESULTS
S~rmatophore
Mass:
Virgin
Males
Male age at mating (in days since eclosion)
and mass at emergence both had significant positive effects on spermatophore mass of virgin
males (multiple regression coefficients ( + 1 SE):
age= 1"855___0"149, P<0.001; m a s s = 0 . 0 4 8 _
0.013, P<0.001; R2=0.722; overall F=86.57,
P < 0-001; N = 67). The effect of male age, the most
important predictor, is illustrated in Fig. 1a. Adult
males weighed 539 + 48 mg (mean + I SE; N = 276) 1
day after eclosion. Spermatophore mass ranged
from about 25 mg in 4-day-old males (younger
6O
55
(o)
50-
4O
35
3O
2520
E 1510 3 i
6
2
I I ; i I I I
5
7
9
II
7
3
I I I I r I I
13 15
17
19
Mole age (days)
45
4O _ ( b )
o~ 3 5 30-
2
25
2O
,5
/I
27 23
/I
I05
~25 44
J
0
I
J
I
I
I
r
2
4
6
Time since last mating (doys)
I
8
Figure 1. Spermatophore weights (wt) plotted against
against their most important predictors. Bars indicate 1 SE
and numbers give sample sizes. Data are from 1985. (a)
Virgin males. (b) Previously mated males.
Animal Behaviour, 36, 5
1386
Table I. Copulations by males with different
mating histories to virgin females
1985
1986
Time since
last mating Number
%
Number
%
(days)
tested Mated tested Mated
0*
1
2
3
4
5
6
7
8
396
49
88
52
40
29
19
21
8
33
57
65
56
63
48
37
62
38
489
36
36
27
42
32
38
27
24
30
31
56
30
40
44
29
30
42
* Virgins.
males did not mate) to about 50 mg in males over 2
weeks old (Fig. la). Thus, spermatophores from
virgin males represent investments of roughly 510% of body mass.
Spermatophore Mass: Previously Mated Males
Three factors had significant effects on spermatophore mass of previously mated males: the
amount of time that had elapsed since the last
mating (in days, log-transformed to linearize data),
the number of previous matings, and mass at
emergence (multiple regression coefficients ( _ !
SE): log time since last mating=26.788_+_l.558,
P < 0-001; number of matings = - 1.479 + 0.325,
P<0-001; mass=0-032+0.010, P<0-001; R2=
0.858; overall F=123-7, P<0.001; N=62). The
effect of time since last mating, the most important
predictor, is illustrated in Fig. lb. The very small
spermatophore mass of males that mated on 2
consecutive days consisted mainly of sperm and
sperm sac, with little or no accessory gland material.
Male Re-matlng
Table I summarizes the results of the first
experiment designed to determine whether males
delay re-mating. A lower percentage of males
mated in 1986 than in 1985 (paired t=3-3, df=8,
P=0-011) because it was an unusually cool summer and fewer matings occur on cold days (personal observation). In neither year did recently mated
males mate less than males with other mating
histories. A chi-squared analysis of the 1985 data
revealed a significant deviation from the null
hypothesis that mating history has no effect on
likelihood to mate (X2= 51-84, df= 8, P < 0.001),
but observed matings by males that had mated 1
and 2 days previously were higher than expected. A
chi-squared test of the 1986 data was not significant
at the 0"05 level of confidence (Z2= 14.43, df=8,
P=0.071).
Male Re-mating and Female Mating History
Thirteen of 15 possible pairs mated in the two
treatments in which virgin and non-virgin males
were combined with virgin females. Two previously
mated females re-mated to virgin males, and four to
non-virgin males. The best loglinear model for
these data is one that includes an interaction
between female type and mating, and none between
male type and mating (G2= 0.85, df= 2, P = 0-659).
The Ioglinear test compares models to find the one
that best fits a data set. Therefore, a low G2 and
high P correspond to the best model. Non-virgin
females were more reluctant to mate than were
virgins, but the two groups of males showed no
differences in propensity to mate.
DISCUSSION
Two interesting features of monarch mating biology are illustrated by this study. (1) Males incur a
non-trivial cost in mating, producing spermatophores that can represent up to 10% of their body
mass. It took males in this experiment approximately 5 days to replenish the accessory gland material
used to make spermatophores. This pattern is
consistent with that reported in several other
Lepidoptera (Rutowski 1979, 1984; Sims 1979;
Boggs 1981; Svard 1985; Rutowski & Gilchrist
1986; Sv/ird & Wiklund 1986). (2) Males do not
delay re-mating until they can produce large spermatophores. This was true whether they were
exposed to virgin or to non-virgin females.
Two aspects of my study make the second
conclusion slightly tentative, and must be considered. It is possible that high butterfly densities or
some other feature of the experiments made males
more likely to mate. Since sample size and space
considerations made lower densities impractical
and the experiments could not be carried out in the
wild, I have assumed the propensity to mate is
similar in the wild and captivity. Second, I com-
Oberhauser: Monarch butterfly mating
pared males that had mated 2 days earlier to virgins
when testing whether female mating history affects
males' propensity to re-mate. At this time, accessory gland material is partially replenished (Fig. I b).
It is possible that males that had mated I day earlier
would have been less likely than virgins to mate
with reluctant females. The most conservative
conclusions that can be drawn from both sets of remating data are: (1) males that produce the smallest
spermatophores (those that mated 1 day previously, Fig. 1) are as likely to mate with virgin
females as males that can produce much larger
spermatophores (Table I), and (2) males that mated
2 days previously are as likely to mate with
reluctant females as are virgin males, and very
likely to mate with virgin females (Table I), despite
the fact that these males produce smaller spermatophores than all but males mated 1 day previously
(Fig. la, b).
Boggs (1981) suggested two ways in which
butterfly spermatophores may benefit males. They
may represent a parental investment, since spermatophore constituents are incorporated in ovaries,
eggs and female somatic tissue (Boggs & Gilbert
1979; Marshall 1980, cited in Marshall 1982; Boggs
1981; Boggs & Watt 1981; Greenfield 1982). However, in polyandrous species such as the monarch it
is not clear that males always benefit by providing
these nutrients, since they may increase the fitness
of other males' progeny (but see Rutowski et al.
1987). The second benefit males may gain by
providing large spermatophores is an increase in
the time before female re-mating. Studies of sperm
precedence in Lepidoptera indicate that the last
male to mate fertilizes most subsequent eggs
(Drummond 1984). Thus, males of polyandrous
species increase their fitness by delaying female remating. Studies of several Lepidoptera (Labine
1964; Boggs 1979; Sugawara 1979; Rutowski 1980;
Rutowski et al. 1981), including monarchs (Oberhauser, unpublished data), have indicated that
larger spermatophores delay female re-mating.
Sv/ird (1985) found lower investment in the monandrous Pararge aegeria than in polyandrous butterflies, arguing that males do not invest as heavily
when there is no advantage to delayed re-mating.
An explanation of the male monarch strategy of
copulating whenever possible can be sought using a
cost-benefit analysis. If a recently mated male
encounters a female, he could attempt to copulate
with her, or wait until he is able to contribute a
larger spermatophore to a future mate. Copulating
1387
with the first female would deplete the small
amount of accessory gland material available, and
may not provide him with many offspring if the
female re-mates after a short time. Waiting could
benefit a male by increasing the number of inseminations gained in a future mating, since a larger
spermatophore should increase the time before this
female re-mates. However, giving up an opportunity to mate in favour of allowing accessory gland
material to be replenished would be a costly
strategy if the male is unlikely to find another mate.
While it is difficult to quantify benefits and costs of
delaying re-mating, their magnitude depends on
several factors, including the effectiveness of large
spermatophores in delaying female re-mating, the
chances of encountering potential mates, the condition of the female encountered by the recently
mated male, and the male's age and condition. If
the conclusions suggested by this study are representative of wild monarch behaviour, it may be that
the chances of obtaining copulations are low
enough that it is always best for a male to mate
whenever he has the opportunity. Low summer
population densities of monarchs may make this
true, especially since Asclepias sp. are characteristically in disturbed habitats, suggesting that summer
densities in which monarchs evolved may have
been even lower.
Dewsbury (1982, page 603) suggested that selection pressures associated with costly ejaculates and
sperm competition should lead males 'not to
inseminate as many females as possible but to
ensure that the number of ejaculates with each
female ensures effective paternity'. While
monarchs deliver only a single spermatophore
during mating, this argument might be extended to
include ejaculate size as well as number, since large
ejaculates should correlate with paternity of more
offspring from each female. The monarch strategy
of mating whenever possible appears to be more
consistent with the viewpoints of Darwin (1871)
and Bateman (1948).
ACKNOWLEDGMENTS
I thank O. R. Taylor for provoking my interest in
butterfly sexual selection, and for suggesting the
monarch as a good study species. P. Abrams, D.
Alstad, D. G. Brown, J. Curtsinger, B. Edinger, W.
Herman, D. F. McKinney, R. Rutowski, O. R.
Taylor, C. Sun and one anonymous reviewer
1388
Animal Behaviour, 36, 5
provided advice on the research a n d / o r m a n u script, a n d D. Alstad, W. H e r m a n , P. O b e r h a u s e r ,
S. O b e r h a u s e r a n d Bachlan Thi Ly provided field
a n d rearing assistance. This research was supported by the James W. Wilkie F u n d for N a t u r a l
History administered by the Bell M u s e u m o f
N a t u r a l History at the University o f M i n n e s o t a .
REFERENCES
Bateman, A. J. 1948. Intra-sexual selection in Drosophila.
Heredity, 2, 349-368.
Boggs, C. L. 1979. Resource allocation and reproductive
strategies in several heliconiine butterfly species. Ph. D.
thesis, University of Texas, Austin.
Boggs, C. L. 1981. Selection pressures affecting male
nutrient investment at mating in Heliconiine butterflies. Evolution, 35, 931-940.
Boggs, C. L. & Gilbert, L. E., 1979. Male contribution to
egg production in butterflies: evidence for transfer of
nutrients at mating. Science, N.Y., 206, 83-84.
Boggs, C. L. & Watt, W. W. 1981. Population structure of
Pierid butterflies IV. Genetic and physiological investment in offspring by male Colias. Oecologia (Berl.), 50,
320-324.
Darwin, C. 1871. The Descent of Man, and Selection in
Relation to Sex. London: John Murray.
Dewsbury, D. A. 1982. Ejaculate cost and male choice.
Am. Nat., 119, 601-610.
Drummond, B. A., III. 1984. Multiple mating and sperm
competition in the Lepidoptera. In: Sperm Competition
and the Evolution of Animal Mating Systems (Ed. by R.
L. Smith), pp. 291-371. New York: Academic Press.
Emlen, S. T. & Oring, L. W. 1977. Ecology, sexual
selection, and the evolution of mating systems. Science,
N.Y., 197, 215-223.
Fienberg, S. E. 1985. The Analysis of Cross-Classified
Categorical Data. Cambridge, Massachusetts: The
MIT Press.
Greenfield, M. D. 1982. The question of paternal investment in Lepidoptera: male-contributed proteins in
Plodia interpunctella. Int. ,I. Invert. Reprod., 5, 323 330.
Knowlton, N. 1979. Reproductive synchrony, parental
investment and evolutionary dynamics of sexual selection. Anim. Behav., 27, 1022-1033.
Labine, P. A. 1964. Population biology of the butterfly
Euphydryas editha. I. Barriers to multiple inseminations. Evolution, 18, 335-336.
Marshall, L. D. 1980. Paternal investment in Colias
philodice-eurytheme butterflies (Lepidoptera: Pieridae).
M.S. thesis, Arizona State University, Tempe.
Marshall, L. D. 1982. Male nutrient investment in the
lepidoptera: what nutrients should males invest? Am.
Nat., 120, 273-279.
Pliske, T. E. 1973. Factors determining mating frequencies in some New World butterflies and skippers. Ann
Entomol. Soc. Am., 66, 164-169.
Rutowski, R. L. 1979. The butterfly as an honest
salesman. Anita. Behav., 27, 1269-1270.
Rutowski, R. L. 1980. Courtship solicitation by females
of the checkered white butterfly, Pieris protodice.
Behav. Ecol. Sociobiol., 7, 113 117.
Rutowski, R. L. 1984. Production and use of secretions
passed by males at copulation in Pieris protodice
(Lepidoptera, Pieridae). Psyche, 91, 141-152.
Rutowski, R. L. & Gilchrist, G. W. 1986. Copulation in
Colias eurytheme (Lepidoptera: Pieridae): patterns and
frequency. J. Zool., 207, 115 124.
Rutowski, R. L., Gilchrist, G. W. & Terkanian, B. 1987.
Female butterflies mated with recently mated males
show reduced reproductive output. Behav. Ecol. Sociobiol., 20, 319-322.
Rutowski, R. L., Long, C. E., Marshall, L. D. & Vetter,
R. S. 1981. Courtship solicitation by Colias females.
Am. Midl. Nat., 105, 334-340.
Sims, S. R. 1979. Aspects of mating frequency and
reproductive maturity in Papilio zelicaon. Am. Midl.
Nat., 102, 36-50.
Sugawara, P. 1979. Stretch reception in the bursa copulatrix of the butterfly, Pieris rapae crucivora, and its role
in behaviour. J. comp. Physiol., 130, 191-199.
Sv/ird, L. 1985. Paternal investment in a monandrous
butterfly, Pararge aegeria. Oikos, 45, 66-70.
Sv~ird, L. & Wiklund, C. 1986. Different ejaculate
delivery strategies in first versus subsequent matings in
the swallowtail butterfly Papilio machaon L. Behav.
Ecol. Sociobiol., 18, 325 330.
(Received 31 August 1987; revised 19 November 1987;
MS. number: A5116)