Behavioral Ecology Vol. 13 No. 4: 511-518
© 2002 International Society for Behavioral Ecology
Male traits under cryptic female choice in the spotted cucumber beetle (Coleoptera: Chrysomelidae)
Delaware Agricultural Experiment Station, Department of Entomology and Applied Ecology, College of Agriculture and Natural Resources, University of Delaware, Newark, DE 19717-1303, USA
Address correspondence to D.W. Tallamy. E-mail: dtallamy{at}udel.edu .
Received 11 January 2001; revised 2 October 2001; accepted 12 October 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Males of the spotted cucumber beetle (Diabrotica undecimpunctata howardi) rhythmically stroke females with their antennae during copulation. Males that stroke quickly have a higher probability of being accepted as a mate. We determined (1) the mechanism by which females prevent unattractive males from passing spermatophores, (2) whether antennal stroking signals to females the likelihood of receiving a nuptial gift, and (3) if other male traits in addition to stroking are subjected to sexual selection from female preference. Dissections of pairs flash-frozen in copula during and after antennal stroking showed musculature that, when contracted, folded the vaginal duct leading to the female's bursa copulatrix in a way that prevented complete penetration by the aedeagus. These muscles were always contracted while males were stroking and always relaxed after stroking had ceased. Males accepted as mates did not differ from males that failed to pass a spermatophore in either absolute or relative body weight, aedeagus length, or the amount of cucurbitacins (potential nuptial gifts) sequestered in their spermatophores. Although 99% of the beetles that came to cucurbitacin-rich Cucurbita fruits in the field were males, males that had sequestered cucurbitacins did not stroke females faster than males with no cucurbitacins, and fast-stroking males were not more likely to find and sequester cucurbitacins than were males that stroked more slowly. Males with a cucurbitacin slurry painted on their antennae had no mating advantage over controls. We conclude that females discriminate among males after copulation has begun on the basis of antennal stroking displays (or some trait correlated with stroking speed) that males perform to entice females to relax their bursal sphincter.
Key words: copulatory courtship, cryptic female choice, Diabrotica undecimpunctata, sexual selection.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Darwin's recognition of female choice as a critical component of sexual selection (Darwin, 1871
) was
followed by a century in which the relative importance of female behavior in
determining paternity was the subject of lively debate
(Huxley, 1938;
Kellogg, 1907;
Lack, 1968;
Maynard Smith, 1976;
Morgan, 1932). The ease with
which male—male competition could be observed in most animals fostered
the conclusion that it was the dominant force regulating paternity. Female
choice, in contrast, was typically subtle and difficult to distinguish from
male domination. Furthermore, females that discriminated among males only on
the basis of indirect benefits from good viability or good attractiveness
genes posed theoretical problems because of the prediction that little genetic
variance should remain in male traits under directional selection from female
mating preferences. If, in fact, males did not differ genetically,
discriminating only on the basis of obtaining good genes for their offspring
would be a wasted exercise for females (Maynard Smith,
1978,
1985;
Parker, 1984).
Two recent advances have put much of this debate to rest. There is now
abundant evidence that (1) females typically do discriminate among males, even
when their only reward for doing so is the acquisition of good genes for
offspring (Andersson, 1994;
Bradbury and Andersson, 1987;
Kirkpatrick and Ryan, 1991;
Welch et al., 1998), and (2)
there is more, rather than less, genetic variance associated with male traits
under female choice when compared to traits under natural selection
(Houle, 1992;
Pomiankowski and Møller,
1995; Wilkinson and Taper,
1999). Thus, students of sexual selection no longer ask why
females are choosy, but instead focus on how and when females exercise such
choice. One of the most important empirical contributions in recent years to
the study of sexual selection has been the recognition that female choice is
not restricted to precopulatory interactions with males as previously thought.
Indeed, the ability of females to discriminate among males during copulation,
after copulation but before fertilization, and/or after fertilization appears
to be commonplace (Birkhead et al.,
1993; Eberhard,
1991,
1996).
Discrimination during or after copulation has been called cryptic female
choice (Thornhill, 1983)
because it occurs after intromission, the event that has heretofore been
considered the hallmark of male acceptance, and is therefore usually difficult
to detect. Dozens of studies (reviewed by Eberhard,
1996,
1997) clearly show that, even
in the absence of sperm competition, intromission by no means guarantees
paternity. In fact, paternity formerly attributed to sperm competition may
more typically be controlled by female behavior, physiology, and morphology
(Birkhead et al., 1993).
Because it sequentially follows intra- and intersexual interactions that occur
before copulation, cryptic female choice has the power to alter or negate all
precopulatory sexual selection that has preceded it. As such, female choice
exercised after intromission may be the dominant form of sexual selection in
many taxa (Eberhard,
1996).
Studies of cryptic female choice, however, present empirical challenges
even more formidable than those encountered in studies of precopulatory
choice. By definition, discrimination through cryptic female choice occurs
within a female's body and is not only difficult to observe, but is even more
difficult to distinguish from male incompetence or male—male competition
via interactions among sperm (Birkhead,
1998). Aggravating these difficulties is the growing consensus
that, although they may be prone to pleiotropic effects
(Hall, 1994), the male traits
upon which cryptic female discrimination is based frequently seem to be
arbitrary, subtle courtship maneuvers performed by males in copula without any
apparent link to male vigor (Eberhard,
1993a,b,
1994;
Rodriguez, 1994b;
van der Assem and Werren,
1994; Weislo et al.,
1992). Consequently, male traits under selection from cryptic
female choice often go unrecognized. This has hampered progress in determining
how pervasive this type of female choice is and how such traits convey to
females a measure of male quality.
In previous studies we determined that spotted cucumber beetle females (Diabrotica undecimpunctata howardi Barber) cryptically discriminate among males and that male mating success is correlated with the quality of ritualized copulatory courtship (Darlington et al., manuscript submitted). Females either use the speed with which males stroke them with their antennae during copulation or a trait correlated with stroking speed as an honest signal of male genetic quality. Males that stroke quickly father offspring with a higher probability of surviving to adulthood and father sons that are more attractive to females than do males that stroke slowly (Darlington et al., manuscript submitted). All males stroke females from the initiation of copulation, but males that succeed in passing a complete spermatophore to their mates abruptly cease stroking at some point during copulation, while males that do not pass a spermatophore stroke until they dismount.
It is clear that copulatory antennal stroking is correlated with female acceptance of males, but several important questions regarding this behavior remain unanswered. In this study we examined the mating behavior of the spotted cucumber beetle in detail to determine (1) the mechanism by which females prevent unattractive males from passing spermatophores to their bursa copulatrix, (2) whether stroking signals male genetic quality only or also indicates the likelihood of receiving a tangible nuptial gift, and (3) if other male traits in addition to antennal stroking are subject to sexual selection from female choice.
Study species
The spotted cucumber beetle (hereafter SCB) is a Nearctic member of the
Luperini (Coleoptera; Chrysomelidae), a large tribe of galerucine leaf beetles
comprising more than 3950 species (Wilcox,
1972a,b).
All luperine larvae eat plant roots exclusively, but adults switch to leaves
and pollen for their nutrition (Branson
and Krysan, 1981). Despite its common name, SCB is not a cucurbit
specialist and favors plant species from the Poaceae and Fabaceae over
Cucurbitaceae for larval development. SCB and a number of New and Old World
Luperini in the subtribe Diabroticina and Aulacophorina have attracted
academic attention for more than 100 years
(Webster, 1895) because of
their pharmacophagous behavior (sensu
Boppré, 1984) toward
cucurbitacins, the bitter, tetracyclic triterpenes produced by all members of
the Cucurbitaceae (Guha and Sen,
1975). Although they are either toxic or distasteful to most
vertebrate and invertebrate herbivores
(Metcalf et al., 1980;
Nielsen et al., 1977;
Tallamy et al., 1997b;
Watt and Breyer-Brandwijk,
1962;), cucurbitacins are phagostimulants for many luperine adults
(reviewed by Metcalf, 1994;
Tallamy and Krischik, 1989)
and larvae (DeHeer and Tallamy,
1991) and have been shown to impart antibiotic
(Tallamy et al., 1998) and
antipredator (Ferguson and Metcalf,
1985) benefits to beetles that consume them. Of interest here is
that males of species or populations that do not develop as larvae on
cucurbits readily leave their host plants in search of cucurbitacin sources
(Tallamy et al., 2000). In the
case of SCB, males sequester in their spermatophores 89% of the cucurbitacins
that are not excreted. These are transferred to females during copulation
which, in turn, sequester 79% of the cucurbitacins received from males in
developing eggs (Tallamy et al.,
2000). Such behavior is ecologically similar to the
pharmacophagous response of a number of insects toward noxious pyrrolizidine
alkaloids (Boppré, 1990;
Krasnoff and Dussourd, 1989;
Pliske, 1975) and suggests
that, like the females of those species, SCB females may select mates on the
basis of their ability to find and sequester cucurbitacins in seminal nuptial
gifts.
Mating behavior
Pharmacophagous luperines are often treated as a uniform group because of
the similarities in the response of many species toward cucurbitacins
(Metcalf et al., 1980;
Tallamy and Halaweish, 1993;
Tallamy et al., 1997a).
However, preliminary reports on the mating behavior of different luperines
suggest that any similarities among them end there. For example, unlike in the
western corn rootworm (D. virgifera virgifera:
Lew and Ball, 1979), there is
no evidence for any precopulatory female choice in SCB. Males approach females
from behind and quickly mount. A typical male will clasp the lateral edges of
the female's elytra with his prothoracic and mesothoracic legs, while his
metathoracic legs are extended behind to serve as stabilizers when they drag
on the substrate. As soon as a male has secured his position, he begins to
probe with his aedeagus and often achieves partial intromission within
seconds. Simultaneously he exhibits a rhythmical behavior that we call
antennal stroking (Darlington et al., manuscript submitted); by quickly
shaking his head three times from side to side while his antennae project
forward, a courting male can alternately stroke in distinct triplets the
lateral edges of both of the female's antennae, eyes, and prolegs. Males in
copula repeatedly stroke females in this way from seconds to hours, but all
successful males (i.e., those that pass a spermatophore) stop stroking and
move both of their antennae posteriorly over their elytra where they hold them
motionless while beginning a slow, rhythmic pulsing of the abdomen.
Most females attempt to dislodge males as soon as they mount, either by
brushing against the substrate, by sweeping with their two hind legs, or by
rocking their abdomens violently from side to side. Males that persist despite
this harassment and achieve complete entry into a female's bursa copulatrix
transfer a liquid spermatophore comprising nearly 7% of male mass
(Tallamy et al., 2000).
Although SCB females that accept a complete spermatophore become unreceptive
to subsequent males almost immediately, females will copulate with up to 15
males before accepting a complete spermatophore
(Tallamy et al., 2000). Males
are highly polygynous in the laboratory; they are capable of producing a new
spermatophore every 2 days and can successfully pass up to 15 spermatophores
before death (Tallamy et al.,
2000).
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METHODS |
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Experiments were conducted on laboratory colonies that were derived from beetles collected on the University of Delaware Agricultural Experiment Station farm in Newark, Delaware, USA. Standard cucumber beetle rearing techniques were used throughout the study (Branson et al., 1988
;
Cuthbert et al., 1968;
Tallamy and Pesek, 1996), with
larvae reared on corn seedlings (Pioneer No. 3397) and adults reared on
lettuce and an artificial pollen diet
(Rose and McCabe, 1973;
BioServe No. F9760, Frenchtown, New Jersey). In experiments examining the
effects of dietary cucurbitacins on female choice and male quality, the
bitterness or degree to which beetles had sequestered cucurbitacins before
testing was manipulated by adding to or withholding from the diet slices of
bitter Cucurbita andreana fruit. Gourds of this cucurbit are
excellent sources of cucurbitacins B and D
(Halaweish and Tallamy, 1993;
Metcalf et al., 1982) and were
grown for use in this study through hand pollination of greenhouse plants.
Males were made bitter by allowing them to eat cucurbitacins for 2 days. Once
bitter, we removed males from C. andreana fruit and gave them 2
additional days to sequester cucurbitacins before being used in mating
experiments. Control diets for these studies were amended with nonbitter
zucchini slices (C. pepo). Unless otherwise stated, means ±
SEs are reported.
Recognizing female choice
To determine whether females discriminate among males after copulation has
begun, it was critical to recognize with certainty which males succeeded in
passing a complete spermatophore and which did not, without destructive
dissections of the female after copulation. We determined this by measuring
weight changes in both males and females during copulation, after adjusting
for weight loss in both sexes from defecation and dehydration during lengthy
periods of pairing (up to 6 h). To quantify the relationship between beetle
mass and weight loss during copulation, both sexes were weighed repeatedly to
the nearest 0.1 mg on a Mettler balance during simulated mating conditions.
Accurate weights of live beetles were obtained without anesthesia by confining
beetles individually in 1.8-ml Ependorf tubes during the measurement. The
tubes completely stabilized beetle movement while on the scale. After initial
weights were recorded, each beetle was placed in a 4-cm diam plastic cup
fitted with an aerated lid. All cups were then enclosed within a clear plastic
box 40 x 30 x 6 cm lined with damp paper towels and fitted with an
airtight lid. Every half hour for 10 h, each beetle was removed from its
container, reweighed, and returned to its container. Beetles typically
defecated during the first hour of confinement, after which weight loss was
from dehydration alone. For this reason a multiple regression model using
initial beetle mass and time in the mating chamber as predictors of weight
lost during copulation confinement was generated from data gathered from hours
2-10, and subsequent mating experiments were initiated only after beetles had
been confined in plastic containers for 1 h. Our regression model included
linear terms and the simple interaction term between the two predictors; its
predictive value was excellent (r2 =.9604, N =
100). Beetle sex and age had no effect on weight loss over time.
Copulations were staged in 5-cm diam plastic cups with aerated lids. Each cup was fitted with a section of pipe cleaner propped against the inner wall to provide substrate that females could use to brush off suitors if they so chose. We weighed both males and females immediately before and after copulation. The weight gained by each female during copulation plus the weight lost by the female from dehydration (estimated from the regressions described above using the initial female mass and the length of the copulation as parameters) represented the fresh weight of the passed spermatophore and thus the degree to which the copulation was successful. This was confirmed from the male's perspective by subtracting the weight lost from dehydration by each male during copulation from the total weight lost by the male during copulation.
Because dissections of recently mated females have shown that males pass
spermatophores that weigh on average 1.12 ± 0.39 mg (SD) fresh weight
(Tallamy et al., 2000), males
that increased female weight by this much were considered to have successfully
passed a complete spermatophore. We considered all males that did not increase
their mate's weight at all as having been rejected, regardless of the length
of time they copulated. Males that increased female weight by some factor less
than 1 SD below the mean mass of dissected spermatophores were considered to
have successfully passed a partial spermatophore, possibly against the
female's will.
We know from previous experiments that approximately 55% of SCB females
copulate with more than one male during their brief mating period and that
females that have accepted a spermatophore completely lose subsequent
receptivity (Tallamy et al.,
2000). To determine whether females that copulate with more than
one male accept more than one spermatophore, we paired 126 virgin 8-day-old
males with virgin 4- to 5-day-old females in plastic cups as described above.
After they copulated, we determined female weight gain as described above and
replaced the male with a new virgin male to monitor subsequent female
receptivity, regardless of whether the first male had passed a spermatophore.
If the female copulated again, her weight gain during the second copulation
was compared by paired t test to weight gained during her first
copulation.
Male traits under selection
We examined how body weight, body weight in relation to female size,
aedeagus length, and amount of sequestered cucurbitacins influence mating
success in SCB males. We also were interested in whether antennal stroking
conveys a message about the cucurbitacin load a male has sequestered at the
time of courtship or about the likelihood that a particular male is carrying
cucurbitacins. Thus, we quantified whether antennal stroking is an accurate
index of male size, male bitterness (that is, the amount of cucurbitacins
already sequestered), or a male's propensity to acquire cucurbitacins.
Finally, we asked whether males stroke a female's antennae to enhance her
ability to detect his bitterness. In all mating manipulations females were
between 3 and 5 days old and males were between 7 and 10 days old.
Morphological traits
We examined the effect of male size (body mass) and aedeagus length on
female choice by comparing these traits by t test between accepted
and rejected males. The aedeagus in SCB males is a curved structure composed
of three sclerotized sections. After surgically removing the aedeagus from
each male, an index of size was created by measuring the length of each
section with an ocular micrometer and then adding all three measurements
together. We also measured the relationship between male size and aedeagus
length to determine if these two traits are correlated.
Antennal stroking
Because males that were not dislodged by female resistance fell into two
groups, those that stroked the female throughout copulation and those that
abruptly stopped stroking, we sought to determine whether the cessation of
antennal stroking marked complete penetration by the male and the beginning of
spermatophore transfer. First, we compared by t test weight gained by
females that copulated with males that never stopped stroking during
intromission with weight gained by females whose partner stopped stroking at
some point before dismounting. We also flash-froze five copulating pairs from
each group by pouring ethanol, supercooled by dry ice, directly on copulating
pairs. This instantly fixed both sexes and permitted dissections to quantify
the degree to which males had penetrated females before and after the
cessation of stroking.
To determine the degree to which antennal stroking might provide discriminating females with a measure of male stamina, we compared by paired t test stroking rate in males during their first and tenth minute of copulation. We also examined the effect of male size on stroking rate because it can be argued that large males may be genetically superior and/or deliver to females larger nuptial gifts via their spermatophores and thus be more desirable mates than small males. We quantified the rate at which male SCBs stroke the females they are courting by high-resolution VHS video recordings of copulating pairs within 25 x 150 mm test tubes (Sony Color Video Camera—DXC-107A fitted with a Navitar 18-108 mm macrozoom F-2.5 lens; JVC-super VHS HRS5300U VCR). Resolution of behavior is excellent with this equipment, and we were able to accurately count the number of antennal strokes from male mounting until female acceptance or rejection.
Male bitterness
If the acquisition of cucurbitacins is an important component of male
mating success, males may seek cucurbitacins in the environment more
aggressively than females. We tested this hypothesis by placing five C.
andreana fruits about 5 m apart from each other in a corn field on 5
consecutive days in July 1997. Each day all SCB were aspirated off each fruit,
sexed, and released at least 5 m from the fruit on which they were collected.
We then compared the number of males at fruits to the number of females by
t test each day. To compare the sex ratio at fruits with the sex
ratio away from fruits, we calculated the sex ratio of beetles collected off
of vegetation with a sweep net at least 5 m from any fruit source on the last
day of the study.
We examined how prior cucurbitacin consumption affected male mating success when paired with females that had no prior access to cucurbitacins by comparing the time elapsed before copulation, rate of acceptance, and the length of time females evaluated males (that is, length of stroking period) when females were paired with bitter versus nonbitter males.
Relationship between stroking and male bitterness
If copulatory stroking evolved in males to signal good attractiveness or
survival genes, females accepting males that antennate properly do so to
receive the indirect benefit of such genes for their offspring (Darlington et
al., manuscript submitted). If, however, stroking in some way signals the
acquisition of cucurbitacins by the courting male, the spermatophores of such
males would provide direct phenotypic benefits to accepting females in the
form of protection from predators
(Ferguson and Metcalf, 1985)
and pathogens (Tallamy et al.,
1998). We did three experiments to determine if stroking conveys a
message to females about the state of male bitterness. First, using the video
protocol discussed above, we measured whether bitter males stroke at the same
rate as nonbitter males. Antennal strokes per minute during the first 5 min of
copulation were compared by t test between bitter and nonbitter
males. Next, we lightly painted the antennae of nonbitter males that had just
started to copulate with nonbitter females with the juice from a slice of
C. andreana fruit. We then compared the acceptance rate of these
males with that of males on whose antennae only water was painted. Finally, we
determined whether males that are able to stroke rapidly are also able to
locate and/or sequester cucurbitacins better than males that cannot stroke
rapidly by allowing males to forage for a small slice of C. andreana
fruit placed in the center of a 1-m3 screen cage. The cage was
supplied with six 10-g slices of fruit, one of which was bitter C.
andreana and the other five nonbitter C. pepo, as well as pollen
diet and water. After 48 h of exposure to the fruit, we removed all males from
the cage and fed them a nonbitter diet for 2 additional days to allow any male
that had eaten cucurbitacins to sequester them within his spermatophore. Each
male was then paired with a female and videotaped to quantify his stroking
rate during the first 5 min of copulation. Five minutes after penetration each
male was teased apart from his female and freeze-dried for later cucurbitacin
quantification. In this way males were separated from females well before any
seminal material (and thus cucurbitacins) were passed. We then regressed
stroking rate against total sequestered cucurbitacins as quantified via high
performance thin layer chromatography as per Tallamy et al.
(2000).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cryptic female choice
Female discrimination among males in copula is obvious in SCB; all females refusing a spermatophore from their first mate remained receptive to the second mate, whereas all females that accepted their first mate's spermatophore did not entice the second male to show any interest in copulation. Rejected males (i.e., those that did not terminate subsequent female receptivity) failed to inject measurable amounts of spermatophore into their mate's bursa (-0.28 ± 0.11 mg; n = 20). In contrast, males that terminated the receptivity of their mate did so by filling the bursa with spermatophore material (2.12 ± 0.10 mg; n = 20; paired t test, t = -17.61, p =.0001).
Male traits under selection
Morphological traits
Males accepted as mates (20.6 ± 0.4 mg, n = 50) did not
differ from males that failed to pass a spermatophore (21.1 ± 0.4 mg,
n = 50) in either absolute body weight (t test, t =
-0.82, p =.4134) or relative body weight (male weight in relation to
the weight of the female that was courted, 0.87 ± 0.02 for accepted
males; 0.85 ± 0.03 for rejected males; t test: t =
0.58, p =.5642). The length of the sclerotized portion of the
aedeagus was positively correlated with male weight (r2
=.25, y = 1.62x + 59.7, p =.0154) but did not differ
between accepted and rejected males (t test; t = -0.27,
p =.7880).
Antennal stroking
Several lines of evidence support our assertion (Darlington et al.,
manuscript submitted) that copulatory stroking is one trait which influences
female mating decisions in SCB. If stroking is critical to achieving complete
penetration, it should only be expressed by males that have not yet gained
access to the bursa. Thus, only males that stop stroking at some point during
copulation should pass complete spermatophores. This hypothesis was supported
by comparing female weight gain (a measure of spermatophore mass received) in
copulations during which males stroked until dismounting with weight gained by
females whose mates stopped stroking well before dismounting. Females
copulating with males that never stopped stroking gained significantly less
weight (-0.20 ± 0.08 mg, n = 31) than females whose mates held
their antennae over their elytra for an extended period (2.15 ± 0.08
mg, n = 50; t test: t = 21.34; p =.0001).
All dissections of pairs flash-frozen in copula while the male was still
stroking showed that the aedeagus had penetrated up to but not past a fold in
the vaginal duct leading to the bursa copulatrix
(Figure 1a). The fold was
maintained by several muscle strands running between the posterior end of the
bursa and the ninth tergite. Only when these muscles are relaxed by the female
does the vaginal duct straighten enough to permit penetration by the aedeagus.
In all pairs in which the male had stopped stroking, the sclerotized portions
of the aedeagus had moved past the fold in the vaginal duct and into the bursa
and the genital sac had been inflated to form the spermatophore
(Figure 1b).
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All males, regardless of the outcome of their mating attempt, stroked females significantly faster in the first minute of copulation than in the 10th minute of copulation (Figure 2; n = 16; paired t test: t = 6.2; p =.0001), suggesting that stroking exacts a measurable energetic drain on courting males. Male size, however, had no effect on stroking rate (Figure 3; n = 34; F1,32 = 0.35; p =.5586).
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Male bitterness
Over the course of 5 consecutive days, 99.0 ± 1.5% of the 56 SCB
adults collected at C. andreana fruits were males
(Table 1). The sex ratio of
beetles swept from vegetation away from bitter fruits on the 5th day of the
experiment was 57.9% male (n = 19), suggesting that these results
reflect biased male foraging rather than skewed population sex ratios.
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Despite male efforts to obtain them, having cucurbitacins sequestered within the spermatophore does not improve male mating success and may even reduce a male's competitive ability. Both the success rate (68.2% for nonbitter males; 65.0% for bitter males) and the length of time acceptable males were evaluated by females (29.5 ± 5.5 min and 42.7 ± 9.7 min for nonbitter and bitter males, respectively) were similar for bitter (n = 103) and nonbitter (n = 107) males (t test, t = 1.18, p =.2408). Bitter males, however, took marginally more time to mount females than did nonbitter males (9.0 ± 2.0 min for nonbitter males; 20.0 ± 5.1 min for bitter males; t test, t = 1.71, p =.0903).
Relationship between stroking and male bitterness
None of our experiments supported the hypothesis that antennal stroking by
courting males conveys information to females about the likelihood of
obtaining cucurbitacin nuptial gifts. Males that had sequestered cucurbitacins
(n = 23) did not stroke females during copulation any faster (67.9
± 15.6 strokes/min) than did males without cucurbitacins (68.4 ±
12.5 strokes/min, n = 23; t test: t = -0.11,
p =.9139). Males with a bitter slurry painted on their antennae
(n = 35) were not as acceptable to females (54% acceptance rate) as
were males with water applied to their antennae (n = 44; 79.5%
acceptance rate). Finally, there was no relationship between stroking speed
and total sequestered cucurbitacins in males that had the opportunity to
forage for and sequester cucurbitacins
(Figure 4; n = 26,
F1,25 = 0.17, p =.6877), confirming that
cucurbitacin consumption does not enhance copulatory stroking, nor does fast
stroking indicate a greater likelihood of locating or sequestering
cucurbitacins. The lack of a relationship between stroking speed and
cucurbitacin load was not because all males tested had sequestered large
amounts of cucurbitacins due to the high probability of locating fruits within
the test cage. There was, in fact, a great deal of variation in cucurbitacin
load among males (10.7 ± 4.8 SD µg/beetle; range = 1.8-25.1
mg/beetle).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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All of the data gathered in this study support the hypothesis that female SCBs discriminate among potential mates either on the basis of the speed of antennal stroking that males display during the early stages of copulation or on the basis of an as-yet unidentified trait that is correlated with stroking speed. Neither absolute male size, male size in relation to the size of the female he is courting, nor aedeagus length influence a female's decision to accept or reject a particular male's spermatophore. Dissections of copulating pairs flash-frozen before and after the cessation of stroking indicate that a male strokes a female with his antennae only until the sclerotized terminus of his aedeagus has moved past a constricted fold in the female's vaginal duct. Stroking appears to be a ritualized form of copulatory courtship designed to induce a female to relax the large muscles that keep the duct folded. When constricted, these can effectively prevent unattractive males from inflating their genital sac within the bursa, a prerequisite for spermatophore transfer. SCB appears to be an excellent example of Eberhard's (1997
) "selective
cooperation" by females; a female resists the advances of all males by
virtue of her bursal sphincter until one male wins her cooperation (that is,
entices her to relax the sphincter) by stroking her in an appropriate manner.
Musculature that is capable of obstructing complete penetration has been
previously noted in some scarab (Macrodactylus spp.:
Eberhard, 1993a) and
chrysomelid beetles (Macrohaltica jamaicensis:
Eberhard and Kariko, 1996;
Chelymorpha alternans: Rodriguez,
1994a), the tsetse fly (Glossina pallidipes:
Jaenson, 1979) and also in
some bees (Roig-Alsina,
1993).
Insect spermatophores can bear nutrients used by females in egg production
or body maintenance (reviewed by Vahed,
1998), but earlier measures of female performance after mating
with either fast- or slow stroking SCB males suggested that no direct
phenotypic benefits are derived by females that prefer fast males (Darlington
et al., manuscript submitted). The absence of a relationship between male size
and stroking speed supports this result. If SCB spermatophores contained
nutritional gifts that females could use directly or pass on to their
offspring, females should prefer males with the largest gifts. Presumably
these would come from the largest males, yet large males do not stroke faster
than small males and consequently have no mating advantage over them.
Although we found no evidence that SCB males pass nutritional gifts within
their spermatophores to females (Darlington et al., manuscript submitted), the
possibility remains that (1) nuptial gifts in the form of noxious
cucurbitacins influences female choice in this species
(Tallamy et al., 2000) and (2)
antennal stroking somehow conveys a message about the quality of the
cucurbitacins carried by the courting male. SCB males pass sequestered
cucurbitacins in their spermatophores to females that in turn sequester the
cucurbitacins in their eggs (Tallamy et
al., 2000) as a defense against soil predators and pathogens
(Tallamy et al., 1998). Our
field counts of SCB sex ratios at C. andreana fruits, which are rich
sources of cucurbitacins, support these hypotheses. Nearly all of the beetles
that responded to volatile cues from these fruits were males, suggesting that
females of pharmacophagous Diabrotica may rely entirely on male
spermatophores as their source of cucurbitacins. Because SCB females only
accept one spermatophore in their lifetime, it is logical that they might
judge potential mates at least in part by the quantity of their cucurbitacin
gift. This, however, does not seem to be the case.
Surprisingly, having cucurbitacins sequestered within the spermatophore or
present on the body surface does not improve male mating success. Furthermore,
bitter males do not stroke females with their antennae more rapidly than
nonbitter males, nor do more vigorous males that stroke females faster locate
and sequester more cucurbitacins. In short, there is no evidence that male
sequestration of cucurbitacins is a sexually selected trait. Instead,
male-biased cucurbitacin sequestration may be a naturally selected mechanism
for males to improve survivorship of themselves, their mates, and their
offspring. This conclusion is not without precedent. Males of the arctiid
moth, Utethesia ornatrix, also pass sequestered allelochemicals to
their mates via their spermatophores (in this case, pyrrolizidine alkaloids).
Utethesia females, however, base mate selection entirely on
spermatophore size (LaMunyon and Eisner,
1994).
Copulatory courtship through ritualized antennal stroking may not be an
arbitrary trait; our data suggest that the rapid stroking preferred by females
is physically tiring and may provide females with a reliable measure of male
endurance and, by extrapolation, male health, vigor, and even genetic quality.
Antennal stroking is a highly heritable trait and fast-stroking males do, in
fact, produce more attractive sons and both sons and daughters with higher
survivorship than slow males (Darlington et al., manuscript submitted). It is
not surprising, then, that at least seven other unrelated chrysomelid beetles
in four subfamilies other than Galerucinae [Varicoxa sp., Altica
cyanea, Disonycha quinquelineata (Alticinae;
Eberhard, 1994; Duckett C,
personal communication), Cryptocephalus cordiger (Cryptocephalinae),
Gastrophysa polygoni (Chrysomelinae;
Medvedev and Pavlov, 1987),
Donacia marginata (Donaciinae;
Medvedev and Pavlov, 1987),
and D. aguatica (Michelsen,
1963), Macrohaltica jamaicensis (Alticinae;
Eberhard and Kariko, 1996)] as
well as a number of insects in other widely divergent taxa [Vespidae
(Ross, 1983;
West-Eberhard, 1969),
Sphecidae (Coville and Coville,
1980; Hook and Matthews,
1980; Longair et al.,
1987), Anthophoridae (Alcock
and Buchmann, 1985), Halictidae
(Barrows, 1975), Curculionidae
(Le Cato and Pienkowski, 1970;
Wojcik, 1969), Brentidae
(Johnson, 1982), Tenebrionidae
(Wocjik, 1969), Cerambycidae (Michelsen,
1963), Cicindellidae, Buprestidae, Pyrrhocoridae
(Eberhard, 1994), Cantharidae
(Chauliognathus pennsylvanicus, Tallamy et al., personal
observations)] have converged upon similar means of copulatory courtship.
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ACKNOWLEDGEMENTS |
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We are grateful for the cooperation of K. Stoops (Stine-Haskell Lab, DuPont Co., Newark, Delaware), the technical assistance of K. Shropshire, the statistical advice of J. Pesek, and the helpful criticism of J. Hough-Goldstein and anonymous reviewers. This article was published as Contribution Paper No. 1712 of the Department of Entomology and Applied Ecology in the Journal Series of the Delaware Agricultural Experiment Station, University of Delaware, Newark. This research was partially supported by U.S. Department of Agriculture-NRI grant 9301684 to D.W.T.
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