Differential sperm expenditure reveals a possible role for post-copulatory sexual selection in a lekking moth.

0.87 MB·English

by  Cordes, Nils

Differential sperm expenditure reveals a possible role for
post-copulatory sexual selection in a lekking moth
Nils Cordes1, Arzu Yig(cid:1)it1,2, Leif Engqvist1 & Tim Schmoll1
1EvolutionaryBiology,BielefeldUniversity,Morgenbreede45,33615Bielefeld,Germany
2InstituteofScience,DepartmentofBiology,OrduUniversity,CumhuriyetCampus,52200Ordu,Turkey
Keywords Abstract
Achroiagrisella,eupyrenesperm,mating
Male reproductive success in the lesser wax moth Achroia grisella is strongly
history,sexualselection,spermallocation,
spermcompetition. determined by pre-copulatory mate choice, during which females choose among
males aggregated in small leks based on the attractiveness of ultrasonic songs.
Correspondence Nothing is known about the potential of post-copulatory mechanisms to affect
NilsCordes,BielefeldUniversity,Evolutionary male reproductive success. However, there is evidence that females at least
Biology,Morgenbreede45,33615Bielefeld,
occasionally remate with a second male and that males are unable to produce
Germany.Tel:[phone];
ejaculates quickly after a previous copulation. Here we investigated the effects of
Fax:[phone];E-mail:ncordes@
mating history on ejaculate size and demonstrate that the number of transferred
uni-bielefeld.de
sperm significantly decreased from first (i.e., virgin) to second (i.e., nonvirgin)
FundingInformation copulation within individual males. For males of identical age, the number of
ThisresearchwasfundedbyBielefeld sperm transferred was higher in virgin than in nonvirgin copulations, too, dem-
University. onstrating that mating history, is responsible for the decrease in sperm numbers
transferred and not the concomitant age difference. Furthermore, the number
Received:7September2012;Revised:21
of transferred sperm was significantly repeatable within males. The demon-
November2012;Accepted:27November
strated variation in ejaculate size both between subsequent copulations as well
2012
as among individuals suggests that there is allocation of a possibly limited
EcologyandEvolution2013;3(3):503–511 amount of sperm. Because female fecundity is not limited by sperm availability
in this system, post-copulatory mechanisms, in particular sperm competition,
doi:10.1002/ece3.458 may play a previously underappreciated role in the lesser wax moth mating
system.
are positively correlated and seem to reinforce each other
Introduction
(Lewis and Austad 1994; Bangham et al. 2002; Evans
Sexual selection, which is responsible for the evolution of et al. 2003; Hosken et al. 2008) whereas other studies
many male reproductive characters (Andersson 1994), can have revealed a negative relationship (Warner et al. 1995;
be separated into two major components. Pre-copulatory Danielsson 2001; Fu et al. 2001; Preston et al. 2001;
sexual selection acts on traits affecting mating success, Demary and Lewis 2007; Engqvist 2011). Very different
whereas post-copulatory sexual selection will influence the evolutionary dynamics will result depending on the sign
evolution of traits associated with male fertilization suc- of this relationship. More studies addressing sperm com-
cess under sperm competition and/or cryptic female petition in species with a well-established pre-copulatory
choice (Andersson and Simmons 2006). Variation in male component of sexual selection and vice versa are therefore
mating success caused either by female preferences or by essential in order to resolve this important evolutionary
male-male competition has long been documented, problem.
whereas inter-male variation in sperm competitiveness The lesser wax moth Achroia grisella (Fig. 1a) is a study
has been revealed more recently (Lewis and Austad 1990; organism in which so far only pre-copulatory mechanisms
Gage and Morrow 2003; Engqvist et al. 2007; Sherman of sexual selection have been investigated, although in
et al. 2009). However, male reproductive success will be great detail and very successfully. In fact, this mating
affected by both processes (Andersson and Simmons system represents a model system for studying the evolu-
2006) and the precise relationship between these episodes tionary ecology of pre-copulatory female mate choice
of sexual selection is not yet clear. In some species, they and the evolution of male secondary sexual characters
ª2013TheAuthors.PublishedbyBlackwellPublishingLtd.ThisisanopenaccessarticleunderthetermsoftheCreative 503
CommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,provided
theoriginalworkisproperlycited.
SpermExpenditureinaLekkingMoth N.Cordesetal.
(Greenfield and Coffelt 1983; Jang and Greenfield 1996, species, eupyrene spermatogenesis usually ends at pupa-
1998; Reinhold et al. 1998; Jia et al. 2001; Brandt and tion (Friedl€ander 1997) and adult males will typically
Greenfield 2004; Brandt et al. 2005; Limousin and Green- emerge with a restricted amount of fertilizing sperm.
field 2009; Zhou et al. 2011). Males gather in leks to Therefore, a relevant factor in determining the amount of
perform ultrasonic courtship songs and attract females eupyrene sperm per copulation is mating history, as
(Spangler et al. 1984). Females then choose partners on sperm reserves may become depleted with every successful
the basis of several song components (Jang and Greenfield copulation and ejaculate size may decline. This has been
1998) and allow copulation. Adult wax moths are short- shown across different taxa including Lepidoptera (Sv€ard
lived; females live 4–7 days, males approximately 14 days and Wiklund 1986; Pierce et al. 1990; Pitnick and Mar-
in the laboratory (Ku€nike 1930; Greenfield and Coffelt kow 1994; Damiens and Boivin 2005; Ro€nn et al. 2008;
1983). During this time, they neither drink nor feed Teng and Zhang 2009). This can either be interpreted as
(Greenfield and Coffelt 1983), which means that much of a constraint due to sperm usage in a recent mating, or it
their activity, e.g. male courtship behaviour (Brandt and could also be considered a strategic decision: males
Greenfield 2004), female fecundity (Danielson-Fran(cid:1)cois should invest more sperm in initial copulations because
et al. 2009), and possibly also the amount of sperm avail- each copulation can be a male’s last and conserving larger
able for copulations rely heavily on accumulated reserves amounts of sperm for future matings might be disadvan-
from larval development. Sperm is transferred in a single tageous (Reinhold et al. 2002). In any case, a reduction in
spermatophore during copulations typically taking around ejaculate size in successive matings has some important
15 min, and males resume signaling shortly after the end consequences. First, males with high mating success might
of copulation. Should they successfully attract another risk running out of an adequate sperm supply and
female soon after the initial copulation, they will occupy become ineffectual in sperm competition (Warner et al.
the female for several hours without transferring a sper- 1995; Preston et al. 2001). Because A. grisella females do
matophore, a behaviour that is hypothesized to function occasionally remate (Greenfield and Coffelt 1983),
as male pre-copulatory mate guarding (Greenfield and successful males would therefore invariably become less
Coffelt 1983). It thus seems likely that males are unable effective competitors in later copulations. Second, if
to transfer a second spermatophore immediately after the female fecundity is constrained by sperm availability, pref-
first copulation. erences for fertile males should evolve, and possibly
Lepidoptera have two types of sperm; eupyrene, nucle- females will benefit by avoiding multiply mated males. In
ate sperm used for fertilization of the eggs, and apyrene both these scenarios, any post-copulatory processes will
anucleate sperm whose function is not known but have the tendency to reduce the intensity of pre-mating
hypothesized to possibly play a role in sperm competition sexual selection. Studies on the effect of male mating
(Silberglied et al. 1984; Cook and Wedell 1999; Swallow history on ejaculate size in general, and the amount of
and Wilkinson 2002). While apyrene sperm are produced eupyrene sperm in particular, might therefore contribute
exclusively at later developmental stages and may be substantially to our understanding of sexual selection,
produced throughout the adult’s lifespan in some moth even in those mating systems where pre-copulatory
(a) (b)
Figure1. Photographs of the lesser wax moth Achroia grisella and its sperm: (a) Male lesser wax moth on honey comb (photograph by Nils
Cordes). (b) Light microscopic picture of lesser wax moth eupyrene sperm bundles as retrieved from the female reproductive tract immediately
afternaturalterminationofcopulation(4009magnification).Inthebackground,notethefilamentaryapyrenespermatozoawhichdonotcome
inbundles(photographbyArzuYig(cid:1)it).
504 ª2013TheAuthors.PublishedbyBlackwellPublishingLtd.
N.Cordesetal. SpermExpenditureinaLekkingMoth
processes are supposedly or evidently prevailing. Here we 2008); here we focused solely on eupyrene sperm. Within
therefore investigate whether A. grisella males transfer 5 min after copulation we dissected the female to collect
similar or differing amounts of eupyrene sperm in virgin the spermatophore. We chilled the female on ice for
versus subsequent nonvirgin matings, and if this potential 2 min, killed the animal by decapitation with a razor
effect is indeed a consequence of male mating history. blade and then cut open the ventral side, starting at the
anterior end and continuing along the thorax and abdo-
men until the bursa copulatrix was exposed. We excised
Materials and Methods
the bursa copulatrix containing the male spermatophore,
transferred it to a microscopic glass slide and carefully
Rearing of experimental animals
disrupted remaining tissue with a needle tip. By adding a
We obtained experimental animals from a laboratory cover slip and applying gentle pressure we evenly spread
population of A. grisella, derived from approximately 100 the contents of the spermatophore over the slide. Using
individuals that were collected from a wild population in standard light microscopy at 4009 magnification, we
bee hives near Bielefeld, Germany, in October 2009. We counted all eupyrene sperm bundles (see Fig. 1b) present
reared the moths in 30 mL plastic cups under 12:12 h on the slide twice, recording the mean value of both
light:dark photoperiod, at 25°C, 40% relative humidity, counts.
using a breeding design which minimizes inbreeding.
Larvae were supplied ad libitum with a food mixture
Statistical analysis
modified from Jang and Greenfield (1996) and consisting
of wheat, oat and maize flour (16% each), brewer’s yeast All statistical tests were two-tailed and the null hypothe-
(8%), glycerine (12%), honey (12%), water (12%) and sis was rejected at P < 0.05. We used R 2.14.1 (R Devel-
used wax combs from bee hives (8%). Experimental males opment Core Team 2011) for all computations. Linear
and females were isolated into individual cups during sec- mixed effects models (LME, R function lme in library
ond to last instar to ensure virginity. Eclosion was moni- nlme, Pinheiro et al. 2006) were used to test for (1)
tored on a daily basis and we define the age of an differences between virgin and nonvirgin copulations of
individual at the day of its eclosion to be zero days. the same males; and (2) effects of copulation duration
on sperm expenditure. Male identity was included as a
random effect to account for the non-independence of
Experimental setup
measurements obtained from the same individuals and
In a first step, we staged experimental matings between to estimate between-male variation in the number of
one-day-old virgin males and randomly chosen two- sperm bundles transferred. P values in the context of
day-old virgin females during a period of 4 h preceding LME analysis refer to the increase in model deviance
scotophase (dark photoperiod). In a second step, we re- (compared against a v2 distribution) when a term is
mated these males 24 h later to different randomly cho- removed from the current model and the significance of
sen two-day-old virgin females. We chose an interval of random effects was tested by removing it from the mini-
24 h between matings, because male wax moths are able mal adequate model after refitting this model using
to successfully transfer a spermatophore approximately restricted estimation of maximum likelihood (REML).
8 h after a previous successful copulation (Greenfield and Repeatability in the number of sperm bundles transferred
Coffelt 1983). To tease apart male mating history from was calculated as the between-male variance relative to
effects of the inevitable age difference, we finally mated total random effects variance as estimated from the
two-day-old virgin males to randomly selected two-day- minimal adequate REML LME (Nakagawa and Schielzeth
old virgin females. We weighed individuals to the nearest 2010). To obtain 95% confidence intervals for the
0.01 mg with a Kern 770 electronic scale (Kern & Sohn repeatability estimate, we used parametric bootstrapping
GmbH, Balingen, Germany) immediately before every with 10,000 iterations as described in Faraway (2006).
copulation and measured copulation duration to the Due to constraints in the availability of unrelated males,
nearest 10 sec. the total number of 49 experimental males originated
from 24 different families with on average 2.0 (cid:1) 1.5
(SD; range: one to six) brothers per family. When test-
Sperm bundle counts
ing for effects of familial background on sperm expendi-
In the lesser wax moth like in many other Lepidoptera, a ture, we found no effects in any of the main analyses
spermatophore consists of solitary apyrene sperm and (P = 0.68, 1.0 and 1.0 for the random effect of family)
eupyrene sperm aggregated in bundles (256 per bundle; and we therefore did not include the respective random
see Fig. 1b) (Fernandez-Winckler and da Cruz-Landim effects in the final models.
ª2013TheAuthors.PublishedbyBlackwellPublishingLtd. 505
SpermExpenditureinaLekkingMoth N.Cordesetal.
100
80
d
d e 80
erre 60 sferr
es transf 40 dles tran 60
bundl m bun 40
perm 20 Sper 20
S
0
0
Virgin Nonvirgin Virgin Nonvirgin
Mating history Mating history
Figure2. Within-individualdecreaseinthenumberofspermbundles Figure3. Number of sperm bundles transferred for male lesser wax
transferred from first (i.e., virgin) to second (i.e., nonvirgin) lifetime moths of identical age (2days after eclosion) during virgin (N=17)
copulation for N=28 male lesser wax moths (linear mixed effect and nonvirgin (N=28) copulations (linear model: F1,43=18.05,
model with male identity as random effect: v2=18.0, df=1, P<0.001). Box plots show medians, interquartile ranges (boxes) and
P<0.001). Virgin copulations took place on day one, nonvirgin datawithin1.5timestheinterquartileranges(whiskers).
copulationsondaytwoaftereclosion.
100
Results
d
e 80
Number of sperm bundles transferred err
sf
n
Overall, the number of eupyrene sperm bundles trans- a
ferred during copulation ranged from one to 101 with a s tr 60
e
mean ((cid:1)SD) of 36.6 (cid:1) 20.5 (N = 77 observations across ndl
male mating histories and male ages). bu 40
m
Within individual males, the number of sperm bundles er
transferred decreased from 41.7 (cid:1) 3.1 (SE) to 24.5 (cid:1) 3.1 Sp
20
fromtheirfirst(i.e.,virgin)tosecond(i.e.,nonvirgin)life-
time copulation (linear mixed effect model LME:
v2 = 18.0, df = 1, P < 0.001, Fig. 2). Conditional on the 0
effect of mating history, we found significant between- One Two
malevariationinthenumberofspermbundlestransferred Male age at virgin mating (days)
(LME: v2 = 4.1, df = 1, P = 0.04, see also Fig. 2). The
repeatability for the number of sperm bundles transferred Figure4. Number of sperm bundles transferred during virgin
was0.38witha95%confidenceintervalof0.02–0.65. copulations for male lesser wax moths one (N=32) or twodays old
(N=17) at virgin mating (linear model: F =0.68, P=0.41; note
For males of identical age (i.e., 2 days after eclosion), 1,47
that sample size for one-day-old virgin males is larger than in the
the number of sperm bundles transferred was higher in
paired test shown in Fig.2 due to four males lacking a second,
virgin (46.9 (cid:1) 4.2) than in nonvirgin copulations nonvirgin copulation). Box plots show medians, interquartile ranges
(24.5 (cid:1) 3.2; linear model LM: F = 18.05, P < 0.001, (boxes)anddatawithin1.5timestheinterquartileranges(whiskers).
1,43
Fig. 3). Furthermore, there was no difference in the num-
ber of sperm bundles transferred between virgin copula- bundles transferred between virgin and nonvirgin copula-
tions of one-day old (41.6 (cid:1) 3.8) versus two-day old tions of the same males.
males (46.9 (cid:1) 5.1; LM: F = 0.68, P = 0.41, Fig. 4), Including male or female mass prior to copulation did
1,47
indicating that indeed mating history, not the inevitable not improve any of the models presented above (all
difference in age, is responsible for the decrease in sperm P > 0.32 or P > 0.24 for the effects of male or female
506 ª2013TheAuthors.PublishedbyBlackwellPublishingLtd.
N.Cordesetal. SpermExpenditureinaLekkingMoth
mass, respectively, data not shown), indicating that sperm lations decreased by over 40% from the virgin copulation
number does not depend on male size and that males do to the subsequent (nonvirgin) copulation. The number of
not allocate sperm in response to female quality. sperm bundles varied considerably and consistently
among males with 38% of the variance attributable to dif-
ferences between individuals.
Copulation duration and sperm bundles
There are several possible explanations for the observed
transferred
decrease in transferred sperm over subsequent matings.
Overall, copulation duration ranged from 120 to 2200 sec Our experimental design allows us to exclude an effect of
with a mean ((cid:1)SD) of 898 (cid:1) 236 (N = 77 observations the age difference between virgin and nonvirgin copula-
across male mating histories and male ages). tions of the same males, because two-day-old nonvirgin
The number of sperm bundles transferred increased males transferred significantly less sperm than two-day-old
with increasing copulation duration (LME: v2 = 16.9, virgin males, while one-day-old virgin males did not dif-
df = 1, P < 0.001, solid line in Fig. 5). This effect fer from two-day-old virgin males. One could argue that
remained significant after excluding two data points with males are constrained in their ability to produce another
high leverage resulting from extreme values in copulation equally large spermatophore within the (experimentally
duration (LME: v2 = 4.2, df = 1, P = 0.04, dashed line in controlled) period of 24 h between subsequent matings.
Fig. 5). There was no evidence that the effects of copula- However, when given the opportunity, males can success-
tionduration onthenumberofspermbundlestransferred fully transfer a further spermatophore already 8 h after an
differed for virgin versus nonvirgin copulations, indicating initial copulation (Greenfield and Coffelt 1983). In the
there were no differences in sperm transfer rate (LME: present study, males had three times longer than this to
v2 = 0.24 and 0.18, df = 1, P = 0.63 and 0.67 for the manufacture another spermatophore, so we consider it
interaction between mating history and copulation dura- unlikely that time constraints are causing the patterns
tionforthefullandthereduceddataset,respectively). observed. Importantly, spermatophores transferred 8 h
after an initial copulation are sufficiently large to guaran-
tee fertilization of all of a female’s eggs (Greenfield
Discussion
and Coffelt 1983). Thus, even if time constraints should
We have demonstrated that the amount of eupyrene exist, an explanation based on such constraints alone
sperm transferred by male lesser wax moths during copu- cannot provide a satisfactory answer as to why spermato-
phores from virgin copulations are substantially larger
than those from nonvirgin copulations (and thereby
100
noticeably larger than necessary for fertilization of a
female’s eggs).
d
e 80 We argue that the detected difference is probably best
sferr explained by strategic sperm allocation. Sperm production
n
a costs and/or constraints will select for male ability to pru-
s tr 60 dently allocate sperm across matings (Wedell et al. 2002;
e
dl Parker and Pizzari 2010). However, increasing sperm
n
u
b 40 number over the level necessary for fertilization of all of a
m
er female’s eggs can only be adaptive in one of two ways.
Sp Either sperm number directly affects female fecundity
20
(e.g., Rutowski et al. 1987; Milonas et al. 2011), or sperm
number is an important factor in guaranteeing male
0 fertilization success under sperm competition. In both
these scenarios, it is advantageous to enlarge ejaculates in
0 500 1000 1500 2000 2500
Copulation duration (s) initial matings compared to subsequent ones because
males can never be certain of future mating opportunities
Figure5. Number of sperm bundles transferred in relation to (see Reinhold et al. 2002). This is especially relevant in a
copulation duration for N=77 virgin and nonvirgin copulations. The short-lived animal like the lesser wax moth. Which of
solid line represents the linear regression of the number of sperm
these scenarios is the more likely in this case? As men-
bundlesoncopulationdurationforalldatapoints(linearmixedeffect
tioned above, female fecundity in the lesser wax moth is
model with male identity as random effect: v2=16.9, df=1,
P<0.001) and the dashed line the regression when excluding the not affected by whether she mates with a virgin or a non-
twodatapointswithhighleverageresultingfromextremecopulation virgin male (Greenfield and Coffelt 1983), so the amount
durationsof120and2200sec(v2=4.2,df=1,P=0.04). of sperm transferred by nonvirgin males does not seem to
ª2013TheAuthors.PublishedbyBlackwellPublishingLtd. 507
SpermExpenditureinaLekkingMoth N.Cordesetal.
be a limiting factor for female offspring production. If, tors (Parker et al. 1996, 1997; Reinhold et al. 2002). For
however, females mate more than once, the number of eupyrene sperm this would lead to a picture similar to
offspring a male produces depends on either quantity or what we found in the lesser wax moth (Figs. 2, 3).
quality of his sperm, setting the stage for sperm competi- However, directional selection for optimal sperm invest-
tion as a major factor to influence male fitness (Parker ment should lead to a decrease in phenotypic variation
1970; Simmons 2001). Reliable data on female re-mating as well (Parker 1993), similar to the reduction in addi-
probability in natural populations of the lesser wax moth tive genetic variance expected to occur for behavioural
are still lacking, but a laboratory experiment by Green- traits from strong precopulatory sexual selection gener-
field and Coffelt (1983) demonstrated that over 10% (4 ated by female preferences (Tomkins et al. 2004). Con-
out of 30) of females remated if given the chance and, trary to these expectations, A. grisella males vary
upon dissection, contained two spermatophores – one persistently in courtship song (Jang and Greenfield
from each male. Based on these data, which at the 1998; Brandt and Greenfield 2004; Danielson-Fran(cid:1)cois
moment provide our best estimate, sperm competition et al. 2009). We demonstrate that individual male
risk (i.e., the probability that a male’s ejaculate will face moths differed considerably in their ability to transfer
competition against another male’s sperm) could be as high numbers of sperm during copulation as well, sug-
high as 20–25% (~8/34, see Parker et al. 1997). Although gesting differences in sperm competitiveness (Lewis and
these results stem from laboratory-reared populations, it Austad 1990; Simmons and Parker 1992; Dziuk 1996;
is likely that the opportunity to remate occurs in the field Radwan 1996; Gage and Morrow 2003; Engqvist et al.
as well, where males aggregate in leks in close proximity 2007; Sherman et al. 2009). Thus, the between-male dif-
to the bee hive (Greenfield and Coffelt 1983). Sperm ferences in transferred sperm bundles reported here may
competition may therefore play an important but previ- well translate into differences in male fertilization suc-
ously underappreciated role in the mating system of the cess. Whether males actually differ in sperm competi-
lesser wax moth. tiveness cannot be determined from our data, and
Transferring an excessive sperm amount could thus be thorough paternity testing is necessary to detect and
a way to outcompete other males’ sperm and could quantify the effect of sperm number on sperm competi-
explain the differences in sperm bundle number between tion success in the lesser wax moth. Nevertheless, given
subsequent matings observed here. This is the case in the ubiquitous importance of sperm number on sperm
other Lepidoptera, in which the amount of sperm avail- competition success (e.g., Parker 1982; Dickinson 1986;
able has been shown to be allocated based on female Parker et al. 1990; Eady 1995; Dziuk 1996; Simmons
quality, the level of sperm competition and, importantly, 2001; Gage and Morrow 2003; Engqvist et al. 2007),
male mating history (Sv€ard and Wiklund 1986; Cook our results suggest that attractiveness and mating suc-
and Gage 1995; Cook and Wedell 1996; Proshold 1996; cess alone may not guarantee fertilization success for
Wedell and Cook 1999; McNamara et al. 2007; Teng and some males, and that sperm competition may be a
Zhang 2009; Xu and Wang 2009). Hitherto, there have non-trivial source of variation in male lifetime repro-
been no studies on post-copulatory sexual selection in ductive success. The lesser wax moth could thus poten-
the lesser wax moth; indeed, sperm competition studies tially be a valuable study system for a better
in lekking insects are rare in general (but see Demary understanding of the interplay between pre- and post-
and Lewis 2007; South and Lewis 2012). In the lesser copulatory components of sexual selection.
wax moth, most research has been devoted to pre-
copulatory sexual selection, and between-male variation
Acknowledgments
in attractiveness is well established (Jang and Greenfield
1996, 1998; Reinhold et al. 1998; Jia et al. 2001; Brandt We would like to thank Hu¨lya O¨nal for assistance with
and Greenfield 2004; Brandt et al. 2005; Limousin and taking mass data and Klaus Reinhold and Michael Green-
Greenfield 2009; Zhou et al. 2011). It might be expected field for stimulating discussion and helpful comments on
that highly attractive males must allocate their sperm a previous version of this manuscript. We acknowledge
reserves across more matings (Warner et al. 1995; Pres- support for the Article Processing Charge by the Deutsche
ton et al. 2001; Engqvist 2011), and thus sperm limita- Forschungsgemeinschaft and the Open Access Publication
tion might affect selection on traits affecting male Funds of Bielefeld University Library.
attractiveness.
If sperm competition occurs, theory predicts that
Ethical standards
males have evolved the ability to optimize sperm pro-
duction and allocation in order to be able to transfer All experiments comply with the current laws of Ger-
the amounts needed to guarantee success over competi- many.
508 ª2013TheAuthors.PublishedbyBlackwellPublishingLtd.
N.Cordesetal. SpermExpenditureinaLekkingMoth
Conflict of Interest Engqvist,L.2011.Maleattractivenessisnegativelygenetically
associatedwithinvestmentincopulations.Behav.Ecol.
None declared. 22:345–349.
Engqvist,L.,G.Dekomien, T.Lippmann,J.T.Epplen,and
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