puet

Received 29 July 2008

idopm a

theory predicts that males should allocate these materials strategically based on the risk and intensity of

number was also correlated with female mating history, with males transferring more sperm to females

ionallyw dec

a mate (Parker 1990; Parker et al. 1996, 1997; Engqvist & Reinhold2006, 2007; Ball & Parker 2007). Recent models distinguishbetween sperm competition risk (i.e. the probability of spermcompetition occurring) and intensity (i.e. the number of competing

more in sperm when the intensity of sperm competition ismoderate (e.g. a female has mated once), but not high (e.g. a femalemated multiple times; Parker et al. 1996). However, more recentmodels developed for internally fertilizing animals predict morecomplicated outcomes (Engqvist & Reinhold 2006, 2007). Maleresponse to the risk and intensity of sperm competition can varywith the degree of polyandry (Yamane & Miyatake 2005), malesperm reserves (Engqvist & Reinhold 2007), sperm precedencepatterns (Engqvist & Reinhold 2006; Engqvist 2007) and sperm

* Correspondence: M. J. Solensky, Department of Biology, The College of Wooster,931 College Mall, Wooster, OH 44691, U.S.A.

E-mail address: msolensky@wooster.edu (M.J. Solensky).

Contents lists availab

Animal Be

.e l

Animal Behaviour 77 (2009) 4654721 K. S. Oberhauser is at the Department of Fisheries, Wildlife and Conservationrecognized that both female choice and male competition continueafter copulation (Parker 1970; Eberhard 1996). Sperm competitionoccurs when the ejaculates of two or more males are simulta-neously available to a female for fertilization. This competitionoften approximates a rafe in which males delivering more spermare likely to fertilize more eggs (Parker 1990; Gage & Morrow2003). However, the energetic demands of producing large quan-tities of sperm can limit male reproductive success (Dewsbury1982; Wedell et al. 2002) and males are consequently predicted toallocate ejaculates strategically based on male condition, risk andintensity of sperm competition and relative reproductive value of

such as situations inwhich females have alreadymated or are likelyto remate (Parker et al. 1997; Engqvist & Reinhold 2006; but see Ball& Parker 2007). Support for a positive relationship between the riskof sperm competition and investment in sperm has been docu-mented in many taxa (Gage 1991; Cook & Gage 1995; Birkhead &Mller 1998; Wedell & Cook 1999a; Wedell et al. 2002; Evans et al.2003; Zbinden et al. 2003; delBarco-Trillo & Ferkin 2004; Garca-Gonzalez & Gomendio 2004; Pound & Gage 2004; Ramm& Stockley2007; Thomas & Simmons 2007). Predictions of the effect of theintensity of sperm competition on male investment are lessstraightforward. Early models predicted that males should investMS. number: A08-00497R

Keywords:apyrene spermDanaus plexippuseupyrene spermmate assessmentmonarch butterysperm competitionsperm transferspermatophorestrategic mating effort

Studies of sexual selection tradittory processes, but in the past feBiology, 1980 Folwell Ave., University of Minnesota, S

0003-3472/$38.00 2008 The Association for the Studoi:10.1016/j.anbehav.2008.10.026that had larger amounts of spermatophore material stored from previous mates, regardless of whetherthis came from one or three mates. This result suggests that males use stored ejaculates to assess femalemating history and increase eupyrene sperm investment under increased sperm competition intensity.Male monarchs appear to be capable of independently manipulating the different components of theirejaculates. Ejaculate allocation patterns suggest that males benet by maximizing spermatophore sizeand apyrene sperm number, possibly to delay future female remating. However, males allocate moreeupyrene sperm to females when sperm competition is more intense, which is consistent with predic-tions from recent sperm competition models. 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

focused on precopula-ades researchers have

sperm or ejaculates). Theory predicts that males should typicallyinvest more sperm when the risk of sperm competition is high,Final acceptance 17 October 2008Published online 11 December 2008material allocation by male monarch butteries, Danaus plexippus. Males that had waited longer betweenmatings transferred larger spermatophores and more apyrene and eupyrene sperm. Eupyrene spermInitial acceptance 1 September 2008 sperm competition. We studied the relationship between behavioural and physiological cues andMale monarch butteries, Danaus plexipin response to intensity of sperm comp

M.J. Solensky a,*, K.S. Oberhauser b,1

aDepartment of Biology, The College of WoosterbDepartment of Fisheries, Wildlife and Conservation Biology, University of Minnesota

a r t i c l e i n f o

Article history:

During mating, male Lepeupyrene (nucleated) sper

journal homepage: wwwt Paul, MN 55108, U.S.A.

dy of Animal Behaviour. Publishes, adjust ejaculatesition

tera transfer spermatophores that consist of accessory gland material,nd apyrene sperm that is incapable of fertilizing eggs. Sexual selection

le at ScienceDirect

haviour

sevier .com/locate/yanbelimitation in females (Ball & Parker 2007).

d by Elsevier Ltd. All rights reserved.

nimSperm precedence patterns vary considerably across taxa, fromcomplete rst-male precedence to complete last-male precedence,with mixed paternity found in a number of species (reviewed inBirkhead & Mller 1998). When the last male to mate has a strongfertilization advantage, selection should favour males that preventor delay female remating, in some cases by chemical deposition(Andersson et al. 2000, 2003; Grillet et al. 2006) or increasedejaculate expenditure (Oberhauser 1989, 1992; Cook & Wedell1999). Selection can also favour male competition against previousmates, in some cases by increased male sperm investment inmatings with previously mated females (Engqvist & Reinhold2006). Mixed paternity can selectively favour any combination ofthese male strategies.

We studied patterns of ejaculate allocation in monarch butter-ies, Danaus plexippus. Malemonarchs use internal fertilization andobtain mates through coercion rather than courtship (Frey 1999;Oberhauser & Frey 1999), which limits opportunities for precopu-latory sexual selection. Regardless of the time of day at whichcopulation begins, mating pairs typically remain coupled until afterdusk, and copulation ends only when the male releases the female(Oberhauser & Frey 1999). During copulation, monarch malestransfer large spermatophores (up to 10% of bodymass: Oberhauser1988) that contain sperm and accessory gland material. Malestransfer accessory gland material throughout the day, but do notinitiate sperm transfer until after dusk (Svard & Wiklund 1988).Copulation generally ends between 2200 and 0400 hours (M. Sol-ensky, unpublished data). Shortly after copulation ends, spermmove from the bursa copulatrix, where the spermatophore isdeposited, through the sperm duct and into the spermatheca,where they are in position to fertilize eggs. The rest of the sper-matophore remains in the bursa copulatrix for several days, whereit is partially digested by the female and causes a delay in rematingthat is positively correlated with its size (Oberhauser 1989, 1992).Because the last male to mate usually has a fertilization advantage(Solensky 2003; Solensky & Oberhauser, in press), males benetfrom delivering a large spermatophore by delaying female remat-ing and consequently preserving last-male sperm advantage.Spermatophore size depends primarily on male mating history;spermatophore size in males increases with time since lastcopulation (Oberhauser 1988).

Male Lepidoptera transfer both eupyrene sperm, capable offertilizing eggs, and apyrene sperm, which are smaller and lacka nucleus. Males eclose with a nearly complete complement ofsperm (Seth et al. 2002), although sperm transfer from the testes toa storage organ accessible during copulation occurs throughout themales lifetime and appears to follow a daily rhythm (Bebas et al.2001). Males appear to have some control over how many spermthey transfer during mating: they may adjust sperm transfer basedon their own condition or their female mates condition (Wedell &Cook 1999a) or their perceived risk of sperm competition (Cook &Gage 1995; Wedell & Cook 1999a, b).

While there is abundant evidence that males can often adjusttheir ejaculates in sophisticated ways (e.g. Pizzari et al. 2003;Thomas & Simmons 2007), the mechanisms that males use toassess sperm competition risk and intensity and to adjust ejaculatesoften remain unclear (Wedell et al. 2002; Gage 2003). Males mayuse female behaviour, male- or female-derived pheromones(Siva-Jothy & Stutt 2003; Carazo et al. 2004), the presence of rivalmales (Evans et al. 2003; Zbinden et al. 2003; Pound & Gage 2004;Ramm & Stockley 2007), female mating history (Cook & Gage 1995;Wedell 1998; Wedell & Cook 1999a) or characteristics of rivalejaculates (Cook & Gage 1995; Wedell & Cook 1999a; Engqvist2007) to assess the risk and intensity of sperm competition.

We investigated three categories of cues that might inuence

M.J. Solensky, K.S. Oberhauser / A466monarch sperm allocation. We measured behavioural cues (matingattempt duration, copulation duration, timing of initiation andtermination of copulation) as potential indicators of female resis-tance and therefore mating history (Frey 1999), female conditioncues (size and mating history) and male condition cues (size andmating history). To further investigate the effect of perceivedintensity of sperm competition on male ejaculate characteristics,we manipulated female mating history such that females hadeither small or large quantities of spermatophore material fromeither one or three previous mates. If males use female bursacopulatrix content as an indicator of female mating history andsubsequent intensity of sperm competition from previous mates,then males should adjust eupyrene sperm transfer in response tothe quantity of spermatophore material stored, regardless of thenumber of a females previous mates.

METHODS

Study Organisms

The adult monarchs used were the offspring of eggs and larvaecollected from the wild in Missouri, Arkansas and Ohio, U.S.A.Laboratory colonies were derived from at least 20 foundingindividuals. We reared larvae in screen cages (0.5 0.3 0.6 m;50150 larvae per cage) on amixture of potted Asclepias curassavicaand cut stems of A. syriaca. Larvae developed at room temperature(roughly 24 C) under natural light conditions (roughly 18:6 hlight:dark cycle). On eclosion, we checked adults for infection byOphryocystis elektroscirrha (Altizer et al. 2000), labelled uninfectedadults on the hindwings using a permanent marker, and measuredforewing length or area. Adults were fed a 20% honeywatersolution to satiation every other day before being released intocages. In the cages, they had continuous access to potted A. cura-ssavica or cut stems of A. syriaca and a 20% honeywater solution.Some females were stored in a cold chamber (12 C) for 510 daysbefore being used in the experiment. This treatment does not affecttheir mating propensity or fecundity (K. Oberhauser, unpublisheddata).

Observational Study: Mating History, Mating Behaviourand Ejaculate Characteristics

We released an equal number of virgin males and virgin females(510 days old, 30 of each sex) into each of six outdoor mesh cages(1.8 m3) in St Paul, MN, and monitored mating activity for 10 daysstarting on 10 July 2003. In 2006, we released an approximatelyequal number of virgin males and virgin females (515 days old,1020 of each sex) into each of seven mesh cages (0.6 m3) nearlarge windows in a warm laboratory (29 C) in Wooster, OH andmonitored mating activity for 19 days starting on 26 June.

In 2003, we observed the mating behaviour of most matingattempts that led to the transfer of the ejaculates whose charac-teristics we measured. On 6 of the 10 days of the experiment, wemade detailed observations of themating behaviour of the adults ina focal cage: each cage was observed only once, and the order ofobservation was randomly assigned. For each mating attemptobserved, we recorded the identity of the individuals involved, starttime, attempt duration, and whether the attempt resulted incopulation. We recorded resistance behaviours (Frey 1999) usedby the female, including arc (female exes abdomen dorsally, wingshorizontal or folded), inverted (dorsal surface of female facing theground), sheath (females wings folded, usually the female liestilted to one side against substrate) and curl (females abdomencurled forward under thorax, female often wraps her legs aroundher curled abdomen).

To measure male ejaculate transfer, we dissected all mated

al Behaviour 77 (2009) 465472females in the focal cage (2003) or in all cages (2006). Measuringthe ejaculate transferred to an individual female was a destructive

the models (P > 0.05), so we report models with linear terms only.

nimaprocess, which ensured that no females were sampled twice. Whenthe ejaculates of individual males were collected from more thanone female, we used only the rst observation of each male in ouranalyses; thus, all ejaculates were independent samples.

Experimental Study: Manipulated Female Mating Historyand Ejaculate Characteristics

We generated three groups of females that differed in thenumber and size of spermatophores received from mates prior tomating with a focal male; females had received either one small (S)spermatophore, one large (L) spermatophore, or three small (SSS)spermatophores. Small spermatophores were delivered by malesthat had mated 1 day prior, while large spermatophores weredelivered by males that had waited 4 days since last mating. Thisdifference in male mating history generates a three- to four-folddifference in size between small and large spermatophores (Ober-hauser 1988), which suggests that females in the L and SSS treat-ments should have stored similar total amounts of spermatophorematerial prior to their focal matings. Males were randomly assignedto deliver either a small or a large spermatophore, and on the dayafter mating were either used to deliver a small (S) spermatophoreor removed from females for 4 days before being released into a cagewith experimental females to deliver a large (L) spermatophore.

Virgin females were randomly assigned to one of the threemating history treatments, and then released into mesh cages(0.6 m3) near large windows in a warm laboratory (29 C) inWooster, OH, that contained 1020 females from the sametreatment group. Females had at least 1 day without copulation inbetween each mating, during which time they were removed toa female-only holding cage. Upon receiving the appropriatenumber and size of spermatophore based on the pre-assignedtreatment, females were held for 1 day without access to males andthenmoved to a cage that housedmales that hadmated 1 day prior.These were the focal males whose ejaculate characteristics wemeasured in response to manipulated female mating history. Thiscage housed all females from all three treatments that had previ-ously received one or three large or small spermatophores. We keptthe sex ratio in this cage equal or male biased to maximize thelikelihood of all experimental females mating soon after they hadcompleted the preliminary matings, before the spermatophorematerial would begin to break down (Oberhauser 1992).

Measuring Sperm Transfer

To measure the spermatophore size and number of spermtransferred, we modied methods described by Oberhauser (1988)and Cook & Wedell (1996). We monitored the cages every 15 minfrom 2100 hours until the last pair had separated. When a pairseparated, we immediately collected the female and decapitatedand dissected it in insect saline within 10 min, before sperm hadleft the spermatophore. We measured spermatophore mass bydissecting out the spermatophore from the bursa copulatrix,blotting it on tissue paper to uniform dryness andweighing it to thenearest 0.1 mg on a Mettler semi-micro analytical balance. Whenmore than one spermatophore was present, we identied thenewest spermatophore by its light coloration and the presence ofsperm. In 2006, we also weighed the old spermatophore materialrecovered from the females bursa copulatrix. We then dissectedthe sperm sac from the newest spermatophore, transferred it toa depression slide, ruptured it into a 10 ml drop of insect saline usingne-point forceps, and counted the number of eupyrene spermbundles under 40 magnication. Among the Lepidoptera, eacheupyrene sperm bundle results from eight cell divisions (Virkki

M.J. Solensky, K.S. Oberhauser / A1969), so we multiplied the number of bundles by 256 (28) todetermine the number of eupyrene sperm transferred.Although male and female mating history did not conform toa normal distribution, we included them in the regression models;when either was identied as a signicant predictor, we assessedthe signicance of the pattern further using a nonparametricSpearman rank correlation test. Within the female mating historytreatment groups, all three ejaculate components were normallydistributed (KolmogorovSmirnov test: P > 0.25). We used one-way ANOVA models to measure differences in each ejaculatecomponent between treatment groups. Because the three ANOVAsmeasured different ejaculate characteristics (spermatophore mass,eupyrene and apyrene sperm number), we applied a Bonferroniadjustment and concluded statistical signicance when P < 0.017(0.05/3) to maintain an experiment-wise error rate of P < 0.05(Sokal & Rohlf 1995) for analyses that were repeated for these threeWe rinsed the sperm solution from the depression slide andcover slip with deionized water into a tared 100 ml glass jar. Wediluted the sperm solution to approximately 60 ml with deionizedwater and recorded the exact volume. After adding 10 ml oftoluidene blue protein stain, we gently agitated the solution andtransferred 10 ml to each of ve clean microscope slides. Afterallowing the slides to dry, we dipped each in deionized water todissolve salt crystals remaining from the insect saline solutionwithout removing sperm (Cook & Wedell 1996). We scanned eachslide at 100 magnication using a compound light microscope tocount the number of apyrene sperm, then calculated the totalnumber of apyrene sperm by multiplying the average number ofsperm subsampled by its dilution factor.

Statistical Analyses

Three of the independent variables (male and female wing area,and copulation duration) were normally distributed (KolmogorovSmirnov test: P > 0.5). Mating attempt duration was not normallydistributed, so we used the natural log of attempt duration in ouranalyses, which did not differ statistically from a normal distribu-tion (KolmogorovSmirnov test: P 0.489). Male time since lastmating (days) and female mating history (number of mates) werenot normally distributed (KolmogorovSmirnov test: P < 0.001),and because both were measured as ordinal categories with a largenumber of observations in the rst category, neither could betransformed to approximate a normal distribution. Bursa copulatrixcontent (the mass of all old spermatophores) was also not normallydistributed (KolmogorovSmirnov test: P 0.002) and could not betransformed to approximate a normal distribution because of thelarge number of observations of virgin females in the observationalstudy, for which this measure equalled zero. Two of the dependentvariables (spermatophore mass and the number of apyrene sperm)were not normally distributed (KolmogorovSmirnov test:P < 0.05), so we used the square root of spermatophore mass andapyrene sperm number; these and eupyrene sperm number wereall normally distributed (KolmogorovSmirnov test: P > 0.15).

We used multiple linear regression to test for relationshipsbetween ejaculate components and the four independent variablesthat were measured in both years of this study (male and femalemating history and wing area) and separately for the behaviouralvariables measured in 2003 only. Sperm competition modelspredict that males should invest maximally in ejaculate transfer atintermediate levels of sperm competition intensity (Parker et al.1996; Engqvist & Reinhold 2007), which predicts a quadratic effectof female mating history on ejaculate transfer, so we initiallyincluded the quadratic term of female mating history in ourregression models. The quadratic termwas not signicant in any of

l Behaviour 77 (2009) 465472 467dependent variables. Unless otherwise noted, all estimates arereported as mean SE.

RESULTS

Observational Study: Mating Behaviour

Mating attempts typically began with the male on top of thefemale and both butteries dorsal side up (46 of 55 attempts forwhich starting positionwas recorded). Of the four female resistancebehaviours recorded, the arc behaviour was the most common,although it was only used in 16 of 51 mating attempts for whichbehaviour was recorded. Only 4 of 51 females used the curlbehaviour, and all of these females had mated previously. In twomating attempts, females used the inverted behaviour, and 4 of 51females used the sheath behaviour. Because resistance behavioursoccurred so infrequently, they were not included as potentialpredictors of spermatophore size or sperm transfer in the statisticalanalyses. Mating attempts were longer when at least one resistancebehaviour was used (retransformed mean (95% condence interval,

1.240.8, N 156). On average, males transferred 81082 2683

Experimental Study: Female Mating History Manipulations

As expected, females in the three treatment groups differed inthe amount of old spermatophore material stored from previouscopulations (ANOVA: F2,39 10.295, P < 0.001; Fig. 4a). Femalesthat had previously received one small (S) spermatophore had lessold spermatophore material stored than females that had previ-ously received one large (L) spermatophore (Tukey post hoc test:P < 0.001) or three small (SSS) spermatophores (Tukey post hoc:P 0.051). Females in the L group had marginally more oldspermatophore material stored than females in the SSS group(Tukey post hoc: P 0.079).

Males transferred signicantly more eupyrene sperm to femalesin the L and SSS groups than to females in the S group (ANOVA:F2,37 5.28, P 0.010; Fig. 4b). Males transferred marginally moreapyrene sperm (ANOVA: F2,41 3.19, P 0.052; Fig. 4c) and smallerspermatophores (ANOVA: F2,37 3.76, P 0.033; Fig. 4d) tofemales in the SSS treatment than to females in the S treatment(using the Bonferroni corrected P < 0.017 to conclude statisticalsignicance), while females in the L treatment received interme-diate amounts of apyrene sperm and spermatophore sizes.

DISCUSSION

M.J. Solensky, K.S. Oberhauser / Anim468(range 1280251136, N 146) eupyrene sperm and 1561693 65939 (range 2904005426419, N 144) apyrene sperm.

Nonvirgin male time since last mating was the only variablethat affected spermatophore size (Spearman rank: rS 0.430,N 132, P < 0.001; Table 1, Fig. 2a) and apyrene sperm transfer(rS 0.398, N 123, P < 0.001; Table 1, Fig. 2c). Both male andfemale mating history affected eupyrene sperm transfer (Table 1).Males that had waited longer since the last mating transferredlarger spermatophores, and more eupyrene and apyrene sperm(Fig. 2). Males transferred more eupyrene sperm to females thathad previously mated more frequently (Fig. 3a). Male and female

0

1

2

3

0 1 2 3 4Number of female's previous mates

Log

(att

emp

t d

ura

tion

) (s

)CI): with resistance 84 (51,139) s; without resistance 25(16, 37) s; t49 4.014, P< 0.001). Female mating history had onlyamarginally signicant effect onwhether a resistance behaviourwasused (logistic regression: N 51; model c12 2.671, P 0.102), butpreviously mated females had longer mating attempts than virgins(retransformed mean (95% CI): mated 55 (35, 84) s; virgin 29(18, 46) s; t55 2.032, P 0.047). Females with more previousmatings had longer mating attempts (Spearman rank: rS 0.284,N 57, P 0.032; Fig.1), although this patternmaybe driven by fourfemales with three and four previous mates (Spearman rankomitting potential outliers: rS 0.186, N 53, P 0.182).

Observational Study: Ejaculate Characteristics

In both years, mating pairs separated between 2140 and 0610hours. The average spermatophore mass was 15.6 0.9 mg (rangeFigure 1. Relation between female mating history and mating attempt duration.Potential outliers shown as open circles.wing area, the time at which a pair coupled or separated, matingattempt duration and copulation duration had no signicanteffects on any of the three ejaculate characteristics measured, andfemale mating history had no effect on spermatophore mass orapyrene sperm transfer (multiple linear regression: all predictorsP > 0.05).

The same patterns emerged from regression analyses using thetotal mass of old spermatophores from previous copulations asa measure of female mating history rather than the number offemale matings: spermatophore mass and apyrene sperm numberwere correlated only with male mating history (multiple linearregression: P < 0.001), while eupyrene sperm transfer was corre-lated with both male mating history (P < 0.001) and the total massof old spermatophores in the female (P 0.009; Fig. 3b).

Table 1Regression models of ejaculate characteristics for square root of spermatophoremass (N 131), square root of the number of apyrene sperm transferred (N 122)and number of eupyrene sperm transferred (N 124)

Coefcient (SE) P

Spermatophore massConstant 0.071 (0.037)Male mating history 0.008 (0.001)

nima5

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(a)

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M.J. Solensky, K.S. Oberhauser / Acompetition. Males appear to assess the intensity of spermcompetition using bursa copulatrix content as a measure of femalemating history, transferring more eupyrene sperm to females thathad stored larger amounts of spermatophore material fromprevious males. It is possible that there is an additional effect of thenumber of mates; females with three small spermatophorescontained marginally less spermatophore material than those withone large spermatophore, but they received a statistically similarnumber of eupyrene sperm.

In the observational study, it is possible that female matinghistory was confounded with female quality, and that high-qualityfemales mated more often and received more sperm. However, inour experimental study, females were pre-assigned to matinghistory treatments, and these data support female mating historyas the cause of increased sperm transfer; we observed the samepattern of eupyrene sperm allocation when male and femalemating histories were randomly pre-assigned as when they wereallowed to vary naturally.

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Figure 2. Relation between male time since last mating and (a) spermatophore size,(b) eupyrene sperm and (c) apyrene sperm transferred. X axes show the number ofdays since a nonvirgin males last mating. Although virgin males were not included inthe regression models, the means (SEs) of this group are shown by large shadedcircles at the right of each graph for comparison.0

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l Behaviour 77 (2009) 465472 469There was no effect of female mating history on apyrene spermnumber or spermatophore size in the observational study, and onlya marginal effect in the experimental study, which featured onlythree possible femalemating histories (S, L or SSS spermatophores).Males delivered marginally more apyrene sperm and smallerspermatophores to SSS females. If males do allocate these ejaculatecomponents strategically, these experiments did not detect strongallocation patterns.

The different spermatophore components serve different rolesin inuencing male reproductive success. Only the eupyrenesperm are capable of fertilizing eggs, yet they are far out-numbered by apyrene sperm (e.g. Gage & Cook 1994; Wedell &Cook 1999a, b; this study). Although the function of apyrenesperm remains unclear, recent research has shown that apyrenesperm storage delays female remating in the green-veined whitebuttery (Pieris napi) (Cook & Wedell 1999), thus preserving thelast males sperm precedence. Spermatophore size also delaysfemale remating in monarch butteries (Oberhauser 1989, 1992),and nutrients in the spermatophore are used by the female forboth somatic maintenance and egg production (Boggs & Gilbert1979; Oberhauser 1989). Mixed paternity is common amongmultiply mated female monarchs, with last-male advantagebeing more common than rst-male advantage (Solensky 2003;Solensky & Oberhauser, in press). A strong last-male fertilizationadvantage should selectively favour male ejaculate allocationstrategies that delay female remating, such as increased apyrenesperm transfer or increased spermatophore size.

There was a marginal effect of female mating history on sper-matophore size in the experiment inwhich wemanipulated femalemating history, with males transferring less material to femalesthat had already mated. If this effect is real, it would result in

Figure 3. Relation between female mating history, as measured by (a) the number ofprevious copulations completed by a female and (b) the total mass of old spermato-phores stored by a female from previous copulations, and the number of eupyrenesperm transferred by a male.

nim5

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M.J. Solensky, K.S. Oberhauser / A470a delay in female remating similar to that generated by a maledelivering a large spermatophore to a virgin female. Alternatively,the pattern could be the result of increased nutrient investmentwhen females are virgins and a males paternity is assured, at leastuntil the next mating.

If the marginal effect of mating history on apyrene spermnumber is real, males invested more in matings with females thathad mated more times, perhaps because the apyrene sperm serveto push a previous males sperm further back in the spermatheca. Ifthe marginal effect is not real, males may maximize apyrene spermtransfer based only on their own mating history. This is consistentwith the cheap ller hypothesis, which proposes that males mayuse the small, energetically less expensive apyrene sperm to lla females spermatheca and consequently delay remating(Silberglied et al. 1984; Cook & Wedell 1999).

Theoretical models predict that males should increase spermexpenditure with increasing risk of sperm competition (Parkeret al. 1997; Engqvist & Reinhold 2006, 2007). Early models pre-dicted a decrease in sperm expenditure with increasing intensity(i.e. number of a females prior mates) of sperm competition

0S L SSS

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Figure 4. Mean (a) mass of old spermatophores stored by females from previous copulaspermatophore transferred by the experimental male to females that had previously receivedifferent letters indicate signicant differences between groups (Bonferroni corrected Tuke2

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al Behaviour 77 (2009) 465472(Parker et al. 1996). However, more recent models have shownthat the reverse can occur (i.e. increased sperm investment whensperm competition intensity is high) when males have largesperm reserves (Engqvist & Reinhold 2007), or when rematingrates are high and last-male sperm precedence is prevalent(Engqvist & Reinhold 2006). Our results support the predictions ofEngqvist & Reinhold (2006). Males allocated more eupyrenesperm to females when the intensity of sperm competition washigher, as predicted for species like monarchs, in which femalesmate multiple times (Hill et al. 1976; Brower et al. 1977; Ober-hauser 1989; Frey 1999) and last-male sperm precedence iscommon (Solensky 2003).

Males of other species use a variety of cues to assess the risk orintensity of sperm competition, including pheromones (Siva-Jothy& Stutt 2003; Carazo et al. 2004), the presence of rival males (Evanset al. 2003; Zbinden et al. 2003; Pound & Gage 2004; Ramm &Stockley 2007), female mating history (Cook & Gage 1995; Wedell1998; Wedell & Cook 1999a), or characteristics of rival ejaculates(Cook & Gage 1995; Wedell & Cook 1999a; Engqvist 2007). Thecoercive mating system of monarchs, accompanied by an apparent

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tions, (b) number of eupyrene sperm, (c) number of apyrene sperm and (d) mass ofd one small (S), one large (L) or three small (SSS) spermatophores. Within each graph,y post hoc: P < 0.017). Error bars show 1 SE.

overwintering monarchs mate at densities that are many orders of

under high-intensity sperm competition (i.e. fewer sperm to

nimafemales with more sperm stored from a previous mating). Oneexplanation offered for these opposite patterns was the differencein sperm precedence patterns (fair rafe in scorpionies versuslast-male precedence in the two Lepidoptera; Engqvist 2007),which is consistent with this study, as monarch butteries alsoshow last-male sperm precedence (Solensky 2003; Solensky &Oberhauser, in press).

We found no evidence of behavioural inuences on ejaculatetransfer. Although female resistance behaviours were too infre-quent to be statistically analysed, we found that attempt duration(another behavioural correlate of female mating history) had noeffect on eupyrene sperm transfer. Frey (1999) reported a correla-tion between female resistance behaviours and bursa copulatrixcontent, but Solensky (2004) found that monarch mating behav-iour and attempt duration were strongly inuenced by the initialstarting positions and the vegetation on which an attemptoccurred, neither of which correlate with female mating history.This nding suggests that mating attempt behavioural cues maynot signal female mating history as reliably as the physiological cueof bursa copulatrix content.

Males in many taxa strategically allocate ejaculates, usinga variety of cues to assess sperm competition (Birkhead & Mller1998; Wedell et al. 2002; Pizzari et al. 2003; Zbinden et al. 2003;delBarco-Trillo & Ferkin 2004; Ramm & Stockley 2007; Thomas &Simmons 2007). Here, we show that male monarch butteriesassess the relative intensity of sperm competition based on theamount ejaculate material stored by a female from previousmatings, and tailor their ejaculates as predicted by sperm compe-tition models to maximize lifetime reproductive success.

Acknowledgments

We thank Lynette Batte, Anja Brunet-Rossinni, Jennifer Brophy,Reba Batalden, Bruce Leventhal, Sara Brinda, Amy Alstad, LeahAlstad, Andy Regan, Kevin Spragg, Beth DeLong, Amy Smith andEvan Slanczka for their assistance in rearing and observing mon-archs and counting sperm. Andy Davis provided valuable advice ondigitally measuring adult buttery size. This work was supportedby Monarchs in the Classroom and the Life Science SummerUndergraduate Research Program at the University of Minnesotaand by the Sophomore Research Program and Howard HughesMedical Institute Summer Research Scholar Program at The Collegeof Wooster.

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M.J. Solensky, K.S. Oberhauser / Animal Behaviour 77 (2009) 465472472

Male monarch butterflies, Danaus plexippus, adjust ejaculates in response to intensity of sperm competitionMethodsStudy OrganismsObservational Study: Mating History, Mating Behaviour and Ejaculate CharacteristicsExperimental Study: Manipulated Female Mating History and Ejaculate CharacteristicsMeasuring Sperm TransferStatistical Analyses

ResultsObservational Study: Mating BehaviourObservational Study: Ejaculate CharacteristicsExperimental Study: Female Mating History Manipulations

DiscussionAcknowledgmentsReferences