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Animal Behaviour 92 (2014) 125e131

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Animal Behaviour

journal homepage: www.elsevier .com/locate/anbehav

Alternative male mating behaviour in the two-spotted spider mite:dependence on age and density

Yukie Sato a,b,c,*, Maurice W. Sabelis a, Martijn Egas a

a Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The NetherlandsbNational Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, JapancResearch Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan

a r t i c l e i n f o

Article history:Received 26 August 2013Initial acceptance 3 September 2013Final acceptance 10 March 2014Published onlineMS. number: 13-00706R

Keywords:alternative mating tacticsconditional strategyfightermaleemale competitionmatingsneakerTetranychus urticae

* Correspondence: Y. Sato, Institute for BiodiversiUniversity of Amsterdam, 1090GE Amsterdam, The N

E-mail address: uchietan@gmail.com (Y. Sato).

http://dx.doi.org/10.1016/j.anbehav.2014.03.0320003-3472/� 2014 The Association for the Study of A

Alternative male mating tactics occur in many taxa, and usually include sneaking behaviour. Typically,sneaking behaviour is expressed by less competitive males to avoid fighting over females guarded bystronger males and to increase their mating chances, but in theory it can be expressed by any male aslong as it improves his future reproduction. Recently, we characterized fighting and sneaking behaviourin males of the two-spotted spider mite that guard females during their preadult moulting stage.Sneaking behaviour involves not responding to rival males to avoid eliciting attack, whereas fightingbehaviour involves challenging all rivals. To understand these alternative precopulatory behaviours froma life history perspective, we investigated (1) their heritability, and how they are affected by (2) maledensity, (3) male age and (4) competition between males differing in age. We established genetic lines forfighting or sneaking by crossing a male that displayed fighting or sneaking behaviour with one of hisdaughters. In fourth-generation males from these lines, however, the proportions of fighting andsneaking behaviour were not significantly different. The frequency of sneaking behaviour increased withmale density, yet decreased with male age. Old males always displayed fighting and rarely lost femalesthey guarded to young males, whereas young fighting males lost females to old rival males morefrequently than young sneaking males. Of the young fighting males that did not lose the female theyguarded to old males, about 40% switched to sneaking. We conclude that alternative male mating be-haviours in this mite are not maintained as a genetic polymorphism, but arise from phenotypic plasticityin response to the male’s environment (male density) and its own condition (age). We hypothesize thatyoung males opt for sneaking behaviour because fighting jeopardises their future reproduction.� 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Males generally compete for access to females in order toreproduce (Andersson, 1994). They usually do this by fighting withrival males and driving them off, but they may also display non-fighting behaviour, such as sneaking and satellite behaviours, so asto increase their mating opportunities (e.g. reviewed in Brockmann,2001; Gross,1996; Oliveira, Taborsky, & Brockmann, 2008; Radwan,2009; Tomkins & Hazel, 2007). Sneaking and satellite behavioursare considered to be alternative mating tactics for males that arelikely to lose fights over females against stronger males (e.g.Eberhard, 1982), but in theory these tactics can be adopted by anymale as long as they sufficiently reduce injury from maleemalefights, thereby promoting future reproduction. Hence, to maximizetheir lifetime reproductive success, males may display sneaking

ty and Ecosystem Dynamics,etherlands.

nimal Behaviour. Published by Els

and satellite behaviours regardless of their current competitive-ness. This life history perspective has been investigated theoreti-cally with respect to the evolution of age-dependent male matingtactics (Kemp, 2006; Kokko, 1997). However, few empirical papershave addressed flexible male mating tactics from a life history pointof view (Bertram, 2000; Brockmann, Colson, & Potts, 1994;Candolin & Vlieger, 2013; Heckel and von Helversen, 2002; Jang,2011; Kemp, 2002), even though alternative male mating tacticshave been studied in many animals (e.g. reviewed in Brockmann,2001; Gross, 1996; Oliveira et al., 2008; Tomkins & Hazel, 2007).One reason may be that it is not easy to isolate the effect of theresidual reproductive value (the likelihood of future reproduction;Roff, 2001) from the effect of the resource-holding potential (suchas energy reserves, strength and body size, which directly affect theprobability that males win fights; Parker, 1974) on lifetime repro-ductive success. For example, fighting or risky mating tactics in oldmales have been observed inmany animals, especially inmammals,birds and reptiles (Kemp, 2006). This phenomenon is consistent

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Y. Sato et al. / Animal Behaviour 92 (2014) 125e131126

with predictions from a life history perspective, because residualreproductive value decreases with age. However, in all these ani-mals, males are likely to grow and become larger even after sexualmaturation, indicating that resource-holding potential increaseswith age. In this case, fighting or risky mating tactics in old malescan be explained by resource-holding potential, for example whenold males are competitive and tend to win fights. Hence, to test forthe effect of a trade-off between current and future reproduction onthe evolution of alternative male mating tactics, it would beimportant to investigate flexiblemalemating tactics in animals thatdo not show an apparent increase in resource-holding potentialwith age (Kemp, 2006).

In this study, we investigated flexibility in male mating behav-iour and the effects of male age on alternative male mating be-haviours in the two-spotted spider mite, Tetranychus urticae Koch.This mite is a serious pest in agriculture, which is why its ecologyand behaviour have been studied relatively well (e.g. Helle &Sabelis, 1985). Recently, we found that male spider mites exhibitthree alternative precopulatory behaviours: fighting (territorial),sneaking and opportunistic behaviour (Sato, Sabelis, Egas, & Faraji,2013). These males guard a female in her final moulting stage(termed ‘teleiochrysalis’) by staying on the female’s dorsum, leav-ing this position only to fight off rival males (Potter, Wrensch, &Johnston, 1976a,b). The fighting behaviour is characterized by themale positioning himself directly in front of the rival male andtaking up a fighting posture with his main weapon, the cheliceralstylets (needle-like mouthparts normally used for piercing hostplant cells) extended and with the first pair of legs lifted up (Potteret al., 1976a,b; see supplementary video in Sato, Sabelis, et al.,2013). We previously called this behaviour ‘territorial’ because ithelps the male to monopolize the area with the female (see sup-plementary video in Sato, Sabelis, et al., 2013). Other males displaysneaking behaviour: they also guard females but do not initiatefights with rival males, evenwhen provoked, nor are sneaker malesattacked by rivals (Sato, Sabelis, et al., 2013). It is important for themale to keep this position close to the female until she moults to anadult because proximity determines mating success (Potter et al.,1976a,b) and the first mating virtually ensures paternity of the(female) offspring (Boudreaux, 1963; Helle, 1967). Nevertheless, wealso observed nonguarding behaviour in some males: theycontinually search for virgin females just emerging from the lastmoulting stage. Thus, these males do not guard females and theirbehaviour is called ‘opportunistic’ (Sato, Sabelis, et al., 2013).Because we have not yet been able to establish a reliable method toassess opportunistic behaviour, we focus here on fighting andsneaking behaviour.

In the mite under study, larger males are more likely to winfights (Potter et al., 1976a,b). However, we did not find any apparentdifferences in body size (body length and body width) or weaponsize (lengths of the first legs, stylets and pedipalps) betweenfighting males and sneaking males (Sato, Sabelis, et al., 2013). Thisindicates that alternative male mating behaviour cannot beexplained only by male competitiveness, and also that male matingbehaviours can be flexible because there is no morphologicalconstraint in displaying each of the possible behaviours. In addi-tion, males of the mite mate with females during their entire adultlifetime as generations overlap (Helle & Sabelis, 1985) which meansthat the residual reproductive value of young males is much higherthan that of older males. In addition, maleemale contests areintense and can result in injury or even death (Potter et al., 1976a,b).Taking both these aspects into consideration, youngmalesmight bebetter off by adopting sneaking behaviour.

To investigate how flexible the alternative male mating behav-iour of the mite is, we measured (1) the heritability of male matingbehaviour and (2) the effect of male density on male mating

behaviour in the mite. To investigate life history effects, wemeasured (3) the effect of male age on male mating behaviour and(4) male mating behaviour when males compete with malesdifferent in age.

METHODS

Life History of the Two-spotted Spider Mite

Based on Helle and Sabelis (1985), a brief review is presented onsome features of the life history of the two-spotted spider mite,T. urticae, relevant to this paper. As with the majority of spidermites, this species is haplodiploid: females develop from fertilizedeggs (2n) and males develop from unfertilized eggs (n). Its devel-opmental rate, oviposition rate and longevity strongly depend onthe host plant they feed on and on the temperature they experi-ence. At 21 �C on beans, Phaseolus vulgaris L., egg-to-adult devel-opment takes 2 weeks; a few days after maturation (shortpreoviposition period), females lay 10e15 eggs per day and thenslow down egg deposition more gradually over a period of ca. 4weeks (relatively long oviposition period) until they stop a few daysbefore death (short postoviposition period). Usually females pro-duce the sexes in a ratio of two to three daughters to one son. Maledevelopment is somewhat faster than that of females and theirlongevity is about half that of the females. Adult males can mate upto four times per day before their sperm store is depleted.

Two-spotted spider mites aggregate in colonies and live insideself-made webs of silk on leaves of their host plants. Coloniesusually start from one or a few foundresses and develop until mitesoverexploit the host plant, at which time the young and matedadult females disperse in search of new host plants to establish newcolonies. The duration of this colonization cycle depends on thelocal conditions for population growth and usually lasts severaloverlapping generations.

Mite Culture

To start our culture of T. urticae, a sample population was ob-tained from a commercial producer of biological control agents(Koppert Biological Systems, Berkel en Rodenrijs, The Netherlands).Mites were reared on detached common bean leaves, P. vulgaris, onwet cotton wool in a plastic box under constant climatic conditions(25 � 1 �C, 60% relative humidity and 16:8 h light:dark photope-riod). Both for rearing mites and for the experiments describedbelow, we always used the first leaves of 1e3-week-old commonbean plants. Because the experiments required a large number ofmales, we prepared a separate culture for males, as follows. Werandomly collected 20 females in the last moulting stage from theculture and introduced them together onto a 5 � 5 cm fragment ofbean leaf placed on wet cotton wool in a plastic box. The virginadult females that developed from these females were allowed tolay unfertilized eggs (which all develop intomales) for 1week, afterwhich the females were removed and the eggs were left to develop.From this culture we collected males in the final moulting stage tobe used in the experiments after they moulted.

Heritability of Mating Behaviour

We established one genetic line for fighting behaviour and onefor sneaking behaviour, by crossing a father with one of hisdaughters (Fig.1). After randomly collecting 30males from themiteculture, we introduced them onto a small leaf arena (bean leaf disc1.5 cm in diameter) on which was a female in the last moultingstage.Whenwe observed amale sitting on the female’s dorsum (i.e.guarding), we identified his mating behaviour by using an artificial

Mother(??)

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25%?, 75%F 25%?, 75%S

Figure 1. Experimental design to establish fighter and sneaker genetic lines of the spider mite Tetranychus urticae. This mite has a haplodiploid genetic system: females developfrom fertilized eggs (2n) and males develop from unfertilized eggs (n). Assuming that male mating behaviour would be determined by one genetic locus, ‘F’ and ‘S’ indicate thealleles expressing fighting and sneaking behaviour, respectively, and ‘?’ indicates unknown allele.

Y. Sato et al. / Animal Behaviour 92 (2014) 125e131 127

disturbance test whereby we brought the guarding male intocontact with another male that was fixed to the tip of a fine wetbrush. If the guardingmale responded actively towards themale onthe brush and showed a fighting posture (extended stylets and firstpair of legs lifted up), the behaviour was classified as fighting. If hedid not show any response to the male on the brush, the behaviourwas classified as sneaking (for details of this classification proce-dure and proof of its reliability, see Sato, Sabelis, et al., 2013). Weintroduced one male, either one that displayed fighting or one thatdisplayed sneaking (randomly selected from the males with iden-tified mating behaviour) onto a large leaf arena (5 � 5 cm bean leafsquare) on which was a female in the last moulting stage andallowed the pair to copulate after the adult female had moulted.After the male had copulated, we removed him from the arena andplaced him alone on a small leaf arena. Then, we allowed the femaleto oviposit for 1 week. Once her daughters had reached the finalmoulting stage, we randomly picked one daughter and, after shehad matured, allowed her to copulate with her father in a largearena. We then removed the father and allowed the daughter tooviposit for 1 week in the large arena. We reared the offspring and,once the inbred granddaughters (F ¼ 0.5) had developed into thefinal moulting stage, we randomly picked 33 granddaughters andintroduced each onto a single large leaf arena. These granddaugh-ters were allowed to lay unfertilized (male) eggs for 1 week and wereared the eggs until they developed into adult males. Werandomly picked one 1e7-day-old great-grandson from eachgranddaughter and placed him onto a small leaf arena (a bean leafdisc 1.5 cm in diameter) together with a female in the last moultingstage to assess the male’s behaviour. One hour after introduction ofthe male, we checked whether the male guarded the female and, ifso, assessed his mating behaviour using the artificial disturbancetest described above (Sato, Sabelis, et al., 2013). If the male did notshow guarding behaviour, we did not subject him to the test andclassified the behaviour as nonguarding (i.e. not sneaking orfighting as we defined these behaviours for guarding males). Forthree great-grandsons of the ‘fighter’ genetic line, we failed toidentify the male mating behaviour because the female moultedbefore the end of the test and had copulated with the male beforehis mating behaviour could be assessed. These three males wereremoved from the data set. We compared the ‘fighter’ genetic lineand the ‘sneaker’ genetic line with respect to the fraction of

guarding males and the fraction of males displaying fightingbehaviour within the category of guarding males (see below fordetails on the statistics used).

Mating Behaviour at Different Male Densities

To assess the effect of male density on the sneaking behaviour offocal males, we randomly collected males in the last moulting stagefrom the male culture and put them individually onto bean leafdiscs, 1.5 cm in diameter, placed on wet cotton wool in petri dishes(bean leaf arenas), one disc per dish. One day after they moultedinto adult males, we placed one male (treatment without rivalmale), two males (treatment with one rival male), five males(treatment with four rival males) or 10 males (treatment with ninerival males) onto a leaf arena. Each arena also contained one femalein the last moulting stage, which was selected beforehand from ourmite culture on the condition that she was accompanied by one ormore guarding males. Two hours after introducing the males, wechecked whether the female was guarded by a new male, afterwhich we identified the mating behaviour of the guarding maleusing the artificial disturbance test (Sato, Sabelis, et al., 2013).Because we aimed to assess the mating behaviour of an equalnumber of guarding males per treatment, we set up replicates untilwe had obtained 65 observations on guarding males. However,males did not always display guarding and the proportion ofguarding males varied with the treatment. Hence, the number ofreplicates differed per treatment: 182 in the treatment without arival male, 118 in the treatment with one rival male, 103 in thetreatment with four rival males and 84 in the treatment with ninerival males.

We compared the proportion of replicates in which one of themales guarded a female (the number of leaf arenas in which one ofthe males guarded a female divided by the total number of leafarenas) among treatments differing in male density. Because malesdo not always display guarding behaviour, the probability that oneof themales guards a female increases with the number of males onthe arena, even if there is no effect of male density on individualmale guarding behaviour. To detect an effect of male density on theobserved guarding behaviour, we tested the null hypothesis thatindividual male behaviour is not affected by male density. To do so,we calculated the expected probability m(x) that at least one of a

Y. Sato et al. / Animal Behaviour 92 (2014) 125e131128

group of x males guards a female by assuming that the guardingbehaviour of each male is independent of the presence of othermales. Hence, using the observed proportion of replicates in whicha single male guards a female (M) in the treatment without rivalmales as the estimated probability of guarding per male, the ex-pected probability m(x) that at least one of the males guards a fe-male in treatments with xmales is calculated asm(x) ¼ 1e(1 �M)x.To estimate the effect of the presence of (x � 1) rival males on theprobability of guarding, we compared the proportion of replicatesin which guarding was observed with the expected probabilitiesm(x) (see below for details on the statistics used).

We compared the proportion of replicates inwhich the guardingmales displayed sneaking behaviour (the number of leaf arenas inwhich the guarding male was classified as a sneaker divided by thenumber of leaf arenas in which one of the males guarded a female)among treatments differing in male density. To detect an effect ofmale density on the probability of individual males displayingsneaking behaviour, we tested the proportion of sneakers classifiedin the different treatments (see below for details on the statisticsused).

Mating Behaviour at Different Ages

We assessed the effect of male age on the probability ofsneaking behaviour, by randomly collecting males in the lastmoulting stage from themale culture (see above for a description ofthe procedure) and putting them individually onto a small bean leafarena (bean leaf disc 1.5 cm in diameter). While checking theirmoulting status on a daily basis, we selected five freshly maturedmales (i.e. that had moulted within a day; 0-day-old males; wescored male age here in days since moulting to adulthood) andintroduced them onto a leaf arena with a female in the lastmoulting stage, which was selected from our mite culture on thecondition that she was guarded there by one or more males. Twohours after mite introduction, we checked whether one of themales guarded the female, after which we used the artificialdisturbance test (Sato, Sabelis, et al., 2013) to identify the matingbehaviour of the guardingmale.We carried out the same procedureusing 3-day-old males and 6-day-old males and replicated thistreatment 148 times in the treatment using 0-day-old males, 148times in the treatment using 3-day-old males and 152 times in thetreatment using 6-day-old males.

To investigate the effect of male age on guarding and malemating behaviour, we compared the proportion of replicates inwhich one of themales guarded a female (the number of leaf arenasin which one of the males guarded a female divided by the totalnumber of leaf arenas) and the proportion of replicates inwhich theguarding male displayed sneaking behaviour (the number of leafarenas in which the mounting male was classified as a sneakerdivided by the number of leaf arenas in which one of the malesguarded a female) among treatments different in male age (seebelow for details on the statistics used).

Mating Behaviour Towards Differently Aged Males

To test for plasticity in mating behaviour of individual males, wecharacterized the mating behaviour of guarding males, confrontedthemwith groups of young or old males, recorded the frequency ofmales that maintained their guarding position and characterizedtheir mating type again (following the method by Sato, Sabelis,et al., 2013). We randomly collected males in the last moultingstage from the male culture (see above for the procedure) and putthem separately onto bean leaf arenas. After checking theirmoulting status on a daily basis, we used the males that moultedwithin a day (0-day-old males; again counting male age in days

since moulting into adulthood) as young males, and used the 6-day-old males as old males. We marked the dorsum of all youngand all old males by using a very small droplet of water-based paintwith different colours to discriminate between younger and oldermales. To rule out an effect of colour on the behaviour of the mites,we randomized the colour coding in each separate experiment. Fiveyoung males and a female in the last moulting stage were intro-duced onto a leaf arena. One hour later, we checked for the pres-ence of a guarding male and identified his mating behaviour usingthe artificial disturbance test (Sato, Sabelis, et al., 2013). Then, weremoved the youngmales except for the guardingmale (focal male)and introduced four old males (intruder) onto the arena. One hourafter the old-male introduction, we checked for the presence of aguarding male, his age (young or old) and his mating behaviourbased on the artificial disturbance test (Sato, Sabelis, et al., 2013).We also conducted the same procedure using old males as the focalmale and young males as the intruders. We replicated this 73 timesin the treatment in which the focal males were young males clas-sified as fighters, 67 times in the treatment inwhich the focal maleswere young males classified as sneakers, and 66 times in thetreatment in which the focal males were old males classified asfighters.

Treatments were compared with respect to the frequency withwhich the focal male succeeded in keeping his guarding position.Some of the males that succeeded in keeping their guarding posi-tion were found to have changed their mating behaviour afterexposure to intruders. We compared the frequency of such changesamong treatments (see below for details on the statistics used).

Statistics

The analyses were carried out with the statistical package Rversion 2.14.2 (The R Foundation for Statistical Computing, Vienna,Austria, http://www.r-project.org). We used Fisher’s exact test (twotailed) to compare the fighter and sneaker genetic lines withrespect to the proportion of replicates in which at least one of themales guarded the female, and the proportion of males displayingfighting behaviour among these guarding males. We calculated thepowers of these Fisher’s exact tests (‘power.fisher.test’ in thepackage ‘statmod’, a ¼ 0.05, nsim ¼ 100), since the number ofreplicates was not large.

To test the effects of male density and male age on guardingbehaviour and on male mating behaviour, we constructed gener-alized linear models with a binomial error distribution. For analysisof the effect of male density on guarding behaviour, we used maledensity as an explanatory variable. Since overdispersion occurred,we used a quasibinomial distribution as the error distributioninstead of a binomial distribution. In the comparison betweenobserved and expected proportions of males displaying guardingbehaviour, we used male density (the number of males introducedonto a leaf arena), data set (observed data from treatments withrival males or expected data calculated from the probability that amale was guarding in the treatment without rival males and thenumber of replicates) and the interaction as explanatory variablesin the model. For analysis of the effect of male density on sneakingbehaviour, we considered squared male density in addition to maledensity as explanatory variables in the model, since a nonlinearrelation between the proportion of sneaking males and male den-sity was observed. For analysis of the effects of male age onguarding behaviour and sneaking behaviour, we used male age (thenumber of days after moulting into adulthood) as an explanatoryvariable and used the binomial error distribution. The effect of eachexplanatory variable was tested by likelihood ratio tests to comparethe model with and without the explanatory variable. We followed

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Figure 2. (a) The proportion of replicates in which one of the available males guardeda female (female in the last moulting stage) and (b) the proportion of sneakers amongthe guarding males, as a function of male density. Filled circles indicate the observedproportion and vertical bars indicate 95% confidence intervals of the observed pro-portions. Open circles indicate the estimated proportions that were calculated basedon observed data in the treatment lacking rival males.

Y. Sato et al. / Animal Behaviour 92 (2014) 125e131 129

Faraway (2006) in these model constructions and likelihood ratiotests.

To compare the frequencies of focal males that succeeded inkeeping their guarding position, and the frequencies of change inmating behaviour between the treatments in which focal maleswere initially classified as young fighting males, young sneakingmales or old fighting males, we used Fisher’s exact probability test(two tailed) with sequential Bonferroni correction for multiplecomparisons.

RESULTS

Heritability of Mating Behaviour

Of 30 males of the fighter genetic line, 19 showed guardingbehaviour; 18 of these were characterized as fighters and one as asneaker. Of 33 males of the sneaker genetic line, 18 showedguarding behaviour; 16 of these were characterized as fighters andtwo as sneakers. There was no significant difference betweenfighter and sneaker genetic lines in the proportion of guardingmales (Fisher’s exact test: P ¼ 0.605) nor in the proportion ofguarding males characterized as fighters (Fisher’s exact test:P ¼ 0.604). The estimated powers of these tests were weak (7%chance of detecting a difference in the proportion of guardingmales, and 5% chance of detecting a difference in the proportion ofguarding males characterized as fighters). However, if the malemating behaviour is determined by one gene locus, the behaviourof males from the fighter or sneaker genetic line is expected to beclassified as fighting or sneaking, respectively, with a probability ofat least 0.75 (Fig. 1). In both genetic lines, only a few males dis-played sneaking behaviour, indicating that sneaking is not commonin the absence of rival males, regardless of genetic differences.

Mating Behaviour at Different Male Densities

As the number of rival males increased, the proportion of rep-licates in which at least one of the males guarded the femaleincreased (Fig. 2a), although the effect of male density on theproportion was not significant (likelihood ratio test: F1 ¼12.706,P ¼ 0.070). However, the observed proportions were significantlylower than the expected proportions, especially whenmale densitywas higher (likelihood ratio test: density*data set: c2

1 ¼ 22:57,P < 0.001; Fig. 2a). When there were no rival males (male density isone), sneaking behaviour was not observed (Fig. 2b). As the numberof rival males increased, the proportion of guarding males dis-playing sneaker behaviour increased with male density in anonlinear way (Fig. 2b): the effects of male density and (maledensity)2 on the proportion of sneakers were significant (likelihoodratio test: (male density)2: c2

1 ¼ 23:377, P < 0.001; male density:c21 ¼ 29:205, P < 0.001).

Mating Behaviour at Different Ages

The proportion of replicates in which at least one of the malesguarded the female slightly increased with male age (Fig. 3a), butthis effect was not significant (likelihood ratio test: c2

1 ¼ 3:029,P ¼ 0.082). As male age increased, the proportion of guardingmalesdisplaying sneaker behaviour decreased significantly (likelihoodratio test: c2

1 ¼ 33:827, P < 0.001; Fig. 3b).

Mating Behaviour Towards Differently Aged Males

The success rate of young fighter males at keeping theirguarding position in the presence of old rival males was signifi-cantly lower than that of old fighter males in the presence of young

rival males (Fig. 4). Note that abandoning the guarding position canalso be seen as losing the contest, because in the absence of rivalsvirtually no males ever did this (Y. Sato, personal observation).Together, this indicates that young males are inferior to old males.However, when young guarding males displayed sneaker behav-iour, the success rate in the presence of old rival males was higherthan that of young fighter males, and similar to the success rate ofold fighter males (Fig. 4).

In young males, changes were observed in mating behaviourafter exposure to old intruders: 40% of the young fighter males thatkept their guarding position changed their mating behaviour tosneaking (Fig. 5). A change inmating behaviour was also sometimesobserved in young sneaker males (to become a fighter), but thefrequency was significantly lower than that in young fighter males(Fig. 5). Changes in mating behaviour were not observed in oldfighter males, that is, all males maintained their fighting behaviour(Fig. 5).

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Figure 3. Relations between male age and (a) the proportion of replicates inwhich oneof the males guarded a female, and (b) the proportion of sneakers among the guardingmales. Filled circles indicate the observed proportion and vertical bars indicate 95%confidence intervals of the observed proportions.

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Figure 4. Proportion of males that succeeded in keeping the guarding position (blackbars), that lost the guarding position to intruders (four rival males; grey bars) and thatstopped guarding in the presence of intruders (i.e. absence of guarding by the focalmale, but also by the intruders; white bars), for young fighter males, young sneakermales and old fighter males. N is number of replicates per treatment; P values indicatethe results of a two-sided Fisher’s exact probability test with sequential Bonferronicorrection in the frequencies of males that succeeded in keeping the guarding position.

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Figure 5. The proportional change in mating behaviour of males that succeeded inkeeping the guarding position (black bars), when there were four intruders as rivals,for young fighter males, young sneaker males and old fighter males. N is number ofreplicates per treatment; P values indicate the results of a two-sided Fisher’s exactprobability test with sequential Bonferroni correction.

Y. Sato et al. / Animal Behaviour 92 (2014) 125e131130

DISCUSSION

We did not find any apparent differences in the proportion offighting males between the genetic lines, nor in the proportion ofmales guarding a female. What is more, in both genetic lines, only afew males displayed sneaking behaviour, indicating that sneakingis not common in the absence of rival males, regardless of geneticdifferences. The display of fighting and sneaking behaviour bymales does depend on the number of rivals present and on the ageof males. When there were no rival males present, all focal malesdisplayed fighting behaviour. However, as the number of rivalmales increased, the proportion of sneakers among guarding malesincreased. We also found that young guarding males displayedsneaking behaviour more frequently than old guarding males.Together with the results showing the absence of apparentmorphological differences (Sato, Sabelis, et al., 2013), these resultssuggest that the occurrence of alternative male mating behavioursin the two-spotted spider mite is caused by switching of matingbehaviour depending on the presence of rivals and on their ownage.

Y. Sato et al. / Animal Behaviour 92 (2014) 125e131 131

Age-dependent and density-dependent male mating behaviourare forms of plasticity that create a life history perspective on malemating decisions. In two-spotted spider mites young males wouldhave a much higher reproductive value than old males becausetheir life history is characterized by relatively long adult lifetimeand overlapping generations. In addition, maleemale contests areintense and can result in injury or even death (Potter et al., 1976a,b;Sato, Egas, Sabelis, & Mochizuki, 2013). Hence, young males mightbe better off by adopting sneaker behaviour, especially when maledensity is high, whereas old males might be better off by adoptingfighting behaviour as only their current reproduction matters toindividual fitness, and especially so if their reproductive success inthe past was low (or absent, as in our experiments with virginmales).

Our experiments also showed that young males could increasethe mating success rate by keeping their guarding position anddisplaying sneaking behaviour. Young males classified as fightersoften failed to keep or gave up their guarding position when oldmales were introduced as intruders, whereas most of the old malesclassified as fighters did succeed in keeping their guarding positionwhen young males were introduced (Fig. 4). However, young malesdisplaying sneaker behaviour were almost as successful as oldmales displaying fighting behaviour (Fig. 4). Furthermore, 40% ofthe youngmales classified as fighters that succeeded in keeping theguarding position switched their mating behaviour from fighting tosneaking when exposed to old males (Fig. 5). These results clearlyshow that alternative male mating behaviour in the two-spottedspider mite is a plastic trait. The mechanism underlying the plas-tic responses of young males to rivals may well be classified asmutual assessment (Arnott & Elwood, 2009), but this needs furtherwork.

For future studies on male mating tactics in the two-spottedspider mite various lines of enquiry present themselves. First, weneed to investigate whether males have higher lifetime reproduc-tive success by displaying sneaker behaviour when young andconfrontedwith old (as opposed to young) rival males.We assumedthat for males sneaking behaviour is less risky than fightingbehaviour. Hence, we predicted that the proportion of sneakermales would increase as the density of male rivals increases. In ourexperiment we indeed found an increase in the proportion ofsneakers among guarding males with an increasing number of ri-vals, but this proportion decreased significantly at the highestnumber of rivals we used (Fig. 2b). We also found that guardingsneaker males can be detected by old males likely to displayfighting behaviour (Fig. 4), and this could explain the decline insneaking behaviour at high male densities. That is, if the number ofaggressive males (i.e. males that would display fighting if theyguarded a female) increases with an increasing number of rivals,the probability that one of them discovers the sneaking male anddrives him off increases rapidly. This prediction remains to betested to assess the risks and costs of sneaking behaviour. Second,there is a need to test other possible explanations of age-dependentmalemating tactics in the two-spotted spider mite. In our study, weassumed that fighting ability does not differ between fighter andsneaker males because there was no significant difference in bodysize or weapon size between them (Sato, Sabelis, et al., 2013).However, the success rate of young, fighter males in keeping theirguarding position was significantly lower than that of old, fightermales (Fig. 4). This may be explained by a low persistence forkeeping the guarding position in youngmales, but possibly also by alow fighting ability in young males. Finally, there may be otherfactors determining winners of fights than the expression of

external morphological features, and this requires scrutiny to testwhether resource-holding potential does not increase with age, aswe assumed. For example, the quality of the cuticle (outer skin) canbe related to competitiveness in maleemale fights. The cuticle of ayoung male may be more easy to penetrate by the opponent’sstylets, since the cuticle of individuals just after emergence isgenerally softer and hardens over time, as in all invertebrates. It isunknown howmuch time it takes until the cuticle gets hard enoughto risk a fight, but it is worth investigating whether or not youngmales are less competitive: their armour may still be too weak.

Acknowledgments

We thank Dr Juan Manuel Alba for the line of spider mites usedfor our study and Professor Robert W. Elwood and Dr IsabelSmallegange for valuable comments on the manuscript. Y.S. wassupported by Grant-in-Aid for JSPS Fellow Grant Number 09J01045.

References

Andersson, M. (1994). Sexual selection. Princeton: NJ Princeton University Press.Arnott, G., & Elwood, R. W. (2009). Assessment of fighting ability in animal contests.

Animal Behaviour, 77, 991e1004.Bertram, S. M. (2000). The influence of age and size on temporal mate signalling

behaviour. Animal Behaviour, 60, 333e339.Boudreaux, H. B. (1963). Biological aspects of some phytophagous mites. Annual

Review of Entomology, 8, 137e154.Brockmann, H. J. (2001). The evolution of alternative strategies and tactics. Advances

in the Study of Behavior, 30, 1e51.Brockmann, H. J., Colson, T., & Potts, W. (1994). Sperm competition in horseshoe

crabs (Limulus polyphemus). Behavioral Ecology and Sociobiology, 35, 153e160.Candolin, U., & Vlieger, L. (2013). Should attractive males sneak: the trade-off be-

tween current and future offspring. PLoS ONE, 8, e57992.Eberhard, W. G. (1982). Beetle horn dimorphism: making the best of a bad lot.

American Naturalist, 119, 420e426.Faraway, J. J. (2006). Texts in statistical science, extending the linear model with R:

Generalized linear, mixed effects and nonparametric regression models. BocaRaton, FL: Chapman & Hall/CRC.

Gross, M. R. (1996). Alternative reproductive strategies and strategies: diversitywithin sexes. Trends in Ecology & Evolution, 11, 92e98.

Heckel, G., & von Helversen, O. (2002). Male tactics and reproductive success in theharem polygynous bat Saccopteryx bilineata. Behavioral Ecology, 13, 750e756.

Helle, W. (1967). Fertilization in the two-spotted spider mite (Tetranychus urticae:Acari). Entomologia Experimentalis et Applicata, 10, 103e110.

Helle, W., & Sabelis, M. W. (1985). Spider mites. Their biology, natural enemies andcontrol (Vol. 1A). Amsterdam, The Netherlands: Elsevier.

Jang, Y. (2011). Male responses to conspecific advertisement signals in the fieldcricket Gryllus rubens (Orthoptera: Gryllidae). PLoS ONE, 6, e16063.

Kemp, D. J. (2002). Sexual selection constrained by life history in a butterfly. Pro-ceedings of the Royal Society B: Biological Sciences, 269, 1341e1345.

Kemp, D. J. (2006). Ageing, reproductive value, and the evolution of lifetime fightingbehaviour. Biological Journal of the Linnean Society, 88, 565e578.

Kokko, H. (1997). Evolutionarily stable strategies of age-dependent sexual adver-tisement. Behavioral Ecology and Sociobiology, 41, 99e107.

Oliveira, R. F., Taborsky, M., & Brockmann, J. H. (2008). Alternative reproductivetactics: An integrative approach. Cambridge, U.K.: Cambridge University Press.

Parker, G. A. (1974). Assessment strategy and the evolution of fighting behaviour.Journal of Theoretical Biology, 47, 223e243.

Potter, D. A., Wrensch, D. L., & Johnston, D. E. (1976a). Guarding, aggressive behavior,and mating success in male twospotted spider mites. Annals of the Entomo-logical Society of America, 69, 707e711.

Potter, D. A., Wrensch, D. L., & Johnston, D. E. (1976b). Aggression and matingsuccess in male spider mites. Science, 193, 160e161.

Radwan, J. (2009). Alternative mating tactics in Acarid mites. Advances in the Studyof Behavior, 39, 185e208.

Roff, D. A. (2001). Life history evolution. Sunderland, MA: Sinauer.Sato, Y., Egas, M., Sabelis, M. W., & Mochizuki, A. (2013). Maleemale aggression

peaks at intermediate relatedness in a social spider mite. Ecology and Evolution,3, 2661e2669.

Sato, Y., Sabelis, M. W., Egas, M., & Faraji, F. (2013). Alternative phenotypes of malemating behaviour in the two-spotted spider mite. Experimental and AppliedAcarology, 61, 31e41.

Tomkins, J. L., & Hazel, W. (2007). The status of the conditional evolutionarily stablestrategy. Trends in Ecology & Evolution, 22, 522e528.