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Alternative male mating behaviour in the two-spotted spider mite: dependence on age and density
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 Netherlands National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, Japan c Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan b
a r t i c l e i n f o Article history: Received 26 August 2013 Initial acceptance 3 September 2013 Final acceptance 10 March 2014 Available online 6 May 2014 MS. number: 13-00706R Keywords: alternative mating tactics conditional strategy fighter maleemale competition mating sneaker Tetranychus urticae
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 by stronger males and to increase their mating chances, but in theory it can be expressed by any male as long as it improves his future reproduction. Recently, we characterized fighting and sneaking behaviour in 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 fighting behaviour involves challenging all rivals. To understand these alternative precopulatory behaviours from a life history perspective, we investigated (1) their heritability, and how they are affected by (2) male density, (3) male age and (4) competition between males differing in age. We established genetic lines for fighting or sneaking by crossing a male that displayed fighting or sneaking behaviour with one of his daughters. In fourth-generation males from these lines, however, the proportions of fighting and sneaking behaviour were not significantly different. The frequency of sneaking behaviour increased with male density, yet decreased with male age. Old males always displayed fighting and rarely lost females they guarded to young males, whereas young fighting males lost females to old rival males more frequently than young sneaking males. Of the young fighting males that did not lose the female they guarded to old males, about 40% switched to sneaking. We conclude that alternative male mating behaviours in this mite are not maintained as a genetic polymorphism, but arise from phenotypic plasticity in response to the male’s environment (male density) and its own condition (age). We hypothesize that young males opt for sneaking behaviour because fighting jeopardises their future reproduction. Ó 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 to reproduce (Andersson, 1994). They usually do this by fighting with rival males and driving them off, but they may also display nonfighting behaviour, such as sneaking and satellite behaviours, so as to increase their mating opportunities (e.g. reviewed in Brockmann, 2001; Gross, 1996; Oliveira, Taborsky, & Brockmann, 2008; Radwan, 2009; Tomkins & Hazel, 2007). Sneaking and satellite behaviours are considered to be alternative mating tactics for males that are likely to lose fights over females against stronger males (e.g. Eberhard, 1982), but in theory these tactics can be adopted by any male as long as they sufficiently reduce injury from maleemale fights, thereby promoting future reproduction. Hence, to maximize their lifetime reproductive success, males may display sneaking
* Correspondence: Y. Sato, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, 1090GE Amsterdam, The Netherlands. E-mail address: [email protected] (Y. Sato).
and satellite behaviours regardless of their current competitiveness. This life history perspective has been investigated theoretically with respect to the evolution of age-dependent male mating tactics (Kemp, 2006; Kokko, 1997). However, few empirical papers have addressed flexible male mating tactics from a life history point of view (Bertram, 2000; Brockmann, Colson, & Potts, 1994; Candolin & Vlieger, 2013; Heckel and von Helversen, 2002; Jang, 2011; Kemp, 2002), even though alternative male mating tactics have 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 the residual reproductive value (the likelihood of future reproduction; Roff, 2001) from the effect of the resource-holding potential (such as energy reserves, strength and body size, which directly affect the probability that males win fights; Parker, 1974) on lifetime reproductive success. For example, fighting or risky mating tactics in old males have been observed in many animals, especially in mammals, birds and reptiles (Kemp, 2006). This phenomenon is consistent
http://dx.doi.org/10.1016/j.anbehav.2014.03.032 0003-3472/Ó 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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with predictions from a life history perspective, because residual reproductive value decreases with age. However, in all these animals, males are likely to grow and become larger even after sexual maturation, indicating that resource-holding potential increases with age. In this case, fighting or risky mating tactics in old males can be explained by resource-holding potential, for example when old males are competitive and tend to win fights. Hence, to test for the effect of a trade-off between current and future reproduction on the evolution of alternative male mating tactics, it would be important to investigate flexible male mating tactics in animals that do not show an apparent increase in resource-holding potential with age (Kemp, 2006). In this study, we investigated flexibility in male mating behaviour and the effects of male age on alternative male mating behaviours in the two-spotted spider mite, Tetranychus urticae Koch. This mite is a serious pest in agriculture, which is why its ecology and behaviour have been studied relatively well (e.g. Helle & Sabelis, 1985). Recently, we found that male spider mites exhibit three 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, leaving this position only to fight off rival males (Potter, Wrensch, & Johnston, 1976a,b). The fighting behaviour is characterized by the male positioning himself directly in front of the rival male and taking up a fighting posture with his main weapon, the cheliceral stylets (needle-like mouthparts normally used for piercing host plant cells) extended and with the first pair of legs lifted up (Potter et al., 1976a,b; see supplementary video in Sato, Sabelis, et al., 2013). We previously called this behaviour ‘territorial’ because it helps the male to monopolize the area with the female (see supplementary video in Sato, Sabelis, et al., 2013). Other males display sneaking behaviour: they also guard females but do not initiate fights with rival males, even when provoked, nor are sneaker males attacked by rivals (Sato, Sabelis, et al., 2013). It is important for the male to keep this position close to the female until she moults to an adult 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, we also observed nonguarding behaviour in some males: they continually search for virgin females just emerging from the last moulting stage. Thus, these males do not guard females and their behaviour is called ‘opportunistic’ (Sato, Sabelis, et al., 2013). Because we have not yet been able to establish a reliable method to assess opportunistic behaviour, we focus here on fighting and sneaking behaviour. In the mite under study, larger males are more likely to win fights (Potter et al., 1976a,b). However, we did not find any apparent differences in body size (body length and body width) or weapon size (lengths of the first legs, stylets and pedipalps) between fighting males and sneaking males (Sato, Sabelis, et al., 2013). This indicates that alternative male mating behaviour cannot be explained only by male competitiveness, and also that male mating behaviours can be flexible because there is no morphological constraint in displaying each of the possible behaviours. In addition, males of the mite mate with females during their entire adult lifetime as generations overlap (Helle & Sabelis, 1985) which means that the residual reproductive value of young males is much higher than that of older males. In addition, maleemale contests are intense and can result in injury or even death (Potter et al., 1976a,b). Taking both these aspects into consideration, young males might be better off by adopting sneaking behaviour. To investigate how flexible the alternative male mating behaviour of the mite is, we measured (1) the heritability of male mating behaviour and (2) the effect of male density on male mating
behaviour in the mite. To investigate life history effects, we measured (3) the effect of male age on male mating behaviour and (4) male mating behaviour when males compete with males different in age. METHODS Life History of the Two-spotted Spider Mite Based on Helle and Sabelis (1985), a brief review is presented on some features of the life history of the two-spotted spider mite, T. urticae, relevant to this paper. As with the majority of spider mites, this species is haplodiploid: females develop from fertilized eggs (2n) and males develop from unfertilized eggs (n). Its developmental rate, oviposition rate and longevity strongly depend on the host plant they feed on and on the temperature they experience. At 21 C on beans, Phaseolus vulgaris L., egg-to-adult development takes 2 weeks; a few days after maturation (short preoviposition period), females lay 10e15 eggs per day and then slow down egg deposition more gradually over a period of ca. 4 weeks (relatively long oviposition period) until they stop a few days before death (short postoviposition period). Usually females produce the sexes in a ratio of two to three daughters to one son. Male development is somewhat faster than that of females and their longevity is about half that of the females. Adult males can mate up to four times per day before their sperm store is depleted. Two-spotted spider mites aggregate in colonies and live inside self-made webs of silk on leaves of their host plants. Colonies usually start from one or a few foundresses and develop until mites overexploit the host plant, at which time the young and mated adult females disperse in search of new host plants to establish new colonies. The duration of this colonization cycle depends on the local conditions for population growth and usually lasts several overlapping generations. Mite Culture To start our culture of T. urticae, a sample population was obtained 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, on wet cotton wool in a plastic box under constant climatic conditions (25 1 C, 60% relative humidity and 16:8 h light:dark photoperiod). Both for rearing mites and for the experiments described below, we always used the first leaves of 1e3-week-old common bean plants. Because the experiments required a large number of males, we prepared a separate culture for males, as follows. We randomly collected 20 females in the last moulting stage from the culture and introduced them together onto a 5 5 cm fragment of bean leaf placed on wet cotton wool in a plastic box. The virgin adult females that developed from these females were allowed to lay unfertilized eggs (which all develop into males) for 1 week, after which the females were removed and the eggs were left to develop. From this culture we collected males in the final moulting stage to be used in the experiments after they moulted. Heritability of Mating Behaviour We established one genetic line for fighting behaviour and one for sneaking behaviour, by crossing a father with one of his daughters (Fig. 1). After randomly collecting 30 males from the mite culture, we introduced them onto a small leaf arena (bean leaf disc 1.5 cm in diameter) on which was a female in the last moulting stage. When we observed a male sitting on the female’s dorsum (i.e. guarding), we identified his mating behaviour by using an artificial
Y. Sato et al. / Animal Behaviour 92 (2014) 125e131
Fighter line Mother (??)
Son (?)
127
Sneaker line X
Father (F )
Daughter (?F)
Granddaughters (?F or FF )
Mother (??)
Son (?)
X
Grandsons (? or F )
Great-grandsons (? or F ) 25%?, 75%F
X
Father (S)
Daughter (?S)
Granddaughters (?S or SS )
X
Grandsons (? or S )
Great-grandsons (? or S ) 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 develop from 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 the alleles expressing fighting and sneaking behaviour, respectively, and ‘?’ indicates unknown allele.
disturbance test whereby we brought the guarding male into contact with another male that was fixed to the tip of a fine wet brush. If the guarding male responded actively towards the male on the brush and showed a fighting posture (extended stylets and first pair of legs lifted up), the behaviour was classified as fighting. If he did not show any response to the male on the brush, the behaviour was classified as sneaking (for details of this classification procedure and proof of its reliability, see Sato, Sabelis, et al., 2013). We introduced one male, either one that displayed fighting or one that displayed sneaking (randomly selected from the males with identified mating behaviour) onto a large leaf arena (5 5 cm bean leaf square) on which was a female in the last moulting stage and allowed the pair to copulate after the adult female had moulted. After the male had copulated, we removed him from the arena and placed him alone on a small leaf arena. Then, we allowed the female to oviposit for 1 week. Once her daughters had reached the final moulting stage, we randomly picked one daughter and, after she had matured, allowed her to copulate with her father in a large arena. We then removed the father and allowed the daughter to oviposit for 1 week in the large arena. We reared the offspring and, once the inbred granddaughters (F ¼ 0.5) had developed into the final moulting stage, we randomly picked 33 granddaughters and introduced each onto a single large leaf arena. These granddaughters were allowed to lay unfertilized (male) eggs for 1 week and we reared the eggs until they developed into adult males. We randomly picked one 1e7-day-old great-grandson from each granddaughter and placed him onto a small leaf arena (a bean leaf disc 1.5 cm in diameter) together with a female in the last moulting stage to assess the male’s behaviour. One hour after introduction of the male, we checked whether the male guarded the female and, if so, assessed his mating behaviour using the artificial disturbance test described above (Sato, Sabelis, et al., 2013). If the male did not show guarding behaviour, we did not subject him to the test and classified the behaviour as nonguarding (i.e. not sneaking or fighting as we defined these behaviours for guarding males). For three great-grandsons of the ‘fighter’ genetic line, we failed to identify the male mating behaviour because the female moulted before the end of the test and had copulated with the male before his mating behaviour could be assessed. These three males were removed from the data set. We compared the ‘fighter’ genetic line and the ‘sneaker’ genetic line with respect to the fraction of
guarding males and the fraction of males displaying fighting behaviour within the category of guarding males (see below for details on the statistics used). Mating Behaviour at Different Male Densities To assess the effect of male density on the sneaking behaviour of focal males, we randomly collected males in the last moulting stage from the male culture and put them individually onto bean leaf discs, 1.5 cm in diameter, placed on wet cotton wool in petri dishes (bean leaf arenas), one disc per dish. One day after they moulted into adult males, we placed one male (treatment without rival male), two males (treatment with one rival male), five males (treatment with four rival males) or 10 males (treatment with nine rival males) onto a leaf arena. Each arena also contained one female in the last moulting stage, which was selected beforehand from our mite culture on the condition that she was accompanied by one or more guarding males. Two hours after introducing the males, we checked whether the female was guarded by a new male, after which we identified the mating behaviour of the guarding male using the artificial disturbance test (Sato, Sabelis, et al., 2013). Because we aimed to assess the mating behaviour of an equal number of guarding males per treatment, we set up replicates until we had obtained 65 observations on guarding males. However, males did not always display guarding and the proportion of guarding males varied with the treatment. Hence, the number of replicates differed per treatment: 182 in the treatment without a rival male, 118 in the treatment with one rival male, 103 in the treatment with four rival males and 84 in the treatment with nine rival males. We compared the proportion of replicates in which one of the males guarded a female (the number of leaf arenas in which one of the males guarded a female divided by the total number of leaf arenas) among treatments differing in male density. Because males do not always display guarding behaviour, the probability that one of the males guards a female increases with the number of males on the arena, even if there is no effect of male density on individual male guarding behaviour. To detect an effect of male density on the observed guarding behaviour, we tested the null hypothesis that individual male behaviour is not affected by male density. To do so, we calculated the expected probability m(x) that at least one of a
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group of x males guards a female by assuming that the guarding behaviour of each male is independent of the presence of other males. Hence, using the observed proportion of replicates in which a single male guards a female (M) in the treatment without rival males as the estimated probability of guarding per male, the expected probability m(x) that at least one of the males guards a female in treatments with x males is calculated as m(x) ¼ 1e(1 M)x. To estimate the effect of the presence of (x 1) rival males on the probability of guarding, we compared the proportion of replicates in which guarding was observed with the expected probabilities m(x) (see below for details on the statistics used). We compared the proportion of replicates in which the guarding males displayed sneaking behaviour (the number of leaf arenas in which the guarding male was classified as a sneaker divided by the number of leaf arenas in which one of the males guarded a female) among treatments differing in male density. To detect an effect of male density on the probability of individual males displaying sneaking behaviour, we tested the proportion of sneakers classified in the different treatments (see below for details on the statistics used). Mating Behaviour at Different Ages We assessed the effect of male age on the probability of sneaking behaviour, by randomly collecting males in the last moulting stage from the male culture (see above for a description of the procedure) and putting them individually onto a small bean leaf arena (bean leaf disc 1.5 cm in diameter). While checking their moulting status on a daily basis, we selected five freshly matured males (i.e. that had moulted within a day; 0-day-old males; we scored male age here in days since moulting to adulthood) and introduced them onto a leaf arena with a female in the last moulting stage, which was selected from our mite culture on the condition that she was guarded there by one or more males. Two hours after mite introduction, we checked whether one of the males guarded the female, after which we used the artificial disturbance test (Sato, Sabelis, et al., 2013) to identify the mating behaviour of the guarding male. We carried out the same procedure using 3-day-old males and 6-day-old males and replicated this treatment 148 times in the treatment using 0-day-old males, 148 times in the treatment using 3-day-old males and 152 times in the treatment using 6-day-old males. To investigate the effect of male age on guarding and male mating behaviour, we compared the proportion of replicates in which one of the males guarded a female (the number of leaf arenas in which one of the males guarded a female divided by the total number of leaf arenas) and the proportion of replicates in which the guarding male displayed sneaking behaviour (the number of leaf arenas in which the mounting male was classified as a sneaker divided by the number of leaf arenas in which one of the males guarded a female) among treatments different in male age (see below for details on the statistics used). Mating Behaviour Towards Differently Aged Males To test for plasticity in mating behaviour of individual males, we characterized the mating behaviour of guarding males, confronted them with groups of young or old males, recorded the frequency of males that maintained their guarding position and characterized their mating type again (following the method by Sato, Sabelis, et al., 2013). We randomly collected males in the last moulting stage from the male culture (see above for the procedure) and put them separately onto bean leaf arenas. After checking their moulting status on a daily basis, we used the males that moulted within a day (0-day-old males; again counting male age in days
since moulting into adulthood) as young males, and used the 6day-old males as old males. We marked the dorsum of all young and all old males by using a very small droplet of water-based paint with different colours to discriminate between younger and older males. To rule out an effect of colour on the behaviour of the mites, we randomized the colour coding in each separate experiment. Five young males and a female in the last moulting stage were introduced onto a leaf arena. One hour later, we checked for the presence of a guarding male and identified his mating behaviour using the artificial disturbance test (Sato, Sabelis, et al., 2013). Then, we removed the young males except for the guarding male (focal male) and introduced four old males (intruder) onto the arena. One hour after the old-male introduction, we checked for the presence of a guarding male, his age (young or old) and his mating behaviour based on the artificial disturbance test (Sato, Sabelis, et al., 2013). We also conducted the same procedure using old males as the focal male and young males as the intruders. We replicated this 73 times in the treatment in which the focal males were young males classified as fighters, 67 times in the treatment in which the focal males were young males classified as sneakers, and 66 times in the treatment in which the focal males were old males classified as fighters. Treatments were compared with respect to the frequency with which the focal male succeeded in keeping his guarding position. Some of the males that succeeded in keeping their guarding position were found to have changed their mating behaviour after exposure to intruders. We compared the frequency of such changes among treatments (see below for details on the statistics used).
Statistics The analyses were carried out with the statistical package R version 2.14.2 (The R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org). We used Fisher’s exact test (two tailed) to compare the fighter and sneaker genetic lines with respect to the proportion of replicates in which at least one of the males guarded the female, and the proportion of males displaying fighting behaviour among these guarding males. We calculated the powers of these Fisher’s exact tests (‘power.fisher.test’ in the package ‘statmod’, a ¼ 0.05, nsim ¼ 100), since the number of replicates was not large. To test the effects of male density and male age on guarding behaviour and on male mating behaviour, we constructed generalized linear models with a binomial error distribution. For analysis of the effect of male density on guarding behaviour, we used male density as an explanatory variable. Since overdispersion occurred, we used a quasibinomial distribution as the error distribution instead of a binomial distribution. In the comparison between observed and expected proportions of males displaying guarding behaviour, we used male density (the number of males introduced onto a leaf arena), data set (observed data from treatments with rival males or expected data calculated from the probability that a male was guarding in the treatment without rival males and the number of replicates) and the interaction as explanatory variables in the model. For analysis of the effect of male density on sneaking behaviour, we considered squared male density in addition to male density as explanatory variables in the model, since a nonlinear relation between the proportion of sneaking males and male density was observed. For analysis of the effects of male age on guarding behaviour and sneaking behaviour, we used male age (the number of days after moulting into adulthood) as an explanatory variable and used the binomial error distribution. The effect of each explanatory variable was tested by likelihood ratio tests to compare the model with and without the explanatory variable. We followed
RESULTS Heritability of Mating Behaviour Of 30 males of the fighter genetic line, 19 showed guarding behaviour; 18 of these were characterized as fighters and one as a sneaker. Of 33 males of the sneaker genetic line, 18 showed guarding behaviour; 16 of these were characterized as fighters and two as sneakers. There was no significant difference between fighter and sneaker genetic lines in the proportion of guarding males (Fisher’s exact test: P ¼ 0.605) nor in the proportion of guarding 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 guarding males, and 5% chance of detecting a difference in the proportion of guarding males characterized as fighters). However, if the male mating behaviour is determined by one gene locus, the behaviour of males from the fighter or sneaker genetic line is expected to be classified as fighting or sneaking, respectively, with a probability of at least 0.75 (Fig. 1). In both genetic lines, only a few males displayed sneaking behaviour, indicating that sneaking is not common in 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 replicates in which at least one of the males guarded the female increased (Fig. 2a), although the effect of male density on the proportion was not significant (likelihood ratio test: F1 ¼ 12.706, P ¼ 0.070). However, the observed proportions were significantly lower than the expected proportions, especially when male density was higher (likelihood ratio test: density*data set: c21 ¼ 22:57, P < 0.001; Fig. 2a). When there were no rival males (male density is one), sneaking behaviour was not observed (Fig. 2b). As the number of rival males increased, the proportion of guarding males displaying sneaker behaviour increased with male density in a nonlinear way (Fig. 2b): the effects of male density and (male density)2 on the proportion of sneakers were significant (likelihood ratio test: (male density)2: c21 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 males guarded the female slightly increased with male age (Fig. 3a), but this effect was not significant (likelihood ratio test: c21 ¼ 3:029, P ¼ 0.082). As male age increased, the proportion of guarding males displaying sneaker behaviour decreased significantly (likelihood ratio test: c21 ¼ 33:827, P < 0.001; Fig. 3b). Mating Behaviour Towards Differently Aged Males The success rate of young fighter males at keeping their guarding position in the presence of old rival males was significantly lower than that of old fighter males in the presence of young
1
129
(a)
0.8
64 / 103 64 / 118
0.6
63 / 84 68 / 182
0.4
0.2
0 0
Fraction of males displaying sneaker behaviour
Faraway (2006) in these model constructions and likelihood ratio tests. To compare the frequencies of focal males that succeeded in keeping their guarding position, and the frequencies of change in mating behaviour between the treatments in which focal males were initially classified as young fighting males, young sneaking males or old fighting males, we used Fisher’s exact probability test (two tailed) with sequential Bonferroni correction for multiple comparisons.
Fraction of replicates a male guarded a female
Y. Sato et al. / Animal Behaviour 92 (2014) 125e131
0.5
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(b) 19 / 64
0.4 12 / 63 0.3
0.2 3 / 64 0.1 0 / 68 0 0
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10
Male density (no. of males per leaf disc) Figure 2. (a) The proportion of replicates in which one of the available males guarded a female (female in the last moulting stage) and (b) the proportion of sneakers among the guarding males, as a function of male density. Filled circles indicate the observed proportion and vertical bars indicate 95% confidence intervals of the observed proportions. Open circles indicate the estimated proportions that were calculated based on observed data in the treatment lacking rival males.
rival males (Fig. 4). Note that abandoning the guarding position can also be seen as losing the contest, because in the absence of rivals virtually 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 behaviour, the success rate in the presence of old rival males was higher than that of young fighter males, and similar to the success rate of old fighter males (Fig. 4). In young males, changes were observed in mating behaviour after exposure to old intruders: 40% of the young fighter males that kept their guarding position changed their mating behaviour to sneaking (Fig. 5). A change in mating behaviour was also sometimes observed in young sneaker males (to become a fighter), but the frequency was significantly lower than that in young fighter males (Fig. 5). Changes in mating behaviour were not observed in old fighter males, that is, all males maintained their fighting behaviour (Fig. 5).
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1
P < 0.001
(a) P < 0.001
0.8 77 / 148
N = 73
67
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79 / 152
Young fighter Old
Young sneaker Old
Old fighter Young
0.6 62 / 148
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Fraction of males displaying sneaker behaviour
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Fraction of replicates a male guarded a female
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Focal male Intruder
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0
Male combination Figure 4. Proportion of males that succeeded in keeping the guarding position (black bars), that lost the guarding position to intruders (four rival males; grey bars) and that stopped guarding in the presence of intruders (i.e. absence of guarding by the focal male, but also by the intruders; white bars), for young fighter males, young sneaker males and old fighter males. N is number of replicates per treatment; P values indicate the results of a two-sided Fisher’s exact probability test with sequential Bonferroni correction in the frequencies of males that succeeded in keeping the guarding position.
0.3 7 / 77
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1 / 79 P < 0.001
0 0
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4 Male age (days)
P < 0.05
6 N = 29
NS 45
60
Young sneaker Old
Old fighter Young
1
Figure 3. Relations between male age and (a) the proportion of replicates in which one of the males guarded a female, and (b) the proportion of sneakers among the guarding males. Filled circles indicate the observed proportion and vertical bars indicate 95% confidence intervals of the observed proportions.
DISCUSSION We did not find any apparent differences in the proportion of fighting males between the genetic lines, nor in the proportion of males guarding a female. What is more, in both genetic lines, only a few males displayed sneaking behaviour, indicating that sneaking is not common in the absence of rival males, regardless of genetic differences. The display of fighting and sneaking behaviour by males does depend on the number of rivals present and on the age of males. When there were no rival males present, all focal males displayed fighting behaviour. However, as the number of rival males increased, the proportion of sneakers among guarding males increased. We also found that young guarding males displayed sneaking behaviour more frequently than old guarding males. Together with the results showing the absence of apparent morphological differences (Sato, Sabelis, et al., 2013), these results suggest that the occurrence of alternative male mating behaviours in the two-spotted spider mite is caused by switching of mating behaviour depending on the presence of rivals and on their own age.
Fraction
0.8
0.6
0.4
0.2
0 Focal male Young fighter Intruder Old
Male combination Figure 5. The proportional change in mating behaviour of males that succeeded in keeping 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 of replicates per treatment; P values indicate the results of a two-sided Fisher’s exact probability test with sequential Bonferroni correction.
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Age-dependent and density-dependent male mating behaviour are forms of plasticity that create a life history perspective on male mating decisions. In two-spotted spider mites young males would have a much higher reproductive value than old males because their life history is characterized by relatively long adult lifetime and overlapping generations. In addition, maleemale contests are intense and can result in injury or even death (Potter et al., 1976a,b; Sato, Egas, Sabelis, & Mochizuki, 2013). Hence, young males might be better off by adopting sneaker behaviour, especially when male density is high, whereas old males might be better off by adopting fighting behaviour as only their current reproduction matters to individual fitness, and especially so if their reproductive success in the past was low (or absent, as in our experiments with virgin males). Our experiments also showed that young males could increase the mating success rate by keeping their guarding position and displaying sneaking behaviour. Young males classified as fighters often failed to keep or gave up their guarding position when old males were introduced as intruders, whereas most of the old males classified as fighters did succeed in keeping their guarding position when young males were introduced (Fig. 4). However, young males displaying sneaker behaviour were almost as successful as old males displaying fighting behaviour (Fig. 4). Furthermore, 40% of the young males classified as fighters that succeeded in keeping the guarding position switched their mating behaviour from fighting to sneaking when exposed to old males (Fig. 5). These results clearly show that alternative male mating behaviour in the two-spotted spider mite is a plastic trait. The mechanism underlying the plastic responses of young males to rivals may well be classified as mutual assessment (Arnott & Elwood, 2009), but this needs further work. For future studies on male mating tactics in the two-spotted spider mite various lines of enquiry present themselves. First, we need to investigate whether males have higher lifetime reproductive success by displaying sneaker behaviour when young and confronted with old (as opposed to young) rival males. We assumed that for males sneaking behaviour is less risky than fighting behaviour. Hence, we predicted that the proportion of sneaker males would increase as the density of male rivals increases. In our experiment we indeed found an increase in the proportion of sneakers among guarding males with an increasing number of rivals, but this proportion decreased significantly at the highest number of rivals we used (Fig. 2b). We also found that guarding sneaker males can be detected by old males likely to display fighting behaviour (Fig. 4), and this could explain the decline in sneaking behaviour at high male densities. That is, if the number of aggressive males (i.e. males that would display fighting if they guarded a female) increases with an increasing number of rivals, the probability that one of them discovers the sneaking male and drives him off increases rapidly. This prediction remains to be tested to assess the risks and costs of sneaking behaviour. Second, there is a need to test other possible explanations of age-dependent male mating tactics in the two-spotted spider mite. In our study, we assumed that fighting ability does not differ between fighter and sneaker males because there was no significant difference in body size or weapon size between them (Sato, Sabelis, et al., 2013). However, the success rate of young, fighter males in keeping their guarding position was significantly lower than that of old, fighter males (Fig. 4). This may be explained by a low persistence for keeping the guarding position in young males, but possibly also by a low fighting ability in young males. Finally, there may be other factors determining winners of fights than the expression of
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external morphological features, and this requires scrutiny to test whether resource-holding potential does not increase with age, as we assumed. For example, the quality of the cuticle (outer skin) can be related to competitiveness in maleemale fights. The cuticle of a young male may be more easy to penetrate by the opponent’s stylets, since the cuticle of individuals just after emergence is generally softer and hardens over time, as in all invertebrates. It is unknown how much time it takes until the cuticle gets hard enough to risk a fight, but it is worth investigating whether or not young males are less competitive: their armour may still be too weak. Acknowledgments We thank Dr Juan Manuel Alba for the line of spider mites used for our study and Professor Robert W. Elwood and Dr Isabel Smallegange for valuable comments on the manuscript. Y.S. was supported 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 between 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. Boca Raton, FL: Chapman & Hall/CRC. Gross, M. R. (1996). Alternative reproductive strategies and strategies: diversity within sexes. Trends in Ecology & Evolution, 11, 92e98. Heckel, G., & von Helversen, O. (2002). Male tactics and reproductive success in the harem 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 and control (Vol. 1A). Amsterdam, The Netherlands: Elsevier. Jang, Y. (2011). Male responses to conspecific advertisement signals in the field cricket Gryllus rubens (Orthoptera: Gryllidae). PLoS ONE, 6, e16063. Kemp, D. J. (2002). Sexual selection constrained by life history in a butterfly. Proceedings of the Royal Society B: Biological Sciences, 269, 1341e1345. Kemp, D. J. (2006). Ageing, reproductive value, and the evolution of lifetime fighting behaviour. Biological Journal of the Linnean Society, 88, 565e578. Kokko, H. (1997). Evolutionarily stable strategies of age-dependent sexual advertisement. Behavioral Ecology and Sociobiology, 41, 99e107. Oliveira, R. F., Taborsky, M., & Brockmann, J. H. (2008). Alternative reproductive tactics: 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 Entomological Society of America, 69, 707e711. Potter, D. A., Wrensch, D. L., & Johnston, D. E. (1976b). Aggression and mating success in male spider mites. Science, 193, 160e161. Radwan, J. (2009). Alternative mating tactics in Acarid mites. Advances in the Study of 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 male mating behaviour in the two-spotted spider mite. Experimental and Applied Acarology, 61, 31e41. Tomkins, J. L., & Hazel, W. (2007). The status of the conditional evolutionarily stable strategy. Trends in Ecology & Evolution, 22, 522e528.
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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 Netherlands National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, Japan c Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan b
a r t i c l e i n f o Article history: Received 26 August 2013 Initial acceptance 3 September 2013 Final acceptance 10 March 2014 Available online 6 May 2014 MS. number: 13-00706R Keywords: alternative mating tactics conditional strategy fighter maleemale competition mating sneaker Tetranychus urticae
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 by stronger males and to increase their mating chances, but in theory it can be expressed by any male as long as it improves his future reproduction. Recently, we characterized fighting and sneaking behaviour in 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 fighting behaviour involves challenging all rivals. To understand these alternative precopulatory behaviours from a life history perspective, we investigated (1) their heritability, and how they are affected by (2) male density, (3) male age and (4) competition between males differing in age. We established genetic lines for fighting or sneaking by crossing a male that displayed fighting or sneaking behaviour with one of his daughters. In fourth-generation males from these lines, however, the proportions of fighting and sneaking behaviour were not significantly different. The frequency of sneaking behaviour increased with male density, yet decreased with male age. Old males always displayed fighting and rarely lost females they guarded to young males, whereas young fighting males lost females to old rival males more frequently than young sneaking males. Of the young fighting males that did not lose the female they guarded to old males, about 40% switched to sneaking. We conclude that alternative male mating behaviours in this mite are not maintained as a genetic polymorphism, but arise from phenotypic plasticity in response to the male’s environment (male density) and its own condition (age). We hypothesize that young males opt for sneaking behaviour because fighting jeopardises their future reproduction. Ó 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 to reproduce (Andersson, 1994). They usually do this by fighting with rival males and driving them off, but they may also display nonfighting behaviour, such as sneaking and satellite behaviours, so as to increase their mating opportunities (e.g. reviewed in Brockmann, 2001; Gross, 1996; Oliveira, Taborsky, & Brockmann, 2008; Radwan, 2009; Tomkins & Hazel, 2007). Sneaking and satellite behaviours are considered to be alternative mating tactics for males that are likely to lose fights over females against stronger males (e.g. Eberhard, 1982), but in theory these tactics can be adopted by any male as long as they sufficiently reduce injury from maleemale fights, thereby promoting future reproduction. Hence, to maximize their lifetime reproductive success, males may display sneaking
* Correspondence: Y. Sato, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, 1090GE Amsterdam, The Netherlands. E-mail address: [email protected] (Y. Sato).
and satellite behaviours regardless of their current competitiveness. This life history perspective has been investigated theoretically with respect to the evolution of age-dependent male mating tactics (Kemp, 2006; Kokko, 1997). However, few empirical papers have addressed flexible male mating tactics from a life history point of view (Bertram, 2000; Brockmann, Colson, & Potts, 1994; Candolin & Vlieger, 2013; Heckel and von Helversen, 2002; Jang, 2011; Kemp, 2002), even though alternative male mating tactics have 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 the residual reproductive value (the likelihood of future reproduction; Roff, 2001) from the effect of the resource-holding potential (such as energy reserves, strength and body size, which directly affect the probability that males win fights; Parker, 1974) on lifetime reproductive success. For example, fighting or risky mating tactics in old males have been observed in many animals, especially in mammals, birds and reptiles (Kemp, 2006). This phenomenon is consistent
http://dx.doi.org/10.1016/j.anbehav.2014.03.032 0003-3472/Ó 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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with predictions from a life history perspective, because residual reproductive value decreases with age. However, in all these animals, males are likely to grow and become larger even after sexual maturation, indicating that resource-holding potential increases with age. In this case, fighting or risky mating tactics in old males can be explained by resource-holding potential, for example when old males are competitive and tend to win fights. Hence, to test for the effect of a trade-off between current and future reproduction on the evolution of alternative male mating tactics, it would be important to investigate flexible male mating tactics in animals that do not show an apparent increase in resource-holding potential with age (Kemp, 2006). In this study, we investigated flexibility in male mating behaviour and the effects of male age on alternative male mating behaviours in the two-spotted spider mite, Tetranychus urticae Koch. This mite is a serious pest in agriculture, which is why its ecology and behaviour have been studied relatively well (e.g. Helle & Sabelis, 1985). Recently, we found that male spider mites exhibit three 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, leaving this position only to fight off rival males (Potter, Wrensch, & Johnston, 1976a,b). The fighting behaviour is characterized by the male positioning himself directly in front of the rival male and taking up a fighting posture with his main weapon, the cheliceral stylets (needle-like mouthparts normally used for piercing host plant cells) extended and with the first pair of legs lifted up (Potter et al., 1976a,b; see supplementary video in Sato, Sabelis, et al., 2013). We previously called this behaviour ‘territorial’ because it helps the male to monopolize the area with the female (see supplementary video in Sato, Sabelis, et al., 2013). Other males display sneaking behaviour: they also guard females but do not initiate fights with rival males, even when provoked, nor are sneaker males attacked by rivals (Sato, Sabelis, et al., 2013). It is important for the male to keep this position close to the female until she moults to an adult 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, we also observed nonguarding behaviour in some males: they continually search for virgin females just emerging from the last moulting stage. Thus, these males do not guard females and their behaviour is called ‘opportunistic’ (Sato, Sabelis, et al., 2013). Because we have not yet been able to establish a reliable method to assess opportunistic behaviour, we focus here on fighting and sneaking behaviour. In the mite under study, larger males are more likely to win fights (Potter et al., 1976a,b). However, we did not find any apparent differences in body size (body length and body width) or weapon size (lengths of the first legs, stylets and pedipalps) between fighting males and sneaking males (Sato, Sabelis, et al., 2013). This indicates that alternative male mating behaviour cannot be explained only by male competitiveness, and also that male mating behaviours can be flexible because there is no morphological constraint in displaying each of the possible behaviours. In addition, males of the mite mate with females during their entire adult lifetime as generations overlap (Helle & Sabelis, 1985) which means that the residual reproductive value of young males is much higher than that of older males. In addition, maleemale contests are intense and can result in injury or even death (Potter et al., 1976a,b). Taking both these aspects into consideration, young males might be better off by adopting sneaking behaviour. To investigate how flexible the alternative male mating behaviour of the mite is, we measured (1) the heritability of male mating behaviour and (2) the effect of male density on male mating
behaviour in the mite. To investigate life history effects, we measured (3) the effect of male age on male mating behaviour and (4) male mating behaviour when males compete with males different in age. METHODS Life History of the Two-spotted Spider Mite Based on Helle and Sabelis (1985), a brief review is presented on some features of the life history of the two-spotted spider mite, T. urticae, relevant to this paper. As with the majority of spider mites, this species is haplodiploid: females develop from fertilized eggs (2n) and males develop from unfertilized eggs (n). Its developmental rate, oviposition rate and longevity strongly depend on the host plant they feed on and on the temperature they experience. At 21 C on beans, Phaseolus vulgaris L., egg-to-adult development takes 2 weeks; a few days after maturation (short preoviposition period), females lay 10e15 eggs per day and then slow down egg deposition more gradually over a period of ca. 4 weeks (relatively long oviposition period) until they stop a few days before death (short postoviposition period). Usually females produce the sexes in a ratio of two to three daughters to one son. Male development is somewhat faster than that of females and their longevity is about half that of the females. Adult males can mate up to four times per day before their sperm store is depleted. Two-spotted spider mites aggregate in colonies and live inside self-made webs of silk on leaves of their host plants. Colonies usually start from one or a few foundresses and develop until mites overexploit the host plant, at which time the young and mated adult females disperse in search of new host plants to establish new colonies. The duration of this colonization cycle depends on the local conditions for population growth and usually lasts several overlapping generations. Mite Culture To start our culture of T. urticae, a sample population was obtained 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, on wet cotton wool in a plastic box under constant climatic conditions (25 1 C, 60% relative humidity and 16:8 h light:dark photoperiod). Both for rearing mites and for the experiments described below, we always used the first leaves of 1e3-week-old common bean plants. Because the experiments required a large number of males, we prepared a separate culture for males, as follows. We randomly collected 20 females in the last moulting stage from the culture and introduced them together onto a 5 5 cm fragment of bean leaf placed on wet cotton wool in a plastic box. The virgin adult females that developed from these females were allowed to lay unfertilized eggs (which all develop into males) for 1 week, after which the females were removed and the eggs were left to develop. From this culture we collected males in the final moulting stage to be used in the experiments after they moulted. Heritability of Mating Behaviour We established one genetic line for fighting behaviour and one for sneaking behaviour, by crossing a father with one of his daughters (Fig. 1). After randomly collecting 30 males from the mite culture, we introduced them onto a small leaf arena (bean leaf disc 1.5 cm in diameter) on which was a female in the last moulting stage. When we observed a male sitting on the female’s dorsum (i.e. guarding), we identified his mating behaviour by using an artificial
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Fighter line Mother (??)
Son (?)
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Sneaker line X
Father (F )
Daughter (?F)
Granddaughters (?F or FF )
Mother (??)
Son (?)
X
Grandsons (? or F )
Great-grandsons (? or F ) 25%?, 75%F
X
Father (S)
Daughter (?S)
Granddaughters (?S or SS )
X
Grandsons (? or S )
Great-grandsons (? or S ) 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 develop from 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 the alleles expressing fighting and sneaking behaviour, respectively, and ‘?’ indicates unknown allele.
disturbance test whereby we brought the guarding male into contact with another male that was fixed to the tip of a fine wet brush. If the guarding male responded actively towards the male on the brush and showed a fighting posture (extended stylets and first pair of legs lifted up), the behaviour was classified as fighting. If he did not show any response to the male on the brush, the behaviour was classified as sneaking (for details of this classification procedure and proof of its reliability, see Sato, Sabelis, et al., 2013). We introduced one male, either one that displayed fighting or one that displayed sneaking (randomly selected from the males with identified mating behaviour) onto a large leaf arena (5 5 cm bean leaf square) on which was a female in the last moulting stage and allowed the pair to copulate after the adult female had moulted. After the male had copulated, we removed him from the arena and placed him alone on a small leaf arena. Then, we allowed the female to oviposit for 1 week. Once her daughters had reached the final moulting stage, we randomly picked one daughter and, after she had matured, allowed her to copulate with her father in a large arena. We then removed the father and allowed the daughter to oviposit for 1 week in the large arena. We reared the offspring and, once the inbred granddaughters (F ¼ 0.5) had developed into the final moulting stage, we randomly picked 33 granddaughters and introduced each onto a single large leaf arena. These granddaughters were allowed to lay unfertilized (male) eggs for 1 week and we reared the eggs until they developed into adult males. We randomly picked one 1e7-day-old great-grandson from each granddaughter and placed him onto a small leaf arena (a bean leaf disc 1.5 cm in diameter) together with a female in the last moulting stage to assess the male’s behaviour. One hour after introduction of the male, we checked whether the male guarded the female and, if so, assessed his mating behaviour using the artificial disturbance test described above (Sato, Sabelis, et al., 2013). If the male did not show guarding behaviour, we did not subject him to the test and classified the behaviour as nonguarding (i.e. not sneaking or fighting as we defined these behaviours for guarding males). For three great-grandsons of the ‘fighter’ genetic line, we failed to identify the male mating behaviour because the female moulted before the end of the test and had copulated with the male before his mating behaviour could be assessed. These three males were removed from the data set. We compared the ‘fighter’ genetic line and the ‘sneaker’ genetic line with respect to the fraction of
guarding males and the fraction of males displaying fighting behaviour within the category of guarding males (see below for details on the statistics used). Mating Behaviour at Different Male Densities To assess the effect of male density on the sneaking behaviour of focal males, we randomly collected males in the last moulting stage from the male culture and put them individually onto bean leaf discs, 1.5 cm in diameter, placed on wet cotton wool in petri dishes (bean leaf arenas), one disc per dish. One day after they moulted into adult males, we placed one male (treatment without rival male), two males (treatment with one rival male), five males (treatment with four rival males) or 10 males (treatment with nine rival males) onto a leaf arena. Each arena also contained one female in the last moulting stage, which was selected beforehand from our mite culture on the condition that she was accompanied by one or more guarding males. Two hours after introducing the males, we checked whether the female was guarded by a new male, after which we identified the mating behaviour of the guarding male using the artificial disturbance test (Sato, Sabelis, et al., 2013). Because we aimed to assess the mating behaviour of an equal number of guarding males per treatment, we set up replicates until we had obtained 65 observations on guarding males. However, males did not always display guarding and the proportion of guarding males varied with the treatment. Hence, the number of replicates differed per treatment: 182 in the treatment without a rival male, 118 in the treatment with one rival male, 103 in the treatment with four rival males and 84 in the treatment with nine rival males. We compared the proportion of replicates in which one of the males guarded a female (the number of leaf arenas in which one of the males guarded a female divided by the total number of leaf arenas) among treatments differing in male density. Because males do not always display guarding behaviour, the probability that one of the males guards a female increases with the number of males on the arena, even if there is no effect of male density on individual male guarding behaviour. To detect an effect of male density on the observed guarding behaviour, we tested the null hypothesis that individual male behaviour is not affected by male density. To do so, we calculated the expected probability m(x) that at least one of a
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group of x males guards a female by assuming that the guarding behaviour of each male is independent of the presence of other males. Hence, using the observed proportion of replicates in which a single male guards a female (M) in the treatment without rival males as the estimated probability of guarding per male, the expected probability m(x) that at least one of the males guards a female in treatments with x males is calculated as m(x) ¼ 1e(1 M)x. To estimate the effect of the presence of (x 1) rival males on the probability of guarding, we compared the proportion of replicates in which guarding was observed with the expected probabilities m(x) (see below for details on the statistics used). We compared the proportion of replicates in which the guarding males displayed sneaking behaviour (the number of leaf arenas in which the guarding male was classified as a sneaker divided by the number of leaf arenas in which one of the males guarded a female) among treatments differing in male density. To detect an effect of male density on the probability of individual males displaying sneaking behaviour, we tested the proportion of sneakers classified in the different treatments (see below for details on the statistics used). Mating Behaviour at Different Ages We assessed the effect of male age on the probability of sneaking behaviour, by randomly collecting males in the last moulting stage from the male culture (see above for a description of the procedure) and putting them individually onto a small bean leaf arena (bean leaf disc 1.5 cm in diameter). While checking their moulting status on a daily basis, we selected five freshly matured males (i.e. that had moulted within a day; 0-day-old males; we scored male age here in days since moulting to adulthood) and introduced them onto a leaf arena with a female in the last moulting stage, which was selected from our mite culture on the condition that she was guarded there by one or more males. Two hours after mite introduction, we checked whether one of the males guarded the female, after which we used the artificial disturbance test (Sato, Sabelis, et al., 2013) to identify the mating behaviour of the guarding male. We carried out the same procedure using 3-day-old males and 6-day-old males and replicated this treatment 148 times in the treatment using 0-day-old males, 148 times in the treatment using 3-day-old males and 152 times in the treatment using 6-day-old males. To investigate the effect of male age on guarding and male mating behaviour, we compared the proportion of replicates in which one of the males guarded a female (the number of leaf arenas in which one of the males guarded a female divided by the total number of leaf arenas) and the proportion of replicates in which the guarding male displayed sneaking behaviour (the number of leaf arenas in which the mounting male was classified as a sneaker divided by the number of leaf arenas in which one of the males guarded a female) among treatments different in male age (see below for details on the statistics used). Mating Behaviour Towards Differently Aged Males To test for plasticity in mating behaviour of individual males, we characterized the mating behaviour of guarding males, confronted them with groups of young or old males, recorded the frequency of males that maintained their guarding position and characterized their mating type again (following the method by Sato, Sabelis, et al., 2013). We randomly collected males in the last moulting stage from the male culture (see above for the procedure) and put them separately onto bean leaf arenas. After checking their moulting status on a daily basis, we used the males that moulted within a day (0-day-old males; again counting male age in days
since moulting into adulthood) as young males, and used the 6day-old males as old males. We marked the dorsum of all young and all old males by using a very small droplet of water-based paint with different colours to discriminate between younger and older males. To rule out an effect of colour on the behaviour of the mites, we randomized the colour coding in each separate experiment. Five young males and a female in the last moulting stage were introduced onto a leaf arena. One hour later, we checked for the presence of a guarding male and identified his mating behaviour using the artificial disturbance test (Sato, Sabelis, et al., 2013). Then, we removed the young males except for the guarding male (focal male) and introduced four old males (intruder) onto the arena. One hour after the old-male introduction, we checked for the presence of a guarding male, his age (young or old) and his mating behaviour based on the artificial disturbance test (Sato, Sabelis, et al., 2013). We also conducted the same procedure using old males as the focal male and young males as the intruders. We replicated this 73 times in the treatment in which the focal males were young males classified as fighters, 67 times in the treatment in which the focal males were young males classified as sneakers, and 66 times in the treatment in which the focal males were old males classified as fighters. Treatments were compared with respect to the frequency with which the focal male succeeded in keeping his guarding position. Some of the males that succeeded in keeping their guarding position were found to have changed their mating behaviour after exposure to intruders. We compared the frequency of such changes among treatments (see below for details on the statistics used).
Statistics The analyses were carried out with the statistical package R version 2.14.2 (The R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org). We used Fisher’s exact test (two tailed) to compare the fighter and sneaker genetic lines with respect to the proportion of replicates in which at least one of the males guarded the female, and the proportion of males displaying fighting behaviour among these guarding males. We calculated the powers of these Fisher’s exact tests (‘power.fisher.test’ in the package ‘statmod’, a ¼ 0.05, nsim ¼ 100), since the number of replicates was not large. To test the effects of male density and male age on guarding behaviour and on male mating behaviour, we constructed generalized linear models with a binomial error distribution. For analysis of the effect of male density on guarding behaviour, we used male density as an explanatory variable. Since overdispersion occurred, we used a quasibinomial distribution as the error distribution instead of a binomial distribution. In the comparison between observed and expected proportions of males displaying guarding behaviour, we used male density (the number of males introduced onto a leaf arena), data set (observed data from treatments with rival males or expected data calculated from the probability that a male was guarding in the treatment without rival males and the number of replicates) and the interaction as explanatory variables in the model. For analysis of the effect of male density on sneaking behaviour, we considered squared male density in addition to male density as explanatory variables in the model, since a nonlinear relation between the proportion of sneaking males and male density was observed. For analysis of the effects of male age on guarding behaviour and sneaking behaviour, we used male age (the number of days after moulting into adulthood) as an explanatory variable and used the binomial error distribution. The effect of each explanatory variable was tested by likelihood ratio tests to compare the model with and without the explanatory variable. We followed
RESULTS Heritability of Mating Behaviour Of 30 males of the fighter genetic line, 19 showed guarding behaviour; 18 of these were characterized as fighters and one as a sneaker. Of 33 males of the sneaker genetic line, 18 showed guarding behaviour; 16 of these were characterized as fighters and two as sneakers. There was no significant difference between fighter and sneaker genetic lines in the proportion of guarding males (Fisher’s exact test: P ¼ 0.605) nor in the proportion of guarding 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 guarding males, and 5% chance of detecting a difference in the proportion of guarding males characterized as fighters). However, if the male mating behaviour is determined by one gene locus, the behaviour of males from the fighter or sneaker genetic line is expected to be classified as fighting or sneaking, respectively, with a probability of at least 0.75 (Fig. 1). In both genetic lines, only a few males displayed sneaking behaviour, indicating that sneaking is not common in 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 replicates in which at least one of the males guarded the female increased (Fig. 2a), although the effect of male density on the proportion was not significant (likelihood ratio test: F1 ¼ 12.706, P ¼ 0.070). However, the observed proportions were significantly lower than the expected proportions, especially when male density was higher (likelihood ratio test: density*data set: c21 ¼ 22:57, P < 0.001; Fig. 2a). When there were no rival males (male density is one), sneaking behaviour was not observed (Fig. 2b). As the number of rival males increased, the proportion of guarding males displaying sneaker behaviour increased with male density in a nonlinear way (Fig. 2b): the effects of male density and (male density)2 on the proportion of sneakers were significant (likelihood ratio test: (male density)2: c21 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 males guarded the female slightly increased with male age (Fig. 3a), but this effect was not significant (likelihood ratio test: c21 ¼ 3:029, P ¼ 0.082). As male age increased, the proportion of guarding males displaying sneaker behaviour decreased significantly (likelihood ratio test: c21 ¼ 33:827, P < 0.001; Fig. 3b). Mating Behaviour Towards Differently Aged Males The success rate of young fighter males at keeping their guarding position in the presence of old rival males was significantly lower than that of old fighter males in the presence of young
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Faraway (2006) in these model constructions and likelihood ratio tests. To compare the frequencies of focal males that succeeded in keeping their guarding position, and the frequencies of change in mating behaviour between the treatments in which focal males were initially classified as young fighting males, young sneaking males or old fighting males, we used Fisher’s exact probability test (two tailed) with sequential Bonferroni correction for multiple comparisons.
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Male density (no. of males per leaf disc) Figure 2. (a) The proportion of replicates in which one of the available males guarded a female (female in the last moulting stage) and (b) the proportion of sneakers among the guarding males, as a function of male density. Filled circles indicate the observed proportion and vertical bars indicate 95% confidence intervals of the observed proportions. Open circles indicate the estimated proportions that were calculated based on observed data in the treatment lacking rival males.
rival males (Fig. 4). Note that abandoning the guarding position can also be seen as losing the contest, because in the absence of rivals virtually 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 behaviour, the success rate in the presence of old rival males was higher than that of young fighter males, and similar to the success rate of old fighter males (Fig. 4). In young males, changes were observed in mating behaviour after exposure to old intruders: 40% of the young fighter males that kept their guarding position changed their mating behaviour to sneaking (Fig. 5). A change in mating behaviour was also sometimes observed in young sneaker males (to become a fighter), but the frequency was significantly lower than that in young fighter males (Fig. 5). Changes in mating behaviour were not observed in old fighter males, that is, all males maintained their fighting behaviour (Fig. 5).
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Male combination Figure 4. Proportion of males that succeeded in keeping the guarding position (black bars), that lost the guarding position to intruders (four rival males; grey bars) and that stopped guarding in the presence of intruders (i.e. absence of guarding by the focal male, but also by the intruders; white bars), for young fighter males, young sneaker males and old fighter males. N is number of replicates per treatment; P values indicate the results of a two-sided Fisher’s exact probability test with sequential Bonferroni correction in the frequencies of males that succeeded in keeping the guarding position.
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Figure 3. Relations between male age and (a) the proportion of replicates in which one of the males guarded a female, and (b) the proportion of sneakers among the guarding males. Filled circles indicate the observed proportion and vertical bars indicate 95% confidence intervals of the observed proportions.
DISCUSSION We did not find any apparent differences in the proportion of fighting males between the genetic lines, nor in the proportion of males guarding a female. What is more, in both genetic lines, only a few males displayed sneaking behaviour, indicating that sneaking is not common in the absence of rival males, regardless of genetic differences. The display of fighting and sneaking behaviour by males does depend on the number of rivals present and on the age of males. When there were no rival males present, all focal males displayed fighting behaviour. However, as the number of rival males increased, the proportion of sneakers among guarding males increased. We also found that young guarding males displayed sneaking behaviour more frequently than old guarding males. Together with the results showing the absence of apparent morphological differences (Sato, Sabelis, et al., 2013), these results suggest that the occurrence of alternative male mating behaviours in the two-spotted spider mite is caused by switching of mating behaviour depending on the presence of rivals and on their own age.
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Male combination Figure 5. The proportional change in mating behaviour of males that succeeded in keeping 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 of replicates per treatment; P values indicate the results of a two-sided Fisher’s exact probability test with sequential Bonferroni correction.
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Age-dependent and density-dependent male mating behaviour are forms of plasticity that create a life history perspective on male mating decisions. In two-spotted spider mites young males would have a much higher reproductive value than old males because their life history is characterized by relatively long adult lifetime and overlapping generations. In addition, maleemale contests are intense and can result in injury or even death (Potter et al., 1976a,b; Sato, Egas, Sabelis, & Mochizuki, 2013). Hence, young males might be better off by adopting sneaker behaviour, especially when male density is high, whereas old males might be better off by adopting fighting behaviour as only their current reproduction matters to individual fitness, and especially so if their reproductive success in the past was low (or absent, as in our experiments with virgin males). Our experiments also showed that young males could increase the mating success rate by keeping their guarding position and displaying sneaking behaviour. Young males classified as fighters often failed to keep or gave up their guarding position when old males were introduced as intruders, whereas most of the old males classified as fighters did succeed in keeping their guarding position when young males were introduced (Fig. 4). However, young males displaying sneaker behaviour were almost as successful as old males displaying fighting behaviour (Fig. 4). Furthermore, 40% of the young males classified as fighters that succeeded in keeping the guarding position switched their mating behaviour from fighting to sneaking when exposed to old males (Fig. 5). These results clearly show that alternative male mating behaviour in the two-spotted spider mite is a plastic trait. The mechanism underlying the plastic responses of young males to rivals may well be classified as mutual assessment (Arnott & Elwood, 2009), but this needs further work. For future studies on male mating tactics in the two-spotted spider mite various lines of enquiry present themselves. First, we need to investigate whether males have higher lifetime reproductive success by displaying sneaker behaviour when young and confronted with old (as opposed to young) rival males. We assumed that for males sneaking behaviour is less risky than fighting behaviour. Hence, we predicted that the proportion of sneaker males would increase as the density of male rivals increases. In our experiment we indeed found an increase in the proportion of sneakers among guarding males with an increasing number of rivals, but this proportion decreased significantly at the highest number of rivals we used (Fig. 2b). We also found that guarding sneaker males can be detected by old males likely to display fighting behaviour (Fig. 4), and this could explain the decline in sneaking behaviour at high male densities. That is, if the number of aggressive males (i.e. males that would display fighting if they guarded a female) increases with an increasing number of rivals, the probability that one of them discovers the sneaking male and drives him off increases rapidly. This prediction remains to be tested to assess the risks and costs of sneaking behaviour. Second, there is a need to test other possible explanations of age-dependent male mating tactics in the two-spotted spider mite. In our study, we assumed that fighting ability does not differ between fighter and sneaker males because there was no significant difference in body size or weapon size between them (Sato, Sabelis, et al., 2013). However, the success rate of young, fighter males in keeping their guarding position was significantly lower than that of old, fighter males (Fig. 4). This may be explained by a low persistence for keeping the guarding position in young males, but possibly also by a low fighting ability in young males. Finally, there may be other factors determining winners of fights than the expression of
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external morphological features, and this requires scrutiny to test whether resource-holding potential does not increase with age, as we assumed. For example, the quality of the cuticle (outer skin) can be related to competitiveness in maleemale fights. The cuticle of a young male may be more easy to penetrate by the opponent’s stylets, since the cuticle of individuals just after emergence is generally softer and hardens over time, as in all invertebrates. It is unknown how much time it takes until the cuticle gets hard enough to risk a fight, but it is worth investigating whether or not young males are less competitive: their armour may still be too weak. Acknowledgments We thank Dr Juan Manuel Alba for the line of spider mites used for our study and Professor Robert W. Elwood and Dr Isabel Smallegange for valuable comments on the manuscript. Y.S. was supported 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 between 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. Boca Raton, FL: Chapman & Hall/CRC. Gross, M. R. (1996). Alternative reproductive strategies and strategies: diversity within sexes. Trends in Ecology & Evolution, 11, 92e98. Heckel, G., & von Helversen, O. (2002). Male tactics and reproductive success in the harem 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 and control (Vol. 1A). Amsterdam, The Netherlands: Elsevier. Jang, Y. (2011). Male responses to conspecific advertisement signals in the field cricket Gryllus rubens (Orthoptera: Gryllidae). PLoS ONE, 6, e16063. Kemp, D. J. (2002). Sexual selection constrained by life history in a butterfly. Proceedings of the Royal Society B: Biological Sciences, 269, 1341e1345. Kemp, D. J. (2006). Ageing, reproductive value, and the evolution of lifetime fighting behaviour. Biological Journal of the Linnean Society, 88, 565e578. Kokko, H. (1997). Evolutionarily stable strategies of age-dependent sexual advertisement. Behavioral Ecology and Sociobiology, 41, 99e107. Oliveira, R. F., Taborsky, M., & Brockmann, J. H. (2008). Alternative reproductive tactics: 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 Entomological Society of America, 69, 707e711. Potter, D. A., Wrensch, D. L., & Johnston, D. E. (1976b). Aggression and mating success in male spider mites. Science, 193, 160e161. Radwan, J. (2009). Alternative mating tactics in Acarid mites. Advances in the Study of 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 male mating behaviour in the two-spotted spider mite. Experimental and Applied Acarology, 61, 31e41. Tomkins, J. L., & Hazel, W. (2007). The status of the conditional evolutionarily stable strategy. Trends in Ecology & Evolution, 22, 522e528.