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Metabolic efficiency in courtship favors males with intermediate mass in the Australian redback spider, Latrodectus hasselti
Journal of Insect Physiology 72 (2015) 35–42
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Metabolic efficiency in courtship favors males with intermediate mass in the Australian redback spider, Latrodectus hasselti Paul A. De Luca ⇑, Jeffrey A. Stoltz, Maydianne C.B. Andrade, Andrew C. Mason Integrative Behaviour and Neuroscience Group, University of Toronto at Scarborough, Toronto, Ontario M1C 1A4, Canada
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Article history: Received 24 October 2014 Received in revised form 20 November 2014 Accepted 22 November 2014 Available online 29 November 2014 Keywords: Web-borne vibrations Metabolic efficiency Duty cycle Capital breeder Signal energy
a b s t r a c t Recent studies have suggested that metabolic efficiency may be an important factor in male mating success when females require vigorous and/or prolonged courtship. In capital breeding animals in which a male’s resource pool is fixed at adulthood the relationship between energy expenditure and courtship performance may be especially important, as males are expected to utilize their finite resources efficiently when soliciting mates. Males may benefit from being efficient, i.e., achieving a sufficiently high level of courtship signaling at low energetic cost, if it enables them to acquire mates before their limited energy reserves are depleted. We investigated the relationship between metabolic efficiency and courtship vibrational signaling in the Australian redback spider, Latrodectus hasselti, a semelparous capital breeder where males invest heavily in courtship to secure a mating. We assessed metabolic rate in a sample of males and measured two courtship components (duty cycle and amplitude) that reflected the energy content of web-borne vibrations. We then calculated two indices of metabolic efficiency for these courtship properties. There was a quadratic relationship between mass and duty cycle such that the highest duty cycle signals were performed by males having intermediate mass. Furthermore, intermediate-mass males were also the most metabolically efficient. Prolonged courtship is necessary in L. hasselti for successful mating, and the results of this study suggest that intermediate-mass males are superior courters because they utilize their finite resource pool most efficiently to produce high energy vibrational signals. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Courtship is an essential precursor to mating for many species (Andersson, 1994). A substantial body of evidence shows that high courtship activity is often associated with increased mating success largely because females are more likely to mate after a vigorous courtship (Carranza and Trucios, 1993; Cook et al., 2013; De Luca and Cocroft, 2008; Knapp and Kovach, 1991; Kotiaho, 2002; Parker, 1974; Stapley, 2008). Much research has focused on examining physiological traits that influence courtship activity, to better understand links between male performance and female choice. One trait that has been the focus of some recent research is the active metabolic rate (AMR), which reflects the amount of energy a male expends while soliciting mates (Gillooly and Ophir, 2010; Kasumovic and Seebacher, 2013; Reinhold, 1999; Stoddard and Salazar, 2011). Generally, high AMR is positively correlated with the level of courtship activity ⇑ Corresponding author at: Department of Biology, Ithaca College, Ithaca, NY 14850, USA. Tel.: +1 607 274 1086; fax: +1 607 274 1131. E-mail address: [email protected] (P.A. De Luca). http://dx.doi.org/10.1016/j.jinsphys.2014.11.004 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
because displays that are louder, longer, or both, require more energy to produce, and females tend to prefer males that display at these higher levels (Bailey et al., 1993; Halliday, 1987; Kotiaho et al., 1998; Prestwich, 1994; Reinhold et al., 1998; Wells and Taigen, 1989). Related to AMR is metabolic efficiency, which measures energy expenditure per unit of courtship activity (Bailey et al., 1993; Watson and Lighton, 1994). In a recent review, Hill (2011) suggested that the most metabolically efficient males (i.e., those achieving a sufficiently high level of courtship signaling at low energetic cost), are likely to represent superior courters because such males demonstrate a capacity to optimize energy expenditure between advertisement and somatic maintenance (Stoddard and Salazar, 2011; Thomson et al., 2014; Watson and Lighton, 1994; Watt, 1986). Uncovering how factors such as AMR, metabolic efficiency, and courtship performance interact can be challenging, though, because male energetic rates during courtship may fluctuate over a male’s reproductive lifetime due to changes in body condition or energy reserves (Sadd et al., 2000; White et al., 2013), reproductive status (Clutton-Brock, 1984; Clutton-Brock and Albon, 1979), or social conditions (Candolin, 2000; Stoltz et al., 2012).
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Some of these complexities may be simplified in taxa where males have a fixed resource pool at adulthood that is not replenished (capital breeders, Stearns, 1992), and limits on the availability of females predicts maximal performance with no trade-offs for future mating opportunities (e.g., monogyny, Fromhage et al., 2005). In such systems, because males will usually encounter only one female in their lifetime (Andrade, 2003), all their energy reserves should be expended, if necessary, to secure a mating. At the same time, however, a finite resource pool necessitates efficient allocation of energy reserves throughout the mate acquisition process (i.e., searching, courtship, and mating) in order to ensure that resources are not depleted before a male has performed sufficient courtship to be accepted by the female (Hill, 2011). Accordingly, this predicts a positive relationship between energy reserves and metabolic efficiency for males most likely to be successful at obtaining a mate. For example, in the Sierra dome spider, Linyphia litigiosa, body mass is positively correlated with two complementary measures of courtship metabolic efficiency, and large mass, high efficiency males are successful in aggressive encounters with other males and at siring more offspring (Watson and Lighton, 1994). Here, we examine links between metabolic rate, male phenotype, and courtship performance in monogynous, capital-breeding male redback spiders (Latrodectus hasselti). Upon reaching sexual maturity, male L. hasselti abandon their webs in search of females. During this time they cease foraging and must rely on energy reserves accumulated during development, which severely limits their energetic budget and contributes to a mate-search mortality rate exceeding 80% (Andrade, 2003). Field-caught adult males vary considerably in size and mass (Andrade, 2003), as do males reared in the laboratory under uniform conditions and fed the same diet (Stoltz et al., 2008), which suggests that inherent differences among males in their ability to accumulate and use energy reserves may be an important factor influencing variability in mate searching success. When a female is found, a male will not abandon her in search of another (Andrade, 2003; Andrade and Kasumovic, 2005), and males that succeed in mating are eaten by the female and so will not have another opportunity to mate (Forster, 1992). Male fitness therefore depends entirely on a single mating event, and thus males are expected to expend their total energy reserve towards securing that sole reproductive opportunity. However, following mate-searching males must engage in a lengthy courtship display that involves producing web-borne vibrational signals for a minimum of 100 min to secure a mating (Stoltz and Andrade, 2010). Even if males mate successfully, courtship duration determines the female’s likelihood of remating with another male (Stoltz et al., 2009). This is perhaps why courtship in this species often includes up to 5 h of vibrational signaling and movement on the female’s web (Forster, 1995). Vibrational signaling is expected to be an energetically demanding form of communication (Kotiaho et al., 1998; Lighton, 1987; Randall, 2014), and the extended courtship of male L. hasselti suggests performance may be limited by energetic expenditure. Consistent with this, a continuous 3-h bout of courtship significantly decreases male longevity, and males initially in poor condition suffer the highest longevity cost (Kasumovic et al., 2009). While duration is indeed an important courtship trait in this species, the role of signal energy is less well understood, yet we expect it to also comprise an important courtship feature in this species. High energy signals are expected to be more attractive to females because they provide more stimulation to her sensory system (Morris et al., 1978; Stoddard and Salazar, 2011), and that is likely to be critical here. Latrodectus females’ webs vary considerably in size, with some that are very large relative to the size of the spiders (Szlep, 1965; M. Andrade, personal communication). A mate searching male entering a female’s web is unlikely to know
her exact position and therefore would be expected produce high energy signals in order to elicit and maintain her attention until she can be found (Bailey et al., 1990; Brenowitz, 1986; Maklakov et al., 2003). This is not a foregone conclusion though, as a recent study of a congener suggested that males may produce low energy ‘whispers’ during the initial phase of courtship to distinguish them from struggling prey (Vibert et al., 2014). Clearly, more data is needed to adequately assess the role of signal energy on courtship performance in Latrodectus spiders. In this study, we investigate the relationships between metabolic rate, male phenotype and courtship vibrational signaling in L. hasselti using stop-flow gas respirometry and laser Doppler vibrometry. We first examine the metabolic expenditure of males during courtship compared to resting to determine if courtship vibrational signaling is energetically demanding. We then quantify courtship activity by measuring two components of web-borne vibrations related to the energy content of signals: (1) duty cycle (proportion of time spent producing vibrational signals during a courtship bout), and (2) amplitude (intensity or ‘loudness’ of courtship vibrations). We then use these properties to calculate a male’s metabolic efficiency (amount of energy expended per unit of courtship activity). Finally, we assess potential associations between metabolic efficiency and two aspects of male phenotype (body size and mass) to test the hypothesis that in L. hasselti larger males represent superior courters because they are most metabolically efficient at producing high energy vibrational signals. 2. Materials and methods 2.1. Spider rearing and maintenance We used spiders that were derived from an outbred laboratoryreared population established with individuals collected in the field from New South Wales, Australia in 2007, and replenished with additional wild-caught spiders in 2009. Spiderlings were held with siblings in clear plastic cages (9 9 11 cm, Amac Plastics) in a temperature-controlled room at the University of Toronto Scarborough at 25 °C on a 12:12 light:dark cycle. We removed spiderlings at the 4th instar to ensure they remained virgins (males become sexually mature at the 5th instar and females around the 7–8th) and kept them individually in plastic containers (9 9 11 cm). We fed spiderlings with flies (Drosophila melanogaster) twice a week, and fed adult females immature house crickets (Acheta domesticus) once per week. 2.2. Metabolic rate measurements We used expired CO2 as a measure of metabolic rate (Stoltz et al., 2012). For each male in our experiment, we measured metabolic rates during rest and during courtship in two separate sessions performed on the same day. We conducted experiments in a laboratory at the University of Toronto Scarborough from January to March, 2010. All measurements were taken in a temperature controlled room (25 °C) under red light during the central 8 h of a 12 h scotophase. We took metabolic measures using stop-flow gas respirometry with a Qubit systems (Kingston, Ontario, Canada) 8 channel gas controller (G245), a Qubit systems 8 channel gas switcher (GS244) and a Li-Cor (Nebraska, USA) CO2 analyzer (LI-6252) where incoming CO2 was removed by filtering the air through soda lime. The test chamber consisted of a cylindrical glass tube (12 2.5 cm) sealed at both ends with steel plugs and rubber o-rings. Briefly, for each trial, air was flushed into the chamber and kept there for a sampling period that lasted either 70 s (courtship) or 3 min (resting). At the end of each sampling period the air was flushed from the chamber and flowed through a magnesium
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perchlorate plug to remove moisture before entering the CO2 analyzer. We configured the system to measure CO2 concentration within the test chamber at regular intervals (approximately every 4 min) while a male was performing courtship behavior. The flow rate was set to 160 ml/min and this did not appear to disturb the spiders (also see Stoltz et al., 2012). On each trial day, we took a baseline CO2 measurement from the empty test chamber, randomly selected a sample of males from our large population, and then measured each male’s CO2 production during courtship (i.e., AMR, see below for our method of eliciting courtship in these males). During courtship males will also walk or rest in addition to producing vibrational signals (Forster, 1995), and thus we did not include CO2 production from a sampling period if a male happened to stop courting for the entire 70 s, as this data point contained no metabolic data relevant to its courtship activity. Accordingly, although the system measured CO2 production several times for each male (between 4 and 11 times, depending on the duration of a male’s courtship bout), not all of the measurements coincided with the male performing courtship activity. Those measurements that did not were ignored, and thus we obtained 4.3 ± 1.4 (mean ± SD) usable courtship metabolic measurements from each male (range: 1–6 measurements per male, N = 25 males). Courtship bouts lasted an average of 28.4 ± 7.1 min (range: 17–47 min, N = 25 males). Although male L. hasselti will court for longer durations, these shorter bouts are not unnatural, as some males attempt mating within 50 min (Stoltz et al., 2008). We ended a trial when a male stopped all activity and remained motionless for at least 15 consecutive minutes. We measured resting metabolic rates several hours later after males had returned to their typical sedentary posture. From each male we obtained three consecutive resting measurements, during which we observed the males to ensure they remained motionless. In all subsequent analyses, for each male we utilize a single measure of its average courtship metabolic rate and average resting metabolic rate (Note: for one male there was only a single measurement of CO2 production that coincided with courtship activity, and thus we could not calculate an average courtship metabolic rate for this male). Prior to courtship measurements males were weighed on an electronic balance (Ohaus Explorer balance accurate to 0.01 mg), and following trials we measured the length of each male’s first pair of legs, using a Zeiss Stemi 2000-C dissecting microscope fitted with a Nikon digital camera and measurement software (Simple PCI, Compix Inc. Imaging systems, 2002). When experiments were completed we subtracted the baseline CO2 measurement from the average measurement for each male to yield expired CO2 estimates (CO2 lmol/h) for resting and courtship. In subsequent analyses, we use mass-corrected AMR values (CO2 lmol/h/mg). This is appropriate for our study since the log courtship metabolic rate regressed on log body mass is y = 0.58x 1.455 (N = 25), and a test assessing whether the slope differed from 1.0 was not significant (t23,2 = 1.967, P = 0.06) (Lighton, 2008). Accordingly, since the increase in courtship metabolic rate with mass is constant for our sample of males we follow the convention of using mass-corrected AMR values, which allows for comparisons of the ‘‘per unit mass’’ metabolic efficiency of males (Bailey et al., 1993; Kotiaho et al., 1998; Stoltz et al., 2012; Watson and Lighton, 1994). 2.3. Courtship vibrational signal measurements We recorded vibratory courtship signals on natural intact webs in conjunction with CO2 production. We constructed rectangular wood platforms (length = 6 cm, width = 1 cm) that contained 2 metal rings (height = 2 cm) affixed at each end. We placed virgin females singly on these platforms for 24 h and the rings provided anchor points from which females constructed webs. Upon their completion webs measured 12 cm3 (6 1 2 cm) and contained
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dozens of web strands. We then removed the female and placed a male on the web, and then the entire apparatus was inserted into the respirometry chamber. Latrodectus males, including redbacks, will spontaneously begin courtship behavior when placed on a female’s web even if the female is not present (Forster, 1992, 1995; Ross and Smith, 1979). Although these webs were smaller than those females typically construct in nature, they were sufficient to elicit male courtship behavior and enable recording of web-borne vibrations. We did not detect any observable differences in the behavior of males on these webs compared to males on naturally-occurring webs, nor were there any significant differences in vibrational properties (S. Sivalinghem and A. Mason, personal communication). We recorded courtship trials with a video camera (Panasonic BP-330) connected to a VHS tape recorder (JVC SR-TS1U), and simultaneously made recordings of courtship vibrations using a Polytec (Tustin, CA, USA) PDV100 portable laser vibrometer. We affixed a small piece of reflective tape (1 mm2) to the top surface of the web midway along its long axis to increase reflectance of the laser beam prior to inserting the platform into the respirometry chamber. Laser sensitivity was set to 25 mm/s/V, and signals were low-pass filtered at 5 kHz prior to being input into the audio channel of the videotape recorder. This enabled us to synchronize vibratory with visual components of courtship for later analysis. When making recordings we monitored signals visually with an oscilloscope and audibly with a speaker to ensure laser recordings were not clipped. During the trials we recorded a 2.8 VRMS test signal (generated by the PDV100) using the same recording system, and later used this signal file in order to calibrate vibration amplitude values. When courtship trials were completed we viewed the videos for each male and calculated the proportion of time within each 70 s CO2 sampling period that a male was involved in any of three distinct activities: courting, walking without courting, and motionless. We used Etholog v.2.2.5 (Ottoni, 2000) to score the duration of each activity. Male courtship is characterized by stereotyped actions that are easily differentiated from walking movements. These include ‘‘strumming’’ of the web with the rear two pairs of legs, and a ‘‘bouncy walk’’ in which the male moves his body upand-down in a conspicuous bobbing fashion (S Sivalinghem and AC Mason, personal communication). Accordingly, for each CO2 sampling period in which courtship signaling occurred, we were able to determine a male’s duty cycle, i.e., time spent producing vibrations/70 s (expressed as a percentage), and then match this value to his corresponding CO2 production for that period. To measure vibration amplitude we calculated the root mean square (RMS; Peterson and Gross, 1974) amplitude level of recorded segments of male courtship vibrations. An advantage of using RMS is that values are time-independent and thus can be used to compare the intensities of signals having different durations. We used Adobe Audition v.3.0 (Adobe Systems, Inc.) to extract audio segments (as 16 bit, 48 kHz sampling rate wave files) from the videos, and we used a custom program written in Matlab v.7.5 (The Mathworks Inc., Natik, MA, USA) to calculate RMS amplitude levels, which we express in velocity units (mm/s). We calculated two indices of courtship metabolic efficiency, one for each signal variable we measured. Duty cycle and RMS amplitude have both been used as measures of the energy content of acoustic mating signals (Andersson, 1994; Ewing, 1989; Gerhardt and Huber, 2002), and our goal here was to assess which trait was a better reflection of signal energy when considered in conjunction with metabolic rate. We decided to use both variables to calculate metabolic efficiency as they were uncorrelated (Pearson r = 0.18, F1,23 = 0.76, P = 0.39, N = 25), and we had no a priori reason to assume one property was a better reflection of signal energy content than the other in this system. Accordingly, for each
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male we took the ratios of mass-corrected AMR/duty cycle, and mass-corrected AMR/RMS amplitude, to calculate metabolic efficiency. As these indices reflect the amount of energy expended per unit of courtship activity, smaller values indicate higher efficiency, i.e., less energy is expended per unit of courtship (Bailey et al., 1993; Watson and Lighton, 1994). 2.4. Statistical analyses Statistical analyses were performed with JMP v.7 (SAS Institute Inc., 2007) and descriptive statistics are reported as the mean ± SE. We used least-squares regression to determine the relationship between metabolic rate (resting and courting) and body mass, and then we evaluated whether the slopes of the lines were different. Since resting and courting metabolic measurements for each male were not independent we compared the slopes using a repeated-measures MANCOVA, and tested for a significant interaction between treatment level (i.e., resting vs. courting) and body mass (Quinn and Keough, 2002). We used multiple regression analyses to measure the relationships between two aspects of male phenotype (body size and mass) and our two measures of courtship signal energy, and our two indices of courtship metabolic efficiency. We decided to use size and mass as separate predictors rather than combining them into a single condition index (e.g., Cotton et al., 2004) because this allowed us more freedom to investigate the independent contributions of each phenotypic trait on courtship performance. We therefore assume that bigger males are in better condition and possess more stored energy reserves than smaller males, regardless of any allometric scaling relationship between size and mass. We analyzed each response variable separately. We standardized predictor variables (mean = 0, SD = 1), and transformed each response variable to achieve normality before proceeding as follows: duty cycle (arcsine transform), Shapiro–Wilk W = 0.943, P = 0.174; RMS amplitude (log transform), Shapiro–Wilk W = 0.936, P = 0.122; duty cycle efficiency (log transform), Shapiro– Wilk W = 0.94, P = 0.15; RMS amplitude efficiency (log transform), Shapiro–Wilk W = 0.93, P = 0.07. We detected no significant collinearity between predictor variables as the correlation was less than 0.5 (0.45), and variance inflation was low (1.3). Exploratory data analysis of response variables using box-plots and calculating Cook’s D statistics revealed no outliers in the data that warranted exclusion (Quinn and Keough, 2002). Although we hypothesized a positive linear relationship between mass/size and courtship signal traits, and metabolic efficiency, we used a statistical model that simultaneously estimated linear and curvilinear effects by incorporating all standardized predictors along with their squares and cross-products. We used the ‘response surface’ macro in JMP to build each model which included quadratic terms, to explore curvilinear relationships between predictor and response variables, and cross-product terms, to explore potential interactions between predictor variables. In all analyses, the cross-product term (i.e., size * mass) was not significant, but removing it from the models and rerunning the analyses did not alter the results, and so we retained it and include it in the results we report below.
between treatment level and body mass (repeated-measures MANCOVA: F1,23 = 0.0004, P = 0.98), which indicated that the rate of increase in metabolic rate with mass did not depend on a male’s activity level. Average courtship AMR was approximately four times greater than resting metabolic levels (courtship AMR: 0.265 ± 0.013 lmol CO2/h; resting: 0.074 ± 0.004 lmol CO2/h; paired t-test: t24 = 18.73, P 0.01). When we examined the relationship between mass-specific metabolic rate and body mass we found that heavier males expended less energy per unit of body mass when at rest (r = 0.41, F1,23 = 4.71, P = 0.04). There was a similar relationship with courtship but the trend was not significant (r = 0.38, F1,23 = 3.87, P = 0.06, Fig. 1b). Average mass-specific AMR was 0.075 ± 0.004 lmol/h/mg, and average mass-specific resting metabolic rate was 0.021 ± 0.001 lmol/h/mg. 3.2. Courtship signal parameters 3.2.1. Duty cycle Duty cycle averaged 68 ± 4% (range: 16–94%, N = 25). There was a significant negative quadratic relationship with body mass, such that the highest duty cycle signals were associated with males having intermediate mass (t = 4.07, P < 0.01; Table 1 and Fig. 2). 3.2.2. RMS amplitude The RMS amplitude level of courtship vibrations averaged 10.69 ± 1.61 mm/s (range: 1.16–26.97, N = 25). There were no significant linear or non-linear influences of body size or mass (Table 1). 3.3. Courtship metabolic efficiency 3.3.1. Duty cycle Mass-corrected metabolic efficiency for duty cycle averaged 0.126 ± 0.012 lmol CO2/h/mg per unit of duty cycle (range: 0.068–0.31, N = 25). There was a significant positive quadratic relationship with body mass, such that males with intermediate mass expended the lowest amounts of energy per unit of duty cycle, while lighter and heavier males were similar in expending greater amounts of energy (t = 3.27, P < 0.01; Table 2 and Fig. 3a). For comparison we also show the relationship between body mass and absolute values of metabolic efficiency (i.e., uncorrected for body mass, lmol CO2/h) in Fig. 3b. Here, intermediate mass males are still most efficient; however, the heaviest males are expending the greatest absolute amounts of energy per unit of courtship performed compared to other males. 3.3.2. RMS amplitude Mass-corrected metabolic efficiency for RMS amplitude averaged 0.018 ± 0.005 lmol CO2/h/mg per amplitude unit (range: 0.002–0.102, N = 25). There were no significant linear or non-linear influences of body size or mass on this measure of courtship efficiency (Table 2). Since duty cycle and duty cycle efficiency were both strongly correlated with body mass compared to RMS amplitude and its efficiency index, this suggests duty cycle is a more relevant indicator of signal energy content than RMS amplitude in L. hasselti.
3. Results 4. Discussion 3.1. Metabolic rate measurements As mass increased there was an increase in the resting rate and courtship AMR (resting: r = 0.52, F1,23 = 8.57, P < 0.01; courtship AMR r = 0.49, F1,23 = 7.41, P < 0.01, Fig. 1a). The slopes of the two lines were equal as revealed by a non-significant interaction
Our results indicate that courtship signaling is energetically costly. Male courtship in L. hasselti increases metabolic rate nearly four times over resting levels, which lies within the range reported from other arthropod taxa that show courtship increases of 0.22–22 times over resting levels (Prestwich, 1994; Stoddard and
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Fig. 1. Relationship between body mass and (a) absolute CO2 production, and (b) mass-corrected CO2 production, during rest (circles) and courtship (squares). Courtship activity increases metabolic rate nearly four times over resting levels. Note: log scale used for both axes.
Table 1 Results of a multiple regression analysis of standardized body size and mass, on duty cycle (arcsine transformed) and RMS amplitude (log10 transformed). Significant results are in bold. Term
Duty cycle model R2 = 0.62 Estimate
Size Mass Size2 Mass2 Size * mass
0.04 0.09 0.04 0.14 0.07
SE 0.04 0.04 0.03 0.03 0.06
RMS amplitude model R2 = 0.39 t
P 0.94 2 1.45 4.07 1.18
Fig. 2. Relationship between body mass and duty cycle. Intermediate mass males produce courtship signals having the highest duty cycles. Plotted are the transformed values. Regression equation: y = 10.75x2 0.21x + 1.2.
Salazar, 2011). In contrast to our prediction, the largest males were not the most efficient at courtship signaling. Rather, males having intermediate mass produced the highest duty cycle signals and were also the most efficient per unit of duty cycle performed. Although these results indicate that intermediate mass males are superior courters, whether their courtship signals are in fact preferred by females remains to be determined with additional studies. For example, an experiment that offers females simultaneous
Estimate
0.36 0.06 0.16 <0.01 0.25
0.02 0.13 0.05 0.03 0.18
SE 0.09 0.09 0.06 0.07 0.13
t
P 0.25 1.41 0.77 0.37 0.14
0.81 0.17 0.45 0.71 0.19
choice of males of different mass (e.g., light, medium, heavy) would directly test the outcome of male courtship performance on female choice, and thus reveal which type of male indeed represents a preferred mating partner in this species. The fact that duty cycle was significantly correlated with body mass suggests an important role for this courtship trait in L. hasselti. Consequently, duty cycle may provide useful information to females for evaluating which males are most metabolically efficient (Hill, 2011; Watson and Lighton, 1994; Watt, 1986). Our finding that an intermediate body mass is associated with high duty cycle signals suggests a disadvantage to lighter and heavier males during courtship. As mass reflects stored energy reserves, lighter males may simply not have a sufficient resource pool to sustain signaling for extended periods following a lengthy and risky period of mate searching. This is reinforced by the size-dependence of relative metabolic rates – lighter males expend the most energy per unit of body mass whether resting or courting (Fig. 1b), and thus their overall rate of energy expenditure may be too high to sustain long periods of courtship signaling. For heavier males, although their energy expenditure per unit of body mass was lowest across the sample of males (Fig. 1b), and therefore these males might be expected to be most metabolically efficient (Watson and Lighton, 1994), when their AMR was evaluated in conjunction with courtship signaling the amount of energy expended per unit of duty cycle was relatively high (Fig. 3a). Therefore, simply having a large energy reserve may not be enough to guarantee successful courtship. Rather, it is the interaction between mass and metabolic expenditure that determines which males are most efficient at high duty cycle signaling, and thus which males represent superior courters. In wolf spiders (Hygrolycosa rubrofasciata), males drum the substrate during courtship in an extremely energetically expensive
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Table 2 Results of a multiple regression analysis of standardized body size and mass, on (log10 transformed) duty cycle efficiency and RMS amplitude efficiency. Significant results are in bold. Term
Duty cycle efficiency model R2 = 0.49 Estimate
Size Mass Size2 Mass2 Size * mass
0.004 0.048 0.03 0.072 0.018
SE 0.02 0.02 0.02 0.02 0.03
RMS amplitude efficiency model R2 = 0.16 t
P 0.18 2.04 1.29 3.27 0.61
0.86 0.06 0.21 <0.01 0.55
Estimate 0.001 0.061 0.209 0.023 0.225
SE 0.11 0.11 0.11 0.1 0.14
t
P 0.01 0.55 1.85 0.23 0.16
0.99 0.59 0.07 0.82 0.13
Fig. 3. Relationship between body mass and (a) mass-corrected metabolic efficiency, and (b) absolute metabolic efficiency, for duty cycle. When corrected for body mass, both heavy and light males are equal in the amount of energy expended, while intermediate mass males expend the lowest amount of energy per unit of courtship performed. Regression equation: y = 5.78x2 0.2x 1.05. In (b) the heaviest males expend the highest absolute amount of energy per unit of courtship performed, while light and intermediate mass males are more similar in energy expenditure. Regression equation: y = 9.28x2 8.98x + 1.67. Plotted are the transformed values.
display (22 times higher than resting, Kotiaho et al., 1998), rapid drumming results in increased mortality (Mappes et al., 1996), and larger males pay higher metabolic costs for drumming (Kotiaho et al., 1998). However, males do not drum continuously as in L. hasselti, but in short one-second bursts given in bouts that last only a few minutes, which may allow some males to achieve the extremely high AMR that makes high energy drumming a reliable indicator of male quality in this species (Parri et al., 2002). In L. hasselti, although the difference between AMR and resting metabolic rate is much lower, courtship is a lengthy process lasting several hours, and thus endurance is as important as power when producing vibrational signals. Males must utilize their finite energy reserves efficiently to ensure they can last for the extended periods required by females, and thus the maximum AMR that can be obtained is likely to be much lower than for species such as wolf spiders, in which courtship occurs in brief bursts that may permit a greater maximum expenditure of energy (Peterson et al., 1990). In another web-building spider species (Stegodyphus lineatus) prolonged courtship using web-borne vibrations functions to stimulate a female into mating (Maklakov et al., 2003). Similarly, the minimum courtship threshold of 100 min in L. hasselti (Stoltz and Andrade, 2010) reveals that females here also require a lengthy period of stimulation before accepting a mate. What is the benefit to males of producing high duty cycle signals during such a prolonged bout of courtship? Recall that higher duty cycles indicate more signal is being produced relative to silent intervals and thus more energy is being broadcast (Jang and Greenfield, 1998; Mason, 1996; Reinhold et al., 1998). Because naturally-occurring
female L. hasselti webs sometimes extend over an area of several square meters and contain hundreds of web strands (Brunet, 2000; MCB Andrade, personal observation), a mate searching male that reaches a female’s web is probably uncertain of her precise location when he begins courtship. High duty cycle signals are therefore more likely to increase and maintain a female’s sensory stimulation (Stoddard and Salazar, 2011) while the male searches for her on the web (but see Vibert et al., 2014). Furthermore, high duty cycle signals are likely to propagate better through the environment and be less adversely affected by attenuation and reverberation along the web (Brenowitz, 1986; Casas et al., 2007; Elias et al., 2004; Schul and Patterson, 2003). Minimizing these effects is important regardless of web size in L. hasselti because females build ‘cobwebs’ with a complex, irregular three-dimensional structure that is likely to degrade transient, low duty cycle vibrational signals (Barth, 1998; Cocroft and Rodrìguez, 2005; Zevenbergen et al., 2008). Independent of these mechanistic advantages, females may simply prefer males that produce high duty cycle signals if such signals reflect the amount of energy a male can afford to allocate towards expensive activities (Bailey et al., 1993; Bennett, 1983; Dearborn et al., 2005; Halliday, 1987; Reinhold et al., 1998; Rivero et al., 2000; Salazar and Stoddard, 2008). This may be especially important in L. hasselti where males are expected to invest maximally in a single mating opportunity with no allocation trade-offs favoring future matings, immediately following a lengthy and risky period of mate searching. Thus, the ability to produce high duty cycle signals at low metabolic cost will be a reflection of a male’s entire energy reserve and the efficiency of
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metabolic expenditure throughout its life, which includes fitnessrelated traits expressed as a juvenile (e.g., foraging success), and as an adult (e.g., mate searching and courtship performance). Although RMS amplitude was not correlated with mass or size, this signal component could still have an important role in facilitating detection of a male by a female once he begins courting on her web. Although transmission effects of amplitude on cobwebs has not been thoroughly examined (Vibert et al., 2014), higher amplitude signals generally propagate farther in the environment regardless of substrate (Bradbury and Vehrencamp, 1998). For L. hasselti males, high RMS amplitude signals may be especially beneficial when several males are competing simultaneously for a female’s attention, which commonly occurs in this species (Andrade and Kasumovic, 2005; Stoltz and Andrade, 2010; Stoltz et al., 2008). To the extent that amplitude can provide directional information on a cobweb, a male that is able to signal ‘loudest’ is likely to elicit and maintain a female’s attention over any rivals that may also be present. In the western black widow, L. hesperus, high amplitude vibrations can result in a male sometimes being mistaken for prey (Vibert et al., 2014), however, the benefits of winning a female’s attention when many males are present may outweigh this risk in L. hasselti due to the intense competition for mates that occurs in this species. 5. Conclusion Recent studies suggest that metabolic efficiency is likely to be an important influence on the outcome of female choice (Hill, 2011; Kasumovic and Seebacher, 2013; Stoddard and Salazar, 2011; Thomson et al., 2014). In this study, we show that in a capital breeding animal that has only a single mating opportunity, metabolic efficiency and body mass are both strongly related to male courtship effort. Our findings reveal that courtship vibrational signaling in L. hasselti is energetically expensive and not all males are capable of producing high duty cycle signals. Furthermore, intermediate mass males may represent superior courters because they are best able to balance energy reserves and metabolic expenditure to be most efficient at producing high duty cycle signals. Acknowledgments We thank members of the Integrative Behaviour and Neuroscience Group at UTSC for their helpful input, the Andrade lab undergraduates for assistance rearing spiders, and K. Brochu for comments on an earlier version of this manuscript. We are especially grateful to C. Wellstood at Qubit Systems who provided valuable technical advice regarding operation of the respirometry equipment. L. Bussière and K. Judge provided much appreciated statistical advice. This work was supported by a Natural Science and Engineering Research Council of Canada (NSERC) post graduate scholarship to JAS, Discovery Grants to MCBA (229029-12) and ACM, and grants from the Canada Research Chairs program, the Canada Foundation for Innovation, and Research & Innovation Ontario (203764) to MCBA. This manuscript was greatly improved from comments provided by two anonymous reviewers. References Andersson, M., 1994. Sexual Selection. Princeton University Press, New Jersey. Andrade, M.C.B., 2003. Risky mate search and male self-sacrifice in redback spiders. Behav. Ecol. 14, 531–538. Andrade, M.C.B., Kasumovic, M.M., 2005. Terminal investment strategies and male mate choice: extreme tests of Bateman. Integr. Comp. Biol. 45, 838–847. Bailey, W.J., Cunningham, R.J., Lebel, L., 1990. Song power, spectral distribution and female phonotaxis in the bushcricket Requena verticalis (Orthoptera: Tettigoniidae): active female choice or passive attraction? Anim. Behav. 40, 33–42.
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Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Metabolic efficiency in courtship favors males with intermediate mass in the Australian redback spider, Latrodectus hasselti Paul A. De Luca ⇑, Jeffrey A. Stoltz, Maydianne C.B. Andrade, Andrew C. Mason Integrative Behaviour and Neuroscience Group, University of Toronto at Scarborough, Toronto, Ontario M1C 1A4, Canada
a r t i c l e
i n f o
Article history: Received 24 October 2014 Received in revised form 20 November 2014 Accepted 22 November 2014 Available online 29 November 2014 Keywords: Web-borne vibrations Metabolic efficiency Duty cycle Capital breeder Signal energy
a b s t r a c t Recent studies have suggested that metabolic efficiency may be an important factor in male mating success when females require vigorous and/or prolonged courtship. In capital breeding animals in which a male’s resource pool is fixed at adulthood the relationship between energy expenditure and courtship performance may be especially important, as males are expected to utilize their finite resources efficiently when soliciting mates. Males may benefit from being efficient, i.e., achieving a sufficiently high level of courtship signaling at low energetic cost, if it enables them to acquire mates before their limited energy reserves are depleted. We investigated the relationship between metabolic efficiency and courtship vibrational signaling in the Australian redback spider, Latrodectus hasselti, a semelparous capital breeder where males invest heavily in courtship to secure a mating. We assessed metabolic rate in a sample of males and measured two courtship components (duty cycle and amplitude) that reflected the energy content of web-borne vibrations. We then calculated two indices of metabolic efficiency for these courtship properties. There was a quadratic relationship between mass and duty cycle such that the highest duty cycle signals were performed by males having intermediate mass. Furthermore, intermediate-mass males were also the most metabolically efficient. Prolonged courtship is necessary in L. hasselti for successful mating, and the results of this study suggest that intermediate-mass males are superior courters because they utilize their finite resource pool most efficiently to produce high energy vibrational signals. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Courtship is an essential precursor to mating for many species (Andersson, 1994). A substantial body of evidence shows that high courtship activity is often associated with increased mating success largely because females are more likely to mate after a vigorous courtship (Carranza and Trucios, 1993; Cook et al., 2013; De Luca and Cocroft, 2008; Knapp and Kovach, 1991; Kotiaho, 2002; Parker, 1974; Stapley, 2008). Much research has focused on examining physiological traits that influence courtship activity, to better understand links between male performance and female choice. One trait that has been the focus of some recent research is the active metabolic rate (AMR), which reflects the amount of energy a male expends while soliciting mates (Gillooly and Ophir, 2010; Kasumovic and Seebacher, 2013; Reinhold, 1999; Stoddard and Salazar, 2011). Generally, high AMR is positively correlated with the level of courtship activity ⇑ Corresponding author at: Department of Biology, Ithaca College, Ithaca, NY 14850, USA. Tel.: +1 607 274 1086; fax: +1 607 274 1131. E-mail address: [email protected] (P.A. De Luca). http://dx.doi.org/10.1016/j.jinsphys.2014.11.004 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
because displays that are louder, longer, or both, require more energy to produce, and females tend to prefer males that display at these higher levels (Bailey et al., 1993; Halliday, 1987; Kotiaho et al., 1998; Prestwich, 1994; Reinhold et al., 1998; Wells and Taigen, 1989). Related to AMR is metabolic efficiency, which measures energy expenditure per unit of courtship activity (Bailey et al., 1993; Watson and Lighton, 1994). In a recent review, Hill (2011) suggested that the most metabolically efficient males (i.e., those achieving a sufficiently high level of courtship signaling at low energetic cost), are likely to represent superior courters because such males demonstrate a capacity to optimize energy expenditure between advertisement and somatic maintenance (Stoddard and Salazar, 2011; Thomson et al., 2014; Watson and Lighton, 1994; Watt, 1986). Uncovering how factors such as AMR, metabolic efficiency, and courtship performance interact can be challenging, though, because male energetic rates during courtship may fluctuate over a male’s reproductive lifetime due to changes in body condition or energy reserves (Sadd et al., 2000; White et al., 2013), reproductive status (Clutton-Brock, 1984; Clutton-Brock and Albon, 1979), or social conditions (Candolin, 2000; Stoltz et al., 2012).
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Some of these complexities may be simplified in taxa where males have a fixed resource pool at adulthood that is not replenished (capital breeders, Stearns, 1992), and limits on the availability of females predicts maximal performance with no trade-offs for future mating opportunities (e.g., monogyny, Fromhage et al., 2005). In such systems, because males will usually encounter only one female in their lifetime (Andrade, 2003), all their energy reserves should be expended, if necessary, to secure a mating. At the same time, however, a finite resource pool necessitates efficient allocation of energy reserves throughout the mate acquisition process (i.e., searching, courtship, and mating) in order to ensure that resources are not depleted before a male has performed sufficient courtship to be accepted by the female (Hill, 2011). Accordingly, this predicts a positive relationship between energy reserves and metabolic efficiency for males most likely to be successful at obtaining a mate. For example, in the Sierra dome spider, Linyphia litigiosa, body mass is positively correlated with two complementary measures of courtship metabolic efficiency, and large mass, high efficiency males are successful in aggressive encounters with other males and at siring more offspring (Watson and Lighton, 1994). Here, we examine links between metabolic rate, male phenotype, and courtship performance in monogynous, capital-breeding male redback spiders (Latrodectus hasselti). Upon reaching sexual maturity, male L. hasselti abandon their webs in search of females. During this time they cease foraging and must rely on energy reserves accumulated during development, which severely limits their energetic budget and contributes to a mate-search mortality rate exceeding 80% (Andrade, 2003). Field-caught adult males vary considerably in size and mass (Andrade, 2003), as do males reared in the laboratory under uniform conditions and fed the same diet (Stoltz et al., 2008), which suggests that inherent differences among males in their ability to accumulate and use energy reserves may be an important factor influencing variability in mate searching success. When a female is found, a male will not abandon her in search of another (Andrade, 2003; Andrade and Kasumovic, 2005), and males that succeed in mating are eaten by the female and so will not have another opportunity to mate (Forster, 1992). Male fitness therefore depends entirely on a single mating event, and thus males are expected to expend their total energy reserve towards securing that sole reproductive opportunity. However, following mate-searching males must engage in a lengthy courtship display that involves producing web-borne vibrational signals for a minimum of 100 min to secure a mating (Stoltz and Andrade, 2010). Even if males mate successfully, courtship duration determines the female’s likelihood of remating with another male (Stoltz et al., 2009). This is perhaps why courtship in this species often includes up to 5 h of vibrational signaling and movement on the female’s web (Forster, 1995). Vibrational signaling is expected to be an energetically demanding form of communication (Kotiaho et al., 1998; Lighton, 1987; Randall, 2014), and the extended courtship of male L. hasselti suggests performance may be limited by energetic expenditure. Consistent with this, a continuous 3-h bout of courtship significantly decreases male longevity, and males initially in poor condition suffer the highest longevity cost (Kasumovic et al., 2009). While duration is indeed an important courtship trait in this species, the role of signal energy is less well understood, yet we expect it to also comprise an important courtship feature in this species. High energy signals are expected to be more attractive to females because they provide more stimulation to her sensory system (Morris et al., 1978; Stoddard and Salazar, 2011), and that is likely to be critical here. Latrodectus females’ webs vary considerably in size, with some that are very large relative to the size of the spiders (Szlep, 1965; M. Andrade, personal communication). A mate searching male entering a female’s web is unlikely to know
her exact position and therefore would be expected produce high energy signals in order to elicit and maintain her attention until she can be found (Bailey et al., 1990; Brenowitz, 1986; Maklakov et al., 2003). This is not a foregone conclusion though, as a recent study of a congener suggested that males may produce low energy ‘whispers’ during the initial phase of courtship to distinguish them from struggling prey (Vibert et al., 2014). Clearly, more data is needed to adequately assess the role of signal energy on courtship performance in Latrodectus spiders. In this study, we investigate the relationships between metabolic rate, male phenotype and courtship vibrational signaling in L. hasselti using stop-flow gas respirometry and laser Doppler vibrometry. We first examine the metabolic expenditure of males during courtship compared to resting to determine if courtship vibrational signaling is energetically demanding. We then quantify courtship activity by measuring two components of web-borne vibrations related to the energy content of signals: (1) duty cycle (proportion of time spent producing vibrational signals during a courtship bout), and (2) amplitude (intensity or ‘loudness’ of courtship vibrations). We then use these properties to calculate a male’s metabolic efficiency (amount of energy expended per unit of courtship activity). Finally, we assess potential associations between metabolic efficiency and two aspects of male phenotype (body size and mass) to test the hypothesis that in L. hasselti larger males represent superior courters because they are most metabolically efficient at producing high energy vibrational signals. 2. Materials and methods 2.1. Spider rearing and maintenance We used spiders that were derived from an outbred laboratoryreared population established with individuals collected in the field from New South Wales, Australia in 2007, and replenished with additional wild-caught spiders in 2009. Spiderlings were held with siblings in clear plastic cages (9 9 11 cm, Amac Plastics) in a temperature-controlled room at the University of Toronto Scarborough at 25 °C on a 12:12 light:dark cycle. We removed spiderlings at the 4th instar to ensure they remained virgins (males become sexually mature at the 5th instar and females around the 7–8th) and kept them individually in plastic containers (9 9 11 cm). We fed spiderlings with flies (Drosophila melanogaster) twice a week, and fed adult females immature house crickets (Acheta domesticus) once per week. 2.2. Metabolic rate measurements We used expired CO2 as a measure of metabolic rate (Stoltz et al., 2012). For each male in our experiment, we measured metabolic rates during rest and during courtship in two separate sessions performed on the same day. We conducted experiments in a laboratory at the University of Toronto Scarborough from January to March, 2010. All measurements were taken in a temperature controlled room (25 °C) under red light during the central 8 h of a 12 h scotophase. We took metabolic measures using stop-flow gas respirometry with a Qubit systems (Kingston, Ontario, Canada) 8 channel gas controller (G245), a Qubit systems 8 channel gas switcher (GS244) and a Li-Cor (Nebraska, USA) CO2 analyzer (LI-6252) where incoming CO2 was removed by filtering the air through soda lime. The test chamber consisted of a cylindrical glass tube (12 2.5 cm) sealed at both ends with steel plugs and rubber o-rings. Briefly, for each trial, air was flushed into the chamber and kept there for a sampling period that lasted either 70 s (courtship) or 3 min (resting). At the end of each sampling period the air was flushed from the chamber and flowed through a magnesium
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perchlorate plug to remove moisture before entering the CO2 analyzer. We configured the system to measure CO2 concentration within the test chamber at regular intervals (approximately every 4 min) while a male was performing courtship behavior. The flow rate was set to 160 ml/min and this did not appear to disturb the spiders (also see Stoltz et al., 2012). On each trial day, we took a baseline CO2 measurement from the empty test chamber, randomly selected a sample of males from our large population, and then measured each male’s CO2 production during courtship (i.e., AMR, see below for our method of eliciting courtship in these males). During courtship males will also walk or rest in addition to producing vibrational signals (Forster, 1995), and thus we did not include CO2 production from a sampling period if a male happened to stop courting for the entire 70 s, as this data point contained no metabolic data relevant to its courtship activity. Accordingly, although the system measured CO2 production several times for each male (between 4 and 11 times, depending on the duration of a male’s courtship bout), not all of the measurements coincided with the male performing courtship activity. Those measurements that did not were ignored, and thus we obtained 4.3 ± 1.4 (mean ± SD) usable courtship metabolic measurements from each male (range: 1–6 measurements per male, N = 25 males). Courtship bouts lasted an average of 28.4 ± 7.1 min (range: 17–47 min, N = 25 males). Although male L. hasselti will court for longer durations, these shorter bouts are not unnatural, as some males attempt mating within 50 min (Stoltz et al., 2008). We ended a trial when a male stopped all activity and remained motionless for at least 15 consecutive minutes. We measured resting metabolic rates several hours later after males had returned to their typical sedentary posture. From each male we obtained three consecutive resting measurements, during which we observed the males to ensure they remained motionless. In all subsequent analyses, for each male we utilize a single measure of its average courtship metabolic rate and average resting metabolic rate (Note: for one male there was only a single measurement of CO2 production that coincided with courtship activity, and thus we could not calculate an average courtship metabolic rate for this male). Prior to courtship measurements males were weighed on an electronic balance (Ohaus Explorer balance accurate to 0.01 mg), and following trials we measured the length of each male’s first pair of legs, using a Zeiss Stemi 2000-C dissecting microscope fitted with a Nikon digital camera and measurement software (Simple PCI, Compix Inc. Imaging systems, 2002). When experiments were completed we subtracted the baseline CO2 measurement from the average measurement for each male to yield expired CO2 estimates (CO2 lmol/h) for resting and courtship. In subsequent analyses, we use mass-corrected AMR values (CO2 lmol/h/mg). This is appropriate for our study since the log courtship metabolic rate regressed on log body mass is y = 0.58x 1.455 (N = 25), and a test assessing whether the slope differed from 1.0 was not significant (t23,2 = 1.967, P = 0.06) (Lighton, 2008). Accordingly, since the increase in courtship metabolic rate with mass is constant for our sample of males we follow the convention of using mass-corrected AMR values, which allows for comparisons of the ‘‘per unit mass’’ metabolic efficiency of males (Bailey et al., 1993; Kotiaho et al., 1998; Stoltz et al., 2012; Watson and Lighton, 1994). 2.3. Courtship vibrational signal measurements We recorded vibratory courtship signals on natural intact webs in conjunction with CO2 production. We constructed rectangular wood platforms (length = 6 cm, width = 1 cm) that contained 2 metal rings (height = 2 cm) affixed at each end. We placed virgin females singly on these platforms for 24 h and the rings provided anchor points from which females constructed webs. Upon their completion webs measured 12 cm3 (6 1 2 cm) and contained
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dozens of web strands. We then removed the female and placed a male on the web, and then the entire apparatus was inserted into the respirometry chamber. Latrodectus males, including redbacks, will spontaneously begin courtship behavior when placed on a female’s web even if the female is not present (Forster, 1992, 1995; Ross and Smith, 1979). Although these webs were smaller than those females typically construct in nature, they were sufficient to elicit male courtship behavior and enable recording of web-borne vibrations. We did not detect any observable differences in the behavior of males on these webs compared to males on naturally-occurring webs, nor were there any significant differences in vibrational properties (S. Sivalinghem and A. Mason, personal communication). We recorded courtship trials with a video camera (Panasonic BP-330) connected to a VHS tape recorder (JVC SR-TS1U), and simultaneously made recordings of courtship vibrations using a Polytec (Tustin, CA, USA) PDV100 portable laser vibrometer. We affixed a small piece of reflective tape (1 mm2) to the top surface of the web midway along its long axis to increase reflectance of the laser beam prior to inserting the platform into the respirometry chamber. Laser sensitivity was set to 25 mm/s/V, and signals were low-pass filtered at 5 kHz prior to being input into the audio channel of the videotape recorder. This enabled us to synchronize vibratory with visual components of courtship for later analysis. When making recordings we monitored signals visually with an oscilloscope and audibly with a speaker to ensure laser recordings were not clipped. During the trials we recorded a 2.8 VRMS test signal (generated by the PDV100) using the same recording system, and later used this signal file in order to calibrate vibration amplitude values. When courtship trials were completed we viewed the videos for each male and calculated the proportion of time within each 70 s CO2 sampling period that a male was involved in any of three distinct activities: courting, walking without courting, and motionless. We used Etholog v.2.2.5 (Ottoni, 2000) to score the duration of each activity. Male courtship is characterized by stereotyped actions that are easily differentiated from walking movements. These include ‘‘strumming’’ of the web with the rear two pairs of legs, and a ‘‘bouncy walk’’ in which the male moves his body upand-down in a conspicuous bobbing fashion (S Sivalinghem and AC Mason, personal communication). Accordingly, for each CO2 sampling period in which courtship signaling occurred, we were able to determine a male’s duty cycle, i.e., time spent producing vibrations/70 s (expressed as a percentage), and then match this value to his corresponding CO2 production for that period. To measure vibration amplitude we calculated the root mean square (RMS; Peterson and Gross, 1974) amplitude level of recorded segments of male courtship vibrations. An advantage of using RMS is that values are time-independent and thus can be used to compare the intensities of signals having different durations. We used Adobe Audition v.3.0 (Adobe Systems, Inc.) to extract audio segments (as 16 bit, 48 kHz sampling rate wave files) from the videos, and we used a custom program written in Matlab v.7.5 (The Mathworks Inc., Natik, MA, USA) to calculate RMS amplitude levels, which we express in velocity units (mm/s). We calculated two indices of courtship metabolic efficiency, one for each signal variable we measured. Duty cycle and RMS amplitude have both been used as measures of the energy content of acoustic mating signals (Andersson, 1994; Ewing, 1989; Gerhardt and Huber, 2002), and our goal here was to assess which trait was a better reflection of signal energy when considered in conjunction with metabolic rate. We decided to use both variables to calculate metabolic efficiency as they were uncorrelated (Pearson r = 0.18, F1,23 = 0.76, P = 0.39, N = 25), and we had no a priori reason to assume one property was a better reflection of signal energy content than the other in this system. Accordingly, for each
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male we took the ratios of mass-corrected AMR/duty cycle, and mass-corrected AMR/RMS amplitude, to calculate metabolic efficiency. As these indices reflect the amount of energy expended per unit of courtship activity, smaller values indicate higher efficiency, i.e., less energy is expended per unit of courtship (Bailey et al., 1993; Watson and Lighton, 1994). 2.4. Statistical analyses Statistical analyses were performed with JMP v.7 (SAS Institute Inc., 2007) and descriptive statistics are reported as the mean ± SE. We used least-squares regression to determine the relationship between metabolic rate (resting and courting) and body mass, and then we evaluated whether the slopes of the lines were different. Since resting and courting metabolic measurements for each male were not independent we compared the slopes using a repeated-measures MANCOVA, and tested for a significant interaction between treatment level (i.e., resting vs. courting) and body mass (Quinn and Keough, 2002). We used multiple regression analyses to measure the relationships between two aspects of male phenotype (body size and mass) and our two measures of courtship signal energy, and our two indices of courtship metabolic efficiency. We decided to use size and mass as separate predictors rather than combining them into a single condition index (e.g., Cotton et al., 2004) because this allowed us more freedom to investigate the independent contributions of each phenotypic trait on courtship performance. We therefore assume that bigger males are in better condition and possess more stored energy reserves than smaller males, regardless of any allometric scaling relationship between size and mass. We analyzed each response variable separately. We standardized predictor variables (mean = 0, SD = 1), and transformed each response variable to achieve normality before proceeding as follows: duty cycle (arcsine transform), Shapiro–Wilk W = 0.943, P = 0.174; RMS amplitude (log transform), Shapiro–Wilk W = 0.936, P = 0.122; duty cycle efficiency (log transform), Shapiro– Wilk W = 0.94, P = 0.15; RMS amplitude efficiency (log transform), Shapiro–Wilk W = 0.93, P = 0.07. We detected no significant collinearity between predictor variables as the correlation was less than 0.5 (0.45), and variance inflation was low (1.3). Exploratory data analysis of response variables using box-plots and calculating Cook’s D statistics revealed no outliers in the data that warranted exclusion (Quinn and Keough, 2002). Although we hypothesized a positive linear relationship between mass/size and courtship signal traits, and metabolic efficiency, we used a statistical model that simultaneously estimated linear and curvilinear effects by incorporating all standardized predictors along with their squares and cross-products. We used the ‘response surface’ macro in JMP to build each model which included quadratic terms, to explore curvilinear relationships between predictor and response variables, and cross-product terms, to explore potential interactions between predictor variables. In all analyses, the cross-product term (i.e., size * mass) was not significant, but removing it from the models and rerunning the analyses did not alter the results, and so we retained it and include it in the results we report below.
between treatment level and body mass (repeated-measures MANCOVA: F1,23 = 0.0004, P = 0.98), which indicated that the rate of increase in metabolic rate with mass did not depend on a male’s activity level. Average courtship AMR was approximately four times greater than resting metabolic levels (courtship AMR: 0.265 ± 0.013 lmol CO2/h; resting: 0.074 ± 0.004 lmol CO2/h; paired t-test: t24 = 18.73, P 0.01). When we examined the relationship between mass-specific metabolic rate and body mass we found that heavier males expended less energy per unit of body mass when at rest (r = 0.41, F1,23 = 4.71, P = 0.04). There was a similar relationship with courtship but the trend was not significant (r = 0.38, F1,23 = 3.87, P = 0.06, Fig. 1b). Average mass-specific AMR was 0.075 ± 0.004 lmol/h/mg, and average mass-specific resting metabolic rate was 0.021 ± 0.001 lmol/h/mg. 3.2. Courtship signal parameters 3.2.1. Duty cycle Duty cycle averaged 68 ± 4% (range: 16–94%, N = 25). There was a significant negative quadratic relationship with body mass, such that the highest duty cycle signals were associated with males having intermediate mass (t = 4.07, P < 0.01; Table 1 and Fig. 2). 3.2.2. RMS amplitude The RMS amplitude level of courtship vibrations averaged 10.69 ± 1.61 mm/s (range: 1.16–26.97, N = 25). There were no significant linear or non-linear influences of body size or mass (Table 1). 3.3. Courtship metabolic efficiency 3.3.1. Duty cycle Mass-corrected metabolic efficiency for duty cycle averaged 0.126 ± 0.012 lmol CO2/h/mg per unit of duty cycle (range: 0.068–0.31, N = 25). There was a significant positive quadratic relationship with body mass, such that males with intermediate mass expended the lowest amounts of energy per unit of duty cycle, while lighter and heavier males were similar in expending greater amounts of energy (t = 3.27, P < 0.01; Table 2 and Fig. 3a). For comparison we also show the relationship between body mass and absolute values of metabolic efficiency (i.e., uncorrected for body mass, lmol CO2/h) in Fig. 3b. Here, intermediate mass males are still most efficient; however, the heaviest males are expending the greatest absolute amounts of energy per unit of courtship performed compared to other males. 3.3.2. RMS amplitude Mass-corrected metabolic efficiency for RMS amplitude averaged 0.018 ± 0.005 lmol CO2/h/mg per amplitude unit (range: 0.002–0.102, N = 25). There were no significant linear or non-linear influences of body size or mass on this measure of courtship efficiency (Table 2). Since duty cycle and duty cycle efficiency were both strongly correlated with body mass compared to RMS amplitude and its efficiency index, this suggests duty cycle is a more relevant indicator of signal energy content than RMS amplitude in L. hasselti.
3. Results 4. Discussion 3.1. Metabolic rate measurements As mass increased there was an increase in the resting rate and courtship AMR (resting: r = 0.52, F1,23 = 8.57, P < 0.01; courtship AMR r = 0.49, F1,23 = 7.41, P < 0.01, Fig. 1a). The slopes of the two lines were equal as revealed by a non-significant interaction
Our results indicate that courtship signaling is energetically costly. Male courtship in L. hasselti increases metabolic rate nearly four times over resting levels, which lies within the range reported from other arthropod taxa that show courtship increases of 0.22–22 times over resting levels (Prestwich, 1994; Stoddard and
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Fig. 1. Relationship between body mass and (a) absolute CO2 production, and (b) mass-corrected CO2 production, during rest (circles) and courtship (squares). Courtship activity increases metabolic rate nearly four times over resting levels. Note: log scale used for both axes.
Table 1 Results of a multiple regression analysis of standardized body size and mass, on duty cycle (arcsine transformed) and RMS amplitude (log10 transformed). Significant results are in bold. Term
Duty cycle model R2 = 0.62 Estimate
Size Mass Size2 Mass2 Size * mass
0.04 0.09 0.04 0.14 0.07
SE 0.04 0.04 0.03 0.03 0.06
RMS amplitude model R2 = 0.39 t
P 0.94 2 1.45 4.07 1.18
Fig. 2. Relationship between body mass and duty cycle. Intermediate mass males produce courtship signals having the highest duty cycles. Plotted are the transformed values. Regression equation: y = 10.75x2 0.21x + 1.2.
Salazar, 2011). In contrast to our prediction, the largest males were not the most efficient at courtship signaling. Rather, males having intermediate mass produced the highest duty cycle signals and were also the most efficient per unit of duty cycle performed. Although these results indicate that intermediate mass males are superior courters, whether their courtship signals are in fact preferred by females remains to be determined with additional studies. For example, an experiment that offers females simultaneous
Estimate
0.36 0.06 0.16 <0.01 0.25
0.02 0.13 0.05 0.03 0.18
SE 0.09 0.09 0.06 0.07 0.13
t
P 0.25 1.41 0.77 0.37 0.14
0.81 0.17 0.45 0.71 0.19
choice of males of different mass (e.g., light, medium, heavy) would directly test the outcome of male courtship performance on female choice, and thus reveal which type of male indeed represents a preferred mating partner in this species. The fact that duty cycle was significantly correlated with body mass suggests an important role for this courtship trait in L. hasselti. Consequently, duty cycle may provide useful information to females for evaluating which males are most metabolically efficient (Hill, 2011; Watson and Lighton, 1994; Watt, 1986). Our finding that an intermediate body mass is associated with high duty cycle signals suggests a disadvantage to lighter and heavier males during courtship. As mass reflects stored energy reserves, lighter males may simply not have a sufficient resource pool to sustain signaling for extended periods following a lengthy and risky period of mate searching. This is reinforced by the size-dependence of relative metabolic rates – lighter males expend the most energy per unit of body mass whether resting or courting (Fig. 1b), and thus their overall rate of energy expenditure may be too high to sustain long periods of courtship signaling. For heavier males, although their energy expenditure per unit of body mass was lowest across the sample of males (Fig. 1b), and therefore these males might be expected to be most metabolically efficient (Watson and Lighton, 1994), when their AMR was evaluated in conjunction with courtship signaling the amount of energy expended per unit of duty cycle was relatively high (Fig. 3a). Therefore, simply having a large energy reserve may not be enough to guarantee successful courtship. Rather, it is the interaction between mass and metabolic expenditure that determines which males are most efficient at high duty cycle signaling, and thus which males represent superior courters. In wolf spiders (Hygrolycosa rubrofasciata), males drum the substrate during courtship in an extremely energetically expensive
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Table 2 Results of a multiple regression analysis of standardized body size and mass, on (log10 transformed) duty cycle efficiency and RMS amplitude efficiency. Significant results are in bold. Term
Duty cycle efficiency model R2 = 0.49 Estimate
Size Mass Size2 Mass2 Size * mass
0.004 0.048 0.03 0.072 0.018
SE 0.02 0.02 0.02 0.02 0.03
RMS amplitude efficiency model R2 = 0.16 t
P 0.18 2.04 1.29 3.27 0.61
0.86 0.06 0.21 <0.01 0.55
Estimate 0.001 0.061 0.209 0.023 0.225
SE 0.11 0.11 0.11 0.1 0.14
t
P 0.01 0.55 1.85 0.23 0.16
0.99 0.59 0.07 0.82 0.13
Fig. 3. Relationship between body mass and (a) mass-corrected metabolic efficiency, and (b) absolute metabolic efficiency, for duty cycle. When corrected for body mass, both heavy and light males are equal in the amount of energy expended, while intermediate mass males expend the lowest amount of energy per unit of courtship performed. Regression equation: y = 5.78x2 0.2x 1.05. In (b) the heaviest males expend the highest absolute amount of energy per unit of courtship performed, while light and intermediate mass males are more similar in energy expenditure. Regression equation: y = 9.28x2 8.98x + 1.67. Plotted are the transformed values.
display (22 times higher than resting, Kotiaho et al., 1998), rapid drumming results in increased mortality (Mappes et al., 1996), and larger males pay higher metabolic costs for drumming (Kotiaho et al., 1998). However, males do not drum continuously as in L. hasselti, but in short one-second bursts given in bouts that last only a few minutes, which may allow some males to achieve the extremely high AMR that makes high energy drumming a reliable indicator of male quality in this species (Parri et al., 2002). In L. hasselti, although the difference between AMR and resting metabolic rate is much lower, courtship is a lengthy process lasting several hours, and thus endurance is as important as power when producing vibrational signals. Males must utilize their finite energy reserves efficiently to ensure they can last for the extended periods required by females, and thus the maximum AMR that can be obtained is likely to be much lower than for species such as wolf spiders, in which courtship occurs in brief bursts that may permit a greater maximum expenditure of energy (Peterson et al., 1990). In another web-building spider species (Stegodyphus lineatus) prolonged courtship using web-borne vibrations functions to stimulate a female into mating (Maklakov et al., 2003). Similarly, the minimum courtship threshold of 100 min in L. hasselti (Stoltz and Andrade, 2010) reveals that females here also require a lengthy period of stimulation before accepting a mate. What is the benefit to males of producing high duty cycle signals during such a prolonged bout of courtship? Recall that higher duty cycles indicate more signal is being produced relative to silent intervals and thus more energy is being broadcast (Jang and Greenfield, 1998; Mason, 1996; Reinhold et al., 1998). Because naturally-occurring
female L. hasselti webs sometimes extend over an area of several square meters and contain hundreds of web strands (Brunet, 2000; MCB Andrade, personal observation), a mate searching male that reaches a female’s web is probably uncertain of her precise location when he begins courtship. High duty cycle signals are therefore more likely to increase and maintain a female’s sensory stimulation (Stoddard and Salazar, 2011) while the male searches for her on the web (but see Vibert et al., 2014). Furthermore, high duty cycle signals are likely to propagate better through the environment and be less adversely affected by attenuation and reverberation along the web (Brenowitz, 1986; Casas et al., 2007; Elias et al., 2004; Schul and Patterson, 2003). Minimizing these effects is important regardless of web size in L. hasselti because females build ‘cobwebs’ with a complex, irregular three-dimensional structure that is likely to degrade transient, low duty cycle vibrational signals (Barth, 1998; Cocroft and Rodrìguez, 2005; Zevenbergen et al., 2008). Independent of these mechanistic advantages, females may simply prefer males that produce high duty cycle signals if such signals reflect the amount of energy a male can afford to allocate towards expensive activities (Bailey et al., 1993; Bennett, 1983; Dearborn et al., 2005; Halliday, 1987; Reinhold et al., 1998; Rivero et al., 2000; Salazar and Stoddard, 2008). This may be especially important in L. hasselti where males are expected to invest maximally in a single mating opportunity with no allocation trade-offs favoring future matings, immediately following a lengthy and risky period of mate searching. Thus, the ability to produce high duty cycle signals at low metabolic cost will be a reflection of a male’s entire energy reserve and the efficiency of
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metabolic expenditure throughout its life, which includes fitnessrelated traits expressed as a juvenile (e.g., foraging success), and as an adult (e.g., mate searching and courtship performance). Although RMS amplitude was not correlated with mass or size, this signal component could still have an important role in facilitating detection of a male by a female once he begins courting on her web. Although transmission effects of amplitude on cobwebs has not been thoroughly examined (Vibert et al., 2014), higher amplitude signals generally propagate farther in the environment regardless of substrate (Bradbury and Vehrencamp, 1998). For L. hasselti males, high RMS amplitude signals may be especially beneficial when several males are competing simultaneously for a female’s attention, which commonly occurs in this species (Andrade and Kasumovic, 2005; Stoltz and Andrade, 2010; Stoltz et al., 2008). To the extent that amplitude can provide directional information on a cobweb, a male that is able to signal ‘loudest’ is likely to elicit and maintain a female’s attention over any rivals that may also be present. In the western black widow, L. hesperus, high amplitude vibrations can result in a male sometimes being mistaken for prey (Vibert et al., 2014), however, the benefits of winning a female’s attention when many males are present may outweigh this risk in L. hasselti due to the intense competition for mates that occurs in this species. 5. Conclusion Recent studies suggest that metabolic efficiency is likely to be an important influence on the outcome of female choice (Hill, 2011; Kasumovic and Seebacher, 2013; Stoddard and Salazar, 2011; Thomson et al., 2014). In this study, we show that in a capital breeding animal that has only a single mating opportunity, metabolic efficiency and body mass are both strongly related to male courtship effort. Our findings reveal that courtship vibrational signaling in L. hasselti is energetically expensive and not all males are capable of producing high duty cycle signals. Furthermore, intermediate mass males may represent superior courters because they are best able to balance energy reserves and metabolic expenditure to be most efficient at producing high duty cycle signals. Acknowledgments We thank members of the Integrative Behaviour and Neuroscience Group at UTSC for their helpful input, the Andrade lab undergraduates for assistance rearing spiders, and K. Brochu for comments on an earlier version of this manuscript. We are especially grateful to C. Wellstood at Qubit Systems who provided valuable technical advice regarding operation of the respirometry equipment. L. Bussière and K. Judge provided much appreciated statistical advice. This work was supported by a Natural Science and Engineering Research Council of Canada (NSERC) post graduate scholarship to JAS, Discovery Grants to MCBA (229029-12) and ACM, and grants from the Canada Research Chairs program, the Canada Foundation for Innovation, and Research & Innovation Ontario (203764) to MCBA. This manuscript was greatly improved from comments provided by two anonymous reviewers. References Andersson, M., 1994. Sexual Selection. Princeton University Press, New Jersey. Andrade, M.C.B., 2003. Risky mate search and male self-sacrifice in redback spiders. Behav. Ecol. 14, 531–538. Andrade, M.C.B., Kasumovic, M.M., 2005. Terminal investment strategies and male mate choice: extreme tests of Bateman. Integr. Comp. Biol. 45, 838–847. Bailey, W.J., Cunningham, R.J., Lebel, L., 1990. Song power, spectral distribution and female phonotaxis in the bushcricket Requena verticalis (Orthoptera: Tettigoniidae): active female choice or passive attraction? Anim. Behav. 40, 33–42.
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