Courtship and male ornaments as honest indicators of immune function

Animal Behaviour 117 (2016) 97e103

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Courtship and male ornaments as honest indicators of immune function Rachel Gilbert*, George W. Uetz Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, U.S.A.

a r t i c l e i n f o Article history: Received 14 December 2015 Initial acceptance 18 February 2016 Final acceptance 20 March 2016 Available online 29 May 2016 MS. number: A15-01045R Keywords: courtship immunocompetence infection Schizocosa ocreata sexual signalling wolf spider

Having an effective immune system can be very costly, sometimes at the expense of other important life history traits, including reproduction. This trade-off can be exaggerated in males of species that have costly sexual signalling, where condition-dependent components of the signalling system reflect the health status of the bearer. It is therefore vital for a male to be able to adequately balance the costs of activating the immune system successfully while also expressing high-quality sexual signals. We examined males of the brush-legged wolf spider Schizocosa ocreata to see whether static condition-dependent components of sexual signalling in adult males are indicative of health status (immune stress response, encapsulation) and whether female preference for these traits is influenced by infection. After experimental ingestion of a bacterial pathogen (Pseudomonas aeruginosa), symmetry of male foreleg tuft (a secondary sexual trait) was found to predict the intensity of the subsequent infection, such that males with more asymmetrical tufts had higher levels of bacteria in the haemolymph. Females were equally likely to mate with infected and uninfected males in mating trials, but females that mated with infected males had bacteria in their haemolymph and on their body surface. Males that engaged in courtship had significantly lower encapsulation responses than males that did not engage in courtship, but among those males that courted, larger tuft size indicated a higher encapsulation response even after energetically costly courtship. These results indicate that females may be able to use static sexual signalling traits to examine a male's overall health, but females do not appear to discriminate against males who are actively infected, even though there is a direct cost to the female via the transfer of male infection. © 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Organisms across all taxa must cope with assault from a variety of parasites and pathogens. While it is advantageous for an individual to have a broadly effective immune system, there is often a complex balance between the ability to successfully execute an immune response and simultaneously express costly sexually selected traits, such as male secondary sexual characters. A broad generalization of the immunocompetence handicap hypothesis (ICHH) predicts that only high-quality males will be able to allocate resources to both immune system activation and the development of these condition-dependent traits (Folstad & Karter, 1992; Simmons, 2011). These types of sexual traits are therefore thought to enforce signalling honesty, since low-quality males cannot withstand the costs of both mounting an immune response and investing resources into sexual signalling (Folstad & Karter, 1992; Grafen, 1990; Zahavi, 1975). Consequently, females that

* Correspondence: R. Gilbert, Department of Biological Sciences, University of Cincinnati, 614 Rieveschl Hall, Cincinnati, OH 45221-0006, U.S.A. E-mail address: [email protected] (R. Gilbert).

select males with more exaggerated signalling traits may receive indirect fitness benefits via successful offspring (Sheldon & Verhulst, 1996; Westneat & Birkhead, 1998). While there is evidence that females gain indirect fitness benefits by selecting for a trait that indicates heritable immunocompetence (Cotter & Wilson, 2002; Fellowes, Kraaijeveld, & Godfray, 1998; Ryder & Siva-Jothy, 2001), there may also exist direct benefits to the female as well. If a condition-dependent trait is indicative of an individual's immune capacity, then it could signal a male's current state of infection, such that a female may be able to avoid an infected male and hence the risk of infection (Able, 1996; Houde & Torio, 1992; Loehle, 1997; Milinski & Bakker, 1990). Many studies in both vertebrates (Duffy & Ball, 2002; Garvin, Dunn, Whittingham, Steeber, & Hasselquist, 2007; Griggio, pez & Martín, 2005; Møller, 2002; Zanollo, & Hoi, 2009; Lo Mougeot, 2008) and invertebrates (Ahtiainen, 2004; Lawniczak et al., 2007; Rantala, Jokinen, Kortet, Vainikka, & Suhonen, 2002; Ryder & Siva-Jothy, 2000; Simmons, Zuk, & Rotenberry, 2005) have found a positive correlation between sexually selected traits and some aspect of immune function, suggesting that female mate

http://dx.doi.org/10.1016/j.anbehav.2016.04.013 0003-3472/© 2016 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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preference may be acting as a selective pressure that influences the ability of males to withstand such trade-offs between sexual signalling and immunity. However, some species have sexual displays that are complex, sometimes consisting of multiple signalling modalities that may each reflect different types of information (Candolin, 2003; Johnstone, 1996; Møller & Pomiankowski, 1993). In some cases, static (fixed) and dynamic traits may correlate with separate aspects of male quality (Møller & Pomiankowski, 1993). While each signal may convey information on different temporal aspects of male condition, all traits combined should be expected to reflect overall male quality, since females would be receiving a more complete assessment of males rather than just relying on a single trait (Doucet, 2003; Loyau, Saint Jalme, Cagniant, & Sorci,  pez, 2008; Møller, 2002). 2005; Martín, Amo, & Lo The brush-legged wolf spider Schizocosa ocreata has a multimodal sexual signalling system, with both visual (foreleg ornaments and leg-tapping displays) and vibratory (stridulation and percussion) components. Females prefer males with larger foreleg tufts, greater courtship display rates and higher-amplitude vibratory signals. The foreleg ornaments (tufts of bristles) are fixed at sexual maturity and are indicative of past condition (Uetz, McClintock, Miller, Smith, & Cook, 1996; Uetz, Papke, & Kilinc, 2002; Uetz, Roberts, Wrinn, Polak, & Cameron, 2009) whereas behavioural displays (courtship vigour) are more dynamic and prone to fluctuate with current male condition (Gibson & Uetz, 2012). There is also some evidence in S. ocreata that past male condition reflected in these traits may include exposure to parasites and pathogens. Males infected in the laboratory as juveniles had more asymmetrical foreleg tufts at maturity, as well as decreased courtship vigour and lower mating success than control males, indicating that infection is causing some level of developmental stress that results in lower-quality condition-dependent traits (Gilbert, Karp, & Uetz, 2016). Additionally, males infected as juveniles had higher encapsulation rates (one measure of immune function) at maturity than control males, suggesting that resources may be allocated away from investment in sexual signals and into immune function (Gilbert et al., 2016). While these results suggest that there is some type of life history trade-off occurring in at least one condition-dependent modality expressed by this species, it remains to be examined whether infection as an adult has an impact on male signalling quality and effort. Because foreleg tufts are fixed after moulting to sexual maturity, infection as an adult would be expected to affect only the more dynamic components of the signalling system, such as courtship vigour. As a consequence, females may not be able to assess male infection status as accurately, since it is unlikely that active infection (exposure as an adult) would be reflected in fixed traits (i.e. leg tufts). It is currently unknown whether dynamic signalling modalities can potentially indicate to a female that the male signaller is currently infected. In this study, we examined relationship(s) between immunity and multimodal sexual signalling in several ways: (1) by infecting adult males and looking at the impact of infection on courtship, performance and mating success; (2) by investigating the potential for transfer of infection during copulation to assess the potential fitness consequences of mating with an infected male and (3) by testing whether the expression of an adult indicator trait (foreleg tuft size) is related to immune response (Fig. 1). METHODS

U.S.A.) in August 2014. Spiders were housed individually in opaque deli dish containers on a 13:11 h light:dark cycle, provided access to water ad libitum and fed on a consistent schedule of two to three crickets (Acheta domesticus, approximately 3.2 mm in length) twice per week. Spiders were kept in separate opaque containers except during mating trials. We examined all individuals postmortem for the presence of parasites (nematodes, insect larvae) and excluded infected individuals from our analysis to rule out the possibility of immunosuppression outside of the experimental treatment. All spiders used in the following experiments were 7e12 days postmaturity. Experimental Infection Infection methods were modified from Gilbert et al. (2016). One week after moulting to sexual maturity, males were subjected to oral ingestion of the bacterial pathogen Pseudomonas aeruginosa (strain PA-14). This pathogen occurs naturally in the environment in which these spiders are found and has been found in the haemolymph of a few individuals at very low levels (Gilbert & Uetz, n.d.). All stocks were kept in Copan Cryovials at
80  C and grown on Luria broth media (1.0% Tryptone, 0.5% yeast extract, 1.0% NaCl, 1.5% agar). All plates containing bacteria were cultured daily approximately 18 h prior to use in the experiments and were discarded within 24 h after culturing. Spiders (N ¼ 60) were withheld water for 24 h to encourage complete consumption of a 1 ml droplet of sterile water containing 600 colony forming units (CFUs) of bacteria as determined by McFarland turbidity standards (McFarland, 1907). Any spider not observed drinking the full amount of water was dismissed from further experiments. Control groups (N ¼ 50 males) were withheld water for 24 h, then given a 1 ml droplet of sterile water only. Following exposure, spiders were returned to a clean housing container and resumed a normal diet and ad libitum access to water. Mating Trials We placed infected males (N ¼ 20, within 1 h of infection) and control males (N ¼ 20) individually into a round plastic arena (diameter: 15.5 cm, height 7.2 cm) lined with filter paper. After a 2 min acclimation period, we placed a virgin adult female (N ¼ 40) in the centre of the arena and allowed each pair 5 min to begin mating. Pairs that did not mate within 5 min were removed from the arenas (N ¼ 8). In cases of successful mating (N ¼ 32), copulation was allowed to proceed until natural separation of mating pairs (2e10 h). Courtship vigour and mating success All behaviour trials were recorded using a Sony camcorder (model HDV-XR260V) and scored blindly at a later date for male courtship displays (leg waves, leg taps, body bounce) to get an approximation of male courtship vigour (number of courtship displays per second) and overall mating success (Delaney, Roberts, & Uetz, 2007; Kaston, 1936; Montgomery, 1903). Transfer of infection After allowing mated pairs to separate naturally, we immediately quantified (within 10 min) P. aeruginosa CFUs on the body surface and in the haemolymph of surviving (noncannibalized) males (N ¼ 25 total, 15 infected and 10 control) and females (N ¼ 25 total).

Study Species and Care All spiders were captured as juveniles from a deciduous leaf litter forest at the Cincinnati Nature Center (Clermont County, OH,

Quantification of bacterial presence To determine levels of P. aeruginosa infection on the body surface and in the haemolymph of treated S. ocreata at the conclusion

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60 Males infected at 1 week postmaturity Water withheld 24 h, given 1 µl sterile water containing 600 CFU Pseudomonas aeruginosa

50 Control males at 1 week postmaturity

Mating trials N=16 Infected males N=16 Control males N=32 Females

Intensity of infection and tuft size N=13 Males 1 h postinfection N=13 Males 3 h postinfection N=13 Males 5 h postinfection N=25 Control males

Courtship and encapsulation N=18 Noncourting males N=18 Noncourting males with tuft measurements

N=12 Courting males N=10 Courting males with tuft measurements

Transfer of infection N=15 Infected mating pairs N=10 Control mating pairs

Figure 1. Visual representation of the methods used for all experiments. Top box represents initial sample sizes; bottom boxes represent actual sample sizes of spiders that survived to be included in statistical analysis. Spiders were also removed from experiments if they were found to have parasites. CFU: colony forming units.

of the experiment, we anaesthetized the spiders in CO2, then subsequently dipped them in 200 ml of 1X PBS buffer and vortexed them at low power for 60 s. We then removed the spiders and immediately surface-sterilized them by dipping them in both 95% ethanol (EtOH) and a sterilizing solution (5% NaClO, 10% EtOH, 85% sterile DI H2O; Yoder et al., 2003). After allowing 60 s for the spiders to dry, we harvested 3 ml of haemolymph by removing the first and second legs at the coxal joints. We used a capillary tube to transfer the haemolymph into an Eppendorf tube containing 200 ml of sterile 1X PBS buffer. We spread both solutions of buffer (one collected from the body surface and one from the haemolymph) onto an LB plate and allowed it to incubate for 18 h at 35  C. We counted individual colonies using ImageJ. Intensity of Infection and Tuft Size and Symmetry To examine whether male tuft size or symmetry varies with the intensity of infection, we counted the number of CFUs from the haemolymph of adult virgin males 1 h (N ¼ 25), 3 h (N ¼ 25) and 5 h (N ¼ 25) after oral ingestion of P. aeruginosa (see Quantification of Bacterial Presence). We then assessed the size and symmetry of each male's tufts postmortem. We measured male tufts using ImageJ. We examined haemolymph of control males (N ¼ 25) using the same methods to compare levels of infection between treatment groups.

Courtship, Tuft Size and Encapsulation We also examined whether energetically costly courtship influences immune responses of uninfected males and whether immune response (encapsulation) is correlated with foreleg tuft size. Males used in this experiment were exposed to female chemical cues only (i.e. silk cues, rather than live females) to reduce variation in the amount of time that each male spent courting. We collected female silk cues by placing a female (2 weeks postmaturity) on filter paper and allowing her to deposit silk for 24 h. We used silk from a different female for each male tested. We allowed males to court for 5 min. Then, 24 h later, we evaluated encapsulation responses of males that courted in response to silk cues and males that were not exposed to silk cues and thus not given an opportunity to court. We used the encapsulation response in this experiment as an approximation of immune function (Ahtiainen, 2004; Ahtiainen, Alatalo, Kortet, & Rantala, 2005). Methods used for measuring encapsulation rate were modified from Ahtiainen et al. (2005). We anaesthetized spiders (N ¼ 45) using CO2 and immobilized them by taping them upside down on a glass slide. We used a sterile needle (Hamilton, 26 gauge) to puncture the underside of the abdomen, through which we introduced a sterile nylon monofilament (Stroft, 0.5  0.08 mm) into the body cavity. After 180 min, we dissected out the filament (a lethal process) and digitally imaged it. We

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measured encapsulation melanization by taking the total greyscale average between 0 and 200 (ImageJ, National Institutes of Health, Bethesda, MD, U.S.A., http://rsbweb.nih.gov/ij/). Higher greyscale average values indicate lower density of melanization. In the present study, we report the inverse function of these values to make them more intuitive (where higher values reflect higher densities of melanization). We measured tufts for these same individuals by removing both forelegs (postmortem) and using ImageJ to find tuft area (in mm2). We quantified tuft symmetry by taking the absolute value of the left tuft area minus the right tuft area. We expressed tuft size as the average of the area for both tufts. We quantified relative tuft size by scaling to a fixed measure of body size (cephalothorax width). Individuals were removed from the analysis if they died before the 180 min encapsulation period had concluded, if the male had not courted for the full 5 min, if the filament was not able to be found or had been forced out through the abdomen prematurely, or if foreleg tufts were not able to be accurately assessed.

100 90 80 % Successful copulation

100

70 60 50 40 30 20 10 0

Ethical Note Although there are no state or federal regulations (Ohio, U.S.A.) and no institutional requirements (University of Cincinnati) for care and maintenance of our study species, S. ocreata, we made every effort to comply with ASAB/ABS Guidelines for the treatment of animals in behavioural research and teaching. We infected spiders with a proven sublethal dose of P. aeruginosa, a common arthropod pathogen, to which they are likely exposed in the wild. We know of no published materials concerning pain or discomfort caused by ingestion and subsequent infection of this pathogen. At the end of the study, spiders were humanely euthanized with CO2 anaesthetization and freezing, then placed in 70% ethanol.

Statistical Analysis We used a ShapiroeWilk test for normality on every response variable for the data. All distributions were found to fit a normal distribution with a P value greater than 0.05. We used a Grubb's outlier test to determine statistical outliers in the data. We then removed outliers from the data set and re-evaluated the data for normality. Outlier removal only occurred for the tuft symmetry test, in which the outliers most likely occurred because tuft dissection in three documented cases involved accidental removal of some bristles, which influenced the results unnecessarily. We used Dunnett's (1955) post hoc analysis for the haemolymph assays because we performed multiple t tests to compare treatment groups to one control. For encapsulation data, we used the inverse function simply to make the data easier to interpret (higher numbers in the raw data reflected lower encapsulation; thus, we used the inverse to indicate higher values as higher encapsulation).

Control

Infected

Figure 2. Overall percentage of successful mating attempts by control and infected males.

Transfer of infection After natural separation of copulating pairs, there was a significant correlation between total CFUs on the infected male's body surface and total CFUs on the (previously uninfected) female's body surface (R2 ¼ 0.446, F1,10 ¼ 6.02, P ¼ 0.034; Fig. 3a), indicating transfer during copulation. Likewise, significant correlations were found between total CFUs in the male's haemolymph and total CFUs on the female's body surface (R2 ¼ 0.510, F1,14 ¼ 16.46, P ¼ 0.0014; Fig. 3b) and between total CFUs on the male's body surface and total CFUs in the female's haemolymph (R2 ¼ 0.545, F1,10 ¼ 12.11, P ¼ 0.0059; Fig. 4). There was no significant correlation between number of CFUs transferred and duration of copulation for either female body surface (F1,14 ¼ 0.113, P ¼ 0.743) or female haemolymph (F1,14 ¼ 1.206, P ¼ 0.3006). No culturable bacteria were seen in any of the control mated pairs, nor in the pairs with treated males where the female was not mounted and had not successfully copulated. Infection Intensity and Tuft Size and Symmetry Overall, there was a significant correlation between the number of CFUs in male haemolymph and tuft asymmetry (F1,43 ¼ 15.224, P ¼ 0.0003). Subsequent analyses revealed a significant correlation between tuft asymmetry and CFUs in haemolymph at 1 h (R2 ¼ 0.694, F1,13 ¼ 29.497, P ¼ 0.0001) and 3 h (R2 ¼ 0.39, F1,13 ¼ 8.312, P ¼ 0.0128) postinfection, but not at 5 h postinfection (R2 ¼ 0.224, F1,13 ¼ 3.753, P ¼ 0.074; Fig. 5). Courtship, Tuft Size and Encapsulation

RESULTS Mating Trials Male courtship vigour and mating success For males that were exposed to live females during mating trials, there was no significant difference between control males and infected males in the amount of time taken to successfully copulate with a female (F1,34 ¼
1.129, P ¼ 0.266). There was also no statistically significant difference in courtship vigour (t1,34 ¼ 1.40, P ¼ 0.17) or overall mating success (c21 ¼ 0.805, P ¼ 0.369; Fig. 2).

For uninfected males exposed to female silk cues, there was a significant correlation between encapsulation rate and tuft size, such that males with larger tufts had a higher rate of encapsulation (R2 ¼ 0.219, F1,18 ¼ 5.073, P ¼ 0.037; Fig. 6). In addition, following exposure to female silk cues, males that performed 5 min of courtship had significantly lower encapsulation than males that did not perform courtship (F1,30 ¼ 4.74, P ¼ 0.037; Fig. 7). Among those males that courted, males with larger relative tuft size had significantly higher encapsulation rate than those with smaller relative tuft size (R2 ¼ 0.365, F1,10 ¼ 5.193, P ¼ 0.0487).

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100

100

80 70 60 50 40 30 20 10 0

90

CFUs on female body surface

(a)

90

CFUs on female body surface

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100 200 300 CFUs on male body surface

(b)

80 70 60 50 40 30 20 10

400

0

5 10 15 20 CFUs in 3 µl male haemolymph

25

Encapsulation (1/greyscale density)

Figure 3. (a) Number of colony forming units (CFUs) found on the body surface of uninfected females after copulation compared to the number on the male's body surface. (b) Number of CFUs found on the female's body surface postcopulation compared to the total CFUs found in infected male haemolymph.

CFUs in 3µl female haemolymph

20 18 16 14 12 10 8 6 4 2 0

100

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300

0.0095 0.009 0.0085 0.008 0.0075 0.007 0.0065 0.006 0.0055 0.005 1.5

400

CFUs on male body surface Figure 4. Number of colony forming units (CFUs) found in female haemolymph after copulation compared to CFUs found on the infected male's body surface.

1.7

1.9 2.1 2.3 Average tuft size (mm2)

2.5

2.7

Figure 6. Encapsulation rate of males relative to average male foreleg tuft size. The inverse relationship of greyscale density was used to make the relationship clearer.

0.0068

250 200

0.0066

1h 0.0064

3h

One/encapsulation

CFU/3µl haemolymph

300

5h

150 100 50

0.0062

0.006 0.0058 0.0056 0.0054

0

0.2

0.4 0.6 0.8 Tuft asymmetry (|L-R|) (mm2)

1

1.2

Figure 5. Number of colony forming units (CFUs) found in circulating haemolymph of adult male S. ocreata compared to tuft asymmetry. Separate lines indicate samples grouped by the time the bacteria were measured (latency postinfection).

0.0052 0.005

Courting

Noncourting

Figure 7. Encapsulation rates of males that courted for 5 min when exposed to female silk cues (no female present) and of noncourting males that were not exposed to female silk cues.

DISCUSSION The results of this study show that, overall, there is strong evidence that male foreleg tufts may be indicators of a male's ability to resist pathogenic infection, as well as the ability to cope with the

high energetic costs of courtship while still maintaining a higher level of immune function relative to males with smaller foreleg tufts. This supports the idea that static sexual traits can be indicative of different and distinct temporal aspects of male quality and

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may potentially provide a choosy female with indirect benefits, such as more fit offspring (Ali & Tallamy, 2010; Head, Hunt, Jennions, & Brooks, 2005; Hoikkala, Aspi, & Suvanto, 1998). Because this experiment required sacrifice of mated females, fecundity and offspring quality and other fitness measures could not be evaluated. Future experiments will involve a more in-depth examination of heritable fitness. While females choosing a male with larger and more symmetrical tufts may accrue indirect benefits by having more attractive or fit offspring, there are more immediate and direct benefits of female choice. In this study, we found that males with more symmetrical foreleg tufts had a lower intensity of infection after consuming a bacterial pathogen. We also found that males with a higher intensity of infection transferred more bacteria to the body surface and haemolymph of females during copulation. These findings suggest that a female who chooses a male with more symmetrical foreleg tufts could be less susceptible to transmittable diseases or parasites (Able, 1996; Milinski & Bakker, 1990). The transmission of parasites and pathogens during copulation has been shown in some insects (Knell & Webberley, 2004; Mann, PelzStelinski, Hermann, Tiwari, & Stelinski, 2011; Miest & Bloch-Qazi, 2014; Reinhardt, Naylor, & Siva-Jothy, 2005; Webberley, Buszko, Isham, & Hurst, 2006), but prior to the present study, had not previously been examined in spiders. Transmission during copulation could be occurring through a number of modes, including seminal fluids (Lung, Kuo, & Wolfner, 2001; Otti, Naylor, Siva-Jothy, & Reinhardt, 2009), surface exposure (Miest & Bloch-Qazi, 2014) or cuticular puncture by males using their fangs to restrain females (Johns, Roberts, Clark, & Uetz, 2009). Since the results our experiment only provide sufficient evidence for surface exposure, other potential modes of pathogen transmission remain to be examined in S. ocreata. Previous studies of S. ocreata have shown that infection at the juvenile stage significantly reduces male tuft symmetry, courtship vigour and overall mating success after sexual maturity (Gilbert et al., 2016). Males that were exposed to bacterial infection as a juvenile ultimately had higher encapsulation response as adults than males that were not exposed to pathogens, but in this study, infection as an adult did not have an impact on any aspect of mating success. This suggests that the impact of infection on reproduction in males may depend on age or life stage (terminal investment), such that males choose to invest more resources in courtship and mating rather than fighting off infection when exposed to pathogens as an adult, whereas males infected before sexual maturity choose to invest in fighting off infection (and thus surviving to sexual maturity (Jacot, Scheuber, Kurtz, & Brinkhof, 2005; Polak & Starmer, 1998)). Because female S. ocreata only mate once (usually within 1e2 weeks postmaturity; Norton & Uetz, 2005), there could be even higher selection pressure for mature males to invest resources into mating instead of immune activation, since mating opportunities are limited and drop severely as the mating season progresses (Roberts & Uetz, n.d.). While this study only examined male foreleg tufts and courtship vigour, there are other signalling modalities in this sexual signalling system that may reflect infection status. For example, males in this species have a complex vibratory component, where males use their pedipalps to create a complex and condition-dependent vibration through the leaf litter (Gibson & Uetz, 2012). The amplitude of this vibratory courtship is correlated with male size (static) as well as male mass (dynamic), so it is possible that the signal quality may be reduced for infected males and would therefore influence female mate choice for this modality (Gibson & Uetz, 2008). Future studies should examine the impact of infection on the remaining components of the multimodal sexual signalling system in S. ocreata.

This experiment demonstrates the first evidence of the transmission of pathogenic bacteria during copulation in spiders, as well as compelling evidence that the secondary sexual trait in this species potentially reflects both direct and indirect benefits to females. By discriminating against males whose smaller or more asymmetrical leg tufts may signal lower immunity and lower resistance to parasites, females reduce the chance of infection and/ or might pass stronger immune traits on to their offspring. Further work will examine the heritability of these properties as well as further potential fitness consequences to females and offspring. Acknowledgments This research was supported by National Science Foundation grant IOS-1026995 (to G.W.U.), the University of Cincinnati Sigma Xi Chapter (to R.G.) and the University Research Council. This research was conducted for the requirements of the Ph.D. program in the Department of Biological Sciences at the University of Cincinnati. We thank the Cincinnati Nature Center for providing access to their property to collect the spiders used in this project. Thanks to J. Benoit and B. Stoffer for providing feedback on early drafts of the manuscript. Thanks also to Julie Stacey for assisting with microbiological materials and to C. Combs for providing research assistance. References Able, D. J. (1996). The contagion indicator hypothesis for parasite-mediated sexual selection. Proceedings of the National Academy of Sciences of the United States of America, 93(5), 2229e2233. http://dx.doi.org/10.1073/pnas.93.5.2229. Ahtiainen, J. J. (2004). Sexual advertisement and immune function in an arachnid species (Lycosidae). Behavioral Ecology, 15(4), 602e606. http://dx.doi.org/ 10.1093/beheco/arh062. Ahtiainen, J. J., Alatalo, R. V., Kortet, R., & Rantala, M. J. (2005). A trade-off between sexual signalling and immune function in a natural population of the drumming wolf spider Hygrolycosa rubrofasciata. Journal of Evolutionary Biology, 18(4), 985e991. http://dx.doi.org/10.1111/j.1420-9101.2005.00907.x. Ali, J. G., & Tallamy, D. W. (2010). Female spotted cucumber beetles use own cuticular hydrocarbon signature to choose immunocompatible mates. Animal Behaviour, 80, 9e12. http://dx.doi.org/10.1016/j.anbehav.2010.03.014. Candolin, U. (2003). The use of multiple cues in mate choice. Biological Reviews, 78(4), 575e595. http://dx.doi.org/10.1017/S1464793103006158. Cotter, S. C., & Wilson, K. (2002). Heritability of immune function in the caterpillar Spodoptera littoralis. Heredity, 88(4), 229e234. http://dx.doi.org/10.1038/ sj.hdy.6800031. Delaney, K. J., Roberts, J. A., & Uetz, G. W. (2007). Male signaling behavior and sexual selection in a wolf spider (Araneae: Lycosidae): a test for dual functions. Behavioral Ecology and Sociobiology, 62(1), 67e75. http://dx.doi.org/10.1007/ s00265-007-0438-7. Doucet, S. M. (2003). Multiple sexual ornaments in satin bowerbirds: ultraviolet plumage and bowers signal different aspects of male quality. Behavioral Ecology, 14(4), 503e509. http://dx.doi.org/10.1093/beheco/arg035. Duffy, D. L., & Ball, G. F. (2002). Song predicts immunocompetence in male European starlings (Sturnus vulgaris). Proceedings of the Royal Society B: Biological Sciences, 269(1493), 847e852. http://dx.doi.org/10.1098/rspb.2002.1956. Dunnett, C. W. (1955). A multiple comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association, 50(272), 1096e1121. Fellowes, M. D. E., Kraaijeveld, A. R., & Godfray, H. C. J. (1998). Trade-off associated with selection for increased ability to resist parasitoid attack in Drosophila melanogaster. Proceedings of the Royal Society B: Biological Sciences, 265(1405), 1553e1558. http://dx.doi.org/10.1098/rspb.1998.0471. Folstad, I., & Karter, A. J. (1992). Parasites, bright males, and the immunocompetence handicap. American Naturalist, 139(3), 603e622. Garvin, J. C., Dunn, P. O., Whittingham, L. A., Steeber, D. A., & Hasselquist, D. (2007). Do male ornaments signal immunity in the common yellowthroat? Behavioral Ecology, 19(1), 54e60. http://dx.doi.org/10.1093/beheco/arm099. Gibson, J. S., & Uetz, G. W. (2008). Seismic communication and mate choice in wolf spiders: components of male seismic signals and mating success. Animal Behaviour, 75, 1253e1262. http://dx.doi.org/10.1016/j.anbehav.2007.09.026. Gibson, J. S., & Uetz, G. W. (2012). Effect of rearing environment and food availability on seismic signalling in male wolf spiders (Araneae: Lycosidae). Animal Behaviour, 84, 85e92. http://dx.doi.org/10.1016/j.anbehav.2012.04.010. Gilbert, R., & Uetz, G.W. (n.d.). [Survey of bacteria in juvenile spiders and their environment using quantification and descriptive assays]. Unpublished raw data.

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