No experimental effects of parasite load on male mating behaviour and reproductive success

Animal Behaviour 82 (2011) 673e682

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No experimental effects of parasite load on male mating behaviour and reproductive success Shirley Raveh a, *, Dik Heg b,1, F. Stephen Dobson c, d, 2, David W. Coltman e, 4, Jamieson C. Gorrell e, 4, Adele Balmer d, 3, Simon Röösli f, 5, Peter Neuhaus a, g, 6 a

University of Neuchâtel, Institute of Biology, Eco-Ethology Department of Behavioral Ecology, University of Bern Centre d’Ecologie Fonctionnelle et Evolutive, Centre National de la Recherche Scientifique d Department of Biological Sciences, Auburn University e Department of Biological Sciences, University of Alberta f University of Neuchâtel, Institute of Biology, Parasitology g Department of Biological Sciences, University of Calgary b c

a r t i c l e i n f o Article history: Received 7 May 2010 Initial acceptance 16 September 2010 Final acceptance 10 June 2011 Available online 17 August 2011 MS. number: 10-00318R Keywords: Columbian ground squirrel manipulation parasite infestation paternity reproductive success Urocitellus columbianus

Parasites can negatively affect their host’s physiology and morphology and render host individuals less attractive as mating partners. The energetic requirements of defending against parasites have to be traded off against other needs such as feeding activity, territoriality, thermoregulation or reproduction. Parasites can affect mating patterns, with females preferentially mating with parasite-resistant or parasite-free partners. We tested experimentally whether removal of both ectoparasites and endoparasites on free-living, male Columbian ground squirrels, Urocitellus columbianus, affected male mating behaviour, reproductive success and seasonal and posthibernation weight gain compared to control males. We predicted that experimental males would lose less body mass and mate more often than control males. In addition, we predicted experimental males would copulate earlier than control males in the mating sequences of receptive females and sire more offspring, because this species exhibits a strong first-male paternity advantage. Parasite treatment significantly reduced the parasite loads of experimental males. None of these males had ectoparasites at the end of the season, compared to 70% infestation of the control males. However, contrary to our expectations, the experimental treatment did not affect male reproductive behaviour (mating frequency, mating position, consort duration and mate-guarding duration), did not increase male reproductive success, and did not influence male body mass. We conclude that parasite infestation plays a minor role in affecting male reproductive behaviour, maybe because of the overall low infestation rates. Alternatively, males may be able to compensate for any costs associated with moderate loads of parasites. Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

* Correspondence and present address: S. Raveh, Konrad-Lorenz-Institute of Ethology, Department of Integrative Biology and Evolution, University of Veterinary Medicine, 1160 Vienna, Austria. E-mail address: [email protected] (S. Raveh). 1 D. Heg is now at the Institute of Social and Preventive Medicine, University of Bern, 3012 Bern, Switzerland. 2 F. S. Dobson is at the Centre d’Ecologie Fonctionnelle et Evolutive - Unité Mixte de Recherche 5175, Centre National de la Recherche Scientifique, 1919 Route de Mende, Montpellier 34293, France. 3 A. Balmer is at the Department of Biological Sciences, 331 Funchess Hall, Auburn University, AL 36849, U.S.A. 4 D. W. Coltman and J. C. Gorrell are at the Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. 5 S. Röösli is at the University of Neuchâtel, Institute of Biology, Parasitology, Rue Emile-Argand 11, Case postale 158, 2009 Neuchâtel, Switzerland. 6 P. Neuhaus is at the Department of Biological Sciences, University of Calgary, 2500 University Dr NW, Calgary, Alberta T2N 1N4, Canada.

Parasites may have detrimental effects on their hosts (Thompson & Kavaliers 1994; Sheldon & Verhulst 1996; Møller et al. 1999). For example, an infection may lead to a reduction in host fertility (Lockhart et al. 1996), alter an animal’s relative attractiveness to potential mates (Hamilton & Zuk 1982; Møller et al. 1999; Verhulst et al. 1999) or affect whether and when to start breeding (Buchholz 2004). Studies in several taxa have also shown that parasites may affect mate choice in both sexes (Freeland 1976; Birkhead et al. 1993; Møller et al. 1999; Barber 2002; Moore & Wilson 2002; Altizer et al. 2003). Frequent contact with conspecifics increases the likelihood of parasite transmission; thus parasites are expected to create a ‘cost’ of sociality (Alexander 1974; Hoogland & Sherman 1976; Hoogland 1995). In addition, males are usually more parasitized than females (Poulin 1996; Schalk & Forbes 1997; Zuk & Johnsen 2000; Moore & Wilson 2002; Morand et al. 2004; Perez-Orella & Schulte-Hostedde

0003-3472/$38.00 Ó 2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2011.06.018

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2005; Gorrell & Schulte-Hostedde 2008). Larger home ranges (Greenwood 1980; Ims 1987; Brei & Fish 2003; Nunn & Dokey 2006) and androgenic hormones suppressing the immune system (Folstad & Karter 1992; Mougeot et al. 2006) may both increase risk of infection and thus may explain this male-biased parasitism (Ferrari et al. 2004). Parasites and resistance to parasites play a prominent role in sexual selection theory (Hamilton & Zuk 1982; Clayton 1991; Zuk 1992; Zuk & Johnsen 2000). Females cannot increase their reproductive output simply by increasing their number of mating partners because their output is limited by their egg production (Bateman 1948). However, females can optimize their reproductive success by acquiring resistant genes for their offspring from the sire (Zeh & Zeh 1996; Jennions & Petrie 1997). According to the theory of Hamilton & Zuk (1982), females may discriminate against parasitized males by considering costly secondary sexual traits indicative of parasite burden. This theory has frequently been tested by relating conspicuous visual or acoustic displays in male birds and fish to their parasite load or resistance (Clayton 1991; Zuk 1992). Hence, females can increase their fitness both directly by reducing their own risk of parasite transmission and indirectly by enhancing the parasite and/ or disease resistance of their offspring (Hamilton & Zuk 1982; Zuk et al. 1995). Parasite-mediated sexual selection assumes that a genetic advantage is conferred by the ‘resistant’, uninfected male and that parasite resistance is heritable (Clayton 1991). In laboratory experiments, avoidance of infected conspecifics has been demonstrated in rodents, fish and birds (Milinski & Bakker 1990; Kavaliers & Colwell 1995; Zuk et al. 1995, 1998; Penn & Potts 1998; Barber 2002; Ehman & Scott 2002; Kavaliers & Colwell 2003; Kavaliers et al. 2003, 2004, 2005b; Deaton 2009). However, few studies have conducted parasite manipulations on free-living mammals and birds, mainly because of the difficulties of manipulation and observation in the field (Richner et al. 1993; Neuhaus 2003; Charmantier et al. 2004; Madden & Clutton-Brock 2009; Hillegass et al. 2010). We studied the relationships between parasite load, reproductive behaviour and reproductive success of free-ranging male Columbian ground squirrels, Urocitellus columbianus, by manipulating male parasite load. Columbian ground squirrels are diurnal, allow reliable observations of mating behaviour, and are tolerant of experimental manipulations in the wild (Murie et al. 1998; Neuhaus 2000; Nesterova 2007). Furthermore, females are in oestrus for only a few hours (<12 h) on a single day each year (Murie 1995), which makes it feasible to obtain complete mating observations on focal females in oestrus. Although mating mainly occurs in underground burrows, copulations or ‘consortships’ are readily detected using established behavioural criteria (Hanken & Sherman 1981; Hoogland & Foltz 1982; Sherman 1989; Boellstorff et al. 1994; Murie 1995). Females mate with up to eight different males while in oestrus, with mating order predicting siring success, indicating that maleemale competition and sperm competition play a major role in generating variation in male reproductive success (Raveh et al. 2010a, b). In the present study we removed ectoparasites and endoparasites on half of the reproductive males in three different colonies using chemical agents (experimental males). Control males were also caught, and treated with a sham solution. We compared these two groups of males to identify the impact of parasites on male mating behaviour, male reproductive success and changes in male body mass, during the 2e3 weeks of the mating season. This is a critically important period for male reproductive success and perhaps fitness, since males give no parental care to their offspring. We predicted that (1) experimental males should show an increase in reproductive behaviours known to translate into reproductive success, such as a higher mating frequency, a higher likelihood of obtaining the first mating position, longer consorts and increased

mate-guarding durations compared to control males. Mate guarding is considered a costly postcopulatory behaviour as a result of increased visibility to predators, energy investment in chasing females, fighting with opponents and missed mating opportunities with other females (Martín & López 1999; Plaistow et al. 2003; Cothran 2004). If parasites have an impact on ejaculate quality or quantity, an increase in time spent mate guarding for parasitized males could be an alternative explanation for differences in mate-guarding duration. We also predicted that (2) experimental parasite-free males should have higher siring success and seasonal reproductive success than control males. Finally, we predicted that (3) experimental males should lose less weight throughout the breeding season and after hibernation than control males. METHODS Study Species We studied Columbian ground squirrels in the Sheep River Provincial Park, Alberta, Canada (110
W, 50
N, and 1500 m elevation). Data on the ground squirrels were obtained from April to mid-July in 2007 and 2008 on three neighbouring colonies (‘meadow’ A, B, C). Columbian ground squirrels are diurnal, inhabiting subalpine and alpine meadows where they live in groups of a dozen to a few hundred individuals (Dobson & Oli 2001). On our study meadows, adult males emerge first from hibernation around mid-April, followed by females a few days to a week later (Murie & Harris 1982; Raveh et al. 2010a). Females breed on average 4 days after emergence from hibernation (Murie 1995). The mating season lasts about 2e3 weeks, depending on emergence dates of adult females (Murie 1995; Raveh et al. 2010a). About 24 days later, females give birth to a litter averaging three (one to seven) naked, blind juveniles in a specially constructed nest burrow (Murie et al. 1998). The offspring emerge above ground when they are approximately 27 days old (Murie & Harris 1982). Experimental Procedure Ground squirrels were caught within the first 2 days of emergence from hibernation with live traps baited with peanut butter (15  15 cm and 48 cm high and 13  13 cm and 40 cm high; National Live Trap Corp., Tomahawk, WI, U.S.A.) and weighed with a Pesola spring scale to the nearest 5 g. This first body mass measurement for each individual male and year combination was entered in the remainder of the analyses. Thereafter, animals were retrapped weekly to obtain body weight. Individually numbered fingerling fish tags (National Band & Tag Company Monel no. 1, Newport, KY, U.S.A.) were attached in both ears for permanent identification. In addition, each ground squirrel was uniquely marked with hair dye on the dorsal fur (Clairol, Hydriance black pearl No. 52, Proctor and Gamble, Stamford, CT, U.S.A.) for visual identification from a distance. All reproductive males were randomly separated into two treatment groups (experimental or control) in each colony separately. The experimental group (abbreviated with an E) was treated with a spot-on solution (Stronghold, Pfizer Animal Health, Montreal, Canada) and flea powder (Zodiac, Wellmark International, Dallas, TX, U.S.A.) to remove ectoparasites and endoparasites (N ¼ 33 males). Stronghold treats against both endoparasites and ectoparasites, and was applied between the shoulders on the skin, using one drop per 100 g of body mass. The flea powder was applied from a shaker, which had several holes on top, and the dosage was three shakes on the back and two shakes on the belly, with the powder applied by rubbing it into the male’s fur. To ensure that mate choice by females was not the result of secondary treatment effects (i.e. handling or odour cues), control animals

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(abbreviated with a C, N ¼ 32) were handled similarly by simulating the flea powder treatment with a massage and by applying a sham of isopropyl-alcohol (the alcohol in the Stronghold solution). First treatments were applied either directly at emergence after hibernation or before the mating period started; further applications were applied after the day’s consortships were over to ensure an undisturbed sequence of mating behaviours. Thus, considerable time elapsed between treatments and subsequent matings, giving males the opportunity to dustbathe and for volatile odours to dissipate. Both the spot-on solution and the sham treatment were reapplied every 17 days, while the flea powder application and massage were repeated every 6 days during the mating season. Control and experimental groups from 2007 were reversed in 2008, so that treated males became controls and vice versa. Four males of the control and three males of the experimental group did not re-emerge in 2008; however, 14 males (six experimental, eight sham treatment) were added in 2008 (either from immigration from a different colony or recruitment into reproductive age). In 2007, a total of 29 males were studied (Nexperimental ¼ 14; Ncontrol ¼ 15) and in 2008 a total of 36 males were included (Nexperimental ¼ 19; Ncontrol ¼ 17); 43 different individual males were thus studied during one or both field seasons (in total 65 males treated, 22 males present in both seasons). Parasite Load We counted, but did not remove, the ectoparasites on every male ground squirrel from both treatments at recaptures. During this procedure we detected only fleas, but no mites, ticks or other ectoparasites. Counting was done visually by combing (using a flea comb) and finger stroking through the fur over the whole body. In total, four flea load categories were defined (called ‘parasite load’ throughout): (0) ¼ no parasites detected; (1) ¼ one to two fleas; (2) ¼ three to five fleas; (3) ¼ more than five fleas (range 6e15) detected on the animal. Parasite load was determined at three time periods for each male on meadow A: time period 1 was directly after hibernation, before the treatment started; time period 2 was 12 days later; and time period 3 was another 12 days later. Complete flea counts were available from colony A in 2007 and 2008 (repeated measures of N ¼ 13 control and N ¼ 14 experimental males) over all three time periods 1e3. We counted endoparasites in faeces collected in 2007 from 10 control males and nine treated males, every 5e11 days throughout the field season (mean  SD ¼ 8.63  1.8 samples per male; for more details see Röösli 2007). Endoparasites were categorized as (1) larvae of hatched helminths, including larvated eggs, (2) coccidial parasites (Eimeria spp.), and (3) helminth eggs (which could not be identified to the species). We excluded the endoparasite count on the first capture day (when the males were not yet sham treated or treated with Stronghold/Revolution). The endoparasite count per day was averaged over all samples per individual male before analyses to remove the apparent high day-to-day variation. Observations of Mating Associations Animals were observed from 2e3 m high observation towers with binoculars. Columbian ground squirrels in our colonies usually mated underground (Murie 1995; Manno et al. 2007). We captured unmated, preoestrous females daily (three to seven times) to evaluate their reproductive status until they had mated. The degree of swelling and the openness of the vulva indicate the upcoming day of mating (for more details see Murie 1995). Observations of mating behaviours were recorded on the annual day of oestrus of each female.

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During a female’s oestrus, mating activity began between 0700 and 1000 hours, and lasted until 1400 to 1700 hours. Although we are confident that the behavioural criteria allowed us to identify correctly when mating occurred (e.g. Hanken & Sherman 1981; Hoogland & Foltz 1982; Sherman 1989; Boellstorff et al. 1994; Murie 1995; Lacey et al. 1997), they did not allow us to determine precisely the number or duration of copulations, or the interval between successive copulations with a single male while underground. Another population of U. columbianus where aboveground copulations were often observed demonstrated that copulations can last 35 min on average (range 1e90 min; Murie 1995). We assumed that underground copulations took place when the oestrous female and a male went down the same burrow system and remained there for at least 5 min (Raveh et al. 2010a). We therefore use the term ‘consort’ to refer to behavioural evidence that mating occurred (Hoogland 1995; Lacey et al. 1997). Some males exhibited mate guarding right after having copulated with an oestrous female by chasing her into a burrow, sitting on that burrow, fighting with other males, and giving mateguarding calls (Manno et al. 2007). We considered that a female’s oestrus had ended when she increased her feeding activity and avoided and chased away potential mating partners and other conspecifics (Murie 1995). One yearling female was observed consorting; however, we never observed yearling males engaged in sexual activities with oestrous females (Murie & Harris 1982). Sampling of Litters All nest burrows were marked and the female using the burrow was identified by observing the fur colour-marked female (1) carrying dry grass into the burrow and/or (2) emerge from the burrow in the morning and/or (3) enter the burrow in the evening. In two colonies, pregnant females were brought to a field laboratory 2 days prior to parturition (Murie & Harris 1982) and housed in polycarbonate cages (48  27 cm and 20 cm high) with wood chip bedding and newspaper for nesting material (Murie et al. 1998). These females received fresh apple and lettuce twice daily, and ad libitum horse breeder feed (a mixture of molasses, grains and vegetable pellets). Within 12 h of parturition, all neonates were weighed, sexed and marked individually by removing a small tissue biopsy from an outer hind toe (see Ethical Note). The tissue samples were stored in 95% ethanol and later used for paternity analysis. Females and their litters were released back into the colony the following day close to their nest burrow (Murie et al. 1998). In the third colony ‘C’, a small amount of tissue biopsy from the ear of juveniles (at first emergence from the natal burrow, age 27 days) was collected with sterile scissors and preserved in 95% ethanol for paternity analysis (Raveh et al. 2010a). Tissues from all adults that were not sampled as young were also taken in this way. Only offspring that emerged from their nest burrows at weaning were included in analyses, to standardize our measures of reproductive success among the three colonies. Hence, reproductive success for males and females was estimated based on number of juveniles at weaning. Offspring were caught within the first 2 days after emergence, with either unbaited National live traps (13  13 cm and 40 cm high) or with multicapture traps (Murie et al. 1998). Juveniles were marked and weighed, and their sex was determined or confirmed if born in captivity. Only females with known mating sequences were included in the mating sequence analyses (N ¼ 67 litters), whereas all litters were tested for total sired offspring and male consortships (N ¼ 80 litters). Paternity Analyses DNA was extracted from preserved tissue using DNeasy Tissue extraction kits (Qiagen, Venlo, The Netherlands), and polymerase

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chain reaction (PCR) amplification was performed for a panel of 13 microsatellite loci using primer pairs already developed for U. columbianus (GS12, GS14, GS17, GS20, GS22, GS25 and GS26: Stevens et al. 1997), Marmota marmota (BIBL18: Goossens et al. 1998; MS41 and MS53: Hanslik & Kruckenhauser 2000) and Marmota caligata (2g4, 2h6: Kyle et al. 2004; 2h4 GenBank accession no. GQ294553; for more details see Raveh et al. 2010a). PCR conditions and cycling parameters were similar to those described in Kyle et al. (2004) except for an annealing temperature of 54
C. We tested for deviations from HardyeWeinberg equilibrium (HWE) at each locus within cohorts, and for linkage disequilibrium between pairs of loci within cohorts using exact tests. Maternity was certain for all the offspring born in captivity and in the wild and paternity was assigned at 95e99% confidence using CERVUS 3.0 (Marshall et al. 1998; Kalinowski et al. 2007). Analyses were conducted for each colony and year (2007 and 2008) separately. Ethical Note In our studies of behaviours and life histories of Columbian ground squirrels we monitor their body condition throughout their life by evaluating reproductive condition and body weight on a weekly basis. Most animals have been regularly caught throughout life starting at the age of 27 days and become habituated to the traps. Weekly trapping sessions took place only on dry days. In this experiment the control and treated males were trapped over the whole season on average  SE 12.9  3.8 times (range 2e18) in 2007 and 8.12  2.8 times (range 3e14) in 2008. The tops of the traps were covered with cardboard to provide shade and set early in the morning before daily emergence to prevent overheating. Ground squirrels were examined and released within 60 min of capture or less. We found no trapping effect on pregnant or lactating females and could not detect any influence on their litters (active females spent up to 7 h away from their nest burrow). Because the animals lose the hair dye during the moult in late summer, we used ear tags (1.8 mm) for permanent identification over the years. In the rare event when an animal lost one of these ear tags (e.g. after a fight), the individual received a new one. For visual identification, distinctive hair dye marks were applied. The colour of these marks, black, occurs in the animals’ fur. There was no indication that hair dye marks increased predation risk, since the major predators of the ground squirrels were badgers, which hunt underground and thus do not see the black dye markings. Predation by visual hunters, such as raptors, foxes and coyotes, were rare according to thousands of hours of observations. Even though we may have increased visibility of ground squirrels for these latter predators, it was essential that we distinguished individuals for our behavioural studies. Females of two study sites were brought into the laboratory to give birth so that we could measure litter size, growth rate and paternity of every individual born. We take the utmost precaution to avoid negative impacts on the females and their offspring. To do this we follow the protocol of Murie et al. (1998) who could not find any negative effect of the procedures used. Females that were brought to the laboratory to give birth (room temperature 17e19
C, Murie & Harris 1982; Murie et al. 1998) were kept in polycarbonate cages (48  27 cm and 20 cm high) with wood chip bedding (to absorb urine and waste odours). All females constructed nests from the newspaper that we provided (Murie et al.1998) and showed no signs of stress. The females were provided with horse feed (EQuisine Sweet Show Horse Ration), lettuce and apples ad libitum. Toe clipping is commonly used for field studies involving rodents (e.g. McGuire et al. 2002; Gannon & Sikes 2007), and numerous studies have found no detrimental effects on survival or

body weight of small mammals (e.g. Ambrose 1972; Korn 1987; Wood & Slade 1990; Braude & Ciszek 1998). None the less, we used a modified procedure that did not involve clipping whole toes. We collected tissue samples of neonates using sharp, sterile scissors, by removing a small amount (1 mm2) of skin tissue from an outer hind toe or the tail. This sampling resulted in either a hind claw not developing or a small knot forming at the end of the tail, and young could thus be identified at weaning from their sex and these marks. These small wounds normally did not bleed, so we did not apply septic powder. This method was effective in identifying individuals in a litter. It was not suitable for long-term identification, since it resulted in many repeats among litters. Therefore, ear tagging at weaning was necessary. Additionally, we collected tissue samples from adult males and females (and weanlings at one meadow) by clipping a slim sliver of ear tissue from the outer pinnae (1 mm2) with sterile sharp scissors. This procedure normally did not cause bleeding and the animals showed no behavioural evidence of pain during or after the procedure. Once these procedures were completed, females and their offspring were released back into the colony close to their nest burrow. After the females entered the nest burrow, they either retrieved their offspring or the neonates were placed inside the nest burrow (Murie et al. 1998). Behavioural observations show that all adult females re-established their territories and foraging areas within a few hours of release. The housing of females 2 days before they gave birth and the processing of the neonates, as well as all field methods, were in accordance with the Institutional Animal Care and Use Committees of Auburn University (no. 418CN; no. 23172CN; no. 25054CN), as well as the Alberta Sustainable Resource Development Organization (no. 16167GP; no. RC-06-05; no. RC-07-09; no. 27047GP) and the Life and Environmental Sciences Animal Resource Centre, University of Calgary Animal, Canada (BI 2007-55).

Statistical Analyses All analyses were performed in SPSS 15 (SPSS Inc., Chicago, IL, U.S.A.). The majority of analyses were conducted using generalized estimating equations (GEE), which allows for the analyses of repeated measurements of the same subjects, which in our case were individual males (individual identifier entered as subject). Results were corrected for breeding season (‘year’, 2007 or 2008) and colony effects (meadow A, B, C) throughout. We used Kendell’s sc to test for differences in ectoparasite loads between the two groups (E and C) before the treatment was applied (at time period 1). Whether the change in male ectoparasite loads over the season (time periods 1e3) depended on the treatment was analysed using ordinal regression with treatment, time period and treatment*time period as fixed factors. Ectoparasite loads (all summed and averaged per individual male before analysis) were compared between C and E males using a one-tailed ManneWhitney U test. We analysed the effects on mating order, consort duration and mate-guarding duration (all three Poisson distributions with a loglink function) using GEE with individual male identifier as subject, including treatment, year and colony as fixed factors, and adding mating position as a covariate for the two analyses of durations (see Raveh et al. 2010b for the strong effect of mating order on both durations). The number of offspring sired per male (binomial distribution with a probit-link function) and the total seasonal reproductive success (which is the total number of offspring sired, as a Poisson distribution with a log-link function) were analysed using GEE with individual male identifier as subjects, with treatment, year and colony as fixed factors and mating order as a covariate. We added the interaction between treatment and mating order to test

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whether the treatment affected the relative success of the males in the different mating positions. To evaluate whether males differed in their body mass and age at the start of the experiment, experimental and control males were compared using independent t tests. Independent t tests were also used to test whether the treatment affected the within-breeding season and posthibernation weight gain. Experimental and control males did not differ in their body mass and age before the treatment (mass after hibernation: control males: mean  SE ¼ 555.0  11.06 g, N ¼ 31; experimental males: 549.67  8.49 g, N ¼ 31; t test: t1 ¼ 0.38, P ¼ 0.70; age: control males: 4.27  0.39 years, N ¼ 22; experimental males: 4.47  0.39 years, N ¼ 21; t test: t1 ¼ 0.36, P ¼ 0.72).

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1.5

(a)

1 31

Consort rate

0.5 0

31

−0.5 −1 −1.5

RESULTS

0.3 (b) Paternity Assignment

Percentage

100

Parasite load (no. of fleas):

80 60

0 1−2 3−5 >5

40 20

0 Time period: 1

2 Control

3

1

2

3

0

31

−0.1

Residual

−0.2 −0.3 8 (c) 6 Consort duration

We determined the parasite load of 27 males, both before and after the treatment in 2007 and 2008; load was measured on an ordinal scale from 0 to 3 (see Methods). Parasite loads did not differ between experimental and control groups prior to the antiparasitic treatment (time period 1; Kendall’s sc ¼ 0.20, N ¼ 27, P ¼ 0.29; Fig. 1). An ordinal regression showed a significant reduction in parasite load over time (from time period 1 & 2 to 3) for the experimental males, but not for the control males (treatment: df ¼ 1, P < 0.001; time period 1: df ¼ 1, P < 0.001; time period 2: df ¼ 1, P < 0.001; treatment*time period 1: df ¼ 1, P < 0.001; treatment*time period 2: df ¼ 1, P < 0.001; time period 3 is the reference category; Fig. 1). The significant accompanying parallelism test showed a different reaction over time for the two treatments (c210 ¼ 43:6, P < 0.001), supporting the result that the decrease in the experimental males was substantial and different from the changes in the control males. All 14 treated males were parasite free at time period 3, while 70% of the 13 control males were still infested.

31

0.1

4 2

31

0 31 −2 −4 (d) 6

Mate-guarding duration

Treatment Effect on Parasite Load

Mating position

0.2 In total, 43 adult males, 55 adult females and 240 offspring were successfully genotyped. Our genotyping success rate was 98%, with 85% of the ground squirrels genotyped at all 13 loci (N ¼ 338). We retained all 13 loci in our analyses, as there was no significant deviation from HWE or linkage disequilibrium within the colonies. All 240 offspring were successfully assigned to both parents: 98% of offspring had 99% trio-confidence, while the remaining 2% had 95% trio-confidence. In 236 of 240 cases (98%) offspring had zero mismatches with both parents.

4 2 0

31

−2

31

−4 C

E Treatment

Experimental

Figure 1. Repeated measures of ectoparasite loads of control (N ¼ 10) and experimental males (N ¼ 10). Each male was measured during the three time periods, from emergence at hibernation (time period 1) until the end of the mating season (time period 3). Percentages of the different parasite loads are presented as a stacked bars graph.

Figure 2. Reproductive behaviour of control males (C; white circles) and experimental males (E; black circles). (a) Consort rate (number of females copulated per season), (b) mating order (1e8), (c) consort duration (min) and (d) mate-guarding duration (min). Mean  SE residuals from the predicted values derived from all fixed effects in the models depicted in Table 1 are shown, without the treatment effect. Number of males is given above or below each symbol.

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Endoparasite counts were significantly lower in treated than control males (ManneWhitney U test: Z ¼ 1.71, one-tailed P ¼ 0.04). Control males had an average  SD of 2118.41  1883.41 endoparasites (range 266e6743, N ¼ 10), whereas treated males had on average 974.65  834.87 endoparasites (range 211e2783, N ¼ 9).

in parasites), to determine whether their reproductive characteristics were influenced by parasite load. Contrary to our expectations, our experimental reductions in parasites during the mating season did not lead to a significant change in male mating behaviour, male reproductive success or body mass. This suggests that the outcome of maleemale competition was not affected by our treatments. We can think of four main reasons why we did not detect an effect of our treatments on male reproductive success. First, the antiparasitic treatment was not effective enough or did not target those parasites that influence male reproduction. Second, natural parasite loads were too low to impact our control males. Third, the antiparasitic treatment was swamped by other factors influencing male reproductive success, such as other traits of the males. Fourth, if male reproductive success were strongly mediated by female mate choice, our treatment would need to influence female choice. We discuss these four potential reasons in more detail below. First, was our treatment actually effective and did it target all important parasites? We think it is safe to assume that both the ectoparasites and the endoparasites were substantially reduced by our treatment. Nevertheless, it would have been ideal for the statistical analyses if all control males were infested at the end of the mating season and none of the treated males were infested, but this was not the case: 30% of the control males were free of ectoparasites at the end of the season (compared to 100% of the treated males); and endoparasites were reduced by half in the treated males compared to the control males, but not completely eradicated. Of course, we can never be sure that our treatment affected all important parasites influencing male reproduction, in particular if effects become apparent after a certain threshold level of infection of a particular parasite species or combination of several parasite species (Hamilton et al. 1990; Penn et al. 2002). Second, were the natural levels of parasites high enough to detect any difference between the control and the treated males? Males with fewer parasites are expected to be in better condition and therefore have more energy to invest in searching for females and in reproduction. In a study on golden hamsters, Mesocricetus auratus, intense male copulatory activities had an immunosuppressive effect (Kress et al. 1989; Ostrowski et al. 1989). Thus, mating effort is assumed to be costly for males; for example, infected male red flour beetles, Tribolium castaneum, exhibited a reduced mating vigour and consequently inseminated fewer females than uninfected males (Pai & Yan 2003). Conversely, our study did not find an association between copulation rate and the different treatments in males. One possible explanation for such a result might be that control males could either cope with the infestation, or the parasite load was not severe enough to be costly. This is a difficult point to answer without further experiments. In any case, the number of males carrying substantial numbers of fleas was very low in our study population, in both the control and

Parasites and Male Behaviour There were no significant effects of the treatment on male consort rate, male mating position, consort duration or mate-guarding duration (Fig. 2, Table 1). In contrast, consort rates differed significantly between colonies and male mating position differed significantly between years (Table 1). Finally, mating order determined both male consortship and mate-guarding durations independently from the treatments (Table 1). Parasites and Male Reproduction In total, 217 offspring were weaned during 2007 and 2008. The treatment did not affect the number of offspring sired per litter (siring success; Fig. 3a, Table 2) or the total number of offspring sired (Fig. 3b). The seasonal reproductive success (uncorrected) was ca. 18% higher for the experimental males (mean  SE ¼ 3.8  3.5, range 0e14, N ¼ 33) compared to the control males (mean  SE ¼ 3.4  2.7, range 0e10, N ¼ 33), but this difference was not significant. Furthermore, the treatment did not influence siring success when we considered only mating positions 1e3 (Table 3), which are the most promising positions for fertilizing females. Parasites and Changes in Male Body Mass Treatment did not influence within-season body mass change (mass at end of mating season minus mass after hibernation; t test: t21 ¼ 0.29, P ¼ 0.78; Fig. 3c). However, the parasite treatment in 2007 might have affected the change in male body mass over hibernation, which would indicate a long-lasting effect of the parasite treatment on male body mass acquisition and/or loss. This was not the case: the treatment in 2007 did not affect the change in body mass over hibernation to emergence in 2008 (change in control males 2007 to experimental males 2008; t test: t11 ¼ 1.06, P ¼ 0.31; change in experimental males 2007 to control males 2008: t test: t10 ¼ 1.70, P ¼ 0.12; Fig. 3d). DISCUSSION Several studies have shown that parasites impact their hosts’ mating behaviour and reproductive success (Milinski & Bakker 1990; Poulin 1994; Rosenqvist & Johansson 1995; Sparkes et al. 2006; Deaton 2009). We experimentally removed parasites from male Columbian ground squirrels (resulting in significant declines Table 1 Treatment effects on male reproductive behaviour Parameter

Mating position (1e8)

Mate guarding (min)

N¼264 from 40 males

Constant Treatment Year Colony Mating order

Consort duration (min)

N¼264 from 40 males

Consort rate

N¼211 from 40 males

N¼62 from 40 males

Wald c2

df

P

Wald c2

df

P

Wald c2

df

P

Wald c2

df

P

1291.886 0.859 6.105 1.777

1 1 1 2

<0.001 0.354 0.013 0.411

102.627 1.112 0.291 1.153 24.232

1 1 1 2 1

<0.001 0.292 0.589 0.562 <0.001

2364.753 0.375 0.399 4.986 20.569

1 1 1 2 1

<0.001 0.540 0.528 0.083 <0.001

658.992 1.406 1.679 34.039

1 1 1 2

<0.001 0.236 0.195 <0.001

The table shows results for mating position, mate-guarding duration, consort duration and consort rate, depending on the treatment (control or treated), corrected for year, colony and mating order effects (in the second and third analysis), using three separate GEEs with male identifier as subjects (N ¼ 67 litters of known mating sequence). Mating position, durations and consort rate were fitted as Poisson distributions with a log-link, the scaling parameter adjusted using the deviance method. The interactions between treatment and mating order were nonsignificant.

S. Raveh et al. / Animal Behaviour 82 (2011) 673e682

0.06 (a)

Siring success

0.04 31

0.02 0

31

−0.02 −0.04

Residual

−0.06 1 (b)

31

Total sired offspring

33 0.5 33 0

31

−0.5

−1

40

(c) 12

Change in body mass

Within season

30

11

20 10 0 −10

Between seasons

40

(d)

30

10 12

20 10 0 −10

C

679

treated males. This is important because studies conducted in the laboratory may not reflect the parasite levels that commonly occur in nature. At least in some years, the reproduction of male Columbian ground squirrels does not seem to be strongly impacted by parasite load, but any impact might become apparent under higher levels of parasites. Our results suggested that parasite load imposed few costs on male Columbian ground squirrels and that loads were generally low. First, control males did not lose more weight than parasitefree animals during the mating season. However, parasite infections can raise energetic costs (Arnold & Lichtenstein 1993; Delahay et al. 1995; Fitze et al. 2004; Scantlebury et al. 2007; Hillegass et al. 2010) and may decrease the motivation to feed which may lead to a reduction in physical activities (Delahay et al. 1995; Mercer et al. 2000). When emerging from hibernation only a few males were heavily infested with fleas. Throughout the season we found very few fleas on adult male and female Columbian ground squirrels, and only yearlings and newly emerging offspring were often heavily infested (S. Raveh, personal observations). Third, did we fail to detect an effect of our treatment because male variation in reproductive success depends more on other factors than parasite load? Indeed, there are some reasons to believe this might be a major reason. Treated males sired on average 3.8 offspring per season compared to 3.4 offspring per season for control males (after we corrected for other effects the difference was 0.6 offspring per season, or 18% difference), particularly because they tended to be more successful in siring offspring in the first mating position. This is a substantial effect of our treatment, but was completely swamped by the high withintreatment variation in male reproductive success and thus failed to reach significance. Male reproductive success is known to depend on male age in this species, and male body condition may affect the likelihood of mating first with a female (Raveh et al. 2010a). Nevertheless, both male traits did not differ between our treatment groups, so cannot explain the absence of a treatment effect. However, previous results indicate male reproductive success is highly variable, depending on the likelihood of mating in the first mating position (Raveh et al. 2010a, b). Thus, male reproductive success is intrinsically highly variable in this species, and this might make it difficult to detect any treatment effect on male reproduction. Tellingly, the difference in male reproductive success (average of the treated minus control males) varied substantially from colony to colony and year to year (data not shown). Fourth, could female mate choice have affected our results in an unexpected way? Previous studies have confirmed that female rodents are capable of choosing nonparasitized males over infested males under standardized laboratory conditions (reviewed in Kavaliers et al. 2005a). Raveh et al. (2010a) showed that mating order plays a key role in male reproductive success in Columbian ground squirrels, with first males siring substantially more offspring than subsequent partners. Thus, we expected parasite-free male ground squirrels to mate first with oestrous females, either because these males are preferred by the females or because they are more successful in maleemale competition. Contrary to our expectation, we found no evidence that experimental males were more successful at consorting in the early (first, second or third) positions, compared to control males. Only the

E Treatment

Figure 3. Reproductive success and body mass change of control males (C; white symbols) and experimental males (E; black symbols). (a) Residual siring success (offspring sired/litter size) and (b) residual total sired offspring produced (circles: N ¼ 62 cases of 40 males, based on paternity in 67 litters) and the seasonal reproductive success (squares: N ¼ 66 cases of 43 males, based on paternity in 80 litters, i.e.

including males not mating at all). Body mass change (c) within the season (end of season minus after hibernation) and (d) between seasons (after hibernation year tþ1 minus after hibernation year t, where t is the year of the treatment). Mean  SE residuals from the predicted values derived from all fixed effects in the models depicted in Table 2 are shown, without the treatment effect. Number of males is given above each symbol.

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Table 2 Treatment effects on male reproductive success Parameter

Sired offspring/litter*

Total sired offspringy

Seasonal sired offspringz

N¼264 of 40 males

N¼62 of 40 males

N¼66 of 43 males

67 litters

Constant Treatment Year Colony Mating order

67 litters

80 litters

Wald c2

df

P

Wald c2

df

P

Wald c2

df

P

19.125 1.858 0.058 0.065 96.650

1 1 1 2 1

<0.001 0.173 0.810 0.968 <0.001

87.943 0.961 0.017 8.549

1 1 1 2

<0.001 0.327 0.898 0.014

87.943 0.273 0.457 5.144

1 1 1 1

<0.001 0.601 0.499 0.076

The table shows results for the number of sired offspring per litter and the total sired offspring (both only for litters with complete mating sequence observations) and the seasonal reproductive success (includes all litters and males not mating at all) depending on the treatment (control or treated), corrected for year, colony, and also mating position for sired offspring/litter effects. Results of three separate GEEs with male identifier as subjects are shown. * Sired offspring fitted as a weighted binomial distribution, the scaling parameter adjusted using the deviance method. The interaction treatment*mating order was not significant (c21 ¼ 0:365, P ¼ 0.546) and was removed from the model. y Total number of sired offspring, fitted as a Poisson distribution with a log-link. z Seasonal reproductive success, fitted as a Poisson distribution with a log-link.

strong mating order effect was important and explained the variation in reproductive success while the treatment had no impact. Likewise, durations of both consortship and mate guarding were not affected by the treatment; again, however, male investment in these behaviours decreased within the mating order (see Raveh et al. 2010b). Similarly, female mate preference did not depend on male infestation rate in several other animal species (red flour beetles: Pai & Yan 2003; Drosophila sp.: Kraaijeveld et al. 1997; pipefish, Syngnathus typhle: Mazzi 2004; pied flycatchers, Ficedula hypoleuca: Dale et al. 1996). Since Columbian ground squirrels commonly engage in sniffing and gaping (kissing) behaviour before and during the mating season, it is likely that odours are important for communicating and exchanging information such as kinship (Raynaud & Dobson 2010) and genetic compatibility for mate choice rather than the degree of parasite infestation. Therefore, female preferences for certain mates among both the control and the experimental males might have swamped our treatment effects, rendering them nonsignificant. In rodents, urine and other odorous secretions, such as the major histocompatibility complex, are considered important for mate detection and selection (Brown 1979; Egid & Brown 1989; Potts et al. 1991; Brown & Eklund 1994; Kavaliers & Colwell 1995; Penn & Potts 1998, 1999; Ehman & Scott 2002; Mougeot et al. 2004). The anabolic and behavioural effects of androgens carry an energetic cost, and high levels of androgens may suppress immune function resulting in an increased susceptibility to diseases and parasites (Grossman 1985; Folstad & Karter 1992; Zuk & McKean 1996; Hillgarth & Wingfield 1997; Mougeot et al. 2004). Folstad & Karter (1992) postulated that these costly effects of exposure to high androgen levels would handicap the expression of androgen-dependent sexual characters, resulting in only

high-quality individuals producing these characters and rendering them honest indicators of quality. Females may sense testosterone levels in urine to detect the presence of parasites in potential partners (Olsson et al. 2000; Mougeot et al. 2004). Willis & Poulin (2000) showed that parasitized male rats, Rattus norvegicus, had a lower testosterone level in their blood and suggested that females used this as a cue to avoid these males and thus to secure resistance genes for their offspring. Neuhaus (2003) showed that female Columbian ground squirrels weaned bigger litters and gained more weight during lactation when treated with flea powder. A study of African ground squirrels, Xerus inauris, found that parasites had a strong impact on female reproductive success (Hillegass et al. 2010). In our study, a spot-on solution was additionally used to create not only ecto- but also endoparasite-free males, whereas in the study by Neuhaus (2003) only flea powder against ectoparasites was applied. Even though this is a customary agent for domestic pets, we cannot exclude a negative effect through light toxicity or by killing useful intestinal flora (see Van Oers et al. 2002 for a negative effect of an Ivermectin antiendoparasitic treatment on the fledging rate of oystercatchers, Haematopus ostralegus). For future experiments, we suggest study of the role of female mating preferences in generating variation in male reproductive success. For instance, our treatment might not have affected the hormonal and odour profiles of males, and therefore did not alter their attractiveness to the females. Or changes might have made experimental males more attractive to some females, but less attractive to others. Testosterone could experimentally be increased (by injection or implantation) or decreased (by blocking the receptors) to test for testosterone-mediated changes in health and infestation rates (Klein et al. 2002). Another interesting

Table 3 Treatment effects on male reproductive output Parameter

Constant Treatment Year Colony

Number of sired offspring Mating position 1e3

Mating position 1 only

N¼180 of 38 males

N¼61 of 26 males

Wald c2

df

P

Wald c2

df

P

0.003 0.197 0.319 4.571

1 1 1 2

0.955 0.657 0.572 0.102

56.988 2.116 1.121 6.167

1 1 1 2

<0.001 0.146 0.290 0.046

The number of sired offspring per litter in first to third position and first position only depending on the treatment (control or treated), corrected for year and colony effects, using two separate GEEs with male identifier as subjects. Sired offspring were fitted as a Poisson distribution with a log link. The scaling parameter was adjusted using the deviance method.

S. Raveh et al. / Animal Behaviour 82 (2011) 673e682

approach would be to apply the treatment before hibernation, since this might ensure that males are parasite free at first emergence (but also throughout hibernation), and test the effects on maleemale competition and female preference. In this study, our main focus was on the host’s behaviour. A next step should be to identify and determine the role of parasites themselves to learn more about their influence on their squirrel hosts. Acknowledgments For help in the field we thank C. Heiniger, N. Brunner, M. Binggeli, L. Hofmann, V. Viblanc, B. M. Fairbanks and A. Skiebiel. Thanks to E. Kubanek, who genotyped tissue samples at the University of Alberta, D. W. Coltman lab. R. Bergmüller helped with statistics. S. G. Kenyon and A. Nesterova, F. Trillmich and B. König provided insightful comments on the manuscript. The study was funded by a Swiss National Science Foundation grant to P.N. (SNF 3100AO-109816). D.H. was supported by SNF grant 3100A0-108473, F.S.D. by a U.S.A. National Science Foundation research grant to DEB-0089473 and J.C.G. by an Alberta Conservation Association Biodiversity grant. Thanks to Pfizer Animal Health Canada for generously providing the project with their product Revolution/ Stronghold. The University of Calgary’s Biogeosciences Institute provided housing at the R. B. Miller Field Station during the field season; we thank the Station Manager, J. Buchanan-Mappin, the Institute Director, E. Johnson and the field station responsible K. Ruckstuhl for their support. References Alexander, R. 1974. The evolution of social behavior. Annual Review of Ecology, Evolution, and Systematics, 5, 325e383. Altizer, S., Nunn, C. L., Thrall, P. H., Gittleman, J. L., Antonovics, J., Cunningham, A. A., Dobson, A. P., Ezenwa, V., Jones, K. E. & Pedersen, A. B., et al. 2003. Social organization and parasite risk in mammals: integrating theory and empirical studies. Annual Review of Ecology, Evolution, and Systematics, 34, 517e547. Ambrose, H. W. 1972. Effect of habitat familiarity and toe-clipping on rate of owl predation in Microtus pennsylvanicus. Journal of Mammalogy, 53, 909e912. Arnold, W. & Lichtenstein, A. V. 1993. Ectoparasite loads decrease the fitness of alpine marmots (Marmota marmota) but are not a cost of sociality. Behavioral Ecology, 4, 36e39. Barber, I. 2002. Parasites, maleemale competition and female mate choice in the sand goby. Journal of Fish Biology, 61, 185e198. Bateman, A. J. 1948. Intra-sexual selection in Drosophila melanogaster. Heredity, 2, 349e368. Birkhead, T. R., Møller, A. P. & Sutherland, W. J. 1993. Why do females make it so difficult for males to fertilize their eggs. Journal of Theoretical Biology, 161, 51e60. Boellstorff, D. E., Owings, D. H., Penedo, M. C. T. & Hersek, M. J. 1994. Reproductive behaviour and multiple paternity of California ground squirrels. Animal Behaviour, 47, 1057e1064. Braude, S. & Ciszek, D. 1998. Survival of naked mole-rats marked by implantable transponders and toe-clipping. Journal of Mammalogy, 79, 360e363. Brei, B. & Fish, D. 2003. Comment on ‘Parasites as a viability cost of sexual selection in natural populations of mammals’. Science, 300, 55. Brown, J. L. & Eklund, A. 1994. Kin recognition and the major histocompatibility complex: an integrative review. American Naturalist, 143, 435e461. Brown, R. E. 1979. Mammalian social odours. Advances in the Study of Behavior, 10, 103e109. Buchholz, R. 2004. Effects of parasitic infection on mate sampling by female wild turkeys (Meleagris gallopavo): should infected females be more or less choosy? Behavioral Ecology, 15, 687e694. Charmantier, A., Kruuk, L. E. B. & Lambrechts, M. M. 2004. Parasitism reduces the potential for evolution in a wild bird population. Evolution, 58, 203e206. Cothran, R. D. 2004. Precopulatory mate guarding affects predation risk in two freshwater amphipod species. Animal Behaviour, 68, 1133e1138. Clayton, D. H. 1991. The influence of parasites on host sexual selection. Parasitology Today, 7, 329e334. Dale, S., Kruszewicz, A. & Slagsvold, T. 1996. Effects of blood parasites on sexual selection and natural selection in the pied flycatcher. Journal of Zoology, 238, 373e393. Deaton, R. 2009. Effects of a parasitic nematode on male mate choice in a livebearing fish with a coercive mating system (western mosquitofish, Gambusia affinis). Behavioural Processes, 80, 1e6.

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