No experimental effects of parasite load on male mating behaviour
and reproductive success
, Dik Heg
, F. Stephen Dobson
, David W. Coltman
, Jamieson C. Gorrell
, Simon Röösli
, Peter Neuhaus
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
Department of Biological Sciences, Auburn University
Department of Biological Sciences, University of Alberta
University of Neuchâtel, Institute of Biology, Parasitology
Department of Biological Sciences, University of Calgary
Received 7 May 2010
Initial acceptance 16 September 2010
Final acceptance 10 June 2011
Available online 17 August 2011
MS. number: 10-00318R
Columbian ground squirrel
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 endopar-
asites 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 experi-
mental males. None of these males had ectoparasites at the end of the season, compared to 70% infes-
tation 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.
Ó2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Parasites may have detrimental effects on their hosts (Thompson&
Kavaliers 1994; Sheldon & Verhulst 1996; Møller et al. 1999). For
example, an infectionmay lead toa reduction inhost fertility(Lockhart
et al. 1996), alter an animal’s relative attractiveness to potential mates
(Hamilton & Zuk 1982;Møller et al.1999; Verhulst etal. 1999)oraffect
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
*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: firstname.lastname@example.org (S. Raveh).
D. Heg is now at the Institute of Social and Preventive Medicine, University of
Bern, 3012 Bern, Switzerland.
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.
A. Balmer is at the Department of Biological Sciences, 331 Funchess Hall,
Auburn University, AL 36849, U.S.A.
D. W. Coltman and J. C. Gorrell are at the Department of Biological Sciences,
University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
S. Röösli is at the University of Neuchâtel, Institute of Biology, Parasitology, Rue
Emile-Argand 11, Case postale 158, 2009 Neuchâtel, Switzerland.
P. Neuhaus is at the Department of Biological Sciences, University of Calgary,
2500 University Dr NW, Calgary, Alberta T2N 1N4, Canada.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/anbehav
0003-3472/$38.00 Ó2011 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animal Behaviour 82 (2011) 673e682
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 repro-
ductive output simply by increasing their number of mating part-
ners 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 parasit-
ized 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 manipu-
lation 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 betweenparasite 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 competi-
tion and sperm competitionplay a major role in generating variation
in male reproductive success (Raveh et al. 2010a, b).
In the present study we removed ectoparasites and endopara-
sites 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 guard-
ing 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.
We studied Columbian ground squirrels in the Sheep River
Provincial Park, Alberta, Canada (110
N, and 1500 m eleva-
tion). 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).
Ground squirrels were caught within the ﬁrst 2 days of emer-
gence from hibernation with live traps baited with peanut butter
(15 15 cm and 4 8 cmhigh and 13 13cm 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 sepa-
rately. The experimental group (abbreviated with an E) was treated
with a spot-on solution (Stronghold, Pﬁzer Animal Health, Mon-
treal, Canada) and ﬂea powder (Zodiac, Wellmark International,
Dallas, TX, U.S.A.) to remove ectoparasites and endoparasites
(N¼33 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
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682674
(abbreviated with a C, N¼32) 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 hiber-
nation 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 (N
¼15) and in 2008 a total of 36 males were included
¼17); 43 different individual males were
thus studied during one or both ﬁeld seasons (in total 65 males
treated, 22 males present in both seasons).
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) ¼no parasites detected; (1) ¼one to two ﬂeas;
(2) ¼three to ﬁve ﬂeas; (3) ¼more 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 N¼13 control and N¼14 experi-
mental 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 ¼8.63 1.8 samples per male; for
more details see Röösli 2007). Endoparasites were categorized as
(1) larvae of hatched helminths, including larvated eggs, (2) coc-
cidial 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
During a female’s oestrus, mating activity began between 0700
and 1000 hours,and lasted until 1400 to 1700 hours. Although we are
conﬁdentthat 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 succes-
sive 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(range1e90 min; Murie 1995). We assumed that under-
ground copulations took place when the oestrous female and a male
went down the same burrow system and remained there for at least
5min(Raveh et al. 2010a). We therefore usethe term ‘consort’to refer
to behavioural evidence that mating occurred (Hoogland1995; 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 mate-
guarding 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 con-
sorting; 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 labora-
tory 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 individuallyby 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 repro-
ductive 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 (N¼67 litters), whereas all litters were tested for total
sired offspring and male consortships (N¼80 litters).
DNA was extracted from preserved tissue using DNeasy Tissue
extraction kits (Qiagen, Venlo, The Netherlands), and polymerase
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682 675
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 Mar-
mota 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
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)
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 habitu-
ated to the traps. Weekly trapping sessions took place only on dry
days. In this experiment the control and treated males were trap-
ped 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 over-
heating. 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 indi-
viduals 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 precautionto
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 scis-
sors, by removing a small amount (1 mm
) 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 identiﬁca-
tion, 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 mm
) with sterile sharp scissors. This procedure nor-
mally did not cause bleeding and the animals showed no behav-
ioural 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 processingof 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).
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
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 aver-
aged per individual male before analysis) were compared between
C and E males using a one-tailed ManneWhitney Utest.
We analysed the effects on mating order, consort duration and
mate-guarding duration (all three Poisson distributions with a log-
link 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
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
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682676
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 ttests. Independent ttests 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 ¼555.0 11.06 g,
N¼31; experimental males: 549.67 8.49 g, N¼31; ttest:
¼0.38, P¼0.70; age: control males: 4.27 0.39 years, N¼22;
experimental males: 4.47 0.39 years, N¼21; ttest: t
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 (N¼338). 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.
Treatment Effect on Parasite Load
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
¼0.20, N¼27, P¼0.29;
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 ¼1, P<0.001; time period 1: df ¼1, P<0.001; time period 2:
df ¼1, P<0.001; treatment*time period 1: df ¼1, P<0.001;
treatment*time period 2: df ¼1, P<0.001; time period 3 is the
reference category; Fig. 1). The signiﬁcant accompanying paral-
lelism test showed a different reaction over time for the two
¼43:6, 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.
Time period: 1 2 3 1 2 3
(no. of fleas):
Figure 1. Repeated measures of ectoparasite loads of control (N¼10) and experi-
mental males (N¼10). 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
Consort rateMating positionConsort
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.
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682 677
Endoparasite counts were signiﬁcantly lower in treated than
control males (ManneWhitney Utest: Z¼1.71, one-tailed
P¼0.04). Control males had an average SD of 2118.41 1883.41
endoparasites (range 266e6743, N¼10), whereas treated males had
on average 974.65 834.87 endoparasites (range 211e2783, N¼9).
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 ¼3.8
3.5, range 0e14, N¼33) compared to the control males
(mean SE ¼3.4 2.7, range 0e10, N¼33), 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; ttest:
¼0.29, P¼0.78; 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; ttest: t
P¼0.31; change in experimental males 2007 to control males
2008: ttest: t
¼1.70, P¼0.12; Fig. 3d).
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
in parasites), to determine whether their reproductive character-
istics were inﬂuenced by parasite load. Contrary to our expecta-
tions, our experimental reductions in parasites during the mating
season did not lead to a signiﬁcant change in male mating behav-
iour, 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 ecto-
parasites 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 eradi-
cated. 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 infec-
tion 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 immunosup-
pressive 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
Treatment effects on male reproductive behaviour
Parameter Mating position (1e8) Mate guarding (min) Consort duration (min) Consort rate
N¼264 from 40 males N¼264 from 40 males N¼211 from 40 males N¼62 from 40 males
df P Wald
df P Wald
df P Wald
Constant 1291.886 1 <0.001 102.627 1 <0.001 2364.753 1 <0.001 658.992 1 <0.001
Treatment 0.859 1 0.354 1.112 1 0.292 0.375 1 0.540 1.406 1 0.236
Year 6.105 1 0.013 0.291 1 0.589 0.399 1 0.528 1.679 1 0.195
Colony 1.777 2 0.411 1.153 2 0.562 4.986 2 0.083 34.039 2 <0.001
Mating order 24.232 1 <0.001 20.569 1 <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 (N¼67 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) 673e682678
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 parasite-
free animals during the mating season. However, parasite infec-
tions 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
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), partic-
ularly 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 within-
treatment 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 repro-
ductive 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 experi-
mental males were more successful at consorting in the early (ﬁrst,
second or third) positions, compared to control males. Only the
Between seasons Within season
Change in body mass
Total sired offspring Siring success
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:
N¼62 cases of 40 males, based on paternity in 67 litters) and the seasonal repro-
ductive success (squares: N¼66 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 tis 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.
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682 679
strong mating order effect was important and explained the vari-
ation 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 ener-
getic 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 squir-
rels 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,
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
Treatment effects on male reproductive success
Parameter Sired offspring/litter*Total sired offspringySeasonal sired offspringz
N¼264 of 40 males N¼62 of 40 males N¼66 of 43 males
67 litters 67 litters 80 litters
df P Wald
df P Wald
Constant 19.125 1 <0.001 87.943 1 <0.001 87.943 1 <0.001
Treatment 1.858 1 0.173 0.961 1 0.327 0.273 1 0.601
Year 0.058 1 0.810 0.017 1 0.898 0.457 1 0.499
Colony 0.065 2 0.968 8.549 2 0.014 5.144 1 0.076
Mating order 96.650 1 <0.001
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
¼0:365, P¼0.546) and was removed from the model.
Total number of sired offspring, ﬁtted as a Poisson distribution with a log-link.
Seasonal reproductive success, ﬁtted as a Poisson distribution with a log-link.
Treatment effects on male reproductive output
Parameter Number of sired offspring
Mating position 1e3 Mating position 1 only
N¼180 of 38 males N¼61 of 26 males
df P Wald
Constant 0.003 1 0.955 56.988 1 <0.001
Treatment 0.197 1 0.657 2.116 1 0.146
Year 0.319 1 0.572 1.121 1 0.290
Colony 4.571 2 0.102 6.167 2 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
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682680
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 mal-
eemale 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.
For help in the ﬁeld we thank C. Heiniger, N. Brunner, M. Bing-
geli, 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.
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