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


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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.
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No experimental effects of parasite load on male mating behaviour
and reproductive success
Shirley Raveh
, Dik Heg
, F. Stephen Dobson
, David W. Coltman
, Jamieson C. Gorrell
Adele Balmer
, Simon Röösli
, Peter Neuhaus
University of Neuchâtel, Institute of Biology, Eco-Ethology
Department of Behavioral Ecology, University of Bern
Centre dEcologie Fonctionnelle et Evolutive, Centre National de la Recherche Scientique
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
article info
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
Columbian ground squirrel
parasite infestation
reproductive success
Urocitellus columbianus
Parasites can negatively affect their hosts 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
rst-male paternity advantage. Parasite treatment signicantly 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 inuence 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 animals 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 conspecics 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: (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 dEcologie Fonctionnelle et Evolutive - Unité Mixte
de Recherche 5175, Centre National de la Recherche Scientique, 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
Animal Behaviour
journal homepage:
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 sh to their
parasite load or resistance (Clayton 1991; Zuk 1992). Hence, females
can increase their tness 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 conspecics
has been demonstrated in rodents, sh 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 difculties of manipu-
lation and observation in the eld (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 consortshipsare 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 tness, 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 rst 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, ghting 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.
Study Species
We studied Columbian ground squirrels in the Sheep River
Provincial Park, Alberta, Canada (110
W, 50
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
(meadowA, 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 rst 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 rst 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 rst 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 ngerling sh
tags (National Band & Tag Company Monel no. 1, Newport, KY, U.S.A.)
were attached in both ears for permanent identication. 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 identication 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, Pzer Animal Health, Mon-
treal, Canada) and ea 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 ea 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 males 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 ea 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 days 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 ea 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
¼19; N
¼17); 43 different individual males were
thus studied during one or both eld 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 eas, but no mites, ticks or other
ectoparasites. Counting was done visually by combing (using a ea
comb) and nger stroking through the fur over the whole body. In
total, four ea load categories were dened (called parasite load
throughout): (0) ¼no parasites detected; (1) ¼one to two eas;
(2) ¼three to ve eas; (3) ¼more than ve eas (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 ea 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 eld 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 identied to the species). We excluded the endoparasite
count on the rst 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.
During a females oestrus, mating activity began between 0700
and 1000 hours,and lasted until 1400 to 1700 hours. Although we are
condentthat 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 consortto 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, ghting with other males, and giving mate-
guarding calls (Manno et al. 2007). We considered that a females
oestrus had ended when she increased her feeding activity and
avoided and chased away potential mating partners and other
conspecics (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 identied 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 eld 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 rst 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 rst 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 conrmed 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).
Paternity Analyses
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) amplication 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% condence using
CERVUS 3.0 (Marshall et al. 1998; Kalinowski et al. 2007). Analyses
were conducted for each colony and year (2007 and 2008)
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 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 inuence 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 identication
over the years. In the rare event when an animal lost one of these
ear tags (e.g. after a ght), the individual received a new one. For
visual identication, distinctive hair dye marks were applied. The
colour of these marks, black, occurs in the animalsfur. 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 nd 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 eld 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 modied 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 identied 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 identica-
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 eld
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 identier entered as subject).
Results were corrected for breeding season (year, 2007 or 2008)
and colony effects (meadow A, B, C) throughout. We used Kendells
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 xed 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 identier as subject,
including treatment, year and colony as xed 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 identier as subjects, with treatment, year and
colony as xed 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:
¼0.38, P¼0.70; age: control males: 4.27 0.39 years, N¼22;
experimental males: 4.47 0.39 years, N¼21; ttest: t
Paternity Assignment
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 signicant
deviation from HWE or linkage disequilibrium within the colonies.
All 240 offspring were successfully assigned to both parents: 98% of
offspring had 99% trio-condence, while the remaining 2% had 95%
trio-condence. 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; Kendalls
¼0.20, N¼27, P¼0.29;
Fig. 1). An ordinal regression showed a signicant 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 signicant accompanying paral-
lelism test showed a different reaction over time for the two
treatments (
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
Control Ex
Parasite load
(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 xed 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 signicantly lower in treated than
control males (ManneWhitney Utest: Z¼1.71, 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 signicant 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 signicantly between colonies and male mating position
differed signicantly 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 signicant. Furthermore, the treatment did not inuence 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 inuence within-season body mass change
(mass at end of mating season minus mass after hibernation; ttest:
¼0.29, 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
¼1.70, 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 signicant declines
in parasites), to determine whether their reproductive character-
istics were inuenced by parasite load. Contrary to our expecta-
tions, our experimental reductions in parasites during the mating
season did not lead to a signicant 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 inuence male reproduction.
Second, natural parasite loads were too low to impact our control
males. Third, the antiparasitic treatment was swamped by other
factors inuencing 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
inuence 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 inuencing 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 our beetles, Tribolium castaneum, exhibited
a reduced mating vigour and consequently inseminated fewer
females than uninfected males (Pai & Yan 2003). Conversely, our
study did not nd 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 difcult point to answer without further experiments. In
any case, the number of males carrying substantial numbers of eas
was very low in our study population, in both the control and
Table 1
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
df P
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 identier as subjects (N¼67 litters of known mating sequence). Mating
position, durations and consort rate were tted as Poisson distributions with a log-link, the scaling parameter adjusted using the deviance method. The interactions between
treatment and mating order were nonsignicant.
S. Raveh et al. / Animal Behaviour 82 (2011) 673e682678
treated males. This is important because studies conducted in the
laboratory may not reect 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 eas. Throughout the
season we found very few eas 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 rst 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 signicance. Male reproductive success is known to
depend on male age in this species, and male body condition may
affect the likelihood of mating rst 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 rst mating position (Raveh et al. 2010a, b). Thus, male repro-
ductive success is intrinsically highly variable in this species, and
this might make it difcult 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 conrmed 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 rst males siring substantially more
offspring than subsequent partners. Thus, we expected
parasite-free male ground squirrels to mate rst 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 (rst,
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 xed 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 our
beetles: Pai & Yan 2003;Drosophila sp.: Kraaijeveld et al. 1997;
pipesh, Syngnathus typhle:Mazzi 2004; pied ycatchers, Ficedula
hypoleuca:Dale et al. 1996).
Since Columbian ground squirrels commonly engage in snifng
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 nonsignicant. 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 & Wingeld 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 ea 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 ea 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
ora (see Van Oers et al. 2002 for a negative effect of an Ivermectin
antiendoparasitic treatment on the edging 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 proles 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 2
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
df P
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 identier as subjects are shown.
Sired offspring tted as a weighted binomial distribution, the scaling parameter adjusted using the deviance method. The interaction treatment*mating order was not
signicant (
¼0:365, P¼0.546) and was removed from the model.
Total number of sired offspring, tted as a Poisson distribution with a log-link.
Seasonal reproductive success, tted as a Poisson distribution with a log-link.
Table 3
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
df P
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 rst to third position and rst position only depending on the treatment (control or treated), corrected for year and colony effects,
using two separate GEEs with male identier as subjects. Sired offspring were tted 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) 673e682680
approach would be to apply the treatment before hibernation,since
this might ensure that males are parasite free at rst 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 hosts behaviour. A next step should be to identify
and determine the role of parasites themselves to learn more about
their inuence on their squirrel hosts.
For help in the eld 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 Pzer Animal Health Canada for
generously providing the project with their product Revolution/
Stronghold. The University of Calgarys Biogeosciences Institute
provided housing at the R. B. Miller Field Station during the eld
season; we thank the Station Manager, J. Buchanan-Mappin, the
Institute Director, E. Johnson and the eld station responsible
K. Ruckstuhl for their support.
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... This species is naturally parasitized by both ecto-(ticks, mites, botflies) and endo-parasites (helminths, coccidia, trypanosomes). The most visible of these are fleas (Raveh et al., 2011(Raveh et al., , 2015 that seem to follow the aggregated 80:20 distribution. As fleas are often localized to individual hosts and burrows, natural parasite dispersal is low, thus allowing enhanced isolation of parasite effects on hosts. ...
... Prior studies on parasite effects in U. columbianus have resulted in variable outcomes (little to no effect: Raveh et al., 2011Raveh et al., , 2015negative effect: Neuhaus, 2003). These studies applied experimental reductions of fleas, in a species that naturally has relatively low levels of infestation, to assess fitness consequences on individuals. ...
... We attempted to quantify the previously variable costs of parasitism in U. columbianus by discriminating between short-and long-term effects (Asghar et al., 2015). However, it became clear that the tendency of U. columbianus to prioritize immediate grooming of parasites was likely the reason for the initial low level of fleas (Raveh et al., 2011(Raveh et al., , 2015 and the lack of seasonal effects of our experimental parasite manipulation. This strong grooming response coupled with oxidative shielding likely resulted in the dampening of any physiologically detectable parasite effects, even during the energetically demanding reproductive period. ...
Parasites affect many aspects of host physiology and behavior, and thus are generally thought to negatively impact host fitness. However, changes in form of short-term parasite effects on host physiological markers have generally been overlooked in favor of fitness measures. Here, we studied flea (Oropsylla idahoensis and Oropsylla opisocroistis tuberculata) parasitism on a natural population of Columbian ground squirrels (Urocitellus columbianus) in Sheep River Provincial Park, AB, Canada. Fleas were experimentally added to adult female U. columbianus at physiologically demanding times, including birth, lactation and weaning of their young. The body mass of adult females, as well as their oxidative stress and immunity were recorded multiple times over the active season under flea-augmented and control conditions. We also measured the prevalence of an internal parasite (Trypanosoma otospermophili). Doubly labeled water (DLW) was intra-peritoneally injected at peak lactation to examine energy expenditure. Effects of parasites on oxidative stress were only observed after offspring were weaned. There was no direct effect of experimentally heightened flea prevalence on energy use. A short-term 24 h mass loss (-17 g) was detected briefly after parasite addition, likely due to U. columbianus preferentially allocating time for grooming. Our parasite augmentation did not strongly affect hosts and suggested that short-term physiological effects were unlikely to culminate in long-term fitness consequences. Columbian ground squirrels appear to rapidly manage parasite costs, probably through grooming.
... The results of the parasite removal experiment did not support our hypothesis that ectoparasites affect the ability of adult males to maintain their body mass. Raveh et al. (2011) found no effect of ectoparasite removal on male body mass in Columbian ground squirrels (Urocitellus columbianus). A lack of any observable effect of ectoparasite removal on male body mass, however, does not mean that ectoparasites have no effect on males in this red squirrel population. ...
... As mate search tactics are under positive sexual selection pressure in male red squirrels (Lane et al., 2009), parasite removal prior to and during the mating period may allow males to invest more energy into mating effort, thereby leading to improved mating success. However, male red squirrels appear to trade-off reproductive investment and parasite infection (Gooderham and Schulte-Hostedde, 2011) and male Columbian ground squirrels did not experience increased reproductive success when ectoparasites were experimentally removed (Raveh et al., 2011). Variability in host and parasite genetics, parasite virulence, parasite coinfection, host demography, and environmental conditions may influence the effects of parasites on host reproductive success, body mass, and transmission dynamics, and these potential relationships require further study. ...
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Within many species, males are often more heavily parasitised than females. Several hypotheses have been proposed to explain this phenomenon, including immunocompetence handicaps, sexual size dimorphism and behavioural differences. Here we set out to test the latter two hypotheses and make inferences about the former by assessing patterns of ectoparasitism across various life-history stages in a population of North American red squirrels (Tamiasciurus hudsonicus). We also conducted an ectoparasite removal experiment to investigate the effects of ectoparasites on male body condition. We found that males were more intensely parasitized than females, but only during the mating period. There was no difference in ectoparasite intensity between male and female juveniles at birth or at emergence, suggesting that ectoparasites do not exploit male red squirrels for longer-range natal dispersal. Male red squirrels in our population were slightly heavier than females, however we did not find any evidence that this dimorphism drives male-biased ectoparasitism. Finally, we could not detect an effect of ectoparasite removal on male body mass. Our results lend support to the hypothesis that ectoparasites exploit their male hosts for transmission and that male red squirrels are important for the transmission dynamics of ectoparasites in this population; however, the mechanisms (i.e., immunocompetence, testosterone) are not known.
... However, the parasite load might better represent the host's ability to control the infection (resistance) and might be a finer correlate of its physiological or energetical costs (Stjernman et al., 2008;Risely et al., 2018;Sánchez et al., 2018;Methling et al., 2019). A negative association between infection intensity and reproductive success was shown in several host-parasite systems (Madsen et al., 2005;Asghar et al., 2011;Gooderham & Schulte-Hostedde, 2011;Hicks et al., 2019;Schoepf et al., 2022) but some studies also show an absence or a positive correlation (Siikamäki et al., 1997;Edler et al., 2004;Raveh et al., 2011;Kulma et al., 2014;Delefortrie et al., 2022). ...
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Although avian haemosporidian parasites are widely used as model organisms to study fundamental questions in evolutionary and behavorial ecology of host-parasite interactions, some of their basic characteristics, such as seasonal variations in within-host density, are still mostly unknown. In addition, their interplay with host reproductive success in the wild seems to depend on the interaction of many factors, starting with host and parasite species and the temporal scale under study. Here, we monitored the intensities of infection by two haemosporidian parasites (i.e. Plasmodium relictum and P. homonucleophilum) in two wild populations of great tits (Parus major) in Switzerland over three years, to characterize their dynamics. We also collected data on birds reproductive output (i.e. laying date, clutch size, fledging success) to determine whether they were associated with infection intensity before (winter), during (spring) and after (autumn) breeding season. Both parasite species dramatically increased their within-host density in spring, in a way that was correlated to their parasitaemia in winter. Infection intensity before and during breeding season did not explain reproductive success. However, the birds which fledged the more chicks had higher parasite burdens in autumn, which were not associated with their parasite burden in previous spring. Our results tend to indicate that high haemosporidian parasite loads do not impair reproduction in great tits, but high resource allocation into reproduction can leave birds less able to maintain low parasitaemia over the following months.
... A. sagrei, male home range size is positively associated with female encounter rate, and the proportion of a female's offspring sired (Kamath & Losos, 2018). Parasites have been shown to reduce reproductive success and offspring production in other species (Newey & Thirgood, 2004;Patterson et al., 2013;Worden et al., 2000; however, see Raveh et al., 2011), but further work involving genetic parentage assignment will be necessary to determine whether the effects of parasite removal on juvenile growth and male mating success that we observed translate into increased reproductive success. ...
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Parasites interact with nearly all free-living organisms and can impose substantial fitness costs by reducing host survival, mating success, and fecundity. Parasites may also indirectly affect host fitness by reducing growth and performance. However, experimentally characterizing these costs of parasitism is challenging in the wild because common antiparasite drug formulations require repeated dosing that is difficult to implement in free-living populations, and because the extended-release formulations that are commercially available for livestock and pets are not suitable for smaller animals. To address these challenges, we developed a method for the long-term removal of nematode parasites from brown anole lizards (Anolis sagrei) using an extended-release formulation of the antiparasite drug ivermectin. This treatment eliminated two common nematode parasites in captive adult males and dramatically reduced the prevalence and intensity of infection by these parasites in wild adult males and females. Experimental parasite removal significantly increased the sprint speed of captive adult males, the mating success of wild adult males, and the growth of wild juveniles of both sexes. Although parasite removal did not have any effect on survival in wild anoles, parasites may influence fitness directly through reduced mating success and indirectly through reduced growth and performance. Our method of long-term parasite manipulation via an extended-release formulation of ivermectin should be readily adaptable to many other small vertebrates, facilitating experimental tests of the extent to which parasites affect host phenotypes, fitness, and eco-evolutionary dynamics in the wild.
... With regard to reproductive costs, female mammals make large energetic investments in gestation and lactation, which might be constrained by helminth infection (Khokhlova et al., 2002;McFalls et al., 1984). In support, the experimental removal of ecto-and endoparasites sometimes leads to increased reproductive success (Hillegass et al., 2010;Neuhaus, 2003;Patterson and Ruckstuhl, 2013, but see Gooderham and Schulte-Hostedde, 2011;Raveh et al., 2015Raveh et al., , 2011. In primates, previous research by Nguyen et al. (2015) found that female gelada baboons infected by Taenia species exhibit longer interbirth intervals (a measure of fertility) than those not infected. ...
Helminth parasites can have wide‐ranging, detrimental effects on host reproduction and survival. These effects are best documented in humans and domestic animals, while only a few studies in wild mammals have identified both the forces that drive helminth infection risk and their costs to individual fitness. Working in a well‐studied population of wild baboons ( Papio cynocephalus ) in the Amboseli ecosystem in Kenya, we pursued two goals, to (a) examine the costs of helminth infections in terms of female fertility and glucocorticoid hormone levels and (b) test how processes operating at multiple scales—from individual hosts to social groups and the population at large—work together to predict variation in female infection risk. To accomplish these goals, we measured helminth parasite burdens in 745 faecal samples collected over 5 years from 122 female baboons. We combine these data with detailed observations of host environments, social behaviours, hormone levels and interbirth intervals (IBIs). We found that helminths are costly to female fertility: females infected with more diverse parasite communities (i.e., higher parasite richness) exhibited longer IBIs than females infected by fewer parasite taxa. We also found that females exhibiting high Trichuris trichiura egg counts also had high glucocorticoid levels. Female infection risk was best predicted by factors at the host, social group and population level: females facing the highest risk were old, socially isolated, living in dry conditions and infected with other helminths. Our results provide an unusually holistic understanding of the factors that contribute to inter‐individual differences in parasite infection, and they contribute to just a handful of studies linking helminths to host fitness in wild mammals.
... Although there have been many studies examining parasitism effects on number and quality of offspring in small mammals, the results are mixed. For instance, despite that several studies found negative parasitism effects on host reproduction in squirrels (e.g., Xerus inauris, Hillegass, Waterman, & Roth, 2010; Tamiasciurus hudsonicus, Gooderham & Schulte-Hostedde, 2011;Patterson, Neuhaus, Kutz, & Ruckstuhl, 2013), both negative and neutral effects have been reported for the same host species (Urocitellus columbianus, Neuhaus, 2003;Raveh et al., 2011;Raveh, Neuhaus, & Dobson, 2015). ...
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Food and parasitism can have complex effects on small mammal reproduction. In this study, we tested the effects of sex, food, and parasitism on reproductive performance of the Taiwan field mouse (Apodemus semotus). In a field experiment, we increased food availability for a portion of the mice in the population by providing sorghum seeds to a set of food stations. We reduced parasite intensity of randomly chosen mice through ivermectin treatment. We determined the number and quality of offspring for the mice using paternity analysis. We quantified seed consumption with stable carbon isotope values of mouse plasma and parasite intensity with fecal egg counts of intestinal nematodes and cestodes (FEC). In a laboratory experiment, we reduced parasite intensity of randomly chosen mice through ivermectin treatment. We quantified their immune functions by total white blood cell count, percent granulocyte count, and percent lymphocyte count through hematological analyses. We measured the FEC and energy intake of the mice. From the field experiment, the number of offspring in A. semotus increased with increasing seed consumption. Due to the trade‐off between number and quality of offspring, the offspring quality decreased with increasing seed consumption for the females. The ivermectin treatment did not affect offspring number or quality. However, the FEC was positively correlated with number of offspring. In the laboratory experiment, the percent lymphocyte/granulocyte count changed with parasite intensity at low energy intake, which was relaxed at high energy intake. This study demonstrated positive effects of food availability and neutral effects of parasitism on A. semotus reproduction. However, the benefits of food availability for the females need to take into account the offspring number–quality trade‐off, and at high infection intensity, parasitism might negatively affect offspring quality for the males. We suggest that food availability could mediate the relationships between parasite intensity and immune responses.
Parasites often have profound effects on the survival and evolution of their hosts, and hence on the structure and health of entire ecosystems. Yet basic questions, such as the degree of virulence of a given parasite on its host, and factors influencing which hosts in a population are at the greatest risk of infection, are vexingly difficult to resolve. The western blacklegged tick-western fence lizard (Ixodes pacificus-Sceloporus occidentalis) system is important, primarily because I. pacificus, a vector of the Lyme disease spirochete Borrelia burgdorferi, is dependent on S. occidentalis for blood meals in its subadult stages, and this lizard possesses an innate immune response that removes the Lyme disease pathogen from attached ticks. My study focused on two aspects of the I. pacificus-S. occidentalis interaction. In Chapter 1, I investigated factors correlating with the intensity of I. pacificus infestations on S. occidentalis. Infection intensity (parasites per host) is often highly variable within a host population, though certain individuals, such as males, tend to be more heavily infected. Previous work in the I. pacificus-S. occidentalis system suggests that differences in behavior, such as the frequency of territorial patrols, may contribute to variation in tick intensity among lizards. I therefore hypothesized that lizard traits that correlate with dominance would also correlate with infestation intensity. Specifically, I predicted that larger and more colorful males would have higher infestation intensities than less impressive animals. In this chapter, I also focused on site selection by ticks infesting S. occidentalis. Skin folds on the necks of these lizards (nuchal pockets) may function to divert ectoparasites away from eyes, ears, and other potentially vulnerable structures. I therefore also looked for factors correlating with tick attachment in these pockets. I sampled ticks on adult male S. occidentalis in the spring and summer, which is the seasonal peak for both S. occidentalis territorial behavior and subadult I. pacificus abundance. After determining the site of infestation and intensity of ticks on these lizards, I re-infested lizards with laboratory-reared I. pacificus larvae, and again quantified tick intensity and attachment location. Contrary to expectation, no host traits correlated with tick intensity among ticks naturally infesting lizards, and lab-reared larval intensity was negatively correlated with lizard body size. As expected, ticks acquired by lizards naturally concentrated inside nuchal pockets, and I also observed this site preference among ticks in lab-based experimental infestations. Although the general pattern, lab-reared ticks were more varied in the sites on which they fed. There was a negative correlation between infestation intensity and the proportion of ticks attached in nuchal pockets. Unsurprisingly, the most reliable predictor of tick intensity and site selection was the season. In Chapter 2, I explored how tick attachment affects male S. occidentalis contest behavior. I. pacificus infestation has been shown to have negative physiological impacts on S. occidentalis, but mechanisms linking physiological changes to ultimate fitness consequences have been largely underexplored. I hypothesized that tick infestation reduces male S. occidentalis fighting ability by reducing O2 carrying ­­capacity­, or by obstructing or damaging vulnerable structures on their hosts. I held fifty half-hour trials between pairs of size- and ventral badge-matched male S. occidentalis, with one male in each pair infested with lab-reared I. pacificus larvae. I found that tick infestation negatively correlated with aggressive behavior in these staged contests. In support of reduced O2 capacity as the mechanism of reduced aggression, my ecologically relevant infestation intensities seemed to cause significant declines in hematocrit among experimentally infested lizards relative to controls. However, the site at which ticks attached did not significantly correlate with the aggressiveness of their lizard hosts. This is one of only a handful of studies to address the direct effect of I. pacificus on S. occidentalis. My study demonstrates that tick infestation can be detrimental to the fitness of their lizard hosts even without the transmission of pathogens. This insight may prove informative in future work on the ecology of I. pacificus-borne diseases in the western United States. This study is also one of only a few to use parasite infection to induce an asymmetry in fighting ability in intrasexual contests.
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Permanent marks such as ear tagging and passive integrated transponder tags have been successfully used in small-mammal mark–recapture research. However, permanent marks can influence small mammal behavior and promote infection. Hair dyes may be favored for short-term mark–recapture studies because they are low-cost, can be applied easily, and are less damaging to marked individuals. Our objective was to determine the longevity of an animal hair dye marker placed on small mammals. Our study was conducted at the Millersville Biological Preserve in Millersville, PA and consisted of two transect lines, each with 10 Sherman traps checked twice a week over 3 months. We captured 27 individuals and marked each of them once with hair dye. Hair dye longevity was monitored via photodocumentation of recaptured individuals. Results showed that the hair dye can be used to distinguish individuals for up to 60 days in short-term mark–recapture studies of small mammals.
Fleas (Insecta: Siphonaptera) are hematophagous ectoparasites that can reduce the fitness of vertebrate hosts. Laboratory populations of fleas decline under dry conditions, implying that populations of fleas will also decline when precipitation is scarce under natural conditions. If precipitation and hence vegetative production are reduced, however, then herbivorous hosts might suffer declines in body condition and have weakened defenses against fleas, so that fleas will increase in abundance. We tested these competing hypotheses using information from 23 yr of research on 3 species of colonial prairie dogs in western USA: Gunnison’s prairie dogs (Cynomys gunnisoni, 1989–1994), Utah prairie dogs (C. parvidens, 1996–2005), and white-tailed prairie dogs (C. leucurus, 2006–2012). For all 3 species, flea-counts per individual varied inversely with the number of days in the prior growing season with >10 mm of precipitation, an index of the number of precipitation events that might have caused a substantial, prolonged increase in soil moisture and vegetative production. Flea-counts per Utah prairie dog also varied inversely with cumulative precipitation of the prior growing season. Further, flea-counts per Gunnison’s and white-tailed prairie dog varied inversely with cumulative precipitation of the just-completed January and February. These results complement research on black-tailed prairie dogs (C. ludovicianus) and might have important ramifications for plague, a bacterial disease, transmitted by fleas, that devastates populations of prairie dogs. In particular, our results might help to explain why, at some colonies, epizootics of plague, which can kill >95% of prairie dogs, are more likely to occur during or shortly after periods of reduced precipitation. Climate change is projected to increase the frequency of droughts in the grasslands of western North America. If so, then climate change might affect the occurrence of plague epizootics among prairie dogs and other mammalian species that associate with them.
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Fleas (Insecta: Siphonaptera) are haematophagous ectoparasites that feed on vertebrate hosts. Fleas can reduce the fitness of hosts by interfering with immune responses, disrupting adaptive behaviors, and transmitting pathogens. The negative effects of fleas on hosts are usually most pronounced when fleas attain high densities. In lab studies, fleas desiccate and die under dry conditions, suggesting that populations of fleas will tend to decline when precipitation is scarce under natural conditions. To test this hypothesis, we compared precipitation versus parasitism of black-tailed prairie dogs (Cynomys ludovicianus) by fleas at a single colony during May and June of 13 consecutive years (1976–88) at Wind Cave National Park, South Dakota, USA. The number of fleas on prairie dogs decreased with increasing precipitation during both the prior growing season (April through August of prior year) and the just-completed winter-spring (January through April of current year). Due to the reduction in available moisture and palatable forage in dry years, herbivorous prairie dogs might have been food-limited, with weakened behavioral and immunological defenses against fleas. In support of this hypothesis, adult prairie dogs of low mass carried more fleas than heavier adults. Our results have implications for the spread of plague, an introduced bacterial disease, transmitted by fleas, that devastates prairie dog colonies and, in doing so, can transform grassland ecosystems.
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Guidelines for use of wild mammal species are updated from the American Society of Mammalogists (ASM) 2007 publication. These revised guidelines cover current professional techniques and regulations involving mammals used in research and teaching. They incorporate additional resources, summaries of procedures, and reporting requirements not contained in earlier publications. Included are details on marking, housing, trapping, and collecting mammals. It is recommended that institutional animal care and use committees (IACUCs), regulatory agencies, and investigators use these guidelines as a resource for protocols involving wild mammals. These guidelines were prepared and approved by the ASM, working with experienced professional veterinarians and IACUCs, whose collective expertise provides a broad and comprehensive understanding of the biology of nondomesticated mammals in their natural environments. The most current version of these guidelines and any subsequent modifications are available at the ASM Animal Care and Use Committee page of the ASM Web site (
Publisher Summary This chapter discusses the information contained in the odorous secretions of mammals and provides a classification system based on the behavioral and chemical analyses. This classification divides social odors into two groups: identifier and emotive. Identifier odors are defined as those produced through the regular metabolic processes of the animal, without specific stimulation. The emotive odors are those produced as the result of some transient emotional state or external stimulus. The chapter categorizes nine different types of information contained in mammalian social odors: species, age, sex, colony membership, individuality, social status, reproductive state, maternal state, and stress odors. Social odors are modified by diet and hormone levels and by bacterial action. When the chemicals and bacteria responsible for producing the social odors have been identified, the responses of test animals show large individual differences. Responses to olfactory stimuli depend on hormonal and experiential factors. Theoretical models in the study of population regulation, sexual selection, kinship recognition, altruism, parental care, and territoriality infer that animals recognize particular individuals and specific relationships, and such recognition may depend to some extent on the information contained in olfactory signals.
We compared survival of free-living naked mole-rats, Heterocephalus glaber, marked by toe-clipping and implantable transponder chips. Although survival was marginally higher for toe-clipped animals than for those with transponder chips in five of six colonies, no significant differences were found between the two marking techniques. Comparison of the costs and benefits of the two marking techniques suggests that toe-clipping is preferable for marking small fossorial mammals in remote areas.