Is there sex-biased resistance and tolerance in Mediterranean wood mouse (Apodemus sylvaticus)...

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Oecologia ISSN 0029-8549 OecologiaDOI 10.1007/s00442-012-2300-5

Is there sex-biased resistance and tolerancein Mediterranean wood mouse (Apodemussylvaticus) populations facing multiplehelminth infections?

Frédéric Bordes, Nicolas Ponlet, JoëlleGoüy de Bellocq, Alexis Ribas, BorisR. Krasnov & Serge Morand

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POPULATION ECOLOGY - ORIGINAL RESEARCH

Is there sex-biased resistance and tolerance in Mediterraneanwood mouse (Apodemus sylvaticus) populations facing multiplehelminth infections?

Frederic Bordes • Nicolas Ponlet • Joelle Gouy de Bellocq •

Alexis Ribas • Boris R. Krasnov • Serge Morand

Received: 9 August 2011 / Accepted: 1 March 2012

� Springer-Verlag 2012

Abstract Sex-biased parasitism is rarely investigated in

relation to host tolerance and resistance, which are two

defense strategies hosts can adopt when challenged by

parasites. Health or fitness deteriorations in less tolerant

individuals with increasing parasite burden would be faster

than those in more tolerant ones. Hence, the body condition

and reproductive potential of an infected individual host

can be considered proxies for tolerance to parasitism. We

studied Mediterranean populations of the wood mouse

(Apodemus sylvaticus) and its helminth parasites. We

assessed their resistance using the phytohemagglutinin test

and spleen size, and their tolerance using body condition in

males and females and testes mass in males. In order to

avoid spurious correlations, we took into account the

phylogeographic structure of the Mediterranean wood

mouse populations. We used a mixed model adapted from

the animal model used in quantitative genetics. While

helminth infection did not differ between the two sexes,

females and males differed in their measured defenses.

Females seem to invest more in immune defense with

increasing risk of parasite diversity, but also appear to be

potentially more tolerant of parasitic diversity. These

results suggest the existence of sexual differences in

resistance and tolerance, and that measurements of para-

sitic loads alone could be insufficient to detect any

underlying sexual differences in the two strategies that

have evolved in response to multiple parasitic attacks.

Keywords Apodemus sylvaticus � Body condition �Testes mass � Spleen � PHA � Helminths � Sexual

dimorphism � Parasites � Resistance � Tolerance

Introduction

Males are often more prone to infection than females in

terms of parasite abundance and/or parasite species rich-

ness (Poulin 1996; Moore and Wilson 2002; Morand et al.

2004; Krasnov et al. 2005, 2012; Perez-Orella and Schulte-

Hostedde 2005; Hoby et al. 2006; Cowan et al. 2007).

However, this pattern does not seem to be a universal rule,

as many counterexamples exist. For example, host sex can

sometimes weakly influence the prevalence and abundance

of helminths or ectoparasites in rodents (Behnke et al.

1999; Ferrari et al. 2004; Fuentes et al. 2004; Harrison

Communicated by Janne Sundell.

F. Bordes � N. Ponlet � S. Morand (&)

Institut des Sciences de l’Evolution, CNRS-IRD-UM2, CC65,

Universite de Montpellier 2, 34095 Montpellier, France

e-mail: [email protected]

J. G. de Bellocq

Evolutionary Ecology Group, University of Antwerp,

Groenenborgerlaan 171, 2020 Antwerp, Belgium

J. G. de Bellocq

Department of Population Biology, Institute of Vertebrate

Biology, Academy of Sciences of the Czech Republic,

Brno, Czech Republic

A. Ribas

Laboratory of Parasitology, Faculty of Pharmacy, University

of Barcelona, Avda Diagonal s/n, 08028 Barcelona, Spain

B. R. Krasnov

Mitrani Department of Desert Ecology, Institute for Dryland

Environmental Research, Jacob Blaustein Institutes for Desert

Research, Ben-Gurion University of the Negev, Sede-Boqer

Campus, 84990 Midreshet Ben-Gurion, Israel

S. Morand

UR22 AGIRs, CIRAD, Campus International de Baillarguet,

34398 Montpellier, France

123

Oecologia

DOI 10.1007/s00442-012-2300-5

Author's personal copy

et al. 2010; Kiffner et al. 2011), bats (Dick et al. 2003;

Kanuch et al. 2005; Patterson et al. 2008), and ungulates

(Munyeme et al. 2011). These contradictory examples

stress that the processes that may explain sex-biased par-

asitism are still poorly understood (Roberts et al. 2004;

Nunn et al. 2009). Fundamentally, the classical conceptual

framework explaining male-biased parasitism refers to

heterogeneity in exposure and/or susceptibility to parasites.

Males can be more exposed to parasites than females due to

differences in their behavior (Harrison et al. 2010). The

larger home ranges often observed for males (Attuquayefio

et al. 1986) could increase their exposure to parasite

infection (Krasnov et al. 2005). Higher male exposure to

parasites may also be linked to the larger body sizes

of males, which makes them better targets for parasites

(Arneberg 2002; Harrison et al. 2010). Two non-mutually-

exclusive hypotheses have been proposed to explain sex

differences in susceptibility. The first hypothesis is related

to a sex-specific immune handicap, where males may

experience reduced immunity to parasites due to higher

levels of testosterone; this known as the ‘‘immunocompe-

tence handicap hypothesis’’ (ICHH) (Folstad and Karter

1992; Klein 2004; Roberts et al. 2004). The second

hypothesis is that the sex differences observed in life his-

tories and sexual behavior are likely reflected in immune

function (Rolff 2002; Zuk 2009; Nunn et al. 2009).

According to this hypothesis, males maximize fitness via

mating rates, while females invest more into progeny

(Bateman 1948). Male intrasexual competition for access

to females should favor bigger males in polygynous sys-

tems that, under energy constraints, may result in a trade-

off between growth/size and immunity (Moore and Wilson

2002; Zuk 2009). On the contrary, females are expected to

invest in longevity and then to select for higher investment

in immunity (Rolff 2002). Importantly, when Bateman’s

principle (1948) is applied to immunity it refers to direct

investment in immunity, which may be not correlated with

infection levels owing to differences in parasite exposure

(Skorping and Jensen 2004). Just as field studies have

stressed classically expected male-biased parasitism,

numerous studies have cast doubt on the universal validity

of this classical conceptual framework. Some studies failed

to detect positive effect of body size on parasitic loads

(Patterson et al. 2008; Scantlebury et al. 2010), while

others have stressed the validity of the ICHH (Roberts et al.

2004) or the positive link between home range size and

parasitic loads (Wilson et al. 2003).

Here we propose to use a new conceptual framework

originating from plant research that has only recently been

applied to wild animal systems: the tolerance/resistance

concept (Raberg et al. 2007, 2009). Briefly, the core of this

conceptual framework infers that hosts can adopt two

approaches when facing infections and challenges by

parasites. The first is to attack parasites directly in order to

prevent parasite invasion, thus limiting their population

growth and/or ultimately eliminating them. This type of

defense refers to resistance (Raberg et al. 2007; Baucom

and de Roode 2011). However, behavioral and immune

defenses may be energetically costly and are therefore

limited by trade-offs with other host tasks such as repro-

duction, growth, or survival (Lochmiller and Derenberg

2000). Moreover, resistance may also be dangerous, due

to self-targeted immune responses (i.e., autoimmunity)

(Limaye et al. 2008; Graham et al. 2010). Due to its

potential negative effects on host fitness, host resistance is

thus only expected to evolve when its costs are outweighed

by its benefits. The second way to approach parasite

infection is to limit the harm caused by parasites without

reducing the parasitic load. This ability to limit the

severity of the disease induced by a given parasitic load

refers to tolerance (Raberg et al. 2007, 2009; Schneider

and Ayres 2008). Although both defense strategies have

the same goals—maintaining health and improving the

fitness of a host—they can fundamentally differ in their

associated mechanisms. These two defense strategies

have been fully investigated by plant ecologists (Clarke

1986), while studies on animal defense strategies have

mainly focused on laboratory models (Ma et al. 1998;

Raberg et al. 2007; Lefevre et al. 2011 but see Blanchet

et al. 2010). Nevertheless, these studies have improved

our knowledge of the immune mechanisms of resistance,

the high genetic variability of resistance and tolerance,

and the potential trade-off between the two ways of

responding (Raberg et al. 2007, 2008; Blanchet et al.

2010; Lefevre et al. 2011). On the other hand, the spe-

cific mechanisms involved in tolerance have been largely

ignored to date.

From the perspective of sex-biased parasitism, studies

related to tolerance/resistance have not considered poten-

tial differences between the sexes in the way that hosts

manage parasitism, despite the fact that differences

between the sexes in terms of immune response and sus-

ceptibility to infection are often observed (Moore and

Wilson 2002; Zuk 2009).

Using Mediterranean populations of the wood mouse

(Apodemus sylvaticus) and their helminth parasites

(Trematoda, Nematoda, and Cestoda), the study reported in

this paper had several aims. The first was to detect sex-

biased parasitism. We also wished to investigate difference

between the sexes in the way that they deal with parasitism

using the resistance and tolerance conceptual framework.

We used body condition to assess tolerance and two clas-

sical immune measures—the PHA test and spleen size—to

assess resistance in the two sexes. We also used testes mass

as a proxy to assess tolerance to helminth parasitism in

males.

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Following the classical conceptual framework, as the

male A. sylvaticus is bigger and has a larger home range

than the female (Attuquayefio et al. 1986), we expected

higher helminth loads in males (Moore and Wilson 2002).

We also expected that males would face stronger trade-offs

between immunity and other tasks (Zuk 2009). Following

both the classical framework and the tolerance resistance

framework, we expected higher investment in immunity

(resistance) in females due to a sex-specific pattern in

steroid hormones, life history, and/or investment in repro-

duction (Klein 2004; Nunn et al. 2009). Predictions related

to tolerance are more difficult to draw. The existence of a

trade-off between resistance and tolerance would lead us to

expect higher resistance in females with a reduced toler-

ance than a reduced condition. On the contrary, if the

mechanisms involved in resistance and tolerance are

linked, we should expect females to both resist and tolerate.

Females should then harbor fewer parasites and be in better

condition than males.

Materials and methods

Study sites and rodent trapping

Two hundred twenty-two A. sylvaticus were trapped using

‘‘Manufrance’’ or ‘‘Sherman’’ live-traps in eight different

localities in the Mediterranean area, including four island

(Majorca, Minorca, Porquerolles, and Sicily) and four

mainland (Montpellier, Py, Montseny, and Calabria) pop-

ulations in two consecutive years (see Gouy de Bellocq

et al. 2003) (Fig. 1).

Animals were live trapped using Sherman traps. Traps

were set in the evening and checked in the early morning.

An initial trapping session lasting 5–7 nights was

performed to catch 30 individuals, with around 100 traps

set per night. The trapping sessions occurred between April

(Porquerolles) and the start of October (Calabria, Sicily,

Py), when wood mice are active and can be trapped in the

Mediterranean environment (see Gouy de Bellocq et al.

2003).

After investigating phytohemagglutinin skin test

responses (PHA, see below), the rodents were euthanized

by cervical dislocation and weighed to the nearest 0.01 g

using a cylindrical mechanical dynamometer (BLET SA,

France). Viscera and liver were removed for further para-

sitological examination. The ages of individuals were

determined using the method proposed by Adamczewska-

Andrezejwska (1967), which utilized three age classes

(2–5 months, 5–9 months, and [9 months). Only adult

animals ([5 months; 85 females and 120 males) were

included in the analysis (Table 1). Reproductive status was

checked by examining the genital organs (developed testis

and seminal glands, open vulva). Although not all of the

adults bred, due to numerous factors such as social pres-

sures, it was still assumed that such individuals wanted to

mate and reproduce.

Infestation data and metrics to assess multiple parasite

pressure

The wood mouse can play host to numerous microparasites

(viruses, bacteria, fungi, protozoans) and macroparasites

(helminths, arthropods), but for practical purposes we

focused on helminth parasites (i.e., cestodes, trematodes,

nematodes), which are responsible for chronic infections

and tend to force their hosts to evolve strong adaptive

coevolutionary processes (Fumagalli et al. 2009, 2010).

The methods used to collect and identify the parasites are

described in Gouy de Bellocq et al. (2003). To assess the

responses of host individuals to multiparasitism, three

metrics were used: individual parasite load (IndPL; total

number of all endoparasites of all species in each indi-

vidual host), individual parasite species richness (IndPSR;

total number of endoparasite species encountered in each

individual host), and population parasite species richness

(PopPSR; total number of parasite species found in the

wood mouse population at each locality). For the latter

metric, we assumed a positive relationship between species

richness in each locality, and a higher probability of mul-

tiple infections at the host individual level.

Assessing tolerance and resistance

Resistance

As noted above, resistance typically refers to defense

mechanisms that are used by animals to prevent infections

Fig. 1 Map of the geographical locations of the wood mice

(Apodemus sylvaticus) sampled on the mainland: Montseny (1),

Py (2), Montpellier (3), Calabria (4), and on the islands of Majorca

(5), Minorca (6), Porquerolles (7), and Sicily (8)

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or limit parasitic burdens. Consequently, resistance has

usually been estimated using parasitic burden (Raberg et al.

2007; Lefevre et al. 2011; Blanchet et al. 2010). For

example, Rohr et al. (2010) estimated the resistance of

frogs to trematode larvae using the inverse proportion of

cercariae that successfully encysted, with the most resistant

frog species being those with the fewest encysted cercariae.

Given that immune defense is the main defense against

helminth parasites, we assumed that measures of various

immune parameters should be a good proxy for immune

investment and thus resistance. To assess immune activity,

ecological immunology requires techniques that allow easy

and reliable measurements of immune investment in the

field (Boughton et al. 2011). The high complexity of the

immune system makes it extremely difficult to assess

protective immunity (which enhances host fitness) per se in

the field. As a result, and following Bradley and Jackson

(2008) and Pedersen and Babayan (2011), field ecologists

commonly identify an ‘‘immune phenotype,’’ which is a

context-dependent state of the immune system at a given

time. Using this approach, ecologists can sample wild

animals and measure their immune phenotypes with vari-

ous immune measures.

In our study, we used two classical and practical mea-

sures, the phytohemagglutinin (PHA) skin test and the

spleen size. We understand the inherent limits of such

simple metrics, but we postulated that they could at the

very least allow a new hypothesis to be tested in an open

area of research.

The PHA test (Smits et al. 1999) has commonly and

repeatedly been used to assess immune function in wild

vertebrates (lizards, birds, and mammals) in vivo (Tschir-

ren et al. 2003; Gouy de Bellocq et al. 2007; Tella et al.

2008; Cox et al. 2010). This test involves subcutaneous

injection of a vegetal lectin, PHA, which induces a series of

cellular responses, including local influxes of lymphocytes,

neutrophils, and macrophages, which, in turn, collectively

manifest as localized swelling at the site of injection (Smits

et al. 1999). We measured the left footpad thickness with a

screw micrometer (Mituyoyo digimatic thickness gauge,

547–301, Japan) to ±0.01 mm and immediately injected

0.15 mg (individuals from Porquerolles and 20 individuals

from Montpellier population) or 0.1 mg (individuals from

all other localities) of PHA dissolved in 0.03 mL of saline

subcutaneously into the middle of the footpad. No differ-

ence in PHA response was recorded for 10 individuals

receiving 0.1 mg of PHA and 20 individuals receiving

0.15 mg of PHA from the Montpellier population (Mann–

Whitney test: z = -0.068, P = 0.946). We measured the

footpad thickness again after 24 h. We defined the PHA

response as the difference between pre- and postinjection

measurements, and assumed it to be proportional to the

intensity of T-cell-mediated immune investment. To stan-

dardize measurements, we recorded the thickness 3 s after

applying the micrometer. Normally, the opposite footpad

should receive a saline buffer solution (phosphate buffer

solution, PBS) injection to check that the T-cell prolifera-

tion is a response to PHA itself and not the vehicle (PBS).

Table 1 Various data categorized by sex for the Apodemus sylvaticussamples, including geographic origin (numbers next to localities refer

to the corresponding numbers on the map in Fig. 1), sampling size

(n), parasite species richness in the population (PopPSR), mean body

mass, mean spleen mass, mean PHA response, and mean parasite

abundance

Locality (see map) Sex (n) TotPSR Body mass

(g ± SD)

Spleen mass

(g ± SD)

PHA

(mm ± SD)

Parasite abundance

(mean ± SD)

Montseny (1) F (12) 12 21.58 (3.06) 0.041 (0.015) 1.49 (0.31) 11.17 (13.95)

M (20) 12 27.00 (3.60) 0.052 (0.012) 1.81 (0.36) 15.0 (19.5)

Py (2) F (17) 11 19.39 (5.84) 0.063 (0.038) 1.29 (0.64) 19.4 (26.0)

M (19) 11 17.23 (4.75) 0.57 (0.044) 1.66 (0.65) 10.8 (16.6)

Montpellier (3) F (10) 9 19.70 (3.74) 0.033 (0.012) 1.40 (0.25) 2.7 (2.7)

M (19) 9 25.03 (3.68) 0.046 (0.018) 1.74 (0.32) 12.9 (24.4)

Calabria (4) F (13) 6 19.80 (4.62) 0.045 (0.032) 1.88 (0.54) 10.7 (13.2)

M (19) 6 18.68 (2.74) 0.041 (0.024) 1.82 (0.50) 12.7 (28.0)

Majorca (5) F (8) 7 18.38 (1.38) 0.029 (0.010) 1.52 (0.32) 7.3 (7.3)

M (17) 7 21.47 (3.92) 0.039 (0.019) 1.47 (0.40) 5.2 (7.4)

Minorca (6) F (5) 7 18.80 (3.96) 0.019 (0.007) 1.25 (0.27) 8.6 (9.5)

M (6) 7 27.70 (4.46) 0.041 (0.014) 1.25 (0.49) 14.3 (17.1)

Porquerolles (7) F (4) 6 26.25 (2.63) 0.035 (0.016) 1.53 (0.14) 112.3 (208.0)

M (6) 6 26.50 (6.38) 0.044 (0.022) 1.78 (0.39) 65.0 (46.0)

Sicily (8) F (17) 14 22.59 (6.12) 0.048 (0.031) 1.85 (0.65) 31.0 (40.8)

M (14) 14 25.04 (5.75) 0.047 (0.028) 2.07 (0.44) 41.1 (69.7)

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We adopted the simplified protocol proposed by Smits

et al. (1999), which avoids the injection of PBS into the

opposite experimental site because no swelling was

observed in several control individuals from various rodent

species (including A. sylvaticus) that were injected in the

opposite footpad with PBS (Gouy de Bellocq, unpublished

data).

Doubts have emerged in recent years about the true

nature of the immune reaction induced by PHA (Kennedy

and Nager 2006). However, the magnitude of swelling has

been shown to correlate positively with infection intensity

(Sak et al. 2006) and to reflect acquired T-cell-mediated

immunity (Tella et al. 2008), or at least a certain level of

immune activation (Boughton et al. 2011). These studies

suggest that different magnitudes of swelling in different

individuals may reflect different levels of immune system

activity.

To assess spleen size, the spleens were removed and

weighed to an accuracy of 0.001 g using a precision

electronic balance (AR1530 AdventurerTM, Ohaus Corp.,

Pine Brook, NJ, USA). Spleen size has often been used as a

proxy for immune investment in mammals and other ver-

tebrates in many field studies (Brown and Brown 2002;

Lefebvre et al. 2004; Horak et al. 2006; Gouy de Bellocq

et al. 2007; Vicente et al. 2007; Cowan et al. 2009, Malo

et al. 2009; Navarro-Gonzalez et al. 2010; Schulte-Host-

edde and Elsasser 2011), as its enlargement is directly

linked to ongoing helminth infestation and parasitic loads

(Ali and Behnke 1985; Horak et al. 2006; Cowan et al.

2009; Ponlet et al. 2011; Schulte-Hostedde and Elsasser

2011). Although spleen size may be linked to body size,

hormonal profile (Navarro-Gonzalez et al. 2010), body

condition, or red blood cell count (Malo et al. 2009), we

assumed—as also assumed in previous studies—that its

enlargement may reflect host immune system activation

(and thus potential resistance), possibly due to the expan-

sion of the splenic B-cell pool, even if it does not indicate

clearance ability per se (Horak et al. 2006).

We were aware of the inherent limits of the metrics

used, but we postulated that they could allow us to test

some predictions and put forward new hypotheses.

Tolerance

Operationally, tolerance can be defined as the change in

host fitness with a change in parasitic burden (i.e., the slope

of the relationship between fitness and parasitic burden)

(Raberg et al. 2007, 2009). In other words, tolerance allows

the host to endure damage caused by a parasite (Schneider

and Ayres 2008; Raberg et al. 2009). Consequently, a less

tolerant individual would show a faster decline in health or

fitness with increasing parasite burden than a more tolerant

one (Raberg et al. 2007). From this perspective, if we want

to detect more tolerant individuals, we must study the links

between indicators of host fitness (survival, reproduction)

and parasitic loads. The body condition of an animal refers

to its energetic state. An animal in good condition is then

assumed to have more energy reserves than an animal in

poor condition, as well as to have better fitness. For

instance, individuals with larger energy reserves may have

better reproductive success than individuals with smaller

ones (Wauters and Dhondt 1995). From the same per-

spective, testis size may be a good proxy for reproductive

investment and host fitness.

Hence, we used body condition and testes mass as two

proxies for host fitness in males and body condition as a

proxy for fitness in females. We are aware of the difficulty

involved in assessing body condition per se (Peig and

Green 2010), so we used the ratio of the body mass to the

body length as a reliable proxy for body condition. Testis

size is a good proxy to use to assess reproductive fitness

in males in a polygynous system, which is the case for

A. sylvaticus (Waterman 2007). Larger testes presumably

produce more testosterone (Parapanov et al. 2009), and

higher concentrations of testosterone are correlated with

increased mobility in male mammals (e.g., Mills et al.

2009).

If better tolerance implies a limited negative change in

host fitness when parasitic loads increase, we may expect

limited parasite-related impact on host body condition in

the more tolerant sex.

Statistical approach

We first conducted statistical analyses on the raw data. We

employed a GLM to show the effect of host sex and

localities on the variability of body mass and individual

parasite load (IndPL), with a negative binomial response

for this latter variable. We performed general linear

regression to show the effect of host sex and locality on the

relationship between body condition (body mass/body

length ratio; see above) and individual parasite load

(IndPL).

These preliminary results may suggest that different

populations of the same species are not statistically inde-

pendent because they are connected by an evolutionary

history depicted by their phylogeography. In other words,

populations should be treated as species in comparative

analyses, by taking the phylogeographic structure into

account. The method of phylogenetically independent

contrasts (Felsenstein 1985) is commonly used to resolve

the lack of data independence (e.g., traits measured across

different species or populations). However, this method

deals with mean values estimated at the species level

without taking individuals into account, so it cannot be

easily adapted to populations belonging to the same

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species. Instead, we use the mixed model of Lynch (1991),

which is a variation of the animal model used in quanti-

tative genetics in which the pedigrees of individuals are

taken into account as covariation among relatives. Blom-

berg (2008, https://stat.ethz.ch/pipermail/r-sig-phylo/2008-

November/000206.html) has proposed that this approach

can be extended by account for not only species (popula-

tions here) but also individuals through the addition of

branches of length zero to individuals observed in the

phylogenetic (here phylogeographic) structure. The vari-

ance–covariance (VCV) matrix calculated from this new

phylogeny (phylogeography here) gives the covariance for

conspecifics. Covariance values decrease with increasing

distance between taxa (individuals here), eventually

reaching null covariance for taxa/individuals that are suf-

ficiently distant that they can be considered independent.

This method uses individual data and is therefore able to

consider an error in the average while controlling for

phylogeographical relationships. The model that can

incorporate the structure of the phylogeography is an

extension of the mixed model with an error term (i.e., a

random variable for the population level). To estimate this

model, we used the function lmekin (package kinship)

(Pinheiro and Bates 2000) implemented in R (R Develop-

ment Core Team 2010). The models were fitted by maxi-

mum likelihood. We used the phylogeographic information

for A. sylvaticus from Nieberding et al. (2004) to calculate

the VCV matrix.

To estimate the variation of resistance as estimated by

PHA and spleen size, we analyzed males and females

separately using a mixed model incorporating phylogeo-

graphic structure (VCV matrix). The fixed effects of the

models were host body mass (log units), body condition,

IndPSR, IndPL, and PopPSR.

To estimate the variation in tolerance assessed via body

condition, we analyzed male and female body condition

separately using the same mixed models with IndPSR,

IndPL, and TotPSR as fixed effects.

Finally, variation in male tolerance assessed via testes

mass was analyzed using host body mass, body condition,

spleen mass, and PHA as fixed effects.

Results

Parasites and parasite loads

A total of 28 helminth species were recorded in the eight

populations of A. sylvaticus (8 cestode species, 5 trematode

species, and 15 nematode species). The Sicilian population

harbored the richest parasite assemblage (14 helminth

species), whereas the Porquerolles population harbored the

poorest parasite assemblage (6 helminth species). The

Italian peninsular population showed low parasite richness

compared to other mainland samples (Table 1). Mean

individual parasite load (IndPL) was 18 ± 34 (range

0–251) for males and 19 ± 50 for females (range 0–424),

while individual parasite species richness (IndPSR) was

1.7 ± 1.4 (range 0–5) for males and 1.58 ± 1.27 (range

0–5) for females.

Effect of locality on host body mass, host condition,

and parasitism

The GLM for the raw data showed that host body mass was

significantly affected by both host sex (with males bigger

than females) and locality (Table 2), whereas individual

parasite load (IndPL) was affected by locality but not by

host sex (Table 2) (but see also Michaux et al. 2002 for the

variation in body mass among insular Mediterranean pop-

ulations of A. sylvaticus).

Host body condition was significantly influenced by host

sex, locality, and individual parasite load, as well as by

interactions between sex and locality and between indi-

vidual parasite load (IndPL) and locality (Table 3). The

sexual size dimorphism in body condition varied among

wood mouse populations.

The significant interactions of locality with host body

mass, host condition, and parasitism suggest that popula-

tions of A. sylvaticus are not independent replicates, which

is confirmed by their phylogenetic structure. This neces-

sitated the use of a comparative method based on phylog-

eographic information between individuals and among

populations.

Variation in resistance as assessed using PHA

and spleen size

PHA values of wood mouse individuals were explained by

body mass and sex (log likelihood = 115.28, df = 6,

P = 0.002) and spleen mass values were explained by

body mass and body condition, with no statistically sig-

nificant influence of sex (log likelihood = 59.39, df = 6,

P \ 0.0001) (Table 4). We investigated the variations in

PHA values and spleen mass separately in males and in

females.

PHA values of females were not explained by any vari-

able (log likelihood = 60.86, df = 5, P = 0.39, Table 4),

whereas spleen size was significantly positively explained

by both host body mass and population parasite species

richness (PopPSR), and negatively by host body condition

(log likelihood = 21.02, df = 5, P \ 0.0001, Table 5).

PHA values of males were only positively related to host

body mass (log likelihood = 50.24, df = 5, P = 0.001,

Table 4). Spleen size was significantly and positively

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https://stat.ethz.ch/pipermail/r-sig-phylo/2008-November/000206.html

https://stat.ethz.ch/pipermail/r-sig-phylo/2008-November/000206.html

explained by host body mass, and negatively explained by

body condition (log likelihood = 42.99, df = 5, P \ 0.0001;

Table 5).

Variation in tolerance as assessed using body condition

Body condition was positively related to individual parasite

species richness (IndPSR) in females only (log likeli-

hood = 34.62, df = 3, P \ 0.0001, Table 6), whereas no

parasitological indices were related to body condition in

males (Table 6).

Variation in tolerance in males as assessed using testes

mass

Testes mass was significantly explained positively by body

mass and negatively by PHA (log likelihood = 15.12,

df = 6, P \ 0.0001, Table 7). Testes mass was also

explained by population parasite species richness (Pop-

PSR) (Table 7, P \ 0,001) (Fig. 2).

Discussion

No significant sex-biased parasitism

Our results cast doubts on classical male-biased parasitism

in mammalian hosts. Instead, the results are in agreement

with numerous other field studies that have shown a weak

influence of sex on helminth loads in rodents, notably in

Apodemus spp. (Behnke et al. 1999; Fuentes et al. 2004;

Ferrari et al. 2004; Milazzo et al. 2010); or, on the contrary,

have revealed higher helminth loads in females (Hillegass

et al. 2008; Sanchez et al. 2011). Our results support the

idea that the influence of sex on parasitic load may depend

largely on the parasitic taxa and/or host–parasite associa-

tions (Klein 2004; Krasnov et al. 2005, 2012).

One of the explanations for this pattern may be related

to host exposure to helminth infective stages. Differences

Table 2 General linear

modeling test of the significance

of the effects of sex and locality

on Apodemus sylvaticus body

mass (g in log units), using a

Gaussian response, and on

individual parasitic load (IPL),

using a negative binomial

response

P values less than 0.05 are in

bold

Dependent variable Independent variable Estimate t value P value Deviance (P value)

Body mass Intercept 1.39 13.6 \0.0001

Sex: male vs. 0.09 (0.003)

female 0.04 2.53 0.011

Locality: Calabria vs. 0.5 (<0.0001)

Mallorca 0.03 1.08 0.29

Menorca 0.08 2.23 0.03

Montpellier 0.08 2.92 0.004

Montseny 0.11 3.82 0.0002

Porquerolles 0.14 3.90 0.0001

Py -0.04 -1.32 0.19

Sicily -0.09 3.47 0.0006

IPL Intercept 2.32 7.54 \0.0001

Sex: male vs. 0.0 (0.98)

female 0.25 1.07 0.28

Locality: Calabria vs. 41.7 (<0.0001)

Mallorca -0.70 -1.66 0.10

Menorca -0.02 -0.04 0.97

Montpellier -0.30 -0.76 0.45

Montseny 0.34 0.79 0.43

Porquerolles 2.00 3.59 \0.0001

Py 0.47 1.15 0.25

Sicily 1.10 2.81 0.005

Table 3 General linear modeling test of the significance of the effects

of sex and locality on the linear relationship between body condition

and individual parasite load (IndPL) of Apodemus sylvaticus

Dependent variable df Mean square F P value

Sex 1 2.089 20.712 <0.001

Locality 7 2.072 20.544 <0.0001

IndPL 1 2.325 23.051 <0.0001

Locality 9 sex 7 0.520 5.157 <0.001

Sex 9 IndPL 1 0.135 1.343 0.25

Locality 9 IndPL 7 0.360 3.573 0.001

Sex 9 locality 9 IndPL 7 0.165 1.634 0.13

Residuals 157 0.101

P values less than 0.05 are in bold

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in exposure to parasites due to behavioral differences

between males and females are often invoked as an

explanation for male-biased parasitism (Klein 2004; Mo-

rand et al. 2004; Krasnov et al. 2005). From this perspec-

tive, it is often assumed that larger home ranges in male

mammals (notably linked to mating success) could be

associated with greater exposure to parasites (Klein 2004;

Krasnov et al. 2005; Nunn et al. 2009). However, this is not

necessary the case, as shown for mammals and their hel-

minth parasites in a comparative study (Bordes et al. 2009).

It appeared that a larger home range could, in contrast, be

linked to a negative effect on parasite success (estimated

via parasite species diversity). As male wood mice have

larger home ranges than females (Attuquayefio et al. 1985),

we can then predict (in contrast to the classical assumption)

that males have fewer parasitic encounters. As a result, and

despite a potential greater susceptibility of males to para-

sites (due to lower immune investment), their reduced

exposure to parasites due to their larger home ranges could

actually result in similar parasitic loads in both sexes.

Another explanation may deal with susceptibility, and

specifically with differences in immune investment between

components of immunity between the two sexes. The use of

Table 4 Results of mixed models incorporating the variance and

covariance matrix of phylogeographic distances between individuals

among wood mouse populations

Dependentvariable (n)

Independentvariable

Estimate Standarderror

t value P value

PHA (189) Body mass 2.24 0.94 2.38 0.02

Bodycondition

-0.28 0.23 -1.23 0.22

IndPSR -0.03 0.04 -0.84 0.40

PopPSR 0.02 0.02 0.88 0.38

IndPL -0.03 0.08 0.38 0.70

Sex -0.15 0.07 -2.17 0.03

Spleen (189) Body mass 2.65 0.37 7.11 <0.0001

Bodycondition

-0.31 0.09 -3.27 0.001

IndPSR -0.02 0.02 -1.36 0.17

PopPSR 0.02 0.01 1.59 0.11

IndPL 0.04 0.03 1.19 0.23

Sex 0.01 0.03 0.30 0.76

The models used fixed effects with host body weight (in log), indi-vidual parasite species richness (IndPSR), individual parasite load(IndPL), total parasite species richness (PopPSR), and host sex in orderto explain PHA and spleen mass variations (models were fitted bymaximum likelihood, with locality treated as a random effect)


Table 5 Results of mixed models incorporating the variance and covariance matrix of phylogeographic distances between individuals among

wood mouse populations according to host sex

Sex Dependent variable (n) Independent variable Estimate Standard error t value P value

Female PHA (85) Body mass 1.69 1.55 1.09 0.28

Body condition -0.18 0.40 -0.46 0.65

IndPSR -0.12 0.07 -1.67 0.09

PopPSR 0.01 0.03 0.34 0.73

IndPL 0.08 0.14 0.60 0.55

Female Spleen (85) Body mass 3.42 0.56 6.08 <0.0001

Body condition -0.60 0.14 -4.30 <0.0001

IndPSR -0.01 0.03 -0.37 0.71

PopPSR 0.02 0.01 2.13 0.03

IndPL 0.06 0.05 1.16 0.25

Male PHA (104) Body mass 3.00 1.12 2.68 0.009

Body condition -0.37 0.26 -1.42 0.16

IndPSR 0.02 0.04 0.49 0.62

PopPSR 0.03 0.02 1.46 0.15

IndPL -0.11 0.09 -1.30 0.20

Male Spleen (104) Body mass 2.61 0.47 5.54 <0.0001

Body condition -0.32 0.11 -2.96 0.003

IndPSR -0.04 0.02 -1.93 0.06

PopPSR 0.01 0.01 0.35 0.73

IndPL 0.02 0.04 0.67 0.51

The models used fixed effects with host body weight (in log units), individual parasite species richness (IndPSR), individual parasite load

(IndPL), and total parasite species richness (PopPSR) in order to explain PHA and spleen mass variations in males and females (models were

fitted by maximum likelihood, with locality treated as a random effect)


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two measures of immune investment (PHA and spleen size)

does not allow us to make inferences about the functioning of

the entire immune system (Demas et al. 2011). It is plausible

that while one sex has higher immune responses on one

immune parameter, their responses may be lower on another

measurement of immunity; in other words, sex-dependent

trade-offs may exist between components of immune func-

tion (Klein and Nelson 1998). While we detected smaller

investments in immunity in males, they may have invested in

other components of their immune defense (that were not

detected with our tools) in order to limit parasitic loads.

Consequently, similar parasitic loads could then be observed

for the two sexes.

Different patterns of resistance for males and females

Investment in immunity was only observed in females

when spleen size was used as a measure of resistance, and

only in relation to population parasite species richness (Pop

PSR) (i.e., local parasite richness). In contrast, only body

mass was linked to the PHA test in males, whereas body

mass and body condition were linked to spleen size in the

two sexes.

Several studies have demonstrated immunological dif-

ferences between the sexes (Klein 2004; see Zuk 2009 for a

recent review). Females typically have stronger immune

responses than males in both birds (Leitner et al. 1989;

Tschirren et al. 2003; Pap et al. 2010) and mammals (Klein

2004; Pinzan et al. 2010). Our results support this idea,

because females but not males increased their immune

responses (at least, in terms of one component of immune


populations

Sex N Independent variable Estimate Standard error t value P value

Female body condition 85 IndPSR 0.16 0.05 3.15 0.002

IndPL 0.01 0.09 0.08 0.94

PopPSR -0.02 0.02 -0.81 0.42

Male body condition 104 IndPSR 0.05 0.04 1.20 0.23

IndPL 0.09 0.07 1.26 0.21

PopPSR 0.02 0.05 -0.38 0.71

The models used all parasitological parameters— individual parasite species richness (IndPSR), individual parasite load (IndPL), and total

parasite species richness (PopPSR)—as fixed effects in order to explain body condition variations in males and females. Models were fitted by

maximum likelihood, with locality treated as a random effect (N = sample size)

P value less than 0.05 is in bold


populations

Dependent variable (n) Independent variable Estimate Standard error t value P value

Testes mass (104) Body mass 3.90 0.37 10.71 <0.0001

IndPSR 0.01 0.03 0.25 0.81

PopPSR 0.04 0.01 3.60 <0.001

IndPL -0.01 0.06 -0.19 0.03

Spleen 0.01 0.18 0.21 0.97

PHA -0.23 0.07 -3.26 0.002

The models used host body mass, individual parasite species richness (IndPSR), individual parasitic load (IndPL), population parasite species

richness (PopPSR), spleen mass, and PHA as fixed effects to explain testes mass variations in males (models were fitted by maximum likelihood,

with locality treated as a random effect)


Fig. 2 Partial relationship between testes mass and PHA response

in males (using residuals from the mixed model in Table 7; slope =

-0.47 ± 0.13, F1,102 = 13.49, P = 0.0004)

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function), as indicated by the significant positive correla-

tion between spleen size and Pop PSR. Due to the strong

impact of parasites on reproductive success in female

rodents (Hillegas et al. 2010); it is not surprising that they

have evolved a greater investment in immunity in order to

limit the parasitic burden. These results should, however,

be approached with caution, because we did not investigate

the immune system in its entirety. Moreover, a larger

spleen size does not allow us to infer ‘‘stronger immunity’’

and resistance per se. We can, however, assume at the very

least that larger spleens in females do indicate greater

investment in immunity.

We have only investigated helminths, ignoring other

macroparasites (arthropods) and microparasites (viruses,

bacteria, fungi). The observed higher investment in

immunity may then be linked to differential parasitic loads

between the two sexes due to other parasites that were not

monitored. Moreover, different immunological back-

grounds and responses to helminth parasites and micro-

parasites may have led to different patterns. This major

limitation calls for new studies that should investigate a

broader range, if not all, parasite taxa to infer stronger

patterns.

Even given this limitation, however, our results may aid

our understanding of evolutionary mechanisms for sex

differences in immunity sexual dimorphism. Sex differ-

ences in immunity have been commonly investigated in the

light of differences in the endocrine system, especially in

relation to sex steroid hormones (Klein 2004; Hoby et al.

2006; but see Nunn et al. 2009). From an evolutionary

perspective, and given that immune function strongly

varies among animal populations as well as between sea-

sons and with reproductive effort (Lindstrom et al. 2004;

Martin et al. 2008; French et al. 2007; Cox et al. 2010), it

appears that immunity is mainly driven by trade-offs.

Immune defenses may then be enhanced only if they result

in a fitness reward. These trade-offs may ultimately explain

sex differences in immunity. Due to the high costs of

reproduction, females are expected to invest in survival and

other reproductive events (unlike males) and then in

immune defenses if this increases survival probability. In

contrast, males tend to invest less in immunity. In terms of

expected trade-offs between immunity and other tasks, we

may have detected a trade-off between immunity and

reproductive investment in males (i.e., a negative correla-

tion between PHA response and testis size). While previous

studies linked higher investment in immunity and reduced

reproductive investment to a trade-off, our study is one of

the few that show a negative association between an

immune measure and testis mass in the field (but see Ponlet

et al. 2011). We can therefore postulate that higher testis

mass should be linked to a higher level of testosterone, and

thus to a partial downregulation of the immune system.

Different patterns of tolerance for males and females

Parasites, by definition, extract resources from their host,

and thus individual hosts that are heavily parasitized are

expected to have fewer energy reserves and to be in worse

condition than individuals with few parasites. It appears,

however, that the links between parasitism and host con-

dition are more complex. First, parasites may not always

negatively impact body condition, as is often observed in

field studies (Khokhlova et al. 2002; Perez-Orella and

Schulte-Hostedde 2005; Schulte-Hostedde and Elsasser

2011). Second, parasite transmission may also depend on

host vulnerability. Recent studies in rodents have high-

lighted that individuals in poor health are the likely to be

the ones infected by viruses or hemoprotozoans (Bel-

domenico et al. 2008, 2009a, b). From this perspective,

infection and condition may act as a vicious circle: poor

condition facilitates host infection, which then deteriorates

host condition (Beldomenico et al. 2008). Finally, host

condition may be crucial to host colonization, and a study

stressed that the most profitable hosts for parasites may be

those in intermediate condition rather those in poor con-

dition (Bize et al. 2008).

Our results suggest a positive link between parasitism,

specifically individual parasite species richness (IndPSR),

and body condition in females only. For the parasites with

complex life styles considered in this study (i.e., hel-

minths), one explanation for this may be related to the

feeding behavior of the hosts and host exposure. As most of

the helminths encountered are taken in by consuming prey,

we can expect that hosts in good condition (i.e., larger

hosts) feed more, and are thus exposed to more parasites,

leading to higher parasite loads. A second explanation may

be linked to social organization and hierarchy, as some

individuals may feed more because of their higher rank in

the social hierarchy. As suggested before, a third expla-

nation may be related to the parasites themselves, because

hosts in poor condition may be a bad choice for parasites

(Bize et al. 2008). This hypothesis may be more spurious

because it implies that parasites actively choose hosts,

which, as we may know, has not been demonstrated in

helminths. Finally, another explanation may be a difference

in the tolerances of the two sexes. Undergoing multiple

parasitic exposures and still being in good condition strongly

suggests that wood mouse females are far more tolerant of

multiparasitism than males, due to better immune investment

or some other as-yet unexplored physiological reason.

Interestingly, for males, even though body condition is

not affected by parasitism, investment in testes mass (and

thus sperm production) is enhanced in populations that are

subjected to a high risk of infection (as estimated from the

local total parasite species richness). This may reflect an

adaptive reproductive strategy by males when facing

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123


multiple parasite infection, as testes mass is potentially

linked to fitness. This statement is strengthened by the

observed negative correlation between PHA response and

testes mass in males (see above).

In summary, our study has produced four main results.

First, sexual differences in tolerance and resistance may exist

in a wild rodent challenged by multiple helminth parasite

species. Second, higher investment in resistance under

increased parasite risk in females could make them more

tolerant of multiple parasite attacks as compared to males.

Third, the reproductive strategy of males may strongly be

driven by trade-offs between investment in reproduction and

immune defense. Finally, our results stress that measuring

parasitic load alone may not be sufficient to detect sex-biased

resistance and tolerance. However, the results found in this

study should be further investigated using new immuno-

logical tools (Jackson et al. 2011; Pedersen and Babayan

2011; Demas et al. 2011) and other indices of body condition.

These results must also be approached with caution due to the

limited taxa of parasites investigated, considering the great

diversity of parasites that infest wild hosts in natural systems

(Bordes and Morand 2009).

Acknowledgments We thank two anonymous referees for their

helpful comments. This is publication no. 758 of the Mitrani

Department of Desert Ecology. A. Ribas was partially supported by

‘‘Generalitat de Catalunya’’ 2009SGR403. JGB is presently a post-

doctoral fellow with FWO.

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