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ARTICLE
Parasitic infection increases risk-taking in a social,
intermediate host carnivore
Connor J. Meyer
1,2,3
, Kira A. Cassidy
1,3
, Erin E. Stahler
1
, Ellen E. Brandell
1
, Colby B. Anton
1
,
Daniel R. Stahler
1
& Douglas W. Smith
1
Toxoplasma gondii is a protozoan parasite capable of infecting any warm-blooded species and
can increase risk-taking in intermediate hosts. Despite extensive laboratory research on the
effects of T. gondii infection on behaviour, little is understood about the effects of tox-
oplasmosis on wild intermediate host behavior. Yellowstone National Park, Wyoming, USA,
has a diverse carnivore community including gray wolves (Canis lupus) and cougars (Puma
concolor), intermediate and denitive hosts of T. gondii, respectively. Here, we used 26 years
of wolf behavioural, spatial, and serological data to show that wolf territory overlap with areas
of high cougar density was an important predictor of infection. In addition, seropositive
wolves were more likely to make high-risk decisions such as dispersing and becoming a pack
leader, both factors critical to individual tness and wolf vital rates. Due to the social hier-
archy within a wolf pack, we hypothesize that the behavioural effects of toxoplasmosis may
create a feedback loop that increases spatial overlap and disease transmission between
wolves and cougars. These ndings demonstrate that parasites have important implications
for intermediate hosts, beyond acute infections, through behavioural impacts. Particularly in a
social species, these impacts can surge beyond individuals to affect groups, populations, and
even ecosystem processes.
https://doi.org/10.1038/s42003-022-04122-0
OPEN
1
Yellowstone Wolf Project, Yellowstone Center for Resources, P.O. Box 168 Yellowstone National Park, WY 82190, USA.
2
Wildlife Biology Program,
Department of Ecosystem and Conservation Sciences, W. A. Franke College of Forestry and Conservation, University of Montana, Missoula, MT 59812, USA.
3
These authors contributed equally: Connor J. Meyer, Kira A. Cassidy.
email: Connor.meyer@umontana.edu
COMMUNICATIONS BIOLOGY | (2022) 5:1180 | https://doi.org/10.1038/s42003-022-04122-0 | www.nature.com/commsbio 1
1234567890():,;
T
oxoplasma gondii is a ubiquitous multihost protozoan
parasite capable of infecting any warm-blooded species
and requires a felid denitive host to sexually reproduce
1
.
Infection spreads either through the ingestion of oocysts shed in
the environment by a denitive host (e.g., environmentally
mediated transmission via water or vegetation), the ingestion of
infected tissue cysts in denitive or intermediate hosts
1,2
, or,
if the fetus survives infection, vertically through congenital
transmission
2
.
Once an intermediate host is exposed, the infection spreads from
the intestinal lining to form cysts in the brain and muscle tissue and
acute toxoplasmosis occurs
2
. If acute infection occurs during
pregnancy it can lead to birthing complications, spontaneous
abortions, and stillbirths; and in young or immunosuppressed
individuals can cause fatal encephalitis
3,4
. Immunocompetent
individuals generally exhibit no clinical symptoms but will have a
chronic lifetime infection due to the presence of cysts
2
.Experi-
mental studies have shown that chronic infections, even in healthy
individuals, can lead to increased dopamine
5,6
and testosterone
production
7,8
. These hormone changes can cause increased
aggression
9,10
and risk-taking behaviour such as increased hyper-
active movement, failure to avoid olfactory predator cues (i.e.,
seeking out instead of avoiding felid urine), and decreased
neophobia
7,1113
.
Considering the effects that T. gondii infection can have on
intermediate host reproduction and behaviour, T. gondiis role in
wild ecosystem processes are understudied. One of the few studies
focused on infection impacts on behavior in a wild mammal,
Gering et al. (2021) found that toxoplasmosis was associated with
increased boldness in hyena (Crocuta crocuta) cubs and that
seropositive hyenas of all ages were more likely to be killed by
African lions (Panthera leo)
14
. That study demonstrated a
mechanistic link between toxoplasmosis and an individuals t-
ness through behaviour and decision-making.
Gray wolves (Canis lupus) in Yellowstone National Park (YNP)
have been the subject of extensive research over several decades,
primarily focused on predator-prey dynamics, population
dynamics, genetics, behaviour, and canine pathogens
15
. YNP is a
complex multi-carnivore system, where wolves and a denitive T.
gondii host, cougars (Puma concolor), overlap spatially due to
high landscape heterogeneity and prey movements
16
. Thus,
similar multispecies T. gondii transmission pathways as those
found between spotted hyenas and lions could be present between
wolves and cougars in North American systems, where wolves
that spatially overlap with cougars may have increased T. gondii
transmission risk via direct or indirect contact with cougars. T.
gondii has been documented in the YNP gray wolf
17
and we seek
to understand T. gondiis role in this social, intermediate
host carnivore using 26 years of gray wolf serological and
observational data.
Our rst aim was to determine which demographic and eco-
logical factors affect T. gondii infection in wolves in YNP. We
tested individual demographic factors, including age, sex, social
status at the time of capture, and coat color due to their potential
variation in disease susceptibility. Previous research has found the
risk of T. gondii infection increases with age due to accumulating
risk of exposure with time
17,18
. The other three wolf demographic
factors were included because of their links to certain hormones,
which may inuence an animals susceptibility to infection
19
. Sex
hormones play a role in infection risk and, once infected, hor-
mone production may be altered
19
; however, other studies found
no link between T. gondii seroprevalence and sex
14,17,18
. Due to
natural variations in hormone levels (testosterone, progesterone,
estrogen, etc.) between the sexes
20
, there may be differing risks
and subsequent behavioral responses to infection. Previous
research has found social status (e.g., pack leaders)
21
and coat
color (gray coat color wolves have higher cortisol levels and
increased behavioral aggression)
22
linked to varying hormone
levels and immune defense
23,24
. To determine if seroprevalence is
affected by the amount of spatial overlap with a T. gondii de-
nitive host (i.e., cougars), we included an overlap index for each
wolf and areas of high cougar density.
Our second aim was to determine if T. gondii infection inu-
ences wolf behavior. We identied three wolf behaviours asso-
ciated with greater risk-taking: (1) dispersing from a pack, (2)
achieving dominant social status (referred to as becoming a lea-
der), (3) approaching people or vehicles (referred to as habitua-
tion), and two causes of death associated with increased risk:(a)
intraspecic mortality (i.e., death by other wolves through
interpack ghts), or (b) anthropogenic mortality (i.e., death by
humans due to decreased proximity to humans or human
structures). As behavior can be inuenced by many factors, we
controlled for certain variables in each of the behavior models:
sex can inuence behaviors such as dispersal, and age can
inuence the probability of a certain behavior occurring
25
.
Northern YNP has very high wolf density, the roads are open
year-round, the elevation is lower and provides winter range for
ungulates and opportunities for wolf hunters just outside the park
boundary. All these factors may affect wolf behavior as the wolves
there may have increased opportunities to disperse, to die, and
may be more susceptible to habituation. Therefore, we controlled
for YNP system (northern or not) as well. In controlling for these
factors that may inuence wolf behavior, we aim to isolate the
inuence of T. gondii infection on behavior. We tested if ser-
ostatus inuenced the odds of a wolf performing these behaviors
or dying of one of these causes. We discuss the ndings from both
of our aims, factors inuencing T. gondii seroprevalence and
determining if toxoplasmosis affects wolf behavior, with respect
to interspecic disease dynamics and how behavioural changes
can impact gray wolves at multiple scales.
Here we found that T. gondii infection in wolves was predicted
by pack overlap with a denitive host, cougars, and that wolves
seropositive for T. gondii changed their behaviour to take greater
risksbeing more likely to disperse and to become pack leaders
than seronegative wolves. Due to a wolf packs social structure,
these behaviour changes may cause a feedback loop that leads to
pack-level increases in risk-taking with important implications
for further disease transmission, interspecic competition with
cougars, and wolf survival.
Results
Serology. Of the 62 cougars tested for T. gondii, 51.6% were
seropositive. Seroprevalence in cougars increased from 45%
during the rst sampling time (n = 47, 1999 to 2004) to 73%
during the second sampling time (n = 15, 2016 to 2020). This test
conrmed the presence of T. gondii in YNPs most-abundant
denitive host.
Between 1995 and 2020, an average of 11.8 sera samples were
collected each year (range = 422) to test for T. gondii antibodies.
All 50 tests from 1995 through 1999 were negative, then three
wolves tested seropositive in 2000. Thereafter, between one and
eight wolves were seropositive each year. Seventeen equivocal
samples were detected using the ELISA and were then rerun using
the MAT assay, which allowed us to distinguish eleven
seropositive and six seronegative samples. The pooled seropre-
valence was 0.0% from 1995 to 2000, 24.5% from 2000 to 2004,
18.7% from 2005 to 2009, 42.9% from 2010 to 2014, and 36.5%
from 2015 to 2020. Using samples collected from 2000 to 2020,
we ran 273 tests on 256 samples. Prevalence was 27.1% (n = 74)
with 61.9% negative (
n = 169) and 11.0% equivocal (n = 30).
Twenty-ve individuals were tested more than once throughout
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their life due to multiple captures, and therefore have multiple
samples, spaced at least eleven months apart. Eight males were
tested twice, 15 females were tested twice, and two females were
tested three times. Accounting for multiple tests, 229 individuals
were tested: 116 males, 112 females, and one hermaphrodite.
Females (31.25%) had slightly higher seroprevalence than males
(25.00%), but these proportions were not different (z-score =
1.05, p = 0.15).
Wolf age was recorded both as a continuous variable and
categorical variable with 100 pups, 53 yearlings, 88 adults (aged
2.05.9), and 15 old adults (aged 6.0 and older). Seroprevalence
was similar between the three younger categories (pup = 29.00%,
yearling = 28.30%, adult=26.14%) and only increased with old
adults (46.67%). A test comparing pup seroprevalence to all other
age categories was not signicant (z-score=0.346, p = 0.36). The
biggest difference was between old adults and all other age
categories pooled (z-score = 1.40, p = 0.08). We also tested for
differences in T. gondii exposure between gray and black coat
colors and found no difference: gray wolves (n = 115) had 25.22%
and black wolves (n = 114) had 31.58% seropositivity (z-score =
1.07, p = 0.14). Similarly, we tested social status at the time of
sampling and found no difference: subordinates (n = 197) had
30.45% and leaders (n = 59) had 23.73% seropositivity.
Wolves that had at least 42.1% overlap with cougar density
1.8/100 km
2
(HCO) had a higher proportion of seropositive
tests than wolves with MCO (5.142.0%), which was higher than
wolves with LCO (0 to 5% overlap). Twelve wolves (n = 83,
14.46%) with LCO were positive, 30 wolves (n = 83, 36.14%) with
MCO were positive, and 31 wolves (n = 84, 36.90%) with HCO
were positive. The proportion of seropositive wolves in a pooled
MCO and HCO was greater than wolves with LCO (z-score =
3.65, p = <0.001). To visualize cougar density and overlap with
different wolf pack territories we pooled seropositive tests in nine
general wolf use areas and plotted them on a map of YNP with
high cougar density highlighted (Fig. 1).
Demography analysis results. The full model testing the prob-
ability of seropositivity, with a w
i
= 0.99, included SEX, AGE IN
YEARS, SOCIAL STATUS, COAT COLOR, and COUGAR
OVERLAP INDEX (Table 1). The NULL model performed
poorly, with a w
i
= 0.01.
The COUGAR OVERLAP index (β = 1.089, 95% CIs:
0.1762.003) was an important factor in the odds a wolf was
seropositive for T. gondii. An increase from LCO to MCO to
HCO was associated with a higher likelihood of testing positive.
The odds ratio of COUGAR OVERLAP was 2.97 (exp[1.089]),
meaning the odds an MCO wolf was seropositive was nearly three
times higher than an LCO wolf. The odds an HCO wolf was
seropositive was almost 9 times higher odds of being seropositive
than a wolf in LCO. Predicted probabilities for seropositivity
(Fig. 2), based on the full model, showed that seropositivity
increased non-linearly with cougar overlap: wolves living in areas
with LCO had a predicted 4.7% prevalence, whereas wolves living
in MCO had a predicted 12.5% prevalence, and wolves in HCO
had a predicted 28.4% prevalence.
Unexpectedly, AGE did not have an effect on T. gondii
infection (β = 0.296, 95% CIs: 0.1580.751) and the 95%
condence intervals overlapped zero. The full model included
SEX, but this variable was nonsignicant and the condence
interval overlapped zero (β = 0.769, 95% CIs:0.49842.022).
SOCIAL STATUS at the time of testing was non-signicant
(β = 0.836, 95% CIs:1.9820.311) as was COAT COLOR
(β = 0.516, 95% CIs:0.7261.757).
Behaviour analysis results. Wolves classied as dispersers had
nearly double the T. gondii seroprevalence of non-dispersers:
36.26% for dispersers and 18.42% for non-dispersers (z-
score=3.11 p < 0.001). The model (DISP
1
) that included TOXO
performed better (w
i
= 0.92; Table 2) than the model without
TOXO (DISP
2
). All four variables were signicant with p values <
0.05 and none of the condence intervals overlapped zero. Males
were more likely to disperse than females, wolves living in
northern YNP were more likely to disperse than wolves in the
interior of YNP, wolves were more likely to disperse with
increasing time monitored, and seropositive wolves were more
likely to disperse than seronegative wolves (β = 2.459, 95% CIs:
0.2984.620). The odds ratio for TOXO was 11.69 (exp[2.459]),
meaning the odds a seropositive wolf disperses was 11 times
higher than the odds a seronegative wolf disperses.
Fig. 1 Map of cougar density and T. gondii seroprevalence in wolves in Yellowstone National Park (YNP). a Map of cougar density and T. gondii
seroprevalence in wolves in Yellowstone National Park (YNP). Yellow indicates cougar density <1.8/100 km
2
and purple indicates cougar density 1.8/
100 km
2
. Pie charts show the T. gondii seroprevalence (seropositive=black; seronegative=white/transparent) from wolves living in nine general areas
throughout YNP, pooled across years 20002020. b A sample year (2015) of wolf pack territory minimum convex polygons in YNP along with each packs
cougar overlap index level (LCO, MCO, or HCO) based on percentage of overlap with cougar density 1.8/100 km
2
(purple).
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Using the best-performing model to predict dispersal, we
found that seropositive male wolves were most likely to disperse,
followed by seronegative male wolves, seropositive females, then
seronegative females (Fig. 3). This result conrms previously
reported evidence of sex-biased dispersal in YNP wolves
25
and
indicates that T. gondii infection inuences the decision to
disperse in both sexes. A seropositive male has a 50% probability
of dispersing by six months monitored and seronegative males by
21 months monitored. Seropositive females have a 25% prob-
ability of dispersing by 30 months monitored whereas seronega-
tive females reach the same probability at 48 months. These
differences (15 months for males to reach 50% dispersing and
18 months for females to reach 25% dispersing) suggest
seropositive wolves disperse at much higher rates than serone-
gative wolves.
Seropositive wolves were more likely to become pack leaders
than seronegative wolves (z = 4.1705, p < 0.001). The model
including TOXO (LEAD
1
) performed better (w
i
= 1.0, Table 3)
than the model without TOXO (LEAD
2
) and SYSTEM was the
only variable that overlapped zero. The effect of TOXO was
positive and the condence intervals just reached zero (β = 3.83,
95% CIs: 0.0047.664). With an odds ratio of 46.06 (exp[3.83]),
the odds that a seropositive wolf becomes a pack leader is more
than 46 times higher than a seronegative wolf becoming a pack
leader.
Using the best-performing model to predict leadership we
plotted the probability of becoming a leader for wolves with and
Table 1 Full GLMM for the probability that an individual wolf tests positive for T. gondii.
Parameter β SE zP95% condence interval for
β
SEX 0.769 0.639 1.20 0.229 0.484 2.022
AGE (in years) 0.296 0.232 1.28 0.201 0.158 0.751
SOCIAL STATUS 0.836 0.585 1.43 0.153 1.982 0.311
COAT COLOR 0.516 0.633 0.81 0.415 0.726 1.757
COUGAR OVERLAP 1.089 0.466 2.34 0.019 0.176 2.003
intercept 4.371 1.624 2.69 0.007 7.553 1.189
The reference sex is male, social status is subordinate and coat color is gray. Individual wolf identication number was included as a random intercept.
Fig. 2 Gray wolf T. gondii serostatus results and predicted probability of infection given cougar density overlap. Gray wolves with T. gondii serology
results were divided into one of three categories relative to their average annual overlap with cougar density 1.8/100 km
2
(top bar): Low Cougar Overlap
(LCO in yellow) indicates wolves living in areas with 0.0 to 5.0% overlap, Moderate Cougar Overlap (MCO in orange) indicates 5.142.0% overlap, and
High Cougar Overlap (HCO in red) indicates 42.1100% overlap. This results in three categories of nearly equal sample size. The lower bars show the
predicted probabilities, with 95% condence intervals, of a seropositive T. gondii test for gray wolves living in LCO (yellow), MCO (orange), or HCO (red).
Predicted probabilities are based on the full model.
Table 2 Best-t GLMM for the probability that a wolf
disperses.
Parameter β SE zP95% condence
interval for β
SEX 5.251 1.837 2.86 0.004 8.852 1.649
SYSTEM 4.590 1.744 2.63 0.008 8.007 1.173
TIME
AVAILABLE
0.005 0.002 3.10 0.002 0.002 0.008
TOXO 2.459 1.103 2.23 0.026 0.298 4.620
intercept 2.058 0.947 2.17 0.030 3.914 0.202
The reference sex is male, reference system is northern YNP, and reference T. gondii status is
negative. Individual wolf identication number was included as a random intercept.
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without T. gondii and found that seropositive wolves were more
likely to become a leader than seronegative wolves and this effect
increased with time monitored (Fig. 4).
T. gondii infection did not explain habituated behaviour
(z = 1.604, p = 0.055) although this sample size was very small
with only 27 wolves classied as habituated and only four of those
seropositive (13.79%). The model without TOXO (HAB
2
; w
i
=
0.69) performed better than the model with TOXO (HAB
1
). This
behavior measure was very course and limited by sample size.
We tested for differences in T. gondii seroprevalence for wolves
with different cause-specic mortality and found no differences
with several comparisons of the proportions: human-caused
(n = 56, 32.14% T. gondii positive) versus natural-caused (n = 99;
31.31%) was not different (z = 0.107, p = 0.456). Wolf-caused
(n = 63, 31.75%) versus any other known cause (n = 137; 33.78%)
was not different (z = 0.253, p = 0.401). And wolf-caused versus
only known (non-wolf) natural mortalities (n = 81; 38.90%) was
not different (z = 0.567, p = 0.284). For both causes of death
examined through GLMMs, the models without TOXO per-
formed better (INTRA-MORT
2
: w
i
= 0.72 and ANTHRO-
MORT
2
: w
i
= 0.75) than models including TOXO (INTRA-
MORT
1
& ANTHRO-MORT
1
).
Discussion
This study provides insights into the relationship between para-
site infection and intermediate host behaviour in a wild system.
Infection with the parasite Toxoplasma gondii has been linked to
increased risk-taking in rodents
7,11,12
, chimpanzees (Pan
troglodytes)
13
, hyenas
14
, and now gray wolves. Wolf territory
overlap with cougars was a major ecological predictor of T. gondii
infection, while wolf demographics (e.g. sex or age) were not
informative. T. gondii seropositivity impacted wolf behaviour in
two of the three measures of risky behaviour that we tested:
seropositive wolves were more likely to disperse and to become
pack leaders than seronegative wolves (Fig. 5). These results
support not only the historical laboratory work, but also the
recent work on hyenas
14
conrming toxoplasmosis can affect
behaviour and decision-making in wild intermediate host
species
5,11,14
.
Gray wolves and cougars are competitors that evolved con-
currently in North America
16
. These two carnivores generally
prey on the same species, yet interspecic conict is mitigated
through partitioned time and space use, as well as active avoid-
ance of each other
16,26
. Our study corroborates Gering et al.
(2021) ndings that parasite transmission is an important factor
in the dynamic between sympatric carnivores
14
.
Due to the strong impact of overlap with cougars on gray wolf
T. gondii seroprevalence, gray wolves are likely contracting the
parasite through direct contact with infected cougars or their shed
oocysts, and not through the consumption of alternative inter-
mediate host species. Large ungulates in YNP migrate seasonally
to all areas of the park, overlapping all the sampled wolf pack
territories. If these ungulates had high T. gondii seroprevalence,
we would likely record all wolf packs having similar ser-
oprevalence levels. Elk, the primary prey of wolves in YNP, have
been tested for T. gondii (n = 155) and none were clearly ser-
opositive and only ve (3.2%) were suspected seropositive
(unpublished data, Yellowstone Center for Resources Wildlife
Health).
During winter, migrating ungulates in YNP move to lower
elevations
2628
and, as both wolves and cougars hunt ungulates,
this subsequently increases overlap between the two predators
16
.
This increase in overlap occurs during the wolf breeding, gesta-
tion, and whelping seasons (February to June
20
) from mid-winter
into early spring. Assuming wolves are more likely to be exposed
to T. gondii during this time, there is potential for negative
impacts on their reproduction. Specically, acute T. gondii can
cause complications in embryonic and fetal development and
result in fetal or newborn mortality, as shown in domestic dogs
4
.
Table 3 Best-t GLMM for the probability that a wolf
becomes a pack leader.
Parameter β SE z P 95% condence
interval for β
TIME
AVAILABLE
0.006 0.002 2.59 0.009 0.002 0.011
SYSTEM 1.453 1.056 1.38 0.169 3.522 0.617
TOXO 3.83 1.956 1.96 0.05 0.004 7.664
intercept 7.696 3.07 2.51 0.012 13.713 1.68
The reference system is northern YNP, and reference T. gondii status is negative. Individual wolf
identication number was included as a random intercept.
Fig. 4 Predicted probabilities of wolf leadership given the amount of time
monitored and T. gondii serostatus. Predicted probabilities of becoming a
pack leader for gray wolves with a seropositive (solid blue line) or
seronegative (dashed lime line) T. gondii test. The shaded areas indicate
95% condence intervals. The gray line indicates the average number of
months (24.9) a wolf is monitored in YNP.
Fig. 3 Predicted probabilities of wolf dispersal given sex, amount of time
monitored, and T. gondii serostatus. Predicted probabilities of dispersing
for male (blue) and female (orange) gray wolves with a seropositive (solid
lines) or seronegative (dashed lines) T. gondii test. The shaded areas
indicate 95% condence intervals. The gray line indicates the average
number of months (24.9) a wolf is monitored in YNP.
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In addition, wolf-to-wolf transmission might be possible during
this time (the winter wolf breeding season) because T. gondii can
be sexually transmitted in canid species during acute infection
4
.
In congruence with previous T. gondii wildlife studies,
including wolves, sex was not an important predictor of T. gondii
infection
14,17,18
. Contrary to other studies
14,17
, model results
showed that wolf infection did not vary with age. These effects
could possibly be masked by spatial differences in exposure
whereby a young wolf living in areas with many cougars has a
higher risk of infection than an older wolf living for years in a
territory with little cougar overlap.
This study is the r st to examine the relationship between
T. gondii infection and wolf behaviour and decision-making,
nding a link between parasitic infection and wolf ecology.
Dispersal is an important function of wolf population dynamics,
but represents a risk y decision as th ose that disperse suffer highe r
mortality rates
25,29,30
. However, if an individual survives the
dispersal process, they often nd increased opportunities for
reproduction
31
. As seropositive wolves are more l ikely to dis-
perse, this presents a potential for wolves with T. gondii to ll
gaps in unoccupied t erritories or attempt to establish p opulations
in new areas.
Additionally, seropositive wolves were almost twice as likely to
become pack leaders compared to seronegative wolves. Having
T. gondii may increase testosterone levels
7,8
leading to heightened
aggression
9,10
and preferential sexual selection (as was found in
rats [Rattus norvegicus])
7,32
. If seropositive wolves are more
aggressive during intra-pack interactions, they may more easily
become pack leaders. Increased aggression and dominance,
combined with possible preferential sexual selection, may explain
the mechanism between toxoplasmosis and leadership. Ulti-
mately, obtaining a dominant leadership position is critical for
increased tness through reproductive success
33,34
and is pre-
sumably under strong selection.
Due to the group-living structure of the gray wolf pack, the
pack leaders have a disproportionate inuence on their pack
mates and on group decisions (Fig. 6). If the lead wolves are
infected with T. gondii and show behavioural changes (e.g.,
seeking out felid scent or novel situations, as in rats or
chimpanzees)
12,13
this may create a dynamic whereby behaviour,
triggered by the parasite in one wolf, inuences the rest of the
wolves in the pack. If pack leaders seek out felid scent, this could
increase the likelihood that uninfected individuals encounter
infected cougars or their shed T. gondii oocysts in the environ-
ment and then become infected as well. An intermediate host
seeking out felid scent helps enable the parasite to complete its
lifecycle if the host was killed and consumed by the felid
12,13
.
Additionally, through social learning, pack leader behaviour (i.e.,
seeking out riskier situations) may be observed and emulated by
uninfected individuals, creating a more assertive, risk-embracing
pack culture even though only a few key individuals are actually
infected. Both pathways could increase wolf T. gondii infections
through increases in spatial overlap and perhaps even direct
interactions with cougars.
There are almost certainly evolutionary limits in place to
moderate this feedback loop. Acute infection during p regnancy
can result in litter mortality
4
. This aspect of acute infection
would severely decrease reproductive success and be evolutio-
narily disadvantageous for infected individuals. Furthermor e, it
is rare that a wolf dies of a cause that has little or no risk. The
three leading causes of death for wolves in YNP are intraspecic
ghts, anthropogenic (e.g., hunted by humans, hit by vehicle),
and fatal injuries incurred while hunting large prey
25
. If infected
wolves (or those that have learned from infected leaders) take
greater risks, it is likely their survival will be lower than those
avoiding risks. This hypothetical T. gondii-cougar-wolf-beha-
viour feedbac k loop depends on the balance between risks
resulting in an evolutionary advantage (e.g., leadership and
Fig. 5 Visual depiction of predicted probabilities of wolf serostatus and behavior. Schematic of results from both the demographic and the behaviour
analysis. Displayed at the top are three sample packs with different cougar overlap categories and their corresponding predicted probabilities of T. gondii
infection (seronegative in black; seropositive in red) based on the best-t demographic model. Red-lled wolves indicate the expected percent of infected
wolves out of 100% (e.g., total number of wolves in the pack). Cougar density 1.8/100 km
2
is depicted with hatch-marks. Cougar density below 1.8/
100 km
2
is all the area outside of the hatch-marks. At the bottom are the predicted probabilities with 95% condence intervals (gray lines) based on the
best-t behaviour models, of two risky behaviours: dispersing and becoming a pack leader for seronegative and seropositive wolves at 24.9 months
monitored (the average number of months wolves in this study were monitored).
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increased reproductive opportunities) and risks resul ting in
prema ture death. Regardless, the effects of T. gondii infection
clearly ripple beyond our ndings in this study with implications
for gray wolf survival and reproduction, and inters pecic com-
petit ion and disease dynamics.
This study is a rare demonstration of a parasite infection
inuencing behaviour in a wild mammal population. We iden-
tied a substantial increase in the odds of dispersal and of
becoming a pack leader, both risky behaviours, in wolves ser-
opositive for Toxoplasma gondii. These two life history beha-
viours represent some of the most important decisions a wolf can
make in its lifetime and may have dramatic impacts on gray wolf
tness, distribution, and vital rates. Dispersing wolves often
explore new habitats and are the individuals expanding current
gray wolf range. Dispersers that survive to establish a territory
often gain breeding positions. Pack leaders are the individuals
most likely to reproduce which has important implications on
population growth rate and may also affect pack behaviour and
culture. Additionally, our results suggest that wolves contract T.
gondii directly from cougars or their shed oocysts, not through an
intermediate host. This study demonstrates how community-level
interactions can affect individual behaviour and could potentially
scale up to group-level decision-making, population biology, and
community ecology. Incorporating the implications of parasite
infections into future wildlife research is vital to understanding
the impacts of parasites on individuals, groups, populations, and
ecosystem processes.
Method
Ethics statement. All wildlife were handled in accordance with recommendations
from the American Society of Mammalogists
35
. Wolf capture protocols are
approved by National Park Service veterinarians and University of Montana
IACUC #AUP 046-21. Cougar capture protocols are approved by National Park
Service veterinarians under IACUC numbe rs IMR_YELL_Stahler_cougar_2018.A1
and 1988-YCW-502.
Study area. YNP is a National Park managed by the United States Department of
the Interior. It is visited by over 4 million people each year, and over 98% of the
parks area (8,991 km
2
) is undeveloped and managed as wilderness. Northern YNP
(995-km
2
) differs from the interior of YNP (see Houston 1978 for detailed
description and map with boundaries)
27
as it has lower elevation and is used by elk
(Cervus canadensis), bison (Bison bison), and mule deer (Odocoileus hemionus)as
primary winter range. Consequently, this area has relatively high wolf and cougar
densities. The wolf population, after being extirpated in the early 1900s and then
reintroduced in 1995 to 1997, generally numbered between 90 to 120 wolves in
eight to twelve packs
25
, with approximately half the total in northern YNP. Three
different felid species that serve as denitive hosts to T. gondii have been found in
YNP. Cougars are the focus of this study because bobcats (Lynx rufus) occur at very
low densities (pers. communication K. Gunther) and Canada lynx (Lynx cana-
densis) are extremely rare
36
. Cougars were largely eradicated from the area by the
1930s, but naturally recolonized YNP by the 1980s, creating a resident population
primarily in northern YNP
37
.
Sample collection. Since wolf reintroduction to YNP in 1995, biologists captured
and radio collared 1220 wolves each year to understand wolf movement and
ecology. The wolves were anesthetized with a 10 mg/kg dose of Telazol® (tiletamine
& zolazepam), categorized as male or female based on external reproductive
organs, then tted with a VHF (Very High Frequency) or GPS (Global Positioning
System) radio-collar (Telonics inc. Mesa, AZ; Vectronic Aerospace Berlin, Ger-
many). Approximately 830 ml of blood was drawn from the cephalic vein into
vacutainer tubes for genetic sampling and disease screening. Between 315 ml of
the blood was centrifuged for 15 minutes and the serum was extracted and stored at
80 °C in 1.8 ml plastic cryovials. Age estimates were based on tooth wear, pelage
fading, and known pack composition. Coat color was recorded as either gray
or black.
Serological screening. Sera from wolves were tested for antibodies to Toxoplasma
gondii by the Cornell Animal Health Diagnostic Center (Ithaca, NY). Samples were
tested using either enzyme-linked immunosorbent assay (ELISA) or modied-
agglutination tests (MAT). For ELISA tests, any optical density measure less than
0.90 was considered negative, 0.911.09 was equivocal, and greater than 1.10 was
considered positive; for MAT tests, any result with a detectable antibody titer level
of 1:25 or greater dilution was considered positive. To be conservative, we treated
equivocal tests as negative for analysis. We report prevalence as the percent of
seropositive wolves out of the total number of wolves tested. As the rst T. gondii
infection in a gray wolf in YNP was conrmed in 2000, ve years after testing
began, the data used in the analyses were from 2000 and after.
To conrm that T. gondii was present in the cougar population, sera from 62
individuals captured (methods described in Ruth et al. 2010, Anton 2020)
38,39
in
two phases, from 1999 to 2004 and 2016 to 2020, and was tested using the same
MAT protocols described above.
Cougar overlap index. Cougars and wolves generally select for different habitats
due to their disparate hunting strategies
16
. However, due to YNPs high landscape
heterogeneity and common use of the same prey populations, the two species
exhibit variable overlap and interspecic encounters, including mortalities, occur
16
.
Cougars and wolves range throughout YNP at low densities except for northern
YNP where both occur at higher densities
3941
. This spatial overlap with a de-
nitive host species has been hypothesized to increase transmission to intermediate
hosts
18
. Anton (2020) used non-invasive genetic surveys to sample northern YNP
Fig. 6 Hypothesized wolf-cougar-T. gondii feedback loop. Schematic of the possible feedback loop involving gray wolves, cougars, and T. gondii. Red
gures indicate seropositive animals and black indicates seronegative animals. Thick, purple arrows indicate links supported by this or other published
literature. Thin, gray lines indicate hypothesized relationships.
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COMMUNICATIONS BIOLOGY | (2022) 5:1180 | https://doi.org/10.1038/s42003-022-04122-0 | www.nature.com/commsbio 7
and constructed a spatial model of cougar density within the sampled area
42
,
identifying cougar density per 3.2 km
2
cell (for specic methods see Anton 2020)
39
.
We used this spatial model to determine areas within the northern range of YNP
where cougar density was 1.8 cougars/100 km
2
. This served as the cutoff between
above and below-average cougar density
39,41
. The remainder of YNP, including
areas outside of Anton 2020s sampling area, were classied as below average
cougar density (less than 1.8/100 km
2
) based on a paucity of cougar sightings and
trail camera captures (usually <1 per year) and little to no use by GPS-collared
cougars. In addition, the area was not conducive to year-round cougar occupancy
because of the relativel y at, treed terrain with little relief and few canyons, and
prey species only using the area from approximately June to September due to high
elevation and severe winters
16,41
. It is possible there were a few small areas that
were classied as below average density that have seasonal cougar densities
approaching average levels. We calculated an index for each wolfs overlap with
cougar density 1.8/100 km
2
by plotting each wolf territory, measured as a 95%
minimum convex polygon (MCP) each biological year (April 1 to March 31) from
aerial locations of wolf packs from 2000 to 2021, with the cougar spatial model.
Although Antons spatial model was built on individual genetic detections in
winter (January-March) from 20142017, cougar use of northern YNP has
remained relatively consistent throughout nearly three decades of wolf research
conducted in YNP
16,39,42
. For each wolf tested, we averaged the annual overlap
with cougar density 1.8 cougars/100 km
2
across years from birth to the time of the
T. gondii test. Therefore, each wolf was assigned an average cougar overlap percent
(mean = 31.53, range 0100). As the individual wolf overlap percentages with
cougar density were not evenly distributed, this index was divided into three
categories: Low Cougar Overlap (LCO, 83 wolves with 0.05.0% overlap), Mod-
erate Cougar Overlap (MCO, 83 wolves with 5.142.0% overlap), and High Cougar
Overlap (HCO, 84 wolves with 42.1100% overlap). This method ensured that the
wolf overlap index with cougars was a metric of relative overlap compared to other
wolves, not absolute wolf overlap with cougar density.
Behavioural data collection . Wolf packs were directly observed at varying fre-
quency (approximately 20 to 250 times per year) depending on sightability within a
packs territory
43
from 1995 to 2021. Individual wolves were scored as a pack leader
after meeting both of the following criteria: repeated observations (at least ve
times per year) of dominance over same-sex pack members and evidence of a pair
bond (double scent-marking)
44
with the opposite-sex dominant individual. Wolves
were recorded as habituated (1) if they approached or travelled near people or
vehicles to within 10 meters. If a wolf was never recorded approaching within 10 m
during the study period, it was coded 0. Dispersal was assessed post hoc when a
wolf was tracked separately from its pack mates and away from the packs territory
and did not return. Cause of death was determined through necropsies in the eld
or at a laboratory.
Statistics and reproducibility
Demographic analysis. We report seroprevalence for specic demographic cate-
gories (e.g., male versus female) and compare these proportions using z-scores. To
examine individual variation in T. gondii infection, we used a generalized linear
mixed model (GLMM) with a binomial distribution using T. gondii infection as the
response variable. We compared a null model to a full model and report model
performance based on DAIC and model weights (w
i
). We used the program
STATA for all analyses.
The full model included sex (either male or female, based on external
reproductive organs), coat color (black or gray), and social status (leader or
subordinate) at time of capture as categorical variables and age as a continuous
variable. Studies on other species suggest that T. gondii seroprevalence and
susceptibility may be inuenced by certain hormones
19
and we included the
variables sex, coat color, and social status because they are associated with certain
hormonal patterns
45
. Gray wolves have higher cortisol levels compared to black
wolves
23,24
. Social status inuences, and is possibly inuenced by,
glucocorticoids
21,46
. Age was included to account for accumulating exposure risk
throughout a wolfs life. We also included a cougar overlap index for each wolf. The
cougar overlap index was averaged across years per individual, from birth until the
time of the wolfs sample was taken. To account for re-tested individuals, and for
unmeasured variables associated with individual wolves, we included wolf identity
(WOLF_ID) as a random intercept. Year was not included as a random intercept
because T. gondii exposure causes a chronic infection and it was unknown when
seropositive wolves were exposed to T. gondii. Accounting for variation in year,
relating to serostatus, would be only a reection of capture effort in a given year
rather than wolf exposure to T. gondii.
We compared a NULL model (testing if seroprevalence is best explained by the
random intercept only) to a full model which included SEX, AGE, COAT COLOR,
SOCIAL STATUS, and COUGAR OVERLAP. We tested for correlations between
these variables and found none to be signicant with p-values less than 0.05
(Supplementary Table 1). Variables from the best-p erforming model were
signicant if the p-value was 0.05 or less and the 95% condence interval did not
overlap zero. We constructed tted value plots for the predicted probability that a
wolf was seropositive for T. gondii antibodies based on the best-performing model.
Behaviour analysis. We identied three wolf behaviours considered risky: (1)
dispersing, (2) becoming a pack leader, and (3) showing habituated behaviour
around humans. We also identied two causes of death associated with known
types of risk: (a) intraspecic mortality and (b) anthropogenic mortality which
included deaths from hunting, poaching, vehicle strikes, lethal removal, and
capture-related mortalities. We rst report seroprevalence for each behavior or
cause of death (e.g., percent of dispersers versus non-dispersers that were ser-
opositive) and compare these proportions using z-tests. We then used generalized
linear mixed models (GLMMs) with a binomial distribution where wolves dis-
playing the behavior of interest were coded as a 1 and those that did not display the
behavior were coded as a 0 (e.g., 1=dispersed or 0=did not disperse). We devel-
oped a base model for each behaviour and included covariates associated with the
specic wolf behaviour and decision-making
15
. We then compared this to a model
that included all the same variables plus T. gondii seroprevalence. We report model
performance based on DAIC and model weights (w
i
). We included wolf identity
(WOLF_ID) as a random intercept and used the program STATA for all analyses.
To test whether T. gondii infection affected dispersal, we compared two models.
DISP
1
included SEX, SYSTEM, TIME AVAILABLE, and TOXO status. DISP
2
included all the same variables minus TOXO. This method allowed us to determine
the effect of T. gondii while controlling for other important factors, described
below. The same method was used to test for T. gondii effects on leadership: LEAD
1
included SYSTEM, TIME AVAILABLE, and TOXO and was compared to LEAD
2
including only SYSTEM and TIME AVAILABLE. The models testing for effects on
habituation included: HAB
1
SEX, TIME AVAILABLE, SYSTEM, and TOXO and
HAB
2
SEX, TIME AVAILABLE , and SYSTEM. For both GLMMs related to cause
of death we compared INTRA-MORT
1
and ANTHRO-MORT
1
including SEX,
AGE CLASS, SYSTEM, and TOXO to INTRA-MORT
2
and ANTHRO-MORT
2
including SEX, AGE CLASS, and SYSTEM. We included individual wolf ID as a
random intercept for all models except the habituation analysis (HAB
1
and HAB
2
)
which were restricted by sample size and models attempting to account for
repeated measures did not converge.
To account for the duration a wolf was monitored while infection status was
known, we used the contin uous variable TIME AVAILABLE. Temporal measure
TIME AVAILABLE was the total number of days each wolf was monitored (mean:
746, range: 33800). The focal time period for the response (i.e., risky behaviour)
was dependent on the time period of a wolfs known T. gondii serostatus. Wolves
that were seronegative had a TIME AVAILABLE from the time of their test
backwards to their birth, and wolves that were seropositive had a TIME
AVAILABLE from their test date forward to death, or to present time if still alive.
Restricting observed behaviours to the time period for which we knew a wolfs
infection status ensured that we accurately accounted for parasite infection when
assessing behaviour. For wolves that tested negative, we subtracted 180 days from
their time available because the rst six months of a wolfs life are spent as
dependents at a den or rendezvous site, and they are not old enough to disperse or
become leaders.
SEX (male or female) and AGE CLASS (pup, yearling, adult, or old) were
included in a subset of the behavior models to contro l for their known effects on
wolf life history, particularly related to dispersal, where males are more likely to
disperse and dispersal rates increase with age, and survival
25
.
Relatively high wolf density in northern YNP may affect wolf behavior. For
example, wolves that lived in northern YNP had closer selection distance to roads
47
and this may correlate with habituated behavior. High wolf density may also affect
behavior such as dispersal given the greater access to potential mates. To account
for this dynamic, we included SYSTEM (northern YNP or non-northern YNP) in
the behavior models.
Variables from the best-performing model were signicant if the p-value was
0.05 or less and the coefcient 95% condence interval did not overlap zero. If the
best-performing model for each behavior included TOXO, we constructed tted
value plots to visualize the predicted probability of the behavior given serostatus
and the other signicant variables.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The datasets generated and analyzed during the current study are available from the
corresponding author upon request. Source data are available in Supplementary Data.
Code availability
The code used during the analysis of the current study are available from the
corresponding author upon request.
Received: 24 November 2021; Accepted: 17 October 2022;
ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-04122-0
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Acknowledgements
We thank Rick McIntyre and the many volunteers and visitors to Yellowstone National
Park who helped make this project possible. We are deeply appreciative to Dr. Mark
Hebblewhite and Dr. Doug Emlen for their valuable insight and feedback on this
manuscript. We additionally want to thank Brenna Cassidy and Wes Binder for their
perspective and input on the topic. This work could not be done without the safe piloting
of Mark Packila from Wildlife Air, Jim Pope from Leading Edge, Bob Hawkins from
Hawkins and Powers, Inc., Roger Stradley and Steve Ard from Gallatin Flying Service,
and Stephan Robinson and Gr ayson Sperry of Ridgeline Aviation. We would like to
thank Toni Ruth, Hornocker Wildlife Institute, and Wildlife Conservation Society for
providing cougar serum samples from northern Yellowstone during 19992004. We also
thank signicant donors to the Yellowstone Wolf Project: Valerie Gates, Annie and Bob
Graham, and Frank and Kay Yeager. Thanks to the Cornell University Animal Health
Diagnostic Center, as well as the U. S. National Park Service, U. S. Geological Survey,
Yellowstone Forever, and the National Science Foundation (DEB- 0613730 and DEB-
1245373) for their support of this work. Any mention of trade, rm, or product names is
for descriptive purposes only and does not imply endorsement by the U.S. government.
Author contributions
C.J.M., K.A.C., and E.E.S. conceived of the research questions. E.E.B. and C.B.A. provided
conceptual advice and D.W.S. and D.R.S. provided conceptual advice and supervised the
project. D.W.S., D.R.S., E.E.S., K.A.C., C.J.M, and C.B.A. collected physical samples while
K.A.C., E.E.S., C.B.A., D.R.S., D.W.S., and E.E.B. collected observational data. K.A.C.
analyzed data and created the schematics and gures. C.J.M. and K.A.C. wrote the paper
with input from the other authors.
Competing interests
The authors declare no competing interests.
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Supplementary information The online version contains supplementary material
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Peer review information Communications Biology thanks Sarah Marshall-Pescini and
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Discussion

>>Here we found that T. gondii infection in wolves was predicted by pack overlap with a definitive host, cougars, and that wolves seropositive for T. gondii changed their behaviour to take greater risks—being more likely to disperse and to become pack leaders than seronegative wolves. Due to a wolf pack’s social structure, these behaviour changes may cause a feedback loop that leads to pack-level increases in risk-taking with important implications for further disease transmission, interspecific competition with cougars, and wolf survival. Great content on the gray wolves at Yellowstone. [Gray Wolf - Yellowstone National Park (U.S. National Park Service)](https://www.nps.gov/yell/learn/nature/wolves.htm#:~:text=An%20estimated%20528%20wolves%20resided,%2D2019%20average%20%3D%2094.5).) Toxoplasma-infected hyenas will venture closer to lions, which increases the likelihood that they will be killed. From the paper: “Toxoplasma gondii is hypothesized to manipulate the behavior of warm-blooded hosts to promote trophic transmission into the parasite’s definitive feline hosts. A key prediction of this hypothesis is that T. gondii infections of non-feline hosts are associated with costly behavior toward T. gondii’s definitive hosts; however, this effect has not been documented in any of the parasite’s diverse wild hosts during naturally occurring interactions with felines. Here, three decades of field observations reveal that T. gondii-infected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality. We discuss these results in light of 1) the possibility that hyena boldness represents an extended phenotype of the parasite, and 2) alternative scenarios in which T. gondii has not undergone selection to manipulate behavior in host hyenas. Both cases remain plausible and have important ramifications for T. gondii’s impacts on host behavior and fitness in the wild” [Toxoplasma gondii infections are associated with costly boldness toward felids in a wild host | Nature Communications](https://www.nature.com/articles/s41467-021-24092-x) >> This study is a rare demonstration of a parasite infection influencing behaviour in a wild mammal population. We identified a substantial increase in the odds of dispersal and of becoming a pack leader, both risky behaviours, in wolves seropositive for Toxoplasma gondii. These two life history behaviours represent some of the most important decisions a wolf can make in its lifetime and may have dramatic impacts on gray wolf fitness, distribution, and vital rates. Dispersing wolves often explore new habitats and are the individuals expanding current gray wolf range. Dispersers that survive to establish a territory often gain breeding positions. Pack leaders are the individuals most likely to reproduce which has important implications on population growth rate and may also affect pack behaviour and culture. Additionally, our results suggest that wolves contract T. gondii directly from cougars or their shed oocysts, not through an intermediate host. This study demonstrates how community-level interactions can affect individual behaviour and could potentially scale up to group-level decision-making, population biology, and community ecology. Incorporating the implications of parasite infections into future wildlife research is vital to understanding the impacts of parasites on individuals, groups, populations, and ecosystem processes. Toxoplasmosis is also linked to increased risk behavior in humans as well. One highly-cited study from 2002 found that there was increased risk of traffic accidents in individuals with latent toxoplasmosis. From their paper: “The parasite /Toxoplasma gondii/ infects 30–60% of humans worldwide. Latent toxoplasmosis, I️.e., the life-long presence of /Toxoplasma/ cysts in neural and muscular tissues, leads to prolongation of reaction times in infected subjects. It is not known, however, whether the changes observed in the laboratory influence the performance of subjects in real-life situations….. The subjects with latent toxoplasmosis have significantly increased risk of traffic accidents than the noninfected subjects. Relative risk of traffic accidents decreases with the duration of infection. These results suggest that ‘asymptomatic’ acquired toxoplasmosis might in fact represent a serious and highly underestimated public health problem, as well as an economic problem.” Article is here: [Increased risk of traffic accidents in subjects with latent toxoplasmosis: a retrospective case-control study - PMC](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC117239/) Toxoplasma can make infected rodents lose their fear of predators. From the study: “Toxoplasma gondii exerts a strange sort of mind control on rodents: Once infected with the brain parasite, they seem to lose their fear of cats and become more likely to get eaten. When they are, the microbe can make its way into the feline intestine to reproduce. But a new study argues that T. gondii’s effects on rodents aren’t cat specific; instead, the parasite simply makes mice more eager to explore and less fearful of any species that might gobble them up.” [Brain parasite may strip away rodents’ fear of predators—not just of cats | Science | AAAS](https://www.science.org/content/article/brain-parasite-may-strip-away-rodents-fear-predators-not-just-cats) This study is enabled by joining two disparate data sets- when the authors combined infection data with past field observations, they were also able to discover that infected wolves were more likely to become pack leaders... Toxoplasma gondii, otherwise known as the mind control parasite can infect the brains of animals, and alter their behavior to improve the chances that the parasite spreads (even if it means the host will be killed along the way). While most known effects of toxoplasmosis have been negative (I️.e. making prey approach their predators, reckless behavior in humans), this study finds a benefit to wolves of an infection- here - it makes wolves more bold and more likely to become an alpha pack leader (and thereby increasing the likelihood that they can reproduce). Nice summary of this article by Science: https://www.science.org/content/article/parasite-makes-wolves-more-likely-become-pack-leaders