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Abstract

Prey animals can assess the risks predators present in different ways. For example, direct cues produced by predators can be used, but also signals produced by prey conspecifics that have engaged in non-lethal predator-prey interactions. These non-lethal interactions can thereby affect the physiology, behavior, and survival of prey individuals, and may affect offspring performance through maternal effects. We investigated how timing of exposure to predation-related cues during early development affects offspring behavior after weaning. Females in the laboratory were exposed during pregnancy or lactation to one of three odor treatments: (1) predator odor (PO) originating from their most common predator, the least weasel, (2) odor produced by predator-exposed conspecifics, which we call conspecific alarm cue (CAC), or (3) control odor (C). We monitored postnatal pup growth, and we quantified foraging and exploratory behaviors of 4-week-old pups following exposure of their mothers to each of the three odour treatments. Exposure to odors associated with predation risk during development affected the offspring behavior, but the timing of exposure, i.e., pre-vs. postnatally, had only a weak effect. The two non-control odors led to different behavioral changes: an attraction to CAC and an avoidance of PO. Additionally, pup growth was affected by an interaction between litter size and maternal treatment, again regardless of timing. Pups from the CAC maternal treatment grew faster in larger litters; pups from the PO maternal treatment tended to grow faster in smaller litters. Thus, in rodents, offspring growth and behavior are seemingly influenced differently by the type of predation risk perceived by their mothers.
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ORIGINAL RESEARCH
published: 14 December 2021
doi: 10.3389/fevo.2021.709207
Edited by:
Peter Banks,
The University of Sydney, Australia
Reviewed by:
Burt P. Kotler,
Ben-Gurion University of the Negev,
Israel
Gabriel Francescoli,
Universidad de la República, Uruguay
*Correspondence:
Thorbjörn Sievert
thorbjoern.sievert@gmx.net
ORCID:
Thorbjörn Sievert
orcid.org/0000-0002-4242-3779
Kerstin Bouma
orcid.org/0000-0002-3366-1003
Marko Haapakoski
orcid.org/0000-0003-0901-6319
Kevin D. Matson
orcid.org/0000-0002-4373-5926
Hannu Ylönen
orcid.org/0000-0001-6935-1464
These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Behavioral and Evolutionary Ecology,
a section of the journal
Frontiers in Ecology and Evolution
Received: 13 May 2021
Accepted: 24 November 2021
Published: 14 December 2021
Citation:
Sievert T, Bouma K,
Haapakoski M, Matson KD and
Ylönen H (2021) Pre- and Postnatal
Predator Cues Shape Offspring
Anti-predatory Behavior Similarly
in the Bank Vole.
Front. Ecol. Evol. 9:709207.
doi: 10.3389/fevo.2021.709207
Pre- and Postnatal Predator Cues
Shape Offspring Anti-predatory
Behavior Similarly in the Bank Vole
Thorbjörn Sievert1*†‡, Kerstin Bouma2†‡, Marko Haapakoski1, Kevin D. Matson2and
Hannu Ylönen1
1Department of Biological and Environmental Science, Konnevesi Research Station, University of Jyväskylä, Jyväskylä,
Finland, 2Wildlife Ecology and Conservation, Environmental Sciences Group, Wageningen University, Wageningen,
Netherlands
Prey animals can assess the risks predators present in different ways. For example,
direct cues produced by predators can be used, but also signals produced by
prey conspecifics that have engaged in non-lethal predator-prey interactions. These
non-lethal interactions can thereby affect the physiology, behavior, and survival of
prey individuals, and may affect offspring performance through maternal effects. We
investigated how timing of exposure to predation-related cues during early development
affects offspring behavior after weaning. Females in the laboratory were exposed during
pregnancy or lactation to one of three odor treatments: (1) predator odor (PO) originating
from their most common predator, the least weasel, (2) odor produced by predator-
exposed conspecifics, which we call conspecific alarm cue (CAC), or (3) control odor
(C). We monitored postnatal pup growth, and we quantified foraging and exploratory
behaviors of 4-week-old pups following exposure of their mothers to each of the three
odour treatments. Exposure to odors associated with predation risk during development
affected the offspring behavior, but the timing of exposure, i.e., pre- vs. postnatally, had
only a weak effect. The two non-control odors led to different behavioral changes: an
attraction to CAC and an avoidance of PO. Additionally, pup growth was affected by an
interaction between litter size and maternal treatment, again regardless of timing. Pups
from the CAC maternal treatment grew faster in larger litters; pups from the PO maternal
treatment tended to grow faster in smaller litters. Thus, in rodents, offspring growth and
behavior are seemingly influenced differently by the type of predation risk perceived by
their mothers.
Keywords: alarm pheromone, conspecific alarm cue, predation risk, odor cues, cross-generational effects,
rodents
INTRODUCTION
Lethal and non-lethal effects of predators influence the life history of prey (Sih, 1994;Ylönen and
Ronkainen, 1994;Werner and Peacor, 2003;Nelson et al., 2004;Ylönen and Brown, 2007;Sheriff
et al., 2009). Over the last two decades, increasing attention has been paid to non-lethal effects that
relate to fear in the face of high predation risk. Overall, this research suggests that non-lethal effects
of predators mediated by fear in prey can have population level effects that are similar in magnitude
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Sievert et al. Bank Vole Early Life Fear
to the direct effects of predators killing prey (Schmitz et al., 1997;
Nelson et al., 2004;Preisser et al., 2005;Pangle et al., 2007;Creel
and Christianson, 2008).
Predation risk can be detected by prey in several ways:
visually, acoustically, olfactorily, or tactilely. Via olfaction, prey
can either detect odors produced directly by a predator (predator
odor, PO) or odors produced by conspecifics that engaged
in non-lethal interactions with predators (conspecific alarm
cue, CAC) (Haapakoski et al., 2018). Conspecific alarm cues
are also sometimes referred to as “alarm secretions” (Brand
et al., 1989) or “alarm pheromones” (Breed et al., 2004;Gomes
et al., 2013). Irrespective of terminology, mechanisms and
consequences associated with CAC production and release are
well-documented (Gomes et al., 2013), including in anthozoa
(Howe and Sheikh, 1975), insects (Bowers et al., 1972;Beale et al.,
2006), amphibians (Hempel et al., 2009), and mammals (Boissy
et al., 1998;Gutiérrez-García et al., 2007). In several social species,
CAC serve as signals to protect at-risk families, colonies, or other
groups (Breed et al., 2004;Kiyokawa et al., 2004;Gomes et al.,
2013). While the structure of CAC remains unresolved for most
mammals, it has been identified in, for example, aphids (Bowers
et al., 1972), sea anemones (Howe and Sheikh, 1975), and several
insects (Heath and Landolt, 1988;Kuwahara et al., 1989). Work
in laboratory rodents has led to analyses of CAC from Wistar
rats (Inagaki et al., 2014) and C57BL/6J and OMP-GFP mice
(Brechbühl et al., 2013).
After encountering PO or CAC, prey are expected to adjust
behaviorally in ways that minimize their chances of being
preyed upon. Such changes include increased vigilance (Périquet
et al., 2012) and freezing (Wallace and Rosen, 2000;Sundell
and Ylönen, 2004), avoidance of risky areas (Kikusui et al.,
2001), and altered space use and activity peaks (edrzejewska
and J˛edrzejewski, 1990;edrzejewski and J˛edrzejewska, 1990;
Sundell et al., 2008). In addition to the foraging-related
repercussions of the behavioral changes listed above (Brown,
1999), decreased foraging can also be a direct result (Ylönen,
1989;Sundell and Ylönen, 2004;Bleicher et al., 2018). Ultimately,
behavioral adjustments that enhance short-term survival of
predators may lead to trade-offs with future reproduction
(Ylönen and Ronkainen, 1994;Fuelling and Halle, 2004;Love and
Williams, 2008;Haapakoski et al., 2012;Sheriff and Love, 2013;
Voznessenskaya, 2014).
Stress responses, and in particular corticosterone production,
represent another mechanism through which PO or CAC can
affect prey reproduction and fitness. Exposure to either type of
olfactory cue of predation risk can be transmitted from mother
to offspring, influencing offspring growth (Berghänel et al.,
2017), development (Hayward and Wingfield, 2004), behavior
(St-Cyr et al., 2017;Sievert et al., 2020), and survival (Chin
et al., 2009). A mother and fetus are connected prenatally
via the placenta and postnatally via lactation; both serve as
transmission routes for hormones, and thus information about
the environment (Sullivan et al., 2011;Sheriff et al., 2017;Kuijper
and Johnstone, 2018). Since maternal care can play a role in
programming the hypothalamic-pituitary-adrenal axis (HPA-
axis) of offspring, the stress responsiveness of offspring may
itself be altered (Liu, 1997). Mothers might also communicate
information about predation risk and stress via odors they
produce (Koyama et al., 2015).
While predation risk in small mammals can affect the behavior
and future reproduction of offspring (Ylönen and Ronkainen,
1994;Ylönen, 2001;Bestion et al., 2014;Sheriff et al., 2015),
the role of the timing of exposure to predation risk remains
unclear. Prenatal and postnatal exposure, which are governed
by different mechanisms, might differ in their consequences. In
this study, we exposed mothers to predator odor, CAC, or a
control cue during pregnancy or during lactation. We observed
the growth rate of their respective offspring and, once weaned,
their offspring were tested in an experimental set-up where they
encountered these odors again. Exploratory behavior as well as
foraging behavior was then observed. We made several specific
predictions. First, we predicted that the offspring of mothers
exposed prenatally to either PO and CAC would grow more
slowly than those of mothers exposed postnatally or to mothers
exposed to neither, due to either increased stress metabolites
in utero or a reduced maternal investment as an reaction to
the perceived increased predation pressure (Bian et al., 2005;
Dunn et al., 2010;Coslovsky and Richner, 2011). Second, we
predicted that the offspring of mothers exposed postnatally to
either PO or CAC would show greater avoidance of the odor to
which their mother was exposed compared to offspring exposed
prenatally (Dias and Ressler, 2014). This assumes that the odors
are carried into the nest in the fur of the mother and the
combination of the odor and potential changes in maternal care
and/or increased stress hormone levels in the milk will trigger
aversive behavior. Third, we predicted that offspring would forage
less in the presence of PO and CAC, regardless of their mother’s
treatment (Brown, 1988;Sievert et al., 2019). Fourth, we predicted
increased latencies to investigate foraging options, reduced time
spent in foraging chambers, and fewer foraging chamber visits
in chambers with PO or CAC compared to the control chamber
(Apfelbach et al., 2015;Sievert and Laska, 2016;Parsons et al.,
2018;Sievert et al., 2019). Prediction three and four assume that
PO and CAC carries information about an increased risk and
therefore these compartments will be largely avoided and not
used for foraging (Brown, 1988).
MATERIALS AND METHODS
Study Species
The bank vole (Myodes glareolus) is a small granivorous rodent
that is common in forested areas in boreal and temperate regions
from Europe to Siberia. It is often used as a model species that
is easy to keep and breed in captivity. The bank vole is killed
by various predators, including the least weasel (Mustela nivalis
nivalis). Vole populations are known to cycle multi-annually in
Scandinavia, and the least weasel and other specialist predators
play an important role in driving these cycles (Hanski et al., 2001).
In central Finland, bank vole breed from April to September.
During this time, female bank voles are strictly territorial, and
each male’s territory overlaps with several female territories
(Bujalska, 1973). After a gestation of about 20 days, 3–6 pups are
born. Pups mature after 30 days.
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The predator-prey interaction between boreal voles and their
specialist predators has been well studied (Korpimäki et al.,
1991;Norrdahl and Korpimäki, 1995;Ylönen et al., 2019). Least
weasels and bank voles share the same habitat. The weasel
is able to hunt in tunnels and burrows throughout the year,
due to its small size, leaving only a few areas safe for voles
(Norrdahl and Korpimäki, 1995, 2000;Sundell et al., 2008). Like
all mustelids, the least weasel uses strong odors for intraspecific
communication (Brinck et al., 1983), giving the prey a cue to
detect the predator’s presence.
Experimental Animals
The experiments were conducted at the Konnevesi Research
Station in Central Finland (62370N, 26200E). Bank voles
were trapped from the wild and brought into captivity, where
they were allowed to acclimatize for 3 weeks before the
experiment commenced. All individuals were ear tagged for
identification (#1005-1L1, National Band and Tag Company,
Newport, Kentucky, United States). In the laboratory, the
voles are kept in climate- and light-controlled (16L:8D daily
cycle) husbandry rooms. Voles were housed individually in
42 ×26 ×15 cm transparent cages with wire mesh lids and
wood shavings and hay as bedding, and they were provided
with ad libitum water and food. Males and female voles were
maintained in the same room.
Weasels for the odor treatment were housed individually in
60 ×160 ×60 cm cages in an outdoor shelter. Each cage had
a nest box and wood shaving and hay as bedding. During the
experiment, weasels were fed only with dead bank voles.
Experimental Design
Voles were weighed and divided randomly into three treatments:
control (C), CAC and predator odor (PO). These three groups
were then divided into prenatal (early) and postnatal (late)
treatment groups, so that the mass distribution was similar across
treatments (mean ±standard deviation: C early 17.7 g ±4 g; C
late 17.4 ±3.2 g; CAC early 17.8 g ±4.1 g; CAC late 17.6 g ±3.9 g;
PO early 17.2 g ±3.4 g; PO late 16.7 g ±4.2 g). The prenatal
and postnatal control groups each consisted of 40 individuals; the
other four treatment groups each consisted of 60 individuals. In
all groups, the sex ratio was 50:50. Voles were randomly paired
within treatment groups for mating; sibling couples were avoided.
To allow for reproduction, the couples were kept in the same
cage for 7 days.
During the treatment phase, females were exposed to one
of the three odor treatments. The control treatment consisted
of clean wood shavings. The CAC treatment consisted of wood
shavings used by predator-exposed male voles. Specifically, four
males that were not otherwise used in the experiment were placed
individually in a weasel cage every weekday (Monday to Friday)
for 3 min per day. During this exposure, the weasel was unable to
actually attack the vole. The bedding from the cages of the four
exposed voles was collected each day after exposure. Males were
used for CAC production to avoid possible confounding effects of
estrus. The PO treatment consisted of weasel bedding materials,
including feces.
Female voles were exposed to the short bursts of the
treatments to mimic encountering the odors outside the nest, for
example during foraging trips, both pre- and postnatally. Females
in the prenatal group were treated from mating until parturition
(i.e., for 18–25 days depending on the timing of parturition);
females in the postnatal group were treated from parturition until
the pups were 21 days old. All the females received the treatment
following the same protocol: three times per week (Monday,
Wednesday, and Friday) for 3 min per session, females were
placed individually in a small cage with a separate compartment
holding the treatment materials (Supplementary Figure 1). The
pups remained in the home cage during the treatments. The
separation from the mothers took place in all six treatment
groups to exclude it as a confounding factor.
Eighteen days after initiating mating, females were checked for
signs of pregnancy, and the cages were checked twice a day (in
the morning and in the evening) for the presence of pups. On
the seventh day after birth, pups were toe marked for individual
recognition. Pups were weighed three times a week until the
21 days of age. During this same 21 day period, the mothers in the
postnatal group received the treatment. In total, 113 experimental
pups were produced in 29 litters across the three treatment
groups. After 21 days, the pups were separated, ear tagged, and
placed in individual cages.
One week after separation, pup behavior was assayed in a
foraging choice arena with three arms (Supplementary Figure 2).
Four hours before being tested, pups had their food removed to
incentivize foraging in the novel environment. The test arena
was used to measure giving up density, GUD, as an indicator
of foraging effort (Brown, 1988). The test arena was Y-shaped
with one central compartment and three surrounding boxes.
Each of the surrounding boxes contained one of the treatment
materials (control (C) bedding, CAC bedding, or predator odor
(PO) bedding). Each of the three boxes also contained a GUD tray
with 1 liter sand mixed with 3.85 g of millet seeds. The experiment
started by releasing a pup in the central compartment. The
orientation of release was consistent over the different trials, but
the orientation of the treatment boxes varied to avoid spatial bias.
All trials, which were recorded from a birds-eye-view with Go
Pro cameras, lasted 3 h. Upon trial completion, the remaining
seeds were collected and quantified and GUD was determined
for each treatment box. The sand from GUD boxes was sieved
to collect the remaining millet. The sand was dried and aired
for at least 5 days before it was reused in the experiments in
order to minimize odor contaminations. To control for variation
in moisture content, the millet was dried for 12 h at 30C
and then equilibrated to ambient indoor humidity for 24 h
before weighing.
Data Analyses
A single observer (KB) analyzed all videos by recording (1) the
time intervals the voles spent in each GUD tray with the millet
seeds, (2) the latency to enter each treatment box, (3) and the
latency to enter each GUD tray. A vole was defined as being in
a treatment box upon first sight of movement in the box and as
being in a GUD tray when wholly in the tray, ignoring its tail.
We used the latencies to each GUD tray to determine the first
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choice (C, CAC or PO GUD tray) of each vole, and we counted
the number of visits to each treatment box.
It was not possible to record data blind because the three
different treatments (C, CAC, and PO) are visually and olfactorily
easily distinguishable.
Statistical analyses were conducted using R version 3.5.1
(R Core Team, 2020). We used linear mixed models (Bates
et al., 2015) to assess differences among treatments in terms of
giving up density (GUD), foraging speed (grams foraged per
second), time spent in boxes, number of box visits, and the
two latency variables (Supplementary Appendix 1 provides all
models and summary tables). To analyze first choice (i.e., non-
numeric: C, CAC, or PO) data, we used the multinomial function
(Venables and Ripley, 2002). Offspring body mass data were log-
transformed prior to fitting linear mixed models. We used Akaike
information criterion (AICc) for purposes of model selection: all
models <2 AICc units away from the best model were averaged
by AICc weight to determine model estimates (Barton, 2020).
A Tukey HSD post hoc test was carried out for the number of
offspring between the treatments. No other post hoc tests were
used in the analyses. The fixed variables in the full starting
model were maternal treatment, timing of the maternal treatment
(i.e., prenatal or postnatal), box treatment, box entry latency,
sex, and age on test day. The variables maternal treatment and
box treatment were retained in all of the compared models.
To account for repeated measures within individuals and for
possible effects of the litter, the random effects pup-identity vs.
mother-identity over pup-identity were fitted in a full starting
model for each response variable and compared by AICc. The
better performing random effect was then used in all the other
models for a given response variable. In the case of significant
interactions between timing and either maternal treatment or
box treatment, data were split to further analyze the effect. The
same was done with interactions between sex and any other
categorical variable.
RESULTS
Birth Mass and Growth Rate
There were no differences in litter size between maternal
treatments [n= 35 (nC= 9, nCAC = 16, nPO = 10 and nearly = 14,
nlate = 21), early vs. late p= 0.680, C vs. CAC p= 0.664, C vs. PO
p= 0.801, Table 1 and Supplementary Table 1]. An additional
Tukey HSD post hoc test did not show any significant differences
either (p>0.05 for all comparisons). The treatments did not
TABLE 1 | Distribution of pups over the six different maternal treatments.
Prenatal Postnatal
Treatment C CAC PO C CAC PO
Litters (n) 4 5 3 5 8 4
Pups (n) 13 20 13 26 25 16
Three prenatal groups: C, CAC and PO. Three postnatal groups: C, CAC, and PO.
C, Control, CAC, Conspecific Alarm Cue, PO, Predator Odor.
affect birth mass, but litter size did: each additional pup in a litter
decreased the individual pup birth body mass by 0.07 g (n= 141,
p<0.001, weighted average, Supplementary Table 2).
Pup body mass was influenced by a three-way interaction
between age in days, litter size, and maternal treatment, while the
timing of the treatments (pre- or postnatal) had no significant
effect on its own nor showed significant interactions with the
treatments (n= 35 litters and n= 1316 observations, daylitter
sizematernal CAC p= 0.023, daylitter sizematernal PO
p= 0.00048, Figure 1,Table 2,Supplementary Table 3). A subset
analysis showed that pups from the maternal C treatment from
larger litters started at a disadvantage compared to smaller litters
from this treatment, but this was compensated for over the first
21 days of growth (log values: day = 0.08, litter size = –0.03,
daylitter size = 0.001, Figure 1). In contrast, pups from the
maternal CAC treatment started heavier and showed slightly
faster growth in larger litters (log values: day = 0.08, litter
size = 0.04, daylitter size = 0.0005, Figure 1). In the maternal PO
treatment, pups from larger litters were smaller at birth and grew
more slowly than pups from smaller litters (log values: day = 0.08,
litter size = –0.03, daylitter size = –0.002, Figure 1).
First Choice of the Treatment Box and
the Giving Up Density Tray
Pups from CAC-treated mothers were more likely to enter first
into the CAC-treated box compared to the control box (54%
vs. 14%; n= 113, p= 0.005, weighted average, Supplementary
Table 4). Similarly, pups from CAC-treated mothers were more
likely to enter first into the CAC GUD tray compared to the
control GUD tray (50% vs. 22%; n= 113, p= 0.0194, weighted
average, Supplementary Table 5). No other treatment showed
significant preferences. Treatment timing and sex did not have
a measurable effect in either case.
Visits to the Treatment Boxes
Overall, pups visited the PO treatment box less frequently than
the C treatment box (ca. 7 fewer visits, n= 113, p<0.001,
weighted average, Figure 2 and Supplementary Table 6).
Additionally, pups from CAC-treated mothers made fewer visits
to any treatment box compared to pups from control mothers (ca.
10 fewer visits, n= 113, p= 0.002, weighted average, Figure 2 and
Supplementary Table 6). The number of visits to any treatment
box decreased by 0.43 per day of age (n= 113, p= 0.026,
weighted average, Supplementary Table 6). Female pups made
more visits to any treatment box compared to males (ca. 7.8
visits more, n= 113, p= 0.014, weighted average, Supplementary
Table 6). Visit frequency was unaffected by the timing of the
maternal treatments.
Latency to Enter the Treatment Box and
the Giving Up Density Tray, and the Time
Spent in the Giving up Density Tray
The results for the differences in latency to enter the treatment
box are inconclusive as all models produce singular fit errors.
There were no significant differences in latency times between
PO, CAC, or control GUD trays (Supplementary Table 7).
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FIGURE 1 | Individual pup mass per day and litter size. Different treatments are in facets.
Overall, pups spent less time in the PO GUD tray compared to
the control tray (ca. 492 s less, n= 113, p<0.001, Figure 3
and Supplementary Table 8). Pups from PO-treated mothers
spent more time in any GUD tray compared to pups from
control mothers (ca. 310 s, n= 113, p= 0.039, Figure 3 and
Supplementary Table 8). Time spent in any GUD tray decreased
by 18 s per day of age (n= 113, p= 0.004, Supplementary
Table 8). Treatment timing and sex did not have statistically
significant effects on time spent in GUD trays.
Giving-Up-Densities and Foraging Speed
The interaction between maternal treatment and treatment
timing significantly influenced GUD in the CAC and PO
treatment, although not in the controls (Figure 4). Pups
from prenatal control mothers left 3.65 g of millet per
liter of sand. Compared to pups from mothers exposed
prenatally to the same treatment, pups from mothers treated
postnatally with CAC foraged 0.11 g more, and pups from
mothers treated postnatally with PO foraged 0.10 g more
(i.e., lower GUD following post-natal exposure, Figure 4,
n= 113, p= 0.006 and p= 0.029, respectively, weighted
model average, Supplementary Table 9). Among pups from
postnatally-treated mothers, pups from CAC-treated mothers
foraged significantly more (by ca. 0.09 g) than pups from
control mothers, which left ca. 3.69 g of millet per liter of
sand (n= 67, p= 0.021, weighted model average, Figure 4 and
TABLE 2 | Model summary of the offspring growth.
Growth (g) of the offspring
(Model averages)
Predictors Estimates (log) Std. error p
(Intercept) 0.881 0.098 <0.001
Treatment (CAC) 0.192 0.129 0.136
Treatment (PO) 0.055 0.136 0.684
Day 0.073 0.004 <0.001
Litter size 0.046 0.019 0.015
Day: Treatment (CAC) 0.010 0.006 0.076
Day: Treatment (PO) 0.022 0.007 0.001
Litter size: Treatment (CAC) 0.075 0.027 0.005
Litter size: Treatment (PO) 0.020 0.026 0.451
Day: Litter size 0.003 0.001 0.001
Day: Litter size: Treatment (CAC) 0.003 0.001 0.023
Day: Litter size: Treatment (PO) 0.005 0.001 <0.001
Timing (postnatal) 0.017 0.046 0.716
Nmother 35
Observations 1,316
Results represent averaged (by AICc) estimates on the log-scale.
P-values in bold are significant at p <0.05.
Supplementary Table 10) while no difference was observed for
pups from PO-treated mothers (n= 67, p= 0.021, weighted
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FIGURE 2 | Number of visits in the treatment boxes, split by maternal treatments. Asterisks indicate a significant difference from C at p<0.01 (**) and p<0.001 (***).
model average, Figure 4 and Supplementary Table 10). Among
pups from prenatally-treated mothers, no significant differences
in GUD were observed between the maternal treatments (n= 46,
p>0.05, Supplementary Table 11). Overall, foraging box
treatment did not significantly influence GUD. The foraging
speed, grams per second, does not differ significantly between
any treatments (n= 64, p>0.05 for all comparisons,
Supplementary Table 12).
DISCUSSION
The current study contributes to a growing understanding
about how olfactory cues related to predation risk can
have transgenerational impacts on behavior and physiology
(Sievert et al., 2019, 2020). Our main focus here was to investigate
to what extent pre- vs. postnatal exposure to predator odor
and CAC affects offspring differently. While we found only
limited evidence for such differential effects in bank voles,
our study suggests that odor cue origin (i.e., from predators
vs. from conspecifics) matters in ways that interact with
other factors that are unrelated to exposure timing. For
example, pups from predator-odor-treated mothers grew faster
in smaller litters, while the opposite (faster growth in bigger
litters) was found for pups from mothers treated with CAC.
Additionally, we found that vole pups showed an innate
avoidance of predator odor regardless of maternal exposure,
but pups from mothers treated with CAC showed signs of
preference for that cue. This apparent mismatch shows that
both physiological and behavioral adaptations need to be
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FIGURE 3 | Time spent in the GUD trays, split by maternal treatments. Asterisks indicate a significant difference from C at p<0.01 (**) and p<0.001 (***).
considered together as only looking at one aspect might skew
the interpretations.
We found limited differences between in anti-predator
behavior in relation to the timing of predation odor cue exposure
of mothers. In fact, only foraging was affected by treatment
timing. However, the increased consumption by pups from
postnatally exposed mothers compared to those from prenatally
treated mothers, ca. 19 millet seeds (ca. 0.0052 g per seed), likely
represents an ecologically meaningful change in the foraging
behavior. Interestingly we did not see a similar pattern when it
comes to the time spent in the GUD trays. There pups from PO-
treated mothers spent about 310 s more time in them, compared
to pups from control-treated mothers. We further found that
foraging speed is independent of any of our treatments. Previous
studies have explored different aspects of early life exposure to
a potential stressor, highlighting possible mechanisms such as
maternal care (Bauer et al., 2015) and differences in the milk
composition (Brummelte et al., 2010), or the adaptive aspects
of the resulting changes in offspring physiology and behavior
(Patacchioli et al., 2002;Dias and Ressler, 2014). However, the
timing during which the early life stressor has to occur to trigger
these changes remains largely obscure.
Pups in bigger litters from mothers treated with CAC and pups
in smaller litters from mothers treated with predator odor both
gained mass faster compared to the control. These drastically
opposite result hint that different reproductive strategies are
associated with different perceived risks that are communicated
by different odor cues, as previously suggested (Sievert et al.,
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Sievert et al. Bank Vole Early Life Fear
FIGURE 4 | Boxplots showing the GUD in gram split by the different maternal treatments and treatment timing (pre- and postnatal). Asterisks indicate a significant
difference at p<0.05 (*) and p<0.01 (**).
2019). CAC exposure might trigger a form of terminal investment
with a focus on larger litters; predator odor might trigger
investment in a few high quality offspring. Specifically, we saw
that offspring in the largest litters from the maternal CAC
treatment weighted on average 14 g, while individuals from
the smallest litters were only about 11 g (Figure 1). This
difference was even more pronounced in the maternal predator
odor treatment where now individuals from the smallest litters
were about 15.5 g and pups in the biggest litters weighted
only 9 g. Only male voles were used to generate the CAC,
and this sex specificity may underlie the cue’s mechanism
of action. For example, exposure might communicate more
about the unfamiliar conspecific male (Eccard et al., 2017)
than about the predator that male encountered. While this
possibility cannot be ruled out in the current study, previous
work in bank voles revealed different reactions to the CAC
produced by predator exposed male voles and the “normal”
odors produced by unexposed male voles (Haapakoski et al.,
2018;Sievert et al., 2019). Furthermore, we have recently
analyzed possible molecular candidates for alarm substances in
the bank vole (Sievert, 2020) showing that differences in chemical
odor composition are only caused by the treatments (i.e., no
handling, handling, and weasel exposure) and show no significant
differences based on the sex of the individual.
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A strong tendency to avoid boxes treated with predator odor
compared to boxes treated with the CAC was found for pups from
both maternal treatments (i.e., PO and CAC) at both time points
(i.e., pre- and postnatally). This avoidance took the form of fewer
visits to and less time spent in boxes treated with predator odor.
Because this effect was independent of the maternal treatments,
voles seemed to innately avoid areas that were possibly
dangerous. Other species exhibit similar avoidance responses
when predator-naïve individuals are confronted with predator
odors, e.g., fathead minnows (Pimephales promelas) (Mathis and
Smith, 2008), European rabbits (Oryctolagus cuniculus) (Monclús
et al., 2005), mice (Sievert and Laska, 2016), and Microtus voles
(Calder and Gorman, 1991;Bolbroe et al., 2000;Borowski, 2011).
However, these studies and our current results contradict the
conclusions that areas with predator odor only are not avoided
by foraging individuals because odor alone might not be a
sufficiently reliable cue (Orrock et al., 2004;Powell and Banks,
2004). Furthermore, some studies suggest that predator odor cues
are actually attractive and trigger intensive investigative behavior
(Garvey et al., 2016;Parsons et al., 2018). These contradictory
outcomes might result from mismatched predator odors and prey
species, as not all predator odors might evoke the same reaction
in prey species, or from differences in individual sensitivity to
the odor (Apfelbach et al., 2005); however, in general odors are
important and reliable chemosensory cues for assessing predation
risk as predator odors not only reveal predator presence but can
also signal predator activity patterns and diet (Kats and Dill,
1998). However, for this study a mismatch between predator and
prey can be excluded.
Opposite to avoidance of predator odor, pups from mothers
treated with CAC tended to go first into CAC-treated box.
Early exposure to odors can alter development of the olfactory
bulb and induce preferences for biologically important odors
(Todrank et al., 2011). For example, mice exhibit enhanced
sensitivity to olfactory cues experienced by their parents (Dias
and Ressler, 2014), and a variety of young mammals (e.g., mice,
rats, and humans) show preferences for individually distinctive
odors associated with their mothers and their mother’s diet
(Todrank et al., 2011). While these studies did not consider
odors related to predation, they identify potential mechanisms
linking maternal olfactory experience with offspring perception
and response. Our study hints that these mechanisms might
show a degree of odor specificity when it comes to risk
cues, thereby highlighting differences between odors produced
directly by predators and those odors produced by conspecifics
having experienced predator threat (i.e., “second-hand” cues;
Bian et al., 2005). While the predator-produced odor may
carry a direct signal of danger, odors produced by conspecifics
may need to be inspected more thoroughly, perhaps due
to the complexity of their information content composition.
Despite these differences, exposure to either odor may well
influence the fitness of cue recipients (Bestion et al., 2014;
Koyama et al., 2015).
Our study documents the relative strength of postnatal cues;
this strength may result from the fact these cues can work via
several mechanisms that may be additive. For example, if a
mother is exposed to cues indicating predator presence (i.e.,
either predator odor or CAC) while outside of her nest (e.g.,
while foraging), she communicates that risk to her pups through
changes in her body odors, her behavior, her milk, or some
combination of these. Both the direct predator odor cue and the
indirect CAC seem to shape the behavior of offspring, but the
timing of exposure, pre- vs. postnatal, played only a minor role.
While our results are mixed, our work highlights the need for
more fine-tuned approaches to investigate how and when cross-
generational effects are triggered in wild animals and in what
ways these effects influence fitness. Moreover, the conditions
under which the CAC becomes attractive, rather than repellant,
warrants further study.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be
found in online repositories. The names of the
repository/repositories and accession number(s) can be
found below: doi: 10.6084/m9.figshare.11185868 and doi:
10.6084/m9.figshare.11186600.
ETHICS STATEMENT
The animal study was reviewed and approved by the Etelä-
Suomen aluehallintovirasto (ESAVI/6370/04.10.07/2014).
AUTHOR CONTRIBUTIONS
TS, MH, and HY designed and planned the study. TS and KB
collected the data and analyzed it. TS, KB, MH, KM, and HY
wrote the manuscript. All authors contributed to the article and
approved the submitted version.
FUNDING
This study was supported by the Finnish Academy Research grant
for HY (Grant No. 288990, 11.5.2015).
ACKNOWLEDGMENTS
We would like to thank the staff of the Konnevesi Research
Station for their technical help, and Olga Ylönen and Teemu
Käpylä for assistance in the laboratory.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fevo.2021.
709207/full#supplementary-material
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Thesis
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Predator-prey interactions are a major evolutionary driver, affecting not only the direct mortality of prey species, but also their behaviours and reproduction. Prey species behavioural adaptations aim to mitigate the effects of predation and to maximise survival and individual fitness. These adaptations include the ability to signal a threat to conspecifics, e.g. via alarm calls or alarm secretions, or to detect predator presence via odours. In this thesis, I studied the effects of predator odours and conspecific alarm secretions on behaviour and reproduction bank voles (Myodes glareolus), a small mammal species inhabiting boreal forests. My work focused on three major points in comparing the direct predator cue and indirect conspecific cue: first, how the reproductive behaviour is affected by the predator odour or alarm pheromone, second, whether there are transgenerational effects and how they are exhibited in offspring, and third, what the chemical nature of these alarm secretions is. I conducted four experiments, which included both trials in semi-natural enclosures and under controlled laboratory conditions. I found evidence that exposure to conspecific alarm secretions causes a shift in voles’ reproductive behaviour, switching towards terminal investment. This became apparent with an increase in parturitions and an increased growth rate in larger litters, which did not occur when exposed to predator odour. I also found evidence of transgenerational effects, which affect aspects of the offspring’s exploratory and foraging behaviour. Additionally, I discovered that these behavioural effects are context-dependent and do not occur in every environment. Lastly, I identified a group of chemicals from voles’ alarm secretion, which are likely to be responsible for the observed effects. The results of my thesis fill a knowledge gap concerning chemical communication in mammals, and help to further understand the implications of predator presence on prey behaviour and reproduction.
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In the predator–prey arms race, survival-enhancing adaptive behaviors are essential. Prey can perceive predator presence directly from visual, auditory, or chemical cues. Non-lethal encounters with a predator may trigger prey to produce special body odors, alarm pheromones, informing conspecifics about predation risks. Recent studies suggest that parental exposure to predation risk during reproduction affects offspring behavior cross-generationally. We compared behaviors of bank vole (Myodes glareolus) pups produced by parents exposed to one of three treatments: predator scent from the least weasel (Mustela nivalis nivalis); scent from weasel-exposed voles, i.e., alarm pheromones; or a control treatment without added scents. Parents were treated in semi-natural field enclosures, but pups were born in the lab and assayed in an open-field arena. Before each behavioral test, one of the three scent treatments was spread throughout the test arena. The tests followed a full factorial design (3 parental treatments × 3 area treatments). Regardless of the parents’ treatment, pups exposed to predator odor in the arena moved more. Additionally, pups spend more time in the center of the arena when presented with predator odor or alarm pheromone compared with the control. Pups from predator odor–exposed parents avoided the center of the arena under control conditions, but they spent more time in the center when either predator odor or alarm pheromone was present. Our experiment shows that cross-generational effects are context-sensitive, depending on the perceived risk. Future studies should examine cross-generational behavioral effects in ecologically meaningful environments instead of only neutral ones. Significance statement We exposed bank voles to odors signaling predation risk to assess the effects parental predation exposure on the behavior of their offspring. Besides predator odor, we also assessed the role of a conspecific alarm cue as a novel way of spreading the predation risk information. Pup behaviors were assessed in the open-field arena, a standard way of assessing animal behavior in a wide range of contexts. We found that also alarm pheromone increased the time pups spend in the center of the arena similarly to predator odor. While previous studies suggested that offspring would be more fearful, our results indicate that the cross-generational effects are very context-dependent; i.e., they differ significantly depending on which scent cue is presented in the open-field arena. This shows the need for better tools or measurements to translate laboratory results into ecologically meaningful frameworks.
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Risk recognition by prey is of paramount importance within the evolutionary arms race between predator and prey. Prey species are able to detect direct predator cues like odors and adjust their behavior appropriately. The question arises whether an indirect predation cue, such as the odor of scared individuals, can be detected by conspecifics and subsequently affects recipient behavior. Parents may also transfer their experience with predators to their offspring. In two experiments, we assessed how direct and indirect predation cues affect bank vole (Myodes glareolus) foraging behavior, reproduction, and pup fitness. Weasel (Mustela nivalis) odor served as the direct cue, whereas the odor of weasel‐scared conspecifics, alarm pheromones, was used as an indirect cue and both of those were compared to a control odor, clean wood shavings. Alarm pheromones attracted female voles, measured as time in proximity to the treatment and foraging. Both predator odor and alarm pheromones enhanced reproduction compared to the control odor. Females treated with alarm pheromone had significantly higher pregnancy rates, and pups from predator‐treated mothers were significantly heavier at birth. Our study provides two novel ideas. First, the impact of a predator can be socially transmitted. Second, predation risk likely triggers terminal investment in reproduction.
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Abstract Predation involves more than just predators consuming prey. Indirect effects, such as fear responses caused by predator presence, can have consequences for prey life history. Laboratory experiments have shown that some rodents can recognize fear in conspecifics via alarm pheromones. Individuals exposed to alarm pheromones can exhibit behavioural alterations that are similar to those displayed by predator-exposed individuals. Yet the ecological and evolutionary significance of alarm pheromones in wild mammals remains unclear. We investigated how alarm pheromones affect the behaviour and fitness of wild bank voles (Myodes glareolus) in outdoor enclosures. Specifically, we compared the effects of exposure of voles living in a natural environment to a second-hand fear cue, bedding material used by predator-exposed voles. Control animals were exposed to bedding used by voles with no predator experience. We found a ca. 50% increase in litter size in the group exposed to the predator cue. Furthermore, female voles were attracted to and males were repelled by trap-associated bedding that had been used by predator-exposed voles. Movement and foraging were not significantly affected by the treatment. Our results suggest that predation risk can exert population-level effects through alarm pheromones on prey individuals that did not encounter a direct predator cue.
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Maternal stress can prenatally influence offspring phenotypes and there are an increasing number of ecological studies that are bringing to bear biomedical findings to natural systems. This is resulting in a shift from the perspective that maternal stress is unanimously costly, to one in which maternal stress may be beneficial to offspring. However, this adaptive perspective is in its infancy with much progress to still be made in understanding the role of maternal stress in natural systems. Our aim is to emphasize the importance of the ecological and evolutionary context within which adaptive hypotheses of maternal stress can be evaluated. We present five primary research areas where we think future research can make substantial progress: (1) understanding maternal and offspring control mechanisms that modulate exposure between maternal stress and subsequent offspring phenotype response; (2) understanding the dynamic nature of the interaction between mothers and their environment; (3) integrating offspring phenotypic responses and measuring both maternal and offspring fitness outcomes under real-life (either free-living or semi-natural) conditions; (4) empirically testing these fitness outcomes across relevant spatial and temporal environmental contexts (both pre- and post-natal environments); (5) examining the role of maternal stress effects in human-altered environments-i.e., do they limit or enhance fitness. To make progress, it is critical to understand the role of maternal stress in an ecological context and to do that, we must integrate across physiology, behavior, genetics, and evolution.
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Pregnancy termination after encountering a strange male, the Bruce effect, is regarded as a counterstrategy of female mammals towards anticipated infanticide. While confirmed in caged rodent pairs, no verification for the Bruce effect existed from experimental field populations of small rodents. We suggest that the effect may be adaptive for breeding rodent females only under specific conditions related to populations with cyclically fluctuating densities. We investigated the occurrence of delay in birth date after experimental turnover of the breeding male under different population composition in bank voles (Myodes glareolus) in large outdoor enclosures: one-male–multiple-females (n = 6 populations/18 females), multiple-males–multiple-females (n = 15/45), and single-male–single-female (MF treatment, n = 74/74). Most delays were observed in the MF treatment after turnover. Parallel we showed in a laboratory experiment (n = 205 females) that overwintered and primiparous females, the most abundant cohort during population lows in the increase phase of cyclic rodent populations, were more likely to delay births after turnover of the male than year-born and multiparous females. Taken together, our results suggest that the Bruce effect may be an adaptive breeding strategy for rodent females in cyclic populations specifically at low densities in the increase phase, when isolated, overwintered animals associate in MF pairs. During population lows infanticide risk and inbreeding risk may then be higher than during population highs, while also the fitness value of a litter in an increasing population is higher. Therefore, the Bruce effect may be adaptive for females during annual population lows in the increase phases, even at the costs of delaying reproduction. Electronic supplementary material The online version of this article (doi:10.1007/s00442-017-3904-6) contains supplementary material, which is available to authorized users.
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Maternal effects can provide offspring with reliable information about the environment they are likely to experience, but also offer scope for maternal manipulation of young when interests diverge between parents and offspring. To predict the impact of parent-offspring conflict, we model the evolution of maternal effects on local adaptation of young. We find that parent-offspring conflict strongly influences the stability of maternal effects; moreover, the nature of the disagreement between parents and young predicts how conflict is resolved: when mothers favor less extreme mixtures of phenotypes relative to offspring (i.e., when mothers stand to gain by hedging their bets), mothers win the conflict by providing offspring with limited amounts of information. When offspring favor overproduction of one and the same phenotype across all environments compared to mothers (e.g., when offspring favor a larger body size), neither side wins the conflict and signaling breaks down. Only when offspring favor less extreme mixtures relative to their mothers (something no current model predicts), offspring win the conflict and obtain full information about the environment. We conclude that a partial or complete breakdown of informative maternal effects will be the norm rather than the exception in the presence of parent-offspring conflict. This article is protected by copyright. All rights reserved.
Article
Significance Maternal stress during gestation causes numerous effects on infant physiology that extend well into adulthood. We contribute to the ongoing debate on whether these effects are adaptive outcomes or merely the product of energetic constraints by presenting an integrated hypothesis that predicts the diversity of observed maternal effects on offspring growth, incorporating both theoretical explanations into one coherent framework. Empirical tests of this hypothesis across mammals suggest that the timing of the stressor during gestation and a simultaneous consideration of maternal investment and adaptive growth plasticity effects are crucial for a full comprehension of prenatal stress effects on offspring growth. The results support an adaptive life history perspective on maternal effects that is relevant for evolutionary biology, medicine, and psychology.