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Thorbjörn Sievert
JYU DISSERTATIONS 211
Indirect and Transgenerational
Effects of Predation Risk
Predator Odour and Alarm
Pheromones in the Bank Vole
JYU DISSERTATIONS 211
Thorbjörn Sievert
Indirect and Transgenerational
Effects of Predation Risk
Predator Odour and Alarm
Pheromones in the Bank Vole
Esitetään Jyväskylän yliopiston matemaas-luonnoneteellisen edekunnan suostumuksella
julkises tarkasteavaksi huhkuun 23. päivänä 2020 kello 14.
Academic dissertaon to be publicly discussed, by permission of
the Faculty of Mathemacs and Science of the University of Jyväskylä,
on April 23, 2020 at 14 o’clock.
JYVÄSKYLÄ 2020
Editors
Jari Haimi
Department of Biological and Environmental Science, University of Jyväskylä
Ville Korkiakangas
Open Science Centre, University of Jyväskylä
ISBN 978-951-39-8135-8 (PDF)
URN:ISBN:978-951-39-8135-8
ISSN 2489-9003
Cover picture by Kersn Sievert.
Copyright © 2020, by University of Jyväskylä
Permanent link to this publicaon: hp://urn./URN:ISBN:978-951-39-8135-8
ABSTRACT
Sievert, Thorbjörn
Indirect and Transgenerational Effects of Predation Risk: Predator Odour and
Alarm Pheromones in the Bank Vole
Jyväskylä: University of Jyväskylä, 2020, 44 p.
(JYU Dissertations
ISSN 2489-9003, 211)
ISBN 978-951-39-8135-8 (PDF)
Yhteenveto: Petoriskin epäsuorat ja sukupolvien väliset vaikutukset: pedon haju
ja hälytysferomonit metsämyyrä–lumikko –suhteessa
Diss.
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.
Keywords: Behaviour; chemical communication; cross-generational effect; fear
effect; predator-prey-interaction; terminal investment.
Thorbjörn Sievert, University of Jyväskylä, Department of Biological and Environmental
Science, P.O. Box 35, FI-40014 University of Jyväskylä, Finland
TIIVISTELMÄ
Sievert, Thorbjörn
Petoriskin epäsuorat ja sukupolvien väliset vaikutukset: pedon haju ja hälytys-
feromonit metsämyyrä–lumikko –suhteessa
Jyväskylä: Jyväskylän yliopisto, 2020, 44 p.
(Jyväskylä Studies in Biological and Environmental Science
ISSN 2489-9003, 211)
ISBN 978-951-39-8135-8 (PDF)
Yhteenveto: Petoriskin epäsuorat ja sukupolvien väliset vaikutukset: pedon haju
ja hälytysferomonit metsämyyrä–lumikko –suhteessa
Diss.
Pedon ja saaliin evolutiivinen kilpajuoksu ilmenee kahtaalla, saaliin kuollei-
suutena sekä selvinneen saaliin käyttäytymisen ja lisääntymisen muutoksina, jotka
parantavat sen hengissä säilymistä ja kelpoisuutta. Saalis pystyy välttämään
pedon, jos se havaitsee sen. Saaliseläin voi aistia pedon suoraan sen jättämistä
ärsykkeistä, kuten äänistä tai hajuista, tai epäsuorasti varoituksena muilta oman
lajin yksilöiltä. Väitöstutkimukseni keskittyi petoriskisignaalien, pedon hajun ja
petoriskille altistuneen oman lajin yksilön hajun, vaikutuksiin saaliin käyttäyty-
miseen ja lisääntymiseen. Tutkimuslajini olivat maamme metsien yleinen jyrsijä,
metsämyyrä, ja sen merkittävä spesialistipeto, lumikko. Tutkimukseni neljä osa-
tutkimusta keskittyivät kolmeen pääkysymykseen: miten suora pedon haju tai
epäsuora, lajitoverin kautta tuleva hälytyshaju vaikuttaa metsämyyrän lisään-
tymiseen; onko vanhempien, etenkin äidin, altistumisella korkeaan petoriskiin
vaikutuksia raskausajan tai imetyksen kautta poikasten pedonvälttämiskäyttäy-
tymiseen; sekä mitkä kemialliset yhdisteet olisivat hälytysferomonin välittämän
informaation taustalla. Metsämyyrän lisääntyminen ja käyttäytyminen muuttui-
vat suoran ja epäsuoran petoärsykkeen vaikutuksesta. Hälytysferomoni näytti
muuttavan myyrän lisääntymisstrategiaa siten, että naaras lisääntyi tehokkaam-
min. Tämä näkyi niin lisääntyvien yksilöiden määrässä kuin poikasten kasvu-
nopeudessakin, etenkin isoissa poikueissa. Ilmiö on yhteensopiva niin sanotun
”terminaalivaiheen investointi” -hypoteesin kanssa. Tutkimukseni vahvisti myös
mahdollisten sukupolvien välisten petoriskivaikutusten olemassaolon. Lisäksi
osoitin, että erityisesti kahdella yhdisteellä, 2- ja 1-oktanolilla, saattaisi olla mer-
kittävä rooli petoriskin kemiallisessa signaloinnissa, sekä todensin, että oletus-
temme mukaisesti hälytysferomonin vaikutus luonnossa oli lyhytaikainen.
Avainsanat: Hajuainekommunikaatio; hälytysferomoni; käyttäytyminen; lisäänty-
misstrategiat; peto-saalis –suhde; sukupolvien väliset vaikutukset.
Thorbjörn Sievert, Jyväskylän yliopisto, Bio- ja ympäristötieteiden laitos PL 35, 40014
Jyväskylän yliopisto
Author’s address Thorbjörn Sievert
Department of Biological and Environmental Science
P.O. Box 35
FI-40014 University of Jyväskylä
Finland
thorbjorn.t.sievert@jyu.fi
Supervisors Professor Hannu Ylönen
Department of Biological and Environmental Science
P.O. Box 35
FI-40014 University of Jyväskylä
Finland
Docent Marko Haapakoski
Department of Biological and Environmental Science
P.O. Box 35
FI-40014 University of Jyväskylä
Finland
Reviewers Assistant Professor Michael Sheriff
Department of Biology
285 Old Westport Road
MA 02747-2300, Dartmouth
United States of America
Associate Professor Zbigniew Borowski
Department of Forest Ecology
3, Braci Leśnej Str. Sękocin Stary
05-090 Raszyn
Poland
Opponent Professor Anders Angerbjörn
Department of Zoology
Zoologiska institutionen: Ekologi
106 91 Stockholm
Sweden
CONTENTS
LIST OF ORIGINAL PUBLICATIONS ........................................................................ 7
1 INTRODUCTION ................................................................................................... 9
1.1 The importance of predator-prey interactions ........................................... 9
1.2 Detecting predator presence ......................................................................... 9
1.3 Adapting to increased risk of predation ................................................... 10
1.3.1 Adaptations: an overview ................................................................ 10
1.3.2 Signalling conspecifics ...................................................................... 11
1.3.3 Behavioural adaptations ................................................................... 12
1.3.4 Transgenerational effects .................................................................. 12
1.4 The boreal model-system: Bank voles and weasels ................................. 13
1.5 Aim of the thesis ........................................................................................... 14
2 METHODS ............................................................................................................. 15
2.1 Husbandry ..................................................................................................... 15
2.2 Field enclosures ............................................................................................. 16
2.3 Odour cues ..................................................................................................... 16
2.3.1 Odour preparation ............................................................................. 16
2.3.2 Presentation ........................................................................................ 16
2.4 Laboratory behaviour trials ........................................................................ 17
2.5 Experimental procedures ............................................................................ 17
3 RESULTS AND DISCUSSION ............................................................................ 19
3.1 An overview .................................................................................................. 19
3.2 Reproductive adaptations to weasel odour and alarm pheromone
(chapters I and III) ................................................................................................. 20
3.3 Transgenerational effects (chapters II and III) .......................................... 21
3.4 Timing of the exposure (study III) ............................................................. 22
3.5 Identification of vole alarm pheromone (study IV) ................................. 22
4 CONCLUSIONS .................................................................................................... 24
Acknowledgements .......................................................................................................... 26
YHTEENVETO (RÉSUMÉ IN FINNISH) .................................................................. 28
ZUSAMMENFASSUNG (RÉSUMÉ IN GERMAN)................................................. 30
REFERENCES ................................................................................................................ 33
LIST OF ORIGINAL PUBLICATIONS
The thesis is based on the following original papers, which will be referred to in
the text by their Roman numerals I–IV.
I Sievert T, Haapakoski M, Palme R, Voipio H, Ylönen H 2019. Secondhand
horror: effects of direct and indirect predator cues on behavior and
reproduction of the bank vole. Ecosphere 10:e02765.
https://doi.org/10.1002/ecs2.2765
II Sievert T, Kerkhoven A, Haapakoski M, Matson KD, Ylönen O, Ylönen H
2020. In utero behavioral imprinting to predation risk in pups of the bank
vole. Behavioural Ecology and Sociobiology 74:13.
https://doi.org/10.1007/s00265-019-2791-8
III Sievert T, Bouma K, Haapakoski M, Matson KD, Ylönen H 2020. Pre- and
postnatal fear shape offspring anti-predatory behaviour in the Bank Vole.
Submitted manuscript.
IV Sievert T, Ylönen H, Blande J, Saunier A, van der Hulst D, Ylönen O,
Haapakoski, M 2020. Mammalian alarm pheromone chemistry and effects
in the field. Manuscript.
The table shows the contributions to the original papers.
Chapter I II III IV
Original idea HY, MH, TS MH, TS, HY TS, HY, MH TS, MH, HY, JB
Data collection TS, HV TS, AK, OY TS, KB TS, OY, DH
Analyses TS, HV, RP TS, AK TS, KB TS, AS
Writing TS, HY, MH TS, HY, MH, TS, HY, MH, TS, MH, HY, AS
KM, AK KM, KB
TS = Thorbjörn Sievert, MH = Marko Haapakoski, HY = Hannu Ylönen, HV = Helinä Voipio,
RP = Rupert Palme, AK = Arjane Kerkhoven, OY = Olga Ylönen, KM = Kevin Matson,
KB = Kerstin Bouma, JB = James Blande, DH = Dave van der Hulst, AS = Amélie
Saunier
1.1 The importance of predator-prey interactions
Research on the interplay between predators and their prey has intrigued
biologists for decades, leading to a constant refinement of models and theories
about population dynamics and evolutionary adaptations throughout the 20th
century (Berryman 1992). Earlier work focused on direct mortality, i.e. prey
animals being consumed by predators and how this shapes population dynamics
and fluctuations (Paine 1966, Taylor 1984b, Krebs et al. 1995). A secondary focus
has been how and to what extent the evolutionary arms race, i.e. the coevolution
of prey to escape and the predators means to increase successful hunts, is a major
driver of both predator and prey evolution (Abrams 1986, 2000, Yoshida et al.
2003). However, in the last decades, a stronger focus was put on predation-risks
effects, as opposed to mere survival, e.g. reduced fitness or reproduction in prey
after an increase in perceived predation risk (Taylor 1984a, Lima 1998, Creel and
Christianson 2008). The research on these indirect effects became an intensely
studied topic in the last decades, leading to a variety of catchy names such as
“cost of fear” (Stankowich and Blumstein 2005) or “ecology of fear” (Brown
1999). While direct consumption by a predator seems like the major effect at first
glance, experiments have shown that predation-risk effects can have similar
effects on prey survival and fitness (Schmitz et al. 1997, Nelson et al. 2004, Pangle
et al. 2007).
1.2 Detecting predator presence
It is essential for a prey species to reliably detect the presence of a predator, as a
wrong choice might be fatal. A failure to recognize a predator can lead to the
individual’s deaths but interpreting an unrelated cue as an increase in predation
risk can affect e.g. foraging and long-term fitness. Predator cues can be
categorized into two main groups, “direct” and “indirect”. However, depending
1 INTRODUCTION
10
on the study, it varies what these terms refer to exactly. As an example, some
studies refer to the environment, e.g. availability of cover or moonlight, as an
indirect effect (Orrock et al. 2004), while others refer to conspecific cues (Barrera
et al. 2011). In order to avoid confusion, I will use a definition based on the origin
of the cue: direct cues originate from the predator, while indirect cues originate
from the prey, as a reaction to a direct cue.
Several studies have concentrated on how reliable different cues are. There
is no consensus, as the reliability seems to vary between systems and research
questions. The main points of disagreement are how reliable chemical cues are
(Bourdeau 2010, Barrera et al. 2011, Parsons et al. 2018), and the reliability of direct
compared to indirect cues (Hare and Atkins 2001, Orrock et al. 2004, Morrison
2011, Barrera et al. 2011). The latter is complicated further by the aforementioned
differing definitions.
Prey species can employ all their senses to detect these cues, and for most
senses both direct and indirect cues have been studied. A direct and very reliable
cue of predation risk is a tactile cue, i.e. a direct attack by a predator. Depending
on the predator-prey pairing, the prey’s survival chances can increase with prior
information about the risk (Mirza and Chivers 2001, Chivers et al. 2002), being
distasteful (Halpin and Rowe 2016), or by signalling its own fitness, e.g. by loudly
singing during a pursuit (Cresswell 1994). Visual cues can either be direct, i.e.
seeing the approaching predator, or indirect. Indirect cues include the flight
response of conspecifics (Lima 1994, Wong et al. 2005) or more aggressive
responses such as mobbing (Dominey 1983, Graw and Manser 2007). Auditory
cues include predator calls or noise while moving through the environment, both
a direct cue (Morrison 2011), but also alarm calls of con- or heterospecifics, an
indirect cue (Schmidt et al. 2008, Forti et al. 2017). Direct chemical cues can consist
of the body odour, faeces, urine or marking secretions of predators (Apfelbach et
al. 2005, Parsons et al. 2018). Indirect cues include an array of alarm secretions
originating from the prey (von Frisch 1938, Verheggen et al. 2010), which will be
further explained in the next section.
1.3 Adapting to increased risk of predation
1.3.1 Adaptations: an overview
Predation can be observed across all taxa. Due to its importance in shaping
populations and driving evolutionary adaptations, a variety of antipredator
defences can be found in prey (Freeland 1991, Caro 2005). Antipredator
adaptations can be morphological, physiological or behavioural. These
adaptations can be constitutive, e.g., quills in porcupines (Cho et al. 2012, Mori et
al. 2014) or thorns in plants (Hanley et al. 2007), or inducible when predation risk
is high, e.g., morphological changes in daphnia (Grant and Bayly 1981), growth
and morphology in some tadpoles (Relyea 2004), and a wide range of behavioural
adaptations (Sih 1992, Lima 1995, 2009, Bell 2004, Valeix et al. 2009). In
11
environments with consistently elevated risk levels or when the fitness costs are
negligible, constitutive defences are favoured. The opposite applies for induced
defences (Tollrian and Harvell 1999, Trussell and Nicklin 2002, Heil 2002, Riessen
2012). The cost of these investments can be in the form of negatively affected
breeding (Mappes and Ylönen 1997, Fuelling and Halle 2004, Creel et al. 2007),
chronically elevated stress levels (Sheriff et al. 2009), or decreased fitness in the
individuals or their offspring (Pangle et al. 2007, Sheriff et al. 2009, Trebatická et
al. 2012, Dudeck et al. 2018). In extreme conditions, prey can show chronic stress
induced disorders, with similar consequences as post‐traumatic stress disorders
(Clinchy et al. 2013). For example, chronically elevated stress levels can negatively
affect immunoresponses (Clinchy et al. 2013).
1.3.2 Signalling conspecifics
Prey can also signal to conspecifics the presence of a predator, often via alarm
calls or chemical secretions (Wisenden et al. 2004, Collier et al. 2017). Alarm calls
have been studied in a variety of vertebrates (Schmidt et al. 2008, MacLean and
Bonter 2013, Collier et al. 2017, Barati and McDonald 2017). Their information
content can vary from basic indication of predator presence (Blanchard et al.
1991), or indicating the level of urgency (Townsend et al. 2012) or basic category
of the predator (Manser 2001, Slobodchikoff et al. 2009), to more detailed predator
identification (Ouattara et al. 2009). Alarm calls can not only be used by
conspecifics but also by eavesdropping heterospecifics (Fichtel 2004, Schmidt et
al. 2008).
Indirect scent cues excreted by the prey, often called alarm secretion, alarm
cue, or alarm pheromone, are a well-studied phenomenon, especially in
invertebrates (von Frisch 1938, Bowers et al. 1972, Howe and Sheikh 1975, Heath
and Landolt 1988). Among the first discovered alarm secretions, called
“Schreckstoff” at the time, are those in fish (von Frisch 1938). Fish and other
aquatic species differ in the way how alarm secretions are released from most
terrestrial species, as they are not released via glands but rather are a result of
tissue damage (Smith 2000, Wisenden 2000, Wisenden et al. 2001, Ferrari et al.
2010). While examples of alarm secretions in insects are abundant and well
established (Crewe and Blum 1970, Bowers et al. 1972, Heath and Landolt 1988,
Collins et al. 1989), examples in mammals are from the last few decades and
heavily focused on laboratory rodents (Kiyokawa et al. 2004, Inagaki et al. 2009,
2014, Brechbühl et al. 2013), but see Gomes et al. (2013) with wild Cabrera’s vole
(Microtus cabrerae, Thomas 1906).
Behavioural responses to indirect conspecific alarm cues can differ
drastically from those to direct predator cues. As an example, cat fur odour
commonly elicits avoidance behaviour in rats (Dielenberg and McGregor 2001,
McGregor et al. 2002), but there are several studies where alarm secretions do not
elicits similar behaviour (Kiyokawa et al. 2006, 2013). A possible explanation is
the difference in information or urgency transferred by each cue. While an alarm
secretion does not convey the identity of a predator, a direct predator cue does
(Kiyokawa et al. 2013). However, an avoidance response has been found in
12
Cabrera voles (Gomes et al. 2013). Other physiological and behavioural changes
in response to conspecific alarm cues include changes in analgesic responses
(Kavaliers et al. 2005), and changes in the reproductive strategy (Haapakoski et
al. 2018).
Many assumptions arise surrounding the term ‘pheromone’ . For this work,
I will use a very clear definition: Pheromones allow semiochemical
communication between individuals of the same species, as opposed to
allelochemicals, which facilitate communication between different species (Dicke
and Sabelis 1988, Sbarbati and Osculati 2006). To determine whether the
pheromones used in this work also possess allelochemical properties goes
beyond the scope of the thesis. From here on, the term ‘alarm pheromone’ solely
refers to this definition of intraspecific communication.
1.3.3 Behavioural adaptations
Behavioural adaptations in prey include simple mechanisms such as escape,
freezing, avoidance or heightened vigilance (Wallace and Rosen 2000, Lung and
Childress 2006, Wang and Zou 2017). More complex adaptations are also
observable such as mobbing (Dominey 1983, Graw and Manser 2007) or adaptive
changes in mating behvaviour (Sih 1994, Creel et al. 2007, Adamo and McKee
2017).
While reduced reproduction is often considered a cost, there is an ongoing
debate about its adaptive value and effects on population dynamics (Ruxton and
Lima 1997, Kokko and Ruxton 2000). Until recently, results concerning
reproductive efforts under an increased predation risk have shown a reduced
reproductive investment. This can been seen in a decrease of offspring fitness
(Feng et al. 2015, Owen et al. 2018), reduced breeding (Ylönen 1989, Ylönen and
Ronkainen 1994, Fuelling and Halle 2004, Haapakoski et al. 2012), or both (Sheriff
et al. 2009). An alternative reproductive strategy is to maximize reproductive
efforts when faced with a high-risk situation (henceforth terminal investment).
Individuals following this strategy disregard individual fitness cost in order to
increase or accelerate reproductive outputs. If any offspring reaches maturity, it
compensates the parental loss of fitness or death (Kokko and Ranta 1996, Kokko
and Ruxton 2000). Terminal investment has been shown to occur in both
vertebrates and invertebrates, such as passerine birds and crickets under high
predation risk (Mönkkönen et al. 2009, Adamo and McKee 2017). Furthermore, it
can also be triggered by infections, for example in ants and sparrows (Bonneaud
et al. 2004, Giehr et al. 2017).
1.3.4 Transgenerational effects
Generally, transgenerational effects manifest in offspring after birth, as a result
of changes in the parents’ environment during gestation and/or lactation. In the
case of predator-prey interactions pups whose mothers experienced high
predation risks, exhibit different development or antipredator behaviours
compared to pups whose mothers experienced low risk. In practice, it could
13
mean that being chased or even attacked by a predator while gravid can cause
differences in offspring development or behaviour. A possible mechanism for
such transgenerational effects are maternal hormones, whose production can be
altered by stressful events. Maternal hormones can exert an in utero influence on
the physiology, behaviour, and life history traits of offspring (Caldji et al. 1998,
Love and Williams 2008, Sheriff and Love 2013). This has been demonstrated in
response to a wide variety of external stimuli or stressors, including foot-shocks
(Archer 1973), impoverished environments (Dell and Rose 1987), varied food
quality and social environments (Van Cann et al. 2019b, a), and scent cues
(Champagne and Meaney 2006). Much less is known regarding the role of
paternal factors. However, some studies discovered transgenerational effects
solely mediated by paternal factors (Rodgers et al. 2013, Van Cann et al. 2019a).
A growing body of literature has been accumulated in recent years about
transgenerational effects caused by direct and indirect predation cues. The
transmission of maternal information to in utero offspring has only received
more attention in the last decade. Early work in this field showed how direct
exposure, for instance via injection into the amniotic sac, to a chemical cue shows
later signs of imprinting or conditioning in offspring (Stickrod et al. 1982,
Smotherman 1982a, b). Recent studies found altered learning behaviour in
stickleback offspring (Roche et al. 2012, Feng et al. 2015), altered stress reaction in
the offspring of C57BL/6 mice and Long Evans rats (St-Cyr and McGowan 2015,
St-Cyr et al. 2017), or changed foraging strategies in Sprague Dawley rat offspring
(Chaby et al. 2015).
1.4 The boreal model-system: Bank voles and weasels
Several decades’ worth of research exist on bank voles (Myodes glareolus, Schreber
1780) and on the interaction between voles and the least weasel (Mustela nivalis
nivalis, Linnaeus 1766).
The bank vole is one of the most common small rodents living in a variety
of northern temperate and boreal European forest habitats west of the Urals
(Stenseth 1985). The species is granivorous-omnivorous, with their diet
consisting mainly of seeds and buds, but also of other plant materials or
invertebrates (Hansson 1979, Eccard and Ylönen 2006). The gestation period of
the bank vole is 19–20 days, and the weaning period is three weeks. Vole pups
sexually mature at around 30 days of age. Litter size averages at five to six pups
but ranges between 2 and 10. In Central Finland, where this work was conducted,
bank voles breed three to five times per season, which lasts from May until
September (Mappes et al. 1995a, Koivula et al. 2003). In the wild, bank voles can
live up to two years, but due to high predation rates, estimated survival is one
breeding season (Ostfeld 1985, Macdonald 2006). Bank voles in Fennoscandia
have typically a three- to four-year population density cycle (Hanski et al. 1991,
Hansen et al. 1999). While the importance of food limitation and maternal effects
14
has been shown, predation pressure is considered the strongest driver of these
cycles (Norrdahl and Korpimäki 1995, Boonstra et al. 1998, Huitu et al. 2003).
Bank voles are preyed upon by a diverse predator assemblage, including
least weasels and stoats (Mustela ermine, Linnaeus 1758) (Ylönen 1989, Meri et al.
2008). The least weasel is an especially effective vole hunter due to their size and
excellent hunting skills, least weasels are likely able to kill bank voles whenever
the two species come into direct contact (Tidhar et al. 2007, Haapakoski et al.
2013). Due to their role as specialist predator, the least weasel is considered the
main reason of boreal vole mortality (Korpimäki et al. 1991, Norrdahl and
Korpimäki 1995, 2000). The capabilities of bank voles to detect their mustelid
predators is well established, and voles show a range of behavioural adaptations.
Bank voles shift activity patterns and spatial use in the vicinity of weasels
(Jędrzejewska and Jędrzejewski 1990, Jędrzejewski and Jędrzejewska 1990,
Sundell et al. 2008), decrease their movement and foraging efforts (Ylönen 1989,
Sundell and Ylönen 2004, Bleicher et al. 2018), and use arboreal escape routes
when chased by a weasel (Jędrzejewska and Jędrzejewski 1990, Mäkeläinen et al.
2014).
1.5 Aim of the thesis
The aim of this thesis is to examine the effects of the recently discovered indirect,
conspecific alarm pheromones in bank voles compared to direct predator odour
cues. With a combination of enclosure and laboratory experiments, I hope to
solidify the idea that alarm pheromones in mammals are an important part of the
complex interplay that predator-prey interactions represent. As of today, there
are only a few studies on this subject. While a meta-analysis in search of common
features in vertebrate body odour called for a wider range of study animals (Apps
et al. 2015), the majority of new studies is focused on traditional laboratory
animals. In four chapters, I explore the pheromone’s effects on reproduction (I),
its effect on growth and behaviour of offspring (II, III and IV), the importance of
timing when the parents are exposed to high predation risk (III), and lastly an
isolation and identification of the alarm pheromone (IV).
Chapter I lays the foundation of the present work by verifying the effects of
alarm pheromones in voles, which have just recently been discovered
(Haapakoski et al. 2018), and explores how the new results can be incorporated
into existing findings. Chapters II and III explore different aspects of
transgenerational effects with testing offspring in standardized laboratory
environments to assess their exploratory and foraging decisions. After three
chapters focusing on the behavioural aspects, chapter IV analyses the body
odours of bank voles in order to identify alarm pheromones and provides
additional insights on the effect of alarm pheromones compared to predator
odour in the field (IV).
2.1 Husbandry
All experiments were conducted at the Konnevesi Research Station in Central
Finland (62° 37′ N, 26° 20′ E).
In the laboratory, voles were housed in husbandry rooms under a 16L:8D
light regime with a constant temperature (22 °C ± 1 °C), males and females were
maintained in the same room. All animals were kept individually in 42 cm × 26
cm × 15 cm transparent cages with wire mesh lids and an ad libitum water and
food supply. The bedding materials in each cage consisted of wood shavings and
hay. The breeding adults used in the study were the F1 generation of wild-caught
individuals that were housed in the laboratory during the winter months
preceding the study periods. Winter colonies are formed from the last cohort of
voles of the previous summer. Thus, their age when paired for the first breeding
is around 7 months. The winter population is housed on a short photoperiod
(8L:16D) at around 17 °C throughout the winter and male voles’ testes are
abdominal and female vaginas are closed. Only after adjusting the photoperiod
to long day in spring to prepare for breeding, our voles become reproductive
again. This is done starting from February when the first voles start to breed also
in the field (Haapakoski et al. 2012). All animals were individually marked with
ear tags (#1005-1L1, National Band & Tag Company, Newport, KY, USA). In
experiments where vole pups needed to be individually identified, toe markings
were applied.
Weasels for the odour treatment were housed individually in 60 × 160 × 60
cm cages in an outdoor shelter. Each cage had a nest box, and wood shavings and
hay as bedding. During the experiments, weasels were fed dead bank voles.
2 METHODS
16
2.2 Field enclosures
Large (0.25 ha) outdoor enclosures are situated near Konnevesi municipality. The
enclosures are made of galvanised steel sheet (125 cm height), reaching 50 cm
underground. This prevents the escape of the experimental animals and
intrusion of mammalian predators, while it allows avian predation. Twenty-five
live traps (Ugglan special, Grahnab AB, Hillerstorp, Sweden) were distributed
evenly in a 5 x 5 grid in each enclosure. All traps were covered with trap
chimneys (40 x 40 x 50 cm) made of sheet metal, protecting the traps from the
seasons. Sunflower seeds were used as bait. All enclosures were emptied of
remaining rodents prior to each experiment.
2.3 Odour cues
2.3.1 Odour preparation
A range of odour treatments were used for this thesis. The preparation of the
three most common ones, alarm pheromone, predator odour and control
(Chapters I-III), is detailed in this section.
The predator odour was obtained by collecting used bedding materials,
including faeces, urine, and body odour, from least weasels kept at the research
station.
The alarm pheromone cue was obtained by collecting bedding materials
that were used by male bank voles that were exposed to the weasels on a daily
basis. During the exposure, alarm pheromone “donor voles” were placed inside
a wire mesh live trap, which was placed inside the weasel cage for 60 s. After the
initial exposure, donor voles were placed individually in clean cages with fresh
bedding materials to allow their scents to infuse into the bedding materials. The
scents produced by the weasel-exposed voles were allowed to accumulate in
used bedding materials following successive exposures.
The control odour cue was clean bedding materials without any added cues
from voles or weasels.
In order to minimize variation in odour source, the beddings were
thoroughly mixed before taking samples of bedding with urine and/or faecal
matter. The same was done for the clean bedding. All odour cues were used
within 3 h after being taken from its source. No odour cue was stored for later
use.
2.3.2 Presentation
In in the first experiment, the odour cues were applied directly into a vole’s cage,
where it accumulated (I). As this was not feasible for the following experiments,
which included the odour presentation in the field, we deigned a special
17
treatment chamber, allowing individual manipulation of each captured vole (Fig.
1). The treatment chamber consisted of two compartments, separated by a
perforated wall and could accommodate a live trap. This allowed a more
controlled presentation of the odour cue in both a field and laboratory setting (II
and III).
FIGURE 1 Treatment chamber. The odour cue is separated from the trap compartment by
a mesh to avoid odour contamination. Closing mechanism not shown. Taken
from Chapter II.
2.4 Laboratory behaviour trials
Chapters I – III include behavioural tests conducted in the laboratory. Each test
was recorded with an overhead camera and later analysed by either a human
observer (I and III) or automated software (II) (Noldus et al. 2001).
Chapter II used a standard open field arena containing a single odour cue,
while chapter I and III use either a four-armed (I) or three-armed (III) maze. In
these complex mazes, each arm of the maze was equipped with a different odour
cue and means to assess the foraging behaviour.
2.5 Experimental procedures
Chapter I focuses on the reproductive behaviour of voles presented with different
odour cues. A complementary behavioural experiment served to assess innate
reaction and foraging decision in a forced-choice setting.
Chapter II combines natural mating behaviour and controlled odour cue
treatments in our enclosures in order to assess their offspring. The resulting vole
pups were tested in an open field arena where they encountered the same odour
18
cues. The emphasis of this chapter is on how the treatment of the parents
interacted with the treatment of the offspring.
In chapter III, the focus is on the importance of the timing of the odour
treatment. For this, half the animals were treated from mating until parturition
and the other half from parturition until weaning. The growth of the pups was
monitored, and their exploratory and foraging behaviour was tested.
Chapter IV involves a gas-chromatographical and mass-spectrometric
analysis of the volatile odour compounds excreted by voles in order to identify
their alarm pheromone. For this, voles were either presented to a weasel (similar
with how the alarm pheromone was obtained in the other chapters), handled or
left unstressed.
For this, an individual vole was enclosed in a class chamber for 20 minutes,
directly after each treatment. Two pumps created an airflow of filtered air around
the animals. This airflow was direct through thermal desorption tubes, which
collected all volatile components.
A complementary field test was done in order to assess the effect and
longevity of the alarm pheromone in a natural environment.
3.1 An overview
Many results and conclusions are summarised in this thesis. For the ease of the
reader, I condensed and simplified the main results and conclusions into the table
below (Table 1).
TABLE 1 Simplified overview of the experiments (I – IV), their main results and
conclusions.
Chapter I II III IV
Place Laboratory Field/laboratory Laboratory Field/laboratory
Analysis of alarm
Subject Effect of alarm Transgenerational Timing of pheromone
pheromone effects exposure (pre- or Effects in the
postnatally field
Measured Reproductive Behaviour in the Foraging Volatiles
output open field arena behaviour Foraging
Growth rate behaviour
Alarm
Timing has only pheromone
minor effect candidates
Increased Transgenerational identified
Main results parturitions effects exist Alarm Alarm
Pheromone pheromone
favours big litters short-lived
in the field
Main Terminal Effects are More support for Potential alarm
Conclusions investment context terminal pheromones
dependant investment identified
3 RESULTS AND DISCUSSION
20
3.2 Reproductive adaptations to weasel odour and alarm
pheromone (chapters I and III)
The ability of prey individuals to inform conspecifics has been well established.
A large focus lays on acoustic signals, i.e. alarm calls (Blanchard et al. 1991, Forti
et al. 2017, Barati and McDonald 2017). In some species, these can be elaborate
and not only signal threat, but also the predator identity (Manser et al. 2002,
Ouattara et al. 2009, Collier et al. 2017, Barati and McDonald 2017). The aspect of
added information translates to alarm pheromones, as they are only secreted as
a reaction to an acute predator presence, as opposed to predator odours, which
may remain in the environment after the acute danger has passed. Prey species
can as a result gain more detailed insight of the threat level, e.g. whether
heightened vigilance is required as predator is somewhere in the area, or whether
more drastic measures need to be taken, as a conspecific barely survived an
attack.
In chapter I, I discovered that bank voles, as a reaction to a treatment of
conspecific alarm pheromone, showed an increase in parturitions. In fact, while
in the control group 36.8% of the females gave birth to a litter, this rate was at
84.5% for those treated with alarm pheromone. No other significant difference
from control was observed. In addition, a treatment with predator odour affected
the reproductive output. Pups, one day after birth, were significantly heavier
when the mother were treated with predator odour.
Chapter II shows an increased mass gain in bigger litters in the alarm
pheromone treatment, but increased mass gain in smaller litters in the predator
odour treatment. Combined with chapter I, this solidifies the idea that different
odour cues carry different information, which in turn allows prey species or
individuals to alter their behaviour. Consequently, alarm pheromone exposure
causes a change towards terminal investment, with a focus on larger litters, while
exposure to predator odours might trigger an investment in a few high-quality
offspring.
Contrary to previous studies on bank voles, I did not observe any decrease
in reproductive output (Ylönen and Ronkainen 1994, Fuelling and Halle 2004,
Haapakoski et al. 2012), but rather the opposite, which is in accordance with a
more recent study which discovered increased litter sizes in bank voles after an
alarm pheromone exposure (Haapakoski et al. 2018).
The ambiguity of these results might seem troubling at first glance, but
recent work has proposed a new idea for adaptive reproductive strategies
(Duffield et al. 2017). At low or medium risk levels, a decrease of reproductive
output is assumed, as prey individuals focus on their short-term individual
fitness. As the perceived risk level passes a threshold, the reproductive strategy
may change towards terminal investment, as a single offspring surviving the
high risk period and reaching sexual maturity compensates the parental
mortality (Kokko and Ranta 1996, Kokko and Ruxton 2000), which is a more long-
term fitness investment. This is similar to the insurance hypothesis, in which
21
reproductive investment increases in expectation of an unfavourable
environment (Promislow and Harvey 1990, Forbes 1991, Houston et al. 2012).
The majority of mammalian studies focusing on terminal investment cover
senescence (Ericsson et al. 2001, Hoffman et al. 2010, Weladji et al. 2010), therefore
it is challenging to assess the adaptive and evolutionary aspects of terminal
investment in mammals as a response to predator cues. However, a very recent
study found similar results in Brandt’s voles (Lasiopodomys brandtii, Radde 1861)
(Gu et al. 2020), so this could be a more widespread phenomenon than assumed
at the moment.
Signalling conspecifics immediate threat is most often seen in social species
such as suricates (Manser et al. 2002) or bees (Johnson et al. 1985). However, bank
voles are commonly not considered a social species. Nevertheless, despite the fact
that females are very territorial during breeding season (Bujalska 1973), during
winter, bank voles tend to form communal nests for better thermoregulation
(Ylönen and Viitala 1985, 1991). Additionally, young female offspring are
tolerated in their mothers territory for a prolonged time (Mappes et al. 1995b).
These indications of temporarily close social groups could be the basis of alarm
pheromone communication in bank voles.
3.3 Transgenerational effects (chapters II and III)
In the current literature on transgenerational effects, the consensus is that
predator exposure (or predator odour exposure) causes increased anxiety-like
behaviour in offspring (Abe et al. 2007, Brunton and Russell 2010, Brunton 2013,
St-Cyr et al. 2017). The majority of the studies in this area use an open-field arena
(or similar neutral environments), which is a neutral, blank environment without
any kind of stimuli. As the range of behavioural adaptations to predation are
vast, an open field arena certainly is a simplification of the natural environment.
In an attempt to bridge this gap, the voles in chapter II were put in such an
arena, but one of the odour cues was present. While this is still a simplified
environment, it presents the animal with an ecologically relevant stimulus,
unlike a completely empty environment. However, the assumption of an open-
field arena is that spending time next to its outer limits, i.e. walls, can be
interpreted as a proxy for anxiety (Treit and Fundytus 1988).
In chapter I, the focus was on proportion of time spent in the centre zone. I
was able to replicate previous experiments, i.e. increased anxiety in pups, whose
parents encountered a predator odour, in a neutral environment. Interestingly, I
also found interactions between the parents’ treatment and the odour
encountered in the arena, meaning that the combination of prenatal predator
odour with either predator odour or alarm pheromone in the testing
environment resulted in offspring reacting less fearfully. Thus, transgenerational
effects are highly context-dependent.
Chapter III used a more complex testing environment. Vole offspring were
observed in a three-armed maze. Each arm contained a foraging patch along with
22
an odour cue. In this set-up, no transgenerational effects were found when it
came to foraging or investigating the predator odour arm. Voles still showed an
innate avoidance of areas treated with a predator, but independent of any
parental treatment. In contrast to this, pups with a parental treatment of alarm
pheromone also explored the area of the maze with alarm pheromone first.
An increase in boldness, resulting from in utero imprinting, can be adaptive
when triggered in a high-risk environment. During the short life span of a vole,
a significant change in predation pressure might not occur. As predator densities
follow prey densities with a time lag, a high predator-to-prey ration outlasts a
prey generation (Hanski et al. 2001, Sundell et al. 2013). In that scenario, bold
individuals are more likely explore, forage or mate (Ylönen et al. 2002, Korpela et
al. 2011, Mella et al. 2015) instead of waiting for an safer environment.
These results also show the importance of the test environment, as some
transgenerational effects become only apparent under specific conditions.
3.4 Timing of the exposure (study III)
Several studies have shown how predation risk affects prey offspring’s fitness
and behaviour (Bestion et al. 2014, Sheriff et al. 2015). To date, an overarching
consensus on when the cue has to be encountered to trigger an effect is still
lacking. Nevertheless, explanations for the different forms of information
transmission exist. In utero transmission of the information is likely to occur via
hormone transmission in the placenta (Sheriff et al. 2017, Kuijper and Johnstone
2018). Postnatally the difference in e.g. behaviour can be explained by different
hormone contents during lactation (Brummelte et al. 2010, Sullivan et al. 2011), or
with differences in maternal care (Bauer et al. 2015).
In contrast to my expectations, only negligible differences in the offspring
behaviour were explained by the timing of parental treatment. The only affected
aspect was the foraging. There, vole pups from mothers treated with alarm
pheromone after parturition foraged 0.1 g more compared to animals from the
control group. The difference of 0.1 g needs to be put into context, as in this
chapter millet seeds were used. Thus, the difference in foraging is about 19 millet
seeds with diminishing returns, which require a significant time investment to
find from the substrate.
Similar to the conclusion drawn in the previous section, this is possibly an
adaptation to a longer lasting high-risk environment, in which individual are
forced to forage in order to survive.
3.5 Identification of vole alarm pheromone (study IV)
The work on invertebrate alarm pheromones is already extensive (e.g. Bowers et
al. 1972, Howe and Sheikh 1975, Heath and Landolt 1988), however literature on
23
terrestrial vertebrate alarm pheromones are lacking. To be precise, “extensive”
solely refers to the number of studies on invertebrates compared to vertebrates,
since in relation to the number of invertebrate species, the numbers become less
impressive. In terrestrial vertebrates, there is a clear overrepresentation of studies
on laboratory rodents (Kiyokawa et al. 2005, Inagaki et al. 2009, 2014, Brechbühl
et al. 2013). On the other hand, there are only a few studies on body odours of any
kind in wild animals (Charpentier et al. 2008, Apps et al. 2015), and very few on
alarm pheromones in wild terrestrial vertebrates (Gomes et al. 2013). This is
partly due to a historical lack of non-invasive sampling techniques, which have
just been developed in recent years (Birkemeyer et al. 2016, Weiß et al. 2018).
Chapter IV aimed at identifying possible alarm pheromones in bank voles.
I was able to narrow down the possible candidates to three potential chemicals.
Of these three, one is an unknown compound, while the other two, 2-octanone
and 1-octnol, have previously been found in alarm secretions of other species.
However, 1-octanol has solely been found in insects (Johnson et al. 1985, Collins
et al. 1989, Hunt et al. 2003, Yamashita et al. 2016) and 2-octanone mostly in ants
(Crewe and Blum 1970, Dumpert 1972, Brand et al. 1989), with the exception of
two loris species (Hagey et al. 2007).
In the aquatic environment, examples of chemical cross-phyla
communication exist (Kaliszewicz and Uchmański 2009), however for terrestrial
vertebrates, a communication across taxonomic classes has only been shown with
alarm calls (Vitousek et al. 2007, Lea et al. 2008). Despite the evidence that our
identified compounds are secreted in invertebrates, my results do not allow
drawing conclusions about a common underlying structure or chemical features
of alarm pheromones. A previous meta-analysis looking for a common
denominator in chemical communication of terrestrial vertebrates concluded that
the current studies are merely scratching the surface of the topic and data on
more species is needed to draw meaningful conclusions (Apps et al. 2015).
I supplemented this analysis with a field experiment on the effect and
longevity of alarm pheromones in the field compared to direct predator odour.
The experiment showed a drastic increase in foraging in the foraging patches
treated with the alarm pheromone after just one day. This can be explained in
two ways. Either, as I expected, the volatile alarm pheromone only efficiently
carries the information on immediate risk short-term. The remaining odours,
after the vanishing of the fear signal, merely indicates the presence of a
conspecific, in turn sending safety signal, as only odours of conspecifics remain,
which increases investigation and foraging. Alternatively, the alarm pheromone
is still present on the second day, but in such low concentrations that that triggers
increased investigation and intensified use of the foraging patch (Parsons et al.
2018), which in turn results in the discovery of the foraging patch. Both options
indicate a minimal information value concerning predator presence on the
second day. It also supports the role of alarm pheromones to indicate an acute
and immediate risk, as argued in chapter I.
Chemical communication in predator-prey relations, between conspecifics
sharing the same risky environment, is a slowly growing field that only has
gained more traction in the last decade. Especially pheromones, which allow
chemical communication between conspecifics, have been mostly ignored in
terrestrial mammals until recently. Even though there has been a call for more
extensive work on semiochemicals, there has only been little progress made in
the last decade on identifying the nature and effects of body odours in terrestrial
vertebrates.
In this thesis, I investigated the predator-prey interaction between bank
voles and least weasels, with a focus on direct and indirect chemical cues of
predation. I also studied how these cues affect the reproductive behaviour,
offspring growth and behaviour, and finally the chemical nature of vole alarm
pheromones. I approached the research questions with a combination of
enclosure and laboratory studies.
Firstly, I showed how voles associated different odour cues with different
risk levels. While the direct odour of a predator represents a medium threat level,
as it is constantly in the vole’s environment, alarm pheromones represent an
acute and high risk (chapter I – III). This leads to my second point, alarm
pheromone triggers terminal investment on several levels in bank voles (chapters
I and III). Specifically, alarm pheromone triggers an increase in parturitions (I),
an increase in litter sizes (Haapakoski et al. 2018), and an accelerated growth in
big litters (III). Thirdly, I showed that both predator odour as well as alarm
pheromones trigger transgenerational effects in bank voles. However, these
effects are highly context-dependent, e.g. they depend on the test environment
and stimuli provided. This in turn has potential consequences on existing
literature, as often-used neutral and simplified environments trigger different
behaviours, compared to enriched environments with odour stimuli. However, I
also showed that the timing of when parents encounter these cues only has minor
effects. Fourthly and lastly, I identified a group of chemicals that are likely to act
as alarm pheromones in bank voles (IV). In the process, using enclosure
experiments, I also confirmed the only short-lasting effect of alarm pheromone
4 CONCLUSIONS
25
as a foraging deterrent in the field, which vanished after just one day. Thus, the
concept of different threat levels or different threat immediacy associated with
different odours is verified.
In this thesis, I have gathered new information on the importance of alarm
pheromones for the assessment of predation risk in small mammals, as well as
how it shapes a new range of behavioural adaptations. Terminal investment, as
a result of high predation risk, is currently a rarely researched topic but
pheromone communication in prey is also often overlooked. I hope that this
thesis helps to develop further the field of predator-prey research by providing a
fresh aspect to it. I am confident that pheromone communication occurs in a
wider range of terrestrial mammals than is currently known. Further, I
recommend that the next generation of research on chemical cues in antipredator
adaptations should strongly focus on conspecific odour cues.
26
Acknowledgements
So, this is it. Four years of research compressed into a couple of pages. Even
though it is the customary to write the thesis in the first person, it was very much
a team effort and I am thankful to quite a few people who helped throughout the
process.
First and foremost, I would like to thank my supervisors Hannu Ylönen and
Marko Haapakoski. When I applied to the position, I was almost certain I would
not get it, but with a stroke of luck (and new exciting results), they decided to
pivot towards an aspect I had more experience in. I am thankful for the
opportunity and all the fruitful discussions and arguments, as they led to four
years of exciting work.
I am also thankful to my opponent, Anders Angerbjörn, who agreed on a
very short notice to act as my opponent and to go through with a remote defence.
Additionally, I would like to thank Peter Banks, who had agreed a while ago to
be my opponent, but due to travel restrictions and time zones could not make it.
I would also like to thank my pre-examiners Michael Sheriff and Zbigniew
Borowski for their comments, feedback and their time to read my thesis.
Thanks go also to my follow-up group, Raine Kortet, Janne Sundel, and
Matthias Laska, who, especially in the beginning, made sure I stayed focused and
would not pick up too many interesting side projects.
I would not be here today without three master students who were busy
wrangling voles and analysing videos: Helinä, Arjane, and Kerstin. Thank you
very much!
But there were also so many helpers during the long summer months in
Konnevesi. We kept each other (more or less) sane with board games, sauna, and
swims. Many thanks go to (in no specific order): Olga, Joni, Teemu, Arjane,
Patricia, Dave, Karoliina, and Hanna. I would like to especially thank Olga, who
helped in each and every experiment of my thesis.
All of my experiments were done at the Konnevesi Research Station. And
most of them involved some weird piece of equipment that first needed to be
built, modified or maintained. A thousand thanks to Janne, Jyrki, and Risto, who
patiently dealt with my weird wishes for equipment. Staying at Konnevesi would
have been half as amazing without Helinä N. Suvi, Miia, Taija, Tarja, and Kirsti.
Thank you all.
A big thank you to my other co-authors Kevin, Rupert, Amélie, and James,
who helped and advised with my manuscripts.
I would like to thank all the amazing PhD students at the department, the
Sauna and Support meetings, the journal club, coffee breaks etc. Thank you Anne,
Seba, Aigi, Liam, Andreas, and so many more.
Thank you, you crazy people in my DnD group. So many hours of
distraction and messing around. Thanks you Arttur, Teemu, Arjana, Santeri, and
Andreas.
27
A big thank you to the AoS Slack for distraction, statistical advice, party
parrots, and discussions about data visualization. A special thanks goes to Edwin
Khoo and Cooper Hodges, who were kind enough to proof-read my thesis.
I would like to thank the Academy of Finland for funding me, and the
Department of Biological and Environmental Science for financially supporting
the last months of my PhD and two of my conference trips.
I am thankful for all the help guiding me through the style and form
requirements provided by Jari Haimi.
A big hug and thanks goes to my parents. Throughout the four years, they
provided mental support and made sure I got regularly “care packages”. Thank
you so very much!
Finally, thank you Helinä. You were with me from my (our) very first
experiment. You have seen and dealt with all the stages of trying to make sense
of statistics. You have put up with me when I felt blue because something did not
work out as it should or when I spent hours staring at my screen, trying to put
my thoughts into coherent sentences. There are not enough words to describe
how thankful I am and how much you mean to me.
I probably forgot a few people, so thank you to all of you who helped and
supported me. I am so sorry for not mentioning you by name.
Kiitos paljon kaikille!
28
YHTEENVETO (RÉSUMÉ IN FINNISH)
Petoriskin epäsuorat ja sukupolvien väliset vaikutukset: pedon haju ja häly-
tysferomonit metsämyyrä–lumikko –suhteessa
Peto–saalis-suhteen evoluutio ja ilmentymät populaatioissa ja yhteisöissä ovat
kiinnostaneet tutkijoita jo vuosisadan ajan. Evolutiivinen kilpajuoksu pedon ja
saaliin välillä on johtanut moniin morfologisiin ja fysiologisiin sopeumiin sekä
käyttäytymisen muutoksiin. Varhaisempi peto–saalis-suhteen tutkimus keskit-
tyi petojen aiheuttamaan kuolleisuuteen ja sen populaatiovaikutuksiin. Viime
aikoina tutkimuskohteina ovat olleet yhä useammin pedon epäsuorat vaikutuk-
set saaliin käyttäytymiseen ja tämän adaptiiviset seuraukset saaliin hengissä
säilymiselle ja lisääntymiselle ja näin ollen lajin populaatioille. Pedon vält-
tämisen edellytys on se, että saalis havaitsee pedon. Saaliseläin voi aistia pedon
läsnäolon suoraan pedon jättämistä ärsykkeistä, kuten äänistä tai hajuista, tai
se voi saada epäsuorasti viestin muilta oman lajin yksilöiltä. Havaitessaan
pedon saaliseläin muuttaa käyttäytymistään siten, että sen selviytyminen para-
nee ja kelpoisuus lisääntyy. Saalis saattaa puolustautua morfologisesti, kuten
ahven, joka nostaa selkäevänsä piikit kohtisuoraan ylös petokalan, esimerkiksi
hauen havaitessaan. Saaliin yleisimpiä vasteita kohonneeseen petoriskiin ovat
käyttäytymisen muutokset, kuten jähmettyminen, jotta peto ei havaitse saalista,
tai pakeneminen. Pedon läsnäolo voi myös muuttaa saaliin lisääntymiskäyt-
täytymistä.
Saalis voi myös viestittää petoriskistä lajitovereilleen pelkoreaktion kautta
erittyvillä hajuilla, niin sanotuilla hälytysferomoneilla. Nämä kemialliset viestit
saattavat sisältää tarkempaa, akuutimpaa informaatiota petoriskistä kuin pe-
don haju. Suorien käyttäytymisvasteiden lisäksi kohonneella petoriskillä saat-
taa olla sukupolvienvälisiä vaikutuksia. Vanhempien, varsinkin äidin, kokema
riski saattaa välittyä kehittyviin poikasiin niin raskauden kuin imetyksenkin
aikana ja vaikuttaa poikasten kykyyn reagoida petoärsykkeisiin pesästä lähdön
jälkeen.
Selkärankaisilla on tehty vain vähän tutkimuksia hälytysferomoneista ja
niiden merkityksestä saaliseläinten keskinäisessä kommunikaatiossa. Väitös-
tutkimukseni keskittyi petoriskiä viestittävien signaalien, pedon hajun ja oman
lajin toisen, petoriskille altistuneen yksilön erittämän hajun, vaikutuksiin saa-
liin käyttäytymiseen ja lisääntymiseen. Tutkimuslajini olivat maamme metsissä
yleinen jyrsijä, metsämyyrä, ja sen merkittävä spesialistipeto, lumikko. Lumik-
ko pystyy pienenä ja pitkulaisena saalistamaan myyriä niiden käytävissä,
pesissä ja myös lumen alla.
Tutkimukseni keskittyi kolmeen pääkysymykseen: miten suora pedon
haju tai epäsuora, lajitoverin erittämä hälytyshaju vaikuttaa metsämyyrän li-
sääntymiseen; onko vanhempien, etenkin äidin, altistumisella korkeaan peto-
riskiin vaikutuksia raskausajan tai imetyksen kautta poikasten pedonvälttämis-
käyttäytymiseen; sekä mitkä kemialliset yhdisteet olisivat hälytysferomonin
29
välittämän informaation taustalla. Tutkin näitä kysymyksiä neljässä osatutki-
muksessa, joista osa tehtiin laboratoriossa ja osa luonnonmukaisissa ulkotar-
hoissa.
Tutkimukseni todensi sekä saaliseläimen lisääntymisen että käyttäytymi-
sen muuttuvan suoran ja epäsuoran petoärsykkeen vaikutuksesta. Hälytysfe-
romoni näytti muuttavan myyrän lisääntymisstrategiaa siten, että naaras li-
sääntyi tehokkaammin petoriskistä huolimatta. Tämä näkyi niin lisääntyvien
yksilöiden määrässä kuin poikasten kasvunopeudessakin, etenkin isoissa poi-
kueissa hälytysferomonille altistuttaessa. Ilmiö on yhteensopiva niin sanotun
”terminaalivaiheen investointi” -hypoteesin kanssa, jossa yksilö pistää kaiken
yhden kortin varaan vaikka henki menisi. Yksikin lisääntymisikään selviävä
poikanen kompensoi mahdollisen oman kuoleman.
Laajemmassa mittakaavassa tulokseni näyttävät olevan ristiriidassa aiem-
pien tutkimusten kanssa, joissa on osoitettu, että korkea petoriski saa aikaan
lisääntymisen siirtymisen tai estymisen. Oletan kuitenkin, että toisen yksilön
erittämä hälytyssignaali on luonteeltaan erilainen ja tuo viestin akuutista,
korkeasta vaarasta, johon reagointi voi olla erilaista kuin elinympäristöön jää-
nyt pedon haju. Molemmilla lisääntymisstrategioilla saattaa olla vaikutusta yk-
silön tai yksilön jälkeläisten selviämiseen, ja reaktio korkean petoriskin signaa-
leihin saattaa olla joustava elinkierron eri tilanteissa.
Tutkimukseni vahvisti myös mahdollisten sukupolvien välisten petoriski-
vaikutusten olemassaolon. Vaikutukset näkyivät yksilön liikkumisessa, riskin-
otossa ja ruokailukäyttäytymisessä, mutta tulokset eivät olleet yksiselitteisiä.
Tutkimukseni toi uutta tietoa eläinten käyttäytymistutkimuksen metodiikkaan
ja suosittaa luonnonmukaisempia testausympäristöjä yksilön säilymiseen liitty-
vien käyttäytymispiirteiden tutkimukseen laboratoriossa.
Viimeinen tutkimus selvitti yhdisteitä, jotka saattaisivat toimia hälytyssig-
naaleina pelästyneen yksilön erittämissä hajuissa. Tulokseni osoittivat, että eri-
tyisesti kahdella yhdisteellä, 2-oktanonilla ja 1-oktanolilla saattaisi olla merkit-
tävä asema petoriskin kemiallisessa signaloinnissa, koska nämä yhdisteet on
löydetty myös muiden lajien pelkovälitteisissä hajuaineissa. Havaitsin myös,
että oletustemme mukaisesti hälytysferomonin vaikutus luonnossa oli lyhyt-
aikainen.
Yhteenvetona totean, että tutkimukseni tuotti uutta tietoa nisäkkäiden hä-
lytysferomonien koostumuksesta ja niiden vaikutuksesta saaliseläimen käyt-
täytymiseen ja lisääntymiseen. Tutkimukseni vertasi näitä vaikutuksia pedon
hajun suoriin vaikutuksiin. Tulokset osoittivat yksilöiden lisääntymisstrate-
gioiden mahdollisen joustavuuden. Varsinkin tulokseni lisääntymispanostuk-
sen kasvusta korkean petoriskin aikana on harvinainen nisäkäsekologiassa,
vaikka se onkin havaittu useilla muilla lajeilla selkärangattomista lintuihin.
30
ZUSAMMENFASSUNG (RÉSUMÉ IN GERMAN)
Indirekte und Transgenerationale Effekte des Prädationsrisikos:
Raubtiergeruch, Alarmpheromone und deren Auswirkungen auf Rötelmäuse
Das Zusammenspiel von Beutegreifern und ihrer Beute fasziniert Forscher bereits
seit Jahrzehnten. Einer der vielen Gründe für diese Faszination ist, dass diese
Interaktionen einer der größten evolutionären Treiber ist und dadurch zu einer Art
„Wettrüsten“ mittels physiologischen Adaptionen und Verhaltensänderungen
führt. Frühe Forschung war stark auf die direkte Mortalität der Beutetiere. Dies
führte zu einer Fokussierung auf die resultierenden Veränderungen in der
Populationsdynamik. Heutzutage liegt der Fokus vermehrt auf nicht
konsumptiven Effekten, wie verändertes Verhalten und dessen Konsequenzen.
Beutetiere können ihre Fressfeinde anhand von direkten, ausgehend von
dem Beutegreifer, oder indirekten, ausgehend von Artgenossen, Signalen
erkennen. Die bereits erwähnten Veränderungen in Verhalten oder Physiologie
der Beutetiere ermöglichen eine maximale Überlebenschance und eine erhöhte
individuelle Fitness. Die Anpassungen können permanent sein, wie zum Beispiel
Stacheln bei Stachelschweinen, oder auch induziert, das heißt nur auftretend wenn
benötigt. Letzteres kann sowohl morphologische wie auch Verhaltensänderungen
umfassen. Das Spektrum der Verhaltensänderungen bei Beutetieren reicht von
einfachen Anpassungen wie Flucht, erstarren, oder vermeiden von Orten, bis hin
zu komplexeren Veränderungen, zum Beispiel in der Fortpflanzung. Eine weitere
komplexe Anpassung sind Effekte, die erst bei den Nachkommen auftreten,
sogenannte transgenerationale Effekte. Diese werden entweder in utero oder
während des Säugens ausgelöst, und führen zu einer Reihe von Veränderungen
wie zum Beispiel verändertem Lernverhalten, veränderten Stressreaktionen oder
Veränderungen bei der Futtersuche. Weiterhing haben Beutetiere die Möglichkeit
Artgenossen vor Gefahr zu warnen, entweder mittels Alarmrufen oder
Alarmsekretion, zum Beispiel Alarmpheromone. Diese konspezifischen Signale
können einen höheren Informationsgehalt haben, als Signale von Fressfeinden. Sie
können Auskunft geben über die Identität des Prädators oder über das aktuelle
Risikoniveau informieren.
Bislang hat nur eine Handvoll von wissenschaftlichen Studien die
Alarmsekretionen und die durch sie ausgelösten Adaptionen in Wirbeltieren
untersucht. In meiner Dissertation untersuchte ich die Auswirkungen von
Raubtiergeruch und konspezifischen Alarmpheromonen auf das Verhalten und
die Fortpflanzung von Rötelmäusen (Myodes glareolus), eine Wühlmaus der
borealen Wälder. Als Prädator für meine Experimente diente das Mauswiesel
(Mustela nivalis nivalis), einer der kleinsten Vertreter der Marder und sehr
effektiver Nagetierjäger. Bedingt durch seinen schlanken Körperbau ist das
Mauswiesel fähig den Wühlmäusen in ihren Tunneln zu folgen und ist dadurch
einer der Hauptursachen der borealen Wühlmaussterblichkeit.
Meine Arbeit fokussiert sich auf drei Hauptpunkte um die Auswirkungen
von Prädatorgeruch und konspezifischen Alarmpheromonen zu vergleichen:
31
erstens, wie wird das Fortpflanzungsverhalten verändert; zweitens, gibt es
transgenerationale Effekte und wie äußern sie sich; und drittens, was genau sind
die Alarmpheromone in Rötelmäusen.
In meinen Studien habe ich Hinweise gefunden, dass die Konfrontation mit
konspezifischen Alarmpheromon einen Wechsel der Fortpflanzungsstrategie zu
„terminal investment“ verursacht. Dies wurde durch einen Geburtenanstieg nach
einer längeren Alarmpheromonbehandlung deutlich. Zusätzlich war auch ein
beschleunigtes Wachstum der Jungtiere bei größeren Würfen zu beobachten.
„Terminal investment“ beschreibt die Idee, dass im Falle eines extremen Risikos
Individuen ihre Fitness steigern, indem alle Ressourcen für die Fortpflanzung
verwendet werden, unabhängig von den eigenen Nachteilen. Sobald ein einziger
Nachkomme geschlechtsreif wird, kompensiert dies jegliche Investition der Eltern.
Ein zusätzlicher Aspekt ist, dass Rötelmäuse ihre Fortpflanzungsstrategie an
die implizierten Informationen eines Geruchs anpassen können. Frühere Studien
mit Raubtiergeruch beschreiben meist eine Verminderung der Fortpflanzung, was
in Kontrast zu meinen Ergebnissen steht. Allerdings, unter der Annahme, dass
Alarmpheromone, im Gegensatz zu Raubtiergeruch, eine sehr akute und zeitlich
begrenzte Gefahr signalisieren, zeigt der Wechsel der Fortpflanzungsstrategie zu
„terminal investment“ eine Plastizität des Verhaltens der Rötelmäuse.
Weiterhin habe ich Hinweise auf transgenerationale Effekte gefunden, die
sich auf das Erkundungs- und Futtersuchverhalten auswirken. Während frühere
Studien die Nachkommen in neutralen Umgebungen, zum Beispiel „open field
arena“, testen, untersuchte ich das Verhalten des Nachwuchses unter dem Einfluss
von Gerüchen. Dies zeigte, dass das veränderte Verhalten abhängig von der
Umgebung ist und nicht unter jeden Umständen auftritt. Im Gegensatz zu
früheren Studien, die Anzeichen für erhöhte Ängstlichkeit gefunden haben, zeigen
meine Ergebnisse, dass dies nur in neutralen Umgebungen zutrifft, und dass
Nachwuchs weniger ängstlich agiert sobald Raubtiergeruch zugegen ist.
Schlussendlich habe ich eine Gruppe von chemische Verbindungen aus den
Alarmsekretionen der Rötelmäuse identifiziert, die mit hoher Wahrscheinlichkeit
für die beschriebenen Ergebnisse verantwortlich sind. Besonders zwei chemische
Verbindungen, 2-Octanon und 1-Octanol, sind wahrscheinliche Kandidaten, da sie
bereits in den Alarmpheromonen anderer Arten nachgewiesen wurden. Ein
Vergleich mit anderen Säugetieren gestaltet sich schwierig, da bisher nur einige
wenige Studien die chemische Natur der Alarmpheromone in Wirbeltieren
untersucht haben. Weiterhin hat sich gezeigt, dass die vergrämende Wirkung von
Alarmpheromonen lediglich einen Tag unter natürlichen Bedingungen anhält.
Dies untermauert die Relevanz von Alarmpheromonen für zeitlich begrenzte
Informationen.
In meiner Dissertation habe ich neue Informationen zu der Relevanz von
Alarmpheromonen für die Beurteilung des Risikos durch Prädatoren für
Kleinsäuger gesammelt. Sowohl „terminal investment“, ausgelöst durch
Raubtiere, wie auch Alarmpheromone bei Säugetieren, sind bisher wenig
erforschte Phänomene. Meine Ergebnisse füllen eine Wissenslücke zur chemischen
Kommunikation bei Säugetieren und helfen das Verständnis von dem
Zusammenspiel zwischen Beutegreifern und deren Auswirkungen auf die
32
Fortpflanzung und das Verhalten der Beutetiere zu vertiefen. Zukünftige Studien
werden intraspezifische Kommunikation in ihre Ergebnisse einbeziehen müssen,
um die Gesamtheit der Räuber-Beute-Interaktionen zu verstehen.
33
REFERENCES
Abe H., Hidaka N., Kawagoe C., Odagiri K., Watanabe Y., Ikeda T., Ishizuka Y.,
Hashiguchi H., Takeda R., Nishimori T. & Ishida Y. 2007. Prenatal
psychological stress causes higher emotionality, depression-like behavior,
and elevated activity in the hypothalamo-pituitary-adrenal axis. Neurosci.
Res. 59: 145–151.
Abrams P.A. 1986. Is predator-prey coevolution an arms race? Trends Ecol. Evol.
1: 108–110.
Abrams P.A. 2000. The Evolution of Predator-Prey Interactions: Theory and
Evidence. Annu. Rev. Ecol. Syst. 31: 79–105.
Adamo S.A. & McKee R. 2017. Differential effects of predator cues versus
activation of fight-or-flight behaviour on reproduction in the cricket Gryllus
texensis. Anim. Behav. 134: 1–8.
Apfelbach R., Blanchard C.D., Blanchard R.J., Hayes R.A. & McGregor I.S. 2005.
The effects of predator odors in mammalian prey species: A review of field
and laboratory studies. Neurosci. Biobehav. Rev. 29: 1123–1144.
Apps P.J., Weldon P.J. & Kramer M. 2015. Chemical signals in terrestrial
vertebrates: search for design features. Nat. Prod. Rep. 32: 1131–1153.
Archer J. 1973. Tests for emotionality in rats and mice: A review. Anim. Behav. 21:
205–235.
Barati A. & McDonald P.G. 2017. Nestlings reduce their predation risk by
attending to predator-information encoded within conspecific alarm calls.
Sci. Rep. 7: 11736.
Barrera J.P., Chong L., Judy K.N. & Blumstein D.T. 2011. Reliability of public
information: predators provide more information about risk than
conspecifics. Anim. Behav. 81: 779–787.
Bauer C.M., Hayes L.D., Ebensperger L.A., Ramírez-Estrada J., León C., Davis
G.T. & Romero L.M. 2015. Maternal stress and plural breeding with
communal care affect development of the endocrine stress response in a
wild rodent. Horm. Behav. 75: 18–24.
Bell A.M. 2004. Behavioural differences between individuals and two
populations of stickleback (Gasterosteus aculeatus). J. Evol. Biol. 18: 464–473.
Berryman A.A. 1992. The Orgins and Evolution of Predator-Prey Theory. Ecology
73: 1530–1535.
Bestion E., Teyssier A., Aubret F., Clobert J. & Cote J. 2014. Maternal exposure to
predator scents: offspring phenotypic adjustment and dispersal. Proc. Biol.
Sci. 281: 20140701.
Birkemeyer C.S., Thomsen R., Jänig S., Kücklich M., Slama A., Weiß B.M. &
Widdig A. 2016. Sampling the Body Odor of Primates: Cotton Swabs
Sample Semivolatiles Rather Than Volatiles. Chem. Senses 41: 525–535.
Blanchard R.J.J., Blanchard D.C.C., Agullana R. & Weiss S.M. 1991. Twenty-two
kHz alarm cries to presentation of a predator, by laboratory rats living in
visible burrow systems. Physiol. Behav. 50: 967–972.
34
Bleicher S.S., Ylönen H., Käpylä T. & Haapakoski M. 2018. Olfactory cues and the
value of information : voles interpret cues based on recent predator
encounters. Behav. Ecol. Sociobiol. 72: 187–199.
Bonneaud C., Mazuc J., Chastel O., Westerdahl H. & Sorci G. 2004. Terminal
investment induced by immune challenge and fitness traits associated with
major histocompatibility complex in the house sparrow. Evolution 58: 2823–
2830.
Boonstra R., Krebs C.J. & Stenseth N.C.H.R. 1998. Population cycles in small
mammals: The problem of explaining the low phase. Ecology 79: 1479–1488.
Bourdeau P.E. 2010. Cue reliability, risk sensitivity and inducible morphological
defense in a marine snail. Oecologia 162: 987–994.
Bowers W.S., Nault L.R., Webb R.E. & Dutky S.R. 1972. Aphid Alarm Pheromone:
Isolation, Identification, Synthesis. Science 177: 1121–1122.
Brand J.M., Page H.M., Lindner W.A. & Markovetz A.J. 1989. Are ant alarm-
defense secretions only for alarm defense ? Naturwissenschaften 76: 277–277.
Brechbühl J., Moine F., Klaey M., Nenniger-Tosato M., Hurni N., Sporkert F.,
Giroud C. & Broillet M.-C.M.-C. 2013. Mouse alarm pheromone shares
structural similarity with predator scents. Proc. Natl. Acad. Sci. U.S.A. 110:
4762–4767.
Brown J.S. 1999. Vigilance, patch use and habitat selection: foraging under
predation risk. Evol. Ecol. Res. 1: 49–71.
Brummelte S., Schmidt K.L., Taves M.D., Soma K.K. & Galea L.A.M. 2010.
Elevated corticosterone levels in stomach milk, serum, and brain of male
and female offspring after maternal corticosterone treatment in the rat. Dev.
Neurobiol. 70: 714–725.
Brunton P.J. 2013. Effects of maternal exposure to social stress during pregnancy:
consequences for mother and offspring. Reproduction 146: R175–R189.
Brunton P.J. & Russell J.A. 2010. Prenatal social stress in the rat programmes
neuroendocrine and behavioural responses to stress in the adult offspring:
Sex-specific effects. J. Neuroendocrinol. 22: 258–271.
Bujalska G. 1973. The role of spacing behavior among females in the regulation
of reproduction in the bank vole. J. Reprod. Fertil. Suppl. 19: 465–474.
Caldji C., Tannenbaum B., Sharma S., Francis D., Plotsky P.M. & Meaney M.J.
1998. Maternal care during infancy regulates the development of neural
systems mediating the expression of fearfulness in the rat. Proc. Natl. Acad.
Sci. U.S.A. 95: 5335–5340.
Cann J. Van, Koskela E., Mappes T., Mikkonen A., Mokkonen M. & Watts P.C.
2019a. Early life of fathers affects offspring fitness in a wild rodent. J. Evol.
Biol. 32: 1141–1151.
Cann J. Van, Koskela E., Mappes T., Sims A. & Watts P.C. 2019b.
Intergenerational fitness effects of the early life environment in a wild
rodent. J. Anim. Ecol. 88: 1355–1365.
Caro T. 2005. Antipredator Defenses in Birds and Mammals. University of
Chicago Press, Chicago, IL.
35
Chaby L.E., Sheriff M.J., Hirrlinger A.M. & Braithwaite V.A. 2015. Does early
stress prepare individuals for a stressful future? Stress during adolescence
improves foraging under threat. Anim. Behav. 105: 37–45.
Champagne F.A. & Meaney M.J. 2006. Stress During Gestation Alters Postpartum
Maternal Care and the Development of the Offspring in a Rodent Model.
Biol. Psychiatry 59: 1227–1235.
Charpentier M.J.E., Boulet M. & Drea C.M. 2008. Smelling right: the scent of male
lemurs advertises genetic quality and relatedness. Mol. Ecol. 17: 3225–3233.
Chivers D., Mirza R. & Johnston J. 2002. Learned recognition of heterospecific
alarm cues enhances survival during encounters with predators. Behaviour
139: 929–938.
Cho W.K., Ankrum J.A., Guo D., Chester S.A., Yang S.Y., Kashyap A., Campbell
G.A., Wood R.J., Rijal R.K., Karnik R., Langer R. & Karp J.M. 2012.
Microstructured barbs on the North American porcupine quill enable easy
tissue penetration and difficult removal. Proc. Natl. Acad. Sci. U.S.A. 109:
21289–21294.
Clinchy M., Sheriff M.J. & Zanette L.Y. 2013. Predator-induced stress and the
ecology of fear Boonstra R. (ed.). Functional Ecology 27: 56–65.
Collier K., Radford A.N., Townsend S.W. & Manser M.B. 2017. Wild dwarf
mongooses produce general alert and predator-specific alarm calls. Behav.
Ecol. 28: 1293–1301.
Collins A.M., Rinderer T.E., Daly H. V., Harbo J.R. & Pesante D. 1989. Alarm
pheromone production by two honeybee (Apis mellifera) types. J. Chem. Ecol.
15: 1747–1756.
Creel S. & Christianson D. 2008. Relationships between direct predation and risk
effects. Trends Ecol. Evol. 23: 194–201.
Creel S., Christianson D., Liley S. & Winnie J.A. 2007. Predation Risk Affects
Reproductive Physiology and Demography of Elk. Science 315: 960–960.
Cresswell W. 1994. Song as a pursuit-deterrent signal, and its occurrence relative
to other anti-predation behaviours of skylark (Alauda arvensis) on attack
by merlins (Falco columbarius). Behav. Ecol. Sociobiol. 34: 217–223.
Crewe R.M. & Blum M.S. 1970. Alarm pheromones in the genus Myrmica
(Hymenoptera: Formicidae). Z. Vgl. Physiol. 70: 363–373.
Dell P.A. & Rose F.D. 1987. Transfer of effects from environmentally enriched
and impoverished female rats to future offspring. Physiol. Behav. 39: 187–
190.
Dicke M. & Sabelis M.W. 1988. Infochemical Terminology: Based on Cost-Benefit
Analysis Rather than Origin of Compounds? Funct. Ecol. 2: 131.
Dielenberg R.A. & McGregor I.S. 2001. Defensive behavior in rats towards
predatory odors: a review. Neurosci. Biobehav. Rev. 25: 597–609.
Dominey W.J. 1983. Mobbing in Colonially Nesting Fishes, Especially the
Bluegill, Lepomis macrochirus. Copeia 1983: 1086–1088.
Dudeck B.P., Clinchy M., Allen M.C. & Zanette L.Y. 2018. Fear affects parental
care, which predicts juvenile survival and exacerbates the total cost of fear
on demography. Ecology 99: 127–135.
36
Duffield K.R., Bowers E.K., Sakaluk S.K. & Sadd B.M. 2017. A dynamic threshold
model for terminal investment. Behav. Ecol. Sociobiol. 71: 185.
Dumpert K. 1972. Alarmstoffrezeptoren auf der Antenne von Lasius fuliginosus
(Latr.) (Hymenoptera, Formicidae). Z. Vgl. Physiol. 76: 403–425.
Eccard J.A. & Ylönen H. 2006. Adaptive food choice of bank voles in a novel
environment: choices enhance reproductive status in winter and spring.
Ann. Zool. Fennici 43: 2–8.
Ericsson G., Wallin K., Ball J.P. & Broberg M. 2001. Age-Related Reproductive
Effort and Senescence in Free-Ranging Moose, Alces alces. Ecology 82: 1613.
Feng S., McGhee K.E. & Bell A.M. 2015. Effect of maternal predator exposure on
the ability of stickleback offspring to generalize a learned colour–reward
association. Anim. Behav. 107: 61–69.
Ferrari M.C.O., Wisenden B.D. & Chivers D.P. 2010. Chemical ecology of
predator–prey interactions in aquatic ecosystems: a review and prospectus.
Can. J. Zool. 88: 698–724.
Fichtel C. 2004. Reciprocal recognition of sifaka (Propithecus verreauxi verreauxi)
and redfronted lemur (Eulemur fulvus rufus) alarm calls. Anim Cogn 7: 45–
52.
Forbes L. 1991. Insurance offspring and brood reduction in a variable
environment: the costs and benefits of pessimism. Oikos 62: 325–332.
Forti L.R., Forti A.B.B.S., Márquez R. & Toledo L.F. 2017. Behavioural response
evoked by conspecific distress calls in two neotropical treefrogs. Foster S.
(ed.). Ethology 123: 942–948.
Freeland W. 1991. Plant secondary metabolites: biochemical coevolution with
herbivores. In: Palo R.T. & Robbins C.T. (eds.), Plant defenses against
mammalian herbivory, CRC Press, Boca Raton, FL, pp. 61--81.
Frisch K. von. 1938. Zur Psychologie des Fisch-Schwarmes. Naturwissenschaften
26: 601–606.
Fuelling O. & Halle S. 2004. Breeding suppression in free-ranging grey-sided
voles under the influence of predator odour. Oecologia 138: 151–159.
Giehr J., Grasse A. V., Cremer S., Heinze J. & Schrempf A. 2017. Ant queens
increase their reproductive efforts after pathogen infection. R. Soc. Open Sci.
4: 170547.
Gomes L.A.P., Salgado P.M.P., Barata E.N. & Mira A.P.P. 2013. Alarm scent-
marking during predatory attempts in the Cabrera vole (Microtus cabrerae
Thomas, 1906). Ecol. Res 28: 335–343.
Grant J.W.G. & Bayly I.A.E. 1981. Predator induction of crests in morphs of the
Daphnia carinata King complex. Limnol. Oceanogr. 26: 201–218.
Graw B. & Manser M.B. 2007. The function of mobbing in cooperative meerkats.
Anim. Behav. 74: 507–517.
Gu C., Liu Y., Huang Y., Yang S., Wang A., Yin B. & Wei W. 2020. Effects of
predator-induced stress during pregnancy on reproductive output and
offspring quality in Brandt’s voles (Lasiopodomys brandtii). Eur. J. Wildl. Res.
66: 1–9.
37
Haapakoski M., Hardenbol A.A. & Matson K.D. 2018. Exposure to Chemical
Cues from Predator-Exposed Conspecifics Increases Reproduction in a
Wild Rodent. Sci. Rep. 8: 17214.
Haapakoski M., Sundell J. & Ylönen H. 2012. Predation risk and food: opposite
effects on overwintering survival and onset of breeding in a boreal rodent:
Predation risk, food and overwintering. J. Anim. Ecol. 81: 1183–1192.
Haapakoski M., Sundell J. & Ylönen H. 2013. Mammalian predator–prey
interaction in a fragmented landscape: weasels and voles. Oecologia 173:
1227–1235.
Halpin C.G. & Rowe C. 2016. The effect of distastefulness and conspicuous
coloration on the post-attack rejection behaviour of predators and survival
of prey. Biol. J. Linn. Soc. Lond. 120: 236–244
Hanley M.E., Lamont B.B., Fairbanks M.M. & Rafferty C.M. 2007. Plant structural
traits and their role in anti-herbivore defence. Perspect. Plant Ecol. Evol. Syst.
8: 157–178.
Hansen T.F., Stenseth N.C. & Henttonen H. 1999. Multiannual Vole Cycles and
Population Regulation during Long Winters: An Analysis of Seasonal
Density Dependence. Am. Nat. 154: 129–139.
Hanski I., Hansson L. & Henttonen H. 1991. Specialist Predators, Generalist
Predators, and the Microtine Rodent Cycle. J. Anim. Ecol. 60: 353–367.
Hanski I., Henttonen H., Korpimäki E., Oksanen L. & Turchin P. 2001. Small-
Rodent Dynamics and Predation. Ecology 82: 1505–1520.
Hansson L. 1979. Condition and Diet in Relation to Habitat in Bank Voles
Clethrionomys glareolus: Population or Community Approach? Oikos 33: 55.
Hare J. & Atkins B. 2001. The squirrel that cried wolf: reliability detection by
juvenile Richardson’s ground squirrels (Spermophilus richardsonii). Behav.
Ecol. Sociobiol. 51: 108–112.
Heath R.R. & Landolt P.J. 1988. The isolation, identification and synthesis of the
alarm pheromone of Vespula squamosa (Drury) (Hymenoptera: Vespidae)
and associated behavior. Experientia 44: 82–83.
Heil M. 2002. Ecological costs of induced resistance. Curr. Opin. Plant Biol. 5: 345–
350.
Hoffman C.L., Higham J.P., Mas-Rivera A., Ayala J.E. & Maestripieri D. 2010.
Terminal investment and senescence in rhesus macaques (Macaca mulatta)
on Cayo Santiago. Behav. Ecol. 21: 972–978.
Houston A.I., Trimmer P.C., Fawcett T.W., Higginson A.D., Marshall J.A.R. &
McNamara J.M. 2012. Is optimism optimal? Functional causes of apparent
behavioural biases. Behav. Processes 89: 172–178.
Howe N. & Sheikh Y. 1975. Anthopleurine: a sea anemone alarm pheromone.
Science 189: 386–388.
Huitu O., Koivula M., Korpimäki E., Klemola T. & Norrdahl K. 2003. Winter food
supply limits growth of northern vole populations in the absence of
predation. Ecology 84: 2108–2118.
Hunt G.J., Wood K. V., Guzmán-Novoa E., Lee H.D., Rothwell A.P. & Bonham
C.C. 2003. Discovery of 3-methyl-2-buten-1-yl acetate, a new alarm
38
component in the sting apparatus of Africanized honeybees. J. Chem. Ecol.
29: 453–463.
Inagaki H., Kiyokawa Y., Tamogami S., Watanabe H., Takeuchi Y. & Mori Y.
2014. Identification of a pheromone that increases anxiety in rats. Proc. Natl.
Acad. Sci. U.S.A. 111: 18751–18756.
Inagaki H., Nakamura K., Kiyokawa Y., Kikusui T., Takeuchi Y. & Mori Y. 2009.
The volatility of an alarm pheromone in male rats. Physiol. Behav. 96: 749–
752.
Jędrzejewska B. & Jędrzejewski W. 1990. Antipredatory behaviour of bank voles
and prey choice of weasels - enclosure experiments. Ann. Zool. Fennici. 27:
321–328.
Jędrzejewski W. & Jędrzejewska B. 1990. Effect of a predator’s visit on the spatial
distribution of bank voles: experiments with weasels. Can. J. Zool. 68: 660–
666.
Johnson L.K., Haynes L.W., Carlson M.A., Fortnum H.A. & Gorgas D.L. 1985.
Alarm substances of the stingless bee, Trigona silvestriana. J. Chem. Ecol. 11:
409–416.
Kavaliers M., Choleris E. & Pfaff D.W. 2005. Recognition and avoidance of the
odors of parasitized conspecifics and predators: Differential genomic
correlates. Neurosci. Biobehav. Rev. 29: 1347–1359.
Kiyokawa Y., Kikusui T., Takeuchi Y. & Mori Y. 2004. Alarm pheromones with
different functions are released from different regions of the body surface
of male rats. Chem. Senses 29: 35–40.
Kiyokawa Y., Kikusui T., Takeuchi Y. & Mori Y. 2005. Alarm pheromone that
aggravates stress-induced hyperthermia is soluble in water. Chem. Senses 30:
513–519.
Kiyokawa Y., Kodama Y., Kubota T., Takeuchi Y. & Mori Y. 2013. Alarm
Pheromone Is Detected by the Vomeronasal Organ in Male Rats. Chem.
Senses 38: 661–668.
Kiyokawa Y., Shimozuru M., Kikusui T., Takeuchi Y. & Mori Y. 2006. Alarm
pheromone increases defensive and risk assessment behaviors in male rats.
Physiol. Behav. 87: 383–387.
Koivula M., Koskela E., Mappes T. & Oksanen T.A. 2003. Cost of Reproduction
in the Wild: Manipulation of Reproductive Effort in the Bank Vole. Ecology
84: 398–405.
Kokko H. & Ranta E. 1996. Evolutionary Optimality of Delayed Breeding in voles.
Oikos 77: 173.
Kokko H. & Ruxton G.D. 2000. Breeding suppression and predator-prey
dynamics. Ecology 81: 1178.
Korpela K., Sundell J. & Ylönen H. 2011. Does personality in small rodents vary
depending on population density? Oecologia 165: 67–77.
Korpimäki E., Norrdahl K. & Rinta-Jaskari T. 1991. Responses of stoats and least
weasels to fluctuating food abundances: is the low phase of the vole cycle
due to mustelid predation? Oecologia 88: 552–561.
39
Krebs C.J., Boutin S., Boonstra R., Sinclair A.R.E., Smith J.N.M., Dale M.R.T.,
Martin K. & Turkington R. 1995. Impact of Food and Predation on the
Snowshoe Hare Cycle. Science 269: 1112–1115.
Kuijper B. & Johnstone R.A. 2018. Maternal effects and parent–offspring conflict.
Evolution 72: 220–233.
Lea A.J., Barrera J.P., Tom L.M. & Blumstein D.T. 2008. Heterospecific
eavesdropping in a nonsocial species. Behav. Ecol. 19: 1041–1046.
Lima S.L. 1994. Collective Detection of Predatory Attack by Birds in the Absence
of Alarm Signals. J. Avian Biol. 25: 319.
Lima S.L. 1995. Back to the basics of anti-predatory vigilance: the group-size
effect. Anim. Behav. 49: 11–20.
Lima S.L. 1998. Nonlethal Effects in the Ecology of Predator-Prey Interactions.
BioScience 48: 25–34.
Lima S.L. 2009. Predators and the breeding bird: behavioral and reproductive
flexibility under the risk of predation. Biol. Rev. Camb. Philos. Soc. 84: 485–
513.
Love O.P. & Williams T.D. 2008. The Adaptive Value of Stress‐Induced
Phenotypes: Effects of Maternally Derived Corticosterone on Sex‐Biased
Investment, Cost of Reproduction, and Maternal Fitness. Am. Nat. 172:
E135–E149.
Lung M.A. & Childress M.J. 2006. The influence of conspecifics and predation
risk on the vigilance of elk (Cervus elaphus) in Yellowstone National Park.
Behav. Ecol. 18: 12–20.
Macdonald D.W. (ed.). 2006. The Encyclopedia of Mammals. Oxford University
Press, Oxford.
MacLean S.A. & Bonter D.N. 2013. The sound of danger: Threat sensitivity to
predator vocalizations, alarm calls, and novelty in gulls. PLoS ONE 8: 1–7.
Mäkeläinen S., Trebatická L., Sundell J. & Ylönen H. 2014. Different escape tactics
of two vole species affect the success of the hunting predator, the least
weasel. Behav. Ecol. Sociobiol. 68: 31–40.
Manser M.B. 2001. The acoustic structure of suricates’ alarm calls varies with
predator type and the level of response urgency. Proceedings of the Royal
Society of London. Series B: Biological Sciences 268: 2315–2324.
Manser M.B., Seyfarth R.M. & Cheney D.L. 2002. Suricate alarm calls signal
predator class and urgency. Trends in Cognitive Sciences 6: 55–57.
Mappes T., Koskela E. & Ylönen H. 1995a. Reproductive costs and litter size in
the bank vole. Proc. R. Soc. Lond., B, Biol. Sci. 261: 19–24.
Mappes T. & Ylönen H. 1997. Reproductive effort of female bank voles in a risky
environment. Evol. Ecol. 11: 591–598.
Mappes T., Ylönen H. & Viitala J. 1995b. Higher Reproductive Success among
Kin Groups of Bank Voles (Clethrionomys Glareolus). Ecology 76: 1276–1282.
McGregor I.S., Schrama L., Ambermoon P. & Dielenberg R.A. 2002. Not all
‘predator odours’ are equal: cat odour but not 2,4,5 trimethylthiazoline
(TMT, fox odour) elicits specific defensive behaviours in rats. Behav. Brain
Res. 129: 1–16.
40
Mella V.S.A., Ward A.J.W., Banks P.B. & McArthur C. 2015. Personality affects
the foraging response of a mammalian herbivore to the dual costs of food
and fear. Oecologia 177: 293–303.
Meri T., Halonen M., Mappes T. & Suhonen J. 2008. Younger bank voles are more
vulnerable to avian predation. Can. J. Zool. 86: 1074–1078.
Mirza R.S. & Chivers D.P. 2001. Chemical Alarm Signals Enhance Survival of
Brook Charr (Salvelinus fontinalis) During Encounters with Predatory Chain
Pickerel (Esox niger). Ethology 107: 989–1005.
Mönkkönen M., Forsman J.T., Kananoja T. & Ylönen H. 2009. Indirect cues of nest
predation risk and avian reproductive decisions. Biol. Lett. 5: 176–178.
Mori E., Maggini I. & Menchetti M. 2014. When quills kill: the defense strategy of
the crested porcupine Hystrix cristata L., 1758. Mammalia 78.
Morrison E.B. 2011. Vigilance behavior of a tropical bird in response to indirect
and direct cues of predation risk. Behaviour 148: 1067–1085.
Nelson E.H., Matthews C.E. & Rosenheim J.A. 2004. Predators reduce prey
population growth by inducing changes in prey behavior. Ecology 85: 1853–
1858.
Noldus L.P.J.J., Spink A.J. & Tegelenbosch R.A.J. 2001. EthoVision: A versatile
video tracking system for automation of behavioral experiments. Behav. Res.
Methods Instrum. Comput. 33: 398–414.
Norrdahl K. & Korpimäki E. 1995. Mortality Factors in a Cyclic Vole Population.
Proc. Biol. Sci. 261: 49–53.
Norrdahl K. & Korpimäki E. 2000. The impact of predation risk from small
mustelids on prey populations. Mamm. Rev. 30: 147–156.
Orrock J.L., Danielson B.J. & Brinkerhoff R.J. 2004. Rodent foraging is affected by
indirect, but not by direct, cues of predation risk. Behav. Ecol. 15: 433–437.
Ostfeld R.S. 1985. Limiting Resources and Territoriality in Microtine Rodents.
Am. Nat. 126: 1–15.
Ouattara K., Lemasson A. & Zuberbuhler K. 2009. Campbell’s monkeys
concatenate vocalizations into context-specific call sequences. Proc. Natl.
Acad. Sci. U.S.A. 106: 22026–22031.
Owen D.A.S., Robbins T.R. & Langkilde T. 2018. Trans-generational but not early
life exposure to stressors influences offspring morphology and survival.
Oecologia 186: 347–355.
Paine R.T. 1966. Food Web Complexity and Species Diversity. Am. Nat. 100: 65–
75.
Pangle K.L., Peacor S.D. & Johannsson O.E. 2007. Large nonlethal effects of an
invasive invertebrate predator on zooplankton population growth rate.
Ecology 88: 402–412.
Parsons M.H., Apfelbach R., Banks P.B., Cameron E.Z., Dickman C.R., Frank
A.S.K., Jones M.E., McGregor I.S., McLean S., Müller-Schwarze D., Sparrow
E.E. & Blumstein D.T. 2018. Biologically meaningful scents: a framework for
understanding predator-prey research across disciplines. Biol. Rev. Camb.
Philos. Soc. 93: 98–114.
41
Promislow D.E.L. & Harvey P.H. 1990. Living fast and dying young: A
comparative analysis of life‐history variation among mammals. J. Zool. 220:
417–437.
Relyea R.A. 2004. Fine-tuned phenotypes: Tadpole plasticity under 16
combinations of predators and competitors. Ecology 85: 172–179.
Riessen H.P. 2012. Costs of predator-induced morphological defences in
Daphnia. Freshw. Biol. 57: 1422–1433.
Roche D.P., McGhee K.E. & Bell A.M. 2012. Maternal predatorexposure has
lifelong consequences for offspring learning in threespined sticklebacks.
Biol. Lett. 8: 932–935.
Rodgers A.B., Morgan C.P., Bronson S.L., Revello S. & Bale T.L. 2013. Paternal
Stress Exposure Alters Sperm MicroRNA Content and Reprograms
Offspring HPA Stress Axis Regulation. J. Neurosci. 33: 9003–9012.
Ruxton G.D. & Lima S.L. 1997. Predator-induced breeding suppression and its
consequences for predator–prey population dynamics. Proc. Biol. Sci. 264:
409–415.
Sbarbati A. & Osculati F. 2006. Allelochemical communication in vertebrates:
Kairomones, allomones and synomones. Cells Tissues Organs 183: 206–219.
Schmidt K.A., Lee E., Ostfeld R.S. & Sieving K. 2008. Eastern chipmunks increase
their perception of predation risk in response to titmouse alarm calls. Behav.
Ecol. 19: 759–763.
Schmitz O.J., Beckerman A.P. & O’Brien K.M. 1997. Behaviorally Mediated
Trophic Cascades: Effects of Predation Risk on Food Web Interactions.
Ecology 78: 1388.
Sheriff M.J., Bell A., Boonstra R., Dantzer B., Lavergne S.G., McGhee K.E.,
MacLeod K.J., Winandy L., Zimmer C. & Love O.P. 2017. Integrating
Ecological and Evolutionary Context in the Study of Maternal Stress. Integr.
Comp. Biol. 57: 437–449.
Sheriff M.J., Krebs C.J. & Boonstra R. 2009. The sensitive hare: sublethal effects of
predator stress on reproduction in snowshoe hares. J. Anim. Ecol. 78: 1249–
1258.
Sheriff M.J. & Love O.P. 2013. Determining the adaptive potential of maternal
stress Clobert J. (ed.). Ecol. Lett. 16: 271–280.
Sheriff M.J., McMahon E.K., Krebs C.J. & Boonstra R. 2015. Predator-induced
maternal stress and population demography in snowshoe hares: the more
severe the risk, the longer the generational effect. J. Zool. 296: 305–310.
Sih A. 1992. Prey Uncertainty and the Balancing of Antipredator and Feeding
Needs. Am. Nat. 139: 1052–1069.
Sih A. 1994. Predation risk and the evolutionary ecology of reproductive
behaviour. J. Fish Biol. 45: 111–130.
Slobodchikoff C.N., Paseka A. & Verdolin J.L. 2009. Prairie dog alarm calls
encode labels about predator colors. Anim. Cogn. 12: 435–439.
Smith M.E. 2000. Alarm response of Arius felis to chemical stimuli from injured
conspecifics. J. Chem. Ecol. 26: 1635–1647.
Smotherman W.P. 1982a. In utero chemosensory experience alters taste
preferences and corticosterone responsiveness. Behav. Neural Biol. 36: 61–68.
42
Smotherman W.P. 1982b. Odor aversion learning by the rat fetus. Physiol. Behav.
29: 769–771.
St-Cyr S., Abuaish S., Sivanathan S. & McGowan P.O. 2017. Maternal
programming of sex-specific responses to predator odor stress in adult rats.
Horm. Behav. 94: 1–12.
St-Cyr S. & McGowan P.O. 2015. Programming of stress-related behavior and
epigenetic neural gene regulation in mice offspring through maternal
exposure to predator odor. Front. Behav. Neurosci. 9: 145.
Stankowich T. & Blumstein D.T. 2005. Fear in animals: a meta-analysis and
review of risk assessment. Proc. Biol. Sci. 272: 2627–2634.
Stenseth N.C. 1985. Geographic distribution of Clethrionomys species. Ann. Zool.
Fennici 22: 215–219.
Stickrod G., Kimble D.P. & Smotherman W.P. 1982. In utero taste/odor aversion
conditioning in the rat. Physiol. Behav. 28: 5–7.
Sullivan E.C., Hinde K., Mendoza S.P. & Capitanio J.P. 2011. Cortisol
concentrations in the milk of rhesus monkey mothers are associated with
confident temperament in sons, but not daughters. Dev. Psychobiol. 53: 96–
104.
Sundell J., O’Hara R.B., Helle P., Hellstedt P., Henttonen H. & Pietiäinen H. 2013.
Numerical response of small mustelids to vole abundance: delayed or not?
Oikos 122: 1112–1120.
Sundell J., Trebatická L., Oksanen T., Ovaskainen O., Haapakoski M. & Ylönen
H. 2008. Predation on two vole species by a shared predator: antipredatory
response and prey preference. Popul. Ecol. 50: 257–266.
Sundell J. & Ylönen H. 2004. Behaviour and choice of refuge by voles under
predation risk. Behav. Ecol. Sociobiol. 56: 263–269.
Taylor R.J. 1984a. Predation. Springer Netherlands, Dordrecht.
Taylor J.D. 1984b. A partial food web involving predatory gastropods on a Pacific
fringing reef. J. Exp. Mar. Biol. Ecol. 74: 273–290.
Tidhar W., Bonier F. & Speakman J.R. 2007. Sex- and concentration-dependent
effects of predator feces on seasonal regulation of body mass in the bank
vole Clethrionomys glareolus. Horm. Behav. 52: 436–444.
Tollrian R. & Harvell C. 1999. The evolution of inducible defenses: current ideas.
In: Tollrian R. & Harvell C. (eds.), The ecology and evolution of inducible
defenses, Princeton University Press, Princeton, NJ, pp. 306–321.
Townsend S.W., Rasmussen M., Clutton-Brock T. & Manser M.B. 2012. Flexible
alarm calling in meerkats: the role of the social environment and predation
urgency. Behav. Ecol. 23: 1360–1364.
Trebatická L., Suortti P., Sundell J. & Ylönen H. 2012. Predation risk and
reproduction in the bank vole. Wildl. Res. 39: 463–468.
Treit D. & Fundytus M. 1988. Thigmotaxis as a test for anxiolytic activity in rats.
Pharmacol. Biochem. Behav. 31: 959–962.
Trussell G.C. & Nicklin M.O. 2002. Cue sensitivity, inducible defense, and trade-
offs in a marine snailL. Ecology 83: 1635–1647.
Valeix M., Loveridge A.J., Chamaillé-Jammes S., Davidson Z., Murindagomo F.,
Fritz H. & Macdonald D.W. 2009. Behavioral adjustments of African
43
herbivores to predation risk by lions: Spatiotemporal variations influence
habitat use. Ecology 90: 23–30.
Verheggen F.J., Haubruge E. & Mescher M.C. 2010. Alarm Pheromones—
Chemical Signaling in Response to Danger. In: Vitamins & Hormones,
Academic Press, pp. 215–239.
Vitousek M.N., Adelman J.S., Gregory N.C. & Clair J.J.H.S. 2007. Heterospecific
alarm call recognition in a non-vocal reptile. Biol. Lett. 3: 632–634.
Wallace K.J. & Rosen J.B. 2000. Predator odor as an unconditioned fear stimulus
in rats: Elicitation of freezing by trimethylthiazoline, a component of fox
feces. Behav. Neurosci. 114: 912–922.
Wang X. & Zou X. 2017. Modeling the Fear Effect in Predator–Prey Interactions
with Adaptive Avoidance of Predators. Bull. Math Biol.: 1–35.
Weiß B.M., Marcillo A., Manser M., Holland R., Birkemeyer C. & Widdig A. 2018.
A non-invasive method for sampling the body odour of mammals
Freckleton R. (ed.). Methods Ecol. Evol. 9: 420–429.
Weladji R.B., Holand Ø., Gaillard J.-M., Yoccoz N.G., Mysterud A., Nieminen M.
& Stenseth N.C. 2010. Age-specific changes in different components of
reproductive output in female reindeer: terminal allocation or senescence?
Oecologia 162: 261–271.
Wisenden B.D. 2000. Olfactory assessment of predation risk in the aquatic
environment Collin S.P. & Marshall N.J. (eds.). Philos. Trans. R. Soc. Lond. B
Biol. Sci. 355: 1205–1208.
Wisenden B.D., Pohlman S.G. & Watkin E.E. 2001. Avoidance of conspecific
injury-released chemical cues by free-ranging Gammarus lacustris
(Crustacea: Amphipoda). J. Chem. Ecol. 27: 1249–1258.
Wisenden B.D., Vollbrecht K.A. & Brown J.L. 2004. Is there a fish alarm cue?
Affirming evidence from a wild study. Anim. Behav. 67: 59–67.
Wong B.B.M., Bibeau C., Bishop K.A. & Rosenthal G.G. 2005. Response to
perceived predation threat in fiddler crabs: trust thy neighbor as thyself?
Behav. Ecol. Sociobiol. 58: 345–350.
Yamashita K., Isayama S., Ozawa R., Uefune M., Takabayashi J. & Miura K. 2016.
A pecky rice-causing stink bug Leptocorisa chinensis escapes from volatiles
emitted by excited conspecifics. J. Ethol. 34: 1–7.
Ylönen H. 1989. Weasels Mustela Nivalis Suppress Reproduction in Cyclic Bank
Voles Clethrionomys Glareolus. Oikos 55: 138.
Ylönen H., Jacob J., Davies M.J. & Singleton G.R. 2002. Predation risk and habitat
selection of Australian house mice, Mus domesticus, during an incipient
plague: desperate behaviour due to food depletion. Oikos 99: 284–289.
Ylönen H. & Ronkainen H. 1994. Breeding suppression in the bank vole as
antipredatory adaptation in a predictable environment. Evol. Ecol. 8: 658–
666.
Ylönen H. & Viitala J. 1985. Social organization of an enclosed winter population
of the bank vole Clethrionomys glareolus. Ann. Zool. Fennici 22: 353–358.
Ylönen H. & Viitala J. 1991. Social overwintering and food distribution in the
bank vole Clethrionomys glareolus. Ecography 14: 131–137.
44
Yoshida T., Jones L.E., Ellner S.P., Fussmann G.F. & Hairston N.G. 2003. Rapid
evolution drives ecological dynamics in a predator–prey system. Nature
424: 303–306.
ORIGINAL PAPERS
I
SECONDHAND HORROR: EFFECTS OF DIRECT AND
INDIRECT PREDATOR CUES ON BEHAVIOR AND
REPRODUCTION OF THE BANK VOLE
by
Thorbjörn Sievert, Marko Haapakoski, Rupert Palme, Helinä Voipio, Hannu Ylönen
2019
Ecosphere 10: e02765.
Licensed under Creative Commons 3.0
© The authors
https://doi.org/10.1002/ecs2.2765
Secondhand horror: effects of direct and indirect predator cues on
behavior and reproduction of the bank vole
THORBJ
€
ORN SIEVERT ,
1,
MARKO HAAPAKOSKI ,
1
RUPERT PALME ,
2
HELIN
€
AVOIPIO,
3
AND HANNU YL
€
ONEN
1
1
Department of Biological and Environmental Science, Konnevesi Research Station, University of Jyv€
askyl€
a, P.O. Box 35, 40014 Jyv€
askyl€
a,
Finland
2
Department of Biomedical Sciences, University of Veterinary Medicine, Veterin€
arplatz 1, Vienna, Austria
3
Faculty of Biological and Environmental Sciences, University of Helsinki, Viikinkaari 1, P.O. Box 65, Helsinki, Finland
Citation: Sievert, T., M. Haapakoski, R. Palme, H. Voipio, and H. Yl€
onen. 2019. Secondhand horror: effects of direct and
indirect predator cues on behavior and reproduction of the bank vole. Ecosphere 10(6):e02765. 10.1002/ecs2.2765
Abstract. 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 con-
specifics, 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 ter-
minal investment in reproduction.
Key words: alarm pheromone; ecology of fear; Mustela nivalis; Myodes glareolus; odor; stress response; terminal
investment.
Received 2 April 2019; revised 30 April 2019; accepted 2 May 2019. Corresponding Editor: Robert R. Parmenter.
Copyright: ©2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
E-mail: thorbjorn.t.sievert@jyu.fi
INTRODUCTION
Predators decrease an individual’s survival
probability (Sih et al. 1985, Murdoch et al. 2003).
Predation, and the indirect effects of predator
presence, has been recognized as strong life-his-
tory determinants across different taxa (Sih 1994,
Yl€
onen and Ronkainen 1994, Werner and Peacor
2003, Nelson et al. 2004, Yl€
onen and Brown 2007,
Sheriff et al. 2009). Historically, ecological
research has focused on the aforementioned
direct predation effects (Paine 1966, Taylor 1984,
Krebs et al. 1995). However, in the last decades,
the focus has shifted more and more toward the
indirect effects of predation (see reviews by Lima
1998, Creel and Christianson 2008), and it has
been recognized that perceived predation risk
alone can have large fitness or survival effects on
the population level as direct mortality by preda-
tors (Schmitz et al. 1997, Nelson et al. 2004,
Preisser et al. 2005, Pangle et al. 2007).
Co-evolution of predator and prey species sug-
gests prey evolved a number of sensory and
behavioral adaptations in order to recognize and
avoid predators. In many mammalian prey spe-
cies, this includes behavioral changes such as
❖www.esajournals.org 1June 2019 ❖Volume 10(6) ❖Article e02765
freezing, avoidance, and heightened vigilance,
but also the ability to detect and correctly recog-
nize the odors emitted by predators, from here
on predator odor (PO), which serve as triggers
for the adaptive behaviors previously mentioned
(Kats and Dill 1998, Dielenberg and McGregor
2001, Sundell and Yl€
onen 2004, Conover 2007,
Osada et al. 2014, Apfelbach et al. 2015, Sievert
and Laska 2016). Indirect effects of predation, for
example, decreased reproduction (Yl€
onen and
Ronkainen 1994, Sheriff et al. 2009, 2015), as well
as the interaction of risk and competition, are
drawing increasing attention in current literature
(e.g., Apfelbach et al. 2005, Parsons et al. 2017).
In a natural environment, the odor of a preda-
tor might be abundant in the form of excrement
or markings. It is therefore not surprising that
several studies have reported rapid habituation
to predator-born odor in a natural environment
(Cox et al. 2010, Elmeros et al. 2011, Bytheway
et al. 2013). This leads to the assumption that
prey, while detecting PO, considers it as an ambi-
ent risk (Brown et al. 2015). A study by Bleicher
et al. (2018) showed that vole’s reaction to preda-
tor odor returns to baseline levels after being
confronted with a live predator. This indicates
that actual predator presence outweighs the
information content of an olfactory cue alone
and that there is no increase in perceived risk
toward a predator odor cue. Prey species then
need different means to convey actual threaten-
ing or acute predation risk, allowing them to
dynamically adjust their behavior to differ-
ent threat levels (Duffield et al. 2017). This
role is most likely covered by intra-species
communication.
Intra-species communication and signaling
about increased risk, for instance, through
Schreckstoff (Frisch 1938) or alarm pheromones
(henceforth AP), are evolutionarily widespread
in many taxa (Bowers et al. 1972, Howe and
Sheikh 1975, Stowe et al. 1995, Boissy et al. 1998,
Beale et al. 2006, Guti
errez-Garc
ıa et al. 2007). In
several social species of fish, insects, and mam-
mals, AP secretions are recognized as a signal to
protect their colony, group, or family when in
danger (Breed et al. 2004, Kiyokawa et al. 2004a,
Gomes et al. 2013). Despite some papers raising
concern about the categorization of APs, arguing
that these chemicals cannot be classified as real
pheromones (Magurran et al. 1996, Viney and
Franks 2004), the behavioral response is the
same, given the correct context (Magurran et al.
1996). While for most mammals, the chemical
structure of APs is still unknown, it has been
identified in, for example, aphids (Bowers et al.
1972, Beale et al. 2006), sea anemones (Howe
and Sheikh 1975), and several insects (Crewe and
Blum 1970, Heath and Landolt 1988, Kuwahara
et al. 1989). To fulfill their sensory warning role,
APs should be volatile or hydrophilic (Kiyokawa
et al. 2005, Inagaki et al. 2009). Given the major-
ity of experiments on mammalian APs have been
done on lab animals, their chemical structure has
been described only for mice (C57BL/6J and
OMP-GFP strains; Brechb€
uhl et al. 2013) and
Wistar rats (Inagaki et al. 2014). Brechb€
uhl et al.
(2013) state that both mouse APs and mam-
malian predator olfactory cues share structural
similarities, specifically sulfur-containing mole-
cules. In this paper, we utilize the concept of APs
similarly as in previous studies, although we
acknowledge that in most studies the chemical or
biological nature of the different odors of stress
is not yet properly determined.
High predation risk affects mating behaviors
and reproductive success (Sih 1994, Ruxton and
Lima 1997, Kokko and Ruxton 2000). There is
strong support for the notion that predation risk
negatively affects breeding success (Sih 1994).
This is manifested as delayed breeding in bank
voles and gray-sided voles (Myodes rufocanus;
Mappes and Yl€
onen 1997, Fuelling and Halle
2004), hindering copulations in bank voles
(Myodes glareolus; Ronkainen and Yl€
onen 1994),
elevating stress levels in snowshoe hares (Lepus
americanus; Sheriff et al. 2009), or decreasing
weights of breeding individuals or their off-
spring in snowshoe hares and bank voles (Sheriff
et al. 2009, Trebatick
a et al. 2012). However, the
mechanisms and adaptive value of delayed or
suppressed breeding under risk are not clear and
continue to be debated (Ruxton and Lima 1997,
Kokko and Ruxton 2000). Several publications
have already explored the effects of increased
risk of predation in parents’environment on off-
spring behavior and fitness, finding altered
learning behavior in three-spine sticklebacks
(Gasterosteus aculeatus; Roche et al. 2012, Feng
et al. 2015), altered stress reaction in C57BL/6
mice and Long Evans rats (St-Cyr and McGowan
2015, St-Cyr et al. 2017), or changed foraging
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SIEVERT ET AL.
strategies in Sprague Dawley rats (Chaby et al.
2015).
An alternative explanation suggests that par-
ents will maximize reproductive efforts at all
costs in risky conditions (henceforth terminal
investment). In this scenario, individuals breed-
ing in a risky environment will enhance, or speed
up, reproduction in order to maximize fitness by
producing a number of strong offspring despite
the high costs for the parents’or mother ’s sur-
vival. If offspring survive and reach a fertile age,
this then compensates for parental disappear-
ance from the reproductive pool (Kokko and
Ranta 1996, Kokko and Ruxton 2000). This strat-
egy of bet-hedging or terminal investment has
been shown in experimental studies in passerine
birds breeding under increased predation risk
(M€
onkk€
onen et al. 2009) as well as in crickets
(Adamo and McKee 2017). Additionally, it has
been shown as a reaction to infections in ants
and sparrows (Bonneaud et al. 2004, Giehr et al.
2017).
The relationship between weasels and voles
has been intensively studied as the weasel is a
specialist predator of rodents and is the major
cause of mortality in boreal voles, especially dur-
ing a population’s decline (Korpim€
aki et al. 1991,
Norrdahl and Korpim€
aki 1995, 2000). As an
adaptation against the dramatic predation pres-
sure by weasels, voles are able to detect the odor
of mustelids as an antipredator measure and
change their behavior accordingly. Bank voles
decrease their movement and foraging when
exposed to weasel odor (Yl€
onen 1989, Sundell
and Yl€
onen 2004, Bleicher et al. 2018). They shift
their activity times and spatial use to avoid wea-
sels (Je
zdrzejewska and Je
zdrzejewski 1990, Je
zdrze-
jewski and Je
zdrzejewska 1990, Sundell et al.
2008) and use more arboreal escape under preda-
tion risk (Je
zdrzejewska and Je
zdrzejewski 1990,
M€
akel€
ainen et al. 2014). In the study by
M€
akel€
ainen et al. (2014), weasels rarely followed
bank voles into a tree, if the bank vole climbed
one, showing the efficiency of bank voles’
antipredator responses.
Here, we studied in two experiments how
increased predation risk, either direct risk in the
form of least weasels (Mustela nivalis nivalis)
odor, or indirect risk in the form of odor emitted
by weasel-scared conspecifics, influenced behav-
ior and reproductive investment in bank voles.
The effect was assessed in both behavioral trials
and a breeding experiment with cue exposure of
parents and monitoring subsequent offspring
performance. Social cues, such as pheromones,
have previously been shown to be sufficient to
trigger cross-generational changes (Koyama
et al. 2015). In order to differentiate between the
effects of PO, AP, and mere social odor, we also
used non-stressed conspecific bedding as a sec-
ond control in addition to clean wood shavings.
In the behavioral experiment, we predicted
that:
1. Voles would feel safer in control and social
odor treatments and spend more time in
boxes containing those treatments. This
would lead to increased foraging in those
treatments and foraging to be lowest in PO
treatment and second lowest in AP treat-
ment. This would be in accordance with
previous studies (Osada et al. 2014,
S
anchez-Gonz
alez et al. 2017).
In the fitness experiment, we predicted, based
on the existing body of research, that predation
cues have a detrimental effect on reproduction.
Specifically, we predicted that:
2. The direct predation cue, PO, would
decrease the breeding success of parent
voles (measured as number of breeding
females and litter size) more than AP.
3. Both predation cues, PO and AP, will
decrease the number of breeding females
and cause the production of smaller litters
(Kokko and Ruxton 2000, Fuelling and Halle
2004).
4. Both PO and AP treatments will cause pups
to be smaller (Sheriff et al. 2009, 2015).
5. There would be no effect of social odor or
control odor on condition, breeding of par-
ent voles, or size of offspring.
MATERIALS AND METHODS
Study species
Bank voles are common rodents in boreal for-
est areas. Vole populations cycle in Scandinavia
and specialist predators have a large role in caus-
ing this cyclicity (Hanski et al. 2001). Regular
high predation pressure in the wild maintains
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SIEVERT ET AL.
bank vole antipredator behavior at a high level.
The breeding season of the bank vole in central
Finland usually begins at the end of April and
lasts until September. During the breeding sea-
son, breeding female bank voles are strictly terri-
torial and male territories overlap with several
female territories (Bujalska 1973). The gestation
period is about 20 d, after which 3–6 pups are
born. These pups mature after 30 d.
The least weasel is a specialist predator of
small mammals and lives in the same habitat as
its prey. Due to its small size, the weasel is able
to hunt in tunnels and burrows of voles during
both summer and winter, leaving only a few safe
places for the voles (Norrdahl and Korpim€
aki
1995, 2000). Weasels are adapted to the harsh
winter conditions by a coat change in late
autumn. The weasel, like all small mustelids,
uses strong odors in its intraspecific communica-
tion, giving the prey a means to evaluate the cur-
rent predation risk.
The studies were conducted in the laboratory
at Konnevesi Research Station in Central Finland
(62°370N, 26°200E). In the laboratory, the voles
are kept in light and climate-controlled
husbandry rooms with a 12-L:12-D daily cycle.
The animals were kept individually in
42 926 915 cm transparent cages with wire
mesh lids with ad libitum water and food supply.
Each cage had wood shavings and hay as bed-
ding. Males and females were kept in the same
room. Study animals were the F1 generation of
individuals housed in the lab during the winter
months. The average initial weight of the voles
was 16.3 g 2.8 g (mean SD). All animals
were individually marked with ear tags (#1005-
1L1, National Band & Tag Company, Newport,
Kentucky, USA).
Weasels for the odor treatment were housed
individually in 60 9160 960 cm cages in an
outdoor shelter. Each cage had a nest box and
wood shavings and hay as bedding. During the
experiment, weasels were fed dead bank voles.
Odor cues
For this experiment, the following odor cues
were used:
Predator odor (PO): 1 mL of odor solution.
The PO was obtained by collecting 6 dL of wea-
sel bedding (wood shavings soiled with urine
and fecal matter) and mixing it with 6 dL of
diethyl phthalate (CAS 84-66-2), a solvent for a
broad variety of chemical substances and often
used for fragrances (Api 2001). The mix was left
overnight in a refrigerator, and the liquid phase
was extracted after 24 h (2 h). The odor solu-
tion was renewed every 7 d and stored in a
stable temperature of +4°C(0.2°C) in a refriger-
ator in-between application. The use of extracted
olfactory cues allowed for even exposure to all
animals and reduced the stress to our captive
weasels. Alarm pheromone (AP): 1 dL of vole
beddings from individuals directly exposed to a
predator. To obtain AP, two male voles were
individually exposed to a weasel for 1 min every
other day. Each individual was placed in a wire
mesh cage, which was then put directly into the
weasel cage. The animal was immediately
returned to its cage afterward. When the treat-
ments were applied, all the bedding of both ani-
mals was thoroughly mixed together. If the voles
were scared on the same day the treatments were
applied, the bedding was collected at the earliest
1 h after the animal returned to its cage. Social
odor (SO): 1 dL of vole beddings collected from
two male voles that were not handled before col-
lection nor exposed to weasel. The bedding of
both animals was carefully mixed before applica-
tion. The control (C) odor consisted of clean vole
bedding, that is, fresh wood shavings changed
between each trial. The odor cues were renewed
for each trial.
Experimental design—behavioral assays
For the first experiment, we used 50 bank voles
(28 males, 22 females). We applied two behav-
ioral measures to study the response to olfactory
cues in the voles: The first was a test measuring
the individual’s perceived risk using optimal
patch use (Brown 1988, Lima and Dill 1990) and
the other investigating spatial avoidance or pref-
erence.
Brown (1988) framed the harvest rate an ani-
mal makes at a given patch as a balance of the
energetic gains and costs attributed to foraging
effort, predation, and missed opportunity costs.
The density of food remaining in a patch after
the forager stops foraging is called a giving-up
density (GUD; Brown 1999) and reflects the point
where the energy remaining in the patch is equal
to or outweighed by the combined costs to the
forager. The GUD, as a method, has been
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SIEVERT ET AL.
adapted to test a large variety of elements affect-
ing the strategic decisions animals take (Bedoya-
Perez et al. 2013) and has been widely applied as
a measure for habitat use (Yl€
onen et al. 2002,
Orrock et al. 2004, Bleicher 2017, Bleicher et al.
2018).
Each individual was placed in a 190 9190 cm
cross-shaped system (Appendix S1: Fig. S1) for
three hours. At the center of the cross is a release
cage (20 920 cm). Going outward in the four
horizontal directions, the odor chamber is con-
nected via an opaque tube (10 cm long, 4 cm
diameter) to an antechamber (30 920 cm) with
a metal grid lid. This prevented the odors enter-
ing and mixing in the central area of the maze
and minimized the chances of an odor contami-
nation. From there, going outward, an opaque
tube (5 cm long, 4 cm diameter) led to a closed
and opaque odor chamber (40 925 cm). This
tube was considered as part of the odor chamber
for the analysis. Each odor chamber contained
one of four odor treatments together with a box
acting as a foraging patch (henceforth patch).
The odor cues were attached to the lid of the
chamber to avoid contaminations by the vole
and renewed for each trial. PO was applied to fil-
ter paper (article no. 120002, grade 1001; Munk-
tell Filter AB, Falun, Sweden). The spatial
orientation of the odors was randomly changed
for each trial to avoid a spatial bias. Two mazes
were used simultaneously, both were located in a
dimly lit room 2 m apart. The ventilated experi-
mental room was 7.5 97.5 m with the height of
4 m, allowing a large overhead space to dilute
escaping odors from the systems. The experi-
ment was performed during day time. Two trials
were run simultaneously for a total of four to six
trials per day. After each trial, every segment of
the maze was cleaned with denatured ethanol
(70%) and dried, to avoid odor contamination
between trials.
The design of the patches was a lidless box
(19 919 96 cm) containing 8 dL of sand into
which 20 husked sunflower seeds were mixed.
Each animal was allowed to forage in the system
for three hours (henceforth trial). The optimal
trial length was determined beforehand with
pilot trials. After each trial, the sand was sieved
and the remaining untouched seeds were
counted to obtain the GUD. To avoid cross-con-
tamination of olfactory treatments, the sand was
left to air out for 3 d between trials. Bytheway
et al. (2013) showed that even though predator
odor still elicited increased investigative behav-
ior after 24 h, it no longer elicited a change in for-
aging behavior. Based on this, it seems
reasonable to assume that if the voles were still
able to detect the odor after 72 h, the information
conveyed drastically changed. To encourage for-
aging in the novel systems, the animals were
starved for three hours prior to each trial.
Each trial was recorded using a GoPro4 for
later analysis. During the video analysis, the fol-
lowing parameters were measured for each of
the four arms: choice of the first odor box
entered, time spent in the connection tubes, and
time spent in the odor box. The first hour of each
trial was analyzed separately from the whole
duration to account for a possible habituation
effect.
Experimental design—trans-generational effects
The 240 bank voles (120 males, 120 females)
were divided equally into four treatment groups
for the second experiment. Prior to grouping the
animals, every individual was weighed and the
dominance of the male individuals was assessed
following the urine marking of males as
described by Horne and Yl€
onen (1996) and
Klemme et al. (2006). The males were placed in
the urine marking arena for 4 h and had access
to a small amount of food and water. The urine
markings were analyzed twice by two observers
independently and the average score was
recorded. Each individual received a dominance
score from 1 (no marking, a subordinate male) to
6 (markings all over the arena, a dominant male).
During the group assignment, we made sure
that all treatment groups consisted of an equal
number of males and females, the weight distri-
bution for each sex was similar and that the dom-
inance distribution for each treatment was
similar. Within these constraints, the animals
were assigned randomly into four different hus-
bandry rooms.
The voles were kept in the rooms for seven
days to acclimate to their new husbandry rooms.
The treatments consisted of the following four
odor cues (measurements per cage). Predator
odor (PO): 1 mL of odor solution on filter paper
(article no. 120002, grade 1001; Munktell Filter
AB, Falun, Sweden), Alarm pheromone (AP):
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SIEVERT ET AL.
1 dL of male vole beddings from scared individ-
uals, social odor (SO): 1 dL of male vole bed-
dings and control (C): 1 dL of dry, aired wood
shavings. Each treatment was directly applied
through the lid of the cage, without handling the
animal or the cage itself. Treatments were
applied three times per week for a total of seven
weeks. The treatments were collected and pre-
pared identically to what was outlined before.
All animals were moved to clean cages after the
mating phase. This is the standard procedure in
our laboratory. It allows the pregnant female to
build a nest in a cage free of the odor of a male
conspecific. Furthermore, it reduces the need to
disturb the female to clean its cages during preg-
nancy/lactation.
After the first week, the animals within the
treatment were randomly paired for mating,
avoiding pairing of first-degree siblings. For pair-
ing, the animals were housed in a joined cage for
seven days. From 18 d on after the beginning of
the pairing, female cages were checked for pups
twice per day. When litters were found each pup
was weighed one day after birth and the size of
the litter was recorded. The treatments were
stopped as soon as all pregnant individuals had
given birth. All individuals were weighed again
and the dominance of the males was reassessed.
The females were weighed again 5 d after giving
birth. The experiment and all measurements
ended at this point.
At the end of the habituation, prior to the odor
treatment, fecal samples were collected from all
voles for stress analysis. The voles were put indi-
vidually in smaller cages without bedding for a
maximum of three hours, after which all fecal
pellets not contaminated with urine were col-
lected into Eppendorf tubes then stored at
20°C. This procedure was repeated for all indi-
viduals, including nursing females, after the
treatment was stopped. Corticosterone metabo-
lites in the samples were analyzed following the
method outlined by Sipari et al. (2017) at the
University of Veterinary Medicine in Vienna.
Statistical analyses
All statistical analyses were performed in R (R
Core Team 2018). Plots were generated with
ggplot2 (Wickham 2009) and ggsignif (Ahlmann-
Eltze 2017). To analyze the directional choice of
voles as they entered the behavioral assays, a
multinomial log-linear regression (MLM), pack-
age nnet (Venables and Ripley 2002), was run.
This was combined with a Wald z-test to deter-
mine P-values, package AER (Kleiber and Zeileis
2008). In order to analyze not only the distribution
of litter sizes between treatments but also the dif-
ferences in successful pregnancies, and GUDs,
zero augmented generalized linear models, from
the package pscl (Zeileis et al. 2008) were used.
The time spent in each compartment, the differ-
ences in weight, the weight of the pups, and the
difference in stress metabolites were analyzed
with a linear model (LM) or linear mixed model
(LMM) for repeated measurements, packages
lme4 (Bates et al. 2014) and lmerTest (Kuznetsova
et al. 2017). Other measurements were analyzed
with linear or generalized (mixed) models,
depending on the measurement in question. Data
points with missing observation were excluded
from the data set, resulting in an effective sample
size for the statistical tests of 93 breeding pairs for
the breeding success part of the experiment.
For each analysis, the most complex model
included an interaction between Treatment and
Sex. Other factors, such as litter size and weight,
were added to the most complex model if appro-
priate, but never in interaction with other factors.
To achieve the best model fit, first the interaction
was removed, then other factors, only leaving
Treatment for the simplest model. The individual
animal was always included as a random factor
in analyses with repeated measurements. Each
treatment was compared to the C (control) treat-
ment. For each analysis, the most fitting distribu-
tion and model were chosen based on AICc,
package MuMIn (Barton 2018). A model was
considered the best if the difference in AICc from
the next model was greater than 2.5. In the cases
where there was no clear best model, all models
within a DAICc of 2.5 were weighed based on
their differences to the best fitting model and
weighed averages of the parameter estimates
were reported. The tables with all fitted models
for each statistical test can be found in
Appendix S1: Tables S1–S14.
RESULTS
Foraging behavior and giving-up densities
The first choice of animals did not show a sig-
nificant preference for or avoidance of entering
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SIEVERT ET AL.
any specific odor compartment. However, we
found a tendency (MLM, P=0.056, df =3,
n=46) that voles were 2.25 times more likely to
enter the SO compartment first. Otherwise, it
seems likely that the sex of the individual had a
negligible role in the decision to enter either odor
compartment of the maze, as it was not included
in the best model.
In contrast to the first choice for odors, we
found that over the full experimental duration of
three hours there was a significant interaction
between the sex of the vole and the time spent in
the AP tube (LMM, P=0.031, df =10, n=50,
Fig. 1). When both sexes were analyzed together,
female voles spent on average 2.5 min (147.5 s)
longer in the AP tubes compared to the males.
When the two sexes were analyzed separately,
male voles did not show a preference or avoid-
ance for the tubes (LMM, P>0.05, df =6,
n=28), but females spent two minutes (121.4 s)
longer in the tubes connecting the AP compart-
ment (LMM, P=0.027, df =6, n=22) compared
to the tubes leading to C compartment (close to
four minutes, 225.2 s). For the time spent in the
odor compartment, there are no significant dif-
ferences for the whole trial (LMM, P>0.05,
df =10, n=50).
The analysis of the GUD showed that about
1.1 seeds more (weighted average) were
harvested from the AP compartment compared
to control independent of the animal’s sex
(GLMM, Poisson, P=0.019, df =5, 6, n=50,
Fig. 2).
Effect on parents and offspring
Weight change in parental generation.—On aver-
age