Fungal-Media ed Mul i ophic In e ac ions - Do G ass
Endophy es in Die P o ec Voles om P eda o s?
Susanna Saa i
1,2
*, Janne Sundell
3,6
, O so Hui u
4
, Ma jo Helande
2
, Elise Ke oja
5
, Hannu Ylo
¨nen
3
, Ka i
Saikkonen
1
1Plan P oduc ion Resea ch, MTT Ag i ood Resea ch Finland, Jokioinen, Finland, 2Depa men o Biology, Uni e si y o Tu ku, Tu ku, Finland, 3Depa men o Biological
and En i onmen al Science, Konne esi Resea ch S a ion, Uni e si y o Jy a
¨skyla
¨, Konne esi, Finland, 4Suonenjoki Resea ch Uni , Finnish Fo es Resea ch Ins i u e,
Suonenjoki, Finland, 5Se ices Uni , MTT Ag i ood Resea ch Finland, Jokioinen, Finland, 6Lammi Biological S a ion, Uni e si y o Helsinki, Lammi, Finland
Abs ac
Plan -associa ed mic o-o ganisms such as myco oxin-p oducing endophy es commonly ha e di ec nega i e e ec s on
he bi o es. These e ec s may be ca ied o e o na u al enemies o he he bi o es, bu his has been a ely explo ed. We
examined how eeding on Neo yphodium endophy e in ec ed (E+) and endophy e ee (E2) meadow yeg ass (Sche odonus
p a ensis) a ec s body mass, popula ion size and mobili y o sibling oles (Mic o us le is), and whe he he die media es he
ulne abili y o oles o leas weasel (Mus ela ni alis ni alis) p eda ion. Because leas weasels a e known o be ol ac o y
hun e s, we also examined whe he hey a e able o dis inguish ol ac o y cues o oles ed on E+and E2die s. Nei he body
mass o oles no popula ion size di e ed be ween die s. Howe e , con a y o ou p edic ion, leas weasels p eyed mo e
o en on oles ed wi h E2g ass han on oles ed wi h E+g ass. The mobili y o oles ed on E+g ass was educed
compa ed o oles ed on E2g ass, bu his e ec was un ela ed o isk o p eda ion. Leas weasels appea ed unable o
dis inguish be ween exc emen odou s o oles be ween he wo ea men s. Ou esul s sugges ha consump ion o
endophy ic g ass is no di ec ly dele e ious o sibling oles. Wha ’s mo e, consump ion o endophy es appea s o be
ad an ageous o oles by educing isk o mammalian p eda ion. Ou s udy is hus he i s o demons a e an e ec o
plan -associa ed mic obial symbion s on he bi o e-p eda o in e ac ions in e eb a e communi ies.
Ci a ion: Saa i S, Sundell J, Hui u O, Helande M, Ke oja E, e al. (2010) Fungal-Media ed Mul i ophic In e ac ions - Do G ass Endophy es in Die P o ec Voles
om P eda o s? PLoS ONE 5(3): e9845. doi:10.1371/jou nal.pone.0009845
Edi o : Jon Moen, Umea Uni e si y, Sweden
Recei ed No embe 9, 2009; Accep ed Ma ch 1, 2010; Published Ma ch 24, 2010
Copy igh : ß2010 Saa i e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This s udy is pa o Endophy ic Fungi in Ecological Plan P oduc ion P og amme no. 110658, unded by he Academy o Finland, Niemi Founda ion,
Ma ja a and Eino Kolli Founda ion and Ol i Founda ion. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion
o he manusc ip . h p://www.aka. i/ i/A/, h p://www.ol isaa io. i/web/ol isaa io/, h ps://www.kollinsaa io.com/. The e is no URL o Niemi Founda ion.
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: [email p o ec ed]
In oduc ion
Al hough mic obial in e ac ions wi hin and be ween ophic
le els a e ubiqui ous, hei oles ha e emained la gely igno ed in
communi y scale s udies [1]. Mic obial symbion s ha e p o ound
pheno ypic e ec s on hei hos s which may cascade upwa d
h ough ood webs [2]. Fo ins ance, plan s hos mul iple symbio ic
mic obes, including myco hizal ungi, endophy ic ungi and
bac e ia ha may a ec he pe o mance o he bi o es h ough
symbion -p oduced oxins which accumula e in he bi o e issue
and he eby di ec ly ha m p eda o s ollowing inges ion o he
p ey. In addi ion, e ec s on highe ophic le els may mani es ,
e.g., ia changes in he bi o e densi ies, popula ion dynamics, body
size o beha iou o he bi o es [3], [4].
One g oup o mic obial symbion s ha a e known o a ec
mul i ophic in e ac ions a e ungal g ass-endophy es in he genus
Neo yphodium. They a e known o in ec 20–30% o all g ass species
[5], o ming sys emic and asymp oma ic in ec ions h oughou he
ae ial pa s o he hos plan , including he seeds, and he eby
allowing e ical dispe sal o he endophy e om one plan
gene a ion o ano he [6].
G ass-endophy es may ha e mul i a ious e ec s on he bi o e
communi ies [7]. Consump ion o Neo yphodium endophy e o igin
myco oxins has long been known o cause se e e li es ock diso de s,
including symp oms anging om embling o s agge ing and
se e e muscle spasms ha cause animals o collapse [8], [9]. Also
smalle e eb a e he bi o es, such as oden s, a e commonly
nega i ely a ec ed by endophy e inges ion. These e ec s include,
e.g., dec ease in popula ion densi y [10], lowe ed body mass [11],
inc eased oxici y-induced mo ali y [12] and supp ession o
ep oduc ion and g ow h [13]. The alkaloids p oduced by
endophy es may also ha e nega i e e ec s on he na u al enemies
o in e eb a e he bi o es [14], [15]. Howe e , expe imen al
s udies on endophy es and hei e ec s on highe ophic le els
a e s ill sca ce [2], [16], and no s udy has examined he cascading
e ec s o ungal symbion s o g asses on e eb a e ood chains.
He e, we examined how eeding on endophy e (Neo yphodium
uncina um (Gains, Pe ini & Schmid ) Glenn, Bacon, P ice &
Hanlin) in ec ed (E+) o endophy e ee (E2) meadow yeg ass
(Sche odonus p a ensis (Huds.) P. Beau ex. Lolium p a ense) a ec s
body mass and popula ion size o sibling oles (Mic o us le is Mille
ex M. ossiaeme idionalis Ogne ) and whe he he E+die in luences
he ulne abili y o oles o p eda ion by hei mos impo an
na u al enemy, he leas weasel (Mus ela ni alis ni alis L.). Because
leas weasels a e known o be ol ac o y hun e s [17], we also
examined whe he leas weasels a e able o dis inguish ol ac o y
PLoS ONE | www.plosone.o g 1 Ma ch 2010 | Volume 5 | Issue 3 | e9845
cues o oles ed on E+and E2die s. Based on p e ious s udies we
p edic ed ha i) consump ion o myco oxic E+g ass has di ec
nega i e e ec s on oles and ha ii) hese e ec s in luence he
ulne abili y o oles o hei mos impo an na u al enemy, he
leas weasel (Mus ela ni alis ni alis L.). Fu he mo e, i endophy e-
con aining die a ec s he chemical composi ion o ole u ine, as
demons a ed by Hui u e al. (2008), we p edic ha iii) ol ac o y
hun ing leas weasels migh be able o disc imina e and p e e he
scen o weake ole p ey ed on E+g ass om hose ed on E2
g ass.
Resul s
Consump ion o E+g ass did no ha e nega i e e ec s on ole
popula ion size o body mass. In he ole popula ion expe imen
he es ima ed median di e ence in he minimum numbe o oles
ali e was only 0.5 indi iduals (95% CI o di e ence = 26–+7;
p = 1.0), in a ou o he E2g ass popula ions (means and
s anda d de ia ions, oles ali e: E+13.269.07, E21365.15) In
he ole body biomass expe imen , emales ed on E+g ass had on
a e age 0.2 g (95% CI o di e ence = 21.0 – +0.7; p = 0.66)
lowe and males 0.3 g (95% CI = 20.4 – +1.1; p = 0.35) highe
body mass compa ed o oles ed on E2g ass (means and
s anda d de ia ions, ole body mass: E+ emale 21.3263.10, E2
emale 21.4962.75, E+male 25.6663.64 and E2male
25.3163.66). Voles main ained on E+g ass exhibi ed lowe
mobili y in he p eda ion expe imen han E2 oles (es ima ed
median di e ence in he ac i i y o oles = 13.3 bel c ossings/
hou in a ou o E2 oles, 95% CI = 2.7–35.4; Fig. 1).
Leas weasels we e mo e likely o cap u e oles ed on E2g ass
han oles ed on E+g ass (14 oles ed on E2cap u ed e sus
h ee oles ed on E+cap u ed in he 17 success ul ials; Fig. 2).
Howe e , ac i i y o oles did no explain he suscep ibili y o oles
o leas weasel p eda ion, no did ole sex, leng h o he eeding
pe iod o body mass di e ence be ween he oles ed on E+and
E2die s in he beginning o he expe imen (Table 1). Leas
weasels appea ed unable o dis inguish be ween he ol ac o y cues
o oles ed on he wo g ass ypes; 10 leas weasels chose he
bedding o a ole ed on E+g ass and 11 chose he bedding o a
ole ed on E2g ass (Fig. 2).
Discussion
Al hough E+g asses a e commonly hough o be chemically
p o ec ed agains he bi o es [7], [16], ou esul s wi h sibling oles
and meadow escue do no suppo his no ion unanimously.
Con a y o ou p edic ions, we did no ind nega i e e ec s o an
E+die ei he on popula ion size o body mass o sibling oles. I
appea s ob ious ha Neo yphodium-in ec ed meadow yeg ass is no
pa icula ly oxic o sibling oles, a leas wi hin a ime ame o a
ew mon hs. Endophy e-in ec ed meadow yeg ass has p e iously
been shown o dec ease body mass in a closely ela ed ole species,
he ield ole (Mic o us ag es is L.) in labo a o y condi ions [11]. I is
he e o e plausible ha ole ance o loline myco oxins a ies
among di e en e eb a e he bi o e species. The disc epancy
be ween his and ea lie s udies may also s em om a ia ion in
myco oxin p oduc ion, which is known o be dependen on
en i onmen al condi ions [7], [16].
Con a y o ou p edic ions, leas weasels p eyed mo e o en on
oles ed wi h E2g ass han on oles ed wi h E+g ass. Voles ha
had consumed E2g ass we e also mo e mobile han oles ha
had consumed E+g ass. Al hough high mobili y is o en associa ed
wi h inc eased p eda ion isk [18], he deg ee o ole mobili y was
un ela ed o he p ey selec ion beha iou o leas weasels in ou
expe imen . Reduced mobili y migh be expec ed i myco oxins
had educed he physiological well-being o oles o a poin o
apa hy. Howe e , his is no plausible in he ligh o ou
expe imen s, as oles did no lose body mass o show educed
popula ion g ow h.
Voles exhibi an a ay o beha iou s in hei a oidance o
p eda o s. Many o hese a e ela ed o mobili y, o example
leeing and eezing [19]. The la e beha iou was equen ly
Figu e 1. Mobili y o oles. Di e ences in mobili y o sibling oles as de ined by numbe s o bel c ossings pe hou o oles ed on endophy e
in ec ed (E+) o endophy e ee (E2) g asses. The uppe and lowe bounda ies o he box indica e he uppe and lowe qua iles, espec i ely. The
ho izon al line deno es he median. Ve ical ba s ep esen he ails o he dis ibu ion. Medians o he g oups a e connec ed wi h do ed line. Filled
ci cles ep esen mean alues. Mild ou lie s a e ma ked wi h open ci cles. The numbe o eplica es is 24.
doi:10.1371/jou nal.pone.0009845.g001
G ass Endophy es in Food Chain
PLoS ONE | www.plosone.o g 2 Ma ch 2010 | Volume 5 | Issue 3 | e9845
obse ed in encoun e s be ween E+ oles and weasels, so i is
plausible ha educed mobili y was ela ed o eezing unde
p eda ion isk. Fu he mo e, as weasels did no di e en ia e
be ween odo s o oles main ained on he di e en die s, we ega d
di e ences in he oles’ an ip eda o y beha iou he mos
pa simonious explana ion o he obse ed pa e ns in weasel
p ey selec ion. Howe e , his easoning is indica i e a bes , as
un o una ely speci ics o ole a oidance beha iou we e no
eco ded. We also canno conclude how ole mobili y o e all,
ega dless o ea men , was a ec ed by he p esence o p eda o s,
since mobili y was no measu ed in he absence o p eda o s in he
sys em. Fu he mo e, eezing may ha e a ec odou compounds
in bedding and we a e indeed unawa e o whe he he oxic
compounds we e ansmi ed o he u ine.
Conclusions
In ou s udy we we e able o demons a e indi ec posi i e
e ec s o mic obial plan symbion s on a e eb a e he bi o e ia
educed p eda ion. Simila e ec s ha e been p e iously demon-
s a ed wi h in e eb a e p eda o s and pa asi es as na u al
enemies in ood webs whe e he bi o es eed on endophy e
in ec ed plan s [14], [15], [20]. In cases whe e species can ole a e
myco oxins p oduced by he endophy e and a e less a isk o
p eda ion due o endophy e consump ion, he ne e ec o he
endophy e on he hos g ass will be nega i e. The e o e he
adi ional iew o endophy es as de ensi e plan mu ualis s may
no hold i a hi d ophic le el is included. Thus, ou esul s
p o ide e idence ha he na u e o he ela ionship be ween g ass
endophy es and hei hos s may ange om mu ualism o
pa asi ism depending on he complexi y o he ood web.
Ma e ials and Me hods
E hics s a emen
All p ocedu es in ol ing oles we e ca ied ou in acco dance
wi h he Ac on he Use o Animals o Expe imen al Pu poses
Figu e 2. P eda ion and ole odou p e e ence o weasel. The e ec s o endophy e in ec ed (E+) and endophy e ee (E2) g ass die s on
p eda ion and ole odou p e e ence o leas weasel. Es ima ed pe cen ages o cap u ed oles (n = 17) and odou p e e ence (n = 21).
doi:10.1371/jou nal.pone.0009845.g002
Table 1. Resul s o i ing logis ic eg ession models o he da a o leas weasel p eda ion.
Explana o y a iable Coe icien ^
bb P- alue o H
0
:b~~0Odds Ra io ORðÞ*
)
95% CI o OR{
)
Di e ence be ween E- and E+
oles in he numbe o bel c oss-
ings pe hou ( = ac i i y
E2
2ac i i y
E+
)
0.01 0.25 1.01 0.99–1.04
Sex o ole 20.60 0.65 0.55 0.00–6.04
Leng h o he eeding pe iod (days) 0.14 0.19 1.15 0.95–1.40
Di e ence be ween E2and E+ oles
in weigh ( = weigh
E2
2weigh
E+
)
0.12 0.81 1.13 0.42–3.22
*)The es ima ed odds a io OR~exp ^
bb

:Fo quan i a i e explana o y a iables 100 OR{1ðÞindica es he pe cen change in he odds o E+ ole being cap u ed o
each 1-uni inc ease in he explana o y a iable. Fo example, o e e y one day inc ease in he leng h o he eeding pe iod, he odds o E+ ole being cap u ed
inc eases by 15%. Fo ca ego ical a iable sex he OR o 0.55 implies ha o emales he odds o E+ ole being cap u ed is 0.55 imes he odds o males, i.e. 45% lowe .
{)The 95% con idence in e al o he OR indica es he ange o alues wi hin which he odds a io om 95 o 100 simila s udies would be expec ed o all. The 95% CI
also indica es he p ecision o he es ima ed OR.
doi:10.1371/jou nal.pone.0009845. 001
G ass Endophy es in Food Chain
PLoS ONE | www.plosone.o g 3 Ma ch 2010 | Volume 5 | Issue 3 | e9845
es ablished by he Minis y o Ag icul u e and Fo es y, Finland.
The s udy was app o ed and supe ised by he Animal
Expe imen Commi ee o Finland (License numbe : STH393A).
Species
Meadow yeg ass, he expe imen al plan species, is one o he
mos impo an o age g asses in Finland. I is a na i e g ass
species in Eu ope which occu s commonly ou side o ag onomic
use in meadows, oadsides and was elands in Finland [21]. Se e al
widely used meadow yeg ass cul i a s in Finland a e commonly
in ec ed Neo yphodium uncina um endophy e [22], which g ows
sys emically in all pa s o he hos plan . N. uncina um p oduces
lolines which may cause a iable esponses in in e eb a es and
small e eb a es [7], [11] bu he loline appea s o be non- oxic o
la ge mammal he bi o es [23].
The sibling ole is a common and widely dis ibu ed species in
sou he n and wes e n Finland. The indi iduals used in he
expe imen we e labo a o y-bo n indi iduals, whose pa en s we e
apped om na u al popula ions in nea by ields o MTT
Ag i ood Resea ch Finland, Jokioinen (60u489150N, 23u299
100E), in au umn 2006. P io o he expe imen , oles we e
housed in ca. 60640640 cm
3
cages (3–5 same sex indi iduals in a
cage) and p o ided wi h ad libi um po a oes, wa e and wice a week
wi h endophy e ee esh g ass cu om he wild. Bedding was
p o ided in he o m o wood sha ings and hay. Tempe a u e in
he labo a o y was ca. 20uC and pho ope iod 16 h ligh : 8 h da k.
The leas weasel is a common specialis p eda o o oles and
hei single mos impo an sou ce o mo ali y in na u al
popula ions [24], [25]. The leas weasels used in he expe imen
we e ei he i s gene a ion labo a o y-bo n indi iduals om he
Konne esi Resea ch S a ion o he Uni e si y o Jy a¨skyla¨,
Finland, o apped om he wild bu kep unde simila
condi ions like lab-bo n ones o weeks be o e he expe imen .
P io o he p eda ion and ol ac o y expe imen s, he leas weasels
we e housed indi idually in 60680660 cm
3
cages in an ou doo
shel e and p o ided wi h a oos e chick pe day wi h one as ing
day a week, and occasionally oles o he gene a Mic o us and
Myodes. Bedding was p o ided in he o m o wood sha ings and
hay.
Vole popula ion size
The expe imen al ield was es ablished o s udy he impo ance
o endophy es on he popula ion de elopmen o oles. Seeds
(cul i a ‘Kaspe ’) we e ob ained in a ious seed lo s om seed
p oduc ion a ms ia he Plan P oduc ion Inspec ion Cen e,
Loimaa, Finland. As samples, we s ained 50 seeds pe seed lo and
examined hem mic oscopically o endophy e s a us [26]. We
chose wo seed lo s o he expe imen : one unin ec ed (E2,0%
endophy e equency) and he o he in ec ed (E+, 79% endophy e
equency). We sowed E+and E2seeds in a ield in i e plo pai s
(each plo 39625 m
2
) so ha we andomized E+and E2
ea men s sepa a ely wi hin each pai . The ield was es ablished
in Jokioinen in May 2006. Each plo was su ounded wi h a shee
me al ence in o de o keep he expe imen al oles inside and
oles o na u al popula ions and small mammal p eda o s ou o
he expe imen al a eas. The shee me al was embedded 60 cm
below g ound while 60 cm emained abo e g ound. Be o e
sowing, he ield was e ilized wi h cow manu e (30 000 kg/ha)
and again in June 2007 wi h a comme cial e ilize [16:9:22
(N : P : K) wi h mic onu ien s, Kemi a, p oduc numbe :
0647334]. All o he plan s excep o meadow yeg ass we e
egula ly oo ed up om he ield du ing he expe imen .
Endophy e in ec ion s a us o nine plan indi iduals pe ield
plo was e i ied be o e he expe imen using immunoplo assay o
de ec monoclonal an ibodies speci ic o Neo yphodium (Phy osc een
Immunoplo Ki #ENDO7973, Ag inos ics, Wa kins ille, Geo -
gia, USA). Alkaloid ex ac s o he plan s we e analysed [27], [28]
and E+plan s we e de ec ed o ha bo ac i e endophy e in ec ions
p oducing loline alkaloids.
Vole indi iduals (50 males and 50 emales) we e andomly
selec ed om a la ge pool o males and emales, all o which we e
sexually ma u e and had a body mass o .20 g. Indi iduals we e
andomly assigned ei he o an E+o an E2 ea men .
The ea e , in Augus , i e male and i e emale oles we e
eleased in o each o he en enclosu es. Fou and a hal mon hs
la e , which app oxima es he annual leng h o he ep oduc i e
pe iod o oles in Finland, ole popula ion sizes we e es ima ed
using Ugglan mul iple li e cap u e aps (G ahnab, Sweden). Fi e
aps we e placed in each enclosu e unde plywood shel e boxes
o educe exposu e o he elemen s. The aps we e bai ed wi h
ca o s and checked wice a day o h ee days. Popula ion size was
es ima ed o each enclosu e wi h he minimum numbe ali e
–me hod [29].
Vole body mass
Voles (72 indi iduals) we e selec ed om a la ge pool o males
and emales, all o which had a body mass o .16 g, and housed
singly in cages. The selec ed oles we e assigned in o 36 pai s (15
emale and 21 male pai s) based on simila i y in body mass. A his
s age, oles we e p o ided ad libi um po a o and wa e . On he
ollowing day, we assigned he ole indi iduals andomly ei he o
an E+o an E2die ea men wi hin each pai and eco ded
hei body mass o he nea es 0.1 g wi h elec onic scales. The
mean di e ences in he body mass (body mass
E2
2body mass
E+
)
o emales and males (and s anda d de ia ions o he di e ences)
we e 0.01 g (0.73 g) and 0.03 g (0.44 g), espec i ely. A e his,
we emo ed po a oes om he cages and p o ided he co e-
sponding expe imen al die s, namely ad libi um esh meadow
yeg ass h ee imes a day cu om espec i e E+o E2plo s in
he ield (see ‘‘Vole popula ion size’’). The body mass o he oles was
again eco ded when he oles had been main ained on he
expe imen al die o se en days.
P ey p e e ence o weasel
P io o he p eda ion expe imen six emale and 18 male ole
pai s (one ed on E+g ass and one ed on E2g ass wi hin each
pai ) we e ed wi h he expe imen al die s as in he body mass
bioassay o 7–30 days. The leng h o he eeding pe iod a ied
be ween ole pai s due o logis ical easons bu was eco ded o
use a co a ia e in he analyses.
The expe imen was conduc ed a he Konne esi Resea ch
S a ion o he Uni e si y o Jy a¨skyla¨ (62u379400N, 26u179150
E), Finland, in an expe imen al enclosu e (10610 m
2
) on a ield
na u ally ege a ed by meadow plan species ( he ield did no
include meadow yeg ass). The enclosu e was di ided in o six
sec o s sepa a ed by ca. 30 cm wide sho -g ass bel s (cu 2 cm
abo e g ound su ace) o enable moni o ing o ole mobili y. One
expe imen al ial consis ed o exposing bo h a ole ed on E+and
a ole ed on E2g ass om he same eeding pai o leas weasel
p eda ion in he enclosu e. Thus, a eplica e consis ed o a pai o
oles ha we e bo h he same sex, simila in weigh in he
beginning o he expe imen and had been on he expe imen al
die o he same pe iod o ime. The oles we e ma ked wi h ibe
s ips o di e en colo s (15 cm long, 2 cm wide) bound a ound
he pel is, whe e i does no hinde mo emen s, and eleased in o
he cu middle bel be ween he sec o s o he enclosu e. We
andomized he s ip colo be ween oles ed on E+and E2g ass
in e e y eplica e. Simul aneously wi h eleasing oles, we placed a
G ass Endophy es in Food Chain
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leas weasel in an Ugglan li e ap in he same middle bel whe e
he oles we e eleased. The leas weasel was eleased om he
ap i e minu es a e eleasing he oles. We obse ed and
eco ded he numbe o bel c ossings o he oles in he enclosu e
un il one o he oles was cap u ed by a leas weasel. A single ial
las ed om 15 min o 9 hou s. Se en ou o he 24 ials had o be
e mina ed o ex e nal easons (da kness a nigh , hea y ain e c.)
be o e he weasel had cap u ed a ole. Di e en oles and leas
weasels we e used in e e y eplica e. A e each ial we apped
he su i ing ole (o bo h oles i a eplica e had o be e mina ed)
and emo ed hem om he enclosu e back o he labo a o y.
Odou p e e ence o weasel
We s udied whe he leas weasels, ha a e known o be ol ac o y
hun e s [19], dis inguish and p e e ol ac o y cues o oles ed wi h
E+g ass om oles ed wi h E2g ass. As a sou ce o odou we
used u ine- and aeces-soaked ole bedding ma e ial om cages o
oles ha had been eeding on ei he E2o E+g ass o he
weasel p eda ion expe imen s (see abo e). The collec ed bedding
ma e ial was s o ed in ai - igh plas ic bags a 222uC o ca. h ee
mon hs. The bedding was hawed in he bags a oom empe a u e
be o e he expe imen .
The ole odou expe imen was ca ied ou in a Y-maze a ena
[19], which consis ed o h ee anspa en Pe spex (Pe spex,
Ro e dam, The Nea he lands) plas ic ubes o 80 mm inside
diame e and 80, 60 and 60 cm leng h, o ming a Y. The weasel
en e ed he 80 cm long ube and came o a bi u ca ion o ,60u.
The e i had o choose o con inue in o ei he o he 60 cm ubes
un il i eached a a ge ‘‘nes box’’ con aining ole odou a he
end o he ube. The nes boxes we e small labo a o y cages
(25610610 cm
3
) co e ed wi h a Pe spex oo . The ends o he
Pe spex ubes we e sepa a ed om he nes box by a Pe spex doo
wi h 12 holes o 4-mm diame e o allow ai low.
In each nes box we placed a 2610610 cm
3
wi e mesh baske
illed wi h ole bedding om ei he he E+o he E2 ea men .
We andomized E+and E2bedding om he same ole eeding
pai o ei he end o he Y-maze p io each ial. Thus, we
collec ed he E+and E2bedding in each eplica e om oles ha
had been on he expe imen al die o he same amoun o ime.
The en ance o he ube was a wooden box, in which we
acclima ized he weasel o i e minu es p io o he ial. The box
was sepa a ed om he ube by a Pe spex doo ha could be
opened om ou side he expe imen a ion oom wi h a mono il-
amen line. The doo had holes o allow ai low om he a ena o
he weasel box du ing he acclima iza ion pe iod. Abo e he a ena
we moun ed in a ed ligh sou ces and a came a ha was
connec ed o a moni o in an adjacen oom whe e he beha io
o he weasel was moni o ed on sc een. Al oge he , we es ed nine
emale and 12 male weasels. All weasels slowly app oached he
ube and selec ed one o he ubes a he bi u ca ion. The es
ended when he weasel eached he end o ei he b anch o he Y-
ube and sni ed he holes a he doo sepa a ing he ube om he
nes box. A e each ial we cleaned all pa s o he a ena wi h
wa e and ca. 50% e hanol.
S a is ical me hods
The esponse a iables used in he s a is ical analyses and o he
de ails o he expe imen s a e summa ized in Table 2.
We analyzed he body mass o oles sepa a ely o emales and
males because a ia ion in male body mass was highe han in
emales. The E+and E2die ea men s we e compa ed using a
pai ed - es and 95% con idence in e al (CI) o he mean
di e ence [30]. The analyses we e pe o med by he MIXED
p ocedu e in e sion 9.1.3 o he SAS/STAT so wa e. The es o
he da a we e analyzed h ough exac s a is ical me hods because
he da a we e small and non-no mally dis ibu ed. We based he
s a is ical analyses o leas weasel p eda ion on logis ic eg ession
models o bina y da a. Le Yi~1i E+ ole was cap u ed and
Yi~0i E2 ole was cap u ed in pai i. Each bina y ou come
a iable Yiis assumed o ha e a Be noulli dis ibu ion wi h
pa ame e pi, whe e piis he p obabili y o E+ ole being cap u ed
in a pai i. The alue pi~0:5indica es an equal p obabili y o
cap u e o E+and E2 oles. When examining whe he leas
weasels p e e E+ oles o e E2 oles as p ey, he logis ic
eg ession model had he ollowing o m:
log pi=1{pi
ðÞ½~að1Þ
whe e ais a cons an and he a io pi=1{pi
ðÞis he odds o E+
Table 2. Summa y o he de ails o he expe imen s.
Expe imen Numbe o pai s Numbe o oles
Males Females C i e ia o pai ing
Response a iable in he s a is ical
analysis
Vole popula ion size 5 25 25 Same sex, app oxima ely
he same ini ial body mass.
Di e ence in he numbe o oles
be ween E+and E2in a enclosu e
pai a e ou and a hal mon hs.
Vole body mass 36 42 30 Same sex, app oxima ely he
same ini ial body mass.
Di e ence in body mass (g)
be ween E+and E2in a ole
pai a e eeding he oles se en days.
P ey p e e ence o weasel 24 36 12 Same sex and leng h o he
eeding pe iod, app oxima ely
he same ini ial body mass.
Di e ence in he numbe o bel
c ossings pe hou be ween E+
and E2in a ole pai
P ey p e e ence o weasel 17
*)
24 10 Same sex and leng h o he
eeding pe iod, app oxima ely
he same ini ial body mass.
E+ ole cap u ed in a pai
o oles ed on E+and
E2(yes, no)
Odou p e e ence o weasel 21
*)
42 0 Same sex and leng h o he
eeding pe iod, app oxima ely
he same ini ial body mass.
Bedding o E+ ole chosen in a
E+and E2bedding pai (yes, no)
*)Also he numbe o leas weasels.
doi:10.1371/jou nal.pone.0009845. 002
G ass Endophy es in Food Chain
PLoS ONE | www.plosone.o g 5 Ma ch 2010 | Volume 5 | Issue 3 | e9845

ole being cap u ed in a pai i. On i ing he model o he da a, an
es ima e o a,^
aa, is ob ained, and he es ima ed cap u e p obabili y
(p opo ion) ^
pp€
ii ~exp ^
aaðÞ=1zexp ^
aaðÞ
g
. The 95% con idence
limi s o piwe e compu ed simila ly om he es ima ed limi s
o a. When examining he dependence o pion he alues xio
each po en ial explana o y a iable (one a a ime), he model was
o he ollowing o m:
log pi=1{pi
ðÞ½~azbxið2Þ
whe e aand bwe e he unknown pa ame e s es ima ed by he
da a. We i ed he models by using he app oach o condi ional
exac in e ence [31], and pe o med he analyses wi h e sion 8 o
he LogXac so wa e. The analysis o he bina y choice da a o
ole odou was based on he co esponding model han he model
(1) abo e. We es ed he di e ence in he amoun o cap u ed oles
pe enclosu e be ween E+and E2g asses and also he di e ence
in he bel c ossings o oles/hou by using he exac Wilcoxon
signed anks es , and es ima ed he median di e ence and i s 95%
con idence in e al (CI) by he Hodges-Lehmann p ocedu e [32].
We pe o med he analysis wi h e sion 8 o he S a Xac so wa e.
Acknowledgmen s
We hank Pa¨i i Leh onen, Maija Sinisalo, Tanja Jyla¨nki, Lenka T eba icka
and Ma ko Haapakoski o hei ield assis ance and Ag i ood Resea ch
Finland Jokioinen and Konne esi Resea ch S a ion o use o labo a o y
and ou doo enclosu e acili ies o he expe imen s.
Au ho Con ibu ions
Concei ed and designed he expe imen s: SS JS OH MH HY KS.
Pe o med he expe imen s: SS JS. Analyzed he da a: EK. Con ibu ed
eagen s/ma e ials/analysis ools: SS JS HY. W o e he pape : SS JS OH
MH HY KS.
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