Himanene al. BMC Ecol (2015) 15:16
DOI 10.1186/s12898-015-0048-6
RESEARCH ARTICLE
U ilizing associa ional esis ance
o biocon ol: impac ed by empe a u e,
suppo ed byindi ec de ence
Sa i J Himanen1*, Thuy Nga T Bui2, Mengis u M Maja2 and Ja mo K Holopainen2
Abs ac
Backg ound: Associa ional he bi o e esis ance is po en ia ed by neighbou ing he e ogenic plan species ha
impac a ocal plan ’s a ac ion o he bi o es o he damage ha hey cause. One mechanism o con e associa ional
esis ance is belie ed o be exposu e o neighbou -emi ed ola iles, he ecei e s o which ange om in a- and
in e speci ic neighbou plan s o highe - ophic-le el insec s. In p e ious s udies he passi e adso p ion o neighbou -
emi ed semi ola iles has been epo ed, bu li le is known ega ding he mechanisms and ecological consequences
on he ecei e plan and i s associa ed bio a. To u ilize ola ile-based associa ional esis ance o ag icul u al applica-
ions, i is impe a i e o know i s e ec i eness unde a ying diu nal empe a u es and whe he he bi o e na u al
enemies, p o iding biological con ol, a e impac ed. Mimicking a ying diu nal empe a u es in a labo a o y se -up,
we assessed how he i ophic model sys em B assica ole acea a . i alica (b occoli)–Plu ella xylos ella (c uci e special-
is he bi o e)–Co esia es alis (endopa asi oid o P. xylos ella) is in luenced by exposu e o he na u al semi ola ile
emi e plan Rhododend on omen osum Ha maja.
Resul s: Rhododend on omen osum-exposed B. ole acea was less suscep ible o P. xylos ella o iposi ion a bo h
nigh - ime (12°C) and day- ime (22°C) empe a u es and less a ou ed and damaged by P. xylos ella la ae a 12°C.
Exposu e did no in e e e wi h indi ec de ence, i.e. a ac ion o he na u al enemy C. es alis on hos -damaged, R.
omen osum-exposed B. ole acea unde 22°C, while he e was a educ ion in a ac ion (ma ginal p e e ence owa ds
hos -damaged B. ole acea) unde 12°C.
Conclusions: The abili y o R. omen osum exposu e o ende associa ional esis ance agains an ag icul u ally impo -
an B assica he bi o e P. xylos ella wi hou se e ely comp omising he specialis pa asi oid C. es alis hos loca ion
encou ages u he s udies on he po en ial o using his na u ally abundan plan o biocon ol. The gene ali y o ou
inding on empe a u e as a po en ial egula ing mechanism o he e icacy o semi ola ile emi e -based associa ional
esis ance owa ds specialis pes la al damage should be u he s udied in na u al and ag icul u al associa ions. Ou
s udy emphasizes he need o de elop echniques o compa e ola iles a he lea e sus ai in e ace and associa e hei
appea ance and ecological ole wi h imes o ac i i y and le el o specialisa ion o he bi o es and hei na u al enemies.
Keywo ds: Associa ional esis ance, He bi o y, Hos loca ion, Indi ec de ence, Pa asi oids, Semi ola iles, Tempe a u e
© 2015 Himanen e al. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License
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Backg ound
Associa ional esis ance, he p o ec ion o a ocal plan
om he bi o y ia neighbou ing a he e ogenic species
[1, 2], is an example o how plan –plan in e ac ions
Open Access
*Co espondence: [email p o ec ed]
1 Na u al Resou ces Ins i u e Finland (Luke), Managemen and P oduc ion
o Renewable Resou ces, P oduc ion Sys ems, Lönn o inka u 5,
FI-50100 Mikkeli, Finland
Full lis o au ho in o ma ion is a ailable a he end o he a icle
migh a ec plan de ence agains bio ic a acke s. Such
neighbou -media ed esis ance is belie ed o ope -
a e ia mul iple s a egies: he “ esou ce concen a ion
hypo hesis” is based on lowe hos plan densi y and
educed accessibili y o hos plan s by a acking he -
bi o es in mixed communi ies [3], and he “enemies
hypo hesis” p edic s ha na u al enemy abundance is
inc eased when ood sou ces and shel e a e p o ided
by neighbou s, he eby educing he bi o y [4]. The
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Himanene al. BMC Ecol (2015) 15:16
“semiochemical di e si y hypo hesis” elies on ola iles
dis u bing he bi o e ol ac ion, which is used in hos
loca ion and ep oduc ion [1]. Ai bo ne plan ola iles
a e impo an in ochemical cues o associa ed spe-
cialis species bio a in pa icula [5]. The hos loca ion
by insec s [6] and he ac i a ion o a plan ’s inducible
de ences o a ac na u al enemies o he bi o e a ack-
e s [7] o p o ide indi ec de ence a e pa ly egula ed
by ola ile signals. The complexi y o ola ile signal-
ling in mul i ophic sys ems is illus a ed by nume ous
ecen indings sugges ing no el mechanisms o ola-
ile-media ed in e ac ions. Such examples include he
passi e adso p ion and e- elease o neighbou s’ de en-
si e ola iles wi h semi ola ile cha ac e is ics, i.e. low
apo iza ion a e o he gaseous phase, on he su ace o
in e speci ic oliage [8].
Associa ional esis ance as media ed by plan ola iles
has ecen ly been demons a ed o a numbe o associa-
ions [8–11]. Associa ional esis ance in gene al has been
u ilised o decades in ag icul u e in he o m o mixed
c opping wi h he aim o educing pes damage [12, 13].
Only in app oxima ely 50% o cases, has pes p o ec-
ion by neighbou s been e ec i e in mixed cul u es [14].
Speci ic a oma ic plan ola iles ha e o en been es ed
wi h success o he pu pose o epelling o masking hos
cues (i.e., ol ac o y camou lage) owa ds he bi o es in
a ious ypes o ag oecosys ems (e.g., [15, 16]), al hough
he ac i e ole o he chemical componen has also been
doub ed, and opinions ha e been aised in a ou o
companion c ops ac ing as pu e physical ba ie s [17].
Va ia ion in he en i onmen al condi ions and speci ic-
i y by he s udied plan -he bi o e sys em can complica e
e ealing any gene al unde lying mechanisms p o iding
associa ional esis ance alone o in conce wi h o he
simul aneously ac ing in e ac ions [9]. Much emains o
be e ealed ega ding mul i ophic in e ac ions ha a e
media ed by ola iles, pa icula ly on hei en i onmen-
al pe sis ence [18–21], po en ial ade-o s be ween he
many ecological e ec s o which hey con ibu e [22],
and hei co-e olu ion and adap i e alue [23]. The u i-
liza ion o associa ional esis ance o biocon ol in he
o m o ap c ops [24, 25], epellen o hos odou -mask-
ing companion plan s [16], in e c opping [15], o syn-
he ic compounds eleased om dispense s [26] migh
also bene i g ea ly om an imp o ed unde s anding o
he mechanisms egula ing ola ile-media ed associa-
ional esis ance.
Unde s anding he concu en oles and in e play
be ween di e en ola ile-media ed in e ac ions (wi h
he same compound changes po en ially impac ing
mul iple in e ac ions simul aneously) is essen ial when
aiming o u ilize ola iles in pes p o ec ion. Fo exam-
ple, a ade-o migh exis i he adso bed compounds,
which a e emi ed by he neighbou and con e associa-
ional esis ance, in e e e wi h he a ac ion o na u al
enemies o supp ess he bi o e p essu e [22]. Thus, when
designing biocon ol applica ions based on ola iles,
impac s on bo h he he bi o es [16] and hei na u-
al enemies [27–29] and, op imally, he pe sis ence o
impac s unde abio ic a ia ion as encoun e ed in ield
en i onmen s should be conside ed. Neighbou ola-
iles migh a iably impac na u al enemies, as ecen ly
shown o ladybi ds on po a o exposed o onion ola iles:
TMTT was an a ac an , whe eas (E)-ne olidol ac ed as
a epellen [30]. I he neighbou -emi ed compounds a e
semi ola ile in na u e, he impac migh be e en s onge
o mo e p olonged.
The s abili y and unc ional dis ance o associa ional
esis ance is ano he puzzling issue also when conside -
ing ai bo ne plan ola iles in ield condi ions [21, 31].
Because insec ol ac ion is belie ed o be ema kably
sensi i e [6], small changes in ola ile signals may e eal
o disguise a g ea deal o in o ma ion, making ola ile-
based de ences ulne able o in e e ence [19]. Associa-
ional esis ance occu s in na u e ac oss a ange o abio ic
a ia ion, including diu nally and seasonally a ying
empe a u e and high p ecipi a ion as well as d ough
episodes and changes in a mosphe ic cons i uen s, such
as ca bon dioxide. O hese, he p e ailing empe a u e
is well known o i s ole in he con ol o di usion and
ola ili y o ola ile compounds and hei longe i y in
he a mosphe e [32, 33]. The majo C5 ola ile isop ene
is syn hesized and emi ed in g ea e amoun s unde
inc easing empe a u es [34]. Fo o he plan ola iles,
such as mono e penes and sesqui e penes, he empe a-
u e dependence o emission is less ob ious. Bo h physi-
ological and physicochemical egula ion akes place in
plan oliage and a he lea -a mosphe e in e ace [35].
In addi ion, he abili y o s o e hese compounds o
hei p ecu so s in specialized lea s uc u es (e.g. sec e-
o y ca i ies o ichomes) and hei inducibili y upon
abio ic and bio ic s esses c ea es a iance om di ec
esponsi eness o inc easing empe a u e o modelling
hei species-speci ic emission po en ial [20, 35]. In he
case o sesqui e penoids, he e m semi ola ile has been
used o desc ibe hei less ola ile na u e and s ickiness
o su aces [36, 37]. Fo such compounds, empe a u e
in luences hei e- elease om su aces [37]. Despi e he
ole o empe a u e in ola ile emissions, li le is known
ega ding how empe a u e in luences he ac ual in o -
ma ion-ca ying capaci y o plan ola iles; o example,
could associa ional esis ance as media ed by ola iles
be subs an ially imp o ed o hampe ed by empe a u e
changes?
B assica plan s hos a ple ho a o economically damag-
ing he bi o es [12, 38]. Thus, we chose B assica ole acea
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Himanene al. BMC Ecol (2015) 15:16
a . i alica (b occoli, B. ole acea om he e on)–Plu ella
xylos ella–Co esia es alis (Haliday) as he ag icul u al
i ophic model sys em o be s udied along wi h he
well-de ended pe ennial e e g een sh ub Rhododen-
d on omen osum Ha maja (p e iously Ledum palus e
L.). Rhododend on omen osum is na i e o no he n
la i udes and emi s speci ic semi ola iles [palus ol
(C15H26O), ledol (C15H26O) and ledene (C15H24)] ha a e
capable o being adso bed o neighbou ing oliage [8];
hus, his species ep esen s a s ong semi ola ile emi -
e ha is abundan in no he n en i onmen s, making i
in e es ing o es o i s po en ial o p o iding associa-
ional esis ance o use in biocon ol. The diamond-back
mo h P. xylos ella L. (Lepidop e a: Yponomeu idae) is a
majo ag icul u al pes o B assica wi h a global dis ibu-
ion [38]. The mo h mass mig a es by wind cu en s o
no he n egions and is a acked by nume ous p eda-
o s and pa asi oids, o which he la al pa asi oids, e.g.,
Co esia sp. and Diadegma sp. pa asi oid wasps, a e he
mos e ec i e o limi ing mo h popula ion g ow h [39].
Co esia es alis (Haliday) (Hymenop e a: B aconidae),
a specialis endopa asi oid pa asi izing all o he la al
ins a s o P. xylos ella, uses plan -emi ed ola iles in hos
sea ching [40].
Al hough we we e in e es ed in ad ancing knowledge
on he ecological mechanism and empe a u e sensi i i y
o associa ional esis ance, ou p ima y aim was o assess
he po en ial o using R. omen osum o biocon ol in
his s udy. The speci ic objec i es o ou s udy we e o
assess (1) whe he associa ional esis ance owa ds P.
xylos ella is con e ed by exposu e o R. omen osum in
B. ole acea; (2) whe he R. omen osum-media ed asso-
cia ional esis ance is e ec i e bo h a nigh - ime and
day- ime empe a u es (12 and 22°C upon es ing); (3)
whe he exposu e o R. omen osum in e e es wi h indi-
ec de ence, i.e., he a ac ion o he C. es alis pa a-
si oid by hos -induced ola iles, o B. ole acea; and (4)
whe he he impac o R. omen osum exposu e on indi-
ec de ence is a ec ed by empe a u e (12 e sus 22°C)
upon exposu e. Expe imen al se -ups o he s udy a e
shown in Figu e1 (see “Me hods” o de ails).
Resul s
Ve i ying he adso p ion o R. omen osum ola iles
on oneighbou ing B. ole acea
We i s e i ied ha R. omen osum (RT om he e
on) exposu e esul ed in semi ola iles being adso bed
and e- eleased om B. ole acea oliage, as p e iously
epo ed o bi ch oliage [8]. RT b anch emissions
we e domina ed by he sesqui e pene alcohol palus ol,
which comp ised 43% o he o al ola ile emissions (Fig-
u e2a). The o he main compounds ha we e de ec ed
we e β-my cene, a omadend ene and ledol (wi h 34, 10
and 9% o he o al emission, espec i ely). Palus ol was
de ec ed om emission p o ile o RT-exposed B. ole acea
a bo h exposu e empe a u es: 6 and 22°C (Figu e2b).
RT-o igina ing ledol and ledene we e emi ed only om
RT-exposed B. ole acea plan s ha we e exposed a 22°C.
No RT-speci ic compounds we e emi ed om he co -
esponding con ol B. ole acea plan s (Figu e2b). The e
we e no s a is ically signi ican di e ences be ween
con ol and RT-exposed B. ole acea in he emissions o
ola iles de ec ed om bo h ea men s, i.e., α-pinene,
δ-ca ene and 1,8-cineole (Figu e2b, P>0.05).
Responses o P. xylos ella oR. omen osum‑exposed
B. ole acea
O iposi ion by P. xylos ella was lowe on RT-exposed
han con ol B. ole acea plan s (Figu e3, F1,36=13.5,
P<0.001). A 22°C, he e we e 38% and, a 12°C, 36%
less o iposi ion on RT-exposed plan s. A highe num-
be o eggs we e laid when he es ing empe a u e du -
ing o iposi ion was 22°C compa ed o 12°C (Figu e3,
F1,36=131.6, P<0.001).
Plu ella xylos ella la ae p e e ed o eed on he con-
ol a he han he RT-exposed B. ole acea lea es a
12°C (Figu e4a, P< 0.05). This p e e ence was main-
ained h oughou all o he obse a ion ime poin s
om 30min o 24h a e elease. When he exposu e
and es ing ook place a 22°C, no signi ican di e ence
in he choice o he la ae be ween he con ol and he
RT-exposed B. ole acea lea es was de ec ed a any o he
obse a ion ime poin s (Figu e4b, P>0.05).
The lea a ea ha was consumed by P. xylos ella la -
ae was highe in he con ol han in he RT-exposed
B. ole acea lea es a 12°C (Figu e5a, =5.75, d =41,
P< 0.001). The e was no di e ence in he amoun o
lea damage be ween he ea men s a 22°C ( =1.61,
d =38, P=0.116). The numbe o eeding holes on RT-
exposed B. ole acea was smalle han ha on con ol B.
ole acea lea es a 12°C (Figu e5b, =−3.26, d =41,
P=0.002), whe eas no di e ence be ween ea men s
was ound when he es ook place a 22°C ( =−0.95,
d =39, P=0.347).
Hos loca ion byC. es alis: ole o R. omen osum exposu e
Co esia es alis emales showed no p e e ence o
in ac B. ole acea o e RT ola iles (Figu e6, χ2=0.27,
P=0.602), and only 33% o he pa asi oids made a choice
in his beha iou al es compa ison. The unc ioning o
he B. ole acea–P. xylos ella–C. es alis i ophic in e -
ac ion was e i ied by he g ea e a ac ion o C. es alis
emales owa ds hos -damaged a he han in ac B. ole -
acea ola iles (χ2=3.90, P=0.048). The pa asi oids also
p e e ed hos -damaged, RT-exposed B. ole acea o e
in ac plan s when he exposu e ook place a day- ime
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Himanene al. BMC Ecol (2015) 15:16
empe a u e (22°C) (χ2=4.83, P=0.028). No p e e ence
by C. es alis was obse ed be ween hos -damaged and
hos -damaged RT-exposed B. ole acea a his empe a-
u e (χ2=1.11, P=0.292). The pa asi oid choosing a es
we e g ea e han 60% in all h ee assays wi h hos -dam-
aged B. ole acea as an odou sou ce.
When using plan s ha we e exposed o RT a 12°C, C.
es alis p e e ed hos -damaged RT-exposed B. ole acea
plan s o in ac RT-exposed plan s wi h a ma ginal s a-
is ical signi icance (Figu e6, χ2=3.81, P=0.067). The
pa asi oid had no p e e ence o hos -damaged o e hos -
damaged RT-exposed B. ole acea (χ2=0.24, P=0.743).
Discussion
Ou esul s e ealed ha RT exposu e educed he o i-
posi ion and la al eeding o a key pes he bi o e o c u-
ci e ous plan s: he diamondback mo h P. xylos ella on
B. ole acea. A colde empe a u e (12°C) upon exposu e
was in eg al o he inc ease in la al esis ance. In addi-
ion, indi ec de ence, e alua ed he e as he a ac ion o
he specialis pa asi oid C. es alis by hos -induced ola-
iles, emained pe sis en despi e RT exposu e, al hough
indica ions o a educed esponse we e ound when he
exposu e ook place a a colde empe a u e (12°C).
Associa ional esis ance iaR. omen osum: mechanisms
o ac ion andpo en ial o biocon ol
The p o ision o associa ional esis ance by RT ola iles,
as p e iously sugges ed o a na u al neighbou (Be ula
sp.) [8], was con i med in his s udy on a B assica c op
species and owa ds a damaging global pes : he dia-
mondback mo h. In ex ac s o RT, wi h ledene, ledol and
palus ol o ming he majo i y o he composi ion, epel-
lence has been epo ed owa ds a ious a h opods [41,
42]. Ou inding ha exposu e ia ai alone was su icien
o ende inc eased esis ance owa ds bo h adul and
2. He bi o e esponses
1. Ve i ying adso p ion o R. omen osum ola iles on o B. ole acea
3. Pa asi oid esponses
R. omen osum
ola iles
R. omen osum –
exposed
B. ole acea
ola iles
Con ol
B. ole acea
ola iles
Exposu e o 24h a 6°C and
22°C
Vola iles collec ed a 22°C
Exposu e o 8 h a 22°C
ollowed by 16 h a 6°C
Exposu e o 48h
a 12°C and 22°C Co esia es alis a acon es ed a 22°C
Combina ions es ed o 22°C exposu es: Combina ions es ed o 12°C exposu es:
Expe imen al se -ups o he s udy
RT CC
Analysis o
Plu ella xylos ella
o iposi ion
O iposi ion o 24h a
12°C and 22°C
P. xylos ella la al choice and
eeding
Exposu e o 24 h a 12°C and 22°C
C RT
La al es ing o 24h
s s
s
C+Px
C RT
RT+Px
C RT
s
RT
s
s
a 12°C and 22°C
Figu e1 Expe imen al se -ups o he s udy. The s udy consis ed o h ee pa s es ing plan , he bi o e and pa asi oid esponses owa ds R. omen-
osum (RT) exposu e. Tempe a u es used in each pa a ied acco ding o he esponse o be es ed, and we e ep esen a i e o bo eal en i on-
men a ia ion in ea ly summe nigh - ime and day- ime empe a u es. In Pa 1 ( ola ile analysis), a 6°C nigh - ime minimum empe a u e e sus
day- ime 22°C was used in o de o e eal whe he a empe a u e-dependen adso p ion–deso p ion p ocess o RT semi ola iles occu s on B.
ole acea. He bi o e o iposi ion in Pa 2 was compa ed a a e age day- ime (22°C) e sus a e age nigh - ime (12°C) empe a u es, wi h equal ini ial
RT exposu e (day- ime 22°C ollowed by minimum nigh - ime empe a u e o 6°C). La al choice and eeding was es ed a s abile 12 and 22°C em-
pe a u es o allow compa ison o a 24 h pe iod o eeding a nigh - ime e sus day- ime empe a u es along wi h empe a u e- ep esen a i e RT
exposu es. In Pa 3, he day-ac i e pa asi oids we e es ed a 22°C, wi h he p io RT exposu e conduc ed a a e age day- ime (22°C) and nigh - ime
(12°C) empe a u es. Px = P. xylos ella. Pho os o P. xylos ella and C. es alis: Ja mo Holopainen.
Page 5 o 12
Himanene al. BMC Ecol (2015) 15:16
la al s ages o P. xylos ella, wi h no need o he physical
applica ion o he compounds, sugges s ha RT could be
a pes -supp essi e in e c op o B assica.
Ou s udy is no able o de e mine whe he he
inc eased esis ance ac s as an ol ac o y, as an con ac
cue o bo h. Fu he mo e, he mode o ac ion o RT
exposu e leading o associa ional esis ance on B. ole a-
cea also emains an open ques ion: he obse ed impac s
could s em om RT ola iles ha a e adso bed on o
and e- eleased om B. ole acea lea es o om B. ole a-
cea ola iles induced by RT exposu e. Because B. ole a-
cea in insic ola ile emission was low and he e we e
no indica ions o o he compounds being al e ed in he
p o ile a e RT exposu e han he RT-speci ic semi ola-
iles, which can be adso bed on o oliage [8], he la e
Figu e2 Vola ile emission om Rhododend on omen osum (a)
and con ol and R. omen osum-exposed B assica ole acea (b). The
mean ± SEM emission o indi idual ola ile compounds de ec ed a e
shown. B assica ole acea plan s we e exposed o R. omen osum o
24 h a 6 o 22°C, and ola ile emissions we e collec ed pos -exposu e
a 22°C. n = 3 in (a) and n = 4 in (b) o bo h exposu e empe a u es.
0
50
100
150
200
250
12°C 22°C
O iposion pe plan
Tempe a u e
Con ol
RT-exposed
P ea <0.001
P emp<0.001
P ea x emp=0.620
Figu e3 O iposi ion by Plu ella xylos ella on con ol and R.
omen osum-exposed B assica ole acea a 12 and 22°C. The alues
ep esen he mean numbe o P. xylos ella eggs pe plan ± SEM
based on h ee independen expe imen s (n = 4 in each expe imen ).
P alues o he main e ec s o ea men and empe a u e and hei
in e ac ion a e shown.
80 60 40 20 020406
08
0
30 min
1h
2h
4h
24h
% P. xylos ella la ae
Con ol RT-exposed
12°C
**
**
**
**
**
80 60 40 20 020406
08
0
30 min
1h
2h
4h
24h
% P. xylos ella la ae
Con ol RT-exposed
22°C
a
b
Figu e4 Plu ella xylos ella la al choice owa ds con ol e sus R.
omen osum-exposed B assica ole acea a 12 and 22°C. The pe cen -
age dis ibu ion (mean ± SEM) o la ae in choice es s pe o med
a 12°C (a) and 22°C (b) is shown. Each eplica e includes he choices
(posi ioning on ei he lea ) o i e indi idual la ae. n = 20–21 in bo h
o he independen eplica e expe imen s o which he esul s a e
summa ized he e. S a is ically signi ican di e ences in la al choice
a e indica ed by as e isks (**P < 0.01).

Page 6 o 12
Himanene al. BMC Ecol (2015) 15:16
hypo hesis is no suppo ed. The e o e, RT ola iles a e
likely candida es o passi ely p o iding inc eased esis -
ance. These pe sis en compounds, which a e emi ed in
la ge amoun s by RT, could disguise hos loca ion, o ipo-
si ion o eeding s imula ion cues, such as glucosinola e
b eak-down p oduc s o he specialis pes P. xylos ella
[39]. Al e na i ely, hese compounds migh cause di ec
epellence [41, 42]. Thus, ollow-up labo a o y wo k wi h
syn he ic palus ol, ledol and ledene is needed o e eal
he ac ing mechanism o RT-exposu e-media ed asso-
cia ional esis ance.
The po en ial impo ance o RT semi ola iles is u he
suppo ed by he obse ed empe a u e dependence o
he esis ance owa ds la al eeding: a 12°C, he RT spe-
ci ic semi ola iles a e s uck on su aces. In [37], an e o
o es ablish a i icial b occoli lea es, i.e., a wax laye on
a mic oscope slide, o de ec ing adso p ion o induced
ola iles, showed how induced sesqui e penes in pa -
icula we e adso bed on o he wax and hen e- eleased
upon inc easing empe a u e. As epicu icula wax is he
su ace encoun e ing RT ola iles unde ae ial exposu e,
hei mode o ac ion could in ol e an in e ac ion wi h
wax chemicals. Plu ella xylos ella p e e s o o iposi
on glossy e sus waxy lea es, and high epicu icula wax
bloom makes cabbage mo e esis an o he pes [43].
The chemical composi ion o he wax a ies be ween he
ypes, i.e., he p opo ion o alkanes and ke ones is lowe ,
while ha o a y acids and i e penoids is la ge on
glossy wax ype lea es [44]. In addi ion, also P. xylos ella
la ae a e impac ed by he wax p ope ies: hey spend
mo e ime sea ching and less ime eeding on glossy cab-
bages [44]. Thus, one hypo hesis o he mechanism o
he pa ly empe a u e-dependen RT-media ed esis -
ance in ou s udy is he chemical composi ional changes
ha a e induced by RT ola iles in B. ole acea lea wax.
In biocon ol, companion c ops wi h epo ed impac s
on ag icul u ally impo an he bi o es ha e o en
included a oma ic- ola ile-emi ing he bs [45, 46], indi-
ca ing ha he po en ial semi ola ile cha ac e is ics ha
hei main ola iles possess migh be mo e gene ally
impo an o he e ec i eness o associa ional esis ance.
Howe e , he e ec i eness o he b in e c opping impac -
ing he bi o e abundance a ies [12, 46]. Addi ional a i-
abili y complica ing unde s anding o he ole o ola iles
o associa ional esis ance is c ea ed by he di e en ial
beha iou al pa e ns ha a e likely obse ed o gene al-
is e sus specialis he bi o es [12]. In addi ion, he e is
ample a ia ion by species in u ilising ola iles in hos
loca ion [6] in compa ison o o he cues, such as pheno-
ypic di e ences ac ing as physical ba ie s o isual cues
[17, 47]. Ou inding ha empe a u e impac s he e ec-
i eness o associa ional esis ance emphasizes he need
o include a mo e de ailed analysis o changes in ola ile
emissions unde na u al empe a u e a ia ions conside -
ing hei p ope ies a ec ing hei ola ili y o adso p ion
on o oliage [36, 37]. The ola ile p o ile o a plan species
is a gene ically egula ed p ope y ha is impo an o be
assessed when de eloping ag icul u al applica ions based
on associa ional esis ance [15, 16].
Pe sis en indi ec de ence: does high ol ac o y speci ici y
a uppe ophic le els p o ec agains in e e ence?
Indi ec de ence, assessed he e o he a ac ion o a spe-
cialis pa asi oid C. es alis o hos -damaged B. ole acea
ola iles, was no in e e ed wi h by RT exposu e a 22°C.
The a ac ion was sligh ly educed ollowing he 12°C
exposu e, indica ing ha RT exposu e migh become
mo e meaning ul a mo ning and la e e ening hou s
when he empe a u e dec eases. The g ea e endency
o he s udied he bi o e o be in luenced by RT expo-
su e a he han i s na u al enemy is suppo ed by a ield
s udy in which he e was no di e ence in pa asi ism, bu
0
0.5
1
1.5
2
2.5
12°C 22°C
A ea ed (cm2)
Con ol
RT-exposed
***
0
2
4
6
8
10
12
14
12°C 22°C
Numbe o eeding holes
Con ol
RT-exposed
**
b
a
Figu e5 Lea a ea ed and eeding holes on con ol and R. omen-
osum-exposed B assica ole acea. The amoun o lea a ea ed a 12°C
and a 22°C (a) and he numbe o eeding holes made a 12°C and
a 22°C (b) (mean ± SEM) by i e Plu ella xylos ella la ae o e 24 h
on con ol and R. omen osum (RT)-exposed B. ole acea lea es a e
shown. n = 20–21 in bo h o he independen eplica e expe imen s
o which he esul s a e summa ized he e. S a is ically signi ican
di e ences be ween ea men s a e indica ed by as e isks (**P < 0.01,
***P < 0.001).
Page 7 o 12
Himanene al. BMC Ecol (2015) 15:16
densi ies o la al and pupal s ages o P. xylos ella we e
educed in he mixed c opping o cabbage and co iande
[47]. The abili y o a non-hos scen o ac owa ds bo h a
he bi o e and i s na u al enemy, has been epo ed o a
Yeheb (Co deauxia edulis) plan ex ac [48] which a he
same ime a ac ed C. es alis and epelled P. xylos ella.
We ound no such a ac ing e ec om RT owa ds C.
es alis ha could ha e led o syne gy in P. xylos ella
biocon ol.
To ou knowledge, he passi e adso p ion o he e ospe-
ci ic species emi ed ola iles, and he esul ing associa-
ional esis ance has no been conside ed in he con ex
o dis u bing indi ec de ence ea lie . Howe e , he abun-
dance and o aging e iciency o pa asi oids ha e been
s udied in complex e sus less complex en i onmen s
[49–51], wi h chemical complexi y hypo hesized o be
one in luence o a h opod abundance [50]. The pa asi-
oid Diadegma semiclausum en e ed a Sinapis–B assica
in e c op as e han a Ho deum–B assica in e c op, bu
ook mo e ime o ind hos he e [49], indica ing ha
species composi ion is meaning ul, e en hough he co-
c op ola iles we e no analysed, pe mi ing a compa i-
son o ola ile-media ed and o he cues. In [51], as he
habi a complexi y inc eased, s onge hos cues o C.
glome a a hos inding we e concluded o be equi ed. In
addi ion, in [52], C. glome a a, wi h a wide hos ange
compa ed o ha o he mo e specialised Co esia ubec-
ula, was less e ec i e in hos inding in mixed cul u es o
B. ole acea and po a o. In a labo a o y s udy wi h ola ile
in e e ence in oduced by isop ene, ano he pa asi oid-
speci ic esponse was ound: isop ene in e e ed wi h he
hos loca ion in D. semiclausum bu no in C. ubecula
[22]. The mixing o non-hos he bi o e-induced cab-
bage ola iles and hos -induced ola iles o bean did no
in e e e wi h he a ac ion o specialis p eda o y mi es
o p ey [53]. In [54], non-hos ola iles we e e i ied o
be sensed by a p edaceous bee le, sugges ing he abili y
o non-hos ola iles in pa icula o hampe i ophic
in e ac ions. I could be ha mo e specialised pa asi-
oids, such as C. es alis in ou s udy, a e less sensi i e o
dis u bance han he bi o e na u al enemies wi h a wide
hos ange and hus a highe endency o adap a ion.
Thus, i will be in e es ing o es in he u u e whe he
he specializa ion o he i ophic in e ac ion plays a
key ole in he sensi i i y o neighbou plan semi ola ile
in e e ence.
Tempe a u e asa media o o associa ional esis ance
Tempe a u e a ies g ea ly in na u e and in ag oecosys-
ems, diu nally, seasonally and egionally, impac ing plan
phenology and insec ac i i y and, hus, hei in e ac ions
[55]. Nigh - ime empe a u es o en all close o 10°C in
100755025025 50 75 100
% C. es alis pa asi oids
n= 100 B. ole acea
n=50 In ac
n=100 In ac
n=100 Hos -damaged
n=80 RT-exposed a 12°C
n=60 Hos -damaged a 12°C
RT
Hos -damaged
Hos -damaged +
RT-exposed
Hos -damaged +
RT-exposed
Hos -damaged +
RT-exposed a
12°C
Hos -damaged+
RT-exposed a
12°C
*
*
18 15
10 21
11 24
32 41
22 37
17 21
Figu e6 O ien a ion o pa asi oid Co esia es alis in Y- ube ol ac ome e assays. The selec ion o he indi idually es ed pa asi oids (% o pa a-
si oids making a selec ion) owa ds ei he o he odou sou ces in a wo-a m ol ac ome e is shown. In ac , hos -damaged and R. omen osum
(RT)-exposed e e o ea men s o B. ole acea plan s. Hos -damaged plan s expe ienced 48 h o Plu ella xylos ella L. la al eeding and RT-exposed
plan s neighbou ed a po ed R. omen osum plan o 48 h be o e es ing a he empe a u e shown. The o al numbe o pa asi oids es ed (n)
and numbe o indi iduals making a selec ion owa ds he odou sou ces a e also epo ed. S a is ically signi ican di e ences in he selec ions a e
indica ed by as e isks (*P < 0.05).
Page 8 o 12
Himanene al. BMC Ecol (2015) 15:16
bo eal egions ( he na u al habi a s o RT) in he sum-
me . Thus, noc u nal and day-ac i e he bi o es migh be
a iably a ec ed by exposu e o RT semi ola iles and hei
p esence in ai e sus oliage. Plu ella xylos ella mo hs
a e nigh -ac i e, and hei o iposi ion peaks a dusk, bu
ligh du ing nigh (as ypical o bo eal summe ) does no
inhibi o iposi ion [39]. Inc easing empe a u e has ens
he du a ion o he li e cycle o P. xylos ella as well as i s
o iposi ion and la al eeding ac i i y [39]. The epo ed
equal educ ion in o iposi ion on RT-exposed plan s
unde bo h s udied empe a u es sugges s a diu nally pe -
sis en inc ease in esis ance owa ds P. xylos ella. O ipo-
si ion is ag onomically impo an because i de e mines
he ini ial accep ance o he hos plan . Plu ella xylos ella
does no o iposi on non-hos plan s as i elies on a a i-
e y o chemical and physical hos ecogni ion cues [56]; RT
exposu e seems o in e e e wi h one o se e al o hese.
Ou esul on esis ance owa ds P. xylos ella la al
eeding eme ging a a lowe empe a u e is less bene icial
o he plan as P. xylos ella eeding inc eases by empe a-
u e [39]. Howe e , he inding is in e es ing wi h ega ds
o hypo hesizing a egula ing ole o empe a u e in
semi ola ile-media ed associa ional esis ance. The em-
pe a u e dependence o he ola iles ac ing migh o e
one hypo he ical explana ion o why a oma ic he bs ha
a e ich in sesqui e penes ha e o en shown success, bu
also ailu es in p o iding esis ance in mixed c opping
[12, 14–16]. To e eal whe he his is he case, he lea -ai
in e ace dynamics o he speci ic ola iles ac ing should
be s udied in de ail unde empe a u e egimes.
Ano he conside a ion in he long- e m e ec i eness o
associa ional esis ance as con e ed by he neighbou ’s
ola iles is ha he non-s a ic na u e o he signal, medi-
a ed by empe a u e, migh also help slow bo h he adap-
a ion o he bi o es o he scen [57] and he e olu ion
o b eaking hos plan esis ance by he bi o es. Tempe a-
u e con ol ende s he signal mos e ec i e a ce ain
ime pe iods only, pa ly esembling he mode o unc-
ion o inducible de ences [23]. Bo h mechanisms migh
be ecologically and e en e olu iona ily suppo ed by he
dynamic na u e o signal appea ance in addi ion o sa ing
esou ces om he ecei e .
The o e all e ec o empe a u e a ia ions on he
plan -he bi o e dynamics in a mul i ophic sys em
depends on he compa a i e esponses o he bi o es and
hei na u al enemies. The empe a u e sensi i i y o di -
e en ophic-le el insec s a ies [58]; pa asi oids and
p eda o s a e gene ally assumed o be mo e esponsi e
han hei p ey, as hey ely mo e on mobili y in p ey
sea ching and a e physiologically mo e esponsi e o
empe a u e. Upon empe a u e changes, he bi o es, on
he o he hand, ace bo om-up changes in plan hos
quali y, show di ec physiological and ep oduc ional
esponses [55], plus a e con on ed by al e ed op-down
con ol om na u al enemies [59]. The pa asi oid s udied
he e, C. es alis, o iposi s upon pho ophase wi h op i-
mal empe a u e o pa asi ism a 20–35°C [60]. Thus,
ou inding ha he hos loca ion by C. es alis was no
in e e ed wi h by RT exposu e a 22°C sugges s ha
using RT o P. xylos ella biocon ol should no comp o-
mise he abili y o C. es alis o loca e hos on B. ole acea.
The sligh ly educed a ac ion owa ds plan s ha we e
exposed o RT a 12°C sugges s, howe e , ha exposu e
o RT migh a ec he in e ac ion a low empe a u es
such as du ing ea ly mo ning.
Conclusions
The abili y o RT exposu e, ea lie disco e ed o ende
associa ional esis ance in na u e, o in luence B assica
he bi o e o iposi ion and la al eeding encou ages u -
he es ing o i s po en ial o biocon ol. The gene ali y
o ou inding on he egula o y ole o empe a u e on
associa ional esis ance should be es ed in he u u e on
na u al and ag icul u al plan sys ems a ying in plan
ola ile composi ion (acknowledging he di e en ial
ole o semi ola ile e sus mo e ola ile compounds).
Addi ionally, he le el o specialisa ion and he use o
he isual, ol ac o y and chemo ecep o cues o a ge ed
he bi o es should be s udied oge he o dis inguish he
ole o ola ile-based and o he neighbou ing po en ia ed
de ences. Finally, ou s udy emphasizes ha o de elop-
ing biocon ol applica ions based on associa ional esis -
ance, mo e a en ion needs o be paid o he compa ibili y
o plan de ences wi h he bi o e diu nal beha iou and
imes o ac i i y as well as ha o hei na u al enemies.
Me hods
Plan s, he bi o es andna u al enemies
B assica ole acea a . i alica (b occoli) c . Lucky seed-
lings we e g own om seed in 0.66 l po s (4 plan s
in each, mix u e 2:1:1 o e ilized compos , NPK
100:30:200mgl−1: B2 pea NPK 110:40:220mgl−1: sand)
in a g eenhouse ( empe a u e 20/16°C day/nigh ) and
used a app oxima ely 3weeks o age o expe imen s.
R. omen osum (RT) b anches ha we e collec ed om a
na u al habi a in Suonenjoki, Finland (62°38′42.683″N,
27°3′55.383″E) and s o ed a 6°C p io o use i neces-
sa y, we e used in he exposu e ea men s. Po ed g een-
house-g own RT plan s (seed o igin: na u al habi a in
Neulaniemi, Kuopio, g own in 3:1 pea :sand) o ci ca 20
cm in heigh we e used in he Y- ube ol ac ome e es s.
The P. xylos ella la ae and adul s and C. es alis pa asi-
oids ha we e used in he expe imen s o igina ed om
labo a o y popula ions main ained on B. ole acea a .
i alica (20–25°C empe a u e and 16L:8D pho ope iod)
a he Uni e si y o Eas e n Finland.
Page 9 o 12
Himanene al. BMC Ecol (2015) 15:16
Vola ile collec ion andanalysis
Fo exposu e, ou B. ole acea po s (wi h ou
21-days-old plan s/po ) we e enclosed in insec cages
(33×33×60cm, wi h wo sides co e ed wi h a ab ic
mesh) wi h 15 cu hibe na ing RT b anches (collec ed
igh be o e he expe imen s om he na u al habi a )
ha had hei s ems in wa e - illed 250-ml decan e bo -
les. Con ol cages had an equal se -up bu hos ed no RT
b anches. The cages we e placed a 6°C (cold oom) and
22°C (a labo a o y ume hood) unde low ligh (app oxi-
ma ely 150µmol m−2s−1). The colde nigh empe a-
u e was used o mimic minimum bo eal ea ly summe
empe a u es. A e 24h, he plan s we e emo ed om
exposu e, and he ola iles we e collec ed a 22°C using
a dynamic bag enclosu e me hod. The RT b anch emis-
sions we e collec ed om h ee b anches a 22°C o
de e mine hei cha ac e is ic emission p o ile.
Fo ola ile collec ion, he en i e shoo o a i e-lea
s age b occoli plan o RT b anch was enclosed inside
p e-cleaned (hea ing o 1h a 120°C) mul ipu pose cook-
ing bags (polye hylene e eph hala e (PET), 25×55cm
in size, Look, Te inex L d, UK), and he bag opening was
ied a ound he base o he s em wi h a piece o hin ga -
den wi e. A e he enclosu e o he plan , ai ha had
been il e ed h ough cha coal and a MnO2 sc ubbe
we e pumped in o bags h ough an opening in one o he
op co ne s o he PET bags a a low a e o 600mlmin−1
o 10min o lush ou esidual con aminan s. The sam-
pling line was inse ed in o ano he co ne o he bag
and ied wi h a wi e. A e ixing he lines, he in low a e
was educed o 300mlmin−1, and he ola ile emissions
we e sampled in s ainless s eel ubes ha we e illed wi h
150mg o Tenax TA adso ben (Supelco, Belle on e, PA
USA) o 30min a he a e o abou 200mlmin−1 wi h
a acuum pump (Thomas 5002 12V DC). An equal ligh
le el (app oxima ely 300µmolm−1s−1) was p o ided by
placing wo lamps (Li al Shu le Plus, Li al Oy, Sipoo,
Finland wi h Os am Delux F, 24W luo escen lamps,
Os am AG, Munich, Ge many) on bo h sides o each
plan . Sample ubes we e sealed wi h Te lon-coa ed b ass
caps immedia ely a e collec ion and s o ed in he e ig-
e a o un il analysis. The plan biomass was o en-d ied
(60°C) o de e mine he d y weigh s o he plan s.
The plan ola ile samples we e analysed by a gas
ch oma og aph-mass spec ome e (GC ype 6890,
MSD 5973: Hewle Packa d; Wilming on, DE, USA) as
desc ibed in [61]. The apped compounds we e deso bed
wi h a he mal deso p ion uni (Pe kin-Elme ATD400
Au oma ic The mal Deso p ion sys em) a 250°C o
10min, c yo ocused a −30°C and injec ed on o a HP-5
capilla y column (50m×0.2mm i.d.×0.5μm ilm
hickness, Hewle -Packa d) wi h helium as a ca ie gas.
The o en empe a u e p og amme was held a 40°C o
1min and hen inc eased o 210°C a a a e o 5°C min−1
and inally o 250°C a a a e o 20°C min−1. The com-
pounds we e iden i ied and quan i ied by compa ing he
spec a o ex e nal s anda ds o a ailable compounds
and he Wiley lib a y (John Wiley & Sons, L d, Chiches-
e , UK). The emission a es we e calcula ed as emission
pe d y weigh (DW) pe hou .
O iposi ion byP. xylos ella mo hs
RT exposu e was iden ical in he se -up as used o he
plan s o ola ile analysis. He e, he exposu e was main-
ained (as well as he co esponding con ol plan s wi h
no RT b anches inside cages) in ume hoods a 22°C o
he p e ious day (8h) ollowed by 16h a 6°C (cold oom,
ep esen ing ypical ea ly summe nigh - ime minimum
empe a u e) un il he s a o he o iposi ion expe i-
men . All o he plan s we e equally exposed o assu e
equal ini ial amoun s o po en ially adso bed semi ola-
iles by exposu e, e ealing solely day- ime e sus nigh -
ime empe a u e di e ences o o iposi ion.
The o iposi ion in con ol and RT-exposed B. ole a-
cea was expe imen ed in 2.6m3 empe a u e-con olled
g ow h chambe s (desc ibed in [62]) a empe a u es o
12 and 22°C (mimicking a e age bo eal nigh - ime and
day- ime empe a u es, espec i ely) and a 22L:2D pho-
ope iod wi h app oxima ely 250µmolm−2s−1 ligh le el.
One con ol and one RT-exposed B. ole acea po we e
enclosed in each o he ou eplica e insec cages unde
bo h o he empe a u e ea men s. Thi y P. xylos ella
mo hs we e andomly selec ed by he expec a ion o he
50:50 emale:male a io (popula ion well s abilised o
assu e ha bo h sexes we e p esen and allowing ma -
ing as in na u e) [48] and eleased in o each cage. A e
24h, he B. ole acea plan s we e emo ed om cages and
s o ed a 6°C un il egg coun ing. Th ee independen ep-
lica e expe imen s we e ca ied ou .
Hos choice and eeding byP. xylos ella la ae
Fo he bi o e choice es s, he exposu e o B. ole acea
lea es o RT ola iles was conduc ed in 1.5-l glass con-
aine s holding wo 30-ml glass ials app oxima ely 3cm
apa , as in [8]. Ten o ele en independen sys ems we e
buil pe eplica e expe imen ( wo independen epli-
ca e expe imen s we e conduc ed o bo h empe a u es),
each ha ing wo B. ole acea lea es in one ial and ei he
an RT b anch (RT-exposu e ea men ) o a B. ole acea
lea (con ol) in he o he ial. The exposu es and he ol-
lowing choice es s ook place o 24h o each pa in
a labo a o y ume hood (22°C) and a cold oom (12°C),
mimicking a e age bo eal ea ly summe day- ime and
nigh - ime empe a u es, espec i ely.
Fo choice es s, he pe ioles o B. ole acea lea es
(one each om con ol and exposed sys em se -ups)