Fundamental host range of Trissolcus japonicus in Europe

Abstract

The brown marmorated stink bug, Halyomorpha halys, native to East Asia, is an invasive alien pest that arrived in Europe in the early 2000s and poses an imminent threat to a wide variety of crops. Adventive populations of the Asian egg parasitoid Trissolcus japonicus, the most promising agent for classical biological control of H. halys, have recently been detected in Italy and Switzerland. Its prospective fundamental host range in Europe was evaluated in behavioural no-choice tests, followed by large-arena choice tests presenting host plants with naturally laid egg masses of target and non-target hosts. Developmental
suitability of European non-target host species for T. japonicus was demonstrated, via no-choice tests, by offspring emergence (successful parasitism) from eleven out of thirteen non-target species tested (85%). Whereas successful parasitism of most non-target species was significantly lower, acceptance of Arma custos, Palomena prasina, Pentatoma rufipes, and Rhaphigaster nebulosa was not significantly different from H. halys controls. When eggs of H. halys and non-target species were exposed in a semi-natural situation in large-arena choice tests, the degree of non-target parasitism was substantially
reduced for three out of four tested species, whereas parasitism of Pa. prasina eggs was not. It remains unclear if there are behavioural barriers to parasitism that may exist and preclude excessive parasitism of potentially threatened species in the field, but field data from the invaded areas in Switzerland and Italy could contribute to a risk–benefit evaluation of releasing or re-locating adventive T. japonicus populations into other parts of Europe.

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Journal of Pest Science (2020) 93:171–182
https://doi.org/10.1007/s10340-019-01127-3
ORIGINAL PAPER
Fundamental host range ofTrissolcus japonicus inEurope
TimHaye1 · SilviaT.Moraglio2· JudithStahl1· SaraVisentin2· TommasoGregorio3· LucianaTavella2
Received: 4 March 2019 / Revised: 23 May 2019 / Accepted: 29 May 2019 / Published online: 3 June 2019
© The Author(s) 2019
Abstract
The brown marmorated stink bug, Halyomorpha halys, native to East Asia, is an invasive alien pest that arrived in Europe in
the early 2000s and poses an imminent threat to a wide variety of crops. Adventive populations of the Asian egg parasitoid
Trissolcus japonicus, the most promising agent for classical biological control of H. halys, have recently been detected in Italy
and Switzerland. Its prospective fundamental host range in Europe was evaluated in behavioural no-choice tests, followed by
large-arena choice tests presenting host plants with naturally laid egg masses of target and non-target hosts. Developmental
suitability of European non-target host species for T. japonicus was demonstrated, via no-choice tests, by offspring emer-
gence (successful parasitism) from eleven out of thirteen non-target species tested (85%). Whereas successful parasitism
of most non-target species was significantly lower, acceptance of Arma custos, Palomena prasina, Pentatoma rufipes, and
Rhaphigaster nebulosa was not significantly different from H. halys controls. When eggs of H. halys and non-target species
were exposed in a semi-natural situation in large-arena choice tests, the degree of non-target parasitism was substantially
reduced for three out of four tested species, whereas parasitism of Pa. prasina eggs was not. It remains unclear if there are
behavioural barriers to parasitism that may exist and preclude excessive parasitism of potentially threatened species in the
field, but field data from the invaded areas in Switzerland and Italy could contribute to a risk–benefit evaluation of releasing
or re-locating adventive T. japonicus populations into other parts of Europe.
Keywords Biological control· Egg parasitoids· Non-target effects· Risk assessment· Halyomorpha halys
Key message
• The Asian egg parasitoid Trissolcus japonicus, the main
antagonist of the invasive Halyomorpha halys, was
recently discovered in Europe (i.e. Italy and Switzer-
land).
• We investigated its fundamental host range in Europe
in behavioural no-choice and large-arena choice experi-
ments.
• In no-choice tests, eleven out of thirteen non-target spe-
cies were suitable for development and acceptance of
four species was not significantly different from H. halys.
• In choice tests, non-target parasitism was substantially
reduced for three out of four tested species.
• Field data from the invaded areas in Europe could con-
tribute to a risk-benefit evaluation of releasing or re-
locating adventive T. japonicus populations.
Introduction
The brown marmorated stink bug, Halyomorpha halys (Stål)
(Hemiptera: Pentatomidae), native to East Asia (China, Tai-
wan, Japan, and Korea), is an invasive alien pest that poses
an imminent and serious threat to a wide variety of tree
fruit, nut, vegetable, and field crops in Europe, due to its
polyphagous behaviour (Leskey and Nielsen 2018). Invasive
Communicated by M. Traugott.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1034 0-019-01127 -3) contains
supplementary material, which is available to authorized users.
* Tim Haye
t.haye@cabi.org
1 CABI, Rue Des Grillons 1, 2800Delemont, Switzerland
2 Dipartimento di Scienze Agrarie, Forestali e Alimentari
(DISAFA), Entomologia Generale e Applicata, University
ofTorino, Largo P. Braccini 2, 10095Grugliasco, TO, Italy
3 Hazelnuts Company Division, Ferrero Trading Luxembourg,
Rue de Trèves, 2632Findel, Luxembourg
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172 Journal of Pest Science (2020) 93:171–182
1 3
populations of H. halys were first detected in North America
and Europe in the 1990s and 2000s, respectively (Hoebeke
and Carter 2003; Haye etal. 2015a), and since its arrival
in Europe, it has spread to 27 countries (Claerebout etal.
2018). Economic damage to agricultural crops occurs five
to 10years after establishing in locations where the pest
is univoltine, whereas severe damage is often observed in
less than 5years after establishing in locations where the
pest is bivoltine (e.g., Italy, Republic of Georgia). In the lat-
ter regions, it has had a particularly devastating economic
impact in tree fruit (e.g., apples, peaches, pears), and nuts
(hazelnuts) (Maistrello etal. 2017; Bosco etal. 2018). If
spring and summer temperatures continue to increase due to
climate change, H. halys has the potential to further expand
its range throughout Europe (Kriticos etal. 2017) and
become bivoltine in regions where it used to be univoltine,
as observed in north-western Switzerland in 2018 (Haye,
personal observation).
To date, the management of H. halys in Europe still relies
on the application of broad-spectrum insecticides, but more
environmentally friendly and self-sustaining control meas-
ures, such as biological control, are urgently needed for an
area-wide control (Haye etal. 2015a). Native European
egg parasitoids, such as Anastatus bifasciatus (Geoffroy)
(Hymenoptera: Eupelmidae) and Ooencyrtus telenomicida
(Vassiliev) (Hymenoptera: Encyrtidae) can successfully
develop on viable eggs of H. halys (Haye etal. 2015b; Rov-
ersi etal. 2016; Stahl etal. 2018), but their impact following
inundative mass releases is currently considered insufficient
to effectively suppress the pest. Other native European egg
parasitoids in the genera Trissolcus Ashmead and Telenomus
Haliday (Hymenoptera: Scelionidae) have been reported to
oviposit in H. halys eggs, but their offspring are unable to
develop on the exotic host (Haye etal. 2015b; Abram etal.
2017).
Whereas the impact of native natural enemies on invasive
H. halys populations in Europe and North America is generally
low (Abram etal. 2017; Costi etal. 2018), classical biological
control using native natural enemies from the pest’s origin
seems to be more promising. In Asia, H. halys is attacked by
more than ten species of parasitoids, mostly egg parasitoids,
among which Trissolcus japonicus (Ashmead) (Hymenoptera:
Scelionidae) was identified as the most promising biocontrol
candidate, with reported parasitism levels ranging from 50 to
90% (Yang etal. 2009; Lee etal. 2013; Zhang etal. 2017). Its
natural geographic range comprises Japan, China, Taiwan, and
South Korea, but adventive populations have been discovered
in the eastern and western USA (Talamas etal. 2015; Milnes
etal. 2016; Hedstrom etal. 2017), and more recently, in Can-
ada (Abram etal. 2019a), southern Switzerland (Stahl etal.
2018), and northern Italy (Sabbatini Peverieri etal. 2018). Bio-
climatic envelope models suggest that T. japonicus will follow
its host H. halys, spreading naturally throughout Europe with
the most suitable regions located in northern Italy, Georgia,
northern Turkey, south-western France, Catalonia, and Croatia
(Avila and Charles 2018).
In many countries, regulatory requirements have become
more proscriptive, and approval for release of any classical
biological control agent is based on a thorough risk assessment
determined from a petition providing detailed information on
the biology and ecology of the agent, and particularly its host
range (Hunt etal. 2008). Ecological risk assessment in clas-
sical biological control estimates the likelihood that negative
effects, such as the reduction of non-target populations, will
occur after releases as well as the dimension and consequences
of these effects (Heimpel and Mills 2017). The likelihood of
potential negative effects is usually evaluated in a series of lab-
oratory host specificity tests in which target versus non-target
parasitism is compared under choice or no-choice conditions
(van Lenteren etal. 2006). In the case of T. japonicus, previous
laboratory host range studies on stink bugs in North America
(Hedstrom etal. 2017) and from its native range in China
(Zhang etal. 2017) indicate that this parasitoid has a broad
fundamental host range within the family Pentatomidae. These
studies were exclusively performed as black box experiments
in small choice arenas, without direct observations of parasi-
toid behaviour to determine the relationship between levels of
acceptance and levels of parasitoid development. In addition,
the experiments lacked host findings cues, such as chemical
footprints left behind by the stink bugs on plant surfaces and
synomones emitted by plants due to oviposition and feeding
by the bugs, which play an essential role in host egg location
of Trissolcus parasitoids (Colazza etal. 2004, 2007, 2009).
Here, we present results from a thorough study which aimed
to assess the prospective fundamental (physiological) host
range of T. japonicus in Europe. To evaluate target (H. halys)
versus non-target parasitism under more realistic conditions
than previous laboratory host range studies, we conducted
behavioural no-choice tests, followed by large-arena choice
tests presenting host plants with naturally laid egg masses of
target and non-target hosts (van Lenteren etal. 2006). We use
these results to predict the ecological host range and possible
non-target impact of T. japonicus. These predictions are par-
ticularly relevant due to the recent adventive establishment of
T. japonicus in two European countries (i.e., Italy and Swit-
zerland) as they provide a unique opportunity to validate esti-
mates of fundamental host range with realized (ecological)
‘post arrival’ host range as it manifests over time.
Materials andmethods
Selection, source, andrearing ofstink bug species
Non-target species were selected according to the infor-
mation on T. japonicus hosts available from the literature,
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173Journal of Pest Science (2020) 93:171–182
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phylogenetic relatedness, and sympatry of target and non-
target species, phenology, safeguard considerations (benefi-
cial species), and availability (Kuhlmann etal. 2006). In
total, thirteen species were selected, including eleven pen-
tatomids (same family as the target H. halys), one scutellerid
and one coreid (outgroup species from different families)
(Table1).
The H. halys colony was originally established in 2017
from about 500 individuals collected in Basel, Switzerland.
The insects were maintained in groups of 50 individuals
in polyester cages (BugDorm-4090 Insect Rearing Cage
47.5 × 47.5 × 47.5cm, MegaView Science Co. Ltd., Tai-
chung, Taiwan) at 26°C, 70% RH, and a 16L/8D photo-
period. Both adults and nymphs were fed with corn, beans,
and peanuts that were replaced twice weekly. In winter and
spring, bugs were provided with fruit-bearing branches
of common ivy (Hedera helix L., Araliaceae) and later in
the season with a variety of seasonal plants (e.g., Cornus
sanguinea L., Cornaceae and Sorbus aucuparia L., Prunus
avium L., Rosaceae).
Overwintered, non-target species (Hemiptera: Pentato-
midae, Scutelleridae, Coreidae) were collected from sun-
exposed house walls in early spring, by visual inspection
or plant beating from their host plants (crop and non-crop,
herbaceous and arboreous) throughout summers 2017–2018
in Piedmont, NW Italy; the Jura mountains, NW Switzer-
land; north of Lake Constance, S Germany; Samegrelo, W
Georgia (Table1). Species were identified using the keys by
Wyniger and Kment (2010), Derjanschi and Péricart (2005),
and Moulet (1995). Non-target stink bugs were reared in the
type of cage as used for H. halys and kept at 24 ± 1°C, 60%
RH, and a 16L/8D photoperiod. Adults of most species were
provided with potted broad bean plants, bramble branches,
apples, hazelnuts, and green beans, which were replaced
once per week. Adults of Eurygaster maura (L.) were pro-
vided with wheat ears instead, and adults of Arma custos
(F.) were fed with adults of Plodia interpunctella (Hübner)
(Lepidoptera: Pyralidae) or larvae of Tenebrio molitor L.
(Coleoptera: Tenebrionidae). Newly laid egg masses of tar-
get and non-target species were collected on a daily basis.
Parasitoid rearing
Trissolcus japonicus were originally collected from H.
halys eggs near Beijing, China (N40°02′06″; E116°12′41″)
in 2013, and maintained on fresh H. halys egg masses in
the CABI quarantine facility. Parasitoids (mated, ≥ 2days
old) were held in a clear plastic container (10cm diam-
eter, 5cm height) with 10% honey water solution as a food
source and 8–10 fresh H. halys egg masses provided once
per week. Parasitized egg masses were kept at 26°C, 60%
RH, and 16L/8D photoperiod. Upon the initial establishment
of the laboratory colony, specimens of T. japonicus were
taxonomically identified by E. Talamas (Systematic Ento-
mology Laboratory, USDA) and confirmed molecularly by
M.C. Bon (USDA-ARS-EBCL, Montferrier le Lez, France)
(Stahl etal. 2018). Reference specimens are located in the
Natural History Museum of Bern, Switzerland.
No‑choice tests
No-choice black box tests performed in China (Zhang etal.
2017) and North America (Hedstrom etal. 2017) indicated
that non-target parasitism of European non-target species
seems likely, so we conducted no-choice behavioural tests,
as suggested by van Lenteren etal. (2006), including direct
observations of the parasitoid oviposition behaviour during
the time of egg exposure. In contrast to black box tests, this
method allowed us to follow the fate of each parasitized
egg and relate parasitoid emergence directly to the observed
oviposition behaviour of the wasps. The advantage of this
method is that false conclusions regarding the parasitoid
behaviour can be avoided, which may be drawn if parasitoids
have non-reproductive effects on their hosts (Abram etal.
2019b). Such effects may occur when the non-target test
list includes species that function as an ‘evolutionary trap’
(emergence of host nymphs despite parasitoid oviposition)
(Abram etal. 2014; Haye etal. 2015b), or die due to para-
sitism but fail to produce parasitoid offspring (parasitoid-
induced host egg abortion, Abram etal. 2016).
Egg masses of H. halys and non-target species were col-
lected from rearing cages on a daily basis and typically used
for tests on the day they were collected. If they could not be
used the same day, eggs were stored at 10°C for no longer
than three days in order to prevent development. Since aver-
age egg mass sizes of H. halys and non-target species can
vary significantly, for each test we standardized the egg mass
size by separating egg masses into smaller clusters (10eggs/
mass) and attaching them to 4cm2 pieces of flat cardboard
with small amount of clear glue (Cementit, merz + benteli
Kolma AG, Wabern, Switzerland). In the case of Gonocerus
acuteangulatus (Goeze), the eggs were left on the leaves
they were laid on, and variable numbers of eggs (3–10) were
used for testing since this species only lays single eggs in
small clusters. Egg masses were then transferred into small
(5cm) Petri dishes.
In each experimental setup, similar numbers of randomly
selected, naïve, mated T. japonicus females were tested
simultaneously on egg masses of the target H. halys (con-
trol) and the non-target species listed in Table1 (between
14 and 46 replicates per non-target species). Since daily off-
spring production of the synovigenic females peaks within
the first week after emergence (Qui 2007), females were
4–7days old when used for experiments. All wasps were
fed with fresh honey water the morning before the experi-
ments. Single T. japonicus females were added to each Petri
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174 Journal of Pest Science (2020) 93:171–182
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Table 1 Non-target test list for Trissolcus japonicus
Test species Host plants Collecting period Selection criteria Origin of laboratory cultures
Family: Pentatomidae
Acrosternum heegeri Fieber Cryptomeria japonica May–June Habitat and host plant overlap, close
relatedness
Zugdidi (Georgia)
Arma custos (F.) Predatory species (Acer spp., Fraxinus
excelsior, Tilia spp.)
August Beneficial species, habitat and host plant
overlap, close relatedness
Cavour, Chieri, Grugliasco (Italy)
Carpocoris fuscispinus (Boheman) Centaurea centaurium, Brassica napus May–June Close relatedness Delémont (Switzerland)
Dolycoris baccarum (L.) Lamium purpureum, Medicago sativa,
Taraxacum officinale March–April Literature host record, close relatedness Chieri, Grugliasco (Italy); Delémont (Swit-
zerland)
Graphosoma lineatum (L.) various Apiaceae, Cornus sanguinea,
Sambucus nigra May–July Close relatedness Chieri, Moretta (Italy); Liesberg, Delémont
(Switzerland)
Halyomorpha halys (Stål) Catalpa bignonioides, Ilex aquifolium April Target Basel (Switzerland)
Nezara viridula (L.) Acer sp., C. sanguinea, Crataegus spp.,
Morus nigra, S. nigra, Rosa spp.
March; May–July Habitat and host plant overlap, close relat-
edness, invasive in Europe
Cavour, Chieri, Grugliasco (Italy)
Palomena prasina (L.) Acer spp., C. sanguinea, Corylus avellana,
Crataegus spp., Prunus avium, Prunus
persica, S. nigra, Tilia spp.
May–July Habitat and host plant overlap, close
relatedness
Bosia, Cavour, Chieri, Grugliasco,
Nichelino, Sanfré, Villar Dora (Italy);
Delémont (Switzerland)
Pentatoma rufipes (L.) Malus spp., Acer spp. August Habitat and host plant overlap, close
relatedness
Cavour, Prunetto (Italy); Schiggendorf
(Germany)
Peribalus strictus (F.) Prunus laurocerasus March Close relatedness Grugliasco (Italy)
Piezodorus lituratus (F.) Spartius junceum March; May–July Close relatedness Grugliasco, Italy
Rhaphigaster nebulosa (Poda) Acer spp., Platanus sp., Populus sp. March; May–July Habitat and host plant overlap, close
relatedness
Bosia, Cavour, Grugliasco, Torino (Italy)
Family: Scutelleridae
Eurygaster maura (L.) Triticum aestivum May–July Outgroup Brozolo (Italy)
Family: Coreidae
Gonocerus acuteangulatus (Goeze) C. avellana, C. sanguinea, Crataegus spp.,
Prunus mahaleb, Rosa spp., S. nigra May–July Outgroup, habitat and host plant overlap Cavour, Chieri, Magliano Alfieri, Nichelino,
Villar Dora (Italy)
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175Journal of Pest Science (2020) 93:171–182
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dish and observed under a stereomicroscope until they had
at least one contact with the egg mass. If females had no
interest in oviposition following the first contact, observa-
tions were continued for another 10min, and egg masses
were counted as ‘rejected’ if no oviposition behaviour was
observed. Females that started ovipositing were observed
until they either had parasitized all 10 eggs, as indicated by
marking behaviour (‘acceptance’), or abandoned the par-
tially parasitized egg mass for more than 10min.
The individual handling time of the egg masses was
recorded for each T. japonicus female. All tests were con-
ducted at 26 ± 1°C, 60–70% RH. In addition, unexposed
controls of target and non-target egg masses were kept at the
same conditions to evaluate baseline host mortality and to
assess if T. japonicus females induced additional host mor-
tality even in cases of failed development, a non-reproduc-
tive non-target effect, which is rarely considered explicitly
in risk assessment of biological control agents (Abram etal.
2019b). After the tests, the wasps were removed, and both
the non-target and H. halys egg masses were incubated under
the above rearing conditions until emergence of stink bug
nymphs and/or wasp adults (‘host suitability’). The number
of emerged parasitoids, nymphs and dead eggs (= no emer-
gence) was recorded as well as the sex ratio of the parasitoid
offspring. Finally, egg dissections were performed to deter-
mine whether any parasitoids or nymphs developed partially.
Paired choice tests
To evaluate target (H. halys) versus non-target parasitism
under more realistic conditions than previous laboratory host
range studies (Zhang etal. 2017; Hedstrom etal. 2017),
large-arena choice tests were conducted (Van Lenteren
etal. 2006) where individual T. japonicus females foraged
on plants where bugs had fed and laid eggs. This proce-
dure was followed because studies by Colazza etal. (2007,
2009) had shown that in a similar system, Trissolcus basalis
(Wollaston) (Hymenoptera: Scelionidae) perceived chemi-
cal footprints left behind by its host Nezara viridula (L.)
(Hemiptera: Pentatomidae) as contact kairomones, which
induced foraging by gravid females. In addition, T. basalis
also responded to synomones emitted by bean plants induced
by feeding and oviposition activity of its host (Colazza etal.
2004).
The following four species were selected as representa-
tive hosts that were accepted frequently or less frequently
in no-choice tests: Acrosternum heegeri Fieber, Ar. cus-
tos, Graphosoma lineatum (L.), and Palomena prasina
(L.). In the case of H. halys, Pa. Prasina, and Ar. custos,
potted broad bean plants (Vicia faba L., Fabaceae, about
20cm high) were placed inside the stink bug rearing cages
described above for 24h. Since Ac. heegeri and G. lineatum
refused to lay eggs on broad bean plants, alternatively cut,
fresh fruit-bearing branches of common ivy (H. helix) placed
in a container with water were used instead. Accordingly,
ivy branches were also offered to H. halys for oviposition
to exclude potential effects of different host plants. After
24h, the plants were removed and inspected for egg masses.
Plants carrying single egg masses were selected for testing.
Since these five species lay egg masses of variable size, it
was not possible to control for the number of eggs per plant.
However, in this way the outcome of choice tests may repre-
sent parasitism in the field more realistically than no-choice
tests, despite the nonstandardized egg masses.
Testing arenas consisted of fine gauze cages
(47.5 × 47.5 × 47.5cm), in which two plants were placed in
the far left and right corners, each carrying a single egg mass
of H. halys or non-target species, respectively. Plants did not
touch the cage walls or each other. At the top of the cages,
small drops of honey were placed in each corner as a food
source for the parasitoids. Single, naïve, mated 4–7days old
T. japonicus females were removed from the rearing cage,
and individually transferred into glass pipettes (10cm long,
diameter 5mm) closed with a cotton wick. These tubes were
then put into a small open plastic cup, which was placed in
the middle of the front side of the cage, equidistant (30cm)
to the two test plants. The cotton wicks were removed, so
the wasps could crawl up to the opening of the tubes and
enter the test arena. All tests were conducted at 26 ± 1°C,
60–70% RH, and a 16L/8D photoperiod. After 24h, the
wasps were removed and both the H. halys and non-target
egg masses were incubated under the above rearing condi-
tions until emergence of stink bug nymphs and wasp adults.
Each combination of target and non-target species was rep-
licated between 19 and 40 times (Table3).
Statistical analysis
In no-choice tests, acceptance (oviposition and marking
behaviour), host suitability (mean offspring emergence per
egg mass), and sex ratio (percentage female parasitoid off-
spring per host species) were compared pairwise between
each non-target species and its respective H. halys control
using generalized linear models (GLMs) with a binomial
error distribution and a logit link function. Similarly, the
host exploitation (number of eggs parasitized within an egg
mass) and egg mass handling time were compared using a
GLM with Poisson (log-link function) and gamma (inverse
link function) error distributions, respectively. Replicates
in which wasps did not parasitize all 10 eggs were excluded
from the handling time analysis. The unattributed mortality
(‘dead eggs’ = no parasitoid emergence or dead parasitoids
in dissected eggs, Table2) of non-target eggs exposed to T.
japonicus was compared pairwise with unattributed mor-
tality in the respective rearing controls using a GLM with
a quasibinomial error distribution (logit link function) to
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176 Journal of Pest Science (2020) 93:171–182
1 3
account for overdispersion. All GLMs were carried out with
R version 3.2.3 (Team 2017) using the development environ-
ment RStudio (Team 2016).
For large-arena paired choice tests, the proportion of
females parasitizing either target or non-target eggs were
compared using a Pearson Chi-square test. The percentage of
parasitoids emerging per parasitized egg mass was compared
between species using a Wilcoxon’s signed-rank test. Sta-
tistical tests were carried out with the SPSS® 20.0 software
package (IBM Corp. 2013).
Results
No‑choice tests
Host acceptance In total, twelve out of thirteen non-target
hosts were accepted by T. japonicus females (Table2). The
proportion of females accepting non-target egg masses, as
indicated by oviposition behaviour and marking of eggs, was
not significantly different for four non-target species, Pa.
prasina, Rhaphigaster nebulosa (Poda), Pentatoma rufipes
(L.), and Ar. custos, than for the target host H. halys (Table2,
ESM 1). All other species were significantly less accepted.
Gonocerus acuteangulatus, belonging to the outgroup fam-
ily Coreidae, was the only species that was not accepted at
all. The proportion of T. japonicus females accepting the
target H. halys (controls) was generally high, ranging from
89% to 100% (average 96%). The number of eggs para-
sitized within accepted egg masses (host exploitation) was
89% or higher for all accepted non-target species and not
significantly different from H. halys controls (Table2). The
handling time (= time spent to parasitize an egg mass of
10 eggs) was significantly longer than in H. halys control
when T. japonicus parasitized egg masses of Pa. prasina
(p ≤ 0.05), and the less accepted species Dolycoris baccarum
(L.), N. viridula, Peribalus strictus (F.), and G. lineatum
(p < 0.001) (Table2). The handling time was longest for the
non-indigenous N. viridula (Table2).
Host suitability Of the twelve non-target species accepted
by T. japonicus, eleven were suitable for parasitoid develop-
ment (Table2). Levels of suitability (= proportion of para-
sitoids successfully emerging from parasitized eggs) were
not significantly different for eight non-target species, Pa.
prasina, Carpocoris fuscispinus (Boheman), Pe. strictus, Ar.
custos, D. baccarum, R. nebulosa, Ac. heegeri and E. maura,
than for the target host H. halys. Suitability of the target H.
halys (controls) was generally high, varying between 87%
and 94% (average 92%). Suitability of Pen. rufipes eggs
(99% emergence) was even higher than for the target host H.
halys. For most non-target species, unattributed egg mortal-
ity (‘dead eggs’, Table2) was higher in eggs masses exposed
to T. japonicus than in the respective rearing controls (ESM
1). Particularly, egg mortality in G. lineatum and N. viridula
increased due to parasitization from 14 and 54 (rearing con-
trols) to 27 and 100%, respectively (Table2).
In general, for H. halys and all non-target species the sex
ratio was female biased (> 60%). However, parasitized eggs
of Per. strictus (61%) produced significantly fewer females
than eggs of H. halys (Table3).
Paired choice tests
The incubation of egg masses exposed in choice tests
showed that some T. japonicus females were able to para-
sitize both egg masses within 24h, and thus, for these rep-
licates it was not possible to state which host the parasitoid
had chosen first. The number of females parasitizing both
egg masses is given in Table3, but these replicates were
not included in the statistical analysis. Results of paired-
choice tests and no-choice tests were generally similar, and
the four non-target test species were parasitized in both
scenarios (Table3). Egg masses of Ar. custos, G. lineatum
and Ac. heegeri were significantly less parasitized when
paired with H. halys egg masses (Pearson Chi-square test,
p ≤ 0.001; Table3; ESM 1), whereas levels of parasitism
of Pa. prasina (47%) were not significantly different than
for the target (53%) (χ2 = 0.267, p = 0.606). The proportion
of females that did not parasitize any egg mass within 24h
was low, ranging from 0 to 13%. Percentages of emerging
T. japonicus per parasitized non-target egg mass were not
significantly different than for paired H. halys egg masses
(Table3, column ‘emerged parasitoids’).
Discussion
Developmental suitability of European non-target host
species for T. japonicus was demonstrated by successful
offspring emergence from eleven out of thirteen non-target
species tested (85%), which is consistent with the results
of earlier host range studies in China (7/8; 88%) and North
America (7/10; 70%) (Zhang etal. 2017; Hedstrom etal.
2017). Parasitoid emergence from successfully parasitized
non-target eggs was generally high (˃ 70%), except for G.
lineatum. Similar high emergence was observed for Asian
non-target species (Zhang etal. 2017), whereas emer-
gence from North American non-target species (and H.
halys controls) was much lower (Hedstrom etal. 2017;
Botch and Delfosse 2018). By relating parasitoid emer-
gence directly to the observed oviposition behaviour and
keeping unexposed rearing controls, we were able to
demonstrate that T. japonicus caused non-reproductive
parasitoid-induced mortality in G. lineatum (55.9%) and
the invasive N. viridula (100%), the only non-indigenous
species included in the test list. Since H. halys and N.
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177Journal of Pest Science (2020) 93:171–182
1 3
Table 2 Outcomes of no-choice tests of single T. japonicus females when exposed to H. halys, or one of thirteen non-target species
Species No. of
females
tested (n)
Host acceptance and exploitation Host suitability % Egg mortality
(mean ± SE) per
egg mass in rear-
ing controls (n)
Fate of parasitized eggs (mean ± SE) %b
Total no. of egg
masses para-
sitized (n)
Mean ± SE
handling time
(min) per egg
mass (n)a
Mean propor-
tion of eggs
parasitized
within egg mass
(± SE)
Parasitoids
emerged
Undeveloped/
dead parasitoids
(dissection)
Dead eggsc
(undefined
content) (dis-
section)
Nymphs
emerged
Sex ratio
(proportion of
females)
Family: Pentatomidae
A. heegeri 25 16** 35.2 ± 1.4 (14) 99.4 ± 0.6 81.8 ± 7.0 6.3 ± 6.3 12.0 ± 4.5 0.0 ± 0.0 66.6 ± 8.3 22.2 ± 5.9 (18)
H. halys 25 24 39.5 ± 2.0 (22) 100.0 ± 0.0 93.3 ± 2.2 2.1 ± 1.7 4.6 ± 1.7 0.0 ± 0.0 74.7 ± 4.7 15.3 ± 5.7(12)
A. custos 30 30 45.9 ± 2.0 (27) 99.0 ± 0.6 87.9 ± 3.2 2.0 ± 2.0 10.1 ± 2.3 0.0 ± 0.0 75.6 ± 3.9 31.1 ± 5.9 (24)
H. halys 30 30 43.7 ± 2.9 (25) 100.0 ± 0.0 93.3 ± 2.5 3.5 ± 1.9 3.0 ± 1.8 0.0 ± 0.0 77.2 ± 4.3 13.3 ± 4.5 (10)
C. fuscispinus 28 21** 31.1 ± 1.4 (18) 97.1 ± 2.0 94.4 ± 3.5 2.1 ± 1.6 3.2 ± 1.4 1.4 ± 1.0 75.2 ± 5.8 19.5 ± 4.9 (9)
H. halys 23 23 30.2 ± 0.9 (19) 95.5 ± 3.3 91.7 ± 4.1 0.9 ± 0.6 8.0 ± 3.6 0.5 ± 0.5 80.1 ± 4.6 9.7 ± 4.4 (11)
D. baccarum 37 25** 36.4 ± 1.6
(19)***
93.2 ± 4.0 76.4 ± 7.3 6.4 ± 4.2 16.8 ± 6.6 0.0 ± 0.0 84.3 ± 1.7 31.7 ± 9.6 (12)
H. halys 23 21 29.6 ± 1.3 (17) 97.1 ± 2.4 91.8 ± 2.1 1.0 ± 0.7 6.4 ± 2.1 0.5 ± 0.5 85.0 ± 1.9 6.4 ± 1.7 (11)
G. lineatum 31 17*** 43.5 ± 5.7
(13)***
93.5 ± 5.4 41.8 ± 10.7*** 29.1 ± 7.6 26.8 ± 7.4 2.4 ± 1.8 76.5 ± 11.6 13.8 ± 4.8 (28)
H. halys 31 31 27.7 ± 1.0 (29) 97.1 ± 2.9 87.4 ± 4.7 1.0 ± 0.7 7.7 ± 3.5 3.9 ± 3.3 84.4 ± 1.8 13.0 ± 4.3 (10)
N. viridula 30 20** 52.3 ± 5.3
(13)***
89.0 ± 4.4 0.0 ± 0.0*** 0.0 ± 0.0 100.0 ± 0.0 0.0 ± 0.0 N/A 54.2 ± 9.7 (9)
H. halys 14 14 31.5 ± 2.5 (9) 100.0 ± 0.0 93.6 ± 2.5 0.7 ± 0.7 5.7 ± 2.5 0.0 ± 0.0 90.2 ± 1.7 7.9 ± 3.4 (12)
P. prasina 36 34 31.0 ± 2.2 (27)* 92.9 ± 2.9 92.1 ± 2.9 0.7 ± 0.5 7.2 ± 2.9 0.0 ± 0.0 78.7 ± 4.5 17.4 ± 4.5 (24)
H. halys 20 20 25.7 ± 1.3 (20) 100.0 ± 0.0 94.0 ± 1.3 2.0 ± 0.9 3.0 ± 1.3 1.0 ± 0.7 87.3 ± 2.0 10.5 ± 3.1 (21)
P. rufipes 30 30 31.6 ± 1.0 (24) 100.0 ± 0.0 99.0 ± 0.6*** 0.0 ± 0.0 1.0 ± 0.6 0.0 ± 0.0 85.5 ± 1.4 4.3 ± 2.9 (5)
H. halys 23 23 31.5 ± 1.0 (23) 93.5 ± 4.3 90.6 ± 2.9 3.5 ± 1.8 5.1 ± 1.7 0.8 ± 0.8 82.4 ± 3.8 11.4 ± 4.6 (12)
P. strictus 16 10* 37.8 ± 3.3
(8)***
94.4 ± 5.7 97.8 ± 1.5 0.0 ± 0.0 2.2 ± 1.5 0.0 ± 0.0 61.0 ± 12.5* 13.4 ± 8.0 (11)
H. halys 14 13 28.9 ± 0.7 (12) 99.2 ± 0.8 93.7 ± 2.5 2.3 ± 1.7 4.0 ± 2.2 0.0 ± 0.0 85.6 ± 4.0 7.3 ± 2.5 811)
P. lituratus 46 1*** 48.5 (1) 100.0 ± 0.0 90.0 0 10 0 55.6 15.3 ± 5.4 (20)
H. halys 21 19 30.5 ± 1.1 (13) 100.0 ± 0.0 94.2 ± 2.4 1.5 ± 0.8 4.0 ± 2.0 0.0 ± 0.0 75.7 ± 5.6 10.4 ± 3.5 (11)
R. nebulosa 17 16 37.1 ± 5.4 (11) 93.8 ± 5.6 70.8 ± 9.0 7.5 ± 3.2 21.0 ± 8.8 0.0 ± 0.0 91.6 ± 2.0 nd
H. halys 12 11 30.8 ± 2.6 (10) 100.0 ± 0.0 89.0 ± 6.7 1.0 ± 1.0 10.0 ± 6.8 0.0 ± 0.0 81.8 ± 9.3 10.5 ± 4.2 (13)
Family: Scutelleridae
E. maura 28 7*** 27.6 ± 1.8 (7) 100.0 ± 0.0 94.9 ± 2.0 1.3 ± 1.3 3.9 ± 1.9 0.0 ± 0.0 70.5 ± 0.3 10.5 ± 4.0 (22)
H. halys 28 25 28.2 ± 1.0 (23) 98.4 ± 1.3 89.0 ± 4.3 1.2 ± 0.9 8.6 ± 3.9 1.2 ± 0.7 82.6 ± 4.2 9.6 ± 2.6 (11)
Family: Coreidae
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178 Journal of Pest Science (2020) 93:171–182
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Table 2 (continued)
Species No. of
females
tested (n)
Host acceptance and exploitation Host suitability % Egg mortality
(mean ± SE) per
egg mass in rear-
ing controls (n)
Fate of parasitized eggs (mean ± SE) %b
Total no. of egg
masses para-
sitized (n)
Mean ± SE
handling time
(min) per egg
mass (n)a
Mean propor-
tion of eggs
parasitized
within egg mass
(± SE)
Parasitoids
emerged
Undeveloped/
dead parasitoids
(dissection)
Dead eggsc
(undefined
content) (dis-
section)
Nymphs
emerged
Sex ratio
(proportion of
females)
G. acuteangu-
latus 34 0*** nd nd nd nd nd nd nd 2.5 ± 1.9 (18)
H. halys 34 34 30.3 ± 1.2 (31) 98.4 ± 0.9 92.8 ± 2.2 1.2 ± 0.7 4.6 ± 1.4 1.5 ± 1.0 79.2 ± 4.4 6.9 ± 1.7(10)
Asterisks indicate significant differences between treatment and control (GLM, see ESM 1) (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). No data collected are indicated with ‘nd’
a Wasps that did not parasitize the full egg mass were excluded
b Parasitized eggs = eggs that females drilled and marked
c No emergence and no signs of parasitism (eggs, larvae or pupae) when dissected
Table 3 Outcomes of paired-choice tests of single T. japonicus females when simultaneously exposed to H. halys and one of four non-target Pentatomidae from Europe
Asterisks indicate differences (Pearson Chi Square test, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0001; ns = not significant; ESM 1) for a given developmental outcome of each non-target species versus
H. halys
a Replicates with both egg masses parasitized were not included
Species Mean (± SE)
egg mass
size
No. of
females
tested (n)
Total no. of
egg masses
parasitized
(%)
No. of females parasitizing Percentage (mean ± SE) per egg massa
None NT + H.
halys Only NT (%) Only H.
halys (%) pEmerged
parasitoids
Undevel-
oped/dead
parasitoids
Total no. of
eggs para-
sitized
Emerged
nymphs
Dead eggs
(undefined
content) (dis-
section)
A. custos 17.1 ± 1.0 38 14 (36.8) 5 9 5 (20.8) *** 90.4 ± 3.3 2.2 ± 1.2 92.8 ± 2.4 8.9 ± 7.2 5.5 ± 2.1
H. halys 22.2 ± 0.9 28 (73.7) 19 (79.2) 81.5 ± 4.4 6.2 ± 1.6 87.6 ± 3.6 1.9 ± 1.4 10.4 ± 3.2
A. heegeri 12.4 ± 0.9 19 8 (42.1) 0 6 2 (15.4) *** 93.9 ± 3.6 3.2 ± 1.9 97.0 ± 2.0 0.0 ± 0.0 3.0 ± 2.0
H. halys 21.4 ± 1.1 17 (89.5) 11 (84.6) 92.8 ± 2.3 1.1 ± 0.6 93.9 ± 1.9 0.0 ± 0.0 6.1 ± 1.9
G. italicum 17.3 ± 1.2 23 9 (39.1) 2 6 3 (20.0) *** 67.0 ± 10.9 15.8 ± 8.9 82.8 ± 10.1 0.4 ± 0.3 11.6 ± 5.2
H. halys 21.2 ± 1.2 18 (78.3) 12 (80.0) 86.1 ± 4.3 3.5 ± 1.8 89.6 ± 4.2 5.6 ± 5.6 10.0 ± 4.1
P. prasina 22.6 ± 1.1 40 22 (55.0) 2 8 14 (46.7) ns 87.4 ± 5.4 0.8 ± 0.5 88.3 ± 5.2 5.5 ± 4.3 6.1 ± 2.8
H. halys 24.5 ± 1.0 24 (60.0) 16 (53.3) 87.6 ± 4.3 2.1 ± 0.7 89.7 ± 4.3 4.5 ± 3.9 5.3 ± 2.1
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179Journal of Pest Science (2020) 93:171–182
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viridula often co-occur in the same crops, T. japonicus
may have a positive non-target effect by contributing to the
mortality of N. viridula eggs masses, which are often not
fully exploited by T. basalis, one of the main egg parasi-
toids associated with N. viridula (Colazza and Bin 1995).
However, the aggressive host guarding behaviour of T.
japonicus could also exclude T. basalis from N. viridula
egg masses, lowering the overall biological control of the
pest. On the other hand, N. viridula could also operate
as an ‘evolutionary trap’, because eggs were frequently
accepted but not suitable for the development of T. japoni-
cus (Abram etal. 2014). The unusually long handling time
in no-choice tests may indicate that N. viridula eggs are
less attractive for T. japonicus and it remains to be veri-
fied if T. japonicus indeed attacks N. viridula egg masses
in the field.
Whereas most non-target species were significantly less
attacked than the target host, acceptance of four suitable
non-target species, Pa. prasina, Pen. rufipes, Ar. custos, and
R. nebulosa, was not significantly different from H. halys
controls. In Europe, these species mainly live on trees,
resulting in habitat overlap with H. halys. Based on the out-
come of the no-choice tests, these species could serve as
alternative hosts for T. japonicus in the field and population-
level impacts are more likely for these species than for any
of the other tested non-targets.
Laboratory no-choice tests in simple structured arenas are
usually the first step in risk assessment of biological control
agents, providing conditions where the maximal fundamen-
tal host range is likely to be expressed (van Lenteren etal.
2006). Since non-targets were consistently attacked in no-
choice tests, step large-arena choice tests were subsequently
conducted to determine if non-targets are attacked when eggs
of the target, H. halys, and non-target species are simultane-
ously present in a semi-natural situation on their host plants.
Presenting eggs of Ar. custos, G. lineatum and Ac. heegeri in
choice tests showed that introducing additional complexity
(host plants with naturally laid eggs, feeding damage, and
stink bug footprints) can reduce the degree of non-target
host parasitism substantially, which agrees with studies by
Hedstrom etal. (2017) and Botch and Delfosse (2018). Com-
pared to small-arena choice tests, movement of T. japonicus
from one egg mass to another within the testing period was
reduced by using larger cages and adding more complexity.
However, the emergence data showed that within 24h some
females were still able to parasitize both egg masses. After
parasitizing the first egg mass, females would have been in
a ‘no-choice situation’ when locating the second egg mass
and we thus did not include these replicates. In a preliminary
study, T. japonicus females guarded parasitized egg masses
for at least 12h, and after 24h 65% of females had left the
egg masses (Haye, unpublished data). Accordingly, in future
choice tests it would be advantageous to reduce the length of
the testing period to 12h or less; however, this would also
increase the proportion of females that are not responsive in
the given time.
Host range studies in Europe, China, and North America
showed that some closely related, ecological equivalents
(i.e., predatory species in the subfamily Asopinae) Ar. custos
(Europe), Arma chinensis (Fallou) (China) and Podisus mac-
uliventris (Say) (North America) are highly suitable hosts
for T. japonicus. Since these species fall into the category
of beneficial ‘safeguard species’ (Kuhlmann etal. 2006),
potential non-target impacts would cause more concern than
effects on other non-beneficial, herbivore stink bug species.
However, the outcome of our choice tests suggests that the
potential risk of substantial parasitism of Ar. custos might be
much lower in the field than initially indicated by no-choice
tests. In addition, experiments measuring fitness-related
phenotypic parameters (e.g., size) of T. japonicus offspring
emerging from H. halys eggs suggest that non-target host
use, especially on predatory pentatomids with smaller eggs,
may carry a significant fitness penalty for parasitoid off-
spring (Botch and Delfosse 2018).
In contrast to Ar. custos, parasitism of Pa. prasina eggs
was not reduced in the presence of H. halys eggs. The reason
why Ar. custos, but not Pa. prasina, was much less para-
sitized in choice tests is unknown. In a similar system, bean
plants (V. faba) damaged by feeding activity of N. viridula,
and onto which an egg mass had been laid, produced vola-
tiles that attracted the egg parasitoid T. basalis (Colazza
etal. 2004). Similarly, V. faba plants that were attacked by
H. halys produced volatiles that were attractive for the native
European egg parasitoids A. bifasciatus and O. telenomicida
(Rondoni etal. 2017). Accordingly, it seems highly likely
that feeding and oviposition of Pa. prasina on V. faba plants
induced plant volatiles that were exploited by T. japonicus
for host egg location. This may also explain why plants car-
rying eggs of the predatory species Ar. custos, which rarely
feeds on plants, were less attractive for T. japonicus in large-
arena choice experiments.
In terms of ecological risk assessment, it remains
unclear if there are behavioural barriers to parasitism,
such as habitat preferences, competition with native egg
parasitoids, or oviposition periods of non-target species,
which may exist and inhibit parasitism of Pa. prasina,
Pen. rufipes, and R. nebulosa in the field. For example,
the oviposition period of the forest bug, Pen. rufipes,
is starting from the end of August (Peusens and Beliën
2012) when the oviposition period (May–August) of H.
halys ends (Costi etal. 2017). Little is known about the
activity of T. japonicus before and after the oviposition
period of H. halys in April and September, respectively.
Further field studies are thus needed to clarify if in late
summer Pen. rufipes escapes parasitism by T. japonicus or
is attacked more frequently due to larger population size
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180 Journal of Pest Science (2020) 93:171–182
1 3
of T. japonicus and the limited availability of H. halys
eggs. Similarly, R. nebulosa is one of the first species to
become active in spring, and it remains unknown if T.
japonicus could use this species as an alternative host in
April before H. halys eggs are present in the field. Fac-
tors such as habitat associations, host location, life cycle
synchrony, and searching capacity of parasitoids are not
easily reproduced in laboratory tests (Cameron etal. 2013)
and some uncertainty may remain even after more complex
host range testing. This is aggravated by the fact that there
is often a lack of knowledge on the phenology and ecology
of the non-target species themselves.
Regardless of the future impact on non-target species
in the field, the arrival of T. japonicus in Europe is now
irreversible, and bioclimatic envelope models (Avila and
Charles 2018) suggest that it will spread throughout Europe,
following the invasion routes of its primary host, H. halys.
Ecological host range studies in China (Zhang etal. 2017)
suggest that ‘host use’ (minor use of a non-target species
for reproduction) of some European non-target species is a
likely scenario, but on the other hand this may also increase
the chances of establishment and survival of T. japonicus
populations. Field data from the invaded areas in Switzer-
land and Italy may help to determine if the re-location of T.
japonicus into other areas in Europe affected by H. halys
poses any additional risk and could contribute to a risk–ben-
efit evaluation of releases of T. japonicus.
In the USA, where adventive populations of T. japoni-
cus were first discovered in 2014 (Talamas etal. 2015), the
wasps are currently being relocated and released in agricul-
tural locations within state boundaries where T. japonicus
has spread naturally (Jentsch 2017). Studies of non-target
parasitism in the field are ongoing, but to our best knowl-
edge severe non-target parasitism has not yet been observed.
Based on a risk–benefit analysis considering scientific data,
economic analysis, contemporary evidence, and cultural
and broader social impacts, an application for release of
T. japonicus was recently approved (with controls) in New
Zealand to support an eradication programme in the event
of a H. halys incursion (https ://www.epa.govt.nz). Follow-
ing the New Zealand example, a risk–benefit analysis could
help European countries affected by H. halys to decide if
releases of T. japonicus are justified. In response to H. halys
outbreaks, the use of broad-spectrum insecticides, espe-
cially pyrethroids, has increased greatly in the USA and
Italy, which has disrupted existing integrated pest manage-
ment programmes and caused outbreaks of secondary pests
(Leskey and Nielsen 2018). Therefore, bearing in mind
the worldwide decline of the entomofauna, some of which
may be linked to the use of insecticides (Sánchez-Bayoa
and Wyckhuys 2019), the small risk of potential non-target
effects on some native stink bug species, in comparison with
the environmental risks of continuous use of insecticides,
should be considered when evaluating the option of re-locat-
ing and releasing T. japonicus in Europe.
Author contribution statement
TH, LT, TG conceived and designed research. TH and JS
conducted experiments and analysed data. TH wrote the
manuscript. STM and SV collected and reared non-target
species for establishing laboratory colonies. All authors read
and approved the manuscript.
Acknowledgements The research was carried out with the coopera-
tion and contribution of the Hazelnut company division of the Ferrero
Group. We would like to thank Chelsey Blackman, Darren Blackburn,
Taylor Kaye, Jessica Fraser, Lindsay Craig, Christie Laing, Anna Grun-
sky, Mariah Ediger for technical assistance in the laboratory, and Lara
Bosco, Gabriele Castelli, Marco G. Pansa for their help in bug collec-
tion. We like to thank Paul Abram for constructive comments on an
earlier version of the manuscript. CABI is an international intergovern-
mental organisation, and we gratefully acknowledge the core financial
support from our member countries (and lead agencies) including the
United Kingdom (Department for International Development), China
(Chinese Ministry of Agriculture), Australia (Australian Centre for
International Agricultural Research), Canada (Agriculture and Agri-
Food Canada), Netherlands (Directorate-General for International
Cooperation), and Switzerland (Swiss Agency for Development and
Cooperation). See http://www.cabi.org/about -cabi/who-we-work-with/
key-donor s/ for full details.
Compliance with ethical standards
Conflict of interest The authors declared that they have no conflict of
interest.
Informed consent Informed consent was obtained from all individual
participants included in the study.
Research involving human participants and/or animals This article
does not contain any studies with human participants or animals (ver-
tebrates) performed by any of the authors.
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creat iveco
mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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