First discovery of adventive populations of Trissolcus japonicus in Europe

Abstract

The brown marmorated stink bug, Halyomorpha halys (Stål), native to East Asia, emerged as an invasive pest in Europe in the 2000s. In its native range, Trissolcus japonicus (Ashmead) is the dominant egg parasitoid of H. halys, and thus it has been considered for classical biological control in countries invaded by the pest. A survey of native egg parasitoids conducted in 2017 and 2018 with frozen, sentinel egg masses of H. halys revealed that T. japonicus was already present in apple orchards in the Canton Ticino, Switzerland. Trissolcus japonicus was recovered in both years and from three different sites. In total, 10 egg masses were recovered from which 29 adult parasitoids emerged. A genetic analysis using the barcode mitochondrial DNA confirmed the morphological identification of T. japonicus and evidenced a best match of the “Ticino populations” to Japanese populations, but the pathways of entry remain unknown.

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Vol.:(0123456789)
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Journal of Pest Science
https://doi.org/10.1007/s10340-018-1061-2
RAPID COMMUNICATION
First discovery ofadventive populations ofTrissolcus japonicus
inEurope
JudithStahl1,2· FrancescoTortorici3· MariannaPontini3· Marie‑ClaudeBon4· KimHoelmer5· CristinaMarazzi6·
LucianaTavella3· TimHaye1
Received: 26 September 2018 / Revised: 23 October 2018 / Accepted: 27 October 2018
© The Author(s) 2018
Abstract
The brown marmorated stink bug, Halyomorpha halys (Stål), native to East Asia, emerged as an invasive pest in Europe in
the 2000s. In its native range, Trissolcus japonicus (Ashmead) is the dominant egg parasitoid of H. halys, and thus it has been
considered for classical biological control in countries invaded by the pest. A survey of native egg parasitoids conducted in
2017 and 2018 with frozen, sentinel egg masses of H. halys revealed that T. japonicus was already present in apple orchards
in the Canton Ticino, Switzerland. Trissolcus japonicus was recovered in both years and from three different sites. In total,
17 egg masses were recovered from which 42 adult parasitoids emerged. A genetic analysis using the barcode mitochondrial
DNA confirmed the morphological identification of T. japonicus and evidenced a best match of the “Ticino populations” to
Japanese populations, but the pathways of entry remain unknown.
Keywords Biological control· Egg parasitoids· Halyomorpha halys· Scelionidae
Key message
• Surveys for egg parasitoids of Halyomorpha halys were
conducted in south-eastern Switzerland.
• For the first time, the Asian parasitoid T. japonicus was
recovered from sentinel H. halys egg masses in Europe.
• Trissolcus japonicus is established in Switzerland and
was found in two consecutive years at three different
sites.
• Parasitism levels by T. japonicus are currently low, with
a maximum of 2%.
Introduction
The brown marmorated stink bug, Halyomorpha halys (Stål)
(Hemiptera: Pentatomidae), is native to East Asia (China,
Japan, and the Korean peninsula) and has emerged as an
invasive pest in North America and Europe in the 1990s and
2000s, respectively (Hoebeke and Carter 2003; Haye etal.
2015). It is highly polyphagous, feeding on over 170 plant
species in at least 12 families (Rice etal. 2014; Leskey and
Nielsen 2018), causing economic losses to a wide variety of
crops in invaded areas, and having a particularly devastating
Communicated by D.C. Weber.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s1034 0-018-1061-2) contains
supplementary material, which is available to authorized users.
* Judith Stahl
j.stahl@cabi.org
1 CABI, Rue des Grillons 1, 2800Delémont, Switzerland
2 Institute ofEcology andEvolutionary Biology, University
ofBremen, Leobener Str. NW2, 28359Bremen, Germany
3 Dipartimento di Scienze Agrarie, Forestali e Alimentari
(DISAFA), ULF Entomologia Generale e Applicata,
University ofTorino, Largo P. Braccini 2, 10095Grugliasco,
TO, Italy
4 European Biological Control Laboratory, USDA Agricultural
Research Service, Campus International de Baillarguet,
MontferrierleLez, France
5 Beneficial Insects Introduction Research Unit, USDA
Agricultural Research Service, Newark, DE, USA
6 Dipartimento delle finanze e dell’economia, Servizio
fitosanitario cantonale, Sezione dell’agricoltura viale
S. Franscini 17, 6501Bellinzona, Switzerland

Journal of Pest Science
1 3
economic impact in tree fruit (e.g. apples, peaches, pears),
and hazelnuts in the USA, Italy, and Georgia (United States
Apple Association 2010; Maistrello etal. 2017; Bosco etal.
2018). As the application of broad-spectrum insecticides
is the most widely used strategy for managing H. halys in
Europe, more environmentally friendly and self-sustaining
control measures, such as classical or augmentative biologi-
cal control, are urgently needed for an area-wide control of
H. halys.
Throughout its native range, H. halys is heavily attacked
by a wide variety of hymenopteran egg parasitoids in the
genera Trissolcus Ashmead, Telenomus Haliday (Scelio-
nidae), Ooencyrtus Ashmead (Encyrtidae) and Anastatus
Motschulsky (Eupelmidae) (reviewed in Lee etal. 2013).
Surveys in north-eastern China have shown that the domi-
nant parasitoid of H. halys is Trissolcus japonicus (Ash-
mead), with parasitism levels often ranging from 50 to 90%
(Qiu etal. 2007; Yang etal. 2009; Zhang etal. 2017) and
thus, this species has been considered as a classical bio-
logical control agent in invaded areas in North America and
Europe.
The native geographic range of T. japonicus widely over-
laps with that of H. halys, including Japan, China, Taiwan,
and the Republic of Korea (Qiu etal. 2007; Yang etal. 2009;
Zhang etal. 2017). Bioclimatic envelope models predict that
T. japonicus could establish in H. halys infested areas in
Europe and North America (Avila and Charles 2018), and
in fact adventive populations have already been discovered
in the USA [Beltsville, Maryland in 2014 (Talamas etal.
2015); Vancouver, Washington in 2015 (Milnes etal. 2016);
Portland, Oregon in 2016 (Hedstrom etal. 2017)] and have
since been reported in 10 states (Morrison etal. 2018).
The pathway(s) of entry for T. japonicus in North America
remains unknown, and is presumed to be the same as for the
introduction of H. halys (Talamas etal. 2015).
In Europe, several native species have been reared from
sentinel H. halys egg masses, including Anastatus bifascia-
tus (Geoffroy) (from both viable and frozen eggs), Trissolcus
cultratus (Mayr) (frozen eggs) (Haye etal. 2015; Abram
etal. 2017), Ooencyrtus telenomicida (Vassiliev) (frozen
eggs) (Roversi etal. 2016), and some Trissolcus and Teleno-
mus spp. (viable eggs) (LT, FT, unpublished data). Most of
the native European Trissolcus species have been reported
to oviposit in H. halys eggs, but their offspring are unable
to develop, and thus, the exotic host is considered as evolu-
tionary trap for native scelionid parasitoids (Schlaepfer etal.
2005; Haye etal. 2015). This phenomenon has also been
observed in North American scelionids (Abram etal. 2014).
Surveys for egg parasitoids in Europe have been restricted
to western Switzerland (Haye etal. 2015) and northern Italy
(Roversi etal. 2016; Costi 2018) and to date no adventive
populations of T. japonicus have been detected. Halyomor-
pha halys has been introduced several times into Europe,
with evidence of multiple invasions from different source
populations (Gariepy etal. 2015; Cesari etal. 2018). As T.
japonicus may use the same pathways of entry as its host
H. halys, we considered that this species may eventually
arrive in Europe as well. Therefore, the exposure of sentinel
H. halys egg masses was extended to include the Ticino in
south-eastern Switzerland to ensure more complete coverage
of the areas where populations of H. halys are established.
Materials andmethods
Exposure ofsentinel eggs
In 2017, a total of 226 H. halys sentinel egg masses were
exposed between August and September in a convention-
ally managed apple orchard in the municipality of Bellin-
zona, Canton of Ticino, Switzerland (site 1, 46°09′42.1″N
8°58′12.2″E). Sentinel egg masses used for exposure were
collected from host plants and mesh parts of cages of the
laboratory rearing at CABI in Delémont, Switzerland, with-
out the use of water when they were less than 24h old.
They were immediately frozen at −80°C for a maximum
of 1month and thawed no more than 2days before the expo-
sure. Rearing methods for H. halys have been described in
detail in Stahl etal. (2018). All eggs were counted before
field exposure, and only egg masses consisting of at least 20
eggs were used for exposure. Egg masses were glued directly
on the underside of tree leaves 50cm to 180cm above
ground, using ‘Cementit’ (merz + benteli AG, Niederwan-
gen, Switzerland). Leaves of various host plants taken from
the H. halys laboratory rearing cages were placed next to the
egg masses and fixed with twist ties to potentially increase
the chance of parasitism by presenting an array of chemical
cues. Exposure lengths varied between 4 and 6days. After
field exposure, recovered eggs were counted and predation
by chewing and sucking predators was assessed under a
Leica routine stereo microscope M50 with a magnification
of up to 40 × (see Morrison etal. 2016). All egg masses were
then kept at 26°C and monitored for parasitoid emergence
for the following 6weeks. Newly emerged parasitoids were
provided with fresh (unfrozen) egg masses to test whether
their offspring could complete development. All parasitoids
that emerged from egg masses, both exposed in the field and
in the laboratory, were stored in 99% ethanol.
In 2018, surveys for parasitoids were extended, and a
total of 710 H. halys egg masses were exposed between
May and August at five sites around Bellinzona, including
the same site as in 2017 (site 1) and four additional sites in
the same municipality (Table1). The new sites included
two conventionally managed mixed fruit orchards (apple,
persimmon, pear, cherry, plum) (site 2, 46°09′34.2″N
8°56′03.3″E, and site 3, 46°09′57.1″N 8°56′37.9″E), one

Journal of Pest Science
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conventional orchard with apple and pear trees (site 5,
46°13′12.4″N 9°03′11.4″E), and one private garden with
peach trees (site 4, 46°09′30.0″N 8°59′21.0″E). Once
per month, 15–135 frozen egg masses were exposed at
every site on apple or peach trees and five additional
egg masses were exposed in the closest available natu-
ral site nearby (shrubs, hedges, etc.). Additionally, egg
masses (in total 191) were exposed three times in an
organic apple orchard located near the Italian border in
the municipality of Manno, Canton of Ticino, Switzerland
(site 6, 46°01′52.8″N 8°55′20.4″E) (Table1).
Morphological identication ofparasitoids
Ethanol-stored specimens were dried and glued on card-
points for morphological analyses. A Leitz large-field stereo
microscope TS with magnification up to 160 × and a spot
light Leica CLS 150X were used for morphological diag-
nosis. For Scelionidae, Telenomus species were determined
Table 1 Parasitism and predation of frozen H. halys egg masses exposed at various sites in the Canton Ticino, Switzerland, in 2017 and 2018
a Egg masses that contained at least one egg that had not been attacked by either chewing or sucking predators after recollection
b Parasitism measured by parasitoid offspring emergence of recovered eggs
Exposure date Location Egg masses
(eggs)
exposed
Egg masses
(eggs)
recovereda
Egg masses (eggs)
attacked by chew-
ing predators
Egg masses (eggs)
attacked by suck-
ing predators
Egg masses
(eggs)
parasitizedb
% Para-
sitism
(total)
% Parasit-
ism by T.
japonicus
2017
3–10 Aug 1 25 (637) 20 (416) 12 (49) 2 (2) 1 (16) 3.9 0
10–17 Aug 1 146 (3767) 141 (3004) 95 (546) 6 (18) 17 (77) 2.6 0.2
28 Aug–4 Sep 1 55 (1387) 53 (774) 48 (391) 0 (0) 1 (5) 0.7 0.7
2018
17– 22 May 1 20 (541) 19 (427) 10 (103) 0 (0) 1 (1) 0.2 0
2 20 (543) 15 (320) 2 (8) 0 (0) 0 (0) 0 0
3 20 (531) 19 (434) 4 (7) 0 (0) 0 (0) 0 0
4 20 (540) 17 (373) 9 (60) 0 (0) 1 (16) 4.3 0
5 20 (496) 16 (328) 7 (32) 1 (1) 3 (13) 4.0 0
Total 100 (2651) 86 (1882) 32 (210) 1 (1) 5 (30) 1.6 0
20–25 Jun 1 50 (1343) 46 (1127) 11 (42) 0 (0) 1 (4) 0.4 0
2 20 (526) 11 (172) 8 (80) 0 (0) 0 (0) 0 0
3 20 (542) 13 (226) 14 (170) 0 (0) 2 (38) 16.8 0
4 20 (535) 10 (227) 13 (175) 0 (0) 1 (12) 5.3 0
5 20 (550) 12 (231) 6 (80) 0 (0) 3 (20) 8.7 0
total 130 (3496) 93 (2018) 52 (547) 0 (0) 7 (74) 3.7 0
19–25 Jul 1 75 (1985) 64 (1555) 12 (46) 6 (36) 0 (0) 0 0
2 20 (508) 16 (355) 13 (42) 0 (0) 3 (37) 10.4 0
3 20 (500) 21 (202) 9 (94) 0 (0) 4 (35) 17.3 0
4 20 (496) 6 (118) 14 (234) 0 (0) 1 (7) 5.9 0
5 20 (501) 7 (129) 11 (137) 0 (0) 1 (16) 12.4 0
Total 155 (3990) 104 (2373) 59 (553) 6 (36) 9 (95) 4.0 0
25–30 Jul 1 140 (3717) 137 (3489) 12 (19) 2 (2) 7 (38) 1.1 0.06
6 30 (757) 27 (612) 14 (85) 0 (0) 0 (0) 0 0
Total 170 (4474) 164 (4101) 26 (104) 2 (2) 7 (38) 0.9 0.06
30 Jul–4 Aug 6 98 (2507) 85 (1797) 38 (246) 0 (0) 12 (27) 1.5 0.2
08–13 Aug 1 105 (2697) 95 (2125) 48 (292) 3 (4) 2 (21) 1.0 0
13–17 Aug 2 20 (516) 18 (383) 16 (107) 0 (0) 0 (0) 0 0
3 20 (537) 17 (335) 11 (161) 0 (0) 0 (0) 0 0
4 20 (517) 16 (290) 12 (152) 1 (10) 1 (6) 2.1 2.1
5 20 (541) 10 (240) 7 (115) 0 (0) 0 (0) 0 0
6 63 (1677) 57 (1356) 28 (146) 5 (18) 11 (22) 1.6 1.3
Total 185 (4808) 118 (2604) 74 (681) 6 (28) 11 (22) 0.8 0.5

Journal of Pest Science
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using the keys of Kozlov and Kononova (1983) and Johnson
(1984), and Trissolcus species were identified using the keys
by Talamas etal. (2017). Moreover, Trissolcus specimens
were compared with pictures of holotype and paratypes in
Hymenoptera Online (HOL) and in Talamas etal. (2017).
Anastatus individuals were identified using the key by
Askew and Nieves-Aldrey (2004). All the specimens used
for morphological analysis were deposited in the Natural
History Museum of Bern, Switzerland (NMBE) and Diparti-
mento di Scienze Agrarie, Forestali e Alimentari (DISAFA)
(ESM1).
Molecular conrmation
Morphological identification of T. japonicus was confirmed
molecularly at the DISAFA as a routine procedure. In total,
two males and three females of T. japonicus from the Ticino
and, as a positive control, one female from the CABI col-
ony (China) were processed (ESM1). Genomic DNA was
extracted according to Gariepy etal. (2014), and the bar-
code region of the cytochrome oxidase I (COI) gene was
amplified using universal PCR primers for insects [LCO-
1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′)
and HCO-2198 (5′-TAA ACT TCA GGG TGA CCA AAA
AAT CA-3′) (Folmer etal. 1994)]. The PCR was performed
in a 50µl reaction volume: 2µl of DNA, 37.9µl molecu-
lar grade water, 5µl 10X Qiagen PCR buffer, 3µl dNTPs
(25mM each), 1.5µl MgCl2, 0.2µl of each primer (0.3µM
each), 0.2µl Taq DNA Polymerase (Qiagen, Hilden, Ger-
many). Thermocycling conditions were optimized to shorten
reaction times and included initial denaturation at 94°C for
300s, followed by 35 cycles of 94°C for 30s, annealing at
52°C for 45s and extension at 72°C for 60s; then further
600s at 72°C for final extension. All PCR products were
purified using a commercial kit (QIAquick PCR Purifica-
tion Kit, Qiagen GmbH, Hilden, Germany) following the
manufacturer’s instructions and sent for sequencing in one
direction using the forward primer to an external service
(Genechron S.r.l., Rome, Italy).
The same sequence of 612bp in length was obtained in
all samples and compared with sequences present in the
GenBank database by similarity search using the Basic
Local Alignment Search Tool (http://www.ncbi.nlm.nih.
gov/BLAST n) confirming the taxonomy of all morphologi-
cally identified specimens. All residual DNA is archived at
DISAFA.
Genetic matching withAsian populations
ofTrissolcus japonicus
We used the barcode approach to identify and exclude
Asian T. japonicus populations that were highly diver-
gent from the “Ticino population.” Mounted specimens
(three males and three females) recovered from three para-
sitized egg masses from Ticino were shipped to EBCL
along with 10 females from the laboratory colony kept
in the quarantine at CABI Switzerland, which originated
from the Beijing Province, China (ESM1). Genomic DNA
from all specimens was extracted according to Gariepy
etal. (2014), except those from mounted specimens which
were non-destructively isolated as described in Giantsis
etal. (2015). Amplification of all barcode sequences and
their analysis were done as described in Ganjisaffar etal.
(2018). All sequences of 674bp in length generated from
this study are deposited in the GenBank (Table2), and all
residual DNA extracts are archived at EBCL (Table2). Of
note, when this study was initiated, only 24 barcodes were
deposited in the GenBank database. Twenty-three of the
24 are from Japan and most are published by Matsuo etal.
(2014). For this study, we had access to a not yet published
EBCL database of 127 barcodes of T. japonicus mostly
from laboratory colonies derived from natural populations
collected in China, Korea, Japan and USA (EBCL custom
database). Searches for sequence similarity against this
database confirmed the close similarity of the “Ticino
sequence” with those of Japanese origin. Therefore, the 26
sequences generated from this study were aligned with 10
“Japanese T. japonicus” sequences from this custom data-
base and 22 sequences retrieved from GenBank following
the procedure described in Ganjisaffar etal. (2018). Two
sequences from GenBank were not considered in our data
set as the sequences were not of adequate length. The final
alignment of 58 barcode sequences of 423bp in length
revealed a total of six haplotypes. The phylogenetic rela-
tionships among these haplotypes were depicted using
statistical parsimony in TCS as implemented in PopART
(Leigh and Bryant 2015). This approach also enabled us
to display the geographical distribution of all haplotypes
analysed.
Results
Exposure ofsentinel eggs
Between August and September 2017 and from May to
August 2018, more than 48,000 sentinel frozen H. halys
eggs were exposed at six sites in the Canton Ticino
to retrieve native egg parasitoids (Table1). Overall

Journal of Pest Science
1 3
parasitism was highly variable among sites and exposure
dates, ranging from 0 to 17.3% (Table1). Predation by
sucking or chewing predators ranged from 0 to 2.2% and
from 1.6 to 66.5%, respectively (Table1).
Morphological identication ofparasitoids
Based on morphological analyses, Trissolcus individuals
were identified as T. cultratus, T. semistriatus (Nees van
Esenbeck) [syn. Trissolcus grandis (Thomson); Talamas
etal. 2017], and the exotic T. japonicus (Fig.1). In par-
ticular, T. japonicus was identified according to the follow-
ing characters: vertex with hyperoccipital carina uniform
and robust; area between hyperoccipital carena and medial
ocellus coriaceous; longitudinal groove below preocel-
lar pit; clypeus with 4 setae; orbital furrow expanded with
medial margin well defined at intersection with malar sulcus;
genal carina absent; mesoscutum with distinct notauli and
without median mesoscutal carina; median lobe of mesos-
cutum without oblique rugulae; mesopleuron with episternal
foveae forming a continuous line of cells from dorsal apex
of postacetabular sulcus to mesopleural pit; laterotergite I
without setae; T1 without sublateral setae; T2 with striae
present throughout anterior half of tergite (Talamas etal.
2017). Other parasitoids were identified as A. bifasciatus and
Telenomus turesis Walker (Fig.1). In total, 12 T. japonicus
adults were reared from three egg masses exposed at two
dates in August 2017 in an apple orchard near Bellinzona,
Canton of Ticino, Switzerland (site 1). In 2018, 17 individu-
als were reared from 7 egg masses at three different sites
(site 1, 4, 6) (Table1).
Molecular conrmation
The morphological identification of T. japonicus was con-
firmed by the molecular analysis performed by DISAFA. A
Table 2 Sampling information, GenBank Accession Numbers and haplotypes for the T. japonicus included in this study (*)
a EBCL DNA collection
b Name of collectors: KH, Kim Hoelmer (USDA-ARS), TM, Toshiharu Mita, KM, Kazunori Matsuo, JS, Judith Stahl (CABI), TH, Tim Haye
(CABI)
c Not available
d Matsuo etal. (2014)
e Matsuo and Hirose unpublished
Collection code and sex Country Region Year of collection,
name of collectorbHost GenBank Accession Number Barcode
haplo-
type
GBIFCH 00543446, ♀Switzerland Ticino 2017, JS Halyomorpha halys MH919753* H1
GBIFCH 00543447, ♀Switzerland Ticino 2017, JS H. halys MH919754* H1
GBIFCH 00543448, ♀Switzerland Ticino 2017, JS H. halys MH919755* H1
GBIFCH 00543449, ♂Switzerland Ticino 2017, JS H. halys MH919756* H1
GBIFCH 00543450, ♂Switzerland Ticino 2017, JS H. halys MH919757* H1
GBIFCH 00543451, ♂Switzerland Ticino 2017, JS H. halys MH919758* H1
CABI colony, ♀ (n = 10) China Beijing 2018, TH H. halys MH919759* H2
Tsp1 EBCLa, nacJapan Tsukuba 2012, KH na MH919743 H1
Tsp77, EBCLa na Japan Tsukuba 2012, KH na MH919744 H3
Tsp78, EBCLa na Japan Tsukuba 2012, KH na MH919745 H3
Tsp79, EBCLa na Japan Tsukuba 2012, KH na MH919746 H3
Tsp88, EBCLa na Japan Tsukuba 2012, KH na MH919747 H3
Tsp90, EBCLa na Japan Tsukuba 2012, KH na MH919748 H3
Tsp91, EBCLa na Japan Tsukuba 2012, KH na MH919749 H3
Tsp93, EBCLa na Japan Tsukuba 2012, KH na MH919750 H3
Tsp223, EBCLa ♀Japan Kanagawa 2015, KH H. halys MH919751 H3
Tsp226 EBCLa,♀Japan Kanagawa 2015, KH Plautia stali MH919752 H3
na, ♀Japan Kanagawa 2012, TM P. stali AB847131-32,36dH4
na, ♀Japan Fukuoka 2012, KM P. stali AB847144-145dH5
na, ♀Japan Fukuoka 2012, KM H. halys AB908179-182dH5
na, na Japan na na na AB894834-35, AB894838-39eH5
na, ♀Japan Fukuoka 2012, KM P. stali AB847129,130, 137,143,146dH6
na, na Japan na na na AB894836,837,840,841eH6

Journal of Pest Science
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BLAST search showed the best similarity score (100%) of
our barcode sequence (612bp in length) with T. japonicus
(Accession No. AB971832).
Genetic matching withAsian populations
ofTrissolcus japonicus
The six analysed specimens recovered from the field
yielded a single haplotype, H1. This haplotype was also
found in Tsukuba in Japan in 2012 (Fig.2, Table2) and
is distant by one mutation from the two haplotypes (H3
and H4) uncovered in Tsukuba and in Kanagawa in Japan
(Fig.2). The 10 specimens from the CABI colony yielded
a single haplotype (H2) which is one mutation away from
H1, indicating that the “Ticino populations” of T. japoni-
cus did not escape accidentally from the CABI colony.
Fig. 1 Species composition of parasitoids reared from sentinel egg
masses of Halyomorpha halys exposed at six sites in the Canton
Ticino, Switzerland, in 2017 and 2018. Numbers of exposure sites
correspond with locations listed in Table1; total number of emerged
parasitoids per site is displayed above bars
Fig. 2 Barcode haplotype net-
work of the 58 T. japonicus ana-
lysed in this study. Each circle
corresponds to one haplotype;
circle size gives the propor-
tion of individuals belonging
to the haplotype. The colour
inside each circle represents the
geographical origin. Numbers
correspond to the haplotype
numbers. Hatch marks symbol-
ize the number of mutations
between haplotypes. The H2
haplotype from Beijing, China,
represents the CABI colony

Journal of Pest Science
1 3
Discussion
Parasitism and predation of sentinel H. halys eggs by
native European egg parasitoids and predators in south-
western Switzerland were generally low, which is consist-
ent with previous studies in Europe and North America
(e.g. Jones etal. 2014; Haye etal. 2015; Abram etal. 2017;
Dieckhoff etal. 2017). Parasitoids recorded in the present
study included two species, T. cultratus and A. bifasciatus,
which have been recorded in earlier surveys in western and
northern Switzerland (Haye etal. 2015), whereas T. semi-
striatus, Tel. turesis, and the exotic T. japonicus have not
been previously reported from H. halys in Europe. To our
best knowledge, this is also the first record of T. japonicus
parasitizing H. halys eggs in apple orchards.
Recently, an increasing number of unintended intro-
ductions of biological control agents have been recorded
(Servick 2018); however, it remains difficult to trace the
origin of such introductions. Presumably, the majority are
accidentally transported from their native range along with
the pest. The actual pathways of entry for T. japonicus
are unknown, but it is likely that either plants carrying
parasitized egg masses of Asian Pentatomidae (H. halys
or other hosts, see Zhang etal. 2017) or diapausing adults
were introduced. As a result of population size bottlenecks
and genetic drift in small founding populations as likely
experienced by the T. japonicus “Ticino population,” the
level of haplotype diversity is reduced to a unique hap-
lotype. Our haplotype analysis found a best match of the
“Ticino populations” to Japanese populations so far, pos-
sibly indicating an introduction from Japan. Although our
custom database is quite representative of the geographic
distribution of T. japonicus, we cannot entirely exclude
that the haplotype H1 may also be present in other parts
of T. japonicus’ native range, e.g. China or Korea, but
has thus far remained unsampled. Tracing the source of
introduction necessitates obtaining meaningful popula-
tion structure information (Gariepy etal. 2015), requir-
ing sometimes to use more than one locus. To add better
resolution, a comprehensive phylogeographic study with
the barcode is currently being undertaken, as well as with
the microsatellite loci recently developed de novo for T.
japonicus. Whether Switzerland was the actual country
of introduction or whether T. japonicus was accidentally
introduced into the climatically highly suitable northern
Italy (Avila and Charles 2018) and is now spreading north-
wards into Switzerland remains unclear. However, consid-
ering that in 2016 Italy imported goods of more than twice
the value from the region of China, Japan and the Republic
of Korea than Switzerland (World Bank 2018), an intro-
duction into Italy seems more likely, but future surveys in
both regions may help to clarify the invasion pathways.
Field and laboratory studies in China and Japan showed
that the host range of T. japonicus is not restricted to H.
halys (Ryu and Hirashima 1984; Matsuo etal. 2016; Yang
etal. 2009; Zhang etal. 2017), and fundamental host range
studies conducted in Europe suggest that some native Euro-
pean Pentatomidae are suitable hosts for development (TH,
LT, unpublished data). Accordingly, T. japonicus has the
potential to directly impact native non-target stink bug spe-
cies. However, the extent to which attacks may result in
significant reductions in native stink bug populations will
depend on various factors, such as habitat overlap, com-
petition with native egg parasitoids, size and suitability of
non-target host species. The adventive establishment of T.
japonicus in Switzerland provides the opportunity to study
the establishment and spread of this species in Europe, as
well as to assess potential risks to native biodiversity under
natural conditions. In particular, it will enable us to deter-
mine whether results from host range studies in Asia (Zhang
etal. 2017) are able to predict the impact of this parasitoid
outside of its native range.
It is too early to evaluate the impact of adventive T.
japonicus populations on invasive H. halys populations in
Switzerland as it has likely only recently arrived. However,
based on the very high parasitism levels of H. halys eggs
observed in Asia, its establishment may have the potential to
reduce invasive stink bug densities below economic thresh-
olds. Current T. japonicus populations are likely very low,
which may explain why in both years it was only recovered
in the second half of summer. In China, T. japonicus para-
sitizes H. halys eggs over the course of the entire season,
from May to September, but parasitism is usually highest
in August (Zhang etal. 2017). Continued studies exposing
sentinel eggs and collecting natural egg masses will be nec-
essary to determine the current distribution and spread of
T. japonicus and to evaluate how egg mortality may affect
populations of H. halys and non-target pentatomids in the
near future.
Authors’ contribution
JS, CM and TH conceived and designed research. LT, FT,
MP and MCB identified the parasitoids. KH provided para-
sitoids from Asia for molecular analysis. All authors con-
tributed to writing the manuscript and approved the final
version.
Acknowledgements This project has received funding from the
European Union’s Horizon 2020 research and innovation programme
under the Marie Sklodowska-Curie Grant Agreement No. 641456.
Additional funding was provided by the Phytosanitary Service of the
Canton Ticino, and the Associazione frutticoltori ticinesi. We would
like to thank Darren Blackburn, Jessica Fraser, Chelsey Blackman,
Taylor Kaye, Anna Grunsky, Christie Laing, Mariah Ediger, Lindsay

Journal of Pest Science
1 3
Craig, and Giorgia Mattei for technical assistance. We are grateful to
Cesare Bassi for allowing us to use his orchard for our studies. CABI
is an international intergovernmental organization, and we gratefully
acknowledge the core financial support from our member countries
(and lead agencies) including the UK (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 declare that they have no conflict of
interest.
Human and animal rights This article does not contain any studies
with human participants or animals (vertebrates) performed by any
of the authors.
Informed consent Informed consent was obtained from all individual
participants included in the study.
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.
References
Abram PK, Gariepy TD, Boivin G, Brodeur J (2014) An invasive stink
bug as an evolutionary trap for an indigenous egg parasitoid. Biol
Invasions 16:1387–1395
Abram PK, Hoelmer KA, Acebes-Doria A etal (2017) Indigenous
arthropod natural enemies of the invasive brown marmorated stink
bug in North America and Europe. J Pest Sci 90:1009–1020
Askew RR, Nieves-Aldrey JL (2004) Further observations on Eupelmi-
nae (Hymenoptera: Chalcidoidea, Eupelmidae) in the Iberian pen-
insula and Canary Islands, including descriptions of new species.
Graellsia 60:27–39
Avila GA, Charles JG (2018) Modelling the potential geographic dis-
tribution of Trissolcus japonicus: a biological control agent of
the brown marmorated stink bug, Halyomorpha halys. Biocontrol
63:505–518
Bosco L, Moraglio ST, Tavella L (2018) Halyomorpha halys, a
serious threat for hazelnut in newly invaded areas. J Pest Sci
91(2):661–670
Cesari M, Maistrello L, Piemontese L, Bonini R, Dioli P, Lee W, Park
CG, Partsinevelos GK, Rebecchi L, Guidetti R (2018) Genetic
diversity of the brown marmorated stink bug Halyomorpha halys
in the invaded territories of Europe and its patterns of diffusion
in Italy. Biol Invasions 20:1073–1092
Costi E (2018) Biologia e monitoraggio in campo della cimice invasiva
Halyomorpha halys in Italia e indagini su potenziali antagonisti
naturali autoctoni. PhD thesis. Università Emilia Romagna
Dieckhoff C, Tatman KM, Hoelmer KA (2017) Natural biological con-
trol of Halyomorpha halys by native egg parasitoids: a multi-year
survey in northern Delaware. J Pest Sci 90(4):1143–1158
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA
primers for amplification of mitochondrial cytochrome c oxidase
subunit I from diverse metazoan invertebrates. Mol Mar Biol Bio-
technol 3:294–299
Ganjisaffar F, Talamas EJ, Bon MC, Gonzalez L, Brown BV, Perring
TM (2018) Trissolcus hyalinipennis Rajmohana & Narendran
(Hymenoptera, Scelionidae), a parasitoid of Bagrada hilaris
(Burmeister) (Hemiptera, Pentatomidae) emerges in North
America. J Hymenopt Res 65:111–130
Gariepy TD, Haye T, Zhang J (2014) A molecular diagnostic tool
for the preliminary assessment of host–parasitoid associations
in biological control programmes for a new invasive pest. Mol
Ecol 23:3912–3924
Gariepy TD, Bruin A, Haye T, Milonas P, Vétek G (2015) Occur-
rence and genetic diversity of new populations of Halyomorpha
halys in Europe. J Pest Sci 88:451–460
Giantsis I, Chaskopoulou A, Bon MC (2015) Mild-vectolysis: a non-
destructive DNA extraction method for vouchering sand flies
and mosquitoes. J Med Entomol 53:692–695
Haye T, Fischer S, Zhang J, Gariepy T (2015) Can native egg para-
sitoids adopt the invasive brown marmorated stink bug, Halyo-
morpha halys (Heteroptera: Pentatomidae), in Europe? J Pest
Sci 88:693–705
Hedstrom C, Lowenstein D, Andrews H, Bai B, Wiman N (2017) Pen-
tatomid host suitability and the discovery of introduced popula-
tions of Trissolcus japonicus in Oregon. J Pest Sci 90:1169–1179
Hoebeke ER, Carter ME (2003) Halyomorpha halys (Stǻl) (Heterop-
tera: Pentatomidae): a polyphagous plant pest from Asia newly
detected in North America. Proc Entomol Soc Wash 105:225–237
Johnson NF (1984) Systematics of Nearctic Telenomus: classification
and revisions of the podisi and phymatae species groups (Hyme-
noptera: Scelionidae). Bull Ohio Biol Surv 6:1–113
Jones AL, Jennings DE, Hooks CR, Shrewsbury PM (2014) Sentinel
eggs underestimate rates of parasitism of the exotic brown mar-
morated stink bug, Halyomorpha halys. Biol Control 78:61–66
Kozlov MA, Kononova SV (1983) Telenominae of the fauna of the
USSR. Nauka, Leningrad
Lee D-H, Short BD, Joseph SV, Bergh JC, Leskey TC (2013) Review
of the biology, ecology, and management of Halyomorpha halys
(Hemiptera: Pentatomidae) in China, Japan, and the Republic of
Korea. Environ Entomol 42:627–641
Leigh JW, Bryant D (2015) POPART: full-feature software for hap-
lotype network construction. Methods Ecol Evol 6:1110–1116
Leskey TC, Nielsen AL (2018) Impact of the invasive brown marm-
orated stink bug in North America and Europe: history, biology,
ecology, and management. Annu Rev Entomol 63:599–618
Maistrello L, Vaccari G, Caruso S etal (2017) Monitoring of the inva-
sive Halyomorpha halys, a new key pest of fruit orchards in north-
ern Italy. J Pest Sci 90:1231–1244
Matsuo K, Hirose Y, Johnson NF (2014) A taxonomic issue of two
species of Trissolcus (Hymenoptera: Platygastridae) parasitic on
eggs of the brown-winged green bug, Plautia stali (Hemiptera:
Pentatomidae): resurrection of T. plautiae, a cryptic species of
T. japonicus revealed by morphology, reproductive isolation and
molecular evidence. Appl Entomol Zool 49:385–394
Matsuo K, Honda T, Itoyama K, Toyama M, Hirose Y (2016) Dis-
covery of three egg parasitoid species attacking the shield bug
Glaucias subpunctatus (Hemiptera: Pentatomidae). Jpn J Appl
Entomol Zool 60:43–45
Milnes JM, Wiman NG, Talamas EJ, Brunner JF, Hoelmer KA, Buff-
ington ML, Beers EH (2016) Discovery of an exotic egg parasitoid
of the brown marmorated stink bug, Halyomorpha halys (Stål)
in the Pacific Northwest. Proc Entomol Soc Wash 118:466–470
Morrison WR III, Mathews CR, Leskey TC (2016) Frequency, effi-
ciency, and physical characteristics of predation by generalist

Journal of Pest Science
1 3
predators of brown marmorated stink bug (Hemiptera: Pentato-
midae) eggs. Biol Control 97:120–130
Morrison WR III, Blaauw BR, Nielsen AL, Talamas E, Leskey TC
(2018) Predation and parasitism by native and exotic natural
enemies of Halyomorpha halys (Stål) (Hemiptera: Pentatomidae)
eggs augmented with semiochemicals and differing host stimuli.
Biol Control 121:140–150
Qiu LF, Yang ZQ, Tao WQ (2007) Biology and population dynamics
of Trissolcus halyomorphae. Sci Silvae Sin 43:62–65
Rice KB, Bergh CJ, Bergmann EJ, Biddinger DJ, Dieckhoff C, Dively
G, Fraser H, Gariepy T, Hamilton G, Haye T, Herbert A (2014)
Biology, ecology, and management of brown marmorated stink
bug (Hemiptera: Pentatomidae). J Integr Pest Manag 5:A1–A13
Roversi PF, Marianelli L, Costi E, Maistrello L, Sabbatini PG (2016)
Searching for native egg-parasitoids of the invasive alien species
Halyomorpha halys Stål (Heteroptera Pentatomidae) in Southern
Europe. Redia 99:63–70
Ryu J, Hirashima Y (1984) Taxonomic studies on the genus Trissolcus
Ashmead of Japan and Korea (Hymenoptera, Scelionidae). J Fac
Agric Kyushu Univ 29:35–58
Schlaepfer MA, Sherman PW, Blossey B, Runge MC (2005) Introduced
species as evolutionary traps. Ecol Lett 8(3):241–246
Servick K (2018) Control freaks. Science 361:542–545
Stahl J, Babendreier D, Haye T (2018) Using the egg parasitoid Anasta-
tus bifasciatus against the invasive brown marmorated stink
bug in Europe—can non-target effects be ruled out? J Pest Sci
91:1005–1017
Talamas EJ, Herlihy MV, Dieckhoff C, Hoelmer KA, Buffington M,
Bon MC, Weber DC (2015) Trissolcus japonicus (Ashmead)
(Hymenoptera, Scelionidae) emerges in North America. J Hyme-
nopt Res 43:119
Talamas EJ, Buffington ML, Hoelmer K (2017) Revision of Palearctic
Trissolcus Ashmead (Hymenoptera, Scelionidae). In: Talamas EJ,
Buffington ML (Eds) Advances in the systematics of Platygas-
troidea. J Hymenopt Res 56:3–185
United States Apple Association (2010) Asian pest inflicting substan-
tial losses, raising alarm in eastern apple orchards. Apple News
41:488
World Bank (2018) Data retrieved October 15, 2018, from World Inte-
grated Trade Solution. UNSD Commodity Trade (COMTRADE)
database
Yang Z-Q, Yao Y-X, Qiu L-F, Li Z-X (2009) A new species of Trissol-
cus (Hymenoptera: Scelionidae) parasitizing eggs of Halyomor-
pha halys (Heteroptera: Pentatomidae) in China with comments
on its biology. Ann Entomol Soc Am 102:39–47
Zhang J, Zhang F, Gariepy T, Mason P, Gillespie D, Talamas E, Haye
T (2017) Seasonal parasitism and host specificity of Trissolcus
japonicus in northern China. J Pest Sci 90:1127–1141