# First detection of Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) in southwestern France

Guillaume Martel, Alexandre Bout, Francesco Tortorici, Rachid Hamidi, Luciana Tavella, Maud Thomas

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Guillaume Martel, Alexandre Bout, Francesco Tortorici, Rachid Hamidi, Luciana Tavella, et al. First detection of Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) in southwestern France. Journal of Hymenoptera Research, 2024, 97, pp.1123-1139. 10.3897/jhr.97.132433. hal-04797628

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# First detection of *Trissolcus japonicus* (Ashmead) (Hymenoptera, Scelionidae) in southwestern France

Guillaume Martel¹, Alexandre Bout², Francesco Tortorici³,
Rachid Hamidi¹, Luciana Tavella³, Maud Thomas¹

1. ANPN, Association Nationale des Producteurs de Noisettes, Lieu-dit Louberie, 1500 route de Monbahus, 47290 Cancon, France 2. INRAE, UMR INRAE 1355 CNRS 7254, Institut Sophia Agrobiotech, Université Côte d'Azur, 400 route des Chappes, 06903 Sophia Antipolis, France 3. DISAFA, Dipartimento di Scienze Agrarie, Forestali e Alimentari, University of Torino, Largo P. Braccini 2, 10095 Grugliasco, Italy

Corresponding author: Guillaume Martel (gmartel@anpn.eu)

Academic editor: Elijah Talamas | Received 18 July 2024 | Accepted 3 October 2024 | Published 4 November 2024

https://zoobank.org/8B42E7A2-41CD-4DE7-A075-00AA4B9412B0

**Citation:** Martel G, Bout A, Tortorici F, Hamidi R, Tavella L, Thomas M (2024) First detection of *Trissolcus japonicus* (Ashmead) (Hymenoptera, Scelionidae) in southwestern France. Journal of Hymenoptera Research 97: 1123–1139. https://doi.org/10.3897/jhr.97.132433

## Abstract

We report the first detection of *Trissolcus japonicus* in southwestern France. A total of almost 3000 sentinel and 700 field-laid egg masses of *Halyomorpha halys* were exposed or collected in the administrative region of Nouvelle-Aquitaine in 2022 and 2023. A total of 12 and 44 specimens of *T. japonicus* emerged from one and two egg masses in 2022 and 2023, respectively. Morphologic analysis confirmed the identification of this egg parasitoid, while molecular analysis of COI matched the haplotype of the French population to the population of *T. japonicus* released in Italy. *Trissolcus japonicus* may have arrived in Nouvelle-Aquitaine from introduction pathways similary to *T. mitsukurii*, detected in 2020, although these pathways cannot be clearly defined. The co-occurrence of these two major parasitoids of BMSB should now be surveyed as possible competition can arise. In the frame of the biological control program currently running in France against BMSB, the use of one or two parasitoid species is also considered.

## Keywords

Biological control, brown marmorated stink bug, DNA barcoding, exotic species
**Introduction**

Herbivorous stink bugs are a good example of increasing economic loss by invasive insects; many of these species are agricultural pests in their native and invasive range (McPherson 2018; Conti et al. 2021). The Brown Marmorated Stink Bug (BMSB), *Halyomorpha halys* (Stål) (Hemiptera: Pentatomidae) is one of the main invasive stink bug species in recent decades. Native to eastern Asia, BMSB arrived in North America in the mid-1990's (Leskey and Nielsen 2018) and in Europe in the 2000's where it spread quickly in Switzerland, Georgia, Italy and, more recently, France (Bosco et al. 2018; Kereselidze et al. 2022). Like many invasive Pentatomidae species, BMSB is highly polyphagous, feeding on more than one hundred host plants (Lee et al. 2013; Bergmann et al. 2016) from wild and ornamental to cultivated species, including peach, peer, apple, grape and hazelnut. Thus, it can incur considerable damages on a wide range of agricultural systems, like in northern Italy (Maistrello et al. 2017; Bosco et al. 2018) where outbreaks in 2019 caused more than € 356 million damage on pear, peach, and nectarine productions, with up to 80–100% yield losses (Centro Servizi Ortofrutticoli 2019). In hazelnut orchards, the absence of insecticide treatment can lead to 23–40% of damaged hazelnuts (de Benedetta et al. 2023). In France, the presence of BMSB is increasing since first identified in 2012, and this species represents an important risk for many French crops, according to the French Agency for Food, Environmental, and Occupational Health and Safety (Haye et al. 2014). In the case of hazelnut orchards, average bug damage increased continuously from 0.2% in 2015 to 2% in 2018, with peaks that reached 14% and up to 30% in 2022 (unpublished data, Unicoque). To control BMSB, the use of broad-spectrum insecticides can no longer be considered due to the ecological and sanitary risks, the growing societal demand that results in the progressive suppression of authorized chemicals, and the possibility of resistance in BMSB populations, as has happened in other pentatomid species (Sosa-Gomez et al. 2001). Classical biological control (CBC) appears as a possible alternative strategy to the use of insecticides. CBC aims at restoring ecosystem balance by introducing exotic natural enemies to better control an invasive pest, which often lacks efficient natural enemies in its invasive range. In Europe for instance, BMSB can be parasitized by parasitoid wasps in the genera *Trissolcus* Ashmead, *Telenomus* Haliday, *Ooencyrtus* Ashmead or *Anastatus* Motchoulsky, but parasitism often leads to egg abortion, incomplete development of parasitoids or low parasitism rates (Haye et al. 2016; Roversi et al. 2016). Among the natural enemies of the BMSB in its native range, *Trissolcus japonicus* (Ashmead) (Hymenoptera: Scelionidae) is considered as one of the most promising candidates for CBC (Yang et al. 2009; Zhong et al. 2017). Numerous studies have been conducted to assess the parasitoid's efficiency and biosafety as part of the requirements for its legal introduction in the USA (Hedstrom et al. 2017), Canada (Abram et al. 2019), New Zealand (Charles et al. 2019), and Europe (Haye et al. 2020). *Trissolcus mitsukurii* (Ashmead) is also known as a natural enemy of the BMSB in Japan
(Arakawa and Namura 2002) and is considered as a valid candidate for its control in Italy together with *T. japonicus* (Sabbatini Pevereri et al. 2020). Since *T. mitsukurii* is absent in North America, Europe (especially Italy) has been the only known place where the control of BMSB by both parasitoid species could be studied (Moraglio et al. 2020).

Legislative restrictions in the USA and Europe may be a limiting factor for the introduction of exotic species by focusing on perceived risks for non-targets organisms rather than the benefits of pest reduction (van Lenteren et al. 2006; Rondoni et al. 2021). However, unintentional introduction of exotic natural enemies is likely to happen more often than expected (Mason et al. 2017). Parasitoids often move along the same routes as their hosts, and this phenomenon can lead to unintentional biological control as defined by Beers et al. (2022) and has been documented several times, for instance in the USA, Germany and Italy: *Lepto-pilina japonica* Novković and Kimura (Hymenoptera: Figitidae) against *Drosophila suzukii* (Matsumura) (Diptera: Drosophilidae) (Puppato et al. 2020; Beers et al. 2022; Martin et al. 2023); as well as Sclelionidae species, *Paratelenomus saccharalis* (Hym. Platygastridae) (Dodd) (Gardner et al. 2013) against *Megacopta cribraria* (F.) (Hem. Plataspidae), with *Trissolcus hyalinipennis* Rajmohana and Narendran and *Gryon aetherium* Talamas (Ganjisaffar et al. 2018; Hogg et al. 2021) against *Bagrada hilaris* (Burmeister) (Hem. Pentatomidae). Fortuitous populations of *T. japonicus* were found in the USA (Talamas et al. 2015; Milnes et al. 2019), Canada (Abram et al. 2019), Switzerland, Italy (Sabbatini Peverieri et al. 2018; Stahl et al. 2019; Moraglio et al. 2020), Germany (Dieckhoff et al. 2021) and Slovenia (Rot et al. 2021), while *T. mitsukurii* was detected in Italy (Sabbatini Peverieri et al. 2018), France (Bout et al. 2021) and Serbia (Konjević et al. 2024).

The discovery of adventive populations of *T. japonicus* in the USA and Italy probably led to lower constraints for releases (Talamas et al. 2015). In France, the discovery of an adventive population of *T. mitsukurii* (Bout et al. 2021) led to the obtainment of a release permit that opened the way to a biological control program against BMSB in 2022. The detection of *T. japonicus* in France should help in the same way and represents a unique opportunity to study possible interactions of both parasitoid species. Despite surveys conducted in southeastern and southwestern France since 2017 and multiple releases in northern Italy since 2020, *T. japonicus* has not been detected yet in France. Meanwhile, the geographical proximity with the Piedmont region (Italy) where *T. japonicus* is released and the important commercial and human connection between northern Italy and southeastern France could lead to unintentional dispersion of this parasitoid. This dispersion is also expected to reach southwestern France where an adventive population of *T. mitsukurii* already established, likely from the Italian population (Bout et al. 2021). In the region Nouvelle-Aquitaine (France), where the current study took place, releases of *T. mitsukurii* were initiated in 2022 in the frame of the project RIPPOSTE and field monitoring was conducted with a focus on *T. mitsukurii* and *T. japonicus*.
# Materials and methods

## Field surveys

Exposure of BMSB sentinel egg masses and collection of field-laid egg masses of Pentatomidae and Coreidae were used to assess the diversity of egg parasitoids in France. The field surveys took place in 2022 and 2023 and covered 11 sampling sites in Nouvelle-Aquitaine (southwestern France) in three departments: Lot-et-Garonne, Dordogne and Gironde. In this sampling area, opportunistic collection of egg masses from *H. halys* and other pentatomid species on localities that are not part of the 11 chosen sites were also included in the samples.

## Laboratory colony of the BMSB

The monitoring of egg parasitoids of BMSB requires the use of sentinel egg masses that can be produced in laboratory conditions. To obtain these egg masses, a laboratory colony of BMSB was constituted from adult individuals collected in Cancon (France) during fall 2021 using pheromone traps (Trece®) and transferred to laboratory conditions (T: 25 ± 2 °C, RH: 50 ± 10%, L:D: 16:8 h). They were reared in nylon net cages (47.5 × 47.5 × 47.5 cm, Bugdorm®, MegaView Science Co., Ltd., Taichung, Taiwan) containing fresh beans and apples, complemented with fresh maize and hazelnuts. Egg masses were collected daily and stored under the same laboratory conditions until hatching of the first instars. These were transferred to new cages so that each cage was homogeneous in developmental stage.

## Field parasitoid monitoring

Sentinel egg masses were obtained from fresh BMSB egg masses (< 24 h) collected from the laboratory colony and were directly exposed in the field or stored in a fridge (c.a. 8 °C) for up to 2 days. Frozen eggs were not used as they can be less detectable or acceptable for egg parasitoids and lead to sampling bias (Jones et al. 2014). Each egg mass was glued on a cardboard piece (1 × 3 cm) and stapled to the under-side of the leaves of a wide variety of host plants, mostly woody trees (e.g., *Prunus* spp., *Acer* spp., *Catalpa bigninoides* Walt., *Paulownia tomentosa* (Thunb.), and *Corylus avellana* L.). Sentinel egg masses were removed from the field three days after deployment. They were exposed in the field every two weeks on each monitored site from June 10th to September 16th in 2022 and every week from April 28th to September 15th in 2023. Field-laid egg masses from BMSB and other heteropteran species (Pentatomidae, Coreidae) were collected in the sampling area in N-A from May 18th to September 16th, 2022, and from May 10th to September 8th, 2023. During the surveys, egg masses of stink bug species were visually located on various host plants and collected by hand. All egg masses, sentinel and field-laid, were kept in the laboratory under controlled
conditions (22 ± 1 °C; RH 50 ± 5%; L:D: 16:8 h) until the emergence of bug nymphs or adult parasitoids. Egg masses were individually stored in Petri dishes and checked daily for emergence since the first observation of external indication of parasitoid development (grey or black coloration of the egg). Emerged parasitoids were stored in 70% ethanol while non-emerged eggs were dissected under a stereomicroscope (Nikon SMZ 1270) to determine whether they were parasitized or not (presence of an identifiable larvae, nymph, or adult parasitoid). When the egg content was not identifiable, the egg was considered as non-parasitized.

## Molecular analysis

DNA-barcoding characterization consisted in a double identification based on the Cytochrome Oxydase I (COI) amplification and sequencing, and morphological identification of the corresponding voucher (exoskeleton of the specimen). Molecular analyses were performed as described in Bout et al. (2021) for *T. mitsukurii* identification. Extraction of DNA was performed using the DNA kit extraction MA150E – QuickExtract – DNA, following company specifications. This non-destructive method allowed the vouchers to remain intact for morphological identifications. PCR amplifications were performed on a portion of the Cytochrome Oxydase I subunit (COI) locus using the LCO-HCO primer: HCO2198 (5'-TAAA CTT CAG GGT GAC CAA AAA ATC A-3'), LCO1490 (5'-GGTC AAC AAA TCA TAA AGA TAT TGG-3') (Folmer et al. 1994), allowing amplification of an approximately 600–700 bp portion of DNA on this locus. The product was sent to Genewiz (Leipzig, Germany) for a double, single read sequencing with the HCO2198 primer. All residual DNAs are archived at INRAE Sophia-Antipolis (France). Correction, annotation and alignment were performed manually using BioEdit Geneious R10 software. The comparison of nucleotide sequences with sequences available in the NCBI database (GenBank) was performed using Blastn (Altschul et al. 1990) with standard settings. From GenBank (NCBI), all COI sequences (72) clearly identified as *T. japonicus* species were downloaded. Two sequences from CREA-Italy were added to the study and compiled with the ten sequences obtained from French population of *T. japonicus* collected in 2022 (7 sequences) and 2023 (3 sequences) (Table 2), resulting in a final table of 84 sequences (Suppl. material 2). Analysis of sequences data was done with the MegaX software (Tamura et al. 2013), using the neighbor joining (NJ) method (Saitou et al. 1987), with bootstrap values based on 500 replications. Nucleotide distances in NJ trees were estimated by the Kimura's two-parameters method (Kimura, 1980). Some sequences were significantly shorter than the others. Hence, analyses exclude the 22 shorter sequences and work only on the common part of the 62 other sequences (fragment of 575 pb). A fast alignment file was used with DnaSP6 to create all haplotypes of *T. japonicus* sequences. The resulting nexus file was analyzed with PopArt software (Otago University, Dunedin, New Zealand) to organize haplotype networks.
## Morphological identification

All parasitoid wasps emerged were morphologically identified using a Wild M5 stereo microscope with the appropriate taxonomic key. Species of Scelionidae were identified using Kozlov and Kononova (1983), Javahery (1968), Moraglio et al. (2021), Talamas et al. (2017), Tortorici et al. (2019). Eupelmidae wasps were identified using Askew and Nieves-Aldrey (2004) and Peng et al. (2020). Images of the specimens were taken using a Canon 90D camera (Canon Inc., Tokyo, Japan) equipped with extension tube, 10× and 20× LWD microscope lenses mounted on a macro-rail. The final pictures were processed with Zerene Stacker (PMax algorithm, Zerene Systems LLC, Richland, WA, USA) and with Photoshop (Adobe Systems Inc, USA). The specimens used for morphological analysis were deposited in the collection of the Dipartimento di Scienze Agrarie, Forestali e Alimentari, University of Turin, Italy.

## Results

### Parasitism on BMSB egg masses

Various species of native egg parasitoids belonging to Scelionidae (*Trissolcus* spp. and *Tel-enomus* spp.), Eupelmidae (*Anastatus bifasciatus* (Geoffroy)), and Encyrtidae (*Ooencyrtus* spp.) emerged from egg masses of BMSB collected during the surveys, as well as sentinel egg masses (data not shown). The parasitism rate of sentinel egg masses was lower than naturally-laid ones in 2022 and 2023 (Table 1). In early August 2022, one parasitized egg mass of BMSB laid on *Ailanthus altissima* L. was collected in Castillonnès (N-A, France) and produced 12 specimens of *T. japonicus*. In early July and late August 2023 respectively, two sentinel egg masses were found parasitized by *T. japonicus* in Sainte Foy la grande (N-A, France; 42 km far from Castillonnès) and produced together 44 specimens. However, no specimen was further recovered from 2023 survey in Castillonnès.

## Morphological identification

Females and males of *T. japonicus* specimens detected in Castillonnès and Sainte Foy la grande had typical morphological features of this species as described by Talamas

<table><caption>**Table 1.** Parasitism rates of sentinel and naturally laid egg masses of BMSB in 2022 and 2023 in Nouvelle-Aquitaine (France). (1) All parasitism occurrences; (2) *Trissolcus japonicus* occurrences.</caption><thead><tr><th>Monitoring period (in Nouvelle-Aquitaine, France)</th><th>Type of egg mass</th><th>Number of collected egg masses</th><th>Number of parasitized egg masses</th><th>(1) % of egg masses parasitized</th><th>(2) % of egg masses parasitized</th></tr></thead><tbody><tr><td>June–September 2022</td><td>&lt; 24 h sentinel</td><td>1169</td><td>40</td><td>3.4</td><td>0</td></tr><tr><td>May–September 2022</td><td>Natural</td><td>155</td><td>26</td><td>19.2</td><td>0.6</td></tr><tr><td>April–September 2023</td><td>&lt; 24 h sentinel</td><td>1822</td><td>83</td><td>4.6</td><td>0.1</td></tr><tr><td>May–September 2023</td><td>Natural</td><td>181</td><td>18</td><td>9.9</td><td>0</td></tr></tbody></table>
et al. (2017): a uniform, well-defined hyperoccipital carina on the vertex between lateral ocelli (Figs 1B, C, 2) and females had a clypeus with four setae and well-defined episternal foveae that extend from the postacetabular sulcus to the mesopleural pit (Fig. 1A). In addition, the absence of rugae on the mesoscutum and the absence of a smooth area below the median ocellus (Fig. 1A, B) confirm that it is neither of the Palearctic species closest to *T. japonicus*, *Trissolcus kozlovi* Rjachovskij and *Trissolcus plautiae* (Watanabe).

## Molecular identification

Molecular characterization of the specimens collected in Nouvelle-Aquitaine, resulted in one specific haplotype: haplotype Hap02 (Fig. 3; Suppl. material 2). These sequences match with *T. japonicus* cluster (Fig. 3; Suppl. material 1). The haplotype analyses highlights that these specimens are the same haplotype that the Asian

<figure><img src="image_3.png" /><figcaption>**Figure 1.** *Trissolcus japonicus* female, voucher n° DISAFA-FT HYM651 **A** head and mesopleuron **B** habitus in dorsal **C** habitus in lateral view; of = orbital furrow; cs = clypeal setae; eps = episternal foveae; ats = postacetabular sulcus; mpp = mesopleural pit; hoc = hyperoccipital carina; A7–A11 = Antenomeres 7–11 (clavomeres).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** *Trissolcus japonicus* male, voucher n° DISAFA-FT HYM652 **A** habitus in latero-dorsal view **B** habitus in dorsal view.</figcaption></figure>

<table><caption>**Table 2.** GenBank accession number and sample information for COI sequences of French *Trissolcus japonicus* presented in this study.</caption><thead><tr><td>Collection Code</td><td>Department country</td><td>Year of collection</td><td>GPS coordinates (DMS)</td><td>GenBank accession number</td></tr></thead><tbody><tr><td>ISA45651</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766189</td></tr><tr><td>ISA45652</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766190</td></tr><tr><td>ISA45653</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766191</td></tr><tr><td>ISA45654</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766192</td></tr><tr><td>ISA45671</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766193</td></tr><tr><td>ISA45672</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766194</td></tr><tr><td>ISA45673</td><td>Lot et Garonne, France</td><td>2022</td><td>44.65474, 0.5883531</td><td>PP766195</td></tr><tr><td>ISA47499</td><td>Gironde, France</td><td>2023</td><td>44.8405337, 0.212578</td><td>PP766196</td></tr><tr><td>ISA47500</td><td>Gironde, France</td><td>2023</td><td>44.8405337, 0.212578</td><td>PP766197</td></tr><tr><td>ISA47501</td><td>Gironde, France</td><td>2023</td><td>44.8405337, 0.212578</td><td>PP766198</td></tr></tbody></table>

strain labelled “Beijing USDA”, recently released in Italy for the official CBC program (Suppl. material 2). In addition, translation of all COI sequences into protein resulted in homogeneous protein sequences, with no stop codons. The few changes in amino acid composition observed are between amino acids of the same structural group (Suppl. material 3). Two sequences, corresponding to haplotype Hap01, show a change of amino acid at position 92 *i.e.* Alanine instead of Serine, which belong to two different structural groups (Alanine being the simplest amino acid, while Serine which derived from alanine, has an alcohol function).

## Discussion

The sampling of egg parasitoids of BMSB using natural and fresh sentinel egg masses has been effective for the detection of exotic *Trissolcus* species in several countries including France (Talamas et al. 2015; Milnes et al. 2019; Stahl et al. 2019; Moraglio et al. 2020; Bout et al. 2021; Dieckhoff et al. 2021). In Nouvelle-Aquitaine, where
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Haplotype network obtained from the 62 COI sequences (575 bp) of *Trissolcus japonicus*. Countries of origin were indicated by colors. POP ART program (Leigh and Bryant 2015).</figcaption></figure>
*T. mitsukurii* was previously collected from natural egg masses, *T. japonicus* was equal-
ly collected from natural and fresh sentinel egg masses. This indicates that both are
physiologically suitable for the parasitoid, although the low general parasitism rate on
sentinel egg masses suggests they are more difficult to detect or exploit. Regarding the
geographic range of our sampling, we likely detected the first non-intentional intro-
duction of *T. japonicus* in France. A migration of the two exotic species *T. japonicus*
and *T. mitsukurii* in southwestern France has been expected as (i) they are widespread in
Italy and Switzerland, and (ii) the growing density of BMSB provides an invasion
opportunity for its native parasitoids (Zapponi et al. 2020). The shared distribu-
tion of *T. mitsukurii* and *T. japonicus* in Asia, as well as their co-occurrence in Italy
(Zapponi et al. 2020; Falagiarda et al. 2023), reveals similar ecological preferences.
French climatic conditions, as those of Europe, are favorable for widespread establish-
ment of *T. japonicus* following the presence of BMSB (Avila and Charles 2018). The
detection of *T. japonicus* in Nouvelle-Aquitaine also matches with its modelized
distribution proposed by Tortorici et al. (2023). On the other hand, *T. japonicus*
has not been collected in the French region of Alpes-Maritimes which is close to
Italy, or the regions close to Switzerland as no sampling was performed along an East-
West gradient. Thus, it cannot be concluded whether the presence of *T. japonicus* in
Nouvelle-Aquitaine results from human activities or natural dispersion, although it is
likely to have used similar dispersal ways than *T. mitsukurii*.

Very low recorded parasitism levels are characteristic of the initial detections in
other areas where adventive *T. japonicus* populations have since been confirmed and
spread (Talamas et al. 2015; Abram et al. 2019; Milnes et al. 2019). Detections of
*T. japonicus* and *T. mitsukurii* in France always occurred in urban areas or in proxim-
ity to ornamental trees. This was also the case in the USA and Italy (Hedstrom et al.
2017; Sabbatinni Peverieri et al. 2018; Lowenstein et al. 2019). While *T. mitsukurii*
was first discovered in France peri-urban area BMSB egg masses laid on cherry laurel,
*T. japonicus* was collected in a residential area where BMSB established at high densi-
ties on preferred host plants *A. altissima* and *C. bigninoides*. In North America, BMSB
is highly associated with *A. altissima*, as well as in Italy (Zapponi et al. 2020; Dyer et al.
2022) so that these tree species are relevant sampling spot (Quinn et al. 2019).

All of the samples of *T. japonicus* collected between 2022 and 2023 in Nouvelle-
Aquitaine present the same haplotype Hap02. This haplotype corresponds to strains
of *T. japonicus* present in China previously collected by USDA and introduced in
Europe i.e. Cabi quarantine (Switzerland) and CREA-DC (Consiglio per la ricerca
in agricoltura e l'analisi dell'economia agraria – Difesa e Certificazione, Italy). In the
same way as for *T. mitsukurii* (Bout et al. 2021) the most probable origin of the French
population of *T. japonicus* appears to be Italy, likely from material or plants moved
from Italy to France which would support the common introduction way. The col-
lection of specimens between 2022 and 2023 would indicate that this population of
*T. japonicus* is established, although it was collected on two different sites. Hence, the
question of multiple introductions remains. Molecular sequences used in this study are
of good quality: translation of sequences into proteins are preserved and homogeneous,
without stop codon. These sequences reflect a genetic polymorphism present within
the species. The two sequences representing the haplotype Hap01, with a more sig-
nificant amino acid replacement at position 92, could reflect a DNA amplification (or
correction) error. These sequences could probably be part of the haplotype Hap06.

**Conclusion**

The detection of *T. japonicus* in 2023, even on a different site than 2022, encour-
ages new prospects and may indicate establishment of the parasitoid. How popu-
lations of *T. japonicus* will manage to establish and spread will depend on their
ability to survive and reproduce after winter, as well as finding suitable hosts when
temperatures rise. In Oregon (USA), *T. japonicus* was able to survive average winter
lows of -3 °C (Lowenstein et al. 2019) and it could establish in Italy despite winter
reaching -5 °C (Falagiarda et al. 2023). With similar thermal conditions in south-
western France, it is likely to overcome winter, which is currently under investiga-
tion. Regarding possible hosts, in addition to BMSB, surveys should continue to
include non-target host species. In Italy, surveys one year after releases of *T. japoni-
cus* indicated that *Pentatoma rufipes* (L.), *Graphosoma lineatum* (L.) and *Palomena
prasina* (L.) were parasitized (up to 26% parasitized eggs for *G. lineatum*) (Fala-
giarda et al. 2023). The monitoring of non-target pentatomid species also provides
data on egg parasitoid diversity and prevalence, with some of species like *A. bifas-
ciatus* or *Trissolcus belenus* (Walker) able to apply a “natural” regulation of BMSB.
Finally, the co-occurrence of *T. japonicus* and *T. mitsukurii* should be investigated
both in terms of parasitism rate and geographical dispersion, since monitoring in
Italy highlighted that the natural spread of *T. japonicus* was of lower importance than
*T. mitsukurii* (Falagiarda et al; 2023).

Acknowledgements

This study took place in the frame of the project RIPPOSTE with the financial
support of the Nouvelle-Aquitaine Region (FR). We acknowledge Emeric Poret,
Léa Tournier and Amandine Jagueneau for their involvement in the project and
the field monitoring during summers 2022 and 2023. This study was also carried
out within the Agritech National Research Center and received funding from the
European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E
RESILIENZA (PNRR)—MISSIONE 4 COMPONENTE 2, INVESTIMEN-
TO 1.4—D.D. 1032 17/06/2022, CN00000022). We acknowledge the reviewers
for their comments and language revision that improved the quality of the manu-
script. This manuscript reflects only the authors’ views and opinions; neither the
European Union nor the European Commission can be considered responsible
for them.
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## Supplementary material 1

### Phylogenetic tree based on molecular clustering of worldwid *Trissolcus japonicus* vouchers including French samples

Authors: Guillaume Martel, Alexandre Bout, Francesco Tortorici, Rachid Hamidi, Luciana Tavella, Maud Thomas

Data type: pdf

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.97.132433.suppl1

## Supplementary material 2

### Sequences references of *Trissolcus japonicus* vouchers used to build the Haplotype network

Authors: Guillaume Martel, Alexandre Bout, Francesco Tortorici, Rachid Hamidi, Luciana Tavella, Maud Thomas

Data type: xlsx

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.97.132433.suppl2

## Supplementary material 3

### COI proteine translation from *Trissolcus japonicus* sequences used in the study

Authors: Guillaume Martel, Alexandre Bout, Francesco Tortorici, Rachid Hamidi, Luciana Tavella, Maud Thomas

Data type: pdf

Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Link: https://doi.org/10.3897/jhr.97.132433.suppl3