Physiological Host Range of Trissolcus Mitsukurii, A Candidate Biological Control Agent of Halyomorpha Halys

doi:10.21203/rs.3.rs-362649/v1

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Physiological Host Range of Trissolcus Mitsukurii, A Candidate Biological Control Agent of Halyomorpha Halys

https://doi.org/10.21203/rs.3.rs-362649/v1

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published 05 Aug, 2021

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The invasive stink bug Halyomorpha halys is a severe agricultural pest of worldwide importance, and chemical insecticides are largely sprayed for the control of its populations. Negative impacts and several failures in chemical pest management led to consider classical biological control as one of the most promising methods in a long-term perspective. The Asian egg parasitoid Trissolcus japonicus is the main candidate biocontrol agent of H. halys, but more recently a second Asian egg parasitoid, Trissolcus mitsukurii, is getting attention after adventive populations were found on H. halys egg masses in Europe. Before recommending the use of T. mitsukurii for biological control of H. halys, a risk analysis is necessary and therefore here we present the first study on the fundamental physiological host range of this parasitoid in Europe. Tests conducted with T. mitsukurii on different hemipterans, using three different experimental designs, revealed a broad physiological host range, comparable with the host range displayed by T. japonicus under similar laboratory conditions. Specifically, in addition to its coevolved host H. halys, T. mitsukurii successfully parasitized the majority of tested pentatomid species and one scutellerid, although with highly variable emergence rates. Host egg sizes positively affected parasitoid size and female egg load. Further studies, testing more complex systems that involve olfactory cues from host and host plants, will simulate different aspects of the parasitoid host location behavior under field conditions, allowing in-depth evaluation of the possible risks associated with the use of T. mitsukurii as a biocontrol agent of H. halys.

Agricultural Engineering

classical biological control

invasive alien species

non-target risk assessment

brown marmorated stink bug

Pentatomidae

Scelionidae egg parasitoid

1) Trissolcus mitsukurii is a potential biological control agent (BCA) of Halyomorpha halys

2) Recently, adventive populations were found in Italy parasitizing H. halys

3) A risk analysis is mandatory for the evaluation of biological control programs

4) Trissolcus mitsukurii displayed in black box tests a host range comparable with that of the main BCA Trissolcus japonicus

5) More complex experimental designs (e.g., tri-trophic system) are needed for more detailed outputs

The Brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), is a polyphagous invasive pest native from Eastern Asia and currently present in several countries of the Americas and Europe, where it causes severe damage to many agricultural crops (Leskey and Nielsen 2018). The low efficacy and high environmental impact of insecticide treatments, combined with the lack of effective native natural enemies, led to consider classical biological control with egg parasitoids a promising method for long-term management of H. halys in the invaded areas (Abram et al. 2017). Although recent reviews indicated limitations in the success of classical biological control of stink bugs, research in this field needs to highlight any potential approach to find effective pest control solutions (Abram et al. 2020; Conti et al. 2021). These solutions could emerge from the integration of classical biological control with techniques targeting different instars of the pest (Abram et al. 2020), like plant resistance (Rondoni et al. 2018), exploitation of plant volatiles to improve parasitoid efficacy (Rondoni et al. 2017), manipulation of stink bug behavior and parasitoid conservation (reviewed by Conti et al. 2021).

The introduction of an exotic organism, including a candidate biological control agent, is strictly regulated in order to avoid potential negative and permanent ecological consequences (van Lenteren et al. 2006; EPPO 2014; FAO-IPPC 2017). Therefore, non-target risk studies of the candidate biological control agent are mandatory and precede any further step for the petition and approval of its release in the field. Laboratory investigations are the first step for risk analysis of the exotic natural enemy and allow to explore its fundamental physiological host range. Then, several different testing methods need to be integrated in a multiple step workplan, including behavior and chemical ecology. Such studies are necessary for moving from a physiological host range outcome to an ecological host range perspective, which allows to prevent or minimize the risk of a negative impact caused by a voluntary human intervention in the ecosystem (van Lenteren et al. 2006).

The Asian egg parasitoid Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) is considered so far the most valid candidate for biological control of H. halys (Zhang et al. 2017; Conti et al. 2021). Its host range, outside the natural one, was investigated in the USA, Europe and New Zealand, confirming its oligophagous habitus but also showing its host preference versus H. halys (Hedstrom et al. 2017; Botch and Delfosse 2018; Charles et al. 2019; Lara et al. 2019; Haye et al. 2019; Sabbatini Peverieri et al., 2021). Adventive populations of T. japonicus were discovered recently in Europe and the USA (Talamas et al. 2015; Sabbatini Peverieri et al. 2018; Stahl et al. 2019). Surprisingly, a second Asian egg parasitoid of H. halys, Trissolcus mitsukurii (Ashmead) (Hymenoptera, Scelionidae), was also found in Italy, as the first record outside its native range (Sabbatini Peverieri et al. 2018). However, while T. japonicus ability to attack H. halys is well recognized, little is known so far about the preferences and life traits of T. mitsukurii on H. halys (Zhang et al. 2017; Arakawa and Namura 2002; Arakawa et al. 2004). This parasitoid species is known mainly from Japan, where it has been recorded parasitizing in the field eggs of about ten species of Pentatomidae (Yasumatsu and Watanabe 1964; Hokyo et al. 1966; Ryu and Hirashima 1984; Arakawa and Namura 2002). Additionally, in laboratory test conducted in Japan, T. mitsukurii was also able to parasitize another pentatomid species in the genus Plautia (Arakawa and Namura 2002). Therefore, T. mitsukurii seems to be an oligophagous egg parasitoid. In Japan it was reported in the past as one of the most active natural enemies of Nezara viridula L. (Hemiptera, Pentatomidae) (Kiritani and Hokyo 1962; Hokyo and Kiritani 1963), although more recent data from the same country reported this parasitoid as commonly associated to H. halys (Arakawa and Namura 2002; Arakawa et al. 2004). Instead, the high parasitization rate reported in China for T. mitsukurii on H. halys (Chu et al., 1997), due to an original misidentification should be assigned to T. japonicus as stated by Yang et al. (2009).

Field data from Northern Italy showed high rates of parasitism of H. halys eggs by T. mitsukurii, suggesting that this egg parasitoid could be a valid candidate for biological control of the invasive stink bug in addition to T. japonicus (Benvenuto et al. 2020; Scaccini et al. 2020; Zapponi et al. 2020). Because no specific and recent information is available on the host range of T. mitsukurii, a risk analysis workplan needs to be initiated before implementation of this egg parasitoid as a biological control agent is considered (van Lenteren et al. 2006). The present work is the first contribution to the exploration of the physiological host range of T. mitsukurii, conducted comparing its performance on H. halys, the target pest of biological control, and other Hemipteran non-target species that are common in Europe.

2.1 Field sampling, insect collection and rearing

Halyomorpha halys adults were collected in Northern Italy in 2020, both in overwintering sites and in the field during the growing season, whereas adults of non-target Hemiptera species were collected in H. halys habitats and surrounding areas, of North and Central Italy, during the 2020 growing season. Non-target Hemiptera species were chosen based on the potential host range of the parasitoid and the information mined from the literature, and according to the centrifugal phylogenetic approach (Kuhlmann and Mason 2003; Kulmann et al. 2006). Beside H. halys, the following non-target herbivorous Hemipteran species were investigated: the pentatomids Acrosternum heegeri (L.), Aelia acuminata (L.), Ancyrosoma leucogrammes (Gmelin), Carpocorispudicus (Poda), Dolycoris baccarum (L.), Eurydema ventralis Kolenati, Eurydema oleracea (L.), Graphosma italicum (Muller), N. viridula, Palomena prasina (L.), Piezodorus lituratus (F.), and Sciocoris sp., the scutellerid Eurygastermaura (L.), and the coreid Gonocerus juniperi Herrich-Schäffer. The two predator species Arma custos (Fabricius) (Hemiptera, Pentatomidae) and Rhynocoris iracundus (Poda) (Hemiptera, Reduviidae) were also considered (see Table 1).

Laboratory colonies were established and maintained using rearing cages (BugDorm 4F4545, Insect MegaView Science Co. Ltd., Taichung, Taiwan) in rearing rooms at 26 ± 1°C and 16:8 L:D. For the phytophagous species, a variety of plant sources was used as food: seeds (Arachis hypogaea), fresh vegetables (Daucus carota, Phaseolus vulgaris, Brassica oleracea, Capparis spinosa), fresh fruits (Malus domestica, Actinidia deliciosa). Food was purchased from local markets. Plants were self-produced and were used when at least 10cm tall. The predator species were reared on Acanthoscelides obtectus Say (Coleoptera, Chrysomelidae) adults, N. viridula nymphs and Tenebrio molitor L. (Coleoptera, Tenebrionidae) pupae. For all species, food and water (provided with wet cotton) were replenished three times per week. Paper towels were hung inside the rearing cages as oviposition substrates, and eggs were collected daily. As a measure of standard quality of laboratory hemipteran rearing, the viability of eggs was occasionally recorded (Tab. 1). At this scope, batches of egg masses produced by target and non-target species were reared in climatic chamber (HPP750, Memmert GmbH + Co. KG, Schwabach, Germany) at 26 ± 1°C, 60 ± 5% RH and 16:8 L:D up to egg hatching.

Trissolcus mitsukurii was obtained from H. halys egg masses collected in 2018 and 2019 in infested sites of Northern Italy. These egg masses were reared in the laboratory and the emerged T. mitsukurii individuals (several dozens of specimens) were used to start a laboratory colony. Adults of the egg parasitoid were fed with pure honey droplets. Periodically, fresh egg masses (<24h old) of H. halys were offered as host for parasitization and juvenile development. The permanent colony of the egg parasitoid was established and maintained in climatic chamber at 26 ± 1°C, 60 ± 5% RH and 16:8 L:D; such conditions were considered as standards for the present work and were used for all experiments. Adults of T. mitsukurii and parasitized egg masses were housed in glass tubes (15cm length, 2cm diam), closed on both ends by a plastic net (250 μm mesh). Establishment and maintenance of the egg parasitoid colony and all experiments (see below) were conducted under quarantine conditions at CREA facilities. Quarantine laboratories are authorized for studies on exotic beneficial organisms in the framework of H. halys biological control program (MiPAAF, DG/DISR/DISR05/0013647-19/04/2018 and SFR Regione Toscana 203304 – 12/04/2018).

2.2 No-choice black box tests

A total of three different experimental conditions were considered for the no-choice black box tests. In the first and second experiment, host egg masses were handled as little as possible to avoid the introduction of external contaminants, and any adjustment of size (egg number per mass) was avoided, as to simulate the natural number of host eggs, per egg batch, that female parasitoids would likely encounter in the field (Hedstrom et al. 2017; Botch and Delfosse 2018). For G. juniperi, which lays individual eggs, ten eggs were aligned and glued (using non-toxic clear school Elmer’s® glue, Newell Office Brands) on a cardboard (15mm x 80mm), each egg distanced 5 mm from the other. For the tests, one egg mass (or a row of ten single eggs) of a given host species was placed in a glass tube (15cm length, 2cm diam.), and one parasitoid female, 7-d old, was introduced for two different exposure times. Specifically, in the first experiment, females were kept for 24h and a small drop of honey was added on the glass internal surface as food source. In the second experiment, the exposure time was reduced to 2h and no food was provided.

In the third type of black box no-choice experiment, only one egg was provided to the female for a total time of 2h. In this experimental condition, a small glass vial (7cm length, 0.5cm diam.) was used and closed with a cotton ball to prevent the parasitoid from escaping. This assay is an extreme simplified exposure condition that has been previously shown to provide reliable results for no-choice tests with egg parasitoids (Sabbatini Peverieri et al. 2021). Tested eggs were glued (Elmer’s® glue) on the cardboard to permit a sufficient stability.

In all no-choice experiments, only T. mitsukurii females originating from H. halys eggs were tested, and only fresh eggs (< 24h old) of H. halys and non-target species were employed. Before use, eggs and egg masses were inspected under a stereo microscope to assess their suitability for the tests. In case of, e.g., non-characteristic color of the eggs, unusual egg arrangement in the batch, unswollen eggs in the mass, or unusual low number of eggs per mass, the egg or the egg mass was discarded. Parasitoids were removed at the end of the allotted time (24h or 2h). Eggs were maintained in the glass vials (single eggs) or tubes (egg masses) and were reared in climatic chamber at standard conditions until nymph hatching or parasitoid emergence. Eggs that did not hatch nor produced a parasitoid after three weeks from tests, were classified as “dead eggs”. These were dissected and inspected under a stereomicroscope, and the content was eventually classified as: dead parasitoid (pupae or pharate adult), dead Hemiptera nymph, or undetermined content.

2.3 Effect of host egg size on parasitoid size and egg load

The effects of host eggs on the parasitoid size and egg load was evaluated using T. mitsukurii females emerged from target and non-target hosts of the no-choice black box tests. For this purpose, the volume (mm³) of eggs of host species was calculated by the parabolic barrel formula V=πH(3r² + 4Rr + 8R²)/15, where “H” is the egg height, “r” is the radius of the operculum and “R” is the radius at the center of the egg (Abram et al. 2016; Botch and Delfosse 2018). The head and thorax width and the length of the hind tibia were measured from females emerged from target and non-target hosts. Additionally, parasitoid females from target and non-target host eggs were reared individually for 7 days at standard conditions in climatic chamber. At the seventh day of rearing, the females were dissected and the egg load in their ovary was counted after staining with 1% toluidine bleu (Sabbatini Peverieri et al. 2020).

2.4 Paired choice black-box tests using parasitoids reared on different hosts

In a first paired-choice experiment, seven-days old T. mitsukurii females reared on H. halys eggs were singly used in dual-choice tests in Petri dishes (9cm diam.). A female was allowed to exploit two fresh egg masses (< 24h), one of H. halys and one of a non-target species (n=12). Arma custos or D. baccarum were chosen as non-target species because of the high parasitization rates that were recorded in the no-choice tests (over 50% of successful parasitization). Arma custos was also chosen as this is a key species for the safeguarding approach in risk analysis. The number of eggs of tested egg masses was not adjusted in order to maintain the similar egg numbers that T. mitsukurii would encounter in the field (Haye et. al 2019). The two egg masses were placed at the bottom of the Petri dish and at the opposite side of the arena. The position of the egg species was inverted at each replicate. The parasitoid female was placed at the center of the arena and tested for a 2h time span in climatic chamber at standard condition. This reduced time of egg mass exposure was adopted because in the previous no-choice experiment the parasitoid females displayed to parasitize host egg masses within this short time interval, and because this would limit the opportunity for multiple parasitization, which is likely to occur in a 24h exposure test (Haye et al. 2019). At the end of the exposure, the females were removed from the arena and the egg masses were reared in the climatic chamber at standard condition until the eggs hatched or the parasitoids emerged. Eggs that did not hatch nor produced a parasitoid after three weeks from the experiment were dissected to determine the contents as described previously.

A second paired-choice experiment was conducted to evaluate possible effects of the rearing host on host preferences by T. mitsukurii. For this purpose, different colonies of T. mitsukurii were established and maintained on egg masses of the non-target species, D. baccarum or A. custos, with the same method used when reared on H. halys as host. The same experimental design as described above was applied using T. mitsukurii parasitoid females originated from host eggs of either D. baccarum or A. custos. When females did not make a choice, the replicate was excluded from the analysis.

2.5 Statistical analysis

In no-choice tests with host egg masses exposure for 24h and 2h, the percentages of hatched Hemiptera eggs, successfully parasitized eggs (emergence of the parasitoid) and dead eggs were analyzed by Kruskall-Wallis and Dunn’s multiple comparison post-hoc test using H. halys as control species. Data were analyzed only for the species showing at least five replicates (although the entire dataset was reported). Pairwise comparison among species in single host egg exposure for 2h was performed using Chi-square test with Yates’ correction. Pooled data on parasitoid female dimensions (head width, thorax width, hind tibia length) were analyzed with Pearson correlation coefficient. The functional relationship between the egg size of host species, emerged parasitoid female size and ovaries egg load were analyzed through linear regression model. In choice-test the percentages of hatched Hemiptera eggs, successfully parasitized eggs (emergence of the parasitoids) and dead eggs were compared using the Mann-Whitney U-test. Ovarian egg load between target and non-target species were compared with Kruskall-Wallis and Dunn’s multiple comparison post-hoc test. Statistics were performed using Graphpad Prism 8.

3.1 No-choice black box tests

When Hemiptera egg masses were exposed to T. mitsukurii for 24h, most eggs were suitable or partially suitable for parasitoid development, with significantly different emergence rates from the diverse tested species (χ²=112.7; P<0.0001) (Table 2). Trissolcus mitsukurii was able to parasitize and develop in 12 pentatomid species out of the 14 that were tested (85.71 % of tested species), and in the only tested scutellerid, E. maura, although with highly variable success. Conversely, it was not able to exploit the remaining pentatomids A. leucogrammes and E. ventralis, the coreid G. juniperi and the reduviid R. iracundus (Table 2). The emergence rate of T. mitsukurii from H. halys egg masses was very high, close to 100%, and not significantly different compared to emergence from the pentatomids A. heegeri, A custos, C. pudicus, D. baccarum, G. italicus, P. prasina, P. lituratus, Sciocoris sp. (8 out of 13 tested non-target pentatomid species) and the scutellerid E. maura (Table 2; see Table S1 for statistics). Instead, emergences were significantly lower, compared to H. halys, when eggs of Aelia acuminata, E. oleracea and N. viridula were tested (Table 2; see Table S1 for statistics). Sex ratios were strongly female-biased in most of the species tested (Table 2).

The percentages of Hemiptera eggs that hatched after exposure to T. mitsukurii were significantly different (χ²=80.04; P<0.0001) and partially complementary to parasitoid emergence rates. Almost none of the eggs of H. halys, A. heegeri, D. baccarum and P. prasina hatched, while hatching percentages were relevant for the other pentatomid species, although only A. leucogrammes and E. ventralis showed significantly higher rates compared to H. halys. The percentages of dead eggs (no hatching and no parasitoid emergence) were significantly different among the tested species (χ²=73.50; P<0.0001). Dead eggs where present in all Hemiptera species except H. halys and P. prasina (but only one egg mass of the latter could be tested), and percentages were notably higher in A. acuminata, E. oleracea, E. ventralis and N. viridula than in H. halys (Table 2; see Table S1 for statistics). Dead specimens of T. mitsukurii (pupae and pharate adults) were detected in eggs of suitable and partially suitable pentatomid host species, with the only exceptions of H. halys, D. baccarum and P. prasina. No dead parasitoid specimens were recorded in eggs of the scutellerid E. maura, although several eggs died for unknown reason (undetermined content). While no T. mitsukurii emerged from A. leucogrammes and E. ventralis, dissection of dead eggs revealed the presence of high numbers of dead parasitoid specimens (adults and pupae) in both species. Instead, dissections of dead eggs of the coreid G. juniperi and the reduviid R. iracundus never reported the presence of dead pupae or adults.

When the Hemiptera egg masses were exposed to T. mitsukurii for 2h, results were similar to the 24h exposure experiment and the emergence rates were significantly different among the tested species (χ²=77.38; P<0.0001) (Table 3; see Table S1 for statistics). Trissolcus mitsukurii was able to parasitize and develop in 6 out of the 9 pentatomid species that were tested (66.67% of tested species) and in the scutellerid E. maura (Table 3). Specifically, the emergence rates of the pentatomids A. heegeri, A custos, C. pudicus, D. baccarum, and G. italicum (5 out of 8 non-target pentatomid species) and the scutellerid E. maura where not significantly different from that of H. halys (Table 3; see Table S1 for statistics). Sex ratios were strongly female-biased in most of the species tested (Table 3). Hatching rates of Hemiptera eggs were significantly different (χ²=46.52; P<0.0001) and complementary with parasitoid emergence rates and dead eggs. Percentages of dead eggs were also significantly different (χ²=74.06; P<0.0001) and consistent with previous data. Dead pupae and adults of the parasitoid were observed in most of the tested species, but not in H. halys, A. heegeri and D. baccarum. Specifically, eggs of A. acuminata showed lower parasitization success compared to H. halys, with significantly lower parasitoid emergence and higher dead eggs containing dead parasitoids. Similarly, eggs of N. viridula showed no parasitoid emergence and significantly higher dead eggs containing dead parasitoids. Instead, a high rate of nymphs emerged from E. ventralis eggs and no parasitoid emerged, while some dead parasitoids were found in the eggs (Table 3; see Table S1 for statistics).

When a single Hemiptera egg was exposed to T. mitsukurii for 2h, six out of seven pentatomid species (85.71% of tested species), and the scutellerid E. maura were suitable for T. mitsukurii development (Table 4). Percentages of parasitoid emergence from eggs of non-target species were significantly lower compared to those from H. halys (Table 4; see Table S2 for statistics), except for P. prasina where 100% of emergence success was recorded, like in H. halys. No parasitoids emerged from eggs of N. viridula and no parasitoid pupae or pharate adults were found in dead eggs.

3.2 Effect of host egg size on parasitoid size and egg load

The three body dimensions measured on T. mitsukurii females (head width, thorax width and hind tibia length, see Table S3) revealed to be highly correlated (head width vs. thorax width: r=0.96, P<0.0001; head width vs. hind tibia length: r=0.81, P<0.0001; thorax width vs. hind tibia length: r=0.81, P<0.0001). Within each Hemiptera host species, the female head width was used as main parameter to analyze in the linear regression model as a function of host egg volume. Regression analysis displayed a positive trend between the volume of host eggs and the head width of the emerged parasitoid females (r²=0.7673, P=0.0002) (Fig. 1).

The egg loads (see Table S4) of T. mitsukurii females originated from the different Hemiptera host species were significantly different (χ²=65.75; P<0.0001). Parasitoid females that developed in H. halys exhibited a higher number of eggs in their ovaries compared to other non-target pentatomid species (see Table S4 for statistics), except for C. pudicus and A. heegeri and the scutellerid E. maura. Regression analysis displayed a positive trend between host egg volume and the ovarian egg load (r²=0.8053, P<0.0001) (Fig. 2).

3.3 Paired choice black-box tests using parasitoids reared on different hosts

In the H. halys vs. D. baccarum choice tests, T. mitsukurii females when reared on H. halys, always performed a choice for parasitization. In H. halys vs. A. custos choice-tests, T. mitsukurii females failed to interact with host eggs in 1 out of total 12 egg masses when the parasitoid females originated from H. halys, and in 2 out of total 12 egg masses when the females originated from A. custos.

When reared on H. halys, emergence of the progeny of T. mitsukurii females showed no significant differences between H. halys and each of the two non-target host species (H. halys vs. D. baccarum, U=48, P=0.1398; H. halys vs. A. custos, U=38.50, P=0.1339). Percentages of dead eggs were not significantly different neither (H. halys vs. D. baccarum, U=58, P=0.3811; H. halys vs. A.custos, U=45.50, P=0.3342) (Fig. 3). A lower percentage of nymphs hatched from H. halys compared to A. custos eggs (U=30, P=0.0434), while similar percentages emerged from H. halys and D. baccarum eggs (U=65, P=0.6924).

When reared on a non-target host (D. baccarum or A. custos), the emergence of the progeny of T. mitsukurii females showed no significant differences in the case of H. halys vs. D. baccarum (U=72, P>0.9999) and in that of H. halys vs. A. custos (U=30.50, P=0.1214) (Fig. 4). Additionally, no differences were observed in the percentage of dead eggs (H. halys vs. D. baccarum, U=57, P=0.8444; H. halys vs. A. custos,U=28, P=0.0927) nor in the percentage of hatched nymphs (H. halys vs. D. baccarum, U=65, P=0.6924; H. halys vs. A. custos, U=37.50, P=0.3541).

The egg parasitoid T. mitsukurii has been poorly investigated till now as a candidate biological control agent of H. halys, especially when compared to other egg parasitoid species like T. japonicus and the non-coevolved Anastatus bifasciatus (Geoffroy) (Hymenoptera: Eupelmidae), on which a large literature is available from China, Europe, USA and New Zealand. Field data from Japan indicate for T. mitsukurii an oligophagous habitus (Yasumatsu and Watanabe 1964; Hokyo et al., 1966; Ryu and Hirashima, 1984; Arakawa and Namura 2002), and data from the invaded areas in Europe (Northern Italy) evidenced the ability of this parasitoid to locate and successfully parasitize H. halys egg masses in the field (Sabbatini Peverieri et al. 2018; Benvenuto et al. 2020; Scaccini et al. 2020; Zapponi et al. 2020). Furthermore, laboratory studies confirm that the reproductive biology of this parasitoid on H. halys is similar to that of T. japonicus on the same host (Sabbatini Peverieri et al. 2020).

In our experiments under laboratory conditions and 24 hours of egg mass exposure, T. mitsukurii displayed the ability to parasitize and develop successfully in 8 (61.54%) out of 13 tested non-target pentatomid species, with no significant differences compared to the coevolved host H. halys, while additional 3 species were partially suitable although at a significantly lower level compared to H. halys. These results were largely confirmed when H. halys and non-target species were exposed to the parasitoid only for 2 hours, as T. mitsukurii successfully parasitized 5 (62.50%) out of 8 non-target pentatomid species with similar rates than when parasitizing H. halys. Additionally, the scutellerid E. maura was successfully parasitized in both the 24h and 2h tests, whereas the coreid G. juniperi and the reduviid R. iracundus were not. This can be at least partially explained if we consider that scutellerids belong to the same superfamily of pentatomids, i.e., Pentatomoidea. In previous laboratory investigations, E. maura was revealed to be physiologically suitable also for T. japonicus (Haye et al. 2019). However, because scutellerids were never found as hosts of neither T. mitsukurii nor T. japonicus in the field, it is possible to hypothesize that other ecological factors, e.g., habitat preferences, interspecific competition or oviposition periods, may limit the access to this host. Laboratory host range of T. mitsukurii can be considered similar to that of T. japonicus, which showed comparable results in previous host specificity tests on European non-target species, as 13 pentatomids were suitable for parasitoid development (Haye et al. 2019; Sabbatini Peverieri et al. 2021).

The highly variable rates of T. mitsukurii emergence from non-target eggs, both in the 24h and 2h exposure experiments, could be discussed in terms of host egg recognition/acceptance and suitability, based on data on parasitoid emergence, egg eclosion, egg death and the presence of dead parasitoids in eggs. Thus, H. halys, A. heegeri and D. baccarum appear to be easily recognized by T. mitsukurii and highly suitable hosts for this parasitoid under laboratory conditions, as emergence rates were always very high, egg hatching low, and almost no dead eggs were recorded. Similar results were also observed in P. prasina, although this species was tested with only one egg mass due to the poor rearing success in the laboratory. Most of the other non-target species seemed to be less suitable, although statistically not different from H. halys, because of the high presence of dead eggs, often containing a dead parasitoid pupa or a pharate adult (A. custos, C. pudicus, G. italicum, P. lituratus, Sciocoris sp.). Still, rates of successful parasitism of this species were rather high. High rates of parasitization successes under laboratory conditions were observed also for T. japonicus in China (Zhang et al., 2017), while other studies in the USA and New Zealand reported lower rates both on the target, H. halys, and no targets, probably due to the different physiological status of tested females (Hedstrom et al. 2017; Botch and Delfosse 2018; Charles et a. 2019; Lara et al. 2019). In our study, a few non-target pentatomid hosts showed significantly lower T. mitsukurii emergence rates compared to emergence from H. halys. This was due either to low recognition and parasitization, as indicated by high egg hatching rates (A. leucogrammes), or to low suitability to parasitoid development, as indicated by the high rates of dead eggs (A. acuminata, E. oleracea, N. viridula), or to both reasons (E. ventralis). The occurrence of dead T. mitsukurii pupae or adults in several Hemiptera host eggs further reflects a low physiological suitability. Remarkably, dead parasitoids were found also in the eggs of pentatomid species that did not allow any parasitoid adult emergence, i.e., E. ventralis and A. leucogrammes. In the field, such cases might act as an evolutionary trap, as was stated for T. japonicus in similar cases, and unsuitable biochemical contentment of hemipteran eggs can here play a key role (Abram et al. 2014; Haye et al. 2019).

Surprisingly, as discussed above, the eggs of N. viridula showed a very low suitability rate for T. mitsukurii. This is a rather unexpected result because, although not coevolved due to the different geographical origin, N. viridula was addressed in the past as a main host for T. mitsukurii in its area of origin (Kiritani and Hokyo 1962; Hokyo and Kiritani 1963; Hokyo et al. 1966; Arakawa and Namura 2002; Arakawa et al. 2004). In our experiment, a very low parasitoid emergence rate and a high rate of dead eggs was detected, especially if compared with the high hatching rate observed in unexposed N. viridula egg masses. Moreover, in only about one tenth of the dead eggs it was possible to find dead parasitoid pupae or adults, whereas the egg content could not be determined in the other cases. We can hypothesize that parasitization occurred also in these cases, but the parasitoid failed to survive to late instars, further suggesting that N. viridula eggs are not suitable for larval development of T. mitsukurii. A similar result was observed in T. japonicus, as the parasitizing females caused 100% mortality in N. viridula eggs, but no emergence of adults were observed, nor parasitoid presence could be clearly identified from egg dissection (Haye et al. 2019).

The extremely simplified experiment conducted using a single host egg, exposed to T. mitsukurii for 2h, showed that among the seven pentatomid and one scutellerid species tested, most of them were detected as physiologically suitable, failing only in the case of N. viridula, which confirms its extremely low physiological suitability for T. mitsukurii. Remarkably, in such kind of test, only H. halys and P. prasina allowed 100% parasitism success by T. mitsukurii, while parasitoid emergence from all other non-target host species was significantly lower. Comparing the three methods of host egg exposure to females of T. mitsukurii adopted in the present study (exposure of egg masses for 24h and for 2h and exposure of single eggs for 2h) and considering only the Hemiptera species that were tested in all three experiments, the 2 h test with single egg exposure failed to detect host physiological suitability in one case, the less suitable host species N. viridula. Additionally, a high number of hatched eggs was observed in all species except H. halys and P. prasina. These results indicate that a single host egg was not easily recognized and probed by the parasitoid. Therefore, by exposing a single egg, the degree of host acceptance and physiological suitability might be more difficult to define since the effect of clustered eggs is not taken into account. However, the use of single host egg exposure was already adopted for host specificity tests with T. japonicus and the output of physiological host suitability range was comparable with the results from experiments conducted using egg mass exposed for 24h using similar host species (Haye et al. 2019; Sabbatini Peverieri et al. 2021). This simple method of exposure might permit to filter a first list of suitable non-target species to investigate successively in more complex systems, optimizing resources and time.

In future tests, the time of egg exposure to parasitoids can be reduced (depending on the size of arena and on complexity of tests) as a few hours are adequate rather than a 24h experimental design. Here, considering outputs of the present work, three hours might act as optimal exposure time. The reduction of time might be a relevant factor in both choice and no-choice tests since this would increase parasitoid selectivity by reducing the number of encounters with egg masses, thus preventing host acceptance as a consequence of multiple contacts (Haye et al., 2019). Under field conditions, a first rejection of a potential host species might turn out in the parasitoid searching for more suitable hosts, while this is not permitted in the constrained environments under laboratory studies (Botch and Delfosse 2018; Boyle et al. 2020).

In our experiments we also aimed at comparing the host preference of T. mitsukurii in black-box choice tests under 2h exposure and the effect of rearing host, using H. halys vs. either one of two suitable non-target species, D. baccarum and A. custos. No significant evidence of host preference by T. mitsukurii females was observed. Additionally, the host species used to rear T. mitsukurii females did not appear to play a significant role in further host selection. Conversely, in two-choice tests, T. japonicus was shown to prefer H. halys more frequently when tests were conducted in small arenas, but only partially in more complex systems using large cages and plants as ovipositing substrates (Hedstrom et al. 2017; Botch and Delfosse 2018; Haye et al. 2019).

The last experiment was conducted to evaluate the effect of host egg size on the size and egg load of emerged T. mitsukurii. The results indicated that host egg volume significantly affected the size of emerging adult females and the number of the eggs in their ovaries, i.e., larger host eggs produced larger females with higher egg loads, confirming previous findings by Arakawa and Namura (2004). Since many non-target host species are smaller in size than H. halys, it can be assumed that a population of T. mitsukurii originating from non-target species results in females with lower reproductive ability. This was already speculated for T. japonicus by Botch and Delfosse (2018) and observed successively in laboratory tests (Sabbatini Peverieri et al. 2021).

Trissolcus mitsukurii displayed in laboratory tests no host specialization, showing capability to successfully parasitize most of tested Pentatomoidea species, including A. custos, an Asopinae predator, as was previously assessed also for T. japonicus. However, while in our laboratory experiments T. mitsukurii showed similar host preference toward more than one pentatomid species, T. japonicus preferred its coevolved host H. halys both in laboratory and field conditions (Milnes and Beers 2019; Botch and Delfosse 2018). It is well known that the testing conditions in the laboratory simplified environment can be misleading in non-target risk analysis and the results must be carefully evaluated (Haye et al. 2019). Thus, more complex systems are needed to better simulate field environmental factors, and experiments should adopt multidisciplinary and integrated approaches. These were considered in the case of T. japonicus and the large literature on host range tests in the laboratory evidenced, on a global scale, that the physiological host range of this parasitoid embraces more than two dozens of species among tested pentatomids (Zhang et al. 2017; Hedstrom et al. 2017; Botch and Delfosse 2018; Charles et a. 2019; Haye et al. 2019; Lara et al. 2019). Meanwhile, by means of olfactometer bioassays and testing volatiles from hosts and host-induced plant volatiles, things appear to be different and a higher level of species selection can be put in evidence, as these experiments allow to predict what cues from which host species the parasitoids are able to exploit in the field during host location (Bertoldi et al. 2019).

Considering the topical interest of classical biological control of H. halys, new field data on host preference by T. mitsukurii in Europe are expected to appear soon in the literature. Combined with the information we provide here, this will help understand the actual parasitoid host range of this exotic parasitoid in the novel area of its establishment.

Acknowledgement

We are grateful to Iris Bernardinelli, Giorgio Malossini and Luca Benvenuto of ERSA Friuli Venezia Giulia, and to Giacomo Vaccari and Stefano Caruso of the Consorzio Fitosanitario Modena for the help in the field collection of H. halys in infested sites in northern Italy. We thank Riccardo Frosinini, Andrea Rocchini, Paolo Toccafondi, Tiziano Fabbricatore and Luca Madonni for assistance in H. halys and non-target species rearing in the laboratory. This work was supported by the Ministero per le Politiche Agricole, Alimentari e Forestali (MiPAAF), projects “Salvaolivi” (grant number DM 0033437 21/12/2017) and “Proteggo 1.2”.

Abram PK, Gariepy TD, Boivin G, Brodeur J (2014) An invasive stink bug as an evolutionary trap for an indigenous egg parasitoid. Biol Invasion 16:1387–1395
Abram PK, Parent JP, Brodeur J, Boivin J. (2016) Size-induced phenotypic reaction norms in a parasitoid wasp: an examination of life-history and behavioural traits. Biol J Linn Soc, 117, 620–632.
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.
Abram PK, Mills NJ & Beers EH (2020) Review: classical biological control of invasive stink bugs with egg parasitoids – what does success look like? Pest Manag Sci 76: 1980– 1992.
Arakawa R, Miura M, Fujita M (2004) Effects of host species on the body size, fecundity, and longevity of Trissolcus mitsukurii (Hymenoptera: Scelionidae), a solitary egg parasitoid of stink bugs. Appl Entomol Zool 39:177–181.
Arakawa R, NamuraY (2002) Effects of temperature on development of three Trissolcus spp. (Hymenoptera: Scelionidae), egg parasitoids of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae). Entomol Sci 5: 215–218.
Benvenuto L, Bernardinelli I, Malossini G, Sabbatini Peverieri G, Marianelli L, Roversi PF (2020) Monitoraggio di ovature parassitizzate di Halyomorpha halys in un actinidieto biologico in Friuli Venezia Giulia nel biennio 2018-2019. Atti Giornate Fitopatologiche 1, 161–166.
Bertoldi V, Rondoni G, Brodeur J, Conti E (2019) An egg parasitoid efficiently exploits cues from a coevolved host but not those from a novel host. Front Physiol 10: 746. https://doi.org/10.3389/fphys.2019.00746
Botch PS, Delfosse ES (2018) Host-acceptance behavior of Trissolcus japonicus (Hymenoptera: Scelionidae) reared on the invasive Halyomorpha halys (Heteroptera: Pentatomidae) and nontarget species. Environ Entomol 47: 403–411.
Boyle SM, Weber DC, Hough-Goldstein J, Hoelmer KA (2020) Host kairomones influence searching behavior of Trissolcus japonicus (Hymenoptera: Scelionidae), a parasitoid of Halyomorpha halys (Heteroptera: Pentatomidae). Environ Entomol 49 (1): 15–20. https://doi.org/10.1093/ee/nvz155
Charles JG, Avila GA, Hoelmer KA, Hunt S, Gardner-Gee R, MacDonald F, Davis V (2019) Experimental assessment of the biosafety of Trissolcus japonicus in New Zealand, prior to the anticipated arrival of the invasive pest Halyomorpha halys. BioControl 64:367-379. https://doi.org/10.1007/s10526-019-09949-x.
Chu, F.-J., Zhou Z.-F., Li R.-P., Liu X.-C. (1997) Study on control and observation of the bionomics characteristics of Halyomorpha picus Fabricius. J. Agric. Univ. Hebei 20: 12-17.
Conti, E., Avila, G., Barratt, B., Cingolani, F., Colazza, S., Guarino, S., Hoelmer, K., Laumann, R. A., Maistrello, L., Martel, G., et al. (2021). Biological control of invasive stink bugs: review of global state and future prospects. Ent. Exp. Appl. 169(1): 28-51.
EPPO (2014) PM 6/2 (3) Import and release of non-indigenous biological control Agents. Bulletin OEPP/EPPO Bulletin 44(3): 320–329.
FAO-IPPC (2017) ISPM 3 Guidelines for the export, shipment, import and release of biological control agents and other beneficial organisms. www.ippc.int
Haye T, Moraglio ST, Stahl J, Visentin S, de Gregorio T, Tavella L (2019) Fundamental host range of Trissolcus japonicus in Europe. J Pest Sci 93: 171–182. https://doi.org/10.1007/s10340-019-01127-3
Hedstrom C, Lowenstein D, Andrews H, Bai B, Wiman N (2017) Pentatomid host suitability and the discovery of introduced populations of Trissolcus japonicus in Oregon. J Pest Sci 90: 1169–1179.
Hokyo N, Kiritani K., Nakasuji F., Shiga M. (1966) Comparative biology of the two scelionid egg parasites of Nezara viridula L. (Hemiptera: Pentatomidae). Jpn J Appl Entomol Z 1: 94–102.
Hokyo N., Kiritani K. (1963) Two species of egg parasites as contemporaneous mortality factors in the egg population of the southern green stink bug, Nezara viridula. Jpn J Appl Entomol Z 7: 214–226.
Kiritani K., Hokyo N. (1962) Studies on the life table of the southern green stink bug, Nezara viridula. Jpn J Appl Entomol Z 6: 124–140.
Kuhlmann U, Mason PG (2003) Use of field host range surveys for selecting candidate non-target species for physiological host specificity testing of entomophagous biological control agents. In:1st International Symposium on Biological Control of Arthropods, Honolulu, Hawaii, USA, January 14-18, 2002. USDA Forest Service FHTET-03-05, pp. 370–377.
Kuhlmann U, Schaffner U, Mason PG (2006) Selection of non-target species for host specificity testing. In: Bigler F, Babendreier D, Kuhlmann U (Eds.) Environmental impact of invertebrates for biological control of arthropods: methods and risk assessment. CAB International, pp 15–37.
Lara JR, Pickett CH, Kamiyama MT, Figueroa S, Romo M, Cabanas C, Bazurto V, Strode V, Briseno K, Lewis M, Oliva J, Hernandez G, Hoddle MS (2019) Physiological host range of Trissolcus japonicus in relation to Halyomorpha halys and other pentatomids from California. BioControl 64: 513–528.
Leskey TC, Nielsen AL (2018) Impact of the invasive brown marmorated stink bug in North America and Europe: History, biology, ecology, and management. Ann Rev Entomol 63: 599–618.
Milnes J, Beers E (2019) Trissolcus japonicus (Hymenoptera: Scelionidae) causes low levels of parasitism in three north american pentatomids under field conditions. J Insect Sci,19(4): 15. doi: 10.1093/jisesa/iez074.
Rondoni, G., Bertoldi, V., Malek, R., Djelouah, K., Moretti, C., Buonaurio, R., & Conti, E. (2018). Vicia faba plants respond to oviposition by invasive Halyomorpha halys activating direct defences against offspring. J Pest Sci 91(2), 671-679.
Rondoni, G., Bertoldi, V., Malek, R., Foti, M. C., Peri, E., Maistrello, L., Haye, T., & Conti, E. (2017). Native egg parasitoids recorded from the invasive Halyomorpha halys successfully exploit volatiles emitted by the plant–herbivore complex. J Pest Sci 90(4), 1087-1095.
Ryu J., Hirashima Y. (1984) Taxonomic studies on the genus Trissolcus Ashmead of Japan and Korea (Hymenoptera, Scelionidae). Journal of the Faculty of Agriculture Kyushu University 29: 35-58.
Sabbatini Peverieri G, Boncompagni L, Mazza G, Paoli F, Dapporto L, Giovannini L, Marianelli L, Hoelmer K, Roversi PF (2021). Combining physiological host range, behavior and host characteristics for predictive risk analysis of Trissolcus japonicus. J Pest Sci. https://doi.org/10.1007/s10340-020-01311-w
Sabbatini Peverieri G, Talamas E, Bon MC, Marianelli L, Bernardinelli I, Malossini G, Benvenuto L, Roversi PF, Hoelmer K (2018) Two Asian egg parasitoids of Halyomorpha halys (Stal) (Hemiptera, Pentatomidae) emerge in northern Italy: Trissolcus mitsukurii (Ashmead) and Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae). J Hym Res 67: 37–53. https://doi.org/10.3897/jhr.67.30883
Sabbatini Peverieri G., Dieckhoff C., Giovannini L., Marianelli L., Roversi P.F., Hoelmer K., 2020. Rearing Trissolcus japonicus and Trissolcus mitsukurii for Biological Control of Halyomorpha halys. Insects 11, 787. https://doi.10.3390/insects11110787
Scaccini D, Falagiarda M, Tortorici F, Martinez-Sañudo I, Tirello P, Reyes-Domínguez Y, Gallmetzer A, Tavella L, Zandigiacomo P, Duso C, Pozzebon A (2020) An Insight into the Role of Trissolcus mitsukurii as Biological Control Agent of Halyomorpha halys in Northeastern Italy. Insects. https://doi.10.3390/insects11050306
Stahl J, Tortorici F, Pontini M, Bon MC, Hoelmer K, Marazzi C, Tavella L, Haye T (2019) First discovery of adventive populations of Trissolcus japonicus (Ashmead) in Europe. J Pest Sci 92:371–379. https ://doi.org/10.1007/s1034 0-018-1061-2
Talamas EJ, Herlihy MV, Dieckhoff C, Hoelmer KA, Buffington ML, Bon M-C, Weber DC (2015) Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) emerges in North America. J Hym Res 43: 119–128. https://doi.org/10.3897/ JHR.43.4661
van Lenteren JC, Bale J, Bigler F, Hokkanen HMT, Loomans AJM (2006) Assessing risks of releasing exotic biological control agents of arthropod pests. Annu Rev Entomol 51: 609–34.
Yang Z-Q, Yao Y-X, Qiu L-F, Li Z-X (2009) A new species of Trissolcus (Hymenoptera: Scelionidae) parasitizing eggs of Halyomorpha halys (Heteroptera: Pentatomidae) in China with comments on its biology. Ann Entomol Soc Am 102:39–47.
Yasumatsu K, Watanabe C (1964) A Tentative Catalogue of Insect Natural Enemies of Injurious Insects in Japan - Part 1. Parasite-Predator Host Catalogue. Fukuoka, Japan: Entomological Laboratory, Faculty of Agriculture Kyushu University
Zapponi L, Bon MC, Fouani JM, Anfora G, Schmidt S, Falagiarda M, 2020. Assemblage of the Egg Parasitoids of the Invasive Stink Bug Halyomorpha halys: Insights on Plant Host Associations. Insects. doi:10.3390/insects11090588
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

Table 1 List of the European native and exotic Hemiptera tested for Trissolcus mitsukurii physiological host suitability and relative egg hatching rates in the rearing facility (n.a. = not assessed)

Target and non-target species	Family, subfamily, tribe	Egg mass hatching rate in the rearing facility
Acrosternum heegeri Fieber	Pentatomidae, Pentatominae, Pentatomini	n.a. (94.0%^*)
Aelia acuminata (L.)	Pentatomidae, Pentatominae, Aelini	n.a.
Ancyrosoma leucogrammes (Gmelin)	Pentatomidae, Podopinae, Graphosomatini	n.a.
Arma custos (F.)	Pentatomidae, Asopinae, Asopini	77.67%
Carpocoris pudicus (Poda)	Pentatomidae, Pentatominae, Carpocorini	95.7% (76.61%^*)
Dolycoris baccarum (L.)	Pentatomidae, Pentatominae, Carpocorini	88.6% (88.0%^*)
Eurydema oleracea (L.)	Pentatomidae, Pentatominae, Strachiini	n.a.
Eurydema ventralis Kolenati	Pentatomidae, Pentatominae, Strachiini	93.1% (96.67%^*)
Graphosma italicum (Muller)	Pentatomidae, Pentatominae, Graphosomatini	83.33%
Halyomorpha halys (Stål)	Pentatomidae, Pentatominae, Cappaeiini	93.35% (90.39%^*)
Nezara viridula L.	Pentatomidae, Pentatominae, Pentatomini	92.34% (85.58%^*)
Palomena prasina (L.)	Pentatomidae, Pentatominae, Carpocorini	n.a. (97.56%^*)
Piezodorus lituratus (F.)	Pentatomidae, Pentatominae, Piezodorini	n.a. (88.0%^*)
Sciocoris sp. Wollaston	Pentatomidae, Pentatominae, Sciocorini	n.a.
Eurygaster maura	Scutelleridae, Eurygastrinae, Eurygastrini	90.15%
Gonocerus juniperi Herrich-Schäffer	Coreidae, Coreinae, Gonocerini	n.a. (100%^*)
Rhynocoris iracundus (Poda)	Reduviidae, Harpactorinae, Harpactorini	n.a. (96.67%^*)

* Data from Sabbatini Peverieri et al., (2020) using the same rearing condition are reported within parentheses

Table 2 Physiological suitability of Hemiptera eggs for Trissolcus mitsukurii in 24h black box tests. Asterisks within each column indicate significant differences among the non-target species and Halyomopha halys (Kruskall-Wallis followed by Dunn’s Multiple Comparison test, * P < 0.05; ** P < 0.01; *** P < 0.001; n.a. = not assessed) (see Table S1 for statistics)

Species (replicates)	n. eggs/egg mass (mean ± SE)	% of eggs with parasitoid emergence/egg mass (mean ± SE)	% of females (mean ± SE)	% of hatched eggs / egg mass (mean ± SE)	% of dead eggs (mean ± SE)	n. of parasitized egg masses /with parasitoid emergence/total n. of eggs exposed	total n. of dead eggs
Species (replicates)	n. eggs/egg mass (mean ± SE)		% of females (mean ± SE)	% of hatched eggs / egg mass (mean ± SE)	% of dead eggs (mean ± SE)		with undetermined content	with dead hemipteran nymphs	with dead parasitoid
Halyomorpha halys (12)	27.42 ± 0.29	99.40 ± 0.60	90.22 ± 3.29	0.60 ± 0.60	0 ± 0.00	12 / 12 / 329	0	0	0
Acrosternum heegeri (12)	13.25 ± 0.46	98.32 ± 1.24	89.78 ± 1.37	0 ± 0.00	0.49 ± 0.49	12 / 12 / 159	0	0	1 (adult)
Aelia acuminata (12)	11.83 ± 0.24	7.20 ± 3.57***	72.00 ± 12.64	25.00 ± 13.06	67.80 ± 12.26***	7 / 4 / 142	93	0	13 (all adults)
Ancyrosoma leucogrammes (10)	11.50 ± 0.45	0 ± 0.00***	-	90.00± 10.00***	10.00 ± 10.00	1 / 0 / 115	3	0	9 (all adults)
Arma custos (12)	14.33 ± 0.63	66.32 ± 12.51	90.65 ± 1.54	25.00 ± 13.06	8.67 ± 5.05	9 / 9 / 172	0	0	14 (all adults)
Carpocoris pudicus (12)	14.08 ± 0.87	75.00 ± 13.06	92.22 ± 0.60	9.22 ± 7.37	15.78 ± 9.95	12 / 9 / 169	0	0	29 (28 pupae, 1 adult)
Dolycoris baccarum (12)	13.92 ± 0.34	98.29 ± 0.89	92.08 ±1.02	0.55 ± 0.55	0.59 ± 0.59	12 / 12 / 167	1	0	0
Eurydema oleracea (10)	10.10 ± 0.38	21.38 ± 11.09**	82.22 ± 5.35	25.00 ± 11.33	55.21 ± 11.50***	4 / 4 / 101	42	0	12 (all adults)
Eurydema ventralis (12)	11.83 ± 0.32	0 ± 0.00***	-	63.81 ± 11.49**	36.19 ± 11.49*	5 / 0 / 142	12	0	41 (6 pupae, 35 adults)
Graphosma italicum (12)	14.58 ± 0.45	78.57 ± 8.05	84.52 ±2.85	15.16 ± 8.07	6.28 ± 2.74	11 / 11 / 175	1	0	10 (all adults)
Nezara viridula (12)	28.25 ± 0.97	1.90 ± 1.57***	100	29.04 ± 10.45	68.36 ± 10.89***	4 / 2 / 339	207	0	22 (all adults)
Palomena prasina (1)	12	100 ^n.a.	75 ^n.a.	0 ^n.a.	0 ^n.a.	1 / 1 / 12	0	0	0
Piezodorus lituratus (5)	10.60 ± 1.29*	68.24 ± 18.39	68.37± 7.29	22.97 ± 19.33	8.79 ± 5.77	4 / 4 / 53	0	0	6 (all adults)
Sciocoris sp. (8)	10.38 ± 0.57	81.60 ± 12.25	83.07± 2.72	10.94 ± 9.28	6.07 ± 3.29	8 / 7 / 83	1	0	5 (all adults)
Eurygaster maura (12)	11.67 ± 0.53	87.36 ± 8.32	90.74 ±1.34	9.03 ± 8.30	3.61 ± 2.51	11 / 11 / 140	6	0	0
Gonocerus juniperi (12)	10.00 ± 0.00	0 ± 0.00***	-	97.50 ± 1.31***	2.51 ± 1.30	0 / 0 / 100	3	0	0
Rhynocoris iracundus (3)	32.22 ± 0.88	0 ± 0.00^n.a.	-	74.33 ± 21.16 ^n.a.	25.67 ± 21.16^n.a.	0 / 0 / 97	26	0	0

Table 3 Physiological suitability of Hemiptera eggs for Trissolcus mitsukurii in 2h black box tests. Asterisks within each column indicate significant differences among the non-target species and Halyomorpha halys (Kruskall-Wallis followed by Dunn’s Multiple Comparison test, * P < 0.05; ** P < 0.01; *** P < 0.001) (see Table S1 for statistics)

Species (replicates)	n. eggs/egg mass (mean ± SE)	% of eggs with parasitoid emergence/egg mass (mean ± SE)	% of females (mean ± SE)	% of hatched eggs / egg mass (mean ± SE)	% of dead eggs (mean ± SE)	n. of parasitized egg masses /with parasitoid emergence/total n. of eggs exposed	total n. of dead eggs
Species (replicates)	n. eggs/egg mass (mean ± SE)		% of females (mean ± SE)	% of hatched eggs / egg mass (mean ± SE)	% of dead eggs (mean ± SE)		with undetermined content	with dead hemipteran nymphs	with dead parasitoid
Halyomorpha halys (12)	27.58 ± 0.74	81.69 ± 3.41	94.76 ± 0.44	18.31 ±3.41	0.00	12/12/331	0	0	0
Acrosternum heegeri (12)	14.50 ± 0.68	91.11 ± 8.30	89.68 ± 1.13	8.89 ± 8.30	0.00	11/11/174	0	0	0
Aelia acuminata (12)	10.92 ± 0.34	3.19 ± 2.53*	66.67 ± 13.61	23.41 ± 11.78	73.39 ± 11.46***	6/1/131	82	0	18 (all adults)
Arma custos (12)	15.33 ± 0.49	40.52 ± 14.47	92.84 ± 0.11	58.33 ± 14.87	1.15 ± 0.77	5/5/184	0	0	2 (all adults)
Carpocoris pudicus (12)	12.33 ± 0.33	88.46 ± 8.65	88.37 ± 1.09	8.97 ± 8.30	2.56 ± 2.56	11/11/160	0	0	4 (all adults)
Dolycoris baccarum (12)	14.00 ± 0.24	83.28 ± 4.41	85.85 ± 3.44	5.40 ± 3.53	11.40 ± 4.26	10/10/175	11	0	0
Eurydema ventralis (12)	11.67 ± 0.19	0 ± 0.00**	-	90.66 ± 3.78	9.28 ± 3.77	1/0/140	9	0	4 (all adults)
Graphosoma italicum (12)	14.75 ± 0.31	77.12 ± 5.84	86.85 ± 2.30	18.87 ± 6.36	4.00 ± 2.72	12/12/177	0	0	7 (all adults)
Nezara viridula (12)	34.25 ± 1.16	0 ± 0.00**	-	22.85 ± 10.62	77.15 ± 9.38***	5/0/411	260	0	27 (all adults)
Eurygaster maura (12)	13.58 ± 0.26	88.64 ± 8.13	92.48 ± 0.93	8.33 ± 8.33	3.02 ± 1.08	11/11/163	0	0	5 (all adults)

Table 4 Physiological suitability of Hemiptera eggs for Trissolcus mitsukurii in 2h exposure of a single host egg. Among brackets number of females tested; asterisks within each column indicate significant differences among the non-target species and Halyomorpha halys (Chi-square test with Yates’ correction, *** P < 0.001) (see Table S2 for statistics)

species	% of emerged parasitoids	% of hatched eggs	% of dead eggs
species	% of emerged parasitoids	% of hatched eggs	with undetermined content or a dead hemipteran nymph	containing pupae or adults
Halyomorpha halys (31)	100	0	0	0
Acrosternum heegeri (30)	46.67***	53.33	0	0
Arma custos (43)	30.23***	55.81	9.31	4.65
Dolycoris baccarum (30)	20.00***	63.33	16.67	0
Graphosma italicum (30)	26.67***	16.67	0	56.67
Nezara viridula (30)	0***	56.67	43.33	0
Palomena prasina (26)	100	0	0	0
Eurygaster maura (34)	2.94***	26.47	61.77	8.82

SupplementarymaterialTablesS1S2S3S4.docx

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published 05 Aug, 2021

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Editorial decision: Major revisions
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Physiological Host Range of Trissolcus Mitsukurii, A Candidate Biological Control Agent of Halyomorpha Halys

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