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

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 rst 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. Specically, 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 eld conditions, allowing in-depth evaluation of the possible risks associated with the use of T. mitsukurii as a biocontrol agent of H. halys. 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


Introduction
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 e cacy 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 eld needs to highlight any potential approach to nd effective pest control solutions ( 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 eld. Laboratory investigations are the rst 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, con rming its oligophagous habitus but also showing its host preference versus H. halys (Hedstrom et  . Surprisingly, a second Asian egg parasitoid of H. halys, Trissolcus mitsukurii (Ashmead) (Hymenoptera, Scelionidae), was also found in Italy, as the rst 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 eld 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 misidenti cation 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 speci c 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 rst 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.
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 bene cial 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).

No-choice black box tests
A total of three different experimental conditions were considered for the no-choice black box tests. In the rst 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 eld (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 O ce 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. Speci cally, in the rst 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 simpli ed 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 su cient 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 nontarget 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 classi ed as "dead eggs". These were dissected and inspected under a stereomicroscope, and the content was eventually classi ed as: dead parasitoid (pupae or pharate adult), dead Hemiptera nymph, or undetermined content.

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 (mm 3 ) of eggs of host species was calculated by the parabolic barrel formula V=πH(3r 2 + 4Rr + 8R 2 )/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).

Paired choice black-box tests using parasitoids reared on different hosts
In a rst 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 eld (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 . 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.

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 ve 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 coe cient. 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 Utest. 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.

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 signi cantly different emergence rates from the diverse tested species (χ 2 =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 signi cantly 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 signi cantly 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 signi cantly different (χ 2 =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 signi cantly higher rates compared to H. halys. The percentages of dead eggs (no hatching and no parasitoid emergence) were signi cantly different among the tested species (χ 2 =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 signi cantly different among the tested species (χ 2 =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). Speci cally, 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 signi cantly 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 signi cantly different (χ 2 =46.52; P<0.0001) and complementary with parasitoid emergence rates and dead eggs. Percentages of dead eggs were also signi cantly different (χ 2 =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. Speci cally, eggs of A. acuminata showed lower parasitization success compared to H. halys, with signi cantly lower parasitoid emergence and higher dead eggs containing dead parasitoids. Similarly, eggs of N. viridula showed no parasitoid emergence and signi cantly 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 signi cantly 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.

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 (r 2 =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 signi cantly different (χ 2 =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 (r 2 =0.8053, P<0.0001) (Fig. 2).

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 a non-target host (D. baccarum or A. custos), the emergence of the progeny of T. mitsukurii females showed no signi cant 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).

Discussion
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. 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 signi cant differences compared to the coevolved host H. halys, while additional 3 species were partially suitable although at a signi cantly lower level compared to H. halys. These results were largely con rmed 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 ). However, because scutellerids were never found as hosts of neither T. mitsukurii nor T. japonicus in the eld, it is possible to hypothesize that other ecological factors, e.g., habitat preferences, interspeci c 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 speci city tests on European non-target species, as 13 pentatomids were suitable for parasitoid development (Haye et  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 nontarget 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. In our study, a few nontarget pentatomid hosts showed signi cantly 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 re ects 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 eld, 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 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 nd 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 identi ed from egg dissection ).
The extremely simpli ed 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 con rms 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 signi cantly 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 di cult to de ne since the effect of clustered eggs is not taken into account. However, the use of single host egg exposure was already adopted for host speci city 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 Sabbatini Peverieri et al. 2021). This simple method of exposure might permit to lter a rst 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 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 signi cant 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 signi cant 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  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 signi cantly 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, con rming previous ndings 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).

Conclusions
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 eld conditions (Milnes and Table 3 Physiological suitability of Hemiptera eggs for Trissolcus mitsukurii in 2h black box tests. Asterisks within each column indicate signi cant 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)  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 signi cant differences among the non-target species and Halyomorpha halys (Chi-square test with Yates' correction, *** P < 0.001) (see Table S2 Figure 1 Regression analysis of Trissolcus mitsukurii females head width as a function of egg volume of Hemiptera host species Figure 2 Regression analysis of Trissolcus mitsukurii female ovaries egg load (at 7 days in age) as a function of egg volume of Hemiptera host species Figure 3 Outputs of choice tests using Trissolcus mitsukurii females, originated from Halyomorpha halys, that were exposed for 2 h to egg masses of target and non-target species in pairwise comparisons (A = H. halys vs. Dolycoris baccarum; B = H. halys vs. Arma custos)