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.