Selection, source, and rearing of stink bug species
Non-target species were selected according to the information on T. japonicus hosts available from the literature, phylogenetic relatedness, and sympatry of target and non-target species, phenology, safeguard considerations (beneficial species), and availability (Kuhlmann et al. 2006). In total, thirteen species were selected, including eleven pentatomids (same family as the target H. halys), one scutellerid and one coreid (outgroup species from different families) (Table 1).
Table 1 Non-target test list for Trissolcus japonicus The H. halys colony was originally established in 2017 from about 500 individuals collected in Basel, Switzerland. The insects were maintained in groups of 50 individuals in polyester cages (BugDorm-4090 Insect Rearing Cage 47.5 × 47.5 × 47.5 cm, MegaView Science Co. Ltd., Taichung, Taiwan) at 26 °C, 70% RH, and a 16L/8D photoperiod. Both adults and nymphs were fed with corn, beans, and peanuts that were replaced twice weekly. In winter and spring, bugs were provided with fruit-bearing branches of common ivy (Hedera helix L., Araliaceae) and later in the season with a variety of seasonal plants (e.g., Cornus sanguinea L., Cornaceae and Sorbus aucuparia L., Prunus avium L., Rosaceae).
Overwintered, non-target species (Hemiptera: Pentatomidae, Scutelleridae, Coreidae) were collected from sun-exposed house walls in early spring, by visual inspection or plant beating from their host plants (crop and non-crop, herbaceous and arboreous) throughout summers 2017–2018 in Piedmont, NW Italy; the Jura mountains, NW Switzerland; north of Lake Constance, S Germany; Samegrelo, W Georgia (Table 1). Species were identified using the keys by Wyniger and Kment (2010), Derjanschi and Péricart (2005), and Moulet (1995). Non-target stink bugs were reared in the type of cage as used for H. halys and kept at 24 ± 1 °C, 60% RH, and a 16L/8D photoperiod. Adults of most species were provided with potted broad bean plants, bramble branches, apples, hazelnuts, and green beans, which were replaced once per week. Adults of Eurygaster maura (L.) were provided with wheat ears instead, and adults of Arma custos (F.) were fed with adults of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) or larvae of Tenebrio molitor L. (Coleoptera: Tenebrionidae). Newly laid egg masses of target and non-target species were collected on a daily basis.
Parasitoid rearing
Trissolcus japonicus were originally collected from H. halys eggs near Beijing, China (N40°02′06″; E116°12′41″) in 2013, and maintained on fresh H. halys egg masses in the CABI quarantine facility. Parasitoids (mated, ≥ 2 days old) were held in a clear plastic container (10 cm diameter, 5 cm height) with 10% honey water solution as a food source and 8–10 fresh H. halys egg masses provided once per week. Parasitized egg masses were kept at 26 °C, 60% RH, and 16L/8D photoperiod. Upon the initial establishment of the laboratory colony, specimens of T. japonicus were taxonomically identified by E. Talamas (Systematic Entomology Laboratory, USDA) and confirmed molecularly by M.C. Bon (USDA-ARS-EBCL, Montferrier le Lez, France) (Stahl et al. 2018). Reference specimens are located in the Natural History Museum of Bern, Switzerland.
No-choice tests
No-choice black box tests performed in China (Zhang et al. 2017) and North America (Hedstrom et al. 2017) indicated that non-target parasitism of European non-target species seems likely, so we conducted no-choice behavioural tests, as suggested by van Lenteren et al. (2006), including direct observations of the parasitoid oviposition behaviour during the time of egg exposure. In contrast to black box tests, this method allowed us to follow the fate of each parasitized egg and relate parasitoid emergence directly to the observed oviposition behaviour of the wasps. The advantage of this method is that false conclusions regarding the parasitoid behaviour can be avoided, which may be drawn if parasitoids have non-reproductive effects on their hosts (Abram et al. 2019b). Such effects may occur when the non-target test list includes species that function as an ‘evolutionary trap’ (emergence of host nymphs despite parasitoid oviposition) (Abram et al. 2014; Haye et al. 2015b), or die due to parasitism but fail to produce parasitoid offspring (parasitoid-induced host egg abortion, Abram et al. 2016).
Egg masses of H. halys and non-target species were collected from rearing cages on a daily basis and typically used for tests on the day they were collected. If they could not be used the same day, eggs were stored at 10 °C for no longer than three days in order to prevent development. Since average egg mass sizes of H. halys and non-target species can vary significantly, for each test we standardized the egg mass size by separating egg masses into smaller clusters (10 eggs/mass) and attaching them to 4 cm2 pieces of flat cardboard with small amount of clear glue (Cementit, merz + benteli Kolma AG, Wabern, Switzerland). In the case of Gonocerus acuteangulatus (Goeze), the eggs were left on the leaves they were laid on, and variable numbers of eggs (3–10) were used for testing since this species only lays single eggs in small clusters. Egg masses were then transferred into small (5 cm) Petri dishes.
In each experimental setup, similar numbers of randomly selected, naïve, mated T. japonicus females were tested simultaneously on egg masses of the target H. halys (control) and the non-target species listed in Table 1 (between 14 and 46 replicates per non-target species). Since daily offspring production of the synovigenic females peaks within the first week after emergence (Qui 2007), females were 4–7 days old when used for experiments. All wasps were fed with fresh honey water the morning before the experiments. Single T. japonicus females were added to each Petri dish and observed under a stereomicroscope until they had at least one contact with the egg mass. If females had no interest in oviposition following the first contact, observations were continued for another 10 min, and egg masses were counted as ‘rejected’ if no oviposition behaviour was observed. Females that started ovipositing were observed until they either had parasitized all 10 eggs, as indicated by marking behaviour (‘acceptance’), or abandoned the partially parasitized egg mass for more than 10 min.
The individual handling time of the egg masses was recorded for each T. japonicus female. All tests were conducted at 26 ± 1 °C, 60–70% RH. In addition, unexposed controls of target and non-target egg masses were kept at the same conditions to evaluate baseline host mortality and to assess if T. japonicus females induced additional host mortality even in cases of failed development, a non-reproductive non-target effect, which is rarely considered explicitly in risk assessment of biological control agents (Abram et al. 2019b). After the tests, the wasps were removed, and both the non-target and H. halys egg masses were incubated under the above rearing conditions until emergence of stink bug nymphs and/or wasp adults (‘host suitability’). The number of emerged parasitoids, nymphs and dead eggs (= no emergence) was recorded as well as the sex ratio of the parasitoid offspring. Finally, egg dissections were performed to determine whether any parasitoids or nymphs developed partially.
Paired choice tests
To evaluate target (H. halys) versus non-target parasitism under more realistic conditions than previous laboratory host range studies (Zhang et al. 2017; Hedstrom et al. 2017), large-arena choice tests were conducted (Van Lenteren et al. 2006) where individual T. japonicus females foraged on plants where bugs had fed and laid eggs. This procedure was followed because studies by Colazza et al. (2007, 2009) had shown that in a similar system, Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) perceived chemical footprints left behind by its host Nezara viridula (L.) (Hemiptera: Pentatomidae) as contact kairomones, which induced foraging by gravid females. In addition, T. basalis also responded to synomones emitted by bean plants induced by feeding and oviposition activity of its host (Colazza et al. 2004).
The following four species were selected as representative hosts that were accepted frequently or less frequently in no-choice tests: Acrosternum heegeri Fieber, Ar. custos, Graphosoma lineatum (L.), and Palomena prasina (L.). In the case of H. halys, Pa. Prasina, and Ar. custos, potted broad bean plants (Vicia faba L., Fabaceae, about 20 cm high) were placed inside the stink bug rearing cages described above for 24 h. Since Ac. heegeri and G. lineatum refused to lay eggs on broad bean plants, alternatively cut, fresh fruit-bearing branches of common ivy (H. helix) placed in a container with water were used instead. Accordingly, ivy branches were also offered to H. halys for oviposition to exclude potential effects of different host plants. After 24 h, the plants were removed and inspected for egg masses. Plants carrying single egg masses were selected for testing. Since these five species lay egg masses of variable size, it was not possible to control for the number of eggs per plant. However, in this way the outcome of choice tests may represent parasitism in the field more realistically than no-choice tests, despite the nonstandardized egg masses.
Testing arenas consisted of fine gauze cages (47.5 × 47.5 × 47.5 cm), in which two plants were placed in the far left and right corners, each carrying a single egg mass of H. halys or non-target species, respectively. Plants did not touch the cage walls or each other. At the top of the cages, small drops of honey were placed in each corner as a food source for the parasitoids. Single, naïve, mated 4–7 days old T. japonicus females were removed from the rearing cage, and individually transferred into glass pipettes (10 cm long, diameter 5 mm) closed with a cotton wick. These tubes were then put into a small open plastic cup, which was placed in the middle of the front side of the cage, equidistant (30 cm) to the two test plants. The cotton wicks were removed, so the wasps could crawl up to the opening of the tubes and enter the test arena. All tests were conducted at 26 ± 1 °C, 60–70% RH, and a 16L/8D photoperiod. After 24 h, the wasps were removed and both the H. halys and non-target egg masses were incubated under the above rearing conditions until emergence of stink bug nymphs and wasp adults. Each combination of target and non-target species was replicated between 19 and 40 times (Table 3).
Statistical analysis
In no-choice tests, acceptance (oviposition and marking behaviour), host suitability (mean offspring emergence per egg mass), and sex ratio (percentage female parasitoid offspring per host species) were compared pairwise between each non-target species and its respective H. halys control using generalized linear models (GLMs) with a binomial error distribution and a logit link function. Similarly, the host exploitation (number of eggs parasitized within an egg mass) and egg mass handling time were compared using a GLM with Poisson (log-link function) and gamma (inverse link function) error distributions, respectively. Replicates in which wasps did not parasitize all 10 eggs were excluded from the handling time analysis. The unattributed mortality (‘dead eggs’ = no parasitoid emergence or dead parasitoids in dissected eggs, Table 2) of non-target eggs exposed to T. japonicus was compared pairwise with unattributed mortality in the respective rearing controls using a GLM with a quasibinomial error distribution (logit link function) to account for overdispersion. All GLMs were carried out with R version 3.2.3 (Team 2017) using the development environment RStudio (Team 2016).
Table 2 Outcomes of no-choice tests of single T. japonicus females when exposed to H. halys, or one of thirteen non-target species For large-arena paired choice tests, the proportion of females parasitizing either target or non-target eggs were compared using a Pearson Chi-square test. The percentage of parasitoids emerging per parasitized egg mass was compared between species using a Wilcoxon’s signed-rank test. Statistical tests were carried out with the SPSS® 20.0 software package (IBM Corp. 2013).