First Report of Native Parasitoids of Halyomorpha halys (Hemiptera: Pentatomidae) in Greece

Simple Summary The brown marmorated stink bug (BMSB) is a polyphagous species that causes severe damage to tree fruit, small fruit, vegetables, ornamental crops, and field crops. Classical biological control is one potential long-term and low-cost strategy to control the BMSB, using natural enemies. However, no natural enemy native to Greece that infects BMSB has been reported yet. Herein, we report the occurrence of two native hymenopteran egg parasitoids—Anastatus bifasciatus and Ooencyrtus telenomicida. Both egg parasitoids were collected from egg masses of the BMSB in the region of Thessaloniki, northern Greece. The total parasitism rate was 8.5%. Furthermore, A. bifasciatus was collected in mid-June and mid-August on egg masses of BMSB that were laid on green beans, apricots, and olives. On the other hand, O. telenomicida was only collected in mid-June, on one egg mass of BMSB that was laid on apricots. This first record could actually facilitate, for future tasks, the biological control of H. halys in Greece. Abstract Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) is an endemic species of East Asia; it was introduced into Europe in 2007. It has a wide range of hosts as it feeds on over 170 host plant species and significantly impacts crop production. In Greece, H. halys causes significant losses in the production of kiwi, peaches, and green beans; thus, control of this species (including biological control) is essential. Here, we focus on the potential impact of native natural enemies of H. halys in Greece. From June to October 2020, we sampled naturally field-laid H. halys egg masses to recover native parasitoids. A total of 20 egg masses of H. halys were collected from infested fields from different locations in northern Greece. Out of 529 eggs, 45 parasitoids managed to hatch successfully. The overall parasitism rate was 8.5%. We found two species of Hymenopteran egg parasitoids attacking H. halys eggs—Anastatus bifasciatus (Geoffrey) (Hymenoptera: Eupelmidae) and Ooencyrtus telenomicida (Vassiliev) (Hymenoptera: Encyrtidae), with the former comprising 58% of all parasitoids that were recovered. These results contribute to the knowledge about the natural enemy community that attacks H. halys in Greece, and the use of these native egg parasitoids in biological control programs may be a viable H. halys management strategy.


Introduction
The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a highly polyphagous pest that is native of Asia; it causes severe damage to a wide variety of fruit and vegetable crops by piercing the surface of the plant and fruit tissues [1,2].
H. halys, apart from being a major agricultural pest, is also considered a nuisance problem, because massive numbers of adults often invade in the fall and winter in residential and commercial buildings, to overwinter [3]. The earliest confirmed sighting in the United States was in 1996 in Allentown, Pennsylvania; since then, it has spread to 46 additional states and 4 Canadian provinces [3,4]. In Europe, H. halys was first discovered in 2007, in particular in Switzerland; within a few years, it managed to spread throughout all of Europe. Nowadays, it is considered a major threat to crops worldwide [5][6][7]. Halyomorpha halys was first mentioned in Greece in the fall of 2011, causing nuisance inside houses in several neighborhoods of Athens [8]. In 2017, in two different kiwi orchards in Northern Greece, located in the regional unit of Imathia and Pieria, respectively, significant damage on kiwi fruits was observed due to infestation by H. halys, and rendered them non-marketable [9]. In Greece, H. halys has two distinct generations [Andreadis, unpublished data].
Control of H. halys in the newly invaded areas, such as Greece, relies mainly on the use of broad-spectrum chemical insecticides [9]. However, chemical insecticides have met with mixed success in controlling H. halys due to the fact that they often lack efficacy [5,10,11], are harmful to the environment [12], incompatible with integrated pest management (IPM) strategies [10], and only provide a short-term solution. A potential long-term and low-cost strategy to reduce populations of the invasive agricultural pest H. halys is the classical biological control using natural enemies [13]. Nevertheless, no information is available regarding the potential for biological control through native parasitoids in Greece.
In North America, field surveys and studies that have been conducted in the past decades have led to the identification of several natural enemies of H. halys, mainly egg parasitoids; however, it was revealed that native egg parasitoids have not been effective at controlling newly laid H. halys eggs [6,[20][21][22][23]. In fact, parasitism rates were typically low and highly variable among locations (and years) [5,6,[24][25][26].
In Greece, regarding native natural enemies, neither parasitoids nor predators of H. halys have been reported. In the present study, carried out in Northern Greece, our main aim was to assess the occurrence of native parasitoids of H. halys, their parasitism rates, and relative abundance for potential application in biological control programs under local environmental conditions.

Description of the Study Area
This study was conducted in the areas of Preveza (Kalamitsi), Chalkidiki (Nea Moudania), Veria (Makrochori), and Thessaloniki (Thermi, Ardameri) in Greece. Among the crops inspected were apricots (Prunus armeniaca) and magnolias (Magnolia virginiana) in Preveza; olives (Olea europaea) and apricots in Nea Moudania; peaches (Prunus persica) in Makrochori; apricots, industrial hemp (Cannabis sativa), tomatoes (Solanum lycopersicum), and beans (Phaseolus vulgaris) in Thermi; and olives in Ardameri ( Figure 1). Apricots in Thermi and magnolias in Kalamitsi were mainly cultivated for family consumption or use, and no fertilizers or chemicals were applied at all. The surrounding environment was mainly dominated by pine trees. Tomatoes and beans in Thermi were located in small plots (less than 1 hectare) and grown next to other vegetables, such as peppers and cucumbers. The sizes of the rest fields ranged from 10 to 20 hectares and fertilizers and pesticides were used according to the farmers under different cultivation systems. and beans (Phaseolus vulgaris) in Thermi; and olives in Ardameri ( Figure 1). Apricots in Thermi and magnolias in Kalamitsi were mainly cultivated for family consumption or use, and no fertilizers or chemicals were applied at all. The surrounding environment was mainly dominated by pine trees. Tomatoes and beans in Thermi were located in small plots (less than 1 hectare) and grown next to other vegetables, such as peppers and cucumbers. The sizes of the rest fields ranged from 10 to 20 hectares and fertilizers and pesticides were used according to the farmers under different cultivation systems.

Collection of Egg Masses from the Field
A field collection of H. halys egg masses was carried out from June to October 2020 (Table 1). A total of 12 fields were inspected, including 5 fields in Thessaloniki, 2 fields in Chalkidiki, 2 fields in Imathia, 2 fields in Preveza, and 1 field in Kavala. Areas were selected based on previous reports of the presence of H. halys. As soon as the presence of H. halys in a field was confirmed, we started to inspect that field weekly. If the presence of H. halys was not confirmed for two consecutive weeks, then the inspection in the specific field stopped, resulting in unequal sampling per field. Both upper and lower surfaces of plant leaves were thoroughly inspected for egg masses. The number of plants that were randomly inspected varied among fields, depending on the severity of the infestation. When located, leaves with naturally laid egg masses were removed and placed individually in 50 mL falcon tubes. After collection, all egg masses were transferred to the Entomology Laboratory of the Institute of Plant Breeding and Genomic Resources (IPBGR), Thermi, Thessaloniki, Greece. Table 1. Data on field survey from June to October, 2020, in Greece.

Collection of Egg Masses from the Field
A field collection of H. halys egg masses was carried out from June to October 2020 (Table 1). A total of 12 fields were inspected, including 5 fields in Thessaloniki, 2 fields in Chalkidiki, 2 fields in Imathia, 2 fields in Preveza, and 1 field in Kavala. Areas were selected based on previous reports of the presence of H. halys. As soon as the presence of H. halys in a field was confirmed, we started to inspect that field weekly. If the presence of H. halys was not confirmed for two consecutive weeks, then the inspection in the specific field stopped, resulting in unequal sampling per field. Both upper and lower surfaces of plant leaves were thoroughly inspected for egg masses. The number of plants that were randomly inspected varied among fields, depending on the severity of the infestation. When located, leaves with naturally laid egg masses were removed and placed individually in 50 mL falcon tubes. After collection, all egg masses were transferred to the Entomology Laboratory of the Institute of Plant Breeding and Genomic Resources (IPBGR), Thermi, Thessaloniki, Greece.

Laboratory Handling of Field-Collected Egg Masses
In the laboratory, H. halys eggs were counted separately by crop, region, and date of collection, and then individually transferred to clear plastic containers (460 mL), of glass vials (5 mL) filled with water. Egg masses, with the help of a fine brush (Artist's Loft TM , MSPCI, Irving, TX, USA), were carefully placed on the upper surface of a green bean leaf from plants grown in the greenhouse at IPBGR, soaked in the glass vial. The containers where then covered with a plastic lid with mesh and placed in a room with a controlled environment at 26 • C, 65% RH, and a L16:D8 photoperiod. Egg masses were monitored daily and the number of parasitoids that hatched was recorded. Newly emerged parasitoids (<24 h) were placed in new clear plastic containers with a small cotton ball soaked in 10% honey-water solution, and a small plate with egg masses of H. halys from the laboratory rearing on the top of a filter paper. Emerged adult parasitoids from the laboratory-exposed egg masses of H. halys were preserved in small glass vials (5 mL), with 80% alcohol, and frozen at −20 • C. Vials with parasitoids were sent for morphological identification to the Natural History Museum of London (NHMUK). Voucher specimens were preserved at NHMUK.
Both parasitoids were sampled only in the regional unit of Thessaloniki. with A. bifasciatus present in two different locations, whereas O. telenomicida was only in the area of Thermi (Table 2).
Insects 2021, 12, x FOR PEER REVIEW 5 of 11 2B). Both parasitoids were sampled only in the regional unit of Thessaloniki. with A. bifasciatus present in two different locations, whereas O. telenomicida was only in the area of Thermi (Table 2).

Morphology of Parasitized Eggs
Detailed observation of the parasitized eggs revealed distinct differences in the exit holes produced by A. bifasciatus and O. telenomicida in accordance with findings of a previous study by Sabbatini-Peverieri et al. [33]. Circular exit holes that are produced by males of A. bifasciatus ( Figure 3A) are noticeably smaller than those of the larger females ( Figure 3B). Ooencyrtus telenomicida, regardless of sex, produced exit holes in H. halys eggs similar in size or slightly smaller compared to that of male A. bifasciatus, but with much more irregular margins ( Figure 3C,D).
Eggs of H. halys, in which we observed A. bifasciatus females insert their ovipositor, showed a characteristic dark spot on the egg chorion ( Figure 4A). In the case of O. telenomicida, one or multiple egg stalks [34] protruding outside the host chorion were visible ( Figure 4B).

Discussion
In our present field study, we observed two chalcid wasps, namely A. bifasciatus and O. telenomicida, to parasitize H. halys eggs. This is the first report of any natural enemy of the invasive species H. halys in Greece, exactly ten years since its first detection [8]. While O. telenomicida was observed only once in the season during mid-June, A. bifasciatus was observed thrice. It was first observed in mid-June in low numbers and again in the second half of August, in two different locations. This discovery adds important knowledge about the natural enemy community composition of H. halys in its native range, and could potentially be used for biological control in northern Greece.
In Europe, the generalist egg parasitoid A. bifasciatus has been reported on, with more than thirty-five hosts in the orders of Hemiptera, Lepidoptera, and Orthoptera [35,36]. It is among the most widespread native egg parasitoids of H. halys, and due to its ability to develop successfully on viable H. halys eggs, it can be considered a potential candidate for biological control [7,23,27,31,32,37,38]. Furthermore, Anastatus sp. is also part of the natural parasitoid community of H. halys in its native range in China [19]. In Greece, A. bifasciatus is a well-known egg parasitoid of the pine processionary caterpillar Thaumetopoea pityocampa (Denis & Schiffermueller) (Lepidoptera: Notodontidae) from the late 1980s [39]. Moreover, A. bifasciatus is part of a complex of primary egg parasitoids of T. pityocampa; however, infestation of T. pityocampa eggs by A. bifasciatus has been reported to be generally low [39][40][41][42][43]. Likewise, in most surveys, egg parasitism rates of A. bifasciatus on both naturally present and sentinel egg masses of H. halys were found to be less than 10% [27,37,44]. Its impact on H. halys is considered low, although it is frequently found throughout the survey region [32]. This is in accordance with our findings, as parasitism rates of A. bifasciatus, especially against naturally laid eggs of H. halys, in mid-June, was as low as 2.1%, while later, during the second half of August, it reached up to 8.0%. Interestingly, in our study, the emerged individuals of A. bifasciatus were of the female-based sex ratio (2.1:1), contrary to the ones that emerge from egg batches of T. pityocampa, where male individuals were the predominant or even the only ones [39][40][41][42][43]. This observation is of particular importance, contributing to the understanding of the sexual reproduction of A. bifasciatus.
The second egg parasitoid species that we detected was O. telenomicida, which is also considered a generalist egg parasitoid, attacking mainly heteropteran as well as lepidopteran species [45][46][47]. Ooencyrtus telenomicida is a dominant species in the Mediterranean climatic regions whose abundance is positively correlated with annual rainfall [48]. Recently, Roversi et al. [23] reported O. telenomicida from sentinel eggs of H. halys from a non-infested area in Tuscany, Italy. However, although O. telenomicida is able to complete development to the adult stage within its host H. halys, such as A. bifasciatus, abundance of this species in the field is generally low. In Western Slovenia, only one egg mass that was exposed in an agricultural area was parasitized by O. telenomicida [49], while in another study, in the region of Emilia-Romagna, Italy, none of the 11,841 sentinel eggs that were exposed were ever parasitized by this species [32]. This may be explained by the climatic and environmental differences between the surveyed regions or the local availability and abundance of the natural host(s) of O. telenomicida [32]. As in the case of A. bifasciatus, the sex ratio of O. telenomicida was female biased (1.86:1), in accordance with that recorded in a laboratory study in Italy, when O. telenomicida was reared on Graphosoma lineatum (Hemiptera: Petatomidae) eggs at temperatures above 26 and 30 • C [50]. According to the literature available, the host species does not affect sex ratio in O. telenomicida [47,48]; however, temperature does play a role in influencing sex ratio with a decrease in the number of females associated with lower temperatures [50]. Interestingly, O. telenomicida seems to be more frequent in mid-June, while A. bifasciatus appears to be in higher numbers during the second half of August, which is in contrast to the findings of a previous study conducted in Central Italy [23].
During the present study, a total of nine H. halys hosts were confirmed (Table 1). Prunus armeniaca, Olea europea, Cannabis sativa, and Phaseolus vulgaris were the most common hosts in the agricultural area. Apricot was the main oviposition host from early June to late July, with seven egg masses collected, almost two-thirds of all H. halys egg masses collected during that period. On the other hand, green beans were the main hosts from early August to early October, with eight egg masses collected during that period. Moreover, H. halys oviposited, based on the relative abundance of potential host plants. Both apricot and green beans are reported among the most acceptable hosts of H. halys in the invaded areas, suitable for oviposition and nymphal development [6,51]. Anastatus bifasciatus was collected in mid-June and mid-August on egg masses of H. halys that were laid on green beans, apricots, and olives. On the other hand, O. telenomicida was collected only on mid-June on one egg mass of H. halys that was laid on apricots. Although both species are considered as generalist egg parasitoids, A. bifasciatus is likely more widespread and present in more habitats compared to O. telenomicida.
The search for native natural enemies as an alternative solution against invasive pests in field crops has been considered recently, particularly for H. halys [7,23]. Their success strongly depends on a thorough understanding of the biology and ecology of both the pest and natural enemy [2,52,53]. Thus, the identification of native natural enemies and egg parasitoids, particularly associated with H. halys, and the evaluation of their impact, is needed, prior to any biological control implementation.

Conclusions
The present study provides basic information on the presence and species diversity of egg parasitoids that are associated with H. halys in Northern Greece. More specifically, the data obtained during this study provide a solid basis for future tasks regarding the biological control of H. halys in Greece. Hence, a better understanding of the specific roles of A. bifasciatus and O. telenomicida as biological control agents of H. halys in field crops, as well as of the interactions between these two species, is essential for providing further tools, in terms of a robust and sustainable IPM strategy.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.