First record in Belgium of Trissolcus basalis (Hymenoptera, Scelionidae), an egg parasitoid of economically important stink bugs (Hemiptera, Pentatomidae)

The scelionid parasitoid Trissolcus basalis (Wollaston,1858) has been detected in Belgium for the first time based on specimens reared from a parasitized egg mass of Nezara viridula (Linnaeus,1758) collected in an urban garden at Sint-Amandsberg, Ghent. Identification was based on adult morphology and DNA barcoding. This is presently believed to be the northernmost record in Europe of T. basalis and could be the consequence of a northward expansion of this species due to climate change. This first record may be of economic importance for the biological control of stink bug pests in Belgian vegetable and fruit production.


Introduction
Parasitoid wasps in the family Scelionidae (Hymenoptera: Platygastroidea) are gaining increasing attention for biological control due to their parasitism of eggs of key insect pests in agriculture. Trissolcus basalis (Wollaston, 1858) (Hymenoptera: Scelionidae) belongs to the basalis-species group of the genus Trissolcus (sensu JoHnson 1987;talamas et al. 2017) and is a solitary egg parasitoid of stink bugs with an assumed worldwide distribution (Colazza & Bin 1995). The taxonomy of the superfamily Platygastroidea was recently revised, reaffirming the Scelionidae as a valid family (CHen et al. 2021).
Trissolcus basalis is a generalist and is known to parasitize the eggs of a wide range of pentatomid hosts (Jones 1988). However, the most common host is the southern green stinkbug Nezara viridula (Linnaeus, 1758) (Hemiptera: Pentatomidae) (Colazza et al. 1995;mCPHerson & mCPHerson 2000). Due to increased global transportation and climate change, non-native species can establish populations beyond their natural distribution (ziska et al. 2011). When non-native herbivores such as N. viridula invade cropping systems, they can quickly become serious pests. Nezara viridula is a highly polyphagous herbivore and due to its habitat expansion, it has become one of the most economically important species of Hemiptera causing damage to crops in the open field and greenhouses (Panizzi 2000;mCPHerson & mCPHerson 2000). Its geographic origin is uncertain but is assumed to be eastern Africa or the Mediterranean (Hokkanen 1986

Material and methods
A single mass of 73 N. viridula eggs was collected from a Stevia rebaudiana leaf (Asteraceae) on 28 August 2020 in an urban garden at Sint-Amandsberg (Ghent), Belgium (51°3′20.666″ N, 3°44′53.297″ E). The egg mass was placed in a climatic chamber in the laboratories of the Department of Plants and Crops, Ghent University, set at 24°C, 18:6 h L:D and 70% RH (PHCBI MLR-352H-PE, Japan) and held for emergence of stink bug nymphs or adult parasitoids. Over 50 parasitoid adult wasps emerged from this egg mass with a sex ratio of 1:9 male to female.
A culture was started from a single isolated mated female wasp from the field-collected egg mass and kept at the above-mentioned climatic conditions by offering fresh (< 24 h old) N. viridula egg masses, from a culture maintained at the Department of Plants and Crops, Ghent University. The culture of the parasitoid was kept in polystyrene insect breeding dishes (100 × 40 mm; SPL Life Sciences Co., Korea). The adults of the F1 generation and all subsequent generations were fed with a drop of honey placed directly on the breeding dish. Water was provided via moistened synthetic cotton (Roltasoft Hartmann, Germany).
The parental specimen and four F1 specimens were morphologically identified using the key of talamas et al. (2017). Specimens were deposited at the Ghent University Museum, Zoology Collections with collection numbers: UGMD_104422 and UGMD_104423. Furthermore, twenty frozen adult individuals of both sexes from the F2 generation, originating from a single mated female were pooled for DNA extraction and mitochondrial cytochrome c oxidase I (COI) fragment sequencing. DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen). The DNA samples were quantified using a NanoDrop2000 spectrophotometer (Thermo Scientific). At least 20 ng of genomic DNA was used per PCR. The 5'-COI region was PCR-amplified using the primers [LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO-2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) (Folmer et al. 1994). The PCR was performed in a 50 µl reaction volume: 1 µl DNA, 29.5 µl molecular grade water, 10 µl 5X Green GoTaq Flexi PCR buffer, 2.5 µl dNTPs (25 mM each), 5 µl MgCl 2 , 1.5 µl of each primer (1 µM each), 0.2 µl GoTaq G2 Flexi DNA polymerase (5 u/µl) (Promega Corp.,Wisconsin, USA). Thermocycling conditions were optimized to shorten reaction times and included initial denaturation at 94 °C for 300 s, followed by 35 cycles of 94 °C for 30 s, annealing at 41 °C for 45 s and extension at 72° C for 60 s; then further 600 s at 72°C for   final extension. All PCR products were purified using the E.Z.N.A Cycle Pure Kit (Omega Bio-tek Inc, Georgia, USA) following the manufacturer's instructions and sent for sequencing to an external service (LGC Genomics GmbH, Berlin, Germany). The obtained forward and reverse sequences were inspected and a consensus sequence was made using the BioEdit Software ver. 7.2.0. The consensus sequence was compared with sequences present in the GenBank database by similarity search using the Basic Local Alignment Search Tool (http://ncbi.nlm.nih.gov/BLASTn), to confirm the taxonomic identity. The COI sequence generated was deposited in GenBank (MZ087751).
We checked collections at the Royal Belgian Institute of Natural Sciences, The Entomological Conservatory at Gembloux and online databases including fauna-eu.org (De JonG et al. 2014) (accessed 20 October 2021) and gbif.org (GBIF, accessed 20 October 2021) to confirm that our record is in fact the first record in Belgium.

Results
Both the morphological and molecular identifications with barcoding converged to the same species: Trissolcus basalis. The BLASTn query returned over 10 sequences of T. basalis with a percent identity of 100% and E-values of 0.0, therefore showing high similarity with other T. basalis sequences. According to talamas et al. (2017), T. basalis can be identified by the combination of the following characters: vertex without hyperoccipital carina, netrion sulcus incomplete, mesopleuron with episternal foveae shallowly impressed, metapleuron without setation and without well-defined paracoxal sulcus, mesoscutal humeral sulcus present as a smooth furrow and second metasomal tergite with longitudinal striation (Figs 1-4).

Discussion
The parasitized egg mass collected at Sint-Amandsberg (Ghent) represents the first record of T. basalis in Belgium and indicates the presence of an established breeding population. There have been no commercial releases of this species for biological control nor any known laboratory cultures in the area, excluding introduction from these sources.
Although T. basalis is considered to be globally distributed (Jones 1988; Colazza 1995; talamas 2017), its distribution within Europe has been rather sparsely documented, with records from Cyprus, Montenegro, Portugal, Spain, Italy, Hungary, France and Germany (awan et al. 1990;Colazza 1995;tortoriCi et al. 2019;awaD et al. 2021). These European specimens were collected in more southern countries or regions. Records from France were from the Aquitaine and Provence-Alpes-Côte-d'Azur, two southern regions (USMENT00896070-00896071, 00896037-00896040, 00896055-0089604060, and 0089629, examined by talamas et al. (2017)). The specimens from Germany (SMNS_Hym_ Sce_000805-000806, examined in awaD et al. (2021)) were collected in the most southern state, Baden-Württemberg. Our record from Belgium would therefore constitute the northernmost record of T. basalis at present in Europe. It is possible that only in recent decades, T. basalis has colonized Belgium and other parts of northwestern Europe or has become ubiquitous enough to be detected in this area due to the northward habitat expansion of its prime host N. viridula. Additionally, the warming of the climate in northwestern Europe could have played a role in the gradual northward habitat expansion of T. basalis following its main host N. viridula.
In order to aid further research on the distribution of this parasitoid wasp in Europe, we have mapped all European records (Fig. 5).
More extensive studies on egg parasitoids of Pentatomidae in Belgium and Western Europe will greatly increase our knowledge of biological control of the various stink bug species currently causing economic damage in the area.
Given the economic importance of some of their pentatomid hosts, it will likely be beneficial to support or attract these parasitic wasps to agricultural fields and commercial fruit orchards in a conservation biological control approach (raHat et al. 2005). In this context, it may be warranted to further investigate the physical and chemical cues by which these parasitoid wasps locate and recognize their hosts (Bin et al. 1993;mattiaCCi et al. 1993) or plant resources (martorana et al. 2017). Moreover, the interactions between populations of T. basalis and other species of egg parasitoids deserve attention. This knowledge will be instrumental in designing lntegrated Pest Management (IPM) strategies against stink bug pests in Belgium and its neighbouring countries.