JHR 67: 37-53 (2018) ge, JOURNAL OF eeninntmnscaan doi: 10.3897/jhr.67.30883 DATA PAPER (ME Hymenoptera http://jhr.pensoft.net The Inerational Society of Hymenoptersts. RESEARCH Two Asian egg parasitoids of Halyomorpha halys (Stal) (Hemiptera, Pentatomidae) emerge in northern Italy: Trissolcus mitsukurii (Ashmead) and Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) Giuseppino Sabbatini Peverieri', Elijah Talamas”, Marie Claude Bon’, Leonardo Marianelli', Iris Bernardinelli*, Giorgio Malossini*, Luca Benvenuto’, Pio Federico Roversi', Kim Hoelmer? | CREA — Research Centre for Plant Protection and Certification, Florence, Italy 2 Florida State Collection of Arthropods, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, FL 32608, USA 3 USDA-ARS European Biological Control Laboratory, 810 Avenue du Campus Agropolis, 34980 Montferrier le Lez, France 4 ERSA — Regional Agency for Rural Development, Plant Health Service, Udine, Italy 5 USDA-ARS Beneficial Insects Introduction Research Unit, Newark, DE, USA Corresponding author: Elijah Talamas (talamas.1@osu.edu) Academic editor: G. Broad | Received 27 October 2018 | Accepted 4 December 2018 | Published 31 December 2018 Attp://zoobank. org/2B206556-8 1 18-4E60-AC52-42C827FO0B26 Citation: Sabbatini Peverieri G, Talamas E, Bon MC, Marianelli L, Bernardinelli I, Malossini G, Benvenuto L, Roversi PE, Hoelmer K (2018) Two Asian egg parasitoids of Halyomorpha halys (Stal) (Hemiptera, Pentatomidae) emerge in northern Italy: Trissolcus mitsukurii (Ashmead) and Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae). Journal of Hymenoptera Research 67: 37-53. https://doi.org/10.3897/jhr.67.30883 Abstract Halyomorpha halys (Stal) is a severe agricultural pest that is spreading worldwide from its original distribu- tion in Asia. Ege parasitoids from Asia, which play a key role in the population dynamics of H. halys, are following its host along global pathways. We present the first records of Trissolcus mitsukurii in Europe, and of Trissolcus japonicus in Italy. Both discoveries were made in northern Italy, where H. halys is widely present and has reached extremely high population densities in some areas. Given the availability of their host, the distributions and populations of these exotic egg parasitoids are expected to expand, even in the absence of human intervention. Keywords Egg parasitoid, brown marmorated stink bug, exotic species Copyright Giuseppino Sabbatini Peverieri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 38 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) Introduction The invasive stink bug, Halyomorpha halys (Stal) (Hemiptera: Pentatomidae), also known as the brown marmorated stink bug, is now a cosmopolitan pest. From its na- tive range in East Asia, it first became established in North America in the mid-1990s (Hoebeke and Carter 2003), followed by Europe in the mid-2000s (Wermelinger et al. 2008), and is now established in South America (Fatindez and Rider 2017). It has been intercepted numerous times in New Zealand but has not yet established a permanent population there (Ormsby 2018). Halyomorpha halys is a highly polyphagous pest, and severe damages are recorded on forest, urban and agricultural trees, and on the fruits and seeds of crops and horticultural plants (Lee et al. 2013, Leskey and Nielsen 2018). Worldwide, the control of H. /alys currently relies mainly on pesticides (Kuhar and Kamminga 2017). However, because only broad-spectrum pesticides are effec- tive, beneficial insects are also killed and integrated pest management programs are negatively impacted. Thus, much attention has been given to alternative methods for a long-term solution, including biological control. Among natural enemies of H. /alys, considerable emphasis has been placed on egg parasitoids, which appear to be the most effective enemies in the native Asian range (Yang et al. 2009, Lee 2015). Several studies explored the ability of parasitoids to attack H. halys eggs, both in its native distribution and in the newly invaded areas worldwide (Haye et al. 2015, Talamas et al. 2015, Ro- versi et al. 2016, Herlihy et al. 2016, Abram et al. 2017, Dieckhoff et al. 2017). Pres- ently, only Asian native species of egg parasitoids appear to be promising candidates as biological control agents, primarily Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae) (Zhang et al. 2017). The spread of H. halys has also provided an invasion opportunity for its parasi- toids. In 2015 T’ japonicus, commonly known as the samurai wasp, was detected in the eastern United States (Talamas et al. 2015), followed shortly by discovery of a second, independently established population in the Pacific Northwest (Milnes et al. 2016). In 2017 and again in 2018, 7! japonicus was recorded in Switzerland, its first reported recovery in Europe (Stahl et al. 2018). We present here the discovery of two adventive populations of exotic parasitoids, 7’ mitsukurii (Ashmead) and T! japonicus, that are parasitizing 1. Aalys eggs in the wild in northern Italy. Methods Field survey and recovery of Trissolcus mitsukurii in northeastern Italy During the 2018 brown marmorated stink bug monitoring campaign in fruit or- chards in the Region of Friuli-Venezia-Giulia (northeastern Italy), personnel of ERSA on August 7, and jointly with personnel of CREA on August 8, noted the presence of darkly colored H. /alys egg masses, which is an indicator of parasitism (see Results for site descriptions). During the surveys, egg masses of other stink bug Two Asian egg parasitoids of Halyomorpha halys (Stal)... a? species were also collected when found. A portion of the egg masses collected in the field were reared in climatic chambers (26 °C, 65%RH, 16:8 L:D) until adult para- sitoids emerged. For further study, adults were kept alive in glass tubes and provided with pure honey droplets as food. The remaining field collected egg masses were reared in a laboratory room in Petri dishes until parasitoids emerged and specimens were stored in ethanol for further studies. All emerged specimens were counted, identified to species and sexed. Field survey and recovery of Trissolcus japonicus in northwestern Italy During routine research activities of CREA personnel on July 27 and August 3, 2018, several H. halys egg masses with dark coloration were observed on Acer campestris L. trees in a parking lot near Lodi, a site close to the town of Milan (see Results for site description). All collected egg masses were reared in climatic chambers as described previously, and adult parasitoids were kept alive in rearing tubes with pure honey for further studies. Emerged specimens were counted, identified to species and sexed. Reared specimens of Trissolcus were identified using the key to Palearctic Trissol- cus provided in Talamas et al. (2017). Image Z-stacks were taken with a Canon EOS 80D camera attached to an Olympus BX51 compound scope and a Macropod im- aging system from Macroscopic Solutions and were rendered using Helicon Focus (Helisoft). Voucher specimens are deposited in the Florida State Collection of Ar- thropods (Gainesville, FL), the USDA-ARS European Biological Control Laboratory (EBCL) (Montpellier, France), and CREA-DC (Florence, Italy). Collection data as- sociated with a subset of these specimens are deposited in the Hymenoptera Online Database (hol.osu.edu). Morphological terminology follows Miké et al (2007). Character annotations ats _ postacetabular sulcus (Fig. 7) cs clypeal setae (Fig. 9) eps _ episternal foveae (Fig. 7) hoc hyperoccipital carina (Figs 3, 6, 8) lo lateral ocellus (Figs 3, 6) Iptl lateral setal patch on mediotergite of T1 (Fig. 3) mp mesopleural pit (Fig. 7) ms malar sulcus (Figs 4, 7) mtps metapleural sulcus (Fig. 2) not notauli (Fig. 8) of orbital furrow (Figs 4, 7) pof preocellar furrow (Fig. 9) sltl__setal patch on laterotergite of T1 (Fig. 3) 40 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) Molecular analysis DNA extraction, PCR amplification and sequencing Following their morphological examination, all specimens were preserved in 95% ethanol and shipped to EBCL. Tables 3, 4 list the specimens and voucher informa- tion included in the analysis. Prior to DNA extraction, individual specimens were bathed three times at room temperature in molecular grade water for five minutes. Genomic DNA was nondestructively isolated from the entire specimen using the Qia- gen DNeasy kit (Hilden, Germany) as published in Taekul et al. (2014) with minor modification. In Step 7, the elution buffer, warmed to 55 °C, was allowed to sit on the membrane during 15 min before centrifugation. The collected flow-through was reloaded onto the spin column to increase the DNA yield. A negative control (no insect tissue) was included in each extraction to detect potential contamination. ‘The barcode region of the mitochondrial Cytochrome Oxidase Subunit I (CO/) was ampli- fied using the universal barcoding primers LCO1490 (5’-GGTCAACAAATCATAAA- GATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). Amplification and barcode editing and analysis were done as described in Ganjisaffar et al. (2018). All sequences generated from this study and those from our custom barcode database are deposited in Genbank (Tables 3 and 4) and all residual DNAs are archived at EBCL (Tables 3 and 4). The sequences obtained were compared with sequences present in Genbank by similarity search using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLASTn). Although tracing the source of the Italian populations was not the scope of the present study, we used this barcode approach to provide better insights into the mito- chondrial diversity of these two Trissolcus species, identify and exclude Asian popula- tions that were highly divergent from Italian populations, and tentatively find similar populations. Of note, 7’ mitsukurii barcodes are poorly represented in Genbank data- base and BOLD as there is only one record published by Mita et al. (2014). For this study we had access to an unpublished EBCL database of 12 barcodes of 77 mitsukurii collected in China, Korea and Japan and obtained within the framework of an on- going USDA biocontrol program. Two sequences generated from the present study were aligned using Clustal W with the sequence from Mita et al. (2014) and the 12 sequences of our custom database. For J! japonicus, the five sequences generated from this study were aligned with Clustal W with all barcode sequences retrieved from Genbank and BOLD (which con- tained some sequences not present in Genbank). Only sequences from Asian and Swiss samples were included in the dataset. For both taxa, the phylogenetic relationships among haplotypes were depicted using statistical parsimony in TCS as implemented in PopART (Leigh and Bryant 2015). This approach enabled us to display the geographi- cal distribution of all haplotypes. Two Asian egg parasitoids of Halyomorpha halys (Stal)... 4] Field surveys Trissolcus mitsukurii recoveries in northeastern Italy Site descriptions for recovery sites are given in Table 1. A total of 31 1. halys egg masses were collected at three survey sites in northeastern Italy on August 7-8, 2018 (Fig. 1, Table 2). Of these, 4 egg masses were not parasitized, and nymphs hatched at a rate of 98.23%; 25 egg masses were parasitized by 7’ mitsukurii; and 2 egg masses were parasitized by Anastatus bifasciatus (Geoffroy) (Eupelmidae) and a species of Pt- eromalidae. From the 25 egg masses parasitized by 7’ mitsukurii, a total of 424 adults emerged of which 91.75% were females. Depending on locality, the parasitism rate of eggs within single egg masses ranged from 32.14% to 100% and number of para- sitized egg masses out of the total number of egg masses detected ranged among sites from 50.00% to 84.21%. During surveys at the Codroipo site, an egg mass of a predatory stink bug be- longing to the subfamily of Asopinae was collected which was also parasitized by 7’ mitsukurii. From this egg mass, 83.87% of the eggs (31 eggs in total) were parasitized (emergence rate 96.15%; 80.00% females); only one egg of the cluster produced a stink bug nymph, and four apparently unparasitized eggs did not hatch. Trissolcus japonicus findings in northwestern Italy The recovery site of 7’ japonicus in northwestern Italy was located at 45.3031N, 9.4794E (Fig. 1). Parasitized egg masses were found on Acer campestris L. trees in a parking lot surrounded by a multi-host patchy landscape (crop fields, uncultivated fields, hedgerows), and industrial and urban areas with ornamental plants. Table |. Descriptions of Trissolcus mitsukurii recovery sites in northeastern Italy. Locality Coordinates Main culture Surrouding cultures Surrounding environment (plant species and management) Cordenons site 1 46.0089N, Kiwi orchard Vineyards, maize and soybean Hedgerows, apple and kiwi 12.6824E (Actinidia chinensis), crops managed by integrated _ orchards, vineyards, maize organic farming pest management and soybean fields Cordenons site 2 46.0082N, Hedgerow (Robinia Apple orchard, vineyards, maize Hedgerows, apple and kiwi 12.6713E pseudoacacia) and soybean crops managed by _ orchards, vineyards, maize integrated pest management and soybean fields Codroipo 45.9675N, Kiwi orchard Apple and pear orchards, Hedgerows, apple and pear 13.0251E (Actinidia deliciosa), vineyards, maize and soybean __ orchards, vineyards, maize integrated pest crops managed by integrated and soybean fields management pest management 42 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) Cordenons (sites 1 & 2) A Codroipo A Trissolcus mitsukurii O Trissolcus japonicus Figure |. Recovery sites of Trissolcus mitsukurii and Trissolcus japonicus in northern Italy. Table 2. Parasitism of Halyomorpha halys eggs by Trissolcus mitsukurii collected in field surveys of August 7-8, 2018, in northeastern Italy. Unparasitized Parasitized egg masses egg masses n. of parasitized egg masses/total egg masses detected Mean n. of eggs/egg mass * hatched:unhatched Mean sex ratio (% eggs/egg mass Mean % of parasitized eggs/ Mean emergence rate (%)* of females) * Mean % of Hatching rate (n. of egg masses) 87.04 94.22 92.31 (32.14-100) | (57.89-100) | (88.99-100) S Oo S a N ™—S NN 100% (3) Cordenons site 1 Cordenons site 2 : 100 92.86 not assessed (0) 0.00:3.59 100% (1) (78.57-100) | (65.38-100) | (84.21-95.45) * one egg mass was entirely parasitized by other parasitoids. *minimum and maximum values in parentheses Field collections were made on July 27 and August 3, 2018, and a total of 45 H. halys egg masses were collected (with a mean of 25.77 eggs/egg mass). On July 27, only one egg mass was detected and was found to be successfully parasitized by 7’ japonicus, with the emergence of 8 specimens, all of which were males. Among the 44 egg masses collected on August 3, 21 egg masses were not parasitized and eggs hatched into nymphs with a mean rate of 82.44%, and 22 egg masses were parasitized by A. bifasciatus. Only Two Asian egg parasitoids of Halyomorpha halys (Sta)... 43 Table 3. Sampling Information, GenBank Accession numbers and haplotypes for Trissolcus mitsukurii included in this study. Collection code and Sex Country Site Year of Collection, Host GenBank Barcode _ Isolate Name of Collector » Accession Haplotype (EBCL) Number (617bp) FSCA00033071, 2 Italy Cordenons, 2018, 1B, LB &GM _ Halyomorpha halys MK097189 H5 Tsp270 Friuli-Venezia- (this study) Giulia FSCA00033072, 2 Italy Cordenons, 2018, IB, LB & GM H. hatlys MKO097190 H5 Tsp269 Friuli-Venezia- (this study) Giulia Tm1-EBCL:, 3 Japan Tsukuba, 2007, KH H. halys MKO097191 H1 Tml (NARO)<* (this study) USNMENT01197989, 2 South Gochang 2015, KH unidentified host MK097192 H3 Tsp202 Korea eggs (not H. halys) (this study) USNMENT01197242, 2 South Jeju 2012, ET& IM na MK097193 H4 Tsp233 Korea (this study) USNMENT01197243, 2 South Jeju 2012, ET& IM na MK097194 H4 Tsp234 Korea (this study) USNMENT01197244, 2 South Jeju 2012, ET& IM na MK097195 H4 Tsp235 Korea (this study) USNMENT00977533, 2 China Yunnan Prov., 2013, KH Erthesina fullo |MKO097196 H2 Tsp39 Kunming (this study) USNMENT01059335, 9. = China Yunnan Prov., 2013, KH E. fullo MK097197 H2 Tsp60 Kunming (this study) USNMENT01197294, 2 Japan Tsukuba, 2012, KH LH. halys MK097198 H1 Tsp149 (NARO)°* (this study) USNMENT01197295, © Japan Tsukuba, 2012, KH H. hatlys MKO097199 H1 Tsp 150 (NARO)<* (this study) Tsp151- EBCL’, na Japan Tsukuba, 2012, KH H. hatlys MK097200 H1 Tsp151 (NARO)<° (this study) Tsp152- EBCL*, na Japan Tsukuba, 2012, KH EH. hatlys MkKO097201 H1 Tsp152 (NARO)<° (this study) Tsp153- EBCL*, na Japan Tsukuba, 2012, KH H. halys MK097202 H1 Tsp153 (NARO)<° (this study) na, na Japan Fukuoka na, na Nezara viridula’ AB971831 H1 (Mita et al. 2014) * EBCL DNA collection. ® name of collectors: IB: Iris Bernardinelli, LB: Luca Benvenuto, GM: Giorgio Malossini, KH: Kim Hoelmer, ET: Elijah Talamas, IM: Istvan Mik6. * NARO: National Agriculture and Food Research Organization. one egg mass collected on August 3 was parasitized by 7! japonicus. All eggs that were ap- parently parasitized (35.71% of eggs in the mass) produced adult parasitoids (9 females and 2 males), 46.43% of eggs were unhatched and 17.86% of the eggs hatched into stink bug nymphs. Ninety percent of the emerged 7’ japonicus were females. Taxonomy Trissolcus mitsukurii Trissolcus mitsukurii is a straightforward species to identify and is separated early in the key to Palearctic Trissolcus (Talamas et al. 2017). We thus do not consider it necessary to produce an updated key to European Trissolcus that includes this species, and instead 44 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) Table 4. Sampling Information, GenBank Accession numbers and haplotype for Trissolcus japonicus included in this study. Collection code and = Country Site Year of Host GenBank Accession Number/ Barcode _Isolate Sex Collection, Bold Accession number Haplotype (EBCL) Collector ° FSCA 00033060, 2 Italy Lodi, 2018, PFR Halyomorpha MKO097 184 (this study) H1 Tj406 Lombardy halys FSCA 00033065, 9 Italy Lodi, 2018, PFR H. hatys MKO097 185 (this study) Hl Tj408 Lombardy FSCA0033097, 3 Italy Lodi, 2018, PFR LH. halys MK097186 (this study) H1 Tj421 Lombardy FSCA0033098, 3 Italy Lodi, 2018, PFR 1. hatys MKO097 187 (this study) H1 Tj422 Lombardy FSCA0033096, 3 Italy Lodi, 2018, PFR LH. halys MK097 188 (this study) Hl Tj423 Lombardy GBIFCH00543446, 2 = Switzerland Ticino 2017, JS H. hatys MH919753 (Stahl et al., 2018) H1 Tj388 GBIFCH00543447, 2 Switzerland Ticino 2017, JS H. hatys MH919754 (Stahl et al., 2018) H1 Tj389 GBIFCH00543448, 2 = Switzerland Ticino 2017, JS LH. halys MH919755 (Stahl et al., 2018) H1 Tj390 GBIFCH00543449, 3 Switzerland Ticino 2017, JS H. halys MH919756 (Stahl et al., 2018) H1 Tj391 GBIFCH00543450, 4 Switzerland Ticino 2017, JS H. halys | MH919757 (Stahl et al., 2018) Hl Tj392 GBIFCH00543451, 4 Switzerland Ticino 2017, JS H. halys |= MH919758 (Stahl et al., 2018) H1 T5393 Tsp77- EBCL*, na Japan Tsukuba 2012, KH LH. halys MH919744 (Bon et al., H1 Tsp77 (NARO)* unpublished) Tsp78- EBCL’, na Japan Tsukuba 2012, KH LH. halys MH919745 (Bon et al., H1 Tsp78 (NARO)* unpublished) Tsp79- EBCL’, na Japan Tsukuba 2012, KH EH. hatys MH919746 (Bon et al., H1 Tsp79 (NARO)* unpublished) Tsp88- EBCL’, na Japan Tsukuba 2012, KH LH. halys MH919747 (Bon et al., H1 Tsp88 (NARO)<* unpublished) Tsp90-EBCL’, na Japan Tsukuba 2012, KH H. hatys MH919748 (Bon et al., Hl Tsp90 (NARO)<* unpublished) Tsp91-EBCL’, na Japan Tsukuba 2012, KH H. halys MH919749 (Bon et al., H1 Tsp91 (NARO)* unpublished) Tsp93- EBCL*, na Japan Tsukuba 2012, KH H. hatys MH919750 (Bon et al., H1 Tsp93 (NARO)* unpublished) Tsp226-EBCL?, 9 Japan Kanagawa 2015,KH = Plautia stali MH919752 (Bon et al., H1 Tsp226 unpublished) na, 2 Japan Kanagawa 2012,TM P stali AB847131-32,36 H1 (Matsuo et al., 2014) na, 9 Japan Fukuoka 2012, KM P stali AB847144-145 H2 (Matsuo et al., 2014) na, Japan Fukuoka = 2012, KM LH. halys AB908179-182 H2 (Matsuo et al., 2014) na, na Japan na na, na na AB894834-35, AB894838-39 H2 (Matsuo, K. and Hirose,Y., unpublished na, 9 Japan Fukuoka 2012, KM BP stali AB847129,130, 137,143,146 H3 (Matsuo et al., 2014) na, na Japan na na, na na AB894836,837,840,841 H3 (Matsuo, K. & Hirose, Y. (unpublished), na, na Japan Kanagawa na, na P stali AB971832 (Mita et al., 2014) H1 na, na China H. halys KF303518.1 H7 (Gariepy et al., 2014) USNMENT01059340, China Langfang 2012, KH E. fullo / NSCEL009-18 H4 Tsp61 Q (Gariepy unpublished) USNMENT01197300, China Kunming 2014, KH E. fullo /NSCEL010-18 H5 Tsp155 Q Gariepy unpublished) Tsp1-EBCL?, na Japan Tsukuba 2012, KH H. halys /NSCELO11-18 H1 Tsp1 (NARO)* Collection code and Sex USNMENT00977534, Tjl-EBCL*, na Trj2-EBCL:, na penal 197806, USNMENT01197320, na, na na, na na, na Na, na Na, na Two Asian egg parasitoids of Halyomorpha halys (Stal)... Country S. Korea China China Japan South Korea China China China China China Site Jirisan Park Hebei Hebei Kanagawa Seoul Hebei Hebei Hebei Hebei Hebei Year of Collection, Name of Collector > 2013, KH 2012, KH 2012, KH 2015, KH 2014, KH 2012, TH 2012, TH 2012, TH 2012, TH 2012, TH Host H. halys LH. hatys H. hatys LH. halys LH. hatys LH. halys H. hatys H. hatys H. hatys H. hatys GenBank Accession Number/ Bold Accession number /NSCEL012-18 Gariepy unpublished) /NSCEL013-18 Gariepy unpublished) /NSCEL014-18 Gariepy unpublished) /NSCEL017-18 Gariepy unpublished) /NSCEL018-18 Gariepy unpublished) /PPENT028-12 Gariepy unpublished) /PPENT029-12 Gariepy unpublished) /PPENT030-12 Gariepy unpublished) /PPENT031-12 Gariepy unpublished) /PPENT032-12 Gariepy unpublished) 45 Barcode Isolate Haplotype (EBCL) (373bp) H6 —Tsp53 H7 Tjl H7 Trj2 Hl —Tsp223 H7 —Tsp175 H7 H7 H7 H7 H7 *EBCL DNA collection. *name of collector: PFR: Pio Federico Roversi, JS: Judith Stahl, KH: Kim Hoelmer, KM: Kazunori Matsuo, TM: Toshiharu Mita, TH: Tim Haye. ‘NARO: National Agriculture and Food Research Organization. h : i Figures 2-3. Trissolcus mitsukurii, female (FSCA 00033025) 2 head, mesosoma, metasoma, lateral view 3 head, mesosoma, metasoma, anterolateral view. Scale bars in millimeters. 46 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) Figures 4-6. 7rissolcus mitsukurii, female (FSCA 00033025) 4 head, anterior view 5 habitus, lateral view 6 head, mesosoma, metasoma, dorsolateral view. Scale bars in millimeters. we provide the following diagnosis, with references to illustrations, that will be help- ful for distinguishing 7’ mitsukurii from the European fauna: the clava of the female antenna is 5-merous, and the clavomeres are distinctly larger and darker than preced- ing antennomeres (Fig. 2); the orbital furrow is expanded where it intersects the malar sulcus (Fig. 4); the hyperoccipital carina is often present directly posterior to the lateral ocellus, but is absent between the ocelli (Figs 3, 6); the metapleuron is without setae below the metapleural sulcus (Fig. 3); a setal patch is present on the first laterotergite of the metasoma (slt1, Fig. 3). Care should be taken not to confuse the setal patch of the laterotergite with the setal patch on the mediotergite (Iptl, Fig. 3). Trissolcus japonicus The key to European Trissolcus in Talamas et al. (2017) included T’ japonicus to pro- vide identification of this species given the possibility that it might become estab- Two Asian egg parasitoids of Halyomorpha halys (Stal)... 47 Figures 7-10. Trissolcus japonicus T female (FSCA 00033063), head, mesosoma, metasoma, ventrolat- eral view 8 female (FSCA 00033063), head, mesosoma, metasoma, dorsolateral view 9 female (FSCA 00033063) head, anterior view 10 male (FSCA 00033095), habitus, lateral view. Scale bars in millimeters. lished in Europe. The specimens from northern Italy are fully congruent with the concept of this species presented by Talamas et al. (2017). Trissolcus japonicus can be separated from other species of European Trissolcus by the following diagnosis: four clypeal setae are present below the antennal insertions (Fig. 9); microsculpture is pre- 48 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) sent throughout the frons (Fig. 9); the orbital furrow is expanded at its intersection with the malar sulcus (Fig. 7); the hyperoccipital carina is complete (Fig. 8); episternal foveae extend from the postacetabular sulcus to the mesopleural pit (Fig. 7); and the mesoscutum is without oblique rugae between the notauli (Fig. 8). The preocellar fur- row, which extends ventrally from the median ocellus (Fig. 9), is a useful character for confirming the identity of 7’ japonicus, but it is not always present, and exhibits the greatest variability in males. Anastatus bifasciatus Specimens of A. bifasciatus were identified by GSP using the keys of Kalina (1981) and Askew and Nieves-Aldrey (2014) and the identification was confirmed by Dr. Lucian Fusu (University of Iasi, Romania) who compared them with authoritatively identified specimens. Molecular identification Trissolcus mitsukurii The two voucher specimens recovered from the field in Cordenons (site 1) in the re- gion of Friuli-Venezia-Giula yielded a similar barcode sequence of 666-bp in length. A BLAST search showed the best similarity score (99%) of this barcode sequence with TL. mitsukurii (Accession No. AB971831). From the final alignment of 617-bp of 15 I! mitsukurii barcodes, a total of five haplotypes (denoted H1-H5) were recovered (Table 3). The haplotype H5 found in Italy is a new haplotype, as it did not match any haplotype found so far in Asia (Fig. 11, Table 3). From the network analysis, H5 differed by seven substitutions from the two closest haplotypes H1 and H2 from Japan and China respectively (Fig. 11). Trissolcus japonicus The five voucher specimens recovered from the field in Lodi yielded a unique barcode sequence of 666-bp in length. A BLAST search showed the best similarity score (100%) of this barcode sequence with 7. japonicus (Accession No. AB971832). From the final alignment of 373-bp of 60 7. japonicus barcodes, a total of seven haplotypes (denoted H1 to H7) were recovered (Table 4). The haplotype H1 found in Italy was also found in Tsukuba and Kanagawa in Japan, and in Switzerland (Fig. 12, Table 4). H1 is also the predominant haplotype found in the specimens collected in Japan (45%). Sam- ples from China were the most diverse, displaying 4 haplotypes (H4, H5, H6, H7), although they represent only 22% of the total sampling. From the network analysis, H7 from China and South Korea differed only by one substitution from the haplotype Two Asian egg parasitoids of Halyomorpha halys (Stal)... 49 ) 1 szaple Ttaly Japan China OCO@@O South_Korea Figure 11. COZ haplotype network of the Trissolcus mitsukurii analyzed in this study. Each circle cor- responds to one haplotype; circle size gives the proportion of individuals belonging to the haplotype. The color inside each circle represents the geographical origin. Numbers correspond to the haplotype num- bers. Hatch marks symbolize the number of mutations between haplotypes. He Ha H4 Italy Japan China South Korea Switzerland Figure 12. CO/ haplotype network of the Trissolcus japonicus analyzed in this study. Each circle corre- sponds to one haplotype; circle size gives the proportion of individuals belonging to the haplotype. The color inside each circle represents the geographical origin. Numbers correspond to the haplotype num- bers. Hatch marks symbolise the number of mutations between haplotypes. 50 Giuseppino Sabbatini Peverieri et al. / Journal of Hymenoptera Research 67: 37-53 (2018) H1 (Fig. 12). Although our haplotype analysis evidenced a best match of the Italian populations to Japanese and Swiss populations so far, we cannot entirely exclude that the haplotype H1 may also be present in unsampled populations of other parts of Asia where 7. japonicus is present. Tracing the source of an introduction depends on the availability of information about population structure and may require analysis of more than one locus. To this end, a more comprehensive phylogeography study is underway which includes the CO/ barcode and microsatellite loci recently developed de novo in 7! japonicus. Discussion Since the first detection of adventive 7’ japonicus in 2015, additional recoveries in the USA have shown that the adventive populations have established and are spreading. Given that 7’ japonicus occurs throughout the range of 1. Aalys in its native range of eastern Asia, one potential outcome of the discovery of 7’ japonicus in Italy is that it will also establish and spread wherever H. /alys has established in this region. Its recent discovery in the Ticino region of Switzerland (Stahl et al. 2018) lends support to this possibility. Trissolcus mitsukurii is widespread in Asia, and its distribution extends to the southern limit of eastern Australia johnson 1991). Barcode sequences of Australian specimens of 7! mitsukurii are not yet available and this region must be considered as a possible source of the Italian population. Of note, 7’ mitsukurii was introduced into Hawaii in 1966 as a biological control agent of Nezara viridula (L.) but apparently did not become established (Davis and Krauss 1967). This species has otherwise not been reported as an adventive parasitoid outside of its native range, but it is conceivable that its distribution will follow that of H. /alys, as has occurred with T’ japonicus. Contin- ued surveys throughout the region will be needed to document their establishment and dispersal, in addition to determining their impact on H. halys populations and their interaction with native natural enemies in the region. The phenomenon of parasitoids following in the footsteps of their invasive hosts has become a growing trend, particularly with species of Trissolcus Ashmead. In addi- tion to discoveries of 7! japonicus and T. mitsukurii in the invaded range of H. halys, an adventive population of V7’ hyalinipennis Rajmohana & Narendran was found in California, USA, parasitizing eggs of the invasive bagrada bug, Bagrada hilaris (Burmeister) (Ganjisaffar et al. 2018). In each of these cases, the discovery was made through the cooperative effort of scientists working in the disciplines of biological control, taxonomy, and biological diversity, highlighting the synergy and necessity of collaboration. Expansive ranges have recently been documented in platygastroid wasps that are not known to be of agricultural significance (Masner et al. 2009, Oliveira and Schoe- ninnger 2017, Popovici et al. 2018), but are charismatic and thus more easily recog- nized. As parasitoids are increasingly examined in the context of the world fauna, we expect to discover many more widespread species. Two Asian egg parasitoids of Halyomorpha halys (Stal)... 51 Acknowledgments We are grateful to Rosario Raso and Chiara Zampa for the assistance during field mon- itoring and egg mass sampling respectively in the sites of Codroipo and Cordenons and to Luca Tirinnanzi for assistance in the site of Lodi. We also thank Paride Dioli for the identification of the egg mass of the stink bug of the family Asopinae, Lucian Fusu (University of Iasi, Romania) for the identification of Anastatus bifasciatus, and Mircea Mitroiu (University of Iasi, Romania) for the ongoing analysis of the reared pteromalid specimens. This work was supported by the Florida Department of Agriculture and Consumer Services—Division of Plant Industry and the Italian Ministry of Agricul- tural Food and Forestry Policies (grant projects “Salvaolivi” DM 0033437 21/12/2017 and “Protezpiante’” DM 0034140 29/12/2017). Taxonomic support was funded in part by USDA/APHIS Farm Bill award #18-8130-0798-APHIS-IA to KAH/MCB/ EJT. USDA is an equal opportunity provider and employer. 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