A peer-reviewed open-access journal NeoBiota 93: 63-90 (2024) &) NeoBiota DOI: 10.3897/neobiota.93.121219 Advancing research on alien species and biological invasions Research Article Widespread establishment of adventive populations of Leptopilina japonica (Hymenoptera, Figitidae) in North America and development of a multiplex PCR assay to identify key parasitoids of Drosophila suzukii (Diptera, Drosophilidae) Tara D. Gariepy™®, Paul K. Abram2, Chris Adams?, Dylan Beal*®, Elizabeth Beers*®, Jonathan Beetle’, David Biddinger®, Gabrielle Brind’Amour’, Allison Bruin’, Matthew Buffington®®, Hannah Burrack?, Kent M. Daane”®, Kathleen Demchak"®, Phillip Fanning’?, Alexandra Gillett®©, Kelly Hamby’, Kim Hoelmer"™, Brian Hogg"®, Rufus Isaacs®®, Ben Johnson’ Jana C. Lee’®, Hannah K. Levensen’”®, Greg Loeb’, Angela Lovero’, Joshua M. Milnes"8, Kyoo R. Park’®, Patricia Prade2°, Karly Regan", Justin M. Renkema?'®, Cesar Rodriguez-Saona2™, Subin Neupane2®, Cera Jones”, Ashfaq Sial2?, Peter Smythman’, Amanda Stout", Steven Van Timmeren®®, Vaughn M. Walton’, Julianna K. Wilson®®, Xingeng Wang'*© Agriculture and Agri-Food Canada, London Research and Development Centre, 1391 Sandford Street, London, Ontario, Canada Agriculture and Agri-Food Canada, Agassiz Research and Development Centre, 6947 Highway 7, Agassiz, British Columbia, Canada Mid-Columbia Agricultural Research and Extension Center, 3005 Experiment Station Dr, Hood River, Oregon, USA Washington State University, Tree Fruit Research and Extension Center, 1100 N Western Ave, Wenatchee, Washington, USA NJ Department of Agriculture, Phillip Alampi Beneficial Insect Laboratory, 20 Cosey Road, West Trenton, New Jersey 08628, USA Penn State College of Agricultural Sciences, Department of Entomology, Fruit Research and Extension Center, 290 University Drive, Biglerville, Pennsylvania, USA Cornell University, Department of Entomology, Geneva, New York, USA Systematic Entomology Laboratory, ARS/USDA c/o Smithsonian Institution, National Museum of Natural History, Washington, DC, USA Department of Entomology, Michigan State University, East Lansing, MI 48824, USA Department of Environmental Science, Policy and Management, University of California, Berkeley, California, USA Penn State College of Agricultural Sciences, Department of Plant Science, 102 Tyson Building, University Park, Pennsylvania, USA 12 University of Maine, School of Biology and Ecology, 5722 Deering Hall, Orono, Maine, USA 13 University of Maryland, Department of Entomology, 4291 Fieldhouse Drive, College Park, Maryland, USA 14 USDA-ARS, Beneficial Insects Introduction Research Unit, 501 South Chapel Street, Newark, Delaware, USA 15 USDA-ARS Invasive Species and Pollinator Health, Albany, California, USA 16 USDA ARS Horticultural Crops Disease and Pest Management Research Unit, 3420 NW Orchard Ave, Corvallis, Oregon, USA 17 North Carolina State University, Department of Entomology and Plant Pathology, Campus Box 7613, Raleigh, North Carolina, USA 18 Washington State Department of Agriculture, Plant Protection Division, 21 North 1st Avenue, Suite 103, Yakima, Washington, USA 19 Oregon State University, Department of Horticulture, College of Agricultural Sciences, 2750 SW Campus Way, Corvallis, Oregon, USA 20 Rutgers, RE. Marucci Center, 125A Lake Oswego Drive, Chatsworth, New Jersey, USA 21 Agriculture and Agri-Food Canada, London Research and Development Centre, Vineland Campus, 4890 Victoria Ave N, Vineland Station, Ontario, Canada 22 University of Georgia, 463 Biological Sciences Building, 120 Cedar St., Athens, Georgia, USA Corresponding author: Tara D. Gariepy (tara.gariepy@agr.gc.ca) oO AN DTD oO FP WO YS — _=_ lm —_ oo OPEN @xc ESS Abstract In recent years, there has been an increase in the adventive establishment and spread of parasitoid Academic editor: Victoria Lantschner Received: 19 February 2024 Accepted: 5 April 2024 Published: 8 May 2024 wasps outside of their native range. However, lack of taxonomic tools can hinder the efficient screen- ing of field-collected samples to document the establishment and range expansion of parasitoids on continent-wide geographic scales. Here we report that Leptopilina japonica (Hymenoptera, Figitidae), a parasitoid of the globally invasive fruit pest Drosophila suzukii (Diptera, Drosophilidae), is now widespread in much of North America despite not having been intentionally introduced. Surveys Copyright: Copyright work is made by His Majesty or by an officer or servant of the Crown in the course of his duties. Canadian Province where it was not previously known to occur. In most surveys, L. japonica was the in 2022 using a variety of methods detected L. japonica in 10 of 11 surveyed USA States and one 63 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Citation: Gariepy TD, Abram PK, Adams C, Beal D, Beers E, Beetle J, Biddinger D, Brind'‘Amour G, Bruin A, Buffington M, Burrack H, Daane KM, Demchak K, Fanning P Gillett A, Hamby K, Hoelmer K, Hogg B, Isaacs R, Johnson B, Lee JC, Levensen HK, Loeb G, Lovero A, Milnes JM, Park KR, Prade P. Regan K, Renkema JM, Rodriguez-Saona C, Neupane S, Jones C, Sial A, Smythman P, Stout A, Van Timmeren S, Walton VM, Wilson JK, Wang X (2024) Widespread establishment of adventive populations of Leptopilina japonica (Hymenoptera, Figitidae) in North America and development of a multiplex PCR assay to identify key parasitoids of Drosophila suzukii (Diptera, Drosophilidae). NeoBiota 93: 63-90. https://doi.org/10.3897/ neobiota.93.121219 most common species of D. suzukii parasitoid found. The surveys also resulted in the detection of Ganaspis cf. brasiliensis (Hymenoptera, Figitidae), the recently-released biological control agent of D. suzukii, in six USA States where it had not previously been found. These new detections are likely a result of intentional biological control introductions rather than spread of adventive populations. A species-specific multiplex PCR assay was developed as a rapid, accurate and cost-effective method to distinguish L. japonica, G. cf. brasiliensis, the closely-related cosmopolitan parasitoid Leptopilina het- erotoma (Hymenoptera, Figitidae) and other native parasitoid species. This dataset and the associated molecular tools will facilitate future studies of the spread and ecological impacts of these introduced parasitoids on multiple continents. Key words: Adventive establishment, DNA barcoding, Drosophila suzukii, Ganaspis brasiliensis, Hymenoptera, molecular diagnostics, multiplex PCR, spotted-wing drosophila, unintentional bio- logical control Introduction The rate of global introductions of non-native insect species due to human activ- ities continues to accelerate (Seebens et al. 2017; Seebens et al. 2021). Many of these introductions result in widespread negative environmental, human health and economic impacts (Bellard et al. 2016; Diagne et al. 2021). A minority of these introductions may have some positive consequences or a mixture of positive and negative effects (Schlaepfer et al. 2011; Vimercati et al. 2020; Sax et al. 2022). The potential for desirable or mixed effects is particularly likely with uninten- tionally introduced parasitoids and predators of invasive insects (Roy et al. 2011; Weber et al. 2021). While it is increasingly recognised that unintentional introduc- tions of species occupying higher trophic levels are probably common (Weber et al. 2021), many are likely undetected and, as such, there are relatively few examples that have been well-documented on continental scales. Parasitoid wasps are one of the most biodiverse groups of animals on the planet (Forbes et al. 2018) and are the most common group of biological control agents used to target invasive insects (Heimpel and Mills 2017). Parasitoid wasps have frequently been intentionally introduced as biological control agents worldwide, successfully controlling numerous invasive pest species in forestry, agriculture, and conservation contexts (Mason 2021). Host range is the strongest filter used to select parasitoid species for modern importation biological control programmes. To minimise the potential for non-target ecological impacts, only natural enemies with relatively narrow host ranges are selected from the native range of an invasive pest (Bigler et al. 2006; Hoddle et al. 2021). However, parasitoids from the native range of an invasive pest, even those with broader host ranges that may be less suitable for biological control programmes, can occasionally establish themselves in regions previously invaded by their host. These unintentional introductions can occur outside the scope of the standard biological control regulatory process (e.g. Frewin (2010); Talamas et al. (2015); Mason et al. (2017); Peverieri et al. (2018); Weber et al. (2021)). There is growing interest in documenting the spread of unintentionally introduced parasitoids and assessing the balance of their positive and negative ecological and environmental impacts (Mason et al. 2017; Weber et al. 2021). One major challenge for documenting the spread of introduced parasitoids in their new geographic ranges is a taxonomic impediment. For many parasitoid NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 64 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America groups, the native versus non-native parasitoid fauna itself may not be entirely known and may be difficult to distinguish based on morphology. Further, it may be unfeasible or inefficient for the limited number of taxonomic experts on a giv- en group of parasitoids to provide identification services for numerous research teams conducting large-scale surveys. The use of molecular tools, including spe- cies-specific PCR primers, for parasitoid species identification can help alleviate this problem (Gariepy et al. 2005; Traugott et al. 2006; Gariepy et al. 2007, 2008; Rugman-Jones et al. 2011, 2020; Gariepy and Messing 2012; Shariff et al. 2014; Furlong 2015; Shimbori et al. 2023). However, their development requires DNA sequences that are tied to authoritatively identified specimens (Lue et al. 2021; Shimbori et al. 2023), which is often not the case, given that many of the world’s parasitoid fauna remain undescribed (Forbes et al. 2018). Fortunately, when an economically-important invasive pest is the focus of an importation biological control programme, the specimens and taxonomic knowledge may be available to facilitate the development of molecular diagnostic tools that can be used to docu- ment the spread and impact of unintentionally introduced parasitoids. These dynamics are currently playing out on a global scale for parasitoids of spotted-wing drosophila, Drosophila suzukii Matsumura (Diptera, Drosophilidae), an invasive vinegar fly that has become a significant pest in all major fruit produc- tion areas in the Americas, Europe and parts of Africa (Asplen et al. 2015; Tait et al. 2021). Although the first report of D. suzukii establishment outside of its native range was in Hawaii in 1980 (Kaneshiro 1983), subsequent spread was not report- ed until 2008 when D. suzukii was found in California (Hauser 2011). By 2011, populations of D. suzukii were well-established in fruit-growing areas in western and eastern North America (Tait et al. 2021), presumably as a result of movement and spread of already-established populations from the western US (Fraimout et al. 2017). Surveys for parasitoids soon after the invasion of North America and Europe did not detect any specialised parasitoids of D. suzukii (Lee et al. 2019; Wang et al. 2020). Foreign exploration for parasitoids of D. suzukii in its native range of Asia documented a total of 21 parasitoid species (summarised in Wang et al. 2020), with three dominant parasitoids attacking D. suzukii larvae: Asobara ja- ponica Belokobylskij (Hymenoptera, Braconidae), Ganaspis cf. brasiliensis Thering and Leptopilina japonica Novkovi¢ & Kimura (Hymenoptera, Figitidae) (Mitsui et al. 2007; Daane et al. 2016; Girod et al. 2018a; Giorgini et al. 2019). One strain of G. cf brasiliensis (called “G1”) was selected and approved for intentional biological control releases in 2021 in the USA and Italy because of its specificity to D. suzukii (Biondi et al. 2021; Daane et al. 2021; Lisi et al. 2021; Fellin et al. 2023). Another genetically distinct lineage of G. cf. brasiliensis (referred to as G3) co-occurs with G1 in East Asia; however, the two strains are reproductively incompatible (Seehausen et al. 2020) and will be described as two distinct species (M. Buffington, in prep). Ganaspis cf. brasiliensis G3 has a broader host range that includes other Drosophila species and was, therefore, not considered for intentional release (Girod et al. 2018a; Giorgini et al. 2019; Seehausen et al. 2020; Daane et al. 2021). Similarly, approval for the release of L. japonica and A. japonica was not pursued because their host range included several species of Drosophilidae other than D. suzukii (Girod et al. 2018b; Giorgini et al. 2019; Daane et al. 2021; See- hausen et al. 2022). However, just prior to intentional releases of G. cf. brasiliensis G1, field surveys in the Pacific Northwest of North America revealed the presence of adventive (pre- NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 65 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America sumed to be accidentally introduced) populations of L. japonica (as early as 2016) and G. cf. brasiliensis G1 (in 2019) in British Columbia, Canada (Abram et al. 2020). By 2020, it was clear that both species were well-established in British Co- lumbia, parasitising D. suzukii in diverse habitats (Abram et al. 2022a). Both par- asitoid species were subsequently found in Washington, USA in 2020 and 2021, respectively (Beers et al. 2022). Similarly, adventive populations of L. japonica were discovered in northern Italy in 2019 (Puppato et al. 2020) and Germany in 2021 (Martin et al. 2023). These observations suggest that L. japonica and G. cf. brasil- iensis may be rapidly spreading to areas outside their native range due to uninten- tional introductions. In North America, however, the distribution of adventive populations of these parasitoids outside of the Pacific Northwest was unknown. During 2022, multiple research groups independently initiated new surveys to document the parasitoid community of D. suzuwkii as part of the intentional release programme for G. cf. brasiliensis G1. However, given the large number of samples involved in these surveys, efhciently documenting the presence of L. japonica, in particular, was challenging and time-consuming because it is exceedingly difh- cult to distinguish from related native fauna, especially the cosmopolitan species L. heterotoma \hompson (Abram et al. 2020; Abram et al. 2022b). In fact, before having access to specimens of L. japonica from Asia, the leading taxonomic expert on Figitidae in North America misidentified the first L. japonica found in North America as L. heterotoma (Abram et al. 2020). In this study, we consolidated the findings of research groups surveying for par- asitoids of D. suzukii in 12 USA States and one Canadian Province. We used this combined dataset to assess how geographically widespread L. japonica and G. cf. brasiliensis G1 are in the surveyed areas of North America. In addition, to address the difficulty of parasitoid identification in these and future surveys, DNA bar- codes were generated from field-collected specimens to confirm the identity and to generate an authoritative DNA barcode library for those species likely encoun- tered in D. sugukii parasitoid surveys. Then, a single-step multiplex PCR assay was developed for G. cf. brasiliensis G1, L. japonica and L. heterotoma to facilitate screening of large numbers of field-collected samples for the presence of adventive and intentionally introduced parasitoid species. We anticipate that continent-wide survey data, combined with new molecular diagnostic tools, will help provide in- formation for future strategies to release and re-distribute D. suzukii parasitoids and will serve as a baseline for measuring the balance of positive and negative ecological effects of unintentionally introduced parasitoid species. Methods Geographic occurrence and species composition of parasitoids associated with Drosophila suzukii Surveys were initiated in 2022 using a variety of methodologies throughout west- ern and eastern regions in North America to characterise the community of para- sitoids associated with Drosophilidae, focusing on D. suzukii. In most locations, direct sampling of ripe fruits was the primary collection method in the USA (Cal- ifornia, Delaware, Maryland, Michigan, New Jersey, New York, North Carolina, Oregon, Pennsylvania, Washington) and Canada (Ontario); however, deployment of D. suzukii-baited sentinel fruits in the field was also used to detect the presence NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 66 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America of Asian parasitoids in Georgia, Maine, New Jersey, North Carolina and Oregon. In addition, opportunistic sampling of adult Figitidae retrieved as by-catch from a variety of other traps deployed for monitoring D. suzukii were collected where available (Ontario, North Carolina and Washington); however, these traps are not specific in terms of capturing Asian parasitoids of D. suzukii and are likely to also contain native or cosmopolitan figitids associated with other drosophilids. Fruit collections and deployment of fruit-baited sentinel traps in the USA took place in the vicinity of G. cf. brasiliensis G1 release sites in 2022. Ganaspis cf. brasiliensis G1 was not released in Ontario, Canada. Detailed methods for each collection type are listed below. For each collection event, GPS coordinates (approximated to preserve land-owner privacy) were recorded and associated with individual wasp specimens retrieved so that the distribution of Asian parasitoids of D. suzukii could be assessed (see Suppl. material 1 for collection information). It is important to note that the dataset presented here has been generated by several groups and there are some modest differences in methodology between groups; as such, our intent is to report on the presence or absence of Asian parasitoids of D. suzukii in surveyed locations, as opposed to reporting detailed datasets on temporal parasitism and parasitoid species composition. Direct sampling of fruit from the field Following the recommended methodology described in Abram et al. (2022b), par- asitoid adults were obtained through collection and rearing of fly puparia from ripe fruits from wild and cultivated hosts from one Canadian Province and 10 US States (Table 1). Sampling of fruits from host plants was performed at focal sites in most locations in 2022 at intervals of 7-14 days over a period of approximately 3-4 months, based on the temporal patterns of ripening fruits throughout the growing season. However, intensive sampling over a shorter period (e.g. 1 month) on the same host plant was performed at some locations. Collection information is provided in Suppl. material 3, including locations, dates and host plants sampled. The sampling effort (number of sites, sampling period) and number of specimens obtained from each State/Province varied (Tables 1, 2) and, as such, it was not pos- sible to quantitatively compare the prevalence of each species between locations; however, the data were analysed from a more descriptive standpoint. Fruit was incubated in ventilated plastic containers as described by Abram et al. (2022b) to allow development of drosophilid larvae and parasitoids. Briefly, collected fruits were placed in a plastic container (500-750 ml volume), typically on a raised metal grid made of wire or hardware cloth (with an appropriate-sized grid such that fruit do not fall through the grid, but fly larvae can easily drop out of the fruit into the bottom of the container). Absorbent paper or cotton pads were placed in the bottom of the container to absorb fruit juices as the fruits decayed and to provide a pupation substrate for larvae. Containers were closed with a venti- lated lid (ventilation hole was approx. 5—7 cm in diameter, covered with fine mesh cloth). Samples were stored in a growth chamber at 21—25 °C, 16:8 (L:D) light cycle and 50-55% relative humidity (RH) or kept under air-conditioned room temperatures (20—23 °C) with natural light accessible from the windows. Contain- ers were checked every 2—3 days and drosophilid puparia were collected and reared either in ventilated Petri-dishes or individual 1.5 ml centrifuge tubes. Emergence was monitored every 2—3 days and the number of parasitoid wasps was recorded. NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 67 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Table 1. Overview of parasitoid collections from direct sampling of fruit in North America in 2022, including host plant species from which parasitoids emerged. Country | Province / State #sites | #Parasitoids | Collection Period Host Plant(s) Canada Ontario 7 39 June — September Rubus occidentalis, Sambucus nigra, Cornus obliqua, Rhamnus cathartica USA California 2 34 June — November Rubus idaeus, Rubus ulmifolius Delaware 5) 328 June — October Rubus allegheniensis, Phytolacca americana, Elaeagnus umbellata, Persicaria perfoliata, Prunus serotina, Rubus phoenicolasius Maryland 3 1198 August — October Elaeagnus umbellata, Rubus spp., Rubus idaeus, Lonicera spp., Phytolacca americana Michigan 16 82 July — August Vaccinium corymbosum., Phytolacca americana, Rubus spp., Sambucus canadensis., Lonicera spp. New Jersey Z is July — September Gaylussacia spp., Vaccinium spp. New York 1 3 August Rubus idaeus North Carolina | 4 21 July — October Rubus idaeus, Rubus subgenus Rubus (blackberry), Phytolacca americana, Celastrus orbiculatus Oregon 6 252 July — October Rubus armeniacus; Rubus idaeus Pennsylvania 2 234° August — October Ribes rubrum, Rubus subgenus Rubus (blackberry), Lonicera maackii, Phytolacca americana, Rubus spp., Elaeagnus umbellata, Vaccinium corymbosum Washington 24 794 August Rubus armeniacus, Prunus avium " A total of 234 parasitoids were obtained; however, only 126 were provided for morphological or molecular identification. Table 2. Number of parasitoids collected and identified (based on morphology and/or molecular methods) from ripe fruit collections in North America in 2022. Lj = Leptopilina japonica, Gb = Ganaspis cf. brasiliensis G1, Ar = Asobara cf. rufescens, Pv = Pachycrepoideus vindemiae, Vd = Trichopria drosophilae, No 1D = unidentified. Parasitoid species identification Country Province/State Total # Parasitoids | # Barcoded / PCR Tj Gb Ar Pv Td No ID Canada Ontario 39 39 38 0 0 0 0 1 USA California’ 34 0 0 0 0 34 0 Delaware’ 328 26 260 64 4 0 0 0 Mariana? | 1198 96 1190 8 0 0 0 0 Michigan | 82 82 82 0 0 0 0 0 New Jersey 7 7 s) 2 0 0 0 2 New York 3 3 3 0 0 0 0 0 North Carolina 21 14 21 0 0 0 0 0 Oregon’ 252 45 233 5 0 14 0 0 Pennsylvania‘ 126% 116 110 16 0 0 0 0 Washington‘ 794 34 696 25, 3 0 0 0 Total 2884 462 2636 190 7 14 34 3 * States from which some specimens were identified morphologically, typically with a subsample of Figitidae identified using molecular approaches (DNA barcoding and/or multiplex PCR); ‘An additional 108 specimens were collected, but were not provided for morphological or molecular identification. Containers were also checked every 2—3 days for emergence of parasitoids that pupated within the fruit. Emerged wasps were placed directly into 95% ethanol or killed by freezing at -20 °C prior to placing in 95% ethanol. However, samples from Michigan were typically captured on sticky cards that were placed inside the container and these parasitoids were soaked in Histoclear (National Diagnostics, NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 68 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Atlanta, Georgia) to loosen the glue, carefully removed with a dissecting needle or forceps and then put into 95% ethanol. When many parasitoids were collected, subsamples were DNA barcoded (see Table 2) and the remaining wasps were iden- tified, based on morphological characteristics (as per Abram et al. (2022b)). Drosophila suzukii-baited sentinels The exact set-up of sentinel baits differed somewhat amongst the teams deploying the sentinels, but were based on the recommendations described by Abram et al. (2022b) and are summarised below. In general, 10 blueberries and 2.5 cm thick in-peel slices of banana were dipped in a 5% bleach solution, allowed to off-gas and then sprinkled with yeast to reduce mould growth in the field. The fruit was then exposed to D. suzukii adults for 48 hours to allow infestation to occur. Infest- ed fruit was placed in 250 ml plastic containers (with lids) with 16 holes (5 mm diameter) punched equally around the outside perimeter. The plastic container was placed in a Trécé Pherocon VI Delta Trap (Great Lakes IPM, Vestaburg, Michigan, USA) and suspended from trees along the edges of D. suzukii-susceptible crops, approximately 1.5 m above the ground. Sentinel traps were left in the field for 7 days before being returned to the lab. Fruit from inside the sentinel traps was transferred to 8 x 8 x 2 cm wire mesh berry holders (0.5 cm grid) and placed in a 500 ml plastic container on top of a folded piece of paper towel and dental wicking to absorb moisture as the fruit broke down. Once re-collected from the field, con- tents in the traps were incubated in the lab as described above for field-collected fruit. Emerged wasps were collected, recorded and preserved in 95% ethanol for molecular identification. An overview of sentinel trapping is presented in Table 3 and exposure information is provided in Suppl. material 1. Opportunistic sampling of by-catch from other trap types Apple cider vinegar (ACV) traps were deployed in non-crop habitats bordering commercial berry sites in three locations in south-western Ontario (Suppl. ma- terial 1). Homemade traps were constructed from 1 litre clear plastic containers (Richards Packaging, Mississauga, Ontario, Canada) and eight entry holes (1.8 cm diameter) were drilled around the upper portion of the jar, leaving an area without holes to facilitate emptying of the jar contents. Holes were screened with fibreglass drywall tape (2.5 x 2.5 mm openings) (Sheetrock, CGC Corp., Chicago, Illinois, USA) to allow drosophilid flies and parasitoids to enter, but to exclude larger in- sects. The bottom half of each jar was covered in red tape (Cantech Industries Inc., Johnson City, Tennessee, USA) to increase attraction and a red plastic plate (22 cm diameter) was affixed to the lid of the jar as a rain barrier and an eyelet screw was placed through the jar lid and plate to facilitate hanging using twist ties. Traps were hung from metal rods placed in the ground adjacent to berry crops. Approximate- ly 250 ml of an ACV-ethanol mixture was added (1 part 95% ethanol to 9 parts ACV). Trap contents were collected weekly from 8 July to 18 August 2022 and were visually inspected for the presence of adult figitids; specimens were retrieved and stored in 95% ethanol for molecular identification (Table 3). Wine-vinegar traps were deployed to monitor for D. suzukii in Washington, us- ing the PHEROCON SWD cup trap (Trécé Inc., Adair, Oklahoma, USA) baited with a wine-vinegar bait (Franzia Crisp White Wine and Western Family Apple NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 69 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Table 3. Parasitoids of Drosophilidae collected from apple cider vinegar (ACV) traps, Scentry traps, ACV/wine traps and Drosophila suzukii (Ds)-baited sentinel fruit. All parasitoid identifications were done by sequencing the COI DNA barcode region. Lj = Leptopilina ja- ponica, Gb = Ganaspis cf. brasiliensis G1, Lh = Leptopilina heterotoma, Ll = Leptopilina leipsi, Lm = Leptopilina maia, No 1D = unidentified. Country Province/State Canada USA Ontario Washington North Carolina Oregon New Jersey Maine Georgia Scentry traps ACV/Wine traps Ds-baited sentinels Ds-baited sentinels Collection type | # Parasitoids collected | Collection Period Parasitoid species composition (%) ACV traps 36 Jul-Aug Lj (100%) Scentry traps 15 May-Jul Lj (53%), LI (33%), Lh (7%), Lm (7%) 6 eat Lj (33%), Lh (33%), Gb (33%) 8 Jun—Sep Lj (50%), Lh (50%) Scentry traps 9 Aug—Nov Lj (33%), Lh (33%), Lm (33%)* 3 Jul No identification (failed to amplify and sequence) 3 Jun—Sep Lj (100%) Ds-baited sentinels ig Aug—Sep Lj (94%), Gb (6%) Ds-baited sentinels 94 Sep Lj (100%) Ds-baited sentinels 5 Sep-Oct Lj (67%), Gb (33%) * Percentages reflect three identified specimens. Six specimens did not yield DNA sequences or PCR results and could not be identified. Cider Vinegar, 50:50 mix, plus ~ 1 ml of Palmolive Pure and Clear Unscented dish soap per litre of wine/vinegar mix). Traps were placed in a wild host plant adjacent to cherry orchards and the contents were checked weekly throughout the growing season. Adult Figitidae found in the traps were retrieved and stored in 95% ethanol for subsequent identification using molecular techniques (Table 3). Scentry traps consisted of homemade jar traps (as described above for the ACV traps) baited with a commercial Scentry lure (Scentry Biologicals, Billings, Mon- tana, USA) and a drowning fluid (water, dish soap and sodium benzoate or a 50:50 mix of antifreeze and water). Traps were placed in commercial berry sites (Ontario and North Carolina) or urban parklands with susceptible host plants (Washington) and contents were collected every 4-16 days from 15 May—15 July 2022 (Ontario), 26 July—17 October 2022 (Washington) and from 25 August—3 November 2022 (North Carolina). Trap contents were visually inspected for the presence of adult Figitidae. Specimens were retrieved and stored in 95% ethanol for identification using molecular techniques (Table 3). Molecular identification tools for parasitoids of Drosophila suzukii DNA barcoding of specimens To confirm the identity of specimens collected in the surveys described above, DNA barcoding was implemented to screen Figitidae collected from ripe fruit, sentinel fruit and other baited traps (n = 653). However, when a large number of parasitoids (> 100) was recovered from a given location, morphological character- istics were used to identify most specimens (Abram et al. 2022b) and only subsam- ples were barcoded. In addition to field-collected samples, 64 identified specimens from laboratory colonies maintained at the quarantine facility of the United States Department of Agriculture (USDA) Beneficial Insects Introduction Research Unit (BIIRU; Newark, Delaware, USA) were barcoded to serve as reference sequences. This included: 15 G. cf. brasiliensis G1 (originating from Japan), 10 G. cf. brasil- iensis G3 (originating from China), 10 L. japonica (originating from China), 9 NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 70 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America L. japonica (originating from South Korea) and 20 A. japonica (originating from South Korea). All wasps were freshly killed in 95% ethanol, except L. japonica from China, which were stored dry after the colony had collapsed and only dead dry wasps were available for molecular analysis. Details of DNA extraction, ampli- fication and DNA sequencing methods are outlined in Suppl. material 2. Sequence data and trace files were uploaded to the Barcode of Life Datasystems (BOLD; www.boldsystems.org) in the Project “APSWD, Adventive populations of SWD parasitoids” (for field-collected specimens) and Project “VPDRS, Verified para- sitoids of Drosophila suzukii’ (for identified specimens from laboratory colonies); both of these databases are publicly available. DNA barcodes from field-collected samples were identified using the BOLD Identification Engine by searching the species-level DNA barcode records for a match. In addition, all material that was DNA-barcoded (653 field-collected specimens and 64 specimens obtained from BIIRU) have been deposited in the insect collection of the National Museum of Natural History (USNM; Smithsonian Institution, Washington, DC). Development of multiplex PCR primers To facilitate screening and identification of large numbers of samples for current and future collections, the DNA barcode dataset (generated from colony-reared and field-collected specimens) was used to develop PCR primers that can be used in multiplex to separate L. japonica, G. cf. brasiliensis G1 and L. heterotoma, with- out amplifying the other parasitoid species that may be encountered in similar habitats (e.g. ripe or rotting fruits, baited traps). The DNA barcode sequences that we generated for A. cf. rufescens Foerster (Hymenoptera: Braconidae), A. japonica, L. leipsi Lue, L. maia Lue, L. heterotoma, L. japonica, G. cf. brasiliensis G1 and G. cf. brasiliensis G3 were aligned in CODONCODE ALIGNER version 9.0.1 (Codon-Code Corporation, Centerville, Massachusetts, USA). Based on areas of sequence variation between the different species, a unique forward primer nested within the DNA barcode region was designed for L. heterotoma, L. japonica and G. cf. brasiliensis G1 that, when combined with the reverse primer HCO-2198 (Folmer et al. 1994), generates a unique fragment size diagnostic for each species (Table 4). Using all three forward primers and the reverse primer in a single mul- tiplex PCR reaction would allow simultaneous screening of a sample for multiple species to reduce the number of PCR reactions required to identify a sample. Once putative primers were located, they were imported into Primer3Plus (Untergrasser et al. 2012) to confirm their suitability (i.e. stability, melting temperature, poten- tial for hairpins, primer dimers) and determine the appropriate temperature for amplification of the desired PCR product. Each multiplex PCR reaction was performed in a 25 pl volume containing 0.125 pl of Taq Platinum, 2.5 pl of 10x PCR buffer, 1.25 pl of 50 mM MgCl, 0.125 ul of 10 uM dNTPs (Invitrogen, Carlsbad, California, USA), 0.25 pl of each 10 uM forward primer (Gb1F-353, LjF-46 and LhF-212, respectively), 0.5 ul of 10 uM reverse primer (HCO-2198), 2 ul UltraPure BSA (50 mg/ml; Invitrogen, Carlsbad, California, USA), 16.75 pl ddH20 and 1 ul of template DNA. The BSA was added to enhance the specificity and efficiency of the multi- plex assay to reduce non-specific binding, particularly for regions with moderate to high GC-rich sequences (Nagai et al. 1998; Markoulatos et al. 2002; Strien et al. 2013). Thermocycling conditions included initial denaturation at 94 °C NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 7A Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Table 4. Putative species-specific forward primers for Ganaspis cf. brasiliensis (G1; Gb1F-353), Leptopilina japonica (LjF-46) and Leptopilina heterotoma (LhF-212) and sequence length when used in combination with the universal reverse primer HCO-2198 (number in brackets refers to PCR product length when primer sequences are trimmed from both ends). PCR product length when used with HCO-2198 Species-specific Forward Primer Primer Sequence (5’-3’) Gb1F-353 CTAAATAAGTCCCACCCAGGAATC 332 bp (282 bp) LjF-46 TGGGTTAAGATTCCTTGTTCGTAC 639 bp (589 bp) LhF-212 CTTACAGTTCCTGATATAGCATTTCCA 473 bp (420 bp) for 1 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 40 s and 72 °C for 1 min and a final extension period of 5 min at 72 °C. PCR products were visualised with a QIAxcel Advanced automated capillary electrophoresis system (Qiagen, Hilden, Germany) using the DNA screening cartridge and method AL320. Results were scored with QIAXCEL SCREENGEL Software (version 1.2.0), samples with signal strength exceeding 0.1 relative fluorescent units were scored as positive and species identity was assigned, based on the PCR fragment size generated (332 bp for G. cf. brasiliensis G1, 639 bp for L. japonica and 473 bp for L. heterotoma). The specificity of the multiplex PCR was tested with DNA from five specimens of each of the following species (obtained from field collections and/or laboratory colonies): G. cf. brasiliensis G1; G. cf. brasiliensis G3; L. japonica (South Korea); L. japonica (China); L. heterotoma; L. leipsi; L. maia; A. japonica; and A. cf. rufescens. In addition, all samples that were DNA barcoded (n = 653) were screened using the multiplex PCR assay and the identity was compared to the DNA barcode re- sults to determine whether they were consistent. Results Geographic occurrence and species composition of parasitoids associated with Drosophila suzukii Direct sampling of fruit from the field Across a total of 70 sampling sites, 2884 parasitoids (including 2636 L. japonica and 190 G. cf. brasiliensis G1) were obtained for morphological and/or molec- ular identification from ripe fruit collections in the sampled locations in North America (10 US States and one Canadian Province) (Table 2). Asian parasitoids associated with D. suzukii were detected in all States/Provinces sampled, except California (Figs 1, 2, Table 2). Leptopilina japonica, which represented 91% of all parasitoids identified, was present in 10 of the 11 States/Provinces surveyed, where it was collected from 11 different host plant genera (Table 2; Suppl. ma- terial 2: table S2). Ganaspis cf. brasiliensis G1 represented 7% of all parasitoids identified and was found in six States from a total of six different host plant genera (Table 2; Suppl. material 2: table $2); all G. cf. brasiliensis G1 detections occurred following the intended release of this species in summer 2022 as a part of the biological control programme targeting D. suzukii in these States, except Washington, where it was already present prior to releases (Beers et al. 2022). NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 72 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America @ Collected from sentinel baits and traps @ Reared from fruit collections @ Absent from fruit collections Figure 1. Detections of Leptopilina japonica in Canada and the USA. Grey shading indicates the States (CA = Cal- ifornia, DE = Delaware, GA = Georgia, ME = Maine, MD = Maryland, MI = Michigan, NJ = New Jersey, NY = New York, NC = North Carolina, PA = Pennsylvania, OR = Oregon, WA = Washington) and Province (ON = Ontario) where sampling took place in the present study. Blue circles represent parasitoids obtained from sentinel baits and as by-catch in Drosophila suzukii traps, green circles represent parasitoids reared from ripe fruit collections and red circles show the absence of parasitoids from fruit collections. Note that adventive L. japonica was already previously reported from British Columbia (BC), Canada (Abram et al. 2020) and Washington, USA (Beers et al. 2022) and these previous finding were not included in the present study. Only three additional species of parasitoids were obtained in the fruit collections: A. cf. rufescens emerged from fruit collections in Delaware and Washington (rep- resenting 0.2% of the 2884 parasitoids collected from all sites), Pachycrepoideus vindemiae Rondani (Hymenoptera, Pteromalidae) emerged from fruit collections in Oregon (representing 0.5% of the 2884 parasitoids collected) and Trichopria drosophilae Perkins (Hymenoptera, Diapriidae) emerged from a single collection in California, representing 1.2% of the 2884 parasitoids collected (Table 2; Suppl. material 3). It is likely that the two pupal parasitoids, P vindemiae and T’ drosophi- lae, were present in other locations as well; however, this study was focused on figitid larval parasitoids. Drosophila suzukii-baited sentinels In total, 120 parasitoid specimens were obtained from baited sentinels deployed in Oregon, New Jersey, North Carolina, Maine and Georgia. Using a combination of DNA barcoding and multiplex PCR, 117 specimens were identified at the species NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 73 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America ® Collected from sentinel baits and traps @ Reared from fruit collections @ Absent from fruit collections Figure 2. Distribution of Ganaspis cf. brasiliensis G1 detections after intended releases in 2022 in the USA. Grey shading indicates the States (CA = California, DE = Delaware, GA = Georgia, ME = Maine, MD = Maryland, MI = Michigan, NJ = New Jersey, NY = New York, NC = North Carolina, PA = Pennsylvania, OR = Oregon, WA = Washington) and Province (ON = Ontario) where sampling took place in the present study. Blue circles represent parasitoids obtained from sentinel baits and as by-catch in Drosophila suzukii traps, green circles repre- sent parasitoids reared from ripe fruit collections and red circles show the absence of parasitoids from fruit col- lections. Note that adventive G. cf. brasiliensis was already previously reported from British Columbia, Canada (Abram et al. 2020) and Washington, USA (Beers et al. 2022) and these previous finding were not included in the present study. level; only three samples from North Carolina failed to amplify or sequence, which may have been due to delayed preservation following collection (Suppl. material 1). Two species were detected: L. japonica (98%) and G. cf. brasiliensis G1 (2%). Leptopilina japonica was found in all four States where parasitoids were identified, whereas G. cf. brasiliensis G1 was only detected in New Jersey (one individual, col- lected 03 August 2022) and Georgia (one individual, collected 31 October 2022) (Table 3, Fig. 2). These G. cf. brasiliensis G1 detections occurred after the intended biological control releases of this parasitoid species took place in New Jersey and Georgia in summer 2022. Opportunistic sampling of by-catch from other trap types Seventy-one Figitidae parasitoid specimens were retrieved as by-catch in traps from Ontario, Washington and North Carolina. As these are non-specific traps at- tracting a variety of Drosophilidae and their parasitoids, a more diverse parasitoid NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 74 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Number of specimens 600 900 400 300 200 100 community, represented by five species, was captured. Collectively, the majority were L. japonica (72%), followed by L. heterotoma (9.9%), L. leipsi (7%), L. maia (2.8%) and G. cf. brasiliensis G1 (2.8%); the remaining specimens (5.5%) were unidentified using molecular techniques (DNA barcoding and multiplex PCR), possibly due to DNA degradation caused by prolonged immersion in drowning fluids (e.g. vinegar) that can degrade the quality of DNA. ‘The parasitoid species composition from each State and Province sampled is shown in Table 3. All three jurisdictions were positive for the presence of adventive L. japonica, whereas G. cf. brasiliensis G1 was only detected in samples from Washington, which is consistent with the other types of collections. Molecular identification tools for parasitoids of Drosophila suzukii DNA barcoding of specimens A total of 653 field-collected parasitoid adults (obtained through direct sampling of fruits, sentinels and other traps) were obtained for DNA barcoding, of which 494 produced complete DNA barcode sequences that permitted identification (Fig. 3). Approximately 24% of the samples failed to produce high-quality DNA Species identification Other Figitidae Negative barcode multiplex Molecular identification method Figure 3. Identification of the same set of field-collected parasitoids (n = 653) using DNA barcoding and a mul- tiplex PCR assay for L. japonica (Lj), L. heterotoma (Lh) and G. cf. brasiliensis G1 (Gb). Ar = Asobara cf. rufescens, Ll = Leptopilina leipsi, Lm = Leptopilina maia, Pv = Pachycrepoideus vindemiae. Negative refers to samples which failed to yield a DNA barcode or a PCR product. NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 75 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America sequences, likely due to DNA degradation from delayed preservation or improp- er storage of specimens following emergence. Nonetheless, 76% of the samples were identified using DNA barcoding. The vast majority of parasitoids collected were the Asian parasitoids, L. japonica (65%) and G. cf. brasiliensis G1 (8%); the remaining specimens were infrequently collected and occupied 0.30—1.1% of the species composition (Fig. 3). Of the identified specimens obtained from USDA-ARS BIIRU laboratory col- onies, 53 of the 64 that were used as references [15 G. cf. brasiliensis G1, 10 G. cf. brasiliensis G3, 10 L. japonica (originating from China), 9 L. japonica (originating from South Korea) and 20 A. japonica (originating from South Korea)] yielded DNA barcode compliant sequences (GenBank Accession numbers: OR974845— OR974897). None of the L. japonica from China produced viable barcode se- quences (five contained stop codons and the other five produced poor quality se- quences). In contrast, all L. japonica from South Korea yielded DNA barcodes. Only one G. cf. brasiliensis G3 failed to amplify and sequence. The failure to se- quence from these few specimens is likely due to delayed preservation of wasps prior to DNA extraction. ‘This was the case for specimens of L. japonica from Chi- na, where wasps were not freshly killed, but were stored dry for some time before preservation in 95% ethanol. Development of multiplex PCR primers When used in multiplex, the primers retained their specificity and amplified the correct fragment size for the intended target species when challenged with DNA from G. cf. brasiliensis G1, G. cf. brasiliensis G3, L. japonica (China), L. japonica (South Korea), L. heterotoma, A. cf. rufescens, A. japonica, P vindemiae, L. maia and L. leipsi (Fig. 4). The G. cf. brasiliensis G1 primers only amplified the G1 strain of G. cf. brasiliensis and they did not amplify the G3 strain, which is consistent with how the primers were designed (i.e. to amplify specifically G1 and not G3). The L. japonica primers amplified all specimens of L. japonica from China and South Korea, even though specimens of L. japonica from China did not produce a viable barcode. This suggests that the specimens that failed to produce a barcode were incorrectly preserved, resulting in degraded DNA, which is also supported by a fainter PCR fragment visualised for these specimens (Fig. 4). Pachycrepoideus vindemiae, L. leipsi, L. maia, A. japonica and A. cf. rufescens were not amplified in the multiplex PCR. When applied to the 653 field-collected specimens, all identifications were con- sistent with the barcoding results and the majority of parasitoids were identified as L. japonica (87%) and G. cf. brasiliensis G1 (8%), with a minor contribution from L. heterotoma (1%) (Fig. 3; Suppl. material 1). Only 25 samples (4%) failed to produce a PCR product using the multiplex PCR protocol, thereby allowing identification of 96% of the samples (Fig. 3). Of the specimens that yielded a neg- ative PCR result, eleven also failed to produce a DNA barcode, indicating that the DNA was of insufficient quality for amplification. The remaining 14 samples that yielded a negative PCR result were identified by DNA barcoding as A. cf. rufescens (3), L. leipsi (5), L. maia (2), PR vindemiae (2) and unidentified Figitidae (2); thus, these samples were negative because they are not amongst the species targeted by the multiplex PCR assay. NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 TG Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America AO1 A02 A03 A04 AOS A06 AQ7 A08 AO9 Al0 All Al2 [bp] Gb-1 Gb-3 Ar [bp] 1500 — — 1500 1000 1000 499 00 = ———— ce 500 400 300 — — 300 200 — 200 100 — — 100 4 BOL BO2 BO3 B04 BO5 B06 BO? BO8 BO9 B10 B1l B12 [bp] Lj (China) Lj (Korea) Ar NEG [bp] 1500 — — 1500 1000 1000 4o9 500 — —800— 400 300 — — 300 200 200 100 — — 100 Col co2 c03 c04 cos C06 C07 C08 cog C10 cll C12 [bp] Lh Pv Aj [bp] 1500 — — 1500 1000 1000 400 5) OE ST — 500 400 300 — — 300 200 200 100 — mm DO1 Do2 DO3 DO4 DOS DO6 DO7 DOs DOS D10 D11 D12 [bp] Lm LI Pv [bp] 3000 ——$— 3000 1500 — — 1500 1000 1000 499 —300 — — 500 409 300 — — 300 200 ————— 200 100 — — 100 Figure 4. Multiplex PCR assay challenged with DNA from Ganaspis cf. brasiliensis G1 (Gb-1: AO01—A05) and G3 (Gb-3: A06—A10), Asobara cf. rufescens (Ar: A11, A12, B11), Leptopilina japonica (China: BO1—B05; South Korea: BO6-B10), Leptopilina heterotoma (Lh: C01—C05), Pachycrepoideus vindemiae (Pv: C06, C07, D11, D12), Asoba- ra japonica (Aj: CO8—C12), Leptopilina maia (Lm: D01—D05), Leptopilina leipsi (LI: DO6—D10) and a negative control (NEG) with no DNA. Alignment markers (in green) are shown at 15 bp and 3000 bp for all samples and positive PCR results are indicated by fragment sizes of 332 bp, 639 bp and 473 bp for Gb-1, Lj and Lh (respec- tively). Absence of a fragment between the 15 bp and 3000 bp alignment markers indicates a negative PCR result. NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 77 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Discussion The geographic range of L. japonica was historically restricted to Asia and although this species was not being considered as a candidate agent for intentional biological control introductions, this study reveals that it is now present throughout a large part of North America and appears to be the dominant parasitoid associated with invasive D. suzukii at this point in time. This represents one of few document- ed cases of what appears to be a rapid (< 10 years) continent-wide spread of an unintentionally introduced parasitoid wasp, with the most similar example being the ongoing spread of unintentionally introduced egg parasitoids of Halyomorpha halys Stal (Hemiptera, Pentatomidae) in North America and Europe (Talamas et al. 2015; Stahl et al. 2019a). This study reports a considerable North American range expansion for L. ja- ponica, with new detections of L. japonica in 10 USA States (Delaware, Georgia, Maine, Maryland, Michigan, New Jersey, New York, North Carolina, Oregon and Pennsylvania) and one Canadian Province (Ontario). We also confirmed its es- tablishment in Washington State, where it had already been reported (Beers et al. 2022) following its initial detection in adjacent British Columbia, Canada in 2016 (Abram et al. 2020). Only one USA State surveyed, California, did not detect any L. japonica; however, 2022 collections in this State were focused on a single location. Although the timeline of LZ. japonica’s unintentional introduction and spread in North America is somewhat uncertain, it seems most likely that it has occurred relatively recently, i.e. since the widespread establishment of D. suzukii (~ 2011). This is supported by both published (Thistlewood et al. 2013; Miller et al. 2015; Lue et al. 2016; Wang et al. 2016; Huang et al. 2023) and unpublished survey results (see Suppl. material 4) from several locations in North America that took place between 2011 and 2021 that did not recover any evidence of the pres- ence of L. japonica. The only prior reports of L. japonica were recent; in the Pacific Northwest by Abram et al. (2020) and Beers et al. (2022) and, in Oregon, where parasitoids recovered from an extensive fruit collection in September 2021 were later identified as L. japonica (Suppl. material 4). Similarly, in Europe, where there has been a considerable amount of field research on parasitoids of Drosophila spp. (Rossi Stacconi et al. 2013; Englert and Herz 2016; Mazzetto et al. 2016; Knoll et al. 2017; Kremmer et al. 2017; Shaw et al. 2023), it seems that L. japonica has only arrived since the introduction of D. suzukii (Puppato et al. 2020; Fellin et al. 2023; Martin et al. 2023). These observations support the “receptive bridgehead hypothesis” (Weber et al. 2021), in which unintentional introductions of biolog- ical control agents to new areas may be more likely following invasions of those areas by suitable hosts from their native range. As L. japonica has a host range that includes several native and cosmopolitan species of Drosophilidae other than D. suzukii, including the abundant and widespread Drosophila melanogaster Mei- gen (Girod et al. 2018b; Giorgini et al. 2019; Daane et al. 2021; Fellin et al. 2023), the L. japonica “invasion” has the potential to have both positive economic and environmental impacts [via suppression of D. suzukii populations (and/or other pestiferous Drosophila spp.)] and potential ecological harm (direct and indirect ef- fects of attacking native and cosmopolitan Drosophilidae) that should be evaluated in the coming years. Unintentionally introduced populations of G. cf. brasiliensis G1 (the more spe- cialised parasitoid of D. suzukii recently approved for intentional releases in North NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 78 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America America and Europe) do not appear to be nearly as widespread as L. japonica. However, it is important to note that this species was only recently released and is in the early stages of establishment in the locations surveyed in the present study. In Washington, populations of G. cf. brasiliensis were detected prior to intentional releases and the present study suggests additional spread of adventive populations to new locations within the state. However, the detection of G. cf. brasiliensis in the additional 6 US States (Delaware, Georgia, Maryland, New Jersey, Pennsylvania and Oregon) reported here only occurred in 2022, following intentional releases. There are records of G. cf. brasiliensis from tropical and sub-tropical regions of the Palearctic (Uganda), Nearctic (Mexico), Neotropical (Panama, Brazil) and Ocea- nia (Hawai'i) (Buffington and Forshage 2016), but these represent strains of this species other than G1 that are likely to be described as distinct species (M. Buffing- ton, in prep.). Given the absence of previous records of G. cf. brasiliensis outside of the Pacific Northwest and the very low abundance of G. cf. brasiliensis G1 relative to L. japonica outside of this area, we conclude that most of the G. cf. brasiliensis G1 identified in the present study represent detections of this parasitoid resulting from the intentional releases (and not adventive populations). Although it is possi- ble that the G. cf. brasiliensis G1 detections in Oregon were from adventive popu- lations (given the proximity to Washington where populations were present before intentional releases took place; Beers et al. (2022)), extensive releases of G. cf. brasiliensis G1 took place in Oregon in 2022, with the only detections occurring after release. As the DNA barcodes for both adventive and intentionally-released populations are identical (Abram et al. 2020; Lue et al. 2021), the methods used in the present study cannot distinguish between post-release detections and the spread of already-established adventive populations. Other methods and contin- ued surveys will be needed to tease apart the contribution of released and adventive populations of G. cf. brasiliensis G1 to its range expansion in North America in the coming years. The present study also yielded new information on the distribution of native and cosmopolitan parasitoids associated with Drosophilidae. Native and cosmo- politan Leptopilina species (L. maia, L. leipsi and L. heterotoma) were only found in the vinegar and Scentry traps and not in fruit collections or sentinel baits, as they are not known to parasitise D. suzukii in the field and these traps are not specific to D. suzukii. Leptopilina maia was identified from trap catches in North Carolina (USA) and Ontario (Canada). The occurrence of ZL. maia in North Carolina is consistent with the distribution of this species in eastern North America (Lue et al. 2016); however, this is the first time this species has been reported in Canada. Similarly, although known from New Hampshire, Illinois, Maryland and Virginia (Lue et al. 2016), this is the first report of L. /eipsi in Canada. Both species were re- cently described by Lue et al. (2016) and the present study provides updated distri- bution records. The records of the cosmopolitan species L. heterotoma from Ontar- io are also the first official records of this species in Canada, although re-inspection of historically collected specimens from British Columbia (reported erroneously in Thistlewood et al. (2013) as “Ganaspis sp.”) indicates that it has been present in Canada since at least 2011 (P.K. Abram and D.R. Gillespie, unpublished data). The amount of new information about figitid geographic distributions gathered by this single study highlights the paucity of data on this important group of parasit- oids and the uncertainty associated with inferring recent spread and establishment from survey data on understudied species groups. NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 79 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Although the routes of invasion have been reconstructed for the global move- ment and spread of D. suzukii (Fraimout et al. 2017), the pathways of adventive introduction of L. japonica and G. cf. brasiliensis G1 are currently unknown and it is not yet clear whether they represent a single introduction with subsequent spread of the same introduced population or whether multiple introduction events have occurred. Given the large geographic distance between the initial introduc- tions in the Pacific Northwest and the more recently-discovered establishments in eastern North America, it is possible that more than one introduction event took place and/or that a bridgehead event occurred, in which one introduced popula- tion served as the source for rapid dispersal and establishment of additional popu- lations in North America (Guillemaud et al. 2011). However, further population genetic analysis of native and introduced populations would be necessary to iden- tify the source population(s) and to trace the potential routes of entry and spread. Although the present study generated DNA barcode sequences for adventive pop- ulations in North America, sequences from additional genes and populations from across the native (and recently invaded) range could clarify source populations and infer pathways of entry. Further, additional genetic approaches may provide a higher resolution of population differences. For example, when COI barcode se- quences were not adequate to detect population-level differences, ddRADseq was used to clarify relationships amongst adventive populations of Trissolcus japonicus Ashmead (Hymenoptera, Scelionidae), a parasitoid of H. halys (Abram et al. 2023) and between different populations of D. suzukii in Canada (Nelson et al. 2023). Detection of introduced and adventive parasitoids in an invasive pest population is critical to document the establishment and spread of biological control agents, assess their effectiveness and evaluate their potential for long-term suppression of an invasive pest species (Gariepy et al. 2007; Furlong 2015; Lue et al. 2022). To do this, large numbers of samples may need to be collected and processed to de- tect parasitism and parasitoid species composition. The availability of a molecular diagnostic tool can facilitate the delivery of these data and provide information for subsequent pest management strategies, including continued release and redistri- bution of biological control agents (Gariepy et al. 2007, 2008). Monitoring efforts to detect new adventive populations and to assess the post-release establishment following intended releases can be facilitated using the multiplex PCR approach described here. Our method provides a rapid, accurate and cost-effective option for screening field-collected samples that does not require the additional expense of DNA sequencing. It also facilitates conclusive identification of species that would otherwise be difficult to separate, based solely on morphological features (e.g. L. ja- ponica versus L. heterotoma; G1 versus G3 G. cf. brasiliensis). This is an important consideration when screening large numbers of samples for these species, as the cost of identifying a sample through bidirectional sequencing is approximately tenfold more than identification using the multiplex PCR protocol described here. Fur- thermore, the species-specific primers more reliably provided species identifications than sequencing the entire barcode region (Fig. 3), likely because they generate a shorter fragment of DNA which can improve identification of samples that fail to sequence due to DNA degradation (Strutzenberger et al. 2012; Hebert et al. 2013; Mitchell 2015). This multiplex PCR may also be useful to detect ecological interac- tions that are difficult to address using conventional techniques, including poten- tial competitive interactions between parasitoids, detection of host-parasitoid asso- ciations and evaluation of the host range in the area of introduction (e.g. Gariepy NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 80 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America and Messing (2012); Gariepy et al. (2019); Lomeli-Flores et al. (2019); Stahl et al. (2019b); Hepler et al. (2020)). It is important to note that, in the current context of screening figitid parasitoids that emerge from D. suzukii in ripe and sentinel fruit and species attracted to baited traps, the multiplex PCR protocol is effective and accurate. However, it may be prudent to screen additional verified species of Figitidae with this multiplex PCR protocol to ensure specificity is retained if this protocol is used in a different context with a more diverse parasitoid community (for example, figitids associated with other drosophilid species or for use in other countries with a different parasitoid complex associated with D. suzukii). Conclusions Our findings raise several questions relevant to interpreting the recent trend of unintentionally introduced populations of parasitoid wasps being detected during the course of biological control programmes. For example, is the apparently more widespread establishment of L. japonica compared to G. cf. brasiliensis G1 due to the fact that it has been established in these locations for a longer period of time? Or is it due to its broader host range and/or a broader climatic tolerance? If so, does that mean that parasitoids that are less likely to be approved for intentional biological control releases are more likely to be unintentionally introduced and become established? If the routes of introduction needed to spread L. japonica to multiple areas of the world are present and this parasitoid can attack Drosophilidae species other than D. suzukii, why did it seemingly not establish outside of Asia before the D. suzukii invasion? If a broader host range makes a parasitoid species more likely to be unintentionally introduced to new areas, why is A. japonica, a very common parasitoid of D. suzukii in some areas of its native range and which attacks a wide range of Drosophilidae species (Daane et al. 2021), not yet present in North America or Europe? The relative contributions of several factors to unin- tentional parasitoid establishment (e.g. parasitoid host range; parasitoid prevalence in source areas; climatic tolerance; patterns of global trade driving pest and parasit- oid propagule pressure; host abundance in space and time) should be the focus of future work. Indeed, our study contributes to the increasing recognition that par- asitoid wasps are part of the rising number of non-native insect introductions and the potential for them to have widespread positive, negative and mixed ecological impacts is considerable. However, the factors that contribute to their global spread and what this means for biological control of invasive species and risks to native ecosystems, deserve more attention. Acknowledgements The authors would like to thank the following people for their assistance with sample collection and processing: L. Jocius, A. Weber, J. Collins, E. Desbois, F Drummond, A. Fischer, G. Wiggins, N. Flanagan, N. Oderkirk, C. Purser, C. Dial, N. Lasala, K. Beckwith, S. Hesler, O. McCall, Y. Grynyshyn, M. Freeman, E. Janasov, S. Harrington, I. McEvoy, K. Wallner, S. Olsen, T. Preston, K. Day, S. Ill, C. Lamberty, A. Young, V. Rogers, R. Saroka, M. Brubaker, K. Robel. M. Huiz- ingh, S. Snowden, G. Johnson, J. Klakulak, R. Chave, R. Turner, C. Molokwu, R. Bryan, A. Antelman, A. Kemmerer, K. Kettmann, S. Rivas, A. Yee, B. Diehl, J. Gray, J. Johnson, M. Smytheman, L. Smytheman and R. Czokajlo. We would NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 81 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America also like to thank the following people and organisations for facilitating the work: Maryland grower cooperators and University of Maryland farm staff (D. Price, B. Spielman and L. Simmons); TJ Hafner (Agricare), S. Robbins and D. Hinds-Cook (OSU Lewis Brown Farm); Ontario Ministry of Agriculture, Food, and Rural Af- fairs (OMAFRA) (H. Fraser, E. Pate) and the Berry Growers of Ontario (BGO), blueberry and cherry growers of Washington. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding Further, the authors would like to acknowledge funding from Agriculture and Agri-Food Cana- da (Project J-002839, Alternative Pest Management Strategies); USDA ARS (Areawide Program administered by Stephen Young, National Program Leader; Base funds USDA ARS CRIS 2072- 22000-044-00D; 8010-22000-033-00D); USDA Specialty Crop Research Initiative (SCRI) Award No. 2020-51181-32140, the USDA Crop Protection and Pest Management (CPPM) Award No. 2021-70006-35312; the New Jersey Blueberry and Cranberry Research Council; Michigan Blueber- try Commission; Project GREEEN; USDA APHIS AP20PPQ & T00C133; Oregon State USDA Block grant, Chlorpyrifos alternatives, Oregon Blueberry Commission; USDA National Institute of Food and Agriculture, Hatch project 1016563. The USDA is an equal opportunity provider and employer and does not endorse products mentioned in this publication. Author contributions Conceptualization: BH, PKA, JCL, KAH, PF, KD, EB, CRS, HKL, TDG, HB, XW, KMD, MLB, RI, VMW. Data curation: KH, HKL, SVT, TDG, BJ, BH, AAS, CA, JKW, AB. Formal analysis: JCL, TDG, XW, PKA. Funding acquisition: KAH, KMD, RI, TDG. Investigation: GB, DB, TDG, EB, JCL, BH, KMD, XW, GL, RI, PE AS, KH, BJ, HB, CRS, PP, AG, AL, JB, KD. Methodology: XW, GL, JCL, JKW, CA, KRP, MLB, AS, TDG, VMW, SVT, HB, JMMM, DB, HKL, KR, CRS, PP CJ, KD, SN, PS, DB, JMR, AB. Resources: VMW, TDG, BH, CRS. Validation: TDG. Visual- ization: XW, TDG, PKA. Writing - original draft: XW, PKA, TDG. Writing - review and editing: JCL, RI, EB, AAS, PE MLB, KMD, KAH, KH, VMW, BJ, CA, SVT, BH, TDG, PS, HKL, PKA, OW, DB, GL, IMR;-GRS, DB. Author ORCIDs Tara D. Gariepy © https://orcid.org/0000-0003-2533-3023 Dylan Beal © https://orcid.org/0000-0001-5965-9043 Elizabeth Beers © https://orcid.org/0000-0003-49 17-9518 Matthew Buffington ® https://orcid.org/0000-0003-1900-3861 Kathleen Demchak ® https://orcid.org/0009-0004-2143-1616 Alexandra Gillett ® https://orcid.org/0000-0003-4477-4270 Rufus Isaacs © https://orcid.org/0000-0001-7523-4643 Hannah K. Levensen ® https://orcid.org/0000-0002-1667-0127 Justin M. Renkema ® https://orcid.org/0000-0003-2165-660X Cesar Rodriguez-Saona © https://orcid.org/0000-0002-1030-2753 NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 82 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Subin Neupane ® https://orcid.org/0000-0000-0000-0000 Steven Van Timmeren © https://orcid.org/0000-0003-2555-4950 Julianna K. 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CSIRO Publishing, Clayton South, 110-140. Supplementary material 1 Collection information and molecular identification of parasitoids reared from field-collected ripe fruit (GPS coordinates for the general site vicinity were used to preserve privacy of landowners) Authors: ara D. Gariepy, Paul K. Abram, Chris Adams, Dylan Beal, Elizabeth Beers, Jonathan Beetle, David Biddinger, Gabrielle Brind'Amour, Allison Bruin, Matthew Buffington, Hannah Burrack, Kent M. Daane, Kathleen Demchak, Phillip Fanning, Alexandra Gillett, Kelly Hamby, Kim Hoe- Imer, Brian Hogg, Rufus Isaacs, Ben Johnson, Jana C. Lee, Hannah K. Levensen, Greg Loeb, Angela Lovero, Joshua M. Milnes, Kyoo R. Park, Patricia Prade, Karly Regan, Justin M. Renke- ma, Cesar Rodriguez-Saona, Subin Neupane, Cera Jones, Ashfaq Sial, Peter Smythman, Amanda Stout, Steven Van Timmeren, Vaughn M. Walton, Julianna K. Wilson, Xingeng Wang Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendata- commons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.93.121219.suppl1 Supplementary material 2 DNA barcoding of specimens Authors: ara D. Gariepy, Paul K. Abram, Chris Adams, Dylan Beal, Elizabeth Beers, Jonathan Beetle, David Biddinger, Gabrielle Brind'Amour, Allison Bruin, Matthew Buffington, Hannah Burrack, Kent M. Daane, Kathleen Demchak, Phillip Fanning, Alexandra Gillett, Kelly Hamby, Kim Hoe- Imer, Brian Hogg, Rufus Isaacs, Ben Johnson, Jana C. Lee, Hannah K. Levensen, Greg Loeb, Angela Lovero, Joshua M. Milnes, Kyoo R. Park, Patricia Prade, Karly Regan, Justin M. Renke- ma, Cesar Rodriguez-Saona, Subin Neupane, Cera Jones, Ashfaq Sial, Peter Smythman, Amanda Stout, Steven Van Timmeren, Vaughn M. Walton, Julianna K. Wilson, Xingeng Wang Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendata- commons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.93.121219.suppl2 NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 89 Tara D. Gariepy et al.: Adventive establishment of Leptopilina japonica in North America Supplementary material 3 All fruit samplings (GPS coordinates for the general site vicinity were used to preserve privacy of landowners) Authors: ara D. Gariepy, Paul K. Abram, Chris Adams, Dylan Beal, Elizabeth Beers, Jonathan Beetle, David Biddinger, Gabrielle Brind'Amour, Allison Bruin, Matthew Buffington, Hannah Burrack, Kent M. Daane, Kathleen Demchak, Phillip Fanning, Alexandra Gillett, Kelly Hamby, Kim Hoe- Imer, Brian Hogg, Rufus Isaacs, Ben Johnson, Jana C. Lee, Hannah K. Levensen, Greg Loeb, Angela Lovero, Joshua M. Milnes, Kyoo R. Park, Patricia Prade, Karly Regan, Justin M. Renke- ma, Cesar Rodriguez-Saona, Subin Neupane, Cera Jones, Ashfaq Sial, Peter Smythman, Amanda Stout, Steven Van Timmeren, Vaughn M. Walton, Julianna K. Wilson, Xingeng Wang Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendata- commons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.93.121219.suppl3 Supplementary material 4 Collection data Authors: ara D. Gariepy, Paul K. Abram, Chris Adams, Dylan Beal, Elizabeth Beers, Jonathan Beetle, David Biddinger, Gabrielle Brind'Amour, Allison Bruin, Matthew Buffington, Hannah Burrack, Kent M. Daane, Kathleen Demchak, Phillip Fanning, Alexandra Gillett, Kelly Hamby, Kim Hoe- Imer, Brian Hogg, Rufus Isaacs, Ben Johnson, Jana C. Lee, Hannah K. Levensen, Greg Loeb, Angela Lovero, Joshua M. Milnes, Kyoo R. Park, Patricia Prade, Karly Regan, Justin M. Renke- ma, Cesar Rodriguez-Saona, Subin Neupane, Cera Jones, Ashfaq Sial, Peter Smythman, Amanda Stout, Steven Van Timmeren, Vaughn M. Walton, Julianna K. Wilson, Xingeng Wang Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendata- commons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.93.121219.suppl4 NeoBiota 93: 63-90 (2024), DOI: 10.3897/neobiota.93.121219 90