JHR 87: 493-501 (2021) ge, JOURNAL OF eeert nnn doi: 10.3897/jhr.87.75363 RESEARCH ARTICLE ) I Tymenopter a 4 https://jhr.pensoft.net Thelnternaonl Sciey of Hymenopexriss, RESEARCH Gryon aetherium Talamas (Hymenoptera, Scelionidae): Parasitoid of Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae) Adventive in Chile N. Rocio Rojas-Galvez', Elijah Talamas’, Marta V. Albornoz!?, M. Fernanda Flores*, Wilson Barros-Parada!', Alexandre Bout? I Escuela de Agronomia, Facultad de Ciencias Agronémicas y de los Alimentos, Pontificia Universidad Catélica de Valparaiso, Quillota 2260000, Chile 2 Florida Department of Agriculture and Consumer Services, Gaines- ville, FL, USA 3 Centro Regional de Investigacién e Innovacién para la Sostenibilidad de la Agricultura y los Territorios Rurales, Centro Ceres, Pontificia Universidad Catélica de Valparaiso, Quillota 2260000, Chile 4 Agroadvance SpA. Camino a Melipilla 26200, Penaflor, Chile INRAE, UMR INRAE 1355 CNRS 7254, Institut Sophia Agrobiotech, Université Cote d'Azur, 400 route des Chappes, 06903 Sophia Antipolis, France Corresponding author: Alexandre Bout (alexandre.bout@inrae.fr) Academiceditor: Zachary Lahey | Received 16September2021 | Accepted 24 October2021 | Published23 December2021 Attp://zoobank. org/2B5 D983A-1764-4D2D-8 BBF-7C8BOED6BA72 Citation: Rojas-Galvez NR, Talamas E, Albornoz MV, Flores MF, Barros-Parada W, Bout A (2021) Gryon aetherium Talamas (Hymenoptera, Scelionidae): Parasitoid of Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae) Adventive in Chile. In: Lahey Z, Talamas E (Eds) Advances in the Systematics of Platygastroidea III. Journal of Hymenoptera Research 87: 493-501. https://doi.org/10.3897/jhr.87.75363 Abstract A parasitoid wasp, Gryon aetherium Talamas (Hymenoptera, Scelionidae), was reared from eggs of the invasive stink bug Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae) in Chile. The identification of G. aetherium, which is under study as a biological control agent, was made with morphological and molecular data in the context of a recent taxonomic treatment of this species. The presence of an adventive population of G. aetherium in South America has implications for biological control of B. hilaris in Chile, and other countries on the continent, where this stink bug may become a pest. Keywords biological control, egg parasitoid, invasive species, DNA barcoding, stink bug Copyright N. Rocio Rojas-Gdlvez 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. 494 N. Rocio Rojas-Galvez et al. / Journal of Hymenoptera Research 87: 493-501 (2021) Introduction Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae), also known as bagrada bug, is now a significant pest in the Western hemisphere, having invaded the western Unit- ed States (Palumbo et al. 2016), Mexico (SAnchez-Pefa, 2014) and Chile (Fatindez et al. 2016). In Chile, B. Ailaris has spread rapidly to the north and south of the Metro- politan region where it was initially detected (Fatiindez et al. 2018) and is associated with both brassicaceous crops and natural areas (Alaniz et al. 2021). Current control measures in Chile consist of reiterative applications of conventional insecticides which appear to be ineffective (SAG 2017a, b). Currently, there are no feasible options to control populations in urban or suburban settings, or in natural habitats that serve as refugia for B. hilaris. At present, Centro Ceres, a research institute in Valparaiso, Chile, is investigating alternative solutions to this pest through diversification of the vegetative components of the agroecosystem. By increasing the functional biodiversity and employing push-pull strategies, the aims are to decrease the density of B. hilaris and damage on crops, and to favor the presence of natural enemies. However, knowledge regarding indigenous can- didate agents for biocontrol against stink bugs in general, and B. Ailaris in particular, is poor in Chile. Due to a need for rearing facilities and COVID restrictions, exposure of sentinel eggs of B. hilaris has occurred only opportunistically, but our efforts to study B. hilaris have serendipitously provided a substantive result that we present here. Materials and methods Bagrada rearing Adults and nymphs of B. hilaris were collected in the field in October, 2020, in the region of Valparaiso (Region V) and used to establish a colony in the Centro Ceres laboratory. The colonies were maintained in 1.5 L glass containers covered by a fine mesh at approximately 25 °C during the day with heating at night, when necessary, to prevent the temperature from dropping below 12 °C. The containers were provided with cabbage and fresh water. The relative humidity was 60 + 10% with a L:D cycle of 16:8 h. Nymphs and adults were kept separately. Eggs were collected regularly and transferred to an independent container until the emergence of nymphs or use for exposure in the field to survey for parasitoids. Parasitoid rearing Two parasitoid wasps were found in one of the colony containers of B. hilaris, which we isolated and exposed to 20 freshly-laid B. hilaris eggs. The exposed eggs were kept separately in a Petri dish in an incubator at 25 + 3 ° Cand 60 = 10% RH. The con- First detection of Gryon aetherium, egg Parasitoid of Bagrada hilaris, in Chile 495 tainer where the parasitoids were initially detected was monitored continuously for the appearance of additional specimens. The specimens retrieved were placed in 96% alcohol for further study. DNA barcoding DNA extraction was performed with 30 uL of buffer using the DNA extraction kit LUCIGEN (MAI50E, QuickExtract DNA Extraction Solution, Middleton, WI, USA), following company specifications. This method allows a non-destructive extrac- tion of the DNA, so that the exoskeleton (voucher) remains intact for morphological identification. PCR amplifications were performed on a portion of the Cytochrome C Oxidase, subunit I (COI) locus using the LCO-HCO primers: HCO2198 (5'-TAAA CTT CAG GGT GAC CAA AAA ATC A-3'), LCO1490(5'-GGTC AAC AAA TCA TAA AGA TAT TGG-3') (Folmer et al. 1994), which amplifies a 600-700 bp por- tion of the COI locus. Amplicons were sent to Beckman Coulter Genomics Genewiz (Leipzig, Germany; Essex, UK) for bidirectional sequencing (Sanger method) using both primers. All residual DNA is archived at the Institut national de recherche pour agriculture, lalimentation et environnement (INRAE), Sophia-Antipolis, France. Correction, annotation, and alignment were performed manually using BioEdit Ge- neious R10 software. Sequences were compared to those available in the NCBI database (GenBank) using BLASTN (Altschul et al. 1990) with standard settings and to COI data made available in Talamas et al. (2021). Gryon prisma Mineo (Hymenoptera, Scelionidae) was selected as the closest outgroup to our specimens based on the phylogenetic analysis in Talamas et al. (2021). Three Hadronotus species i.e. H. drunoris, H. ancin- la and H. charon group sp. 2, were added to this analysis because they were originally described under the genus Gryon, until they were synonymized with Hadronotus by Talamas et al. (2021). Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae) |MN615650| was used as the outgroup to root the tree. These data were analyzed with MegaX software (Tamura et al. 2013) using the neighbour joining (NJ) meth- od (Saitou and Nei 1987) with bootstrap values based on 500 replications. Nucleo- tide distances in NJ trees were estimated by the Kimura’s two-parameters method (Kimura 1980). Morphological analysis Ethanol-stored and DNA-extracted specimens were dried and glued on card points for optical observation using a Zeiss Macroscope AxioZoom.v16. Morphological con- firmation of Gryon specimens was performed using the description and diagnosis of Gryon aetherium in Talamas et al. (2021). Voucher specimens are deposited at INRAE UMR 1355 ISA in Sophia-Antipolis, France. 496 N. Rocio Rojas-Galvez et al. / Journal of Hymenoptera Research 87: 493-501 (2021) Results Parasitoid rearing Sixteen parasitoids emerged from the 20 eggs that were exposed to the initial two para- sitoids detected in the B. /ilaris colony, yielding a parasitism rate of 80%. Monitoring of the B. hilaris colony yielded additional specimens: 11 parasitoids were collected 26 days after the initial detection and eight were collected at day 29. DNA barcoding Sequences from two specimens were 607 bp in length and correspond to one haplo- type. Sequences of each specimen were deposited in GenBank (Table 1). The best matches (100% identity) in GenBank were with sequences of G. aethe- rium from Mexico (MK720832.1 and MK720831.1, reported as G. myrmecophilum in Felipe-Victoriano et al. (2019)). Furthermore, they are identical to sequences of G. aetherium from California reported by Talamas et al. (2021) (Figure 1). Morphological analysis According to the diagnosis in Talamas et al. (2021), the specimens clearly belong to the genus Gryon Haliday (Hymenoptera, Scelionidae) based on the glabrous metapleuron (Figure 2A), striate axillula (Figure 2B) and frons without transverse sculpture (Figure 2C). They also match the diagnosis for G. aetherium provided in Talamas et al. (2021), in- cluding the absence of a mesopleural carina (Figure 2A), the acetabular carina and ventral mesopleural carina intersecting ventrally (Figure 2A) and the posterior margin of mesos- cutellum extending posteriorly over the metascutellum and metanotal trough (Figure 2B). Discussion The phenomenon of adventive biological control agents seems to be increasing in fre- quency. For scelionid parasitoids of stink bug eggs, surveys designed to determine their presence and efficacy are undoubtedly accelerating the rate of their detection. In the past decade, these include Trissolcus japonicus (Ashmead) from North America (Tala- mas et al. 2015; Milnes et al. 2016; Abram et al. 2019) and Europe (Stahl et al. 2019; Sabbatini-Peverieri et al. 2018; Dieckhoff et al. 2021), 7’ mitsukurii (Ashmead) in Europe (Sabbatini-Peverieri et al. 2018; Rot et al. 2021; Bout et al. 2021) and 7 hya- linipennis Rajmohana & Narendran from the USA (Ganjisaffar et al. 2018). Our analysis is facilitated by recent studies of the genus Gryon that were conducted to support biological control of B. Ailaris in North America. In North America, adven- tive populations of G. aetherium, a species under study as a biological control agent, have been reared from sentinel or naturally laid eggs of B. hilaris (Felipe-Victoriano First detection of Gryon aetherium, egg Parasitoid of Bagrada hilaris, in Chile 497 Table |. GenBank accession numbers and sample information for COI sequences of Gryon aetherium presented in this study. Species Collection Region, country Year of GPS Coordinates (DMS) Host species GenBank code collection Accession Number Gryon 43070_HCO Valparaiso, Chile 2021 32°52'58.19"S, 71°12'22.99"W — Bagrada hilaris OK104071 aetherium 43071_HCO_ Valparaiso, Chile 2021 32°52'58.19"S, 71°12'22.99"W Bagrada hilaris OK104072 Gryon aetherium MZ513478 FSCA 00091216 Pakistan Punjab Gryon aetherium OK104072 ISA 43071 Chile Region V Gryon aetherium OK104071 ISA 43070 Chile Region V 51 Gryon aetherium MK720832 FSCA 00090443 Mexico Coahuila 70] Gryon aetherium MK720831 FSCA 00090442 Mexico Coahuila Gryon aetherium MZ513479 FSCA 00091217 USA California Gryon aetherium MZ513477 FSCA 00091210 USA California Gryon aetherium MZ513476 FSCA 00033319 USA California Gryon prisma MZ513487 SA PL234 Hadronotus drunoris MT604063.1 FSCA 00094684 Hadronotus ancinila MT604059.1 FSCA 00094671 Hadronotus charon group sp. 2 MT604066.1 SAM-HYM-P093671 Trissolcus basalis MN615660 voucher USNM:ENT-01197237 Figure |. Molecular clustering of 13 sequences including the 2 sequences of Gryon aetherium from Chile. Sequences of G. aetherium from California, Mexico and Pakistan are from the Talamas et al. (2021) study. Gryon prisma Mineo, three Hadronotus species and Trissolcus basalis were selected as outgroups. Trissolcus basalis was used to root the tree based on the topology of Talamas et al. (2021). Numbers at nodes indicate bootstrap support values derived from 500 replicates. 498 N. Rocio Rojas-Galvez et al. / Journal of Hymenoptera Research 87: 493-501 (2021) ate |" * “h $944 Rebs. a