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Research Article
 Gryon aetherium Talamas (Hymenoptera, Scelionidae): Parasitoid of Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae) Adventive in Chile
expand article infoN. Rocío Rojas-Gálvez, Elijah Talamas§, Marta V. Albornoz, M. Fernanda Flores|, Wilson Barros-Parada, Alexandre Bout
‡ Pontificia Universidad Católica de Valparaíso, Quillota, Chile
§ Florida Department of Agriculture and Consumer Services, Gainesville, FL, United States of America
| Agroadvance SpA. Camino a Melipillа, Peñaflor, Chile
¶ Université Côte d’Azur, Sophia-Antipolis, France
Open Access

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

Introduction

Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae), also known as bagrada bug, is now a significant pest in the Western hemisphere, having invaded the western United States (Palumbo et al. 2016), Mexico (Sánchez-Peña, 2014) and Chile (Faúndez et al. 2016). In Chile, B. hilaris has spread rapidly to the north and south of the Metropolitan region where it was initially detected (Faúndez 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 Valparaíso, 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 candidate agents for biocontrol against stink bugs in general, and B. hilaris 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 Valparaíso (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 ° C and 60 ± 10% RH. The container 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 µL of buffer using the DNA extraction kit LUCIGEN (MA150E, QuickExtract DNA Extraction Solution, Middleton, WI, USA), following company specifications. This method allows a non-destructive extraction 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 portion 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 l’agriculture, l’alimentation et l’environnement (INRAE), Sophia-Antipolis, France. Correction, annotation, and alignment were performed manually using BioEdit Geneious 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. ancinla 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) method (Saitou and Nei 1987) with bootstrap values based on 500 replications. Nucleotide 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 confirmation 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.

Results

Parasitoid rearing

Sixteen parasitoids emerged from the 20 eggs that were exposed to the initial two parasitoids detected in the B. hilaris 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 haplotype. Sequences of each specimen were deposited in GenBank (Table 1).

Table 1.

GenBank accession numbers and sample information for COI sequences of Gryon aetherium presented in this study.

Species Collection code Region, country Year of collection GPS Coordinates (DMS) Host species GenBank Accession Number
Gryon aetherium 43070_HCO Valparaiso, Chile 2021 32°52'58.19"S, 71°12'22.99"W Bagrada hilaris OK104071
43071_HCO Valparaiso, Chile 2021 32°52'58.19"S, 71°12'22.99"W Bagrada hilaris OK104072

The best matches (100% identity) in GenBank were with sequences of G. aetherium 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).

Figure 1. 

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.

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), including the absence of a mesopleural carina (Figure 2A), the acetabular carina and ventral mesopleural carina intersecting ventrally (Figure 2A) and the posterior margin of mesoscutellum extending posteriorly over the metascutellum and metanotal trough (Figure 2B).

Figure 2. 

Gryon aetherium, male, ISA30411 A head, mesosoma, metasoma, lateral view B head, mesosoma, metasoma, dorsolateral view C head, anterodorsal view.

Discussion

The phenomenon of adventive biological control agents seems to be increasing in frequency. 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 (Talamas 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), T. mitsukurii (Ashmead) in Europe (Sabbatini-Peverieri et al. 2018; Rot et al. 2021; Bout et al. 2021) and T. hyalinipennis 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. hilaris in North America. In North America, adventive 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 et al. 2019; Hogg et al. 2021). The taxonomy of G. aetherium has been challenging, but a recent treatment by Talamas et al. (2021) corrected prior misidentifications (as G. myrmecophilum (Ashmead) and G. gonikopalense Sharma) and provided morphological and molecular means to identify it. Our study used these data to identify parasitoids reared from the eggs of B. hilaris in the V region, Region of Valparaíso, Chile.

Detection of an adventive population of G. aetherium, a promising biological control agent of the invasive B. hilaris in Chile, is a great opportunity for the country to develop classical biological control programs against this pest. Indeed, B. hilaris displays a specific oviposition behavior among stink bugs by laying part of its eggs in isolation and underground. Nonetheless, G. aetherium can detect buried eggs and overcome the physical barrier constituted by the soil (Martel and Sforza 2021). Other parasitoids of B. hilaris eggs, such as T. hyalinipennis (Hymenoptera, Scelionidae), may not be capable of this (Tofangsazi et al. 2020).

Gryon aetherium was not recovered from sentinel eggs deployed near Centro Ceres. This may be the result of the low number of exposures in the 2020–2021 period (<100) and the normal low rate of parasitism typically observed with this methodology. We have no information yet on G. aetherium in Chile regarding its broader distribution, host range or host preferences. No stink bugs from the tribe Strachiini are recorded from Chile (Prado 2008; Faúndez and Carvajal 2011) except for the invasive B. hilaris. So, there are no species close to B. hilaris or Eurydema species in Chile which could appear as a logical alternative host. It remains unclear if G. aetherium arrived simultaneously with B. hilaris and spread with the stink bug or if it is the result of a separate introduction. The 100% match of COI with adventive populations in Mexico and the United States is noteworthy, and may indicate that the parasitoid arrived in these three places from the same place, perhaps via similar pathways. Additional studies on populations of G. aetherium throughout the world may yield insight into this matter and are ongoing. Future monitoring and study of G. aetherium in Chile is needed to guide efforts for inoculative or augmentation releases that have the potential to regulate B. hilaris both inside and outside of agricultural ecosystems.

Acknowledgments

This work was funded by ANID Regional/Centro Ceres/Projet R18F10004. Elijah Talamas was supported by the Florida Department of Agriculture and Consumer Services-Division of Plant Industry and a USDA-APHIS Farm Bill: Biological Control of Bagrada Bug. Additionally, we acknowledge the Plant-BioS Microscopy Facility of the Institute Sophia AgroBiotech UMR INRA 1355-CNRS7254 – Université Côte d’Azur for access to instruments and technical advice.

References

  • Abram PK, Talamas EJ, Acheampong S, Mason PG, Gariepy TD (2019) First detection of the samurai wasp, Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae), in Canada. Journal of Hymenoptera Research 68: 29–36. https://doi.org/10.3897/jhr.68.32203
  • Alaniz AJ, Vergara PM, Carvajal MA, Fierro A (2021) Recent rapid colonization of the invasive species Bagrada hilaris (Heteroptera: Pentatomidae) in the collapsed ecosystem Aculeo lake, Chile. International Journal of Pest Management. https://doi.org/10.1080/09670874.2021.1915512
  • Bout A, Tortorici F, Hamidi R, Warot S, Tavella L, Thomas M (2021) First Detection of the Adventive Egg Parasitoid of Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) Trissolcus mitsukurii (Ashmead) (Hymenoptera: Scelionidae) in France. Insects 12: e761. https://doi.org/10.3390/insects12090761
  • Dieckhoff C, Wenz S, Renninger M, ReiFsig A, Rauleder H, Zebitz CPW, Reetz J, Zimmermann O (2021) Add Germany to the List – Adventive Population of Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae) Emerges in Germany. Insects 12: e414. https://doi.org/10.3390/insects12050414
  • Faúndez EI, Lüer A, Cuevas ÁG, Rider DA, Valdebenito P (2016) First record of the painted bug Bagrada hilaris (Burmeister, 1835) (Heteroptera: Pentatomidae) in South America. Arquivos EntomolóXicos 16: 175–179.
  • Faúndez EI, Larrea-Meza S, Carvajal MA (2018) High, up and down: Updating the distribution of the painted bug Bagrada hilaris (Burmeister) (Heteroptera: Pentatomidae) in Chile. Review of Chilean Entomology 44: 257–261. https://doi.org/10.1093/jme/tjy091
  • Felipe-Victoriano M, Talamas EJ, Sánchez-Peña SR (2019) Scelionidae (Hymenoptera) parasitizing eggs of Bagrada hilaris (Hemiptera, Pentatomidae) in Mexico. In: Talamas E (Eds) Advances in the Systematics of Platygastroidea II. Journal of Hymenoptera Research 73: 143–152. https://doi.org/10.3897/jhr.73.36654
  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxydase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.
  • Ganjisaffar F, Talamas EJ, Bon MC, Gonzalez L, Brown BV, Perring TM (2018) Trissolcus hyalinipennis Rajmohana and Narendran (Hymenoptera, Scelionidae), a parasitoid of Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae), emerges in North America. Journal of Hymenoptera Research 65: 111–130. https://doi.org/10.3897/jhr.65.25620
  • Hogg BN, Hougardy E, Talamas E (2021) Adventive Gryon aetherium Talamas (Hymenoptera, Scelionidae) associated with eggs of Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae) in the USA. In: Lahey Z, Talamas E (Eds) Advances in the Systematics of Platygastroidea III. Journal of Hymenoptera Research 87: 481–492. https://doi.org/10.3897/jhr.87.73778
  • Kimura MA (1980) Simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111–120. https://doi.org/10.1007/bf01731581
  • Milnes JM, Wiman NG, Talamas EJ, Brunner JF, Hoelmer KA, Buffington ML, Beers EH (2016) Discovery of an Exotic Egg Parasitoid of the Brown Marmorated Stink Bug, Halyomorpha halys (Stål) in the Pacific Northwest. Proceedings of the Entomological Society of Washington 118: 466–470. https://doi.org/10.4289/0013-8797.118.3.466
  • Martel G, Sforza R (2021) Catch me if you can: novel foraging behavior of an egg parasitoid, Gryon gonikopalense, against the stink bug pest, Bagrada hilaris. Journal of Pest Science 94. https://doi.org/10.1007/s10340-020-01325-4
  • Prado CE (2008) Conocimiento actual de Hemiptera - Heteroptera de Chile con lista de especies. Boletín del Museo Nacional de Historia Natural, Chile, 57: 31–75.
  • Rot M, Maistrello L, Costi E, Bernardinelli I, Malossini G, Benvenuto L, Trdan S (2021) Native and Non-Native Egg Parasitoids Associated with Brown Marmorated Stink Bug (Halyomorpha halys [Stål, 1855]; Hemiptera: Pentatomidae) in Western Slovenia. Insects 12: e505. https://doi.org/10.3390/insects12060505
  • Sabbatini-Peverieri G, Talamas E, Bon MC, Marianelli L, Bernardinelli I, Malossini G, Benvenuto L, Roversi PF, Hoelmer K (2018) Two Asian egg parasitoids of Halyomorpha halys (Stål) (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
  • Sánchez-Peña S R (2014) First record in Mexico of the invasive stink bug Bagrada hilaris on cultivated crucifers in Saltillo. Southwestern Entomologist 39: 375–377. https://doi.org/10.3958/059.039.0219
  • SAG - Servicio Agrícola y Ganadero. (2017) a. Resolución N° 1.577/2017. Establece control obligatorio de la plaga Chinche pintada Bagrada hilaris (Burmeister, 1835) (Hemiptera: Pentatomidae).
  • SAG- Servicio Agrícola y Ganadero. (2017) b. Resolución N° 6.491/2017. Establece medidas fitosanitarias de emergencia para la plaga chinche pintado Bagrada hilaris (Burmeister,1835).
  • Stahl J, Tortorici F, Pontini M, Bon MC, Hoelmer K, Marazzi C, Haye T (2019) First discovery of adventive populations of Trissolcus japonicus in Europe. Journal of Pest Science 92: 371–379. https://doi.org/10.1007/s10340-018-1061-2
  • Talamas EJ, Bremer JS, Moore MR, Bon M-C, Lahey Z, Roberts CG, Combee LA, McGathey N, van Noort S, Timokhov AV, Hougardy E, Hogg B (2021) A maximalist approach to the systematics of a biological control agent: Gryon aetherium Talamas, sp. nov. (Hymenoptera, Scelionidae). In: Lahey Z, Talamas E (Eds) Advances in the Systematics of Platygastroidea III. Journal of Hymenoptera Research 87: 323–480. https://doi.org/10.3897/jhr.87.72842
  • Talamas EJ, Herlihy MV, Dieckhoff C, Hoelmer KA, Buffington M, Bon MC, Weber DC (2015) Trissolcus japonicus (Ashmead) (Hymenoptera, Scelionidae) emerges in North America. Journal of Hymenoptera Research 43: 119–128. https://doi.org/10.3897/JHR.43.4661
  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. https://doi.org/10.1093/molbev/mst197
  • Tofangsazi N, Hogg BN, Hougardy E, Stokes K, Pratt PD (2020) Host searching behavior of Gryon gonikopalense and Trissolcus hyalinipennis (Hymenoptera: Scelionidae), two candidate biological control agents for Bagrada hilaris (Hemiptera: Pentatomidae). Biological Control 151: e104397. https://doi.org/10.1016/j.biocontrol.2020.104397