JHR 73: 143-152 (2019) re JOURNAL OF | *0eerrtievedopenacoss ural doi: 10.3897/jhr.73.36654 RESEARCH ARTICLE (ME Hymenoptera http://jhr.pensoft.net The Inerational Society of ymenoptersts. RESEARCH Scelionidae (Hymenoptera) parasitizing eggs of Bagrada hilaris (Hemiptera, Pentatomidae) in Mexico Moisés Felipe-Victoriano', Elijah J. Talamas’, Sergio R. Sanchez-Pefa' | Departamento de Parasitologia Agricola, Universidad Auténoma Agraria Antonio Narro (UAAAN), Saltillo, Coahuila 25315, México 2 Florida State Collection of Arthropods, Division of Plant Industry, Florida De- partment of Agriculture and Consumer Services, Gainesville, Florida, USA Corresponding author: Sergio R. Sdnchez-Pena (sanchezcheco@gmail.com) Academic editor: J. Fernandez-Triana | Received 30 May 2019 | Accepted 30 July 2019 | Published 18 November 2019 http://zoobank.org/590CC4D6-EFFC-4F99-8365-08676A 181271 Citation: Felipe-Victoriano M, Talamas EJ, Sanchez-Pefia 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 Abstract The painted bug or bagrada bug, Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae), is a key pest of crops in the family Brassicaceae. In this work, three species of Scelionidae (Hymenoptera) are reported for the first time as parasitoids of painted bug eggs in Mexico, at Saltillo, state of Coahuila: Gryon myr- mecophilum (Ashmead), Telenomus podisi Ashmead and Trissolcus basalis (Wollaston). This is also the first report of a species of the widespread genus TJé/enomus as an egg parasitoid of B. hilaris outside of India. Total percent parasitism, high resolution images, and CO1 sequences are provided for each species. In the future, research in Mexico should be carried out on parasitoid species presented in this work to determine their potential as biological control agents and the feasibility of augmentative, classical or inoculative biocontrol strategies for integrated pest management. Keywords Heteroptera, stink bug, biological control, parasitoid Introduction Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae), known in Mexico with the com- mon names of bagrada bug or painted bug, is a key pest of cole crops (family Brassicaceae) originally distributed in Africa and Asia (Howard 1906; Ahuja et al. 2008; Kavita et al. Copyright Moisés Felipe-Victoriano 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. 144 Moisés Felipe-Victoriano et al. / Journal of Hymenoptera Research 73: 143-152 (2019) 2014). This pest first invaded California, USA, in 2008 (Palumbo et al. 2016) and in 2014 was detected in Saltillo, southeastern Coahuila, Mexico, causing economic damage in broccoli (Brassica oleracea L. var. italica), cabbage (B. oleracea var. capitata), and cauliflower (B. oleracea var. capitata) (Sanchez-Pefia 2014; Torres-Acosta and Sanchez-Pefia 2016). The family Scelionidae is a cosmopolitan group of parasitoids that attacks the eggs of a variety of arthropods, including Hemiptera. In the Old World, several authors have reported parasitoids of this family attacking painted bug eggs. In India, Gryon karnalense (Chacko and Katiyar) and Télenomus samueli Mani were reported from the eges of B. hilaris (as Bagrada cruciferarum Kirkaldy) (Chacko and Katiyar 1961; Mani and Sharma 1982). In Pakistan, sentinel eggs of B. hilaris placed in the field for four days and subsequently incubated in the laboratory yielded three species of hymenop- teran parasitoids: Trissolcus hyalinipennis Rajmohana & Narendran, Gryon gonikopal- ense Sharma and a species of Ooencyrtus Ashmead (Encyrtidae) (Mahmood et al. 2015; Sforza et al. 2019). In the USA, Ganjisaffar et al. (2018) reported Trissolcus basalis and Tr. hyalinipennis parasitizing painted bug eggs in California. The objective of this work is to determine the presence of parasitoids of painted bug through sentinel eggs in northwestern Mexico and facilitate future work in this line of research. Materials and methods Field site The work was carried out in the experimental fields of the Universidad Autonoma Agrar- ia Antonio Narro (UAAAN) in Saltillo, state of Coahuila, México (25°21'15.80"N, 101°2'17.98"W, 1746 meters above sea level. The specific irrigated field (0.07 hectares) was planted with an assortment of Brassicaceae cultivars in equal numbers of the fol- lowing plants: Arugula (Eruca vesicaria L. ssp. sativa), broccoli, cabbage, cauliflower, kohlrabi (Brassica napobrassica Miller. 1768), mustard (Sinapis alba L. 1753), radish (Raphanus sativus L. 1753) and turnip (Brassica rapa L. 1753 subsp. rapa). Detection of parasitoids of painted bug through sentinel eggs. Eggs were obtained by rearing field-collected mating pairs of painted bugs in the labora- tory. Eight mating pairs were placed in Petri dishes at a temperature of 26—28 °C with diffuse overhead daylight. After 12 hours in the laboratory, mating pairs in the Petri dish- es produced an average of 270 eggs (range of 85-580). The mating pairs were removed and the eggs (on the same uncovered Petri dish bottom they were laid on) were placed on the soil surface at a distance of approximately 5 cm from a broccoli stem. If it was neces- sary to handle the eggs, a soft number 2 brush (Pinceles Rex, Mexico City) was used. The sentinel egg tests were conducted monthly in the field from 25 November 2017 — 20 December 2018. The eggs were exposed 7—8 days in the field, and subse- quently incubated at 24—28 °C and a relative humidity of 60% in the laboratory until Parasitic wasps from painted bug eggs 145 the emergence of parasitoids. The wasps that emerged were placed in 96% ethanol until their subsequent identification. DNA analysis Specimens were softened in 70% ethanol for two hours, then DNA was extracted us- ing a DNeasy Blood and Tissue Kit (Qiagen). DNA extracts were quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific). At least 20 ng of genomic DNA was used per PCR. The 5’-CO1 barcode region was PCR-amplified using the primers LCO1490 and HCO2198 (Folmer et al. 1994). PCRs were performed at 25 ul volumes using HiFi HotStart DNA Polymerase (Kapa Biosystems). PCR thermo- cycle conditions were: 1) initial denaturing at 95 °C for 2:00 minutes followed by 32 cycles of steps 2-4, 2) 98 °C for 30 seconds, 3) 50 °C for 30 seconds, 4) 72 °C for 40 seconds, and 5) final extension at 72 °C for 7:00 minutes. PCR products were verified by gel electrophoresis and cleaned for sequencing with QIAquick Gel Extraction Kits (Qiagen). Purified PCR products were Sanger sequenced in both directions using Big- Dye Terminator v3.1 (Applied Biosystems) chemistry on a SeqStudio Genetic Ana- lyzer (Applied Biosystems). Sequence reads were trimmed and sequence contigs were assembled in Sequencher 5.4.6 (Gene Codes Corporation). CO1 barcodes generated during this study were deposited in GenBank. Accession numbers for these sequences are presented in Table 1. Morphological identification Specimens of G. myrmecophilum and Te. podisi were identified to species using the keys of Masner (1980) and Johnson (1984), respectively. Specimens of 77. basalis were identified using Talamas et al. (2015) and the description by Ganjisaffar et al. (2018) of morphological variation present in individuals that emerge from B. hilaris eggs. Stacks of photographs were taken with a Macropod imaging system and rendered using HeliconFocus. Specimen collection data and host associations are deposited in the Hymenoptera Online Database (htttp://hol.osu.edu). Voucher specimens for all scelionid species are deposited at the Florida State Collection of Arthropods (FSCA), Gainesville, Florida, and the Entomology collection, Universidad Autonoma Agraria Antonio Narro, Saltillo, Mexico. Table |. Accession numbers for specimens of Scelionidae used for DNA sequencing. Species Locality Collection Unit Identifier GenBank Accession Number Trissolcus basalis Saltillo, Mexico FSCA 00090267 MK720829 Telenomus podisi Saltillo, Mexico FSCA 00090266 MK720830 Gryon myrmecophilum Saltillo, Mexico FSCA 00090442 MK720831 Gryon myrmecophilum Saltillo, Mexico FSCA 00090443 MK720832 Gryon myrmecophilum Rutgers, NJ, USA FSCA 00090445 MK937524 146 Moisés Felipe-Victoriano et al. / Journal of Hymenoptera Research 73: 143-152 (2019) Results Scelionid wasps were detected only in November 2017 and June, July and August 2018. In a subsequent paper we will discuss in detail the phenology of the parasitoid complex on painted bug eggs at this location in Mexico. Trissolcus basalis (Wollaston) Figs 1-3 As reported by Ganjisaffar et al. (2018), specimens of 77. basalis that emerge from the eges of B. hilaris have reduced episternal foveae and fainter striation on T2 relative to specimens that emerge from larger stink bug eggs (Figs 2-3). BLAST comparison of the CO1 sequence from specimen FSCA 00090267 resulted in a 100% identity to a 77. basalis sequence in Genbank from the USA (MK188338.1), providing confirmation of the morphological identification. On the collection date of 25 November 2017, a total of 29 77 basalis were col- lected (this date was the only time 77 basalis emerged from sentinel eggs) and the percentage of parasitism was 12.4% (n= 234 eggs). Telenomus podisi (Ashmead) Figs 4—6 The small size of B. hilaris eggs does not influence the diagnostic morphology of 7e. podisi and no relevant differences were found between the specimens in this study and Te. podisi reared from other stink bug eggs. BLAST comparison of the CO1 sequence from specimen FSCA 00090266 resulted in 98.9% sequence identity with Te. podisi sequence KR870961.1 from Genbank. On the monthly collection dates between January-December 2018, a total of 51 Te. podisi was collected in the months of June and July, resulting in 9.2 and 9.9% of parasitism respectively (n= 532 eggs). Gryon myrmecophilum (Ashmead) Figs 7-11 Our identification of this species is based on the revision of the genus by Masner (1980) and the specimens in this study were compared to photographs of the holo- type specimen made available by Talamas et al. (2017). The systematics of Gryon is currently under revision by the second author. Preliminary analysis indicates that G. myrmecophilum belongs to a cosmopolitan cluster of similar species, some of which may have intercontinental distributions. A specimen of G. myrmecophilum from New Parasitic wasps from painted bug eggs 147 0.2 Figures 1-3. Trissolcus basalis female (FSCA 00090267) I head, mesosoma, metasoma, ventrolateral view 2 head, mesosoma, metasoma, lateral view 3 head, mesosoma, metasoma, dorsolateral view. Scale bars: in millimeters. Jersey (FSCA 00090445) was sequenced to provide a comparison with a specimen closer to the type locality (Washington, DC). Comparison of the sequences from Mex- ico and New Jersey showed 88% sequence identity, indicating that G. myrmecophilum 148 Moisés Felipe-Victoriano et al. / Journal of Hymenoptera Research 73: 143-152 (2019) Figures 4-6. Zélenomus podisi 4 female (FSCA 00033549) head, anterior view 5 female (FSCA 00090266) habitus, lateral view 6 female (FSCA 00033275) head, mesosoma, metasoma, dorsolateral view. Scale bars: in millimeters. exhibits a high degree of variability in this gene region, or it is possibly a cryptic species complex. On the monthly collection dates between January—December 2018, a total of 115 G. myrmecophilum were collected in the months of June, July and August, resulting in 3.0, 7.6 and 43.2% of parasitism respectively (n=786 eggs). Discussion and conclusion In 2017, only 77. basalis emerged from eggs, in the month of November (29 speci- mens), for 12.4% of parasitism. This wasp is a near-cosmopolitan parasitoid of stink bug eggs for which one widespread host is the southern green stink bug, Nezara vir- idula (L.) (Powell and Shepard 1982). During the 2018 monthly sampling dates, scelionid wasps were detected only in June—August. The percentages of egg parasitism by all Scelionidae in the months of June, July and August 2018 correspond to 12.2, 17.4 and 49.6% respectively (total of 166 wasp specimens). Out of the total monthly percent parasitism, Gryon myrmeco- philum contributed with 24.2, 43.5 and 100% in June, July and August, respectively (115 specimens total); and Te. podisi contributed with 75.8 and 56.5%, for the months Parasitic wasps from painted bug eggs 149 Figures 7-9. Gryon myrmecophilum T female (FSCA 00090446) head, anterior view 8 female (FSCA 00090447) habitus, lateral view 9 female (FSCA 00090446) head, mesosoma, metasoma, ventral view. Scale bars: in millimeters. of June and July, respectively (51 specimens total). Species of Telenomus have been reported as a parasitoids of other stink bugs, including Euschistus heros (F.), Halyomor- pha halys (Stal), Oebalus insularis Stal, Piezodorus guildinii (Westwood) and Tibraca limbativentris (Stal). In Pakistan, Mahmood et al. (2015) reported that 77. hyalinipennis and G. goniko- palense had a combined parasitism rate of 32-38%. This level is similar to the parasit- ism level obtained in our work. In California, USA, Ganjisaffar et al. (2018) reported that 77. basalis and Tr. hyalinipennis parasitized 4.0—20.0% of B. Ailaris sentinel eggs in January of 2018. We did not observe parasitism by scelionids in January, but it should be noted that our study includes only a small number of sampling dates. Additional sampling is required to describe the phenology of these wasps on Bagrada eggs. To our knowledge, the present work reports the highest percentage of field para- sitism of painted bug eggs in the USA and Mexico. It is also the first report of Ze. podisi parasitizing painted bug eggs. We continue studying the identity, distribution and population fluctuation of beneficial wasps associated with painted bug eggs at selected localities in Mexico. Future research should be carried out on these spe- cies, and possibly others that have yet to be detected, to determine their potential 150 Moisés Felipe-Victoriano et al. / Journal of Hymenoptera Research 73: 143-152 (2019) ee J ing A, = 7 a if Se Figures 10-11. Gryon myrmecophilum, female (FSCA 00090447) 10 head and mesosoma, dorsal view 1 1 habitus, dorsal view. Scale bars: in millimeters. as biological control agents. In particular, there is a need for critical comparative analysis of the different modalities of biological control (classical, augmentative or inoculative) that can utilize parasitic wasps in the integrated pest management of the painted bug in Mexico. Acknowledgments This work was supported by Consejo Nacional de Ciencia y Tecnologia (CONA- CYT) scholarship 300868 to MFV; Direccién de Investigacién, UAAAN, project Parasitic wasps from painted bug eggs 151 2109; USDA APHIS Farm Bill, Biological Control of Bagrada Bug, and the Florida Department of Agriculture and Consumer Services — Division of Plant Industry. We also thank Matthew Moore and Cheryl Roberts (FDACS/DPI) for performing the PCR and sequencing reactions, and to Paul Skelley, Zee Ahmed and Susan Halbert (FDACS/DPI) for reviewing earlier version of the manuscript. References Ahuja B, Kalyan R, Ahuja U, Singh S, Sundria M, Dhandapani A (2008) Integrated manage- ment strategy for painted bug, Bagrada hilaris (Burm.) inflicting injury at seedling stage of mustard (Brassica juncea) in arid western Rajasthan. Pesticide Research Journal 20: 48-51. Chacko MJ, Katiyar RN (1961) Hadrophanurus karnalensis sp. n. 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