Telenomus Haliday (Hymenoptera, Scelionidae) parasitizing Pentatomidae (Hemiptera) in the Palearctic region

Scelionidae) parasitizing Pentatomidae

Telenomus is by far the largest genus in the subfamily and includes a considerable number of species that cannot be reliably identified.This taxonomic challenge has its roots in the diversity and size of the genus, and in what Meier et al. (2022) termed the "superficial description impediment".Descriptions for Palearctic taxa, particularly from the early years of European insect taxonomy, are woefully insufficient for species-level identification.Despite these inauspicious beginnings, there have been notable advancements in the classification of telenomine wasps.Kozlov (1967Kozlov ( , 1968)), Kozlov and Kononova (1983), and Kononova (2014) treated Palearctic species; Johnson (1981) dealt with Nearctic Telenomus with keys to identify the species of the nigricornis group (Johnson 1981), and podisi and phymatae groups (Johnson 1984b).After this, the world fauna was catalogued by Johnson (1992).The podisi species group, which parasitizes the eggs of Pentatomidae and Scutelleridae, was defined by Johnson (1984b) and includes the species treated here.
The taxonomy of Telenomus in the western Palearctic received little attention in the 21 st century until the arrival of the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera, Pentatomidae).The pestiferous nature of this stink bug and the potential risk of dispersal to other countries (Zhu et al. 2012) has led several institutions to deepen the knowledge on biological control strategies (Gariepy et al. 2014b;Maistrello et al. 2017;Bosco et al. 2018;Leskey and Nielsen 2018;Moore et al. 2019).In Integrated Pest Management (IPM) programs, much attention has been given to the ability of BCAs to counter the pest population, and, in the specific case of stink bugs, first to native and then exotic egg parasitoids.
Trissolcus japonicus (Ashmead) and Tr.mitsukurii (Ashmead) (Hymenoptera, Scelionidae) have been shown in Europe to be the most promising BCAs of H. halys in terms of habitat suitability (Yonow et al. 2021;Tortorici et al. 2023), exploitation efficiency, and parasitoid impact (Giovannini et al. 2022).These species were quickly recognized because the Palearctic fauna of Trissolcus has been well-characterized using morphology, molecular data, and mating experiments to resolve cryptic species (Talamas et al. 2017;Tortorici et al. 2019;Ranjbar et al. 2021).However, the same is not true for Telenomus, despite that some species are widespread and are known to attack stink bug eggs.
In Europe, some authors have reported Te. chloropus (Thomson) from their surveys (Haye et al. 2015;Roversi et al. 2017) and numerous studies of biological attributes have also used this name (Orr et al. 1985a, b;Orr 1988;Bulezza 1996;Açikgöz and Gözüaçik 2021), but most Telenomus species reared from large-scale surveys of stink bug egg parasitism have been indicated as "Telenomus spp." (Abram et al. 2017;Moraglio et al. 2020;Bout et al. 2021;Rot et al. 2021;Zapponi et al. 2021;Ozdemir et al. 2022;Ricupero et al. 2022).This reflects the challenge of species-level identification for Telenomus and clearly points to the need for better diagnostic tools.Here, we make progress in meeting this need by providing taxonomic treatments of two Palearctic species in the Te.podisi group, Te. truncatus (Nees von Esenbeck) and Te.turesis Walker, which attack stink bugs in the families Pentatomidae and Scutelleridae (Javahery 1968;Voegelé 1969;Kozlov and Kononova 1983;Johnson 1984b;Graham 1988a) and an overview of Telenomus species found to parasitize eggs of pentatomids and scutellerids in Europe.Telenomus gifuensis Ashmead has been reported as a parasitoid of pentatomids in the eastern Palearctic region.To our knowledge, this species has not been reported from the western Palearctic, but we included it in our identification key because the limits of its distribution are not known and there may be regions where it overlaps with the distributions of Te. truncatus and Te.turesis.
Our work includes examination of historical type specimens, some of which are nearly 200 years old, which is essential for resolving long-standing ambiguity.An updated morphological diagnosis section provides previously unexplored or unused character systems, and we provide simplified descriptions that focus on diagnostic characters.As other Old-World species of the Te.podisi group are treated taxonomically, these descriptions are likely to expand to include characters used for the species group more broadly.We complement our analysis with molecular data that is helpful for establishing which characters are prone to interspecific variation and which are diagnostically stable.For the analyzed species, we provide host associations and biological observations.

Reared specimens
Telenomus specimens were reared from naturally laid egg masses (Pentatomidae and Scutelleridae) collected in different sites in Piedmont, Italy, from 2019 to 2022 during surveys to investigate the egg parasitoid populations of native and exotic bugs.Each egg mass was isolated in a plastic Petri dish (6 cm diameter) and reared in a climate-controlled chamber at 24 ± 1 °C, 65 ± 5% r.h., and L16:D8 photoperiod.All egg masses were examined under a stereomicroscope and identified to the species or family level according to Derjanschi and Péricart (2005), Péricart (2010), Ribes and Pagola-Carte (2013).The eggs were visually inspected daily and emerging bug nymphs or parasitoid adults were examined.Parasitoids were stored in 99% ethanol until species identification, as described below.Additional specimens of Telenomus were collected on November 26, 2022, in Liguria, Italy, hidden in leaf mines of Phyllonorycter viburni (Kumata) (Lepidoptera, Gracillariidae).
Telenomus specimens were also reared from egg masses of Palomena prasina (Linnaeus) (Hemiptera, Pentatomidae) or collected by sweeping in their natural habitats in Moscow Province, Russia, in 2016 for a cytogenetic study (Gokhman and Timokhov 2020).Each egg mass was isolated in plastic tubes (5 cm 3 ) and reared in a thermostatic chamber at 24 ± 1 °C.Female parasitoids (both Te. truncatus and Te.turesis) were then individually transferred to egg masses of a lab host, Graphosoma lineatum Linnaeus (Hemiptera, Pentatomidae), for oviposition.To obtain a proper immature stage of wasps for the cytogenetic study, parasitized host eggs were incubated under thermostatic conditions for a few days (Gokhman and Timokhov 2020).

Morphological analysis
A Wild M5 steromicroscope with 15× oculars and a spotlight were used for biometric diagnosis.Slides were mounted with Eukitt mounting medium (Merck Life Science, Milan, Italy) and examined under a Leitz Dialux 20 EB compound microscope.Male genitalia were prepared by following the protocol of Polaszek and Kimani (1990).Terminology for surface sculpture and morphological terminology follows Harris (1979), Johnson (1984b), Mikó et al. (2007), and Talamas et al. (2017).The morphological identification was performed independently from keys and once the morphometric analysis of characters was complete and confirmed by molecular analysis, species names were assigned by comparison to primary types.

Imaging
Images of primary type specimens were taken with a Macropod imaging system using 10× and 20× Mitutoyo objective lenses (Mitutoyo Corporation, Kawasaki, Japan) and rendered with Helicon Focus (HeliconSoft Limit., Kharkiv, Ukraine).Photographs of non-type specimens were taken using a Canon 90D camera (Canon Inc., Tokyo, Japan) equipped with an extension tube; 5×, 10×, 20×, and 50× LWD microscope lenses mounted on a macro-rail and illuminated with two speedlight flashes.The frames were merged with Zerene Stacker (PMax algorithm, Zerene Systems LLC, Richland, WA, USA).
The ultrastructures of non-type specimens were examined under a Jeol JSM-6380 scanning electron microscope (SEM) after critical point drying (Hitachi HCP-2) of the specimens and sputter coating with gold (Giko JSM-6380).

Molecular analysis
DNA extraction, amplification, and sequencing were performed at multiple institutions.At the Florida State Collection of Arthropods (FSCA) and the European Biological Control Laboratory (EBCL), this was performed as in Talamas et al. (2021).Cytochrome Oxidase subunit I (COI) sequences from French specimens (INRAE UMR ISA) were obtained as in Bout et al. (2021).At the Dipartimento di Scienze Agrarie, Forestali e Alimentari laboratory (DISAFA) of the University of Torino, a non-destructive Chelex DNA extraction method was performed and adapted according to Kaartinen et al. (2010).DNA was extracted from insects by dipping samples in 50 µl of 5% Chelex with 5 µl of 20 mg/ml proteinase K at 37 °C for at least 18 h.The specimens were boiled at 95 °C for 5 min to inactivate proteinase K and then used as templates for PCR.The insects were then removed from the Chelex, washed in 70% ethanol and later mounted on card points.The barcode region of the mitochondrial COI was amplified using the universal Folmer primer LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') (Folmer et al. 1994) and the primer HCOout (5'-CCAGGTAAAATTAAAATATAAACTTC-3') (Carpenter 1999).PCR amplifications were performed on a C1000 Touch™ Thermal Cycler (Bio-Rad, CA, USA) in 25 µl volume containing: 2.5 µl of 10 X Buffer and 10 mM dNTPs, 1.25 µl of MgCl 2 , 0.3 µl of Taq Polymerase, 0.1 µl of 100 µM forward and reverse primer, 16.25 µl of sterile water, and 2 µl of DNA template.Thermocycling conditions were: 95 °C for 15 min, followed by 34 cycles of 95 °C for 30 s, 50 °C for 45 s, and 72 °C for 1 min.After a final extension at 72 °C for 5 min, reactions were held at 4 °C.For the nested PCR, 2 µl of the first PCR was used a template using the reverse primer HCO2189 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') and the forward primer LCO1490, using the same PCR cycling program described above.The fragment size at the end of nested PCR was 700 bp.PCR products were examined by electrophoresis on a 1% agarose gel.Positive samples were purified using a commercial kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), and sequenced by a commercial service (Eurofins Genomics, Germany).
The sequences were compared with the GenBank database using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLASTn).All sequences obtained from this study are deposited in GenBank or BOLD (Ratnasingham and Hebert 2007), and all residual DNAs are achieved at DISAFA, FSCA, INRAE UMR ISA or EBCL.Sequences were used to query GenBank (Altschul et al. 1990) and BOLD for similar sequences, which were downloaded from both databases.The COI barcodes of Trissolcus belenus (Walker) (MN603806) and of Tr. semistriatus (Nees von Esenbeck) (MN603800) (Tortorici et al. 2019) were selected as outgroups for the Maximum Likelihood analysis.All sequences were aligned using MUSCLE with default setting as implemented in MegaX (Kumar et al. 2018), and a phylogenetic tree was created by using the Maximum Likelihood method and Tamura-Nei model (Tamura and Nei 1993).Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value.The resulting phylogenetic tree was exported and redrawn in the Interactive Tree of Life (iTOL) v5 (Letunic and Bork 2021).
Geographic records of specimens used for molecular and morphological analysis were retrieved from GPS latitude and longitude coordinates and from available data on the GenBank and BOLD dataset as obtained above.A distribution map was created using QGIS.org(2023).The data for the examined specimens were uploaded onto the BOLD platform (www.barcodinglife.org),and the list of material examined was generated as a supplementary spreadsheet file (Suppl.material 4).

Taxonomy
Two species were detected in our surveys, Te. truncatus and Te.turesis, which we identified by comparison to type material (Table 1).Doğanlar (2001) simply described morphological characters of Te. chloropus.According to Kozlov and Kononova (1983), the sculpture of the posterior margin of mesoscutum was not clear, and this character is considerably variable in the specimens examined by us.The color of the femora weakly matches with the types of both species, but this is difficult to assess because the types are very old.The description of the shape of A2-A4 of males of both species corresponds with our opinion.The most accurate description for the two species was made by Javahery (1968).According to Graham (1988a), the most detailed description of Te. chloropus was provided by Johnson (1984).Graham, referring to the postocellar furrows behind the lateral ocelli, reported by Johnson (1984), wrote that there are at least two "European forms" of this character: the form, described as Te.sokolovi by Javahery (1968) with short, weak postocellar furrows extending inward behind the lateral ocelli, and the form erroneously described as Te.truncatus Nees by Javahery (1968) with long, marked postocellar furrows extending inward behind the lateral ocelli.Graham associated the character of eyes densely covered with moderately long hairs and eyes sparsely covered with short hairs, with the two forms, respectively.The two forms described by Graham concur respectively with our concepts of Te. turesis and Te.truncatus.
The present study reports a similar composition of hosts for Te.truncatus and Te.turesis as reported by previous authors (Javahery 1968;Kozlov 1968;Samin et al. 2010) but with some new records (Table 2).Some other dubious records from Coleoptera eggs are reported for both species (Kieffer 1926;Samin et al. 2010).

Diagnosis
We identified specimens of Te. truncatus and Te.turesis based on characters in the following species treatments:
From the analysis of the lectotype of Te. heydeni (NHMW-HYM#0005387), the combination of morphological characters (Fig. 4) coincides with the characters of the lectotype of Te. truncatus (OXUM 0011), and the length ratio between the A3 and A2 antennomeres (Fig. 4A) matches with that of the male of Te. truncatus (Fig. 3C).Therefore, Te. heydeni is here considered a junior synonym of Te. truncatus.

Biological information.
Host species associated: Table 2.The specimen DISAFA-FT HYM-0657 -OQ466097 was found overwintering in November in Viburnum leaf mines created by P. viburni; the specimens AVT001908 and AVT001909 were found already dead in egg-mass of Lymantria monacha (Linnaeus) (Lepidoptera, Erebidae), presumably after wintering.
DNA barcoding.Barcode sequences were obtained from 49 specimens of Te. truncatus.Pairwise distance values within species are shown in Suppl.material 3. The genetic distances between the insects identified as the same species were between 0.000 and 0.074 (mean 0.013 +/-0.003).The analysis of COI sequences discovered that Te. truncatus includes the specimen OL631282, previously identified as Telenomus sp.(Ricupero et al., 2022) (Suppl.material 1).

Molecular analysis
The analysis involved 105 nucleotide sequences.All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option).There was a total of 492 positions in the final dataset.Barcode sequences were obtained from 95 Telenomus specimens (Suppl.material 1) from the Palearctic Region.They were compared with eight sequences of specimens from the Nearctic Region identified as Te.cristatus Johnson (n: 4), Te. persimilis Ashmead (n: 3), and Te.sanctivincenti Ashmead (n: 1).

Conclusion
In recent years, researchers have limited the identification of Telenomus to genus level or grouped all of the specimens, referring only to Te. chloropus when they emerged from eggs of pentatomids in western Palearctic region.Despite the setbacks of these misidentifications, the taxonomy of Palearctic species of Telenomus associated with stink bugs has advanced, and we here provide a more solid foundation for continued research.For the first time, the West Palearctic species of the Te.podisi species group associated with the Pentatomidae can be reliably identified, with diagnostic tools based on multiple lines of evidence.The logical next test of our species concepts would be interbreeding studies, as were performed for cryptic species of the genus Trissolcus (Matsuo et al. 2014;Tortorici et al. 2019;Moraglio et al. 2021a).The identification of European Telenomus species that attack stink bugs also provides new prospects for a detailed study of their biology, which may lead to improved pest management.Furthermore, identification of the wasps from new localities and hosts will expand the distributional and biological knowledge that is available from specimens in collections.only the authors' views and opinions; neither the European Union nor the European Commission can be considered responsible for them.Elijah Talamas was supported by the Florida Department of Agriculture and Consumer Services, Division of Plant Industry.Some of the sequence data were produced by Matthew R. Moore, Cheryl G. Roberts and Lynn A. Combee at the Molecular Diagnostics Laboratory (FDACS/ DPI).We are very thankful to the colleagues from the Interdepartmental Laboratory of Electron Microscopy (Faculty of Biology, Lomonosov Moscow State University) for the provided facilities and help during electron microscopic studies.We are also grateful to Silvia Teresa Moraglio, Paolo Navone, and Sara Scovero for collecting specimens used in our analyses.

Table 1 .
Links to images of primary type specimens.

Table 2 .
Host associations."X" indicates an association recorded during the present study.