Eurygaster spp.

2.1. Morphological identification of Eurygaster spp.

The collected samples were meticulously examined for morphological characteristics—such as color, pattern, and sex—and were divided into morphotypes according to Fasulati (2005). These morphotypes were subsequently classified systematically. To ascertain species-specific morphological traits, high-resolution digital images of all specimens were captured with a DFC295 stereomicroscope (Leica, Wetzlar, Germany) equipped with an integrated camera system. The morphological identification of sunn pest species, particularly within the genus Eurygaster , primarily relies on the extraction and examination of male genitalia. Definitive conclusions cannot be drawn for females due to the absence of diagnostic traits, necessitating the dissection and detailed analysis of anatomical structures in male specimens ( Neimorovets, 2020; Syromyatnikov et al., 2017).

Male and female specimens, initially identified as potentially distinct based on morphological examination, were selected for DNA barcoding. For male specimens, genitalia were extracted, and the legs from the same specimens were used for subsequent DNA barcoding analysis. Males were soaked in a 10% potassium hydroxide (KOH) solution for 90 min to soften them. The genitalia were extracted after softening in glycerin and examined under a binocular microscope in the laboratory of Plant Protection Department at Van Yüzüncü Yıl University.

2.2. Identification of Eurygaster spp. with DNA barcoding Total genomic DNA was extracted from morphologically examined male and female specimens using the saltingout technique, as outlined in Supplementary Material 1. DNA was nondestructively extracted from insect samples, with specimens from each morphotype cataloged in an institutional collection and preserved at –20 °C for long-term storage. The COI region in mitochondrial DNA was targeted for amplification using universal and species-specific primers. Universal primers LCO1491 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) were largely ineffective and therefore were unsuitable for barcoding this species. Consequently, HemCOI_F (5’-CCACGWWTAAATAATATWAGWTTYTG-3’)and HemCOI_ R (5’-TATGGWGCWGCTATAWGWCTAAT-3’) primers, previously tested in Hemiptera insects, were used (Budak et al., 2018) 2. Additionally, specific primers for Eurygaster spp. weredesignedbasedonNCBIGenBankdata to verify the results. Separate primers were developed for E.

1 TÜİK (2021). Turkish Stastical Institute [online]. Website https://biruni.tuik.gov.tr/medas/?kn=92&locale=tr [accessed 15 May 2024] .

2 Budak M, Çıplak B, Başıbüyük HH, Kaydan MB, Keskin B et al.(2018).Barkodlamada İkili Belirteç ( COI ve ITS2) Sisteminin Bazı Hemimetabol ve Holometabol Böceklerde Araştırılması. Program Kodu: 1001. Proje No: 113Z753 ( Ankara, Türkiye: TÜBİTAK) [online]. Website https:// search.trdizin.gov.tr/tr/yayin/detay/618443/barkodlamada-ikili-belirtec-coi-ve-its2-sisteminin-bazi-hemimetabol-ve-holometabolboceklerde-arastirilmasi [accessed 18 May 2024]. integriceps (Eint_F: 5’-CCCCTTCACTAACCCTGCTA-3’, Eint_ R: 5’-AATGATGTGTTGAAGTTACGGTC-3’), E. maura (Emau_F: 5’-TTACCACCCTCACTAACCCT-3’, Emau_ R: 5’-GTGGGATTCGTTCAGGTGTTAT-3’), and E. austriaca (Eaust_F: 5’-TACCACCTTCACTGACCCTG-3’, Eaust_ R: 5’-CGTTCAGGTGTTATCCCAACA-3’) and all were confirmed with 100% sequence identity using the Basic Local Alignment Search Tool ( BLAST) from NCBI.

Each PCR reaction was performed in a final volume of 50 µL. Each tube had 10X reaction buffer (100 mM Tris-HCl, pH 8.8, 500 mM KCl, 0.8% Nonidet P-40), 25 mM MgCl 2, 10 mM dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA), 1 µL primer F, 1 µL primer R (each one was diluted to 10 pmol) (Sentebiolab, Ankara, Türkiye), 8 µL template DNA (previously isolated for each reaction), and 0.625 U Taq DNA polymerase (Thermo Fisher Scientific). The PCR reaction started with initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 47–59 °C, and elongation at 72 °C for 1 min. A final elongation step at 72 °C for 5 min completed the reaction.

The genetic material obtained for verifying the occurrence of PCR reactions, confirming the desired length, and assessing the ratio and purity, was analyzed using a 1% agarose gel electrophoresis. The gel was run at 100 V for 60 min, and the resulting band images were recorded under UV light. To determine the sequences, 30 µL of the PCR products were sent for Sanger sequencing (Sentebiolab).