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ŠUMARSKI LIST 3-4/2021 str. 43     <-- 43 -->        PDF

morphological characteristics into presumed insect taxa, which were preserved in 75% (v/v) ethanol solution, each in a separate microcentrifuge tube. As the number of individuals in sampled seeds was generally low, additional 2.000 of narrow-leaved ash seeds from Županja and 800 green ash seeds from Zagreb and Sunja were examined in order to collect as much biological material as possible for species identification by molecular genetic analysis.
Total genomic DNA was extracted from ethanol-preserved specimens with DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The Folmer region of mitochondrial CO1 gene (cytochrome c oxidase 1) was amplified from obtained DNA using primers LCO1490 and HCO2198 (Folmer et al. 1994), in 50 µL PCR reactions containing 25 μL TopTaq Master Mix kit (Qiagen, Hilden, Germany), 1 μL of DNA, 1 μL of 10 μmol/L of each primer, and 22 μL of distilled RNA-free water. Cycling conditions were as follows: an initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 54 °C for 1 min, extension at 72 °C for 1 min and a final extension step at 72 °C for 10 min. The resulting PCR products were purified with MinElute PCR Purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and sequenced at the DNA sequencing facility (Macrogen Europe, Amsterdam, The Netherlands) using PCR amplification primers. The obtained sequences were identified by comparison with reference sequences in NCBI GenBank (Benson et al. 2017) and BoLD (Ratnasingham and Hebert 2007) databases. Species were identified using both morphological and molecular data.
Inspection of 100 seeds per location revealed that 10% or less of narrow-leaved ash seeds were damaged by insects from all locations except Vukovar, whereas more than third of green ash seeds from both locations were occupied by insects (Figure 2). In total, 10.6% of narrow-leaved ash seeds and 42% of green ash seeds displayed insect damage or presence.
Damaged seeds of both ash species contained weevil larvae (Coleoptera: Curculionidae), wasp larvae and adults (Hymenoptera), and moth larvae (Lepidoptera), while wasp pupae and fly larvae (Diptera) where found only in green