Nematode Identification in Ladybirds

Adult beetles and L4 larvae from the greenhouse-reared population (n = 80 each), as well as laboratory-reared beetles (n = 142) and individuals collected around Ober-Mörlen in late October 2017 (n = 324) were dissected immediately after sampling. Nematodes from infected ladybirds were collected in PBS and the species identified using a FLoid® Cell Imaging Station (Thermo Fisher Scientific) to capture images at different life stages. The nematodes were centrifuged at 16,000 × g for 15 min at 4 °C and the pellet was resuspended in nuclease-free water before lysis and amplification of the 18S SSU rRNA gene as previously described^{50 (link)} using GoTaq® G2 DNA Polymerase (Promega). The PCR products were purified and transferred to the vector pGEM®-T Easy (Promega) for sequencing, followed by sequence analysis using Geneious v10.2.2^{51 (link)}. A consensus sequence was generated by the de novo assembly of the three overlapping sequence fragments and a BLAST search against the NCBI nr/nt database was carried out using Megablast with standard parameters.