Phasmarhabditis and Gastropod Survey 2012-2021
Summary of all gastropod collections throughout California between 2012 and 2021
Steps to reproduce
Gastropods were collected from nurseries and garden centers for a total of 1 person hour. The gastropods were collected using clean metal spatulas and were stored in plastic containers lined with moistened paper towels and punctured lids. Containers were placed inside of a cooler and at the end of each sampling day, the gastropods were sorted into 540 ml deli containers with a moistened paper towel and cut organic carrot pieces for food. The gastropods were sorted by species, and the deli containers were labeled accordingly, and kept in the coolers. The deli containers were cleaned and provided with a new moist paper towel and gastropods were given fresh organic carrot pieces every other day. After each survey trip, gastropods were examined again to ensure they were identified correctly. To accurately identify gastropods, we used McDonnell et al. (2009) . Once gastropods were sorted, relevant information was recorded and summarized, tracking the dates of collection, as well as the life course of the gastropods. The gastropods were kept in the lab at room temperature and were given fresh changes of paper towel and organic carrot discs every other day. Each gastropod was given an accession number once it was deceased and immediately transferred to plated 1% plain agar (1L: 10g agar, 900ml H2O) that served as seed culture, as described in Tandingan De Ley et al. (2014). To encourage better growth of nematodes, we modified the method and used nematode growth medium (NGM; 1L: 3g NaCl, 20g Agar, 2.5g Peptone, 975ml deionized H2O, 10ml Uracil (2g/L) were added to a liter of deionized water, autoclaved and let cool, to which were added 25ml filtered KPO4, 1ml filtered MgSO4, 1ml CaCl2, and 1ml Cholesterol (5mg/ml)). As surveys progressed in 2018, and in the interest of time, speed, and laboratory space, we modified the nematode recovery, following the protocol of Wilson et al. (2016) decapitating slugs in batches and immediately placing them on NGM. At least 5 individual nematodes that emerged from slug cadavers were picked from seed culture plates and grown on NGM plates, kept at 17°C. These plates were labeled as single nematode isolations. Each single nematode isolation plate was then designated a unique accession number. Preliminary examination was done through a stereomicroscope, using morphological traits e.g. the presence of large phasmids and vulval body position, to identify suspected Phasmarhabditis. After suspects were identified, at least 2 individual nematodes from each isolate were prepared for PCR and DNA sequencing of the ribosomal RNA (D2-D3 domains of the large subunit or LSU), as described in Tandingan De Ley et al. (2014). When necessary, the small subunit (SSU) was also sequenced following the same protocols. Contigs were assembled and compared by BLAST with published sequences in GenBank using CodonCode Aligner (CodonCode Corp., 58 Beech Street, Dedham, MA, USA) to verify their identity or determine if sequences were unique.