RNA-Seq detection of nematodes in Australian snails

Snails are robust organisms and can travel long distances attached to surfaces, therefore snails are commonly intercepted at border control points. They represent a biosecurity threat, firstly because exotic species may have a direct negative effect on Australian biodiversity and agriculture as pests, and secondly, because they may carry parasites of vertebrates and invertebrates. To fully assess the threat and decide whether more effort should be put into their detection at the border, we need to know what species they may be carrying. There are several potential methods available, but each has limitations. This paper presents a proof-of-concept study of RNA-seq as a complementary approach for parasite surveillance in snails, by investigating detection of nematodes associated with snails from transcripts, representing potential evidence of metabolically active organisms. We describe a modified RNA extraction protocol to process whole-snail tissues and a bioinformatic workflow to analyze the sequencing data. Using these methods on a small data set of multiple species of both native and introduced land snails (n = 60) from locations near major Australian ports, we identified RNA sequences from the nematode Caenorhabditis briggsae within the introduced snail species Paropeas achatinaceum. We also identified candidate sequences aligning to other nematodes using a curated specialized nematode sequence database (18S NemaBase) but adding control sequence alignment against a very broad database (NCBInt) identified these alignments as probable false identifications because there were much closer matches with other organisms. This result highlights the importance of using both high-quality, curated reference databases for assigning identities, and broad databases for checking alternative matches. By collecting native and introduced snails close to ports, we demonstrated that the method can work on species and sites of interest for biosecurity surveillance.