Bastian Egeter

Molecular analysis of gut content is one of the widest used methods to investigate diet in arthropods. Stomach content analysis in some arthropods is particularly difficult, e.g., in arachnids, because they have external digestion and a low foraging frequency. Scorpions have a particularly low feeding frequency, and their diet information is scarce. In this work we explore a DNA metabarcoding approach to detect prey DNA in Vietnamese forest scorpions ( Heterometrus laoticus ) under a controlled diet regime. A different type of prey (crickets, mealworms, and cockroaches) was offered once every 3 weeks for a total of 9 weeks. To assess the most suitable part of the digestive system and extraction method to use for molecular diet analysis, we separately analyzed three different portions of the digestive tract of scorpions (mid‐gut, hepatopancreas, and hindgut) using two different extraction methods (salt‐out method and a customized beads‐based protocol). We calculated the detectability half‐life of the prey DNA for each digestive tract section. We detected all three targeted prey items, showing that in scorpions multiple predation events can be distinguished in the same specimen within its last 9 weeks of foraging activity. The hepatopancreas was the portion of the digestive tract that provided the best prey detection and the longest DNA detectability half‐life (51 days), followed by the mid‐gut (22 days) and the hindgut (16 days). We found no significant difference between the extraction methods used. However, the salt‐out method was less effective in some of the PCRs and is therefore not recommended for molecular diet analysis.

Traditional detection of aquatic invasive species via morphological identification is often time‐consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina‐based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and substantially decrease sample turnaround times. However, nanopore‐sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools (msi package) to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves while comparing it with Illumina‐based sequencing. We sampled water from sites with pre‐existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. Samples were extracted, amplified, and sequenced by the two platforms. The mean agreement between sequencing methods was 69% and the difference between methods was nonsignificant. The lack of detections of some species at some sites could be explained by their known low abundances. This is the first reported use of MinION to detect aquatic invasive species from eDNA samples.