Preserving Shell Integrity of Microgastropods With the Nondestructive HotSHOT DNA Extraction Method

Gastropods rank among the most species-rich animal classes, accounting for 5% (4-8) of all described animal species with about 1/3 of microgastropods (< 5 mm in size). DNA extraction from preserved microgastropods retracted into their shells is challenging because established methods usually damage the shell. This study tests the HotSHOT alkaline lysis method as a nondestructive protocol for small ethanol-preserved gastropods. Using a brief lysis step at high pH and temperature, we achieved effective DNA extraction suitable for COI barcode amplification while preserving shell integrity and fragile periostracal structures. High-quality full-length sequences were obtained from 76% of the samples, and 85% of the sequences were suitable for species identification. The remaining samples (15%) did not yield usable sequence data, possibly due to poor preservation, insufficient buffer access in tightly closed shells, or potential PCR problems. Specimens with fully closed opercula showed reduced success unless buffer access was facilitated. Our results show that HotSHOT is a fast and inexpensive method for DNA extraction that preserves shell vouchers, making it ideal for large-scale barcoding of retracted microgastropods and valuable when working with rare specimens. By its low cost and high speed, it has a high potential to accelerate species discovery and delimitation, diversity assessments and monitoring and thus support conservation.