A protocol for sequential isolation, purification, and collection of Luticola nematophagus eggs to obtain...

Conventional strategies for obtaining planarian tissue for genomic DNA extraction rely on diet-based rearing or amputation-induced regeneration. These approaches are inapplicable to soil-dwelling flatworms that predominantly reproduce by egg-laying and lack regenerative capacity, such as the recently discovered nematode predator Luticola nematophagus. Here, we developed a protocol for the sequential isolation, purification, and collection of L. nematophagus eggs, comprising three steps: (1) physical sieving through sintered meshes of graded pore sizes under recirculating water flow to exclude nontarget eggs; (2) chemical digestion with 8% (w/w, weight/weight) urea or 5% (w/w) hydroxylamine hydrochloride under continuous aeration for 18 h to eliminate contaminating microinvertebrates; and (3) manual sorting under a stereomicroscope. Genomic DNA extracted from purified eggs exhibited A260/A280 ratios of 1.93-2.00 and A260/A230 ratios exceeding 2.0, meeting the quality thresholds for whole-genome sequencing. Successful PCR amplification of 18S and 28S ribosomal DNA (rDNA) fragments confirmed the suitability of the DNA for downstream molecular analyses. Compared with untreated controls, urea treatment (8% urea and manual sorting) reduced the final hatching rate by 5.2% and delayed the half-hatching time by 5.1 days, whereas hydroxylamine hydrochloride treatment (5% hydroxylamine hydrochloride and manual sorting) reduced the final hatching rate by 11.6% and delayed the half-hatching time by 7.9 days. However, the majority of the eggs remained viable. The quality of the extracted DNA was further confirmed by successful whole-genome sequencing, which yielded a chromosome-level genome assembly, indicating its suitability for the most demanding downstream applications.