DNA Integrity of Chironomids and Oligochaetes Is Maintained During Long-Term Storage in Neutral Buffered Formalin:...

Oligochaetes and chironomids are valuable bioindicators but ethanol preservation is problematic: it causes oligochaetes to disintegrate and forces a trade-off in insects between DNA integrity and morphological structures. Aiming to develop genetic oligochaete and chironomid indices based on the high-throughput sequencing (HTS) of genetically tagged specimens, we tested whether long-term specimen storage in neutral buffered formalin affects the success rate of subsequent genetic analyses. We performed PCR amplifications of the COI gene on specimens preserved in neutral buffered formalin for periods up to 6 months. For oligochaetes, DNA quantities (amplicon yields) decreased over time but remained sufficient for sequencing at all tested periods; for chironomids, no temporal trend toward degradation was observed. Storing these tissues in neutral buffered formalin for up to 6 months does not prevent subsequent genetic analyses and provides ample time for logistical processing, such as labor-intensive sieving. These results facilitate the implementation of oligochaete and chironomid indices using a single sediment sample for both groups. More broadly, replacing ethanol with neutral buffered formalin as a fixative simultaneously preserves DNA quality, specimen density, community composition, and morphological structures across macroinvertebrate taxa. This integrated preservation allows for: (1) the refinement of macroinvertebrate indices both morphology- and bulk-DNA-based, by ensuring that all taxa, including soft-bodied organisms, are accurately represented; (2) the perfection of insect barcode reference databases by enabling a dual validation approach where high-quality sequences are directly linked to intact morphological vouchers; and (3) the improvement of quality control in bulk-sample HTS by allowing morphological verification of molecular results on the same specimens.