Introduction. Oligochaetes are recognized as valuable bioindicators of sediment quality in streams and lakes. The development of an oligochaete index based on the identification of specimens using DNA barcodes requires a method for simultaneously preserving the DNA quality and information on the specimen density and oligochaete community composition. Absolute ethanol optimally preserves DNA but fixation of freshwater oligochaetes with this medium can cause disintegration and fragmentation of specimens. Here, we investigated the possibility to preserve oligochaete specimens in low-pH formalin and in neutral buffered formalin for up to four weeks before genetic analyses and tested if the addition of absolute ethanol to formalin-fixed oligochaetes resulted in a loss of specimens and/or species.
Methods. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of a fragment of the cytochrome c oxidase I (COI) gene on tissue fragments preserved in low-pH formalin for up to 3 weeks and in neutral buffered formalin for up to 4 weeks. In addition, we compared the density and taxonomic composition of formalin-fixed oligochaetes of several sieved sediment samples before and after the addition of absolute ethanol.
Results. The COI fragment of all oligochaete specimens preserved in neutral buffered formalin for up to 28 days was successfully amplified by PCR and obtained sequences were complete and of high quality. The amplification success rate for low-pH formalin fixed specimens declined after 7 days of storage. The addition of absolute ethanol to formalin-fixed oligochaete communities did not alter density or diversity estimates.
Discussion. Our results indicate that sediment samples can be stored in neutral buffered formalin for up to 4 weeks and the sieved material can then be transferred to absolute ethanol, without affecting DNA quality, density and community composition of oligochaetes. Based on these results, a protocol for preserving freshwater oligochaetes, describing all the steps from collection of sediments to preservation of the biological material in absolute ethanol, is proposed. This method of fixation/preservation is of relevance for establishing DNA barcode reference databases, inventories of genetic diversity and developing genetically based biological indices.