Simultaneous preservation of the DNA quality, the community composition and the density of aquatic freshwater oligochaetes. Simultaneous preservation of the DNA quality, the community composition and the density of aquatic freshwater oligochaetes for the development of genetically based biological indices

Oligochaetes are recognized as valuable bioindicators of the sediment quality of in streams and lakes sediments. The development of an oligochaete index based on the identification of specimens using DNA barcodes requires a method for simultaneously preserving simultaneously the DNA quality, as well as and information on the specimen density and composition of an oligochaete community composition. Absolute ethanol optimally preserves DNA but fixation of aquatic freswater oligochaetes with this medium can induce 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. We performed guanidine extraction and polymerase chain reaction (PCR) amplification / sequencing of the cytochrome c oxidase I (COI) gene on tissue fragments fixed 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. All oligochaete specimens fixed in neutral buffered formalin for up to 28 days were successfully amplified by PCR and obtained sequences were complete and of high quality. The amplification success 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. 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 aquatic 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 the establishment ofestablishing DNA barcode reference databases, and inventories of genetic diversity as well asand for the development developing of genetically based biological indices.