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

Régis Vivien,Inge Werner,Benoit J.D Ferrari

doi:10.7717/peerj.6050

Abstract

IntroductionOligochaetes 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.MethodsWe 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.ResultsThe 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.DiscussionOur 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.

Highlights

Oligochaetes are recognized as valuable bioindicators of sediment quality in streams and lakes
The c oxidase I (COI) fragment of all specimens preserved in low-pH formalin for periods up to 3 days was successfully amplified by polymerase chain reaction (PCR), but the COI fragment of only a part of the specimens stored in low-pH formalin for 7 to 21 days was amplified, about 50% after 7 days and less than 20% after 14 and 21 days (Fig. 1, Table S3)
We highlight that the use of neutral buffered formalin for fixation/preservation of oligochaete tissues will be suitable for amplifying such small COI fragments and for developing genetic indices based on the analysis of oligochaete samples

Summary

Introduction

Oligochaetes are recognized as valuable bioindicators of sediment quality in streams and lakes. Our results indicate that sediment samples can be stored in neutral buffered formalin for up to 4 weeks and the sieved material can be transferred to absolute ethanol, without affecting DNA quality, density and community composition of oligochaetes. Sanger sequencing of isolated specimens and high throughput sequencing (HTS) of samples composed of genetically tagged specimens (Shokralla et al, 2014) or of pooled specimens (similar quantity of tissue between specimens) constitute possible ways for both identifying the species present in a sample and estimating their abundances Developing such approaches requires a method for simultaneously preserving the DNA quality, the densities and the community structure of oligochaetes. Low-pH formalin causes more degradation of DNA than neutral buffered formalin, especially for long preservation time (Koshiba et al, 1993; Schander & Halanych, 2003)

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