DNA barcoding of formalin‑fixed aquatic oligochaetes for biomonitoring

Background: Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol. Results: The large majority of aquatic oligochaete specimens fixed in formalin for up to 1 week could be successfully amplified and all obtained sequences were of high quality. The amplification and sequencing success rate of formalin-fixed samples and ethanol-fixed samples was similar. These results suggest that formalin fixation of aquatic oligochaete tissues for a short time does not cause serious damages to DNA and inhibit PCR amplification. Conclusion: The possibility to fix aquatic oligochaetes with formalin before genetic analyses is very promising for diversity monitoring, for construction of a comprehensive DNA barcode library and for development of an index based on Next-generation sequencing analysis of samples composed of sorted specimens.

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

Régis Lionel Vivien

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.

2018

Faunistic note on the aquatic oligochaetes of the Geneva area and of Switzerland

Régis Lionel Vivien

Aquatic oligochaetes are used in Switzerland to assess sediment quality of watercourses and lakes. The aim of this note is to mention the presence of new species of aquatic oligochaetes for Switzerland and to update the inventory of this group in the Geneva area (Switzerland). Species diversity was studied in fine/sandy sediments of watercourses (canton of Geneva) and lakes (canton of Bern) as well as in very little studied environments in Switzerland, coarse surficial sediments and the hyporheic zone of watercourses (cantons of Geneva and Valais). Seven new species for Switzerland and twelve new species for the Geneva area were found. The number of taxa in the Geneva area is currently 81. The current development and use of molecular biology (barcoding, high-throughput sequencing technologies) as a tool for aquatic oligochaetes identification will allow researchers to improve significantly future species inventories.

Museum Histoire Naturelle2015

High-throughput DNA barcoding of oligochaetes for abundance-based indices to assess the biological quality of sediments in streams and lakes

Benoît Jean Dominique Ferrari, Régis Lionel Vivien

Aquatic oligochaete communities are valuable indicators of the biological quality of sediments in streams and lakes, but identification of specimens to the species level based on morphological features requires solid expertise in taxonomy and is possible only for a fraction of specimens present in a sample. The identification of aquatic oligochaetes using DNA barcodes would facilitate their use in biomonitoring and allow a wider use of this taxonomic group for ecological diagnoses. Previous approaches based on DNA metabarcoding of samples composed of total sediments or pools of specimens have been proposed for assessing the biological quality of ecosystems, but such methods do not provide precise information on species abundance, which limits the value of resulting ecological diagnoses. Here, we tested how a DNA barcoding approach based on high-throughput sequencing of sorted and genetically tagged specimens performed to assess oligochaete species diversity and abundance and the biological quality of sediments in streams and lakes. We applied both molecular and morphological approaches at 13 sites in Swiss streams and at 7 sites in Lake Geneva. We genetically identified 33 or 66 specimens per site. For both approaches, we used the same index calculations. We found that the ecological diagnoses derived from the genetic approach matched well with those of the morphological approach and that the genetic identification of only 33 specimens per site provided enough ecological information for correctly estimating the biological quality of sediments in streams and lakes.

2020

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

Régis Lionel Vivien

2018