Introduction
The Water Framework Directive (WFD) outlines the requirements of the
European Commission for regular assessment of European surface waters
(Bengtsson et al., 2012), as a measure to monitor and ensure that water
bodies of the member states have a high ecological quality. A large
number of different protocols applicable to the assessment of water
bodies by the member states are based on a diverse array of
morphologically based descriptions of macroinvertebrates to plant life
combined with chemical/physical parameters are currently implemented for
assessment (Birk et al., 2012). This diversity in methodology has shown
the need for cross comparison and standardisation to obey central
directives. Furthermore, current assessment protocols rely heavily on
invasive sampling and fauna identification by taxonomic experts, which
is both time-consuming and costly (Leese et al., 2016). One example of
stream ecosystem assessment protocols is the Danish Stream Fauna Index
(DSFI) (Skriver, Friberg, & Kirkegaard, 2000). This method uses
macroinvertebrate diversity as Biological Quality Elements (BQE)
(Agouridis, Wesley, Sanderson, & Newton, 2015; Elbrecht & Leese,
2017). The water quality of the stream ecosystems are categorised into
one of seven groups, ranging from poor (1), to very good (7) (Skriver et
al., 2000). The DSFI categories translate to the ecological status
classes as described by the WFD (Baattrup-Pedersen et al., 2004).
It has been proposed that bioassessment methods can be improved through
the use of molecular techniques such as metabarcoding (Blackman et al.,
2019; Pawlowski et al., 2018). Next generation sequencing (NGS) has
become an increasingly mainstream and convenient approach to perform
analysis of diverse ecosystems, The use of standardised DNA barcoding
based on universal genetic markers allows for the identification of
species through sequence data (Leese et al., 2016). Previous studies
have yielded generalised protocols for sampling and DNA extraction from
marine sediments (Aylagas, Mendibil, Borja, & RodrÃguez-Ezpeleta,
2016), as well as broad range primer sets targeting invertebrate
biomarkers (Elbrecht & Leese, 2017), laying the foundation for
high-throughput assay development. Recently, an amplicon sequencing
approach based on targeting the cytochrome c oxidase I (COI ) gene
was developed and successfully applied as an alternative method of
performing water quality assessments in streams (Kuntke, de Jonge,
Hesselsøe, & Nielsen, 2020).
The drawback of molecular approaches for bioassessment is that design of
barcoding targets can be problematic. Often not all of the desired
diversity can be captured in a single barcode (i.e. one DNA
amplification). Therefore, no consensus on genes and specific barcodes
has been reached so far (Leese et al., 2016). Samples collected for
fauna indexing according to water quality assessments contain DNA from
all materials present within the ecosystem, including soil, water,
plants and organisms inhabiting the sampling site. By choosing a
universal approach that captures the majority of diversity present in a
sample, it could potentially be possible to report on the state and
dynamics of entire ecosystem, rather than focusing on a single source of
DNA in the sample. Microbial communities of stream water ecosystems have
previously been shown to correlate with land usage, and the status of
the stream environment (Lear et al., 2013). Furthermore, quality
assessment of ground and stream water via detection of selected
microbial indicators such as Escherichia coli andClostridium perfringens has also shown promising results (Francy,
Helsel, & Nally, 2000). Moreover, the superior size of bacterial and
archaeal diversities have previously been linked to changes in ecosystem
quality after an oil spill (Urakawa, Garcia, Barreto, Molina, &
Barreto, 2012). Collectively, these findings suggest that microbial
community studies may present a promising approach for fast, accurate
and cost effective bioassessment of freshwater ecosystems.
In the present study we characterise the complete biome profile of 53
bulk samples collected from freshwater streams across Denmark. The
results are aligned with the pre-determined ecological quality status
based on conventional water quality assessment using macroinvertebrate
composition as BQE. A high-throughput amplicon sequencing approach using
a universal primer set that targets all three domains (Bacteria ,Archaea and Eukaryota ) was chosen for this purpose. The
potential of whole biome analysis for ecosystem quality determination is
assessed. Furthermore, taxa from all three domains with potential as
indicators for ecological status were explored.