Introduction
Biodiversity is decreasing globally due to human alteration and
pollution of terrestrial and aquatic environments (Brondizio, Settele,
Díaz, & Ngo, 2019). Essential ecosystem services affiliated with human
health, such as availability of food, clean water, and recreational
areas are dependent on biodiversity (Cardinale et al., 2012; Pan,
Marcoval, Bazzini, Vallina, & Marco, 2013). In addition to the
provision of ecosystem services, biodiversity losses have also been
linked to a decrease in ecosystem stability (McCann, 2000).
Anthropogenic pressure on coastal aquatic ecosystems by e.g. climate
change, eutrophication and contaminant pollution threatens the diversity
of many organisms in these systems (Pan et al., 2013). Anthropogenic
pressure in coastal ecosystems should be taken seriously because coastal
zones are transitional areas directly adjacent to human settlements
between land and sea, and impacted areas are predicted to increase in
both number and area with a continued climate change scenario (Levin et
al., 2001; Rabalais, Turner, Díaz, & Justić, 2009). It is therefore
essential to understand how the diversity of organisms living in coastal
zones respond to changes in environmental gradients and anthropogenic
pressure (Snelgrove, Thrush, Wall, & Norkko, 2014).
Biodiversity assessments of benthic macrofauna are commonly used in
national monitoring programs, including coastal zones, to determine
various ecological indexes (Pinto et al., 2009). However,
microeukaryotes present in sediment such as meiofaunal nematodes
(< 1 mm body size) are also known to react to e.g.
eutrophication status (Ristau, Spann, & Traunspurger, 2015), and the
composition and quantity of organic carbon (OC) (Ingels, Kiriakoulakis,
Wolff, & Vanreusel, 2009; Pusceddu, Gambi, Zeppilli, Bianchelli, &
Danovaro, 2009). Furthermore, because nematodes are known to have
different feeding behaviours such as bacterivory, detritivory or algal
feeding (Moens, Traunspurger, & Bergtold, 2006; Wieser, 1953) changes
in nematode assemblages are therefore likely to affect food web dynamics
and ecosystem function (e.g. Nascimento et al., 2019; Nascimento,
Karlson, & Elmgren, 2008; Nascimento, Näslund, & Elmgren, 2012). Other
arguments for including meiofauna such as nematodes in national
monitoring systems include their high diversity, short generation time,
and ubiquitous distribution (Kennedy & Jacoby, 1999). However, these
organisms are often neglected in monitoring studies (Kennedy & Jacoby,
1999), likely due to financial reasons derived from time consuming
activities such as sieving, sorting, and microscopic morphological
analyses.
In addition, the protist phyla Foraminifera (henceforth forams)
and Ciliophora (i.e. ciliates) are well-studied as bioindicators
of environmental state of aquatic ecosystems. The diversity and
community composition of forams are known to change with anthropogenic
pollution, fish farming, and decreasing water quality (Damak,
Frontalini, Elleuch, & Kallel, 2019; Frontalini & Coccioni, 2011; Jan
Pawlowski, Esling, Lejzerowicz, Cedhagen, & Wilding, 2014; Raposo et
al., 2018; Uthicke & Nobes, 2008), and similar to nematodes, OC
enrichment of the sediment also influences the diversity of forams
(Martins et al., 2015). Ciliates are used as bioindicators in wastewater
treatment plants (Chen, Xu, Tam, Cheung, & Shin, 2008; Foissner, 2016).
In natural aquatic environments, the diversity and community composition
of ciliates are influenced by e.g. salinity, pH, and anthropogenic
pollution (e.g. Gong et al., 2015; Jiang, Xu, Hu, Warren, & Song,
2013). One of the main merits of assessing the diversity of protists as
bioindicators is their documented rapid change to environmental
conditions (Payne, 2013). The assessment of both meiofaunal and protist
biodiversity is therefore a good proxy in monitoring programmes to study
changes in ecosystems. However, these communities are rarely studied
together and challenges still include being able to investigate multiple
communities from bulk sediment samples without time consuming activities
involved in studying the benthic meiofaunal fraction such as sieving,
sorting, and microscopy.
In the last ten years, environmental DNA (eDNA) metabarcoding studies
using the 18S rRNA marker gene have been extensively conducted to study
microeukaryotes including nematodes, forams, and ciliates (Bik et al.,
2012; Elias Broman et al., 2019; Carugati, Corinaldesi, Dell’Anno, &
Danovaro, 2015; Fonseca et al., 2010; Forster et al., 2019; Lallias et
al., 2014; Lara & Acosta-Mercado, 2012; Nascimento et al., 2019; J.
Pawlowski, Lejzerowicz, & Esling, 2014; Peham, Steiner,
Schlick-Steiner, & Arthofer, 2017). Such tools have drastically reduced
the time needed to taxonomically classify organisms compared to
morphological taxonomic techniques, that also involves sieving and
sorting of organisms (Carugati et al., 2015). However, limitations exist
with DNA metabarcoding such as non-optimized PCR protocols and primer
bias when targeting multiple taxa (Kelly, Shelton, & Gallego, 2019). In
addition to using eDNA that will assess the biodiversity of both living
organisms plus non-degraded DNA from dead organisms, environmental RNA
(eRNA) is targeting only living organisms or RNA derived from organisms
of recent origin in the environment (Cristescu, 2019; S. A. Wood et al.,
2020). With new bioinformatic tools that can taxonomically classify
hundreds of millions of sequences within minutes to hours (e.g. D. E.
Wood, Lu, & Langmead, 2019) and estimate relative abundances at species
or genus level (e.g. Lu, Breitwieser, Thielen, & Salzberg, 2017), it is
valuable to investigate how eRNA combined with the latest sequencing
technology can be used to assess differences in biodiversity of active
multiple communities from highly diverse and dense environments such as
soils and sediments, while avoiding the biases associated with PCR
amplification.
Here we assessed the biodiversity and community composition of three
microeukaryotic groups in sediment samples: nematodes, forams, and
ciliates, along an OC gradient in a coastal archipelago in the Gulf of
Finland, Baltic Sea. The aim was to investigate if eRNA shotgun
sequencing, without any sieving or sorting of samples (i.e. bulk
sediment), could be used to detect differences in biodiversity of
multiple microeukaryotic communities. Additionally, we assessed if
changes in nematode functional ecology (feeding type) as a response to
the OC gradient could be detected. We expected that nematode deposit
feeders would have higher relative abundance in stations with higher OC.
This approach was coupled to the latest sequencing platform (Illumina
NovaSeq S4, yielding ~87 million sequences per sample),
and new bioinformatic tools to estimate taxonomic classifications and
relative abundances from data of this size (Kraken 2 + Bracken 2
combination). The Gulf of Finland is characterised by strong
environmental gradients associated with eutrophication (Andersen et al.,
2015; Villnäs et al., 2019). This contributes to spatially heterogenous
benthic macro-communities in terms of diversity and composition in this
ecosystem (Bonsdorff, Laine, Hanninen, Vuorinen, & Norkko, 2003). The
Gulf of Finland is therefore a well-suited system to investigate if a
similar heterogeneity exists in active microeukaryotic communities.