Introduction
To date, most of the molecular microbial ecology research has focused on
the prokaryotic branch of the microbiota. Yet, previous findings have
linked the eukaryotic communities living in hosts’ guts to several
pathologic conditions in fish (Scheifler et al . 2019) and mammals
(Aivelo and Norberg 2018, Mann et al . 2020) using a metabarcoding
approach. Furthermore, major interactions between species from the three
taxonomic domains have been reported in animal guts (Leung et al .
2018, Kodio et al . 2020). However, this field of study is slowed
by the poor availability of simple and cost-effective molecular methods
to analyse gut eukaryotic communities. While a variety of approaches are
currently available, none are exempt from the introduction of bias nor
are well optimized (Green & Minz 2005, Vestheim & Jarman 2008). For
instance, taxonomic identification using taxonomic keys does not allow
the detection of microscopic organisms and requires a prioriknowledge of the potential biodiversity present. Conversely,
high-throughput DNA sequencing approaches have proven their ability to
identify the taxonomic richness in samples at a higher taxonomic
resolution than visual identification in fish, while also giving
additional information on diet and the unknown diversity present (Berry
et al . 2015, Trujillo-González et al . 2022). The
development of optimised molecular methods to characterise eukaryotic
gut communities is essential to better describe the commensal eukaryotic
microbiota and host-parasite interactions in natural populations of
fish.
The main challenge in analysing the eukaryotic fraction of the
microbiota comes from the homologies between the conserved ribosomal RNA
SSU sequences of the host and their associated eukaryotic organisms.
While universal primers have the advantage of amplifying taxonomically
diverse phyla, this characteristic favors the amplification of unwanted
host DNA. Indeed, DNA amplification with universal primers often leads
to a preferential amplification of the predominant host DNA in gut
samples (Su et al. 2018). This high abundance of host DNA
contamination considerably limits the detection of DNA from other
organisms that are present in lower abundances (Vestheim and Jarman
2008, Liu et al . 2019).
There are multiple methods to reduce the quantity of host DNA amplicons
in samples. Recently, Zhong et al . (2021) developed CRISPR-Cas
Selective Amplicon Sequencing (CCSAS) based on taxon-specific
single-guide RNA to direct Cas9 to cut host 18S rRNA gene sequences.
Using this method, they achieved a near complete blockage of host DNA.
They also designed guide RNAs for approximately 16 000 metazoan and
plant taxa. While, CRISPR is known to recognize the wrong target gene in
certain occurrences (Fu et al . 2013), this major advance to the
field could rapidly become the go-to approach to inhibit the
amplification of specific genes. Also, Green & Mitz (2005) developed a
similar method using a Suicide Polymerase Endonuclease Restriction
(SuPER), in which specific restriction enzymes cut the host’s amplicons
before PCR amplification. However, this method requires a unique cutting
site in the host’s target gene and has only been applied once in another
study (Guedegbe et al . 2009). Another way is to use a cocktail of
multiple group-specific amplification primers to detect a wide range of
organisms in samples (Koester & Gergs 2017). While this method has
proven to be useful, the use of multiple primers can lead to an
amplification bias and their usage requires in-depth a prioriknowledge of the eukaryotic communities under study. Since there is
scarce information about fish eukaryotic gut communities, the usage of a
multiple group-specific primers for highly conserved regions such as the
18S rRNA SSU is suboptimal compared to methods that allow for
explorative and integrative research.
A method of growing interest, developed by Vestheim & Jarman (2008) is
the usage of blocking primers to inhibit the PCR amplification of
undesired DNA in metabarcoding studies. These species-specific primers
are modified with a C3 spacer at the 3’ end, a short three carbon chain
that prevents the elongation of the DNA during PCR amplification. While
it is slowly getting a wide-range acceptance (Boessenkool et al .
2012, Belda et al . 2017, Su et al . 2018, Liu et al .
2019, Mayer et al . 2021, Rojahn et al . 2021, Stewart etal . 2021), the usage of blocking primers to describe
host-parasite interactions could still benefit from additional testing
of the method for other non-referenced species. Moreover, additional
data should help compare this method to the newly developed approach
based on CRISPR-Cas (Zhong et al . 2021). Studies have used
annealing inhibiting blocking primers, primers overlapping with the 3’
end of universal primers and competing directly for annealing, to reduce
host DNA amplification in the krill (Euphausia superba )
(Vestheim and Jarman 2008), mosquitoes (Anopheles gambiae ) (Belda
et al . 2017), fish species (Su et al . 2018) and humans
(Boessenkool et al . 2012). On the contrary, elongation arrest
blocking primers are primers annealing anywhere in the target sequence
and stopping the advancement of the DNA polymerase. This type of
blocking primers is less studied since their effectiveness was seemingly
lower than for annealing inhibition blocking primers (Vestheim and
Jarman 2008). However, the development path for elongation arrest
blocking primer is simpler since they can be developed in ultra-variable
regions of the target gene, facilitating the discovery of unique
sequences in the host target gene.
Here, we successfully developed and tested specific elongation arrest
blocking primers based on the methodology suggested by Vestheim &
Jarman (2008) to block Mesonauta festivus 18S rRNA SSU during PCR
amplification. This integrative and easily implemented method allowed a
significant reduction of host’s DNA amplification, while not interfering
with the amplification of other target sequences. Furthermore, the
blocking primers enhanced the detectability of other species in gut
samples and helped dress a short portrait of the commensal eukaryotic
diversity present in M. festivus gut. Overall, this method
represents a good approach to enhance the detection of parasitic species
and ensure a cost-effective sequencing of samples enriched with host
DNA. Although developed for Mesonauta festivus , a good model
species for ecological and evolution studies in the Amazon basin (Pires
et al . 2015), this approach could potentially be modified for
usage with various other Teleosts.