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.