Discussion
The aim of this study was to find the best possible set of eukaryote 18S rRNA primers to be used in studies based on faeces metagenomics for the simultaneous analysis of diet and parasites in threatened farmland birds. The mini-barcodes developed in this study were found to meet these expectations. The primer set MiniB18S_81F/MiniB18S_81R in particular showed great taxonomic coverage of almost all eukaryotic taxa, as well as good taxonomic resolution of the most interesting phyla associated with the diet and parasitology of birds both through both ”in silico” and real metabarcoding testing.
According to our results, of the three new mini-barcodes, MiniB18S_81 was by far the best although the other two mini-barcodes designed, MiniB18S_41 and MiniB18S_43, did not show poor behaviour. Both achieved quite high taxonomic coverage of Apicomplexa, Arthropoda, Chloroplastica, Nematoda and Platyhelminthes, but also displayed a lower taxonomic coverage and resolution than MiniB18S_81. This was nevertheless expected as the V7-V8 region of the 18S gene amplified by MiniB18S_81 is much more variable than the V3 region amplified by MiniB18S_41 and MiniB18S_43 (Hadziavdic et al., 2014). Moreover, the MiniB18S_81 mini-barcode showed a much wider amplification capacity than the 28S mini-barcode nucLSUDf1/nucLSUDr1 (Sonnenbe et al., 2007) and proved slightly superior to the Short28SF/Short28SR (Vestheim & Jarman, 2008) 28S mini barcode for four of the five target phyla. MiniB18S_81 displayed especially good amplification skills on Arthropoda, Nematoda and Platyhelminthes. In addition, it showed a great taxonomic resolution capacity at the order level in four of five target phyla in the metabarcoding test conducted on bird faeces. Its good taxonomic resolution at the arthropod order level should also be mentioned. Around 83.5% of arthropod OTUs obtained in the metabarcoding test were unambiguously identified. Hence this mini-barcode could provide information on the contribution to the bird’s diet of different arthropod orders, other invertebrates and plants. Although its taxonomic resolution within plants was poor, it did provide an overall picture of the importance of plants in the diet. According to our ”in silico” and metabarcoding tests, the mini-barcode proposed here allowed for the accurate identification of the orders of most Apicomplexa, Nematoda and Platyhelminthes organisms (using SILVA SSU v132 dataset). In fact, this barcode could provide detailed information useful for comparative parasite load studies. The lower taxonomic resolution observed at species level in the metabarcoding test is likely attributable to the fact that many of the OTUs recovered corresponded to organisms that were not included in the reference dataset. Thus according to our ”in silico PCR” analysis, MiniB18S_81 shows good taxonomic resolution at the species level of Arthropoda, Nematoda and Platyhelminthes using a suitable reference database. Although, the percentage of reads obtained for each taxa cannot be considered quantitative of the real contribution of these taxa to the gut microbiota (Evans et al., 2016; Lamb et al., 2019; Piñol et al., 2019), this variable could be useful for comparative studies.
The capacity of MiniB18S_81 to unambiguously identify some pathogens such as the genera Cryptosporidium and Blastocystissuggests its possible use for estimating parasite loads. In effect, there is much interest in Blastocystis spp. because of their impacts on human health (Tan, 2008; Scanlan, 2012) and zoonotic capacity (Greige et al., 2018). Studies of Blastocystis spp. have been often based on presence/absence data and this new mini-barcode could provide information on the proportions of this organism in the host’s gut microbiota especially useful for comparative studies.
All the tested barcodes showed quite short but constant amplicon lengths. While amplicon lengths ranged from 30 to 900 bp for all the studied primers, few were longer than 300 bp. In the case of MiniB18S_81, one of the taxa eliciting amplicons longer than 300 bp was Cestoda, with a mean of 299 bp. This is because Cestoda have an insertion of more than 100 bp in this region. However, this should not be a problem as Cestoda DNA in the samples is not degraded as it is tissue DNA (adults, larvae or eggs).
All barcodes displayed really good coverage of the Fungi kingdom and its phyla Ascomycota and Basidiomycota. This is important as, when working with eDNA, environmental contamination particularly with fungi could be a problem (Bohmann et al., 2014). When animal faeces is sampled from the ground, the time interval between deposition and sampling should be minimal, as a longer interval will mean a greater likelihood of sample colonization by other organisms such as fungi or bacteria. Further, target organisms can also die due to the aerobic conditions and be degraded by bacteria and fungi. Hence, it is essential to collect fresh faeces. Using the broad-spectrum barcodes proposed here, fungal contamination can be identified and contaminated samples discarded.
All things considered, the barcodes tested featured quite good qualities. The Short28SF/Short28SR primer pair designed by Vestheim and Jarman (2008) showed wide taxonomic coverage of all the phyla examined along with an acceptable and constant taxonomic resolution capacity within these phyla. The only drawback of this mini-barcode is that the available reference datasets for LSU are not as long and purified as those for SSU, and without a robust reference database, information is lost. The MiniB18S_81 mini-barcode was also very constant, but was better at detecting the most important diet and parasite phyla than Short28SF/Short28SR, as it showed a high taxonomic resolution capacity for these phyla. In contrast, nucLSUDf1/nucLSUDr1 (Sonnenbe et al., 2007) did not behave adequately for the present purpose. This mini-barcode amplified less than 90% of sequences belonging to 8 of 10 studied phyla, which is far from the values shown by the other primer pairs. Although its taxonomic resolution capacity was also high, its taxonomic coverage (82% of eukaryotic organisms) was the lowest of all the primers tested. We would thus recommend the use of MiniB18S_81 for faecal metagenomics studies. For other target taxa, primers should be first tested for their suitability.
For ecological studies and biomonitoring programmes, high throughput sequencing holds great promise. The optimal primers proposed here were able to generate amplicon sequences with good resolution when applied to bird faeces samples and thus seem exceptional candidates for gut and faeces eDNA metabarcoding. Useful information was obtained about the arthropod orders and plants in the diet. An idea was also provided of the contributions of Nematoda, Platyhelminthes and Apicomplexa parasites to the gut microbiota. MiniB18S_81 could thus be a new tool for simultaneous biomonitoring of diet and intestinal parasites through eDNA metabarcoding of bird faeces samples. Understanding the ecological determinants of animal density is a central question in the field of species conservation, especially if we consider the current rate of human interference. It is in this context that MiniB18S_81 could have important implications offering more and better information on the ecology of farmland birds and on the reasons for their decline.