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Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities.

ABSTRACT: Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single-specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.

SUBMITTER: Schenk J

PROVIDER: S-EPMC7083658 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Understanding interactions between herbivores and parasitoids is essential for successful biodiversity protection and monitoring and for biological pest control. Morphological identifications employ insect rearing and are complicated by insects' high diversity and crypsis. DNA barcoding has been successfully used in studies of host-parasitoid interactions as it can substantially increase the recovered real host-parasitoid diversity distorted by overlooked species complexes, or by species with slight morphological differences. However, this approach does not allow the simultaneous detection and identification of host(s) and parasitoid(s). Recently, high-throughput sequencing has shown high potential for surveying ecological communities and trophic interactions. Using mock samples comprising insect larvae and their parasitoids, we tested the potential of DNA metabarcoding for identifying individuals involved in host-parasitoid interactions to different taxonomic levels, and compared it to standard DNA barcoding and morphological approaches. For DNA metabarcoding, we targeted the standard barcoding marker cytochrome oxidase subunit I using highly degenerate primers, 2*300 bp sequencing on a MiSeq platform, and RTAX classification using paired-end reads. Additionally, using a large host-parasitoid dataset from a Central European floodplain forest, we assess the completeness and usability of a local reference library by confronting the number of Barcoding Index Numbers obtained by standard barcoding with the number of morphotypes. Overall, metabarcoding recovery was high, identifying 92.8% of the taxa present in mock samples, and identification success within individual taxonomic levels did not significantly differ among metabarcoding, standard barcoding, and morphology. Based on the current local reference library, 39.4% parasitoid and 90.7% host taxa were identified to the species level. DNA barcoding estimated higher parasitoid diversity than morphotyping, especially in groups with high level of crypsis. This study suggests the potential of metabarcoding for effectively recovering host-parasitoid diversity, together with more accurate identifications obtained from building reliable and comprehensive reference libraries, especially for parasitoids.

Project description:BackgroundGastrointestinal parasitic nematodes can impact fecundity, development, behaviour, and survival in wild vertebrate populations. Conventional monitoring of gastrointestinal parasitic nematodes in wild populations involves morphological identification of eggs, larvae, and adults from faeces or intestinal samples. Adult worms are typically required for species-level identification, meaning intestinal material from dead animals is needed to characterize the nematode community with high taxonomic resolution. DNA metabarcoding of environmental samples is increasingly used for time- and cost-effective, high-throughput biodiversity monitoring of small-bodied organisms, including parasite communities. Here, we evaluate the potential of DNA metabarcoding of faeces and soil samples for non-invasive monitoring of gastrointestinal parasitic nematode communities in a wild ruminant population.MethodsFaeces and intestines were collected from a population of wild reindeer, and soil was collected both from areas showing signs of animal congregation, as well as areas with no signs of animal activity. Gastrointestinal parasitic nematode faunas were characterized using traditional morphological methods that involve flotation and sedimentation steps to concentrate nematode biomass, as well as using DNA metabarcoding. DNA metabarcoding was conducted on bulk samples, in addition to samples having undergone sedimentation and flotation treatments.ResultsDNA metabarcoding and morphological approaches were largely congruent, recovering similar nematode faunas from all samples. However, metabarcoding provided higher-resolution taxonomic data than morphological identification in both faeces and soil samples. Although concentration of nematode biomass by sedimentation or flotation prior to DNA metabarcoding reduced non-target amplification and increased the diversity of sequence variants recovered from each sample, the pretreatments did not improve species detection rates in soil and faeces samples.ConclusionsDNA metabarcoding of bulk faeces samples is a non-invasive, time- and cost-effective method for assessing parasitic nematode populations that provides data with comparable taxonomic resolution to morphological methods that depend on parasitological investigations of dead animals. The successful detection of parasitic gastrointestinal nematodes from soils demonstrates the utility of this approach for mapping distribution and occurrences of the free-living stages of gastrointestinal parasitic nematodes.

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Comparison of morphological, DNA barcoding, and metabarcoding characterizations of freshwater nematode communities.

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