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Comparative metabarcoding study performed on the mock soil nematode communities, composed of DESS-preserved and freshly extracted nematodes Cover

Comparative metabarcoding study performed on the mock soil nematode communities, composed of DESS-preserved and freshly extracted nematodes

Journal of Nematology
Volume 58 (2026): Issue 1 (February 2026)
By: ,   and    
Open Access
|May 2026
Figures & tables

Figures & Tables

Figure 1

Simplified scheme of (mock) community sample preparation used for metabarcoding analyses. (a) Extracted heat-killed nematodes. (b) DESS preservative treatment or lack of treatment. (c) Transfer of the individual representatives of the segregated and identified nematode taxa into the test tubes. (d) Replicates containing milliQ water (marked in blue) or ATL buffer (yellow). Sets 1 and 2 differed in taxa composition (Table 1). Each replicate within the same set comprised of the same taxa and their amounts. (e) DNA isolation either with “CD” or “Q” method. As a result, 12 DNA samples were prepared and further analysed: 1aCD-1cCD, 1dQ-1fQ, 2aCD-2cCD, and 2dQ-2fQ.

Figure 2

Taxa abundance and composition in two mock nematode community sets. (a–c) Set 1, DESS-pretreated nematodes. (d–f) Set 2, DESS-untreated, freshly extracted nematodes. (a) Exact numbers of nematode taxa included in the samples 1aCD-1cCD and 1dQ-1fQ. (d) Exact numbers of nematode taxa, included into the samples 2aCD-2cCD and 2dQ-2fQ. Taxa initially occurring in the two nematode community sets in the same amounts (Table 1) are marked with an asterisk. Taxa occurring in the two nematode community sets in the different amounts are marked with two asterisks. (b and e) Read numbers and retrieved genera after 18S rDNA sequencing. (c and f) Read numbers and retrieved genera after 28S rDNA sequencing. The obtained false-positive reads were not included into the visualization and the corresponding data are available in the suppl. data S1. “CD” and “Q” samples were isolated with the Clear Detection or Qiagen DNA extraction kits, respectively.

Figure 3

Heatmaps of the most abundant sequences obtained after metabarcoding sequencing of sample replicates (a–f), representing two mock nematode communities (Sets 1 and 2), isolated with two DNA extraction kits (“CD” and “Q”). (a) Most abundant nematode species sequences after 18S rDNA sequencing. (b) Most abundant nematode species sequences after 28S rDNA sequencing.

Figure 4

ZOTUs richness estimated with Chao1 and Shannon indices obtained after 18S (a) and 28S rDNA (b) metabarcoding sequencing of sample replicates (a–f), representing two mock nematode communities (Sets 1 and 2), isolated with two DNA extraction kits (“CD” and “Q”).

Information on the primer combinations used in this metabarcoding study

Primer pair codesGenomic regionNucleotide primer sequences (bolded) including the Illumina adapter (5′–3′)Approx. amplicon length (bp)Reference
NemFopt 18Sr2bRopt18S rDNA5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-GGGGWAGTATGGTTGCAAA-3′5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-TGTGTACAAAKGRCAGGGAC-3′500Waeyenberge et al. (2019)
D2A D228S rDNA5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-ACAAGTACCGTGAGGGAAAGTTG-3′5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-TCCGTGTTTCAAGACGGG-3′400Nunn (1992); Chombard, et al. (1998)

Taxonomical and quantitative composition of the two artificially composed sample sets (Set 1 and 2)

Taxon (order, family, feeding strategy)Set 1 (nematode treated with DESS, replicates: 1aCD-1cCD and 1dQ-1fQ)Set 2 (freshly extracted nematodes, replicates: 2aCD-2cCD and 2dQ-2fQ)
Sampling details: locality, environment, associated plant, sampling year# Individuals per each replicateSampling details: locality, environment, associated plant, sampling year# Individuals per each replicate
1 Meloidogyne hapla (Rhabditida, Meloidogynidae, plant parasite) Solanum lycopersicum L. (culture),a 2019100 Solanum lycopersicum L. (culture),a 2020100
2 Aphelenchoides ritzemabosi (Rhabditida, Aphelenchoididae, fungivore) Brunnera sp. C. Steven (culture),b 201820 Clematis sp. L. (culture 2018),b 202020
3 Acrobeles sp. (Rhabditida, Cephalobidae, bacterivore)Mistrzewice, meadow, Berteroa incana (L.) DC., 201920Mistrzewice, meadow, Berteroa incana (L.) DC., 202020
4 Anaplectus sp. (Plectida, Plectidae, bacterivore)Mistrzewice, Bzura river bank plants, Rubus caesius L., 201910Mistrzewice, Bzura river bank plants, Rubus caesius L., 202010
5 Rotylenchus uniformis (Rhabditida, Hoplolaimidae, plant parasite)Dziekanów Leśny, garden, Ribes sp. L., 201010Dziekanów Leśny, garden, Ribes sp. L., 202010
6 Helicotylenchus sp. (Rhabditida, Hoplolaimidae, plant parasite)Mistrzewice, meadow, Berteroa incana (L.) DC., 201925Mistrzewice, meadow, Berteroa incana (L.) DC., 202020
7Qudsianematidae (Dorylaimida, omnivore)Mistrzewice, forest, Quercus robur L., 201920Mistrzewice, forest, Quercus robur L., 202010
8 Longidorus sp. (Dorylaimida, Longidoridae, plant parasite)Augustów, herbaceus plants near Alnus Mill., 20195Mistrzewice, field cultivation, Zea mays L., 20202
9 Mesocriconema xenoplax (Rhabditida, Criconematidae, plant parasite)Mistrzewice, Bzura river bank plants, Rubus caesius L., 20194Mistrzewice, Bzura river bank plants, Rubus caesius L., 202020
10 Paratylenchus projectus (Rhabditida, Tylenchulidae, plant parasite)Augustów, meadow, Trifolium arvense L., 201920—
11 Tylenchorhynchus sp. (Rhabditida, Dolichodoridae, plant parasite)Mistrzewice, meadow, Linaria vulgaris Mill., 201910—
12 Tripylina cf. arenicola (Enoplida, Trefusiidae, predaceous)Mistrzewice, meadow, Berteroa incana (L.) DC., 201910—
13 Plectus sp. (Plectida, Plectidae, bacterivore)Augustów, herbaceus plants near Alnus Mill., 2019; Mistrzewice, Bzura river bank plants, Rubus caesius L., 201910—
14Aporcelaimidae (Dorylaimida, omnivore)Mistrzewice, Bzura river bank plants, Rubus caesius L., 20195—
15 Tylocephalus auriculatus (Plectida, Plectidae, bacterivore)Augustów, meadow, Trifolium arvense L., 20192—
16 Coomansus parvus (Mononchida, Mononchidae, predaceaus)Mistrzewice, meadow, Berteroa incana (L.) DC., 20192—
17 Helicotylenchus pseudorobustus (Rhabditida, Hoplolaimidae, plant parasite)—Łomna, wasteland, Juncus sp. L., 202020
18 Pratylenchus sp. (Rhabditida, Pratylenchidae, plant parasite)—Łomna, wasteland, Pyrus sp. L., 202020
19 Paratrichodorus pachydermus (Triplonchida, Trichodoridae, plant parasite)—Mistrzewice, forest, Quercus robur L., 202020
20 Rotylenchus goodeyi (Rhabditida, Hoplolaimidae, plant parasite)—Łomna, wasteland, Juncus sp. L., 202010
21 Geocenamus longus (Rhabditida, Dolichodoridae, plant parasite)—Mistrzewice, forest, Quercus robur L., 202010
22Alaimidae (Enoplida, bacterivore)—Mistrzewice, forest, Quercus robur L., 20205
23 Discolaimus major (Dorylaimida, Qudsianematidae, omnivore)—Mistrzewice, field cultivation, Zea mays L., 20205
24 Mylonchulus sp. (Mononchida, Mylonchulidae, predaceaus)—Dziekanów Leśny, garden, Ribes sp. L., 20202
∑273304

PERMANOVA test results for differences in number of reads obtained for various nematode taxa, that were: (A) both, the DESS-treated and DESS-untreated (Fresh), (B) DESS-treated, and (C) DESS-untreated (Fresh)

A
TaxonDESS-treated and DESS-untreated (Fresh)
Influence of all factors: preservation, primers, and isolationJoint influence of preservation and isolationJoint influence of preservation and primersInfluence of preservation
All treatments18S treatments28S treatmentsCD treatmentsQ treatments18S/CD treatments18S/Q treatments28S/CD treatments28S/Q treatments
1 M. hapla <2.2 × 10−16***<2.2 × 10−16***2 × 10−4***<2.2 × 10−16***<2.2 × 10−16***0.001389**0.001389**0.11
2 A. ritzemabosi <2.2 × 10−16***<2.2 × 10−16***<2.2 × 10−16***<2.2 × 10−16***2 × 10−4***0.001389**0.001389**11
3 Acrobeles sp.<2.2 × 10−16***<2.2 × 10−16***<2.2 × 10−16***0.0082**6 × 10−4***0.70140.001389**0.30140.001389**
4 Anaplectus sp.<2.2 × 10−16***8 × 10−4***6 × 10−4***0.003**<2.2 × 10−16***0.001389**0.20140.001389**0.8014
5 R. uniformis <2.2 × 10−16***<2.2 × 10−16***<2.2 × 10−16***<2.2 × 10−16***0.006**0.001389**0.001389**0.001389**0.001389**
6 Helicotylenchus sp.<2.2 × 10−16***0.0172*<2.2 × 10−16***<2.2 × 10−16***<2.2 × 10−16***0.001389**0.001389**0.001389**0.001389**
7Qudsianematidae<2.2 × 10−16***0.0012**6 × 10−4***0.0034**2 × 10−4***0.001389**0.001389**0.001389**0.1014
8 Longidorus sp.0.0036**0.0018**0.0044**0.0078**0.0102*0.50140.70140.20140.5014
9 M. xenoplax <2.2 × 10−16***0.002**0.0034**2 × 10−4***0.011*0.001389**0.40.001389**0.001389**
Total<2.2 × 10−16***0.0048**0.0048**<2.2 × 10−16***2 × 10−4***0.001389**0.20140.40140.1014
DOI: https://doi.org/10.2478/jofnem-2026-0014 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X
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Language: English
Page range: 165 - 185
Submitted on: Aug 5, 2025
Accepted on: Apr 8, 2026
Published on: May 16, 2026
Published by: Society of Nematologists, Inc.
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year
Keywords:
18S and 28S ribosomal DNA,
DESS,
Illumina,
Metabarcoding,
Mock community,
Nematode diversity,
Nematode preservation
Related subjects:
Life sciences,
Life sciences, other

© 2026 Katarzyna Rybarczyk-Mydłowska, Mohammed Ahmed, Grażyna Winiszewska, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 58 (2026): Issue 1 (February 2026)
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