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Quantitative taxonomic compositions of nematode communities help to assess soil environments due to their rich abundance and various feeding habitats. DNA metabarcoding by the 18S ribosomal RNA gene (SSU) regions were preferentially used for analyses of soil nematode communities, but the optimal regions for high-throughput amplicon sequencing have not previously been well investigated. In this work, we performed Illumina-based amplicon sequencing of four SSU regions (regions 1–4) to identify suitable regions for nematode metabarcoding using the taxonomic structures of nematodes from uncultivated field, copse, and cultivated house garden soils. The fewest nematode-derived sequence variants (SVs) were detected in region 3, and the total nematode-derived SVs were comparable in regions 1 and 4. The relative abundances of reads in regions 1 and 4 were consistent in both orders and feeding groups with prior studies, thus suggesting that region 4 is a suitable target for the DNA barcoding of nematode communities. Distinct community structures of nematodes were detected in the taxon, feeding habitat, and life-history strategy of each sample; i.e., Dorylamida- and Rhabditida-derived plant feeders were most abundant in the copse soil, Rhabditida-derived bacteria feeders in the house garden soil, and Mononchida- and Dorylamida-derived omnivores and predators and Rhabditida-derived bacteria feeders in the field soil. Additionally, low- and high-colonizer–persister (cp) groups of nematodes dominated in the house garden and copse soils, respectively, whereas both groups were found in the field soil, suggesting bacteria-rich garden soil, undisturbed and plant-rich copse soil, and a transient status of nematode communities in the field soil. These results were also supported by the maturity indices of the three sampling sites. Finally, the influence of the primer tail sequences was demonstrated to be insignificant on amplification. These findings will be useful for DNA metabarcoding of soil nematode communities by amplicon sequencing. [ABSTRACT FROM AUTHOR]

Nematodes are representative soil metazoans with diverged species that play crucial roles in nutrient recycling in the pedosphere. Qualitative and quantitative information on nematode communities is useful for assessing soil quality, and DNA barcode-mediated taxonomic analysis is a powerful tool to investigate taxonomic compositions and changes in nematode communities. Here, we investigated four regions (regions 1–4) of the 18S small subunit ribosomal RNA (SSU) gene as PCR targets of deep amplicon sequencing for the taxonomic profiling of individual soil nematodes. We determined the sequence variants (SVs) of 4 SSU regions for 96 nematodes (total 384 amplicons) isolated from copse soils and assigned their taxonomy using the QIIME2 software with dada2 or deblur algorithm and the SILVA database. Dada2 detected approximately 2-fold more nematode-derived SVs than deblur, and a larger number of SVs were obtained in regions 1 and 4 than those in other regions. These results and sufficient reference sequence coverage in region 4 indicated that DNA barcoding using a primer set for region 4 followed by dada2-based analysis would be most suitable for soil nematode taxonomic analysis. Eighteen SSU-derived operational taxonomic units (rOTUs) were obtained from 68 isolates, and their orders were determined based on the phylogenetic trees built by four regional sequences of rOTUs and 116 nematode reference species as well as the BLASTN search. The majority of the isolates were derived from three major orders Dorylaimida (6 rOTUs, 51.5% in 68 isolates), Rhabditida (4 rOTUs, 29.4%), and Triplonchida (7 rOTUs, 17.6%). The predicted feeding types of the isolates were fungivores (38.2% in total nematodes), plant feeders (32.4%), and 14.7% for both bacterivores and omnivores/predators. Additionally, we attempted to improve the branch structure of phylogenetic trees by using long nucleotide sequences artificially prepared by connecting regional sequences, but the effect was limited. [ABSTRACT FROM AUTHOR]

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis. [ABSTRACT FROM AUTHOR]

Abstract: The acute toxicities of heavy metals and detergents have been well examined with respect to several endpoints, such as mortality, for application to toxicity tests for environmental assessments. However, chronic influences of these agents on multicellular organisms still need to be determined. Here we studied long-term effects on the lifespan of a free-living nematode, Caenorhabditis elegans, resulting from prolonged exposure to heavy metals or detergents, as well as short-term inhibitory effects on reproduction and growth. These agents inhibited growth of hatched larvae and reproductive capacity in a concentration-dependent manner. They also effectively shortened the lifespan of the adult nematode over the same concentration range. Since toxic effects on both growth and lifespan were observed over similar concentration ranges, where acute toxicities in various endpoints are detected, the shortening of the lifespan can be used as a new endpoint for the assessment of various ecotoxic agents. [Copyright &y& Elsevier]