Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding.
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ABSTRACT:
SUBMITTER: Nakao R
PROVIDER: S-EPMC8333312 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Environmental DNA (eDNA) metabarcoding has been used increasingly to assess biodiversity of aquatic vertebrates. However, there still remains to be developed a sampling design of eDNA metabarcoding that can ensure high detection rates of species with minimum total survey effort, especially for large-scale surveys of aquatic organisms. We here tested whether pooling of eDNA samples can be used to evaluate biodiversity of freshwater fishes in four satellite lakes of Lake Biwa, Japan. Fish communities detected by eDNA metabarcoding of the mitochondrial 12S region were compared between the individual and pooled samples. In the individual samples, 31, 22, 33, and 31 fish lineages (proxies for species) were observed at the respective sites, within which moderate spatial autocorrelation existed. In the pooled samples, 30, 20, 29, and 27, lineages were detected, respectively, even after 15 PCR replicates. Lineages accounting for < 0.05% of the total read count of each site's individual samples were mostly undetectable in the pooled samples. Moreover, fish communities detected were similar among PCR replicates in the pooled samples. Because of the decreased detection rates, the pooling strategy is unsuitable for estimating fish species richness. However, this procedure is useful potentially for among-site comparison of representative fish communities.
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Project description:Environmental DNA (eDNA) techniques refer to utilizing the organisms' DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high-throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein-coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs-AcMDB07, MiFish-U, Ve16S1, and Ve16S3-designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish-U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.
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Project description:Environmental DNA (eDNA) metabarcoding has been increasingly used to monitor the community assemblages of a wide variety of organisms. Here, we test the efficacy of eDNA metabarcoding to assess the composition of Iberian freshwater fishes, one of the most endangered groups of vertebrates in Spain. For this purpose, we sampled 12 sampling sites throughout one of Spain's largest basins, the Duero, which is home to approximately 70% of the genera and 30% of the primary freshwater fish in Spain. We sampled these sampling sites in the summer by using electrofishing, a traditional sampling method, and eDNA metabarcoding of river water samples using the mitochondrial 12S rRNA gene (12S) as a marker. We also resampled four of these sampling sites in autumn by eDNA. We compared the results obtained through eDNA metabarcoding with those of electrofishing surveys (ones conducted for the present study and past ones) and assessed the suitability of 12S as an eDNA metabarcoding marker for this group of freshwater fishes. We found that the 12S fragment, analysed for 25 Iberian species, showed sufficient taxonomic resolution to be useful for eDNA approaches, and even showed population-level differences in the studied populations across the tissue samples for Achondrostoma arcasii. In most cases, a greater number of species was detected through eDNA metabarcoding than through electrofishing. Based on our results, eDNA metabarcoding is a powerful tool to study the freshwater fish composition in the Iberian Peninsula and to unmask cryptic diversity. However, we highlight the need to generate a local genetic database for 12S gene for such studies and to interpret the results with caution when studying only mitochondrial DNA. Finally, our survey shows that the high detection sensitivity of eDNA metabarcoding and the non-invasiveness of this method allows it to act as a detection system for species of low abundance, such as early invasive species or species in population decline, two key aspects of conservation management of Spanish freshwater fishes.
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Project description:Sponges are primitive metazoans that are known to harbour diverse and abundant microbes. All over the world attempts are being made to exploit these microbes for their biotechnological potential to produce, bioactive compounds and antimicrobial peptides. However, the majority of the studies are focussed on the marine sponges and studies on the freshwater sponges have been neglected so far. To increase our understanding of the microbial community structure of freshwater sponges, microbiota of two fresh water sponges namely, Eunapius carteri and Corvospongilla lapidosa is explored for the first time using Next Generation Sequencing (NGS) technology. Overall the microbial composition of these sponges comprises of 14 phyla and on an average, more than 2900 OTUs were obtained from C. lapidosa while E. carteri showed 980 OTUs which is higher than OTUs obtained in the marine sponges. Thus, our study showed that, fresh water sponges also posses highly diverse microbial community than previously thought and it is distinct from the marine sponge microbiota. The present study also revealed that microbial community structure of both the sponges is significantly different from each other and their respective water samples. In the present study, we have detected many bacterial lineages belonging to Firmicutes, Actinobacteria, Proteobacteria, Planctomycetes, etc. that are known to produce compounds of biotechnological importance. Overall, this study gives insight into the microbial composition of the freshwater sponges which is highly diverse and needs to be studied further to exploit their biotechnological capabilities.
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Project description:The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.
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Project description:Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206-L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina-sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.
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