Freshwater Macroinvertebrates and DNA Metabarcoding

Importance of Freshwater Macroinvertebrates

• Indicators of stream health
• Assess human impact on stream ecosystems
• Aim for better stream management and freshwater resource security

Challenges of Taxa Identification

• Routine monitoring uses morphological characteristics for identification
• Difficulty identifying some taxa at the species level
• Time-consuming identification process
• Adults can be identified reliably by experts

Genetic Barcoding Technique

• Use of CO1 barcode for sequencing specimens
• Creation of a reference database for reliable identification
• DNA barcoding allows species-level identification but is expensive for routine monitoring

DNA Metabarcoding Technique

• New technology enables simultaneous identification of all specimens in a sample
• Process involves:
  • DNA extraction from homogenized samples
  • Amplification of barcoding gene using polymerase chain reaction (PCR)
  • High-throughput sequencing
• Generates millions of sequences for analysis

Study Overview

• Presented by Vasco Elbrecht from University Duisburg-Essen
• Co-authors include Edith Wammus, Florian Lese, Christian Meissner, Jukka Arovjeta
• Investigated efficiency of DNA metabarcoding in routine monitoring of macroinvertebrates
• Analyzed 18 samples from Finnish monitoring programs for stream quality

Sample Processing and Sequencing

• Samples dried and homogenized for DNA extraction
• Four different primer combinations used to amplify CO1 barcode
• BF-BR primer sets specifically developed for freshwater macroinvertebrates
• Used Fusion Primer System for tagging and pooling samples
• Sequenced two replicates per sample for a total of 144 samples.
• High sequencing depth achieved: 260 million read pairs, average 1.53 million reads per sample

Data Processing

• Reads demultiplexed and preprocessed for clustering
• Quality control measures applied: trimming, filtering, and dereplication
• Taxonomy assigned using BOLT and NCBI reference databases
• Comparison of DNA-based identifications with morphological identifications

Results

• DNA metabarcoding identified approximately 60% more taxa per sample compared to morphology
• However, around 30% of taxa identified morphologically were not detected by DNA metabarcoding
• DNA metabarcoding can accurately identify taxa, especially challenging larval stages
• Assessment indices based on DNA and morphology showed similar results, indicating viability for ecosystem assessment

Challenges and Limitations

• Primer bias affects amplification and detection of taxa
• Misidentification of taxa can lead to discrepancies
• Limited taxonomic expertise affects database accuracy
• PCR inhibition can impact the efficiency of DNA extraction from mixed samples

Potential Solutions

• Improve reference databases through better curation and expert collaboration
• Use size sorting and ecosystem-specific primers to enhance accuracy
• Standardize and validate protocols for DNA metabarcoding to set quality standards

Conclusion

• DNA metabarcoding is a promising method for macroinvertebrate monitoring and assessing stream health
• Further testing and improvements needed for routine application
• Study results provided in detail in the referenced manuscript
• Questions encouraged regarding the study or DNA metabarcoding in general.