Abstract
The freshwater quality in many Aotearoa – New Zealand catchments is declining due to increased agricultural practices, urbanization, and other anthropogenic effects. The Macroinvertebrate Community Index (MCI) has been the gold standard for assessing the long-term ecological health of freshwater streams and rivers in Aotearoa – New Zealand for decades. However, the MCI approach is limited in several ways. Firstly, it requires significant time, cost, effort, and specialist taxonomic expertise, which limits its usefulness and frequency of monitoring. Secondly, the approach is biased towards certain taxa and may miss rare species, leading to incomplete assessments of freshwater health. Lastly, the traditional approach is costly and not easily scalable for large-scale biomonitoring efforts. Molecular approaches utilizing environmental DNA (eDNA) have the potential to overcome these limitations and revolutionize freshwater macroinvertebrate biomonitoring. By collecting and sequencing eDNA from freshwater samples, it is possible to identify the species present in the waterway without the need for specialist taxonomic knowledge or microscopic analyses; however, biases involved in the methods of these techniques prevent accurate detection of rare species and quantification of abundances.
To assess the potential of new molecular biomonitoring techniques, I compared the accuracy of two methods, metabarcoding and CRISPR-Cas enrichment, using mock communities of seven macroinvertebrate taxa commonly found in local Dunedin streams. Biomass estimates for each taxa in the mock communities were compared to the resulting sequence read counts produced by each molecular method. Metabarcoding uses universal primers to target a gene present in all species and sequencing this identify the taxa present in the sample based on the DNA sequence. CRISPR-Cas enrichment uses the Cas9 enzyme with guide RNAs specific to each taxa to cut the DNA and isolate it for sequencing. As a part of this, seven whole mitochondrial genomes of the taxa studied here were produced so that the optimal guides for each taxa could be designed.
The resulting relationship between sequence read count and biomass estimates was influenced by the taxa type and method type, both metabarcoding and the CRISPR-Cas enrichment approaches could not consistently predict the starting biomass estimates. This could be due to lack of relationship between biomass estimates and total DNA content, or inconsistent guide cutting (Cas- enrichment) or priming (metabarcoding). Given the vast scope of Cas enrichment techniques for improvement and development, Cas enrichment is a promising method of DNA detection.
This finding marks the first successful use of CRISPR-Cas enrichment in bulk-eDNA samples of non-model insects after only one attempt at the CRISPR-Cas enrichment technique. With more time the technique can be refined by improving the sgRNAs for different genes, involving more species, and investigating other Cas enrichment techniques. This advancement enables more efficient, cheaper, and more accurate measures of stream health, and presents an exciting opportunity to expand the field of genomic-based biomonitoring techniques.