Detecting Diatoms through Kick-net DNA Metabarcoding

Post provided by Dr. Chloe Robinson

Diatoms may be the only organisms to live in houses made of glass, but some species of diatom are far from fragile. Certain groups of diatoms are highly tolerant of poorer water quality and therefore their presence can be diagnostic for freshwater health estimates. A recent study, featuring MEE Associate Editor, Chloe Robinson, investigated whether communities of freshwater diatoms can be collected via kick-net methodology, which is an approach currently used for collecting benthic macroinvertebrates. In this post, Chloe highlights how applying previously optimised freshwater methods can result in a more holistic understanding of freshwater health.

History of diatom monitoring

Found across the world in aquatic habitats, diatoms are single-celled algae, with cell walls composed of transparent, opaline silica. They either exist free-flowing in water (planktonic) or attached to sediment/rocks/substrates (benthic). As well as being important sources of food and carbon fixers, they can also tell us a lot about the health of aquatic systems. Diatoms can be found across a wide variety of aquatic habitats, and therefore have different ranges of environmental conditions, such as temperature, pH and the amount of suspended sediment (turbidity) that they can tolerate.

Lead author, Carley Maitland, scrapes diatoms from rocks. Picture credit: Chloe Robinson.

So, we know diatoms are useful for monitoring aquatic systems, but how do we collect them? Conventional methods of collecting diatoms are often limited to the collection of diatoms that live on removable rocks (scraping of periphyton) and involves taxonomic identification via microscopy. This approach is costly in terms of sampling effort, financially for the taxonomic expertise required and timewise in terms of time taken to generate results.

Age of DNA-based monitoring

Until recently, diatoms were identified using solely morphological features, however there is now more of a focus on using DNA-based methods, such as DNA metabarcoding, to identify diatoms down to genus and even species level. This approach identifies based on genetic information as opposed to morphological and has been used across the world to inform freshwater health management actions, including in France, northern Europe and North America.

The copy number of DNA metabarcoding gene, rbcL, has been shown to correlate with diatom cell biovolume in DNA-based research of freshwater diatoms. A recent study published in Methods in Ecology and Evolution, ‘Avoiding quantification bias in metabarcoding: Application of a cell biovolume correction factor in diatom molecular biomonitoring’, features a correction factor for improving taxa quantification of diatoms using this approach. In addition, DNA-based approached can be used as a means of investigating historic diatom assemblages through collection of lake sediment samples, as described in Methods in Ecology and Evolution article, ‘Estimating species colonization dates using DNA in lake sediment’.

Example of diatom photographed under the microscope. Picture credit: CBG Photography Group 2020.

Similar to the DNA-based approach used to identify macroinvertebrates, DNA metabarcoding enables down to species-level identification of diatoms and therefore identification of biological indicator (bioindicator) diatom species. This identification is made possible via highly reproducible bioinformatic pipelines such as MetaWorks, and gene-specific DNA classifiers, such as the rbcL classifier used in this study, which make identifications based on comparing DNA sequences produced to a library of known diatom DNA sequences.

A comparison of current data quality and costs associated with traditional morphology-based taxonomic sample analysis versus DNA metabarcoding analysis for diatoms.

Going back to how diatoms are collected; the DNA pipelines may be working well to produce diatom identifications, however, by just collecting diatoms which live on small rocks, only a portion of the whole diatom community is being represented. For example, planktonic diatom species are often missed when diatoms have been collected via rock scraping only. Bioindicator diatoms do not only exist in on rocks, they can also be found attached to macrophytes (aquatic plants), within the sediment, on submerged boulders and within leaf litter in freshwater ecosystems. So, to capture this diversity of diatoms, we need to rethink how we collect diatoms.

Catch them all approach of kick-net biomonitoring

We already know that standardised benthic kick-net protocols (e.g. CABIN in Canada) work well for acquiring a wide diversity of macroinvertebrates, so the study by Maitland et al. (2020), investigated how effective this method is for also collecting a range of different diatom species. To test this, we sampled at four sites; two of ‘good’ and two of ‘fair’ water quality. In addition to collecting diatoms through benthic kick-netting, we collected diatoms using standardised diatom collection methods for rocks, sediment, macrophytes and leaf litter (referred to as conventional methods) to compare diatoms collected between the kick-net and conventional methods.

Diatom species detected in kick-net samples but not rock samples. Clockwise: Epithemia gibba, Aulacoseira ambigua, Navicula trivialis, Craticula cuspidata and Caloneis lewisii. Picture credit: Chloe Robinson.

Overall, kick-net samples detected the same range of diatom species as the conventional methods combined. When comparing with just rock scraping (which is the most used method for diatom collection), kick-net samples detected more diatom species, particularly planktonic diatom species. We detected 10 planktonic species via kick-net samples, 8 of which were missed in rock scraping samples.

We didn’t see much difference in diatom communities between ‘good’ and ‘fair’ sites, however, kick-net samples consisted of 28 bioindicator species, some of which were indicators of good quality (sensitive diatoms) and others of poorer water quality (tolerant diatoms).

Sensitive and tolerant diatoms detected in the study via DNA metabarcoding. Left panel: Diatoma tenuis (centre), Epithemia gibba and Achanthidium minutissimum (smaller); right panel: Melosira varians (centre), Planothidium lanceolatum (smaller) and Nitzschia palea. Picture credit: Chloe Robinson.

Applications for water quality monitoring

Overall, this study found that kick-net methodology, which is currently widely used for macroinvertebrates, is a suitable collection method for capturing a wide range of diatom species. The results of this study are particularly important for freshwater biomonitoring, as by using a single sample method, we can look at both macroinvertebrate and diatom species within the same sample. This keeps field and labour costs low and combined with the lower price of sample processing using DNA (~$100), DNA metabarcoding of kick-net samples is a win-win for a better understanding freshwater health.

To read the study ‘Freshwater diatom biomonitoring through benthic kick-net metabarcoding’ for free on PLoS ONE, click here.

 To read the Methods in Ecology & Evolution article ‘Avoiding quantification bias in metabarcoding: Application of a cell biovolume correction factor in diatom molecular biomonitoring’, click here.

To read the Methods in Ecology & Evolution article ‘Estimating species colonization dates using DNA in lake sediment’, click here.

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