Overcoming the Challenges of Studying Soil Nematodes: A New Approach with Implications for All (Soil) Organisms

Post provided by Stefan Geisen

(Soil) Nematodes

“…if all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable, and if, as disembodied spirits, we could then investigate it, we should find its mountains, hills, vales, rivers, lakes, and oceans represented by a film of nematodes…” (Cobb 1914)

He may have said it more than a century ago but we now, more than ever, realise that Nathan Augustus Cobb was right. Nematodes are by far the most abundant animals soil, freshwater and marine ecosystems. These tiny worms are barely visible to the human eye (if they’re visible at all), hundreds can inhabit a single gram of soil . Their similar shape might lead you to think that they’re all alike, but that’s not the case. More than 25,000 species have been identified and estimates put their entire species diversity in the 100,000s.

Some common nematode species found in most soils. a) Plectus sp; b) Aphelenchus sp; c) Helicotylenchus sp; d) Thonus sp; e) Mononchus sp; © Wageningen University, Laboratory of Nematology, NL; Hanny van Megen

Some common nematode species found in most soils. a) Plectus sp, b) Aphelenchus sp, c) Helicotylenchus sp, d) Thonus sp, e) Mononchus sp. © Wageningen University, Laboratory of Nematology, NL; Hanny van Megen

This taxonomic and functional diversity has boosted nematodes to become useful bioindicators for soil quality. Nematodes perform many different functions in both terrestrial and aquatic ecosystems. These are mainly defined by what they eat:

  • Bacteria/Fungi: Many nematode groups eat bacteria and fungi. They control the population of these organisms and keep them active.
  • Plants: Plant feeders are the unwanted guests in agricultural systems as well as in our gardens. They can destroy entire harvests by piercing into or infiltrating roots.
  • Omnivores/Predators: Many nematode species prey on other smaller organisms including smaller nematodes and control their abundances.
  • Parasites: These species inhabit other larger organisms and can act as biocontrol agents.

It’s pretty clear to see that nematodes have an important role in soils and other systems, right? But how to we study them?

Challenges of Studying Nematodes

What might be in there? Small soil organisms would be so easy to study if soil was transparent!

What might be in there? Small soil organisms would be so easy to study if soil was transparent!

One of the main challenges when trying to study soil nematodes is just seeing them! Unfortunately for those of us who study these species, soil isn’t transparent. Luckily, there’s a long history of studying soil nematodes and several fancy extraction techniques have been developed.

Most rely on the activity of nematodes as they move through a small mesh that prevents inert particles and organisms from passing in their final extraction phase. This prevents a few taxa and some inactive individuals from being detected, so there is some bias.

But the advantage of this method has to be highlighted: ONLY active organisms are identified! In microbial ecology, we (I am at least 75% microbial ecologist) usually sequence communities based on DNA and get information on a mix of active, inactive and dead organisms (also RNA is NOT the holy grail of molecules when studying active organisms, as nicely illustrated by Noah Fierer). The ability to identify between active and inactive/dead organisms is hugely important.

So we’ve extracted our nematodes, what’s next? Unfortunately, we haven’t come to the hard part yet.

The nice thing about nematodes is that you can roughly group individuals into the main functional groups based on their mouth structure. But, this information isn’t always enough. There are a lot of different soil quality indices and most of these need higher taxonomic resolution of the community structure.

 You can determine the differences in nematodes function in soils by their mouth structure. These images are the mouth structures of the species shown above that represent a) bacterivorous, b)fungivorous, c) herbivorous, d) omnivorous and e) predacious nematodes. © Wageningen University, Laboratory of Nematology, NL; Hanny van Megen

You can determine the differences in nematodes function in soils by their mouth structure. These images are the mouth structures of the species shown above that represent a) bacterivorous, b)fungivorous, c) herbivorous, d) omnivorous and e) predacious nematodes. © Wageningen University, Laboratory of Nematology, NL; Hanny van Megen

Only a few trained experts can identify nematode individuals to family, sometimes genus, very rarely species level under a microscope. And for those few experts (that are arguably not becoming more abundant…), it’s a hell of a job to get through a sample. Even if a subset of 100-200 organisms per sample is identified, it takes an expert at least 90min on the microscope to identify them. Don’t even think about calculating time-costs (personnel as the usually greatest cost factor) for bigger ecological studies…

For main-stream analyses, to reduce labour cost and to increase sample throughput, we need other techniques to dig into nematode community composition. One option for this is high-throughput sequencing, particularly metabarcoding. In this approach, a DNA barcoding region (part of the genome that differs between taxa to allow high-taxonomic identification) of an almost infinite number of organisms is amplified in parallel and this is used to identify the community structure of the sample.

This technique is often applied to study microbes –  bacterial, archaea, fungi and protists – but it’s rarely used to investigate soil animals. In ‘Integrating quantitative morphological and qualitative molecular methods to analyse soil nematode community responses to plant range expansion’ we show that metabarcoding can be applied for in-depth studies of soil nematodes to allow much higher taxonomic community resolution than studies based on morphology.

Methodological Implications Beyond Studies on Soil Nematodes

Overall, metabarcoding and other sequence-based tools might help us more easily study and better understand biodiversity across different groups of animals. This was nicely visualised in ‘The ecologist’s field guide to sequence‐based identification of biodiversity’ (check out the recent Editor Recommendation of the article too). Our study shows some metabarcoding caveats that need to be considered though: absolute abundance data of different functional groups as determined by microscopic counting sometimes resulted in different ecological patterns than obtained by the semi-quantitative metabarcoding. In fact, metabarcoding can never provide absolute abundance data*.

Interestingly, ecological patterns were nearly identical when we compared relative abundances of functional groups based on morphological identification with metabarcoding. It seems possible to combine easy counting of a sample (which takes 3-5 minutes and could be automated using image analysers or flow cytometry in the future) with metabarcoding to get reliable abundances and high-taxonomic community composition in an easy and cost-efficient way. We believe that this method combination is essential to correctly identifying ecological patterns!

Proposed integrated methodology for cumulative and more reliable ecological data interpretation.

Proposed integrated methodology for cumulative and more reliable ecological data interpretation.

We hope that this method combination serves as an example for more thorough studies on other groups of (soil) life and that absolute quantification finds its place back in microbial ecology studies!

To find out more, read our full Open Access Methods in Ecology and Evolution article ‘Integrating quantitative morphological and qualitative molecular methods to analyse soil nematode community responses to plant range expansion’ (No Subscription Required)

*some trials were performed with non-complex communities and there are potentials to simultaneously study abundances and community structures in bacteria. But it will take many years until reliable absolute quantification using metabarcoding might be possible.

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