Jun 24, 2025
Protocol for Extracting Amynthas Species Cocoons
- 1University of Vermont
Protocol Citation: Maryam Nouri-Aiin 2025. Protocol for Extracting Amynthas Species Cocoons. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbqjzvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 31, 2025
Last Modified: June 24, 2025
Protocol Integer ID: 119362
Keywords: Amynthas, cocoon, eathworms, cocoon extraction, soil, protocol for extracting amynthas species cocoon, extracting amynthas species cocoon, extracting cocoon, accuracy in cocoon retrieval, extraction, cocoon retrieval, amynthas species, more bins of water, sieving approach, mesh sieve, water reuse, water conservation, removing large debris, such as wood fragment, minimizing waste, finer sieving, soil, improved efficiency, wood fragment, processing, improving efficiency, reduced water usage, larger debris, benchtop extraction, processing time
Disclaimer
This protocol is intended for research and educational purposes only.
Abstract
This protocol presents an optimized method for extracting cocoons of three Amynthas species—A. tokioensis, A. agrestis, and A. hilgendorfi—building on the techniques described by Ikeda et al. (2015) and Nouri-Aiin & Görres (2019). By incorporating key modifications, this wet-sieving approach enhances accuracy in cocoon retrieval while significantly improving efficiency, ergonomics, and water conservation.
Key Modifications:
- Reduced Water Usage: Instead of using continuous running water in a sink, this method utilizes two or more bins of water (depending on the number of samples), allowing for water reuse and minimizing waste.
- Improved Efficiency: A coarse-mesh sieve (e.g., Sieve No. 8) is introduced to quickly remove larger debris (such as wood fragments and roots) before finer sieving. This significantly reduces processing time, as removing large debris through Sieve No. 12 alone is much more time-consuming.
- Ergonomic Advantage: Unlike previous methods where the person extracting cocoons had to wash soil over a bucket in a sink, this approach allows for a comfortable, seated, benchtop extraction, significantly reducing physical strain.
Guidelines
- Use small soil portions (~1 cup) per batch for higher accuracy.
- Always stack Sieve No. 8 on top of No. 12 to prevent cocoon loss.
- Additional bins can be used as needed based on the number of samples and the amount of soil being processed.
- Perform a final rinse in clean water for better visibility before cocoon extraction.
- A white background can improve visibility and make cocoons easier to identify.
- Using a stereo microscope can help distinguish cocoons from similar-looking debris.
- The water in the bins can be reused for multiple samples; replace it when it becomes heavy with sediment and organic debris, or develops a murky, moldy appearance.
Materials
• Bins filled with water X 2
• Sieve No. 12 (1.7mm)
• Sieve No. 8 (2.36mm)
• Forceps
• Petri dish
• Ruler
Optional:
• Sieve No. 14 (1.4mm) – Alternative to No. 12 for finer soil filtration
Safety warnings
- Do not submerge the soil completely in water, as this may wash away cocoons.
- Too much pressure when handling soil can break cocoons.
- Some seeds and small soil particles resemble cocoons—verify by gently squeezing with forceps (cocoons are elastic, while seeds/soil particles break).
- Extracted cocoons should be kept moist to prevent rapid dehydration.
Before start
- If the soil is excessively dry, gradually moisten it over a period of time appropriate for its dryness. This allows cocoons to rehydrate, preventing shrinkage that could cause them to pass through the sieve mesh.
- Remove large debris, such as sticks and leaves, before sieving to ensure a smoother process.
- Work in a well-lit area to facilitate accurate cocoon identification.
- Use a flat, stable surface to minimize the risk of spills or sample loss.
- Keep a waste container nearby for easy disposal of unwanted debris.
Sieve Setup
Stack Sieve No. 8 on top of Sieve No. 12, or simply just use Sieve No. 12.
Note
- Sieve No. 12 is the standard size for capturing cocoons of the three Amynthas species, ranging from 2.5 ± 0.05mm to 4.5 ± 0.05mm.
- Sieve No. 14 (1.4mm) can be used as an alternative to No. 12; however, its smaller mesh size increases processing time by slowing the removal of soil particles.
- Sieve No. 8 is optional but recommended for removing large debris such as wood fragments and roots.
- If using Sieve No. 8, always ensure it is stacked on Sieve No. 12 to prevent cocoon loss.
Soil Washing
Place approximately one cup of soil into Sieve No. 8 stacked on Sieve No. 12.
- Using smaller soil portions increases accuracy and ease of cocoon extraction.
Submerge the sieves in a bin filled with water.
Note
- Water level should cover the soil but should not fully submerge the sieve.
Gently rub the soil to break down larger particles, allowing soil to pass through Sieve No. 8 onto Sieve No. 12.
- This process disperses aggregates, retaining only coarse soil particles and organic debris on the sieve.
Remove Sieve No. 8 along with the retained debris.
Lift Sieve No. 12 so that water covers the soil but does not submerge it.
Gently rub the soil against the sieve to separate remaining particles.
Note
- Avoid applying too much pressure as it can break cocoons.
After soil removal, perform a final rinse in a second bin filled with clean water.
Cocoon Extraction
Extract cocoons using forceps. Small soil particles and seeds may resemble cocoons. To distinguish them:
Note
- Apply gentle pressure with forceps.
- Cocoons are elastic and bouncy, while seeds and soil particles break apart quickly.
- A white background can improve visibility and make cocoons easier to identify.
- Using a stereo microscope can further aid in distinguishing cocoons from similar-looking debris.
Place extracted cocoons in a petri dish filled with water.
Note
- Cocoons dehydrate quickly if not kept moist.
Cocoon Identification
Cocoon size measurements according to Nouri-Aiin & Görres (2019):
Note
- Amynthas tokioensis: ~2.5 ± 0.05mm
- Amynthas agrestis: ~3.5 ± 0.05mm
- Amynthas hilgendorfi: ~4.5 ± 0.05mm
For specimens that fall between species size ranges, a molecular identification method can be used, as described in Nouri-Aiin et al. (2021).
Protocol references
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Citations
Ikeda, H., Callaham Jr, M.A., O’Brien, J.J., Hornsby, B.S., Wenk, E.S.. Can the invasive earthworm, Amynthas agrestis, be controlled with prescribed fire?
https://doi.org/10.1016/j.soilbio.2014.12.011Nouri-Aiin, M. and Görres, J.H.. Earthworm cocoons: the cryptic side of invasive earthworm populations.
https://doi.org/10.1016/j.apsoil.2019.05.004Nouri-Aiin, M., Schall, J.J., Keough, C.A., Wen, Y. and Görres, J.H.. Identifying the unidentifiable: a PCR multiplex protocol for the diagnosis of invasive pheretimoid earthworm species, verified by morphological and barcode identification.
https://doi.org/10.1016/j.apsoil.2020.103822Nouri-Aiin, M.. Elements of Biocontrol Strategies for Pheretimoid Earthworms
https://www.proquest.com/docview/2703003513?pq-origsite=gscholar&fromopenview=true&sourcetype=Dissertations%20&%20Theses