Oct 24, 2024
Extraction of meiofauna and meioflora from aquatic environmental samples
This protocol is a draft, published without a DOI.
- 1College of Economics, Nihon University
- Daisuke Shimada: Center for Molecular Biodiversity Research, National Museum of Nature and Science
Protocol Citation: Daisuke Shimada 2024. Extraction of meiofauna and meioflora from aquatic environmental samples. protocols.io https://protocols.io/view/extraction-of-meiofauna-and-meioflora-from-aquatic-ceyutfww
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: August 08, 2022
Last Modified: October 24, 2024
Protocol Integer ID: 68340
Keywords: meiobenthos, specimen, field method, DESS, molecular, morphology
Funders Acknowledgement:
JSPS KAKENHI
Grant ID: JP21K06299
Abstract
Meiofauna and microalgae are benthic communities of small animals and algae, respectively, that can pass through a 1-mm sieve but are retained in a 32-µm sieve. This simplified protocol collects meiofauna and microalgae living in or attached to aquatic substrates such as sediments, seagrasses, seaweeds, and sessile animals. Animals and algae are filtered, killed by heating, fixed with DESS solution, and sorted under a stereomicroscope. The advantage of this method is that both the morphological characteristics and DNA can be preserved for a long period (more than ten years).
Materials
Filter
Nylon mesh (32 or 63 μm)
Wide-mouth screw-cap plastic bottle (500 or 1,000 ml)
Rubber bands
DESS solution
Ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt dihydrate (2Na-EDTA)
Dimethyl sulfoxide (DMSO)
Sodium chloride (NaCl)
Distilled water (DW)
Irwin loop
Stainless steel wire (0.2–0.3 mm diameter)
Sampling
Sampling
Collect substrates (sediments, seagrasses, seaweeds, sessile animals, etc.) by hand or by using sampling devices.
Transport entire substrates to the laboratory at low temperature or immediately filter and fix the living organisms in the field by following the steps below. If performing this step is difficult, substrates should be frozen or stored in the DESS solution, despite the risk of injury to organisms.
Filtration
Filtration
Transfer 100–500 ml of substrate into a bucket and wash the substrate well with approximately 10 times volume of tap water. If the substrate is mud, reduce the substrate volume to 50 ml or less. For fragile animals, such as turbellarians, use 3.5% MgCl2 instead of tap water and stir gently. Wait for 10 min or more and stir again gently.
References: Hulings & Gray (1971); Pfannkuche & Thirl (1988); Giere (2009)
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Immediately pass the supernatants through a 10–100 μm (usually 32 or 63 μm) nylon mesh filter. Smaller mesh sizes can collect more organisms but are easily clogged with debris.
How to make a filter
Remove the bottom from a 500 or 1,000 ml wide-mouth screw-cap plastic bottle and cut a piece of nylon mesh larger than the mouth of the bottle.
How to make a filter.
Place the mesh over the mouth of the bottle and fasten tightly with rubber bands.
A completed filiter.
Repeat steps 3 and 4 once or twice.
Carefully remove the nylon mesh from the bottle, turn over, and wash in a Petri dish with 20 ml of filtered seawater. When handling a large substrate amounts or when the mesh appears clogged, the collected material can be transferred to a Petri dish with a dropper after each filtration.
Transfer the whole seawater with collected material to a 25–50 ml sampling bottle. If necessary, divide into several bottles.
Wait 10 min for the organisms to settle at the bottom and remove as much of the supernatant as possible with a dropper.
Fixation
Fixation
Heat the sampling bottle in 80–90°C water for 2 min to kill the organisms and to inactivate the DNase. Several animals, especially nematodes, straighten when killed by heating. When looking for smaller animals, such as tardigrades, which are easier to find when they are alive, the order of heat killing, fixation, and sorting can be switched.
Add 5 to 10 times the volume of DESS solution to the collected material and mix well. The sample can be stored in this state for a long period (more than 10 years).
Reference: Yoder et al. (2006)
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How to make 1 liter of DESS solution
Dissolve 93.0 g of 2Na-EDTA in 400 ml of DW by stirring while gradually adding NaOH. Adjust pH to 7.5–8.0 and add DW until the total volume reaches 500 ml (= 0.5M EDTA).
*Buying a pre-conditioned EDTA solution is less time-consuming (but more expensive).
Mix 500 ml of 0.5 M EDTA with 200 ml (or 200 g) of DMSO and add DW until a final volume of 1,000 ml.
Add a saturated amount of NaCl and stir well (precipitation of NaCl is not a problem).
Sorting
Sorting
Transfer the sample to a Petri dish. Pick up organisms from the collected material using an Irwin loop under a stereomicroscope and store in 2.0-ml screw-cap micro tubes of DESS solution. To prevent rusting, rinse the loop with DW after each use.
References: Shirayama (2004); Schram & Davison (2012)
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How to make an Irwin loop
Cut a stainless-steel wire (0.2–0.3 mm diameter) into 10 cm and 5 cm pieces.
Hook the pieces together and twist them using pliers, leaving small loops.
How to make an Irwin loop.
Remove the shorter piece and fix the longer piece to a suitable handle with water-resistant adhesive.
Bend the loop part to a manageable angle with pliers.
A completed Irwin loop, fixed to a disposable chopstick.
Citations
Step 10
Yoder M, Tandingan De Ley I, King IW, Mundo-Ocampo M, Mann J, Blaxter M, Poiras L, De Ley P. DESS: a versatile solution for preserving morphology and extractable DNA of nematodes
https://doi.org/10.1163/156854106778493448Step 11
Schram MD, Davison PG. Irwin loops—a history and method of constructing homemade loops
https://doi.org/10.1660/062.115.0106Step 3
Hulings NC, Gray JS. A manual for the study of meiofauna
https://doi.org/10.5479/si.00810282.78