Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Using mustard, Lumbricus terrestris earthworm populations can be sampled directly from soil depths without landscape disturbance or toxicity. Earthworms can then be counted for data and statistical analysis using a bar graph and student’s t-test.
Monitoring earthworm populations is a vital technique for environmental scientists, as multiple species of earthworms (most notably those from the suborder Lumbricina) have been invasively spreading throughout North America and South America. Exotic earthworms can be found on nearly every land mass and in nearly every ecosystem on the planet, and where and when these species become invasive has been a focus of international environmental research.1
Ecological invasion typically lowers biodiversity of an ecosystem by directly outcompeting, endangering, or otherwise contributing to the extirpation of native species. As ecosystem engineers, invasive earthworm species alter the cycling of nutrients through decomposition rates of organic matter on the upper horizons of soil, where plant roots mine for nutrients. Invasive Lumbricus species have both extirpated native earthworm species and have been shown to increase the available nitrogen concentration and rates of nitrogen in invaded soils.2 In a positive feedback loop, accelerated levels of nitrogen in turn make the system more hospitable to invasive plant species that are adapted to high levels of nitrogen compared to native plant species, and will outcompete natives in a phenomenon known as “invasion meltdown.” An invasion meltdown relationship has been proposed for invasive earthworm species Lumbricus terrestris (European earthworm) and an invasive plant species Rhamnus cathartica (European Buckthorn).3
A solution is prepared by extracting capsaicin from spicy mustard and then poured directly onto the soil within a sampling quadrat on the ground to sample from each collection site. Collection sites are determined in order to compare three random samples from an area that has been invaded by European buckthorn to three random samples from an uninvaded area. Once poured directly on the ground, the mustard solution can penetrate down through the soil matrix to where earthworms reside. The capsaicin in the mustard causes irritation to mucous membranes. Earthworm bodies exposed to the mustard solution react to the capsaicin irritation by moving away from the mustard solution and coming to the soil surface to expose themselves to oxygen, thereby reducing the irritation. After surfacing, earthworms can be collected and population density analyzed for relationships with European buckthorn. The population means of each collection site is compared with a bar graph to determine if areas with other invasive species have more earthworms, thereby supporting the presence of invasion meltdown. A Student’s T-test is used to determine if the two sites are significantly different enough to support the invasional meltdown hypothesis proposed to exist between European earthworm and European buckthorn.
1. Preparation of Mustard Concentrate Solution
- Turn on balance, place a weigh boat on top, and zero the balance.
- Weigh out 38.1 g of ground oriental mustard into weigh boat and transfer to a plastic container with cap.
- Measure 100 mL of tap water in a graduated cylinder and add to the plastic container with mustard.
- Secure cap on container and shake vigorously until all mustard is mixed off the bottom of the plastic container and dissolved into the tap water.
- Let solution sit for 24 h for maximum capsaicin extraction from mustard.
- Fill two 8 L water carriers halfway with tap water (approximately 4 L of water into each carrier).
- Shake mustard concentrate several times to mix then transfer mustard concentrate solution to water carrier.
- Transfer a small amount of the solution from the water carrier back into the concentrate container and shake vigorously. Pour back into the water carrier to transfer all of concentrate into the diluted solution.
- Seal the water carrier cap, ensure cap valve is in “OFF” position, and invert water carrier three times to mix evenly.
2. Extraction of Earthworms
- Label three sample cups for each collection site.
- Proceed to sampling site with one quadrat, labeled sampling cups with lids, and water carrier with diluted mustard solution.
- At the sampling site, clear away brush, leaves, or mulch as much as possible to clearly expose the ground.
- Place quadrat randomly on the ground in a cleared spot.
- Invert one water carrier three more times to mix.
- Turn the water carrier cap valve to the “ON” position and pour approximately a third (1.3 L) of the diluted mustard solution within the quadrat, concentrating on the center of the quadrat area. If soil becomes saturated and solution pools, stop pouring and wait until pooled solution infiltrates into soil before continuing to pour.
- Observe quadrat area closely for earthworm appearance for five minutes, including the area directly under sides of quadrat.
- Use forceps to collect all earthworms that appear in quadrate area by waiting for worms to completely emerge from ground before transferring to first sample cup. After five minutes lid the sample cup and proceed to next sampling site.
- Repeat collection steps for all sampling sites, with three replicates per collection site (6 replicates total)
3. Comparing Earthworm Population Density Between Collection Sites
- Count the number of earthworms collected for each sample and calculate the mean and standard deviation for each collection site.
- To compare earthworm densities between collection sites, create a bar graph from the means and use the standard deviations to create error bars on the graph.
The monitoring of earthworm populations is vital to environmental scientists, as invasive exotic earthworms can be found in nearly every ecosystem on the planet. Ecological invasion typically lowers biodiversity of an ecosystem by directly outcompeting, endangering, or contributing to the extirpation, or local extinction, of native species.
The Lumbricus terrestris species of European earthworm, also called the nightcrawler, is extremely common in North America, but is not native. As a result, it has greatly extirpated native earthworm species. Lumbricus terrestris alters the cycling of nutrients through decomposition of organic matter in the upper layers of soil, where plant roots mine for nutrients, thereby changing the soil layer structure. In addition, the organic debris layer, containing much of the decomposing material that provides nutrients, is completely lost.
These invasive worms also increase the available nitrogen concentration in invaded soils. In turn, the changing soil layers and high levels of nitrogen make the soil more hospitable to invasive plant species, such as the European Buckthorn, which are more adapted to high levels of nitrogen as compared to native plant species. This phenomenon is known as "invasional meltdown."
The invasional meltdown resulting from invasion of the European earthworm and exotic plants like the European buckthorn is of key concern because it is dramatically decreasing the diversity of forest plant life in North America.
This video will demonstrate the monitoring of European earthworms in various park areas in order to assess their vulnerability for buckthorn invasion.
To determine earthworm populations in invaded areas, worms are directly extracted from soil using a capsaicin solution.
In this experiment, capsaicin is extracted from spicy mustard and poured directly onto the soil in an area defined by a pre-sized square, or quadrat. It then penetrates through the soil matrix to where the earthworms reside.
The capsaicin solution causes irritation to mucous membranes in the earthworm. Earthworms react to the irritation by moving to the soil surface to escape the capsaicin solution. After surfacing, earthworms are collected and the population density analyzed.
The following experiment will demonstrate the extraction of earthworms from soil, and their population analysis.
First, prepare the capsaicin solution at least 24 h in advance by weighing 38 g of ground oriental hot mustard, and transferring it to a plastic container with a cap. Add 100 mL of tap water to the plastic container containing mustard. Secure a cap on the container, and shake vigorously until all of the mustard is dissolved in the water.
Let the solution sit for 24 h for maximum capsaicin extraction from the mustard. When the capsaicin extraction is complete, dilute the mustard solution with 4 L of water in an 8-L water carrier. Shake the mustard solution several times to mix, and transfer it into the water carrier. Rinse any residual mustard using the diluted solution.
Seal the water carrier cap, and ensure that the valve is in the "OFF" position. Invert the water carrier three times to mix evenly. Prepare one container of capsaicin solution for each testing site.
Proceed to the sampling site with a quadrat and the water carrier containing diluted mustard solution. Also bring three sampling cups per site. They should be labeled appropriately for three replicates per sampling site.
Place the quadrat randomly on the ground in a cleared spot. Clear away the brush, leaves, and mulch as much as possible to clearly expose the soil. Mix the dilute solution again, and then switch the cap valve to the ON position.
Pour approximately a third of the diluted mustard solution within the quadrat, concentrating the majority of the liquid at the center of the quadrat area. If the soil becomes saturated and forms pools, stop pouring, and wait until pooled solution infiltrates the soil before continuing.
Observe the quadrat area closely for 5 minutes, looking for earthworm appearance. Be sure to look directly under the sides of the quadrat.
Wait for all earthworms to emerge from the soil within the quadrat area, and then collect them with forceps. After 5 minutes, close the sample cup and proceed to the next sampling site.
Repeat the collection steps for all sampling sites. Return to each site and perform 3 replicates per site. Count the number of earthworms collected for each sample, and then calculate the mean and standard deviation for each collection site.
Create a bar graph to compare the average earthworm population densities between collection sites. Use the standard deviation to create the error bars. Site one is a managed park, and is therefore more hospitable to earthworm populations due to disturbances such as aeration and fertilizers. Site two is unmanaged, and is therefore less hospitable to earthworm populations.
Exotic earthworms and European buckthorn have been implicated as part of an "invasional meltdown" occurring, especially in the mid-western United States. Tracking earthworm populations can help to elucidate relationships between the two invasional species and enable researchers to develop methods to prevent further spreading.
You've just watched JoVE's introduction to the extraction and analysis of earthworm populations. You should now understand the principles of earthworm extraction from soil, and the comparison between sampling sites. Thanks for watching!
Sampling site 1 was a managed park, which sees significant disturbances such as aeration and fertilizers. Sampling site 2 was an unmanaged area, which sees no human interferences. As shown in Figure 1, site 1 has a higher density of earthworm populations, likely due to the increased hospitability due to human disturbances. However, site 1 also has higher variability of sampling, indicating the earthworm population may not be as consistently dense as the average suggests.
Figure 1. Bar graph displaying population results from each collection site.
Applications and Summary
Invasive species are a major threat to biodiversity. Exotic earthworms (eg: Lumbricus terrestris) and European buckthorn (Rhamnus cathartica) have been implicated as part of an “invasional meltdown” occurring in mid-western United States wooded communities. An invasional meltdown is the process where one invasion of a species facilitates the invasion of others. Thus, the rate of loss of ecological health can greatly accelerate as one invasive species makes way for additional ones. As undesired Rhamnus populations currently account for over 90% of vegetative cover in Illinois, the role of Lumbricus populations in landscape management has become critical to understanding and predicting Rhamnus invasion on managed land. Landscape disturbance tends to facilitate Lumbricus invasion and sampling for Lumbricus populations can be an indicator of vulnerability of land areas to likely invasion. Comparing samples of Lumbricus populations can help land management to know where more intensive methods are needed to maintain intended plant diversity and prevent invasion of Rhamnus.
- Belote, R.T., Jones, R.H. Tree leaf litter composition and nonnative earthworms influence plant invasion in experimental forest floor mesocosms. Biological Invasions. 11, 1045-1052 (2009).
- Costello, D.M., Lamberti, G.A. Non-native earthworms in riparian soils increase nitrogen flux into adjacent aquatic ecosystems. Oecologia. 158, 499-510 (2008).
- Nuzzo, V.A., Maerz, J.C., Blossey, B. Earthworm invasion as the driving force behind plant invasion and community change in northeastern north American forests. Conserve Biol.23, 4. 966-974 (2009).