Activity - Invisible Allies: Beneficial Nematodes

E&E IPM Standard: 7C, 10C, 12A
Skills: Observation, Scientific Method, Biocontrol

Introduction:
What goes on in the soil under your feet? Most people don't give it a thought - but if they looked closely, they would discover a fascinating world full of actors, all sizes and shapes, playing many roles. One of the most abundant soil creatures are tiny invertebrates, generally called roundworms or nematodes. They appear "thread-like" under the microscope, hence their name (nema = thread). The vast number of nematode species includes free-living and parasitic forms. The parasitic forms can infect plants, other invertebrates, birds and mammals including people. Those nematodes that infect insects, or entomopathogenic nematodes (ento = insect; pathogenic = causing disease) are the ones we will investigate further. These beneficial parasites are an important component to many IPM programs because they can be applied to the soil to help manage underground insect pests. Let's find out more.

How do the nematodes find and attack their "host" insects? The free-living stage of these nematodes is called an "infective juvenile" or IJ and it is just that-it is the immature stage of the nematode that is capable of infecting an insect. Inside their bodies, these nematodes carry special bacteria that they use to actually kill the insects (but don't worry, these bacteria aren't harmful to people or pets!) The nematode crawls through the insect's mouth, anus, or spiracles (breathing holes) and invades the insects blood cavity (hemocoel). There it releases the bacteria and the insect generally dies within 1 or 2 days. The nematodes then use the dead insect as a site for development and reproduction because the bacteria break the internal tissues down to a nutritious "soup." A couple of weeks after infection, thousands of new infective juveniles exit the dead insect in search of a new host. Thus, the biocontrol agent replicates itself in the soil environment.

There are many species of entomopathogenic nematodes, so to maximize their effectiveness, there should be a proper match between the nematode and the target insect. Some nematodes, referred to as "cruisers," actively move through the soil to locate an insect host. They work better against insects that are generally less active and that are found beneath the soil surface. The species of nematode that we will be working with today, Heterorhabditis bacteriophora, is a "cruiser" nematode. Other nematodes, called "ambushers," are more effective against insects that actively move over the surface of the soil. These nematodes sit and wait for a host instead of actively searching for one in the soil.

In this experiment, the question of nematode infection against different insect stages will be investigated. Nematode infection of mealworm beetle larvae vs. mealworm adults will be compared. This activity is designed to investigate one of many factors that might affect the effectiveness of entomopathogenic nematodes, and the use of biological control agents in general, in IPM systems.

Learning objectives:
1. Learn what entomopathogenic nematodes are and understand their role in IPM.
2. Investigate how insect stage (larvae vs adults) might influence nematode infection.
3. Understand that entomopathogenic nematodes are living organisms and thus should not be dealt with like insecticides.

Materials needed:

• entomopathogenic nematodes
• mealworm larvae and adults
• soil
• plastic containers with lids,
• pipettes
• forceps

Timeline:
30 minutes to set up the experiment; 3 days to run the experiment; 30 minutes to check the experiment at the end.

Procedure:
1. Prepare 4 cups of soil. Fill each cup with 50 ml moistened soil (about halfway full).

2. Add insects to the soil cups. Two of the cups will get 5 mealworm larvae each. The other two cups will each get 5 adults.

3. Add nematodes to one cup of larvae and one cup of adults. The goal is to use about 100 nematodes per insect. We will be using 5 insects per cup, so in this exercise, 500 nematodes should be added to each cup. The nematodes have already been measured out for you with approximately 500 nematodes in each ml of stock solution. Therefore, you should add 1 ml of the nematode stock solution to each cup. Before adding the nematodes, be sure to shake the vial to thoroughly mix the solution-if not, the nematodes will all settle to the bottom of the vial.

4. The other two cups will serve as controls. These will receive 1 ml water only (without nematodes). Be sure to label your cups so that you know which containers received nematodes and which received water only. You should also pierce a hole in each lid because the nematodes, like all aerobic organisms, need oxygen to breathe.

5. It takes about three days to observe results, so for the remainder of today's exercise, we will be using previously prepared cups to simulate the experiment. If you were doing this experiment with a class, the cups should be stored in a dark area (a cabinet drawer is fine) at room temperature for the duration of the study.

Analysis:
1. Inspect each of the soil containers. Count and record the number of dead insects. You may have to dig through the soil with your forceps to find all of the insects. Be on the lookout for nematode-infected mealworm larvae. They will be dead and light red in color (the red pigment is produced by the bacteria). Mealworms dying from natural causes or other pathogens will not appear that way. Adult mealworms that have died from nematode infections will not appear red in color (their cuticle is not as transparent).

• Number dead
• Number alive

Nematodes + mealworm larvae

Nematodes + mealworm adults

Control mealworm larvae

Control mealworm adults

2. Calculate the percentage of dead insects in each treatment. We will pool the data as a class and compare treatments.
Was the mortality in the nematode treatments greater than the controls?
Were the nematodes equally able to infect larvae and adult mealworms?
What are possible reasons if there are differences? If there are not differences?

3. For optional exercises with your class, you could have students compare nematode infection to different insects such wax worms, crickets, aphids, or white grubs. You could also experiment with different concentrations of nematodes, different exposure temperatures, or different soil moistures. All of these factors interact when using biological control organisms.

Discussion:
Why is it important to have a good match between nematodes and their hosts? In other words, why is it important to know about the habits and life cycles of both the insect and nematodes? What other factors might influence the effectiveness of biological control organisms? How does the use of biological control organisms differ from the use of pesticides?

Resources:
A great website about nematodes is: http://www2.oardc.ohio-state.edu/nematodes/
The site discusses the use of these organisms in IPM, has many cool photos, and has links to nematode suppliers. If in PA, you can contact Lane Loya at Saint Francis University, Department of Biology, Loretto, PA 15940 for nematodes.